EGFR/CEP7 high polysomy is separate and distinct from EGFR amplification in glioblastoma as determined by fluorescence in situ hybridization.

EGFR amplification in gliomas is commonly defined by an EGFR/CEP7 ratio of ≥2. In testing performed at a major reference laboratory, a small subset of patients had ≥5 copies of both EGFR and CEP7 yet were not amplified by the EGFR/CEP7 ratio and were designated high polysomy cases. To determine whether these tumors are more closely related to traditionally defined EGFR-amplified or nonamplified gliomas, a retrospective search identified 22 out of 1143 (1.9%) gliomas with an average of ≥5 copies/cell of EGFR and CEP7 with an EGFR/CEP7 ratio of <2 displaying high polysomy. Of these cases, 4 had insufficient clinicopathologic data to include in additional analysis, 15 were glioblastomas, 2 were IDH-mutant astrocytomas, and 1 was a high-grade glial neoplasm, NOS. Next-generation sequencing available on 3 cases demonstrated one with a TERT promoter mutation, TP53 mutations in all cases, and no EGFR mutations or amplifications, which most closely matched the nonamplified cases. The median overall survival times were 42.86, 66.07, and 41.14 weeks for amplified, highly polysomic, and nonamplified, respectively, and were not significantly different (p = 0.3410). High chromosome 7 polysomic gliomas are rare but our data suggest that they may be biologically similar to nonamplified gliomas.

Genomic classifiers and prognosis of localized prostate cancer: a systematic review.

BACKGROUND: Refinement of the risk classification for localized prostate cancer is warranted to aid in clinical decision making. A systematic analysis was undertaken to evaluate the prognostic ability of three genomic classifiers, Decipher, GPS, and Prolaris, for biochemical recurrence, development of metastases and prostate cancer-specific mortality in patients with localized prostate cancer. METHODS: Data sources: MEDLINE, Embase, and Web of Science were queried for reports published from January 2010 to April 2022. STUDY SELECTION: prospective or retrospective studies reporting prognosis for patients with localized prostate cancer. DATA EXTRACTION: relevant data were extracted into a customized database by one researcher with a second overreading. Risk of bias was assessed using a validated tool for prognostic studies, Quality in Prognosis Studies (QUIPS). Disagreements were resolved by consensus or by input from a third reviewer. We assessed the certainty of evidence by GRADE incorporating adaptation for prognostic studies. RESULTS: Data synthesis: a total of 39 studies (37 retrospective) involving over 10,000 patients were identified. Twenty-two assessed Decipher, 5 GPS, and 14 Prolaris. Thirty-four studies included patients who underwent prostatectomy. Based on very low to low certainty of evidence, each of the three genomic classifiers modestly improved upon the prognostic ability for biochemical recurrence, development of metastases, and prostate cancer-specific mortality compared to standard clinical risk-classification schemes. LIMITATIONS: downgrading of confidence in the evidence stemmed largely from bias due to the retrospective nature of the studies, heterogeneity in treatment received, and era in which patients were treated (i.e., prior to the 2000s). CONCLUSIONS: Genomic classifiers provide a small but consistent improvement upon the prognostic ability of clinical classification schemes, which may be helpful when treatment decisions are uncertain. However, evidence from current management-era data and of the predictive ability of these tests is needed.

Histologic patterns in prostatic adenocarcinoma are not predictive of mutations in the homologous recombination repair pathway.

