Molecular characterisation of metanephric adenomas beyond BRAF: genetic evidence for potential malignant evolution

Abstract

Aims: Metanephric adenomas (MAs) have historically been characterized and are generally perceived as rare, benign renal tumors that typically exhibit an indolent clinical course. However, a limited number of individual case reports have emerged in recent literature, describing instances where MAs have displayed aggressive pathological features and clinical behaviors, challenging the conventional understanding of their uniformly benign nature. From a molecular perspective, conventional MAs are well-established to consistently harbor specific hotspot mutations in the BRAF gene, most notably the BRAF V600E mutation. A proposed molecular pathway, known as the BRAF V600E senescence pathway, has been hypothesized to play a crucial role in conferring the typically benign phenotype observed in MAs. This pathway is believed to be mediated, at least in part, by the cyclin-dependent kinase inhibitor 2A (CDKN2A), also known as p16. Despite the known involvement of BRAF, the broader molecular landscape, encompassing other potential genomic alterations in both conventional MAs and, more critically, in those rare cases exhibiting aggressive features, has not yet been comprehensively or fully characterized. Therefore, the primary aim of this study was to undertake a thorough molecular profiling of a distinct series of Metanephric Adenomas. Our specific objective was to investigate whether a direct correlation could be established between the identified genomic findings and the observed clinical outcomes of the patients, thereby seeking to unravel the molecular underpinnings of MA behavior.

Methods and Results: To achieve the stated aims, we conducted a meticulous retrospective examination that encompassed both the histomorphology and the long-term patient outcomes for a cohort of 11 cases diagnosed as conventional Metanephric Adenomas. Additionally, our investigation included one particularly notable case of an MA that exhibited aggressive pathological and clinical features. Each of these tumor samples underwent comprehensive molecular analysis. This involved capture-based next-generation DNA sequencing, a high-throughput technology that allowed for the simultaneous sequencing of 479 meticulously selected cancer-related genes. In parallel, immunohistochemical profiling was performed on each tumor to assess protein expression patterns, which often correlate with underlying genetic alterations. A consistent finding across all tumors analyzed was positive immunostaining for WT1, a protein commonly expressed in MAs. Furthermore, all tumors, without exception, were found to harbor the characteristic BRAF V600E mutation, underscoring its pivotal role in the molecular genesis of these lesions. In one specific case of a conventional MA, an additional somatic pathogenic mutation in the BRCA2 gene was identified, highlighting the possibility of rare co-occurring alterations even in otherwise indolent tumors. The most striking and complex case in our series was the MA with aggressive features. This particular tumor presented with a distinct biphasic appearance, indicating the presence of two morphologically disparate components. One component was predominantly epithelial, exhibiting areas that were histologically consistent with the appearance of a conventional MA, suggesting a possible origin from such a lesion. The second, more concerning component, was distinctly sarcomatous, characterized by areas of aggressive solid growth and, notably, regions displaying angiosarcomatous differentiation, indicative of a highly malignant transformation. To understand the genetic relationship between these two components, differential molecular profiling was performed on both populations. This analysis revealed the presence of identical BRAF, EIF1AX, and TERT promoter hotspot mutations in both the epithelial and the sarcomatous components, strongly suggesting a clonal relationship and evolution from a common progenitor. Crucially, however, a deep deletion of CDKN2A (the gene encoding p16) and amplification of the MYC oncogene were identified exclusively within the sarcomatous component. The absence of these specific alterations in the epithelial component, coupled with their presence in the aggressive sarcomatous component, points to them as likely drivers of malignant progression.

Conclusions: In summary, while the vast majority of Metanephric Adenomas consistently demonstrate an indolent clinical behavior, our study provides compelling evidence that rare but significant pathogenic genetic alterations can indeed occur in conventional MAs, appearing in addition to the universally observed BRAF V600E mutation. More critically, the detailed molecular profiling of a specific case that exhibited aggressive clinical and pathological features yielded profound insights. The identification of a shared set of driver mutations (BRAF, EIF1AX, TERT promoter) across both the conventional epithelial and the aggressive sarcomatous components, coupled with the exclusive presence of CDKN2A deep deletion and MYC amplification in the sarcomatous component, provides compelling genetic evidence for malignant evolution within Metanephric Adenomas. These findings suggest that certain secondary genomic events, particularly those affecting tumor suppressor genes like CDKN2A or oncogenes like MYC, may confer an aggressive phenotype and drive the transformation of an otherwise benign MA into a more malignant entity. This study significantly advances our understanding of the molecular pathogenesis of MAs, particularly in rare cases of aggressive disease.

