Amsterdam Medical Student journal (AMSj) | Volume 7 by Amsterdam Medical Student journal

ARTICLES / SUBJECT 101 / TRIAL & ERROR / SOLVING STATISTICS / OBITUARY / IN RETROSPECT / CLINICAL IMAGE / RADIOLOGY IMAGE

7th Edition December 2016

Amsterdam Medical Student journal

P. 4 ARTICLE

P. 18 ARTICLE

Trombolysis Dose Regimens for Peripheral Arterial Occlusions: a Systematic Review K. Ergin

Human Genome Editing: Cure for Patient, Curse for Doctor? J. Broertjes

P. 10 RADIOLOGY IMAGE S. Spijkers & M. Maas

P. 12 ARTICLE Creutzfeldt-Jakob disease mistaken for malingered symptoms S.E. Dekker

P. 23 SOLVING STATISTICS Should I use Pearson’s or Spearman’s correlation coefficient? N. van de Klundert & R. Holman

P. 24 TRIAL & ERROR The Tortoise M.J.B. van Baar

P. 14 IN RETROSPECT Prof. Dr. S. Middeldorp A. Zwagemaker

P. 16 SUBJECT 101 Polycystic ovarium syndrome (PCOS) M. Kok & F. Dusseldorp

P. 17 CLINICAL IMAGE An abnormal ovary A. Schreurs & J. Bramer

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AMSj #6

EDITORIAL I am staring at a blank page, trying to figure out what my last contribution to AMSj will be. I have the honour of writing this editorial, as it will be my final contribution to this amazing journal. Recently, for the first time in my life, I discovered how hard it is to let go of something that is so close to your heart. In one of the previous editorials I wrote about the first question I was asked while setting up AMSj, ‘Are you not contributing to the Publish or Perish by setting up another journal?’ I was astonished by this question, as this had never occurred to me, but nevertheless a fair thing to ask. As the years passed, I started to realise that it is never what you do but why you do it that counts.

About the cover: A heart-shaped villus was found in human placental villous tissue. Marieke van der Linden (VUmc student) discovered the unique villus while doing pregnancy research at the Department of Obstetrics and Gynaecology in Cambridge (UK). The photo won a prize in a Histology Image Contest this December 2016. 20X magnification. Tissue counterstained with haematoxylin, July 2016

AMSj is certainly a journal but the Why of its existence it not to publish but to serve as a mean that enables the older generation to educate the younger. It is about intrinsic motivation and teamwork. And it is about taking care of each other. As for my contribution to AMSj, it has been as little as bringing intrinsically motivated people together. The content is the hard work of the authors and our reviewers. It makes me proud to be able to say that I have worked with such a wonderful team and I would not be honest if I would not acknowledge that it is still with a little heartache that I pass the baton. But at the same time it is also a pleasure to introduce to you our new AMC student Editor-in-Chief, Michiel Schuijt. Meeting Michiel made me feel reassured that he has the capacity and drive to contribute in the further growth of AMSj. AMSj remains in good hands. Thank you for accompanying me on this pleasant journey. Hassina Bahadurzada

DEC 2016

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ARTICLE

THROMBOLYSIS DOSE REGIMENS FOR PERIPHERAL ARTERIAL OCCLUSIONS: A SYSTEMATIC REVIEW K. Ergin Department of Surgery, VU Medical Centre, Amsterdam, The Netherlands

Results: Eight studies were identified from the Medline search. Together with one article cited by an included study, nine studies were included. Reported total dose of rt-PA ranged from 13.0 to 51.2 mg and the therapy duration from 2 to 25 hours. Lysis was achieved in 41% to 83% of the patients. There was a wide range of incidence of amputation, from no amputations up to 26% during the hospital stay and 30-days follow-up. Reported mortality rates ranged from 2.3% to 35%. Conclusions: High-dose recombinant tissue plasminogen activator with long therapy duration in relatively younger patients reported the best results. Thrombolytic therapy with a prolonged infusion time may be a safer alternative, based on fewer complications and better lysis rates in patients. More bleeding events are reported in patients with an initial bolus injection. Key words: Thrombolytic therapy, fibrinolytic agents, tissue plasminogen activator and peripheral arterial disease.

INTRODUCTION

ABSTRACT Background: Current guidelines recommend treating peripheral arterial occlusions with thrombolytic therapy. Many thrombolytic agents are available. Recombinant tissue plasminogen activator (rt-PA) is the most frequently used agent. There is no consensus about the optimal recombinant tissue plasminogen activator dose regimen. This literature review was performed to analyse the most common dose-regimens of thrombolytic therapy for peripheral arterial occlusions. Methods: The medical database Medline was searched to identify all studies reporting rt-PA dosing and the treatment outcome. Articles related to the included studies were also examined using a snowball search. The publication year, number of patients, mean age, the gender of the patients, dose-regimen, mean total dose and therapy duration were extracted from the included studies. Outcomes were successful lysis, bleeding events, amputations and mortality within 30 days. P. 4

Under pathological conditions, a thrombus can obstruct blood flow in arteries in the arm or leg, a condition termed peripheral arterial occlusion.1 This leads to hypoxia, or worse anoxia, which may cause tissue infarction. Patients with acute limb ischemia usually present with the five Pâ&#x20AC;&#x2122;s of ischemia: paraesthesia (abnormal sensation such as numbness), pain, pallor (pale skin), pulselesness (lack of blood flow) and paralysis (sensory loss). This is a life-threatening condition and immediate treatment is critical.2 Thrombolytic therapy, also known as thrombolysis, is the first choice of treatment. During thrombolysis high doses of thrombolytics are infused through a catheter, which is placed intra-arterially nearby the thrombus to break up the blood clot eventually leading to reperfusion of the artery. Providing intravenous thrombolysis with short door-to-needle time is the result of a complex process that requires specific work standards. Standard protocols are therefore very important during thrombolysis. High doses of thrombolytics can cause bleeding events. Major haemorrhagic complications have been reported up to 3% and minor complications up to 30%. 3

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Fibrinolysis is an important part of the haemostatic system that functions to dissolve fibrin clots. The fibrinolytic system consists of many enzymes, including plasminogen. Tissue plasminogen activator is a protein, which enables plasminogen to be converted to plasmin, an important enzyme involved in clot breakdown.4 Due to recombinant DNA technology recombinant tissue plasminogen activator (rt-PA), a synthetic form of tissue plasminogen activator, is available for clinical use. Rt-PA is one of the most frequently used and the most preferred among the thrombolytic agents. There are a variety of dose-regimens available and there is no consensus on this topic. Over the years a limited amount of clinical trials were published about thrombolysis dosing for peripheral arterial occlusions. The majority of studies on thrombolytic therapy were performed to compare this therapy to surgical revascularization.5,6,7 An optimal dose-regimen is necessary for improved patient outcome. It is therefore important to examine the differences in clinical success, morbidity and mortality rates and patient outcome between the dose-regimens. The primary aim of this review is to investigate the most common thrombolysis dose-regimens for peripheral arterial occlusions.

METHODS A systematic review was performed following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement in a period of four weeks. Search strategy The medical database Medline was searched using the medical subject headings (MeSH): thrombolytic therapy, arterial occlusive diseases, peripheral arterial disease, fibrinolytic agents and tissue plasminogen activator. The MeSH terms were specified using the subheadings: drug therapy, methods, adverse effects and administration and dosage. The following combination of the MeSH terms and subheadings were used: “Tissue Plasminogen Activator/ administration and dosage” AND (((“Fibrinolytic Agents/ administration and dosage”) OR (“Arterial Occlusive Diseases/ drug therapy” OR (“Thrombolytic Therapy/ adverse effects” OR “Thrombolytic Therapy/ methods”)) OR “Peripheral Arterial Disease/ drug therapy”. Relevant DEC 2016

articles were firstly selected based on title and abstract. An additional snowball search of citing and cited articles provided more studies. Study selection Inclusion criteria. Studies reporting on thrombolytic therapy for peripheral arterial occlusions with rt-PA were selected based on abstract review. To be eligible, studies had to investigate thrombolytic therapy with the fibrinolytic agent recombinant tissue plasminogen activator and report their dose-regimen. Selected studies had to report patient demographics, amount of complete lysis and complications within 30 days. Exclusion criteria. Studies reporting venous or central artery occlusions (stroke, myocardial infarct, pulmonary embolism) were excluded. Also excluded were studies investigating different fibrinolytic agents such as urokinase, streptokinase and tissue plasminogen activator. Studies written in a foreign language and without full text availability or studies that could not be obtained within two weeks with the Interlibrary Loan (IBL) were excluded. Review articles, meta-analyses, case reports and technical reports were considered not eligible. Data extraction The study design, publication year, number of patients, mean age and the gender of the patients were extracted from the included studies, in addition to the recombinant tissue plasminogen activator dose regimen and infusion technique, mean total dose and therapy duration in hours. All included studies reported various outcome measures. The outcome measures extracted from the studies were the percentage of patients with successful lysis, bleeding events (minor and major), amputations and mortality within 30 days. The primary endpoint was clinical success, which is the percentage of successful lysis. Secondary endpoints were morbidity and mortality of patients undergoing thrombolysis. Some of the studies compared thrombolysis to surgery or different fibrinolytic agents. Only data concerning treatment with rt-PA were extracted from these studies.