Somatic or germline homologous recombination repair (HRR) pathway gene mutations are commonly detected in prostate cancer, especially in advanced disease, and are associated with response to poly (ADP-ribose) polymerase (PARP) inhibitors. In this study, we evaluated whether histological patterns are predictive of HRR pathway gene mutations. The study population comprised 130 patients with advanced prostate carcinoma who underwent comprehensive genomic profiling (CGP) of tumor tissue at a CLIA-certified laboratory. HRR genes in the study included BRCA1, BRCA2, ATM, BARD1, BRIP, CHEK2, MRE11A, NBN, PALB2, RAD51C, RAD51D, EMSY, ATR, CHEK1, and FAM175A. Overall, 38 patients had mutations in BRCA1/2, 36 in other HRR genes, and 56 were negative for HRR mutations. All cases were re-reviewed and quantified by two genitourinary pathologists blinded to mutational status for the following histological patterns of prostate carcinoma: cribriform, ductal, intraductal carcinoma (IDC), small cell carcinoma, signet ring-like pattern, and lobular carcinoma-like pattern. Discordances were resolved by consensus review. Histologic patterns were analyzed for any correlation with mutations in HRR pathway genes (grouped as BRCA1/2 mutated or non-BRCA1/2 mutated) compared to tumors without mutations in HRR genes by Chi-square testing. Patterns with >20 % and >30 % of tumor volume were additionally evaluated for correlation with mutational status. We found no significant association between HRR pathway mutations and cribriform pattern, IDC, ductal carcinoma, small cell carcinoma, signet ring-like pattern, or lobular carcinoma-like patterns. Tumors with >20 % or >30 % histologic patterns by volume also demonstrated no significant association with mutational status. This study suggests that histopathologic examination alone is insufficient to distinguish prostate cancer with germline or somatic mutations in HRR pathway genes, highlighting the continuing importance of ancillary molecular diagnostics in guiding therapy selection for prostate cancer patients who may benefit from PARP inhibitors.

Incidental Detection of TFEB-Amplified Renal Cell Carcinoma by Colocated Gene Amplification of CCND3 (6p21): A Case Report and Review of the Literature.

TFEB-amplified renal cell carcinoma (RCC), which belongs to the MITF family of RCC, is characterized by genomic amplification at the 6p21.1 locus where the TFEB gene is located. The vascular endothelial growth factor A and cyclin D3 genes are also located at this same locus. When tumors lack classic morphologic features, they may be classified as "RCC not otherwise specified (NOS)." However, it is increasingly important to accurately diagnose the RCC subtype to define the patient's individual prognosis and select the subsequent therapeutic modalities, which now include targeted agents. Therefore, knowledge of the diagnostic features of TFEB-altered RCCs, such as t(6;11) RCCs and TFEB-amplified RCCs, is critical for identifying these tumors. Herein, we present an interesting case of TFEB-amplified RCC that was initially diagnosed as RCC NOS on biopsy of a renal tumor in a community practice setting with available molecular findings demonstrating CCND3 amplification. The genetic abnormality was "accidentally" detected due to the amplification of the colocated CCND3 gene at the 6p21 locus of the TFEB gene on a limited genetic sequencing panel. This case highlights the importance of molecular tests in accurately diagnosing RCC and carefully interpreting molecular findings in the context of histomorphologic features.

Clinicopathologic Features of Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists Fluorescence In Situ Hybridization Group 3 (Human Epidermal Growth Factor Receptor 2 Chromosome 17 Centromere Ratio <2.0 and Average Human Epidermal Growth Factor Receptor 2 Copy Number ≥6.0) Breast Cancers.

CONTEXT.­: The American Society of Clinical Oncology/College of American Pathologists 2018 update of the human epidermal growth factor receptor 2 (HER2) testing guideline includes a fluorescence in situ hybridization (FISH) group with a HER2 to chromosome 17 centromere (CEP17) ratio less than 2.0 and HER2 copy number 6.0 or greater (group 3), which requires integrated review of HER2 immunohistochemistry (IHC). OBJECTIVE.­: To assess the clinicopathologic features of group 3 patients and determine features associated with HER2-positive status after workup. DESIGN.­: Cases submitted for HER2 FISH between January 2019 and June 2022 were identified, and relevant clinicopathologic information was obtained. RESULTS.­: One hundred forty-two HER2 FISH cases (1.6%) were group 3. In 52 cases (36.6%) IHC was negative (0/1+), in 3 (2.8%) IHC was positive (3+), and in 86 (60.6%) IHC was 2+. Annotated IHC 2+ slides were recounted by a second reviewer in targeted areas, where 16 of 86 (18.6%) had a HER2:CEP17 ratio less than 2.0 and a HER2 copy number of 4.0 or greater to less than 6.0 (HER2 negative). After combined IHC/FISH review, 74 of 142 (52.1%) were classified as HER2 positive. HER2 copy number/cell was higher in HER2-positive compared with HER2-negative cases after the workup. The extent and intensity of staining in IHC 2+ cases did not correlate with the level of gene amplification. Twenty percent of HER2-positive patients achieved pathologic complete response. CONCLUSIONS.­: About half of group 3 cases were classified as HER2 positive after additional workup. Pathologic complete response rates in HER2-positive cases were lower than expected for group 1 (HER2:CEP17 ratio ≥2.0; HER2 copy number ≥4.0) patients. IHC targeted FISH recounts may be redundant and may potentially lead to classification of some patients as HER2 negative, resulting in withholding of targeted therapy.

