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Mixed invasive ductal and lobular carcinoma (MDLC) is a rare histologic subtype of breast cancer displaying both E-cadherin positive ductal and E-cadherin negative lobular morphologies within the same tumor, posing challenges with regard to anticipated clinical management. It remains unclear whether these distinct morphologies also have distinct biology and risk of recurrence. Our spatially-resolved transcriptomic, genomic, and single-cell profiling revealed clinically significant differences between ductal and lobular tumor regions including distinct intrinsic subtype heterogeneity (e.g., MDLC with TNBC/basal ductal and ER+/luminal lobular regions), distinct enrichment of senescence/dormancy and oncogenic (ER and MYC) signatures, genetic and epigenetic CDH1 inactivation in lobular, but not ductal regions, and single-cell ductal and lobular sub-populations with unique oncogenic signatures further highlighting intra-regional heterogeneity. Altogether, we demonstrated that the intra-tumoral morphological/histological heterogeneity within MDLC is underpinned by intrinsic subtype and oncogenic heterogeneity which may result in prognostic uncertainty and therapeutic dilemma., Significance: MDLC displays both ductal and lobular tumor regions. Our multi-omic profiling approach revealed that these morphologically distinct tumor regions harbor distinct intrinsic subtypes and oncogenic features that may cause prognostic uncertainty and therapeutic dilemma. Thus histopathological/molecular profiling of individual tumor regions may guide clinical decision making and benefit patients with MDLC, particularly in the advanced setting where there is increased reliance on next generation sequencing.

Objective: Adenoid cystic carcinoma (AdCC) of the Bartholin's gland (AdCC-BG) is a very rare gynecologic vulvar malignancy. AdCC-BGs are slow-growing but locally aggressive and are associated with high recurrence rates. Here we sought to characterize the molecular underpinning of AdCC-BGs., Methods: AdCC-BGs (n = 6) were subjected to a combination of RNA-sequencing, targeted DNA-sequencing, reverse-transcription PCR, fluorescence in situ hybridization (FISH) and MYB immunohistochemistry (IHC). Clinicopathologic variables, somatic mutations, copy number alterations and chimeric transcripts were assessed., Results: All six AdCC-BGs were biphasic, composed of ductal and myoepithelial cells. Akin to salivary gland and breast AdCCs, three AdCC-BGs had the MYB::NFIB fusion gene with varying breakpoints, all of which were associated with MYB overexpression by IHC. Two AdCC-BGs were underpinned by MYBL1 fusion genes with different gene partners, including MYBL1::RAD51B and MYBL1::EWSR1 gene fusions, and showed MYB protein expression. Although the final AdCC-BG studied had MYB protein overexpression, no gene fusion was identified. AdCC-BGs harbored few additional somatic genetic alterations, and only few mutations in cancer-related genes were identified, including GNAQ, GNAS, KDM6A, AKT1 and BCL2, none of which were recurrent. Two AdCC-BGs, both with a MYB::NFIB fusion gene, developed metastatic disease., Conclusions: AdCC-BGs constitute a convergent phenotype, whereby activation of MYB or MYBL1 can be driven by the MYB::NFIB fusion gene or MYBL1 rearrangements. Our observations further support the notion that AdCCs, irrespective of organ site, constitute a genotypic-phenotypic correlation. Assessment of MYB or MYBL1 rearrangements may be used as an ancillary marker for the diagnosis of AdCC-BGs., Competing Interests: Declaration of competing interest B. Weigelt reports research funding from REPARE Therapeutics, outside the submitted work. F. Pareja is a member of the scientific advisory board of MultiplexDx Inc. J.S. Reis-Filho reported receiving personal/consultancy fees from Goldman Sachs, Bain Capital, REPARE Therapeutics, Saga Diagnostics and Paige.AI, membership of the scientific advisory boards of VolitionRx, REPARE Therapeutics and Paige.AI, membership of the Board of Directors of Grupo Oncoclinicas, and ad hoc membership of the scientific advisory boards of AstraZeneca, Merck, Daiichi Sankyo, Roche Tissue Diagnostics and Personalis, outside the submitted work. M. Leitao reports personal fees from Medtronic, Intuitive Surgical, J&J/Ethicon, and Immunogen. N.R. Abu-Rustum reports research funding paid to the institution from GRAIL; Memorial Sloan Kettering Cancer Center also has equity in GRAIL. The remaining authors have no competing interests to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Mesonephric (MA) and mesonephric-like (MLA) adenocarcinomas are rare cancers, and data on clinical behavior and response to therapy are limited. We sought to report molecular features, treatment, and outcomes of MA/MLA from a single institution., Methods: Patients with MA (cervix) or MLA (uterus, ovary, other) treated at Memorial Sloan Kettering Cancer Center (MSK) from 1/2008-12/2021 underwent pathologic re-review. For patients with initial treatment at MSK, progression-free survival (PFS1) was calculated as time from initial surgery to progression or death; second PFS (PFS2) was calculated as time from start of treatment for recurrence to subsequent progression or death. Overall survival (OS) was calculated for all patients. Images were retrospectively reviewed to determine treatment response. Somatic genetic alterations were assessed by clinical tumor-normal sequencing (MSK-Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT])., Results: Of 81 patients with confirmed gynecologic MA/MLA, 36 received initial treatment at MSK. Sites of origin included cervix (n = 9, 11%), uterus (n = 42, 52%), ovary (n = 28, 35%), and other (n = 2, 2%). Of the 36 patients who received initial treatment at MSK, 20 (56%) recurred; median PFS1 was 33 months (95% CI: 17-not evaluable), median PFS2 was 8.3 months (95% CI: 6.9-14), and median OS was 87 months (95% CI: 58.2-not evaluable). Twenty-six of the 36 patients underwent MSK-IMPACT testing, and 25 (96%) harbored MAPK pathway alterations., Conclusion: Most patients diagnosed with early-stage disease ultimately recurred. Somatic MAPK signaling pathway mutations appear to be highly prevalent in MA/MLA, and therapeutics that target this pathway are worthy of further study., Competing Interests: Declaration of competing interest B. Weigelt reports research funding by Repare Therapeutics, outside the current work. N. R. Abu-Rustum reports grant funding from GRAIL paid to the institution. A. Iasonos reports consulting fees from Mylan. C. Aghajanian reports clinical trial funding paid to the institution from AstraZeneca; consulting fees (advisory board) from Eisai/Merck, Roche/Genentech, Abbvie, AstraZeneca/Merck, and Repare Therapeutics; advisory board participation (no fee) for Blueprint Medicine; and leadership/fiduciary roles for the GOG Foundation Board of Directors (travel cost reimbursement) and NRG Oncology Board of Directors (unpaid). D. Chi reports personal fees from Apyx Medical, Verthermia Inc., Biom ‘Up, and AstraZeneca, as well as recent or current stock/options ownership of Apyx Medical, Verthemia, Intuitive Surgical, Inc., TransEnterix, Inc., Doximity, Moderna, and BioNTech SE. R.N. Grisham reports honoraria from GSK, AstraZeneca, Natera, Springworks, Corcept, MJH, and PER. The other authors do not have potential conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Racial disparities in outcomes exist in endometrial cancer (EC). The contribution of ancestry-based variations in germline pathogenic variants (gPVs) is unknown., Methods: Germline assessment of ≥76 cancer predisposition genes was performed in patients with EC undergoing tumor-normal Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets sequencing from January 1, 2015 through June 30, 2021. Self-reported race/ethnicity and Ashkenazi Jewish ancestry data classified patients into groups. Genetic ancestry was inferred from Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets. Rates of gPV and genetic counseling were compared by ancestry., Results: Among 1625 patients with EC, 216 (13%) had gPVs; 15 had >1 gPV. Rates of gPV varied by self-reported ancestry (Ashkenazi Jewish, 40/202 [20%]; Asian, 15/124 [12%]; Black/African American (AA), 12/171 [7.0%]; Hispanic, 15/124 [12%]; non-Hispanic (NH) White, 129/927 [14%]; missing, 5/77 [6.5%]; p = .009], with similar findings by genetic ancestry (p < .001). We observed a lower likelihood of gPVs in patients of Black/AA (odds ratio [OR], 0.44; 95% CI, 0.22-0.81) and African (AFR) ancestry (OR, 0.42; 95% CI, 0.18-0.85) and a higher likelihood in patients of Ashkenazi Jewish genetic ancestry (OR, 1.62; 95% CI; 1.11-2.34) compared with patients of non-Hispanic White/European ancestry, even after adjustment for age and molecular subtype. Somatic landscape influenced gPVs with lower rates of microsatellite instability-high tumors in patients of Black/AA and AFR ancestry. Among those with newly identified gPVs (n = 114), 102 (89%) were seen for genetic counseling, with lowest rates among Black/AA (75%) and AFR patients (67%)., Conclusions: In those with EC, gPV and genetic counseling varied by ancestry, with lowest rates among Black/AA and AFR patients, potentially contributing to disparities in outcomes given implications for treatment and cancer prevention., Plain Language Summary: Black women with endometrial cancer do worse than White women, and there are many reasons for this disparity. Certain genetic changes from birth (mutations) can increase the risk of cancer, and it is unknown if rates of these changes are different between different ancestry groups. Genetic mutations in 1625 diverse women with endometrial cancer were studied and the lowest rates of mutations and genetic counseling were found in Black and African ancestry women. This could affect their treatment options as well as their families and may make disparities worse., (© 2023 American Cancer Society.)

