Comprehensive genomic characterization of HER2-low and HER2-0 breast cancer.

The molecular underpinnings of HER2-low and HER2-0 (IHC 0) breast tumors remain poorly defined. Using genomic findings from 1039 patients with HER2-negative metastatic breast cancer undergoing next-generation sequencing from 7/2013-12/2020, we compare results between HER2-low (n = 487, 47%) and HER2-0 tumors (n = 552, 53%). A significantly higher number of ERBB2 alleles (median copy count: 2.05) are observed among HER2-low tumors compared to HER2-0 (median copy count: 1.79; P = 2.36e-6), with HER2-0 tumors harboring a higher rate of ERBB2 hemideletions (31.1% vs. 14.5%). No other genomic alteration reaches significance after accounting for multiple hypothesis testing, and no significant differences in tumor mutational burden are observed between HER2-low and HER2-0 tumors (median: 7.26 mutations/megabase vs. 7.60 mutations/megabase, p = 0.24). Here, we show that the genomic landscape of HER2-low and HER2-0 tumors does not differ significantly, apart from a higher ERBB2 copy count among HER2-low tumors, and a higher rate of ERBB2 hemideletions in HER2-0 tumors.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

PURPOSE: Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS: In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS: In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). CONCLUSION: The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Deep-penetrating-nevus-like melanoma arising in patients with familial adenomatous polyposis syndrome.

Deep penetrating nevi (DPN) are uncommon but distinctive melanocytic neoplasms that show an epithelioid to spindle cell morphology, prominent pigmentation with melanophages, and a plexiform growth pattern. Molecularly, most DPN are thought to be characterized by dual activation of the mitogen-activated protein kinase and the wingless-related integration site (Wnt) pathways, the latter being most commonly driven by activating ß-catenin mutations. DPN-like melanomas are very rare but can be recognized through their overlapping morphologic and architectural features with DPN. Familial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome associated with multiple tumor types including colorectal carcinoma and desmoid fibromatosis. Like DPN, FAP is also driven by activation of the Wnt pathway, most commonly through loss of function mutations in APC, which is a major negative regulator of ß-catenin. Here we report two cases of DPN-like melanoma arising in FAP patients. While the small number of cases precludes definitive establishment of an etiologic link between these entities, the shared molecular pathogenesis of DPN-like lesions and FAP suggests that FAP patients may be at increased risk for this rare subtype of melanoma.

Programmatic Precision Oncology Decision Support for Patients With Gastrointestinal Cancer.

PURPOSE: With the growing number of available targeted therapeutics and molecular biomarkers, the optimal care of patients with cancer now depends on a comprehensive understanding of the rapidly evolving landscape of precision oncology, which can be challenging for oncologists to navigate alone. METHODS: We developed and implemented a precision oncology decision support system, GI TARGET, (Gastrointestinal Treatment Assistance Regarding Genomic Evaluation of Tumors) within the Gastrointestinal Cancer Center at the Dana-Farber Cancer Institute. With a multidisciplinary team, we systematically reviewed tumor molecular profiling for GI tumors and provided molecularly informed clinical recommendations, which included identifying appropriate clinical trials aided by the computational matching platform MatchMiner, suggesting targeted therapy options on or off the US Food and Drug Administration-approved label, and consideration of additional or orthogonal molecular testing. RESULTS: We reviewed genomic data and provided clinical recommendations for 506 patients with GI cancer who underwent tumor molecular profiling between January and June 2019 and determined follow-up using the electronic health record. Summary reports were provided to 19 medical oncologists for patients with colorectal (n = 198, 39%), pancreatic (n = 124, 24%), esophagogastric (n = 67, 13%), biliary (n = 40, 8%), and other GI cancers. We recommended ≥ 1 precision medicine clinical trial for 80% (406 of 506) of patients, leading to 24 enrollments. We recommended on-label and off-label targeted therapies for 6% (28 of 506) and 25% (125 of 506) of patients, respectively. Recommendations for additional or orthogonal testing were made for 42% (211 of 506) of patients. CONCLUSION: The integration of precision medicine in routine cancer care through a dedicated multidisciplinary molecular tumor board is scalable and sustainable, and implementation of precision oncology recommendations has clinical utility for patients with cancer.