Papillary Renal Cell Carcinoma: A Review of Prospective Clinical Trials.

OPINION STATEMENT: PRCC is a unique histologic entity compared to other forms of renal cell carcinoma, harboring distinct molecular drivers. The WHO 2022 classification is further emphasizing the molecular biology by making molecular classifications of PRCC subclassifications and discontinuing the morphologic type 1 and type 2 classification system. We agree with this functional classification system and encourage all future clinical trials to only include patients with similar diagnosis instead of conducting basket trials (including all nccRCC together) which limits the scientific value of those conclusions. Based on recent disease-specific clinical trial (S1500, PAPMET), the current standard of care for patients with treatment naïve PRCC is cabozantinib. Prospective clinical trials clearly establish that immune checkpoint inhibitor therapy has meaningful activity in PRCC. The data to date include only single-arm clinical trials of combination immune therapy. Despite the positive and encouraging results, we need validation through randomized studies because of the overestimation of effect size seen in single-arm trials. These randomized trials are currently underway and enrolling. We strongly encourage all physicians to support these studies and enroll patients with PRCC to these trials in order to continue improving the standard of care.

Federated learning for 6G-enabled secure communication systems: a comprehensive survey.

Machine learning (ML) and Deep learning (DL) models are popular in many areas, from business, medicine, industries, healthcare, transportation, smart cities, and many more. However, the conventional centralized training techniques may not apply to upcoming distributed applications, which require high accuracy and quick response time. It is mainly due to limited storage and performance bottleneck problems on the centralized servers during the execution of various ML and DL-based models. However, federated learning (FL) is a developing approach to training ML models in a collaborative and distributed manner. It allows the full potential exploitation of these models with unlimited data and distributed computing power. In FL, edge computing devices collaborate to train a global model on their private data and computational power without sharing their private data on the network, thereby offering privacy preservation by default. But the distributed nature of FL faces various challenges related to data heterogeneity, client mobility, scalability, and seamless data aggregation. Moreover, the communication channels, clients, and central servers are also vulnerable to attacks which may give various security threats. Thus, a structured vulnerability and risk assessment are needed to deploy FL successfully in real-life scenarios. Furthermore, the scope of FL is expanding in terms of its application areas, with each area facing different threats. In this paper, we analyze various vulnerabilities present in the FL environment and design a literature survey of possible threats from the perspective of different application areas. Also, we review the most recent defensive algorithms and strategies used to guard against security and privacy threats in those areas. For a systematic coverage of the topic, we considered various applications under four main categories: space, air, ground, and underwater communications. We also compared the proposed methodologies regarding the underlying approach, base model, datasets, evaluation matrices, and achievements. Lastly, various approaches' future directions and existing drawbacks are discussed in detail.

Triple-Negative Apocrine Carcinomas: Toward a Unified Group With Shared Molecular Features and Clinical Behavior.