RESULTS The search strategy identified 2175 articles of which 2167 were excluded based on P. 5

ARTICLE

eligibility criteria. Together with one relevant articles cited in one of the eight selected studies (Grip et al. 2014) a total of nine studies were included. Study flow and reasons for exclusion are presented in Figure 1. Outcomes Outcomes are depicted in Table II. All included studies used various outcome measures. The outcome measures in common were bleeding complications, amputation rate and mortality. Reported total dose of rt-PA ranged widely from 13.0 to 51.2 mg, when the second highest dose was 36.0 mg. Patients received the thrombolytic agents from 2 to 25 hours. KĂźhn et al. 2011 and Ward et al. 1994 stated the total dose and therapy duration as averages in the article.11,12 Complete lysis, which is described as breaking down of the occluding thrombus, was achieved in 83% of patients in Berridge et al. and 41% in Kuoppala et al. Number of amputations and mortality were reported during the hospital stay and 30-days follow-up. Amputation was not reported in Berridge et al. 2009 and Shortell et al. 2001.13,16 A wide range was found, from no amputations up to 26% of amputations. Mortality was reported in all included studies and ranged from 2.3% to 35%.

DISCUSSION Acute peripheral occlusion is a serious condition and the incidence is approximately 1,5 cases per 10,000 persons per year.17 Despite improvements in the revascularization techniques, high morbidity and mortality remain a problem.2 Early diagnosis and treatment are essential for the ischemic extremity to recover. Surgical thromboembolectomy and bypass grafting were the mainstays of therapy for many years.18 Subsequently; thrombolytic therapy has become the treatment of choice. Clinical trials comparing thrombolytic therapy to surgical revascularization conclude thrombolysis is a safe and effective alternative to operation in the initial treatment of patients diagnosed with acute limb-threatening peripheral arterial occlusion.7 However, thrombolysis is associated with a higher frequency of haemorrhagic complications and surgical revascularization is preferred in patients with chronic ischemia P. 6

Tissue plasminogen activator/administration and dosage AND Fibrinolytic Agents/administration and dosage) OR Arterial Occlusive Diseases/drug therapy OR Thrombolitic Therapy/adverse eďŹ&#x20AC;ects OR "Thrombolitic Therapy/methods OP "Peripheral Arterial Disease/drug therphy

2175 articles 2176 excluded after screening of abstracts 8 articles 1 included after snowball search 9 articles

Figure 1. Flow chart illustrating study selection

(>14 days). 5,6 Currently, patients are assigned to a therapy using the Rutherford classification, a clinical staging system for describing peripheral arterial disease.19 Thrombolytic therapy is the treatment of choice for patients of categories I and IIa.20 Recombinant tissue plasminogen activator is a thrombolytic agent produced by recombinant DNA technology. After many trials comparing rt-PA to other agents, such as streptokinase and urokinase, rt-PA is the most frequently used thrombolytic agent. Studies report intra-arterial delivery of the drug is the preferred infusion technique.21 However, it is not possible to draw conclusions regarding the most effective dose-regimen based on the current evidence. There are different infusion techniques, including low-dose, high-dose, bolus injection and pulse-spray (brief high-pressure injection of the drug). It is important to reach a clinical consensus on the most effective thrombolysis dosing and infusion technique to minimize complications and achieve the best degree of lysis. A systematic search revealed nine clinical studies reporting on rt-PA dosing. AMSj #7

FIRST AUTHOR

YEAR

STUDY DESIGN

AGE, MEAN Y

MALE (%)

DOSE-REGIMEN

Plate et al.

2006

RCT

58 63

A: pulse spray (15 mg/h) during 2 hours B: low-dose (0.5mg/h) during 48 hours

Kurtoglu et al.

2001

Clinical trial

Pulse spray: 5 mg bolus followed by 0.05 mg/kg/hr during 12 hours.

Kuoppala et al.

2008

Retrospective cohort study

220

1-2 mg/h first 4 hours followed by 0.5-1 mg/h

Kühn et al.

2011

Retrospective cohort study

129

2.5-5 mg bolus followed by 20mg/1224h

Ward et al.

1994

Clinical trial

27 23

71 69

48 52

A: 1.0 mg/h B: 20 mg bolus followed by 1.0 mg/h

Shortell et al.

2001

Retrospective cohort study

2 mg/h

Decrinis et al.

1993

Clinical trial

210

10 mg over 6 hours

Grip et al.

2014

Clinical trial

232

1-2 mg/h first 4 hours followed by 0.51.0 mg/h

Berridge et al.

1990

Clinical trial

0.5 mg/h

Table I. Dose regimen N: number of patients; RCT: randomized clinical trial; NS: not stated in the article; A/B: two groups of patients receiving diﬀerent doses of rt-PA

FIRST AUTHOR

YEAR

TOTAL DOSE, THERAPY DURATION, MEAN, MG MEAN, HOURS

Plate et al.

2006

A: 36.0 B: 13.0

2 25

Kurtoglu et al.

2001

Kuoppala et al.

2008

Kühn et al.

COMPLETE LYSIS, %

BLEEDING AMPUTAEVENT, % TION, %

MORTALITY %

78.0 67.0

28 24

5.0 3.0

10.0 11.0

65.0

0.0

28.0

20.5

41.0

26.0

35.0

2011

31.9±16.1*

13.3±11.0*

73.6

10.6

2.3

Ward et al.

1994

A 25.0±9.8 B 31.0±7.9

26.7±9.4* 14.4±7.9*

74.0 70.0

4 35

15.0 7.5

4.0 13.0

Shortell et al.

2001

51.2

20.9

82.0

5.0

Decrinis et al.

1993

10.0

59.0

17.0

14.0

Grip et al.

2014

21.0

77.0

14.0

9.0

Berridge et al.

1990

83.0

13.0

Table II. Outcomes within 30 days NS: not stated in the article; A/B: two groups of patients receiving diﬀerent doses of rt-PA; * reported as average range in the article DEC 2016

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ARTICLE

In this group of studies published between 1990 and 2014 different treatment strategies were performed. Thrombolytic therapy had a steep learning curve over the past years. Techniques have branched in several directions, which in turn lead to more experience. Modern imaging techniques such as perfusion CT, diffusion- and perfusion weighted MRI, CT angiography and MR angiography were developed in addition to the conventional Doppler ultrasound and arteriography. Imaging-based decision-making is now an inevitable part of thrombolytic therapy. Plate et al. 2006 compared the pulse-spray method to the low-dose strategy.8 The pulsespray method consists of brief high-pressure injection of tPA and is used to shorten the therapy duration. Patients in the pulse spray group had the shortest (2 hours) therapy duration. However, 66% of the patients were treated with additional low-dose thrombolysis when lysis was not achieved. The mortality was lower in the pulse-spray group, in spite of more bleeding events and amputations. Kurtoglu et al. 2001 also investigated the pulse-spray method.9 The high mortality rate (28%) in this study could be explained by the small population. The short follow-up may have led to no amputations being reported. Therefore it is not possible to conclude that the pulse-spray method is significantly better than the low-dose method. An important advantage could be the shorter hospital stay and lower costs. Two very similar studies were Kuoppala et al. 2008 and Grip et al. 2014.10,16 Both used the same dose-regimen, had the same mean total dose (20.5 and 21 mg) and included more than 220 patients. Grip et al. 2014 had longer therapy duration, but reported less bleeding events, amputations and mortality rate.16 Lysis was also achieved in more patients. The differences

in these outcome measures are important and could possibly be explained by the infusion time (20 versus 25 hours). It has been reported that high-dose protocols shortens the necessary infusion time to achieve complete lysis, though increases the complication risks, therefore lowdose thrombolysis during a prolonged therapy is a safer alternative.22 There was a wide range in reported bleeding events. This variation may be caused by varying haemorrhage classification. Some studies chose to report only major haemorrhages when others also included minor bleedings at puncture site. Nevertheless, an important outcome in Ward et al. 1994 is noted.12 Two dosages were compared, group A received 1.0 mg/h t-PA and a 20 mg bolus was added to group B. Bleeding occurred in 4% of the patients in group A and 35% in group B. Accordingly a large initial bolus of t-PA causes significantly more bleeding events. On the other hand, twice more amputations were performed in group A. Plate et al. 2006 did not use an initial bolus and reports least amputations (3-5%). 8 An extended follow-up period is necessary to investigate the effect of t-PA dosing on amputation rates. All included studies reported mortality rates. KĂźhn et al. 2011 states a minimum mortality of 2.3% and seems preferable compared to Shortell et al. 2001 with a mortality of 5%.11,13 Additionally, the mean total dose Shortell et al. 2001 used is 1.2 mg.13 However the outcome of this study is intriguing. 16% of the patients developed a bleeding event and an impressive 82% achieved complete lysis. Berridge et al. 2009 reports up to 83% lysis, but over twice more deaths among patients.16 Hence Shortell et al. 2001 could be the most effective dose-regimen, due to lower mortality. A factor that needs to be considered is the mean age (64 years) of the patients in this

