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Preferred first-line treatment for patients with metastatic EGFR-mutant lung cancer is osimertinib, yet it is not known if patient outcomes may be improved by identifying and intervening upon molecular markers associated with therapeutic resistance.All patients with metastatic EGFR-mutant lung cancer treated with first-line osimertinib at Memorial Sloan Kettering Cancer Center (n=327) were identified. Available pre-treatment and post-progression tumor samples underwent targeted gene panel sequencing and mutational signature analysis using SigMA algorithm. Progression-free and overall survival were estimated using Kaplan Meier methods.Using multivariate analysis, baseline atypical EGFR (mPFS 5.8 mo, p0.001) and concurrent TP53/RB1 alterations (mPFS 10.5 mo, p=0.015) were associated with shorter progression-free survival on first-line osimertinib. Of 95 patients with post-progression biopsies, acquired resistance mechanisms were identified in 52% (off-target n=24, histological transformation n=14, on-target n=12), with MET amplification (n=9), small cell lung transformation (n=7) and acquired EGFR amplification (n=7) the most frequently identified mechanisms. While there was no difference in post-progression survival based on identified resistance (p=0.07), patients with subsequent second-line therapy tailored to post-progression biopsy results had improved post-progression survival (HR 0.09, p=0.006). Paired post-progression tumors had higher tumor mutational burden (p=0.008) and further dominant APOBEC mutational signatures (p=0.07) compared to pre-treatment samples.Patients with EGFR-mutant lung cancer treated with first-line osimertinib have improved survival with treatment adaptation based on identified mechanisms of resistance at time of progression using tissue-based genomic analysis. Further survival gains may be achieved using risk-based treatment adaptation of pre-treatment genomic alterations.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. Significance: Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.

We describe a case of a 46-year-old woman diagnosed with localized PTC 20 years ago, having already undergone several treatments with iodine-131 and then treatment with lenvatinib for metastatic disease, to which she developed intolerance. In 2020, in addition to pleural, thoracic, and abdominal lymph node metastasis, progression with symptomatic vertebral bone metastasis was detected, which led to the equation of new therapeutic options. In this context, a genetic/molecular test was carried out, which identified the BRAF V600E mutation and enabled the start of treatment with dabrafenib/trametinib since June 2020. This treatment allowed functional gain, symptomatic relief, and stabilization of the disease. It demonstrates how, in rare tumors, the personalized medicine approach can bring new treatment possibilities.

Background: Clinical trials reported 25-30% pathologic complete response (pCR) rates in HER2+ breast cancer (BC) patients 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. Patients and Methods: Baseline HER2+ BC specimens from TBCRC023 and PAMELA trials of neoadjuvant lapatinib+trastuzumab (plus endocrine therapy in ER+ tumors) were used. 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 BCs 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 and %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 de-escalation. 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 to chemotherapy plus dual anti-HER2 therapy in unselected patients.

Artificial intelligence; Precision oncology Intel·ligència artificial; Oncologia de precisió Inteligencia artificial; Oncología de precisión R.P.L. is supported by LaCaixa Foundation, a CRIS Foundation Talent Award (TALENT19-05), the FERO Foundation, the Instituto de Salud Carlos III-Investigacion en Salud (PI18/01395 and PI21/01019), the Prostate Cancer Foundation (18YOUN19) and the Asociación Española Contra el Cancer (AECC) (PRYCO211023SERR, funding C.M.). J.N.K. is supported by the German Federal Ministry of Health (DEEP LIVER, ZMVI1-2520DAT111) and the Max-Eder-Program of the German Cancer Aid (grant #70113864), the German Federal Ministry of Education and Research (PEARL, 01KD2104C; CAMINO, 01EO2101; SWAG, 01KD2215A; TRANSFORM LIVER, 031L0312A; TANGERINE, 01KT2302 through ERA-NET Transcan), the German Academic Exchange Service (SECAI, 57616814), the German Federal Joint Committee (Transplant.KI, 01VSF21048) the European Union’s Horizon Europe and innovation program (ODELIA, 101057091; GENIAL, 101096312) and the National Institute for Health and Care Research (NIHR, NIHR213331) Leeds Biomedical Research Centre. JSR-F is funded in part by the Breast Cancer Research Foundation, a Susan G Komen Leadership grant, an NIH/NCI P50 CA247749 01 grant, and an NIH/NCI grant No. P30CA008748. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care.

Poorly differentiated pancreatic neuroendocrine tumors (PDNEC), are a subtype of pancreatic cancer encompassing both small cell and large cell neuroendocrine carcinoma subtypes, and are characterized as distinct in terms of biology and prognosis compared to the more common pancreatic adenocarcinoma. Until recently, there has been a paucity of data on the genomic features of this cancer type. We describe a male patient diagnosed with PDNEC and extensive metastatic disease in the liver at diagnosis. Genomic analysis demonstrated a germline pathogenic variant in BRCA2 with somatic loss-of-heterozygosity of the BRCA2 wild-type allele. Following a favorable response to platinum-based chemotherapy (and the addition of immunotherapy), the patient received maintenance therapy with olaparib, which resulted in a further reduction on follow-up imaging (Fig. 1). After seventeen months of systemic control with olaparib, the patient developed symptomatic central nervous system metastases, which harboured a BRCA2 reversion mutation. No other sites of disease progression were observed. Herein, we report an exceptional outcome through the incorporation of a personalized management approach for a patient with a pancreatic PDNEC, guided by comprehensive genomic sequencing.

Breast cancer remains a highly prevalent disease with considerable inter- and intra-tumoral heterogeneity complicating prognostication and treatment decisions. The utilization and depth of genomic, transcriptomic and proteomic data for cancer has exploded over recent times and the addition of spatial context to this information, by understanding the correlating morphologic and spatial patterns of cells in tissue samples, has created an exciting frontier of research, histo-genomics. At the same time, deep learning (DL), a class of machine learning algorithms employing artificial neural networks, has rapidly progressed in the last decade with a confluence of technical developments - including the advent of modern graphic processing units (GPU), allowing efficient implementation of increasingly complex architectures at scale; advances in the theoretical and practical design of network architectures; and access to larger datasets for training - all leading to sweeping advances in image classification and object detection. In this review, we examine recent developments in the application of DL in breast cancer histology with particular emphasis of those producing biologic insights or novel biomarkers, spanning the extraction of genomic information to the use of stroma to predict cancer recurrence, with the aim of suggesting avenues for further advancing this exciting field.

