Foundation Medicine Database Research Articles

Molecular characteristics of advanced colorectal cancer and multi-hit PIK3CA mutations.

Approximately 20% of patients living with colorectal cancer (CRC) have activating mutations in their tumors in the PIK3CA oncogene. Two or more activating mutations (multi-hit) for the PIK3CA allele increase PI3K⍺ signaling compared to single-point mutations, resulting in exceptional response to PI3K⍺ inhibition. We aimed to identify the prevalence of PIK3CA multi-hit mutations in metastatic CRC to identify patients who may benefit from PI3K inhibitors. The Foundation Medicine database (Boston, MA, USA) was analyzed for patients with CRC who underwent genomic profiling on tumor DNA isolated during routine clinical care from 2013 to 2021. Molecular and clinical variables were abstracted for patients with PIK3CA mutations. We identified 49051 patients with CRC who underwent Foundation Medicine testing. 710/41154 (1.7%) patients had multi-hit PIK3CA mutations, of which 53% were male (n = 448) with a median age of 60. Microsatellite status was available for 697 patients with multi-hit PIK3CA and 17.6% (123/697) were microsatellite instability-high. Clinically relevant mutations in KRAS and BRAFV600E were seen in 459/710 (64.7%) and 65/710 (9.1%), respectively. The 4 most common PIK3CA variants were H1047R (9.8%), E545K (9.2%), E542K (9.0%), and R88Q (7.1%). The most common variant pair was E542K-E545K (4.7%). Multi-hit mutations in PIK3CA are seen in 1.7% of advanced CRC, a meaningful prevalence given the high burden of CRC worldwide, and may represent a subset of patients that have enhanced sensitivity to PI3K inhibition. Future investigation regarding the clinical utility of PI3K inhibitors is warranted in multi-hit PIK3CA CRC.

Abstract PO5-06-14: Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma

Abstract Background: Triple-negative (TN) invasive lobular carcinoma (ILC) of the breast is a rare entity, comprising roughly 2% of primary ILC. Clinicopathologic studies have shown that TN ILC is associated with more pleomorphism, higher nuclear grade, older patient age, and worse disease-related outcomes compared to estrogen receptor-positive (ER+) ILC. Intrinsic subtyping indicates that TN ILC less frequently expresses basal markers than TN invasive ductal carcinoma (IDC). This prompted investigation of the genomic landscape and clinical management of TN ILC to provide insight into potential therapeutic approaches. Methods: Twenty patients with TN ILC were included in this study. Three patients were treated at a single academic center and underwent comprehensive genomic profiling (CGP) of breast or metastatic tissue; clinicopathologic details were obtained from their electronic health records. Seventeen patients with TN ILC who underwent CGP of breast or metastatic biopsies were included from the Foundation Medicine, Inc. database. PD-L1 testing for all patients was determined by immunohistochemistry (IHC) using the VENTANA SP142 or Dako 22C3 commercial assays. HER2 expression was determined by IHC. Negative HER2 expression was defined as an IHC score of 0, or IHC 1+ or 2+ with non-amplified fluorescence in situ hybridization (FISH). Results: In the Foundation Medicine database of patients with TN ILC, the most frequent genomic alterations were in CDH1 (15/17 patients [88.2%]), TP53 (10/17 patients [58.8%]), and PIK3CA (10/17 patients [58.8%]). Short variant mutations in ERBB2 were found in 3/17 patients (17.6%). Fourteen patients were HER2-low (HER2 IHC 1+ or 2+ with non-amplified FISH). No cases were MSI high or expressed PD-L1 (8 of 17 cases underwent PD-L1 assessment by SP142). Median tumor mutational burden (TMB) was 5 muts/Mb. Among the three patients with TN ILC treated at a single center, the most common somatic mutations were in CDH1 and TP53, and no cases were MSI high or expressed PD-L1 (by SP142 or 22C3). All three patients had pleomorphic ILC, histologic grade 2 or 3, and HER2 IHC 0. One patient with mpT1cN1a(sn) ER+/HER2- ILC of the right breast in 2011 received adjuvant TAC (docetaxel, doxorubicin, cyclophosphamide), breast/axillary radiation, and ten years of adjuvant endocrine therapy. She was diagnosed with TN ILC of the left supraclavicular nodes in 2022; she did not exhibit a response to docetaxel and carboplatin with pembrolizumab, and she subsequently received palliative radiation followed by capecitabine. Another patient with locally advanced TN ILC progressed on the KEYNOTE-522 regimen, immediately developed metastases, and is currently receiving palliative sacituzumab govitecan. The third patient with cT3N1 TN ILC progressed on neoadjuvant therapy (through the I-SPY 2 trial) with oral paclitaxel, encequidar, and dostarlimab, followed by dostarlimab with dose-dense doxorubicin and cyclophosphamide. Her surgical pathology demonstrated ypT3N3a disease with 10 × 7 cm of tumor, and she developed skin metastases on the chest wall within weeks post-operatively. She received comprehensive chest wall and nodal radiation and is currently receiving palliative capecitabine with neratinib given a high variant allele frequency driver somatic ERBB2 mutation. Conclusions: The most common somatic mutations among TN ILC patients included in this study were in CDH1, TP53, and PIK3CA. Currently the management of TN ILC is extrapolated from that of TN IDC, however our patients did not respond to chemoimmunotherapy. The presence of HER2-low status or somatic ERBB2 mutations may provide opportunities for treatment with an anti-HER2 antibody-drug conjugate or tyrosine kinase inhibitor. Studies with larger sample sizes of TN ILC are needed for molecular profiling and assessment of outcomes with specific therapeutic approaches. Citation Format: Hemali Batra-Sharma, Smruthy Sivakumar, Prashanth Ashok Kumar, Ethan Sokol, Rebecca Shatsky, Jeffrey Ross. Genomic Landscape and Clinical Outcomes of Triple-Negative Invasive Lobular Carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-14.