Table III. Rutherford classification CATEGORY

DESCRIPTION/PROGNOSIS

I. Viable

Not immediately threatened

II. Threatened a) Marginally b) Immediately

Salvageable if promptly treated Salvageable with immediate revascularization

III. Irreversible

Major tissue loss or permanent nerve damage inevitable

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study. The mean age of the patients included in Shortell et al. 2001 (64 years) is relatively low.13 It has been reported that thrombolytic therapy in elderly patients should not be avoided, but pursued cautiously, due to poorer outcome. Increased age has been associated with a higher risk for complications such as haemorrhagic events and amputations.23, 24 Several limitations to this review need to be addressed. Only articles published in English and with full text availability were included. Due to this construction, relevant studies could have been missed by our search. As mentioned earlier, studies on thrombolysis dosing are extremely limited. To retrieve more studies an additional publication year filter was not applied to the search. Therefore the included studies have a wide range of publication years. Thrombolytic therapy changed over the years and more recent studies could have been of better use. Furthermore it is difficult to make conclusions based on these data series due to the variations. This review examines the different dose regimens of rt-PA. However, there are many more fibrinolytic agents, including streptokinase and urokinase. Clinical trials have examined and compared these agents. Further research is necessary to investigate various dose-regimens and infusion techniques for an optimal patient outcome. Advanced medical care is reaching towards patient directed therapy. It is suggested that thrombolysis should be adjusted for elderly patients. Studies on thrombolytic therapy for more patient types and groups are important to improve clinical success.

However, these findings come from small studies and in order to reach final conclusions on this topic further research is necessary.

REFERENCES 1.

2. 3.

CONCLUSIONS High-dose recombinant tissue plasminogen activator with long therapy duration in relatively younger patients reported the best results. Thrombolytic therapy with a prolonged infusion time may be a safer alternative, due to fewer complications and more lysis in patients. Some of the studies chose to add an initial bolus injection of rt-PA to the treatment. More bleeding events are reported in patients with an initial bolus injection and the safety and necessity of this addition should be investigated. DEC 2016

10.

11.

12.

Wolberg, A. S., & Campbell, R. A. (2008). Thrombin generation, fibrin clot formation and haemostasis. Transfusion and Apheresis Science : Official Journal of the European Society for Haemapheresis, 38(1), 15–23 Ouriel K. Thrombolytic Therapy for Acute Arterial Occlusion. 2002;7515(01):32–9 Levine MN, Raskob G, Beyth RJ, Kearon C, Schulman S. Hemorrhagic complications of anticoagulant treatment: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest. 2004;126(3 Suppl):287S-310S. Gurman P, Miranda OR, Nathan A, Washington C, Rosen Y, Elman NM. Recombinant tissue plasminogen activators (rtPA): a review. Clin Pharmacol Ther. 2015;97(3):274-85. Results of a prospective randomized trial evaluating surgery versus thrombolysis for ischemia of the lower extremity. The STILE trial. Ann Surg. 1994;220(3):251-66; discussion 266-8. Ouriel K, Veith FJ, Sasahara AA. A comparison of recombinant urokinase with vascular surgery as initial treatment for acute arterial occlusion of the legs. Thrombolysis or Peripheral Arterial Surgery (TOPAS) Investigators. N Engl J Med. 1998;338(16):1105-11. Ouriel K, Shortell CK, DeWeese JA, et al. A comparison of thrombolytic therapy with operative revascularization in the initial treatment of acute peripheral arterial ischemia. J Vasc Surg. 1994;19(6):1021-30. Plate G, Jansson I, Forssell C, Weber P, Oredsson S. Thrombolysis for acute lower limb ischaemia--a prospective, randomised, multicentre study comparing two strategies. Eur J Vasc Endovasc Surg. 2006;31(6):651-60. Kurtoğlu M, Granit V, Necefli A, Güloğlu R. [Thrombolysis of acute arterial occlusion with rt PA]. Ulus Travma Derg. 2001;7(3):158-62. Kuoppala M, Franzén S, Lindblad B, Acosta S. Longterm prognostic factors after thrombolysis for lower limb ischemia. J Vasc Surg. 2008;47(6):1243-50. Kühn J-P, Hoene A, Miertsch M, et al. Intraarterial recombinant tissue plasminogen activator thrombolysis of acute and semiacute lower limb arterial occlusion: quality assurance, complication management, and 12-month follow-up reinterventions. AJR Am J Roentgenol. 2011;196(5):1189-93. Ward AS, Andaz SK, Bygrave S. Peripheral P. 9

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22.

23.

24.

thrombolysis with tissue plasminogen activator. Results of two treatment regimens. Arch Surg. 1994;129(8):861-5. Shortell CK, Queiroz R, Johansson M, et al. Safety and efficacy of limited-dose tissue plasminogen activator in acute vascular occlusion. J Vasc Surg. 2001;34(5):854-9. Decrinis M, Pilger E, Stark G, Lafer M, Obernosterer A, Lammer J. A simplified procedure for intra-arterial thrombolysis with tissue-type plasminogen activator in peripheral arterial occlusive disease: primary and long-term results. Eur Heart J. 1993;14(3):297-305. Grip O, Kuoppala M, Acosta S, Wanhainen A, Åkeson J, Björck M. Outcome and complications after intra-arterial thrombolysis for lower limb ischaemia with or without continuous heparin infusion. Br J Surg. 2014;101(9):1105-12. Berridge DC, Gregson RH, Makin GS, Hopkinson BR. Tissue plasminogen activator in peripheral arterial thrombolysis. Br J Surg. 1990;77(2):179-82. Creager MA, Kaufman JA, Conte MS. Clinical practice. Acute limb ischemia. N Engl J Med. 2012;366(23):2198-206. Yeager RA, Moneta GL, Taylor LM, Hamre DW, McConnell DB, Porter JM. Surgical management of severe acute lower extremity ischemia. J Vasc Surg. 1992;15(2):385-91; discussion 392-3. Rutherford RB, Baker JD, Ernst C, et al. Recommended standards for reports dealing with lower extremity ischemia: revised version. J Vasc Surg. 1997;26(3):517-38. Morrison HL. Catheter-directed thrombolysis for acute limb ischemia. Semin Intervent Radiol. 2006;23(3):258-69. Robertson I, Kessel DO, Berridge DC. Fibrinolytic agents for peripheral arterial occlusion. Cochrane database Syst Rev. 2013;12:CD001099. 22. Falkowski A, Poncyljusz W, Samad RA, Mokrzyński S. Safety and efficacy of ultra-highdose, short-term thrombolysis with rt-PA for acute lower limb ischemia. Eur J Vasc Endovasc Surg. 2013;46(1):118-23. 23. Kim D, Ford GA, Kidwell CS, et al. Intra-Arterial Thrombolysis for Acute Stroke in Patients 80 and Older: A Comparison of Results in Patients Younger than 80 Years. AJNR Am J Neuroradiol. 2007;28(1):159-163. 24. Mishra NK, Ahmed N, Andersen G, et al. Thrombolysis in very elderly people: controlled comparison of SITS International Stroke Thrombolysis Registry and Virtual International Stroke Trials Archive. BMJ. 2010;341(nov23_1):c6046.

RADIOLOGY IMAGE

RADIOLOGY IMAGE S. Spijkers & M. Maas

PATIENT DATA

· · · · ·

Age: 65 years Gender: female Medical history: hypertension, hypothyroidism Initial presentation: fever for four weeks. Dyspnoea, increasing when lying down. Chest pain. Echocardiography: right ventricle dilatation.

CHECKLIST CT 1. 2.

4. 5.

Soft tissues, especially: a. Axillary LNs b. Breast (malignant lesions?) Mediastinum in four regions: a. From the aortic arch cranially (LNs? thymoma/struma?) b. Hilar region (configuration and size of vessels, lobulated and enlarged?) c. Heart and coronary arteries (sclerosis?) Parenchyma of the lung: a. Normal branching pattern and caliber of vessels? Normal contrast distribution? b. Vascular oligemia only at interlobar fissures? Bullae? c. Any suspicious lung foci? Inflammatory infiltrates? Pleura: a. Plaques, calcification, pleural fluid, pneumothorax? Bones (vertebrae, scapula, ribs): a. Normal structure of marrow? b. Degenerative osteophytes? c. Focal lytic or sclerotic processes? d. Stenosis of the spinal canal?

