Nonsynonymous Missense Mutations Research Articles

Comparison of immunohistochemistry and next-generation sequencing results in oncogenic PTEN missense mutations

BackgroundPhosphatase and tensin homolog (PTEN) is one of the most frequently mutated tumor suppressor genes in malignant tumors. Oncogenic PTEN mutations have diagnostic, prognostic, and therapeutic implications. Similar to TP53 mutations, oncogenic PTEN mutations can result from nonsynonymous missense mutations. However, there has been no detailed study on the immunostaining pattern of oncogenic PTEN missense mutations. MethodsWe retrospectively selected 18 cancers (13 endometrial cancers, 2 brain tumors, 1 ovarian cancer, 1 lung cancer, and 1 cancer of unknown origin) harboring oncogenic PTEN missense mutations, which were confirmed by targeted next-generation sequencing. PTEN immunohistochemistry was conducted for all cases, and the results were compared with sequencing results. ResultsThe immunostaining results of PTEN missense mutations revealed a diverse pattern depending on the site of mutation and co-occurring mutation. The most frequent oncogenic PTEN mutations were R130G (4/18, 22.2 %) and R130Q (3/18, 16.7 %). Eleven cases harbored PTEN missense mutations only, whereas the remaining seven cases harbored PTEN truncating mutations and PTEN missense mutations. Complete loss of cytoplasmic expression were found in five cases, of which three had missense mutation only. PTEN R130 residue mutation alone did not showed altered PTEN immunostaining pattern in this study. ConclusionsPTEN missense mutation, which comprises a portion of oncogenic PTEN mutation, can manifest as a diverse immunostaining pattern. Complementary testing using both immunostaining and next-generation sequencing should be conducted to accurately evaluate the PTEN status in malignancy.

Glioma genetic profiles associated with electrophysiologic hyperexcitability.

Distinct genetic alterations determine glioma aggressiveness, however, the diversity of somatic mutations contributing to peritumoral hyperexcitability and seizures over the course of the disease is uncertain. This study aimed to identify tumor somatic mutation profiles associated with clinically significant hyperexcitability. A single center cohort of adults with WHO grades 1-4 glioma and targeted exome sequencing (n = 1716) was analyzed and cross-referenced with a validated EEG database to identify the subset of individuals who underwent continuous EEG monitoring (n = 206). Hyperexcitability was defined by the presence of lateralized periodic discharges and/or electrographic seizures. Cross-validated discriminant analysis models trained exclusively on recurrent somatic mutations were used to identify variants associated with hyperexcitability. The distribution of WHO grades and tumor mutational burdens were similar between patients with and without hyperexcitability. Discriminant analysis models classified the presence or absence of EEG hyperexcitability with an overall accuracy of 70.9%, regardless of IDH1 R132H inclusion. Predictive variants included nonsense mutations in ATRX and TP53, indel mutations in RBBP8 and CREBBP, and nonsynonymous missense mutations with predicted damaging consequences in EGFR, KRAS, PIK3CA, TP53, and USP28. This profile improved estimates of hyperexcitability in a multivariate analysis controlling for age, sex, tumor location, integrated pathologic diagnosis, recurrence status, and preoperative epilepsy. Predicted somatic mutation variants were over-represented in patients with hyperexcitability compared to individuals without hyperexcitability and those who did not undergo continuous EEG. These findings implicate diverse glioma somatic mutations in cancer genes associated with peritumoral hyperexcitability. Tumor genetic profiling may facilitate glioma-related epilepsy prognostication and management.

Insights on monkeypox disease and its recent outbreak with evidence of nonsynonymous missense mutation.

The 2022 monkeypox outbreak has created a new global health threat and pandemic. Monkeypox virus is a descendant of the genus Orthopoxvirus, producing a febrile skin rash disease in humans. Monkeypox iszoonotic transmitted and transmitted from human to human in several ways. Even though this disease is self-limited, it creates important community health worries due to its inconvenience and widespread complications. Herein, we discussed the up-to-date current situation of monkeypox regarding its epidemiology, clinical manifestations, current in-use therapeutics, necessary protective measures, and response to potential occurrences considering the recent pandemic. Also, in this review, a comparative genomic analysis of the recent circulating strains that have been recovered from various countries including, Egypt, USA, Spain, Japanand South Africa has been investigated.

