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A woman presented to medical oncology with almost 4 years of untreated, slowly progressing, triple negative metastatic breast cancer to the lung. About 15 years prior, she was diagnosed with invasive ductal carcinoma of the right breast with ipsilateral chest wall recurrence 6 years later. Comprehensive molecular profiling of a metastatic lesion detected a hotspotETV6-NTRK3fusion, which was not present on circulating tumour DNA or molecular profile performed 4 years prior. A second look pathological examination demonstrated tumour characteristics consistent with secretory breast carcinoma. Identification ofETV6­-NKRT3fusion allowed for treatment with larotrectinib, a tyrosine kinase inhibitor specifically indicated for secretory breast carcinoma. After 3 months, she experienced a partial response.

Background Advanced pelvic squamous cell carcinoma (pSCC) is a broad category of cancers affecting different pelvic organs and usually featuring unfavorable clinical outcomes. Thus, we aimed to assess genomic differences among pSCC cases and learn whether pSCC could potentially benefit from targeted therapies and/or immunotherapy. Materials and Methods A total of 1917 advanced pSCCs, including penile (penSCC), male urethral (murthSCC), male anal (manSCC), female urethral (furthSCC), vulvar (vulSCC), cervical (crvSCC), female anal (fanSCC), and vaginal (vagSCC), underwent comprehensive genomic profiling (CGP). We used hybrid capture-based CGP to evaluate recurrent genomic alterations (GAs). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on up to 95 loci. Programmed cell-death-ligand-1 (PD-L1) expression was determined by immunohistochemistry (IHC; Dako 22C3). Results PIK3CA was the most frequently identified potentially “actionable” GA (22%-43%), followed by mTOR pathway [PTEN (0%-18%), FBXW7 (7%-29%)], and cell-cycle GAs. DNA-damage response (DDR) GAs and receptor-tyrosine kinase (RTK) targeted options were uncommon. NOTCH1 GAs were present in >15% of penSCC and vulvSCC. TMB ≥10 mut/Mb was >15% in manSCC, fanSCC, crvSCC, and vagSCC. PD-L1 high expression was >18% in all pSCC except urthSCC, manSCC, and vagSCC. HPV-16/18 detection was highest in manSCC, fanSCC, and crvSCC. Conclusion Despite similar histology, pSCCs can differ in GAs and HPV status. Overall, PIK3CA is the most frequent potentially “targetable” GA followed by mTOR and cell cycle pathway. RTK and DDR GAs are rare in pSCC. Immunotherapy could be considered for pSCC management based on TMB and PD-L1 expression.

Supplementary Table 4

Supplementary Table 1. This is a table of all mutations found the four InvTCC tumors, there positions, and genotype quality scores.

All supplemental text and 9 supplementary figures

Supplementary Tables 2 and 3. Listing genotypes and clinical data.

Supplementary Table 1

Supplementary Table 3

Background In patients with non-small cell lung cancer (NSCLC), 10%-40% will eventually develop brain metastases. We present the clinicopathologic, genomic, and biomarker landscape of a large cohort of NSCLC brain metastases (NSCLC-BM) samples. Materials and Methods We retrospectively analyzed 3035 NSCLC-BM tested with comprehensive genomic profiling (CGP) during routine clinical care. In addition, we compared the NSCLC-BM to a separate cohort of 7277 primary NSCLC (pNSCLC) specimens. Finally, we present data on 67 paired patients with NSCLC-BM and pNSCLC. Results Comprehensive genomic profiling analysis of the 3035 NSCLC-BMs found that the most frequent genomic alterations (GAs) were in the TP53, KRAS, CDKN2A, STK11, CDKN2B, EGFR, NKX2-1, RB1, MYC, and KEAP1 genes. In the NSCLC-BM cohort, there were significantly higher rates of several targetable GAs compared with pNSCLC, including ALK fusions, KRAS G12C mutations, and MET amplifications; and decreased frequency of MET exon14 skipping mutations (all P < .05). In the subset of NSCLC-BM (n = 1063) where concurrent PD-L1 immunohistochemistry (IHC) was performed, 54.7% of the patients with NSCLC-BM were eligible for pembrolizumab based on PD-L1 IHC (TPS ≥ 1), and 56.9% were eligible for pembrolizumab based on TMB-High status. In addition, in a series 67 paired pNSCLC and NSCLC-BM samples, 85.1% (57/67) had at least one additional GA discovered in the NSCLC-BM sample when compared with the pNSCLC sample. Conclusions Herein, we defined the clinicopathologic, genomic, and biomarker landscape of a large cohort of patients with NSCLC-BM which can help inform study design of future clinical studies for patients with NSCLC with BM. In certain clinical situations, metastatic NSCLC brain tissue or cerebral spinal fluid specimens may be needed to fully optimize personalized treatment.

Background In the current study, we examined the real-world prevalence of highly pigmented advanced melanomas (HPMel) and the clinicopathologic, genomic, and ICPI biomarker signatures of this class of tumors. Materials and Methods Our case archive of clinical melanoma samples for which the ordering physician requested testing for both PD-L1 immunohistochemistry (IHC) and comprehensive genomic profiling (CGP) was screened for HPMel cases, as well as for non-pigmented or lightly pigmented advanced melanoma cases (LPMel). Results Of the 1268 consecutive melanoma biopsies in our archive that had been submitted for PD-L1 IHC, 13.0% (165/1268) were HPMel and 87.0% (1103/1268) were LPMel. In the HPMel cohort, we saw a significantly lower tumor mutational burden (TMB, median 8.8 mutations/Mb) than in the LPMel group (11.4 mut/Mb), although there was substantial overlap. In examining characteristic secondary genomic alterations (GA), we found that the frequencies of GA in TERTp, CDKN2A, TP53, and PTEN were significantly lower in the HPMel cases than in LPMel. A higher rate of GA in CTNNB1, APC, PRKAR1A, and KIT was identified in the HPMel cohort compared with LPMel. Conclusions In this study, we quantified the failure rates of melanoma samples for PD-L1 testing due to high melanin pigmentation and showed that CGP can be used in these patients to identify biomarkers that can guide treatment decisions for HPMel patients. Using this practical clinical definition for tumor pigmentation, our results indicate that HPMel are frequent at 13% of melanoma samples, and in general appear molecularly less developed, with a lower TMB and less frequent secondary GA of melanoma progression.

