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NTHL1-associated tumor syndrome (NATS) is an autosomal recessive condition characterized by an increased risk for colorectal polyposis and colorectal cancer (CRC). Only 46 case reports have been previously published. In a retrospective review, we analyzed the clinical histories of six patients found to have NATS after genetic counseling and testing. NATS appears to be associated with an increased risk for colorectal polyposis, CRC, female breast cancer, meningiomas, and endometrial cancer. Although research is limited, prior publications have reported a multi-tumor predisposition for individuals with biallelic pathogenic or likely pathogenic variants in NTHL1. Additional data are necessary to further define the cancer risks so affected individuals can be appropriately managed.

Gliomas are the most prevalent neurological cancer in the United States and care modalities are not able to effectively combat these aggressive malignancies. Identifying new, more effective treatments require a deep understanding of the complex genetic variations and relevant pathway associations behind these cancers. Drawing connections between gene mutations with a responsive genetic target can help drive therapy selections to enhance patient survival. We have performed extensive molecular profiling of the Capicua gene (CIC), a tumor and transcriptional suppressor gene, and its mutation prevalence in reference to MAPK activation within clinical glioma tissue. CIC mutations occur far more frequently in oligodendroglioma (52.1%) than in low-grade astrocytoma or glioblastoma. CIC-associated mutations were observed across all glioma subtypes, and MAPK-associated mutations were most prevalent in CIC wild-type tissue regardless of the glioma subtype. MAPK activation, however, was enhanced in CIC-mutated oligodendroglioma. The totality of our observations reported supports the use of CIC as a relevant genetic marker for MAPK activation. Identification of CIC mutations, or lack thereof, can assist in selecting, implementing, and developing MEK/MAPK-inhibitory trials to improve patient outcomes potentially.

PDF file - 132K, Cell Cycle Analysis.

PDF file - 187K, Tyrpan Blue Assay.

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9. Mol Cancer Res; 9(12); 1621–31. ©2011 AACR.

Supplementary Data from Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer

Supplementary Table S1 from Gene expression profiling-based identification of cell-surface targets for developing multimeric ligands in pancreatic cancer

Multimeric ligands are ligands that contain multiple binding domains that simultaneously target multiple cell-surface proteins. Due to cooperative binding, multimeric ligands can have high avidity for cells (tumor) expressing all targeting proteins and only show minimal binding to cells (normal tissues) expressing none or only some of the targets. Identifying combinations of targets that concurrently express in tumor cells but not in normal cells is a challenging task. Here, we describe a novel approach for identifying such combinations using genome-wide gene expression profiling followed by immunohistochemistry. We first generated a database of mRNA gene expression profiles for 28 pancreatic cancer specimens and 103 normal tissue samples representing 28 unique tissue/cell types using DNA microarrays. The expression data for genes that encode proteins with cell-surface epitopes were then extracted from the database and analyzed using a novel multivariate rule-based computational approach to identify gene combinations that are expressed at an efficient binding level in tumors but not in normal tissues. These combinations were further ranked according to the proportion of tumor samples that expressed the sets at efficient levels. Protein expression of the genes contained in the top ranked combinations was confirmed using immunohistochemistry on a pancreatic tumor tissue and normal tissue microarrays. Coexpression of targets was further validated by their combined expression in pancreatic cancer cell lines using immunocytochemistry. These validated gene combinations thus encompass a list of cell-surface targets that can be used to develop multimeric ligands for the imaging and treatment of pancreatic cancer. [Mol Cancer Ther 2008;7(9):3071–80]

Supplementary Figure Legends - PDF file 90K, Figure legends for Supplementary Material

Figure S2 - PDF file 2023K, IHC images for HIF-1� and SHH in patient matched tumor TMA cores

Figures S3 to S7 and Tables S1 and S2 - PDF file 532K, S3 Kaplan-Meier patient survival analyses reveals no correlation between survival and PTCH levels. S4 Survival plots based on the univariate table S5 Increased expression of SHH during hypoxia in pancreatic cancer cell lines. S6 A HIF1-�- and SHH-dependent activation of hedgehog signaling in fibroblasts grown in 3D co-cultures with Panc-1 cells. S7, SHH formation by pancreatic cancer cells in hypoxia is mediating the desmoplastic reaction in fibroblasts Table S1 Univariate table showing that high histologic grade Table S2 A pathway array profiling the expression of genes involved in hedgehog signaling in MiaPaca 2 cells transfected with shHIF-1� in normoxia and hypoxia

Background Nuclear protein transport is essential in guiding the traffic of important proteins and RNAs between the nucleus and cytoplasm. Export of proteins from the nucleus is mostly regulated by Exportin 1 (XPO1). In cancer, XPO1 is almost universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm. Currently, there are no studies evaluating XPO1 amplifications and mutations in NSCLC and the impact on outcomes. Methods Tumor samples were analyzed using next-generation sequencing (NGS) (NextSeq, 592 Genes), immunohistochemistry (IHC), and whole transcriptome sequencing (WTS, NovaSeq) (Caris Life Sciences, Phoenix, AZ). Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. Results Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations and 24 had amplifications. XPO1 mutant tumors were more likely to have high TMB (79% vs. 52%, p = 0.007) and less likely to have high PD-L1 (32% vs. 68%, p = 0.03). KRAS co-mutations were seen in 19% (n = 5) and EGFR co-mutations were rare (n = 2). Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant. Comparison of survival between XPO1 mutant and WT showed a negative association with a hazard ratio (HR) of 1.932 (95% CI: 1.144–3.264 p = 0.012). XPO1 amplification was not associated with survival. Conclusions XPO1 pathogenic mutations were associated with a poor survival in NSCLC. Although XPO1 mutations are rare in NSCLC, further studies to assess its associations with treatment responses are warranted.

PURPOSE The adoption of precision medicine (PMed) depends on the critical curation of data and interpretation of genomic results. Herein, we sought to study the effect of a coordinated multidisciplinary program to assess results in a community cancer center clinic. METHODS In a retrospective review from July 2018 to July 2021, we analyzed the implementation of a multidisciplinary PMed program in a tertiary referral community cancer center. Germline genetics test results have been reviewed since 2017. RESULTS A total of 3,131 tumor samples were analyzed by large panel somatic genomic testing through commercial laboratories during the study period. The number of reviewed cases rose from 661 in the first year to 1,532 in year 3. Additional recommendations beyond what was reported by the commercial laboratory were made in 42.9% of cases. Referrals to the hereditary cancer program for germline testing increased by 32% from the 2017 baseline. Process improvement efforts reduced the rate of DNA quantity nonsufficient for testing to 3.3% compared with a national average of 4.89%. The average time from receipt of orders to issuing of a report of the somatic panel was 15.5 days, compared with 19.1 days for other institutions using the same laboratory. The PMed team has been critical in support of clinical research by assisting in trial procurement and feasibility assessment to the identification of patients for clinical trials. Conclusion The use of somatic genomic testing is increasing at our cancer center. Education and in-depth analysis of the data are valued by cancer physicians. The development and implementation of a PMed program has demonstrated improved physicians' understanding of molecular testing, resulting in improved outcomes for patients.