MatchMiner: an open-source platform for cancer precision medicine.

Widespread, comprehensive sequencing of patient tumors has facilitated the usage of precision medicine (PM) drugs to target specific genomic alterations. Therapeutic clinical trials are necessary to test new PM drugs to advance precision medicine, however, the abundance of patient sequencing data coupled with complex clinical trial eligibility has made it challenging to match patients to PM trials. To facilitate enrollment onto PM trials, we developed MatchMiner, an open-source platform to computationally match genomically profiled cancer patients to PM trials. Here, we describe MatchMiner's capabilities, outline its deployment at Dana-Farber Cancer Institute (DFCI), and characterize its impact on PM trial enrollment. MatchMiner's primary goals are to facilitate PM trial options for all patients and accelerate trial enrollment onto PM trials. MatchMiner can help clinicians find trial options for an individual patient or provide trial teams with candidate patients matching their trial's eligibility criteria. From March 2016 through March 2021, we curated 354 PM trials containing a broad range of genomic and clinical eligibility criteria and MatchMiner facilitated 166 trial consents (MatchMiner consents, MMC) for 159 patients. To quantify MatchMiner's impact on trial consent, we measured time from genomic sequencing report date to trial consent date for the 166 MMC compared to trial consents not facilitated by MatchMiner (non-MMC). We found MMC consented to trials 55 days (22%) earlier than non-MMC. MatchMiner has enabled our clinicians to match patients to PM trials and accelerated the trial enrollment process.

Primary cutaneous SMARCA4-deficient undifferentiated malignant neoplasm: first two cases with clinicopathologic and molecular comparison to eight visceral counterparts.

SMARCA4-deficient undifferentiated malignant neoplasms (SD-UMN) comprise a group of aggressive tumors with epithelioid morphology that are characterized by loss of function of SMARCA4, a component of the SWI/SNF chromatin remodeling complex. SD-UMN was first recognized in the thoracic cavity but is now appreciated to occur at multiple anatomic sites. A notable exception has been skin. Here we report the first two cases of primary cutaneous SD-UMN and compare their features to a cohort of eight visceral cases arising in lung, gastrointestinal tract, and gallbladder. Evidence for a bona fide cutaneous origin included extensive clinical, radiologic, and serologic analyses that failed to identify a metastatic source as well as the molecular identification of a UV-associated mutational pattern. The cutaneous cases showed strikingly similar morphologic, immunohistochemical, and molecular features to the visceral cases, strongly suggesting that they belong to this family of tumors. In addition to biallelic inactivation of SMARCA4, both cutaneous tumors also showed biallelic inactivation of TP53 and CDKN2A, findings which also appear common in visceral cases. One patient died of disease at 18 months after diagnosis, consistent with the aggressive nature of this tumor. Our results expand the anatomic spectrum of SD-UMN, adding this entity to an already challenging differential diagnosis that includes melanoma, squamous cell carcinoma, Merkel cell carcinoma, epithelioid sarcoma, and others. Given the potentially aggressive nature of SD-UMN, the timely and accurate diagnosis of this entity may have implications for prognosis and therapy.

Ancestry-driven recalibration of tumor mutational burden and disparate clinical outcomes in response to immune checkpoint inhibitors.

The immune checkpoint inhibitor (ICI) pembrolizumab is US FDA approved for treatment of solid tumors with high tumor mutational burden (TMB-high; ≥10 variants/Mb). However, the extent to which TMB-high generalizes as an accurate biomarker in diverse patient populations is largely unknown. Using two clinical cohorts, we investigated the interplay between genetic ancestry, TMB, and tumor-only versus tumor-normal paired sequencing in solid tumors. TMB estimates from tumor-only panels substantially overclassified individuals into the clinically important TMB-high group due to germline contamination, and this bias was particularly pronounced in patients with Asian/African ancestry. Among patients with non-small cell lung cancer treated with ICIs, those misclassified as TMB-high from tumor-only panels did not associate with improved outcomes. TMB-high was significantly associated with improved outcomes only in European ancestries and merits validation in non-European ancestry populations. Ancestry-aware tumor-only TMB calibration and ancestry-diverse biomarker studies are critical to ensure that existing disparities are not exacerbated in precision medicine.

Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Identification and Management of Pathogenic Variants in BRCA1, BRCA2, and PALB2 in a Tumor-Only Genomic Testing Program.

PURPOSE: Tumor-only genomic testing can uncover somatic and germline pathogenic variants [pathogenic/likely pathogenic (P/LP)] in cancer predisposition genes. We describe the prevalence of P/LPs in BRCA1/2 and PALB2 (B1B2P2) across malignancies and the frequency of clinical germline testing (CGT) in patients with P/LPs in B1B2P2 identified on tumor-only testing. EXPERIMENTAL DESIGN: Among 7,575 patients with cancer tested between 2016 and 2018 with the OncoPanel tumor-only sequencing assay, we characterized P/LP frequencies by tumor type, receipt of CGT prior to or within 12 months after OncoPanel, and factors associated with CGT. RESULTS: 272 (3.6%) patients had OncoPanel-detected P/LPs in B1B2P2: 37.5% of P/LPs were in BRCA-related cancers; the remainder were in non-BRCA tumors. P/LPs were detected in ≥5% of breast, pancreatic, prostate, ovarian, nonmelanoma skin, endometrial, small cell lung, and colorectal cancers. 37.9% of patients with P/LPs received CGT prior to OncoPanel; an additional 10.7% underwent CGT within 12 months of OncoPanel. Among 132 with CGT, 88.6% had ≥1 clinical factor for CGT compared with 47.1% who did not undergo CGT. Patients with BRCA tumors were more likely to have CGT compared with those without (81.4% vs. 29.0%, P < 0.0001). Among patients with CGT, 70.5% (93/132) of P/LPs were germline. CONCLUSIONS: Tumor-only genomic testing identified P/LPs in B1B2P2 in 3.6% of patients. 52.9% of patients with tumor-detected P/LPs and without CGT did not meet personal or family history criteria for CGT. In addition, some patients with tumor-detected P/LPs were not referred for CGT, especially those with non-BRCA tumors. Given implications for treatment selection and familial cancer risk, processes to reliably trigger CGT from tumor-genomic findings are needed.

Germline Testing Data Validate Inferences of Mutational Status for Variants Detected From Tumor-Only Sequencing.

Pathogenic germline variants (PGVs) in cancer susceptibility genes are usually identified through germline testing of DNA from blood or saliva: their detection can affect treatment options and potential risk-reduction strategies for patient relatives. PGV can also be identified in tumor sequencing assays, which, when performed without patient-matched normal specimens, render determination of variants' germline or somatic origin critical. METHODS: Tumor-only sequencing data from 1,608 patients were retrospectively analyzed to infer germline versus somatic status of variants using an information-theoretic, gene-independent approach. Loss of heterozygosity was also determined. Predicted mutational models were compared with clinical germline testing results. Statistical measures were computed to evaluate performance. RESULTS: Tumor-only sequencing detected 3,988 variants across 70 cancer susceptibility genes for which germline testing data were available. We imputed germline versus somatic status for > 75% of all detected variants, with a sensitivity of 65%, specificity of 88%, and overall accuracy of 86% for pathogenic variants. False omission rate was 3%, signifying minimal error in misclassifying true PGV. A higher portion of PGV in known hereditary tumor suppressors were found to be retained with loss of heterozygosity in the tumor specimens (72%) compared with variants of uncertain significance (58%). CONCLUSION: Analyzing tumor-only data in the context of specimens' tumor cell content allows precise, systematic exclusion of somatic variants and suggests a balance between type 1 and 2 errors for identification of patients with candidate PGV for standard germline testing. Although technical or systematic errors in measuring variant allele frequency could result in incorrect inference, misestimation of specimen purity could result in inferring somatic variants as germline in somatically mutated tumor suppressor genes. A user-friendly bioinformatics application facilitates objective analysis of tumor-only data in clinical settings.

Twists and turns from "tumor in tumor" profiling: surveillance of chronic lymphocytic leukemia (CLL) leads to detection of a lung adenocarcinoma, whose genomic characterization alters the original hematologic diagnosis.