Introduction Tumors with alterations in BRCA1 or BRCA2 (BRCAm) are sensitive to poly ADP ribose polymerase inhibitors (PARPi), with significant benefit in ovary, breast, prostate, and pancreatic cancers. Despite strong early responses on PARPi, many patients eventually exhibit relapse. Previous literature has identified BRCA1/2 reversion as a common class of acquired alteration in the resistance setting; however, additional resistance pathways have not been well characterized. Here, we examined 100 BRCAm patients who were profiled with serial biopsy during clinical care to identify possible therapy resistance alterations. Methods Comprehensive genomic profiling (CGP) was carried out in a Clinical Laboratory Improvement Amendments (CLIA)-certified, CAP (College of American Pathologists)-accredited laboratory (Foundation Medicine Inc., Cambridge, MA, USA). Tissue biopsy CGP was performed on FFPE blocks examining at least 324 genes for all classes of alterations (FoundationOne® and FoundationOne® CDx). Liquid biopsy CGP was performed examining at least 62 genes (FoundationACT®, FoundationOne®Liquid).One hundred breast cancer patients with baseline BRCAm were serially profiled with CGP testing during routine clinical care (n=50 tissue then liquid; n=50 tissue for both tests). As a comparator, we examined 1,294 patients without a baseline BRCAm (BRCAwt) profiled with serial biopsy (n = 585 tissue then liquid; n=709 tissue for both tests). Co-occurrence analyses (Fisher’s exact) were run on the full research dataset, including 12,198 breast-biopsied (local) and 16,586 metastatic-biopsied samples. All classes of genomic alterations were included in the analysis. Results Of the serially biopsied samples, 7.2% (100/1,394) had baseline BRCAm. In patients with baseline BRCAm, acquired alterations were frequently observed in BRCA1/2 (17%), ESR1 (16%), TP53 (15%), MYC (12%), CREBBP (10%), RB1 (9%), PIK3CA (9%), and NF1 (5%). To understand if any of these are specific to the BRCAm population, we compared the milieu of acquired alterations to a serially biopsied BRCAwt cohort (n=1,294). While many of the alterations were common and shared across both cohorts, likely due to endocrine therapy use, acquired alterations in BRCA1, BRCA2, and CREBBP were specifically enriched in the BRCAm population (all p0.05). Acquired BRCAm were never observed in cases with baseline deletion of BRCA1/2. The high rate of acquired CREBBP alterations was specific to the BRCAm population (10% BRCAm v 1% BRCAwt; p = 0.0013), suggesting a possible role for CREBBP in platinum or PARPi resistance. Consistent with this, BRCAm and CREBBP significantly co-occur in the metastatic setting (Odds ratio, OR = 1.6; p = 0.016) but not in the local setting (OR = 1.1; p = 0.68). Conclusions Analysis of serially biopsied BRCAm breast cases revealed frequent acquisition of BRCA1/2 reversion mutations and CREBBP alterations that are not frequently observed in BRCAwt samples. Additional studies are warranted to investigate the possible role of CREBBP in PARPi therapy resistance. Citation Format: Ethan Sokol, Smruthy Sivakumar, Brennan Decker, Jeffrey Ross, Priti Hegde. Serially biopsied BRCA1/2 mutant breast tumors frequently acquire alterations in BRCA1, BRCA2, and CREBBP [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 P5-13-02.

Purpose:Oncogenic kinase fusions are targetable with approved and investigational therapies and can also mediate acquired resistance (AR) to targeted therapy. We aimed to understand the clinical validity of liquid biopsy comprehensive genomic profiling (CGP) to detect kinase fusions pan tumor.Experimental Design:CGP was performed on plasma and tissue samples during clinical care. All exons plus selected introns of 16 kinases involved in oncogenic fusions (ALK, BRAF, EGFR, ERBB2, FGFR1/2/3, MET, NTRK1/2/3, PDGFRA/B, RAF1, RET, and ROS1) were sequenced to capture fusions, including well-characterized and novel breakpoints. Plasma circulating tumor DNA (ctDNA) fraction was estimated to inform sensitivity.Results:Of 36,916 plasma cases, 32,492 (88%) had detectable ctDNA. Kinase fusions were detected in 1.8% of ctDNA-positive cases (571/32,492) and were most prevalent in patients with cholangiocarcinoma (4.2%), bladder cancer (3.6%), and non–small cell lung cancer (NSCLC; 3.1%). Of the 63 paired patient samples that had tissue and ctDNA specimens collected within 1 year and with estimated plasma ctDNA fraction >1%, fusions were detected in 47 of 51 (92%) liquid specimens with a fusion in the tissue sample. In 32 patients with fusions detected in liquid but not in tissue, 21 (66%) had evidence of putative acquired resistance.Conclusions:Targetable kinase fusions are identified in ctDNA across cancer types. In pairs with tissue-identified fusions, fusion detection in ctDNA is reliable with elevated ctDNA fraction. These data support the validity of CGP to enable ctDNA-based fusion detection for informing clinical care in patients with advanced cancer.