Purpose:Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC.Experimental Design:To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations.Results:Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation.Conclusions:These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Pancreatic ductal adenocarcinoma (PDAC) incidence is rising worldwide, and most patients present with an unresectable disease at initial diagnosis. Measurement of carbohydrate antigen 19-9 (CA19-9) levels lacks adequate sensitivity and specificity for early detection; hence, there is an unmet need to develop alternate molecular diagnostic biomarkers for PDAC. Emerging evidence suggests that tumor-derived exosomal cargo, particularly micro RNAs (miRNAs), offer an attractive platform for the development of cancer-specific biomarkers. Herein, genomewide profiling in blood specimens was performed to develop an exosome-based transcriptomic signature for noninvasive and early detection of PDAC.Small RNA sequencing was undertaken in a cohort of 44 patients with an early-stage PDAC and 57 nondisease controls. Using machine-learning algorithms, a panel of cell-free (cf) and exosomal (exo) miRNAs were prioritized that discriminated patients with PDAC from control subjects. Subsequently, the performance of the biomarkers was trained and validated in independent cohorts (n = 191) using quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays.The sequencing analysis initially identified a panel of 30 overexpressed miRNAs in PDAC. Subsequently using qRT-PCR assays, the panel was reduced to 13 markers (5 cf- and 8 exo-miRNAs), which successfully identified patients with all stages of PDAC (area under the curve [AUC] = 0.98 training cohort; AUC = 0.93 validation cohort); but more importantly, was equally robust for the identification of early-stage PDAC (stages I and II; AUC = 0.93). Furthermore, this transcriptomic signature successfully identified CA19-9 negative cases (37 U/mL; AUC = 0.96), when analyzed in combination with CA19-9 levels, significantly improved the overall diagnostic accuracy (AUC = 0.99 vs AUC = 0.86 for CA19-9 alone).In this study, an exosome-based liquid biopsy signature for the noninvasive and robust detection of patients with PDAC was developed.

nab-Sirolimus is a novel albumin-bound mTOR inhibitor (mTORi) approved in the US for adult patients with malignant PEComa. In an exploratory analysis of the pivotal AMPECT trial of nab-sirolimus in advanced malignant PEComa (NCT02494570), 8/9 (89%) and 1/5 (20%) patients with inactivating alterations in TSC1 and TSC2, respectively, had confirmed response (Wagner, J Clin Oncol, 2021). TSC1 and TSC2 alterations have been observed in patients with a broad variety of cancers. Most treatment-related adverse events in AMPECT were grade 1/2 (none were grade ≥4) and were consistent with long-term treatment of nab-sirolimus. PRECISION I (NCT05103358) will evaluate efficacy and safety of nab-sirolimus in patients with TSC1 (Arm A) and TSC2 (Arm B) alterations. Eligible patients are ≥12 years old and mTORi-naïve, possess malignant solid tumors with TSC1 or TSC2 inactivating alterations (confirmed by central review of sequencing reports), and have received appropriate standard treatments, as determined by the investigator. nab-Sirolimus 100 mg/m2 will be given weekly intravenously over 30 minutes on Days 1 and 8 of each 21-day cycle. The primary endpoint is overall response rate per independent radiographic review (IRR) using RECIST v1.1. Other endpoints include duration of response, time to response, progression-free survival by IRR, overall survival, patient-reported quality of life, and safety. Enrollment began March 2022. Collaboration with leading next-generation sequencing vendors will expedite the identification of patients with qualifying TSC1 or TSC2 mutations; study access will be facilitated through a “just-in-time” approach to trial location activation. Based on the prevalence of TSC1 or TSC2 inactivating alterations, the most frequent tumor types expected are bladder, hepatobiliary, endometrial, soft tissue sarcoma, ovarian, and esophagogastric (Table). Est incidence of patients with TSC1 or TSC2 alterations available for 1L therapy in 2030 Tumor Type TSC1 Mutations, %a TSC2 Mutations, %a Eligible TSC1 or TSC2 Combined, % Bladder 6.33 1.70 8.03 Hepatobiliary 1.27 3.31 4.58 Endometrial 2.10 1.22 3.32 Soft tissue sarcoma 1.28 1.71 2.99 Ovarian 1.85 0.92 2.77 Esophagogastric 0.65 1.46 2.11 Colorectal carcinoma 0.99 0.39 1.38 Pancreatic 0.57 — 0.57 Note: Estimated incidence of patients in the United States with definite impact TSC1 or TSC2 alterations available for 1L therapy in 2030. All gastrointestinal tumors (bolded) with known incidence of TSC1 or TSC2 and tumor types with combined incidence of TSC1 or TSC2 alterations of >2% are listed. aThe proportion of patients with definite impact mutations (ie, mutations known to have a biological impact, including frameshift, nonsense, and splice-site mutations and deep deletions) was derived from the NIH NCI Genomic Data Commons Data Portal (NIH NCI Genomic Data Commons). 1L, first-line. Citation Format: Jordi Rodon Ahnert, Brian Schulte, Michael J. Demeure, Dustin Deming, Noah Federman, Meredith A. McKean, Elizabeth Lee, Alexander Spira, David J. Kwiatkowski, Maen Hussein, Erlinda Gordon, David Crockett, Kristen Ganjoo, Lee D. Cranmer, Anita N. Schmid, Willis H. Navarro, Loretta M. Itri, Gopa Iyer. Phase 2, multicenter open-label basket trial of nab-sirolimus for patients with inactivating alterations in TSC1 or TSC2 (PRECISION I) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT057.

TPS818 Background: Albumin-bound ( nab)-sirolimus, a novel mTOR inhibitor (mTORi) that utilizes nanoparticle technology to preferentially target tumors, is approved in the US for the treatment of adults with malignant PEComa. In an exploratory analysis of the AMPECT registrational trial of nab-sirolimus in advanced malignant PEComa (NCT02494570), 8/9 (89%) and 1/5 (20%) patients with TSC1 and TSC2 inactivating alterations, respectively, had confirmed response (Wagner, J Clin Oncol. 2021). Importantly, both TSC1 and TSC2 alterations have been observed in patients with various gastrointestinal cancers (Table). Overall, most treatment-related adverse events (TRAEs) in AMPECT were grade 1/2 and manageable for long-term treatment; no grade ≥4 TRAEs occurred. Methods: PRECISION I (NCT05103358) is a phase 2, open-label, multi-institutional basket trial evaluating efficacy and safety of nab-sirolimus in patients with alterations in TSC1 (Arm A) and TSC2 (Arm B). Patients ≥12 years old with malignant solid tumors harboring pathogenic inactivating alterations in TSC1 or TSC2 (confirmed by central review of next generation sequencing reports) who have progressed on standard therapies and are mTORi-naïve are eligible. nab-Sirolimus 100 mg/m2 will be administered weekly as an intravenous infusion over 30 minutes on Days 1 and 8 of each 21-day cycle. The primary endpoint is overall response rate determined by independent review using RECIST v1.1; other endpoints include duration of response, disease control rate, time to response, progression-free survival by independent radiographic review, overall survival, patient-reported quality of life, and safety. Enrollment is ongoing. The most frequent tumor types expected in this tissue-agnostic trial are bladder, hepatobiliary, endometrial, soft tissue sarcoma, ovarian, and esophagogastric based on the prevalence of TSC1 or TSC2 alterations (Table). Clinical trial information: NCT05103358 . [Table: see text]

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.