Triple-negative apocrine carcinomas (TNACs) are rare breast tumors with limited studies evaluating their molecular characteristics and clinical behavior. We performed a histologic, immunohistochemical, genetic, and clinicopathologic assessment of 42 invasive TNACs (1 with a focal spindle cell component) from 41 patients, 2 pure apocrine ductal carcinomas in situ (A-DCIS), and 1 A-DCIS associated with spindle cell metaplastic carcinoma (SCMBC). All TNACs had characteristic apocrine morphology and expressed androgen receptor (42/42), gross cystic disease fluid protein 15 (24/24), and CK5/6 (16/16). GATA3 was positive in most cases (16/18, 89%), and SOX10 was negative (0/22). TRPS1 was weakly expressed in a minority of tumors (3/14, 21%). Most TNACs had low Ki67 proliferation (≤10% in 67%, 26/39), with a median index of 10%. Levels of tumor infiltrating lymphocytes were low (≤10% in 93%, 39/42, and 15% in 7%, 3/42). Eighteen percent of TNACs presented with axillary nodal metastasis (7/38). No patients treated with neoadjuvant chemotherapy achieved pathologic complete response (0%, 0/10). Nearly all patients with TNAC (97%, n = 32) were without evidence of disease at the time of study (mean follow-up of 62 months). Seventeen invasive TNACs and 10 A-DCIS (7 with paired invasive TNAC) were profiled by targeted capture-based next-generation DNA sequencing. Pathogenic mutations in phosphatidylinositol 3-kinase pathway genes PIK3CA (53%) and/or PIK3R1 (53%) were identified in all TNACs (100%), including 4 (24%) with comutated PTEN. Ras-MAPK pathway genes, including NF1 (24%), and TP53 were mutated in 6 tumors each (35%). All A-DCIS shared mutations, such as phosphatidylinositol 3-kinase aberrations and copy number alterations with paired invasive TNACs or SCMBC, and a subset of invasive carcinomas showed additional mutations in tumor suppressors (NF1, TP53, ARID2, and CDKN2A). Divergent genetic profiles between A-DCIS and invasive carcinoma were identified in 1 case. In summary, our findings support TNAC as a morphologically, immunohistochemically, and genetically homogeneous subgroup of triple-negative breast carcinomas and suggest overall favorable clinical behavior.

Phase I Trial of Combination Therapy With Avelumab and Cabozantinib in Patients With Newly Diagnosed Metastatic Clear Cell Renal Cell Carcinoma.

BACKGROUND: Combination immunotherapy is now considered the standard first-line therapy for patients with metastatic clear cell renal cell carcinoma (mccRCC) after multiple clinical trials demonstrated improved overall survival compared with single-agent tyrosine kinase inhibitors. Cabozantinib modulates critical components of the immune system, such as decreasing regulatory T cells and increasing T-effector cell populations, and is approved for the treatment of mRCC. Avelumab is a human IgG1 monoclonal antibody that binds to programmed death-ligand 1 protein and inhibits the interaction with PD-1. This phase I trial assessed the safety and clinical activity of avelumab and cabozantinib combination therapy in mccRCC. METHODS: This study was a phase I, 3+3 dose escalation clinical trial. The primary endpoint was the safety and identification of the recommended phase II dose (RP2D). Secondary endpoints included objective response rate (ORR) and radiographic progression-free survival (rPFS). There were 3 dose cohorts: cabozantinib 20, 40, and 60 mg/day, each combined with avelumab (10 mg/kg intravenously every 2 weeks). An additional 3 patients were included in the final dose cohort as a confirmation of the RP2D. No dose modifications were allowed for avelumab, but dose delays were permitted. Both dose reductions and holds were allowed for cabozantinib. Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, was used to determine ORR, and treatment beyond progression was allowed. RESULTS: Twelve patients with newly diagnosed mccRCC were enrolled from July 2018 until March 2020. Three patients were enrolled in the 20 and 40 mg cohorts each, and 6 were enrolled in the 60 mg cohort. The International Metastatic RCC Database Consortium (IMDC) risk categories for these patients were: 4 patients (favorable risk), 6 patients (intermediate risk), and 2 patients (poor risk). No dose-limiting toxicities (DLTs) were observed in any cohort. Six patients developed serious adverse events related to study treatment after the DLT window period. Immune-related adverse events (iRAEs) were reported in 11 patients; fatigue and diarrhea were the most common (each with n = 4, 33.3%), followed by maculopapular rash and hand-foot syndrome (each with n = 3, 25%). Dose reductions were required in 5 of 6 patients in the cabozantinib 60 mg cohort after the DLT period. One patient discontinued avelumab due to irAE (nephritis), while none discontinued cabozantinib due to toxicity. The ORR was 50%, with one complete response (CR) and 5 partial responses (PR). The disease control rate (CR + PR + stable disease) was noted in 92% of the patients. Radiological PFS survival rate at 6 and 12 months was reported in 67.7% and 33.5% of patients, respectively. CONCLUSION: Combination therapy with avelumab and cabozantinib is safe and showed preliminary clinical activity in mccRCC. Even though the DLT was not met in any of the 3 cohorts, the recommended RP2D dose for the combination is cabozantinib 40 mg/day due to a high incidence of grade 2 toxicity for cabozantinib 60 mg/day after the DLT period. (ClinicalTrials.gov Identifier: NCT03200587).

