Tdt magazine

Tumors of Digestive Tract The Underexposed Facts and Unresolved Questions Edition 1, volume 1, February 2018

In this issue The heterogenecity of pancreatic cancer

The gut microbiome and colorectal cancer

Diet and gastrointestinal cancer

Editor-in-Chief: F. Doubrava-Simmer Assistant-editor: T. Bisseling

Foods or supplements?

Tumors of Digestive Tract

Contents Volume 1 Issue 1 | TDT Feb 2018

The Underexposed Facts and Unresolved Questions Edition 1, volume 1, February 2018

In this issue The heterogenecity of pancreatic cancer

The gut microbiome and colorectal cancer

Diet and gastrointestinal cancer

Editor-in-Chief: F. Doubrava-Simmer Assistant-editor: T. Bisseling

Editor-in-chief Femke Doubrava-Simmer

Assistant Editor Tanya Bisseling

Associate Editors Erik Aarntzen Annemarie Boleij Manon van den Berg Alina Vrieling

Editorial Office Radboudumc Department of Pathology

P.O. Box 9101 6500 HB Nijmegen Internal post 824 Geert Grooteplein Zuid 10 The Netherlands T +31 (0) 24 3614361

Pancreatic Cancer Targeting the tumor-stroma in pancreatic ductal adenocarcinomato improve therapy 2 outcomes D. Draper, B. Vervoort, T. van Wessel

The influence of obesity on the development and prognosis of pancreatic adenocarcinoma 8 Emma Kuiper, Matthijs Snelders, Anouk Stoffels

Immunotherapy as a novel treatment of pancreatic cancer 12 Alex Geerlings, Dylan Jongerius and Jens Jacobs

Biomarkers: The Next Opportunity in Pancreatic Cancer Research?

17 R.M.E. Janssen, J. Lankhof & M. Muller

Treatment of extracellular matrix of pancreatic ductal adenocarcinoma (PDAC) with the vitamin D analog Nab-MART-10 to decrease desmoplasia 22 Gina Gerhorst, Christina Hahnen, Annemarijn Offens

Gut Microbiome The potential role of probiotics in the prevention of colorectal cancer 28 Alex Geerlings, Dylan Jongerius and Jens Jacobs

Microbiome screening for the detection of colorectal cancer 33 D. Draper, B. Vervoort, T. van Wessel

The gut microbiome and colorectal cancer: aspirin as a preventive measure?

38 Emma Kuiper, Matthijs Snelders, Anouk Stoffels

Therapeutic Applications Targeting the Microbiome in the Prevention of Microbiota-Driven Carcinogenesis 46 R.M.E. Janssen, J. Lankhof & M. Muller

Evaluating the amount of Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis and enteropathogenic Escherichia coli in the gut microbiome prior to colorectal cancer 52 development Gina Gerhorst, Christina Hahnen, Annemarijn Offens

Diet and Gastrointestinal Cancer Prevention of gastrointestinal cancers through the diet 57 R.M.E. Janssen, J. Lankhof & M. Muller

Comparison of the use of early enteral nutrition and parenteral nutrition in esophageal 62 carcinoma Gina Gerhorst, Christina Hahnen, Annemarijn Offens

The benefits of nutrition in palliative care: should we care?

67 Emma Kuiper, Matthijs Snelders, Anouk Stoffels

Nutrition and/or physical activity as an addition to current cancer treatment?

71 Alex Geerlings, Dylan Jongerius and Jens Jacobs

Recommended dietary pattern to reduce the risk on colorectal cancer 77 D. Draper, B. Vervoort, T. van Wessel

Targeting the tumor-stroma in pancreatic ductal adenocarcinoma to improve therapy outcomes D. Draper1, B. Vervoort1, T. van Wessel1 Abstract In pancreatic ductal adenocarcinoma the 1 year survival is low with 15%, and the 5 years survival is even negligible. A dense desmoplastic cell mass, called the stroma, is causing 90% of the drugs to fail from tumor entry and thus cure. This concerns a lot of research departments, which are searching for novel treatment strategies to stabilize metastatic pancreatic carcinomas and improve therapeutic outcome. Different strategies, such as targeting SPARC protein with nab-paclitaxel,CD40 immunotherapy, targeting the hedgehog pathway, are explained in this article. These strategies aim to target the stroma of the tumor in combination with a chemotherapeutic agent. However, in theory this strategy is promising, but in trials, only several months extra overall survival was found. Better drugs need to be developed to improve overall survival drastically. Biomarkers should be identified to see whether a tumor with a specific mutation respond to a treatment or not, so that the best treatment option can be provided for each patient.

Introduction Due to increasing knowledge and the development of new treatment strategies incidence rates of a lot of cancer sites are decreasing. Although, the prevalence of pancreatic cancers is still increasing with 40.000 estimated death cases compared to an incidence of 46.000 cases in the US. Additionally, there is a 1 year survival rate around 15% and a 5 year survival rate that is even negligible (1, 2). As the primary tumor usually develops without early symptoms, most patients have already metastatic disease during initial diagnosis and especially within this subgroup treatment options are limited (3). The treatment regimen nowadays includes radiotherapy or chemotherapy. In patients with advanced disease chemotherapeutic treatment with gemcitabine in combination with additional chemotherapeutic agents has had the greatest impact on survival rates. However, improvements in survival outcomes remain only modest (4, 5). For these reasons, novel treatment strategies are of great importance to stabilize metastatic pancreatic carcinomas and improve therapy outcome.

Over 90% of pancreatic cancers are ductal adenocarcinomas (PDAC) which are histologically characterized by a dense desmoplastic cell mass surrounding the pancreatic tumor cells, also called the stroma (6-8). The stroma is responsible for the main tumor bulk and consists of highly proliferative fibrotic tissue composed of extracellular matrix, proteins, blood vessels, fibroblasts and immune-inflammatory

cells. It is thought that this key characteristic of PDAC functions as a mechanical barriers and prevents the delivery of chemotherapeutics to the tumor. A blockade of the tumor blood vessels and inordinately high interstitial fluid pressures limits the perfusion and diffusion of therapeutic agents to the pancreatic tumor cells (9). This could clarify why more than 90% of exciting cancer drugs fail in the treatment of PDAC and that even the effective chemotherapeutic gemcitabine has a very low response rates less than 12% (9, 10). Additionally, the stroma is also thought to be involved in tumor formation, progression and cancer stem cell maintenance (11-13).

Targeting the stroma could be a promising strategy to enhance the sensitivity of pancreatic tumors to existing chemotherapies.

Here we review the recently developed strategies that affect the stroma in PDAC and discuss the most promising therapies.

Treatment techniques Nab-paclitaxel targets a specific stromal protein A promising strategy to affect the pancreatic tumor stroma is by targeting the SPARC (secreted protein acidic and rich in cysteine) protein that is expressed in approximately 80% of all PDAC (14) As SPARC is highly and specifically expressed in the tumor-stroma and only reduced expression is

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found in the epithelial pancreatic tumor cells this could be a promising target. The exact role of SPARC in tumor progression is unknown, however it is suggested that SPARC regulates several cellular processes including migration, adhesion, proliferation and survival (14, 15).

The recently developed drug Nabpaclitaxel is a cytotoxic agent that specifically targets this SPARC protein. Paclitaxel is a cytotoxic nanoparticle that can easily be transported and up taken by the pancreatic tumor cells as it forms a complex with the water soluble plasma protein albumin. Albumin itself has a high affinity for SPARC and therefore the albuminpaclitaxel complex (Nab-paclitaxel) can very efficiently and specifically target the pancreatic stromal cells (12).

Preclinical data already indicated that combination treatment of nab-paclitaxel with the already used chemotherapeutic gemcitabine results in depletion of the stroma but also effects the pancreatic tumor by enhancing tumor vascularization followed by increasing gemcitabine concentrations in the pancreatic tumor cells (16).

Higher concentrations of gemcitabine results in increased toxicity and therefore in tumor regression. Additionally, results of phase I, II and ongoing phase III clinical study suggested that the co-treatment of both drugs resulted in a high level of anticancer activity that significantly improved the overall survival from 6.9 to 11.3 months (1618).

Hedgehog expression and stroma development Another pathway that is being extensively studied for targeting pancreatic cancer is the Hedgehog Signaling in which the ligand Sonic Hedgehog (SHH) plays an important role. This pathway plays a key role in the development of the embryo and several structures such as the pancreas, but also the neural tube for example(19). It has been suggested that this pathway is extra stimulated in pancreatic cancer by the overexpression of SHH and stimulates stroma formation which therefore might be an interesting target for treating pancreatic cancer(20). In a study from Thayer et al. they found that upon histochemical staining for SHH that this ligand was expressed in 70% of the samples(21). The SHH functions by binding to the receptor patched (PTC), that normally inhibits the function of a protein called smoothened (SMO)

which is located in the cell membrane(22). Upon binding with SHH, PTC is inhibited and therefore SMO is activated which in turn leads to the activation of Gli transcription factors. The presence of these factors induces cell proliferation and promotes cell survival of stromal cells.

In one study from Rhim et al. researchers used a mouse model for pancreatic cancer (called PKCY model) in which they deleted the expression of SHH to investigate the presence of this ligand in pancreatic cancer(23). They observed against expectations that pancreatic tumors were present in both the PKCY model and the PKCY model with the SHH deletion. It was a striking result however that mice that did not express SHH even had a significantly shorter survival and a higher rate of metastasis development when compared to the PKCY model.

Additionally, histology showed that there were less stromal cells present in the mouse model without SHH and more blood vessels, which could possibly explain why these tumors developed more metastases.

In another study by Olive et al. researchers investigated a combination therapy of gemcitabine, a chemotherapeutic agent, and the drug IP-926, a drug that interferes with the SHH pathway, in another pancreatic ductal adenocarcinoma mouse model called KPC compared with three other mouse models where a tumor was transplanted in the mice(20). First, they investigated the effect of gemcitabine alone compared with saline in the KPC model and they found that the pancreatic tumors grew at a similar speed as the saline-treated mice in the KPC while a decreasing effect was observed within the gemcitabine-treated mice with a transplanted tumor. Next, they investigated the vasculature of these pancreatic tumors by staining the blood vessels with a CD31 antibody. They found that the tumors of the KPC mice had poorly perfused tumors compared with the transplanted tumors.

This was also investigated by other visualization techniques, including high resolution contrast ultrasound. Another difference they found between the histological coupes of the transplanted tumors and of the tumors of the KPC mice was that there was more distance to the blood vessels and the cancer cells due to the presence of stroma. Lastly, they investigated the combination therapy of IP926 and gemcitabine and they found that the

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tumors of KPC mice showed less stroma formation and increased perfusion. Drug delivery of gemcitabine was more effective with coadministration of IP-926 since the amount of metabolites from gemcitabine was higher and this increased the median survival from 11 to 25 days for the KPC mice.

CD40 immunotherapy CD40 is a member of the tumor necrosis factor family and is expressed on B cells, macrophages, dendritic cells, endothelial and epithelial cells (24).

Mice lacking CD40 cells showed decreased angiogenesis, leading to an impairment of tumor development (25). In the progression of pancreatic cancer stroma formation, a lot of different cell types are involved (26). In the early stage, leukocytes, fibroblasts and macrophages accumulate and together with collagen deposition they form the main components of the stroma. In first attempts to target leukocytes with CD40 mAb, mouse models with induced pancreatic cancer were sacrificed during different timepoints to analyse the contribution of these cells. These mouse models gave a representative insight in how the stroma components are regulated in human pancreatic cancer (27).

In the stroma, especially tumorassociated macrophages, which present CD40 were found (24). Furthermore, prominent infiltration of immunosuppressive cells such as myeloid-derived suppressor cells, and regulatory T cells (CD40 positive cells) were observed, while effector cells were rarely present. IL-10 might play a role in immunosuppression, since it is upregulated in these patients and has immunosuppressive capacities (28).

When CD40 mAb was administered as therapy, mainly macrophages were turned tumoricidal and reacted against the stroma of the tumors (29). This activation can reverse immunosuppression and may thereby be a key regulator in antitumor responses. Macrophages treated with CD40 mAbs were capable of lysing tumor cells in vitro, which was also found in vivo.

CD40 treatment initiated degradation of the tumor and stroma (30). However, for human trials, there are concerns that CD40 monoclonal antibodies may have a significant toxic response or promote tumor growth (25). Nevertheless, when clinical experiments were performed with a combination of CD40 mAb and gemcitabine mild to moderate side

effects were found (29). 4 out of 21 patients benefits partially, 11 patients had a stable disease state and in 4 patients the disease progressed.

The median progression free survival increased from 5.6 months to 7.4 months.

Photodynamic therapy A newly developed promising strategy is photodynamic therapy (PDT) which is an approach that uses a light activatable chemical, called a photosensitizer (31). Light has to be emitted in a specific wavelength to the pancreatic stroma to activate this molecule and induce cytotoxicity through production of reactive oxygen species. Hereby, cells will be cleared via necrosis or through the apoptotic pathway, which is mainly stimulated (31-33). Thus, tissue surrounding the tumor will disappear.

In usual tumor applications, the photosensitizer requires time to accumulate inside the tumor, where then the photosensitizer will be stimulated with light (34). Usually, photosensitizers are coupled to antibodies or other targeting molecules, to target the specific area of interest. In this case, fibroblasts from the stroma of PDAC are targeted (32). This treatment is usually combined with a chemotherapeutic agent (35). The agent itself was not capable to pave its way into the stroma, but by targeting fibroblasts the chemotherapeutics can reach the pancreatic tumor cells.

An advantage of this technique is that photosensitizer molecules themselves are not harmful so distribution throughout the body has no negative impact at all on healthy tissue. One drawback of PDT is that patients will suffer from heavily pain during treatment (36). Several studies have been trying to control this side effect but only a few effective methods were found. However, when PDT is used for pancreatic cancer treatment specifically, the pain that arises appears only mild to moderate (37).

PDT has a positive impact on the drug resistance of gemcitabine. In vitro studies show that combination therapy of PDT and gemcitabine destroys tumor cells effectively and in vivo, combination therapy has a synergistic effect on destroying tumor cells (38). Overall, PDT might be an effective therapy in the treatment of pancreatic cancers. More preclinical and clinical research should be performed, and different therapy combinations should be tested to discover the best

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suitable treatment regimen for patients suffering from PDAC in the future.

Concluding remarks and future perspectives Although the stroma seems to be an obstacle for treatment delivery and has an effect on the tumor formation, it was also found that the stromal tissue has some sort of protective role, since anti-stromal treatment in mice increased the risk of developing metastases. This means that targeting the stroma, present as a treatment for pancreatic cancer, is not enough to improve survival. However, combination therapies of anti-stromal treatment with a chemotherapeutic agent might be the solution, because it increases the likelihood that the treatment will arrive at the site of interest. In this way, the chemotherapeutic agent will be more effective and a successful treatment outcome will be more likely.

However, in theory this strategy is promising, but in trials, only several months extra

overall survival was found. For example CD40 mAb treatment stimulated macrophages and showed promising results in vitro, but in vivo, overall survival was prolonged from 5.6 to 7.4 months. Still, the 5 years survival of patients with pancreatic ductal adenocarcinoma remains very low.

Strategies need to be developed to identify responders from non-responders in the treatment of pancreatic cancer because there is not sufficient time for these patients to try different options. This can be done by comparing different treatments that tackle desmoplasia formation in combination with gemcitabine in different mouse models for pancreatic cancer. For this, researchers could use different mouse models in which the phenotype of the tumors differ from each other to assess if tumors with a certain mutation might respond better to a certain treatment. In this way, biomarkers that could distinguish a non-responder from a responder can be identified and the best treatment option can be provided to the patient.

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The influence of obesity on the development and prognosis of pancreatic adenocarcinoma Emma Kuiper, Matthijs Snelders, Anouk Stoffels

ABSTRACT Introduction: Pancreatic adenocarcinoma (PDAC) is one of the less known and less common cancers.

Even though some risk factors, such as obesity, are known, the overall survival remains poor with only 9% in five years.

Aim: Investigate the influence of obesity on the development and prognosis of PDAC. Results: In the development of PDAC, body fat shows evidence of being able to induce proliferation in pancreatic intraepithelial neoplasia (PanIn) and PDAC cells. Also, adipocytes express proteins like TNF-Îą that induce insulin resistance and inflammation which may be an important promoting factor in cancer development. The possible underlying mechanisms for the influence of obesity on the prognosis are: altered levels of oestrogens and insulin, low grade chronic inflammation and higher weight loss in obese PDAC patients.

Conclusion: Obesity increases the risk of developing PDAC and possible decreases the survival in PDAC patients. However, more research is needed to use this knowledge for prevention, diagnosis or treatment purposes.

Introduction

Pancreatic adenocarcinoma (PDAC) is one of the less known and less common cancers. It contributes for only 2% of all cancer diagnosis in the Netherlands, men and women combined (1). There are a few common risk factors for the development of pancreatic cancer known, such as smoking and alcohol consumption, chronic pancreatitis, pancreatic cancer in the family, diabetes mellitus and obesity (2). Even though these risk factors are acknowledged, it is still difficult to diagnose pancreatic cancers early. A possible reason could be the vague symptoms (i.e. development of diabetes, inflammation of the pancreas, nausea, losing weight and distorted defecation). Due to this prolonged diagnosis phase, cancerous tumours are often in a more advanced stage (i.e. size, metastases) and therefore result in a 5-year survival of only 9% (1).

As said above, obesity is one of the acknowledged risk factors for developing pancreatic cancer. It is a common and worldwide increasing problem. According to the World Health Organisation are 39% of adults (>18 years) overweight (3). Besides causing health related problems (i.e. diabetes, cardiovascular diseases and musculoskeletal disorders) is obesity known to be associated with a number of different cancers, such as:

colon, breast, endometrium, oesophagus and kidney cancer (4). In 2007, the World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) considered that body fatness resulted in an increased risk on pancreatic cancer (5). In short, the increased risk of developing cancer due to obesity can be explained by the circulating endogenous hormones (i.e. insulin and insulin-like growth factors), which can distort the balance between cell proliferation and apoptosis (6).

But also mechanisms including changes in the metabolism of adipokines, localised inflammation, oxidative stress, altered immune response and hypertension can contribute to this increased risk (7). Not only has obesity an effect on the development of PDAC, it might also affect the treatment outcome and therefore the prognosis.

Treatments used for pancreatic cancer are surgery, radiation therapy and chemotherapy (and other drug therapies) (8). However, surgery is often complicated due to the anatomy surrounding the pancreas, which can cause an unresectable tumour (9).

With this study, we aim to combine literature on association between obesity and PDAC to give an overview about the recent knowledge, mainly focussing on the development and prognosis of PDAC and the influence of obesity on it. With combining the knowledge we hope to help with generating a

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better prevention and diagnosis of PDAC and more efficient and successful treatments so the survival of PDAC patients will increase.

The development of PDAC and obesity The pancreas consists of three sections: the head, body and tail. There are two cell types present: endocrine cells, which express hormones into the bloodstream, and exocrine cells, which produce pancreatic enzymes into the pancreatic duct. Pancreatic cancers are in 95% adenocarcinomas of the exocrine cells (10).

There are clear indications that obesity plays a big role in the development of PDAC. Data from the Pancreatic Cancer Cohort Consortium (PanScan) has been studied to determine the association between BMI and pancreatic cancer risk (10). The results were significant: the group of patients with the highest BMI had on average a 33% increased risk of pancreatic cancer compared to the lowest group. The combination of high body fat, increased exposure to carcinogens in foods and reduced physical activity are said to play a role in obesity-mediated PDAC development (10). Several assumptions were made about the underlying mechanisms that could explain the association between obesity

and an increased development of the disease, the most prominently examined being body fat. Body fat, also called adipose tissue, shows evidence of inducing proliferation in pancreatic intraepithelial neoplasia (PanIn) and PDAC cells (11). Meyer et al. has shown that murine adipocytes were able to secrete glutamine to rescue PanIn and PDAC cells in a low-nutrient environment, subsequently increasing proliferation.

Apart from proliferative effects, adipocytes express proteins like TNF-Îą that induce insulin resistance and inflammation (12). A schematic representation of this process can be seen in figure 1.

Meyer et al. concluded that this may just be an important promoting factor in PDAC development, as the tumour microenvironment of PDAC is known to be low on nutrient because of dense desmoplasia; fibroblasts surround the tumour in a dense layer that lowers blood and nutrient supply, as well as drug delivery and immune cell recruitment (11). Adipose tissue also has altering effects on its surroundings. In fact, it is an endocrine organ which can release hormones, cytokines and chemokines. Insulin resistance and inflammation can be the result of adipocyte activity, promoting tumour growth. It is therefore said that new-onset of diabetes mellitus can be an early sign of PC (13).

Figure 1. Adipocyte-secreted proteins involved in inducing insulin resistance and inflammation.