OBJECTIVE: To determine the effect of hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin on the transcriptomic profiles of normal and ovarian cancer (OC) tissues. METHODS: Normal and tumor samples from four OCs were prospectively collected pre- and immediately post-HIPEC treatment and subjected to RNA-sequencing. Differential gene expression, gene ontology enrichment and pathway analyses were performed. Heat shock protein and immune-response protein expression was assessed using protein arrays and western blotting. RESULTS: RNA-sequencing revealed 4,231 and 322 genes significantly differentially expressed between pre- and post-treatment normal and OC tissues, respectively (both adjusted p-value

Background: Germline genetic alterations are established mediators of breast carcinogenesis, often giving rise to specific forms of genomic instability. BRCA1/2 pathogenic variants (PVs) are emblematic of this phenomenon through their induction of homologous recombination deficiency. While specific patterns of genomic instability may sensitize cancers to therapies such as PARP inhibitors (PARPi) or platinum chemotherapy, their implications for lineage-directed therapies such as endocrine therapy (ET) or CDK4/6 inhibitors (CDK4/6i) are unknown. Herein, we systematically investigated the patterns of association of germline alterations with specific somatic alterations and explored the resulting effect on clinical outcomes. Methods: Patients who underwent germline and matched tumor tissue sequencing utilizing MSK-IMPACT from April 2014 to May 2021 and had available germline analysis results were included. The final analysis presented at SABCS will include 6000 tumors from 5,150 patients, anonymized according to established institutional IRB guidelines to allow for germline analysis on the full cohort. We analyzed genomic data to inform the full spectrum of somatic and germline mutations, ploidy, and allele-specific copy number to determine loss of heterozygosity (LOH). We performed gene- and pathway-level enrichment analyses between somatic variants and germline PVs. Univariable and multivariable Cox proportional hazards models were constructed to assess the association of therapy-specific progression-free survival (PFS) with select germline PVs and germline-somatic interactions. Results: The preliminary analysis includes 2,798 tumors from 2,242 patients with germline and somatic sequencing results. The most frequent germline PVs were: BRCA2 (n = 81), BRCA1 (n = 67), CHEK2 (n = 57), ATM (n = 32), PALB2 (n = 19). The cohort robustly confirmed previously established relationships such as mutual exclusivity of gATM and TP53 variants (OR 0.10, 95% CI 0.032 - 0.33, q = 0.005). Alterations of TP53 were seen in 83% (56/67) of gBRCA1 patients; however, this did not achieve significance when adjusted for receptor subtype (OR 3.90, 95% CI 1.34-11.38, q = 0.15). The size of the cohort allowed discovery of several novel relationships. For instance, gBRCA2 loss was associated with alterations in TGF-B pathway components (OR 3.58, 95% CI 1.70 - 7.56, q = 0.002), potentially relevant to metastatic disease progression. PIK3CA mutations were significantly less prevalent in both gBRCA2 (OR 0.52, 95% CI 0.31 - 0.87, q = 0.063) and gBRCA1 PVs (OR 0.21, 95% CI 0.085 - 0.51, q = 0.014). Our analysis uncovered a strong association between gBRCA2 and somatic RB1 pathogenic alterations (OR 3.58, 95% CI 1.70 - 7.56, q = 0.011), with most variants (80%) encountered in metastatic gBRCA2 tumors. Given the essential role of RB1 in CDK4/6i response, we investigated the effect of BRCA2 status on clinical efficacy of CDK4/6i-ET. Strikingly, gBRCA2 PVs were significantly associated with inferior PFS (HR 2.17, 95% CI 1.46-3.22, p < 0.001) on first line treatment with CDK4/6i-ET. We posited the enrichment of somatic RB1 loss as a potential mechanism of resistance to CDK4/6i. Given the proximity of RB1 to BRCA2 on chromosome 13, we hypothesized that co-LOH of BRCA2 and RB1 predisposes the cancer cells to bi-allelic loss under therapeutic pressure of CDK4/6i. Indeed, 18/26 gBRCA2 (69.2%) tumors evaluable for allele-specific copy number had evidence of RB1 LOH. Discussion: Analysis of germline-somatic interactions yielded novel associations relevant to breast cancer progression and treatment resistance. Among these, we demonstrated BRCA2 carriers to have inferior outcomes to first line CDK4/6i-ET with potential implications for optimal first line therapy and sequencing of CDK4/6i vs PARPi in this patient population. Citation Format: Anton Safonov, Chai Bandlamudi, Paulino Tallón de Lara, Emanuela Ferraro, Fatemeh Derakhshan, Marie Will, Mark Donoghue, Pier Selenica, Joshua Drago, Ezra Rosen, Carlos dos Anjos, Elaine Walsh, Elizabeth A Comen, Mehnaj Ahmed, Barbara Acevedo, Ahmet Zehir, Michael F Berger, David Solit, Larry Norton, Ronglai Shen, Zsofia Stadler, Simon Powell, Jorge S Reis-Filho, Sarat Chandarlapaty, Mark Robson, Pedram Razavi. Comprehensive genomic profiling of patients with breast cancer identifies germline-somatic interactions mediating therapy resistance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS4-08.

Background: Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes drive genomic instability in breast cancer (BC) characterized by specific single base substitution mutation signatures (COSMIC S2 and S13). Computational algorithms have the potential to derive signatures from targeted sequencing assays with relatively small genomic footprints. We sought to develop a computational approach to identify APOBEC mutagenesis in samples subjected to targeted sequencing and to characterize the genomic and clinical features of APOBEC-positive BC. Methods: We retrieved clinical and genomic data from 4,595 consecutive BCs that had been subjected to targeted sequencing using the FDA-approved MSK-IMPACT assay. Additional whole exome sequencing (WES) data were retrieved from publicly available datasets, including TCGA (primary BC, n=1019) and Bertucci et al. cohorts (metastatic BC, n=617). APOBEC-related mutational signatures were computed by using the SigMA algorithm (Gulhan, Nat Genet 2019) for MSK-IMPACT samples with at least 5 single nucleotide variants (SNV) (n=2,983). Survival analyses were performed by using Kaplan Meier method with log-rank test and Cox proportional-hazards models. Results: A significant positive correlation between APOBEC exposure calculated by SigMA vs other computational tools (e.g., DeconstructSig, SigProfiler, MutationalPatterns) was found (r=0.99). After down-sampling the WES data from the TCGA BC dataset to the genomic footprint of MSK-IMPACT, SigMA showed high concordance in determining APOBEC exposures (r=0.78). BCs with high APOBEC exposure were enriched for pathogenic somatic mutations affecting several genes, including PIK3CA, CDH1 and GATA3 (q0.05), overall and in different BC subtypes. We also assessed the association between APOBEC and response to endocrine therapy (ET). Patients with metastatic HR+ BC and APOBEC as dominant signature had a statistically significant lower progression-free survival (PFS) on first-line single-agent ET (aromatase inhibitors or SERDs; median PFS of 9.7 versus 14.7 months, p30% of metastatic breast cancers and associated with resistance to ET. Unlike ESR1 mutations, which are acquired as a late event in metastatic HR+ BCs, APOBEC mutagenesis is commonly present in paired primary and metastatic BCs, indicating its relatively early onset and potential role in driving the poor prognosis of these cancers. Citation Format: Antonio Marra, Andrea Gazzo, Pier Selenica, Xin Pei, Avantika Gupta, Fresia Pareja, Giuseppe Curigliano, Reuben Harris, Nadeem Riaz, Jorge S. Reis-Filho, Sarat Chandarlapaty. Apobec mutagenesis is a pervasive feature of poor prognosis breast cancer associating with ESR1 wild type, endocrine resistant disease [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-06.