2019 Background: Gliomagenesis is regulated by dynamic epigenetic modifications of DNA methylation, deregulation of histones and alteration of the human Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. These epigenetic genes are responsible for treatment resistance by inducing stemness of glioma cells and immune cells with in the tumor microenvironement (TME). We evaluated the key chromatin remodeling (CR) genes and their interactions with other regulatory genes that are of prognostic importance. Methods: 1856 HGGs underwent molecular profiling at Caris Life Sciences (Phoenix, AZ). Analyses included next-generation sequencing of DNA (592 Genes, NextSeq or WES, NovaSeq) and RNA (WTS, NovaSeq). Cell infiltration in the TME was estimated by quanTIseq. X2/Fisher’s-exact/Mann-Whitney U tests were used for comparison, and significance was determined as p-value adjusted for multiple comparison by the Benjamini-Hochberg method (q < 0.05). Overall survival (OS) was calculated from the start of temozolomide (TMZ) to last contact using insurance claims data. Results: In a cohort of 1856 HGGs, 181 (9.8%) displayed >=1 mutation of 19 CR genes considered, including mutations (mt) of histone methyltransferases (HM) comprising SETD2 (62, 3.4%), KMT2D (18, 1.0%), KMT2C (11, 0.6%); SWI/SNF complexes (SSNF) including ARID1A (32, 1.74%), ARID2 (15, 0.82%), SMARCA4 (14, 0.76%) and ARID1B (12, 0.66%); and others including ( DNMT3A, 0.94%, ASLXL1: 13, 0.98%). When compared to CR-WT, CR-mt HGGs showed higher prevalence of Tumor Mutational Burden-High (TMB-H) (23% vs. 1.3%), MSI-H/dMMR (13% vs. 0.2%), gLOH (9.5% vs 4.3%), and mts in IDH1/2 (29% vs. 14%), TP53 (55% vs. 36%), MSH6 (8.8% vs. 0.6%), and PIK3CA (18.8% vs. 8.3%) (all q

3071 Background: Targeted therapy of NENs based on the presence of SSTRs fills a unique niche in tumor biology and clinical treatment of patients with solid tumors. SSTRs have multiple isoforms and are collectively expressed in the majority of NENs. However, subtypes are still not routinely tested and thus not assessed for clinical decision-making, especially for patients meriting consideration of targeted radionucleotide therapy. Clarifying the landscape of SSTR subtypes using molecular techniques more sensitive than immunohistochemistry (IHC)-the standard of testing, and identifying associated genomic biomarkers that differ between them, will pave the way for more sophisticated decision-making in the future. Additionally, leveraging transcriptomics to better assess mitotic markers such as Ki-67 to assess tumor grade, would increase diagnostic accuracy. Here we provide initial validation across a spectrum of NENs. Methods:1595 NENs were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U and p-adjusted for multiple comparisons (qs= 0.63) and SSTRs 1 and 2 in LG-NENs (rs= 0.64) were positively correlated. Overall, the prevalence of altered TP53, RB1, PIK3CA, APC, KRAS was higher and MEN1 was lower in HG-vs LG-NENs (q

2630 Background: MDM2/MDM4 are implicated in hyperprogression after immune checkpoint blockade (ICB). Our preclinical studies showed reduced T-cell killing of MDM2-amplified tumor cells that was overcome by an MDM2 antagonist or gene knockdown, and we observed additional tumor killing by T-cells with MDM2 inhibition plus anti-PD1. We hypothesized that MDM2/4 gene amplification/overexpression correlates with resistance to ICB and investigated the association of MDM2/4 alterations to overall survival (OS) following ICB across multiple solid tumors. Methods: Solid tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (NGS) were analyzed. MDM2/4 amplification (amp) was tested by NGS and determined as either amp4 (cutoff of >=4.0 copies) or amp6 (>=6.0) or amp8 (>=8.0). Real-world OS was obtained from insurance claims data and calculated from treatment start or tissue collection to last contact. Kaplan-Meier estimates were calculated for molecularly defined groups. X2/Fisher-Exact were used and significance determined as P-value adjusted for multiple comparisons (q

4524 Background: TERT is a catalytic subunit of telomerase, the unique enzyme that confers immortality to cells and is expressed in >90% of cancer cells. Mutations in the TERT promoter region (pTERTmut) are the most prevalent noncoding mutations in cancer. Recent data suggest pTERTmut are associated with improved outcomes in patients with UC treated with immune checkpoint inhibitors. We evaluated the molecular and immune landscape of UC with and without pTERTmut. Methods: UC tissue samples were analyzed for DNA alterations (NextSeq, 592 Genes; NovaSeq, WES) and mRNA expression (NovaSeq, WTS). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). PD-L1 expression was assessed by immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by fragment analysis, IHC and NGS. TMB-H was based on a cut-off of > 10 mut/MB. We compared alterations between samples with and without detected pathogenic pTERTmut. Significance was determined by Mann–Whitney U, X2, and Fischer-Exact and p adjusted for multiple comparisons (q) was < 0.05 using Benjamini-Hochberg. Results: Overall, 1686 UC samples were analyzed, 1166 from primary lesions and 499 from a lymph node or metastatic site. pTERTmut was present in 68% of primary and 61% of metastatic tumors, and correlated with modest increase of TERT expression (1.18 fold, p=0.015). pTERTmut was associated with less frequent alterations in TP53, KMT2D, CCND1, MYC, KEAP1 and less MSI/dMMR. By contrast, pTERTmut was associated with more frequent alterations in ARID1A, TSC1, PIK3CA and TMB-H (all q