Keywords: BRAF V600E; cancer genetics; metanephric adenoma; p16(INK4a); renal tumour.

Introduction

Metanephric adenoma (MA) is recognized as a comparatively rare type of renal tumor, which is conventionally presumed to exhibit an indolent or benign clinical course. These tumors can manifest across all age groups, though they show a discernible predominance in female patients. Histologically, MA is characterized by its composition of small, uniform epithelial cells. These cells are typically arranged in densely packed acini or tubules, forming intricate glandular structures. Additionally, in some instances, characteristic glomeruloid structures, papillary architecture, and more solid areas may also be observed within the tumor. The morphological features of MA can, at times, overlap with those of other distinct renal tumor entities, posing a diagnostic challenge. These include epithelial-predominant Wilms’ tumor (WT) and papillary renal cell carcinoma, necessitating careful differentiation.

To aid in resolving this crucial differential diagnosis, immunohistochemistry plays a vital role. Metanephric adenomas are commonly characterized by positive immunostaining for WT1 and CD57, while generally showing negative to minimal staining for cytokeratin (CK) 7. From a genetic perspective, a defining molecular hallmark of MAs is the consistent presence of BRAF V600E mutations. These specific mutations are exceedingly rare in other types of renal tumors, making them a highly valuable diagnostic marker for MA. Furthermore, specialized antibodies designed to specifically target the BRAF V600E mutant protein are now clinically available and can significantly assist in the accurate diagnosis of MA, complementing standard histological and immunohistochemical analyses.

The conventionally benign nature of MA has been theoretically attributed to a specific BRAF V600E senescence pathway. This pathway is thought to be mediated, at least in part, by the cyclin-dependent kinase inhibitor 2A (CDKN2A), also known as p16. This proposed mechanism is consistent with observations in other benign BRAF-mutant neoplasms, such as melanocytic nevi, where BRAF-driven proliferation is typically constrained by senescence. Supporting this concept in MAs, increased immunohistochemical expression of p16 has been demonstrably observed in these tumors. However, despite the general understanding of MA’s benignity, rare cases of metanephric tumors exhibiting adverse pathological features, such as the presence of sarcomatous elements or evidence of nodal metastasis, have been sporadically reported in the literature. These unusual cases raise critical questions regarding the precise malignant potential of MA, which remains to be fully and comprehensively defined. Moreover, the broader molecular landscape of MA is still largely incomplete, extending beyond the well-established prevalence of BRAF mutations. Therefore, to address these knowledge gaps and to establish a potential molecular route for malignant transformation in these rare tumors, we undertook a comprehensive approach. This involved performing detailed clinicopathological, immunohistochemical, and advanced molecular analyses on a retrospective cohort of MAs, specifically including one particularly challenging case that presented with adverse pathological features and an aggressive clinical behavior.

Materials and Methods

Case Selection

This comprehensive study was meticulously conducted under the stringent ethical oversight and formal approval of the institutional review boards of both Stanford University and the University of California San Francisco (UCSF), operating under Institutional Review Board no. 18-25999. From the extensive archives maintained at both UCSF and Stanford, a total of 12 Metanephric Adenoma (MA) cases were carefully selected for inclusion in this investigation. The primary selection criterion was the availability of both histological slides and corresponding tissue samples for in-depth analysis (designated as MA1 to MA12). To ensure diagnostic accuracy and consistency, all Hematoxylin and Eosin-stained slides from these cases underwent rigorous review by a minimum of two expert genitourinary pathologists. It is important to note that one particular case, MA11, had been previously included in an earlier study. However, for the purposes of the present investigation, MA11 was subjected to a more extensive and detailed genomic analysis, allowing for deeper molecular insights within this expanded series.

Immunohistochemistry

The specific antibody clones and precise conditions employed for all immunohistochemical analyses performed in this study were meticulously detailed and comprehensively presented in Table S1, which serves as a supplementary resource. This thorough documentation ensures the reproducibility and transparency of our immunohistochemical findings.

Capture-Based Next-Generation DNA Sequencing

For a subset of five cases (specifically MA1 through MA4, and MA12), both matched normal tissue and tumor tissue were carefully selected for comprehensive molecular analysis, allowing for the identification of somatic alterations. For the remaining seven cases (MA5 through MA11), only tumor tissue was available and thus utilized for sequencing. The molecular profiling was conducted using capture-based next-generation DNA sequencing. This advanced sequencing technique was performed with a custom assay, designated as UCSF500, which is specifically designed to target the coding regions of 479 meticulously selected cancer-related genes. Additionally, the assay included selected intronic regions from 41 genes, and, critically, the promoter region of the TERT gene, all of which are relevant to cancer biology. The precise details of the genes targeted by this assay were thoroughly outlined in Table S2, and the methodology has been previously described in detail in a prior publication.