! P. 10

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QUESTION 1 Based on the initial presentation, which diagnosis/diagnoses should not be considered? Multiple answers possibly. A. Pneumonia B. Pneumothorax C. Pulmonary embolism D. Acute coronary syndrome E. Heart failure Hint: consider the fact that the symptoms have been ongoing since four weeks.

QUESTION 2 An ultrasound of the proximal leg veins is performed to rule out DVT. Are there anomalies visible on this ultrasound? A. Yes B. No Hint: do veins normally remain open during compression?

QUESTION 3 What anomalies are visible on the CT-scan? A. Aorta dissection B. Pneumothorax C. Pleural mass D. Pulmonary embolism E. Pleural effusion Hint: do you see an abnormal filling with contrast?

ULTRASOUND Ultrasound of the proximal v. femoralis communis: left without pressure on the leg, right with pressure on the leg

ANSWER ON P. 25

DEC 2016

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ARTICLE

CREUTZFELDTJAKOB DISEASE MISTAKEN FOR MALINGERED SYMPTOMS S.E. Dekker, VU University Medical Center, Amsterdam, The Netherlands

SUMMARY Creutzfeldt-Jakob disease (CJD) is a diagnostic challenge because of a combination of early-state atypical neuropsychiatric symptoms and rareness of the disease. In neuropsychiatric literature there is little documentation of CJD as differential diagnosis for malingering. This case report presents a CJD patient initially suspected of malingering because of bizarre unexplained symptoms, with a false positive outcome on a validated structured inventory for malingered symptoms (SIMS).

CASE PRESENTATION A 47-year-old female was presented at the neurology department with a progressive tremor of her right hand, atypical gait and aphasia. She had previously complained about mild headache, diminished concentration and problems understanding work-related tasks. The patient had a normal physical neurological examination and brain Magnetic Resonance Imaging (MRI) showed no specific abnormalities. At hospital admission, Montreal Cognitive Assessment (MoCa) was 16 (n>26) and mini–mental state examination (MMSE) was 21 (n>17). Because the P. 12

patient showed elevated thyroid stimulating hormone (TSH) levels, positive anti-thyroid peroxidase (TPO) antibodies and signs of encephalopathy on electroencephalography (EEG), the working diagnosis was Hashimoto encephalitis. However, after five days of intravenous methylprednisolone treatment, her condition did not improve and the initial diagnosis was rejected. Other organic causes were considered to be unlikely, because of a lack of any typical pattern and negative results on liquor and blood infection tests. The results of a specific liquor protein test would not be available for one week. Subsequent diagnostic interventions followed a twofold strategy, focusing on both possible organic and psychiatric/psychological etiologies. A psychiatric consultation was requested to determine if the patient exaggerated, imitated or simulated the illness. Heteroanamnesis showed no positive psychiatric or neurologic family history and no external motive to fake an illness. The patient had no history of unexplained symptoms or somatization. The nursing staff observed signs of incongruent behavior (e.g. using her toothpaste as hand cream). In psychiatric assessment, we saw a 47-year-old Caucasian female who rose from her chair, trembling. She had a surprised expression on her face during the entire interview. There was a salient discrepancy in the seriousness of her symptoms and her mild suffering. She missed the hand of the investigator while attempting to shake it. She was friendly in her contact. During the interview, the patient had problems finding her words, but she said she understood the questions correctly. She answered with short sentences or ‘yes’ or ‘no’. She was fully conscious, she showed no apparent delusions and did not report hallucinations. She was able to maintain attention without loss of vigilance. She knew the name of the current month, but repeated the name of the month as an answer to other questions about orientation. Her mood was normal with a normal modulating affect and she had no thoughts of death. As additional questionnaire she took the structured inventory of malingered symptomatology (SIMS). This structured inventory is a self-reporting measure designed to screen for malingering of psychiatric or neuropsychological symptoms.1 The use of SIMS has been validated in a variation of studies, with sensitivity, specificity and positive predictive power AMSj #7

rates of ≥0.90.2-6 SIMS consists of 75 yes- or no questions. Our patient scored 51 points, which is a score three times above cut-off, indicating malingered symptomatology as differential diagnosis. The staff grew increasingly concerned with her condition during the subsequent days. The patient continued to show incongruence of symptoms and had discrepancy in symptom severity. This clinical presentation combined with a SIMS of 51 points, increased the likelihood of malingering. This thought was communicated within the medical team, but not yet communicated to the patient or her relatives, because of the pending results of the previously mentioned specific liquor protein test. After a week, liquor results were unexpectedly positive for 14-3-3 protein, with elevated tau protein and normal levels of phosphorylated-tau. Patient was diagnosed with the sporadic variant of CJD. Two weeks after hospital admission, her MMSE score further decreased to 8 points. The patient developed constant monoclonal activity and died within two months after hospital dismissal in a hospice.

DISCUSSION The patient presented in this case report showed a combination of neurologic and psychiatric symptoms. Patients suspected for neuropsychiatric disorders should have diagnostic interventions focusing on both organic and psychiatric etiologies. For example, Taber et al. identified several clinical indications for brain imaging in psychiatric patients, such as: psychiatric symptoms outside “clinical norms” or with any unusual presentation or course, and focal neurological signs, as presented in this case report.7 MRI may be a valuable diagnostic tool for CJD, with a characteristic finding of the pulvinar sign as defined by increased intensity in the pulvinar relative to the anterior putamen.8 In this unique case, however, the patient did not exhibit typical MRI or EEG abnormalities that were indicative of CJD. Rather, the patient continued to show incongruent symptoms and discrepancies in symptom severity, which led the psychiatric team to employ the SIMS test for malingering. While SIMS may be considered a validated measurement to detect malingering, in this case it failed to differentiate malingering from CJD. The literature reports differential diagnoses such as dementia, mood and psychotic disorders DEC 2016

in subclinical CJD 9-10, but testing for malingering or factitious disorder is unreported. The false positive SIMS score incorrectly suggested a tendency to exaggerate symptoms, indicating that SIMS might not be applicable in patients with yet undiscovered neurodegenerative disorders. Therefore physicians and psychologists should be reluctant to use SIMS in neuropsychiatric populations. Further research for application of SIMS or other screening tools for malingering is necessary.

TAKE HOME MESSAGES

· Creutzfeldt-Jakob disease is a diagnostic challenge due to a combination of early-state atypical neuropsychiatric symptoms and rareness of the disease. · The current case suggests that SIMS might not be applicable in patients with yet undiscovered neurodegenerative disorders. · While clinical tests such as MRI and EEG and screening tools such as SIMS, may help clarify diagnostic questions, these tests alone cannot make or reject a severe neuropsychiatric diagnosis such as Creutzfeldt-Jakob disease. REFERENCES 1.

Smith GP & Burger GK. Detection of malingering: Validation of the Structured Inventory of Malignered Symptomatology (SIMS). Journal of the Academy of Psychiatry and the Law 2007;25: 180-183. Merckelbach, H. & Smith, G.P. Diagnostic accuracy of the Structured Inventory of Malignered Symptomatology (SIMS) in detecting instructed malingering. Archives of Clinical Neuropsychology. 2003;18, 145-152. Wisdom, N.M., Callahan, J.L., Shaw, T.G. Diagnostic utility of the structured inventory of malingered symptomatology to detect malingering in a forensic sample. Archives Clinical Neuropsychology. 2003;25(2):118-25. Edens, J.F., Otto, R.K., Dwyer, T. Utility of the structured inventory of malingered symptomatology in identifying persons motivated to malinger psychopathology. Journal of the American Academy of Psychiatry and the Law. 1999;27(3):387-96. Edens, J.F., Poythress, N.G., Watkins-Clay, M.M. Detection of malingering in psychiatric unit and general population prison inmates: a comparison of the PAI, SIMS and SIRS. Journal of Personality Assessment. 2007;88(1):33-42.

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10.

