Pathogenic Somatic Mutations Research Articles

Two-hit model for the development of aldosterone-producing adenoma: supporting from two new cases.

Recently, a two-hit model for the development of aldosterone-producing adenoma (APA) was proposed but until now, only two cases supporting the model have been reported. Here, we present two new cases of primary aldosteronism (PA), both of which had large functional adenomas with somatic mutations in aldosterone-driving genes. Furthermore, the first patient, who had a history of colorectal cancer, was found to have a germline and an additional somatic mutation in APC, and APC inactivation was confirmed by immunohistochemistry. The other patient had pathogenic somatic mutation inCTNNB1. These pro-proliferation mutations resulted in abnormal activation of the Wnt/β-catenin pathway. Two consecutive events apparent in these patients, namely, the first event leading to cell proliferation and the second driving hormonal hypersecretion, supported the two-hit model of APA development. The two-hit model usually occurs in the larger adenomas, and the driving factors of the first hit that promote cell proliferation still require further research and exploration.

PARP Inhibitors in Pancreatic Cancer with Homologous Recombination Repair Gene Mutations: A Single-Institution Experience.

Limited data are available regarding the anticancer activity of PARP inhibitors (PARPis) in pancreatic cancer with mutations in HRR genes other than BRCA and PALB2. We retrospectively reviewed the clinical characteristics and outcomes of 48 patients with advanced pancreatic cancer harboring pathogenic germline and/or somatic HRR mutations who were treated with PARPis. Thirty patients had germline (g)HRR mutations only, twelve had somatic (s)HRR mutations only, and six had concomitant gHRR and sHRR mutations. The objective response rate (ORR) was 22%. The median progression-free survival (mPFS) and overall survival (mOS) were 6.9 and 11.5 months, respectively. Five patients received olaparib in the front-line setting due to borderline performance status. Their ORR was 20%, and their mPFS and mOS were both 11.3 months. The ORR was higher in patients with BRCA or PALB2 mutations (germline or somatic) than in those with non-BRCA/PALB2 mutations. Patients with somatic non-BRCA/PALB2 variants had a shorter mPFS. Patients with concomitant gHRR/sHRR mutations or gHRR mutations alone had a significantly longer mPFS than those with sHRR mutations only. PARP inhibitors may be considered for patients with advanced pancreatic cancer harboring pathogenic alterations of BRCA who cannot tolerate standard chemotherapy. Maintenance PARPis can be considered in selected patients with non-BRCA/non-PALB2 HRR mutations.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

Wnt/β-Catenin–Activated Nonpilomatrical Carcinoma of the Skin: A Case Series

Among skin epithelial tumors, recurrent mutations in the APC/CTNNB1 genes resulting in activation of the Wnt/β-catenin pathway have been reported predominantly in neoplasms with matrical differentiation. In the present study, we describe the morphologic, immunohistochemical, and genetic features of 16 primary cutaneous carcinomas harboring mutations activating the Wnt/β-catenin pathway without evidence of matrical differentiation, as well as 4 combined tumors in which a similar Wnt/β-catenin–activated carcinoma component was associated with Merkel cell carcinoma (MCC) or pilomatrical carcinoma. Among the pure tumor cases, 6 of 16 patients were women with a median age of 80 years (range, 58-98 years). Tumors were located on the head and neck (n = 7, 44%), upper limb (n = 4, 25%), trunk (n = 3, 18%), and leg (n = 2, 13%). Metastatic spread was observed in 4 cases resulting in death from disease in 1 patient. Microscopically, all cases were poorly differentiated neoplasms infiltrating the dermis and/or subcutaneous tissue. In 13 cases, solid “squamoid” areas were associated with a basophilic component characterized by rosette/pseudoglandular formation resulting in a biphasic appearance. Three specimens consisted only of poorly differentiated carcinoma lacking rosette formation. Immunohistochemical studies showed frequent expression of EMA (100%), BerEP4 (100%), cytokeratin 7 (94%), chromogranin A (44%), synaptophysin (82%), and cytokeratin 20 (69%). Complete loss of Rb expression was observed in all but 1 case. Nuclear β-catenin and CDX2 expressions were detected in all cases. Recurrent pathogenic somatic mutations were observed in APC (60%), CTNNB1 (40%), and RB1 (n = 47%). Global methylation analysis confirmed that cases with rosette formation constituted a homogeneous tumor group distinct from established skin tumor entities (pilomatrical carcinoma, MCC, and squamous cell carcinoma), although the 3 other cases lacking such morphologic features did not. In addition, we identified 4 combined neoplasms in which there was a component showing a similar poorly differentiated rosette-forming carcinoma demonstrating Rb loss and β-catenin activation associated with either MCC (n = 3) or pilomatrical carcinoma (n = 1). In conclusion, we describe a distinctive neoplasm, for which we propose the term “Wnt/β-catenin–activated rosette-forming carcinoma,” morphologically characterized by the association of rosette formation, squamous and/or neuroendocrine differentiation, diffuse CDX2 expression, Rb loss, and mutations in CTNNB1/APC genes.

Somatic and germline aberrations in homologous recombination repair genes among Chinese high-risk breast cancer patients by multi-gene next-generation sequencing.