Quantitative Imaging Analysis Fluorescence In Situ Hybridization Validation for Clinical HER2 Testing in Breast Cancer.

CONTEXT.­: Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines. OBJECTIVE.­: To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline. DESIGN.­: Clinical validation of HER2 FISH was performed using the US Food and Drug Administration-approved dual-probe HER2 IQFISH (Dako, Carpinteria, California) with digital scanning performed on a PathFusion (Applied Spectral Imaging, Carlsbad, California) system. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting. RESULTS.­: The accuracy samples showed a final concordance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review. CONCLUSIONS.­: Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Excluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration.

Predicting IHC staining classes of NF1 using features in the hematoxylin channel.

Immunohistochemistry (IHC) highlights specific cell types in tissues and traditionally involves antibody staining together with a hematoxylin counterstain. The intensity and pattern of hematoxylin staining differs between cell types and reveals morphological characteristics of cells. Here, we propose that features in the hematoxylin stain can be used to predict IHC labels, such as Neurofibromin (encoded by the gene NF1). The dataset consists of 7.2 million cells from benign and kidney cancer cores in a tissue microarray. Morphology and hematoxylin (H&M) features defined within QuPath are subjected to a clustering analysis in CytoMap. H&M features are also used to train 4 different XGBoost models to predict high, low, and negative NF1 stain classes in benign renal tubules, clear cell (ccRCC), papillary (PRCC), and chromophobe (ChRCC) renal carcinoma. The prediction accuracies of NF1 staining classes in benign, ccRCC, ChRCC, and PRCC range between 70% and 90% with areas under the precision recall curve PRAUCNF1-high = 0.82+0.12, PRAUCNF1-low = 0.62+0.25, and PRAUCNF1-negative = 0.83+0.16. The most important feature for predicting the NF1 class involves the minimum cellular hematoxylin staining intensity. Together, these results demonstrate the feasibility to predict NF1 expression solely from features in hematoxylin staining using open source software. Since the hematoxylin features can be obtained from regular H&E and IHC slides, the proposed workflow has broad applicability.

Morphologic Characteristics and Mutational Analysis of Fumarate Hydratase Deficient Kidney and Smooth Muscle Tumors.

OBJECTIVES: Fumarate hydratase (FH)-deficient tumors can occur due to germline or somatic mutations and have distinctive morphologic features. The aims of this study are to refine morphologic criteria and identify mutations in FH-deficient smooth muscle tumors (SMTs). METHODS: The morphology of SMTs and kidney tumors submitted to a national reference laboratory for FH immunohistochemistry (IHC) was reviewed by two gynecologic and two genitourinary pathologists, respectively. Fisher exact test was used for analysis. Fourteen SMTs were sequenced using the Illumina TruSight Oncology 500 Assay. RESULTS: Twenty-two kidney tumors (5 FH deficient) and 51 SMTs (27 FH deficient) were reviewed. FH-deficient kidney tumors exclusively showed cord-like growth, rhabdoid change, and absence of coagulative tumor necrosis and psammoma bodies. FH-deficient SMTs were significantly more likely to have staghorn vessels, eosinophilic cytoplasmic inclusions, schwannoma-like areas, or hereditary leiomyomatosis and renal cell cancer-like nuclei (P < .05 for each). Seven of 14 sequenced SMTs showed mutations of the FH gene and no other driver mutations. CONCLUSIONS: FH-deficient SMTs submitted for FH immunohistochemistry (IHC) showed distinct morphology. Although FH IHC is used for screening of FH-deficient tumors, FH mutations were identified in only 50% of FH-deficient SMTs. This highlights the need for additional exploration of mechanisms of FH protein loss in tumors lacking FH mutations.