Given the tubal origin of high-grade serous ovarian cancer (HGSC), we sought to investigate intrauterine lavage (IUL) as a novel method of biomarker detection. IUL and serum samples were collected from patients with HGSC or benign pathology. Although CA-125 and HE4 concentrations were significantly higher in IUL samples compared to serum, they were similar between IUL samples from patients with HGSC vs benign conditions. In contrast, CA-125 and HE4 serum concentrations differed between HGSC and benign pathology ( P  =.002 for both). IUL and tumor samples from patients with HGSC were subjected to targeted panel sequencing and droplet digital PCR (ddPCR). Tumor mutations were found in 75 % of matched IUL samples. Serum CA-125 and HE4 biomarker levels allowed for better differentiation of HGSC and benign pathology compared to IUL samples. We believe using IUL for early detection of HGSC requires optimization, and current strategies should focus on prevention until early detection strategies improve., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: D.A.H. is a co-founder and officer with equity interest of Lime Therapeutics Inc., co-founder with equity interest of Selectin Therapeutics Inc. and Resident Diagnostics, Inc., and a member of the scientific advisory boards of Concarlo Therapeutics Inc., Nanorobotics Inc., and Mediphage Bioceuticals Inc. B.W. reports research funding by Repare Therapeutics, outside of the submitted work. E.S. reports honoraria from Dilon Technologies, Inc. D.A.L. is a full-time employee of Merck & Co., Inc. (Rahway, NJ, USA) and a co-founder with equity interest of Resident Diagnostics, Inc. N.A.R. reports grant funding from GRAIL paid to the institution. D.S.C. reports personal fees from Apyx Medical, Verthermia Inc., Biom 'Up, and AstraZeneca, as well as recent or current stock/options ownership of Apyx Medical, Verthemia, Intuitive Surgical, Inc., TransEnterix, Inc., Doximity, Moderna, and BioNTech SE. K.L.R. reports travel support from Intuitive Surgical. T.Y.S and M.F. have no conflicts of interest to report., (© 2024 The Authors. Published by Elsevier Inc.)

Objective: To define molecular features of ovarian cancer (OC) with germline pathogenic variants (PVs) in non-BRCA homologous recombination (HR) genes and analyze survival compared to BRCA1/2 and wildtype (WT) OC., Methods: We included patients with OC undergoing tumor-normal sequencing (MSK-IMPACT) from 07/01/2015-12/31/2020, including germline assessment of BRCA1/2 and other HR genes ATM, BARD1, BRIP1, FANCA, FANCC, NBN, PALB2, RAD50, RAD51B, RAD51C, and RAD51D. Biallelic inactivation was assessed within tumors. Progression-free (PFS) and overall survival (OS) were calculated from pathologic diagnosis using the Kaplan-Meier method with left truncation. Whole-exome sequencing (WES) was performed in a subset., Results: Of 882 patients with OC, 56 (6.3%) had germline PVs in non-BRCA HR genes; 95 (11%) had BRCA1-associated OC (58 germline, 37 somatic); and 59 (6.7%) had BRCA2-associated OC (40 germline, 19 somatic). High rates of biallelic alterations were observed among germline PVs in BRIP1 (11/13), PALB2 (3/4), RAD51B (3/4), RAD51C (3/4), and RAD51D (8/10). In cases with WES (27/35), there was higher tumor mutational burden (TMB; median 2.5 [1.1-6.0] vs. 1.2 mut/Mb [0.6-2.6]) and enrichment of HR-deficient (HRD) mutational signatures in tumors associated with germline PALB2 and RAD51B/C/D compared with BRIP1 PVs (p < 0.01). Other features of HRD, including telomeric-allelic imbalance (TAI) and large-scale state transitions (LSTs), were similar. Although there was heterogeneity in PFS/OS by gene group, only BRCA1/2-associated OC had improved survival compared to WT OC (p < 0.01)., Conclusions: OCs associated with germline PVs in non-BRCA HR genes represent a heterogenous group, with PALB2 and RAD51B/C/D associated with an HRD phenotype., Competing Interests: Declaration of Competing Interest YLL reports research funding from AstraZeneca, GSK and Repare Therapeutics outside of this work. KEB reports that an immediate family member is on the Scientific Advisory Board of Emendo Biotherapeutics, Karyopharm Therapeutics, Imago BioSciences, and DarwinHealth; is co-Founder of Isabl Technologies; and has equity interest in Imago BioSciences, Emendo Biotherapeutics and Isabl Technologies. BW reports research funding by Repare Theraputics. CA has received research grants from Abbvie, Clovis, Genentech, and Astra Zeneca and served on advisory boards for Abbvie, AstraZeneca/Merck, Eisai/Merck, Mersana Therapeutics, Repare Therapeutics, and Roche/Genentech. ZS reports an immediate family member who serves as a consultant in Ophthalmology for Adverum, Genentech, Gyroscope Therapeutics Limited, Neurogene, Optos Plc, Outlook Therapeutics, RegenexBio, and Regeneron. RO reports meeting/travel support from the Gynecologic Oncology Foundation, Curio, and Hitech Health; participation in the advisory boards of Tesaro/GlaxoSmithKline (GSK), Regeneron, Seattle Genetics, Fresenius Kabi, Bayer, and CarinaBiotech (non-compensated); non-compensated steering committee participation for Tesaro/GSK and AstraZeneca; and grant support paid to the institution from Bayer/Celgene/Juno, Tesaro/GSK, Merck, the Ludwig Cancer Institute, Abbvie/StemCentrx, Regeneron, TCR2 Therapeutics, Marker Therapeutics, Syndax Pharmaceuticals, Genmab/Seagen Therapeutics, Sellas Therapeutics, Genentech, KitePharma, and the Gynecologic Oncology Foundation. AI reports consulting fees from Mylan. RG reports honoraria from GSK, AstraZeneca, Natera, Springworks, Corcept, MJH, and PER. DC reports personal fees from Apyx Medical, Verthermia Inc., Biom ‘Up, and AstraZeneca, as well as recent or current stock/options ownership of Apyx Medical, Verthemia, Intuitive Surgical, Inc., TransEnterix, Inc., Doximity, Moderna, and BioNTech SE. KO is a founder and shareholder of AnaNeo Therapeutics Incorporated. MFB reports receiving research funding from GRAIL and advisory board activities for Eli Lilly, AstraZeneca, and PetDx. All other authors have no potential conflicts of interest to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aims: Activating somatic mutations or gene amplification of KIT result in constitutive activation of its receptor tyrosine kinase, which is targetable in various solid tumours. Here, we sought to investigate the presence of KIT genetic alterations in breast cancer (BC) and characterise the histological and genomic features of these tumours., Methods: A retrospective analysis of 5,575 BCs previously subjected to targeted sequencing using the FDA-authorised Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Targets (MSK-IMPACT) assay was performed to identify BCs with KIT alterations. A histological assessment of KIT -altered BCs was conducted, and their repertoire of genetic alterations was compared with that of BCs lacking KIT genetic alterations, matched for age, histological type, oestrogen receptor/HER2 status and sample type., Results: We identified 18 BCs (0.32%), including 9 primary and 9 metastatic BCs, with oncogenic/likely oncogenic genetic alterations affecting KIT , including activating somatic mutations (n=4) or gene amplification (n=14). All KIT -altered BCs were of high histological grade, although no distinctive histological features were observed. When compared with BCs lacking KIT genetic alterations, no distinctive genetic features were identified. In two metastatic KIT -altered BCs in which the matched primary BC had also been analysed by MSK-IMPACT, the KIT mutations were found to be restricted to the metastatic samples, suggesting that they were late events in the evolution of these cancers., Conclusions: KIT genetic alterations are vanishingly rare in BC. KIT -altered BCs are of high grade but lack distinctive histological features. Genetic alterations in KIT might be late events in the evolution and/or progression of BC., Competing Interests: Competing interests: JSR-F reports receiving personal/consultancy fees from Goldman Sachs, REPARE Therapeutics and Paige. AI, membership of the scientific advisory boards of VolitionRx, REPARE Therapeutics, Personalis, Bain Capital and Paige. AI, membership of the Board of Directors of Grupo Oncoclinicas, and ad hoc membership of the scientific advisory boards of Roche Tissue Diagnostics, Ventana Medical Systems, Merck, Daiichi Sankyo and Astrazeneca, outside the scope of this study. BW reports ad hoc membership of the scientific advisory board of REPARE Therapeutics, outside the scope of the submitted work. All other authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To assess the clinicopathologic, molecular profiles, and survival outcomes of patients with endometrial carcinomas (ECs) harboring telomerase reverse transcriptase (TERT) hotspot mutations or gene amplification., Methods: ECs harboring somatic TERT promoter hotspot mutations or gene amplification (TERT-altered) were identified from 1944 ECs that underwent clinical tumor-normal sequencing from 08/2016-12/2021. Clinicopathologic variables, somatic mutation profiles, and survival outcomes of TERT-alt and TERT-wild-type EC were assessed., Results: We identified 66 TERT-altered ECs (43 TERT-mutated and 23 TERT-amplified), representing 3% of the unselected ECs across histologic subtypes. Most TERT-altered ECs were of copy number (CN)-high/TP53abn molecular subtype (n = 40, 60%), followed by microsatellite-unstable (MSI-H) or CN-low/no specific molecular profile (NSMP)(n = 13, 20% each). TERT-amplified and TERT-mutated ECs were molecularly distinct, with TERT-amplified ECs being more genomically instable and more frequently harboring TP53 and PPP2R1A alterations (q < 0.1). Compared to TERT-wild-type ECs, TERT-altered ECs were more commonly of CN-H/TP53abn molecular subtype (31% vs 57%, p = 0.001), serous histology (10% vs 26%, p = 0.004), and were significantly enriched for TP53, CDKN2A/B, and DROSHA somatic genetic alterations (q < 0.1). Median progression-free survival was 18.7 months (95% CI 11.8-not estimable [NE]) for patients with TERT-altered EC and 80.9 months (65.8-NE) for patients with TERT-wild-type EC (HR 0.33, 95% CI 0.21-0.51, p < 0.001). Similarly, median overall survival was 46.7 months (95% CI 30-NE) for TERT-altered EC patients and not reached for TERT-wild-type EC patients (HR 0.24, 95% CI 0.13-0.44, p < 0.001)., Conclusion: TERT-altered ECs, although rare, are enriched for CN-high/TP53abn tumors, TP53, CDKN2A/B and DROSHA somatic mutations, and independently predict worse survival outcomes., Competing Interests: Declaration of Competing Interest N.R. Abu-Rustum reports Stryker/ Novadaq and GRAIL grants paid to the institution, outside the current study. B. Weigelt reports a research grant from REPARE Therapeutics paid to the institution, outside the submitted work. C. Aghajanian reports clinical trial funding paid to the institution from AstraZeneca; consulting fees (advisory board) from Eisai/Merck, Roche/Genentech, Abbvie, AstraZeneca/Merck, and Repare Therapeutics; advisory board participation (no fee) for Blueprint Medicine; and leadership/fiduciary roles for the GOG Foundation Board of Directors (travel cost reimbursement) and NRG Oncology Board of Directors (unpaid). The remaining authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although the incidence of endometrial carcinoma (EC) is similar in Black and White women, racial disparities are stark, with the highest mortality rates observed among Black patients. Here, analysis of 1,882 prospectively sequenced ECs using a clinical FDA-authorized tumor-normal panel revealed a significantly higher prevalence of high-risk histologic and molecular EC subtypes in self-identified Black (n = 259) compared with White (n = 1,623) patients. Clinically actionable alterations, including high tumor mutational burden/microsatellite instability, which confer benefit from immunotherapy, were less frequent in ECs from Black than from White patients. Ultramutated POLE molecular subtype ECs associated with favorable outcomes were rare in Black patients. Results were confirmed by genetic ancestry analysis. CCNE1 gene amplification, which is associated with aggressive clinical behavior, was more prevalent in carcinosarcomas occurring in Black than in White patients. ECs from Black and White patients display important differences in their histologic types, molecular subtypes, driver genetic alterations, and therapeutic targets., Significance: Our comprehensive analysis of prospectively clinically sequenced ECs revealed significant differences in their histologic and molecular composition and in the presence of therapeutic targets in Black versus White patients. These findings emphasize the importance of incorporating diverse populations into molecular studies and clinical trials to address EC disparities. This article is featured in Selected Articles from This Issue, p. 2293., (©2023 American Association for Cancer Research.)