Background: Cancer genomes provide a durable record of the genetic alterations that are acquired during normal cell development and carcinogenesis from DNA damage and DNA repair defects. As DNA repair-deficient tumors often become dependent on backup repair pathways, mutational signatures found in such tumors are thought to reflect the absence of a particular repair pathway as well as the activity of the backup repair mechanism responsible for maintaining genome integrity. While homologous recombination (HR) deficiency is primarily a disorder of double-strand break (DSB) repair, the mutation classes most specifically associated with HR-deficiency in cancer are paradoxically small variants, namely single nucleotide variants and short deletions. Mechanistic and cytogenetic studies, however, indicate that HR-deficiency should compromise structural genomic integrity and yield complex rearrangements. Here we elucidate complex structural variants that are specific for HR-deficient cancers and identify rearrangements that differentiate BRCA1 from BRCA2 loss and illuminate divergent backup DNA repair mechanisms. Methods: To investigate the role of complex SVs in HR-deficient cancers, we assembled a cohort of 2,367 WGS profiles from four tumor types (breast, ovarian, prostate, and pancreatic cancer) known to be associated with HR-deficiency. We identified 48 BRCA1-/- and 87 BRCA2-/- cases and called samples lacking (mono- or biallelic) mutations in BRCA1, BRCA2, or any other HR associated gene (e.g. PALB2, RAD51C) as wild-type. To validate observed structural variant patterns associated with homologous recombination deficiency from this larger dataset, a new cohort of 49 cases of invasive breast cancer with known BRCA1 (N = 29) or BRCA2 (N = 20) deficiency was collected as part of a prospective research study at Memorial Sloan Kettering Cancer Center and sequenced with 10X-linked read WGS in addition to standard Illumina short-read WGS. Results: Analysis of nearly 2,400 short-read whole genomes revealed distinct quasi-reciprocal structural variants (SVs) highly enriched in BRCA1-/- versus BRCA2-/- cancers. Applying high physical coverage (>150X) long molecule WGS to 49 tumor-normal pairs from breast cancer patients with inherited BRCA1 or BRCA2 loss-of-function mutations, we show that these SVs are associated with distinct cis or trans somatic allelic phases despite having nearly identical short read WGS footprints. Trans (crossover) outcomes were found to give rise to large-scale chromosomal variants that mediate the loss-of-heterozygosity patterns previously described as a cardinal feature of HR-deficient cancers. We find that these quasi-reciprocal SVs can be explained as distinct template switching outcomes from a shared intermediate arising after replication fork stalling in HR-deficient cancers. Furthermore, our WGS analyses reveal that BRCA2 genomes are highly enriched in deletions that harbor long (50-1000bp) tracts of inexact microhomology. These events, validated by long molecule WGS, indicate that single-stranded annealing serves as an active backup repair pathway in BRCA2-/- but not BRCA1-/- cancers. Conclusions: These results provide direct genomic evidence linking large-scale structural changes in HR-deficient tumors with specific backup repair pathways that suggest novel, therapeutically targetable dependencies. Our findings elucidate backup repair mechanisms responsible for generating structural variation in HR-deficient tumors, demonstrate the genotype-specific divergence of such compensatory DNA repair, and provide genomic features that improve the detection accuracy of HR-deficiency with utility for the optimal selection of treatment. Citation Format: Jeremy Setton, Kevin Hadi, Huasong Tian, Arnaud Da Cruz Paula, Joel Rosiene, Zi-Ning Choo, Julie Behr, Xiaotong Yao, Olivier Elemento, Britta Weigelt, Nadeem Riaz, Jorge S Reis-Filho, Marcin Imielinski, Simon N Powell. Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-04.

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.

Purpose: Lynch syndrome is defined by germline pathogenic mutations involving DNA mismatch repair (MMR) genes and linked with the development of MMR-deficient colon and endometrial cancers. Whether breast cancers developing in the context of Lynch syndrome are causally related to MMR deficiency (MMRd), remains controversial. Thus, we explored the morphologic and genomic characteristics of breast cancers occurring in Lynch syndrome individuals. Experimental Design: A retrospective analysis of 20,110 patients with cancer who underwent multigene panel genetic testing was performed to identify individuals with a likely pathogenic/pathogenic germline variant in MLH1, MSH2, MSH6, or PMS2 who developed breast cancers. The histologic characteristics and IHC assessment of breast cancers for MMR proteins and programmed death-ligand 1 (PD-L1) expression were assessed on cases with available materials. DNA samples from paired tumors and blood were sequenced with Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (≥468 key cancer genes). Microsatellite instability (MSI) status was assessed utilizing MSISensor. Mutational signatures were defined using SigMA. Results: A total of 272 individuals with Lynch syndrome were identified, 13 (5%) of whom had primary breast cancers. The majority of breast cancers (92%) were hormone receptor–positive tumors. Five (42%) of 12 breast cancers displayed loss of MMR proteins by IHC. Four (36%) of 11 breast cancers subjected to tumor-normal sequencing showed dominant MSI mutational signatures, high tumor mutational burden, and indeterminate (27%) or high MSISensor scores (9%). One patient with metastatic MMRd breast cancer received anti-PD1 therapy and achieved a robust and durable response. Conclusions: A subset of breast cancers developing in individuals with Lynch syndrome are etiologically linked to MMRd and may benefit from anti-PD1/PD-L1 immunotherapy.

Pathologists worldwide are facing remarkable challenges with increasing workloads and lack of time to provide consistently high-quality patient care. The application of artificial intelligence (AI) to digital whole-slide images has the potential of democratizing the access to expert pathology and affordable biomarkers by supporting pathologists in the provision of timely and accurate diagnosis as well as supporting oncologists by directly extracting prognostic and predictive biomarkers from tissue slides. The long-awaited adoption of AI in pathology, however, has not materialized, and the transformation of pathology is happening at a much slower pace than that observed in other fields (eg, radiology). Here, we provide a critical summary of the developments in digital and computational pathology in the last 10 years, outline key hurdles and ways to overcome them, and provide a perspective for AI-supported precision oncology in the future.

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.

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.