Recently, genes involved in homologous recombination repair (HRR) pathway have been extensively studied. However, the landscapes of HRR gene mutations remain poorly defined in Chinese high-risk breast cancer (BC) patients. Our study aims to identify the status of germline and somatic HRR gene mutations and their association with clinicopathological features in these patients. A total of 100 high-risk BC patients from our institution who underwent paired peripheral blood germline and BC tissues somatic 26 genes next-generation sequencing (NGS) from January 2018 to July 2023 were enrolled for retrospective analysis. Out of 100 high-risk BC patients, 55 (55%) had at least one germline or somatic mutation in HRR genes. Among them, 22% carried germline pathogenic variants (19 BRCA1/2 and 3 non-BRCA genes), 9% harbored somatic pathogenic mutations (3 BRCA1/2 and 6 non-BRCA genes). Among high-risk factors, family history and early onset BC showed a correlation with HRR gene mutations (p < 0.05). BRCA1 germline and HRR gene somatic mutations showed a correlation with TNBC, but BRCA2 germline mutations were associated with Luminal B/HER2-negative BC (p < 0.05). Patients with HRR gene somatic pathogenic variant more likely had a lympho-vascular invasion and distant metastasis (p < 0.05). The prevalence of HRR gene germline and somatic mutations were higher in Chinese BC patients with high risk factors. We strongly recommend that these high-risk BC patients receive comprehensive gene mutation testing, especially HRR genes, which are not only related to genetic consultation for BC patients and provide a theoretical basis for necessary prevention and individualized treatment.

Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases,<1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Succinate dehydrogenase A deficient renal cell carcinoma: A rare renal tumor distinct from typical Succinate dehydrogenase deficient renal cell carcinoma

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare subtype of RCC classified as a molecularly defined RCC in the fifth edition of the WHO. Most gene alterations in patients with SDH-deficient RCC involve the SDHB subunit, with less involvement of the SDHC, SDHA, and SDHD subunits. Four cases of SDHA-deficient RCC have been reported in the literature, of which one case was associated with an NF2 gene mutation. Herein, we report six novel SDHA-deficient RCC cases, including two cases with NF2 gene mutations. In contrast to the typical morphology of SDH-deficient RCC, the six tumors mainly displayed glandular, sheet-like, or papillary growth patterns with prominent nucleoli (Grades 2–3), among which two cases with NF2 mutations had prominent nucleoli (Grade 3), large transparent vacuoles in the cytoplasm, and a large number of lymphocytes in the stroma. Six tumors showed negative immunohistochemical staining for SDHA and SDHB, and three cases presented with high expression of PD-L1. Second-generation sequencing revealed novel pathogenic somatic SDHA gene mutation and NF2 gene mutations in six and two tumors, respectively. Follow-up data were collected for the six patients with a follow-up time ranging from 7 to 268 months, and all six patients have survived to date. One patient received targeted therapy for tumor metastasis to the lungs after seven months, and another patient with an NF2 gene mutation received immunotherapy for lymph node metastasis revealed during surgery. SDHA-deficient RCCs with NF2 gene mutations have the ability to metastasize but might respond well to immunotherapy. For the first time, we report the largest number of SDHA-deficient RCC cases and comprehensively investigate their clinicopathological and molecular features to provide important guidance for diagnosis and clinical immunotherapy.

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer (MBC) with a somatic BRCA1/2mutation identified in a cell-free DNA or tumor tissue genotyping assay.

TPS1143 Background: PARP inhibitors are approved for germline BRCA1/2mutant MBC but the applicability of these well-tolerated oral targeted therapies is limited, as germline BRCA1/2mutations account for 5% of breast cancer. We demonstrated that pathogenic somatic BRCA1/2mutations are detectable in cell-free DNA and tumor tissue genotyping assays in a subset of HER2- MBC, in patients who are not known germline BRCA1/2carriers. In a circulating tumor cell culture model derived from a patient with a pathogenic somatic BRCA1 mutation, we demonstrated that the growth inhibitory effect of a PARP inhibitor paralleled germline BRCA1/2mutant cultures. We hypothesize that somatic BRCA1/2mutations may be amenable to PARP inhibition. In this study, we are evaluating the efficacy of a PARP inhibitor in somatic BRCA1/2mutant MBC. The results may expand the population of patients who benefit from PARP inhibitors. Methods: In this phase II investigator-initiated clinical trial, 30 patients with HER2- MBC with pathogenic somatic BRCA1/2mutations (in the absence of a known germline BRCA1/2mutation) identified in a CLIA certified cell-free DNA or tumor tissue genotyping assay are being enrolled at 7 academic centers. Mutations are vetted for pathogenicity by genetic counselors using validated genomic databases such as ClinVar. Patients with metastatic triple-negative or hormone receptor positive (HR)+/HER2- breast cancer who received ≥1 prior therapy in the metastatic setting are eligible. Any number of prior therapies are acceptable, including a prior platinum (in absence of disease progression). Patients must have adequate baseline performance status and organ function. Patients are treated with talazoparib 1 mg/day until disease progression. Disease assessments occur at baseline and every 3 months via CT chest, abdomen, and pelvis, and bone scan. The primary endpoint is progression free survival (PFS) via RECIST 1.1. This study has 81% power to demonstrate the 12-week PFS is ≥53% (4% alpha). Secondary endpoints include objective response rate and toxicity (NCI CTCAE v 5.0). Exploratory endpoints, determined using serial cell-free DNA collected at baseline and monthly, include identifying resistance mutations, and evaluating the impact of mutant allele fraction and BRCA1/2reversion mutations on response. The Cancer Risk B (CR-B) assay, a novel method to identify double-strand break repair mutations in circulating blood cells, will be correlated with response. The study (NCT03990896) is open at Massachusetts General Hospital, University of California San Francisco, MD Anderson, Emory, Northwestern, and Vanderbilt and will open at Cornell, with 17 patients enrolled as of 2/2024. Clinical trial information: NCT03990896 .