Purpose: Clinical trials reported 25% to 30% pathologic complete response (pCR) rates in HER2+ patients with breast cancer treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2-"addicted" tumors who may benefit from a chemotherapy-sparing strategy., Experimental Design: Baseline HER2+ breast cancer specimens from the TBCRC023 and PAMELA trials, which included neoadjuvant treatment with lapatinib and trastuzumab, were used. In the case of estrogen receptor-positive (ER+) tumors, endocrine therapy was also administered. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA., Results: In TBCRC023, 72 breast cancer specimens had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio ≥ 4.6 and %3+ IHC staining ≥ 97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (WT). For clinical implementation, the classifier was locked as HER2 ratio ≥ 4.5, %3+ IHC staining ≥ 90%, and PIK3CA-WT and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier's high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment deescalation., Conclusions: Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable with chemotherapy plus dual anti-HER2 therapy in unselected patients., (©2023 American Association for Cancer Research.)

Purpose: Using next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data., Experimental Design: All patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution., Results: Utilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949-0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC., Conclusions: The integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification., Competing Interests: Conflicts of interest N.R. Abu-Rustum reports Stryker/Novadaq and GRAIL grants paid to the institution, outside the current study. C.F. Friedman reports institutional research support from Seagen, Merck, BMS, AstraZeneca, Mersana and Hotspot Therapeutics; consulting fees from BMS, Seagen and Aadi Biosciences; honoraria for lectures from Onclive; meeting/ travel support by Puma; participation on Data Safety Monitoring or Advisory Board of Merck, Genentech and Marengo (all uncompensated). D. Zamarin reports institutional research support from AstraZeneca, Merck, Plexxikon Synthekine and Genentech; consulting fees from AstraZeneca, Synthekine, Astellas, Tessa Therapeutics, Memgen, Celldex, Crown Biosciences, Hookipa Biotech, Kalivir, Xencor and GSK; royalties from Merck; and stock options from Accurius Therapeutics, ImmunOS Therapeutics and Calidi Biotherapeutics, all outside the submitted work. V. Makker reports meeting/travel support by Eisai and Merck; participation on Data Safety Monitoring or Advisory Board of Duality, Merck, Karyopharm, Exelexis, Eisai, Karyopharm, BMS, Clovis, Faeth Immunocore, Morphosys, AstraZeneca, Novartis, GSK, Bayer (all unpaid), and study support to the institution by Merck, Eisai, AztraZeneca, Faeth, Karyopharm, Zymeworks, Duality, Clovis, Bayer and Takeda. Y. Liu reports institutional research funding from Repare Therapeutics, AstraZeneca and GSK; honoraria from Total Health and Sarah Lawrence College; and travel/meeting support by AstraZeneca, outside the submitted work. B. Weigelt reports a research grant from REPARE Therapeutics paid to the institution, outside the submitted work. D.S. Chi reports personal fees from Apyx Medical, Verthermia Inc., Biom ‘Up, and AstraZeneca, as well as recent or current stock/options ownership of Apyx Medical, Verthemia, Intuitive Surgical, Inc., TransEnterix, Inc., Doximity, Moderna, and BioNTech SE. E.L. Jewell reports personal fees from Covidien/Medtronic. M. M. Leitao is an ad hoc speaker for Intuitive Surgical, Inc.; outside the submitted work, he is on the Advisory Board of Ethicon/Johnson & Johnson and Takeda; and reports grants paid to the institution by KCI/Acelity. C. Aghajanian reports clinical trial funding paid to the institution from AstraZeneca; consulting fees (advisory board) from Eisai/Merck, Roche/Genentech, Abbvie, AstraZeneca/Merck, and Repare Therapeutics; advisory board participation (no fee) for Blueprint Medicine; and leadership/fiduciary roles for the GOG Foundation Board of Directors (travel cost reimbursement) and NRG Oncology Board of Directors (unpaid). The remaining authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: We sought to determine the prevalence of germline pathogenic variants (gPVs) in unselected patients with endometrial cancer (EC), define biallelic gPVs within tumors, and describe their associations with clinicopathologic features., Methods: Germline assessment of at least 76 cancer predisposition genes was performed in patients with EC undergoing clinical tumor-normal Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing from January 1, 2015, to June 30, 2021. In patients with gPVs, biallelic alterations in ECs were identified through analysis of loss of heterozygosity and somatic PVs. Clinicopathologic variables were compared using nonparametric tests., Results: Of 1625 patients with EC, 216 (13%) had gPVs, and 15 patients had 2 gPVs. There were 231 gPVs in 35 genes (75 [32%] high penetrance; 39 [17%] moderate penetrance; and 117 [51%] low, recessive, or uncertain penetrance). Compared with those without gPVs, patients with gPVs were younger (P = .002), more often White (P = .009), and less obese (P = .025) and had differences in distribution of tumor histology (P = .017) and molecular subtype (P < .001). Among 231 gPVs, 74 (32%) exhibited biallelic inactivation within tumors. For high-penetrance gPVs, 63% (47 of 75) of ECs had biallelic alterations, primarily affecting mismatch repair (MMR) and homologous recombination related genes, including BRCA1,BRCA2, RAD51D, and PALB2. Biallelic inactivation varied across molecular subtypes with highest rates in microsatellite instability-high (MSI-H) or copy-number (CN)-high subtypes (3 of 12 [25%] POLE, 30 of 77 [39%] MSI-H, 27 of 60 [45%] CN-high, 9 of 57 [16%] CN-low; P < .001)., Conclusions: Of unselected patients with EC, 13% had gPVs, with 63% of gPVs in high-penetrance genes (MMR and homologous recombination) exhibiting biallelic inactivation, potentially driving cancer development. This supports germline assessment in EC given implications for treatment and cancer prevention., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: We sought to determine whether sequencing analysis of circulating cell-free DNA (cfDNA) in patients with prospectively accrued endometrial cancer captures the mutational repertoire of the primary lesion and allows for disease monitoring., Experimental Design: Peripheral blood was prospectively collected from 44 newly diagnosed patients with endometrial cancer over a 24-month period (i.e., baseline, postsurgery, every 6 months after). DNA from the primary endometrial cancers was subjected to targeted next-generation sequencing (NGS) of 468 cancer-related genes, and cfDNA to a high-depth NGS assay of 129 genes with molecular barcoding. Sequencing data were analyzed using validated bioinformatics methods., Results: cfDNA levels correlated with surgical stage in endometrial cancers, with higher levels of cfDNA being present in advanced-stage disease. Mutations in cfDNA at baseline were detected preoperatively in 8 of 36 (22%) patients with sequencing data, all of whom were diagnosed with advanced-stage disease, high tumor volume, and/or aggressive histologic type. Of the 38 somatic mutations identified in the primary tumors also present in the cfDNA assay, 35 (92%) and 38 (100%) were detected at baseline and follow-up, respectively. In 6 patients with recurrent disease, changes in circulating tumor DNA (ctDNA) fraction/variant allele fractions in cfDNA during follow-up closely mirrored disease progression and therapy response, with a lead time over clinically detected recurrence in two cases. The presence of ctDNA at baseline (P < 0.001) or postsurgery (P = 0.014) was significantly associated with reduced progression-free survival., Conclusions: cfDNA sequencing analysis in patients with endometrial cancer at diagnosis has prognostic value, and serial postsurgery cfDNA analysis enables disease and treatment response monitoring. See related commentary by Grant et al., p. 305., (©2022 American Association for Cancer Research.)