Table S1 describes primer sequences and amplicon details. Table S2 summarizes percent callable bases and depth summary for the dataset. Table S3 lists the criteria for the hard filtering of variant calls. Table S4 shows a list of common variants used in the construction of the breast cancer polygenic risk scores. Table S5 shows a summary of 649 protein-truncating variants by gene in 597 unique samples. Table S6 tabulates protein-truncating variants and carrier counts.

Supplementary Data from The Pan-Tumor Landscape of Targetable Kinase Fusions in Circulating Tumor DNA

eMethods describes additional details on the targeted sequencing and data processing methodology. Table S7 shows the associations between breast cancer genetic loci and breast cancer tumor characteristics diagnosed with breast cancer at below age 50. Table S8 shows the associations between breast cancer genetic loci and breast cancer tumor characteristics diagnosed with breast cancer at age 50 years and above. Figure S1 illustrates a flowchart summarizing the data processing work flow. Figure S2 shows a visual summary of the different rare variants called. Figure 3 shows the relationship between polygenic risk score (PRS) and protein-truncating variant carriership illustrated by boxplots. Figures S4-S6 show the results obtained by considering a subset of HBOC genes for the analyses on tumor characteristics, survival and mode of detection, respectively.

Genetic variants that increase breast cancer risk can be rare or common. This study tests whether the genetic risk stratification of breast cancer by rare and common variants in established loci can discriminate tumors with different biology, patient survival, and mode of detection. Multinomial logistic regression tested associations between genetic risk load [protein-truncating variant (PTV) carriership in 31 breast cancer predisposition genes—or polygenic risk score (PRS) using 162 single-nucleotide polymorphisms], tumor characteristics, and mode of detection (OR). Ten-year breast cancer–specific survival (HR) was estimated using Cox regression models. In this unselected cohort of 5,099 patients with breast cancer diagnosed in Sweden between 2001 and 2008, PTV carriers (n = 597) were younger and associated with more aggressive tumor phenotypes (ER-negative, large size, high grade, high proliferation, luminal B, and basal-like subtype) and worse outcome (HR, 1.65; 1.16–2.36) than noncarriers. After excluding 92 BRCA1/2 carriers, PTV carriership remained associated with high grade and worse survival (HR, 1.76; 1.21–2.56). In 5,007 BRCA1/2 noncarriers, higher PRS was associated with less aggressive tumor characteristics (ER-positive, PR-positive, small size, low grade, low proliferation, and luminal A subtype). Among patients with low mammographic density (BRCA1/2 PTV carriers were more often interval than screen-detected breast cancer (OR, 1.89; 1.12–3.21) than noncarriers. In contrast, higher PRS was associated with lower risk of interval compared with screen-detected cancer (OR, 0.77; 0.64–0.93) in women with low mammographic density. These findings suggest that rare and common breast cancer susceptibility loci are differentially associated with tumor characteristics, survival, and mode of detection.Significance: These findings offer the potential to improve screening practices for breast cancer by providing a deeper understanding of how risk variants affect disease progression and mode of detection. Cancer Res; 78(21); 6329–38. ©2018 AACR.

Evidence linking coding germline variants in breast cancer (BC)-susceptibility genes other than BRCA1, BRCA2, and CHEK2 with contralateral breast cancer (CBC) risk and breast cancer-specific survival (BCSS) is scarce. The aim of this study was to assess the association of protein-truncating variants (PTVs) and rare missense variants (MSVs) in nine known (ATM, BARD1, BRCA1, BRCA2, CHEK2, PALB2, RAD51C, RAD51D, and TP53) and 25 suspected BC-susceptibility genes with CBC risk and BCSS. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated with Cox regression models. Analyses included 34,401 women of European ancestry diagnosed with BC, including 676 CBCs and 3,449 BC deaths; the median follow-up was 10.9 years. Subtype analyses were based on estrogen receptor (ER) status of the first BC. Combined PTVs and pathogenic/likely pathogenic MSVs in BRCA1, BRCA2, and TP53 and PTVs in CHEK2 and PALB2 were associated with increased CBC risk [HRs (95% CIs): 2.88 (1.70-4.87), 2.31 (1.39-3.85), 8.29 (2.53-27.21), 2.25 (1.55-3.27), and 2.67 (1.33-5.35), respectively]. The strongest evidence of association with BCSS was for PTVs and pathogenic/likely pathogenic MSVs in BRCA2 (ER-positive BC) and TP53 and PTVs in CHEK2 [HRs (95% CIs): 1.53 (1.13-2.07), 2.08 (0.95-4.57), and 1.39 (1.13-1.72), respectively, after adjusting for tumor characteristics and treatment]. HRs were essentially unchanged when censoring for CBC, suggesting that these associations are not completely explained by increased CBC risk, tumor characteristics, or treatment. There was limited evidence of associations of PTVs and/or rare MSVs with CBC risk or BCSS for the 25 suspected BC genes. The CBC findings are relevant to treatment decisions, follow-up, and screening after BC diagnosis.