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The prognosis of PDAC and obesity As mentioned in the previous section, it is proven that the BMI affects the development of pancreatic cancer. However, the studies about the influence of BMI on the survival rate of pancreatic cancer show contradictory results. Some studies did not find a significant correlation between BMI and survival in PDAC patients. Like Jiang et al. who performed a retrospective analysis to evaluate the association of BMI on the overall survival of PDAC patients and concluded that the survival is not affected by the BMI (14). In addition, Gong et al. used a multivariable cox proportional hazards model to measure the association between pre-diagnostic obesity and pancreatic cancer survival (15). He did also not find a statistically significant difference between the survival in obese and normal BMI patients (15). Another retrospective analysis, which was conducted

by Dandona et al, investigated patients with pancreatic adenocarcinoma who underwent a pancreaticoduodenectomy (16). He also did conclude that obesity has no impact on overall survival in these patients (16).

In contradiction, other studies do report an association between a higher BMI and decreased survival rate in pancreatic cancer patients. McWilliams reported an association between a high BMI and decreased survival in pancreatic cancer using a cox proportional hazard survival analysis (17).

Other studies like Li et al. and Hassan et al. confirmed this (18, 19).

The underlying mechanism that could explain a possible correlation between obesity and the prognosis of pancreatic cancer is still unknown. There are some hypotheses mentioned in literature but none is proven yet. The first possibility is the altered levels of oestrogens and insulin in obese people.

McWilliams et al. investigated the hyperinsulinemia in the pancreatic and obese patients but it did not explain the effect of BMI on the survival (17). In addition, the survival rate of men and women do not differ significantly, therefore the oestrogens are not expected to be the reason as well (17). The second hypothesis is the low grade chronic

inflammation which is caused by obesity. This could result in a shift towards a Th2 immune state, which can cause carcinogenesis and cancer progression. The low grade chronic inflammation can therefore lead to a decreased immune function, which can result in a more rapid tumour progression and therefore a poorer survival (20). A third possibility could be a bias in the treatment determinants, like aggressiveness and doses of the therapy (19). A fourth and last hypothesis is the higher weight loss in obese PDAC patients in comparison to normal BMI PDAC patients. Weight loss is common in PDAC patient, but obese patients show a higher amount of weight loss. This is depicted in a reduction of skeletal muscle and visceral adipose tissue, which might contribute to a poorer survival of pancreatic cancer patients (21).

Discussion

Obesity has an influence on the development and possible also the prognosis of PDAC. The prognosis of PDAC patients is poor due to late diagnosis, propensity to metastasize and its resistance to radiation and chemotherapy due to desmoplasia. In addition, is it hard to operate the pancreas because it is located close to important veins, arteries and organs.

It is known that obesity is a major risk factor for developing PDAC and probably also has a negative influence on the survival. Therefore, it is important that more insight is given into the influences and mechanisms of obesity on PDAC to develop better therapeutic strategies to prevent, diagnose or treat patients with PDAC. In this article we have combined literature to give an overview of the association between obesity and PDAC. Obesity increases the risk of developing PDAC due to an increase in chronic inflammation and insulin resistance caused by the high amount of adipose tissue. The mechanisms for a possible association between obesity and the survival are not studied sufficiently. There are some possible mechanisms proposed: elevated levels of oestrogens, insulin resistance, chronic low grade inflammation and bias in treatment determinants. But more

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studies are needed to further investigate these mechanisms.

Even though there this is an ongoing topic, not enough is known how to increase

the survival in PDAC patients. Therefore, it is important that more studies are performed so we can decrease the risk of developing PDAC and increase the prognosis.

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Immunotherapy as a novel treatment of pancreatic cancer

By Alex Geerlings, Dylan Jongerius and Jens Jacobs

ABSTRACT Pancreatic cancer is one of the most lethal types of cancer which shows a high metastatic tendency, often a severe degree of dissemination at the time of diagnosis and significant resistance to treatment. Only a few treatment options are available. The efficacy of these therapies is suboptimal however, especially in comparison with the treatment options that are available for other types of cancer. As such, only small improvements have been made in improving the survival rates of pancreatic cancer patients. Current research is taking place into the development of therapies that target the microenvironment of the tumour in order to decrease the therapeutic resistance. Several studies have shown promising results in trials. In this review, we discuss the latest advances that have been made in the field of immunotherapy that target the microenvironment, and the therapeutic potential it may have in the fight against pancreatic cancer.

INTRODUCTION Pancreatic cancer is an aggressive and often lethal type of cancer. It is known to be very invasive, heterogeneous in genetic make-up, to have a high metastatic tendency, as well as being fairly resistant to current treatment options. It is being diagnosed approximately 2300 times in Netherlands annually, which accounts for 2% of all cancer diagnoses in the Netherlands. However, the current chances of survival are very slim in the case of a resectable tumour: 5-yr overall survival (OS) = 23%, non-resectable tumour or metastases 5-yr OS = 6%1. For many years, gemcitabine has been the first-line treatment for pancreatic ductal adenocarcinoma (PDAC). The effectiveness of gemcitabine as a chemotherapy is often criticised.

Newer chemotherapies like FOLFIROX and nab-paclitaxel are however, hardly more effective than gemcitabine2-4. The 5-yr survival rates for pancreatic cancer have only increased with a few percent since chemotherapy was introduced. This is why there is an increased interest in other types of therapies such as immunotherapy. This article intends to give a brief overview of the immunotherapies that are currently researched and/or applied in clinical practice and tries to evaluate their effectiveness as a potential new treatment strategy for pancreatic cancer.

IMMUNOTHERAPY IN THE FIGHT AGAINST PANCREATIC CANCER Immune checkpoint inhibitors The main focus of immunotherapy is the use of the hostâ&#x20AC;&#x2122;s own immune system in order to target the tumour. This includes presentation of tumour antigens by the major histocompatibility complex (MHC) to the T-cells. In the normal immune response this is regulated by immune checkpoints, which regulate the release of costimulatory molecules that can either inhibit or stimulate the immune response. These are pivotal in self-tolerance and maintaining immune homeostasis. However, immune checkpoints are

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activated by the cancer microenvironment in order to suppress the anti-tumour immune response.

Because these immune checkpoints are often initiated by ligand-receptor interactions, they can be targeted by antibodies, making it a viable target for novel therapeutic strategies.

Anti-CTLA-4 One of these checkpoints is CTLA-4. It has inducible expression on CD4+ and CD8+ cells and regulates early stages of T-cell activation. CTLA-4 binds to CD80 and CD86 on APC. This causes the displacement of costimulation, inhibiting the activation of effector T-cells and activating regulatory T-cells5. This ultimately leads to reduced proliferation of T-cells6.

The anti-CTLA-4 drug ipilimumab has already been approved for metastatic melanoma. Due to the increased regulatory and effector T-cells in the tumour microenvironment in pancreatic cancer, clinical trials have been done to assess the effectiveness of anti-CTLA-4 in pancreatic cancer as well. In a phase 2 trial ipilimumab was administered in locally advanced or metastasized pancreatic patients. Effectiveness of monotherapy was insignificant but it did delay the response7. In another study they compared the effectiveness of monotherapy with ipilimumab and combination therapy of ipilimumab with a GM-CSF vaccine8. Combination therapy showed an increased overall median survival of 5.7 months and a one year survival of 27% versus 3.6 months survival and a 7% one year survival in monotherapy. Additionally, there are many ongoing studies investigating the effectiveness of combination therapy with CTLA-4 inhibitors (e.g. ipilimumab + Gemcitabine, ipilimumab + imatinib).

Anti-PD-1/PD-L1 Programmed cell death 1 (PD-1) is a co-inhibitory molecule expressed on CD4+ and CD8+ T-cells, regulatory t cells, B-cells, NK cells, monocytes and dendritic cells. When it bound to PD-1 ligand (PD-L1), it inhibits proliferation of T-cells and cytokine release. Chronic antigen exposure in cancer can lead to overexpression of PD-L1 on cancer cells. This results in local downregulation of the immune response. In pancreatic cancer, increased expression of PD-L1 is correlated with worse prognosis. Knockdown of PDL1 by shRNA also inhibited proliferation of pancreatic ductal adenocarcinoma cells9.

Monotherapy with anti-PD-1/PD-L1 drugs has not been found effective in pancreatic cancer. However, there are many ongoing studies that investigate the effectiveness of combination therapy. In an ongoing study they tested nivolumab (anti-PD-1) in combination with dendritic cell vaccines in seven stage IV pancreatic cancer patients. At the time of abstract submission, they found partial remission in two patients with an OS of 13 and 5 months. Additional ongoing anti-PD-1/PD-L1 studies are dual checkpoint inhibitor studies; nivolumab + GM-CSF vaccine; nivolumab + nab-paclitaxel + gemcitabine; pembrolizumab + tyrosine kinase inhibitors10.

Therapeutic vaccine immunotherapy In addition to the use of immune checkpoint inhibitors as a therapeutic option in the treatment of pancreatic cancer, various vaccine immunotherapies are also actively in development. The principle of vaccine immunotherapy is similar to the passive immunity that fights infections. Such vaccines are designed in such a way that they trigger an immune response against a tumour by the administration of certain antigens that are derived from the tumour tissue itself, and generally do not match with antigens in any other tissue of the body. Various different types of vaccine immunotherapies have been developed; these include whole-cell vaccines, dendritic cell (DC) vaccines, DNA vaccines and finally peptide vaccines11.

Whole-cell vaccines Whole-cell vaccines use the entire tumour cells that have a wide range of antigens and actively express epitopes which can be presented to CD8+ and CD4+ T cells. In some cases, allogeneic cell lines are used 2

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instead of autologous cell lines when it proves to be very difficult and/or dangerous to harvest the tumour tissue. Various different types of whole-cell vaccines are currently researched. Algenpantucel-L is a vaccine which has allogenic pancreatic cancer cells that express alpha-1,3-galactosyl transferase which is an enzyme that is required for the synthesis if alpha-galactosyl epitopes12. These specific epitopes are not found in humans, but they do have antibodies against these epitopes which are continuously produced in response to bacteria that are present in the intestines. This type of vaccine exploits this acquired immunity, which leads to an enhanced antitumour response. One open-label, single-arm phase II study (n=70) of algenpantucel-L in combination with gemcitabine and 5-fluorouracil found that the one-year disease free survival was 63% and an overall survival of 86% in comparison with historic controls that found 45% and 65% respectively13.

GVAX GVAX is an allogenic cancer vaccine that is comprised of whole tumour cells that have been genetically altered to induce the secretion of an immune stimulatory cytokine called granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF has been shown to stimulate stem cells to produce monocytes and granulocytes which promote the destruction of cancerous tissue14. It simultaneously delivers antigens that are associated with the tumour to increase the recognition of cancerous cells by the immune system. In addition, it has also been shown to cause an influx of bone-marrow derived dendritic cells that bind and present the tumour specific antigens to CD4+ and CD8+ T cells. In one phase 1 clinical trial, GVAX was used before as well as after chemoradiation therapy in resectable pancreatic cancer (n=14)15. A mean disease free survival of 13 months was found. As these results were found to be relatively favorable, researchers continued to determine the therapeutic potential of GVAX in combination with cyclophosphamide in pancreatic cancer patients that showed no optimal response to gemcitabine therapy. Unfortunately, no significant difference was found in overall survival and 1-year survival between control cohort which only had GVAX and the cohort that was given the combination therapy.

Dendritic cell vaccine Dendritic cells (DC) are known to be a very potent antigen-presenting cell (APC), their main function is to process and present antigens, with MHC, to activate cytolytic as well as regulatory T-cell response16. These DC characteristics are used to develop new vaccines, with their main goal to mount an antitumour response. These vaccines are designed to present tumour-associated antigens (TAAs) like peptides, cDNA, mRNA or fused autologous DC with tumour cells17. DC vaccines are being tested as a standalone treatment, as well as combination treatment with chemotherapy gemcitabine and with chemokine modulatory regimens (CKM). A promising DC based immunotherapy are the recombinant bacterial vaccines, like for instance listeria monocytogenes (LM) immunotherapy.

Recombinant bacterial vaccine and LM immunotherapy LM is a bacterial agent which acts as delivery systems for TAAs, the bacteria are actively phagocytosed by APCs, by macrophages for instance. LM immunotherapy uses weakened LM bacteria to act as a vaccine vector to deliver TAAs to the APC and thereby evoke an immune response.

Although preclinical studies have shown promising results, the development of the vaccine is still in the earlier stages, most clinical studies are currently in phase I or II18,19. The most recent completed phase II study of Le et al., assessed the safety and survival of CRS-207, which is a specific weakened LM vaccine, in combination with GVAX and low-dose cyclophosphamide (CY) for 90 metastatic pancreatic cancer patients20. This GVAX-CY combination inhibits regulatory T-cells and helps the vaccine to induce the innate as well as the adaptive immunity. The OS in this study was 9.7 months with an improvement of 2.2 months (5.6%) when compared with the current first-line therapy, gemcitabine plus nab-paclitaxel.

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CONCLUSION Various positive results have been found in studies that researched the efficacy of immunotherapy in the treatment of pancreatic cancer. In most cases, the combination of a certain type of immunotherapy with a conventional anti-cancer treatment showed an improvement in anti-tumour response. The combination of various immunotherapeutic strategies has also been shown to give favorable outcomes with clinical benefits in some cases. There is a lack of large clinical trials that test the combination of immune checkpoint inhibitors with vaccines and/or other newer personalized therapies however. As pancreatic cancer is still one of the most lethal cancers in current clinical practice with a low degree of overall survival in comparison with other types of cancer, more research is needed in order to find therapeutic strategies that are more effective in slowing down and ultimately eradicating pancreatic cancer tissue.

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Le, Dung T. et al. “Evaluation of Ipilimumab in Combination with Allogeneic Pancreatic Tumor Cells Transfected with a GM-CSF Gene in Previously Treated Pancreatic Cancer.” Journal of immunotherapy (Hagerstown, Md. : 1997) 36.7 (2013): 382–389. PMC. Web. 7 Feb. 2018.

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Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancerspecific antigens and induce antitumor immunity against autologous tumor cells. Koido S, Hara E, Homma S, Torii A, Toyama Y, Kawahara H, Watanabe M, Yanaga K, Fujise K, Tajiri H, Gong J, Toda G. Clin Cancer Res. 2005 Nov 1; 11(21):7891-900.

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Hardacre JM, Mulcahy M, Small W, et al. Addition of algenpantucel-L immunotherapy to standard adjuvant therapy for pancreatic cancer: a phase 2 study. J Gastrointest Surg. 2013;17(1):94-100.

Thomas AM, Santarsiero LM, Lutz ER, et al. Mesothelin-specific CD8(+) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients. J Exp Med. 2004;200(3):297-306.

Jaffee EM, Hruban RH, Biedrzycki B, et al. Novel allogeneic granulocyte-macrophage colonystimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation. J Clin Oncol. 2001;19(1):145-56.

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Biomarkers: The Next Opportunity in Pancreatic Cancer Research?

R.M.E. Janssen, J. Lankhof & M. Muller

Abstract INTRODUCTION. Even after years of research, survival amongst pancreatic cancer patients has not improved significantly. There is a clear need for methods that have the potential to early detect pancreatic cancer to improve the alarmingly poor prognosis. Biomarkers might offer a solution, however biomarkers that are currently available do not meet de requirements. This review aims to assess other possibly potential biomarkers for pancreatic cancer.

RESULTS. The only currently approved biomarker is CA 19-9. However, its sensitivity and specificity vary widely (45-90%). Another possible biomarker is cystatin 1, which was found to have elevated levels in both pancreatic cancer tissue and blood. Not only is research being conducted on blood-derived biomarkers, thousands of cell-bound proteins are being discovered as well. Not only proteins, but also circulating microRNAs can serve as diagnostic biomarkers.

Several microRNAs have been proven to be elevated in blood of patients, with specificities and sensitivities in the range of 60 to 90%.

DISCUSSION. Both CA 19-9 and cystatin 1 might be useful biomarkers for pancreatic cancer, but both are not ideal. A promising set of biomarkers could potentially be found in circulating microRNAs. Even though current tests do not have staggering sensitivities and specificities, these are likely to be increased in the future through combination of existing or discovery of new microRNAs. CONCLUSION. As none of the biomarker discussed seemed to be ideal for the early detection of pancreatic cancer, one should further investigate novel potential biomarkers. A panel, consisting of multiple biomarkers, might be a suitable option for early detection of pancreatic cancer. Combination of biomarkers increase the sensitivity and specificity of the biomarkers.

However, practical and financial feasibility of such a panel should be investigated first.

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1. Introduction Pancreatic cancer is one of the deadliest cancers worldwide and continues to be a major unresolved health problem (1). The prognosis is the worst of any major malignancy, with an average 5-year survival rate of only 3% (2). Even after years of thorough research, not a single treatment approach has been proven to be effective (1). The main problem with this type of cancer is that there are usually no symptoms present in the early stages. Symptoms that are specific enough to indicate pancreatic cancer generally do not develop until the cancer has reached an advanced stage (3). Besides that, pancreatic cancer is often already metastasized at the time of diagnosis (4).

Both the poor prognosis and late appearance of pancreatic cancer in most patients highlight the need for the development and improvement of early detection methods (5).

Nowadays, lots of research is done on biomarkers. According to the NIH Biomarker Working Group, a biomarker is â&#x20AC;&#x2DC;a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic interventionâ&#x20AC;&#x2122; (6).

For pancreatic cancer, the development of simple and non-invasive tests that indicate cancer risks, provide early cancer detection, predict treatment response and monitor disease progression, regression and recurrence are important (7). Unfortunately, biomarkers that are currently available for pancreatic cancer detection in clinical practice lack the sensitivity and specificity required to do this sufficiently (8). The aim of this review is therefore to determine if other potential biomarkers are available for the early diagnosis, prognosis and treatment of pancreatic cancer in clinical practice.

2. Potential biomarkers 2.1 CA 19-9 The only currently approved biomarker for pancreatic cancer is CA 19-9, which is derived from blood serum and it routinely used. CA 19-9 is short for carbohydrate antigen or cancer antigen 19-9 and it is a sialylated Lewis antigen. Even though this biomarker is frequently used, the opinions in literature about the effectiveness of CA 19-9 differs greatly (8). Ballehaninna & Chamberlain (2013) stated that the sensitivity of this biomarker lies between 44 and 90% and the specificity between 45 and 88%. They also found false positive results in 10 to 30% of the patients, which were suffering from another pancreatic disease (9).

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2.2 Cystatin 1 Cystatin 1 (CST1) is currently used as a biomarker for diagnosis of gastric, esophageal and colorectal cancers. CST1 is an inhibitor of cysteine proteases and might also have a value as a diagnostic biomarker for pancreatic cancer. Data from Jiang et al. (2015) revealed that CST1 expression was upregulated in pancreatic cancer tissue in comparison with normal pancreatic tissue. The problem is that translation from isolated tissue to patients is not trivial. Even if the similar result were found in patients, the retrieval of pancreatic tissue is highly invasive (10).

However, it is also showed that the CST1 protein can be detected in serum, and these levels were significantly elevated in pancreatic cancer patients (10).

2.3 Cell surface bound proteins and secreted proteins In 2009, Harsha et al. performed a major systematic review regarding all genes and proteins that were found to be overexpressed in pancreatic cancer. In their review, they provided a list with proteins which are detectable on the cell surface of pancreatic cells or are secreted. This provides candidate biomarkers for early detection of pancreatic cancer, as well as clinical targets and imaging targets when expressed at the cell surface. An advantage of biomarkers that are secreted, is that they can easily be measured in urine, serum or even saliva. Harsha et al. found 930 genes encoding proteins that are excreted through body fluids with up-regulated expression. 567 genes encoded proteins that are present on the plasma membrane and also overexpressed in pancreatic cancer. In total, at most 1497 genes are potential biomarkers for pancreatic cancer, which is of course way too many to describe in this article. However, this list might provide a useful list of biomarkers to be tested for reliability in the future, especially the ones that are excreted, since these biomarkers can be obtained from patients in a noninvasive manner (11).

2.4 MicroRNA MicroRNAs (miRNAs) in the blood can serve as biomarkers. These miRNAs are often released into the circulation by tumor cells, and could therefore be indicative of the presence of pancreatic cancer. Furthermore, because they are present in the blood, testing for elevation of miRNA levels is non-invasive (12).

In a recent study of Wang et al (2017), the expression levels of a number of miRNAs in the blood of pancreatic cancer patients were compared to the levels in healthy controls. Here, it was determined that several miRNAs (miR-155, miR-187, miR-210, miR-221 and miR-223) were significantly elevated in the patientsâ&#x20AC;&#x2122; plasma (13). A study into the diagnostic value of 2

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plasma levels of miR-155, miR-210 and additional miRNAs (miR-21 and miR-196a) concluded that this combination of miRNAs has a specificity of 64% and a sensitivity of 89% (14).

Another study, however, showed that miR-21 alone had a specificity of 77% and a sensitivity of 79% (15). Kawaguchi et al. (2013) assessed the value of the ratio between miR-221 and miR-375 plasma levels, and concluded that the specificity and sensitivity of this approach were 78 and 74%, respectively (16).