Aims: Primary skin mucinous carcinoma is a rare sweat gland neoplasm with a high local recurrence rate after conventional excision but a low distant-metastasis rate. The genetic underpinning of skin mucinous carcinoma is presently unknown. Here, we sought to define whether the repertoire of somatic mutations of a primary mucinous carcinoma of the skin would be similar to that of mucinous breast carcinomas, given the histologic similarities between these tumor types., Methods and Results: The tumor was situated in the dermis and partially involved the subcutaneous fat. Tumor cells were suspended in periodic acid-Schiff diastaseresistant- positive mucin lakes and expressed cytokeratin 7, synaptophysin and estrogen receptor. DNA samples extracted from microdissected tumor and matched normal tissue were subjected to massively parallel sequencing targeting 410 cancer-related genes. The skin mucinous tumor was found to have a low tumor mutation burden, but to harbor a clonal GATA3 frameshift mutation (p. T418Hfs*89) and amplification of FOXA1 , genes not uncommonly altered in breast mucinous carcinomas., Conclusions: In this primary skin mucinous carcinoma, GATA3 and FOXA1 driver genetic events were identified, consistent with a possible developmental relationship between skin and breast mucinous neoplasms., Competing Interests: Declaration of Competing Interest JSR-F reports receiving personal/consultancy fees from Goldman Sachs, Bain Capital, REPARE Therapeutics, Saga Diagnostics and Paige. AI, membership of the scientific advisory boards of VolitionRx, REPARE Therapeutics and Paige.AI, membership of the Board of Directors of Grupo Oncoclinicas, and ad hoc membership of the scientific advisory boards of AstraZeneca, Merck, Daiichi Sankyo, Roche Tissue Diagnostics and Personalis, outside the scope of this study. BW reports research funding from Repare Therapeutics, outside the scope of the submitted work. The remaining authors have no conflicts of interest to declare.

Low-stage, low-grade endometrioid endometrial carcinoma (EEC), the most common histologic type of endometrial cancer, typically has a favorable prognosis. A subset of these cancers, however, displays an aggressive clinical course with early recurrences, including distant relapses. All statistical tests were 2-sided. Using a combination of whole-exome and targeted capture sequencing of 65 FIGO stage IA and IB grade 1 EECs treated with surgery alone, we demonstrate that chromosome 1q gain (odds ratio [OR] = 8.09, 95% confidence interval [CI] = 1.59 to 54.6; P = .02), PIK3CA mutation (OR = 9.16, 95% CI = 1.95 to 61.8; P = .01), and DNA mismatch repair-deficient molecular subtype (OR = 7.92, 95% CI = 1.44 to 87.6; P = .02) are independent predictors of early recurrences within 3 years in this patient population. Chromosome 1q gain was validated in an independent dataset of stage I grade 1 EECs subjected to whole-exome sequencing. Our findings expand on the repertoire of genomic parameters that should be considered in the evaluation of patients with low-stage, low-grade EEC., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Microsatellite instability-high (MSI-H) endometrial carcinomas are underpinned by distinct mechanisms of DNA mismatch repair deficiency (MMR-D). We sought to characterize the clinical and genetic features of MSI-H endometrial cancers harboring germline or somatic mutations in MMR genes or MLH1 promoter hypermethylation (MLH1ph)., Experimental Design: Of > 1,100 patients with endometrial cancer that underwent clinical tumor-normal sequencing, 184 had MSI-H endometrial cancers due to somatic MMR mutations or MLH1ph, or harbored pathogenic germline MMR mutations. Clinicopathologic features, mutational landscape, and tumor-infiltrating lymphocyte (TIL) scores were compared among MMR-D groups using nonparametric tests. Log-rank tests were used for categorical associations; Kaplan-Meier method and Wald test based on Cox proportional hazards models were employed for continuous variables and survival analyses., Results: Compared with patients with germline (n = 25) and somatic (n = 39) mutations, patients with MLH1ph endometrial cancers (n = 120) were older (P < 0.001), more obese (P = 0.001) and had more advanced disease at diagnosis (P = 0.025). MLH1ph endometrial cancers were enriched for JAK1 somatic mutations as opposed to germline MMR-D endometrial cancers which showed enrichment for pathogenic ERBB2 mutations. MLH1ph endometrial cancers exhibited lower tumor mutational burden and TIL scores compared with endometrial cancers harboring germline or somatic MMR mutations (P < 0.01). MLH1ph endometrial cancer patients had shorter progression-free survival (PFS) on univariate analysis, but in multivariable models, stage at diagnosis remained the only predictor of survival. For stage I/II endometrial cancer, two-year PFS was inferior for patients with MLH1ph endometrial cancers compared with germline and somatic MMR groups (70% vs. 100%, respectively)., Conclusions: MLH1ph endometrial cancers likely constitute a distinct clinicopathologic entity compared with germline and somatic MMR-D ECs with potential treatment implications., (©2022 American Association for Cancer Research.)

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland., (© 2022 The Pathological Society of Great Britain and Ireland.)

Background: Gastroesophageal junction (GEJ) adenocarcinoma is a rare cancer associated with poor prognosis. The genetic factors conferring predisposition to GEJ adenocarcinoma have yet to be identified., Methods: We analyzed germline testing results from 23 381 cancer patients undergoing tumor-normal sequencing, of which 312 individuals had GEJ adenocarcinoma. Genomic profiles and clinico-pathologic features were analyzed for the GEJ adenocarcinomas. Silencing of ATM and ATR was performed using validated short-interfering RNA species in GEJ, esophageal, and gastric adenocarcinoma cell lines. All statistical tests were 2-sided., Results: Pathogenic or likely pathogenic ATM variants were identified in 18 of 312 patients (5.8%), and bi-allelic inactivation of ATM through loss of heterozygosity of the wild-type allele was detected in all (16 of 16) samples with sufficient tumor content. Germline ATM-mutated GEJ adenocarcinomas largely lacked somatic mutations in TP53, were more likely to harbor MDM2 amplification, and harbored statistically significantly fewer somatic single nucleotide variants (2.0 mutations/Mb vs 7.9 mutations/Mb; P < .001). A statistically significantly higher proportion of germline ATM-mutated than ATM-wild-type GEJ adenocarcinoma patients underwent a curative resection (10 [100%] vs 92 [86.8%], P = .04; Fisher's exact test.), A synthetic lethal interaction between short-interfering RNA silencing of ATM and ATR was observed in the models analyzed., Conclusions: Our results indicate that germline pathogenic variants in ATM drive oncogenesis in GEJ adenocarcinoma and might result in a distinct clinical phenotype. Given the high prevalence of germline ATM-mutated GEJ adenocarcinomas, genetic testing for individuals with GEJ adenocarcinomas may be considered to better inform prognostication, treatment decisions, and future cancer risk., (© The Author(s) 2022. Published by Oxford University Press.)