BCORL1 is a transcriptional corepressor homologous to BCOR. We describe 12 BCORL1-altered uterine sarcomas with striking resemblance to BCOR-altered endometrial stromal sarcoma (BCOR-ESS), including 5 with BCORL1 rearrangements (JAZF1-BCORL1, EP300-BCORL1, or internal BCORL1 rearrangement), 5 with inactivating BCORL1 mutations (T513fs*22, P600fs*1, R945*, R1196*, or R1265fs*4) and 2 with homozygous BCORL1 deletion. The median patient age was 57.5 years (range 33-79). An association with aggressive clinical behavior was identified. Diagnoses assigned prior to genomic testing varied: 7 tumors were previously diagnosed as ESS, 2 as high-grade uterine sarcomas, 2 as myxoid uterine leiomyosarcomas, and 1 as a uterine spindle cell neoplasm consistent with leiomyosarcoma. Tumors harbored frequent gelatinous, mucomyxoid-like appearance by gross examination and unique histology with morphological overlap with BCOR-ESS. Key microscopic features included (1) a spindle cell appearance, most often with at least focal myxoid stroma, (2) variable amounts of hypocellular fibromyxoid spindle areas with lower grade atypia and/or (3) variable amounts of epithelioid areas with higher grade atypia. Specifically, spindle and epithelioid components were present in 100 and 75% of sarcomas, respectively; myxoid stroma was identified in 83%, collagen plaques or fibrosis in 50%, and high-grade nuclear atypia was present in 42%. Like BCOR-ESS, 50% of BCORL1-altered sarcomas exhibited CDK4 amplification or CDKN2A loss. In contrast, 33% harbored NF1 alterations, while 25% had other alterations in the NF2-mTOR pathway, expanding potential therapeutic targets. In conclusion, inactivating BCORL1 genomic alterations may define a distinct subset of high-grade endometrial stromal sarcomas with biological overlap with BCOR-ESS, both of which may mimic myxoid leiomyosarcomas.

PURPOSE Profiling of circulating tumor DNA (ctDNA) is increasingly adopted in the management of solid tumors, concurrent with increased availability of more comprehensive ctDNA panels. However, variable ctDNA shed can result in variable assay sensitivity. We studied the relationship between ctDNA tumor fraction (TF) and detection of actionable alterations across cancer types. METHODS A total of 23,482 liquid biopsies (LBx) submitted between September 2020 and October 2021 were sequenced using a hybrid capture panel that reports genomic alterations (GAs) and genomic biomarkers across 324 cancer-related genes. The primary end points were the prevalence of targetable GAs by cancer type and detection in relationship to ctDNA TF. Sensitivity of detection in LBx was assessed in 1,289 patients with available tissue results. RESULTS 94% (n = 22,130) of LBx had detectable ctDNA, with a median TF of 2.2%. LBx profiling detected GAs in National Comprehensive Cancer Network category 1 genes in 37% of lung, 30% of prostate, 36% of breast, and 51% of colon cancer cases. Potential germline GAs flagged on clinical reports were detected in genes including BRCA1/2, PALB2, CHEK2, and ATM. Polyclonal mutations in genes associated with resistance such as AR, ESR1, RB1, and NF1 were detected. The sensitivity of LBx to detect driver alterations identified in tissue biopsy from the same patient ranged from 58% to 86% but was consistently at or near 100% in cases with TF ≥ 10%. CONCLUSION Elevated ctDNA shed is associated with both high sensitivity and negative predictive value for detection of actionable GAs. The presence of elevated TF suggests adequate tumor profiling and may reduce the value of subsequent reflex to confirmatory tissue testing in patients with negative LBx results.

BACKGROUND: Pathogenic POLE exonuclease domain mutations (pPOLE) undermine mismatch error correction during DNA replication, causing somatic ultramutation and response to immunotherapy. We examined the pan-cancer landscape of POLE mutations and applied a POLE-specific variant classification model. METHODS: Comprehensive genomic profiling was performed during clinical care. Mutational signature calling was performed via decomposition using the 96-feature single-base substitution COSMIC reference signatures. A POLE-specific classification model encompassing mutation position in the exonuclease domain, TMB, presence of POLE signature, absence of other signatures, germline frequency, and other features was applied to identify pPOLE mutations causative of ultramutation. RESULTS: POLE mutation status was evaluated in 458,437 samples (425,520 tissue biopsies (TB) and 32,917 liquid biopsies (LB)). One or more POLE alterations, including pathogenic alterations and variants of unknown significance (VUS), were detected in 3.8% of samples. 19,470 total alterations were identified, 84.8% of which were missense substitutions. Application of the POLE-specific classification model identified 35 unique pathogenic variants, many of which were VUS prior to this study. 749 samples harbored a pPOLE, more than half (56.6%) of which were either p.P286R (n=245) or p.V411L (n=179). pPOLE were found in 1.4% (199/13,688) of endometrial cancers (EC) and 0.5% (270/55,981) of colorectal cancers (CRC) and were rarer in a long tail of other malignancies. The overall pPOLE rate was significantly lower in LB than TB (0.02% vs 0.17%, P CONCLUSIONS: pPOLE were seen in both TB and LB across cancer types. The high rate of passenger mutations underscores the utility of this POLE-specific variant classification model. Because TMB can be underestimated when tumor purity is near the limit of detection for the assay, accurate detection and classification of pPOLE is critical for identifying patients who may benefit from immunotherapy. Citation Format: Rachel B. Keller, James Haberberger, Tyler Janovitz, Alexa B. Schrock, Hanna Tukachinsky, Lei Zhong, Douglas A. Mata, Lyle V. Lopez, Zoe Fleischmann, Radwa Sharaf, Ethan S. Sokol, Garrett M. Frampton, Nimesh R. Patel, Douglas I. Lin, Geoff R. Oxnard, Erik A. Williams, Julia A. Elvin, Brennan Decker. POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy [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 305.