Results

Clinical, Gross, Microscopic and Immunophenotypic Features

This comprehensive study encompassed a total of twelve identified cases. Of these, eleven cases were categorized as conventional Metanephric Adenomas (designated as cases 1–11), exhibiting typical features consistent with this diagnosis. The remaining single case (case 12) was particularly noteworthy as it presented with distinct malignant features, prompting a more in-depth investigation into its aggressive characteristics. A detailed compilation of the clinical characteristics and morphological features observed across all cases is meticulously presented in Tables S3 and S4, providing a comprehensive overview of the cohort.

For the cohort of eleven conventional Metanephric Adenomas, the age at which patients presented with the tumor ranged from 38 to 72 years, with an average age of 54 years. A notable female predominance was observed, with a female-to-male ratio of 10:1. The size of these tumors varied considerably, ranging from 10 to 60 millimeters, with a mean diameter of 29 millimeters. Encouragingly, all patients within this conventional MA group, for whom follow-up data were available, showed no evidence of residual or recurrent disease, confirming the generally indolent nature of these tumors. At a macroscopic level, the conventional MAs were consistently well-circumscribed, indicating a clear demarcation from the surrounding healthy renal tissue, and remained confined strictly within the kidney. On cut surfaces, they typically exhibited a solid, tan-white to yellow appearance. In some instances, focal areas of hemorrhage or cystic change were also noted, reflecting minor internal variations. Microscopically, these MAs displayed the characteristic histological features that have been previously well-described for this entity. The predominant growth patterns observed were acinar/tubular and papillary structures. Importantly, no significant mitotic activity, which would suggest rapid cell division, or necrosis, indicative of widespread cell death, was seen in these conventional tumors. Immunohistochemical staining further supported the diagnosis, with all conventional MAs consistently showing positive staining for WT1 and CD57. In contrast, they demonstrated negative to only focal staining for CK7, further differentiating them from other renal tumors. A crucial molecular finding was that BRAF V600E immunostaining was strongly positive in eight out of the eleven cases, reinforcing its common presence. Similarly, p16 immunostaining was strongly positive in all cases, aligning with the theory of BRAF V600E-induced senescence. Furthermore, Ki67, a marker of cellular proliferation, consistently showed low proliferation rates, ranging from less than 1% to 3%, again indicative of their benign nature.

Case 12 presented a unique and complex scenario, involving a 76-year-old woman who initially sought medical attention due to gross hematuria, the presence of visible blood in her urine. Diagnostic imaging subsequently revealed a 55-millimeter complex cystic mass located in her right kidney. Following a radical nephrectomy, the patient unfortunately experienced an exceptionally aggressive clinical course. This was characterized by the rapid and extensive regrowth of tumor tissue in the retroperitoneum and intra-abdominal regions, ultimately leading to her succumbing to the disease within a mere four months of the initial surgery. Macroscopic examination of the resected kidney revealed a partially encapsulated, dark brown mass, characterized by significant hemorrhage and necrosis. Additionally, multiple tan satellite nodules were observed, along with clear involvement of the segmental renal veins, all indicative of widespread and aggressive disease. Histological sections obtained from this tumor demonstrated the presence of multiple morphologically distinct components, highlighting its heterogeneous nature. One component was unequivocally epithelial, containing some areas that were histologically compatible with the appearance of a conventional metanephric adenoma. However, other areas within this epithelial component displayed higher-grade features, characterized by nodules of primitive-appearing, densely packed tubules and pseudorosettes. These structures were lined by cells exhibiting elongated, overlapping nuclei, a vesicular chromatin pattern, and high nuclear-to-cytoplasmic ratios, all suggestive of increased cellular atypia and proliferation. The remainder of the tumor was distinctly sarcomatous in nature. This sarcomatous component included anastomosing vasoformative-appearing structures, lined by highly pleomorphic cells, indicative of an aggressive vascular differentiation. Additionally, solid sheets of poorly differentiated neoplastic cells were observed. Critically, significant mitotic activity and tumor-type necrosis, both strong indicators of malignancy, were readily appreciated within both the primitive epithelial and the sarcomatous components, confirming the highly aggressive nature of this particular tumor.