2056 Background: Capicua (CIC) Gene is a tumor suppressor, transcriptional repressor, and a member of the high mobility (HMG)-box protein family. CIC is a negative regulator of MAPK and RTK pathways; inactivating CIC mutations (mut) occur in approximately 40% of oligodendrogliomas (OLIG) and less frequently in other gliomas putatively activating downstream signaling. With a goal to identify potential novel treatment options for various gliomas, we explored key signaling pathways associated with CIC mut. Methods: Consecutive glioma tumors were analyzed using Next-Gen DNA sequencing (NextSeq, 592 genes or NovaSeq, whole-exome), RNA sequencing (NovaSeq, WTS ) and IHC (Caris Life Sciences, Phoenix, AZ). Immune cell fraction was calculated by QuantiSeq; MAPK activation score (MPAS) was evaluated using RNAseq data. A comparison was made using Chi2 or Fisher’s-exact test with correction for multiple-comparison (q) using Benjamini-Hochberg. Results: A total of 196 (3.7%) tumors with CIC mut were seen in 5266 gliomas tumors analyzed, with the highest prevalence seen in OLIG (143 of 285, 50.2%). There was no difference between grade 3 (73 of 142, 51.4%) and grade 2 OLIG (70 of 143, 49%). CIC mut were infrequent in astrocytomas (16 of 829, 1.9%; grade 3, 12/510 or 2.4%; grade 2, 4/261 or 1.5%; grade 1, 0/58). CIC mut were present in glioblastomas (24/2753 or 0.6%), gliosarcomas (1/128 or 0.8%), and other mixed subtypes (12/185 or 6.5%). CIC mut were associated with higher prevalence of IDH1/2 mut (92% in CIC-mut vs. 17% in wild type), MGMT promoter methylation (97% vs. 47%), FUBP1 mut (32% vs. 1%) but lower PTEN mut (1% vs. 25%) and TP53 mut (12% vs. 39%) (all q < 0.05). Significant mutual exclusivity for CIC mut and MAPK pathway drivers observed: EGFR amplification (1.5% vs. 27%) , EGFR mut (0.5% vs. 12.6%), NF1 mut (4% vs. 18%) (all q < 0.05). BRAF mut rate was similar in CIC-mut or wild-type (1% vs. 3%, p = ns). Although associated with a higher tumor mutation burden (cutoff > = 10 mut/MB, 13% vs. 3%), a lower prevalence of PDL1 expression (1% vs. 16%) and lower M1 macrophage infiltration were seen (all q < 0.05). Similar effects were seen when stratifying by oligodendroglial and astrocytic histology. CIC mut were associated with increased MPAS score in OLIG (p = 0.01), particularly when compared to tumors lacking additional MAPK drivers (p = 0.001). This effect was not seen in astrocytic tumors, although EGFR alterations (including CNA, EGFRvIII, EGFR fusion and mut) were independently associated with increased MPAS scores (p < 0.001). Conclusions: CIC mut were frequent in oligodendrogliomas but occurred rarely in other glioma tumors and are associated with favorable prognostic markers. RNA expression analysis suggests CIC mut is associated with MAPK activation in OLIG, as are EGFR alterations in astrocytomas. Targeted inhibition of this pathway in selected gliomas may be a promising therapeutic avenue and should be explored further.

3132 Background: Reversion mutations (RM) in homologous recombination pathway genes including BRCA1/2 have been identified in patients with ovarian, breast, and prostate cancers whose tumors have become refractory to platinum chemotherapy or PARP inhibition. Utilizing a multi-institutional molecular database, we report the prevalence of BRCA1/2 RM in a large cohort across various tumor types. Methods: Primary and/or metastatic tumor samples underwent DNA (underwent NextSeq, 592 genes; NovaSeq, whole-exome) and RNA (NovaSeq, whole transcriptome) sequencing (Caris Life Sciences, Phoenix, AZ). RM were identified by a board-certified molecular geneticist and called only if the patient had been treated with a PARPi or a platinum agent. Baseline clinical and outcomes data were obtained through linked insurance claims data. Results: Among 118,000 solid tumors profiled, RM were observed in 54 tumors samples. RMs were seen most commonly in ovarian cancer (OC), 1.5% (23/1500) of tumors with BRCA1/2 pathogenic mutations (mut), followed by breast cancer (BC) (2.4%, 17/700), endometrial cancer (1.0%, 4/400), pancreatic cancer (1.0%, 2/210), cholangiocarcinoma (2.5%, 2/80), prostate cancer (1.3%, 3/230), cervical cancer (1.4%, 1/70), cancer of unknown primary (1.0%, 1/100), and a neuroendocrine tumor of prostate (1 RM of 9 BRCA mut). Among all RM, we detected 17 in BRCA1 and 6 in BRCA2 in OC. In BC, we identified 7 RM in BRCA1 and 10 in BRCA2. Frameshift mut that restored the reading frame in BRCA1/2 were the most common type of RM. Molecular profiles of 14 high-grade serous ovarian cancers (HGSOC) with RM were compared to 87 control HGSOC with pathogenic BRCA1/2 mut without RM. Tumors with RM had lower ER expression (25% vs. 64%, p = 0.024) and higher KDM6A mut rate (15% vs. 0, p = 0.016). Additionally, TP53 mut rates were similar in RM and control (100% vs. 95%), seen in HGSOC. In patients with RM, 7 of the 14 (50%) TP53 mut were gain-of-function (GOF) while only 19 of 84 (23%) TP53 mut in the control group were GOF (p = 0.048). More detailed clinical data were available for 29 patients with RM (17 BRCA1 & 12 BRCA2). Among these patients, 7 had received prior platinum-based chemotherapy (carboplatin or cisplatin), 7 patients were treated with PARP inhibitors (olaparib or rucaparib), or both (n = 7). Notably, 5 patients had been treated with carboplatin (n = 2, ovarian), olaparib (n = 1, breast), or both agents (n = 2, ovarian and prostate) after the detection of RM. Conclusions: This dataset is one of the largest reporting on the prevalence of BRCA1/2 RM across various tumor types. We demonstrate that the rate of RM was low among BRCA1/2 mutated tumors; this may be because some patients may not have repeat profiling post-treatment. Repeating tumor profiling at times of treatment resistance can help inform therapy selection in the refractory disease setting.

Advances in immuno-oncology over the last several years have led to FDA approvals of novel agents. As our understanding of immune response and its checkpoints has evolved, further advances have been made in treatment for several cancer types. To predict a response to immunotherapy, the initial biomarkers used were expression of the PD-1 receptor and PD-L1, as assessed by immunohistochemistry. More recently, predictive biomarkers have included microsatellite instability, DNA mismatch repair, and tumor mutational burden. Although these markers may be clinically relevant in predicting an immunotherapy response, cancer immunotherapy fails some patients. Improved understanding of the human immune system is necessary, as is a careful evaluation of the methods used to predict and assess response to immuno-oncology treatments. With the application of therapeutic immune-modulating agents, more comprehensive assays, and associated bioinformatics tools to accurately assess the tumor microenvironment, we may better predict responses to immuno-oncology agents and the ever-increasing complexity of their clinical use.

Invasive melanoma is the deadliest type of skin cancer, with 101,110 expected cases to be diagnosed in 2021. Recurrent BRAF and NRAS mutations are well documented in melanoma. Biologic implications of gene fusions and the efficacy of therapeutically targeting them remains unknown. Retrospective review of patient samples that underwent next-generation sequencing of the exons of 592 cancer-relevant genes and whole transcriptome sequencing for the detection of gene fusion events and gene expression profiling. Expression of PDL1 and ERK1/2 was assessed by immunohistochemistry (IHC). There were 33 (2.6%) cases with oncogenic fusions (14 novel), involving BRAF, RAF1, PRKCA, TERT, AXL, and FGFR3. MAPK pathway-associated genes were over-expressed in BRAF and RAF1 fusion-positive tumors in absence of other driver alterations. Increased expression in tumors with PRKCA and TERT fusions was concurrent with MAPK pathway alterations. For a subset of samples with available tissue, increased phosphorylation of ERK1/2 was observed in BRAF, RAF1, and PRKCA fusion-positive tumors. Oncogenic gene fusions are associated with transcriptional activation of the MAPK pathway, suggesting they could be therapeutic targets with available inhibitors. Additional analyses to fully characterize the oncogenic effects of these fusions may support biomarker driven clinical trials.

Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs). Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clinically annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA. TMA-1 (n = 80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n = 51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA-2, 20 (74%) of the ACC tumors demonstrated PTTG1 nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p

Glomus tumors are uncommon perivascular neoplasms.1 Derived from the glomus bodies of the dermis involved in thermoregulation, glomus tumors are found most frequently in areas of the body rich in such glomus bodies, including the subungual areas of the digits and the distal extremities in general. Although they are most common in the superficial tissues, glomus tumors may occur in visceral organs, including the liver, lung, pancreas, and gastrointestinal and genitourinary tracts.2 When glomus tumors do occur in the gastrointestinal tract, they are most often located in the stomach.3 This article is protected by copyright. All rights reserved.

Background At presentation, 21% to 49% of patients with adrenocortical cancer have metastases. Standard chemotherapy has a 23% response rate. We assessed whether next generation sequencing could elucidate additional treatment options in refractory adrenocortical cancer. Methods Retrospective analysis using a commercial, 592-gene DNA-based panel was performed of next generation sequencing data from 94 adrenocortical cancer tumors profiled for clinical care. We compared our data to the adrenocortical cancer database of The Cancer Genome Atlas containing survival data. We evaluated mutations, indels, amplifications, tumor mutation burden, microsatellite instability, and programmed death-ligand 1 protein expression. Results Our cohort included 54 primary neoplasms and 40 metastatic lesions. The most frequently mutated genes were TP53 (36%) and CTNNB1 (19%). Low prevalence mutations were noted in 37 genes including DNA damage repair genes in 15 samples. High tumor mutation burden was seen in 3 patients, and programmed death-ligand 1 was positive in 12. Potential targets to Food and Drug Administration–approved drugs were seen in 16% of cases. Conclusion DNA sequencing panel tests may identify therapeutic options for some patients with adrenocortical cancer. TP53 and mutations were associated with an adverse outcome. An expanded repertoire of drugs and, perhaps, more expansive multi-omic sequencing are needed to advance the treatment of adrenocortical cancer.

Rare cancers pose unique challenges for patients and their physicians arising from a lack of information regarding the best therapeutic options. Very often, a lack of clinical trial data leads physicians to choose treatments based on small case series or case reports. Precision medicine based on genomic analysis of tumors may allow for selection of better treatments with greater efficacy and less toxicity. Physicians are increasingly using genetics to identify patients at high risk for certain cancers to allow for early detection or prophylactic interventions. Genomics can be used to inform prognosis and more accurately establish a diagnosis. Genomic analysis may also expose therapeutic targets for which drugs are currently available and approved for use in other cancers. Notable successes in the treatment of previously refractory cancers have resulted. New more advanced sequencing technologies, tools for interpretation, and an increasing array of targeted drugs offer additional hope, but challenges remain.

Introduction: Oncogenic gene fusions are frequently identified in different cancers; however, the exact incidence of oncogenic fusions in melanoma is not well defined. Several targeted therapies are approved and considered the standard of care for patients whose solid or hematologic tumors harbor particular gene fusions, but their role as targets in melanoma also remains undelineated. We sought to determine the prevalence of oncogenic fusions in metastatic or locally advanced melanoma. Methods: We retrospectively analyzed data from formalin-fixed paraffin-embedded (FFPE) tumor samples sent to a commercial CLIA-certified laboratory (Caris Life Sciences) from February 2019 to July 2020. Samples were profiled by next-generation sequencing of a 592-gene DNA panel, whole transcriptome sequencing, and immunohistochemistry (IHC). Results: Melanoma specimens were analyzed from 1,255 subjects, of whom 478 (38.1%) were female and 777 (61.9%) were male, with a median age of 67 years. Of these specimens, 780 (63.1%) were from metastatic sites. We identified 33 (2.6%) cases with in-frame oncogenic fusions (14 novel) including 21 BRAF fusions and 4 RAF1 fusions, as well as fusions involving PRKCA (n=4), TERT (n=2), AXL (n=1), and FGFR3 (n=1). PD-L1 expression by IHC (SP142 or 28-8 antibody) was detected in 512 (42.5%) specimens, including 10 (32.3%) of the fusion-positive tumors, while 572 (47.4%) specimens were TMB-High (≥10 mutations/Mb), including 11 (33.3%) of the fusion-positive tumors, suggesting these patients may respond to immunotherapy. We identified 796 (63.4%) cases with RAS/RAF pathogenic or likely pathogenic mutations, including 373 (30.0%) BRAF p.V600X mutations. With the exception of a single BRAF p.S467L (Class 3, “kinase-dead”) mutation, the absence of BRAF mutations in BRAF fusion-positive tumors suggests these fusions are oncogenic drivers. However, tumors harboring PRKCA and TERT fusions were each detected with at least one MAPK pathway co-alteration (NRAS, NF1, or BRAF p.V600E mutation). Fusion transcripts with unknown pathogenicity were also detected in 668 (53.2%) cases. RNA expression analysis of key molecular pathways (including Wnt/β-catenin, PI3K/AKT/MTOR, DNA repair, INFG, and JAK/STAT) showed a high degree of variability among fusion-positive tumors and other mutational subgroups, which may reflect the heterogeneity common to melanoma, prior treatments or development of various resistance mechanisms. Conclusions: Oncogenic gene fusions are rare in melanoma when compared with other genetic variants. We identified potentially actionable fusions as well as co-alterations in fusion-positive cases, with notably mutual exclusivity of BRAF fusions and p.V600X mutations. The data suggest that targetable variants, including oncogenic fusions, may be identified in melanoma with comprehensive tumor profiling and provide patients with personalized treatment or clinical trial options. Citation Format: Sourat Darabi, Andrew Elliott, David R. Braxton, Jia Zeng, Kelsey Poorman, Jeffrey Swensen, Geoffrey T. Gibney, Justin C. Moser, Thuy Phung, Michael B. Atkins, Gino K. In, Wolfgang Michael Korn, Burton L. Eisenberg, Michael J. Demeure. Whole transcriptome sequencing reveals oncogenic fusions in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2221.