A novel mutation of DNA2 regulates neuronal cell membrane potential and epileptogenesis.

Mesial temporal lobe epilepsy (MTLE) is one of the most intractable epilepsies. Previously, we reported that mitochondrial DNA deletions were associated with epileptogenesis. While the underlying mechanism of mitochondrial DNA deletions during epileptogenesis remain unknown. In this study, a novel somatic mutation of DNA2 gene was identified in the hippocampal tissue of two MTLE patients carrying mitochondrial DNA deletions, and this mutation decreased the full-length expression of DNA2 protein significantly, aborting its normal functions. Then, we knocked down the DNA2 protein in zebrafish, and we demonstrated that zebrafish with DNA2 deficiency showed decreased expression of mitochondrial complex II-IV, and exhibited hallmarks of epileptic seizures, including abnormal development of the zebrafish and epileptiform discharge signals in brain, compared to the Cas9-control group. Moreover, our cell-based assays showed that DNA2 deletion resulted in accumulated mitochondrial DNA damage, abnormal oxidative phosphorylation and decreased ATP production in cells. Inadequate ATP generation in cells lead to declined Na+, K+-ATPase activity and change of cell membrane potential. Together, these disorders caused by DNA2 depletion increased cell apoptosis and inhibited the differentiation of SH-SY5Y into branched neuronal phenotype. In conclusion, DNA2 deficiency regulated the cell membrane potential via affecting ATP production by mitochondria and Na+, K+-ATPase activity, and also affected neuronal cell growth and differentiation. These disorders caused by DNA2 dysfunction are important causes of epilepsy. In summary, we are the first to report the pathogenic somatic mutation of DNA2 gene in the patients with MTLE disease, and we uncovered the mechanism of DNA2 regulating the epilepsy. This study provides new insight into the pathogenesis of epilepsy and underscore the value of DNA2 in epilepsy.

Abstract PO4-19-06: Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer with a somatic BRCA1/2 mutation present in cell-free DNA or tumor tissue genotyping

Abstract Background: PARP inhibitors improve both progression free survival (PFS) and patient quality of life in germline BRCA1/2 mutant metastatic breast cancer (MBC), which accounts for 5-10% of breast cancer, leading to their approval in this setting. We have previously shown that a subset of patients with MBC who are not germline BRCA1/2 carriers may harbor pathogenic somatic BRCA1/2 mutations that are identified by cell-free DNA and/or tumor tissue genotyping. In our prior work, we developed a circulating tumor cell culture from a patient with MBC harboring a pathogenic somatic BRCA1 mutation, and demonstrated that a PARP inhibitor induced cell growth inhibition similar to germline BRCA1/2 mutant cultures. Thus, we hypothesize that a PARP inhibitor may be effective in treating somatic BRCA1/2 mutant MBC. In this clinical trial, we are studying the efficacy of a PARP inhibitor in somatic BRCA1/2 mutant MBC. Our work may help expand the clinical application of PARP inhibitors in MBC. Trial Design: This phase II investigator-initiated clinical trial is enrolling 30 patients with MBC who have a pathogenic somatic BRCA1/2 mutation identified by a CLIA certified cell-free DNA and/or tumor tissue genotyping assay. Patients are treated with the PARP inhibitor talazoparib 1 mg/day until progression of disease. Imaging (CT chest, abdomen and pelvis, and bone scan) occurs for disease assessment at baseline and every 3 months. Cell-free DNA is collected at baseline and then monthly to evaluate changes in the genomic environment. Patients also undergo the Cancer Risk B (CR-B) assay, a novel flow variant assay to identify double-strand break repair mutations in circulating blood cells, at baseline. Eligibility Criteria: Patients must have MBC with a pathogenic somatic BRCA1/2 mutation identified in a cell-free DNA and/or tumor tissue genotyping assay, with pathogenicity confirmed by a genetics counselor using validated genomic databases such as ClinVar. Patients may have triple-negative (receipt of at least 1 prior chemotherapy) or hormone receptor positive/HER2- MBC (receipt of at least 1 prior hormone therapy). Patients must not be known germline BRCA1/2 carriers. Any number of prior therapies including a prior platinum (in absence of progressive disease on a platinum) are allowed, but patients should not have received a prior PARP inhibitor. Patients must have adequate organ function and performance status. Specific Aims: The primary aim is to determine PFS (RECIST 1.1). Secondary aims include determining the objective response rate and toxicity (NCI CTCAE v 5.0). Exploratory aims include evaluating serial changes in BRCA1/2 mutant allelic frequency in cell-free DNA, understanding the impact of BRCA1/2 reversion mutations in cell-free DNA, comparing pre- and post-treatment cell-free DNA results to understand changes in the genomic environment, studying the CR-B assay positivity rate, and correlating these biomarker analyses with treatment response. Statistical Methods: This study has 81% power to demonstrate that the 12-week PFS is 53% or higher. In contrast, there is a 4% (alpha) probability of concluding that the 12-week PFS is ≥ 53% if the true 12-week PFS is 30% or lower. If 14 or more of 30 total patients achieve PFS &amp;gt; 12 weeks, the null hypothesis (12-week PFS ≤ 30%) will be rejected. Present accrual and target accrual: This study (NCT03990896) is open at Massachusetts General Hospital, MD Anderson, University of California San Francisco, Emory, Northwestern, and Vanderbilt. As of 7/2023, 14/30 patients are enrolled. Funding: This study is funded by a Pfizer ASPIRE award and Conquer Cancer Foundation of ASCO–Breast Cancer Research Foundation- Career Development Award. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu. Citation Format: Neelima Vidula, Senthil Damodaran, Manali Bhave, Hope Rugo, Ami N. Shah, Erica Blouch, Nathan Royce Ruffle-Deignan, Ogadinma Ogbenna, Lisa E. Flaum, Massimo Cristofanilli, Joseph Sparano, Harry Ostrer, Vandana Abramson, Nora Horick, Aditya Bardia. Phase II study of a PARP inhibitor, talazoparib, in HER2- metastatic breast cancer with a somatic BRCA1/2 mutation present in cell-free DNA or tumor tissue genotyping [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-19-06.

