12 minute read
Liquid biopsy in Renal Cell Carcinoma Current and future applications
REVIEW
Alessia Cimadamore 1, Simone Scarcella 2, Erika Palagonia 2, Lucio Dell’Atti 2, Andrea Benedetto Galosi 2 , Rodolfo Montironi 1 .
1 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Ancona, Italy; 2Department of Urology, Marche Polytechnic University, School of Medicine, United Hospitals, Ancona, Italy.
SUMMARY Renal cell carcinoma (RCC) is the seventh most common cancer and the most lethal urological malignancy with 5‐year overall survival of 74%, that decrease to 8% in patients with evidence of distant metastasis upon initial diagnosis. To date, no serum or urine biomarkers are currently available for early diagnosis, for monitoring recurrence and response to therapy. Liquid biopsy is a non-invasive diagnostic assay that can detect tumor molecular alterations at diagnosis, after surgery and during progression. Liquid biopsy includes testing for circulating tumor nucleic acids (DNA and RNA), circulating tumor cells (CTC) and small vesicles. In this paper, we reviewed the current knowledge on circulating tumor DNA and CTC in RCC, focusing on the newly developed and promising techniques, such as the circulating free-DNA methylation analysis, and their potential applications in the future.
KEY WORDS: Renal cell carcinoma, kidney tumors, liquid biopsy, circulating tumor cells, circulating DNA, biomarkers.
INTRODUCTION
Renal cell carcinoma (RCC) is the seventh most common cancer and the most lethal urological malignancy with 5‐year overall survival of 74%, that decrease to 8% in patients with evidence of distant metastasis upon initial diagnosis (1). Around 30% of patients have metastases detected at preoperative screening or surgery, termed synchronous metastases. Up to 50% develop metastases after the removal of the primary tumor (at least 3 months and as late as 30 years after primary surgery), called metachronous metastases. The timing and location of metastases in RCC are difficult to predict, which makes surveillance challenging (2). At present, radiological assessments of renal masses and metastases are insufficient for qualitative characterization of the tumor. Histopathological evaluation is needed for diagnosis, grading and staging of primary tumors; however, in the setting of metastatic disease, no serum or urine biomarkers are currently available to monitor recurrence and response to therapy. During the last decade, multiple genetic alterations of RCC have been associated with prognosis and response to therapy, even though no specific mutations have been associated with sensitivity or resistance to a specific drug. LIQUID BIOPSY IN RENAL CELL CARCINOMA
Liquid biopsy consists in a liquid sample such as whole blood/plasma/urine used for identifying molecular circulating signatures shared with solid tumors. In other words, it can be considered a surrogate material of tissue/cytological sample and can be used as a non-invasive test that allows multiple serial sampling at any stage of disease (3) (Figure 1). Liquid biopsy has been proposed as a potential strategy to improved stratification of patients for adjuvant therapy trials (4, 5). The main goal of liquid biopsy in this scenario would be the detection of minimal residual disease following intended curative nephrectomy. In other malignancies such as lung cancer, colon cancer, breast cancer and prostate cancer, the development of circulating tumor DNA (ctDNA) assays is intended to evaluate of the presence of specific genetic alterations able to predict response to targeted therapy. Contrariwise, in the therapeutic scenario of RCC there is no predictive biomarker able to select patient for a specific therapy. As consequence, many of the studies published in the literature have focused on the quantification of ctDNA and CTCs as measures of tumor burden and on its correlation with prognosis and development of metastasis (6, 7).
Figure 1. Graphic description of the process during liquid biopsy. (Image reproduced under Creative Commons license from reference 28)
CTDNA IN RENAL CELL CARCINOMA
CtDNA has been detected in the plasma and urine of patients with oncocytomas and early-stage clear cell RCCs (8). This finding is noteworthy since liquid biopsy can be potentially implemented to differentiate small renal masses and to guide decision over invasive surgery versus active surveillance. ctDNA has potential as a surveillance biomarker for patients with localized RCC after nephrectomy. In a study of 30 patients with RCC preparing for nephrectomy, ctDNA NGS was used to interrogate 14 commonly mutated genes.(9) Twenty of the 30 patients had detectable somatic mutations in at least one of the 14 genes assessed. This suggests that even the low tumor burdens seen in localized RCC shed detectable quantities of ctDNA. Another study used quantitative real-time PCR to measure the level of ctDNA in 92 patients with clear-cell RCC across different stages of disease (10). Metastatic RCC (mRCC) patients had a higher level of ctDNA compared to localized RCC (6.04 vs. 5.29, p = 0.017). The authors also showed that recurrence of RCC was associated with higher levels of ctDNA (p = 0.024). In the metastatic setting, patients with mRCC are monitored for treatment response with a CT chest/abdomen/pelvis every 3-6 months. Recurrent CT scans are time consuming, costly, and expose cancer patients to high levels of radiation. ctDNA has the potential to act as a surrogate for radiographic disease progression in mRCC. Studies suggest that ctDNA can be routinely monitored at set intervals to monitor for disease recurrence (11, 12). Use of ctDNA could decrease the potential harms associated with screening CT scans, including contrast nephropathy and radiation exposure. Pal and colleagues detected ctDNA in 78.6% of 200 metastatic patients using the Guardant360 plasma assay (Guardant Health), though with a median of one genomic alteration per sample (13). The same authors detected ctDNA in a further 18/34 (53%) metastatic RCC patients and observed a possible correlation between detection and lesion diameter. In a recent study of 34 patients with mRCC, patients with detectable ctDNA had greater radiographic tumor burden than patients with no detectable ctDNA (14). CtDNA-positive mRCC patients had shorter overall survival and progression-free survival on first-line therapy. Among ctDNA-positive patients,
ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables (15). While these findings suggest that ctDNA has the capability to complement or replace frequent CT scans in patients with mRCC, these results are from a small, retrospective study and warrant further evaluation in larger, prospective cohorts. Overall, in contrast with other tumors, low levels of ctDNA has been found in RCC. Thirty-three percent of patients had evidence for RCC-derived ctDNA, significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. The probability of detecting ctDNA rises with increasing size of the primary tumor and in patients with growth of a tumor thrombus into the renal vein or inferior vena cava. However, consensus concerning ctDNA levels in RCC has yet to be reached; data are conflicting and often not comparable because of the use of different techniques for DNA extraction, different sequencing methods and limit of detection.