3. Discussion and Conclusion To assess the possibilities for new potential biomarkers, the biomarkers found will be critically evaluated. The first biomarker mentioned was the already approved CA 19-9. The functionality of this biomarker is doubted and multiple studies were not able to show significant elevated levels of CA 19-9 in pancreatic cancer patients, compared to healthy controls. The sensitivity and specificity rates of this biomarker vary enormously, from which can be concluded that this is not a reliable biomarker to detect pancreatic cancer (9).

CTS1, the second biomarker we discussed, was proven to be elevated in serum of pancreatic cancer patients. This biomarker could thus be measured non-invasively. However, in this study, the researchers were not able to detect elevated levels for early stage patients, one of the main requirements of a potential biomarker. Besides that, this biomarker seems to be also elevated in other types of cancer, lowering the specificity for pancreatic cancer (17-19).

Consequently, CST1 as a biomarker to detect pancreatic cancer is not very specific in general, but might give an indication for closer examination of the patient.

MiRNAs are also considered as potential biomarkers. As miRNA levels can be detected in the blood, this biomarker can be measured non-invasively. A major disadvantage of miRNA, however, is the low specificity and sensitivity (12). If the specificity and sensitivity will be improved, this could be a suitable set of biomarkers to determine early stage pancreatic cancer.

As none of the biomarker discussed seemed to be ideal for the early detection of pancreatic cancer, one should further investigate novel potential biomarkers. One option might be to create a panel of multiple markers, to increase the sensitivity and specificity to detect, predict and eventually treat pancreatic cancer. Multiple genes, proteins and miRNAs could be included (11). Additional research should be done to determine the practical and economic feasibility of testing for biomarker levels with a panel of multiple markers in pancreatic cancer

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Treatment of extracellular matrix of pancreatic ductal adenocarcinoma (PDAC) with the vitamin D analog Nab-MART-10 to decrease desmoplasia Gina Gerhorst, Christina Hahnen, Annemarijn Offens

Abstract This research proposal presents a potential new method for handling the desmoplasia and drug inefficiency in pancreatic ductal adenocarcinoma (PDAC). Desmoplasia decreases drug efficacy in pancreatic cancer. Vitamin D is known to reduce fibrotic reactions. We will test a vitamin D analog bound to albumin-coated nanoparticles, Nab-MART-10, which is expected to result in a more selective delivery of vitamin D to the tumor site. The effect of Nab-MART-10 on vitamin D receptor (VDR) activation and on tumor desmoplasia will be measured in vitro and in vivo, respectively. The expected results are that Nab-MART-10 will still be able to bind the Vitamin D receptor and induce anti-fibrotic effects, which will result in decreased tumor desmoplasia when administered to mice.

The research proposal presented here has profound implications for future studies on PDAC and may result in a treatment that ameliorates.

Keywords: Pancreatic ductal adenocarcinoma (PDAC), Vitamin D analog, Nab-MART-10, Vitamin D receptor (VDR)

Introduction Pancreatic ductal adenocarcinoma (PDAC) are tumors with a high mortality. The 5-year survival is less than 6% since the tumor presents itself in a late state Society (1). Besides, the symptoms of the tumor are diverse and not very specific such as abdominal pain which can have a lot of different causes. Further on, since the pancreatic tumor grows in the abdominal cavity, there is enough space for the tumors to grow before leading to a medical condition. Often, the tumor is not detected until there are already metastases present. The late presentation of the tumor, present metastases and no efficient therapy currently available, together lead to a bad prognosis.

Currently, a relatively effective therapy is a combination of nanoparticle-bound albumin-paclitaxel (Nab-paclitaxel) combined with gemcitabine (2). Gemcitabine is a nucleoside analog, which blocks the DNA replication (3). Paclitaxel achieves a cell cycle arrest in the G2/M phase, leading to apoptosis of the cell (4). Nab-paclitaxel consists of nanoparticles of paclitaxel coated with albumin (5). Albumin binds to SPARC (6), which is increasingly expressed in several tumor types, including PDAC (7). Also, Nab-paclitaxel crosses the endothelium more efficiently due to transcytosis by endothelial cells (8), and albumin is thought to be taken up by tumors as a source of nutrients (9). Therefore, the nanoparticles are taken up more and contained better in pancreatic tumor tissue compared to the regular drug. The mechanism of Nab-paclitaxel is visible in figure 1.

However, the efficacy of this treatment and other treatment is limited by desmoplasia, which causes a decreased drug delivery into the tumor and activates pathways that limit the effect of chemotherapy (10). Desmoplasia plays an important role in tumors of pancreatic cells. The stroma of the tumor gets a more complex structure (11). Typically, there is an altered expression, organization and/or post-translational modification of various components of the stroma such as collagen,

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laminin, fibronectin, hyaluronic acids and molecular growth factors (12-15). Various cell types are involved in PDAC desmoplasia, such as endothelial cells forming blood vessels, immune cells, cancer stem cells, pancreatic stellate cells (PSCs), neural cells and cancer-associated fibroblasts (CAFs) (15).

Figure 1. Transcytosis by endothelial cells and subsequent binding of albumin to SPARC, which is increasingly expressed in tumor tissue.

When paclitaxel dissociates from albumin, it diffuses into the surrounding cells and exerts its effects. From Cortes and Saura, 2010 (5).

All these components together result in a dense stroma and a lowered microvascularity. This is a huge problem for an efficient drug delivery to the tumor.

To overcome the drug delivery problems and the inefficiency of the drugs in vivo, it could therefore be useful to use a substance that can decrease desmoplasia, which could be vitamin D. Not much information is available regarding the mechanism of vitamin D specifically in desmoplasia caused by cancer, but its mechanism has been investigated in several fibrotic conditions induced by TGFβ-1, which is also an important mediator in cancer-associated desmoplasia. In mice, vitamin D supplementation decreases intestinal fibrosis (16). Also, vitamin D inhibits TGFβ-1 induced upregulation of Collagen I, α-smooth muscle actin and other markers of fibrosis in lung and colonic fibroblasts (17). It was found that the inhibiting effect of vitamin D on the effects of TGFβ-1 is mediated by its effect on members of the SMAD family, which are profibrotic transcriptional regulators, activated following TGFβ-1 receptor activation (18). Two proposed mechanisms for this inhibition exist, described in a review by Shany et al. (18) which are genomic competition and direct binding of phosphorylated SMAD. Many regions in the DNA contain SMAD binding sites and TGFβ-1 binding sites in close proximity to each other. The effect of this could be that binding of these site by VDR blocks binding of SMAD, preventing it from activating gene transcription. However, it was also shown that direct binding of the VDR to phosphorylated (activated) SMAD3 by the VDR receptor can inhibit SMAD3 induced gene expression.

There are several other reasons that make vitamin D a good candidate for future combination therapies. Vitamin D has been shown to sensitize pancreatic cancer cells to treatment with gemcitabine (19), although the opposite was also found: in one study, cells with a lowered number of VDRs were more sensitive to gemcitabine resulting from impaired DNA damage repair (20). It also is known to have an anti-proliferative activity on tumor cells. VDR signaling leads to a binding to the expression of critical cell cycle regulators giving a negative effect on the regulation of the cell cycle progression (21). An antiproliferative effect via cell cycle arrest at the G0/G1 can be achieved by the action of VDR (21).

However, a problem of using vitamin D can be that it results in hypercalcemia, since the expression of genes essential for bone mineralization and calcium homeostasis are regulated by the binding of

23

Vitamin D to the VDR (22). Therefore, it would be better to use an analog that causes less hypercalcemia, and use a method that increases delivery to the tumor site to reduce off target effects. MART-10 is a vitamin D analog that is less calcemic (23). In addition, it is a more potent activator of the VDR receptor than vitamin D (23, 24). Since the Nab-paclitaxel method increases targeted delivery to the tumor site, we aim at developing a Nab-MART-10 formula, to increase MART-10 delivery to the tumor.

Therefore, we hypothesize that MART-10 is more effective than vitamin D in activating the VDR receptor and counteracting profibrotic effects, that MART-10 will be more potent in reducing desmoplasia than vitamin D and that Nab-MART-10 particles will be more potent than Nab-vitamin D. Experimental setup The study will be divided in two experiments. First an in vitro experiment will be performed with human PDAC fibroblasts. The purpose of this experiment will be to compare potency of MART-10 to activate the VDR receptor, and if the Nab-bound versions retain their VDR-activating ability. Cells will be treated with Mart-10 as well as vitamin D (calcitriol), both in standard form as well as in a Nabform. As a control situation neither MART-10 nor vitamin D will be added. Various concentrations will be tested, i.e. 1µM, 5µM and 10µM of (Nab-)MART-10 or (Nab-)Vitamin D in the culture medium for 24-48-72 h, similar to a previous study (25). To examine VDR receptor activation in the different conditions, the fibroblasts will be transfected with pVDRE-Luc one day prior to the experiment.

Luciferase will be produced when the VDR is activated, making the amount of produced light by luciferase a measure of VDR activation. Based on the receptor activation, a concentration will be chosen that sufficiently activates the VDR receptor, which will be used for further studies. Here, TGFβ-1 will be added to the cell culture with or without the different forms of vitamin D (or analog).

Fibrosis-inhibiting effects will be assessed by examining the expression of Collagen I and α-smooth muscle actin, which are induced by TGFβ-1 and increased in desmoplasia. Based on a previous report (23, 24), we expect that MART-10 will be a more potent VDR activator than vitamin D. We also expect that it will retain its VDR activating ability and its ability to inhibit TGFβ-1-induced fibrotic proteins. Should this be the case, we will proceed to step 2, the purpose of which is to examine the efficacy of Nab-MART-10 to decrease PDAC desmoplasia in a mouse model.

For the second experiments, nude mice will be used that will be injected with BxPC-3 cells (human primary pancreatic adenocarcinoma cells) to develop pancreatic cancer (23). After 4 weeks tumor size and composition of the tumor will be determined by using MRI and mice will be divided into 4 groups. Per group 10 female and 10 male mice will be needed. All groups will be treated 2 times per week for 4 weeks. The first group will receive intraperitoneal vitamin D (calcitriol) injections in a dose of 0.3 µg/kg, a dose used in a comparable study (23). The second group will receive Nab-vitamin D with a dose that contains an equal number of vitamin D-molecules as the dose of vitamin D, and the third group will receive Nab-MART-10 in an dose equal to Nab-vitamin D. A control group will receive placebo injections. The control group will be used to determine the normal tumor growth without medication. The experimental set up can be seen in figure 2. To display a change in tumor size and a change in the composition of the tumor we will image the tumor once a week using MRI. A T2weighted image should image inhomogeneity within the malignant tumor whereas we expect a homogenous signal in a T1-weighted image (26). The T1-weighted image will be done using a contrast enhancing agent such as Gadolinium to strengthen the signal of the lesion (27). A diffusionweighted image can be used to image the diffusion restrictions within the tumor and the

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organization of the mass. During the procedure the mice will be under anesthesia to reduce the stress for the mice and the motion of the object which will lead to artefacts in the images. The calcium levels and a possibly induced hypercalcemia will be checked once a week by blood sampling.

At the end of week 4, mice will be euthanized and the tumor tissue will be histologically examined for desmoplasia of the matrix and possible inflammations as a result of an immune response. The expectation is that the desmoplasia of the matrix will be reduced due to the treatment with MART10 and vitamin D and that recruited cells will be found, clearing up the dead cells.

The obtained data will be analyzed statistically to see whether the three treatment conditions differ significantly in their results. To examine this, 2-sided t-test with an Îą of 0.05 will be performed. Our expectation is that Nab-MART-10 will be more effective in reducing tumor desmoplasia than standard MART-10, since it is expected to have a higher delivery in the tumor, which should make it more effective at a similar systemically administered dose. Also, we expect a lower calcium-related toxicity compared to vitamin D, based on previous reports comparing MART-10 with vitamin D.

Figure 2: experimental setup of animal experiments on the effect of vitamin D, Nab-vitamin D and Nab-

MART-10 on desmoplasia in PDAC

Discussion The present study aims to answer the question whether PDAC cells will take up Nab-MART-10 and Nab-Vitamin D significantly more than fibroblasts, whether this will lead to Vitamin D receptor activation, and whether the receptor activation will be higher if cells were treated with Nab-MART-10 in comparison Nab-Vitamin D treatment. Furthermore, it will be addressed if there will be a significant reduction in desmoplasia if mice were treated with Nab-MART-10 compared to treatment with Nab-vitamin D and to untreated mice.

If this study shows, that treatment with Nab-MART-10 will result in a significant reduction of tumor dysplasia, chemotherapeutics in future could better reach the tumor following Nab-MART-10 treatment. In a future experiment, mice with pancreatic cancer xenografts could first be treated with Nab-MART-10 to reduce tumor desmoplasia and could after this be treated with the chemotherapeutic nab-paclitaxel-gemcitabine. Similar to the method in our proposed nab-MART10 study, the tumor could be imaged by MRI to display a change in tumor size and a change in the composition of the tumor. Mice could be examined for metastasis using a F-FDG PET/CT scan. The used fluorodeoxyglucose (FDG) is a glucose analog and is labeled with Fluor. Since tumors have an enhanced uptake of glucose, the tumor regions will appear brighter on the scan due to an accumulation of F-FDG within the tumor cells enhancing the signal.

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Limitations A first problem that could occur is that the Nab-MART-10 or Nab-vitamin D particles cannot be prepared. Since Nab-particles are described to be an option for hydrophobic molecules and vitamin D

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is hydrophobic, we think that it is an option but it has not been tried before. Another limitation is that we do not look directly at vitamin-D/MART-10 delivery in various organs in the mouse study.

This could be done by performing mass spectrometry of the organs, including the pancreas and the pancreatic tumor, and determining the relative levels of vitamin D/MART-10, as has been previously done to examine the Nab-paclitaxel delivery. However, we also want to use the pancreatic tumor for histology, so we would need extra mice to perform mass spectrometry of the pancreatic tumor. We have weighed the advantages and disadvantages and decided not to evaluate the delivery directly, but indirectly, since an increased delivery to the pancreas would likely result in an increased effect when the compounds are administered in a comparable dose.

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The potential role of probiotics in the prevention of colorectal cancer

By Alex Geerlings, Dylan Jongerius and Jens Jacobs Abstract Colorectal cancer is the third most common type of cancer worldwide. With the main risk factors including diet and obesity, this type of cancer is more prevalent in first world countries than in developing ones. Some studies suggest that the link between diet and colorectal cancer may be caused by a disturbance of the intestinal microflora. Probiotics are products that contain bacteria that are thought to give a healthy benefit to the host. Several in vivo and molecular studies have shown that probiotics have a preventive effect on the formation of colorectal cancer. This review aims to summarize the main findings that have been made about the preventive role that probiotics may have. In addition, several possible mechanisms will be discussed.

Introduction Colorectal cancer (CRC) is the most common malignancy of cells that occupy the gastrointestinal (GI) tract36. The incidence of CRC is 4 times higher in first world countries than in developing ones37.

This is thought to be mainly because of differences in diet and lifestyle and not due to racial differences. A wide range of sources supports the assumption that there is a link between diet and CRC. For instance, the consumption of red meat has been found to be associated with an increased risk for CRC development38.

There is also evidence that CRC may be a result of dysbiosis in the GI tract39.

Normally, the gut is colonised by a wide range of bacterial species that each have their own characteristics. Their specific location is determined by local conditions such as pH and oxygen supply. Most of the colonic microflora consists of anaerobic bacteria such as Clostridium spp. and others40. Probiotic bacteria are bacteria that are thought to have a health benefit to the patient in case they are administered in sufficient amounts. Lactic acid bacteria (LAB) are most commonly used as probiotics and are present in many types of dairy products41. Some evidence has been found that suggests that the consumption of probiotics has a beneficial effect in the treatment of a wide

variety of disorders which include gastroenteritis and constipation42. More recent evidence however also suggest that probiotics may have a preventive role in the carcinogenesis of CRC43. As CRC is still a big health problem, we intend to provide an overview of the evidence that is available about the role that probiotics may have in preventing CRC. In addition, we will discuss the possible mechanisms by which probiotics may exert their CRC suppressing effect(s).

Mechanistic perspective The exact mechanism of how probiotics act in the prevention of CRC is still largely unknown. However, varies mechanism are suggested, namely; intestinal microflora alterations, carcinogenic compound inactivation, competition with putrefactive and pathogenic microbiota, host immune response improvements and the effects on tyrosine kinase signaling pathways. All these suggested mechanisms will be discussed within this report.

Intestinal microflora alterations The intestines of the human body can be a breeding-ground for several potentially carcinogenic substances. For example, the deconjugation of glucuronides by the b-glucuronidase bacteria can lead to the release of, the potential carcinogenic substance, aglycones1.

Other fecal

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bacterial enzymes, like; nitroreductase and azoreductase can also catalyze the release of pro-carcinogenic substances in the intestine2,3. The alteration the intestinal metabolism, by modulating the activity of these bacterial enzymes, is expected to be a possible mechanism by which probiotics can reduce the risk on CRC4.

In mice with colon cancer it is already shown that feeding of yogurt can reduce levels of nitroreductase and b5 glucuronidase . Goldin ad Gorbach also showed in animal models a decrease in fecal bacterial enzyme activity after the feeding with l. acidophilus4. These authors did such a study also in humans, 21 healthy participants were exposed to l. acidophilus supplements for ten days, the enzyme activities of b-glucuronidase, azoreductase and nitroreductase were monitored6. After ten days significant decreases in all the fecal enzyme activities were seen, after four weeks the fecal enzyme levels returned to normal. The later suggest that a continuous intake of the probiotic supplement is needed to maintain the enzyme reduction effects.

However, discordant results are displayed by other human probiotic supplementation studies7-11. Differences in effects within different strains of l. acidophilus were shown, which may explain some of the discordant results. These findings suggest that strain-specific probiotics may be capable after continues intake to modulate fecal enzyme bacteria activity. However, more and strain-specific studies are needed to further clarify the potential relation of probiotics on intestinal microflora alterations in the prevention of CRC. Carcinogenic compound inactivation Individuals with a higher consumption of red meat have a relative risk of 1.28 for developing CRC compared to individuals with lower consumption of red meat12. It is believed that this is caused by the formation of heterocyclic aromatic amines (HCA) that are formed when red meat is cooked. Intestinal microbiota can activate HCA into carcinogenic compounds13,14.

Lactic acid bacteria (LAB) along with other commensal bacteria have the ability to bind or metabolize known carcinogens like

HCA and N-nitroso compounds. Research has shown that some strains of LAB were able to bind some of the known mutagens formed from cooked red meat15-19.The carcinogenic compounds mostly bind to the cell wall of the bacteria and become inactive.

Other bacteria, like the Lactobacillus plantarum, have extracellular glycoproteins to bind the mutagens20.

Challa et al. found that the B. Longum together with lactulose increases the activity of colonic glutathione Stransferase21.

This enzyme usually detoxifies toxic and carcinogenic metabolites in the liver. Lactobacillus casei has been shown to thrive in an environment containing HCA metabolites and decreasing their concentrations, provided that the pH and other physicochemical conditions are right22.

These examples show the ability of LAB to antagonize the onset of CRC. Competition with putrefactive and pathogenic microbiota The gut flora consist of benign and pathogenic bacteria. Pathogenic bacteria regarding CRC include: bile salt-producing bacteria, sulfate-reducing bacteria and putrefactive intestinal microbiota23-25. While LAB have been found to have cancerpreventing abilities. Rafter et al. found that a combination of specific oligofructoseenriched inulin with probiotics increases the number of LAB in polyp and cancer patients and decreased the number of certain pathogenic bacteria (Clostridium perfringens) in polyp patients26. Oâ&#x20AC;&#x2122;Mahony studied the effect of probiotica on IL-10 deficient mice, which are known to develop enterocolitis and CRC in the presence of enteric bacterial flora27,28.

Modification of the flora with Lactobacillus salivarius resulted in a decreased prevalence of CRC. This gives an indication that the probiotics may counter CRC development by competing with the pathogenic bacteria and letting the benign bacteria thrive.

Host immune response improvements The immune response is a pivotal defence mechanism against cancer. It has been suggested that probiotics can play and important role in the enhancement of the

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intestinal immune system29. Yokura et al saw that the Lactobacillus casei Shirota (LcS) has potent anti-tumor effects in mice30. There was no anti-tumor effect In vitro, so upon further investigation they found that LcS incorporated into microfold cells (M cells)31. Here itâ&#x20AC;&#x2122;s phagocytized by macrophages and dendritic cells which produce several cytokines, but mainly TNF-Îą. Other components of LcS are presented through TLR-2 in APCs which also produce cytokines32. The exact mechanism of how this leads to an tumor preventing effect is not yet known. One theory is that NK cells get activated, which play a role in immune surveillance33.

Another theory is the activation of Th1 cells which inhibit incidence of tumors34.

