Homologous Recombination Deficiency Landscape of Breast Cancers and Real-World Effectiveness of Poly ADP-Ribose Polymerase Inhibitors in Patients With Somatic BRCA1/2, Germline PALB2, or Homologous Recombination Deficiency Signature.

PURPOSE: Poly ADP-ribose polymerase inhibitors (PARPi) are approved for patients with human epidermal growth factor receptor 2-negative metastatic breast cancer (mBC) and germline pathogenic/likely pathogenic variant (hereafter mutation) in the BRCA1/2 genes (gBRCA); however, clinical benefit has also been demonstrated in mBC with somatic BRCA1/2 mutations (sBRCA) or germline PALB2 mutations (gPALB2). This study aims to describe the genomic landscape of homologous recombination repair (HRR) gene alterations in mBC and assess PARPi treatment outcomes for patients with gBRCA compared with other HRR genes and by status of a novel homologous recombination deficiency signature (HRDsig). METHODS: A real-world (RW) clinico-genomic database (CGDB) of comprehensive genomic profiling (CGP) linked to deidentified, electronic health record-derived clinical data was used. CGP was analyzed for HRR genes and HRDsig. The CGDB enabled cohort characterization and outcomes analyses of 177 patients exposed to PARPi. RW progression-free survival (rwPFS) and RW overall survival (rwOS) were compared. RESULTS: Of 28,920 patients with mBC, gBRCA was detected in 3.4%, whereas the population with any BRCA alteration or gPALB2 increased to 9.5%. HRDsig+ represented 21% of patients with mBC. BRCA and gPALB2 had higher levels of biallelic loss and HRDsig+ than other HRR alterations. Outcomes on PARPi were assessed for 177 patients, and gBRCA and sBRCA/gPALB2 cohorts were similar: gBRCA versus sBRCA/gPALB2 rwPFS was 6.3 versus 5.4 months (hazard ratio [HR], 1.37 [0.77-2.43]); rwOS was 16.2 versus 21.2 months (HR, 1.45 [0.74-2.86]). Additionally, patients with HRDsig+ versus HRDsig- had longer rwPFS (6.3 v 2.8 months; HR, 0.62 [0.42-0.92]) and numerically longer rwOS (17.8 v 13.0 months; HR, 0.72 [0.46-1.14]). CONCLUSION: Patients with sBRCA and gPALB2 derive similar benefit from PARPi as those with gBRCA alterations. In combination, HRDsig+, sBRCA, and gPALB2 represent an additional 19% of mBC that can potentially benefit from PARPi. Randomized trials exploring a more inclusive biomarker such as HRDsig are warranted.

Gene partners of the EWSR1 fusion may represent molecularly distinct entities.

EWSR1 fusions are highly promiscuous and are associated with unique malignancies, clinical phenotypes, and molecular subtypes. However, rare fusion partners (RFP) of EWSR1 has not been well described. Here, we conducted a cross-sectional, retrospective study of 1,140 unique tumors harboring EWSR1 fusions. We identified 64 unique fusion partners. RFPs were identified more often in adults than children. Alterations in cell cycle control and DNA damage response genes as driving the differences between fusion partners. Potentially clinically actionable genomic variants were more prevalent in tumors harboring RFP than common fusions. While the data presented here is limited, tumors harboring RFP of EWSR1 may represent molecularly distinct entities and may benefit from further molecular testing to identify targeted therapeutic options.

Tumor sequencing of African ancestry reveals differences in clinically relevant alterations across common cancers.

Cancer genomes from patients with African (AFR) ancestry have been poorly studied in clinical research. We leverage two large genomic cohorts to investigate the relationship between genomic alterations and AFR ancestry in six common cancers. Cross-cancer type associations, such as an enrichment of MYC amplification with AFR ancestry in lung, breast, and prostate cancers, and depletion of BRAF alterations are observed in colorectal and pancreatic cancers. There are differences in actionable alterations, such as depletion of KRAS G12C and EGFR L858R, and enrichment of ROS1 fusion with AFR ancestry in lung cancers. Interestingly, in lung cancer, KRAS mutations are less common in both smokers and non-smokers with AFR ancestry, whereas the association of TP53 mutations with AFR ancestry is only seen in smokers, suggesting an ancestry-environment interaction that modifies driver rates. Our study highlights the need to increase representation of patients with AFR ancestry in drug development and biomarker discovery.

Pan-Cancer Analysis of Copy-Number Features Identifies Recurrent Signatures and a Homologous Recombination Deficiency Biomarker to Predict Poly (ADP-Ribose) Polymerase Inhibitor Response.