A promising reliable biomarker for early detection of RCC is the analysis of CpG island hypermethylation in circulating free DNA (cfDNA). The first study was published in 2013 by Hauser et al. (16) on a small cohort of patients with RCC. In 30 of 35 investigated patients with RCC, at least one of the eight gene tested was methylated within the serum cfDNA. The Area under the receiver operating characteristic (AUROC) curve showed a high specificity for serum cfDNA methylation (range 85-100%) but low sensitivity in single-gene analysis (range 14-54%). Similar results were obtained in 2016 by Skrypkina et al. testing a different set of genes.(17) In 2018, the first genome-wide cfDNA methylation analysis study was conducted by the newly developed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP–seq) method. With this advanced, highly sensitive and costeffective technique, Shen et al. achieved an AUROC curve of approximately 0.9 for detecting and classifying patients with RCC from patients with other tumor types and healthy controls (18). Recently Nuzzo and colleagues (19) validated the cfMeDIP–seq method in a series of RCC and reported the first application of this method on urine samples. Notably, the authors showed an accurate classification of patients across all stages of renal cell carcinoma (RCC) in plasma (AUROC curve 0.99) and an AUROC curve of 0.858 for correctly classifying urine RCC and control samples in a cohort where two-thirds of patients with RCC had localized disease. This assays proved to be capable of detecting early stage tumors, even at lower sequencing depth and ctDNA abundance (20). Of interest is also the analysis of SHOX2 gene methylation which demonstrated to be strongly correlated with an advanced disease stage and risk of death after initial partial or radical nephrectomy (21), so identifying a group of patients who might benefit from an adjuvant treatment or early initiation of a palliative treatment. CTC IN RENAL CELL CARCINOMA
Few studies are present in the literature concerning CTC in RCC. In general, detection of CTCs requires specific techniques able to overcome problems related to identification and isolation of tumors cells from blood. CellSearch system, the only platform approved by FDA, showed a very low detection rate in patients with localized and metastatic RCC. However, in samples collected at baseline/presurgery, despite the extremely low CTC counts, the presence of even one single eCTC was associated with a shorter PFS (22). Presence of CTCs has been associated with more aggressive tumor features and worse outcome (23). A greater number of CTCs was found after open radical nephrectomy than after laparoscopic procedures(24). CTC changes over time and correlation with tumor response have been included as secondary outcomes in ongoing trial (NCT02978118) which also will investigate the immunemarker profile evaluating the expressions of PD-1, PD-L1, CTLA-4, CD27, OX40, or LAG3 on isolated CTCs. Contrarily to Cell Search approach, which detects only cells expressing epithelial markers, marker independent CTC capture approach have been developed (25). Among these new methods, antibody cocktail targeting four RCCCTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80% (26). These newly engineered capture platform outperforms the conventional assay that rely exclusively on epithelial markers and may improve the use of CTCs as potential biomarker (27).
CONCLUSIONS
Liquid biopsy is a non-invasive test that allows multiple serial sampling at any stage of disease. ctDNA has potential as prognostic factor, predicting recurrence after nephrectomy, as a surveillance biomarker for patients in follow-up, and as biomarker of response during therapy. Cancer-specific DNA methylation changes could enable highly sensitive and low-cost detection, classification and monitoring of cancer. Improving CTC detection in mRCC by developing alternative CTC detection approaches will help unlock their informative content and offer important hints in treatments planning of this disease.