Traditional genetic testing for patients with gastrointestinal stromal tumors (GISTs) focus on those with syndromic features. To assess whether expanded genetic testing of GIST patients could identify hereditary cancer predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs over a 6-year period. Germline pathogenic/likely pathogenic (P/LP) variants in GIST-associated genes (SDHA, SDHB, SDHC, NF1, KIT) were identified in 69% of patients with KIT/PDGFRA-wildtype GISTs, 63% of whom did not have any personal or family history of syndromic features. To evaluate the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed 499 de-identified tumor-normal pairs. P/LP variants in certain genes (e.g., BRCA1/2, SDHB) identified in tumor-only sequencing of GISTs were almost exclusively germline in origin. Our results provide guidance for genetic testing of GIST patients and indicate that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs regardless of their history of syndromic features.

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

Breast adenoid cystic carcinoma (AdCC) is a rare subtype of triple negative breast cancer. Two morphologic variants are described, namely classic AdCC (C-AdCC) and solid basaloid (SB-AdCC). Recent studies have shown that the SB-AdCC variant has significantly worse prognosis than C-AdCC. Due to the rarity of SB-AdCC, no standard recommendations are available for its management. Data on the use and benefit of chemotherapy in patients with SB-AdCC are sparse and the response to neoadjuvant chemotherapy has not been reported. We present the clinical and pathologic findings of a patient with SB-AdCC treated with neoadjuvant chemotherapy who achieved a remarkable pathologic response.

Immunotherapy for the treatment of programmed death-ligand 1 (PD-L1) positive locally advanced or metastatic triple negative breast cancer may benefit patients with metaplastic breast cancer (MpBC). Previous study of PD-L1 in MpBC scored tumor cells (TCs), different from Food and Drug Administration-approved scoring methods. We sought to define PD-L1 expression in MpBCs and to evaluate concordance of 3 PD-L1 assays. Primary, treatment naive MpBC treated at our Center from 1998 to 2019 were identified. PD-L1 expression was assessed using SP142, E1L3n, and 73-10. We evaluated PD-L1 expression on tumor infiltrating immune cells (IC) and also in TCs. For each assay, we scored PD-L1 expression using ≥1% IC expression according to the IMpassion130 trial criteria and using combined positive score (CPS) ≥10 according to the KEYNOTE-355 trial cutoff. A total of 42 MpBCs were identified. Most MpBC had PD-L1 positivity in ≥1% IC with all 3 assays (95%, 95%, 86%) in contrast to a maximum 71% with a CPS ≥10. PD-L1 IC expression was comparable between the SP142 and 73-10 assays and was lowest with E1L3n. PD-L1 TC expression was lowest using SP142. The overall concordance for IC scoring was 88% while 62% had concordant CPS. For each assay, the results of the 2 scoring algorithms were not interchangeable. The SP142 assay showed distinct expression patterns between IC (granular, dot-like) and TC (membranous) while 73-10 and E1L3n showed membranous and/or cytoplasmic expression in both IC and TC. Most MpBC in our cohort were positive for PD-L1 indicating eligibility for anti-PD-L1/programmed death-1 immunotherapy.

Small cell carcinoma (SCC) of the uterine cervix is a rare and aggressive form of neuroendocrine carcinoma, which resembles small cell lung cancer (SCLC) in its histology and poor survival rate. Here, we sought to define the genetic underpinning of SCCs of the uterine cervix and compare their mutational profiles with those of human papillomavirus (HPV)-positive head and neck squamous cell carcinomas, HPV-positive cervical carcinomas, and SCLCs using publicly available data. Using a combination of whole-exome and targeted massively parallel sequencing, we found that the nine uterine cervix SCCs, which were HPV18-positive (n = 8) or HPV16-positive (n = 1), harbored a low mutation burden, few copy number alterations, and other than TP53 in two cases no recurrently mutated genes. The majority of mutations were likely passenger missense mutations, and only few affected previously described cancer-related genes. Using RNA-sequencing, we identified putative viral integration sites on 18q12.3 and on 8p22 in two SCCs of the uterine cervix. The overall nonsilent mutation rate of uterine cervix SCCs was significantly lower than that of SCLCs, HPV-driven cervical adeno- and squamous cell carcinomas, or HPV-positive head and neck squamous cell carcinomas. Unlike SCLCs, which are reported to harbor almost universal TP53 and RB1 mutations and a dominant tobacco smoke-related signature 4, uterine cervix SCCs rarely harbored mutations affecting these genes (2/9, 22% TP53; 0% RB1) and displayed a dominant aging (67%) or APOBEC mutational signature (17%), akin to HPV-driven cancers, including cervical adeno- and squamous cell carcinomas and head and neck squamous cell carcinomas. Taken together, in contrast to SCLCs, which are characterized by highly recurrent TP53 and RB1 alterations, uterine cervix SCCs were positive for HPV leading to inactivation of the suppressors p53 and RB, suggesting that these SCCs are convergent phenotypes.

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

Inherited mutations in BRCA1, BRCA2, and PALB2 cause a high risk of breast cancer. Here, we conducted parallel conditional knockout (CKO) of Brca1, Palb2, and Brca2, individually and in combination, along with one copy of Trp53, in the mammary gland of nulliparous female mice. We observed a functional equivalence of the three genes in their basic tumor-suppressive activity, a linear epistasis of Palb2 and Brca2, but complementary roles of Brca1 and Palb2 in mammary tumor suppression, as combined ablation of either Palb2 or Brca2 with Brca1 led to delayed tumor formation. Whole-exome sequencing (WES) revealed both similarities and differences between Brca1 and Palb2 or Brca2 null tumors. Analyses of mouse mammary glands and cultured human cells showed that combined loss of BRCA1 and PALB2 led to high levels of reactive oxygen species (ROS) and increased apoptosis, implicating oxidative stress in the delayed tumor development in Brca1;Palb2 double CKO mice. The functional complementarity between BRCA1 and PALB2/BRCA2 and the role of ROS in tumorigenesis require further investigation.