Squamous cell carcinoma initially occurring on the tongue dorsum: a case series report with molecular analysis

BackgroundSquamous cell carcinoma (SCC) of the dorsum of the tongue is extremely rare, and it clinically resembles various benign lesions. Somatic mutations in TP53 and some driver genes were implicated in the development of SCC; however, the somatic genetic characteristics of dorsal tongue SCC remain unknown. With a detailed analysis of gene mutations in dorsal tongue SCC, we aimed to better understand its biology.MethodsFour cases of SCC initially occurring on the tongue dorsum were evaluated for clinical and histological findings and immunohistochemical expression of p53 and p16. Gene mutations were analyzed using next-generation sequencing with a custom panel of driver genes.ResultsWe retrospectively investigated 557 cases of tongue SCC, and only four cases of SCC initially occurred on the tongue dorsum. The four patients (cases 1–4) were one woman and three men with a mean age of 53.75 years (range: 15–74 years). Histological analysis revealed well-differentiated SCC. Through molecular analysis, we identified pathogenic somatic mutations, namely, TP53 p.C176F (c.527G > T) in case 3 and TP53 p.R282W (c.844 C > T) in case 4. No pathogenic variants were identified in the PI3K/AKT or RAS/RAF pathways. The p53 immunohistochemical examination revealed a wild-type expression pattern in cases 1–3 and strong expression in case 4. The results of p16 immunostaining were negative in all cases.ConclusionsWe described four previously unreported genetic characteristics of dorsal tongue SCC. Somatic TP53 mutations may contribute to the development of a subset of dorsal tongue SCC; however, more cases with genetic analysis need to be accumulated.

Decreased calcium permeability caused by biallelic TRPV5 mutation leads to autosomal recessive renal calcium-wasting hypercalciuria.

Hypercalciuria is the most common metabolic risk factor in people with kidney stone disease. Its etiology is mostly multifactorial, although monogenetic causes of hypercalciuria have also been described. Despite the increased availability of genetic diagnostic tests, the vast majority of individuals with familial hypercalciuria remain unsolved. In this study, we investigated a consanguineous pedigree with idiopathic hypercalciuria. The proband additionally exhibited severe skeletal deformities and hyperparathyroidism. Whole-exome sequencing of the proband revealed a homozygous ultra-rare variant in TRPV5 (NM_019841.7:c.1792G>A; p.(Val598Met)), which encodes for a renal Ca2+-selective ion channel. The variant segregates with the three individuals with hypercalciuria. The skeletal phenotype unique to the proband was due to an additional pathogenic somatic mutation in GNAS (NM_000516.7:c.601C>T; p.(Arg201Cys)), which leads to polyostotic fibrous dysplasia. The variant in TRPV5 is located in the TRP helix, a characteristic amphipathic helix that is indispensable for the gating movements of TRP channels. Biochemical characterization of the TRPV5 p.(Val598Met) channel revealed a complete loss of Ca2+ transport capability. This defect is caused by reduced expression of the mutant channel, due to misfolding and preferential targeting to the proteasome for degradation. Based on these findings, we conclude that biallelic loss of TRPV5 function causes a novel form of monogenic autosomal recessive hypercalciuria, which we name renal Ca2+-wasting hypercalciuria (RCWH). The recessive inheritance pattern explains the rarity of RCWH and underscores the potential prevalence of RCWH in highly consanguineous populations, emphasizing the importance of exploration of this disorder within such communities.

