Differential VHL Mutation Patterns in Bilateral Clear Cell RCC Distinguishes Between Independent Primary Tumors And Contralateral Metastatic Disease

Objective

To evaluate whether bilateral, multifocal clear cell renal cell carcinoma (ccRCC) patients can be differentiated by VHL mutation analysis into cases that represent either multiple independently arising primary tumors or a single primary tumor that has spread ipsilaterally as well as to the contralateral kidney. The nature of kidney cancer multifocality outside of known hereditary syndromes is as yet poorly understood.

Materials and methods

DNA from multiple tumors per patient was evaluated for somatic VHL gene mutation and hypermethylation. A subset of tumors with shared VHL mutations was analyzed with targeted, next-generation sequencing assays.

Results

This cohort contained 5 patients with multiple tumors that demonstrated a shared somatic VHL mutation consistent with metastatic spread including to the contralateral kidney. In several cases, this was substantiated by additional shared somatic mutations in ccRCC-associated genes. In contrast, the remaining 14 patients with multiple tumors demonstrated unique, unshared VHL alterations in every analyzed tumor, consistent with independently arising kidney tumors. None of these latter patients showed any evidence of local spread or distant metastasis.

Conclusion

The spectrum of VHL alterations within evaluated bilateral, multifocal ccRCC tumors from a single patient can distinguish between multiple independent tumor growth and metastasis. This can be performed using currently available clinical genetic tests and will improve the accuracy of patient diagnosis and prognosis, as well as inform appropriate management.

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Introduction

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, accounting for 75% of cases. The prognosis and management of ccRCC patients are dependent upon the clinical stage. If disease is detected early, and is still localized within the kidney, surgical resection is associated with a 95% 5-10-year survival rate. However, if the tumor has spread locally or systemically, the 5-year survival rates drop to 67% or 12% respectively. Clear cell RCC is most often characterized by biallelic loss of the von-Hippel Lindau tumor suppressor gene, VHL. Mechanistically, this is achieved by the loss of one copy of chromosome 3p, including the region encoding VHL, in combination with mutation or promoter hypermethylation of the other copy of VHL. Somatic VHL gene alterations are observed in about 90% of sporadic ccRCC, with gene mutation seen in over 80% of ccRCCs and CpG island promoter hypermethylation observed in approximately 8% of ccRCCs.

While in the majority of sporadic ccRCC cases, there is only a single lesion in one kidney, a fraction presents with multiple lesions in one or both kidneys. These patients exhibit bilateral multifocal (BMF) ccRCC and the nature of these multiple tumors can influence diagnosis and patient management. The presentation could represent a single primary tumor that has spread within the ipsilateral kidney or that has metastasized to the contralateral kidney. Several studies have investigated cases of multifocal ccRCC by comparing either the degree of 3p loss or the spectrum of chromosomal alterations among tumors within the same patient to show that they share a common clonal origin consistent with metastatic spread or that each tumor represents an independently arising event. This susceptibility could be induced by an environmental effect, such as exposure to trichloroethylene (TCE), or due to a germline genetic alteration. A known cause of bilateral multifocal kidney cancer is the germline mutation of an RCC susceptibility gene, such as VHL, MET, BAP1, or FLCN, associated with an inherited tumor predisposition syndrome. The most common of these is von Hippel-Lindau (VHL) disease, caused by germline alteration of the VHL gene, resulting in a predisposition to bilateral multifocal ccRCC. While some BMF ccRCC patients will have VHL syndrome, many patients with multifocal bilateral disease will have no diagnosed RCC susceptibility syndrome.

Evaluating the difference between metastatic or independently arising disease is possible by genetic analysis of the tumors to identify either common shared genetic alterations, in the case of metastasis, or disparate varied genetic changes in the case of independent events. Distinguishing between these 2 clinical presentations is critical for the appropriate management of the patient. If metastasis has occurred, the patient would most likely be treated for systemic disease. In contrast, if all the tumors were multiple independent events within the kidney(s), the patient would likely be treated for localized disease and be investigated for germline genetic disorders with screening of potentially at-risk family members also being considered.

In this study, we utilized VHL gene mutation analysis in ccRCC as a genetic marker to compare tumors within each patient in a cohort of bilateral multifocal ccRCC patients that had no known germline genetic alteration. Two patient populations were identified: those with independently arising tumors and those with metastasis to the contralateral kidney.