Other probiotics that possibly can increase the immune response are the L. acidophilus, L. casei and B. longum35. Itâ&#x20AC;&#x2122;s shown that these probiotica can increase the survival of mice injected with tumor cells. This survival was correlated with an increase in immune cells (CD8+ T-cells, NK cells and APCs). These examples suggest that probiotics have the ability to activate the immune response that reduce cancer incidence.

Effects on tyrosine kinase signaling pathways Cells communicate with their environments through a series of biochemical events that are called signaling pathways.

This includes interactions with other cells, but also with bacteria for instance. One group of pathways that play an essential role in the regulation of cell proliferation are the tyrosine kinase signaling pathways44.

Partly for this reason, they are often targeted in the latest anti-cancer therapies.

One probiotic agent that is currently used to treat human GI disorders is Saccharomyces boulardii or Sb. Sb has

been found to modulate signaling pathways, including several tyrosine kinase pathways, that regulate the inflammatory response of the intestinal mucosa45. One if the pathways is the MAPK-signaling pathway which is located downstream of many growth-factor receptors, which play an important role in carcinogenesis. One study found that the administration of Sb increased apoptosis, reduced cell proliferation that was induced

by EGFR, and prevented formation of cancer cells in an Apc-knockout mouse model46. In addition, a different study found that Bacillus Polyfermenticus suppressed the growth of colon cancer cells both in vitro and in vivo47. In short, the suppressing effect on tyrosine kinase signaling pathways may add to the other mechanisms to prevent the formation of CRC. Conclusion The supporting role of probiotics in CRC has become a widely researched topic in recent years. Although interesting findings have been made, conclusive clinical evidence in the supporting role of probiotics in CRC are still lacking. CRC is simply not an easy practical endpoint to determine in probiotic intervention studies.

Intermediate biomarkers of cancer or preneoplastic lesions are more practical and are thus mostly used in these studies.

There are most likely several mechanisms involved in the supporting role of probiotics in the prevention of CRC. The best known mechanisms have been discussed in this paper. These current findings are in deficient to conclude which mechanisms are most effective. It is likely that distinct strains of certain probiotics act with specific mechanisms. Further research is needed to determine the impact of all the mechanisms involved in probiotic use to CRC prevention.

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Microbiome screening for the detection of colorectal cancer D. Draper 1 , B. Vervoort 1 , T. van Wessel 1

Abstract: Colorectal cancer (CRC) is a prevalent type of cancer occurring in Western countries and is the second most deadly type of cancer. Over the years, several tests have been developed for screening for CRC, such as the fecal immunochemical test (FIT) which tests for the presence of human blood in a stool sample. Disadvantages of this technique are that only bleeding and thus advanced lesions can be detected and that the sensitivity is not high. A new technique that has potential to be used in the future is screening for microbiota present in the gut, since several bacteria have been identified to be more prominently present in the gut of patients with CRC compared to patients without CRC. In this review we will look into the specific bacteria that have been know to be correlated with CRC and what hurdles still need to tackled before it is possible to imply microbiome screening for detection of CRC.

Introduction Colorectal cancer (CRC) is one of the most prevalent cancers occurring in the United States: the incidence between 2009 and 2013 was 40.7 per 100.000 citizens(1). It is also the second most common cancer causing death with a mortality rate of 14.8 per 100.000 citizens between 2010 and 2014(1). Several risk factors have been identified over the years, which include a high BMI, smoking, alcohol consumption and low physical activity and a combination of these factors increases the risk for CRC(2).

To decrease the mortality and increase the detection of CRC, several screening methods have been developed over the years. First of all, two tests are available to detect blood in a stool sample, namely the guaiac fecal occult blood (G-FOBT) test and the newer fecal immunochemical test (FIT). Both these tests pose the advantages that they are low in costs, are non-invasive and have a high specificity (higher than 95%)(3). In one study, researchers compared FIT with G-FOBT and they discussed several differences between the two tests (4). One of these differences is that the FIT has the advantage that it is specific for human blood and G-FOBT not, and therefore FIT does not require adaptations in food intake. It was found that the sensitivity of

these tests is not very high, namely 61% for FIT and 23.8% for G-FOBT(3). This means that not hardly all the patients with CRC will be detected by this test. This could possibly be due to the disadvantage of these tests that they only detect bleeding advanced lesions, while small early-stage lesions will remain undetected. DNA analysis of the stool sample is also possible, however this test is not used much since it is very costly and therefore not cost-effective.

Other invasive techniques exist for detecting CRC, namely flexible sigmoidoscopy and colonoscopy. In the first test, flexible sigmoidoscopy, the distal part of the colon is checked for abnormalities, while in colonoscopy the whole colon is examined(5).

Sigmoidoscopy has the advantage over colonoscopy that it is safer and quicker.

However, abnormalities solely located in the proximal colon remain undetected with sigmoidoscopy: the sensitivity is 75-85% in men and 45-55% in women. This difference is due to the fact that women are more likely to develop cancer in the proximal part of the colon compared to men. The colonoscopy is more thorough than sigmoidoscopy, but is not preferred by the participants due to its invasive nature.

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Ideally, a new test with high sensitivity numbers and that will not be invasive should be developed. For example, a new test in which a stool sample is examined, but is more sensitive than the current tests. In this review we will focus on microbiota and specifically microbiota that can serve as a biomarker for CRC.

Microbiota The human microbiota consist of approximately 100 trillion microbial symbionts. These provide a main role in nutrition, resistance of pathogens and have a role in the immune system (6, 7). In the last decade, it has become more popular to use sequencing methods to characterize the microbiome in humans (8). Biologists found thousands of bacterial species in different compositions, unique to each body site. It is stated that the microbiome has a significant role in human related health and disease.

Especially the diversity of the microbes is an important factor in diseases. For example, low diversity of microbes is associated with obesity and inflammatory bowel diseases (9, 10).

The microbiome also appeared to be of great importance in the development of CRC (11).

Recently, more interest was gained in researching the microbiome as potential factor that could play a part in colorectal cancer. Animal studies have shown that the microbiome interacts with the immune system. Due to this reaction, cancerassociated metabolites can be produced and genotoxic virulent factors can directly promote development of CRC (12). Also in human studies it was found that bacteria produce carcinogens, cocarcinogens and procarcinogens (13). Also, differences in structure of the microbiome were found in patients compared to healthy controls (12, 14).

(11). The study of Moore and Moore (1995) compared healthy adults with 18 patients which recovered from surgical removal of polyps, thus having four times higher risk on CRC. Found was that bacteroides vulgatus, eubacterium spp, ruminococcus, streptococcus, hansenii, Bifidobacterium spp, and faecalibacterium prausnitziiwere were significantly higher abundant in people with high risk of CRC. People with lower risk showed higher levels of lactobacillus s06 and eubacterium aerofaciens (15).

The study of J. Zackular, et al. (2014) searched for potential bacteria as biomarkers by collecting whole stool and comparing results with gFOBT and colonoscopies. Their results revealed that patients with adenomas had relative higher abundances of OTUs related to ruminococcaceae, clostridium (OTU: 60), pseudomonas and porphyromonadaceae.

Lower abundance was found in OTUs linked to bacteroides, lachnospiracaceae, clostridialis and clostridium (OTUs: 20, 97, 99) (11).

It has been found that the human microbiome is an important emerging area for metagenomic biomarkers to early or preidentify CRC (15). The fusobacterium is a common bacterium which was found to be enriched in the gut microbiome of patients after CRC or adenomas are diagnosed (16, 17).

However, some participants had a low prevalence of the fusobacterium, while they had advanced adenoma or cancer in first stage(18). Thereby, it was thought that this bacteria cannot be used as a representative biomarker for CRC, since it is not sensitive enough (19). Nevertheless, a more recent study describes that it is now known that fusobacterium nucleatum can initiate CRC and can be potentially used as diagnosis or a potential target for the treatment of CRC (20, 21).

Operational taxonomic units (OTUs) are couples of sequences which are closely related of bacteria present in the microbiome gut

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Microbiome as a screening method for CRC Sample collection for a microbiome screening is non-invasive and similar to the FOBT and FIT (22). Patients have to collect one whole stool after following their normal dietary and medication restrictions for 24 hours. The microbiome is unequally divided in the stool and therefore several samples are taken from different areas of the stool. The microbial genomic DNA has to be extracted and subsequently the most common housekeeping genetic marker that is present in almost all bacteria can be sequenced. This region is the V4 region of the 16S rRNA gene that is be amplified by PCR and sequenced to determine the species present in the microbiome (23, 24).

Imperiale et al. tested a large cohort of CRC patients and healthy patients to directly compare the sensitivity of the FIT and gut microbiome screenings methods. They showed that multitarget stool DNA testing significantly detected more colorectal cancers that FIT. Testing the DNA of the microbiome resulted in a sensitivity for colorectal cancer of 92.3% compared to 73.8% for FIT. However, the microbiome DNA test has more false positive results (25). But the gut microbiome screening could even distinguish early and late stadium carcinomas without losing its sensitivity (26).

A major advantage of the microbiome screening method would be that also the nonbleeding lesions can be detected (27).

Unfortunately, there are also limitations of this test compared to the FOBT and FIT. One of those limitations is the universally usability of the test. It is known that gender, origin and lifestyle affect the diversity of the gut microbiome (28-30). Therefore the biomarkers of CRC associated microbes are not universal.

However a recently published metagenomics analysis of the faecal microbiome has shown that there are some microbial gene markers shared at least shared between diverse

human communities (Chinese, Danish, Austrian and French community) (12). These four microbial markers might be universally used to screen for CRC. However, screening for markers on particular populations should be encouraged.

Altogether, measuring the fecal microbiome to identify those at risk in CRC seems promising as a novel screening method although it remains questionable if this new method brings us any closer to screening for CRC comparing with the currently used FOBT and FIT screening methods.

Discussion & conclusion Several bacterial species have been implicated in the development of CRC (31). This could provide useful biomarkers for CRC that might even detect earlier carcinoma stages compared to FOBT/FIT since the microbiome screening could also reveal non-bleeding lesions. Therefore the gut microbiome screening could be a better alternative to the currently used FOBT and FIT screening methods. However, in The Netherlands FIT remains the number one used screening method. Therefore, the question arises; what needs to be improved in the gut microbial screening before it could be used during the nationally CRC screenings?

First of all, the diverse compositions of the gut microbiome needs to be better understood.

Increased knowledge about the factors that influence microbiome composition, and the specific species that occur in diverse human populations could result in a higher specificity of this diagnostic tool (32).

Secondly, it would be interesting to investigate the isolated effect of specific organisms present in the microbiome on the cells present in the colorectal tract, since the in vivo situation is complex and the microbiome is diverse. This can be done by conducting in vitro experiments in which colon cells are exposed to bacterial species to

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quantify its specific effect on the proliferation of these cells.

The most important obstacle that needs to be overcome is that the screening for CRC with microbiota present in the gut is still more difficult to interpret than the current FIT and not completely possible. Before we can implement this strategy in a screening program for example, easy and cheap tests should be developed that can be used in a fast manner.

Overall, screening the microbiome for possible biomarkers that can be used seems very promising, but the attribution of several bacteria alone in CRC should still be determined and easy, cheap and fast tests for microbiota should be developed.

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The gut microbiome and colorectal cancer: aspirin as a preventive measure?

Emma Kuiper, Matthijs Snelders, Anouk Stoffels

ABSTRACT Colorectal cancer (CRC) is increasing in incidence in the Netherlands. Because treatment usually involves resection of parts of the colon, it is important to understand the mechanisms of CRC development to find better ways of treatment and possible prevention. Recent studies have discovered that anti-inflammatory drugs, such as aspirin, might have a protective effect on the gut microbiome in relation to CRC. We performed a literature study on the effect of aspirin on the gut microbiome, and whether it could be used as a preventive measure for known risk groups. Our study showed that although aspirin shows signs of reducing risk of CRC, it also comes with its own risks such as gastrointestinal bleeding. We therefore suggest performing more research into the efficacy and of aspirin in treatment of CRC.

The tight junctions between the epithelial Introduction cells along with the mucosal layer prevent the In the Netherlands colorectal cancer (CRC) is bacteria from entering the body (7). the second most diagnosed cancer in men and

women. Partly due to screening programs, the incidence of CRC in 2016 in women was estimated at 44.7 cases per thousand, and 63.3 for men (1). It is therefore a major health concern that we still do not know much about; treatment usually involves resection of part of the colon. Better treatment or prevention would help relieve the burden CRC causes. As of yet, the mechanism of CRC

Figure 1: The inner lining of the intestine. Adapted from development is not fully understood. There Xi et al (7). have been indications that unlike other

cancers, CRC can be caused by the bacteria In disease, the inner lining of the intestine is that reside in our gut (2). compromised. The tight junctions between The gut microbiome is a diverse collection of the epithelial cells are weakened or disappear bacteria, fungi, archaea and viruses (3). It and the mucosa is thinned, allowing starts developing at birth and is ever infiltration of bacteria into the intestine walls. changing, influenced by many factors such as This process of dysbiosis can cause diet and medication. It is present throughout inflammation, which can lead to dysplasia and the gastrointestinal tract with varying eventually can result in adenoma (2). diversity and quantity; more than a trillion Studies have shown that nonsteroidal antibacteria and thousands of heterogeneous inflammatory drugs (NSAID) have a positive species have been identified (4). The effect on the gut microbiome (8). We raised microbiome has several functions; it protects the question whether aspirin (acetylsalicylic against pathogens, helps with the digestion of acid), another NSAID, would have a protective foods, promotes fat storage and intestinal effect on the gut microbiome in relation to angiogenesis, and modulates the central CRC development. We expect that it might nervous system (5). In turn, the microbiome indeed have an effect on the microbiome, requires nutrients, gas and the right which could lower the risk of CRC. We temperature and acidity to function (6). performed a literature study on the subject of The inner lining of the intestine consists of an CRC and the effects of aspirin on its epithelium separated from the microbiota via microbiome. a mucosal layer, as depicted in Figure 1.

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Microbiome in CRC patients Many studies have investigated the microbiome in CRC patients by assessing the tumour tissue in comparison to matched normal tissue. Another prevalent used method is to analyse the faecal samples of those patients. These studies discovered the present of microbial dysbiosis in CRC patients, meaning an imbalance in the microbial community (4). A second common result is an altered microbial diversity and structure (912). In addition, an overabundance of Fusobacteria was reported in the microbiome of the CRC patients in comparison with the controls (10, 12-14). Other studies also stated a decreased butyrate producing bacteria and elevated IL-17 levels (12, 15, 16).

Microbiome and CRC development

Next, we will inform you about some dominant bacteria and metabolites produced by colonic microbiota that are expected to have a leading role in the relation between microbiome and cancer development. Of course, there are a lot more bacteria and metabolites in the microbiome that play a role in the development of CRC. The four most prominent bacteria will be discussed, as depicted in Figure 2.

The first bacteria that is expected to play a role in developing CRC is Escherichia coli. E. coli possess the polyketide synthase (PKS)

Genotoxic Island, which results in more activation. These Islands encode for Colibactin, but also promote CRC via induction of DNA double strand breaks (17).

The second bacteria, Fusobacterium nucleatum, can bind with FadA adhesion molecule to E-cadherin, which leads to activation of beta-catenin signalling and induces pro-oncogenic and inflammatory pathways (18). In addition, Fusobacterium increase infiltration of myeloid cells which also results in a NF-kB-driven proinflammatory response, promoting CRC. Furthermore, the overabundance of Fusobacterium results in an increased cytokines production and oncogenes expression (4).

As third, Enterococcus faecalis play a role in the development of CRC by promoting the release of extracellular superoxide in host cells. The superoxide is converted by hydrogen peroxide and could induce DNA damage, chromosome instability and cancer (19-21).

And last, Clostridium cluster IX, XI, and XVI are capable of metabolizing primary bile acids into secondary bile acids (22). These secondary bile acids are expected to contribute to CRC progression by interacting with host metabolism and immunity (23-26).

Figure 2: Mechanisms of specific bacteria for the development of CRC (4).

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Figure 3: Suspected mechanisms of gut bacterial microbiota in colorectal carcinogenesis (2).

Suspected mechanisms

The prominent microbes that play a role in the development of CRC are described above.

However, there are a lot more microbes that contribute to the CRC progression, suspected to develop CRC by the following four mechanisms. Important mechanisms recently discovered are shown in Figure 3.

The first mechanism is the bacterial-derived genotoxins and bacterial virulence factors.

Bacteria have developed virulence factors that promote pathogenicity. Examples are penetration of the gut mucosal barrier and adhering and invading intestinal epithelial cells (27-29). The pro-carcinogenic effects of pathogens depend on these virulence factors.

Toxins are produced by some bacteria and modulate certain host-derived signalling pathways, which result in the activation of carcinogenesis-promoting pathways (2). In addition, toxins have the ability to induce DNA damage, interfere with the cell cycle and/or apoptosis (17, 30, 31). Gram-negative bacteria produce CDT, a genotoxin, which stimulates the interaction between pathogens and host cells (32). CDT also favours continuous gut colonization and induces the production of pro-inflammatory molecules, which are involved in carcinogenic processes. DNA damage, interference with the cell cycle and modulation of pro-inflammatory pathways can all lead to mutations that are involved in genomic instability in CRC (2).

The second proposed mechanism is the microbial-derived metabolism affecting

carcinogenesis. The metabolic activities may affect colorectal carcinogenesis via the following several processes: regulating the generation of CRC-promoting secondary bile acids; the metabolic activation or inactivation of pro-carcinogenic compounds, dietary phytochemicals and xenobiotics; hormone metabolism; and the modification of inflammation pathways (33).

The third mechanism is a host defence modulation and inflammation. The intestinal mucosa is the first line of defence against gut commensal or pathogen bacteria and related microbial molecules. It is important that the intestinal epithelial cells rapidly detect the presence of pathogens in order to initiate a suitable immune response. However, these cells must also maintain a moderate immune response against or tolerance for nonpathogenic bacteria (34). Innate immunity receptors recognize particular molecular motifs associated with pathogens and help to maintain gut homeostasis. Stimulation of these receptors results in activation of pathways which induce the expression of proinflammatory cytokines and/or antimicrobial peptides. These play a role in the development of inflammatory response. The inflammation is associated with modulation of the microbiota and dysbiosis during CRC. Therefore, people who suffer from inflammation, like inflammatory bowel disease patients, have an increased risk for developing CRC (2).

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The last mechanism is oxidative stress and anti-oxidative defences modulation. Reactive oxygen species (ROS) can be generated by cells during infection and inflammation, or directly by gut microbiota (35). The induction of ROS in infected cells serves as a defence mechanism by elimination of bacteria (36).

An imbalance between the levels of prooxidative molecules and the effectiveness of anti-oxidative defences results in oxidative stress (37). Oxidative stress results in irreversible cell damage, including DNA breaks and damage, protein aggregation or fragmentation, and cellular membranes dysfunction (37, 38). Normally, enzymatic and non-enzymatic anti-oxidative defences help regulate ROS/NOS (nitric oxide synthase) production and repair mechanisms, and thereby balancing the toxic effects of ROS and NOS (39, 40). In bacterial infections this balance is lost, which results in a decreased expression of DNA repair and oxidative response (2).

The effect of aspirin on gut microbiome in CRC patients Aspirin is both a chemopreventive and chemotherapeutic agent for colon cancer and epidemiological and clinical studies supporting its decreased risk on CRC (41, 42).

Nonetheless, aspirin is associated with harms, including gastrointestinal bleeding for which the risk increases with age, as well as dose and duration of use (42, 43).

Three of the earlier described mechanisms induce (in)direct inflammation. Drew et al describe a few mechanisms that are influenced by aspirin (44). One of them is the cytokine MIC-1, also known as growth differentiation factor 15, which may be an important mediator in systemic inflammatory response. Experiments suggest that MIC-1, as a member of the human transforming growth factor-B superfamily, may play a role in carcinogenesis (45). Aspirin was associated in patients with a high MIC-1 with a lower risk of CRC (RR = 0.6 95% CI 0.41-0.88) (44). This could mean that plasma MIC-1 might serve as a biomarker to define patients who may benefit from chemopreventive aspirin.

The first mechanism as mentioned earlier describes the influence on signalling pathways. Drew et al also investigated this pathway for the Wnt signaling genes and proteins (i.e. AXIN-2 and MYC) (44). Wnt signaling pathways help proteins pass signals through the cell surface and induce cell proliferation and cell migration. They say that Wnt plays a critical role in colon tumorigenesis as aspirin is suggested to suppress the Wnt signaling through the PTGSindependent pathway. Also, an interaction between prostaglandin pathways and Wnt signaling may inhibit Wnt signaling through suppression of PTGS-mediated synthesis of PGE2 (44). Therefore, this pathway plays an important role in the inhibition of tumorigenesis.