11555 Background: Leiomyosarcoma (LMS) is a rare group of mesenchymal malignancies found in the uterus, retroperitoneum, skin, or other soft-tissue sites. Treatment for LMS is extrapolated from trials including both uterine (uLMS) and non-uLMS subtypes, although whether they respond similarly and have similar outcomes from treatment is not clear. We examined the molecular composition of LMS by site of origin to better inform future drug development and trial design. Methods: We reviewed 1115 specimens with LMS histology tested by Caris Life Sciences for targeted exome (NextSeq, 592 gene panel), whole exome, and whole transcriptome sequencing (NovaSeq). Specimens were stratified into uLMS, rpLMS (retroperitoneal), and otherLMS (non-uterine/retroperitoneal) subgroups based on tumor origin sites. Genomic data was analyzed for mutations, copy number aberrations, and fusions. RNA expression profiling included evaluation of individual genes and gene set enrichment analysis (GSEA). P-value adjustment performed by the Benjamini-Hochberg procedure. Results: The study cohort was comprised of 62.9% uLMS (n = 701), 14.9% rpLMS (n = 166) and 22.2% otherLMS (n = 248) specimens. Overall, LMS specimens most frequently harbored TP53 (64%, n = 612), ATRX (30%, n = 219), RB1 (22%, n = 156), and MED12 (16%, n = 94) mutations, with these genes accounting for 74.4% (n = 1044) of all observed pathogenic/likely pathogenic mutations. RB1 mutations were significantly less common in uLMS (15%) compared to rpLMS (30%, p < 0.05) and otherLMS (33%, p < 0.01), whereas MED12 mutations were almost exclusive to uLMS (22% vs 1% rpLMS, 3% otherLMS, p < 0.05). MAP2K4 copy number amplification were more common in rpLMS (22%, p < 0.001) and otherLMS (14%, p < 0.182) compared to uLMS (7%), with frequent co-amplification of nearby genes ( FLCN, GID4, SPECC1, GAS7, PER1, and AURKB) located at chr17p11-13. Actionable gene fusions involving ALK (2.1%, n = 11), FGFR1 (0.2%, n = 1), and NTRK1/2 (0.2%, n = 1 each) were rare overall, with similar prevalence across subtypes. Genomic alteration rates were not significantly different between rpLMS and otherLMS subtypes . RNA expression profiling identified significant upregulation of PI3K/AKT/mTOR, DDR, WNT/Beta-Catenin pathway genes in non-uLMS. GSEA indicated several immune-related gene sets were enriched in rpLMS and otherLMS compared to uLMS. Conclusions: Comprehensive molecular profiling suggests that LMS originating from the uterus represents a molecularly distinct disease compared to other primary sites of origin. We identified key genomic patterns which have potential for targeted therapy. These data provide insight for the framework of future clinical trials designed to separate uLMS from non-uLMS histologies, although further subdivision does not appear to be warranted.

e20533 Background: Nuclear protein transport is essential in guiding the organized traffic of important proteins and RNAs between the nucleus and cytoplasm of the cell. Export of proteins from the nucleus is exclusively regulated by Exportin 1(XPO1). In cancer, XPO1 is universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm, leading to their functional inactivation. XPO1 is aberrantly over expressed in NSCLC and this over expression has been linked to poor overall survival. The underlying mechanisms of XPO1 over expression are not known. Currently there are no studies evaluating the impact of XPO1 mutations on NSCLC incidence and therapy resistance. Additionally, there are no studies that examined the XPO1 related pathways in NSCLC harboring co-alterations with other driver mutations such as EGFR or ALK. Methods: Tumor samples were analyzed using next-generation sequencing (NextSeq, 592 Genes), IHC, and whole transcriptome sequencing (WTS ,NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC using 22c3 (Dako) and TPS scores were reported (cutoff > 1%). TMB was measured by totaling somatic mutations (TMB-high cut-off ³ 10 mutations per Megabase). Gene fusions were detected by RNA sequencing using either the Archer FusionPlex panel or WTS. Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. Statistical significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons. Results: Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations. XPO1 mutant tumors were more likely to be TMB High(79% vs. 52%, p = 0.007) and less likely to have high PDL1(32% vs. 68%, p = 0.03). KRAS mutations were seen in 19%(n = 5), EGFR mutation were rare (n = 2), and no targetable fusions were seen. Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant tumors with no histology imbalance observed in mutant vs. wild-type(WT). Comparison of survival in the NSCLC group between XPO1 mutant and WT showed a negative association with a hazard ratio(HR) of 1.932 (95% CI: 1.144- 3.264 p = 0.012). Comparing the survival within the subgroup with confirmed adenocarcinoma histology (9973 XPO1 WT and 14 XPO1 mutant) showed a similar negative correlation in survival with a HR of 2.156 (95% CI: 1.027- 4.525 P = 0.037). Conclusions: Presence of XPO1 pathogenic mutations was associated with a poor survival in both the entire NSCLC cohort and the adenocarcinoma subgroup. Further studies of this negative association at the molecular level along with effect of other co-existing mutations can result in development of novel treatment strategies.

e22522 Background: Pharmacogenetics is the study of genetic variants that define drug metabolism and dosing requirements, response or toxicity. Knowledge regarding drug-drug interactions and individual variations in genes involved in drug metabolism is increasing. Since patients with cancer often take several drugs (polypharmacy), they have a higher risk of severe drug interactions. The FDA lists several pharmacogenetic biomarkers in drug labeling, including genetic variants that impact how certain medications are prescribed. The drugs listed span multiple medical applications, including but not limited to oncology, primary care, psychiatry, neurology, cardiology, and many more. Adverse drug reactions are a significant cause of morbidity and mortality, yet the use of pharmacogenetic testing has not yet become established as a routine in clinical practice. Herein we sought to study the feasibility of applied pharmacogenetics in a pilot drug safety program in patients with advanced cancer. Methods: We enrolled patients in a prospective single-center IRB approved study. Patients with advanced cancer on three or more drugs about to begin chemotherapy at the Hoag Family Cancer Institute were eligible. Informed consent was obtained from the study participants, and their samples were sent to a CLIA laboratory to conduct a 25-gene pharmacogenetics testing panel, which includes “major drug-metabolizing enzymes” CYP2D6, CYP2C19, CYP2C9, CYP3A4, and CYP3A5 as well as other genes with known drug implications. We recorded patients’ demographics and clinical information. In addition, participant’s drug lists were assessed for potential polypharmacy interactions. Results: We analyzed the results from 36 patients, 20 females and 16 males, enrolled between December 2017 and June 2020. Pharmacogenetics results from these patients showed that 61% (22 patients) had a variant with gene drug interaction indication. The most common variants were identified in the CYP2D6 gene, followed by CYP2C19, CYP3A4, and CYP3A5. Of these 22 patients, seven patients had at least one major criterion (drug interactions may lead to severe clinical effects), and eight patients with reported moderate criteria (may lead to substantial clinical effects). Additionally, seven patients had variants with both major and moderate criteria. Patients were taking an average of 10 medications per person ranging from 3-19 drugs. No patient, however, experienced significant drug toxicity during the observation interval. Conclusions: Patients with advanced cancer are at particular risk for polypharmacy and adverse drug reactions. Our data report supports pharmacogenetics testing as being feasible for oncology patients. Therefore, programs to prevent or limit the frequency and severity of drug reactions warrant further exploration as they offer the potential to improve patient safety and decrease healthcare costs.