Pilomyxoid astrocytomas (PMAs) are tumors of infancy occurring in the hypothalamic chiasmatic region, with somewhat worse prognosis than pilocytic astrocytomas (PAs). We report molecular findings in PAs with a dominant pilomyxoid histomorphology across the various age groups and CNS locations. Foundation Medicine comprehensive genomic profiling (CGP) (FoundationOne, FoundationOneCDx, FoundationOneHeme) were performed during routine clinical care. A retrospective cohort analysis was done to evaluate the genomic characteristics of PMAs. 57 PMAs were identified, of which 6 occurred in patients < 12 months old (10.5%); 14 in 1-3 year old (24.6%); 15 in 4-10 y.o. (26.3%); 12 in 11-20 y.o. (21.1%), and 10 occurred in young adults 21-39 y.o. (17.5%). 30/57 (52.6%) were female and 27/57 (47.4%) male. 55 PMAs occurred in the brain and 2 in the spinal cord. Tumors were categorized by notable rearrangements and point mutations. The common fusion, KIAA1549:BRAF, was detected in 23/57 samples (40.4%). BRAF fusions with other gene partners (BRAF, TAX1BP1) occurred in 2, and BRAF V600E mutations in 4 patients. An additional 4 patients had FGFR1 fusions (FGFR1, TACC1) and 14 had FGFR mutations. Other alterations included 3 NF1 mutations, 2 NTRK family receptor tyrosine kinase fusions, 2 RAF gene fusions, 2 other (ATRX, ALK) alterations, and 1 VUS. Tumor Mutational Burden was under 10 mut/Mb for all patients. In this cohort of 57 PMAs, MAPK pathway genetic alterations were dominant drivers, similar to other PAs. We also observed other frequent genetic alterations not involving BRAF, including FGFR and NTRK gene family members that have FDA approved targeted inhibitors. While these also likely cause MAPK pathway activation, these non-BRAF variants are less common in PAs. Our report contributes to growing evidence that PMAs harbor a distinct molecular alteration profile, which is becoming increasingly important in the context of the evolving targeted therapeutics landscape.

Biliary tract cancers (BTCs) are a heterogeneous group of malignancies that make up ~7% of all gastrointestinal tumors. It is notably aggressive and difficult to treat; in fact, >70% of patients with BTC are diagnosed at an advanced, unresectable stage and are not amenable to curative therapy. For these patients, chemotherapy has been the mainstay treatment, providing an inadequate overall survival of less than one year. Despite the boom in targeted therapies over the past decade, only a few targeted agents have been approved in BTCs (i.e., IDH1 and FGFR inhibitors), perhaps in part due to its relatively low incidence. This review will explore current data on PARP inhibitors (PARPi) used in homologous recombination deficiency (HRD), particularly with respect to BTCs. Greater than 28% of BTC cases harbor mutations in genes involved in homologous recombination repair (HRR). We will summarize the mechanisms for PARPi and its role in synthetic lethality and describe select genes in the HRR pathway contributing to HRD. We will provide our rationale for expanding patient eligibility for PARPi use based on literature and anecdotal evidence pertaining to mutations in HRR genes, such as RAD51C, and the potential use of reliable surrogate markers of HRD.

PURPOSE Leiomyosarcoma (LMS) harbors frequent mutations in TP53 and RB1 but few actionable genomic alterations. Here, we searched for recurrent actionable genomic alterations in LMS that occur in the absence of common untreatable oncogenic drivers. METHODS Tissues from 276,645 unique advanced cancers, including 2,570 uterine and soft tissue LMS, were sequenced by hybrid-capture–based next-generation DNA and RNA sequencing/comprehensive genomic profiling of up to 406 genes. We characterized clinicopathologic features of relevant patient cases. RESULTS Overall, 77 LMS exhibited homozygous copy loss of CDKN2C at chromosome 1p32.3 (3.0% of LMS). Genomic alterations (GAs) in TP53, RB1, and ATRX were rare compared with the remainder of the LMS cohort (11.7% v 73.4%, 0% v 54.5%, 2.6% v 24.5%, respectively; all P < .0001). CDKN2C-null LMS patient cases were significantly enriched for GAs in CIC (40.3% v 1.4%) at 19q13.2, CDKN2A (46.8% v 7.0%), and RAD51B (16.9% v 1.7%; all P < .0001). Chromosome arm-level aneuploidy analysis of available LMS patient cases (n = 1,284) found that 81% (58 of 72) of CDKN2C-null LMS exhibited 1p/19q-codeletion, a significant enrichment compared with 5.1% in the remainder of the LMS cohort ( P < .0001). In total, 99% of CDKN2C-null LMS were in women; the median age was 61 years at surgery (range, 36-81 years). Fifty-five patient cases were uterine primary, four were nonuterine, and the remaining 18 were of uncertain primary site. Sixty percent of cases showed at least focal epithelioid variant histology. Most patients had advanced-stage disease, with 62% of confirmed uterine primary LMS at International Federation of Gynecology and Obstetrics stage IVB. We further validated our findings in two publicly available datasets: The Cancer Genome Atlas and the Project GENIE initiative. CONCLUSION CDKN2C-null LMS defines a genomically distinct tumor that may have prognostic and/or therapeutic clinical implications, including possible use of specific cyclin-dependent kinase inhibitors.