3DVizSNP: a tool for rapidly visualizing missense mutations identified in high throughput experiments in iCn3D

BackgroundHigh throughput experiments in cancer and other areas of genomic research identify large numbers of sequence variants that need to be evaluated for phenotypic impact. While many tools exist to score the likely impact of single nucleotide polymorphisms (SNPs) based on sequence alone, the three-dimensional structural environment is essential for understanding the biological impact of a nonsynonymous mutation.ResultsWe present a program, 3DVizSNP, that enables the rapid visualization of nonsynonymous missense mutations extracted from a variant caller format file using the web-based iCn3D visualization platform. The program, written in Python, leverages REST APIs and can be run locally without installing any other software or databases, or from a webserver hosted by the National Cancer Institute. It automatically selects the appropriate experimental structure from the Protein Data Bank, if available, or the predicted structure from the AlphaFold database, enabling users to rapidly screen SNPs based on their local structural environment. 3DVizSNP leverages iCn3D annotations and its structural analysis functions to assess changes in structural contacts associated with mutations.ConclusionsThis tool enables researchers to efficiently make use of 3D structural information to prioritize mutations for further computational and experimental impact assessment. The program is available as a webserver at https://analysistools.cancer.gov/3dvizsnp or as a standalone python program at https://github.com/CBIIT-CGBB/3DVizSNP.

Integrated multi-omic analysis of low-grade ovarian serous carcinoma collected from short and long-term survivors

BackgroundLow-grade serous ovarian cancer (LGSOC) is a rare disease that occurs more frequently in younger women than those with high-grade disease. The current treatment is suboptimal and a better understanding of the molecular pathogenesis of this disease is required. In this study, we compared the proteogenomic analyses of LGSOCs from short- and long-term survivors (defined as < 40 and > 60 months, respectively). Our goal was to identify novel mutations, proteins, and mRNA transcripts that are dysregulated in LGSOC, particularly in short-term survivors.MethodsInitially, targeted sequencing of 409 cancer-related genes was performed on 22 LGSOC and 6 serous borderline ovarian tumor samples. Subsequently, whole-genome sequencing analysis was performed on 14 LGSOC samples (7 long-term survivors and 7 short-term survivors) with matched normal tissue samples. RNA sequencing (RNA-seq), quantitative proteomics, and phosphoproteomic analyses were also performed.ResultsWe identified single-nucleotide variants (SNVs) (range: 5688–14,833 per sample), insertion and deletion variants (indels) (range: 880–1065), and regions with copy number variants (CNVs) (range: 62–335) among the 14 LGSOC samples. Among all SNVs and indels, 2637 mutation sites were found in the exonic regions. The allele frequencies of the detected variants were low (median12%). The identified recurrent nonsynonymous missense mutations included KRAS, NRAS, EIF1AX, UBR5, and DNM3 mutations. Mutations in DNM3 and UBR5 have not previously been reported in LGSOC. For the two samples, somatic DNM3 nonsynonymous missense mutations in the exonic region were validated using Sanger sequencing. The third sample contained two missense mutations in the intronic region of DNM3, leading to a frameshift mutation detected in RNA transcripts in the RNA-seq data. Among the 14 LGSOC samples, 7754 proteins and 9733 phosphosites were detected by global proteomic analysis. Some of these proteins and signaling pathways, such as BST1, TBXAS1, MPEG1, HBA1, and phosphorylated ASAP1, are potential therapeutic targets.ConclusionsThis is the first study to use whole-genome sequencing to detect somatic mutations in LGSOCs with matched normal tissues. We detected and validated novel mutations in DNM3, which were present in 3 of the 14 samples analyzed. Additionally, we identified novel indels, regions with CNVs, dysregulated mRNA, dysregulated proteins, and phosphosites that are more prevalent in short-term survivors. This integrated proteogenomic analysis can guide research into the pathogenesis and treatment of LGSOC.

Abstract 5764: Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations

Abstract Background: Targeted therapies and immunotherapies have significantly improved the survival of patients with cutaneous melanoma. Nevertheless, patients with acral and mucosal melanomas show limited benefit. The proportion of acral and mucosal subtypes are much higher in Chinese populations than Caucasians. Therefore, it is of great interest to detail the genomic features of acral and mucosal melanomas and compare these with cutaneous melanoma. Methods: A total of 111 Chinese patients with melanoma treated in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study, comprising 32 cutaneous(28.8%), 41 acral(36.9%) and 26 mucosal(23.4%) subtypes. NGS-based genomic profiling of major cancer-related genes was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens with matched blood samples. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. Results: The most commonly mutated genes of cutaneous melanoma include BRAF(48%), CDKN2A(41%), CDKN2B(41%), NRAS(22%) and PTEN(22%). In acral melanoma patients, the mutation rate of BRAF was 10% while NRAS(31%), CDKN2A(21%), EMSY(21%), KIT(18%), CCND1(18%)) and NF1(18%) were frequently mutated. Mucosal melanomas were found most commonly to have mutations in CDKN2A(36%), KIT(32%), CDKN2B(28%), PDGFRA(24%), KDR(20%) and BRAF(16%). In acral and mucosal subtypes, the overall mutation rates of BRAF were significantly lower while the relative proportions of non-V600 mutations were higher as compared to cutaneous subtype. These may explain the less significant survival benefit of BRAFi+/-MEKi in acral and mucosal melanomas. In contrast, KIT mutations including SNVs and amplification, were enriched in acral and mucosal melanomas. Acral melanomas had lower mutation load than mucosal melanomas(2.42 Muts/Mb vs 4.84 Muts/Mb, p=0.0002), while cutaneous melanomas(4.03 Muts/Mb) did not differ from other subtypes in mutation burden. Additionally, patients with TERT, APC and FAT1 gene mutations had a significantly higher TMB than patients with wild-type genes respectively, indicating more activated anti-tumor immune profiles and suggesting therapeutic benefit from immune checkpoint inhibitors. Conclusions: We conducted a direction comparison of comprehensive genomic profiles of different melanoma subtypes among Chinese patients. The genomic alterations identified in our study may provide reference to discover potential strategies for targeted therapy and immunotherapy in acral and mucosal melanomas. Citation Format: Jing Chen, Ting Ye, Li Fan, Jieying Zhang, Rubo Cao, Yanshen Liu. Genomic profiling of cutaneous, acral and mucosal melanomas in Chinese populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5764.

Molecular profiling of signet-ring-cell carcinoma (SRCC) from the stomach and colon reveals potential new therapeutic targets

Signet ring cell carcinoma (SRCC) is rare: about 10% of gastric cancer (GC) and 1% of colorectal cancer (CRC). SRCC is associated with poor prognosis, however the underlying molecular characteristics are unknown. SRCCs were analyzed using NGS, immunohistochemistry, and in situ hybridization. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. A total of 8500 CRC and 1100 GC were screened. Seventy-six SRCC were identified from the CRC cohort (<1%) and 98 from the GC cohort (9%). The most frequently mutated genes in CRC-SRCC were TP53 (47%), ARID1A (26%), APC (25%); in GC-SRCC were TP53 (42%), ARID1A (27%), CDH1 (11%). When compared to non-SRCC histology (N = 3522), CRC-SRCC (N = 37) more frequently had mutations in BRCA1 (11% vs 1%, P < 0.001) and less frequently mutations in APC (19% vs 78%, P < 0.001), KRAS (22% vs 51%, P = 0.001) and TP53 (47% vs 73%, P = 0.001). Among the GC cohort, SRCC (N = 54) had a higher frequency of mutations in CDH1, BAP1, and ERBB2, compared to non-SRCC (N = 540). Our data suggest that SRCCs harbor a similar molecular profile, regardless of the tumor location. Tailored therapy may become available for these patients.

Identification and Fine Mapping of the Recessive Gene BK-5, Which Affects Cell Wall Biosynthesis and Plant Brittleness in Maize.

The cellulose of the plant cell wall indirectly affects the cell shape and straw stiffness of the plant. Here, the novel brittleness mutant brittle stalk-5 (bk-5) of the maize inbred line RP125 was characterized. We found that the mutant displayed brittleness of the stalk and even the whole plant, and that the brittleness phenotype existed during the whole growth period from germination to senescence. The compressive strength was reduced, the cell wall was thinner, and the cellulose content was decreased compared to that of the wild type. Genetic analysis and map-based cloning indicated that bk-5 was controlled by a single recessive nuclear gene and that it was located in a 90.2-Kb region on chromosome 3 that covers three open reading frames (ORFs). Sequence analysis revealed a single non-synonymous missense mutation, T-to-A, in the last exon of Zm00001d043477 (B73: version 4, named BK-5) that caused the 951th amino acid to go from leucine to histidine. BK-5 encodes a cellulose synthase catalytic subunit (CesA), which is involved with cellulose synthesis. We found that BK-5 was constitutively expressed in all tissues of the germinating stage and silking stage, and highly expressed in the leaf, auricula, and root of the silking stage and the 2-cm root and bud of the germinating stage. We found that BK-5 mainly localized to the Golgi apparatus, suggesting that the protein might move to the plasma membrane with the aid of Golgi in maize. According to RNA-seq data, bk-5 had more downregulated genes than upregulated genes, and many of the downregulated genes were enzymes and transcription factors related to cellulose, hemicellulose, and lignin biosynthesis of the secondary cell wall. The other differentially expressed genes were related to metabolic and cellular processes, and were significantly enriched in hormone signal transduction, starch and sucrose metabolism, and the plant–pathogen interaction pathway. Taken together, we propose that the mutation of gene BK-5 causes the brittle stalk phenotype and provides important insights into the regulatory mechanism of cellulose biosynthesis and cell wall development in maize.

Effects of Long-Term In Vitro Expansion on Genetic Stability and Tumor Formation Capacity of Stem Cells.

Stem cell therapeutics are emerging as novel alternative treatments for various neurodegenerative diseases based on their regenerative potentials. However, stem cell transplantation might have side effects such as tumor formation that limit their clinical applications. Especially, in vitro expansion of stem cells might provoke genetic instability and tumorigenic potential. To address this issue, we analyzed genomic alterations of adult human multipotent neural cells (ahMNCs), a type of human adult neural stem cells, after a long-term in vitro culture process (passage 15) using sensitive analysis techniques including karyotyping, array comparative genomic hybridization (aCGH), and whole exome sequencing (WES). Although karyotyping did not find any major abnormalities in chromosomal number or structure, diverse copy number variations (CNVs) and genetic mutations were detected by aCGH and WES in all five independent ahMNCs. However, the number of CNVs and genetic mutations did not increase and many of them did not persist as in vitro culture progressed. Although most observed CNVs and genetic mutations were not shared by all five ahMNCs, nonsynonymous missense mutations at MUC4 were found in three out of five long-term cultured ahMNC lines. The genetic instability did not confer in vivo tumorigenic potential to ahMNCs. Collectively, these results indicate that, although genetic instability can be induced by long-term in vitro expansion of stem cells, it is not sufficient to fully exert tumor formation capacity of stem cells. Other functional effects of such genetic instability need to be further elucidated.

Molecular differences between lymph nodes and distant metastases compared with primaries in colorectal cancer patients