Also, Drew at al investigated other mechanisms in which aspirin plays a role, such as that of the inhibition of prostaglandinendoperoxide synthase-2 (PES-2), which acts like a hormone (41, 44). This can be done via i.e. the cyclooxygenases (COX) pathway, in which the formation of prostaglandins is inhibited by aspirin (46). Another pathway is that when inhibition of PES-2 occurs the production of prostaglandin E2 (PGE-M) decreases, which results in lower proliferation, less migration and invasiveness, lower promotion of angiogenesis, less resistance to apoptosis and less modulation of cellular and humoral immunity within the tumour (41, 44). Moreover, aspirin was associated with a reduced risk for women with a high PGE-M (47). Johnson et al already investigated if PGE-M in urine could be used as a biomarker for CRC patients and suggest that it is a potentially useful biomarker (48).

Since PGE-M is rather easy to measure in urine and could therefore be used as a good biomarker, patients can be defined which may benefit from aspirin as chemopreventive treatment.

Furthermore, aspirin strongly induce reactive oxygen species (ROS), also known as oxidative stress. This is said to play a role for NSAIDmediated pro-apoptosis since ROS is linked to signals inducing apoptosis (43). ROS causes an increase in the inhibition of NF-kB and

41

activates stress-related kinases JNK and p38 (46).

Lastly, another mechanism that is influenced by aspirin is that of the apoptosis of cancer cells. Aspirin has pro-apoptotic abilities. Brady et al investigated the long-term use of aspirin for colon cancer patients. They found that the stimulation of nuclear factor kappaB (NF-kB) pathway is a key component of this proapoptotic effect (45). The c-Src tyrosine kinase pathway in CRC cells get activated by aspirin (48). This eventually results in the induction of apoptosis.

Discussion In this study we searched the literature for information on the effects of aspirin on got microbiome in CRC patients. In the first part of this research, we investigated how the microbiome alters the colon and how this can develop into cancer. Four bacteria were named as main factor: E. coli (causes activation for colibactin, which promotes CRC via double strand DNA breaks), F. nucleatum (causes inflammation pathways), E. faecalis (causes DNA damage) and Clostridium cluster IX, XI and XVI (causes secondary bile acids which contribute to CRC progression). Next to bacteria, four suspected mechanisms were explained: 1) bacterial-derived genotoxins and bacterial virulence factors (pro-carcinogenic effect, signalling pathways, induction of DNA damage and pro-inflammatory molecules), 2) metabolism affecting carcinogenesis (metabolic activation of pro-carcinogenic compounds, dietary phytochemicals and

xenobiotics, hormone metabolism, modification of inflammations pathways), 3) defence modulation and inflammation (inflammation is associated with modulation of the microbiota and dysbiosis during CRC) and 4) Oxidative stress and anti-oxidative defences modulation.

In the second part, a few of these mechanisms were studied in combination with the use of aspirin to assess if aspirin has a protective or healing effect. Three of the mechanisms mentioned earlier induce inflammation. It was found that several CRC patients had upregulated MIC-1 (44). In fact, aspirin was thought to influence MIC-1 Wnt-signaling which is an important pathway in the progression of CRC (44). This could indicate that aspirin might have a protective effect on CRC in patients with upregulated MIC-1. In addition to MIC-1, aspirin was found to have a protective effect on PGE-M upregulated patients as well. As PGE-M is easily measured in urine, it can be used as a biomarker for aspirin treatment. Further, aspirin was found to induce apoptosis through ROS formation and downstream regulation of the NF-kB pathway (46, 48).

In conclusion, there is evidence that aspirin can be used as a (preventive) treatment of CRC. There are also some downsides which should be taken into account when considering proscribing aspirin for treatment of CRC. We therefore advise to further investigate the efficacy and safety of using aspirin as a possible treatment of CRC.

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Therapeutic Applications Targeting the Microbiome in the Prevention of Microbiota-Driven Carcinogenesis R.M.E. Janssen, J. Lankhof & M. Muller

Abstract INTRODUCTION. Colorectal cancer is the third most prevalent cancer in the Netherlands.

The microbiome, which is largely influenced by the diet, can become disturbed in such a way, that it promotes the formation of gastrointestinal cancers like colorectal cancer.

THERAPIES. Six possible therapeutic applications that target the microbiome are discussed: Antibiotics, drugs targeting genoxins, drugs targeting inflammation, prebiotics, probiotics and fecal microbiota transplantation. These therapies might play a role in the prevention of carcinogenesis in colon cancer.

CONCLUSION. Most therapies that are currently being developed show promising results, but additional research is required to get them into clinical practice.

1. Introduction Colorectal cancer is the third most prevalent type of cancer in both men and women. In the Netherlands, over 13.000 patients are diagnosed with the disease each year. Five-year survival is relatively high at approximately 60%, but this is heavily dependent on the stage of the disease at the time of diagnosis. To allow for detection at earlier stages, a screening program for people between 55 and 75 years-old has been introduced in the Netherlands (1).

The human gastrointestinal tract contains a vast number of microbiota. These are vital to good digestion, and the composition of the microbiome is mainly influenced by the diet.

When significant changes to the microbiotic composition occur, a state of dysbiosis can arise, which can lead to an increase in the number of harmful microbiota (2).

Dysbiosis is thought to have many devastating effects, potentially causing a multitude of diseases. Evidence is mounting that dysbiosis plays a role in the pathogenesis of, amongst others, Crohnâ&#x20AC;&#x2122;s disease (3).

Likewise, the microbiome, when disturbed, can play a role in the development of cancer. For example, in colorectal cancer, certain microbiotes are present in reduced quantities, whilst others are present in too large numbers (2).

In order to prevent the commencing of colorectal cancer, therapies that are targeted at restoring the microbiotic balance present a good opportunity. This review will give a brief

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overview of the most promising treatments in the prevention of microbiota-driven carcinogenesis.

2. Possible therapeutic applications 2.1 Antibiotics The microbiome consists of various bacterial strains, as was mentioned before. The use of antibiotics might influence the representation of these strains, thereby altering the microbiome (2). Both positive and negative effects of the use of antibiotics on the carcinogenesis of colon cancer will be described in this paragraph.

In rats and in mice, it was found that germ-free animals developed less tumours (4, 5).

To maintain their status of germ-free animals, antibiotics were used. However, antibiotic treatment in animals that were not germ-free also reduced the number of colon cancer lesions (6). Similar results have not been found in studies in humans.

Although the results from the aforementioned animal studies were very promising, many other studies report tumour-promoting effects when an antibiotic treatment was administered (7, 8). As a result of interference with proinflammatory cytokines by the antibiotics, this tumour-reducing effect is diminished, thereby enhancing tumour growth (2).

2.2 Drugs targeting genotoxins The microbiome can both directly and indirectly influence carcinogenesis. The direct way is through the production of toxins which affect the DNA damage response, called genotoxins (9). Targeting these genotoxins with specific drugs might prevent or decrease the risk of carcinogenesis in the colon. Two genotoxins which are produced by various bacteria are cytolethal distending toxin (CDT) and colibactin (9).

Antibodies used for detection of CDT in inflammatory bowel disease can be measured in the serum of patients (10). These antibodies could also be used to reduce the number of free CDT molecules in the blood. However, no research about this option related to colon cancer has been published.

The colibactin toxin was also found to directly promote tumour growth in the colon (11). This toxin can be inhibited by the use of small molecules, thereby decreasing the tumour promoting effects of colibactin (12).

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2.3 Drugs targeting bacterially-induced inflammation Inflammation caused by the gut microbiome also plays an important role in tumorigenesis of colon cancer(2). Targeting this bacterially induced inflammation might prevent tumour promotion. Treatment with antibiotics, as mentioned above, may reduce inflammation.

Furthermore, immune suppressors and anti-inflammatory drugs can be used to treat the inflammation, thereby reducing the risk of developing colon cancer (13).

2.4 Probiotics In the search for microbiota-driven carcinogenesis prevention therapies, there is increasing interest in the effects of probiotics (14). According to the World Health Organisation, probiotics are â&#x20AC;&#x2DC;living microorganisms that, when administered in adequate amounts, confer a health benefit to the hostâ&#x20AC;&#x2122;, and can be ingested either through the diet, as supplements or as drugs (15, 16). Up to now, most of the probiotics used are lactic acid bacteria, including lactobacilli and bifidobacteria (17). Probiotics have the potential to restore the microbiota disbalance of the intestine in chronic disease states by influencing the development and stability of the microbiome, inhibiting pathogens, affecting the mucosal barrier, protecting against physiological stress and stimulating specific and non-specific components of the immune system, thereby possibly prevent the development of cancer (14, 17). The actual effects, however, depend on the properties of the microorganism that is used (9, 17).

2.5 Prebiotics The microbial composition in the gut can also be altered by prebiotics. Prebiotics are indigestible compounds, including insulin, oligosaccharides, lactulose and starch, that stimulate the proliferation of beneficial bacteria in the intestine, mainly bifidobacterium and lactobacillus (14, 16). Prebiotics selectively stimulate these beneficial bacteria to exert antimicrobial effects, to regulate immune responses and to compete with pathogens for receptors (18). Ideally, by altering the microbiome, prebiotics could be beneficial in the prevention of the development of cancer (9).

2.6 Fecal microbiota transplantation With fecal microbiota transplantation (FMT), microbiota from the intestine of a healthy donor are transplanted into the intestine of a patient (19). This technique aims to introduce or

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restore the healthy microbiome balance of the gut and has already been demonstrated to be an effective therapy for several gut infections (20). It is suggested that FMT could also be a potential therapy for the prevention of carcinogenesis by reducing the pathways of genotoxicity, inflammation, proliferation and apoptosis in the gut (9). Nevertheless, due to the lack of high-quality data, more studies need to be performed to further establish the efficacy of FMT in microbiota-driven carcinogenesis (21).

3. Conclusion Disturbances in the microbiome can lead to the development of cancer. However, many therapies that target the microbiome in order to prevent cancer formation are in development.

These include the alteration of the microbiome by the use of antibiotics, probiotics, prebiotics and fecal transplantation and methods that other focus on targeting genotoxins and inflammation which play a role in the tumorigenesis of colon cancer. However, all of the mentioned therapies should be studied more thoroughly to determine their precise effects, and to bring these therapies into clinical practice, additional research is required.

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Tlaskalova-Hogenova H, Stepankova R Fau - Kozakova H, Kozakova H Fau Hudcovic T, Hudcovic T Fau - Vannucci L, Vannucci L Fau - Tuckova L, Tuckova L Fau Rossmann P, et al. The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases. (2042-0226 (Electronic)).

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Xu MQ, Cao HL, Wang WQ, Wang S, Cao XC, Yan F, et al. Fecal microbiota transplantation broadening its application beyond intestinal disorders. World journal of gastroenterology. 2015;21(1):102-11.

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Evaluating the amount of Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis and enteropathogenic Escherichia coli in the gut microbiome prior to colorectal cancer development Gina Gerhorst (s4476352), Christina Hahnen (s4354478), Annemarijn Offens (s4647688) 16-02-2018 Abstract Colorectal cancer (CRC) stays on place number 3 of the most common cancer types in The Netherlands.

The gut microbiome seems to play an important role during the development of CRC. In the process of CRC the bacteria Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis (ETBF) and enteropathogenic Escherichia coli (EPEC) seem to be involved. Therefore, the objective of this proposal is to investigate the association between the mentioned bacteria and the overall microbiome diversity in stool samples of patients with and without CRC. We will perform a retrospective study, where we will use 10 years old stools samples from an earlier study, to evaluate if there is an association between the microbiome and the development of CRC. The extracted DNA will be used for PCR to amplify the 16S rRNA and the PCR products will be sequenced via MiSeq Illumina technology. The sequences will be analysed by different matrices to identify the amount and presence or absence of bacterial species. We hypothesize that the amount of the earlier mentioned bacteria and the number of bacterial species present in the stools of persons who will in future develop CRC is different from persons who will not develop CRC. Keywords: colorectal cancer (CRC), microbiome, Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis, enteropathogenic Escherichia coli

Introduction Colorectal cancer (CRC) is one of the most common cancer types in the world (1). In 2010, approximately 5.000 deaths because of CRC were recorded in The Netherlands (2). According to the ´Integraal Kankercentrum Nederland´ about 13.000 persons in The Netherlands are diagnosed with CRC every year (3). These many new and old cases are of course associated with incredibly high costs.

The most common risk factors for the development of CRC include a CRC family history, red meat consumption, cigarette smoking, a high body mass index (BMI), Inflammatory bowel disease, high consumption of vegetables and fruits and many more (4, 5). During the development of CRC the gut microbiome seems to play an important role (6, 7). Various studies in animals have indicated a causal relationship between the gut microbiome and CRC. For example, mice and rats without bacterial colonization of the gut do not develop CRC, while their colonized counterparts do (8-10). Also gavage of fecal samples from CRC patients was shown to increase intestinal carcinogenesis in mice (11). About 100 trillion microbes can be found living within the human body whereas about 1013-1014 of them belonging to more than 100 different bacterial species are found in the guts (7). Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis (ETBF) and enteropathogenic Escherichia coli (EPEC) are thought to belong to the key species in the process of CRC (7). EPEC seems to play a role in CRC via their action in depleting proteins in DNA mismatch repair (MMR) in patients (12). Also, EPEC seems to drive the inflammation during tumor formation and to modulate host cells via virulence factors in vivo and in patients (12). Via the inflammatory lesions, NO is synthesized. NO belongs to the reactive oxygen species (ROS) that lead to oxidative stress in patients and damages the genome (12).

Fusobacterium nucleatum is shown to induce inflammation via the NF-kB pathway and can therefore also lead to ROS formation and oxidative stress in vivo (12). Besides, F. nucleatum can also adhere to enterocytes or intestinal epithelia cells altering cell signaling (12). ETBF is the third mentioned bacterium species that might play a role in CRC. The gram- negative obligate anaerobe bacteria can be

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found in nearly all humans and is the most often found anaerobe bacteria on infection sites (7).

Infection is followed by inflammation also leading to ROS (7).

Although carcinogenic mechanisms for these bacteria have been established, it is challenging to confirm these relationships in humans. Various cross-sectional studies have been performed, comparing people suffering from colon polyps or CRC to healthy individuals, but the results of these studies strongly differ from each other (13-16). This is likely due to the inclusion of few subjects (mostly less than 30 per group), and a large variability in the microbiome in the general population. In addition, different sampling methods (stool samples vs. mucosal samples) and racial or cultural differences in study populations make it difficult to combine the results of these smaller studies, because they can result in very different detected microbiomes (17, 18). Moreover, cross-sectional studies cannot support a causal relationship, since the possibility exists that a difference in microbiome results from altered conditions caused by the tumor (19). This is by some scientists suspected to be the case for fusobacterium colonization (20).

Therefore, a large study is needed that evaluates the occurrence of CRC several years after assessment of the microbiome. Ideally, a prospective study would be conducted, in which microbiome samples are collected and subsequent CRC incidence is recorded. However, this is a time and money consuming method, which in our eyes is not justified with the available evidence. Therefore, we choose to perform a retrospective study, using stool samples that were collected roughly ten years ago for the purpose of another study (FOCUS) (21). This method will save time and money, since follow-up is not needed and stool samples have already been collected. It will however, still provide essential knowledge about the microbiome and CRC development.

The objective of this proposal is to investigate the association between the risk of CRC and Fusobacterium nucleatum, EPEC, and ETBF in stool samples, and to identify any additional microbiome-related differences present in the stool samples in relation to CRC risk. We hypothesize that Fusobacterium nucleatum, EPEC and ETBF will be increasingly present in stools of subjects who later developed CRC. Variables The first goal of this project is to investigate the relationship between the presence of certain bacteria in stools of patients and the development of CRC five to ten years later. The amount (as a percentage of the total number of bacteria) of Fusobacterium nucleatum, EPEC and ETBF, as well as the diversity (total number of bacterial species), will be compared between the patient and control group. In addition to these specific variables, the total microbiome will be analyzed to be able to detect unforeseen differences between the groups (e.g. differences in other bacterial species, microbiome diversity or combinations/ratios of species). Additional variables that will be recorded are patient characteristics which will be age, sex, BMI, family history of CRC, smoking status, alcohol intake (days per week and glasses per week), and medication. Patients will be asked if major changes in their diet, BMI and/or smoking status occurred between now and the collection of the stool samples (ten years ago). If possible, information about the subjectsâ&#x20AC;&#x2122; medication since the moment of sampling will be requested at their pharmacist. Of the variables mentioned above, the most likely confounding variables would be lifestyle aspects such as diet or alcohol intake, and use of medication, since they can influence the microbiome, but may have other effects on cancer development as well (22-24).

However, these variables may be so strongly intertwined that the sample size of this study may be insufficient to separate them. With regard to diet and physical inactivity, it would be ideal to include a questionnaire. However, the subjects will unlikely be able to provide detailed information about their diet or physical activity at the time of sampling, which is of higher interest. Therefore, questionnaires about diet and physical activity will not be included.

Subjects We will use about 400 to 600 of the originally 10.993 samples that were collected during the FOCUS study by van Rossum et al. (21). Exclusion criteria are a diagnosis of CRC or any other form of cancer (based on medical records) before the start of the study or within five years after sample collection.

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All people developing CRC (based on medical records) later than five years after the FOCUS study (between 2013 and 2018) who are willing to participate will be included in the CRC group. People who have not been diagnosed with CRC or any other cancer at the time of data collection are included in the control group.

Corresponding to the numbers of participating patients with CRC, two healthy individuals will be taken per sample, resulting in a control group with double the size of the patient group. If possible, we will match control patients to CRC patients on age and gender.

We will not calculate sample sizes based on the expected effect, since this is very difficult to do based on existing knowledge, and we will be limited by the number CRC patients from the focus population.

Ethical issues/ Informed consent Since the samples take form the FOCUS study should be used, the participants need to be contacted and asked whether they would give consent for a further use of their FOBT samples and whether they are willing to supply access to their medical records to get further information about their current state of health.

Information about their health is needed to decide whether they developed CRC and at what time. And if not, whether they might be candidates to join the control group. To inform the participants, a patient information letter and the protocol is forwarded. The form of medical ethical assessment and registration is used to justify the method of selection of subjects. Since there are no interventions on the participants and only the old stool sampling from the FOCUS study from 2008 is used, there is no harm for the participants.

Sampling, instruments, data collection, data analysis DNA will be extracted from the FOBT cards by using the PowerSoil DNA isolation kit (25). Furthermore, a bead-beating step will be included, to extract a higher amount of DNA of high quality (26).

The same DNA extraction methods should be used for all samples, to make sure that the results are comparable (26). Via PCR the 16S rRNA will be amplified (25). Therefore, region V3 to V5 (257F/926R primer set) of the in total 9 regions of the 16S rRNA of all bacteria will be amplified, by using the associated forward and reverse primer (25). After purification of the PCR products, MiSeq Illumina technology will be used to sequence the 16S rRNA (25). The MiSeq platform will be used because of its high throughput, long reads and therefore lower costs (26). For the analysis, the sequences will be clustered into Operational Taxonomic Units (OTUs) (27). During the analysis, the weighted and unweighted version of phylogeny-based distance (UniFrac) matrices and the nonphylogeny-based distance (Bray-Curtis) matrices will be used (25).

Unweighted UniFrac metric will give identify the absence or presence of rare lineages or variability within the community (25), whereas the weighted UniFrac will identify the relative abundance of the OTUs (25), thus of the amount of different bacterial species.

The timeframe of the research study can be found in figure 1. To

Figure 1|Timeframe of the research study. The samples will be used from FOCUS from 2008. Our study will take place between 2018-2020.

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compare the relative abundance of Fusobacterium nucleatum, EPEC and ETBF in stool samples of patients and controls, the Wilcoxon-Mann-Whitney test will be used, with α 0.05. Subgroup analyses will be performed based on smoking status and alcohol intake.

Budget Based on the incidence of CRC in the general population of The Netherlands in the age group 55-80 (for the year 2013) and 60-85 (2014-2017), and the number of people within this population, a rough estimate was made of the expected CRC incidence in the study population. The total CRC incidence in the general population, consisting of about 4 million people (28), was 60812 (29). The expected incidence in the proposed study (with 10000 subjects that can be approached) is therefore expected to be around 150 from the year 2013-2017, assuming that the test population is a good reflection of the general population. If 2018 will be included, the incidence would rise to 175. Assuming that we will not be able to reach all subjects and some subjects will refuse participation, we estimate the people in the CRC group at around 130 if the year 2018 is included in the analysis. The control group will therefore include about 260 subjects. The estimated costs per sample range around € 100. This would result in costs of about € 39.000 only for sampling the FOBTs. Additionally, there will be salaries, facilities and administrative costs, material expenses and additional costs for unpredictable expenses.

Significance of the study It is important to investigate the role of the microbiome in the development in CRC as it was done with this study. Only a small amount of observational studies about the topic were found, and the role of single bacterial species of the microbiome is not clear. Some species are considered to play a key role in CRC but they have not been confirmed in a prospective study, so causality is unclear. Our proposal could bring knowledge about element of the microbiome in CRC development a large step further.

These results could provide a basis for prospective observational studies and/or may open up new possibilities to prevent or treat CRC. References 1.

Li SK, Martin A. Mismatch Repair and Colon Cancer: Mechanisms and Therapies Explored.