Reporting Trends, Practices, and Resource Utilization in Neuroendocrine Tumors of the Prostate Gland: A Survey among Thirty-Nine Genitourinary Pathologists.

Background. Neuroendocrine differentiation in the prostate gland ranges from clinically insignificant neuroendocrine differentiation detected with markers in an otherwise conventional prostatic adenocarcinoma to a lethal high-grade small/large cell neuroendocrine carcinoma. The concept of neuroendocrine differentiation in prostatic adenocarcinoma has gained considerable importance due to its prognostic and therapeutic ramifications and pathologists play a pivotal role in its recognition. However, its awareness, reporting, and resource utilization practice patterns among pathologists are largely unknown. Methods. Representative examples of different spectrums of neuroendocrine differentiation along with a detailed questionnaire were shared among 39 urologic pathologists using the survey monkey software. Participants were specifically questioned about the use and awareness of the 2016 WHO classification of neuroendocrine tumors of the prostate, understanding of the clinical significance of each entity, and use of different immunohistochemical (IHC) markers. De-identified respondent data were analyzed. Results. A vast majority (90%) of the participants utilize IHC markers to confirm the diagnosis of small cell neuroendocrine carcinoma. A majority (87%) of the respondents were in agreement regarding the utilization of type of IHC markers for small cell neuroendocrine carcinoma for which 85% of the pathologists agreed that determination of the site of origin of a high-grade neuroendocrine carcinoma is not critical, as these are treated similarly. In the setting of mixed carcinomas, 62% of respondents indicated that they provide quantification and grading of the acinar component. There were varied responses regarding the prognostic implication of focal neuroendocrine cells in an otherwise conventional acinar adenocarcinoma and for Paneth cell-like differentiation. The classification of large cell neuroendocrine carcinoma was highly varied, with only 38% agreement in the illustrated case. Finally, despite the recommendation not to perform neuroendocrine markers in the absence of morphologic evidence of neuroendocrine differentiation, 62% would routinely utilize IHC in the work-up of a Gleason score 5 + 5 = 10 acinar adenocarcinoma and its differentiation from high-grade neuroendocrine carcinoma. Conclusion. There is a disparity in the practice utilization patterns among the urologic pathologists with regard to diagnosing high-grade neuroendocrine carcinoma and in understanding the clinical significance of focal neuroendocrine cells in an otherwise conventional acinar adenocarcinoma and Paneth cell-like neuroendocrine differentiation. There seems to have a trend towards overutilization of IHC to determine neuroendocrine differentiation in the absence of neuroendocrine features on morphology. The survey results suggest a need for further refinement and development of standardized guidelines for the classification and reporting of neuroendocrine differentiation in the prostate gland.

Detection of BRCA1, and BRCA2 Alterations in Matched Tumor Tissue and Circulating Cell-Free DNA in Patients with Prostate Cancer in a Real-World Setting.

BACKGROUND: Poly (ADP-ribose) polymerase (PARP) inhibitors are approved for patients with metastatic castration-resistant prostate cancer harboring deleterious or suspected deleterious BRCA1 and/or 2 mutations. Identifying patients with prostate cancer harboring these mutations may be challenging. Circulating cell-free DNA (cfDNA) provides an avenue for an easier detection of these mutations. Herein, we aimed to evaluate the concordance of BRCA mutations in the tumor tissue and cfDNA in patients with metastatic prostate cancer in the real-world setting. METHODS: Somatic genomic profiling results were obtained from a clinical cohort of patients at our institution who had at least two samples tested. One of the samples needed to be from either primary or metastatic tissue. Concordance was adjusted to not include mutation types that the cfDNA platforms were not designed to detect. RESULTS: The presence or absence of mutations in the BRCA gene was assessed in a total of 589 samples, including 327 cfDNA samples, from 260 patients with metastatic prostate cancer. The median time between the first test and any subsequent test was 22.8 (0.0-232) months. BRCA mutation was present in the patient's original prostate tissue in 23 samples (3.9%) of patients. The adjusted concordance between prostate tumor tissue and cfDNA was 97.9% [95% CI, 95.3-99.1%]. The adjusted concordance between metastatic samples and cfDNA was 93.5% [95% CI, 86.4-97.3%]. Of the patients who had a BRCA mutation detected in their prostate tissue, there was a 70% probability of detecting a BRCA mutation in the patient's cfDNA as well. For patients who did not have a detectable BRCA mutation in their primary prostate tissue, the probability of detecting a subsequent one later in the disease course was less than 0.9%. CONCLUSION: There is a high level of concordance between tissue and blood for BRCA mutations. Testing cfDNA can provide reliable information on BRCA mutational status and is a viable alternative to solid tissue sequencing when unavailable. The development of a new BRCA mutation later in the disease course is a rare event.