e15530 Background: The TP53 gene product is a multifunctional protein and prototypical tumor suppressor. Certain missense mutations occurring in hotspots in the TP53 gene may confer gain of function (GOF) oncogenic properties. A recent mouse model (Kadosh et. al., Nature, 2020) demonstrated that secreted factors in the gut microbiome permit GOF p53 to be oncogenic in lower GI tract cancers (LT). By contrast, the GOF p53 mutants in the upper tract cancers (UT) retained tumor suppressor functions. We aimed to provide evidence of the plasticity of TP53 GOF mutants in human GI cancers. We hypothesized that UT should have a lower rate of TP53 GOF than the LT, and UT cancers with putative GOF TP53 mutations should display a higher degree of co-occurring oncogenic alterations such as dysregulation of CCND1, MYCS, or WNT. We also explored outcome differences for TP53 GOF variants. Methods: Tumors of UT (stomach, esophagus, small intestine) and LT (colorectum, anus) were tested at Caris Life Sciences (Phoenix, AZ) by NGS (NextSeq, 592 or NovoSeq, whole exome). MSI/MMRP status was determined by IHC and NGS. Real-world overall survival (OS) was obtained from payor claims data and Kaplan-Meier estimates were calculated. P values adjusted for multiple correction (q) of < 0.05 was considered significant. Six p53 variants were classified as GOF (p.R175, p.G245, p.R248, p.R249, p.R273, and p.R282). Results: Of the 5311 UT and 14810 LT tumors sequenced, 4799 harbored a TP53 GOF variant. UT had TP53 GOF in 22.9% of cases compared to 27.0% of LT (q < 0.05). 67 genes were enriched for amplification (q < 0.05) in the UT TP53 GOF cancers compared to the LT TP53 GOF, including cell cycle regulators( CCND1, CCNE1, CDK6, CDK12), and oncogenes ( KRAS, ERBB2, EGFR). LT TP53 GOF cancers were enriched for eight gene amplifications, including CDX2, FLT3, and SRC. Differences in SNV/InDel patterns were not remarkable. OS analysis revealed TP53 GOF in UT had shorter OS when compared to TP53 wild-type (WT) (Median OS: 418 vs 515 days; HR: 0.864; 95%CI [0.772-0.966]; p = 0.011), but not when MSI/MMRP status was considered separately. LT TP53 GOF did not show OS difference in aggregate, however, MSI-High TP53 GOF cancers showed a shorter OS (Median OS: 762 vs 1479 days; HR: 1.522; [1.068 - 2.171]; p = 0.019). Sub-analysis of LT MSI-High cancers treated by immune checkpoint blockade showed a non-significant trend toward shorter OS of TP53 GOF versus TP53 wild type cancers (HR: 2.55; [0.90-7.19], p: 0.067), but no such trend was seen in UT (HR: 1.434; [0.304-6.758], p:0.646). Conclusions: Our study is the first to compare putative TP53 GOF variants across UT and LT GI adenocarcinoma. TP53 GOF variants are associated with decreased OS in both UT cancers and MSI-High LT cancers. Our findings support the functional plasticity of TP53 GOF variants in human GI cancers. Therefore, the purported effects of secreted microbiome factors on TP53 GOF variants cannot be ruled out.

11512 Background: Leiomyosarcomas (LMS) have been reported to have immunohistochemical (IHC) and gene expression signatures suggestive of an immune-responsive tumor microenvironment. Despite this, immune checkpoint inhibitors have demonstrated minimal activity in LMS. We examined molecular profiles of LMS specimens from multiple institutions to explore mechanisms of immunotherapy (IO) resistance. Methods: LMS specimens (n = 1115), including 701 uterine (uLMS) and 414 soft tissue site (stLMS) samples, underwent next-generation sequencing (NGS) of DNA (592-gene panel or whole exome) and RNA (whole transcriptome, n = 537) at Caris Life Sciences (Phoenix, AZ). A threshold of 10 mut/Mb was used to identify high tumor mutational burden (TMB-H). IHC was performed for PD-L1 (SP142; 2+|5% positive). Deficient mismatch repair (dMMR)/high microsatellite instability (MSI-H) was tested by IHC and NGS, respectively. RNA expression was analyzed using Gene Set Enrichment Analysis and Microenvironment Cell Populations-counter, with results compared to melanoma (n = 1255) as a representative immunogenic tumor type. P-values were adjusted for multiple hypothesis testing. Results: TMB-H was observed in 3.8% (n = 41) of LMS specimens, with a median of 5 mut/Mb (IQR 3.3-6.7). dMMR/MSI-H was rarely detected (1.5%, n = 17), whereas 8.2% (n = 88) were positive for PD-L1 expression. uLMS and stLMS did not differ in TMB-H (3.4 vs 4.5%, p = 0.277), PD-L1 expression (8.6 vs 7.4%, p = 0.322), or dMMR/MSI-H (2.0 vs 0.7% p = 0.207). stLMS demonstrated upregulation of immune-related gene sets, including interferon γ (p = 0.035) and α (p = 0.033) response, inflammatory response (p = 0.038), interleukin-6/STAT3 signaling (p = 0.030), and TNFα signaling (p = 0.026) compared to uLMS. Immune cell infiltration was increased in stLMS over uLMS, most notably for CD8 T-cell and B-cell abundance ( > 2-fold increase, p < 0.0001). Compared to melanoma, all LMS had lower abundance of CD8 T cells, cytotoxic lymphocytes, and B-cells ( > 2-fold decrease, p < 0.0001). Fibroblasts were more prevalent in LMS relative to melanoma (3.2-fold increase, p < 0.0001). Interestingly, while higher CD8 T-cell infiltration was positively associated with dMMR/MSI-H among LMS specimens (p = 0.032), TMB-H and PD-L1 expression were associated with lower CD8 T-cell infiltration (p < 0.01). Conclusions: Only a small proportion of LMS are TMB-H or MSI-H, suggesting that the neoantigen burden in LMS may be insufficient to promote a robust anti-tumor response, even in the presence of PD-L1 positive tumor cells. Traditional predictive biomarkers of response to IO are unlikely to be useful in LMS. Furthermore, both uLMS and stLMS have an immune microenvironment characterized by a high fibroblast and low T cell abundance relative to melanoma. Future IO trials in LMS should focus on combination therapies that may reverse the observed T-cell exclusion/desmoplastic phenotype.