PURPOSE To estimate age-specific relative and absolute cancer risks of breast cancer and to estimate risks of ovarian, pancreatic, male breast, prostate, and colorectal cancers associated with germline PALB2 pathogenic variants (PVs) because these risks have not been extensively characterized. METHODS We analyzed data from 524 families with PALB2 PVs from 21 countries. Complex segregation analysis was used to estimate relative risks (RRs; relative to country-specific population incidences) and absolute risks of cancers. The models allowed for residual familial aggregation of breast and ovarian cancer and were adjusted for the family-specific ascertainment schemes. RESULTS We found associations between PALB2 PVs and risk of female breast cancer (RR, 7.18; 95% CI, 5.82 to 8.85; P = 6.5 × 10−76), ovarian cancer (RR, 2.91; 95% CI, 1.40 to 6.04; P = 4.1 × 10−3), pancreatic cancer (RR, 2.37; 95% CI, 1.24 to 4.50; P = 8.7 × 10−3), and male breast cancer (RR, 7.34; 95% CI, 1.28 to 42.18; P = 2.6 × 10−2). There was no evidence for increased risks of prostate or colorectal cancer. The breast cancer RRs declined with age ( P for trend = 2.0 × 10−3). After adjusting for family ascertainment, breast cancer risk estimates on the basis of multiple case families were similar to the estimates from families ascertained through population-based studies ( P for difference = .41). On the basis of the combined data, the estimated risks to age 80 years were 53% (95% CI, 44% to 63%) for female breast cancer, 5% (95% CI, 2% to 10%) for ovarian cancer, 2%-3% (95% CI females, 1% to 4%; 95% CI males, 2% to 5%) for pancreatic cancer, and 1% (95% CI, 0.2% to 5%) for male breast cancer. CONCLUSION These results confirm PALB2 as a major breast cancer susceptibility gene and establish substantial associations between germline PALB2 PVs and ovarian, pancreatic, and male breast cancers. These findings will facilitate incorporation of PALB2 into risk prediction models and optimize the clinical cancer risk management of PALB2 PV carriers.

Activation of the tyrosine kinase receptor IGF1R is targetable with existing tyrosine kinase inhibitors (TKIs) and monoclonal antibodies, but mutations in IGF1R have not been systematically characterized. Pan-cancer analysis of 326,911 tumors identified two distinct, activating non-frameshift insertion hotspots in IGF1R, which were significantly enriched in adenoid cystic carcinomas (ACCs). IGF1R alterations from 326,911 subjects were analyzed by variant effect prediction class, position within the gene, and cancer type. 6502 (2.0%) samples harbored one or more alterations in IGF1R. Two regions were enriched for non-frameshift insertions: codons 663-666 at the hinge region of the fibronectin type 3 domain and codons 1034-1049 in the tyrosine kinase domain. Hotspot insertions were highly enriched in ACCs (27.3-fold higher than in the remainder of the pan-cancer dataset; P = 2.3 × 10

New treatment strategies for advanced non-small-cell lung carcinoma (NSCLC) include synthetic lethality targets focused on protein arginine methyl transferases such as PRMT5 that exploit the impact of genomic loss of methylthioadenosine phosphorylase (MTAP).Twenty nine thousand three hundred seventy nine advanced NSCLC cases underwent hybrid-capture based comprehensive genomic profiling between June 1, 2018 and May 31, 2020. PD-L1 expression was determined by immunohistochemistry (Dako 22C3 PharmDx assay).13.4% (3928/29,379) NSCLC cases exhibited MTAP loss distributed in adenocarcinoma (59%), squamous cell carcinoma (22%), NSCLC not otherwise specified (16%), and 1% each for large-cell neuroendocrine, sarcomatoid, and adenosquamous carcinoma. Statistically significant differences in mitogenic driver alterations included more KRAS G12C mutations in MTAP-intact versus MTAP-lost (12% vs. 10%, p = 0.0003) and fewer EGFR short variant mutations in MTAP-intact versus MTAP-lost NSCLC (10% vs. 13%, p 0.0001). Statistically significant differences in currently untargetable genomic alterations included higher frequencies of TP53 (70% vs. 63%, p 0.0001) and RB1 inactivation (10% vs. 2%, p 0.0001) in MTAP-intact compared to MTAP-lost NSCLC. SMARCA4 inactivation (7% vs. 10%, p 0.0001) was less frequent in MTAP-intact versus MTAP-lost NSCLC. Alterations in ERBB2, MET, ALK, ROS1, and NTRK1 did not significantly differ between the two groups. Predictors of immunotherapy efficacy were higher in MTAP-intact versus MTAP-lost NSCLC including tumor mutational burden (9.4 vs. 8.6 mut/Mb, p = 0.001) and low (30% vs. 28%, p = 0.01) and high PD-L1 (32% vs. 30%, p = 0.01) expression. Alterations in biomarkers potentially predictive of immune checkpoint inhibitor resistance (STK11, KEAP1, and MDM2) were similar in the two groups.MTAP loss occurs in 13% of NSCLC, supporting the development of targeted therapies to exploit PRMT5 hyper-dependence. MTAP loss is accompanied by small differences in targeted and immunotherapy options which may impact future combination strategies.