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Materials and methods

1. Patients

Patients were seen at the Urologic Oncology Branch (UOB) of the National Cancer Institute (NCI), the National Institutes of Health (NIH) for clinical assessment. Patients were considered to have a family history if they have at least one first or second-degree relative with a history of clear cell RCC, and early onset if they were diagnosed at age 46 or younger. This study was approved by the Institutional Review Board of the National Cancer Institute and all patients provided written informed consent on Urologic Oncology Branch protocols. All patients were given a laboratory-assigned deidentified 6-digit number for internal referencing.

2. PCR and DNA Sequencing

DNA was extracted from blood and tumor tissue using Promega Maxwell 16 Blood and Tissue DNA Purification Kits (Promega, WI,). DNA sequencing was performed by PCR using a Qiagen Taq PCR Core Kit (Qiagen, MD) according to the manufacturer’s specifications, followed by bidirectional sequencing using the Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, CA) according to the manufacturer’s specifications and run on an ABI 3130xl or 3730 Genetic Analyzer (Applied Biosystems, CA). Sanger Sequencing was conducted at the CCR Genomics Core at the National Cancer Institute, NIH, Bethesda, MD 20892. Forward and reverse sequences were evaluated using Sequencher 5.0.1 (Genecodes, MI).

3. DNA Methylation Analysis

DNAs were subjected to bisulfite treatment using a Qiagen EpiTect Bisulfite Kit (Qiagen, MD) according to the manufacturer’s specifications. Two primer pairs were designed that would be capable of annealing to both methylated and unmethylated/bisulfite-converted DNA in the vicinity of the VHL promoter. PCR was performed as above with the outer primers, followed by a second round of PCR with the inner primers, and the products were subjected to bidirectional sequencing as above.

4. Oncomine and TruSight Oncology 500 Analysis

Select tumors from clinically indicated surgeries were chosen for analysis by the Oncomine Comprehensive Assay v3 (ThermoFisher, MA) or later, the TruSight Oncology 500 (Illumina, CA), next-generation sequencing assays capable of detecting single nucleotide variations, copy number variations, gene fusions and indels from 161 to 523 cancer driver genes, respectively. Formalin-fixed, paraffin-embedded samples were processed by the Molecular Diagnostics Section of the Laboratory of Pathology at NCI.

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Discussion

The ability to better characterize patients with bilateral multifocal renal tumors will enable better patient management and could lead to new insights into the genetic nature of clear cell renal cell carcinoma. In general, there is relatively little known about the evolution of bilateral multifocal ccRCC. The frequency of bilateral and/or multifocal RCC either at presentation or within a year of initial tumor discovery is relatively low and the reported frequency varies greatly from 0.8% to 5.4%. Further studies of larger cohorts with universal diagnostic criteria are required to ascertain the exact frequency of BMF ccRCC. Wiklund et al reported that BMF RCC is more common in younger patients and Syed et al demonstrated increased incidence with longer follow-up. A few studies have investigated whether multifocal RCC represents local metastasis or independently arising events by comparing either the degree of 3p loss or the spectrum of chromosomal alterations between tumors within the same patient. Miyake et al and Junker et al both showed shared patterns of chromosome 3p loss or chromosomal alteration between tumors in the same patient consistent with metastatic spread, while Ji et al showed different degrees of 3p loss among the tumors from 2 different patients consistent with independently arising tumors in both cases. A few case reports show evidence of metastatic spread from 1 kidney to the contralateral kidney, adrenal, or kidney region.

In the current study, all patients developed tumors in both kidneys. The metachronous disease was more common (4/5; 80%) in the group that demonstrated contralateral metastases than in those in which tumors grew independently (3/14; 21%), but both synchronous and metachronous contralateral disease was observed in both cohorts. Analysis of the pattern of VHL mutation among tumors from each patient allowed all patients to be designated as having either concern for metastatic disease or localized bilateral kidney tumors likely to have arisen independently. Patients with metastatic disease shared a common VHL alteration among all tumors, while patients with independently arising kidney tumors had different VHL alterations in different tumors.