Abstract 6127: Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals

Abstract Background: Approximately 50% of high-grade serous ovarian carcinomas (HGSOC) are homologous recombination deficient (HRD). HRD is associated with increased T-cell infiltration and improved survival. This study aimed to describe the distribution of HRD features and cytotoxic T-cell infiltration by gene expression subtype among Black HGSOC cases. Methods: We included Black HGSOC cases from the African American Cancer Epidemiology Study and the North Carolina Ovarian Cancer Study. We used RNASeq data to assign gene expression subtypes (n=160), and whole exome sequencing data from matched blood and tumor formalin-fixed paraffin-embedded specimens (n=206) to characterize HRD features. We defined HRD features as (1) germline variants and somatic mutations in HRD genes, and (2) HRD-associated signatures (SBS3 and ID6) that we obtained from de novo mutational signature analysis for single-base pair substitution, insertion, and deletion. We estimated the proportion of intratumoral cytotoxic T cells using multiplex immunofluorescence (n=125). Results: The subtype distribution in our study was 39% immunoreactive, 29% mesenchymal 27% proliferative, and 4% differentiated. Germline variants and somatic mutations in any HRD gene were observed in 27% and 23% of cases, respectively, and at least one of these was observed in 42% of cases. Pathogenic germline variants and somatic mutations were observed in 7% and 3% of cases, respectively. The prevalence of germline or somatic variants by gene was 16% for BRCA1, 14% for BRCA2, 11% for ATM, 5% for RAD51, and 2% each for BRIP1, PALB2, and BARD1. We observed a COSMIC HRD signature in 43% of cases, with 25% SBS3 only, 5% ID6 only, and 13% with both signatures. The prevalence of any HRD gene variant or signature was higher in immunoreactive cases (52% and 59%, respectively) than in mesenchymal (28% and 43%) or proliferative (44% and 28%) cases. Germline HRD variants were more common in the proliferative subtype (44%) compared with immunoreactive (25%) and mesenchymal (17%). In contrast, somatic HRD mutations were more common in the immunoreactive subtype (33%), compared with proliferative (16%) and mesenchymal (15%). This was due to a higher occurrence of BRCA1 somatic mutations in the immunoreactive subtype (25%) compared with proliferative (9%) and mesenchymal (8%). A higher proportion of cytotoxic T-cells (≥1% vs &amp;lt;1%) was also observed in the immunoreactive subtype (44%) compared with mesenchymal (31%) and proliferative (11%). Conclusion: We observed differences in the distribution of HRD features and cytotoxic T-cells by gene expression subtype. In particular, in the immunoreactive subtype, the most common gene expression subtype in Black HGSOC cases, we observed a higher occurrence of any HRD feature, somatic BRCA1 mutations, and cytotoxic T cell infiltration than in any of the other gene expression subtypes. Citation Format: Katherine Lawson-Michod, Mollie E. Barnard, Natalie Davidson, Lindsay J. Collin, Courtney Johnson, Lucas A. Salas, Casey Greene, Jeffrey R. Marks, Lauren Peres, Joellen M. Schildkraut, Jennifer A. Doherty. Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6127.

Abstract 2443: Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform

Abstract Ovarian and endometrial cancers come within the top-4 for incident cancers as well as deaths in North American women. Cure rates have not improved in 30 years as high-grade subtypes continue to be diagnosed in Stage III/IV. Attempts at early diagnosis have failed because high-grade cancer cells exfoliate and metastasize while the primary cancer is small and undetectable by existing tests based on imaging and blood-based tumor markers. DOvEEgene (Detecting Ovarian and Endometrial cancers Early using genomics) is a genomic uterine pap test developed by a McGill team to screen and detect these cancers while they are confined to the gynecologic organs and curable by surgery. The test identifies pathogenic somatic mutations in uterine brush samples. Here we tested the Onso system, a highly accurate sequencing technology from PacBio in order to potentially increase sensitivity while driving down sequencing costs by reducing required sequencing depth vs the current NGS standard. A highly sensitive, error-correcting capture technology (DOvEEgene-SureSelectHS) utilizing duplex error correction sequencing interrogated the exons of 23 genes involved in the development of sporadic and hereditary ovarian and endometrial cancers. We applied a combination of germline gene panel testing on saliva samples with deep duplex sequencing to detect somatic mutations at &amp;lt;0.1% VAF, interrogation of microsatellite loci for instability, and low coverage WGS for copy number analysis of uterine brush samples. We sequenced 20 duplex Illumina sequencing libraries produced using the DovEE assay at PE 100bp mode and compared Onso data in non- duplex sequencing mode as well as duplex sequencing mode to the original Illumina duplex sequencing method. Here, we present this comparison and highlight the benefits of high accuracy sequencing for the detection of very low frequency (&amp;lt;0.1%) somatic mutations. We observed improved mismatch rates for Onso data compared to Illumina, even after duplex error correction was applied. In addition, we found that more individuals are called as displaying microsatellite instablity from the Onso data, which may be due to improved sequencing performance in repetitive regions for Onso. Finally, we observed fewer potential false positive variant calls in the Onso data, highlighting the value of improved sequencing accuracy for rare variant detection. Citation Format: Timothée Revil, Nairi Pezeshkian, Lucy Gilbert, Alexandra Sockell, Jiannis Ragoussis. Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2443.