Lewis, J.L., Simcox, A.M., Berry, D.T. Screening for feigned psychiatric symptoms in a forensic sample by using the MMPI-2 and the structured inventory of malingered symptomatology. Psychology Assessment. 2002;14(2):170-6. Taber KH, Hurley RA, Yudofsky SC. Diagnosis and treatment of neuropsychiatric disorders. Annu Rev Med. 2010;61:121-33. Zeidler M, Sellar RJ, Collie DA, et al: The Pulvinar Sign on variant Creutzfeldt- Jacob Disease. Lancet 2000;55:1412â&#x20AC;&#x201C;1418 Shekhawat LS, Kumar S, Ramdurg SI. et al. New Variant Creutzfeldt-Jacob Disease Presenting With Catatonia: A Rare Presentation. J Neuropsychiatry Clin Neurosci 2012;24;3:51-53. Wall CA, Rummans TA, Aksamit AJ. et al. Psychiatric Manifestations of Creutzfeldt-Jakob Disease: A 25-Year Analysis. J Neuropsychiatry Clin Neurosci 2005;17(4):489-495

IN RETROSPECT

PROF. DR. SASKIA MIDDELDORP Interview by Anne-Fleur Zwagemaker, medical student, Academic Medical Centre, Amsterdam, The Netherlands

Looking back at my career â&#x20AC;&#x201C; hopefully I can still look ahead as well! At the age of 14, I already knew I wanted to study medicine and since then I luckily never doubted that choice. I studied medicine here in Amsterdam at the AMC. It was during clinical lectures that I found out about my interest in internal medicine and my enthusiasm for this specialty was confirmed during my clinical rotations. My dream was to become a really good internist and work in a large, peripheral hospital, like the OLVG. I was eager to work hard and to see extraordinary patients in a broad specialty like internal medicine, but research was not one of my aspirations. Some people genuinely enjoy unravelling every part of a cell, at a level were they almost become a part of that cell. Although I admire such commitment to basic research, it was not particularly my thing. When I had to do my four-month scientific internship, I took the opportunity to go abroad and I ended up performing a genetics study about Tourette syndrome in the United States, a truly great (and fun) experience. As ANIOS internal medicine at the AMC, I applied for the trainee program. There were three positions available, but we were with eight applicants and I had little research aspirations. When I was asked by head of the residency program (prof. Arisz) if I wanted to obtain a PhD position first, I rejected the offer and I was not selected. However, when a few weeks later a position became vacant, I was the first one they called to offer a residency in internal medicine. Apparently, my honesty and clearly outspoken ambition to become an excellent internist were being appreciated. I believe it is vital to follow your heart, as it would not help you to do some-

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thing that you actually do not like to do. Work as a doctor comprises a substantial part of your life, so your work should give you energy. The first time I really performed research was also more or less obligatory since we had to publish at least one paper during residency. Factor V Leiden had just been discovered and this topic particularly appealed to me as I acknowledged the clinical relevance and importance in this research field. At this point I started to sincerely enjoy research and the good atmosphere between colleagues contributed to my enthusiasm. A research team that is productive and willing to give the right guidance is particularly important in this. Luckily, I was being offered a number of great opportunities, such as speaking at conferences and collaborating with investigators from other centres. I had to work hard, but my work was being appreciated and I saw my results being translated into clinical practice, which was very satisfying. What I like about research, and especially observational and interventional research, is the clinical relevance of such studies. I feel the urgency to answer questions that pregnant women with thrombophilia ask me every day. Although some persistence is needed, it feels great when you are finally able to publish research that is a substantial contribution to the medical field. What are my ambitions for the future? I still have at least 18 years to go and I feel the need to

keep working on clinical trials, to give evidence-based answers that have significant clinical implications. Being able to combine my clinical work with research makes my work very diverse and interesting, although it may seem impossible at first to combine all these things. In my experience, you can design your life in a way that you can have both a successful career and a pleasant private life. Little things like having groceries delivered, having a dishwasher, or investing in a cleaning service can help you spend more time doing the things that you want to do. Who are you? Saskia Middeldorp (1966). When and where did you graduate? - Medicine: University of Amsterdam, 1992 - Registration as internist: AMC, 1999 - PhD thesis: “Clinical and laboratory studies in genetic and acquired thrombotic states”, 2000 - Registration as internist vascular medicine: AMC, 2002 What is your area of expertise? Thrombosis and haemostasis. What is your current position? Professor of vascular medicine at the AMC and co-chair of the department of vascular medicine. What were your previous positions? 1999: Staff member internal medicine, AMC 2006: Staff member clinical epidemiology and internal medicine, LUMC Leiden 2010: Staff member internal and vascular medicine, AMC What are your research interests? Coagulation and pregnancy has my particular interest. Publications? Lots. Awards? She held a research fellowship (20032007) and was Clinical Established Investigator (2008-2010) of the Netherlands Heart Foundation. VIDI grant from the Netherlands Organisation for Scientific Research (NWO) on the topic “Thrombophilia and Reproduction”. !

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SUBJECT 101

POLYCYSTIC OVARIUM SYNDROME (PCOS) M. Kok & F. Dusseldorp Department of gynaecology, Academic Medical Centre, Amsterdam, The Netherlands

PCOS is the most common endocrine disorder in women of child-bearing age and is an important cause of menstrual irregularity, infertility and androgen excess in women.1 PCOS literally means the presence of multiple cysts in the ovarium.1 However, the appearance of polycystic ovaries on ultrasonography is not sufficient to diagnose PCOS and is present in 20-30% of otherwise healthy women as well.2 PCOS is often misunderstood and wrongly diagnosed, therefore this subject 101 is dedicated to polycystic ovarium syndrome.

(MIS)DIAGNOSIS AND ROTTERDAM CRITERIA PCOS is a heterogeneous disorder which is associated with clinical, endocrine and ultrasonographic features that could also be present in several other diseases.3 The overall prevalence of PCOS is between 6-10%.4 Confusion would arise if only the appearance of polycystic ovaries on ultrasound would be enough to make the diagnosis, as this could lead to under- and overestimation of disease. Therefore, the diagnosis of PCOS can be made if at least two of the following three criteria are present: oligo- or anovulation, clinical/biochemical signs of hyperandrogenism and the appearance of polycystic ovaries (PCO) on ultrasonography.4 The clinical image can be diverse and the intensity of symptoms varies between people.5 The symptoms of PCOS could also be present in a range of other disorders such as thyroid disorders, states of prolactin excess and congenital adrenal hyperplasia.3 P. 16

The overall consensus is that PCOS remains a diagnosis of exclusion.6

PATHOPHYSIOLOGY OF PCOS The pathophysiology of PCOS is multifactorial and polygenetic and the cause of PCOS is not fully clear.7 Insulin resistance, which manifests mostly in obese or overweight women but often also in lean PCOS women, is one of the key observations of this complex disorder.8 Hyperinsulinemia has a causal association to all features of the syndrome, such as hyperandrogenism, reproductive disorders, acne and hirsutism.8 Another key observation is that women with PCOS have elevated levels of LH due to increased amplitude or frequency of LH pulses.7 The shorter pulses of GnRH result in promoting LH while FSH secretion is the same or even decreased.9 Androgen production in the ovary is regulated by the theca interna layer of the ovarian follicles, whereas the granulose cells in ovaries have receptors for FSH.9 The result is that the follicles are failing to develop and high levels of androgens appear. Infertility affects 40% of women with PCOS.2 That means that 60% of women with PCOS are not affected with infertility. Even if there is a diminished ovulation rate in most women with PCOS, every ovulation could result in a pregnancy. Therefore, it remains important for women with PCOS to use contraceptives, even if the chance of a pregnancy is smaller than in the healthy population.

CONSEQUENCES There are several short term and long term consequences for women with PCOS.10 Insulin resistance is prevalent in 50-70% of PCOS patients, leading to a number of comorbidities including the metabolic syndrome.6 Lifestyle changes such as weight loss can improve the metabolic syndrome by lowering androgen levels and improve PCOS symptoms as well.6 Mental health disorders including depression, anxiety, bipolar disorders and binge eating disorder also occur more frequently in women with PCOS.6 Besides that, women with PCOS have an increased risk of endometrial hyperplasia and carcinoma due to chronic anovulation that is associated with PCOS.2 This can be prevented by using progesterone to prevent endometrial hyperplasia. AMSj #7

CLINICAL IMAGE

CONCLUSION In conclusion, PCOS is an important endocrine disorder in women which is strongly correlated to insulin dysfunction. PCOS remains a diagnosis of exclusion and other endocrine disorders should be considered. Women with PCOS must be aware of the short term and long term consequences of the disorder and, luckily, PCOS does not mean a fertility problem by definition.

CLINICAL IMAGE: AN ABNORMAL OVARY A. Schreurs & J. Bramer

REFERENCES 1.

10.

Rosenfield RL. Diagnostic evaluation of polycystic ovary syndrome in adolescents. Uptodate (online). [updated 2016 October 14]. Available from: http:// www.uptodate.com/contents/diagnostic-evaluation-of-polycystic-ovary-syndrome-in-adolescents Sirmans SM, Kristen AP. Epidemiology, diagnosis, and management of polycystic ovary syndrome. Clin Epidemiol. 2014; 6:1-13. Kyritsi EM, Diitriadis GK, Kyrou L, Kaltas G, Randeva HS. PCOS remains a diagnosis of exclusion: a concise review of key endocrinopathies to exclude. Clinical endocrinology. In press 2016, September . Bozdag G, Mumusoglu S., Zengin D, Karabulut E, Yildiz BO. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Human reproduction; In press 2016 September. Bachanek M, Abdalla N, Cendrowski K, Sawicki W. Value of ultrasonography in the diagnosis of polycystic ovary syndrome. Journal of clinical endocrinology and metabolism. 2015; 15(63): 410-22. Azziz R, Carmina E, Dewailly D, Diamanti-Kandaraki E, Escobar-Morreale HF, Futterweit W, et al. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen excess society guideline. J Clin Endocrin Metab 2006; 91(11):4237-45 Balen A. The pathophysiology of polycycstic ovary syndrome: trying to understand PCOS and its endocrinology. Best practice & Rresearch clinical obstetrics and gynaecology. 2004; 18(5) 685-706. De Leo V, La Marca A, Felice P. Insulin-Lowering Agents in the management of polycystic ovary syndrome. Endocr Rev. 2003;24:633-67 Cheung A, Cog F. Polycystic Ovary Syndrome: A contemporary view. J Obstet Gynaecol Can. 2010; 3(5):423-5, 426-8. Heineman MJ, Evers JLH, Massuger LFAG, Steegers EAP. Obstetrie en Gynaecologie: De voortplanting van de mens. Amsterdam: Reed Business; 2007.