PURPOSE: Copy-number (CN) features reveal the molecular state of cancers and may have predictive and prognostic value in the treatment of cancer. We sought to apply published CN analysis methods to a large pan-cancer data set and characterize ubiquitous CN signatures across tumor types, including potential utility for treatment selection. METHODS: We analyzed the landscape of CN features in 260,333 pan-cancer samples. We examined the association of 10 signatures with genomic alterations and clinical characteristics and trained a machine learning classifier using CN and insertion and deletion features to detect homologous recombination deficiency signature (HRDsig) positivity. Clinical outcomes were assessed using a real-world clinicogenomic database (CGDB) of comprehensive genomic profiling linked to deidentified, electronic health record-derived clinical data. RESULTS: CN signatures were prevalent across cancer types and associated with diverse processes including focal tandem duplications, seismic amplifications, genome-wide loss of heterozygosity (gLOH), and HRD. Our novel HRDsig outperformed gLOH in predicting BRCAness and effectively distinguished biallelic BRCA and homologous recombination-repair wild-type (HRRwt) samples pan-tumor, demonstrating high sensitivity to detect biallelic BRCA in ovarian (93%) and other HRD-associated cancers (80%-87%). Pan-tumor prevalence of HRDsig was 6.4%. HRRwt cases represented a significant fraction of the HRDsig-positive cohort, likely reflecting a population with nongenomic mechanisms of HRD. In ovarian and prostate CGDBs, HRDsig identified more patients than gLOH and had predictive value for poly (ADP-ribose) polymerase inhibitor (PARPi) benefit. CONCLUSION: Tumor CN profiles are informative, revealing diverse processes active in cancer. We describe the landscape of 10 CN signatures in a large pan-cancer cohort, including two associated with HRD. We trained a machine learning-based HRDsig that robustly identified BRCAness and associated with biallelic BRCA pan-tumor, and was predictive of PARPi benefit in real-world ovarian and prostate data sets.

A pan-sarcoma landscape of telomeric content shows that alterations in RAD51B and GID4 are associated with higher telomeric content.

Tumor cells need to activate a telomere maintenance mechanism, enabling limitless replication. The bulk of evidence supports that sarcomas predominantly use alternative lengthening of telomeres (ALT) mechanism, commonly associated with alterations in ATRX and DAXX. In our dataset, only 12.3% of sarcomas harbored alterations in these genes. Thus, we checked for the presence of other genomic determinants of high telomeric content in sarcomas. Our dataset consisted of 13555 sarcoma samples, sequenced as a part of routine clinical care on the FoundationOne®Heme platform. We observed a median telomeric content of 622.3 telomeric reads per GC-matched million reads (TRPM) across all samples. In agreement with previous studies, telomeric content was significantly higher in ATRX altered and POT1 altered sarcomas. We further observed that sarcomas with alterations in RAD51B or GID4 were enriched in samples with high telomeric content, specifically within uterus leiomyosarcoma for RAD51B and soft tissue sarcoma (not otherwise specified, nos) for GID4, Furthermore, RAD51B and POT1 alterations were mutually exclusive with ATRX and DAXX alterations, suggestive of functional redundancy. Our results propose a role played by RAD51B and GID4 in telomere elongation in sarcomas and open research opportunities for agents aimed at targeting this critical pathway in tumorigenesis.

Genetic Heterogeneity and Tissue-specific Patterns of Tumors with Multiple PIK3CA Mutations.

PURPOSE: To comprehensively characterize tissue-specific and molecular subclasses of multiple PIK3CA (multi-PIK3CA) mutations and assess their impact on potential therapeutic outcomes. EXPERIMENTAL DESIGN: We profiled a pan-cancer cohort comprised of 352,392 samples across 66 tumor types using a targeted hybrid capture-based next-generation sequencing panel covering at least 324 cancer-related genes. Molecularly defined subgroups, allelic configuration, clonality, and mutational signatures were identified and tested for association with PI3K inhibitor therapeutic response. RESULTS: Multi-PIK3CA mutations are found in 11% of all PIK3CA-mutant tumors, including 9% of low tumor mutational burden (TMB) PIK3CA-mutant tumors, and are enriched in breast and gynecologic cancers. Multi-PIK3CA mutations are frequently clonal and in cis on the same allele and occur at characteristic positions across tumor types. These mutations tend to be mutually exclusive of mutations in other driver genes, and of genes in the PI3K pathway. Among PIK3CA-mutant tumors with a high TMB, 18% are multi-PIK3CA mutant and often harbor an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational signature. Despite large differences in specific allele combinations comprising multi-PIK3CA mutant tumors, especially across cancer types, patients with different classes of multi-PIK3CA mutant estrogen receptor-positive, HER2-negative breast cancers respond similarly to PI3K inhibition. CONCLUSIONS: Our pan-tumor study provides biological insights into the genetic heterogeneity and tissue specificities of multi-PIK3CA mutations, with potential clinical utility to guide PI3K inhibition strategies.