Rare germline mutation and MSH2-&MSH6 + expression in a double primary carcinoma of colorectal carcinoma and endometrial carcinoma: a case report

BackgroundMultiple primary malignancies are rare in cancer patients, and risk factors may include genetics, viral infection, smoking, radiation, and other environmental factors. Lynch syndrome (LS) is the most prevalent form of hereditary predisposition to double primary colorectal and endometrial cancer in females. LS, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is a common autosomal dominant condition. Pathogenic germline variants in the DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6, and PMS2, and less frequently, deletions in the 3’ end of EPCAM cause LS. It manifested itself as loss of MMR nuclear tumor staining (MMR protein deficient, dMMR).Case presentationThis case study describes a double primary carcinoma in a 49-year-old female. In June 2022, the patient was diagnosed with highly to moderately differentiated endometrioid adenocarcinoma. The patient’s mother died of esophageal cancer at age 50, and the father died of undefined reasons at age 70. Immunohistochemical stainings found ER (++), PR (++), P53 (+), MSH2 (-), MSH6 (+), MLH1 (+), and PMS2 (+). MMR gene sequencing was performed on endometrial tumor and peripheral blood samples from this patient. The patient carried two pathogenic somatic mutations in the endometrial tumor, MSH6 c.3261dupC (p.Phe1088LeufsTer5) and MSH2 c.445_448dup (p.Val150fs), in addition to a rare germline mutation MSH6 c.133G > C (p.Gly45Arg). Two years ago, the patient was diagnosed with moderately differentiated adenocarcinoma in the left-half colon. Immunohistochemical stainings found MSH2(-), MSH6(+), MLH1(+), and PMS2(+) (data not shown).ConclusionsIn the case of a patient with double primary EC and CRC, a careful evaluation of the IHC and the genetic data was presented. The patient carried rare compound heterozygous variants, a germline missense mutation, and a somatic frameshift mutation of MSH6, combined with a novel somatic null variant of MSH2. Our study broadened the variant spectrum of double primary cancer and provided insight into the molecular basis for abnormal MSH2 protein loss and double primary carcinoma.

Real-world prevalence of homologous recombination repair mutations in advanced prostate cancer: an analysis of two clinico-genomic databases.

Homologous recombination repair mutation (HRRm) status may guide risk-stratification and treatment decisions, including polyadenosine diphosphate-ribose polymerase inhibitor use, in advanced prostate cancer. Although HRRm prevalence has been reported in single-institution studies or clinical trials, real-world HRRm prevalence in diverse populations is unknown. We describe HRRm in the clinical setting using two real-world clinicogenomic databases: the Flatiron Health and Foundation Medicine, Inc. Clinico-Genomic Database (CGDB), a national electronic health record-derived database, and the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE). This cross-sectional analysis included 3757 individuals diagnosed with prostate cancer who had next generation sequencing (NGS) as standard of care. The CGDB included men with advanced/metastatic prostate cancer and genetic data included both germline and somatic pathogenic mutations. The GENIE analysis included men with prostate cancer whose received NGS as standard of care, but the data were filtered to include somatic mutations only. Due to key differences among databases, direct comparisons were not possible. Overall prevalence of HRRm was calculated and stratified by demographic and clinical characteristics. HRRm prevalence (combined germline and somatic) in CGDB (n = 487) was 24.6% (95% CI 20.9-28.7%), with no major differences across demographic and disease characteristic subgroups. HRRm prevalence (somatic) in GENIE (n = 3270) was 11.0% (95% CI 10.0-12.1%), which varied between 9.5% and 18.4% across treatment centers. Approximately one-quarter of patients with advanced/metastatic prostate cancer in the CGDB had germline and/or somatic HRRm, which is consistent with clinical trials such as the PROfound study that used a similar NGS platform and algorithm to define HRRm. In the GENIE database, HRRm prevalence varied by treatment center or NGS platform. More research is needed to understand real-world HRRm prevalence variations.

Biological Factors Associated with Treatment-Free Remission in Clinical Practice: Data from the Canarian Registry of CML