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers., Significance: Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring. See related commentary by Liggett and Sankaran, p. 889. This article is highlighted in the In This Issue feature, p. 873., (©2021 American Association for Cancer Research.)

Background: Genetic testing for Li-Fraumeni syndrome (LFS) is performed by using blood specimens from patients selected based on phenotype-dependent guidelines. This approach is problematic for understanding the LFS clinical spectrum because patients with nonclassical presentations are missed, clonal hematopoiesis-related somatic blood alterations cannot be distinguished from germline variants, and unrelated tumors cannot be differentiated from those driven by germline TP53 defects., Methods: To provide insights into the LFS-related cancer spectrum, we analyzed paired tumor-blood DNA sequencing results in 17 922 patients with cancer and distinguished clonal hematopoiesis-related, mosaic, and germline TP53 variants. Loss of heterozygosity and TP53 mutational status were assessed in tumors, followed by immunohistochemistry for p53 expression on a subset to identify those lacking biallelic TP53 inactivation., Results: Pathogenic/likely pathogenic TP53 variants were identified in 50 patients, 12 (24.0%) of which were clonal hematopoiesis related and 4 (8.0%) of which were mosaic. Twelve (35.3%) of 34 patients with germline TP53 variants did not meet LFS testing criteria. Loss of heterozygosity of germline TP53 variant was observed in 96.0% (95% confidence interval [CI] = 79.7% to 99.9%) of core LFS spectrum-type tumors vs 45.5% (95% CI = 16.8% to 76.6%) of other tumors and 91.3% (95% CI = 72.0% to 98.9%) of tumors from patients who met LFS testing criteria vs 61.5% (95% CI = 31.6% to 86.1%) of tumors from patients who did not. Tumors retaining the wild-type TP53 allele exhibited wild-type p53 expression., Conclusions: Our results indicate that some TP53 variants identified in blood-only sequencing are not germline and a substantial proportion of patients with LFS are missed based on current testing guidelines. Additionally, a subset of tumors from patients with LFS do not have biallelic TP53 inactivation and may represent cancers unrelated to their germline TP53 defect., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Network analysis methods can potentially quantify cancer aberrations in gene networks without introducing fitted parameters or variable selection. A new network curvature-based method is introduced to provide an integrated measure of variability within cancer gene networks. The method is applied to high-grade serous ovarian cancers (HGSOCs) to predict response to immune checkpoint inhibitors (ICIs) and to rank key genes associated with prognosis. Copy number alterations (CNAs) from targeted and whole-exome sequencing data were extracted for HGSOC patients (n = 45) treated with ICIs. CNAs at a gene level were represented on a protein-protein interaction network to define patient-specific networks with a fixed topology. A version of Ollivier-Ricci curvature was used to identify genes that play a potentially key role in response to immunotherapy and further to stratify patients at high risk of mortality. Overall survival (OS) was defined as the time from the start of ICI treatment to either death or last follow-up. Kaplan-Meier analysis with log-rank test was performed to assess OS between the high and low curvature classified groups. The network curvature analysis stratified patients at high risk of mortality with p = 0.00047 in Kaplan-Meier analysis in HGSOC patients receiving ICI. Genes with high curvature were in accordance with CNAs relevant to ovarian cancer. Network curvature using CNAs has the potential to be a novel predictor for OS in HGSOC patients treated with immunotherapy., (© 2021. The Author(s).)

Objective: Since the approval of pembrolizumab for advanced or recurrent PD-L1 positive (CPS > 1%) cervical cancer, the clinical characteristics associated with response have remained undefined. We sought to characterize the clinicopathologic features of patients with advanced cervical cancer at our institution who derived durable clinical benefit from treatment with pembrolizumab., Methods: We conducted a retrospective cohort study of 14 patients with recurrent or metastatic cervical cancer who received pembrolizumab monotherapy from August 2017 to November 2019 and were followed until November 1, 2020. Reviewed clinical data included age, histology, tumor molecular profiling results, stage at diagnosis, treatment history, baseline pattern of metastatic disease at initiation of anti-PD-1 therapy, and outcomes. Treatment response was evaluated by computed tomography using RECIST v1.1 criteria., Results: The objective response rate was 21% (n = 3), including two partial responses and one complete response. Two patients (14%) had stable disease of six months or greater, for an observed durable clinical benefit rate of 36%. When stratified by those who derived clinical benefit, metastatic spread to lung and/or lymph node only at baseline was associated with improved response to pembrolizumab (n = 7, p = 0.02) and associated with significantly improved PFS and OS. Tumor mutational burden was higher in those with durable clinical benefit compared to non-responders (median 12.7 vs. 3.5 mutations/megabase, p = 0.03)., Conclusions: Our findings highlight clinical features that may select for a population most likely to benefit from pembrolizumab monotherapy and underscores the need for identification of additional biomarkers of response., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier Inc.)

Objectives: To determine if the mutational landscapes and genomic features of homologous recombination DNA repair defects (HRD) vary between younger and older patients with high-grade serous ovarian cancer (HGSOC)., Methods: Younger and older women were defined as bottom and top age quartiles, respectively. HGSOCs from 15 younger (median 49 years, range 35-53) and 15 older women (median 72 years, range 70-87) were subjected to whole-exome sequencing (WES). For validation, HGSOC WES data were obtained from The Cancer Genome Atlas (TCGA), including 38 younger (median 45 years, range 34-50) and 30 older women (median 74 years, range 68-84). Mutational profiles, BRCA1/2 status, genomic HRD features, and for TCGA cases RNA-sequencing-based HRD transcriptomic signatures were assessed., Results: In the institutional cohort, pathogenic germline BRCA1/2 mutations were more frequent in younger (5/15) than older women (0/15, p = 0.042). No somatic BRCA1/2 mutations were identified. HGSOCs from older patients preferentially displayed aging-related mutational signatures and, in contrast to younger patients, harbored CCNE1 amplifications (3/15, 20%). In the TCGA cohort, pathogenic germline BRCA1 (younger 8/38, older 0/30, p = 0.007) but not BRCA2 mutations (young 3/38, older 4/30, p = 0.691) were more frequent in younger patients. Again, no somatic BRCA1/2 mutations were identified. HGSOCs from younger women more frequently displayed genomic features of HRD (all, p < 0.05), a significant HRD gene-signature enrichment, but less frequently CCNE1 amplification (p = 0.05). Immunoreactive CLOVAR subtypes were more common in HGSOCs from younger women, and proliferative subtypes in HGSOCs from older women (p = 0.041)., Conclusions: HGSOC patients diagnosed at an older age less frequently harbor pathogenic BRCA1 germline mutations and genomic features of HRD than younger women. Individualized treatment options, particularly pertaining to use of PARP inhibitors, in older women may be warranted., Competing Interests: Declaration of Competing Interest N.R. reports research support/ grants from Pfizer, BMS and REPARE Therapeutics as well as honoraria from REPARE Therapeutics and Illumina, outside the scope of this study. D.S.C. reports membership of the medical advisory boards and stock options of Bovie Medical Co (now Apyx Medical) and Verthermia Inc., Chief Editor and Shareholder of C Surgeries, and ad hoc membership of the medical advisory board of Biom ‘Up, outside the scope of the submitted work. N.R.A.-R. reports institutional grants from GRAIL and Stryker, outside the scope of this study. D.Z. reports personal/consultancy fees from Merck, Genentech, Synlogic Therapeutics, Biomed Valley Discoveries, Trieza Therapeutics, Tesaro, and Agenus, and institutional research support from Genentech, Astra Zeneca, and Plexxikon, all outside of the scope of the submitted work. J.S.R.-F. reports receiving personal/consultancy fees and is a member of the scientific advisory board of Repare Therapeutics. J.S.R.-F. also declares receiving personal/consultancy fees from Goldman Sachs and Paige.AI, membership of the scientific advisory boards of VolitionRx and Paige.AI, membership of the Board of Directors of Grupo Oncoclinicas, and ad hoc membership of the scientific advisory boards of Roche Tissue Diagnostics, Ventana Medical Systems, Novartis, Genentech and InVicro, all outside the scope of this study. B.W. reports membership of the ad hoc scientific advisory board of Repare Therapeutics, outside the scope of the submitted work. The remaining authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER + ) patients with breast cancer. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2, and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacologic inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA -mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance. SIGNIFICANCE: These findings show that genetic lesions of multiple negative regulators of mTORC1 could limit the efficacy of PI3Kα inhibitors in breast cancer, which may guide patient selection strategies for future clinical trials., (©2021 American Association for Cancer Research.)