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.

Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 ­ and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

External Validation of Association of Baseline Circulating Tumor Cell Counts with Survival Outcomes in Men with Metastatic Castration-Sensitive Prostate Cancer.

Approximately 20% of men with metastatic castration-sensitive prostate cancer (mCSPC) progress within 1 year of treatment, and biomarkers to identify them up front are lacking. In a randomized phase III trial in men with mCSPC (SWOG S1216), higher baseline circulating tumor cells (CTCs) were prognostic of inferior outcomes. We aimed to validate these findings and interrogate corresponding tumor genomic profiles. Consecutively seen men with newly diagnosed mCSPC undergoing systemic therapy and baseline CTC enumeration by CellSearch assay were included. Gene alterations were determined by comprehensive genomic profiling of tumor tissue by Clinical Laboratory Improvement Amendments-certified lab. The relationship between categorized CTC counts and both progression-free survival (PFS) and overall survival (OS) was assessed in the context of Cox proportional hazards models, both unadjusted and adjusted for age, Gleason score, PSA at androgen-deprivation therapy initiation, disease volume, de novo status, treatment intensification, and number of altered genes. Overall, 103 patients were included in the analysis. On multivariate analysis high CTCs (≥ 5 vs. 0) were associated with poorer PFS [HR, 4.52; 95% confidence interval (CI), 1.84-11.11; P = 0.001) and OS (HR, 3.59; 95% CI, 0.95-13.57; P = 0.060). Patients with higher CTC counts had a greater number of altered genes and total number of alterations (all P < 0.02). In this article, for the first time, we externally validate the association of higher CTC counts with inferior survival outcomes in men with mCSPC and show a distinct associated tumor genomic landscape. These findings may improve prognostication, patient counseling, and treatment selection in men with mCSPC.

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

Genomic landscape of locally advanced or metastatic urothelial carcinoma with squamous differentiation compared to pure urothelial carcinoma.

BACKGROUND: Urothelial carcinoma with squamous differentiation (UCS) is the most common variant differentiation of urothelial carcinoma (UC). Although treatment is usually similar to pure UC, there is paucity of data regarding its genomic landscape and putative molecular drivers. In this study, we compared the mutational profile of tumors with UCS and UC histology. METHODS: In this IRB-approved retrospective study, patients with advanced UCS and UC undergoing tumor based comprehensive genomic profiling from a CLIA-certified laboratory were included. An independent genitourinary pathologist reviewed all cases. Patients were determined to have UCS based on presence of any component of squamous differentiation. Patients with UC having any other secondary histology variant were excluded. Genes with alterations (GA) in less than 5% of patients and variants of unknown significance were excluded from the analysis. Chi-square test was used to compare gene aberration frequency and the p-values were adjusted for false using Benjamini-Hochberg (BH) correction. RESULTS: Among the 87 eligible patients with UCS (n=31) and UC (n=56), patients with UCS were more likely to be female (32.3% vs. 14.3%, p=0.047) with no significant differences in other clinicopathological features. Most common genomic alterations seen in UCS were TP53 (67.7%), KMT2D (48.4%) and ARID1A (32.3%). KMT2D mutations were significantly enriched in UCS (48.4% vs. 0%, FDR adj p <0.001, p = <0.001) compared to UC. Prevalence of CUL4A mutations was numerically higher in UCS vs. UC (12.9% vs. 1.8%, FDR adj p = 0.43, p = 0.03). Tumor mutation burden and the number of genomic aberrations per patient were not significantly different between the two groups. CONCLUSION: These findings highlight significant enrichment of KMT2D mutations in UCS and potential role of chromatin remodeling genes as drivers and potential therapeutic targets.