On detailed immunohistochemical analysis, the epithelial component of Case 12 demonstrated characteristics supportive of Metanephric Adenoma (MA), exhibiting positive staining for PAX8, WT1, CD57, BRAF V600E, and p16. Importantly, it showed negative staining for CK7, further aligning with the MA immunophenotype. In the sarcomatous portion of the tumor, the vasoformative areas showed positive staining for multiple vascular markers, providing strong support for the presence of an angiosarcoma component. The remaining solid areas within the sarcomatous component were thoroughly tested but found to be negative for an exhaustive list of lineage-specific markers, including those for epithelial, muscle, neural/melanocytic, and other specific lineages, suggesting a poorly differentiated or undifferentiated phenotype. Interestingly, the sarcomatous component also notably showed positive staining for BRAF V600E. This shared BRAF mutation across both the epithelial and sarcomatous components is a crucial finding, strongly suggesting a shared clonal origin between them, rather than representing a chance “collision tumor” where two unrelated malignancies happen to co-exist. In a striking contrast to the epithelial component, p16 immunostaining was entirely negative in the sarcomatous component. Concurrently, Ki67 expression, a marker of cellular proliferation, was markedly increased in the sarcomatous component. This inverse relationship between p16 expression and proliferation, coupled with the shared BRAF mutation, provides compelling evidence for the potential role of p16 loss in driving malignant progression within the context of MA.

Molecular Analysis of Conventional MAs and the MA with Malignant Features

The comprehensive genomic data derived from our molecular analyses are succinctly presented, with further intricate details provided in supplementary figures and tables. Across all twelve tumors analyzed in this study, a consistent and pivotal finding was the presence of a BRAF p.V600E mutation. This underscores the fundamental role of this specific genetic alteration in the pathogenesis of metanephric adenomas. Among the conventional MAs, one particular case (case 4) stood out, exhibiting an additional somatic pathogenic mutation: a BRCA2 p.L2587fs inactivating mutation. Beyond this rare occurrence, no other pathogenic alterations were detected in the remaining conventional MA cases. Copy number analysis of the conventional MAs revealed relatively flat genomes, indicating a general absence of widespread chromosomal gains or losses. However, recurrent chromosomal gains in region 19p were specifically detected in six out of eleven (55%) of these tumors. In four of these cases (4/11, 36%), these 19p gains were accompanied by concurrent gains in region 19q. Importantly, no amplifications, deep deletions, or fusions were identified in this group of conventional MAs, reinforcing their typically stable genetic profile.

In the case of the Metanephric Adenoma (MA) with malignant features (Case 12), a more intricate and revealing molecular profile emerged. To precisely delineate the genetic landscape of its distinct components, the epithelial and angiosarcomatous portions of the tumor were meticulously sequenced separately. Differential profiling of these two components unveiled a number of shared alterations, which provided crucial insights into their clonal relationship. Specifically, identical BRAF p.V600E, EIF1AX p.K10E, and TERT promoter c.-124C>T hotspot mutations were identified in both the epithelial and sarcomatous components. This commonality strongly suggests that these mutations represent early genetic events in the tumor’s pathogenesis and that both components originated from a shared progenitor cell that harbored these initial alterations. However, the analysis also revealed critical differences that illuminate the path of malignant progression. A deep deletion of CDKN2A, the gene encoding the p16 tumor suppressor, was identified exclusively in the sarcomatous component. This finding was entirely consistent with the immunohistochemical data, which showed a complete loss of p16 protein expression in this aggressive part of the tumor. Furthermore, amplification of the MYC oncogene was also found to be specific to the sarcomatous component, suggesting its role in driving the accelerated proliferation and aggressive behavior characteristic of this transformed lineage. Another alteration, a SETD2 p.N1396fs mutation, was found to be exclusive to the epithelial component, indicating a divergent evolutionary path for this less aggressive subpopulation. Despite these specific genomic changes, both the epithelial component and the sarcomatous component otherwise exhibited relatively flat genomes, with only scant chromosomal gains and losses, suggesting that the key drivers of malignancy were highly specific genomic events rather than widespread chromosomal instability. Crucially, no fusions or pathogenic germline alterations were detected, confirming that the observed changes were somatic and related to tumor development rather than inherited predispositions.