3091 Background: Gene fusions often represent critical therapeutic targets across cancer subtypes. Fusions within the ErbB family of receptor tyrosine kinases, including EGFR, ERBB2 ( HER2) and ERBB4 ( HER4), have been previously described and represent potentially actionable alterations. Here, we report the relative incidence and functional characterization of these rare genomic events. Methods: Tumor samples (n = 64,354; representing > 40 tumors types) submitted to Caris Life Sciences (Phoenix, AZ) were molecularly profiled by next-generation sequencing of DNA (NextSeq, 592-gene panel; or NovaSeq, whole exome) and RNA (whole transcriptome). Gene fusion partners, in/out-of-frame status, retention of ERBB kinase domain, and topology of fusion breakpoints were characterized for each ERBB fusion transcript detected. Fusion prevalence was further examined in public data sets (TCGA, MSK-IMPACT and AACR GENIE). Results: From the Caris database, a total of 64 EGFR fusion isoforms were detected in 59 tumors (incidence 0.09%); 83% were in-frame and 91% retained the EGFR kinase domain. 206 ERBB2 fusion isoforms were detected in 114 tumors (0.18%); 37% were in-frame and 34% retained the ERBB2 kinase domain. 131 ERBB4 fusion isoforms were detected in 108 tumors (0.17%); 62% were in-frame and 51% retained the kinase domain. All fusions were detected at low incidence across all tumor types. EGFR fusions were most common in high grade glioma (1.7%, n = 35), largely driven by recurrent EGFR-SEPT14 fusions (n = 20). ERBB2 fusions were most common in esophageal/gastroesophageal junction carcinoma (1.1%, n = 20), with recurrent fusion to PGAP3 observed in multiple tumor types (n = 37). ERBB4 fusions were most common in ovarian (0.7%, n = 40) and bladder (0.7%, n = 15) cancers, which often resulted from recurrent fusion with IKZF2 (n = 36). EGFR and ERBB2 fusions were generated predominantly (44-48%) from inversion events, while ERBB4 fusions arose more frequently and at similar rates (27-32%) from deletions, duplications, or translocations. Mining of public data sets corroborated the prevalence of ERBB gene fusions: the frequency of EGFR fusions was 0.63%, ERBB2 was 0.14% and ERBB4 was 0.04%. TP53 mutations frequently co-occurred with ERBB2 and ERBB4 fusions ( > 60% average across public data sets), with higher co-mutation rates ( > 70%) observed for samples in the Caris database. Conclusions: ERBB gene fusions are detectable at low frequency in various tumor types and may represent a unique genomic subset of cancer. Identification of novel ERBB gene fusions warrants further investigation to determine the potential pathogenicity and actionability of these fusions.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy affecting individuals across a broad age spectrum. Disease rarity, scarcity of pre-clinical models, lack of effective targeted therapy and limited clinical trials have contributed to poor prognosis for patients with ACC. Identifying targetable genetic drivers and pathways to guide precision medicine approaches is therefore critical to improve outcomes. The purpose of this study was to analyze the genomic profile of a large cohort of ACC to identify potential therapeutic targets. FoundationOne (Foundation Medicine Inc.; FMI, Cambridge, MA) is a next-generation sequencing-based platform for somatic genetic testing in solid tumors. The FoundationOne genomic data and limited demographic data through 2018 for 364 unique ACC specimens were analyzed. The cohort of 364 tumors were from 222 females and 141 males (1 gender unknown). The mean age (SD) was 48.6 (13.6) for females and 50.6 (12.20) for males with overall median age of 52 years. A total of 3117 genomic alterations were identified impacting 457 genes. The median number of genomic alterations per tumor was 7 (range 1–56), with single nucleotide variants and indels being the most common alterations (median=4), followed by copy number alterations (median=1) and rearrangements (median=0). The most frequently altered genes were TP53 (38%), CTNNB1 (28%), ZNRF3 (17%), CDKN2A (13%), ATRX (11%), TERT promoter (10%). Several novel recurrent alterations were identified including IL7R (6%), LRP1B (8%), FRS2 (4%), PTCH1 (4%) and KRAS (3%). Pathway enrichment analysis confirmed that tumor suppressor genes (51%) and Wnt signaling pathways (51%) are the most commonly dysregulated in ACC tumors. Epigenetic alterations, including histone modification (38%), SWI/SNF (21%) and DNA methylation (8%), affected upwards of one third of ACC tumors. Mutation signature analysis identified tumors with signatures 6, 15 and 26 associated with defective DNA mismatch repair (MMR), which was not reported previously. In addition, fifty ACCs (13.7%) exhibited 60 genomic alterations in MMR genes, MLH1, MSH2, MSH6 and PMS2, which included 49 SNVs/indels, 10 CNAs and one truncating rearrangement. In addition to MMR gene alterations, potentially actionable (www.oncokb.org) genomic alterations were found in 46 genes in 213 (58.5%) ACCs. In summary, this study represents the largest to date genomic analysis of ACC that showed that over 50% of ACC tumors had potentially actionable genomic alterations. Approximately 13% of tumors had an alteration in MMR pathway, suggesting that immunotherapy is a relevant therapeutic modality in a significant subset of patients with ACC.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.

Rare cancers pose unique challenges for patients and their physicians arising from a lack of information regarding the best therapeutic options. Very often, a lack of clinical trial data leads physicians to choose treatments based on small case series or case reports. Precision medicine based on genomic analysis of tumors may allow for selection of better treatments with greater efficacy and less toxicity. Physicians are increasingly using genetics to identify patients at high risk for certain cancers to allow for early detection or prophylactic interventions. Genomics can be used to inform prognosis and more accurately establish a diagnosis. Genomic analysis may also expose therapeutic targets for which drugs are currently available and approved for use in other cancers. Notable successes in the treatment of previously refractory cancers have resulted. New more advanced sequencing technologies, tools for interpretation, and an increasing array of targeted drugs offer additional hope, but challenges remain.

Purpose Current first-line chemotherapy for patients with metastatic adrenocortical cancer (ACC) includes doxorubicin, etoposide, cisplatin, and mitotane with a reported response rate of only 23.2%. New therapeutic leads for patients with refractory tumors are needed; there is no standard second-line treatment. Methods Samples from 135 ACC tumors were analyzed by immunohistochemistry, in situ hybridization (FISH or CISH), and/or gene sequencing at a single commercial reference laboratory (Caris Life Sciences) to identify markers associated with drug sensitivity and resistance. Results Overexpression of proteins related to demonstrated chemotherapy sensitivity or resistance included topoisomerase 1, progesterone receptor, and topoisomerase 2-alpha in 46%, 63%, and 42% of cases, respectively. Loss of excision repair cross-complementary group 1 (ERCC1), phosophatase and tensin homolog, O(6)-methylguanine-methyltransferase, and ribonucleotide reductase M1 (RRM1) was identified in 56%, 59%, 71%, and 58% of cases, respectively. Other aberrations included overexpression of programmed death-ligand 1 or programmed cell death protein 1 tumor-infiltrating lymphocytes in >40% of cases. In all, 35% of cases had a mutation in the canonical Wnt signaling pathway (either CTNNB1 or APC) and 48% had a mutation in TP53. No other genomic alterations were identified. Conclusion Biomarker alterations in ACC may be used to direct therapies, including recommendations for and potential resistance of some patients to traditional chemotherapies, which may explain the low response rate in the unselected population. Limited outcomes data support the use of mitotane and platinum therapies for patients with low levels of the proteins RRM1 and ERCC1.