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER + ) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response., Competing Interests: Declaration of Interests J.L.V.M. is an employee at Inzen Therapeutics. M.Sallaku has received funds from Puma Biotechnology, AstraZeneca, Daiichi-Sankio, Immunomedics, Targimmune, and Menarini Ricerche, is a cofounder of Medendi.org and is on the advisory board of Menarini Ricerche. J.B. is an employee and shareholder of AstraZeneca, Board of Directors member of Foghorn Therapeutics, and is a past board member of Varian Medical Systems, Bristol-Myers Squibb, Grail, Aura Biosciences, and Infinity Pharmaceuticals. He has performed consulting and/or advisory work for Grail, PMV Pharma, ApoGen, Juno, Lilly, Seragon, Novartis, and Northern Biologics. He has stock or other ownership interests in PMV Pharma, Grail, Juno, Varian, Foghorn, Aura, Infinity, ApoGen, as well as Tango and Venthera, of which he is a co-founder. He has previously received Honoraria or Travel Expenses from Roche, Novartis, and Lilly. C.L.S. serves on the board of directors of Novartis, is a co-founder of ORIC Pharm and co-inventor of enzalutamide and apalutamide. He is a science advisor to Agios, Beigene, Blueprint, Column Group, Foghorn, Housey Pharma, Nextech, KSQ, Petra, and PMV. He was a co-founder of Seragon (purchased by Genentech/Roche, 2014). J.S.R.-F. is a consultant of Goldman Sachs and REPARE Therapeutics, a member of the Scientific Advisory Board of VolitionRx and Paige.AI, and an ad hoc member of the Scientific Advisory Board of Ventana Medical Systems, Roche, Genentech, Novartis, and InviCRO. C.S.V. has been the recipient of research grants from Ipsen Pharmaceuticals, MSD International, and Proctor & Gamble. E.T. has received Honoraria from AstraZeneca for invited lectures. P.R. has received consultation fees from Novartis, AstraZeneca, Foundation Medicine and institutional research funds from Grail, Novartis and Illumina. W.R.K. is a patent holder with KNAW, Organoid Technology shared with Hans Clevers.S.Kannan and C.S.V. are founder directors of SiNOPSEE Therapeutics and Aplomex. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The BRCA1-PALB2-BRCA2 axis, or the BRCA pathway, plays key roles in genome stability maintenance and suppression of breast and several other cancers. Due to frequent p53 mutations in human BRCA1 breast cancers and mouse mammary tumors from Brca1 , Brca2 and Palb2 conditional knockout models, it is often thought that p53 inactivation accelerates BRCA1/2 and PALB2-associated tumorigenesis. Here, we studied tumor development in mice with a mutation in Palb2 that disengages the PALB2-BRCA1 interaction in different Trp53 backgrounds. Rather than mammary tumors, Palb2 and Trp53 compound mutant mice developed, with greatly reduced latencies, lymphomas and sarcomas that are typically associated with germline Trp53 inactivation. Whole exome sequencing failed to identify any significant differences in genomic features between the same tumor types of Trp53 single mutant and Palb2;Trp53 compound mutant mice. These results suggest that loss of the BRCA pathway accelerates p53-associated tumor development, possibly without altering the fundamental tumorigenic processes., (© 2020 Chongqing Medical University. Production and hosting by Elsevier B.V.)

Aims: Metaplastic breast carcinoma (MBC) is a rare type of triple-negative breast cancer that shows vast histological and genetic heterogeneity. Osseous differentiation can be found in different subtypes of MBC. Whether MBCs with osseous differentiation are underpinned by specific genetic alterations has yet to be defined. The aim of this study was to investigate the repertoire of somatic mutations and copy number alterations (CNAs) in three MBCs with extensive osseous differentiation., Methods and Results: Tumour and normal DNA samples from three MBCs with extensive osseous differentiation were subjected to whole-exome sequencing. Somatic mutations, CNAs and mutational signatures were determined by use of a validated bioinformatics pipeline. Our analyses revealed clonal TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele coupled with mutations affecting genes related to the Wnt and/or the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathways in all cases analysed. All cases showed a dominant mutational signature 1, with two cases showing a secondary signature 3 in addition to other features of homologous recombination DNA repair defects. The oncostatin M receptor gene, which plays a role in mesenchymal differentiation and bone formation, was found to be mutated in two MBCs with extensive osseous differentiation and in none of 35 previously published 35 MBCs., Conclusion: Our findings suggest that MBCs with osseous differentiation have somatic mutations similar to those of other forms of MBC., (© 2020 John Wiley & Sons Ltd.)

Purpose: Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease., Experimental Design: Synchronous DCIS ( n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS ( n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome ( n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes ( n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods., Results: DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed., Conclusions: Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case., (©2020 American Association for Cancer Research.)

Purpose: Genomic methods can identify homologous recombination deficiency (HRD). Rigorous evaluation of their outcome association to DNA damage response-targeted therapies like platinum in pancreatic ductal adenocarcinoma (PDAC) is essential in maximizing therapeutic outcome., Experimental Design: We evaluated progression-free survival (PFS) and overall survival (OS) of patients with advanced-stage PDAC, who had both germline- and somatic-targeted gene sequencing. Homologous recombination gene mutations (HRm) were evaluated: BRCA1, BRCA2, PALB2, ATM, BAP1, BARD1, BLM, BRIP1, CHEK2, FAM175A, FANCA, FANCC, NBN, RAD50, RAD51, RAD51C , and RTEL1 HRm status was grouped as: (i) germline versus somatic; (ii) core ( BRCAs and PALB2) versus non-core (other HRm); and (iii) monoallelic versus biallelic. Genomic instability was compared using large-scale state transition, signature 3, and tumor mutation burden., Results: Among 262 patients, 50 (19%) had HRD (15% germline and 4% somatic). Both groups were analyzed together due to lack of difference in their genomic instability and outcome. Median [95% confidence interval (CI)] follow-up was 21.9 (1.4-57.0) months. Median OS and PFS were 15.5 (14.6-19) and 7 (6.1-8.1) months, respectively. Patients with HRD had improved PFS compared with no HRD when treated with first-line (1L) platinum [HR, 0.44 (95% CI: 0.29-0.67); P < 0.01], but not with 1L-non-platinum. Multivariate analysis showed HRD patients had improved OS regardless of their first-line treatment, but most had platinum exposure during their course. Biallelic HRm (11%) and core HRm (12%) had higher genomic instability, which translated to improved PFS on first-line platinum (1L-platinum) versus 1L-non-platinum., Conclusions: Pathogenic HRm identifies HRD in patients with PDAC with the best outcome when treated with 1L-platinum. Biallelic HRm and core HRm further enriched benefit from 1L-platinum from HRD., (©2020 American Association for Cancer Research.)