Patients that demonstrated a shared VHL mutation in multiple kidney tumors could result from germline mosaicism VHL mutations rather than metastasis. In this case, the germline mutation may only be present in kidney cells, or even a fraction of the kidney cells, but not present in the white blood cells evaluated in a blood test or the buccal cells from a cheek swab. The patient would appear germline negative by standard testing but could have the same VHL mutation present in multiple independently arising tumors. This principle has recently been demonstrated in Wilms tumor patients who had mutations early in development that resulted in bilateral multifocal tumors with a shared driver mutation. Importantly, it was also shown that in this scenario the independent tumors would develop unique additional mutations as the tumor evolved. In general, mosaicism is a rare event and unlikely in this study as 3 patients demonstrated tumors with shared additional somatic gene mutations (MTOR and ARID1A for Patient 3; PBRM1, SETD2, and NFE2L2 for Patient 4; PBRM1 and KDM5C for Patient 5) that could only be shared due to metastasis rather than mosaicism. In addition, 2 patients with shared VHL mutations (Patients 2 and 3) also had sequencing performed on the adjacent normal kidney tissue that showed no evidence of VHL mutation. Finally, 4 of 5 patients (Patients 1 through 4) developed metastases outside the kidneys

Of note, patient 5 has not yet demonstrated evidence of distant metastases. While his largest tumor was 4.8 cm and he could be considered at higher risk for metastatic spread, the observation that all 7 of his recently resected tumors share VHL mutations and most share PBRM1 and KDM5C mutations raises the suspicion for distant metastases even higher. As a result, this patient will be followed with particularly careful and close observation going forward.

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Patients with independently arising kidney tumors demonstrated enrichment for both a family history of kidney cancer and early onset disease, both of which are indicators of inherited germline alterations. All patients had screened negative for known kidney cancer gene pathogenic variations, suggesting that alternative unknown genetic causes could be present. Germline translocation involving chromosome 3p has also been shown to increase the risk of ccRCC and is associated with bilateral multifocal disease. Translocations are not detectable by targeted gene arrays and require karyotype analysis. Thus, patients with independently arising kidney tumors that present without mutations in the known RCC susceptibility genes should also be screened for chromosome 3p translocations. Karyotype analysis was available for 12 of 14 of the patients with independently arising kidney tumors in this study, but no relevant translocations were observed. Further evaluation of these patients for additional germline alterations could identify novel RCC susceptibility genes and the VHL mutation spectrum analysis allows for the selection of these patients.

It is becoming increasingly common to perform targeted cancer gene mutation analysis on patient tumors or biopsies, and these analyses frequently include the VHL gene as well as additional ccRCC-associated genes such as PBRM1, BAP1, SETD2, and ARID1A. Therefore, routine bilateral biopsies of bilateral kidney masses may provide an easy and efficient method for distinguishing between contralateral metastasis and independently arising multifocal tumors, as opposed to the chromosomal alteration-based analyses that have been previously performed, allowing for further confirmation in the patients with metastatic disease. Several recent studies have highlighted the intratumoral heterogeneity present within ccRCC and this could confound biopsy analysis as the sampled area may lack a mutation that is present only in some areas of a tumor and provide inaccurate results. The advantage of evaluating VHL mutation is that it is present in most ccRCCs and is an early truncal mutation that would be present throughout the whole tumor, thus biopsies should nearly always be informative.

The importance of being able to distinguish between patients with either metastatic disease or localized independently arising kidney tumors is that they would be managed differently and potentially have distinctly different outcomes. While evidence of metastasis outside of the kidney is conclusive, imaging of patients that have BMF ccRCC does not allow for an accurate designation. In this study, this issue is best exemplified by Patients 4, 9, and 19 as all 3 presented with bilateral disease with a large tumor in one kidney and a smaller tumor or tumors in the contralateral kidney and no synchronous evidence of lesions outside of the kidney. Without VHL mutation analysis, the only noticeable feature is that Patient 19 had the youngest onset at age 36, and Patient 9 had the latest onset at age 62. Evaluation of the VHL mutation spectrum showed that both Patients 9 and 19 had different VHL alterations in different tumors, consistent with independently arising kidney tumors. Conversely, Patient 3 had the same shared VHL mutation in all tumors consistent with a common origin and contralateral metastasis. This was supported by the shared presence of shared additional somatic mutations in MTOR and ARID1A in all of patient 3’s tumors. Thus, Patients 9 and 19 could be treated by surgical excision of the tumors and routine surveillance, while Patient 3 might be treated with systemic therapies appropriate for a patient with metastatic disease.

In conclusion, analysis of VHL alteration in BMF ccRCC can help distinguish between contralateral metastatic kidney tumors and localized, independently arising kidney tumors and this can be used to influence patient management and outcome.

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Cathy D. Vocke, Christopher J. Ricketts, Adam R. Metwalli, Peter A. Pinto, Rabindra Gautam, Mark Raffeld, Maria J. Merino, Mark W. Ball, and W. Marston Linehan