Comprehensive characterization of somatic genomic alterations has led to fundamental shifts in our understanding of tumor biology. In clinical practice, these studies can lead to modifications of diagnosis and/or specific treatment implications, fulfilling the promise of personalized medicine. Herein, we describe a 78-yr-old woman under surveillance for long-standing untreated chronic lymphocytic leukemia (CLL). Molecular studies from a peripheral blood specimen revealed a TP53 p.V157F mutation, whereas karyotype and fluorescence in situ hybridization (FISH) identified a 17p deletion, trisomy 12, and no evidence of IGH-CCND1 rearrangement. Positron emission tomography-computed tomography scan identified multistation intra-abdominal lymphadenopathy and a pulmonary nodule, and subsequent pulmonary wedge resection confirmed the presence of a concurrent lung adenocarcinoma. Targeted next-generation sequencing of the lung tumor identified an EGFR in-frame exon 19 deletion, two TP53 mutations (p.P152Q, p.V157F), and, unexpectedly, a IGH-CCND1 rearrangement. Follow-up immunohistochemistry (IHC) studies demonstrated a cyclin D1-positive lymphoid aggregate within the lung adenocarcinoma. The presence of the TP53 p.V157F mutation in the lung resection, detection of an IGH-CCND1 rearrangement, and cyclin D1 positivity by IHC led to revision of the patient's hematologic diagnosis and confirmed the extranodal presence of mantle cell lymphoma within the lung mass, thus representing a "tumor in tumor." Manual review of the sequencing data suggested the IGH-CCND1 rearrangement occurred via an insertional event, whose size precluded detection by original FISH studies. Thus, routine imaging for this patient's known hematologic malignancy led to detection of an unexpected solid tumor, whose subsequent precision medicine studies in the solid tumor redefined the original hematological diagnosis.

Molecular Characterization and Therapeutic Targeting of Colorectal Cancers Harboring Receptor Tyrosine Kinase Fusions.

PURPOSE: Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion-associated colorectal cancer. EXPERIMENTAL DESIGN: We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results. RESULTS: We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair-deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E-mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy. CONCLUSIONS: RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Hedgehog Pathway Alterations Downstream of Patched-1 Are Common in Infundibulocystic Basal Cell Carcinoma.

ABSTRACT: The infundibulocystic variant of basal cell carcinoma (BCC) is characterized histologically by anastamosing strands of basaloid epithelium with associated small infundibular-type cysts. Since its first description in 1987, this rare entity has generated considerable controversy with some authors classifying it as a benign follicular neoplasm rather than a BCC subtype. Prior studies aiming to settle this issue using immunohistochemical analysis reached opposite conclusions. The defining feature of BCC is activation of the Hedgehog signaling pathway, and mutations in Patched-1 (PTCH1) are the most common molecular finding in both sporadic and inherited forms of BCC. Mutations in other downstream components including Smoothened (SMO) and Suppressor of Fused (SUFU) also occur, but are much less common. Here, we report a molecular genetic analysis of a small series of infundibulocystic BCC using a next-generation DNA sequencing platform. All 4 cases harbored mutations or other genetic alterations in components of the Hedgehog pathway, supporting the classification of this entity as a BCC variant. Interestingly, these tumors were enriched for genetic alterations downstream of PTCH1, involving SUFU, SMO, GLI1, and GLI2. This observation was of particular interest given that rare kindreds of the Multiple Hereditary Infundibulocystic BCC syndrome (MHIBCC), which is related, but possibly distinct from the nevoid BCC syndrome, harbored mutations in SUFU. Our results support the classification of the infundibulocystic variant as a subtype of BCC, and suggest that the level at which genetic alterations occur within the Hedgehog pathway may be an important determinant of the morphologic features in BCC.

Genomic Characterization of de novo Metastatic Breast Cancer.

PURPOSE: In contrast to recurrence after initial diagnosis of stage I-III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS). EXPERIMENTAL DESIGN: Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC. RESULTS: When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2- tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041). CONCLUSIONS: Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.

Molecular Characterization of Neuroendocrine Carcinomas of the Endometrium: Representation in All 4 TCGA Groups.