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2. van Erning FN, van Steenbergen LN, Lemmens VEPP, Rutten HJT, Martijn H, van Spronsen DJ, et al. Conditional survival for long-term colorectal cancer survivors in the Netherlands: who do best?

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Drewes JL, Housseau F, Sears CL. Sporadic colorectal cancer: microbial contributors to disease prevention, development and therapy. British Journal of Cancer. 2016;115(3):273-80.

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Marmol I, Sanchez-de-Diego C, Pradilla Dieste A, Cerrada E, Rodriguez Yoldi MJ. Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer. International journal of molecular sciences. 2017;18(1).

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Li Y, Kundu P, Seow SW, de Matos CT, Aronsson L, Chin KC, et al. Gut microbiota accelerate tumor growth via c-jun and STAT3 phosphorylation in APCMin/+ mice. Carcinogenesis.

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Wong SH, Zhao L, Zhang X, Nakatsu G, Han J, Xu W, et al. Gavage of Fecal Samples From Patients With Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-Free and Conventional Mice. Gastroenterology. 2017;153(6):1621-33.e6.

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Leung A, Tsoi H, Yu J. Fusobacterium and Escherichia: models of colorectal cancer driven by microbiota and the utility of microbiota in colorectal cancer screening. Expert Review of Gastroenterology & Hepatology. 2015;9(5):651-7.

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Drewes JL, White JR, Dejea CM, Fathi P, Iyadorai T, Vadivelu J, et al. High-resolution bacterial 16S rRNA gene profile meta-analysis and biofilm status reveal common colorectal cancer consortia.

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Scanlan PD, Shanahan F, Clune Y, Collins JK, O'Sullivan GC, O'Riordan M, et al. Cultureindependent analysis of the gut microbiota in colorectal cancer and polyposis. Environmental microbiology. 2008;10(3):789-98.

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Shen XJ, Rawls JF, Randall T, Burcal L, Mpande CN, Jenkins N, et al. Molecular characterization of mucosal adherent bacteria and associations with colorectal adenomas. Gut microbes.

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Russo E, Bacci G, Chiellini C, Fagorzi C, Niccolai E, Taddei A, et al. Preliminary Comparison of Oral and Intestinal Human Microbiota in Patients with Colorectal Cancer: A Pilot Study. Frontiers in microbiology. 2017;8:2699.

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Liao M, Xie Y, Mao Y, Lu Z, Tan A, Wu C, et al. Comparative analyses of fecal microbiota in Chinese isolated Yao population, minority Zhuang and rural Han by 16sRNA sequencing. Scientific reports. 2018;8(1):1142.

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Tjalsma H, Boleij A, Marchesi JR, Dutilh BE. A bacterial driver-passenger model for colorectal cancer: beyond the usual suspects. Nature reviews Microbiology. 2012;10(8):575-82.

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Amitay EL, Werner S, Vital M, Pieper DH, Hofler D, Gierse IJ, et al. Fusobacterium and colorectal cancer: causal factor or passenger? Results from a large colorectal cancer screening study.

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Random Comparison of Guaiac and Immunochemical Fecal Occult Blood Tests for Colorectal Cancer in a Screening Population. Gastroenterology. 2008;135(1):82-90.

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Prevention of gastrointestinal cancers through the diet R.M.E. Janssen, J. Lankhof & M. Muller Abstract INTRODUCTION. The World Cancer Research Fund published a report in 2007 about various ways to prevent cancer. In this review, the effects of the diet and the interaction of the diet with the genome on the development of gastrointestinal cancers will be discussed.

DIET. A lot of research has been conducted on the relation between the diet in relation to cancer prevention in the last years. The use of fruit and vegetables, dietary fibres, antioxidants and calcium might play a role in this process.

GENETICS. The field of nutrigenomics has identified possible genetic variants which, when altered, influence the risk of developing cancer. Amongst these are the GSTM1 gene and several polymorphisms.

CONCLUSION. The gastrointestinal tract is continuously being exposed to all kinds of compounds present in the food. Some of these compounds have potential anti-carcinogenic effects. Genetic variance between individuals and the interaction of this with the diet also plays an important role in the development cancer. More research is required to know exactly how these factors influence cancer development and each other.

1. Introduction Tumours of the gastrointestinal (GI) tract have the highest incidence rate of Europe (1). In 2007, the World Cancer Research Fund (WCRF) published a report about the prevention of various types of cancer, including stomach cancer, oesophageal cancer, and colorectal cancer.

In this report the WCRF describes the influences of the diet and physical activity on tumour development (2).

The function of the GI tract is to digest food and take up nutrients. The diet has the biggest impact on the micro-environment of the GI tract, thereby possibly influencing the risk of developing tumours (3).

Moreover, it is very likely that the genetic makeup of an individual determines the response to different dietary factors. The research fields of nutrigenetics and nutrigenomics are trying to answer questions related to the dietary effects on the genome and gene expression (4, 5). These studies can be used to provide information for the design of a personal diet, related to the genetic makeup of a person.

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The WCRF states that around 33% of all tumours is preventable when a healthy diet is followed and when individuals are sufficiently physical active (2). This review gives an overview of how different aspects of the diet and their interaction with nutrigenetics can help to prevent the development of cancers in the GI tract. Although it was found that physical activity also plays an important role in the prevention of cancer (2), this will not be discussed here.

2. Diet 2.1 Fruits and vegetables In recent years, a lot of research has focussed on the relationship between fruit and vegetable intake and the development of cancer. This is because fruits and vegetables contain several potential anti-carcinogenic substances, including folate, antioxidants, carotenoids, flavonoids, vitamins, fibres and trace elements (6, 7). Conceivable cancer prevention mechanisms induced by these substances include modulation of DNA methylation, prevention of DNA adduct formation, induction of phase II carcinogen-metabolizing enzymes, alteration of hormone levels, inhibition of nitrosamine formation and carcinogen-binding (6).

There is convincing evidence that consumption of fruits and vegetables lower the risk of GI cancers (8). Nonetheless, even though certain fruits and vegetables may protect against specific cancers, it is unlikely that there is a general protective effect for all fruits and vegetables (6).

2.2 Dietary fibres According to the report of the WCRF, there is probable evidence that dietary fibres protect against colorectal cancer (2). Sources of dietary fibres include whole grains, fruits, vegetables and supplements (9). There are several proposed underlying mechanisms in the prevention of colorectal cancer by fibres. During bacterial anaerobic fermentation of the dietary fibres in the colon, short chain fatty acids, including butyrate, are produced. Butyrate has in inhibitory and tumour suppressor effect on colon cancer, it reduces cell proliferation and induces apoptosis (10, 11). The consumption of dietary fibres also results in decreased adiposity, improved insulin sensitivity and reduced contact between the intestinal contents and mucosa (10). However, foods that have a high fibre content often contain other substances with potential anti-carcinogenic effects which could also account for the protective effects found (11).

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2.3 Anti-oxidants Antioxidants are substances that are generally found in a standard healthy and varied diet.

Several antioxidants, such as vitamin C, vitamin E, carotenoids and flavonoids, potentially influence the risk of the development of colon cancer by neutralizing reactive oxygen species or free radicals that can damage the DNA (6, 12).

2.4 Calcium Another substance that has the potential to prevent cancer is calcium. It is hypothesised that a relatively high intake of calcium reduces the risk for colorectal cancer, by binding to secondary bile acids and ionised fatty acids in the intestinal lumen to indirectly reduce proliferative stimuli on the mucosa of the intestine. Calcium can also directly reduce cellular proliferation or induce terminal differentiation of intestinal mucosal cells. The optimal dose and form of calcium for the prevention of cancer, however, is not known (6, 13).

3. Genetics 3.1 Nutrigenomics A lot of research has been done in the field of nutrigenomics, which is the relation between the diet and certain genetic variations. It is hypothesised that these variations result in altered proteins, which then go on to have different interactions with consumed products (14). In the following sections, several genomic variations and their interactions will be discussed.

3.2 GSTM1 Glutathione S-transferase Mu 1 (GSTM1) is an enzyme involved in the detoxification of compounds through conjugation of glutathione. Alterations in the functionality of this gene could therefore result in altered interactions with the diet. Gaudet et al. (15) reported mixed results when comparing the diet-gene interactions of active GSTM1 to inactive GSTM1 in 149 head/neck cancer patients and 180 controls. When consuming moderate amounts of vegetables, inactive GSTM1 resulted in a lower odds-ratio (OR), but for high amounts of vegetables, the OR was the same as for active GSTM1. On the other hand, a decrease in OR was found for fruits, regardless of the consumed amount. However, a clear pattern of association could not be established (15).

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3.3 Polymorphisms Rs16892766 is a polymorphism that has been identified to possibly affect the risk on colorectal cancer. A review by Hutter et al. (16) found that the presence of this polymorphism in combination with vegetable consumption lead to an increased OR of 1.17 (16).

Another polymorphism, rs4143094, has been found to interact with processed meat.

In their review, Figueiredo et al. (17) reported that consumption of processed meat by carriers of the TG and TT variant of this polymorphism resulted in an OR of 1.17 for developing colorectal cancer, while no increased OR was found for the GG variant (17).

4. Conclusion As the WCRF stated, about a third of all the cancers is preventable (2). Therefore, we looked especially at preventable cancers in the GI tract. This tract is continuously exposed to dietary factors, which might influence the environment in which a tumour may develop (3).

Furthermore, the interaction of the dietary factors and several genetic variations might contribute to tumorigenesis (4).

Based on current knowledge, the diet should contain sufficient amounts of fruit and vegetables, because of their abundance in antioxidants, fibres and other potential anticarcinogenic compounds. In the future, personal genetic variance should also be taken into account when designing a diet, to keep the risk of developing cancer as low as reasonably possible. To achieve this, however, more research into the links between diet and genetic variants is required, as the relationship between them is not entirely clear yet.

5. References 1.

Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JWW, Comber H, et al. Cancer

incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. Eur J Cancer. 2013;49:1374-403.

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American Institute for Cancer Research, World Cancer Research Fund. Food, Nutrition, Physical

Activity, and the Prevention of Cancer: a Global Perspective. AICR, editor. Washington DC2007.

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Kerr J, Anderson C, Lippman SM. Physical activity, sedentary behaviour, diet, and cancer: an update

and emerging new evidence. The Lancet Oncology. 2017;18(8):e457-e71.

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Fenech M, El-Sohemy A, Cahill L, Ferguson LR, French TA, Tai ES, et al. Nutrigenetics and

nutrigenomics: viewpoints on the current status and applications in nutrition research and practice. J Nutrigenet Nutrigenomics. 2011;4(2):69-89.

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Mariman EC. Nutrigenomics and nutrigenetics: the 'omics' revolution in nutritional science. Biotechnol

Appl Biochem. 2006;44(Pt 3):119-28.

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McCullough ML, Giovannucci EL. Diet and cancer prevention. Oncogene. 2004;23(38):6349-64.

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Abdulla M, Gruber P. Role of diet modification in cancer prevention. BioFactors. 2000;12:45-51.

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Go VL, Wong DA, Wang Y, Butrum RR, Norman HA, Wilkerson L. Diet and cancer prevention:

evidence-based medicine to genomic medicine. J Nutr. 2004;134(12):3513S-6S. 9.

Elleuch M, Bedigian D, Roiseux O, Besbes S, Blecker C, Attia H. Dietary fibre and fibre-rich by-

products of food processing: Characterisation, technological functionality and commercial applications: A review. Food Chem. 2011;124(2):411-21.

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McNabney SM, Henagan TM. Short Chain Fatty Acids in the Colon and Peripheral Tissues: A Focus

on Butyrate, Colon Cancer, Obesity and Insulin Resistance. Nutrients. 2017;9(12).

11.

Bingham SA, Day NE, Luben R, Ferrari P, Slimani N, Norat T, et al. Dietary fibre in food and

protection against colorectal cancer in the european Prospective Investigation into Cancer and Nutrition (EPIC): an observational study. The Lancet. 2003;361(9368):1496-501.

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Bruce WR, Giacca A, Medline A. Possible mechanisms relating diet and risks of colon cancer. Cancer

Epidemiology and Prevention Biomarkers. 2000;9(12):1271-9.

13.

Key TJ, Schatzkin A, Willett WC, Allen NE, Spencer EA, Travis RC. Diet, nutrition and the

prevention of cancer. Public Health Nutr. 2007;7(1a).

14.

Theodoratou E, Timofeeva M, Li X, Meng X, Ioannidis JPA. Nature, Nurture, and Cancer Risks:

Genetic and Nutritional Contributions to Cancer. Annu Rev Nutr. 2017;37:293-320.

15.

Gaudet MM, Olshan AF, Poole C, Weissler MC, Watson M, Bell DA. Diet, GSTM1 and GsTT1 and

head and neck cancer. Carcinogenesis. 2004;25(5):735-40.

16.

Hutter CM, Chang-Claude J, Slattery ML, Pflugeisen BM, Lin Y, Duggan D, et al. Characterization of

gene-environment interactions for colorectal cancer susceptibility loci. Cancer Res. 2012;72(8):2036-44.

17.

Figueiredo JC, Hsu L, Hutter CM, Lin Y, Campbell PT, Baron JA, et al. Genome-wide diet-gene

interaction analyses for risk of colorectal cancer. PLoS Genet. 2014;10(4):e1004228.

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Comparison of the use of early enteral nutrition and parenteral nutrition in esophageal carcinoma

Gina Gerhorst (s4476352), Christina Hahnen (s4354478), Annemarijn Offens (s4647688) 21-02-2018

Abstract Esophageal carcinoma is one of the most lethal cancer types in the world. One of the major issues of esophageal carcinoma is malnutrition, which is caused by decreased food intake because of the disease, pain, decreased appetite, metabolic changes, nutrient loss or obstructions of the digestive tract. Esophageal carcinoma patients therefore often show a poor nutrition status even before surgery which can lead to increased morbidity and mortality. Therefore supportive feeding is considered, which can be given intravenous called parenteral nutrition or via a tube into the stomach or jejunum called enteral feeding. The aim of the article is to discuss whether parenteral nutrition of enteral nutrition should be preferably administered to the patients. The results for the comparison of parenteral nutrition and enteral nutrition show that there are no statistically significant differences in the total complications after surgery and the non-life-threatening surgical complications, but the lifethreatening complications were significantly decreased in patients receiving early enteral nutrition.

Also, postoperative length of hospital stay and hospitality costs were decreased in these patients.

The conclusion therefore is that enteral nutrition should be preferred over parenteral nutrition. A combination of parenteral- and enteral nutrition compared to enteral nutrition usage alone results in better outcome concerning higher positive energy balance, body weight, fat free mass and a better quality of life. This show that maybe a combination of both could even increase the results obtained by enteral nutrition. Striking is that most of the studies are done in Asian cohorts and therefore it might be favorable to do some large cohort studies in Caucasian populations as well to see whether the results can be reproduced.

Keywords: malnutrition, esophageal carcinoma, squamous cell carcinoma, adenocarcinoma, early enteral nutrition (EEN), parenteral nutrition (PN)

Introduction Esophageal carcinoma (EC) is a major health issue, being the sixth most lethal cancer worldwide (1). EC consists of squamous cell carcinoma and adenocarcinoma. Squamous cell carcinoma is more dominant in Asia, the Middle East and Eastern Africa (2). Especially in China it is a major health issue, since half of the total EC mortality in the world takes place there (1). Adenocarcinoma is the most frequent in Western countries, where its incidence has increased strongly over the past two decades (1, 2). EC is generally diagnosed at a late stage.

About 50 % of patients is treated with curative intent, in which case surgery is the essential treatment, often supported by neoadjuvant chemotherapy and radiation (3).

Malnutrition is a major issue in EC and cancer in general. This has many different causes (4

(for a review)). Firstly, food intake is often decreased as a consequence of the disease itself or due to side effects of treatments. For example, a decreased appetite, nausea and vomiting, depression or pain can reduce food intake. In addition, there are metabolic changes caused by agents produced by the tumor directly or resulting from a response to the tumor (such as pro-inflammatory cytokines).

Tissue damage due to surgery or radiation leads to an increased nutritional (protein) demand.

Finally, nutrient loss, for example through wounds, vomiting or diarrhea is an important factor in cancer-associated malnutrition (4).

Besides these general mechanisms of malnutrition, gastrointestinal malignancies, and especially EC, are associated with obstruction of the digestive tract. This is illustrated by the fact that weight loss in EC is higher than in any

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other cancer (2). Esophageal tumors often cause dysphagia (difficulty in swallowing), or odynophagia (pain with swallowing) (5). Usually beginning with a difficulty in swallowing solid foods, the obstruction can get more severe with growth of the tumor, to a point were even liquids become difficult to ingest (6). Therefore, EC patients often present with a poor nutritional status already before surgery (7).

Postoperative hypercatabolism and complications after surgery, which may require a fasting period, further worsen the nutritional status (7). Malnutrition in EC is associated with lower quality of life and increased morbidity and mortality, although it must be said that not all studies confirmed this correlation (2).

The best way to obtain adequate nutrition is considered to be oral food intake (8). Nutrient intake can be improved by using special formulas(9), which usually have high energy and protein content . In EC, however, oral intake is often not sufficient to meet the nutritional requirements. In this case, supportive feeding is considered, which can be enteral or parenteral. Enteral feeding is the delivery of nutrition through a tube, which can be a nasogastric tube (short term) or a percutaneous tube inserted in the stomach or jejunum (long term) (5). Parenteral nutrition is the intravenous administration nutrients (5). In general, it is seen as less suitable as a long-term option compared to enteral nutrition, due to increased risk of infection . However, it can be used when enteral feeding is insufficient or unfeasible and in the postoperative setting (10). Recently, evidence has suggested that early enteral feeding (EEN) is preferable over parenteral feeding in the postoperative setting as well (11). We will discuss in depth the evidence available on parenteral and enteral feeding in the postoperative setting with respect to various clinical outcome measures.

In addition, we will discuss research on the combination of parenteral and enteral feeding, a topic which has not got much attention yet in EC.

Comparison of post operative EEN and PN therapy We selected 4 articles and one position paper describing studies in which PN and early EN

(EEN) were evaluated and compared postoperatively.

The first article we selected from Fujita et al.

(12) was published in 2012 and is an article about a randomized trial done in 154 patients after esophagectomy. 88 Patients received PN whereas 76 received EEN between 2009 and 2010. No significant difference was found in the total surgical complications among the two groups and also no difference was seen in the amount of occurring non-life-threatening surgical complications whereas the lifethreatening surgical complications were lowered in patients receiving EEN seen among others in less catheter- or wound related sepsis.

Non-life-threatening surgical complications contained anastomotic stenosis, delayed gastric emptying, recurrent nerve palsy, and superficial and deep fascial surgical site infections. Life-threatening surgical complications contained anastomotic leakage and pneumonia. The clinical management pathway (CMP) success rate was significantly higher in the EEN group leading to a shorter median postoperative length of hospital stay (PLOS) in this group. The article claims that a small volume of EEN results in an improved pulmonary function due to a reduced sequestration of fluid within the third space.

Other actions occurring after surgery are that the gut barrier function is improved and the immune system is normalized therefore the local tissue immune response is improved as well and the resistance to bacterial contamination (12).

A position paper from the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP) from 2014 advices that patients with all cancers that can be treated with radiotherapy or radiochemotherapy, preferentially should not receive PN (13). If these patients need artificial nutrition, EN is preferable as first-line treatment as long as the small intestines can be accessed. The disadvantage of PN is that the risk of infections during chemotherapy rise significantly (13).

A meta-analysis published in 2016 by Peng et al. analyzed 10 randomized controlled studies that were published between 2001 and 2014 and compare EEN and PN patient groups that underwent esophagectomy (14). The used patient groups compared consisted of between

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24 to 164 participants. PN was defined in most of the studies as feeding via the central vein and EEN as a nasojejunal feeding tube. As indicated earlier by Fujita et al. (12), the combination of non-life-threatening and lifethreatening surgical complications to toal complications did not differ between the two groups, also Peng et al. found these results (14). Also, both studies found a lowered anastomotic leakage which is considered a lifethreatening surgical complication in EEN. Some of the analyzed studies also claimed that there are less anastomotic leakages in EEN, no significant differences in digestive complications between the groups and significantly more postoperative pulmonary complications in patients receiving PN. Consistent findings were also found between Fujita et al. (12) and Peng et al. (14) regarding the preservation of the intestinal mucosa and the immunological function of it. Normally, the esophagectomy will result in intestinal ischemia and intestinal paralysis leading to atrophy. This will be caused by the absent stimulus of oral nutrition. The mucosa will become more permeable leading to migrating gut bacteria and endotoxins and blood stream entrance. But EEN seems to reduce these actions leading to a lowered serum endotoxin level resulting in less infections and inflammations (12, 14).