PRESENTATION A 31-year-old woman consulted the gynecologic outpatient clinic complaining of dysmenorrhea, dyspareunia and daily abdominal discomfort since several months. The pain had been progressive since ceasing the oral contraceptive pill in order to try to conceive. She has a regular cycle, with an average duration of 28 days and a menstruation lasting 3-4 days. Vaginal examination showed a normal cervix, the mobility of the uterus was reduced. Transvaginal ultrasound repeatedly showed an anomaly with variable echogenicity. What is the most likely diagnosis? A.

Persistent follicle

Corpus luteum

Endometrioma

Pregnancy of unknown location

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HUMAN GENOME EDITING: CURE FOR PATIENT, CURSE FOR DOCTOR? J. Broertjes, School of Medical Sciences, Vrije Universiteit Amsterdam, The Netherlands

published and clinical trials are approved. These rapid developments could have a major impact on medicine. Future doctors and medical professionals may be confronted by questions whether technically achievable genetic modifications are appropriate and ethical. Therefore, there is an increased urgency to develop a bioethical framework regarding human genome editing. The aim of this article is not to give an exhaustive list of all developments regarding to human genome editing, but rather a clear and concise overview of genome editing and CRISPR/Cas9, placed in a historical context. This may provide a primer for future doctors and medical professionals to develop their own opinion about human genome editing.

INTRODUCTION

ABSTRACT Human genome editing is gaining momentum. The technology to modify genomes has been available since the seventies and is continuously improving. Using these techniques, many genetically modified organisms have been created, such as: insulin producing bacteria, FLAVR SAVR™ tomatoes with prolonged shelf-life and GloFish®, a fluorescent pet fish. Recently, developments in genetic modification accelerated with the emergence of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/associated protein 9). This revolutionary genetic tool acts as a DNA scissor, guided by RNA. Such proteins, capable of cutting DNA at a specific location, are not new. However, these old proteins were limited to cutting one single location of the genome. If scientists wanted to modify another gene, then a new protein would have to be developed, in a laborious process. By contrast, the novel CRISPR/ Cas9 technique uses a replaceable RNA guide to target the desired gene. This RNA guide allows researchers to rapidly target new genes, by simply ordering a cheap new guide, online. This enables unprecedented flexibility and a low-cost method to edit genomes. The scientific community has rapidly adopted this technique. CRISPR/Cas9 makes human genome editing attainable: the first preliminary human embryo experiments have already been P. 18

The era of human genome editing may be imminent and this could affect medicine as we know it today. The first human embryo experiments have already been published by Chinese scientists.1,2 Currently, these experiments are limited to non-viable embryos. However, human embryo experiments continue as researchers from the United Kingdom, China and Sweden have received permission for new experiments.3 Furthermore, clinical trials have been approved in China 4 and the United States 5. These developments are further discussed below (heading ‘The Cure’). During the past decades, we have seen many milestones in genetic modification accomplished. In 1953, the molecular structure of our genetic information was discovered: DNA appeared to be arranged in a double helix.6 Since then, it took only twenty years for humans to start modifying it. In 1973, molecular scissors known as nucleases were used to cut DNA and to create the first genetically modified (GM) bacteria.7 Within a few years, this technique was commercialized with the founding of the first biotechnology company, Genentech in 1976, now a major pharmaceutical company.8 Using genetic modification, bacteria producing human insulin, used to treat diabetes, were developed. Since the seventies, the genetic toolbox has expanded, for example enabling researchers to read DNA, using sequencing techniques and copy DNA with the polymerase chain reaction (PCR). In 1990, the first GM bovine was born in the Netherlands: a bull named Herman. The aim AMSj #7

was that his female offspring would produce an anti-inflammatory human protein in their milk. Although the technique worked, there was only little protein produced in the milk.9 Humans started eating GM plants in 1994, when FLAVR SAVR™ tomatoes were introduced. These tomatoes contained disabled enzymes, aimed at prolonging shelf life.10 In 2003, the first GM pet (GloFish®) was approved for sale in Taiwan. In the genomes of these zebrafish, DNA is inserted from fluorescent jellyfish. This results in their bright fluorescent phenotype.11 Recently, the first GM animal for human consumption was approved, the fast growing AquAdvantage® salmon.12

proteins apart. The RNA guide acts as a molecular postal code, recognizing complementary target DNA (Figure 1). If a new gene needs to be cleaved, a cheap new RNA guide can simply be ordered online.

THE CRISPR REVOLUTION In 2012, Jennifer Doudna and Emmanuelle Charpentier published their landmark paper describing DNA scissors, guided by RNA.13 This tool is known as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/associated protein 9). These DNA cutting enzymes originate from bacterial immunity. Like humans, bacteria need to protect themselves from viral infection. Viruses infect bacteria by injecting their own viral genome into the bacterial cytoplasm. When successful, the bacterium will transcribe the viral genome and thus produce more virus particles, which will be released following bacteria lysis. However, if the bacterium recognizes the viral genome as non-self, it will cut it into pieces. To prevent future viral infections, viral fragments are stored into a CRISPR sequence. The CRISPR sequence acts as a molecular charm bracelet, archiving viral genomic fragments. This creates a genetic fingerprint of a virus, improving resistance against future infection.14 These genetic fingerprints can be transcribed into RNA guides. If the bacterium is re-infected, the guide RNA enables Cas9 protein to recognize the infectious genome and Cas9 inactivates it by cleaving it. These RNA guided Cas proteins, rather than the CRISPR sequences, are revolutionizing genome editing. The ability to cut DNA at a specific location is not new. As described above, such DNA scissors have been used to edit genomes since 1973.7 However, the major drawback of these older molecular scissors is the fact that they recognize only one unique sequence. To cut DNA at a new location, a new protein must be developed. This is what sets the RNA guided Cas DEC 2016

Figure 1. The CRISPR/Cas9 system: the Cas9 nuclease acts as a pair of molecular scissors, using the replaceable RNA guide to target the gene of interest. The mutated gene can be cut out and replaced with a healthy donor gene. Modified from dharmacon.gelifesciences.com

Using a standardized DNA cutting protein with custom RNA guides enables genetic modification with unprecedented flexibility, at very low costs. The advantage of using a replaceable RNA guide drives the current genetic engineering revolution. This caused a wave of papers describing CRISPR/Cas9, as shown in Figure 2.

Figure 2. Number of PubMed (Medline) publications mentioning CRISPR in the title. Two older DNA scissors, or nucleases, are included: Transcription activator-like eﬀector nucleases (TALEN) and Zinc finger nucleases (ZFN). For comparison, another new high profile technique is included: the creation of induced pluripotent stem cells (iPSC), described in in 2006 for which the authors received the Nobel Prize in 2012. The fact that CRISPR/Cas9 publications outpace iPSC papers is an example of the major impact CRISPR/Cas9 is considered to have on biomedical research. At the moment of data collection, the expectation is that the number of CRISPR publications will increase even further.