Tissue and liquid biopsy profiling reveal convergent tumor evolution and therapy evasion in breast cancer.

Pathological and genomic profiling have transformed breast cancer care by matching patients to targeted treatments. However, tumors evolve and evade therapeutic interventions often through the acquisition of genomic mutations. Here we examine patients profiled with tissue (TBx) and liquid biopsy (LBx) as part of routine clinical care, to characterize the tumor evolutionary landscape and identify potential vulnerabilities in the relapsed setting. Real-world evidence demonstrates that LBx is utilized later in care and identifies associations with intervening therapy. While driver events are frequently shared, acquired LBx alterations are detected in a majority of patients, with the highest frequency in ER+ disease and in patients with longer biopsy intervals. Acquired mutations are often polyclonal and present at lower allelic fractions, suggesting multi-clonal convergent evolution. In addition to well-characterized resistance mutations (e.g., ESR1, NF1, RB1, ERBB2), we observe a diversity of rarer but potentially targetable mutations (e.g., PIK3CA, HRAS/NRAS/KRAS, FGFR1/2/3, BRAF) and fusions (e.g., FGFR1/2, ERBB2, RET), as well as BRCA1/2 reversions through a variety of mechanisms, including splice alterations and structural deletions. This study provides insights on treatment and selection-driven tumor evolution and identifies potential combinatorial treatment options in advanced breast cancer.

PARP Inhibitor Insensitivity to BRCA1/2 Monoallelic Mutations in Microsatellite Instability-High Cancers.

PURPOSE: To examine the overlap of homologous recombination deficiency (HRD) and microsatellite instability high (MSI-H) status, and to dissect driver versus bystander status of BRCA1/2 mutations (BRCAm) in this context. METHODS: A pan-cancer comprehensive genomic profiling cohort (n = 213,199) was examined for overlap between BRCAm and MSI-H status. BRCA1/2 variant zygosity was examined and correlated with MSI-H status, tumor mutational burden, and genome-wide loss of heterozygosity (gLOH). Clinical histories of two patients with prostate cancer with co-occurring BRCAm and MSI-H are described. RESULTS: HRD and MSI-H phenotypes were generally mutually exclusive events (P < .001). BRCAm that co-occurred together with high tumor mutational burden or MSI-H were predominantly monoallelic bystander alterations. In breast, ovarian, and pancreatic cancers, very few BRCAm occurred in the context of MSI-H; however, in prostate cancer, 12.8% of BRCA1 and 3.4% of BRCA2 alterations co-occurred with MSI-H. In these BRCA-associated cancers, co-occurring BRCAm were generally monoallelic and were not associated with elevated gLOH. Two patients with prostate cancer with co-occurring BRCAm and MSI-H showed resistance to poly (ADP-ribose) polymerase inhibition but sensitivity to subsequent anti-programmed cell death protein 1 therapy. CONCLUSION: MSI-H status and HRD are generally mutually exclusive phenomena across cancer types, but may rarely co-occur, especially in prostate cancer. Although MSI-H samples had a higher BRCAm prevalence relative to microsatellite-stable tumors, these BRCA1/2 mutations were generally monoallelic and were not associated with elevated gLOH. Our findings suggest that most BRCAm coexisting with microsatellite instability are likely bystander events that may not result in sensitivity to poly (ADP-ribose) polymerase inhibitors.

Clinical genomic profiling in the management of patients with soft tissue and bone sarcoma.