Context.— Multiple procedural techniques can be used to obtain tissue to create a formalin-fixed, paraffin-embedded specimen for comprehensive genomic profiling (CGP) in lung cancer. The literature is mixed on whether the procedure affects CGP success. Objective.— To examine whether biopsy procedure affects lung cancer CGP success. Design.— This was a cross-sectional study of all patients with lung cancer whose specimens were submitted for CGP between January and February 2020. Multiple quality control metrics were used to determine whether cases were successfully profiled. Results.— In all, 3312 samples were identified. Overall, 67.5% (2236 of 3312) of samples were obtained from biopsies, 13.0% (432 of 3312) from fine-needle aspirations (FNAs), 9.7% (321 of 3312) from resections, 5.3% (174 of 3312) from fluid cytology cell blocks, and 4.5% (149 of 3312) from bone biopsies. Overall, 70.1% (2321 of 3312) of cases passed CGP, 15.4% (510 of 3312) of cases were released as qualified reports, and 14.5% (481 of 3312) of cases failed CGP. Resection samples were the most likely to be successfully sequenced, failing in only 2.8% (9 of 321) of instances, while fluid cytology specimens were the least likely, failing in 23.0% (40 of 174) of instances. Biopsy (14.5% [324 of 2236]), FNA (18.5% [80 of 432]), and bone biopsy (18.8% [28 of 149]) specimens failed at intermediate frequencies. On multivariate logistic regression analysis of CGP success on specimen type, fluid cytology (odds ratio [OR], 0.08; 95% CI, 0.03–0.19), biopsy (OR, 0.25; 95% CI, 0.11–0.52), FNA (OR, 0.14; 95% CI, 0.06–0.32), and bone biopsy (OR, 0.07; 95% CI, 0.03–0.17) specimens had decreased odds of CGP success relative to resection samples. Among patients with successfully sequenced samples, 48.0% were eligible for at least 1 therapy, based on a companion diagnostic or National Comprehensive Cancer Network biomarker. Conclusions.— The method of tissue acquisition was an important preanalytic factor that determined whether a sample would be successfully sequenced and whether a clinically actionable genomic alteration would be detected.

PURPOSE Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

BackgroundAmong patients with breast carcinoma who have metastatic disease, 15%–30% will eventually develop brain metastases. We examined the genomic landscape of a large cohort of patients with breast carcinoma brain metastases (BCBMs) and compared it with a cohort of patients with primary breast carcinomas (BCs).Material and MethodsWe retrospectively analyzed 733 BCBMs tested with comprehensive genomic profiling (CGP) and compared them with 10,772 primary breast carcinomas (not-paired) specimens. For a subset of 16 triple-negative breast carcinoma (TNBC)–brain metastasis samples, programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) was performed concurrently.ResultsA total of 733 consecutive BCBMs were analyzed. Compared with primary BCs, BCBMs were enriched for genomic alterations in TP53 (72.0%, 528/733), ERBB2 (25.6%, 188/733), RAD21 (14.1%, 103/733), NF1 (9.0%, 66/733), BRCA1 (7.8%, 57/733), and ESR1 (6.3%,46/733) (p < .05 for all comparisons). Immune checkpoint inhibitor biomarkers such as high tumor mutational burden (TMB-high; 16.2%, 119/733); high microsatellite instability (1.9%, 14/733); CD274 amplification (3.6%, 27/733); and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like mutational signature (5.9%, 43/733) were significantly higher in the BCBM cohort compared with the primary BC cohort (p < .05 for all comparisons). When using both CGP and PD-L1 IHC, 37.5% (6/16) of patients with TNBC brain metastasis were eligible for atezolizumab based on PD-L1 IHC, and 18.8% (3/16) were eligible for pembrolizumab based on TMB-high status.ConclusionWe found a high prevalence of clinically relevant genomic alterations in patients with BCBM, suggesting that tissue acquisition (surgery) and/or cerebrospinal fluid for CGP in addition to CGP of the primary tumor may be clinically warranted.Implications for PracticeThis study found a high prevalence of clinically relevant genomic alterations in patients with breast carcinoma brain metastasis (BCBM), suggesting that tissue acquisition (surgery) and/or cerebrospinal fluid for comprehensive genomic profiling (CGP) in addition to CGP of the primary tumor may be clinically warranted. In addition, this study identified higher positive rates for FDA-approved immunotherapy biomarkers detected by CGP in patients with BCBM, opening a possibility of new on-label treatments. Last, this study noted limited correlation between tumor mutational burden and PD-L1 immunohistochemistry (IHC), which shows the importance of testing patients with triple-negative BCBM for immune checkpoint inhibitor eligibility with both PD-L1 IHC and CGP.

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.

Endometrial serous carcinoma (EMSC) is an aggressive variant of uterine cancer with limited therapeutic options. We sought to define distinct clinicopathologic and genomic EMSC subgroups.We retrospectively analyzed 2159 EMSC and 2346 endometrioid-type endometrial carcinomas (EEC) tissue specimens that had undergone comprehensive genomic profiling (CGP) via the FoundationOne CDx assay during routine clinical care. High tumor mutational burden (TMB) was defined as ≥10mut/Mb using the FDA-approved CDx cutoff for pembrolizumab. Microsatellite instability (MSI) was determined on 95 loci. Evidence of homologous recombination deficiency (HRD) was determined via genomic loss of heterozygosity (gLOH), a validated HRD detection method for predicting PARP inhibitor effectiveness in ovarian carcinoma. High gLOH was defined as ≥16%.A genomic analysis of 2159 EMSCs revealed a predominance of TP53 mutations, microsatellite stability, low tumor mutational burden (TMB), and recurrent alterations of PIK3CA, PPP2R1A, ERBB2, CCNE1, FBXW7 and MYC. Evidence of HRD via high gLOH was identified in 22% of EMSCs. BRCA1 and BRCA2 alterations, as well as unique SET (solid, pseudo-endometrioid, and transitional cell-like) variant morphology, were enriched in HRD-EMSC. There was an increased frequency of CCNE1 amplification, a lower prevalence of PIK3CA and PPP2R1A alterations, and no differences in HRD, MSI or TMB biomarker frequencies in patients of predicted African ancestry. EMSC exhibited distinct gene mutation frequencies and MSI, TMB and gLOH biomarker signatures compared to a cohort 2346 EEC.Molecularly defined subgroups provide a framework to test the susceptibility of EMSC to targeted therapies in specific genetic settings (e.g. HRD, PIK3CA, PPP2R1A, ERBB2, MYC, CCNE1).