Discussion

This study undertook a comprehensive examination of the genetic profiles across a series of Metanephric Adenomas (MAs), specifically including eleven conventional MAs and one particularly notable MA that exhibited malignant features. The application of next-generation sequencing technology provided crucial insights, confirming the presence of BRAF p.V600E mutations in all eleven conventional MAs. Furthermore, BRAF V600E immunohistochemistry was positive in 73% of these cases, a sensitivity rate that is comparable to findings reported in various previous studies. In one of the conventional MAs (case 4), a rare and interesting finding was the identification of an additional single-copy somatic BRCA2 p.L2587fs inactivating mutation. The precise clinical significance of this co-occurring mutation remains uncertain, as this particular case did not exhibit any distinguishing morphological or clinical characteristics that set it apart from other conventional MAs. However, a recent publication that profiled a cohort of MAs in Chinese patients, utilizing a panel of cancer-related genes, also identified numerous other mutations. In that study, only alterations in NF1 and ASXL1 were deemed pathogenic, with the remainder being synonymous or missense variants of uncertain significance. Nonetheless, these findings, including our own, collectively provide supporting evidence that MAs, even those considered conventional, can indeed acquire additional pathogenic alterations beyond the signature BRAF mutation. Moreover, our observation of recurrent gains in chromosome 19 serves as a confirmatory finding, corroborating a previous report in the literature.

Regarding Case 12, a particularly challenging diagnostic scenario, a diagnosis of adult Wilms’ tumor (WT) was initially strongly considered given its aggressive presentation. However, despite extensive and meticulous tissue sampling, no blastemal or stromal components, which are typical histological hallmarks of WT, were identified. Furthermore, diffuse positivity for CD57 within the epithelial component, the confirmed identification of a BRAF V600E mutation, and critically, the absence of alterations in genes previously reported to be recurrently involved in WT, all collectively provided strong molecular and immunohistochemical support for a diagnosis of MA over WT. We also acknowledge a recent publication that described a series of unusual cases initially diagnosed as epithelial WT but exhibiting areas morphologically resembling MA. In that particular series, four out of nine cases were found to harbor BRAF V600E mutations. However, that study did not perform more detailed molecular profiling to further investigate potential mechanisms of pathogenesis. Interestingly, one case within that series demonstrated progression after traditional chemotherapy but subsequently showed a positive response to BRAF inhibitor therapy, powerfully highlighting the clinical importance of recognizing and identifying BRAF V600E-mutated tumors, regardless of their initial diagnostic label.

The observed pattern of molecular alterations in the Metanephric Adenoma (MA) with aggressive features bears a striking resemblance to the well-established molecular progression described in the transformation from a benign melanocytic nevus to a malignant melanoma. In this widely recognized oncogene-induced senescence model, the expression of BRAF V600E initially leads to a critical cell cycle arrest in benign nevi. This arrest is mediated by the robust induction of the tumor suppressor p16(INK4a), acting as a protective mechanism against uncontrolled proliferation. However, during the process of malignant progression to nevus-associated melanoma, a crucial event involves the functional loss of p16 or its encoding gene, CDKN2A, which then allows uncontrolled growth. Similarly, pathogenic mutations in the TERT promoter and overexpression of the MYC oncogene have also been reported as additional selective steps that contribute to the malignant evolution in this context, providing further drivers for uncontrolled proliferation and invasion. Analogously, in our malignant MA case (case 12), both the epithelial component and the sarcomatous component harbored shared BRAF and TERT promoter mutations. This strongly suggests that these mutations represent early, foundational alterations in the tumor’s pathogenesis, present in its progenitor cells. Crucially, however, the deep deletion of p16/CDKN2A and the amplification of MYC were found exclusively within the sarcomatous component. This differential presence strongly leads us to speculate that these specific genetic events occurred later in the malignant transformation process, acting as key drivers that facilitated the aggressive, sarcomatous phenotype.

In conclusion, our study comprehensively confirms the long-held understanding that the vast majority of conventional Metanephric Adenomas exhibit an indolent and benign clinical behavior. However, our findings also provide crucial new insights, demonstrating that in rare instances, conventional MAs can indeed acquire additional pathogenic genetic alterations beyond the universally recognized BRAF mutation. Furthermore, and consistent with the prevailing theory of BRAF V600E-induced senescence acting as a protective mechanism in MAs, we present compelling evidence for a potential molecular mechanism by which MA can undergo malignant transformation. This transformation, PF-07799933 as exemplified by our single, aggressive case, appears to involve key genetic events such as p16 inactivation and TERT promoter mutation. This groundbreaking work significantly reinforces the importance of conducting additional and more comprehensive molecular characterization of MA-like cases, particularly those that exhibit any signs of malignant transformation. Ultimately, the accurate recognition of these malignant tumors as MAs, combined with the critical need for BRAF mutation testing, carries significant clinical implications. This is especially pertinent given the documented potential for positive therapeutic response to BRAF-targeted therapies in tumors harboring this specific mutation, offering new avenues for precision medicine in these rare and challenging renal malignancies.