There are more than 70 distinct sarcomas, and this diversity complicates the development of precision-based therapeutics for these cancers. Prospective comprehensive genomic profiling could overcome this challenge by providing insight into sarcomas' molecular drivers. Through targeted panel sequencing of 7494 sarcomas representing 44 histologies, we identify highly recurrent and type-specific alterations that aid in diagnosis and treatment decisions. Sequencing could lead to refinement or reassignment of 10.5% of diagnoses. Nearly one-third of patients (31.7%) harbor potentially actionable alterations, including a significant proportion (2.6%) with kinase gene rearrangements; 3.9% have a tumor mutational burden ≥10 mut/Mb. We describe low frequencies of microsatellite instability (<0.3%) and a high degree of genome-wide loss of heterozygosity (15%) across sarcomas, which are not readily explained by homologous recombination deficiency (observed in 2.5% of cases). In a clinically annotated subset of 118 patients, we validate actionable genetic events as therapeutic targets. Collectively, our findings reveal the genetic landscape of human sarcomas, which may inform future development of therapeutics and improve clinical outcomes for patients with these rare cancers.

Association of CD274 (PD-L1) Copy Number Changes with Immune Checkpoint Inhibitor Clinical Benefit in Non-Squamous Non-Small Cell Lung Cancer.

BACKGROUND: We sought to characterize response to immune checkpoint inhibitor (ICI) in non-squamous non-small cell lung cancer (NSCLC) across various CD274 copy number gain and loss thresholds and identify an optimal cutoff. MATERIALS AND METHODS: A de-identified nationwide (US) real-world clinico-genomic database was leveraged to study 621 non-squamous NSCLC patients treated with ICI. All patients received second-line ICI monotherapy and underwent comprehensive genomic profiling as part of routine clinical care. Overall survival (OS) from start of ICI, for CD274 copy number gain and loss cohorts across varying copy number thresholds, were assessed. RESULTS: Among the 621 patients, patients with a CD274 CN greater than or equal to specimen ploidy +2 (N = 29) had a significantly higher median (m) OS when compared with the rest of the cohort (N = 592; 16.1 [8.9-37.3] vs 8.6 [7.1-10.9] months, hazard ratio (HR) = 0.6 [0.4-1.0], P-value = .05). Patients with a CD274 copy number less than specimen ploidy (N = 299) trended toward a lower mOS when compared to the rest of the cohort (N = 322; 7.5 [5.9-11.3] vs 9.6 [7.9-12.8] months, HR = 0.9 [0.7-1.1], P-value = .3). CONCLUSION: This work shows that CD274 copy number gains at varying thresholds predict different response to ICI blockade in non-squamous NSCLC. Considering these data, prospective clinical trials should further validate these findings, specifically in the context of PD-L1 IHC test results.

Drivers of genomic loss of heterozygosity in leiomyosarcoma are distinct from carcinomas.

Leiomyosarcoma (LMS) is a rare, aggressive, mesenchymal tumor. Subsets of LMS have been identified to harbor genomic alterations associated with homologous recombination deficiency (HRD); particularly alterations in BRCA2. Whereas genomic loss of heterozygosity (gLOH) has been used as a surrogate marker of HRD in other solid tumors, the prognostic or clinical value of gLOH in LMS (gLOH-LMS) remains poorly defined. We explore the genomic drivers associated with gLOH-LMS and their clinical import. Although the distribution of gLOH-LMS scores are similar to that of carcinomas, outside of BRCA2, there was no overlap with previously published gLOH-associated genes from studies in carcinomas. We note that early stage tumors with elevated gLOH demonstrated a longer disease-free interval following resection in LMS patients. Taken together, and despite similarities to carcinomas in gLOH distribution and clinical import, gLOH-LMS are driven by different genomic signals. Additional studies will be required to isolate and confirm the unique differences in biological factors driving these differences.

Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1.

Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network.

Circulating Cell-Free DNA Yield and Circulating-Tumor DNA Quantity from Liquid Biopsies of 12 139 Cancer Patients.

BACKGROUND: The amounts of circulating cell-free DNA (cfDNA) and circulating-tumor DNA (ctDNA) present in peripheral blood liquid biopsies can vary due to preanalytic/analytic variables. In this study, we examined the impact of patient age, sex, stage, and tumor type on cfDNA yield, ctDNA fraction, and estimated ctDNA quantity from a large cohort of clinical liquid biopsy samples. METHODS: We performed a retrospective analysis of 12 139 consecutive samples received for liquid biopsy (FoundationOne® Liquid) clinical testing. RESULTS: Significant differences in both cfDNA yield and estimated ctDNA quantity were observed based on the underlying tumor type that initiated the liquid biopsy analysis and the stage of the patient (P < 0.001). In addition, significant differences in ctDNA quantity were present based in both the patient age and sex (P < 0.001). Importantly, we saw a significantly higher success rate of issuing a clinically useful report in patients with higher levels of cfDNA yield and ctDNA quantity (P < 0.001). CONCLUSIONS: In this study, we show that ctDNA quantity varied significantly based on patient age, sex, stage, and tumor type, which could offer an explanation as to why certain liquid biopsy specimens are more likely to fail sequencing or provide clinically meaningful results. In addition, this could affect future clinical decisions on the blood sample volumes required to allow successful liquid biopsy testing.

Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors.

BACKGROUND: The translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. CASE PRESENTATION: Thirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0-4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases - including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. CONCLUSIONS: CGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

The spectrum of tumors harboring BAP1 gene alterations.

Germline mutations in the BRCA1-associated protein (BAP1) gene (MIM # 603089) are associated with a substantially increased risk for developing melanoma, mesothelioma, and renal cell carcinoma. Somatic inactivation of the BAP1 gene was noted in these and other tumors types, including esophageal cancer and cholangiocarcinoma. The favorable response of BRCA1/2-associated tumors to poly (ADP-ribose) polymerase (PARP) inhibitor therapy, raises the possibility that tumors harboring BAP1 mutations may exhibit similar sensitivity to PARP inhibitor therapy. Given the possibility that BAP1 alterations may have therapeutic implications, this study was aimed to describe the spectrum of tumors that harbor BAP1 alterations. The Foundation Medicine database was queried for known or likely pathogenic BAP1 genomic variants through July 2019. Overall, 4982/374,694 (1.81%) tumors harbored pathogenic BAP1 genomic alterations. Highest rates were noted in mesothelioma (45.24%), cholangiocarcinoma (13.37%), renal cell carcinoma (10.52%), thymic cancer (8.16%), salivary gland cancer (6.18%), and melanoma (5.1%). There were 59 unique BAP1 short variants detected in at least 10 samples. More same tissue tumors of squamous cell histology harbored BAP1 alterations than adenocarcinomas. The current study highlights tumor types that display higher than previously appreciated rates of somatic BAP1 genomic alterations.

Comprehensive Genomic Profiling of Carcinoma of Unknown Primary Origin: Retrospective Molecular Classification Considering the CUPISCO Study Design.

BACKGROUND: Carcinoma of unknown primary origin (CUP) accounts for 2%-5% of newly diagnosed advanced malignancies, with chemotherapy as the standard of care. CUPISCO (NCT03498521) is an ongoing randomized trial using comprehensive genomic profiling (CGP) to assign patients with CUP to targeted or immunotherapy treatment arms based on genomic profiling. We performed a retrospective analysis of CUP cases referred for CGP to determine how many were potentially eligible for enrollment into an experimental CUPISCO arm. MATERIALS AND METHODS: Centrally reviewed adenocarcinoma and undifferentiated CUP specimens in the FoundationCore database were analyzed using the hybrid capture-based FoundationOne CDx assay (mean coverage, >600×). Presence of genomic alterations, microsatellite instability (MSI), tumor mutational burden (TMB), genomic loss of heterozygosity (gLOH), and programmed death-ligand 1 (PD-L1) positivity were determined. RESULTS: A total of 96 of 303 patients (31.7%) could be matched to an experimental CUPISCO arm. Key genomic alterations included ERBB2 (7.3%), PIK3CA (6.3%), NF1 (5.6%), NF2 (4.6%), BRAF (4.3%), IDH1 (3.3%), PTEN, FGFR2, EGFR (3.6% each), MET (4.3%), CDK6 (3.0%), FBXW7, CDK4 (2.3% each), IDH2, RET, ROS1, NTRK (1.0% each), and ALK (0.7%). Median TMB was 3.75 mutations per megabase of DNA; 34 patients (11.6%) had a TMB ≥16 mutations per megabase. Three patients (1%) had high MSI, and 42 (14%) displayed high PD-L1 expression (tumor proportion score ≥50%). gLOH could be assessed in 199 of 303 specimens; 19.6% had a score of >16%. CONCLUSIONS: Thirty-two percent of patients would have been eligible for targeted therapy in CUPISCO. Future studies, including additional biomarkers such as PD-L1 positivity and gLOH, may identify a greater proportion potentially benefiting from CGP-informed treatment. Clinical trial identification number. NCT03498521 IMPLICATIONS FOR PRACTICE: The findings of this retrospective analysis of carcinoma of unknown primary origin (CUP) cases validate the experimental treatment arms being used in the CUPISCO study (NCT03498521), an ongoing randomized trial using comprehensive genomic profiling to assign patients with CUP to targeted or immunotherapy treatment arms based on the presence of pathogenic genomic alterations. The findings also suggest that future studies including additional biomarkers and treatment arms, such as programmed death-ligand 1 positivity and genomic loss of heterozygosity, may identify a greater proportion of patients with CUP potentially benefiting from comprehensive genomic profiling-informed treatment.