Association of FGFR2 structural alterations in intrahepatic cholangiocarcinomas with female gender and younger age.

537 Background: FGFR2 fusions and other rearrangements are targetable alterations due to the advent of FGFR2 kinase domain inhibitors, and are considerably more common in intrahepatic cholangiocarcinomas (iCCA) than other cancer types. Understanding clinical factors associated with FGFR2 fusions and other rearrangements may guide precision oncology efforts. Prior studies indicate that FGFR2 alterations are more common in younger patients, but it remains unknown whether other demographics are specifically associated with FGFR2 structural alterations. Here, we reviewed the frequency of FGFR2 structural alterations in 8,898 iCCA patients from the AACR Genie and the Foundation Medicine databases. Methods: We reviewed the Foundation Medicine (n= 7,934) and AACR GENIE database (n=964) for intrahepatic cholangiocarcinomas. We identified iCCAs with FGFR2 structural alterations and mutations in other common driver genes and analyzed their distribution by age and sex. Results: FGFR2 structural alterations were more frequent in female (10.4%) compared to male (6.4%) patients across both cohorts. FGFR2 structural alterations had an association with an odds ratio of 2.5 (95% CI 1.6-3.7) and 1.7 (95% CI-2.0) with the female gender in the AACR GENIE and Foundation Medicine databases, respectively. The frequency of FGFR2 fusions/rearrangement was highest during reproductive years (up to age 40) and subsequently declined with age. This decline was greater in females than males. Mutations in other genes associated with iCCA including non-fusion FGFR2 alterations, and mutations in ARID1A, KRAS, and TP53 were not associated with gender or age. Conclusions: Our results show that FGFR2 structural alterations are significantly enriched in younger female patients with iCCA. Clinical assessment of FGFR2 alterations should be prioritized in this patient population, and future clinical studies in this molecular subtype should ensure inclusive enrollment practices. Further research is warranted to determine if the pathogenesis of FGFR2 structural alterations are driven by female biology including the involvement of sex hormones and the potential impact on the development of acquired resistance to FGFR2 kinase inhibitors.

Abstract 2120: Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development

Abstract Background: Activation of the MAPK-signaling by RAS- (KRAS or NRAS) alterations is an important driver event in colorectal cancer (CRC) tumorigenesis that determines treatment selection (1).Real-world data (RWD) mining is an important tool to inform patient selection and stratification (2,3). Here, RWD genomics data mining was conducted using the Foundation Medicine database to explore RAS and BRAF-alterations in CRC patients and to investigate the general alterations’ landscape - including rare alterations. The prevalence of microsatellite instability (MSI) and tumor mutational burden (TMB) were also explored. Methods: The prevalence of gene alterations and their co-occurrence with RAS/BRAF-alterations in tumor tissue of CRC patients (N=42800) were investigated using the FoundationInsightsTM web platform, which includes harmonized results from the FoundationOneCDx, FoundationOneHeme, and PD-L1 assays from 2012 to March 2022. Data was collected from FMI solid and heme tests from 2012 to March 2022. Alterations that render a particular gene ‘altered’ included single nucleotide variants (SNVs) or insertion-deletion mutations (Indels), collectively referred to as short variants (SVs), rearrangements (RE) and copy number events (CN). TMB (cut-off 10 mut/MB) and MSI status were also characterized. Results: Altogether, 36.9% of CRC samples were RAS/BRAF-WT and 63.1% had a known or likely functional RAS/BRAF-alteration, respectively. Of all KRAS and NRAS alterations (SV, CN and RE regardless of annotation as functional or presence in a hotspot), SVs in exons 2, 3 and 4 constituted 96.1% and 94.2% of all alterations, respectively. Relative to all samples the potentially targetable KRAS p.G12C, p.G12D and p.G12V mutations were present in 3.6%, 15.1% and 10.2% of samples, respectively. The p.G12E variant was present in less than ten samples. The five most frequently altered potentially targetable genes were PIK3CA (19.6%), PTEN (8.7%), ERBB2 (4.7%), EGFR (2.5%) and FGFR1 (2.0%). NTRK-alterations (NTRK1/2/3) were present in 0.9%, ROS1-alterations in 0.4%, RET-alterations in 0.6% and ALK-alterations in 0.5% of samples, respectively. The prevalence of immuno-oncology biomarkers was also explored; overall, 9.6% of samples were TMB-high, 6.1% MSI-high and 0.6% POLE-altered. MSI-high or TMB-high samples were significantly more abundant in the RAS/BRAF-altered subset. In the RAS/BRAF-WT subset 4.6% were MSI-high and 7.9% TMB-high, whereas in the RAS/BRAF-altered subset 7.1% were MSI-high and 10.6% TMB-high. Conclusion: RWD based clinical genomic profiling confirms that the vast majority of RAS-alterations in CRC are SVs in exons 2, 3 and 4. Both TMB-high and MSI-high samples are significantly more abundant in the RAS/BRAF-altered cohort. RWD confirms that PI3K-, ERBB- and FGFR-signaling pathways are frequently altered in CRC. RWD based clinical genomic profiling has the potential to support comprehensive clinical study design as well as patient selection/stratification. Citation Format: Markus Schulze, XiaoZhe (Janet) Wang, Jawad Hamad, Julia F. Hopkins, Julia F. Hopkins, Jürgen Scheuenpflug, Zheng Feng. Exploring the genomic landscape of colorectal cancer for enabling clinical genomics informed and real-world data based clinical development [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 2120.

Activating MET kinase domain mutations define a novel molecular subtype of non–small cell lung cancer that is clinically targetable with the MET inhibitor elzovantinib (TPX-0022).

9124 Background: In non-small cell lung cancer (NSCLC), MET exon 14 skipping (METex14) mutations and MET amplification can be targeted with MET inhibitors. Here, we describe a novel, actionable molecular subtype of NSCLC characterized by activating MET-tyrosine kinase domain (MET-TKD) mutations in the absence of METex14 mutations. Methods: Clinicopathologic and genomic data were abstracted from NSCLC cases included in a multi-institutional cohort of tumors that underwent genomic profiling in the GENIE v10, China PanCancer, and the International Cancer Genome Consortium/ The Cancer Genome Atlas (ICGC/TCGA) datasets. External validation of the prevalence of MET-TKD mutations was performed on an independent cohort of NSCLC tissue and liquid samples from the Foundation Medicine genomic database. Results: Among 14,099 NSCLC samples in the multi-institutional cohort, 71 (0.5%) harbored MET-TKD mutations without concurrent METex14 mutations: 55 of these had uncertain pathogenic significance and 16 had known oncogenic potential, including MET H1094Y/R, D1228H/N/V, N1100S, H1106D, V1188I, and M1250T, in order of decreasing prevalence. In a separate cohort of 91,515 NSCLC samples from the Foundation Medicine database, MET-TKD mutations lacking concurrent METex14 mutations were identified in 799 (0.9%) samples, including H1094Y, L1195V, D1228H/N, M1250T and others. Among 60 NSCLC samples harboring MET-TKD mutations without concurrent METex14 mutations with complete genomic data in the multi-institutional cohort, 36 (60%) had concurrent driver alterations in KRAS, EGFR, ROS1, BRAF, HER2, or RET, while 24 (40%) had no concurrent oncogenic drivers. Among patients with available demographic data in the multi-institutional cohort, those with MET-TKD-mutant NSCLC (N = 70) were significantly younger than patients with METex14-mutant NSCLC (N = 353) (median age 63 [range 30-86] vs 73 [range 44-88], p < 0.0001), and there was no significant difference in sex or self-reported race. Confirmed partial responses to the MET tyrosine kinase inhibitor elzovantinib (TPX-0022) were observed in two patients with MET-TKD-mutant NSCLC and no other detectable driver mutations: a 64-year-old man with MET H1094Y-mutant NSCLC, and an 80-year-old man with MET F1200I-mutant NSCLC. Conclusions: Potentially actionable MET-TKD mutations lacking concurrent METex14 mutations represent a novel genomic subtype in 0.5-0.9% of NSCLC, and occur in the absence of other known drivers in a subset of cases.

Establishment and Characterization of Advanced Penile Cancer Patient-derived Tumor Xenografts: Paving the Way for Personalized Treatments

BackgroundSystemic treatments for penile squamous cell carcinoma (pSCC) are toxic and inefficient. Patient-based preclinical models are essential to study novel treatments. ObjectiveTo establish a library of patient-derived tumor xenograft (PDX) models of human papillomavirus-positive (HPV+) and -negative (HPV−) pSCC and characterize these at the genomic and histological levels. Design, setting, and participantsEighteen tumor samples from 14 patients with recurrent or metastatic pSCC were implanted in nude mice. A biobank of PDX tumors was established after passaging of patient samples (F0) for three generations (F1, F2, F3) and was characterized using histopathology and targeted next-generation sequencing (tNGS). Single-nucleotide polymorphism fingerprinting was used to confirm PDX genealogy. Outcome measurements and statistical analysisThe engraftment rate, overall growth rate, and pSCC histomorphology were checked for each PDX generation. Staining for p40 (a pSCC marker) and p16 (a surrogate for HPV infection) was performed for F0 samples. The mutational profile according to a validated panel of 96 cancer genes was determined for F0 and F3 samples and compared to a larger tNGS database. Results and limitationsIncluding a previously established pilot model, 11 out of 18 tumor samples (61%) successfully engrafted in F1. The mean time from implantation in F1 to completion of F3 was 36 wk (standard deviation 18). Histological fidelity was demonstrated across generations. The patient mutational profiles were preserved in F3 and were representative of 277 pSCC samples in the Foundation Medicine database. The rapid progression of pSCC in patients from our selected high-risk cohort impeded the use of PDXs as avatars. ConclusionsWe successfully established the first library of 11 PDX models of HPV− and HPV+ pSCC. Our PDX models showed high engraftment rates and histological and genomic fidelity to the tumor tissue of origin. These models may help in paving the way towards the development of novel treatments. Patient summaryWe established 11 animal models based on tumor tissue from patients with penile cancer. These models could play a vital role in selection of novel treatments according to genetic mutations. In the future, therapies with confirmed preclinical effects may have a profound impact on the development of personalized treatments in penile cancer.

Intra-patient stability of tumor mutational burden from tissue biopsies at different time points in advanced cancers

BackgroundTumor mutational burden (TMB) may be a predictive biomarker of immune checkpoint inhibitor (ICI) responsiveness. Genomic landscape heterogeneity is a well-established cancer feature. Molecular characteristics may differ even within the same tumor specimen and undoubtedly evolve with time. However, the degree to which TMB differs between tumor biopsies within the same patient has not been established.MethodsWe curated data on 202 patients enrolled in the PREDICT study (NCT02478931), seen at the University of California San Diego (UCSD), who had 404 tissue biopsies for TMB (two per patient, mean of 722 days between biopsies) to assess difference in TMB before and after treatment in a pan-cancer cohort. We also performed an orthogonal analysis of 2872 paired pan-solid tumor biopsies in the Foundation Medicine database to examine difference in TMB between first and last biopsies.ResultsThe mean (95% CI) TMB difference between samples was 0.583 [− 0.900–2.064] (p = 0.15). Pearson correlation showed a flat line for time elapsed between biopsies versus TMB change indicating no correlation (R2 = 0.0001; p = 0.8778). However, in 55 patients who received ICIs, there was an increase in TMB (before versus after mean mutations/megabase [range] 12.50 [range, 0.00–98.31] versus 14.14 [range, 0.00–100.0], p = 0.025). Analysis of 2872 paired pan-solid tumor biopsies in the Foundation Medicine database also indicated largely stable TMB patterns; TMB increases were only observed in specific tumors (e.g., breast, colorectal, glioma) within certain time intervals.ConclusionsOverall, our results suggest that tissue TMB remains stable with time, though specific therapies such as immunotherapy may correlate with an increase in TMB.Trial registrationNCT02478931, registered June 23, 2015.

Comprehensive Genomic Profiling (CGP) Identifies Novel BCL6 Rearrangements in Diverse Subtypes of Non-Hodgkin Lymphoma (NHL) As Well As Known Rearrangements Not Detected Using Standard of Care (SOC) Assays

▪Background: Chromosomal translocations of 3q27 are among the most frequent genomic aberrations in B-cell NHL, particularly large B-cell lymphoma (LBCL, 30%) and follicular lymphoma (FL, 15-45%, grade and subtype dependent). This region harbors the locus for proto-oncogene BCL6, a transcription factor implicated in lymphoma pathogenesis; translocation commonly replaces the 5'UTR of BCL6 with a heterologous promoter. BCL6 rearrangements are disease-defining within the WHO 2016 classification of high grade BCLs. Immunoglobulin (Ig)- BCL6 rearrangement concurrent with Ig-MYC in the presence or absence of Ig-BCL2 is a poor prognostic marker in LBCL. Non-Ig partners of BCL6 (>30 have been described) are less well-characterized, but also confer a poor prognosis in LBCL. Rearranged BCL6 has also been reported to distinguish t(14;18) negative FL, a unique clinicopathologic entity with worse prognosis. Historically, the cadre of BCL6 rearrangements has been assessed by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and karyotype; however, all methodologies have limitations. Given the diagnostic and prognostic relevance of BCL6 rearrangements, we report our experience using CGP to detect rearranged BCL6 and, when feasible, have compared these results to SOC testing.Methods: We interrogated the Foundation Medicine database (>100,000 diverse cancers) for rearrangements of the BCL6 locus in juxtaposition with a partner gene. Median age of the patient cohort was 64 years (range, 16-95 years), including 34 females and 49 males. All samples had been submitted for routine clinical evaluation and sequenced in a CLIA-certified, CAP-accredited, NYS-approved laboratory (Foundation Medicine, Inc). DNA and RNA was extracted from formalin fixed paraffin-embedded tissue or fresh peripheral blood. Hybrid capture of adaptor-ligated sequencing libraries was performed using FoundationOne® Heme, which targets 406 genes for DNAseq and detects gene fusions for 265 genes by RNAseq. Pathology reports were reviewed for FISH results or BCL6 expression by IHC.Results: In 83 cases, 85 BCL6 rearrangements were identified. The majority of rearranged cases were B-cell NHL: large BCL (LBCL, 60), follicular lymphoma (FL, 10), aggressive B-cell lymphoma (BCL, 7), and mantle cell lymphoma (MCL, 1). Unexpectedly, rearrangements were also detected in peripheral T-cell lymphoma (PTCL, 2), lymphoblastic leukemia (ALL, 1), soft tissue sarcoma (1), and lung small cell carcinoma (SCLC, 1). Of the rearrangements detected, 63 involved non-Ig partners.Within the 85 BCL6 rearrangements, 78% (66) involved a previously reported partner gene [‘known’, of which 67% (44) were non-Ig], whereas 22% (19) involved a partner gene not yet described in published reports (‘novel‘). In total, 34 partners were identified: 16 (47%) were known and 18 (53%) were novel (Table 1).FISH results were available in 13/83 cases (16%); 10 (77%) cases were BCL6 rearrangement-positive. Interestingly, using this orthogonal methodology, 3/13 cases (23%) were negative for detection of BCL6 rearrangement. All 3 false-negative FISH cases involved the known partner EIF4A2 .Conclusions: In this study, novel BCL6 rearrangements were identified in 22% of cases examined; notably, these novel partners comprised >50% of all partners detected. In the most recent WHO classification (2016), BCL6 rearrangement has known diagnostic and possible prognostic utility in high-grade BCLs, whereas previously Ig-BCL6 was considered secondary to other immunoglobulin gene partners (e.g. BCL2, MYC). Additionally, the presence of BCL6 rearrangements can distinguish t(14;18)-negative FL, a unique clinicopathologic entity with a worse prognosis. Non-Ig rearrangements may also portend a worse prognosis in other lymphoma subtypes; therefore, both the identification of novel partners and an understanding of their frequency has clinical utility and may lead to improved management for patients. Furthermore, the presence of false-negative results for known partners, using FISH, suggests an opportunity for alternative diagnostic testing via CGP. [Display omitted] DisclosuresVergilio:Foundation Medicine: Employment, Other: Stock. Gay:Foundation Medicine: Employment, Other: stock. Zhong:Foundation Medicine, Inc: Employment, Other: Stock. Rosenzweig:Foundation Medicine, Inc: Employment, Other: Stock. Chudnovsky:Foundation Medicine, Inc: Employment, Other: Stockholder. He:Foundation Medicine, Inc: Employment, Other: Stock. Nahas:Foundation Medicine inc: Employment, Other: stockholder. Ali:Foundation Medicine, Inc: Employment, Other: Stock. Daniel:Foundation Medicine: Employment, Other: Stock. Ramkissoon:Foundation Medicine: Employment, Other: stock. Suh:Foundation Medicine: Employment, Other: Stock. Elvin:Foundation Medicine: Employment, Other: Stock. Mughal:Foundation Medicine, Inc: Employment, Other: Stock. Stephens:Foundation Medicine Inc.: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Other: Stock. Erlich:Foundation Medicine: Employment, Other: stock. Ross:Foundation Medicine Inc: Employment, Other: stockholder. Severson:Foundation Medicine: Employment, Other: Stock. Morley:Foundation Medicine: Employment, Other: Stock.

The spectrum of tumors harboring BAP1 gene alterations

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

Revealing the BRD4-NOTCH3 fusion: A novel hill in the cancer landscape

ObjectivesGene fusions are becoming more evident in cancer scenario for either being the driver alterations, or for the great therapeutic target potential in many cases. Our aim was to characterize the BRD4-NOTCH3 fusion correlating with clinical features, and to determine the frequency of this fusion in the oncological population. Material and methodsOne patient diagnosed with lung adenocarcinoma at Hospital Sírio-Libanês (Brazil) was included. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among 233,804 specimens. ResultsA 76-year-old male patient was diagnosed with lung adenocarcinoma. Molecular assessments demonstrated negative ALK and EGFR, with PD-L1 expression positive by 60 %. He was treated with first line chemotherapy and second line immunotherapy. Subsequent treatments resume re-exposures to chemotherapy with poor responses. A next-generation sequencing (NGS) based assay was performed in the tumor biopsy, revealing mainly mutation in STK11, microsatellite stability, TMB-intermediate, MYC amplification and a BRD4-NOTCH3 fusion. The breakpoint analysis of this fusion indicates that BRD4 active domains are preserved, suggesting that it maintained DNA binding activity, as well as its capacity to be halt by BET inhibitors. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among more than 230-thousand specimens and it was found in 87 new cases in a rate of 0.04 % occurrence in solid tumors, predominately in gynecological cancers. The same rate was found when we analyzed a different dataset. ConclusionIn conclusion, this is the first report of the BRD4-NOTCH3 gene fusion associated with clinical characterization and, although rare, the occurrence of this fusion is constant in different population. Our data suggest that this fusion has great potential to targeted-therapy.

Comprehensive genomic profiling (CGP) utilizing cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

421 Background: PDAC has a propensity for early systemic dissemination and most pts with primary tumors radiographically confined to the pancreas harbor micro-metastases. cfDNA based CGP offers a non-invasive mechanism to identify actionable genomic targets in PDAC and account for serial clonal evolution in response to therapeutic selection pressure. We interrogated the Foundation Medicine database to characterize cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of pts with PDAC who underwent cfDNA based CGP between May 2016 and February 2020. cfDNA based CGP was done utilizing either the FoundationOne Liquid (F1-Liquid) or the Foundation-ACT (F-ACT) testing platform. Samples were interrogated for exonic and select intronic regions of cancer-related genes for both F1-Liquid (Exons: 70 genes; Select Introns: 7 genes) and F-ACT (Exons: 59 genes; Select Introns: 6 genes). Variant zygosity and somatic/germline status (SGZ) for short variant mutations was computationally predicted without matched normal tissue using an investigational method. A Gaussian Mixture Model (GMM) was used to identify latent molecular classes in samples harboring somatic, pathogenic variants (n=613); the Bayesian Information Criteria (BIC) was used for model selection. Results: We identified 1,009 pts with cfDNA based CGP - median age was 65, 55% were male, and median cfDNA concentration was 24 ng/µL. cfDNA yield had an influence on the detection of somatic alterations (p <0.001). 613/1009 (60.8%) of pts had at least one somatic alteration detected on cfDNA based CGP. TP53 (53.7%), KRAS (40.3%) and CDKN2A (6.5%) were the most frequently altered genes (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [somatic and/or germline BRCA2, ATM, and CHEK2 alterations], were detected in 12.3% (124/1009) pts – 11.4% (46/403) in KRAS mutated pts and 12.9% (78/606) in KRAS wildtype (WT) pts. Fusions were detected only in KRAS WT pts (1.5%, 6/403). Pts with cfDNA based CGP could be classified into 4 latent classes – class 1 ( KRAS - 100%, TP53 - 100% and CDKN2A – 33.3%), class 2 ( KRAS - 65.7%), class 3 ( KRAS – 68.2%, CDKN2A/B – 7.7% ) and class 4 ( KRAS – 38.5%, TP53 - 26.7%). Conclusions: The putative role of cfDNA based CGP in monitoring therapeutic response and clonal evolution in pts with PDAC may be predicated on cfDNA yield. In clinical scenarios where pre-treatment tissue availability is limited, cfDNA based CGP may be have utility in leveraging the predictive value of somatic and/or germline HR-DDR gene alterations. The prognostic and/or predictive relevance of the four latent classes needs validation in clinically annotated data sets. [Table: see text]

Association Between Human Papillomavirus Infection and Outcome of Perioperative Nodal Radiotherapy for Penile Carcinoma

BackgroundData on the impact of human papillomavirus (HPV) infection status and outcomes for perioperative treatments for patients with lymph node–involved penile squamous-cell carcinoma (PSCC) are lacking. ObjectiveTo analyze the benefit from perioperative radiotherapy (RT) for PSCC according to HPV infection status. Design, setting, and participantsIn an international multicenter database of 1254 patients with PSCC who received inguinal lymph node dissection (ILND), 507 had suitable clinical information. InterventionILND, with or without chemotherapy or RT for involved lymph nodes. Outcome measurements and statistical analysisKaplan-Meier and restricted mean survival time (RMST) analyses for overall survival (OS) were performed for all patients and after propensity score-matching (PSM; n = 136), for which patient age, histology, type of penile surgical procedure, pathological tumor and nodal stage, ILND laterality, pelvic LND, and perioperative treatment were taken into account when assessing differences between HPV+ and HPV– patients. Finally, we looked at genomic alterations in PSCC using data from the Foundation Medicine database (n = 199) to characterize HPV+ PSCC. Results and limitationsPatients with HPV+ PSCC (n = 86; 17%) had lower clinical N stage (p < 0.001) and inguinal lymph node metastasis density (p < 0.001). Perioperative RT was delivered in 49 patients (9.7%), with the vast majority receiving adjuvant RT (n = 40). HPV+ patients had similar median OS (p = 0.1) but longer RMST than HPV– patients at different time points. Nevertheless, HPV+ patients treated with perioperative RT exhibited longer median OS (p = 0.015) and longer RMST compared to HPV– patients. In the PSM cohorts, HPV+ status remained significantly associated with longer OS after RT. The HPV– PSCC group had a higher frequency of TP53 mutations compared to HPV+ PSCC (75% vs 15%; p < 0.001). The results are limited by the retrospective nature of the data. ConclusionsPerioperative RT was more effective in the HPV+ PSCC subgroup. Reasons for the enhanced radiosensitivity may be related to the lack of TP53 mutations. Patient summaryWe analyzed data from a large multicenter database for patients with penile cancer who had received inguinal lymph node dissection, with or without chemotherapy or radiotherapy. We found that for tumors positive for human papillomavirus (HPV), use of radiotherapy resulted in prolonged survival compared to HPV-negative tumors. On the basis of these results we are inspired to design studies on the use of radiotherapy in HPV-selected patients.

Abstract A17: Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies

Abstract Comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) provides an opportunity to noninvasively monitor a patient’s tumor burden via liquid biopsy. Liquid biopsies can assess a patient’s mutational landscape through time and provide information on treatment response and relapse. It has become increasingly common to have paired genomic data analysis between liquid and tissue biopsies from the same patient. However, the impact of clinical, temporal, and biologic factors on percentage of positive agreement (PPA) of variant detection between these biopsies is unclear. For our study, we leveraged two databases containing CGP data from paired liquid and tissue biopsy specimens tested by Foundation Medicine (FMI): the FMI database (&amp;gt;1700 paired samples) and the Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB). The CGDB is a subset of the FMI database linked with the Flatiron Health nationwide de-identified EHR-derived database, which includes demographic, treatment, and clinical outcomes information (&amp;gt;500 paired samples). We report pan-solid tumor and per-indication prevalence and PPA for variants commonly assayed in both liquid and tissue tests. We found that PPA depended on the tumor DNA concentration in plasma and tissue biopsies, as well as the amount of time between tests. We also investigated the effect of treatments administered in the time between liquid and tissue tests on the detected variants and observed some well-described resistance alterations at a higher frequency in liquid samples, including a higher prevalence of ESR1 point mutations in breast cancer, more EGFR T790M alterations in lung cancer, and frequent KRAS Q61H alterations in colorectal cancer. Liquid biopsies can also contain DNA shed from multiple metastatic sites. We observed evidence of this in the form of polyclonal resistance alterations, which may also account for differences in PPA over time. Our findings indicate that while PPA is generally high between samples, it may be influenced by factors such as intervening therapies, resistance, therapy efficacy, and polyclonality of liquid samples. Citation Format: Zoe June F. Assaf, Smruthy Sivakumar, Dexter X. Jin, Sophia L. Maund, Svetlana Lyalina, Guneet Walia, Ethan S. Sokol, Sally E. Trabucco. Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A17.

Abstract P3-06-09: Incidence of ROS1 genomic alterations in breast cancer

Abstract Background: ROS1 is an important proto-oncogene involved in the development of various cancers and increased expression is a poor prognostic feature in breast cancer. Activation of the ROS1 tyrosine kinase receptor has been reported and is being targeted with several FDA approved small molecule inhibitors, primarily in lung cancer. However, the frequency and type of ROS1 alterations in breast cancer have not been fully explored. The purpose of this study was to identify the incidence and type of ROS1 genomic alterations occurring in breast cancer as a prelude to potentially targeting this protein in the setting of metastatic breast cancers that harbor these changes. Methods: We queried 21,125 breast cancer samples (~50% from distant metastatic sites) from the Foundation Medicine breast cancer database, 5,964 breast cancer samples from five publicly available breast cancer databases (MBC Project, INSERM, METABRIC, TCGA, MSK-IMPACT), and 217 breast cancer samples from Project GENIE v6.5 to identify the incidence of ROS1 alterations in breast cancer. We identified fusion partner genes and classified each alteration type into the following categories: fusion, rearrangement, short variant (missense or indel), or copy number variation. A computational approach was used to distinguish somatic from germline single nucleotide polymorphisms in the Foundation Medicine database (Sun et. al., 2018). PolyPhen2 and in vitro analyses were used to investigate the effect of ROS1 nonsynonymous mutations on the ROS1 protein. Using Foundation Medicine, we made associations with ROS1 alterations and receptor status, histologic subtype, metastatic site, tumor mutational burden, and co-occurring mutated genes. Estrogen receptor (ER) status was available for 2,371 samples. HER2 status and all other analyses were identified from the entire cohort. Results: The incidence of any ROS1 alteration across the Foundation Medicine breast cancer database, including variants of unknown significance, was 4.6% and ranged from 1.67% to 6.9% from the five publicly available breast cancer databases and Project GENIE. Foundation Medicine samples revealed 5 (0.024%) breast cancers with ROS1 fusions, 86 (0.41%) with rearrangements, 733 (3.47%) with short nucleotide variants, and 182 (0.86%) with copy number variations. ROS1 alterations occurred similarly in HER2, TNBC, and ER+ breast cancers, 5.24%, 4.5%, and 4.37%, respectively (ns, all p&amp;gt;0.2). Invasive ductal carcinoma, invasive lobular carcinoma, and metaplastic carcinoma harbored ROS1 alterations at 4.35%, 3.65%, and 4.36%, respectively (ns, all p&amp;gt;0.3). ROS1 alterations were significantly associated with metastatic breast cancer spread to the brain (p=0.0005) and lymph nodes (p=0.017). There were 18 gene mutations significantly co-occurring with ROS1 alterations when controlling for multiple comparisons. The top 5 were TP53 (OR 1.55, p=3.12 × 10−8), RB1 (OR 1.66, p=0.0001), NOTCH3 (2.27, p=0.0001), TAF1 (OR 4.82, p=0.0002), and ARID1A (OR 1.71, p=0.0003). Conclusions: A modest incidence of genomic alterations in ROS1 occurs across all breast cancer subtypes and histologies and may be more actionable than currently recognized. New somatic alterations in the ROS1 gene were identified from Foundation Medicine that were not detected in publicly available databases or Project GENIE. Investigations using PolyPhen2 and in vitro analyses of ROS1 gene activation from these newly discovered somatic alterations and other short variant mutations are currently being investigated in our lab with results to be reported. Co-occurring mutations reveal a unique genotype associated with ROS1 alterations that may play a biologic role in ROS1-mediated pathogenesis and should be explored further. ROS1 alterations occurring in breast cancers might be leveraged in an innovative personalized medicine treatment approach for breast cancer patients. Citation Format: Jeremy Force, Ethan S Sokol, Mary Love Taylor, Dale Huang, Paul K Marcom, Monika Davare, Jeffrey Marks. Incidence of ROS1 genomic alterations in breast cancer [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 P3-06-09.

Urothelial Cancers with Small Cell Variant Histology Have Confirmed High Tumor Mutational Burden, Frequent TP53 and RB Mutations, and a Unique Gene Expression Profile

Although predominantly urothelial, some bladder cancer and upper tract urothelial cancer (BC/UTUC) harbor histologic variants. Small cell BC (SCBC) variants comprised ˜5% of The Cancer Genome Atlas BC cohort, with a poor prognosis. We describe genomic profiles of BC/UTUC with small cell/neuroendocrine features identified in the Foundation Medicine database from June 2012 to September 2018. Of 3368 BC/UTUC samples, 3.92% (132) harbored small cell/neuroendocrine features by immunohistochemistry. Mutations were noted in: TP53 (92%), RB1 (75%), combined TP53/RB1 (72%), and TERT promoter (68%). Of the samples, 6.5% had TMB ≥ 10 mutations/Mb. RNA expression profiling of 24 pure SCBC and 51 urothelial BC (UBC) muscle-invasive samples evaluated from a separate cohort revealed a large number of differentially expressed genes with suppression of several inflammatory pathways in SCBC compared with UBC. This largest reported SCBC dataset to date confirms enrichment of signatures in SCBC similar to small cell lung cancer and describes unique gene expression compared with UBC. These findings may explain aggressive SCBC phenotype. Patient summarySmall cell bladder cancer (SCBC) is an aggressive subtype that microscopically resembles aggressive small cell lung cancer (SCLC). This study confirms that SCBC shares DNA changes similar to SCLC and that SCBC expresses many genes that urothelial bladder cancer does not, possibly explaining aggressive SCBC activity.

Molecular genetic changes in solid pseudopapillary neoplasms (SPN) of the pancreas.

749 Background: Solid Pseudopapillary Neoplasms (SPNs) of the pancreas are rare accounting for 1-2% of all pancreatic tumors. Previous studies had shown that a pathogenic mutation of the CTNNB1 gene is present in over 90% of the SPN tumors with very limited information available on the specific molecular changes present in SPN. Here we report the results of next generation genetic testing of a large series of SPN tumors from the Foundation Medicine database. Methods: Foundation Medicine database from 07/2012 to 04/2019 was reviewed. A total of 31 cases of SPN tumors were identified out of 12,892 cases of pancreatic cancers. Information collected included demographic information on the patients as well as tumor analysis for next generation genetic sequencing of 315 genes associated with cancer with FoundationOne or 324 genes associated with FoundationOneCDx. Microsatellite stability status and tumor mutation burden were determined. Results: Twenty nine out of 31 cases (93%) had a CTNNB1 mutation. Seventy percent of the CTNNB1 mutations were on position 32 (14 cases, 41%) or in position 37 (9 cases, 29%). Other sites of CTNNB1 mutations included position 34 (3 cases, 9.6%), position 2 (2 cases, 6.4%) and position 38 (1 case, 3.2%). Two cases did not have a CTNNB1 mutation, one had a CDKN2A mutation and the other had no detectable mutations. Most cases had additional mutations aside from the CTNNB1, the most common were TP53 (3 cases, 9.6%) and LRP1B (2 cases, 6.4%). Other accompanying mutations were seen just once. Twenty-five percent of these cases had actionable gene mutations, each found in one case including: MSH2, BRCA2, ATM, XRCC3, ATRX, PTEN, ESR1, CDKN2, and PIK3CA. Conclusions: Next generation genetic testing of SPN tumors is of clinical benefit since it identifies actionable mutations in 29% of the cases.

Comprehensive Assessment of Immuno-oncology Biomarkers in Adenocarcinoma, Urothelial Carcinoma, and Squamous-cell Carcinoma of the Bladder

BackgroundIn patients with rare histologies of bladder cancer, including adenocarcinoma of the bladder (ACB) and squamous-cell carcinoma (SCC), there are limited standard therapy options, defining an unmet medical need. ObjectiveIn this comparative comprehensive genomic profiling (CGP) study, genomic alterations (GAs), and immuno-oncology (IO) biomarkers have been analyzed. Design, setting, and participantsWithin the Foundation Medicine database, 143 cases with centrally reviewed pure ACB, 2142 with pure urothelial carcinoma (UC), and 83 with pure SCC were subjected to CGP. All patients developed advanced disease following a primary diagnosis of bladder cancer. InterventionCGP using a hybrid capture–based assay and immunohistochemistry (IHC). Outcome measurements and statistical analysisTumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) was determined on 114 loci. Programmed cell-death ligand-1 (PD-L1) expression was determined by IHC (Ventana SP-142 assay), with >1% tumor cells (TCs) or tumor-infiltrating lymphocytes (TILs) scoring positive. Results and limitationsPure ACB patients were younger and more often female than pure UC and pure SCC patients. UC and SCC had a significantly higher median TMB than ACB (p < 0.001). Rare CD274 (PD-L1) amplification cases were more frequently seen in SCC than in UC (5% vs 1%), and were not seen in ACB. MSI high status was very uncommon in all tumor types (0–1%). The frequencies of PD-L1 expression in both TCs and TILs was higher in UC and SCC (both 30%) than in ACB (18%). The results are limited by their retrospective nature and lack of clinical data annotation. ConclusionsDeep sequencing revealed significant differences in IO biomarkers among the three major subtypes of bladder carcinomas. UC and SCC revealed higher frequencies of PD-L1 expression and higher TMB than ACB, and SCC has the highest frequency of CD274 amplification. The presence of pure SCC features should not disqualify patients for inclusion in IO trials. Patient summaryTumor samples from patients diagnosed with advanced pure adenocarcinoma of the bladder (ACB) or pure squamous-cell carcinoma (SCC) have been analyzed in terms of frequency of putative immunotherapy biomarkers. The results indicated that pure SCC of the bladder was characterized by genomic features that portend similar response possibilities to immunotherapy compared with the classical pure urothelial carcinoma. Conversely, for pure ACB there might be different therapeutic opportunities, such as targeted therapies against peculiar genomic alterations in selected patients.

ROS1 in Squamous Non-Small Cell Lung Cancer—Combined Immunotherapy (PD1/CTLA4) or Targeted Therapy?

ROS1 oncogenic fusion is reported to be 1% - 2% of non-small cell lung cancers (NSCLCs) of the adenocarcinoma subgroup. Meanwhile, there are no records of squamous cell cancer patients with tumors harboring ROS1 fusions. The Foundation Medicine database indicates a frequency of ROS1 rearrangements is 0.2% among squamous NSCLC. Crizotinib is known to be very effective in these patients. Here we present a non-smoker patient who had pure squamous NSCLC that was treated by combinational immunotherapy under a clinical trial and progressed after 2 cycles. Surprisingly, comprehensive genomic profiling detected a rare oncogenic EZR-ROS1 fusion, and the patient was treated by crizotinib with a significant response within 6 weeks. To date, the patient has been on therapy for 42 months and has achieved a complete metabolic response.

Atypical RAS Mutations in Metastatic Colorectal Cancer.

To describe the clinical and molecular features of metastatic colorectal cancers (mCRCs) bearing uncommon atypical RAS (At-RAS) mutations at codons other than 12, 13, 59, 61, 117, and 146. By exploiting five next-generation sequencing sources (Italian collaboration, Memorial Sloan Kettering Cancer Center, Samsung Medical Center, the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics (BREAC) study, and the Foundation Medicine database), we retrieved 175 At-RAS mutated cases. Molecular data were obtained from 163 samples from Memorial Sloan Kettering Cancer Center and the Foundation Medicine database. Clinical data were available for 27 At-RAS-positive and 467 negative cases from the Italian collaboration, Memorial Sloan Kettering Cancer Center, Samsung Medical Center, and the BREAC study. At-RAS mutations were identified in 163 (0.9%) of 18,270 mCRCs. Among 133 with evaluable microsatellite instability status, 11 (8%) were microsatellite instability high. POLE exonuclease domain mutations had higher frequency (7%) than expected and were found only in microsatellite-stable tumors with high tumor mutational burden (TMB). Overall, 17% (28 of 163) of At-RAS cases had TMB greater than 20 mutations/Mb. Co-occurring typical RAS/BRAF V600E mutations and NF1 mutations, presumed to cause RAS activation, were found in 30% and 12% of samples, respectively (up to 43% and 50%, respectively, in TMB-high samples). Patients with RAS/BRAF wild-type mCRC achieved a median overall survival (OS) of 42.1 months, whereas those harboring isolated At-RAS, typical RAS, or BRAF V600E mutations showed a median OS of 32.3, 30.0, and 17.9 months, respectively (P < .001). No significant OS difference (P = .240) was found between patients with At-RAS versus typical RAS-mutated mCRC. Only one of six patients evaluable for primary resistance to anti-epidermal growth factor receptors achieved tumor response. At-RAS mutations may be a marker for RAS pathway activation and can be associated with high co-occurrence of POLE exonuclease domain mutations.

A clinico-genomic analysis of soft tissue sarcoma patients reveals CDKN2A deletion as a biomarker for poor prognosis

BackgroundSarcomas are a rare, heterogeneous group of tumors with variable tendencies for aggressive behavior. Molecular markers for prognosis are needed to risk stratify patients and identify those who might benefit from more intensive therapeutic strategies.Patients and methodsWe analyzed somatic tumor genomic profiles and clinical outcomes of 152 soft tissue (STS) and bone sarcoma (BS) patients sequenced at Stanford Cancer Institute as well as 206 STS patients from The Cancer Genome Atlas. Genomic profiles of 7733 STS from the Foundation Medicine database were used to assess the frequency of CDKN2A alterations in histological subtypes of sarcoma.ResultsCompared to all other tumor types, sarcomas were found to carry the highest relative percentage of gene amplifications/deletions/fusions and the lowest average mutation count. The most commonly altered genes in STS were TP53 (47%), CDKN2A (22%), RB1 (22%), NF1 (11%), and ATRX (11%). When all genomic alterations were tested for prognostic significance in the specific Stanford cohort of localized STS, only CDKN2A alterations correlated significantly with prognosis, with a hazard ratio (HR) of 2.83 for overall survival (p = 0.017). These findings were validated in the TCGA dataset where CDKN2A altered patients had significantly worse overall survival with a HR of 2.7 (p = 0.002). Analysis of 7733 STS patients from Foundation One showed high prevalence of CDKN2A alterations in malignant peripheral nerve sheath tumors, myxofibrosarcomas, and undifferentiated pleomorphic sarcomas.ConclusionOur clinico-genomic profiling of STS shows that CDKN2A deletion was the most prevalent DNA copy number aberration and was associated with poor prognosis.