Purpose: Homologous DNA repair-deficient (HRD) ovarian cancers (OCs), including those with BRCA1/2 mutations, have higher levels of genetic instability, potentially resulting in higher immunogenicity, and have been suggested to respond better to immune checkpoint inhibitors (ICIs) than homologous DNA repair-proficient OCs. However, clinical evidence is lacking. The study aimed to evaluate the associations between BRCA1/2 mutations, HRD, and other genomic parameters and response to ICIs and survival in OC., Methods: This is a single-institution retrospective analysis of women with recurrent OC treated with ICIs. BRCA1/2 mutation status and clinicopathologic variables were abstracted from the medical records. Targeted and whole-exome sequencing data available for a subset of patients were used to assess tumor mutational burden (TMB), HRD, and fraction of genome altered (FGA). ICI response was defined as lack of disease progression for ≥ 24 weeks. Associations of BRCA1/2 status and genomic alterations with progression-free survival (PFS) and overall survival (OS) were determined using Cox proportional hazards models., Results: Of the 143 women treated with ICIs, 134 had known BRCA1/2 mutation status. Deleterious germline or somatic BRCA1/2 mutations were present in 31 women (24%). There was no association between presence of BRCA1/2 mutations and response ( P = .796) or survival. Genomic analysis in 73 women found no association between TMB ( P = .344) or HRD ( P = .222) and response, PFS, or OS. There were also no significant differences in somatic genetic alterations between responders and nonresponders. High FGA was associated with an improvement in PFS ( P = .014) and OS ( P = .01)., Conclusion: TMB, BRCA1/2 mutations, and HRD are not associated with response or survival, cautioning against their use as selection criteria for ICI in recurrent OC. FGA should be investigated further as a biomarker of response to immunotherapy in OC., Competing Interests: The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center. Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments). Alexia IasonosConsulting or Advisory Role: Mylan, Brightpath, IntelligenciaMargaret CallahanEmployment: Bristol Myers Squibb (I), Celgene (I), Kleo Pharmaceuticals (I) Consulting or Advisory Role: AstraZeneca, Moderna Therapeutics, Merck, Immunocore Research Funding: Bristol Myers Squibb (Inst) Other Relationship: Clinical Care Options, Potomac Center for Medical EducationIgnacio Vazquez-GarciaStock and Other Ownership Interests: CRISPR Therapeutics, Editas MedicineAhmet ZehirHonoraria: IlluminaRobert A. BurgerEmployment: Genentech Stock and Other Ownership Interests: Genentech Consulting or Advisory Role: AstraZeneca, Tesaro, Merck, Genentech, Morphotek, Myriad Genetics Research Funding: Incyte (Inst), Astra Zeneca (Inst), and Genzyme (Inst) Travel, Accommodations, Expenses: Tesaro, GenentechDaniel J. Powell JrStock and Other Ownership Interests: Atara Biotherapeutics, InsTIL Bio Consulting or Advisory Role: Neon Therapeutics, Iovance Biotherapeutics, Tmunity Therapeutics, InsTIL Bio, Bellicum Pharmaceuticals Research Funding: Eli Lilly, Tmunity Therapeutics, Incyte, Monojul, AstraZeneca/MedImmune Patents, Royalties, Other Intellectual Property: I hold patents in the field of chimeric antigen receptor (CAR) T-cell therapy in oncology and have received royalties related to their licensing to Novartis; I hold patents in the field of CAR T cell therapy in Oncology and have received royalties related to their licensing to Tmunity Travel, Accommodations, Expenses: Iovance BiotherapeuticsClaire FriedmanConsulting or Advisory Role: AstraZeneca/MedImmune Research Funding: Bristol Myers Squibb (Inst), Arcus Biosciences (Inst), Genentech (Inst), Merck (Inst) Other Relationship: Guidepoint Global Uncompensated Relationships: Genentech, Merck Open Payments Link: https://openpaymentsdata.cms.gov/physician/477023/summaryKaren CadooHonoraria: OncLive Consulting or Advisory Role: GSK Tesaro Research Funding: AstraZeneca (Inst), Syndax (Inst) Travel, Accommodations, Expenses: AstraZenecaRachel GrishamConsulting or Advisory Role: Mateon Therapeutics, Clovis Oncology, Regeneron Research Funding: Context Therapeutics (Inst) Travel, Accommodations, Expenses: EMD Serono Other Relationship: Prime Oncology, MCM Education, OncLiveJason A. KonnerConsulting or Advisory Role: Immunogen, AstraZeneca, Tesaro, AbbVie Research Funding: Genentech Travel, Accommodations, Expenses: AstraZenecaRoisin E. O'CearbhaillConsulting or Advisory Role: Tesaro, GlaxoSmithKline Research Funding: Juno Therapeutics (Inst), Sellas Life Sciences (Inst), Ludwig Institute for Cancer Research (Inst), Stem CentRx (Inst), TapImmune (Inst), TCR2 Therapeutics (Inst), Regeneron (Inst), Genmab (Inst)Carol AghajanianConsulting or Advisory Role: Tesaro, Mersana, Eisai, Roche Research Funding: Genentech (Inst), AbbVie (Inst), Clovis Oncology (Inst), AstraZeneca (Inst), Clovis Oncology (Inst), AstraZeneca (Inst)Jorge S. Reis-FilhoConsulting or Advisory Role: Genentech, Invicro, Ventana Medical Systems, Volition RX, Paige AI, Goldman Sachs, Novartis, Repare TherapeuticsBritta WeigeltConsulting or Advisory Role: Genentech (I), Invicro (I), Ventana Medical Systems (I), Volition RX (I), Page AI (I), Goldman Sachs (I), Repare Therapeutics (I)Dmitriy ZamarinConsulting or Advisory Role: Merck, Synlogic, Western Oncolytics, Tesaro, Agenus, Trieza Therapeutics, ACM Biolabs Research Funding: Genentech (Inst) Patents, Royalties, Other Intellectual Property: I hold a patent regarding the use of recombinant Newcastle disease virus (NDV) for cancer therapy (Inst) No other potential conflicts of interest were reported., (© 2020 by American Society of Clinical Oncology.)

Aims . The aim of this study was to identify potential driver genetic alterations in a dedifferentiated liposarcoma (DDLPS) with rhabdomyosarcomatous differentiation. Methods and Results . A 24-year-old female underwent resection of an abdominal mass, which on a previous biopsy demonstrated rhabdomyosarcomatous differentiation concerning for embryonal rhabdomyosarcoma. Histologically the resected tumor displayed a high-grade sarcoma with rhabdomyosarcomatous differentiation in the background of well-differentiated liposarcoma consistent with DDLPS. Fluorescence in situ hybridization confirmed MDM2 amplification, as did array-based copy number profiling. Whole-exome sequencing revealed a somatic FGFR1 hotspot mutation and RNA sequencing an LMNB2-MAP2K6 fusion only within the dedifferentiated component. Conclusions . This study represents an in-depth examination of a rare DDLPS with rhabdomyosarcomatous differentiation in a young individual. Additionally, it is also instructive of a potential pitfall when assessing for MDM2 amplification in small biopsies. Despite exhaustive analysis, mutation and gene copy number analysis did not identify any molecular events that would underlie the rhabdomyoblastic differentiation. Our understanding of what causes some tumors to dedifferentiate as well as undergo divergent differentiation is limited, and larger studies are needed.

Aims: Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs., Methods and Results: Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n = 21) or Sanger sequencing (n = 5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n = 7) and Q61K (n = 2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n = 17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P < 0.001), necrosis (P < 0.01) and mitotic index in the epithelial (P < 0.05) and myoepithelial (P < 0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2)., Conclusions: IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Forkhead box A1 (FOXA1) is a pioneer factor that facilitates chromatin binding and function of lineage-specific and oncogenic transcription factors. Hyperactive FOXA1 signaling due to gene amplification or overexpression has been reported in estrogen receptor-positive (ER + ) endocrine-resistant metastatic breast cancer. However, the molecular mechanisms by which FOXA1 up-regulation promotes these processes and the key downstream targets of the FOXA1 oncogenic network remain elusive. Here, we demonstrate that FOXA1 overexpression in ER + breast cancer cells drives genome-wide enhancer reprogramming to activate prometastatic transcriptional programs. Up-regulated FOXA1 employs superenhancers (SEs) to synchronize transcriptional reprogramming in endocrine-resistant breast cancer cells, reflecting an early embryonic development process. We identify the hypoxia-inducible transcription factor hypoxia-inducible factor-2α (HIF-2α) as the top high FOXA1-induced SE target, mediating the impact of high FOXA1 in activating prometastatic gene sets and pathways associated with poor clinical outcome. Using clinical ER + /HER2 - metastatic breast cancer datasets, we show that the aberrant FOXA1/HIF-2α transcriptional axis is largely nonconcurrent with the ESR1 mutations, suggesting different mechanisms of endocrine resistance and treatment strategies. We further demonstrate the selective efficacy of an HIF-2α antagonist, currently in clinical trials for advanced kidney cancer and recurrent glioblastoma, in reducing the clonogenicity, migration, and invasion of endocrine-resistant breast cancer cells expressing high FOXA1. Our study has uncovered high FOXA1-induced enhancer reprogramming and HIF-2α-dependent transcriptional programs as vulnerable targets for treating endocrine-resistant and metastatic breast cancer.

Aims: Acinic cell carcinoma (ACC) of the breast is a rare histological form of triple-negative breast cancer (TNBC). Despite its unique histology, targeted sequencing analysis has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here we subjected three breast ACCs to whole-exome and RNA sequencing to determine whether they would harbour a pathognomonic genetic alteration., Methods and Results: DNA and RNA samples from three breast ACCs were subjected to whole-exome sequencing and RNA-sequencing, respectively. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined with state-of-the-art bioinformatics methods. Our analyses revealed TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele in two cases. Mutations affecting homologous recombination DNA repair-related genes were found in two cases, and an MLH1 pathogenic germline variant was found in one case. In addition, copy number analysis revealed the presence of a somatic BRCA1 homozygous deletion and focal amplification of 12q14.3-12q21.1, encompassing MDM2, HMGA2, FRS2, and PTPRB. No oncogenic in-frame fusion transcript was identified in the three breast ACCs analysed., Conclusions: No pathognomonic genetic alterations were detected in the breast ACCs analysed. These tumours have somatic genetic alterations similar to those of common forms of TNBC, and may show homologous recombination deficiency or microsatellite instability. These findings provide further insights into why breast ACCs, which are usually clinically indolent, may evolve into or in parallel with high-grade TNBC., (© 2019 John Wiley & Sons Ltd.)

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.

Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer (BC) characterized by tumor cells floating in lakes of mucin. We assessed the genomic landscape of 32 MCBs by whole-exome sequencing and/or RNA-sequencing. GATA3 (23.8%), KMT2C (19.0%), and MAP3K1 (14.3%) were the most frequently mutated genes in pure MCBs. In addition, two recurrent but not pathognomonic fusion genes, OAZ1-CSNK1G2 and RFC4-LPP, were detected in 3/31 (9.7%) and 2/31 (6.5%) samples, respectively. Compared with ER-positive/HER2-negative common forms of BC, MCBs displayed lower PIK3CA and TP53 mutation rates and fewer concurrent 1q gains and 16q losses. Clonal decomposition analysis of the mucinous and ductal components independently microdissected from five mixed MCBs revealed that they are clonally related and evolve following clonal selection or parallel evolution. Our findings indicate that MCB represents a genetically distinct ER-positive/HER2-negative form of BC., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: Micropapillary variant of mucinous carcinoma of the breast (MPMC) is a rare histological form of oestrogen receptor (ER)-positive invasive carcinoma that is characterised by micropapillary clusters of tumour cells in lakes of extracellular mucin. The aims of this study were to determine the genetic alterations underpinning MPMCs, and to determine whether they overlap with those of mucinous carcinomas and/or invasive micropapillary carcinomas., Methods and Results: DNA from five MPMCs was subjected to whole-exome sequencing. Somatic mutations, copy number alterations and mutational signatures were determined with state-of-the-art bioinformatics methods. No mutations in genes significantly mutated in breast cancer, including TP53, PIK3CA, GATA3, and MAP3K1, were detected. We identified copy number alterations that have been reported in invasive micropapillary carcinomas, such as recurrent gains in 1q, 6p, 8q, and 10q, and recurrent losses in 16q, 11q, and 13q, as well as a recurrent 8p12-8p11.2 amplification encompassing FGFR1. Like mucinous carcinomas, three of the five MPMCs analysed lacked PIK3CA mutations, 1q gains, and 16q losses, which are the hallmark genetic alterations of ER-positive breast cancers, whereas two MPMCs harboured 16q losses and/or a complex pattern of copy number alterations similar to those found in breast-invasive micropapillary carcinomas., Conclusions: MPMCs are heterogeneous at the genetic level; some tumours show a pattern of somatic genetic alterations similar to those of mucinous carcinomas, whereas others resemble invasive micropapillary carcinomas at the genetic level. These findings suggest that MPMCs may not constitute one histological subtype, but rather a convergent phenotype that can stem from mucinous carcinomas or invasive micropapillary carcinomas., (© 2019 John Wiley & Sons Ltd.)

Aims: Melanocytic naevi are benign lesions of the skin or mucosa that may constitute non-obligate precursors of malignant melanoma, particularly when they show lentiginous and dysplastic features. The aim of this study was to investigate the repertoire of somatic genetic alterations in melanocytic naevi., Methods and Results: DNA extracted from 12 melanocytic naevi and DNA from matching normal tissue were separately microdissected and subjected to targeted massively parallel sequencing of ≥300 cancer genes. A median of 5.5 (range 1-12) non-synonymous somatic mutations were detected, with 10 cases harbouring mutually exclusive BRAF V600E (6/12) or NRAS (4/12) clonal hotspot mutations. One of the two cases lacking BRAF and NRAS mutations was a dysplastic naevus harbouring an HRAS Q61L hotspot mutation. Analysis of the laser-capture microdissected components of a naevus synchronously diagnosed with in-situ and invasive malignant melanoma revealed a truncal, clonal BRAF V600E mutation, and the acquisition of a CDKN2A homozygous deletion in the invasive component, in conjunction with additional clonal mutations affecting NF2, FAT4 and KDR in both in-situ and invasive malignant components., Conclusion: Melanocytic naevi harbour recurrent BRAF V600E or NRAS hotspot mutations with low mutational burdens. Our findings also show that progression from naevi to malignant melanoma may be driven by the acquisition of additional genetic alterations, including CDKN2A homozygous deletions., (© 2019 John Wiley & Sons Ltd.)

Pathogenic germline variants in checkpoint kinase 2 ( CHEK2 ), which plays pivotal roles in DNA damage response and cell cycle regulation, confer an increased breast cancer (BC) risk. Here, we investigated the phenotypic and genomic characteristics of 33 BCs from CHEK2 germline mutation carriers (16 high-risk variants and 17 low-risk p.Ile157Thr variants). CHEK2 -associated BCs from patients with high-risk germline variants were largely hormone receptor-positive (87%, 13/15), and 81% (13/16) exhibited loss of heterozygosity (LOH) of the CHEK2 wild-type allele. Conversely, CHEK2 -associated BCs from patients with the low-risk p.Ile157Thr variant displayed less-frequent loss of heterozygosity (5/17, 29%) and higher levels of CHEK2 protein expression than those with high-risk germline variants. CHEK2 -associated BCs lacked a dominant mutational signature 3, a genomics feature of homologous recombination DNA repair deficiency (HRD). Our findings indicate that CHEK2 -associated BCs are generally hormone receptor-positive and lack HRD-related mutational signatures, recapitulating the features of ATM -associated BCs. Specific CHEK2 germline variants may have a distinct impact on tumor biology.

Aims: Low-grade serous carcinomas (LGSCs) and their precursors serous borderline tumours (SBTs) characteristically harbour mutations in BRAF, KRAS or NRAS but rarely in TP53, whereas high-grade serous carcinomas (HGSCs) are characterised by frequent TP53 mutations but rare BRAF, KRAS or NRAS mutations. In a small subset of cases, LGSCs and/or SBTs develop into high-grade tumours, including HGSCs and poorly differentiated carcinomas (PDCs). Here, we sought to define the repertoire of somatic genetic alterations in low-grade serous tumours and synchronous or metachronous high-grade adnexal carcinomas., Methods and Results: DNA extracted from five SBTs/LGSCs and synchronous or metachronous HGSCs/PDCs and matched normal tissue was subjected to massively parallel sequencing targeting all exons and selected non-coding regions of 341 cancer-related genes. The low-grade and high-grade tumours from a given case were related, and shared mutations and copy number alterations. Progression from low-grade to high-grade lesions was observed, and involved the acquisition of additional mutations and/or copy number alterations, or shifts from subclonal to clonal mutations. Only two (an HGSC and a PDC) of the five high-grade tumours investigated harboured TP53 mutations, whereas NRAS and KRAS hotspot mutations were seen in two HGSCs and one HGSC, respectively., Conclusions: Our results suggest that progression from SBT to HGSC may take place in a subset of cases, and that at least some of the rare HGSCs lacking TP53 mutations may be derived from a low-grade serous precursor., (© 2018 John Wiley & Sons Ltd.)

Breast adenomyoepitheliomas (AMEs) are rare epithelial-myoepithelial neoplasms that may occasionally produce myxochondroid matrix, akin to pleomorphic adenomas (PAs). Regardless of their anatomic location, PAs often harbor rearrangements involving HMGA2 or PLAG1 . We have recently shown that the repertoire of somatic genetic alterations of AMEs varies according to their estrogen receptor (ER) status; whilst the majority of ER-positive AMEs display mutually exclusive PIK3CA or AKT1 hotspot mutations, up to 60% of ER-negative AMEs harbor concurrent HRAS Q61 hotspot mutations and mutations affecting either PIK3CA or PIK3R1 . Here, we hypothesized that a subset of AMEs lacking these somatic genetic alterations could be underpinned by oncogenic fusion genes, in particular those involving HMGA2 or PLAG1 . Therefore, we subjected 13 AMEs to RNA-sequencing for fusion discovery ( n  = 5) and/or fluorescence in situ hybridization (FISH) analysis for HMGA2 and PLAG1 rearrangements ( n  = 13). RNA-sequencing revealed an HMGA2 - WIF1 fusion gene in an ER-positive AME lacking HRAS, PIK3CA and AKT1 somatic mutations. This fusion gene, which has been previously described in salivary gland PAs, results in a chimeric transcript composed of exons 1-5 of HMGA2 and exons 3-10 of WIF1 . No additional in-frame fusion genes or HMGA2 or PLAG1 rearrangements were identified in the remaining AMEs analyzed. Our results demonstrate that a subset of AMEs lacking mutations affecting HRAS and PI3K pathway-related genes may harbor HMGA2 - WIF1 fusion genes, suggesting that a subset of breast AMEs may be genetically related to PAs or that a subset of AMEs may originate in the context of a PA., Competing Interests: J.S.R.-F. reports personal/consultancy fees from VolitionRx, Page.AI, Goldman Sachs, Grail, Ventana Medical Systems and Genentech, outside the submitted work. The remaining authors declare no competing interests.

Purpose: Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers. Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods., Results: WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases., Conclusions: Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions., (©2018 American Association for Cancer Research.)

Cyclin dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) are effective in breast cancer; however, drug resistance is frequently encountered and poorly understood. We conducted a genomic analysis of 348 estrogen receptor-positive (ER + ) breast cancers treated with CDK4/6i and identified loss-of-function mutations affecting FAT1 and RB1 linked to drug resistance. FAT1 loss led to marked elevations in CDK6, the suppression of which restored sensitivity to CDK4/6i. The induction of CDK6 was mediated by the Hippo pathway with accumulation of YAP and TAZ transcription factors on the CDK6 promoter. Genomic alterations in other Hippo pathway components were also found to promote CDK4/6i resistance. These findings uncover a tumor suppressor function of Hippo signaling in ER + breast cancer and establish FAT1 loss as a mechanism of resistance to CDK4/6i., (Copyright © 2018 Elsevier Inc. All rights reserved.)