The last articles used are from 2015 and 2018 and are two retrospective cohort studies. The large cohort study from 2017 was done by Han et al. and included 665 patients with esophageal carcinoma of the esophagus or the esophagogastric junction in China (7). The patients were divided into groups receiving EEN (n=403) or PN (n=262) to clarify the validity of EEN compared to PN. The EEN delivery started on day one after surgery. Results showed that the PLOS was significantly shorter in EEN, even after adjustment of the multivariable linear regression analysis for tumor histology and location, type of esophagectomy and postoperative albumin infusion. These findings correspond with the findings of Fujita et al.

(12). Due to the shorter PLOS also the hospital charges for EEN are significantly lower. In contrast to the earlier studies, no significant difference regarding the anastomotic leakage was found between the groups. Patients in the PN treatment group had a higher 1-year

postoperative mortality in percentage than the EEN but the difference was not statistically significant. The possible explanation for this finding might be that different types of esophagectomy are performed leading to introduction of bias. For instance, different numbers of McKeown esophagectomies were found in the two groups. The authors found in the literature of other studies, meta-analyses and guidelines that EN was recommended over PN for critically ill and surgical patients (7). A reason for this is that EN has a lower infection rate and a short PLOS resulting in lower cost.

These findings are also consistent with earlier findings.

The chinese study from Yu et al. from 2015 got to the same results as Han et al. (15). They induced EN within in the first 24-48 hours post operational. Additionally, they found an earlier fecal pass after surgery and a short duration of the systemic inflammatory response syndrome (SIRS) in EEN whereas pneumonia occurred significantly more in PN. These findings are consistent with Fujita et al. and Peng et al. (12, 14).

In summary, nearly all articles had consistent findings indicating that EN should be preferred over PN to achieve better clinical outcomes and a higher quality of life for the patient. One aspect that should be considered is that several of these studies were done in China or at least Asia where almost half of the new cases of esophageal carcinoma occur. Since there are more new cases, the possibilities to set up big trials there are more easily. On the other hand, there may be a difference in susceptibilities for the Asian and Caucasian populations to become diseased by esophageal cancer which can be due to different heritage backgrounds.

The epidemiology in terms of histology, incidence, demographics and risk factors as well as the molecular epidemiology in terms of sensitivity to treatments and genetic suspections of esophageal cancer might differ between the populations (16). This indicates that the Asian populations might react different to the cancer, surgery and treatments than the Caucasian population do. The studies published from Asia should therefore be done in a Caucasian cohort to test whether the results are reproducible for this population. If so, EN

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should also be recommended over PN in Europe.

Comparing the combination of PN and EN therapy with EN solo therapy The previously mentioned articles indicated that EN should be preferred over PN, because of higher quality of life and better clinical outcomes which are achieved after EEN usage (14). However, the question rises, if EN and PN could also be used in combination. In this way, the benefits of both therapies could be combined to achieve even better clinical outcomes for the patient, than after use of EN alone. A further reason to investigate whether EN could be used in combination with PN is, that some patients possibly do not react well to a full-calorie EN which could lead to underfeeding and therefore to complications (17, 18). We found one study where esophageal cancer patients got EN in combination with PN after an esophagectomy.

In this study of Wu et al., a randomized controlled trial was carried out to compare the effect of PN+EN and EN to different patient outcome measurements like fat free mass (FFM), body weight or energy balance (18). In the study, 73 esophageal cancer patients who had undergone esophagectomy were randomized into 2 groups. After surgery one group received EN and one PN+EN in combination with a supplementary PN (SPN) to compensate calorie deficiencies. Primary outcome measurements showed that there were no major changes in the fat free mass (FFM) of the PN+EN patient group. However, in the EN group a decrease in FFM was seen. Also a decrease in body weight was seen in the EN groups, whereas an preservation in body weight was found in the PN+EN group.

Furthermore, an increase in extra- and intracellular fluids was measured in the PN+EN group. For the secondary outcome measurements no significant differences were seen in postoperative complications between the PN+EN and the EN group. There were no significant differences in PLOS between the two groups and no patients died. Patients from both groups showed a systemic inflammation reaction with no significant differences after surgery and also no differences were found in transferrin, prealbumin and retinaldehyde-

binding protein (RBP) concentration. Four days after surgery, the EN group showed a negative energy balance, thus a shortage in calories, whereas in the PN+EN group a positive energy balance was seen. Questionnaires were given to the patients before and 90 days after surgery. The questionnaire asked for mental health, energy/ fatigue, health perception, social and physical functioning, bodily pain and physical and emotional role. No significant differences were found between the two groups, but 90 days after surgery better scores for energy/fatigue and physical functioning were measured in the PN+EN group compared to the EN group (18).

In summary, the PN+EN patient group had a better outcome concerning fat free mass, body weight, positive energy balance and the health related quality of life questionnaires. Therefore, patient who don't react well to full-calorie EN could receive a PN+EN therapy instead. A new trail should be performed with more patients to investigate if PN+EN could even replace EN, if again more positive patient outcome measurements will be seen after treatment with PN+EN (18). Furthermore, this study was conducted in Zhongshan Hospital in China. As said earlier, there can be differences in the Asian and Caucasian population considering genetic factors, risk factors or demographic differences (16). Therefore, also this study should be repeated in future with a bigger Caucasian cohort, to test whether these results can be reproduced for the Caucasian population.

Conclusion

To conclude, the given articles are an indication whether what is better to use, but since a lot of the studies are done in Asian studies, studies with Caucasian cohorts need to be done. Also, there is the need of more research on the effects of the combination of PN en EN in EC since there are only some recent articles about the topic, but these indicate that there might be benefits from using the combination.

Further on, the found article about the combination was only done with a quite small cohort, indicating that there is the need of larger cohort studies to confirm the found results.

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2. Reim D, Friess H. Feeding Challenges in Patients with Esophageal and Gastroesophageal Cancers. Gastrointestinal Tumors. 2016;2(4):166-77.

3. Mawhinney MR, Glasgow RE. Current treatment options for the management of esophageal cancer. Cancer Management and Research. 2012;4:367-77.

4. Van Cutsem E, Arends J. The causes and consequences of cancer-associated malnutrition.

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6. Rubenstein JH, Shaheen NJ. Epidemiology, Diagnosis, and Management of Esophageal Adenocarcinoma. Gastroenterology. 2015;149(2):302-17.e1.

7. Han H, Pan M, Tao Y, Liu R, Huang Z, Piccolo K, et al. Early Enteral Nutrition is Associated with Faster Post-Esophagectomy Recovery in Chinese Esophageal Cancer Patients: A Retrospective Cohort Study. Nutrition and cancer. 2018;70(2):221-8.

8. Mislang AR, Di Donato S, Hubbard J, Krishna L, Mottino G, Bozzetti F, et al. Nutritional management of older adults with gastrointestinal cancers: An International Society of Geriatric Oncology (SIOG) review paper. Journal of geriatric oncology. 2018.

9. Baldwin C, Spiro A, Ahern R, Emery PW. Oral nutritional interventions in malnourished patients with cancer: a systematic review and meta-analysis. Journal of the National Cancer Institute.

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11. Steenhagen E, van Vulpen JK, van Hillegersberg R, May AM, Siersema PD. Nutrition in perioperative esophageal cancer management. Expert review of gastroenterology & hepatology.

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12. Fujita T, Daiko H, Nishimura M. Early enteral nutrition reduces the rate of life-threatening complications after thoracic esophagectomy in patients with esophageal cancer. European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes. 2012;48(2):7984.

13. Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2014;46(8):667-74.

14. Peng J, Cai J, Niu ZX, Chen LQ. Early enteral nutrition compared with parenteral nutrition for esophageal cancer patients after esophagectomy: a meta-analysis. Diseases of the esophagus : official journal of the International Society for Diseases of the Esophagus. 2016;29(4):333-41.

15. Yu HM, Tang CW, Feng WM, Chen QQ, Xu YQ, Bao Y. Early Enteral Nutrition Versus Parenteral Nutrition After Resection of Esophageal Cancer: a Retrospective Analysis. The Indian journal of surgery. 2017;79(1):13-8.

16. Zhang H-Z, Jin G-F, Shen H-B. Epidemiologic differences in esophageal cancer between Asian and Western populations. Chinese Journal of Cancer. 2012;31(6):281-6.

17. Villet S, Chiolero RL, Bollmann MD, Revelly JP, Cayeux RNM, Delarue J, et al. Negative impact of hypocaloric feeding and energy balance on clinical outcome in ICU patients. Clinical nutrition (Edinburgh, Scotland). 2005;24(4):502-9.

18. Wu W, Zhong M, Zhu DM, Song JQ, Huang JF, Wang Q, et al. Effect of Early Full-Calorie Nutrition Support Following Esophagectomy: A Randomized Controlled Trial. JPEN Journal of parenteral and enteral nutrition. 2017;41(7):1146-54.

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The benefits of nutrition in palliative care: should we care?

Emma Kuiper, Matthijs Snelders, Anouk Stoffels

ABSTRACT Esophageal cancer is one of the most common and deadliest cancers worldwide with a survival of only 15-25%. It is proven that proper nutrition can decrease the risk of developing esophageal cancer and can increase the prognosis. However, the role of nutritional support in the palliative state of those cancer patients is not fully investigated. Therefore, we have performed a literature search to study whether nutritional care can improve the Quality of Life (QoL) in esophageal cancer patients with advanced cancer. We concluded that a novel questionnaire specific for esophageal cancer patients could be a more quantitative tool for determining QoL in these patient groups. In addition, the use of progestational agents like ghrelin might prove a beneficial addition to nutrition of esophageal patients in palliative care.

Introduction Esophageal cancer is one of the most common and deadliest cancers worldwide.

The survival is poor, only 15-25%, which is primarily caused by the late diagnosis (1,2).

Screening of esophageal cancer is performed by endoscopy. Ideally, the endoscopic biopsies are taken with high-resolution endoscopes and narrow banding imaging (NBI) (1).

There are two major subtypes of esophageal cancer. The first subtype is squamous carcinoma, which result from squamous cells.

These epithelial cells form the surface of the skin lining of hollow organs, and respiratory and digestive tracts (1). The second subtype is esophageal adenocarcinoma. This type is developed from the glandular cells which are present in the lower part of the esophagus.

These cells are often transformed to intestinal cell type in conditions like Barrett’s esophagus (BE) (1).

A major consequence of esophageal cancer is malignant esophageal obstruction, a condition wherein the tumour obstructs the esophagus, resulting in dysphagia. Patients with dysphagia have difficulty with swallowing solid foods. It is one of the most troubling and unbearable symptoms of this conditions (3).

In worse cases can the patients obtain problems with swallowing semi-solid foods, obtain a poor appetite, may experience significant weight loss and are likely to develop nutritional compromise. A poor nutritional status is a significant prognostic

factor for mortality in patients with esophageal cancer (4).

The risk factors are depended on the esophageal cancer subtype, but are in general: gender and race, gastresophageal reflux disease and Barrett’s esophagus, obesity, drugs, smoking, genetic aspects and alcohol, tobacco and nutritional deficits (1).

An inverse relation between dietary fiber, vitamins, fruit and vegetables and development of this disease has been proven before (5).

Even in the palliative state of cancer, nutrition is very important to improve survival and Quality of Life (QoL). The ESPEN guidelines on nutrition in cancer patients recommend “offering and implementing nutritional interventions in patients with advanced cancer only after considering together with the patient the prognosis of the malignant disease and both the expected benefit on quality of life and potentially survival as well as the burden associated with nutritional care” (6). The most important consideration of nutritional support is the expected survival.

If survival is expected to be several months to years the goal is to ensure an adequate intake of energy and proteins, diminish metabolic disturbances, and to maintain an adequate performance status and subjective QoL. When the life expectancy is only several weeks to months, the intervention should be noninvasive. Nutrition support includes oral nutritional supplements, enteral or parenteral, or combined interventions (6).

1

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However, only few studies have investigated the objective benefit of nutritional support in palliative care. Therefore, we have performed a literature study to answer the following question whether nutritional care can improve the Quality of Life in esophageal cancer patients undergoing palliative care. We expect that providing those cancer patients with palliative care enough energy and protein intake will not only improve their QoL, but can also lengthen their survival.

The effects of nutritional care on QoL in patients with advanced esophageal cancer Patients with esophageal cancer hold many challenges in receiving proper nutrition.

Surgical reconstruction, chemotherapy, radiotherapy and surgical complications can result in dysphagia (cancer related cachexia), reflux, diarrhea or dumping syndrome and may affect the proper nutrition in patients with esophageal cancer (7,8). Advanced esophageal cancer is not curable and palliative treatment is required, with the aim to improve the QoL and reduce dysphagia and poor nutrition (2). Especially patients treated with chemotherapy often suffer from malnutrition, which results in poorer treatment outcomes and a decreased QoL (7,9).

Blazeby et al assessed the impact on QoL for palliative treated patients with the EORTC-30 questionnaire which is widely used to assess the QoL. They found that palliative treated patients had a lower QoL than non-palliative patients (8). But after treatment, QoL scores of palliative patients were similar to those non-palliative patients who died within two years after surgery. A randomized trial by Poulsen et al found that intensive, individual counseling for oral nutrition in patients with esophageal cancer, treated with chemo- or radiotherapy, was associated with a better weight maintenance during treatment but not after treatment, which did not result in a better QoL (10). In patients who cannot receive oral nutrition, Zeng et al discussed the potential of home enteral nutrition (HEN).

HEN provides enteral nutrition support through a nasogastric feeding tube or via a jejunostomy tube for patients who underwent surgical treatment (9). They hypothesized that the increased delivery of nutrients would help the recovery of the patients and improve their QoL, since it was shown by Uitdehaag that the efficacy of HEN is strong in this patient group (11). The QoL was higher in the HEN group compared to the control group 4 and 12 weeks after surgery (respectively p<0.05 and p<0.01), but this effect disappeared after 24 weeks (see figure 1).

Figure 1: QoL scores at 1, 4, 12 and 24 weeks after surgery for HEN and control group. (9)

Miller et al reported that patients which are solely fed via a tube (like is done with HEN), have a worse QoL than those who are able to tolerate some oral nutrition (7). This could be explained by the fact that patients value the taste and sensation of food taken orally, which give them a greater feeling of satisfaction, resulting in a better QoL. Yang et al compared the QoL for two palliative treatments: nutritional palliation with nasogastric (NG) tube, esophageal stenting (2). They found that enteral nutrition and esophageal stenting had similar benefits with regard to survival and QoL. However, enteral feeding via a NG tube is prefered since this is a safe, inexpensive treatment with little complications and therefore a better QoL in comparison with other treatments (2).

Looking at nutrient agents, Miller et al found that progestational agents, such as megastrol (a synthetic derivative of progesterone), have a positive effect on the nutrition of patients with esophageal cancer (7). Even though the mechanism is still unknown, multiple humanbased studies have shown that this drug 2

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stimulates the appetite and therefore weight gain. Unfortunately, this weight gain consisted mainly of adipose tissue, which does not improve performance status nor QoL (7).

Another side-effect are the sedative and hallucinogenic properties of this drug, which do not make it a generally accepted and used drug (7). Hiura et al found that the lack of ghrelin (known as the “hunger hormone”) results in anorexia symptoms in patients undergoing chemotherapy (12). In a later publication by Hiura it was found that an increased intake of ghrelin of 3µg/kg twice a day for one week resulted in significant increased food intake (18.2 kcal/kg/day vs 12.7 kcal/kg/day) and in subjective also an increase in QoL scoring (4). Therefore, ghrelin remains a promising potential therapeutic factor for helping this fragile patient group.

Discussion Nutrition is an important means of improving survival but also quality of life, both in curable as well as in palliative care of esophageal cancer patients. We’ve shown that the effects of various nutrition methods vary. Blazeby et al used the EORTC-30 questionnaire to assess QoL. It is commonly known that questionnaires are a qualitative measure

which can have problems with information bias. As Honda et al explain, the EORTC-30 includes only one item related to appetite and has not many items specific for esophageal cancer patients (13). The QoL improvements shown in studies that rely on such questionnaires may not be representative for all patient groups. And it shows; the use of HEN was shown to improve QoL after treatment for up to 24 weeks (11), however another study found lower QoL compared to control groups (7). It was thought that this might be due to being able to enjoy food rather than be fed through a tube. Whether this was covered in both studies through a questionnaire or other means is unknown.

The data we present here highlights the importance treating patients individually; how patients react to their conditions and their personal needs should be taken into account when making a treatment plan.

In the future, a new questionnaire specific for esophageal cancer patients could be made to allow for a better, more universal way of quantifying QoL in patients. In addition, the use of progestational agents such as ghrelin may be a promising addition to the nutrition in palliative care of esophageal cancer patients.

References 1.

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Arnal MJD, Arenas ÁF, Arbeloa ÁL. Esophageal cancer: Risk factors, screening and endoscopic treatment in Western and Eastern countries. World J Gastroenterol. 2015;21(26):7933–43.

Yang C, Lin H, Hsieh T, Chang W. Palliative enteral feeding for patients with malignant esophageal obstruction: a retrospective study.

BMC Palliat Care [Internet].

2015;14(1):58. Available from: http://bmcpalliatcare.biomedcentral.c om/articles/10.1186/s12904-0150056-5 Bethge N, Sommer A, Vakil N. Palliation of malignant esophageal obstruction due to intrinsic and extrinsic lesions with expandable metal stents. Am J Gastroenterol.

1998;93(10):1829–32.

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Hiura Y, Takiguchi S, Yamamoto K, Takahashi T, Kurokawa Y, Yamasaki M, et al. Effects of ghrelin administration during chemotherapy with advanced esophageal cancer patients: A prospective, randomized, placebocontrolled phase 2 study. Cancer.

2012;118(19):4785–94.

Kubo A, Corley DA, Jensen CD, Kaur R. Barrett’s, Dietary Factors and the Risks of Esophageal Adenocarcinoma and Barrett’s esophagus. Nutr Res Rev. 2011;23(2):230–46.

Arends J, Bachmann P, Baracos V, Barthelemy N, Bertz H, Bozzetti F, et al.

ESPEN guidelines on nutrition in cancer patients. Clin Nutr [Internet].

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Blazeby JM, Farndon JR, Donovan J, Alderson D. A prospective longitudinal study examining the quality of life of patients with esophageal carcinoma.

Cancer [Internet]. 2000;88(8):1781–7.

Available from: http://www.ncbi.nlm.nih.gov/pubmed /10760752 Zeng J, Hu J, Chen Q, Feng J. Home enteral nutrition’s effects on nutritional status and quality of life after esophagectomy. Asia Pac J Clin Nutr. 2017;26(5):804–10.

Poulsen GM, Pedersen LL, Østerlind K, Bæksgaard L, Andersen JR. Randomized trial of the effects of individual nutritional counseling incancer patients. Clin Nutr [Internet].

2014;33(5):749–53. Available from: http://dx.doi.org/10.1016/j.clnu.2013.

10.019 Uitdehaag MJ, Van Putten PG, Van

12.

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Eijck CHJ, Verschuur EML, Van Der Gaast A, Pek CJ, et al. Nurse-led followup at home vs. conventional medical outpatient clinic follow-up in patients with incurable upper gastrointestinal cancer: A randomized study. J Pain Symptom Manage [Internet].

2014;47(3):518–30. Available from: http://dx.doi.org/10.1016/j.jpainsymm an.2013.04.006 Hiura Y, Takiguchi S, Yamamoto K, Kurokawa Y, Yamasaki M, Nakajima K, et al. Fall in plasma ghrelin concentrations after cisplatin-based chemotherapy in esophageal cancer patients. Int J Clin Oncol.

2012;17(4):316–23.

Honda M, Wakita T, Onishi Y, Nunobe S, Miura A, Nishigori T, et al.

Development and Validation of a Disease-Specific Instrument to Measure Diet-Targeted Quality of Life for Postoperative Patients with Esophagogastric Cancer. Ann Surg Oncol. 2015;22(June):848–54.

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Nutrition and/or physical activity as an addition to current cancer treatment?

By Alex Geerlings, Dylan Jongerius and Jens Jacobs Abstract Gastrointestinal cancers are the leading cause of cancer death worldwide. Although the treatment of these cancers has improved over the years, it still has a significant impact on the quality of life of the patients. Current gastrointestinal cancer treatments are associated with various comorbidities and side effects, including diabetes cardiovascular disease, cachexia and fatigue. Optimal nutrition and the physical activity might be key to reduce these comorbidities and side effects. This review explores the potential of nutrition, exercise or a combination of both as a complementary therapy to the current anti-gastrointestinal cancer treatment.

Introduction Even though improvements have been made in extending the overall survival rates of patients that are suffering from gastrointestinal (GI) tumors, GI cancers are still the leading cause of cancer death worldwide. (1) These cancers include oesophageal, gastric, intestinal, colorectal, pancreatic and finally hepatic cancer.

According to the most recent publication of cancer statistics by the World Health Organization, there were 2.808.131 new cases of GI cancer, and 2.030.988 deaths worldwide in 2012. (2) Risk factors that have been found to be associated with GI cancer include smoking, diabetes, obesity and age. (3) It is also important to mention that gastrointestinal cancer has a significant impact on the quality of life. It is significantly associated with various comorbidities including diabetes, cardiovascular disease, and cachexia.