Somatic HLA Class I Loss Is a Widespread Mechanism of Immune Evasion Which Refines the Use of Tumor Mutational Burden as a Biomarker of Checkpoint Inhibitor Response.

Neoantigen presentation arises as a result of tumor-specific mutations and is a critical component of immune surveillance that can be abrogated by somatic LOH of the human leukocyte antigen class I (HLA-I) locus. To understand the role of HLA-I LOH in oncogenesis and treatment, we utilized a pan-cancer genomic dataset of 83,644 patient samples, a small subset of which had treatment outcomes with immune checkpoint inhibitors (ICI). HLA-I LOH was common (17%) and unexpectedly had a nonlinear relationship with tumor mutational burden (TMB). HLA-I LOH was frequent at intermediate TMB, yet prevalence decreased above 30 mutations/megabase, suggesting highly mutated tumors require alternate immune evasion mechanisms. In ICI-treated patients with nonsquamous non-small cell lung cancer, HLA-I LOH was a significant negative predictor of overall survival. Survival prediction improved when combined with TMB, suggesting TMB with HLA-I LOH may better identify patients likely to benefit from ICIs. SIGNIFICANCE: This work shows the pan-cancer landscape of HLA-I LOH, revealing an unexpected "Goldilocks" relationship between HLA-I LOH and TMB, and demonstrates HLA-I LOH as a significant negative predictor of outcomes after ICI treatment. These data informed a combined predictor of outcomes after ICI and have implications for tumor vaccine development.This article is highlighted in the In This Issue feature, p. 211.

First results of the EORTC-SPECTA/Arcagen study exploring the genomics of rare cancers in collaboration with the European reference network EURACAN.

PURPOSE: Rare cancers are defined by an incidence of <6 out of 100 000 cases per year. They are under-represented in clinical research including tumour molecular analysis. The aim of Arcagen is to generate a multinational database integrating clinical and molecular information of patients with rare cancers. PATIENTS AND METHODS: We present the retrospective feasibility cohort of patients with rare cancers, with previously collected tumour samples available from any stage. Molecular analysis was performed using FoundationOne CDx for all histologies except for sarcoma where FoundationOne Heme was used. Clinical data including demographic data, medical history, malignant history, treatment and survival data were collected. RESULTS: Eighty-seven patients from three centres were screened; molecular data were obtained for 77 patients (41 sarcomas, 9 yolk sac tumours, 14 rare head and neck cancers, 13 thymomas). The median age at the time of diagnosis was 48 (range 28-85). Most patients had reportable genomic alterations (89%). The most common alterations were linked to cell cycle regulation (TP53, RB1, CDKN2A/B deletions and MDM2 amplification). Multiple activating single-nucleotide variants (SNVs) could be detected in the RAS/RAF family. The tumour mutational burden status was globally low across all samples with a median of 3 Muts/MB (range 0-52). Only 4 cases (ie, 4.7% of tumours) had direct actionable mutations for a treatment approved in Europe within the patient's tumour type. CONCLUSION: The Arcagen project aims to bridge the gap and improve knowledge of the molecular landscape of rare cancers by prospectively recruiting up to 1000 patients.

Activating genomic alterations in the Gs alpha gene (GNAS) in 274 694 tumors.

Activating point mutations in two codons (R201 and Q227) in the alpha subunit of the stimulatory GTP binding protein (GNAS) gene-coined gsp mutations-were originally reported in growth hormone secreting pituitary adenomas. In these tumor types, gsp activating mutations were associated with uncontrolled intracellular cAMP accumulation leading to cellular proliferation and tumor formation. Since the original description of gsp mutations in pituitary and later thyroid neoplasia, many more tumors were genotyped for these specific activating mutations. In this paradigm, GNAS is an oncogene that can be activated by other molecular mechanisms, such as DNA amplification and translocation. Herein, we describe the largest account to date of tumor types that harbor pathogenic GNAS genomic alterations (GAs) including the "classical" gsp activating point mutations, delineate some common features of these tumors, and speculate as to the possible mechanisms whereby GNAS activating GAs are associated with the various stages of tumorigenesis.

Loss of Slug Compromises DNA Damage Repair and Accelerates Stem Cell Aging in Mammary Epithelium.

DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging.