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Background The status of CDH1, ERBB2, and ESR1 is important for the diagnostic and treatment workup for patients with breast cancer. Most alterations in these genes occur in the form of short variants (eg missense and indel alterations) or copy number alterations (eg amplifications of ERBB2 or deletions in CDH1). The prevalence and characteristics of rearrangement events have been understudied. Materials and Methods: Comprehensive genomic profiling using a hybrid-capture based approach was performed on 44,842 breast carcinomas during the course of routine clinical care (FoundationOne® or FoundationOne®CDx) examining all classes of alterations in up to 395 genes, including CDH1, ESR1, and ERBB2. All rearrangements were included in the analysis (known/likely pathogenic and variants of uncertain significance). Estrogen receptor status was extracted from pathology reports for a subset of samples. Results: Rearrangements in CDH1, ESR1, and ERBB2 were observed in 0.26% (115/44842), 0.34% (153/44842), and 1.33% (598/44842) of breast cancer samples, respectively. As expected, CDH1 rearrangements were most common in invasive lobular carcinoma (ILC) (0.64%; 16/2516) though events were observed in samples originally submitted as invastive ductal carcinoma (IDC) (0.16%; 26/15,836), suggesting possible misdiagnosis. CDH1 rearrangements were predominantly loss of function consisting of large deletions, inversions, and truncation events. ESR1 rearrangements were observed at the highest frequency in ER+/HER2- tumors (0.58%) and were never seen in ER-/HER2- and ER-/HER2+ tumors. ESR1 rearrangements were observed with a variety of partners, with recurrent events with CCDC170, SYNE1, RMND1, PLEKHG1, ARMT1, MTHFD1L, and ZBTB2. Consistent with a possible role in therapy resistance, ESR1 rearrangements were enriched in metastatic samples relative to those biopsied from the breast (OR = 2.25; p = 8E-05). ERBB2 rearrangement events were commonly observed in HER2 amplified tumors (13.4%) and rarely in other subtypes (0.12% in ER+/HER2- and 0.28% in ER-/HER2-). Most of the events were intra-chromosomal and typically represented non-fusion duplication fragments that may have generated the ERBB2 amplification. Fusions were much rarer. Out of the 598 rearrangement events, only 18 were predicted to create in-frame and in-strand fusion products retaining the HER2 kinase domain following manual review of the events. Most fusion events were unique, though a fusion with IKZF3 was seen recurrently (n=2). Conclusions Rearrangement events in diagnostically important breast cancer genes (CDH1, ESR1, and ERBB2) were commonly observed in breast cancer with subtype-specific enrichment. Since these alterations have implications in disease diagnosis and therapy response (eg endocrine therapy resistance), comprehensive genomic profiling can provide value in breast cancer care. Citation Format: Ethan Sokol, Dexter Jin, Jeffrey S. Ross, ADRIAN V. LEE, Steffi Oesterreich. Rearrangements in CDH1, ESR1, and ERBB2 are commonly observed in breast cancer and may influence diagnosis and treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-11.

Background: Breast cancer brain metastasis (BCBM) represents an area of unmet clinical need. We previously demonstrated the genomic differences between primary BC and BCBM. In this study we examined the genomic differences between BCBM by BC subtypes and the potential actionable targets that might be taken forward for each subtype in clinical trials. Material and Methods: A total of 761 BCBMs were analyzed by comprehensive genomic profiling (CGP) for alterations in up to 395 genes (Foundation Medicine, USA). The samples were classified by immunohistochemistry (IHC) of the BCBM to ER+ (ER+/HER2-, ER+/HER2+), ER-/HER2+ and ER-/HER2-. Homologous recombination deficiency (HRD-gLOH; cutoff 16%), tumour mutational burden (TMB; cutoff 10 mutations/Mb), microsatellite instability (MSI) and PD-L1 prevalence and expression by IHC using the VENTANA SP142 assay (Immune Cell Score [IC] cutoff 1%) were also investigated. Results: For all BC subtypes the most enriched gene alterations in BCBM (>20% prevalence) were: TP53, MYC and PIK3CA. ESR1 and ERBB2 were more prevalent in ER+ and HER2+ tumours respectively, with ESR1 alterations significantly enriched in ER+/HER2- BCBMs (p< 0.0001). Frequently altered genes by BCBM subtype were: ER+/HER2-: CDH1 (8%) and BRCA2 (7%); ER+/HER2+: PIK3C2B (11%), MDM4 (11%), TBX3 (9%) and AKT2 (8%); ER-/HER2+: LYN (9%). Significantly enriched genes (p< 0.01) by BCBM subtype were: HER2+: CDK12 (15%); ER-/HER2-: BRCA1 (14%), CCND3 (9%), VEGFA, JAK2 (8% each) and the immune checkpoint inhibition (ICPI) biomarkers PDCD1LG2 (PDL2), CD274 (PDL1) (7% each). HRD-gLOH was high in ER+/HER2-: (43%) and ER-/HER2-: (70%). TMB and MSI were present in 10%-21% and 1-3% of BCBM respectively, whereas PDL1 protein expression by subtypes was: ER+/HER2- 23%, ER+/HER2+ 27%, ER-/HER2+ 57% , ER-/HER2- 48%. Table 1 summarizes the proportion of BCBMs with actionable alterations and selected clinical trials in BCBM. Conclusion: Clinically-relevant genomic alterations were identified across all BCBM subtypes. High HRD-gLOH, TMB and MSI were observed across all the BCBMs subtypes whereas PDL1/PDL2 alterations were a distinctive feature of ER-/HER2- BCBM. These data highlight the need to assess the genomic landscape of BCBM to enable rational treatment decisions with targeted agents, as well as to enable enrichment of relevant populations within clinical trials. Table 1. Selected studies with targeted therapy and/or immunotherapy in breast cancer brain metastases. The percentage (%) of actionable alterations by ER and HER2 status is summarized. ND: Not Detectable. Citation Format: Athina Giannoudis, Ethan Sokol, Shakti Ramkissoon, Talvinder Bhogal, Jeff Ross, Kimberly McGregor, Evangelia Razis, Rupert Bartsch, Richard S. Huang, Carlo Palmieri. Targetable alterations and genomic signatures within breast cancer brain metastases: Data from comprehensive genomic profiling of 761 breast cancer brain metastases [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-14-10.

Background: Malignant phyllodes tumors (MPT) are rare fibroepithelial breast tumors that disproportionately affect young women. Despite high local and distant recurrence rates, treatment is almost exclusively surgical. With no known effective chemotherapy or targeted systemic therapy options, metastatic progression portends dismal prognosis. Current national guidelines do not recommend biomarker testing in the non-metastatic setting. Management of metastatic disease follows principles of soft tissue sarcoma recommending Next-Generation Sequencing on an individual basis for patients who may qualify for clinical trials or are refractory to standard therapies. In the largest genomic profiling effort of MPT to date, we sought to describe the genomic landscape of MPTs through comprehensive genomic profiling (CGP) and immunotherapeutic biomarker analysis. Methods: Cases of sequenced MPT were identified from a CLIA-certified, CAP-accredited laboratory database (Foundation Medicine, Cambridge, MA). Cases were categorized as localized/locally recurrent or metastatic. All cases underwent CGP by DNA extraction from FFPE samples using a hybrid capture, adaptor ligation-based next generation sequencing assay, and all classes of genomic alterations, microsatellite instability (MSI), and tumor mutational burden (TMB) were evaluated. PD-L1 expression by IHC was measured by the DAKO 22C3 assay (scored CPS) or the Ventana SP142 assay (scored as IC). Patient characteristics and results of CGP of MPT were summarized by either N (%) or median (range). Factors from MPT were compared to all cases of breast carcinoma in the lab database. Fisher’s Exact Tests were used to test for differences between groups and analysis of variance was used to test for differences for continuous variables. Results: Of 135 MPT cases identified; 94 (69.6%) were localized/locally recurrent (breast, chest wall, soft tissue, skin, or lymph node) and 41 (30.4%) were metastatic (73% lung, followed by bone, liver, other intraabdominal). All patients were female with a median age of 54 years (range 14-86). The median TMB across samples was 2.5 mut/Mb and only 3 were TMB-high tumors (>10mut/Mb). Over 1/3 (36.8%) of samples were PD-L1+ via Ventana SP142 assay (≥1) and 21.4% were PD-L1+ via Dako 22C3 assay (CPS ≥10). All evaluable cases (N=132) were microsatellite stable. The ten most commonly altered genes included TERT-promoter (69.7%), CDKN2A (45.9%), TP53 (37.8%), NF1 (35.6%), CDKN2B (33.3%), MED12 (28.9%), MTAP (27.7%), KMT2D (22.2%), PIK3CA (20.0%), PTEN (18.5%), and RB1 (18.5%); notably distinct in frequency from the breast carcinoma cohort (Table 1). Alterations in genes affecting cell cycle regulation (i.e. CDKN2A/B and TP53) were mutually exclusive from one another (p< 0.001). Several tumors harbored genomic alterations with FDA-approved indications in other tumor types were found including: NF1, PIK3CA, EGFR Exon 19/20 insertions, and BRAF V600E mutations. There were no statistically significant differences between alterations in localized/locally recurrent versus metastatic specimens. Conclusions: In the largest genomic evaluation of localized/locally recurrent or metastatic MPTs to date, multiple clinically actionable mutations for further exploration were found. TERT-promoter was altered in the majority of cases. PDL-1 was expressed in over 1/3 of cases suggesting that clinical investigation of immunotherapeutic strategies is warranted. Routine sequencing of metastatic MPT may provide additional information to guide treatment decisions and clinical trial enrollment. Table 1. Frequency of Genomic Alterations identified in 135 cases of Malignant Phyllodes Tumors. Citation Format: Laura H. Rosenberger, Richard F. Riedel, Natalie A. Danziger, Ethan Sokol, Jeffrey S. Ross, Sarah L. Sammons. Genomic Evaluation of Malignant Phyllodes Tumors Reveals Multiple Targetable Opportunities [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-03.

Oncocytic histologic features can be seen in a variety of salivary gland carcinomas. We performed a comprehensive molecular profiling of 15 salivary gland malignancies with oncocytic features (diagnosed as oncocytic carcinoma, carcinoma NOS with oncocytic features, or salivary duct carcinoma with oncocytic features). We reveal multiple novel molecular alterations that have not been previously described in other salivary gland malignancies, including, but not limited to, KEL amplification (13.3%, 2/15), PARP1 amplification (13.3%, 2/15), and EPHB4 amplification (13.3%, 2/15). Alterations in KMT2C (13.3%, 2/15), ERBB3 (13.3%, 2/15), CTNNA1 (13.3%, 2/15), and SMAD4 (20%, 3/15) were also found in this series and have been reported in other salivary gland malignancies. Alterations that have been reported in salivary duct carcinoma were also identified, including TP53 (40%, 6/15) , ERBB2 mutations (13.3%, 2/15) , ERBB2 amplification (13.3%, 2/15), PIK3CA (26.7%, 4/15) , PTEN (20%, 3/15), BRCA2 (20%, 3/15), BRAF (20%, 3/15), CDKN2A/B (20%, 3/15), CDH1 (13.3%, 2/15), and HRAS (13.3%, 2/15). Oncocytic salivary gland malignancies are a molecularly heterogenous group of tumors with partial overlap with salivary duct carcinoma subtypes.

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.

Background NTRK1/2/3 kinase fusions are rare oncogenic drivers found in Citation Format: Ethan Sokol, Sophia Maund, Jeffrey Ross, Timothy Wilson. NTRK1/2/3 fusions are observed in both secretory and non-secretory breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-10.

Background: Central nervous system (CNS) disease secondary to breast cancer (BC) is a growing clinical problem and a cause of significant morbidity and mortality. A better understanding of the genomic landscape is key to understanding its cause and to developing novel treatments. In this study, we examined the genomic landscape of a large cohort of breast cancer brain metastases (BMs) and compared them to a cohort of local breast cancers (BCs) and non-CNS metastases (N-CNS). Material and Methods: We analyzed 822 BMs and compared them to 11,988 local, breast-biopsied BCs and 15,516 N-CNS (unpaired samples) with comprehensive genomic profiling (CGP) for all classes of alterations in at least 324 genes (Foundation Medicine, Cambridge, MA, USA). Homologous recombination deficiency detected by genome-wide loss of heterozygosity (HRD-gLOH; cutoff 16%), tumour mutation burden (TMB; cutoff 10 mutations/Mb), microsatellite instability (MSI) and PD-L1 prevalence and expression by VENTANA SP142 immune cell immunohistochemistry (IHC; cutoff 1%) was also investigated within and across the cohorts. Results: As compared to local BCs, 31 genes were significantly enriched for genomic alterations in BMs with BM prevalence >3% and false-discovery rate (FDR) Table 1.Prevalence (%) of the genomic signatures HRD-gLOH, TMB, MSI and PD-L1+ expression.Site% HRD-gLOH% TMB-H% MSI-H% PDL1+ALLlocal BC33.70%. (2573/7636)5.04%. (527/10464)0.37%. (39/10464)54.25%. (1092/2013)BM52.01%. (362/696)15.51%. (116/748)2.01%. (15/748)38.73%. (55/142)N-CNS31.55%. (3237/10260)9.82%. (1395/14210)0.23%. (32/14210)26.21%. (615/2346)ER+/HER2-local BC31.48%. (119/378)3.41%. (19/557)0.36%. (2/557)40.00%. (14/35)BM43.17%. (79/183)11.40%. (22/193)1.04%. (2/193)23.08%. (9/39)N-CNS29.65%. (126/425)8.92%. (51/572)0.17%. (1/572)12.99%. (10/77)ER+/HER2+local BC19.57%. (9/46)2.78%. (2/72)0.00%. (0/72)42.86%. (3/7)BM26.67%. (16/60)9.68%. (6/62)0.00%. (0/62)27.27%. (3/11)N-CNS47.37%. (18/38)19.57%. (9/46)0.00%. (0/46)14.29%. (1/7)ER-/HER2+local BC32.26%. (10/31)8.70%. (4/46)0.00%. (0/46)100.00%. (5/5)BM33.33%. (23/69)21.33%. (16/75)2.67%. (2/75)57.14%. (4/7)N-CNS34.38%. (11/32)4.26%. (2/47)0.00%. (0/47)42.86%. (3/7)ER-/HER2-local BC52.25%. (186/356)2.70%. (14/518)0.19%. (1/518)50.00%. (30/60)BM70.45%. (174/247)17.41%. (47/270)2.59%. (7/270)48.33%. (29/60)N-CNS43.52%. (84/193)10.04%. (27/269)0.74%. (2/269)21.43%. (6/28) Citation Format: Athina Giannoudis, Ethan Sokol, Shakti H Ramkissoon, Talvinder Bhogal, Kimberly McGregor, Allison Clark, Evangelia D Razis, Rupert Bartsch, Richard SP Huang, Carlo Palmieri. Comprehensive assessment of the genomic landscape of breast cancer brain metastases reveals targetable alterations and genomic signatures relevant to immune-checkpoint and PARP inhibitors [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 PD4-09.

Background: APOBEC mutagenesis underlies somatic evolution and accounts for tumor heterogeneity in several cancers, including BC. In our clinical experience, HR+HER2- BC with an APOBEC signature do poorly on standard of care (SOC) first line endocrine therapy (ET) + CDK4/6 inhibitor (CDK4/6i) and need additional treatment options. Here we evaluated the characteristics of a real-world cohort for time-to-treatment discontinuation (TTD) and overall survival (OS) on SOC and immune checkpoint inhibitors (ICI).Methods: Hybrid capture-based comprehensive genomic profiling (CGP) results from 29,833 formalin-fixed paraffin-embedded tissue biopsies of all BC subtypes were analyzed. For outcomes (TTD and OS), this study used a de-identified nationwide (US-based) BC clinico-genomic database (CGDB, ~800 sites of care, 1/2011 - 12/2020). HR+HER2- metastatic BC pts who received first line ET + CDK4/6i were included (CGDB cohort).TTD was defined as the difference between the first and last drug episode within a given line of treatment (LOT). LOT were derived based on FH algorithms. OS was defined as the time from LOT start to the date of death or data cutoff. Log-rank test and Cox model were used to evaluate the difference in outcomes. To reduce the impact of confounding variables (Age at Dx, Stage at Dx, Tumor Type, Metastases sites, TMB group (≥10 vs 6 mo. In Mayo and Duke cohort, there were 6 pts, 5/6 received ICI + chemotherapy. The TTD was 0.9-40.5 months with longest 2 pts receiving 5-FU plus ICI (11 and 40.5 months). To better understand the ICI treatment landscape, TTD in HR+HER2- hTMB MSS APOBEC- CGDB cohort (N=6) was analyzed: 5/6 had evaluable treatment data, 4/5 finished ICI treatment, 1 pt’s TTD was > 3 mo, 0/5 had TTD > 6 mo. Conclusions: APOBEC+ occurs in ~7% of BC and is more common in ILC and metastatic lesions. APOBEC+ HR+HER2- pts had shorter TTD and OS on SOC ET+CDK4/6i relative to APOBEC- pts. However, TTD on ICI tended to be longer in APOBEC+ pts, but our data is limited, and more research is needed. CGDB APOBEC+ vs. APOBEC- with SOC 1st lineAPOBEC+ (N=69)APOBEC- (N=788)p adjusted (FDR)*Age at Dx, Median (IQR)59.0 (53.0, 65.0)56.0 (47.0, 65.0)0.102Stage at Dx0.067- 0-III52 (75.4%)505 (64.1%)- IV11 (15.9%)245 (31.1%)- Not documented6 (8.7%)38 (4.8%)Metastasis free interval, yrs, Median (IQR)5.2 (3.0, 10.5)5.1 (2.8, 9.3)0.734Tumor Grade0.104- Grade 12 (2.9%)49 (6.2%)- Grade 234 (49.3%)259 (32.9%)- Grade 311 (15.9%)163 (20.7%)- Not documented22 (31.9%)317 (40.2%)Tumor Type0*- IDC7 (10.1%)251 (31.9%)- ILC30 (43.5%)138 (17.5%)- Other /Not documented32 (46.4%)399 (50.6%)Metastases sites0.734- Bone-only17 (24.6%)169 (21.5%)- CNS11 (15.9%)104 (13.2%)- Visceral41 (59.4%)514 (65.3%)TMB, Median (IQR)11.3 (8.8, 18.8)2.5 (1.3, 3.8)0*MSI0.946- MSI-H0 (0.0%)4 (0.5%)- MSI-I0 (0.0%)1 (0.1%)- MSS68 (98.6%)728 (97.2%)- Not documented1 (1.4%)16 (2.1%)BRCA5 (7.2%)45 (5.7%)0.734PIK3CA46 (66.7%)341 (43.3%)0.001*Treatments0.005*- AI + CDK4/6i31 (44.9%)510 (64.7%)- Fulvestrant + CDK4/6i38 (55.1%)278 (35.3%) Citation Format: Saranya Chumsri, Kira Raskina, Sarah Sammons, Laura Alder, Natalie Danziger, Alexa B Schrock, Kim McGregor, Ethan Sokol. APOBEC signature, clinical characteristics, and outcome in hormone receptor-positive (HR+) HER2-negative (HER2-) breast cancer (BC) patients (pts) in real-world data (RWD) [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 PD14-09.

Understanding the genomic landscape of breast cancer brain metastases (BCBMs) is key to elucidating their cause and developing novel treatments. In this study, comprehensive genomic profiling was performed on 822 BCBMs, 11,988 local breast cancer (BC) biopsies and 15,516 non-central nervous system (N-CNS) metastases (all unpaired samples). Clinically-relevant genomic alterations were significantly enriched in BCBMs compared to local BCs and N-CNS metastases. Homologous recombination deficiency as measured by BRCA1/2alteration prevalence and loss-of-heterozygosity and immune checkpoint inhibitor (ICI) biomarkers [Tumour mutation burden (TMB)-High, Microsatellite instability (MSI)-High, PD-L1/L2)] were significantly more prevalent in BCBM than local BC and N-CNS. High PD-L1 protein expression was observed in ER-negative/HER2-negative BCBMs (48.3% vs 50.0% in local BCs, 21.4% in N-CNS). Collectively, our data highlights that a high proportion of BCBMs are potentially amenable to treatment with targeted therapeutic agents including PARP inhibitors and ICIs.

Background Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. Materials and methods Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. Results Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. Conclusions Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Purpose: To study associations across tumor types between genome-wide loss of heterozygosity (gLOH) and alterations in homologous recombination repair (HRR)-associated genes beyond BRCA1 and BRCA2. Experimental Design: Genomic profiling using a targeted next-generation sequencing assay examining 324–465 genes (FoundationOne, FoundationOne Heme, and FoundationOne CDx; Foundation Medicine, Inc.) was performed in a cohort of 160,790 samples across different tumor types. Zygosity predictions and gLOH status were calculated and linked with alterations in 18 HRR-associated genes (BRCA1, BRCA2, PALB2, BARD1, ATR, ATRX, ATM, BAP1, RAD51B, RAD51C, RAD51D, BRIP1, NBN, CHEK1, CHEK2, FANCA, FANCC, MRE11) and other genomic features, using Fisher's exact test and Mann–Whitney U tests. Results: We identified a strong correlation between elevated gLOH and biallelic alterations in a core set of HRR-associated genes beyond BRCA1 and BRCA2, such as BARD1, PALB2, FANCC, RAD51C, and RAD51D (particularly in breast, ovarian, pancreatic, and prostate cancer). Monoallelic/heterozygous alterations in HRR-associated genes were not associated with elevated gLOH. gLOH was also independently associated with TP53 loss. Co-occurrence of TP53 loss and alterations in HRR-associated genes, and combined loss of TP53-PTEN or TP53-RB1, was associated with a higher gLOH than each of the events separately. Conclusions: Biallelic alterations in core HRR-associated genes are frequent, strongly associated with elevated gLOH, and enriched in breast, ovarian, pancreatic, and prostate cancer. This analysis could inform the design of the next generation of clinical trials examining DNA repair–targeting agents, including PARP inhibitors.

c-Ros oncogene 1, receptor tyrosine kinase (ROS1) genomic rearrangements have been reported previously in rare cases of colorectal cancer (CRC), yet little is known about the frequency, molecular characteristics, and therapeutic vulnerabilities of ROS1-driven CRC. We analyzed a clinical dataset of 40 589 patients with CRC for ROS1 genomic rearrangements and their associated genomic characteristics (Foundation Medicine, Inc [FMI]). We moreover report the disease course and treatment response of an index patient with ROS1-rearranged metastatic CRC. ROS1 genomic rearrangements were identified in 34 (0.08%) CRC samples. GOPC-ROS1 was the most common ROS1 fusion identified (11 samples), followed by TTC28-ROS1 (3 samples). Four novel 5' gene partners of ROS1 were identified (MCM9, SRPK1, EPHA6, P4HA1). Contrary to previous reports on fusion-positive CRC, ROS1-rearrangements were found exclusively in microsatellite stable (MSS) CRCs. KRAS mutations were significantly less abundant in ROS1-rearranged vs ROS1 wild type cases. The index patient presented with chemotherapy-refractory metastatic right-sided colon cancer harboring GOPC-ROS1. Molecularly targeted treatment with crizotinib induced a rapid and sustained partial response. After 15 months on crizotinib disseminated tumor progression occurred and KRAS Q61H emerged in tissue and liquid biopsies. ROS1 rearrangements define a small, yet therapeutically actionable molecular subgroup of MSS CRC. In summary, the high prevalence of GOPC-ROS1 and noncanonical ROS1 fusions pose diagnostic challenges. We advocate NGS-based comprehensive molecular profiling of MSS CRCs that are wild type for RAS and BRAF and patient enrollment in precision trials.

PURPOSE APOBEC mutagenesis underlies somatic evolution and accounts for tumor heterogeneity in several cancers, including breast cancer (BC). In this study, we evaluated the characteristics of a real-world cohort for time-to-treatment discontinuation (TTD) and overall survival on CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (ET) and immune checkpoint inhibitors. METHODS Comprehensive genomic profiling results from 29,833 BC samples were analyzed for tumor mutational burden and APOBEC signatures. For clinical outcomes, a deidentified nationwide (United States–based) BC Clinico-Genomic Database (CGDB) was evaluated with log-rank and Cox models. Patients with hormone receptor–positive (HR+) human epidermal growth factor receptor 2–negative (HER2–) BC who received first-line ET and CDK4/6i were included. Eligible patients from Mayo Clinic and Duke University were HR+ HER2– BC with sequencing data between September 2013 and July 2020. RESULTS Of 29,833 samples sequenced, 7.9% were APOBEC+ with a high rate in invasive lobular carcinoma (16.7%) and in metastatic tumors (9.7%) relative to locally biopsied BC (4.3%; P < .001). In CGDB, 857 patients with HR+ HER2– BC received ET plus CDK4/6i in the first line. APOBEC+ patients had significantly shorter TTD on ET plus CDK4/6i than APOBEC– patients, 7.8 (95% CI, 4.3 to 14.6) versus 12.4 months (95% CI, 11.2 to 14.1; hazard ratio, 1.6; 95% CI, 1.03 to 2.39; P = .0036). Clinical benefit to immune checkpoint inhibitors was observed in HR+ HER2–, APOBEC+, tumor mutational burden–high patients, with four of nine CGDB patients (TTD 0.3-11.3 months) and four of six patients in Duke/Mayo cohorts (TTD 0.9-40.5 months) with a TTD of ≥ 3 months. CONCLUSION APOBEC+ HR+ HER2– patients had shorter TTD on first-line ET plus CDK4/6i relative to APOBEC– patients. Further research is needed to optimize the treatment of APOBEC+ HR+ HER2– BC and to investigate the efficacy of immunotherapeutic strategies in this population.

Introduction:. Breast cancer (BC) brain metastases (BM) are a growing clinical problem as survival for metastatic BC improves. Genomic analyses of BM are key to developing more targeted therapeutic interventions. In this study we explored the gene rearrangements of BM and compared these to a cohort of non-paired local breast cancers (BCs) and non-CNS metastasis (N-CNS). Methods:. We analyzed 822 BMs and compared them to 11,988 local, breast-biopsied BCs and 15,516 N-CNS (samples unpaired) profiled with comprehensive genomic profiling (CGP) (Foundation Medicine, Cambridge, MA, USA) examining all classes of alterations in at least 324 genes. The prevalence of individual gene rearrangements within the BM was compared to those within BC and N-CNS cases. Further analysis was undertaken by classifying BM, BC and N-CNS specimens with respect to their oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status, available for a subset of samples. Results:. CGP identified 307 rearrangement events in BMs impacting 169 genes. Twenty genes exhibited recurrent rearrangement events with a prevalence greater than 0.5% in either the BM, BC or N-CNS. 10 of these 20 genes were significantly more prevalent within the BM specimens vs. BC and N-CNS (p The prevalence of CDK12 rearrangements within BM, BCs, and other N-CNS in different sub-types of BC.SubtypePrevalenceP-valueBM BCN-CNSBM vs. BCBM vs. . N-CNSAll3.53%. (29/822)0.86%. (103/11,988)0.68%. (105/15,516)3.55x10-91.85x10-11HER2+(irrespective of ER status)14.59%. (27/185)7.80%. (86/1,102)7.87%. (94/1,194)0.00460.0048HER2+, ER-15.29%. (13/85)10.53%. (6/57)1.96%. (1/51)0.460.02HER2+, ER+13.64%. (9/66)7.14%. (6/84)10.20%. (5/49)0.270.77HER2-, ER-0.67%. (2/298)0.17%. (1/585)0.69%. (2/291)0.261.00HER2-, ER+0.00%. (0/212)0.16%. (1/641)0.00%. (0/634)1.001.00 Citation Format: Talvinder Bhogal, Ethan Sokol, Shakti Ramkissoon, Athina Giannoudis, Kimberly McGregor, Allison Clark, Evangelia Razis, Rupert Bartsch, Richard Huang, Carlo Palmieri. Comprehensive analysis of the gene rearrangements within 822 breast cancer brain metastases reveals an enrichment of cyclin dependent kinase 12 rearrangements in HER2-positive brain metastases [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 P5-07-04.

BackgroundAt diagnosis, the majority of patients with intrahepatic cholangiocarcinoma (IHCC) present with advanced disease and a poor prognosis. Comprehensive genomic profiling (CGP) early in the disease course may increase access to targeted therapies and clinical trials; however, unresolved issues remain surrounding the optimal biopsy type to submit for CGP.Patients and MethodsMutational frequencies between primary tumor biopsies (Pbx), metastatic biopsies (Mbx), and liquid biopsies (Lbx) in 1,632 patients with IHCC were compared.ResultsPotentially actionable alterations were found in 52%, 34%, and 35% of patients in the Pbx, Mbx, and Lbx cohorts, respectively. In Pbx, Mbx, and Lbx, FGFR2 rearrangements were found in 9%, 6%, and 4%, and IDH1 mutations were identified in 16%, 5%, and 9% patients, respectively. Moreover, alterations in FGFR2 and IDH1 were significantly associated with distinct ancestries, including 2.1-fold enrichment for FGFR2 rearrangements in patients with African ancestry and 1.5-fold enrichment for IDH1 mutations in patients with admixed American (Hispanic) ancestry. Finally, the publication of biomarker-driven clinical trials in IHCC correlated with changing CGP testing patterns. Significant correlations between patient characteristics and IHCC trial disclosures were observed, including a significant decrease from time between biopsy and CGP testing, and more frequent testing of primary versus metastatic samples.ConclusionOverall, because of the high likelihood of identifying actionable genomic alterations, CGP should be considered for the majority of patients with inoperable IHCC, and Lbx and Mbx can be considered as part of the diagnostic suite.Implications for PracticeComprehensive genomic profiling (CGP) should be considered for all patients with intrahepatic cholangiocarcinoma (IHCC) or suspected IHCC, as actionable alterations were commonly found in multiple genes and a wide variety of FGFR2 fusion partners were identified. The disclosure of IHCC trial data correlated with increased use of CGP, an encouraging trend that moves new therapeutic options forward for rare cancers with a rare biomarker. Although tissue from the primary lesion may identify actionable alterations at higher rates, CGP of a liquid biopsy or metastatic site can be considered, particularly if the primary tissue block is exhausted.

PURPOSE To study the impact of standard-of-care hormonal therapies on metastatic prostate cancer (mPC) clinical genomic profiles in real-world practice, with a focus on homologous recombination-repair (HRR) genes. PATIENTS AND METHODS Targeted next-generation sequencing of 1,302 patients with mPC was pursued using the FoundationOne or FoundationOne CDx assays. Longitudinal clinical data for correlative analysis were curated via technology-enabled abstraction of electronic health records. Genomic biomarkers, including individual gene aberrations and genome-wide loss-of-heterozygosity (gLOH) scores, were compared according to biopsy location and time of sample acquisition (androgen deprivation therapy [ADT]-naïve, ADT-progression and post-ADT, and novel hormonal therapies [NHT]-progression), using chi-square and Wilcoxon rank-sum tests. Multivariable analysis used linear regression. False-discovery rate of 0.05 was applied to account for multiple comparisons. RESULTS Eight hundred forty (65%), 132 (10%), and 330 (25%) biopsies were ADT-naïve, ADT-progression, and NHT-progression, respectively. Later-stage samples were enriched for AR, MYC, TP53, PTEN, and RB1 aberrations (all adjusted P values < .05), but prevalence of HRR-related BRCA2, ATM, and CDK12 aberrations remained stable. Primary and metastatic ADT-naïve biopsies presented similar prevalence of TP53 (36% v 31%) and BRCA2 (8% v 7%) aberrations; 81% of ADT-naïve BRCA2-mutated samples presented BRCA2 biallelic loss. Higher gLOH scores were independently associated with HRR genes ( BRCA2, PALB2, and FANCA), TP53, and RB1 aberrations, and with prior exposure to hormonal therapies in multivariable analysis. CONCLUSION Prevalence of HRR-gene aberrations remains stable along mPC progression, supporting the use of diagnostic biopsies to guide poly (ADP-ribose) polymerase inhibitor treatment in metastatic castration-resistant prostate cancer. gLOH scores increase with emerging resistance to hormonal therapies, independently of individual HRR gene mutations.

Colorectal cancer (CRC) is currently the fourth leading etiology of brain metastasis (BM). Yet, mechanisms supporting the formation of CRC BM are mostly unknown. In order to identify drivers that lead to tropism and adaptation of CRC cells to the brain environment, we analyzed an extensive genomic database, consisting of over 36,000 human CRC primary and metastasis samples. Several genomic alterations specific for BM were noted, among them increased prevalence of insulin receptor substrate 2 (IRS2) amplification, observed in 7.6% of BM, compared to only 2.9% of primary tumors or other metastatic sites (pAccession no GSE203017 and here is the link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE203017

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

258 Background: Tumors harboring 2 or more PIK3CA Short variants (SV) (“Multi-hit”) have been described in breast cancer as linked to enhanced clinical outcome from anti-PIK3CA targeted therapies including alpelisib and investigational agents in clinical trials. The landscape and clinical implications of multi-hit PIK3CA alterations in other tumors, including in CAPC, are underexplored. Methods: 19,978 CAPC samples underwent hybrid capture based comprehensive genomic profiling (CGP) to evaluate all classes of GA and determine tumor mutational burden (TMB), microsatellite instability (MSI), genomic ancestry, signature and loss of heterozygosity (gLOH). Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). Results: 18,741 (93.8%) CAPC were PIK3CA wild type (WT), 1,155 (5.8%) featured a single PIK3CA SV and 82 (0.4%) featured multi-hit PIK3CA SVs. The median ages of CAPC patients with single hit (69.1 yrs) or multi-hit (68.7 yrs) PIK3CA SV were older than the PIK3CA WT (67.6 yrs) CAPC (p

566 Background: Given the recent approval in lung cancer for the first anti-HER2 targeted therapy directed against activating kinase domain exon 20 ERBB2 insertion mutations, considerable interest has arisen in targeting additional locations in the ERBB2 gene in other tumor types. Methods: 5,485 UBC samples were sequenced using a hybrid capture-based comprehensive genomic profiling (CGP) assay to assess all classes of genomic alterations (GA), tumor mutational burden (TMB), microsatellite instability (MSI), genomic loss of heterozygosity (gLOH), trinucleotide signatures and predominant genetic ancestry. Statistical comparisons utilized the Bonferroni correction. Results: 548 (10.0%) of UBC featured ERBB2 gene amplification (amp) and 219 (4.0%) featured an ERBB2 ECD+ sequence mutation (74% S310F and 26% S310Y). ERBB2 ECD+ mutations and ERBB2 amplification were mutually exclusive. 16.4% of ERBB2 ECD+ UBC had more than one E RBB2 sequence mutation. Central pathology review of the 219 ERBB2 ECD+ UBC revealed that 63 (28.8%) featured a micropapillary histologic appearance (MPUC). ERBB2 ECD+ UBC featured more patients with European ancestry than both ERBB2 WT (90.9% vs 84.2%; p=.03) and ERBB2 amp (90.9% vs 83.2%; p=.04). ECD mut+ UBC also featured lower association with African ancestry than ERBB2 amp (2.7% vs 7.8%; p=.04) and lower frequency of gLOH > 16% (9.3% vs 19.1%; p=.04). MSI-High status was similar and rare in all 3 groups (range 0 to 0.9%). ERBB2 ECD+ UBC had a higher frequency of APOBEC signature than ERBB2 amp (82.9% vs 72.4%; p=.03). TMB > 10 mut/mb was higher in ERBB2 amp (49.2% vs 31.4%; p

We examined a large dataset of female metastatic breast cancers (MBCs) profiled with comprehensive genomic profiling (CGP) to identify the prevalence and distribution of immunotherapy responsiveness‐associated biomarkers. DNA was extracted from 3831 consecutive MBCs: 1237 (ERpos/HER2 neg), 1953 ERneg/HER2 amp, and 641 triple‐negative breast cancer (TNBC). CGP was performed using the FoundationOne® or FoundationOne®CDx NGS assay. Tumor mutational burden (TMB) and microsatellite instability (MSI) were determined in a subset of cases. PD‐L1 expression in immunocytes in a subset of cases was determined by immunohistochemistry using the companion diagnostic VENTANA PD‐L1 SP142 Assay. The median age of the cohort was 54 years (range 20–89). Genomic alterations (GAs)/tumor were similar (range: 5.9–7.3). Markers of potential immune checkpoint inhibitor (ICPI) benefit included: CD274 (PD‐L1) amplification (1%–3%), BRAF GA (1%–4%), TMB of ≥10 mutations/Mb (8%–12%), MSI‐high (0.1%–0.4%), PBRM1 GA (1%), and positive PD‐L1 staining of immunocytes ranging from 13% in ERpos/HER2 neg and 33% in ERneg/HER2 amp to 47% in the TNBC group. Potential markers of ICPI resistance included inactivating STK11 GA (1%–2%) and MDM2 amplification (3%–6%). MTOR pathway targets were common with lowest frequency in TNBC. ERBB2 short variant mutations were most frequent ERpos/HER2 neg and absent in TNBC. BRCA1/2 GA were least frequent in ERneg/HER2 amp. The demonstrations of clinical benefit of immunotherapy in MBC support the need for development and utilization of biomarkers to guide the use of ICPIs for these patients. In addition to guiding therapy selection, CGP shows potential to identify GA linked to response and resistance to ICPI in MBC., Both our results and published literature suggest that in addition to guiding targeted therapy selection, comprehensive genomic profiling shows potential to identify genomic alterations linked to response and resistance to immune checkpoint inhibitor (ICPI) therapy in metastatic breast cancer (MBC). The demonstration of clinical benefit of immune checkpoint blockade in MBC supports the need for the development of biomarkers used to guide the use of ICPI drugs for these patients.

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., This article focuses on the ability of comprehensive genomic profiling to identify potentially targetable genetic alterations in cancers of unknown primary, based on the inclusion criteria for the CUPISCO clinical trial and aiming for more effective therapeutic options for patients.

Rare reports of anal carcinoma (AC) describe histologic resemblance to cutaneous cylindroma, but mutations in the tumor suppressor CYLD, the gene responsible for familial and sporadic cylindromas, have not been systematically investigated in AC. Here, we investigate CYLD-mutant AC, focusing on molecular correlates of distinct histopathology. Comprehensive genomic profiling (hybrid-capture-based DNA sequencing) was performed on 574 ACs, of which 75 unique cases (13%) harbored a CYLD mutation. Clinical data, pathology reports, and histopathology were reviewed for each CYLD-mutant case. The spectrum of CYLD mutations included truncating (n = 50; 67%), homozygous deletion (n = 10; 13%), missense (n = 16; 21%), and splice-site (n = 3; 4%) events. Compared with CYLD-wildtype AC (n = 499), CYLD-mutant ACs were significantly enriched for females (88% vs. 67%, p = 0.0001), slightly younger (median age 59 vs. 61 years, p = 0.047), and included near-universal detection of high-risk HPV sequences (97% vs. 88%, p = 0.014), predominantly HPV16 (96%). The CYLD-mutant cohort also showed significantly lower tumor mutational burden (TMB; median 2.6 vs. 5.2 mut/Mb, p

Sarcomas are driven by diverse pathogenic mechanisms, including gene rearrangements in a subset of cases. Rare soft tissue sarcomas containing KMT2A fusions have recently been reported, characterized by a predilection for young adults, sclerosing epithelioid fibrosarcoma-like morphology, and an often aggressive course. Nonetheless, clinicopathologic and molecular descriptions of KMT2A-rearranged sarcomas remain limited. In this study, we identified by targeted next-generation RNA sequencing an index patient with KMT2A fusion-positive soft tissue sarcoma. In addition, we systematically searched for KMT2A structural variants in a comprehensive genomic profiling database of 14,680 sarcomas interrogated by targeted next-generation DNA and/or RNA sequencing. We characterized the clinicopathologic and molecular features of KMT2A fusion-positive sarcomas, including KMT2A breakpoints, rearrangement partners, and concurrent genetic alterations. Collectively, we identified a cohort of 34 sarcomas with KMT2A fusions (0.2%), and YAP1 was the predominant partner (n = 16 [47%]). Notably, a complex rearrangement with YAP1 consistent with YAP1–KMT2A–YAP1 fusion was detected in most cases, with preservation of KMT2A CxxC-binding domain in the YAP1–KMT2A–YAP1 fusion and concurrent deletions of corresponding exons in KMT2A. The tumors often affected younger adults (age 20–66 [median 40] years) and histologically showed variably monomorphic epithelioid-to-spindle shaped cells embedded in a dense collagenous stroma. Ultrastructural evidence of fibroblastic differentiation was noted in one tumor examined. Our cohort also included two sarcomas with VIM–KMT2A fusions, each harboring concurrent mutations in CTNNB1, SMARCB1, and ARID1A and characterized histologically by sheets of spindle-to-round blue cells. The remaining 16 KMT2A-rearranged sarcomas in our cohort exhibited diverse histologic subtypes, each with unique novel fusion partners. In summary, KMT2A-fusion-positive sarcomas most commonly exhibit sclerosing epithelioid fibrosarcoma-like morphology and complex YAP1–KMT2A–YAP1 fusions. Cases also include rare spindle-to-round cell sarcomas with VIM–KMT2A fusions and tumors of diverse histologic subtypes with unique KMT2A fusions to non-YAP1 non-VIM partners.

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy., SMARCA4 is the core catalytic subunit of the mammalian SWI/SNF complex and is known to be mutated in many cancers. Here, the authors detect more than 10,000 SMARCA4 variants across different cancer subtypes and find hotspot mutations throughout the helicase domain, which reduce remodeling activity.

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 Vulvar squamous cell carcinoma (vSCC) encompasses two predominant variants: one associated with detectable high-risk strains of human papillomavirus (hrHPV) and a second form often occurring in the context of chronic dermatitis in postmenopausal women. Genomic assessment of a large-scale cohort of patients with aggressive vSCC may identify distinct mutational signatures. MATERIALS AND METHODS Tumor samples from a total of 280 patients with vSCC underwent hybridization capture with analysis of up to 406 cancer-related genes. Human papillomavirus (HPV) sequences were detected by de novo assembly of nonhuman sequencing reads and aligned to the RefSeq database. Immunohistochemistry for programmed death-ligand 1 (PD-L1) was assessed. RESULTS One hundred two of 280 vSCCs (36%) contained hrHPV sequences, predominantly HPV 16 (88%). The HPV-positive (HPV+) group was significantly younger (median age, 59 v 64 years; P = .001). Compared with HPV-negative (HPV–) vSCCs, HPV+ tumors showed more frequent pathogenic alterations in PIK3CA (31% v 16%; P = .004), PTEN (14% v 2%; P < .0001), EP300 (14% v 1%; P < .0001), STK11 (14% v 1%; P < .0001), AR (5% v 0%; P = .006), and FBXW7 (10% v 3%; P = .03). In contrast, HPV– vSCCs showed more alterations in TP53 (83% v 6%; P < .0001), TERTp (71% v 9%; P < .0001), CDKN2A (55% v 2%; P < .0001), CCND1 amplification (22% v 2%; P < .0001), FAT1 (25% v 4%; P < .0001), NOTCH1 (19% v 6%; P = .002), and EGFR amplification (11% v 0%; P < .0001), as well as a higher rate of 9p24.1 ( PDL1/PDL2) amplification (5% v 1%) and PD-L1 immunohistochemistry high-positive tumor staining (33% v 9%; P = .04). CONCLUSION Comprehensive molecular profiles of vSCC vary considerably with hrHPV status and may inform patient selection into clinical trials. Sixty-one percent of HPV+ vSCCs had a pathogenic alteration in the PI3K/mTOR pathway, whereas HPV– vSCCs showed alterations in TP53, TERTp, CDKN2A, CCND1, and EGFR, and biomarkers associated with responsiveness to immunotherapy.

Immunotherapy has recently been incorporated into the treatment guidelines for metastatic urothelial carcinoma. Nevertheless, the role of prognostic and predictive biomarkers in this setting is not completely defined. To date, PD-L1 expression and a high tumour mutational burden (TMB) seem to predict better responses to immune checkpoint inhibitors, but patients without these biomarkers may still respond to immunotherapy. There are some caveats regarding these biomarkers, such as lack of standardisation of techniques, tumour heterogeneity and other factors influencing the tumour microenvironment. Genomic signatures are other promising emerging strategies. We hereby discuss the management of a 70-year-old man with a metastatic recurrence of urothelial carcinoma within 1 year after neoadjuvant chemotherapy and radical cystectomy. Tumour next-generation sequencing showed a high TMB and a CD274 (PD-L1) amplification. The patient was treated with pembrolizumab and achieved a complete response.

Highlights • NUT carcinoma is a rare but aggressive solid tumor that can be diagnosed by presence of the BRD4-NUT fusion. • This series presents 31 cases of solid tumors that harbor BRD4-NUT but often carry other diagnoses such NSCLC—NOS and NSCLC-SCC. • Despite lack of PD-L1 expression and a low tumor mutational burden, two index cases responded to either nivolumab or atezolizumab+chemotherapy with partial response or better with 4–5 month duration of response. • The unexpected response to checkpoint inhibitors could be explained by a very high affinity of the fusion peptide at the junction of BRD4 and NUT to the MHC complex as recently suggested for an exceptional response to an immune checkpoint inhibitor in a fusion bearing low TMB, low PD-L1 expression head and neck carcinoma., 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 NSCLC—NOS (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.

Objectives Castleman disease (CD) is a heterogeneous group of disorders involving systemic inflammation and lymphoproliferation. Recently, clonal mutations have been identified in unitcentric CD (UCD) and idiopathic multicentric CD (iMCD), suggesting a potential underlying neoplastic process. Methods Patients with UCD or iMCD with next generation sequencing (NGS) data on tissue DNA and/or circulating tumor DNA (ctDNA) were included. Results Five patients were included, 4 with iMCD and 1 with UCD. Four patients (80%) were women; median age was 40 years. Three of five patients (60%) had ≥1 clonal mutation detected on biopsy among the genes included in the panel. One patient with iMCD had a 14q32-1p35 rearrangement and a der(1)dup(1)(q42q21)del(1)(q42) (1q21 being IL-6R locus) on karyotype. This patient also had a NF1 K2459fs alteration on ctDNA (0.3%). Another patient with iMCD had a KDM5C Q836* mutation, and one patient with UCD had a TNS3-ALK fusion but no ALK expression by immunohistochemistry. Conclusions We report 4 novel somatic alterations found in patients with UCD or iMCD. The 1q21 locus contains IL-6R, and duplication of this locus may increase IL-6 expression. These findings suggest that a clonal process may be responsible for the inflammatory phenotype in some patients with UCD and iMCD.

Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are rare oncogenic drivers in solid tumours. This study aimed to interrogate a large real-world database of comprehensive genomic profiling data to describe the genomic landscape and prevalence of NTRK gene fusions. NTRK fusion-positive tumours were identified from the FoundationCORE

Most colorectal (CRC) tumors are dependent on EGFR/KRAS/BRAF/MAPK signaling activation. ARID1A is an epigenetic regulator mutated in approximately 5% of non-hypermutated CRC tumors. Here we show that anti-EGFR but not anti-VEGF treatment enriches for emerging ARID1A mutations in CRC patients. In addition, we find that patients with ARID1A mutations, at baseline, are associated with worse outcome when treated with cetuximab- but not bevacizumab-containing therapies; thus, this suggests that ARID1A mutations may provide both an acquired and intrinsic mechanism of resistance to anti-EGFR therapies. We find that, ARID1A and EGFR-pathway genetic alterations are mutually exclusive across lung and colorectal cancers, further supporting a functional connection between these pathways. Our results not only suggest that ARID1A could be potentially used as a predictive biomarker for cetuximab treatment decisions but also provide a rationale for exploring therapeutic MAPK inhibition in an unexpected but genetically defined segment of CRC patients.

Introduction: The activity of immune checkpoint blockade including anti-cytotoxic T-lymphocyte associated protein-4 and anti-programmed cell death protein-1 monoclonal antibodies in hematologic malignancies is limited outside of classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. Tumor mutational burden (TMB), programmed death ligand-1 (PD-L1) expression, and microsatellite instability-high (MSI-H) are well-established biomarkers predicting response to checkpoint blockade in solid malignancies. In addition, tumors with high TMB, defined as ≥10 mutations/megabase (mut/Mb), and/or MSI-H are Food and Drug Administration (FDA) approved tissue agnostic biomarkers for treatment with pembrolizumab. The frequencies of high TMB, MSI-H, and expression pattern of PD-L1 across specific hematologic malignancies are undefined. Methods: Patients with hematologic malignancies who had next generation sequencing (NGS) performed by Foundation One Heme were identified. TMB and MSI were measured by NGS. TMB was classified as high if ≥10 mut/Mb and low if Results: A total of 390 patients with hematologic malignancies with NGS were identified. Forty eight of the 390 samples (12%) had a high TMB (Table 1). Twenty five of 45 (56%) patients with DLBCL had a high TMB (Table 2). The TMB was low in all myeloid malignancies tested. None of the 302 samples tested were MSI-H. PD-L1 IHC was performed on 86 samples. Eleven (13%) had high expression, 26 (30%) had low expression, and 49 (57%) had no expression of PD-L1 on the tumor cells (Table 1). The majority of samples with PD-L1 expression were mature lymphomas (81%). TMB and PD-L1 score had a significant linear relationship (R = 0.22,p= 0.04, 95% CI 0.01 - 0.41) (Figure 1). Conclusion: This study provides detailed characteristics of TMB, MSI status, and PD-L1 expression for hematologic malignancies. Notably, a subset of lymphomas had high TMB and/or PD-L1 expression. Biomarker driven studies of checkpoint blockade in hematologic malignancies with high TMB and/or PD-L1 expression are warranted. Disclosures Sokol: Foundation Medicine:Current Employment;Roche:Current equity holder in publicly-traded company.Kurzrock:Medimmune:Research Funding;Foundation Medicine:Research Funding;Konica Minolta:Research Funding;IDbyDNA:Current equity holder in private company;Pfizer:Consultancy, Research Funding;Sequenom:Research Funding;Bicara Therapeutics, Inc.:Consultancy;Incyte:Research Funding;Takeda:Research Funding;TopAlliance:Research Funding;Boehringer Ingelheim:Research Funding;CureMatch Inc:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Grifols:Research Funding;Guardant:Research Funding;X Biotech:Consultancy;Neomed:Consultancy;Actuate Therapeutics:Consultancy;Roche:Consultancy;Merck Serono:Research Funding;Genentech:Research Funding;Debiopharm:Research Funding;CureMetrix:Membership on an entity's Board of Directors or advisory committees;OmniSeq:Research Funding;TD2/Volastra:Consultancy;Turning Point Therapeutics:Consultancy.Goodman:EUSA Pharma:Consultancy;Seattle Genetics:Consultancy.

The aim of the present study was to determine cell of origin (COO) from a platform using a DNA-based method, COO DNA classifier (COODC). A targeted exome-sequencing platform that applies the mutational profile of a sample was used to classify COO subtype. Two major mutational signatures associated with COO were identified: Catalogue of Somatic Mutations in Cancer (COSMIC) signature 23 enriched in activated B cell (ABC) and COSMIC signature 3, which suggested increased frequency in germinal center B cell (GCB). Differential mutation signatures linked oncogenesis to mutational processes during B-cell activation, confirming the putative origin of GCB and ABC subtypes. Integrating COO with comprehensive genomic profiling enabled identification of features associated with COO and demonstrated the feasibility of determining COO without RNA.Lay abstract To determine subtypes of diffuse large B-cell lymphoma (DLBCL), a cancer with poor survival, we aimed to identify DLBCL subtypes using DNA mutation-based tools. A targeted gene-sequencing platform, which measures the number and types of DNA mutations in a sample, was used to categorize DLBCL subtypes. Two major patterns of mutations associated with subtypes were identified: Catalogue of Somatic Mutations in Cancer (COSMIC) signature 23 and COSMIC signature 3. Differences in how the subtypes developed suggest a link between tumor developments and B cells being activated normally, confirming where the DLBCL subtypes came from. Combining this information with comprehensive genomic profiling, which determines all of the genes that a person has, allowed identification of features that are associated with DLBCL subtypes and showed that a DLBCL subtype can be determined without using RNA.