Most of these comorbid conditions have been shown to be a result of chemotherapy/treatment, genetic predisposition and/or lifestyle factors. (4) Because GI cancer patients often have a poor prognosis, even with current therapeutic strategies, lifestyle interventions that may support current anticancer treatments have drawn a lot of attention. The main methods are individualized diets that are adjusted to the patientâ&#x20AC;&#x2122;s needs, as well as the addition of an increase in physical exercise to the

Figure 1 Overview of gastrointestinal tract

current treatment regimen. Such lifestyle interventions have been proposed to ameliorate the negative effects on the quality of life of cancer as well as its treatment. The American Cancer Society has issued guidelines for diet and physical exercise that target cancer survivors. (5) These guidelines include a limited consumption of salt, avoidance of sugary

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drinks and at least 30 minutes of moderate-to-vigorous physical activity.

This review aims to give an overview of the evidence that describes the efficacy of the addition of diet and physical exercise interventions to current anti-GI cancer treatment, and considers whether they are beneficial to be used in both curative and palliative settings.

The interplay between nutrition and cancer treatment Cancer is known to cause significant changes in metabolism and physiology that can affect the nutritional needs of a patient. (6) Common side effects of cancer treatment that can severely affect the nutritional intake are changes in taste and smell, nausea, and disturbances of the gastrointestinal tract. (7) More than 50% of cancer patients have been documented to suffer from substantial weight loss and a poor nutritional status at the time of diagnosis. (8) In addition, the same fraction of all cancer patients experience cachexia. (9) Cachexia is a complex metabolic syndrome that is characterized by the loss of muscle which can go along with or without the loss of fat mass.

Systemic inflammation and a negative protein and energy balance are the underlying mechanisms that cause the overall loss of weight. Taken together, both cachexia and cancer treatment have a significant detrimental effect on the nutritional status of the patient. Therefore the nutritional care should be adjusted to the patientâ&#x20AC;&#x2122;s needs to preserve lean body mass, reverse nutrient deficiencies, and to maximize the quality of life.

The use of dietary supplements to support cancer treatment is controversial. Some supplements may for instance affect the metabolism of a chemotherapeutic drug.

This is the case for folate supplements, which partly counteract the function of folate antagonist methotrexate. (10) Most cancer experts also advise against taking higher doses of antioxidant supplements.

Higher concentrations of antioxidants could interfere with the oxidative damage to cancer cells, which is one of the mechanisms that chemotherapy and radiotherapy require to be effective. (11)

Bozzetti and colleagues have found one important point that plays a role in the relationship between diet and cancer. (12) It is widely acknowledged that most of the tumours rely primarily on glucose as their main source of energy. (13) Ketogenic diets that are high in fat content, moderate to low in protein content, and very low in carbohydrates may take advantage of this finding. Evidence from randomized clinical controlled trials is lacking unfortunately, but preclinical trials tend to show an antitumor effect. One review found that 72% of 29 animal studies determined that a ketogenic diet had an anti-tumor effect which was achieved by either slowing down the progression of the tumor or by extending overall survival times. (14) Comparing the data from preclinical evidence led to the conclusion that the effectivity of a ketogenic diet as an adjuvant cancer therapy seems to strongly depend on the type of tumor, and most importantly, its genetic alterations. (15) Human data was not as optimistic. Of 24 studies, 42% provided evidence that ketogenic diets may have an anti-cancer effect. Seven of the studies showed no evidence of either a pro- or anti-cancer effect. Only one found a pro-tumorigenic effect of the ketogenic diet. (16) Upon initial diagnosis, malnutrition may already be identified by the clinician.

Subsequent nutritional support may help patients in their battle against cancer, while supporting their current treatment.

Nutritional support involves the administration of nutrients either in place of or in addition to the patientâ&#x20AC;&#x2122;s diet that aims to improve the nutritional status of the patient. This support can be provided by two different methods: intravenous nutritional liquids that bypass the gut (parenteral), or by oral administration which can be drank or administered by a tube (enteral). In a study among 111 colorectal cancer patients, Ravasco and colleagues sought to find out whether there is any benefit for the patient when they are given individualized nutritional therapy. (17) More than 91% of the patient group that received individualized nutritional counseling and education about regular foods maintained an adequate

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nutritional status compared to none in the control group. In addition, total intake of food was lower in the control group compared to the group that received counseling. Finally, the median survival in the control group (4.9 y (30% died) compared to 7.3 y (only 8% died) ) and quality of life were worse in comparison.

Overall, it could be concluded that nutritional support was very effective at improving the long-term prognosis of colorectal cancer.

A Cochrane review by Ward et determined the effects of any form of either parenteral or enteral nutritional support in patients undergoing chemotherapy. (18). In addition, the nutritional contents were reviewed. A limited amount of trials suggested that parenteral nutrition was more effective in patients undergoing chemotherapy than enteral nutrition. In addition, there was limited evidence that the supplementation of glutamine would decrease the incidence of infections or the length of hospital stay. Unfortunately, the evidence for their other comparisons remained unclear due to a lack of power.

In summary, there is still a lack of large and well designed trials that assess the effectiveness as well as safety of nutritional support in patients with cancer in general. For specific subsets of cancer patients, nutritional support does seem to have a large beneficial effect on prognosis.

The role of exercise during cancer treatment In the past, chronically ill patients were often told to reduce their physical activity.

This advice was based on the idea that rest and comfort leads to healing. This can be true for some chronic diseases, but cancer patients actually benefit from physical exercise. It has been shown that physical exercise during and after cancer treatment are associated with increased physical functioning and quality of life in addition to a decreased cancer specific and overall mortality. (19) Colorectal cancer (CRC) is one of the most common cancers in both men and

women. Common risk factors for CRC are lack of physical activity and obesity.

Despite significant advancements in CRC treatment, recurrence rates are still high.

To reduce the recurrence rates, assessment of factors that contribute to better outcome of CRC is needed. Je et al. conducted a meta-analysis to assess the effects of physical activity on CRCspecific mortality. They included seven prospective cohort studies which all regarded the effects of exercise on CRC outcome. The meta-analysis concluded that physical activity during diagnosis had a dose-dependent positive effect on mortality. They made categories ranging in level of activity (low, moderate, high) and found that CRC patients with moderate and high levels of physical activity had a 24% and 39% mortality risk reduction, respectively, compared to patients with low physical activity. (20) Besides mortality, exercise can also improve quality of life and physical function.

Advanced treatments like chemotherapy are effective in increasing the life span of a cancer patient. However, this goes at a cost. Patients who undergo these advanced therapies often suffer from low quality of life and decreased physical fitness. A meta-analysis by Buffart and colleagues looked at the effect of exercise on quality of life and physical function in patients with cancer. They found a small but significantly increased effect of exercise on quality of life and physical function both during and and following cancer treatment. Additionally, supervised exercise seemed to have a greater increase in quality of life compared to leisure activities. (21) Exercise can roughly be divided into two groups: resistance and aerobic. According to the American College of Sports Medicine (ACSM) resistance training (RT) can be defined as improving muscular fitness by exercising a muscle or a muscle group against external resistance. This type of exercise could be especially effective in patients with muscle loss. androgen deprivation therapy (ADT) is commonly used in prostate cancer and

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results in muscle loss. A study investigated if RT can help patients who are being treated with ADT. They concluded that RT can help reduce adverse effects of ADT treatment and detrimental psychosocial health issues.

(22) This could mean that this treatment can be continued for a longer time which favors the therapy.

Aerobic training (AT) is defined as: any activity that uses large muscle groups, can be maintained continuously, and is rhythmic in nature. (23) A meta analysis

by Li et al. concluded that AT can be effective in battling cancer-related fatigue.

Another study looked at the effects of aerobic exercise on chemotherapyinduced anemia. Here they found increased hemoglobin levels in the group who participated in AT compared to untrained controls. (24) Nutrition and exercise a combination cancer treatment As described previously, nutrition as well as exercise can be useful additions in antiGI cancer treatment. The combination of these interventions has not yet been discussed. Most of the studies which combine nutrition and exercise are focused on cancer prevention, not as a treatment. The prevention studies are, however, showing some promising results.

(25-28) An adjusted diet and physical exercises is generally associated with lower risk of overall cancer incidence and mortality.

Studies which are looking into the combination of nutrition and exercise as a treatment are mainly focused on cancer survivors. (29-31) With the goal to find the optimal complementary therapy to achieve good weight management and health promotion among the cancer survivors.

This improved health and weight can potentially reduce cancer treatment associated morbidity and mortality in cancer survivors and can enhance their quality of life as well. All these studies showed that there is clearly a benefit of healthy nutrition and regular exercise for reducing risk on varies comorbidities, like; other cancers, cardiovascular disease,

osteoporosis and diabetes, as well as on other side effects, like fatigue and depression, which is very common in cancer survivors. Com Studies which are looking solely into gastrointestinal cancers were not found, but it is likely that the described results are applicable for all cancers as it impacts general health.

Clinical studies in which this combinational therapy is given alongside normal treatment in GI cancer patients are currently lacking. This is much needed as it has potential to be beneficial as a complementary therapy alongside active cancer treatment as well. As the preventive and the treatment in cancer survivors results have shown the potential of such a combinational therapy.

Conclusion This review has given a short overview of the available evidence about the effectiveness of the addition of diet and physical exercise interventions to current anti-GI cancer treatment. Some studies have shown that nutrition can play a beneficial role in long-term prognosis of colorectal cancer. The nutritional support has to be personalized to obtain the best results and to not interfere with their primary anti-cancer treatment. If this is being done the weight and health of the patient can be optimized which can reduce the risk on varies comorbidities and side-effects.

Exercise as a complementary treatment has also shown that it can increase the general health of the patient and thereby the long-term prognosis of colorectal cancer, mainly in a lowered mortality rate but also in an improved quality of life. Evidence for the effectivity of the combination of nutrition and exercise as an addition to treatment is lacking. This combinational therapy has the potential to be effective as the individual components have shown to be beneficial for the patient. To conclude, more research is needed to determine the optimal combination of nutrition and exercise to complement the normal anti-GI cancer treatment.

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17. Paula Ravasco, Isabel Monteiro-Grillo, Maria Camilo; Individualized nutrition intervention is of major benefit to colorectal cancer patients: long-term follow-up of a randomized controlled trial of nutritional therapy, The American Journal of Clinical Nutrition, Volume 96, Issue 6, 1 December 2012, Pages 1346– 1353, https://doi.org/10.3945/ajcn.111.018838 18. Ward EJ, Henry LM, Friend AJ, Wilkins S, Phillips RS. Nutritional support in children and young people with cancer undergoing chemotherapy. Cochrane Database of Systematic Reviews 2015, Issue 8. Art. No.: CD003298. DOI: 10.1002/14651858.CD003298.pub3 19. Doyle, C., Kushi, L. H., Byers, T., Courneya, K. S., Demark-Wahnefried, W., Grant, B., McTiernan, A., Rock, C. L., Thompson, C., Gansler, T. and Andrews, K. S. (2006), Nutrition and Physical Activity During and After Cancer Treatment: An American Cancer Society Guide for Informed Choices. CA: A Cancer Journal for Clinicians, 56: 323– 353. doi:10.3322/canjclin.56.6.323 20. Je, Y., Jeon, J. Y., Giovannucci, E. L. and Meyerhardt, J. A. (2013), Association between physical activity and mortality in colorectal cancer: A meta-analysis of prospective cohort studies. Int. J. Cancer, 133: 1905–1913. doi:10.1002/ijc.28208 21. Laurien M. Buffart, Joeri Kalter, Maike G. Sweegers, Irma M. Verdonck-de Leeuw, Johannes Brug, Effects and moderators of exercise on quality of life and physical

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Recommended dietary pattern to reduce the risk on colorectal cancer D. Draper 1 , B. Vervoort 1 , T. van Wessel 1

Abstract Over the years, several factors have been identified to modify the risk of developing colorectal cancer (CRC).This type of cancer is the third most common cancer and the incidence is expected to increase over the years. It is estimated that around 45% of the cases of CRC is preventable by adhering to a healthy lifestyle and diet plays an important factor in this. In this review, we will discuss several nutritional factors that have been investigated if they affect the risk of CRC. For some nutrients, a clear increase or decrease in risk of CRC was identified, but for others the results obtained from different studies were inconsistent. Conducting systematic reviews and meta-analyses could provide us with more insight in the overall real effect and could even identify possible gaps in our knowledge on nutrition and CRC. Lastly, future studies should focus more on complete dietary patterns instead of focusing on a single nutrient.

and low fat containing food, were less likely to Introduction get CRC compared with people eating a low Colorectal cancer (CRC) is a common type of fiber and high fat diet (3). Human enzymes cancer: it is even the third most occurring usually do not have the capacity to digest cancer worldwide with 1.4 million cases dietary fibers (4). The protective role is diagnosed in 2012(1). The incidence will likely thereby not related to the fibers themselves, increase over the years by 60% and 2.2 million but is thought to be initiated by the dilution of new cases by 2030 as predicted in a study carcinogens, the reduction of the fecal conducted by Arnold et al. Prevention of CRC passage time, scavenging of carcinogenic bile could decrease the burden and mortality, but acids and the production of short-chain fatty also the costs of treating patients that are acids. 13 pooled cohort studies showed that diagnosed with CRC. Lifestyle seems to be an eating fibers lowered risk on CRC by 16%. important factor that influences the risk of However, all the significance disappeared developing CRC and it is estimated that out of after correction for other risk factors, except all cases of CRC, around 45% is preventable for grain fibers (5). Nevertheless, it is advised with a healthy lifestyle(2). An important factor to consume more fibers by eating more fruits, of a healthy lifestyle is diet and several vegetables and whole grains. This might also nutritional factors have been identified that lower the risk of heart diseases (6). increase or decrease the risk for CRC over the

years. In this review, we will summarize the Supplements most important dietary factors that have been identified to increase or decrease the risk of Several minerals and vitamins have shown to CRC and pose recommendations for a healthy be protective for CRC. For example, higher diet. calcium intake of dietary or supplemental might have a positive effect on CRC prevention (7). In animals studies, calcium Dietary factors decreasing the administered as supplements seemed to have risk on CRC development a protective role in the development of CRC (8-10). However, in human studies, Fibers inconsistency was found on the preventive The role of fibers on the risk on CRC was first role of calcium on CRC, in several studies (11, described by Burkitt in 1960. He found a 12). A potential protective role of calcium is relation between people who ate high fiber

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seen when dietary fat intake is high. This fat can promote colon cancer by increasing the ionized fatty acid and bile acid levels in the colon. Here, the presence of calcium can reduce the effect of irritating and toxic molecules on epithelial cells by conversion of free acids to insoluble calcium soaps (7).

Another factor which has been studied extensively is the effect of folic acid on the prevention of CRC (13). Folic acid has been of interest due to its important role in nucleotide synthesis, and biological methylation of DNA, RNA, proteins and phospholipids. Therefore it might have a significant role in cancer prevention. Epidemiologic studies showed that low folic acid intake is associated with several cancers and especially CRC (14).

Clinical trials which are researching the chemopreventive effect of folic acid, also showed inconsequent results (15). For example the study of Cole, et al. (2007) showed no effect on CRC risk when 1 mg folic acid per day was administered. Patients were followed up for 6 or 8 years (16). The study of Wu, et al. (2009) found an positive effect of folic acid, but only when baseline levels were low (17). However, the study of Jaszewski, et al. (2008) claimed a positive effect of folic acid when high dose intake is applied (18). In a randomized clinical trial from China, folic acid also showed to have a positive effect on the prevention of CRC (15). Also, several metaanalysis gave inconsequent results (19, 20).

Dietary factors increasing the risk on CRC development Red and Processed meat Nutrition could not only impact the prevention of CRC, as several studies have also suggested a link between nutritional factors and carcinogenesis. Red and processed meat are both well-known carcinogenic products that increase the risk of several cancers including CRC (21, 22). Red meat and processed meat contain important micronutrients such as vitamin B, iron, zinc and proteins on the one hand which makes it biologically valuable. However meat processing and cooking can results in

formation of carcinogenic chemicals including polycyclic aromatic hydrocarbons (PAH) and heterocyclic aromatic amines (HCA) (23). HCAs and PAHs have shown to be potent carcinogens in animal studies (24) as both are thought to affect the intestine mucosa with genotoxicity and metabolic disturbances (25). Additionally, insufficient digestion of proteins, that are a mayor consistent of red meat, is also thought to affect the intestinal epithelial and increase the risk for CRC. A decrease in protein digestibility increases the amount of protein reaching the colon and the rate of protein fermentation by colonic bacteria. The metabolites produced in excess by these colonic bacteria are potentially toxic and may promote DNA damage in intestinal epithelial cells and therefore disrupt the homeostasis and renewal of the mucosa resulting in CRC (26). This correlates with the fact that most CRC are found in the distal colon and rectum where protein fermentation actively occurs (27). Altogether, these studies indicate a positive association between high consumption of red and processed meats and CRC and therefore the intake of red and processed meat should be avoided or reduced.

Alcoholic drinks Another factor that has been investigated thoroughly if it has an impact on developing CRC is the consumption of alcohol. Several meta-analyses have been conducted to investigate the relation between CRC and alcohol consumption. In one meta-analysis conducted by Fedirko et al. they found no correlation with light consumption (1 or less drinks per day), but moderate and heavy drinking was found to be correlated with an increased risk of developing CRC(28). They even found that consuming more than 4 drinks of alcohol per day increases the risk of CRC with 52%. In humans, ethanol is metabolized to acetyldehyde by three different mechanisms: by microbiota in the gut, by cytochrome P450 2E1 (CYP2E1) and most commonly by alcohol dehydrogenase (ADH)(29). Acetyldehyde is then further metabolized to acetate by aldehyde dehydrogenase (ALDH). In the case of chronic

78

alcohol consumption, ethanol is metabolized

by CYP2E1 instead of ADH and reactive oxygen species (ROS) are also created during this process. Acetyldehyde and ROS can form adducts with DNA and eventually cause CRC by this process and in chronic alcohol consumption these products are prominently present.

insight in existing gaps of knowledge that still exist in the field. A disadvantage of conducting these meta-analyses is that often an overestimation of the effect is found, since there is a risk of publication bias because researchers do not always publish their data if they do not find any effect of the specific nutrient.

Discussion

However, research on nutrition is extremely challenging. First of all, people do not consume isolated nutrients but eat complete meals. Meals are complex combinations of specific nutrients that are likely to interact synergistically (30). Traditional diet related studies only focus on isolated nutrients so there is a lack of studies on complete dietary patterns. Therefore future cohort and casecontrol studies should focus on whole diets including combinations on the earlier mentioned nutrients to design diet recommendations on decreasing CRC risk.

Secondly, cancer originates from a combination of genetic, environmental and lifestyle factors while most studies on cancer and diet have not considered interactions of these factors. Genetic mutations and a specific diets are found to interact with each other. For example, Ma et all showed a correlation in man with homozygous mutations in a folate metabolism gene and alcohol consumption. It was found that the risk on CRC development in man harboring the mutations who drink little or no alcohol decreased with 8-fold compared to man without this specific mutation (31). Therefore a personâ&#x20AC;&#x2122;s genotype could be a very important factor in the composition of an optimal diet.

Also physical activity is associated with decreasing risk on the development of several cancers while a sedentary lifestyle and excess weight are thought to account for 25% of globally cancers (32). It should be further elucidated if the effects of a healthy lifestyle could even amplify each other in reducing the risk on CRC. Altogether, the purpose of new studies on diet and specific diseases should incorporate a combination of diet, lifestyle and genetic background to disclose the best suitable lifestyle to reduce the risk on developing CRC.

Many studies have shown that specific nutrients could initiate the development of CRC. Therefore a well-balanced dietary pattern could be important in reducing the risk to develop this commonly occurring tumor. For example, the intake of fiber rich food as fruit and vegetables should be promoted, even while only fibers derived from grains gave an significant result in preventing CRC. Calcium seemed to have a positive effect in animal trials, but inconsistent effects in human studies. However, calcium had a positive protective effect when people consumed a high fat diet. But, because of the inconsistent results, higher calcium intake might only have a very small effect in preventing CRC. However, since there was no negative impact of higher calcium intake, it can be helpful to consume extra calcium, when fat intake is highly. When folic acid was researched, the same inconsistency was found and even meta-analysis were contradictory. A lot of randomized controlled trials and metaanalysis claimed to see an effect or not, thus were not pointing the same direction. No study was found that showed a carcinogenic effect of folic acid. The reason for this inconsistency is not clear and therefore no strict advice can be provided on folic acid intake to prevent CRC. In addition the consumption of alcohol and red and processed meat should be avoided.

Conducting meta-analyses and systematic reviews on the studies that investigate the isolated effect of a specific nutrient might provide us with more insight on the real effect of that specific nutrient and therefore a recommendation for food intake. Also, conducting these systematic reviews and meta-analyses could provide us with an

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