REFERENCES
1. Gupta K, Miller JD, Li JZ, et al. Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): A literature review. Cancer Treatment Reviews. 2008; 34:193-205. 2. Ljungberg B, Bensalah K, Canfield S, et al. EAU guidelines on renal cell carcinoma: 2014 update. Eur Urol. 2015; 67(5):913-24. 3. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid biopsies into the management of cancer. Nature Reviews Clinical Oncology. 2017; 14:531-48. 4. Cimadamore A, Gasparrini S, Massari F, et al. Emerging Molecular
Technologies in Renal Cell Carcinoma: Liquid Biopsy. Cancers (Basel). 2019; 11(2):196. 5. Cimadamore A, Massari F, Santoni M, et al. Molecular characterization and diagnostic criteria of renal cell carcinoma with emphasis on liquid biopsies. Expert Review of Molecular Diagnostics. 2020; 20(2):141-150. 6. Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014; 6(224). 7. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nature Reviews Genetics. 2019; 20(2):71-88. 8. Smith CG, Moser T, Mouliere F, et al. Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors. Genome Med. 2020; 12(1). 9. Al-Qassab U, Lorentz CA, Laganosky D, et al. PNFBA-12 Liquid biopst for renal cell carcinoma. J Urol. 2017; 197(4):e913-4. 10. Wan J, Zhu L, Jiang Z, Cheng K. Monitoring of plasma cell-free DNA in predicting postoperative recurrence of clear cell renal cell carcinoma. Urol Int. 2013; 91(3):273-8. 11. Yamamoto Y, Uemura M, Fujita M, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma. Cancer Sci. 2019; 110(2):617-28. 12. Corrò C, Hejhal T, Poyet C, et al. Detecting circulating tumor DNA in renal cancer: An open challenge. Exp Mol Pathol. 2017; 102(2):255-61. 13. Pal SK, Sonpavde G, Agarwal N, et al. Evolution of Circulating Tumor DNA Profile from First-line to Subsequent Therapy in Metastatic Renal Cell Carcinoma. Eur Urol. 2017; 72(4):557-564. 14. Maia MC, Bergerot PG, Dizman N, et al. Association of Circulating Tumor DNA (ctDNA) Detection in Metastatic Renal Cell Carcinoma (mRCC) with Tumor Burden. Kidney Cancer. 2017; 1(1):65-70. 15. Bacon JVW, Annala M, Soleimani M, et al. Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma. Clin Genitourin Cancer. 2020; 18(4):322-331.e2. 16. Hauser S, Zahalka T, Fechner G, Müller SC, Ellinger J. Serum DNA hypermethylation in patients with kidney cancer: Results of a prospective study. Anticancer Res. 2013; 33(10):4651-6. 17. Skrypkina I, Tsyba L, Onyshchenko K, et al. Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer. Dis Markers. 2016; 2016. 18. Shen SY, Singhania R, Fehringer G, et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature. 2018; 563(7732):579-83. 19. Nuzzo PV, Berchuck JE, Korthauer K, et al. Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med. 2020; 26(7):1041-3. 20. Cimadamore A, Santoni M, Massari F, et al. Liquid biopsies in renal cell carcinoma with focus on epigenome analysis. Annals of translational medicine. China. 2019; 7:S194. 21. Jung M, Ellinger J, Gevensleben H, et al. Cell-free SHOX2 DNA methylation in blood as a molecular staging parameter for risk stratification in renal cell carcinoma patients: A prospective observational cohort study. Clin Chem. 2019; 65(4):559-68. 22. Cappelletti V, Verzoni E, Ratta R, et al. Analysis of single circulating tumor cells in renal cell carcinoma reveals phenotypic heterogeneity and genomic alterations related to progression. Int J Mol Sci. 2020; 21(4):1475. 23. Bluemke K, Bilkenroth U, Meye A, et al. Detection of circulating tumor cells in peripheral blood of patients with renal cell carcinoma correlates with prognosis. Cancer Epidemiol Biomarkers Prev. 2009; 18(8):2190-4. 24. Haga N, Onagi A, Koguchi T, et al. Perioperative Detection of Circulating Tumor Cells in Radical or Partial Nephrectomy for Renal Cell Carcinoma. Ann Surg Oncol. 2020; 27(4):1272-1281. 25. Ye Z, Ding Y, Chen Z, et al. Detecting and phenotyping of aneuploid circulating tumor cells in patients with various malignancies. Cancer Biol Ther. 2019; 20(4):546-51. 26. Bu J, Nair A, Kubiatowicz LJ, et al. Surface engineering for efficient capture of circulating tumor cells in renal cell carcinoma: From nanoscale analysis to clinical application. Biosens Bioelectron. 2020; 162. 27. Cimadamore A, Aurilio G, Nolé F, et al. Update on Circulating Tumor Cells in Genitourinary Tumors with Focus on Prostate Cancer. Cells. 2020; 9(6):1495. 28. Remon J, García-Campelo R, de Álava E, et al. Liquid biopsy in oncology: a consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology. Clin Transl Oncol 2020; 22, 823-834.
CORRESPONDENCE Alessia Cimadamore Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals. Via Conca 71 − 60126 Ancona, Italy. Phone: +390715964805 e-mail: a.cimadamore@staff.univpm.it