THE CURE Editing the human genome requires a deep understanding of our genetic code. Reading P. 19

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the human genome became possible after it was sequenced in 2001 by the Human Genome Project 17 and the Celera corporation.18 The ability to read our entire genetic makeup enhanced the understanding of genetic diseases. The recent development of CRISPR/Cas9 has accelerated the quest for therapeutic genome editing (gene therapy). Using CRISPR/Cas9, genetic diseases can be cured by cutting out pathogenic genes and replacing them with healthy donor DNA. A plethora of genetic diseases are currently being investigated for therapeutic genome editing. An example of a potential target for genome editing is β-thalassemia. This genetic disease is caused by a mutation in the gene encoding for the oxygen carrying β globin. This mutation results in severe anemia. Using CRISPR/Cas9, the mutated gene can be removed and replaced with a donor gene. The first human embryo experiment was aimed at curing this disease. However, this proved to be only partly successful.2 CRISPR/Cas9 could also be utilized to impede the spread of the human immunodeficiency virus (HIV). The virus enters immune cells using the C-C chemokine receptor type 5 (CCR5). Therefore, CCR5 is a potential target for human genome editing. Some humans carry a naturally occurring variant of this receptor, resulting in resistance to HIV infections. Using CRISPR/ Cas9, it was tried to introduce the resistant variant of this receptor into human embryos.1 Again, this experiment encountered several difficulties. Furthermore, cancer immunotherapy can be enhanced using CRISPR/Cas9. Recently, two human clinical trials have been approved in China 4 and the United States 5. In these trials, immune cells will be extracted from cancer patients. These immune cells are genetically enhanced in a laboratory to improve their anti-cancer properties. Using CRISPR–Cas9, their ability to detect and destroy cancer cells will be enhanced. After modification, the immune cells will be reintroduced into the patients’ bloodstream, aiming to cure the patient. For now, human genome editing faces many technical and safety challenges. However, it is conceivable that these issues will be mitigated in the near future. For an extensive overview of the CRISPR/Cas9 capabilities, prospects and limitations, see the extensive review written by Savić et al.19 Apart for the cut-and-paste methods described above, another approach is to correct genomic P. 20

typos. Correcting these single nucleotide polymorphisms (SNPs) enables a more subtle method of genome editing.15,16 By contrast, a more radical approach would be to print completely new genomes. The first bacterial genomes have already been printed.20 As described above, the human genome was sequenced in 2001.17,18 Since then, the Human Genome Project has been renamed as the ‘Genome Project – Read’.21 In 2016, the ‘Genome Project – Write’ was launched, aimed at developing the technology to synthesize complete human genomes. Printing complete human genomes is predicted to be feasible within ten years.21

THE CURSE The development of human genome editing has bioethical consequences and is a double-edged sword. On one side, it enables the permanent curation of genetic diseases, as described above. On the other side, genome editing could theoretically be employed to enhance other aspects of human life, such as athletic ability 22, happiness 23 , and potentially, even intelligence 24. There is no clear line between ‘curation’ and ‘enhancement’. This poses a regulatory conundrum to select diseases eligible for therapeutic genome editing. An apparent solution would be to only consider severe diseases. However, genetic diseases cannot be dichotomously categorized as either severe or negligible. Rather, they form a continuous spectrum ranging from debilitating genetic diseases such as cystic fibrosis, to mostly inconsequential deviations such as color blindness. Concerns regarding bioethical implications may result in an effort to ban human genome editing. However, such a ban would be complicated by the globalized nature of the international community. Air travel and the Internet allow people and information to reach almost the entire globe. However, regulation regarding human genome editing is fragmented.25 Many countries are participating in the development of genome editing. This technology could offer these countries significant advantages in agriculture 26, industrial biotech 27 and the military 28. Given the relative low cost and high prevalence of genome editing techniques, it is conceivable that technologies capable of editing the human genome will be developed further, despite any attempts to ban it.

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DISCUSSION: CURE FOR PATIENT, CURSE FOR DOCTOR? As described above, it is improbable that technological progress will be halted. Although the CRISPR/Cas9 technology is revolutionary, seen in historical context it constitutes a major leap rather than a starting point, towards editing the human genome. There has been a continuous effort to master the genomes of many species, including our own. By engaging in human genome editing, we step onto a slippery slope. It is plausible that future parents want to endow their offspring with favorable genomic characteristics. This could result in genetic inequality. If more affluent groups of society were to routinely give birth to designer babies, this would affect the human gene pool and may have long-term social implications. This could result in new forms of social inequality between genetic engineered offspring and those whose genome is constituted by chance. Another scenario would be the reemergence eugenics (‘well-born’), a pursuit of the Übermensch, associated with the Nazi regime during the Second World War. Although new technologies like human genome editing bear certain risks, hope and inspiration can be drawn from the last milestone in mastering our genomes: sequencing the human genome. This project started out as a profit-driven endeavor to patent genetic information. The Celera corporation, led by Craig Venter 18, initiated the effort to monopolize and commercialize genetic information. However, international intervention transformed it into the freely accessible foundation of modern medicine. To achieve this, the international community launched a parallel sequencing project.17 In 2000, American President Bill Clinton announced in his State of the Union that naturally occurring genes cannot be patented and this information should be openly accessible for research.30 In hindsight, these bioethical threats were well handled, offering hope and inspiration for managing future disruptive technologies, including human genome editing. Again, we may well be on the eve of a new biotechnical revolution: rewriting our genomes. Human genome editing can potentially provide a cure for debilitating genetic diseases of future patients. At the same time, human genome editing may prove to be a curse to future doctors and medical professionals. During their careers, they may be confronted by the dilemma whether DEC 2016

technically achievable genetic modifications are appropriate and ethical. Therefore, it is imperative to develop a rigorous and thoughtfully drafted bioethical framework, regarding human genome editing. The aim of this framework will be to preserve the potential as cure, while preventing it from becoming a curse for doctors and society as a whole. The ‘Genome Project – Write’ provides a basis for such a framework.21 In drafting this bioethical framework, doctors and medical professionals should contribute. Although human genome editing is very much a political and scientific debate, medical professionals act as a link between scientific technologies and public applications. Therefore, medical professionals worldwide can, and should, play an important role in this discussion. Placing it on the public will contribute to discussions as well as inform policy decisions. Such information ranges from practical issues such as patient safety, current developments, and clarifying potential future bioethical implications. Hence it is important that future doctors and medical professionals start thinking about the bioethical concerns of human genome editing and help to direct our future in a positive direction. Although the future remains elusive, we do know the following: humankind is rapidly mastering the techniques capable of reshaping the genetic makeup of various species, including H. sapiens.

REFERENCES 1.

3. 4. 5.

Kang, X. et al. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J. Assist. Reprod. Genet. 33, 581–588 (2016). Liang, P. et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015). Callaway, E. Gene-editing research in human embryos gains momentum. Nature 532, 289–90 (2016). Cyranoski, D. Chinese scientists to pioneer first human CRISPR trial. Nature 535, 476–7 (2016). Reardon, S. First CRISPR clinical trial gets green light from US panel. Nature (2016). doi:10.1038/ nature.2016.20137 Watson, J. D. & Crick, F. H. C. Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature 171, 737–738 (1953). Cohen, S. N., Chang, A. C. Y., Boyert, H. W. & Hellingt, R. B. Construction of Biologically Functional Bacterial Plasmids In Vitro (R factor/restriction

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8. 9.

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11. 12. 13.

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17. 18. 19. 20.

21. 22. 23.

24.

25. 26.

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04-11-16 16:

AMSj #7

SOLVING STATISTICS

systolic blood pressure than those who have a lower body mass index. I know I have to examine the correlation between blood pressure and body mass index. The problem is that I do not know which correlation coefficient I should calculate. I need to choose between Pearson’s correlation coefficient and Spearman’s rank correlation coefficient. Can you help me to figure out which one I should use?

SHOULD I USE PEARSON’S OR SPEARMAN’S CORRELATION COEFFICIENT?

100

Both types are only suitable for describing the association between X and Y if the relationship between them is linear. A linear relationship means that if you were to plot the values of X against Y in a scatterplot, the points representing the data will fall around a straight line. If they fall around a curved or an angled line, the relationship is non-linear. There is a number of differences between the two coefficients. In this paper, we focus on outliers. Outliers are values that for some reason fall a substantial distance from the other values. Pearson’s correlation coefficient should only be used if neither X nor Y have any outliers. Spearman’s rank correlation coefficient can be used even if neither, one or both of X or Y have outliers.

QUESTION I want to determine whether patients, who have a higher body mass index, tend to have a higher

A correlation coefficient is a number that shows if there is an association between variables. It provides an indication of the association between two variables X and Y.1 Two types of correlations are commonly used for continuous, numerical data. These are Pearson’s correlation coefficient and Spearman’s rank correlation coefficient.

BACKGROUND

R. Holman, Clinical Research Unit, Academic Medical Centre, Amsterdam, The Netherlands

N. van de Klundert, master student, Academic Medical Centre, Amsterdam, The Netherlands

ANSWER

Body mass index of healthy subjects

Body mass index of patients

Figure 1. Histograms of body mass index for healthy subjects and patients.

Let us look at some examples. In Figure 1, we present histograms of body mass index for healthy subjects and patients. The histogram for healthy subjects shows that all of the points are fairly close together. We can conclude that body mass index for healthy subjects does not have any outliers. Hence, Pearson’s correlation coefficient is suitable for representing the association between body mass index and systolic blood pressure for healthy subjects. However, keep in mind that there also needs to be a linear relationship between body mass index and systolic blood pressure. If we look at the second histogram for body mass index in patients, it shows that there are three values that are substantially larger than the other values. These values are outliers. Hence, we can conclude that body mass index for patients does not follow a normal distribution and that Pearson’s correlations coefficient is not suitable for representing this association for patients. Spearman’s correlation coefficient is suitable

:15

DEC 2016

P. 23

TRIAL & ERROR

if there is a linear relationship between body mass index and systolic blood pressure for patients.