Introduction: The probability of maintaining treatment-free remission (TFR) after the discontinuation of tyrosine kinase inhibitors (TKI) is affected by clinical factors such as length of TKI treatment and the depth of molecular response. However, some individuals with similar treatment duration and response remain in TFR while others relapse. Thus, patient-specific factors must also play a role. Biological factors suggested to influence TFR include BCR::ABL1 transcript type, blasts at diagnosis, halving time (HT, first 3 months), BCR::ABL1 positivity at stop, and presence of somatic mutations. The aim of this study was to evaluate the biological factors associated with TFR in the clinical practice, using data from the Canarian CML Registry. Methods: The OMOP-standardized data of adult patients with chronic phase CML who discontinued TKI treatment included in the Canarian CML Registry (https://rclmc.es/rclmc/) were reviewed. Criteria to discontinue included TKI treatment ≥5 years and MR4.5 or better ≥3 years. TFR was defined as major molecular response (MMR), or deeper after stop. Molecular relapse was defined as loss of MMR in two consecutive tests. NGS was performed with the 30-gene panel Myeloid Solution (SOPHiA) on the MiSeq (Illumina) platform using 200 ng genomic DNA extracted from peripheral blood at diagnosis. Results: A total of 109 patients were included, with an average age of 50.5 years; 54 were male (49.5%). Median follow-up was 3.3 years. 86 patients discontinued from first-line, 21 from second- and 2 from third-line. 51% of patients (n=56) discontinued from imatinib, 31% from nilotinib (n=34), 16% from dasatinib (n=17), and 1% from bosutinib and ponatinib (1 patient each). The median TFR was 101 months ( Figure 1A; range 1.02-148.4 months). Thirty-two patients had molecular relapse and re-started TKI treatment: 36.7% with imatinib or dasatinib, 26.7% with nilotinib. Median time to regain MMR was 101 days (range 25-276). No difference was observed in the time to regain MMR for imatinib vs. 2G-TKI (106 vs. 144, p=0.419). No patients progressed to an accelerated phase. Differences were observed in duration of MR4 and MR4.5 prior to stop comparing the TFR vs. relapse groups (88.3 vs. 69.9 months, p=0.044; 74.2 vs. 58.6 months, p=0.079), but not total time on TKI (172.6 vs. 102.1, p=0.310), respectively. In terms of biological factors at diagnosis, 44 patients had blasts, 66.7% had a low Sokal risk. 23.9% expressed transcript e13a2 and 76.1% had e14a2. No differences in TFR were observed when blasts, Sokal or transcript were considered in chi-squared analyses. A long HT (&amp;gt;21.9 days, Shanmuganathan et al. Blood 2021;137) was of marginal significance for molecular relapse (p=0.076, OR 3.2) but no difference was found for short HT (&amp;lt;9.4 days). By Kaplan-Meier analysis, an MR3, 4, or 4.5 at stop was predictive of molecular relapse (n=42; Figure 1B) vs. an MR5 or undetectable (limit of detection 0.0001%). In multivariable analysis, only an MR5 or deeper at stop and withdrawal syndrome were predictive for TFR (p=0.003, HR 4.3; p=0.049, HR 0.20, respectively). NGS data was available for 47 patients. Myeloid mutations were detected in 21 patients (44.7%) at diagnosis, of these 11 had an epigenetic mutation (23.9%; in ASXL1, DNMT3A, TET2 or EZH2). 11 patients (23.9%) had a variant that was potentially germline (VAF 40-60%). By chi-squared and Kaplan-Meier analysis, no differences were observed between the median TFR for patients with or without a pathogenic somatic, epigenetic, ASXL1 or germline mutation. However, a greater separation of the curves was observed when considering TFR at 36 months for somatic (p=0.101) but not germline mutation. Conclusions: We confirmed previous observations that the biological factors BCR::ABL1 positivity at stop and longer HT were associated with molecular relapse although no difference was found for short HT. The impact of clinical factors on TFR, including duration of MR4 and MR4.5 prior to stop, were also confirmed in our real-life series with a long follow-up, while total TKI duration was not prognostic. Adnan Awad et al. associated the presence of epigenetic mutations at diagnosis with shorter TFR and higher relapse probability (Blood Cancer J. 2022;12). Here, a significant difference in TFR was not observed for epigenetic mutation at diagnosis; however, a tendency was observed between a pathogenic somatic but not a germline myeloid mutation and TFR at 36 months.

Nonlobular Invasive Breast Carcinomas with Biallelic Pathogenic CDH1 Somatic Alterations: A Histologic, Immunophenotypic, and Genomic Characterization

CDH1 encodes for E-cadherin, and its loss of function is the hallmark of invasive lobular carcinoma (ILC). Albeit vanishingly rare, biallelic CDH1 alterations may be found in nonlobular breast carcinomas (NL-BCs). We sought to determine the clinicopathologic characteristics and repertoire of genetic alterations of NL-BCs harboring CDH1 biallelic genetic alterations. Analysis of 5842 breast cancers (BCs) subjected to clinical tumor-normal sequencing with an FDA-cleared multigene panel was conducted to identify BCs with biallelic CDH1 pathogenic/likely pathogenic somatic mutations lacking lobular features. The genomic profiles of NL-BCs with CDH1 biallelic genetic alterations were compared with those of ILCs and invasive ductal carcinomas (IDCs), matched by clinicopathologic characteristics. Of the 896 CDH1-altered BCs, 889 samples were excluded based on the diagnosis of invasive mixed ductal/lobular carcinoma or ILC or the detection of monoallelic CDH1 alterations. Only 7 of the 5842 (0.11%) BCs harbored biallelic CDH1 alterations and lacked lobular features. Of these, 4/7 (57%) cases were ER-positive/HER2-negative, 1/7 (14%) was ER-positive/HER2-positive, and 2/7 (29%) were ER-negative/HER2-negative. In total, 5/7 (71%) were of Nottingham grade 2, and 2/7 (29%) were of grade 3. The NL-BCs with CDH1 biallelic genetic alterations included a mucinous carcinoma (n = 1), IDCs with focal nested growth (n = 2), IDC with solid papillary (n = 1) or apocrine (n = 2) features, and an IDC of no special type (NST; n = 1). E-cadherin expression, as detected by immunohistochemistry, was absent (3/5) or aberrant (discontinuous membranous/cytoplasmic/granular; 2/5). However, NL-BCs with CDH1 biallelic genetic alterations displayed recurrent genetic alterations, including TP53, PIK3CA (57%, 4/7; each), FGFR1, and NCOR1 (28%, 2/7, each) alterations. Compared with CDH1 wild-type IDC-NSTs, NL-BCs less frequently harbored GATA3 mutations (0% vs 47%, P = .03), but no significant differences were detected when compared with matched ILCs. Therefore, NL-BCs with CDH1 biallelic genetic alterations are vanishingly rare, predominantly comprise IDCs with special histologic features, and have genomic features akin to luminal B ER-positive BCs.