High-grade neuroendocrine carcinomas (NEC) of the endometrium are rare and account for <1% of all endometrial carcinomas. Both small cell neuroendocrine carcinoma (SCNEC) and large cell neuroendocrine carcinoma (LCNEC) morphologies have been reported. Little is known regarding the molecular features of endometrial NEC including how they compare to pulmonary NEC (the most common site for these neoplasms) and the more common endometrial carcinoma histotypes. In this study, we investigated the molecular alterations in a series of endometrial NEC using a targeted next generation sequencing panel (Oncopanel). Fourteen NEC were sequenced; pure NEC (n=4) and mixed (n=10) with endometrioid adenocarcinoma (n=9) or carcinosarcoma (n=1). The NEC components of mixed tumors comprised LCNEC (n=6) and SCNEC (n=4). The 4 pure NEC comprised LCNEC (n=2) and SCNEC (n=2). Molecular analysis classified tumors into the 4 The Cancer Genome Atlas groups: (1) POLE-mutated/ultramutated (1/14; 7%), (2) microsatellite instability/hypermutated (6/14; 43%), (3) TP53 mutated/copy number high (2/14; 14%), or (4) no specific molecular profile (5/14; 36%). Overall, 50% of cases were ultramutated or hypermutated. In 8 cases of mixed carcinomas, the different histologic components were macrodissected and separately sequenced; molecular alterations were nearly identical among the 2 components, with the non-NEC component harboring slightly increased tumor mutational burden. Only 2 carcinomas (both with pure SCNEC morphology) had a molecular profile that would be expected in typical pulmonary SCNEC (RB1 deletion and TP53 mutations). Our findings, similar to data from NECs of other anatomic sites, suggest that the molecular context may be important when selecting therapies for women with endometrial NEC. Immune checkpoint inhibition may be a reasonable approach to treatment of microsatellite instability-NEC and we thus recommend that all endometrial NEC be tested for mismatch repair abnormalities, either molecularly or by mismatch repair protein immunohistochemistry.

Clinical and molecular characterization of virus-positive and virus-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC. METHODS: In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure. RESULTS: Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival. CONCLUSIONS: These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.

ViroPanel: Hybrid Capture and Massively Parallel Sequencing for Simultaneous Detection and Profiling of Oncogenic Virus Infection and Tumor Genome.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Tumor Mutational Burden and PTEN Alterations as Molecular Correlates of Response to PD-1/L1 Blockade in Metastatic Triple-Negative Breast Cancer.

PURPOSE: Few patients with metastatic triple-negative breast cancer (mTNBC) benefit from immune checkpoint inhibitors (ICI). On the basis of immunotherapy response correlates in other cancers, we evaluated whether high tumor mutational burden (TMB) ≥10 nonsynonymous mutations/megabase and PTEN alterations, defined as nonsynonymous mutations or 1 or 2 copy deletions, were associated with clinical benefit to anti-PD-1/L1 therapy in mTNBC. EXPERIMENTAL DESIGN: We identified patients with mTNBC, who consented to targeted DNA sequencing and were treated with ICIs on clinical trials between April 2014 and January 2019 at Dana-Farber Cancer Institute (Boston, MA). Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were correlated with tumor genomic features. RESULTS: Sixty-two women received anti-PD-1/L1 inhibitors alone (23%) or combined with targeted therapy (19%) or chemotherapy (58%). High TMB (18%) was associated with significantly longer PFS (12.5 vs. 3.7 months; P = 0.04), while PTEN alterations (29%) were associated with significantly lower ORR (6% vs. 48%; P = 0.01), shorter PFS (2.3 vs. 6.1 months; P = 0.01), and shorter OS (9.7 vs. 20.5 months; P = 0.02). Multivariate analyses confirmed that these associations were independent of performance status, prior lines of therapy, therapy regimen, and visceral metastases. The survival associations were additionally independent of PD-L1 in patients with known PD-L1 and were not found in mTNBC cohorts treated with chemotherapy (n = 90) and non-ICI regimens (n = 169). CONCLUSIONS: Among patients with mTNBC treated with anti-PD-1/L1 therapies, high TMB and PTEN alterations were associated with longer and shorter survival, respectively. These observations warrant validation in larger datasets.