Background This study assessed the contrasting genomic profiles from the primary tumors (PTs), metastatic (MET) sites, and circulating tumor DNA (ctDNA) of patients with prostate cancer (PC). Methods A total of 1294 PC tissue specimens and 2462 ctDNA specimens underwent hybrid capture-based comprehensive genomic profiling (CGP). Specimens included tissue from PTs; MET biopsies from bone, liver (LIV), lung (LU), brain (BN), lymph node, and soft tissue sites; and ctDNA. Results Differences in alteration frequencies between PT, MET, and ctDNA specimens for selected genes were observed. TMPRSS2:ERG fusion frequencies were similar between PTs and MET sites (35% vs 33%) but varied among MET sites. Genomic alterations (GAs) in AR were lowest in PTs (2%) and highest in MET sites (from 24% in LU to 50% in LIV). BN had the highest genomic alterations/tumor (8) and enrichment for PTEN GAs. The BRCA2 GA frequency varied from 0% in BN to 15% in LIV. ERBB2 amplification was increased in MET sites in comparison with PTs. RB1 GAs were increased in LIV. Biomarkers potentially associated with an anti-PD(L)1 response included CDK12 GAs (16% in LU) and a microsatellite instability-high status (29% in BN). Analyses of ctDNA featured a broad spectrum of GAs similar to those detected across MET sites. Conclusions CGP of PTs, MET sites, and ctDNA in PC exhibited differences most likely associated with tumor progression, clonal evolution, and exposure to systemic therapies; ctDNA can also capture a broad range of potential therapeutic opportunities for patients with PC.

Domestic dog breeds are characterized by an unrivaled diversity of morphologic traits and breed-associated behaviors resulting from human selective pressures. To identify the genetic underpinnings of such traits, we analyze 722 canine whole genome sequences (WGS), documenting over 91 million single nucleotide and small indels, creating a large catalog of genomic variation for a companion animal species. We undertake both selective sweep analyses and genome wide association studies (GWAS) inclusive of over 144 modern breeds, 54 wild canids and a hundred village dogs. Our results identify variants of strong impact associated with 16 phenotypes, including body weight variation which, when combined with existing data, explain greater than 90% of body size variation in dogs. We thus demonstrate that GWAS and selection scans performed with WGS are powerful complementary methods for expanding the utility of companion animal systems for the study of mammalian growth and biology., Being man’s best friend, dogs have been bred and selected for certain morphologic traits and breed-associated behaviours. Here, Plassais et al. analyse 722 canine whole genome sequences including modern breeds, wild canids and village dogs by GWAS and search for signatures of selection.

Background: Genes regulated by breast cancer risk alleles identified through genome-wide association studies (GWAS) may harbor rare coding risk alleles. Methods: We sequenced the coding regions for 38 genes within 500 kb of 38 lead GWAS SNPs in 13,538 breast cancer cases and 5,518 controls. Results: Truncating variants in these genes were rare, and were not associated with breast cancer risk. Burden testing of rare missense variants highlighted 5 genes with some suggestion of an association with breast cancer, although none met the multiple testing thresholds: MKL1, FTO, NEK10, MDM4, and COX11. Six common alleles in COX11, MAP3K1 (two), and NEK10 (three) were associated at the P < 0.0001 significance level, but these likely reflect linkage disequilibrium with causal regulatory variants. Conclusions: There was no evidence that rare coding variants in these genes confer substantial breast cancer risks. However, more modest effect sizes could not be ruled out. Impact: We tested the hypothesis that rare variants in 38 genes near breast cancer GWAS loci may mediate risk. These variants do not appear to play a major role in breast cancer heritability.

Purpose: Oncogenic kinase fusions are targetable with approved and investigational therapies and can also mediate acquired resistance (AR) to targeted therapy. We aimed to understand the clinical validity of liquid biopsy comprehensive genomic profiling (CGP) to detect kinase fusions pan tumor. Experimental Design: CGP was performed on plasma and tissue samples during clinical care. All exons plus selected introns of 16 kinases involved in oncogenic fusions (ALK, BRAF, EGFR, ERBB2, FGFR1/2/3, MET, NTRK1/2/3, PDGFRA/B, RAF1, RET, and ROS1) were sequenced to capture fusions, including well-characterized and novel breakpoints. Plasma circulating tumor DNA (ctDNA) fraction was estimated to inform sensitivity. Results: Of 36,916 plasma cases, 32,492 (88%) had detectable ctDNA. Kinase fusions were detected in 1.8% of ctDNA-positive cases (571/32,492) and were most prevalent in patients with cholangiocarcinoma (4.2%), bladder cancer (3.6%), and non–small cell lung cancer (NSCLC; 3.1%). Of the 63 paired patient samples that had tissue and ctDNA specimens collected within 1 year and with estimated plasma ctDNA fraction >1%, fusions were detected in 47 of 51 (92%) liquid specimens with a fusion in the tissue sample. In 32 patients with fusions detected in liquid but not in tissue, 21 (66%) had evidence of putative acquired resistance. Conclusions: Targetable kinase fusions are identified in ctDNA across cancer types. In pairs with tissue-identified fusions, fusion detection in ctDNA is reliable with elevated ctDNA fraction. These data support the validity of CGP to enable ctDNA-based fusion detection for informing clinical care in patients with advanced cancer.