Clonal Bone Marrow Plasma Cells Are Common in Patients with Vexas Syndrome

Introduction Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder. Its prevalence increases with age, reaching 5.3% at 70 years and older. MGUS progression rate to multiple myeloma (MM) or lymphoplasmacytic malignancies is about 1% per year. Patients with autoimmune diseases are at increased risk for developing MGUS and MM. VEXAS ( vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic syndrome), an autoinflammatory disorder caused by somatic mutations in ubiquitin-activating enzyme, UBA1 gene, has been associated with plasma cell disorders, specifically MGUS, but the rate of progression is unknown. Cyclin D1, important in cell cycle progression, is regulated by ubiquitin-proteasome mediated degradation, and deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. We aim to assess marrow characteristics of plasma cell disorders (MGUS and MM) in VEXAS, and the impact of a defective UBA1 gene product on protein expression of cyclin D1. Methods We assessed 28 patients with VEXAS for the presence of plasma cell disorders, and studied the clinical and laboratory characteristics of those with clonal plasma cells (PC) in the marrow. Bone marrow biopsies and aspirates were evaluated by morphology, immunohistochemistry (IHC) dual staining CD138/cyclin D1, dual staining MUM-1/CD20), in situ hybridization (Kappa and Lambda light chains) and flow cytometry (FC) using antibodies for CD45, CD19, CD20, CD38, CD138, CD56, CD27, CD81, and cytoplasmic Kappa and Lambda immunoglobulins. Serum free kappa and lambda light chains, IgG, IgM, and IgA, and level of M paraprotein by serum protein electrophoresis with immunofixation were obtained. Fluorescent in situ hybridization (FISH) analysis for MM was performed in most patients with clonal PCs. Results Patients' characteristics are shown in Table 1. Eight out of 28 (28.6%) patients with VEXAS syndrome, all males, had clonal plasma cells in their bone marrows: 3 MGUS, and 5 MM (3 smoldering, SMM; 2 active MM). Median age at diagnosis of plasma cell disorder was 69.5 years (range 60-75). All patients had pathogenic UBA1 somatic mutations (5 M41T, 1 each with M41V, M41L, and splice). Clinically, monoclonal protein size was small, with no specific selection of the type of M protein. Strikingly, most (7/8) clonal plasma cells showed cyclin D1 expression by IHC (dual immunohistochemical stain cyclinD1/CD138 shows double-positive plasma cells; figure not shown), displaying nuclear immunoreactivity in 6 cases, and cytoplasmic immunoreactivity in 1 case. FC showed positive CD20 expression of clonal plasma cells in 4 cases, dim to negative CD19 expression in 4 cases, variable CD56 (positive in 3 cases, partial positive in 1 case, negative in 2 cases), dim to negative CD27 in 3 cases, dim to negative CD81 in 3 cases. FISH for MM revealed: t(11;14) in 4 cases, gain of 11q (CCND1) in 2 cases (in one case, CCND1 gain was present in addition to t(11;14); in the second case, CCND1 gain was associated with gains of 1q and 17p(TP53). The cyclin D1-negative MM case was negative for t(11;14) but had t(4;14). All cases with t(11;14) were positive for cyclin D1 by IHC (nuclear staining). The one MM case with negative nuclear staining for cyclin D1 but positive cytoplasmic staining, was negative for t(11;14) but had gain of 11q (CCND1).One patient evolved from SMM to active MM after 4 years. No evolution to MM was seen in patients with MGUS with median follow-up of 30 months. Conclusions Patients with VEXAS have a high prevalence of clonal plasma cell disorders. Clonal plasma cells in VEXAS patients are associated with t(11;14), and with nuclear cyclin D1 expression by IHC. Cytoplasmic cyclin D1 staining in the absence or weak nuclear staining was observed with CCND1 gain and absence of t(11;14). Cytoplasmic cyclin D1 expression may be related to lack of cyclin D1 protein degradation by a defective UBA1 gene product. Our results suggest that defective UBA1 and autoinflammation pose an increased risk for developing MGUS or multiple myeloma.