THE TORTOISE M.J.B. van Baar, master student, VU Medical Centre, Amsterdam, The Netherlands

Figure 2. The relationship between body mass index and systolic blood pressure for students and lecturers.

In Figure 2, we present scatterplots of body mass index against systolic blood pressure for students and lecturers. For students, there is a clear linear relationship between these variables. This suggests that presenting the value of a correlation coefficient is appropriate. Because there are no substantial outliers, both Pearson’s and Spearman’s correlation coefficients would be appropriate to use. You may hope to move on from correlation to linear regression, perhaps with more explanatory variables. In this case, Pearson’s correlation coefficient may be more appropriate, as it is mathematically closely linked to linear regression. When you have calculated either correlation coefficient, the value will be between -1 and +1. If your correlation coefficient is between 0 and 1, it means that when one variable increases, the other increases as well. Your variables are positively correlated. If your correlation coefficient is between -1 and 0, it means that when one variable increases, the other decreases. Your variables are negatively correlated. If your correlation coefficient equals 1 or -1, all points derived from plotting your variables in a scatter plot will fall on a perfect straight line. Your values are than perfectly correlated; this is nearly impossible to find in real life data. 1.

P. 24

Too slow! After a quick look at all the feedback forms I gathered during my rounds, I realized that my main reoccurring error was that I was not quick enough. Somehow all my supervisors came to the same conclusion; this young doctor is not able to sprint. I vividly remember my first feedback form, the pro’s were read and promptly forgotten, but the con’s: oh . . .they stuck in my mind, making my next round a complete joke. Have you ever seen a tortoise trying to sprint? In my effort to be quick, I lost the joy of learning. I had set aside my holistic ideals in an attempt to improve my weakness. In doing so, I tried to change the nature of my character and guess what: I failed! Not only did I fail, the whole process of trying gave me the feeling that I was not competent, not capable, and foremost not good enough to ever succeed in the medical profession. Observing all the hares jumping from one patient to another in an endless winning streak, I lost confidence in myself. However, through trial and error I now know that the goal of negative feedback is not always change. It is your consciousness of time, your style and your character that is far more important than change itself. Once I realized that, I was not doubtful about my speed anymore, I knew it was part of the way I do best. My advice is to stay true to your character, use feedback to understand your talents and shortcomings, and by doing so become quicker than the hares. !

Petrie A, Sabin C. Medical Statistics at a glance. West sussex, UK: Wiley Blackwell; 2009. AMSj #7

CLINICAL IMAGE

RADIOLOGY IMAGE

ANSWERS

A. Schreurs & J. Bramer

S. Spijkers, R. Hemke & M. Maas

Persistent follicle. No, a follicle should variate in size over time and would disappear

QUESTION 1

Corpus luteum. No, a corpus luteum should variate in size over time, and would disappear, but it could look a lot like it. This is why it is important to follow cysts over time.

Pneumothorax

QUESTION 2 A.

Yes

QUESTION 3

Endometrioma. Yes, combined with clinical complaints of endometriosis it is most likely that this cyst is an endometrioma.

Pregnancy of unknown location. There will not be a menstruation during pregnancy, so complaints of recurrent dysmenorrhea does not fit with a P.U.L. A P.U.L. could be an acute and severe problem with severe abdominal pain that should be investigated immediately.

After the initial presentation, especially when considering the echocardiography (not shown in this article), where right ventricle dilatation was visible, pulmonary embolism (PE) is the most likely cause. As about 90% of all pulmonary embolisms are a result of proximal leg deep vein thrombosis (DVTs), an ultrasound of these veins is often made to supplement a CT pulmonary angiography (CTPA) scan. Here the latter visualization method is the most used method for PEs, where contrast fluid is used to visualize the pulmonary arteries (PAs). The technique used for the proximal leg veins is fairly simple. While visualizing the veins, two steps are used: firstly, one analyses whether the content of the vein is hypo-echoic (black) or contains areas of hyper-echogenicity (thrombus). Secondly one put a slight pressure on the leg at the location of the veins. If the compression of the vein is impossible, seen as persisting volume and size, the diagnosis of thrombus is made. Because contrast fluid is used in the CTPA, the blood vessels are especially well visible. This allows for a reliable diagnoses of PE, since the obstructed parts of the will not show complete contrast filling. According to a large study1, the sensitivity of this method is 83% and the specificity was 96%, confirming the reliability of this method. A more recent study shows a sensitivity of 81.7% and a specificity of 93.4%2.

DEC 2016

Pulmonary embolism

REFERENCE

P. 25

EXPLANATION OF THE IMAGES

LITERATURE

Below both the ultrasound of our patient and a normal version. Notice the vein that is non-compressible in our patient.

Stein, P., Fowler, S., Goodman, L., Gottschalk, A., Hales, C., Hull, R., Leeper, K., Popovich, J., Quinn, D., Sos, T., Sostman, H., Tapson, V., Wakefield, T., Weg, J. and Woodard, P. (2006). Multidetector Computed Tomography for Acute Pulmonary Embolism. New England Journal of Medicine, 354(22), pp.2317-2327. He, J., Wang, F., Dai, H., Li, M., Wang, Q., Yao, Z., Lv, B., Xiong, C., He, J., Liu, Z., He, Z. and Fang, W. (2012). Chinese multi-center study of lung scintigraphy and CT pulmonary angiography for the diagnosis of pulmonary embolism. The International Journal of Cardiovascular Imaging, 28(7), pp.1799-1805. Leg DVT Normal - Ultrasoundpaedia. (2016). Ultrasoundpaedia.com. Retrieved 21 September 2016, from http://www.ultrasoundpaedia.com/normaldvt-leg/ Knipe, H. & D'Souza, D. (2016). Pulmonary embolism | Radiology Reference Article | Radiopaedia. org. Radiopaedia.org. Retrieved 21 September 2016, from https://radiopaedia.org/articles/pulmonary-embolism Misra, R., Planner, A., & Uthappa, M. (2007). A-Z of Chest Radiology. New York: Cambridge University Press.

Below are the images from the CTPA, showing the obstructed parts of the PAs with a filling defect within the white contrast.

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AMSj #7

COLOPHON

STUDENT REVIEWERS A. van Aken, M. Arnoldussen, F. Dusseldorp, S. Spijkers, R. Betgem, A.F. Zwagemaker, M. Nazir, S. Hoeﬀelman, T. J. van Trier, D.E.F.W.M. van Toledo, A.R. Schuurman, R.J. Molenaar, N. Wolfhagen, J.V. Veld, A.M.F. Schreurs, J. Saris, E. Leenders, M. van Baar, D.N.H. van Vemde, P.J. Hengeveld, M. de Jong, R.A.L. Beekman GRAPHIC DESIGN Dianne Rijnbende Grafisch Ontwerp

Amsterdam Medical Student journal is a scientific medical journal with the purpose to enable medical students to publish clinical observations, research articles and case reports. The journal was founded by students from the Academic Medical Center (AMC) and the VU Medical Center (VUmc) in Amsterdam with the intention to provide education and development of academic skills for medical students. The entire journal is created and published by staﬀ members and students from both faculties. CORRESPONDANCE info@amsj.nl WEB www.amsj.nl Like us on facebook.com/amsjournal & follow us on twitter.com/amsterdammsj SUBMISSIONS If you would like to publish your research in AMSj, please ckeck out our guidelines on www.amsj.nl EDITORS IN CHIEF H. Bahadurzada, M. Schuijt, A. Farokhi, G.E. Linthorst, F. Daams BOARD R.M. van den Hurk, R.V. Baboeram Panday, R. Rassir, A.J. Hoving, A.W.D. Edridge, M.M. Wennekers CONTENT EDITOR & NATIVE EDITOR T.R. de Back, M.E. Ribbink STAFF REVIEWERS P.P. de Koning, L. Vogt, M. Kok, M. Maas, W.W.L. Li, M.W. Hollman, I.N. van Schaik, M.U. Schafroth, M.J.S. Oosterveld, V.G.M. Geukers, L.M. Hubers, J.W. Wilmink, C.G. Genc, G. de Waard, J.A.M. Bramer, W. Fuijkschot, J.S. ten Kulve, J. Cloos, S. Bossers, N. Rol, J. Tichelaar, M. Best, M.W: Bijlsma, B. de Witte, R. Holman

COVER ILLUSTRATION Marieke van der Linden, VU medical Centre, see Editorial, page 3. PRINT Drukwerkdeal.nl COPYRIGHT & WARRANTY Statements, opinions, and results of studies published in Amsterdam Medical Student journal are those of the authors and do not reflect the policy of the Academic Medical Center, VU University Medical Center, the Editors or Board of AMSj. The Academic Medical Center, VU University Medical Center, Editors andBoard of AMSj provide no warranty as to their accuracy or reliability. This volume of Amsterdam Medical Student journal is licensed under a CC BY-NC-ND 4.0 license. For more information on your rights to share this work, please see the full lincense on bit.ly/1weyPUN

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