Most pancreatic ductal adenocarcinomas (PDAC) are lethal and resistant to immunotherapy. Thus, identifying the immunogenic subgroup (iPDAC) and therapeutic targets can save lives. Herein, we present molecular features of iPDAC. 3 cohorts (A, B, C) from 288 patients whose sequenced tumors (MSK-IMPACT) were classified by homologous recombination deficiency groups. MSI-H were excluded. Survival, tumor mutation burden, genomic instability score, and enriched pathways for each cohort are included in Table 1. Patients in A (BRCA1/2/PALB2) had longer survivals vs B/C. 61 samples were selected for bulk RNAseq analysis for A vs C. Gene Ontology was enriched for upregulated humoral, T cell, and neutrophil immunity. CIBERSORT suggested higher infiltration of gamma delta T (Tgd) cells (p=0.039) and neutrophils (p=0.012), but lower Treg (p=0.001). Multidimensional insights in cellular components of cancer, immune, stroma, and neural genes were obtained by single nuclear RNA (snRNAseq) analysis from 30 biopsies for A vs C. 10x Genomics Chromium platform for library and Scanpy for computational analysis after Cell Ranger pipelines were used. 61,868 nuclei were profiled from 18 (13 baseline and 5 matched longitudinal) samples after quality evaluation. UMAP accurately clustered cells from each patient. Long-term survivors (LTS) had heterogenous baseline immune cell infiltrates of plasma cells, neutrophils, and CD8 (+) cytotoxic T cells. In matched samples of LTS, evolution of more prominent CD8 (+) T cells, macrophage, plasma cell, and neutrophil were observed. Single nucleus T-Cell Receptor sequencing for clonal trajectory inference will be done to determine the associated single cell molecular features contributing to iPDAC and identify novel targets for future intervention. Table 1. Cohort (Total: N=288) A: core HRD (BRCA1/2/PALB2) B: non-core HRD (ATM, BARD1, BLM, CHEK2, RAD50, RAD51C, RTEL1, MUTYH) C: others without HR-gene alterations Number (%) 48 (16.6) 19 (6.5) 221 (76) Median overall survival (95% confidence Interval) 33 months (3.6-64) 16 (11- not reached) 16 (14-18) Tumor Mutation Burden (TMB) 4.4 3.5 3.9 Genomic Instability Score (GIS, HRD score) 26 12 13 Gene Ongology term, enrichment score, adjusted p-value Adaptive immune response, GO:0002250, 0.49, 1.69e-10 Not included Reference to cohort A Humoral immune response, GO:0006959, 0.58, 1.67e-9 T cell activation, GO:0042110, 0.44, 2.75e-8 Neutrophil chemotaxis, GO:0030593, 0.73, 4.3e-10 Citation Format: Wungki Park, Catherine O'Connor, Shigeaki Umeda, Roshan Sharma, Yingjie Zhu, Elias-Ramzey Karnoub, Anna Varghese, Kevin C. Soares, Alejandro Jimemez, Asli Yavas, Kenneth H. Yu, Balachandran P. Vinod, Joanne F. Chou, Danny N. Khalil, Kelsen David, Hulya Sahin Ozkan, Olca Basturk, Marinela Capanu, Tal Nawy, Michael F. Berger, Ghassan K. Abou-Alfa, Jorge S. Reis-Filho, Ronan Chaligne, Nadeem Riaz, Dana Pe'er, Christine Iacobuzio-Donahue, Eileen M. O'Reilly. Molecular profiles and single cell analysis identify immunogenic pancreatic ductal adenocarcinoma (iPDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6421.

Amplification of CCNE1 is a recurrent genetic alteration that has been associated with chemoresistance and poor prognosis in gynecological malignancies. We identified Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) as a novel synthetic lethal target in CCNE1 amplified cancers. In this genomic setting, PKMYT1 inhibition causes premature mitotic entry, mitotic catastrophe and, in turn, cell death. In pre-clinical models, correlation between CCNE1 copy number, cyclin E1 expression and sensitivity to PKMYT1 inhibition is imperfect. Thus, a deep understanding of CCNE1 amplification as a predictive biomarker for PKMYT1 inhibition is critical for refining patient selection strategies and strengthening our understanding of the mechanism of action. These learnings will be applied to correlative biomarker analyses in clinical trials evaluating RP-6306, a first-in-class, potent, and highly selective PKMYT1 inhibitor, in tumors with CCNE1 amplifications (NCT04855656, NCT05147272, NCT05147350). In this study, multiple features of CCNE1 were examined using a combination of whole genome and targeted next generation sequencing (NGS), FISH, and immunohistochemistry (IHC). Cyclin E1 levels were evaluated across tumor tissue microarrays spanning 5 tumor indications: triple-negative breast (n=100), colorectal (n=100), gastric (n=100), uterine (n=49), ovarian (n=100) cancers. The highest cyclin E1 protein levels were found in ovarian and uterine malignancies, consistent with indications with high prevalence of CCNE1 amplification (median H-score ovarian = 125; uterine = 100; gastric = 45; TNBC = 48 and CRC = 24). To explore CCNE1 amplification on a single cell level, we established a novel FISH assay using a subtelomeric q-arm control probe. The subtelomeric q-arm band is more frequently diploid than other regions on chromosome 19. Analytical validation of the FISH assay demonstrated sensitivity of 95%, specificity of 95.2%, and overall concordance of 95.1% when compared to a targeted sequencing panel (SNIPDx®). Within the uterine and ovarian cohorts, cancers harboring CCNE1 amplification were found to display higher cyclin E1 protein levels than non-amplified tumors (p-val=0.0002) and cyclin E1 expression was positively correlated with CCNE1 copy number (Spearman ρ=0.67; p-value=4.1e-05). Substantial intratumoral spatial heterogeneity of CCNE1 copy number when measured by FISH was observed across ovarian and uterine tumors. The potential impact of CCNE1 amplification heterogeneity, cyclin E1 protein levels and CCNE1 amplification fragment size on response to RP-6306 in preclinical models are currently being investigated and will be reported. Citation Format: Adam Petrone, Elia Aguado-Fraile, Sunantha Sethuraman, Adrienne Johnson, Ian Silverman, Gary Marshall, Jorge S. Reis-Filho, John Iafrate, Artur Veloso, Victoria Rimkunas. Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2132.