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.

Simple Summary Clinically advanced pheochromocytomas and paragangliomas are a rare form of endocrine malignancy which can occur in familial and sporadic clinical settings and feature a variety of genomic alterations. Comprehensive genomic profiling (CGP) was performed to characterize the genomic alterations (GA) in clinically advanced disease to enable the search for potential therapy targets. Although the GA/tumor is relatively low for clinically advanced disease, CGP can reveal important potential targets for therapy in the metastatic setting including RET, NF1 and FGFR1. Based on this data, further study of CGP as a method of developing precision therapies for clinically advanced disease appears warranted. Abstract Patients with clinically advanced paragangliomas (CA-Para) and pheochromocytomas (CA-Pheo) have limited surgical or systemic treatments. We used comprehensive genomic profiling (CGP) to compare genomic alterations (GA) in CA-Para and CA-Pheo to identify potential therapeutic targets. Eighty-three CA-Para and 45 CA-Pheo underwent hybrid-capture-based CGP using a targeted panel of 324 genes. Tumor mutational burden (TMB) and microsatellite instability (MSI) were determined. The GA/tumor frequencies were low for both tumor types (1.9 GA/tumor for CA-Para, 2.3 GA/tumor for CA-Pheo). The most frequent potentially targetable GA in CA-Para were in FGFR1 (7%, primarily amplifications), NF1, PTEN, NF2, and CDK4 (all 2%) and for CA-Pheo in RET (9%, primarily fusions), NF1 (11%) and FGFR1 (7%). Germline mutations in known cancer predisposition genes were predicted in 13 (30%) of CA-Pheo and 38 (45%) of CA-Para cases, predominantly involving SDHA/B genes. Both CA-Para and CA-Para had low median TMB, low PD-L1 expression levels and none had MSI high status. While similar GA frequency is seen in both CA-Para and CA-Para, germline GA were seen more frequently in CA-Para. Low PD-L1 expression levels and no MSI high status argue against strong potential for novel immune checkpoint inhibitors. However, several important potential therapeutic targets in both CA-Para and CA-Para are identified using CGP.

Despite recent advances in elucidating molecular pathways underlying adrenocortical carcinoma (ACC), this orphan malignancy is associated with poor survival. Identification of targetable genomic alterations is critical to improve outcomes. The objective of this study was to characterize the genomic profile of a large cohort of patient ACC samples to identify actionable genomic alterations. Three hundred sixty-four individual patient ACC tumors were analyzed. The median age of the cohort was 52 years and 60.9% (n = 222) were female. ACC samples had common alterations in epigenetic pathways with 38% of tumors carrying alterations in genes involved in histone modification, 21% in telomere lengthening, and 21% in SWI/SNF complex. Tumor suppressor genes and WNT signaling pathway were each mutated in 51% of tumors. Fifty (13.7%) ACC tumors had a genomic alteration in genes involved in the DNA mismatch repair (MMR) pathway with many tumors also displaying an unusually high number of mutations and a corresponding MMR mutation signature. In addition, genomic alterations in several genes not previously associated with ACC were observed, including IL7R, LRP1B, FRS2 mutated in 6, 8 and 4% of tumors, respectively. In total, 58.5% of ACC (n = 213) had at least one potentially actionable genomic alteration in 46 different genes. As more than half of ACC have one or more potentially actionable genomic alterations, this highlights the value of targeted sequencing for this orphan cancer with a poor prognosis. In addition, significant incidence of MMR gene alterations suggests that immunotherapy is a promising therapeutic for a considerable subset of ACC patients.

Introduction: De-regulation of the class I phosphoinositide 3’-kinase (PI3K) pathway has long been known to contribute to the development and progression of many tumors. However, the FDA only recently approved the first selective inhibitor of PIK3CA, the p110-alpha catalytic subunit of PI3K, specifically for use in treatment of a subset of PIK3CA-mutant breast carcinomas. Given the emergence of this therapeutic class of agents, we sought to identify other subsets of breast tumors that may be driven largely by PIK3CA mutations and which therefore could be candidates for these therapies. In breast fibroepithelial neoplasms, mutations in PIK3CA have been occasionally reported in borderline and malignant phyllodes tumors. In contrast, mutations in MED12 are common and recurrent across the entire spectrum of benign and malignant breast fibroepithelial tumors, and are enriched in borderline/malignant phyllodes cases that still have benign fibroadenoma-like areas. In the current study, we sought to define the histologic and molecular features of PIK3CA-mutant phyllodes tumors. Methods: From 2014 to 2019, we analyzed clinical tumor samples using comprehensive genomic profiling by a hybrid capture-based DNA sequencing platform. We searched our case archive to find breast phyllodes tumors with known or likely pathogenic alterations in PIK3CA and other known tumor-related genes. All cases were clinically advanced. We reviewed pathology reports, histopathology, and patient clinical data. Results: We identified 12 (16%) of 76 breast phyllodes tumors in our case archive as PIK3CA-mutant. Median patient age for PIK3CA-mutant phyllodes tumors was 56 years. Cases consisted of 6 primary tumors, 2 local recurrences, and 4 lung metastases. Primary tumor size measured from 38 to 220 mm (median 100 mm; mean 114 mm). Digital slides were available for histology review in 9 cases. Cases showed uniformly malignant histology, with no benign or fibroadenoma-like regions. 3 cases showed malignant heterologous elements. Compared to the rest of our breast phyllodes tumor cohort, PIK3CA-mutant cases showed significantly fewer pathogenic genomic alterations in MED12 (8% vs. 55%, p=0.0037) and TP53 (17% vs. 56%, p=0.0245), as well as a trend to fewer mutations in RB1 (0% vs. 22%, p=0.11). The other most frequently mutated genes in the PIK3CA-mutant group were TERTp (70%), CDKN2A (67%), and NF1 (50%). Conclusions: PIK3CA-mutant phyllodes tumors define a unique subset of tumors characterized by aggressive histologic features and largely lacking the MED12 mutations seen in many fibroepithelial neoplasms. These findings provide compelling rationale for comprehensive genomic profiling of advanced cases of this disease in an effort to inform therapeutic options including clinical trials of PI3K-targeted agents in this setting. Citation Format: Erik A Williams, Ethan S Sokol, Dean C Pavlick, Nikunj Shah, Julia A Elvin, Jo-Anne Vergilio, Jonathan K Killian, Nhu Ngo, Douglas Lin, Vincent A Miller, Jeffrey S Ross. PIK3CA-mutant breast phyllodes tumors show a uniformly aggressive histology and significant mutual exclusivity with MED12 mutation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-07.

Purpose Amplifications of receptor tyrosine kinases (RTKS) are therapeutic targets in multiple tumor types (e.g. HER2 in breast cancer), and amplification of the chromosome 4 segment harboring the three RTKs KIT, PDGFRA, and KDR (4q12amp) may be similarly targetable. The presence of 4q12amp has been sporadically reported in small tumor specific series but a large‐scale analysis is lacking. We assess the pan‐cancer landscape of 4q12amp and provide early clinical support for the feasibility of targeting this amplicon. Experimental Design Tumor specimens from 132,872 patients with advanced cancer were assayed with hybrid capture based comprehensive genomic profiling which assays 186–315 genes for all classes of genomic alterations, including amplifications. Baseline demographic data were abstracted, and presence of 4q12amp was defined as 6 or more copies of KIT/KDR/PDGFRA. Concurrent alterations and treatment outcomes with matched therapies were explored in a subset of cases. Results Overall 0.65% of cases harbored 4q12amp at a median copy number of 10 (range 6–344). Among cancers with >100 cases in this series, glioblastomas, angiosarcomas, and osteosarcomas were enriched for 4q12amp at 4.7%, 4.8%, and 6.4%, respectively (all p < 0.001), giving an overall sarcoma (n = 6,885) incidence of 1.9%. Among 99 pulmonary adenocarcinoma cases harboring 4q12amp, 50 (50%) lacked any other known driver of NSLCC. Four index cases plus a previously reported case on treatment with empirical TKIs monotherapy had stable disease on average exceeding 20 months. Conclusion We define 4q12amp as a significant event across the pan‐cancer landscape, comparable to known pan‐cancer targets such as NTRK and microsatellite instability, with notable enrichment in several cancers such as osteosarcoma where standard treatment is limited. The responses to available TKIs observed in index cases strongly suggest 4q12amp is a druggable oncogenic target across cancers that warrants a focused drug development strategy. Implications for Practice Coamplification of the receptor tyrosine kinases (rtks) KIT/KDR/PDGFRA (4q12amp) is present broadly across cancers (0.65%), with enrichment in osteosarcoma and gliomas. Evidence for this amplicon having an oncogenic role is the mutual exclusivity of 4q12amp to other known drivers in 50% of pulmonary adenocarcinoma cases. Furthermore, preliminary clinical evidence for driver status comes from four index cases of patients empirically treated with commercially available tyrosine kinase inhibitors with activity against KIT/KDR/PDGFRA who had stable disease for 20 months on average. The sum of these lines of evidence suggests further clinical and preclinical investigation of 4q12amp is warranted as the possible basis for a pan‐cancer drug development strategy., Matching treatment strategy to tumor biology is central to precision medicine. The co‐amplification of three distinct RTK encoding genes (KIT, KDR, and PDGFRA) on chromosome 4q12 (4q12amp) may be an oncogenic driver and thus a target for therapy. This article assesses the pan‐cancer landscape of 4q12amp and provides clinical support for the feasibility of targeting this amplicon.

