Molecular underpinnings of dedifferentiation and aggressiveness in chromophobe renal cell carcinoma.

Sarcomatoid dedifferentiation is common to multiple renal cell carcinoma (RCC) subtypes, including chromophobe RCC (ChRCC), and is associated with increased aggressiveness, resistance to targeted therapies, and heightened sensitivity to immunotherapy. To study ChRCC dedifferentiation, we performed multiregion integrated paired pathological and genomic analyses. Interestingly, ChRCC dedifferentiates not only into sarcomatoid but also into anaplastic and glandular subtypes, which are similarly associated with increased aggressiveness and metastases. Dedifferentiated ChRCC shows loss of epithelial markers, convergent gene expression, and whole genome duplication from a hypodiploid state characteristic of classic ChRCC. We identified an intermediate state with atypia and increased mitosis but preserved epithelial markers. Our data suggest that dedifferentiation is initiated by hemizygous mutation of TP53, which can be observed in differentiated areas, as well as mutation of PTEN. Notably, these mutations become homozygous with duplication of preexisting monosomes (i.e., chromosomes 17 and 10), which characterizes the transition to dedifferentiated ChRCC. Serving as potential biomarkers, dedifferentiated areas become accentuated by mTORC1 activation (phospho-S6) and p53 stabilization. Notably, dedifferentiated ChRCC share gene enrichment and pathway activation features with other sarcomatoid RCC, suggesting convergent evolutionary trajectories. This study expands our understanding of aggressive ChRCC, provides insight into molecular mechanisms of tumor progression, and informs pathologic classification and diagnostics.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

HIF2 Inactivation and Tumor Suppression with a Tumor-Directed RNA-Silencing Drug in Mice and Humans.

PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that ∼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.

Predicting Oncologic Outcomes in Small Renal Tumors.

BACKGROUND: Most patients diagnosed with renal cancer today present with small renal masses (SRMs). Although these patients have a low risk of dying from their disease and many are followed with active surveillance protocols, a small subset of renal cell carcinomas (RCCs) behave aggressively. Knowledge regarding features of aggressive behavior would enable better adoption of active surveillance strategies among these patients. OBJECTIVE: We sought to improve prognostic models to predict metastasis-free survival after nephrectomy through focused analyses of clinicopathologic characteristics of SRMs associated with adverse outcomes. DESIGN, SETTING, AND PARTICIPANTS: We identified consecutive patients with surgically resected SRMs (≤4 cm) at the University of Texas Southwestern Kidney Cancer Program between 1998 and 2020. In addition, we evaluated the ability of SRMs to form tumors when implanted in mice, an indicator of tumor aggressiveness. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We examined the clinicopathologic factors associated with metastasis including prospectively performed BAP1 immunohistochemistry at our Clinical Laboratory Improvement Amendments laboratory. Multivariable Cox proportional hazard regression was used to predict metastasis-free survival. RESULTS AND LIMITATIONS: A total of 3900 evaluable nephrectomies (from 3674 ethnically diverse patients) were identified, of which 1984 (51%) were SRMs including 1720 RCC. Of these patients with RCC (SRMRCC), 1576 did not have synchronous or metachronous larger RCCs and among these, 37 (2%) developed metastases. SRMRCC that metastasized were significantly enriched for aggressive morphologic phenotypes and engrafted in mice at comparable rates as larger metastatic tumors. BAP1 loss remained significantly associated with metastasis-free survival after accounting for TNM (tumor-node-metastasis) stage and SSIGN (stage, size, grade, and necrosis) score in multivariable analysis. CONCLUSIONS: We identified clinicopathologic features that influence metastasis-free survival for patients with SRMRCC. If validated independently, these data should assist with patient prognosis and help with active surveillance strategies. PATIENT SUMMARY: We report the identification of features of aggressiveness in small renal tumors that influence the likelihood of metastases after surgery.

Germline and sporadic mTOR pathway mutations in low-grade oncocytic tumor of the kidney.

Low-grade oncocytic tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63-86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well-circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos, and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117, and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors. At a median follow-up of 29 months, four patients were alive without recurrence (three patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo, and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation, and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

SARS-CoV-2 Immunohistochemistry In Placenta.

Compared to the parental SARS-CoV-2 virus, infections by the now dominant Delta variant of SARS-CoV-2 appear to be more common and more severe in pregnant women. The need for a robust, cheap, and quick method for diagnosing placental infection by SARS-CoV-2 has thus become more acute. Here, we describe a highly sensitive and specific immunohistochemical assay for SARS-CoV-2 nucleocapsid protein for routine use in placental pathology practice.

Cytoplasmic P53 Immunostaining With N-Terminus P53 Antibody And Absence Of Staining With C-Terminus P53 Antibody: A Report Of Two Pediatric Sarcomas With Distal Truncating TP53 Mutations Affecting Nuclear Localization Domain.

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used.