Intro: Half of all patients with NSCLC develop BM during their clinical course. While modern NSCLC-directed agents yield excellent systemic response, most patients require focal BM treatment. Prior reports of BM genomics have been limited by low numbers and a lack of matched specimens. Here, we report the largest cohort to date of molecularly-profiled NSCLC BM samples with comprehensive clinical follow-up. Methods: Clinical data and outcomes for 244 patients with NSCLC and resected BM were identified. Samples were assessed using MSK-IMPACT, a custom tumor-normal next generation sequencing assay. 51 (20.9%) patients had matched primary site tissue, and 44 (18%) patients had matched tissue from another metastatic site or CSF. Genomic alterations were filtered for driver variants using OncoKB. Publicly available genomic data for NSCLC primary samples was used for comparison against the primary samples from our BM cohort. Results: The most frequently altered genes in BM tumors were TP53 (72%), CDKN2A (34%), KRAS (31%), KEAP1 (26%), and EGFR (21%). CDKN2A was more frequently altered in BM samples compared to NSCLC primary lesions (34% vs 14%, p = 0.003). Additionally, cell cycle pathway alterations were enriched in BM (56% vs 31%, p = 0.002). BM samples also had a significantly higher fraction of genome altered (FGA) relative to primary samples (p < 0.0001). We then compared primary samples from BM patients against primary samples from metastatic NSCLC patients without BM and primary samples from non-metastatic NSCLC patients. We found an enrichment of alterations in TP53 (68.6% vs 27.7%, p < 0.0001), NKX2-1 (11.4% vs 1.7%, p = 0.006), SMARCA4 (11.4% vs 2.1%, p = 0.01), RB1 (11.4% vs 1.7%, p = 0.006), and FOXA1 (11.4% vs 0.9%, p = 0.001) in the primary samples from BM patients compared to non-metastatic patients. Next, we grouped patients based on CNS progression patterns and found that EGFR alterations were enriched in patients with leptomeningeal progression when compared to patients without progression (42% vs 18%, p = 0.03). Conclusions: In our cohort of molecularly-profiled NSCLC BM, we found an enrichment of cell cycle pathway alterations and a higher FGA in BMs compared to their primary tumor controls. Additionally, several genes were enriched in the primary tissue of patients that developed BM compared to primary tissue from non-metastatic patients. EGFR alterations were enriched in patients who develop leptomeningeal disease (LMD). Our work herein characterizes the genomic profiles of NSCLC patients with BM and identifies specific genes enriched in the primary tissue of BM patients compared to primary tissue from both non-BM metastatic patients and non-metastatic patients. Finally, our finding that EGFR alterations were enriched in patients with LMD suggests specific biologic underpinnings driving patterns of CNS progression. Citation Format: Henry Walch, Anna Skakodub, Kathryn R. Tringale, Harish N. Vasudevan, Jordan Eichholz, Daniel W. Kelly, Emily Lebow, Nelson S. Moss, Kenny Kwok Hei Yu, Bob T. Li, Boris Mueller, Atif Khan, Yao Yu, Simon Powell, Jorge S. Reis-Filho, Brandon S. Imber, Pedram Razavi, Daniel R. Gomez, Nikolaus Schultz, Luke R. Pike. Genomic characterization of non-small cell lung cancer (NSCLC) brain metastasis (BM) patients identifies novel alterations associated with tropisms and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6063.

International audience; Aberrant activation of RET is a critical driver of growth and proliferation in diverse solid tumours. Multikinase inhibitors (MKIs) showing anti-RET activities have been tested in RET-altered tumours with variable results. The low target specificity with consequent increase in side-effects and off-target toxicities resulting in dose reduction and drug discontinuation are some of the major issues with MKIs. To overcome these issues, new selective RET inhibitors such as pralsetinib (BLU-667) and selpercatinib (LOXO-292) have been developed in clinical trials, with selpercatinib recently approved by the Food and Drug Administration (FDA). The results of these trials showed marked and durable antitumour activity and manageable toxicity profiles in patients with RET-altered tumours. The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) launched a collaborative project to review the available methods for the detection of RET gene alterations, their potential applications and strategies for the implementation of a rational approach for the detection of RET fusion genes and mutations in human malignancies. We present here recommendations for the routine clinical detection of targetable RET rearrangements and mutations.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Background Patients with human papillomavirus–related oropharyngeal cancers have excellent outcomes but experience clinically significant toxicities when treated with standard chemoradiotherapy (70 Gy). We hypothesized that functional imaging could identify patients who could be safely deescalated to 30 Gy of radiotherapy. Methods In 19 patients, pre- and intratreatment dynamic fluorine-18-labeled fluoromisonidazole positron emission tomography (PET) was used to assess tumor hypoxia. Patients without hypoxia at baseline or intratreatment received 30 Gy; patients with persistent hypoxia received 70 Gy. Neck dissection was performed at 4 months in deescalated patients to assess pathologic response. Magnetic resonance imaging (weekly), circulating plasma cell-free DNA, RNA-sequencing, and whole-genome sequencing (WGS) were performed to identify potential molecular determinants of response. Samples from an independent prospective study were obtained to reproduce molecular findings. All statistical tests were 2-sided. Results Fifteen of 19 patients had no hypoxia on baseline PET or resolution on intratreatment PET and were deescalated to 30 Gy. Of these 15 patients, 11 had a pathologic complete response. Two-year locoregional control and overall survival were 94.4% (95% confidence interval = 84.4% to 100%) and 94.7% (95% confidence interval = 85.2% to 100%), respectively. No acute grade 3 radiation–related toxicities were observed. Microenvironmental features on serial imaging correlated better with pathologic response than tumor burden metrics or circulating plasma cell-free DNA. A WGS-based DNA repair defect was associated with response (P = .02) and was reproduced in an independent cohort (P = .03). Conclusions Deescalation of radiotherapy to 30 Gy on the basis of intratreatment hypoxia imaging was feasible, safe, and associated with minimal toxicity. A DNA repair defect identified by WGS was predictive of response. Intratherapy personalization of chemoradiotherapy may facilitate marked deescalation of radiotherapy.

Triple-negative breast cancers (TNBCs) often have a high Ki-67 proliferation index and respond favorably to neoadjuvant chemotherapy (NACT) with pathologic complete response (pCR) resulting in ~40% of cases. Nevertheless, morbidity/mortality remain high, mostly due to recurrence in patients with residual disease. In contrast, the incidence and clinical features of TNBC with low proliferation (TNLP), defined as TNBC with a Ki-67 index of ≤30% remains unknown. We report 70 cases of TNLP identified at our center from 2008 to 2018, including 18 treated with NACT. TNLP tumors represent

Late-stage relapse (LSR) in patients with breast cancer (BC) occurs more than five years and up to 10 years after initial treatment and has less than 30% 5-year relative survival rate. Long non-coding RNAs (lncRNAs) play important roles in BC yet have not been studied in LSR BC. Here, we identify 1127 lncRNAs differentially expressed in LSR BC via transcriptome sequencing and analysis of 72 early-stage and 24 LSR BC patient tumors. Decreasing expression of the most up-regulated lncRNA, LINC00355, in BC and MCF7 long-term estrogen deprived cell lines decreases cellular invasion and proliferation. Subsequent mechanistic studies show that LINC00355 binds to MENIN and changes occupancy at the CDKN1B promoter to decrease p27Kip. In summary, this is a key study discovering lncRNAs in LSR BC and LINC00355 association with epigenetic regulation and proliferation in BC.

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

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.

Histologic special types of breast cancer (BC) account for ~20% of BCs. Large sequencing studies of metastatic BC have focused on invasive ductal carcinomas of no special type (IDC-NSTs). We sought to define the repertoire of somatic genetic alterations of metastatic histologic special types of BC. We reanalyzed targeted capture sequencing data of 309 special types of BC, including metastatic and primary invasive lobular carcinomas (ILCs; n = 132 and n = 127, respectively), mixed mucinous (n = 5 metastatic and n = 14 primary), micropapillary (n = 12 metastatic and n = 8 primary), and metaplastic BCs (n = 6 metastatic and n = 5 primary), and compared metastatic histologic special types of BC to metastatic IDC-NSTs matched according to clinicopathologic characteristics and to primary special type BCs. The genomic profiles of metastatic and primary special types of BC were similar. Important differences, however, were noted: metastatic ILCs harbored a higher frequency of genetic alterations in TP53, ESR1, FAT1, RFWD2, and NF1 than primary ILCs, and in CDH1, PIK3CA, ERBB2, TBX3, NCOR1, and RFWD2 than metastatic IDC-NSTs. Metastatic ILCs displayed a higher mutational burden, and more frequently dominant APOBEC mutational signatures than primary ILCs and matched metastatic IDC-NSTs. ESR1 and NCOR mutations were frequently detected in metastatic mixed mucinous BCs, whereas PIK3CA and TP53 were the most frequently altered genes in metastatic micropapillary and metaplastic BCs, respectively. Taken together, primary and metastatic BCs histologic special types have remarkably similar repertoires of somatic genetic alterations. Metastatic ILCs more frequently harbor APOBEC mutational signatures than primary ILCs and metastatic IDC-NSTs.