Introduction Presently, programmed death ligand 1 is the most commonly used biomarker to predict response to immune checkpoint inhibitors (ICIs) in NSCLC. Owing to its several limitations, there is continuous search for more precise and reliable markers. Frameshift mutations by insertion or deletion (fsindels) are suggested to induce more immunogenic tumor-specific neoantigens, conferring better response to ICIs. Positive correlation of fsindels with ICI response has been studied in melanoma and renal cell carcinoma. We investigated the implication of fsindels in the clinical outcomes and immune landscape of patients with NSCLC treated with ICIs. Methods We utilized The Cancer Genome Atlas data set to analyze tumor mutational burden, neoantigen burden, and immune landscape in relation to fsindel status. In addition, utilizing the clinical data from 122 patients treated with ICIs, we evaluated the influence of fsindels on disease response rates and survival outcomes. Results A positive correlation between fsindel burden and tumor mutational burden and activated CD4/CD8 T-cell infiltration was shown. Presence of fsindels was also associated with significant prolongation of progression-free survival in patients treated with ICIs (median 6.2 versus 2.7 months [p = 0.01]). In addition, significant differences in the overall response rates (26% versus 12% [p = 0.04]) and disease control rates (68% versus 48% [p = 0.02]) were observed in patients with fsindels. Conclusion Our findings suggest that fsindels may have a predictive role for ICI response in NSCLC.

Purpose: Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous malignancy, which has demonstrated sensitivity to immune checkpoint inhibitor therapy. Here, we perform the largest genomics study in MCC to date to characterize the molecular landscape and evaluate for clinical and molecular correlates to immune checkpoint inhibitor response. Experimental Design: Comprehensive molecular profiling was performed on 317 tumors from patients with MCC, including the evaluation of oncogenic mutations, tumor mutational burden (TMB), mutational signatures, and the Merkel cell polyomavirus (MCPyV). For a subset of 57 patients, a retrospective analysis was conducted to evaluate for clinical and molecular correlates to immune checkpoint inhibitor response and disease survival. Results: Genomic analyses revealed a bimodal distribution in TMB, with 2 molecularly distinct subgroups. Ninety-four percent (n = 110) of TMB-high specimens exhibited an ultraviolet light (UV) mutational signature. MCPyV genomic DNA sequences were not identified in any TMB-high cases (0/117), but were in 63% (110/175) of TMB-low cases. For 36 evaluable patients treated with checkpoint inhibitors, the overall response rate was 44% and response correlated with survival at time of review (100% vs. 20%, P < 0.001). Response rate was 50% in TMB-high/UV-driven and 41% in TMB-low/MCPyV-positive tumors (P = 0.63). Response rate was significantly correlated with line of therapy: 75% in first-line, 39% in second-line, and 18% in third-line or beyond (P = 0.0066). PD-1, but not PD-L1, expression was associated with immunotherapy response (77% vs. 21%, P = 0.00598, for PD-1 positive and negative, respectively). Conclusions: We provide a comprehensive genomic landscape of MCC and demonstrate clinicogenomic associates of immunotherapy response.

Background CDK12 loss-of-function (LOF) genomic alterations are associated with focal tandem duplications (FTDs) in ovarian and prostate cancers. Because these FTDs may produce fusion-induced neoantigens (FINAs), CDK12 alteration is a candidate biomarker for immune checkpoint inhibitor sensitivity. Here we determine the prevalence of CDK12-LOF alterations and their association with FTDs across diverse tumor types. Materials and Methods A total of 142,133 tumor samples comprising 379 cancer types were sequenced (August 2014 to April 2018) by hybrid capture-based comprehensive genomic profiling (Foundation Medicine, Cambridge, MA) as part of routine clinical care. Results were analyzed for base substitutions, short insertions/deletions, rearrangements, and copy number alterations. CDK12-LOF genomic alterations were assessed for zygosity status and association with FTDs/focal copy number gain. Results CDK12 genomic alterations were detected in 1.1% of all cases, most frequently in prostate cancer (5.6%), but were also observed at >1% frequency in 11 cancer types. Across multiple cancer types, including prostate, gastric/esophageal, ovarian, breast, and endometrial cancer, the number of FTDs was significantly increased in CDK12-LOF versus CDK12 wild-type cases. Notably, CDK12-LOF was not consistently associated with a homologous recombination deficiency genomic signature. Quantitative assessment of CDK12-associated FTDs by measurement of single copy number gains identified novel likely deleterious CDK12 kinase-domain mutations in prostate and ovarian cancers. Conclusion Detection of CDK12-LOF genomic alterations and their association with FTDs in a diverse spectrum of malignancies suggests that immunotherapy approaches targeting FINAs derived from CDK12-associated FTDs may be a broadly applicable strategy that could be explored across cancer types in a tumor-agnostic manner. Implications for Practice CDK12 inactivation in ovarian and prostate cancer results in the generation of focal tandem duplications, which can cause fusion-induced neoantigens. In prostate cancer, CDK12 alterations have demonstrated promise as a potential predictive biomarker for response to immune checkpoint blockade. This study evaluated genomic profiling data from >142,000 tumors to determine the prevalence of CDK12 loss-of-function genomic alterations across tumor types and demonstrated that CDK12 alterations are associated with the tandem-duplicator phenotype in cancer types other than ovarian and prostate cancer. The association of CDK12 alterations with focal tandem duplications across broad cancer types suggests that CDK12 inactivation warrants further investigation as a pan-cancer biomarker for immunotherapy benefit.

Background The recent approval of PARP inhibitor, olaparib, in HER2-negative breast cancer expands the therapeutic options for patients with germline BRCA1/2 alterations. The role of somatic BRCA alteration as a predictive biomarker in breast cancer is currently unclear. NCCN guidelines call for germline testing in all young patients ( Methods Hybrid-capture based comprehensive genomic profiling of 395 cancer-related genes using the FoundationOne assay (Foundation Medicine, MA) was performed on 12,508 breast carcinomas. Somatic/germline/zygosity status for BRCA1/2 variants was analyzed as described in Sun, 2018 (PMID: 29415044). High genome-wide loss-of-heterozygosity (gLOH) was classified as ≥16% LOH. Subgroups were analyzed on histological {Invasive Ductal Carcinoma (IDC), Invasive Lobular Carcinoma (ILC)} and molecular subtypes [ER-positive (ER+), HER2-amplified (HER2+), TNBC], patient age [≤45, 46-60, 61+], and gender. Results Consistent with previous reports, the frequency of BRCA1/2 alterations was highest in young patients ( The fraction of somatic BRCA mutations (sBRCA) was 38%, with the lowest fraction of sBRCA in TNBC and young patients (30%, 30% overall, 36%, 42% for BRCA1) and highest in HER2+, ILC, and older patients (46%, 52%, 51%); the absolute frequency of sBRCA is approximately 3.5%. In tumors with ESR1 mutations, we detected concurrent BRCA mutation in 6.4% (85/1319), with 48% predicted somatic. Almost all tumors with BRCA mutations had biallelic inactivation, with LOH of the second allele irrespective of predicted germline status (90% of gBRCA and 86% of sBRCA under LOH). gLOH score was used as a phenotypic measure of homologous recombination deficiency (HRD): patients harboring biallelic BRCA alterations had elevated rates of gLOH with 89% of gBRCA and 84% of sBRCA tumors harboring a high gLOH score, vs 47% with heterozygous BRCA, 34% with no BRCA alteration, and 80% for patients with BRCA deletion. Conclusions Thirty-eight percent of deleterious BRCA alterations in breast are predicted somatic, including 36% of BRCA1 alterations in TNBC. Germline testing would miss these alterations even though they are frequently under LOH and are associated with high gLOH, a biomarker with predictive value in ovarian cancer. While patients with ER+ and HER2+ tumors have low rates of gBRCA alterations, the overall BRCA mutation rate, including somatic alterations, is appreciable at 8.3% and 5.3%. Our findings demonstrate that sBRCA alterations are associated with a comparable HRD phenotype to gBRCA altertions and suggests that PARP inhibitors may have potential value for a wider range of breast cancer patients. Citation Format: Sokol ES, Frampton GM, Ross J, Ali S, Chung J, Oesterreich S. BRCA1/2 alterations are present at significant rates across breast cancer subtypes and are associated with a high genome-wide loss of heterozygosity signature [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-05.