The objective of this study was to evaluate the effect of L-arginine supplementation on the formation of jejunal lesions and micronuclei in Wistar rats following 5-fluorouracil (5-FU) treatment. Fifty rats were separated into five groups: CG served as the control group, G

Background Radiation therapy is one of the most commonly used cancer therapeutics but genetic determinants of clinical benefit are poorly characterized. Pathogenic germline variants in ATM are known to cause ataxia-telangiectasia, a rare hereditary syndrome notable for marked radiosensitivity. In contrast, somatic inactivation of ATM is a common event in a wide variety of cancers, but its clinical actionability remains obscure. Methods We analyzed 20 107 consecutively treated advanced cancer patients who underwent targeted genomic sequencing as part of an institutional genomic profiling initiative and identified 1085 harboring a somatic or germline ATM mutation, including 357 who received radiotherapy (RT). Outcomes of irradiated tumors harboring ATM loss-of-function (LoF) mutations were compared with those harboring variants of unknown significance. All statistical tests were 2-sided. Results Among 357 pan-cancer patients who received 727 courses of RT, genetic inactivation of ATM was associated with improved radiotherapeutic efficacy. The 2-year cumulative incidence of irradiated tumor progression was 13.2% vs 27.5% for tumors harboring an ATM LoF vs variant of unknown significance allele, respectively (hazard ratio [HR] = 0.51, 95% confidence interval [CI] = 0.34 to 0.77, P = .001). The greatest clinical benefit was seen in tumors harboring biallelic ATM inactivation (HR = 0.19, 95% CI = 0.06 to 0.60, P = .005), with statistically significant benefit also observed in tumors with monoallelic ATM inactivation (HR = 0.57, 95% CI = 0.35 to 0.92, P = .02). Notably, ATM LoF was highly predictive of outcome in TP53 wild-type tumors but not among TP53-mutant tumors. Conclusions We demonstrate that somatic ATM inactivation is associated with markedly improved tumor control following RT. The identification of a radio-sensitive tumor phenotype across multiple cancer types offers potential clinical opportunities for genomically guided RT.

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.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial's primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

Background: According to several estimates, African American women are 40% more likely to die from breast cancer than white women in the United States. Proposed explanations for this disparity include differential socioeconomic factors and access to care, however some studies have raised the possibility that variation in tumor biology is a contributing factor. Methods: We prospectively sequenced primary and metastatic breast cancer tumors and their matched normal DNA using the MSK-IMPACT assay. We performed gene enrichment analyses to identify the oncogenic mutations and copy number alterations that were more frequent in patients who self-identified as African American or black (AA/B) compared with patients who self-identified as non-Hispanic white (NHW). Detailed clinicopathologic variables were collected for the full cohort. Mann-Whitney U test, Fisher’s exact test, and multivariate binomial regression models were used for statistical analyses. Results: Genomic profiling was performed on 339 tumors from 301 AA/B patients (44.6% metastatic), and 2,607 tumors from 2,248 NHW patients (48.5% metastatic). Age of AA/B and NHW patients at diagnosis was similar (51.5 vs. 52.8 years, p = 0.065). However, AA/B patients were more likely to be pre- or peri-menopausal at diagnosis (51.6 vs. 44%, p = 0.013), have triple-negative disease (26.5 vs. 13%, p < 0.001), and have higher stage at diagnosis (p = 0.024). Of note, invasive lobular carcinomas were significantly less frequent in AA/B patients compared to NHW (5.9 vs. 14.5%; p < 0.001), a trend that persisted when controlling for receptor subtype. In the unadjusted analysis, AA/B women were more likely to have TP53 mutations (53.4 vs. 36.5%; q < 0.01) and NF1 loss of function mutations (9.9% vs. 3.7%; q < 0.01), and less likely to have CDH1 mutations (6.3% vs. 15.4%; q < 0.01) and PIK3CA mutations (25.7 vs. 35.6%, q = 0.017). However, in a multivariate analysis adjusted for receptor subtype, histology, and sample type (primary vs. metastatic), NF1 was the only gene found to be more commonly mutated in AA/B women (odds ratio: 2.84; 95% CI: 1.73 - 4.08, q < 0.01). Focusing specifically on ER+/HER2- disease, AA/B women were more likely to have mutations in TP53 (31.6% vs. 24.6%), NF1 (7.7% vs. 3.1%) and FGFR1 amplification (21.4% vs. 13.1%), and less likely to have mutations in CDH1 (9.2% vs. 18.8%) or PIK3CA (29.6% vs. 39.6%), however these results did not retain statistical significance when adjusted for multiple comparisons. In triple-negative breast cancer patients, TP53 was mutated at equal rates in AA/B and NHW patients (88.9% vs. 85.9%; p = 0.59), and there was a numerically higher frequency of NF1 mutations in AA/B patients (11.1% vs. 5%). There was no significant difference in tumor mutational burden between AA/B and NHW women (4.24 vs. 4.87; p = 0.19), and no difference in the frequency of microsatellite instability (defined as MSISensor score > 10, 0.9% vs. 0.5%; p = 0.44). Conclusions: In this large clinico-genomic analysis, as previously reported, AA/B patients were more likely to have the clinical hallmarks of aggressive disease, as defined by triple-negative subtype, higher stage, and premenopausal status at diagnosis. Our analysis demonstrated trends towards enrichment for some of the genomic alterations previously identified to be associated with poor outcome in the AA/B population, however, after controlling for the aforementioned clinical factors, breast cancer in AA/B did not differ significantly from breast cancer in NHW in terms of their driver genomic alterations, MSI or tumor mutation burden. Further studies are required to fully characterize the genomics of breast cancer in AA/B women, which may play a role in larger efforts to equalize the disparities observed in this population. Citation Format: Cristian Serna-Tamayo, Joshua Z Drago, Carlos Dos Anjos, Joshua Herbert, Fresia Pareja, Shanu Modi, Komal Jhaveri, Chau Dang, David B Solit, Larry Norton, Mauricio Scaltriti, Jorge S Reis-Filho, Sarat Chandarlapaty, Mark E. Robson, Pedram Razavi. The genomic landscape of breast cancer in African American women [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-01.

Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.

Background: Male breast cancer is rare, with approximately 2,600 cases diagnosed annually in the United States. Data are scant regarding the genomics and pathophysiology of male breast cancer, especially in the metastatic setting, requiring most treatment recommendations in male breast cancer to be made by inference from breast cancer in women. Methods: We performed prospective genomic profiling of primary and metastatic tumor samples from men with breast cancer treated at Memorial Sloan Kettering Cancer Center using the MSK-IMPACT targeted-DNA-sequencing panel for somatic mutations. Comprehensive demographic, clinical, and pathologic data were collected on all included patients. Statistics are descriptive. Results: Genomic sequencing was performed on 45 samples from 41 men (31 primary samples and 14 from metastatic sites). Median age at time of sample collection was 61 years, with a range of 27-92 years. Thirty-seven (90.2%) men had ER+/HER2- breast cancer, 3 (7.3%) had ER+/HER2+ breast cancer and 1 (2.4%) had triple negative disease. Thirty-nine (95.1%) had ductal carcinoma, and no cases of lobular carcinoma were identified. Forty patients underwent germline testing, and 12 (30%) were found to have pathogenic germline mutations (6 BRCA2 mutations, 2 BRCA1 mutations [one of whom had a concurrent CHEK2 mutation], and one mutation each in PALB2, MUTYH, and MSH6). Overall, the pattern of genomic alterations in male breast cancer was similar that in women. Twelve (29.3%) patients had PIK3CA mutations, 9 (22%) had GATA3 mutations, 3 (7.3%) had TP53 mutations, 3 (7.3%) had ARID1A mutations, 3 (7.3%) had KMT2C mutations, 2 (4.9%) had FOX1A mutations, 2 (4.9%) had RB1 mutations, and 2 (4.9%) had TERT promoter hotspot mutations. Eleven (26.8%) patients had CCND1 amplification, 8 (19.5%) had MYC amplification, 6 (14.6%) had FGFR1 amplification, and 5 (12.2%) had MDM2 amplification. All other findings were present in ≤ 1 patient. All included patients had normal mutational burden, and all samples were microsatellite stable. PIK3CA mutations occurred in 33% of primary samples vs. 15% of metastatic samples, CCND1 amplification occurred in 23% of primary samples vs. 38% of metastatic samples, and TERT hotspot promoter mutations were found only in metastatic samples. Of note, we observed a single ESR1 D538G mutation in the metastatic sample of a patient with significant prior exposure to aromatase inhibitors in the adjuvant and metastatic settings. We further found concurrent ERBB2 mutation and amplification in the post-treatment metastatic samples of an ER+/HER2- patient, who was treated with neratinib for 14 weeks with clinical response. Lastly, we report a heavily pretreated patient with metastatic secretory breast carcinoma who was found to have an ETV6-NTRK3 fusion gene. This patient was treated with a first-generation TRK inhibitor and continues to exhibit an ongoing clinical response at 8.6 months. Conclusions: Based on our data, the overall genomic landscape of male breast cancer appears comparable to that of breast cancer in women, as has been previously reported. However, despite the small number of metastatic cases examined, several previously unreported and treatment-informing signatures were discovered, especially in those patients with less common male breast cancer variants. Further study is warranted to confirm these findings in a larger cohort. Citation Format: Joshua Z Drago, Cristian Serna-Tamayo, Carlos H Dos Anjos, David N Brown, Shanu Modi, Komal Jhaveri, David B Solit, Tiffany A Traina, Sarat Chandarlapaty, Jorge S Reis-Filho, Mark E Robson, Ayca Gucalp, Pedram Razavi. Genomic profiling of primary and metastatic breast cancer in men [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-17-01.

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2+ breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear. Experimental Design: Hematoxylin and eosin–stained slides (n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER+ tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides (n = 33). Results: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4+, CD8+, CD20+ s-TILs, and high CD20+ intratumoral TILs, was independently associated with a higher pCR rate (P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20+ TILs. Conclusions: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2+ breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response.

PURPOSE Mitogen-activated protein kinase pathway–activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/ BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. MATERIALS AND METHODS We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. RESULTS Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/ KRAS G12C, 95%; KRAS G12D/ NRAS G12V, 48%; BRAF V600E/ KRAS G12D, 44%; and KRAS G12D/ NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability–high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability–high. CONCLUSION Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.

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.

Studies of targeted therapy resistance in lung cancer have primarily focused on single-gene alterations. Based on prior work implicating apolipoprotein b mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) mutagenesis in histological transformation of epidermal growth factor receptor (EGFR)-mutant lung cancers, we hypothesized that mutational signature analysis may help elucidate acquired resistance to targeted therapies.APOBEC mutational signatures derived from an Food and Drug Administration-cleared multigene panel [Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT)] using the Signature Multivariate Analysis (SigMA) algorithm were validated against the gold standard of mutational signatures derived from whole-exome sequencing. Mutational signatures were decomposed in 3276 unique lung adenocarcinomas (LUADs), including 93 paired osimertinib-naïve and -resistant EGFR-mutant tumors. Associations between APOBEC and mechanisms of resistance to osimertinib were investigated. Whole-genome sequencing was carried out on available EGFR-mutant lung cancer samples (10 paired, 17 unpaired) to investigate large-scale genomic alterations potentially contributing to osimertinib resistance.APOBEC mutational signatures were more frequent in receptor tyrosine kinase (RTK)-driven lung cancers (EGFR, ALK, RET, and ROS1; 25%) compared to LUADs at large (20%, P0.001); across all subtypes, APOBEC mutational signatures were enriched in subclonal mutations (P0.001). In EGFR-mutant lung cancers, osimertinib-resistant samples more frequently displayed an APOBEC-dominant mutational signature compared to osimertinib-naïve samples (28% versus 14%, P = 0.03). Specifically, mutations detected in osimertinib-resistant tumors but not in pre-treatment samples significantly more frequently displayed an APOBEC-dominant mutational signature (44% versus 23%, P0.001). EGFR-mutant samples with APOBEC-dominant signatures had enrichment of large-scale genomic rearrangements (P = 0.01) and kataegis (P = 0.03) in areas of APOBEC mutagenesis.APOBEC mutational signatures are frequent in RTK-driven LUADs and increase under the selective pressure of osimertinib in EGFR-mutant lung cancer. APOBEC mutational signature enrichment in subclonal mutations, private mutations acquired after osimertinib treatment, and areas of large-scale genomic rearrangements highlights a potentially fundamental role for APOBEC mutagenesis in the development of resistance to targeted therapies, which may be potentially exploited to overcome such resistance.

This Viewpoint discusses immunohistochemistry and tissue-hybridization-based diagnostics delivery and compares it with digital pathology.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.