Lymph nodes (LNs) and distant metastases can arise from independent subclones of the primary tumor. Herein, we characterized the molecular landscape and the differences between LNs, distant metastases and primary colorectal cancers (CRCs). Samples were analyzed using next generation sequencing (NGS, MiSeq on 47 genes, NextSeq on 592 genes) and immunohistochemistry. Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. In total, 11,871 samples were examined, comprising primaries (N = 5862), distant (N = 5605) and LNs metastases (N = 404). The most frequently mutated genes in LNs were TP53 (72%), APC (61%), KRAS (39%), ARID1A (20%), PIK3CA (12%). LNs showed a higher mean TMB (13 mut/MB) vs distant metastases (9 mut/MB, p < 0.0001). TMB-high (≥17mut/MB) and MSI-H (8.8% and 6.9% vs 3.7%, p < 0.001 and p = 0.017, respectively) classifications were more frequent in primaries and LNs vs distant metastases (9.5% and 8.8% vs 4.2%, p < 0.001 and p = 0.001, respectively). TMB-high is significantly more common in LNs vs distant metastases and primaries (P < 0.0001), regardless MSI-H status. Overall, LNs showed significantly different rates of mutations in APC, KRAS, PI3KCA, KDM6A, and BRIP1 (p < 0.01) vs primaries, while presenting a distinct molecular profile compared to distant metastases. Our cohort of 30 paired samples confirmed the molecular heterogeneity between primaries, LNs, and distant metastases. Our data support the hypothesis that lymphatic and distant metastases harbor different mutational landscape. Our findings are hypothesis generating and need to be examined in prospective studies.

Molecular characterization of squamous cell carcinoma of the anal canal.

Squamous cell carcinoma of the anal canal (SCCA) is an uncommon malignancy with limited therapeutic options. Nivolumab and pembrolizumab show promising results in patients with SCCA. Human papillomavirus (HPV)-negative tumors are frequently TP53-mutated (TP53-MT) and often resistant to therapy. We present a large molecularly-profiled cohort of SCCA, exploring the underlying biology of SCCA, differences between TP53-wild type (TP53-WT) and TP53-MT tumors, and differences between local and metastatic tumors. SCCA specimens (n=311) underwent multiplatform testing with immunohistochemistry (IHC), in situ hybridization (ISH) and next-generation sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chi-square testing was used for comparative analyses. The most frequently mutated genes included PIK3CA (28.1%), KMT2D (19.5%), FBXW7 (12%), TP53 (12%) and PTEN (10.8%). The expression of PD-1 was seen in 68.8% and PD-L1 in 40.5% of tumors. High TMB was present in 6.7% of specimens. HER2 IHC was positive in 0.9%, amplification by chromogenic in situ hybridization (CISH) was seen 1.3%, and mutations in ERBB2 were present in 1.8% of tumors. The latter mutation has not been previously described in SCCA. When compared with TP53-WT tumors, TP53-MT tumors had higher rates of CDKN2A, EWSR1, JAK1, FGFR1 and BRAF mutations. PD-1 and PD-L1 expression were similar, and high TMB did not correlate with PD-1 (P=0.50) or PD-L1 (P=0.52) expression. Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

Assessment of immune biomarkers and establishing a triple negative phenotype in gynecologic cancers

ObjectiveImmuno-oncology (IO) has rapidly evolved, with many IO therapies either approved or under investigation for multiple malignancies. Biomarkers exist that can predict response to IO therapies including PD-L1 expression, microsatellite instability (MSI), and total mutation burden (TMB). This paper serves to analyze the presence of these biomarkers across gynecologic cancers. MethodsA total of 16,300 gynecologic cancer specimens submitted for molecular profiling to Caris Life Sciences were reviewed. Immunohistochemistry was performed using the SP142 anti-PD-L1 clone and assessed for intensity. Next-generation sequencing, immunohistochemistry, and fragment analysis were used to determine MSI status. TMB was measured by counting all non-synonymous missense mutations found per tumor not previously described as germline alterations. Chi-Square, Fisher Exact, and the Kruskal-Wallis test were used to compare cohorts. ResultsOf 16,300 specimens, 54.1% were ovarian, 37.2% uterine, 7.2% cervical, 0.3% vulvar, 1.2% vaginal, with 0.1% unspecified. MSI-H was most frequent in uterine cancer (17.7%) and only 1% of ovarian cancers. PD-L1 expression was present in 38.3% of cervical and 62.5% of vulvar cancers, but less than 8% of ovarian and uterine cancers. TMB-H was present in 21.1% cervical, 19.7% uterine, and 5% ovarian cancers. Few specimens exhibited a “triple positive” phenotype - 0.3% ovarian, 1.5% uterine, and 1.5% cervical. Associations were seen between MSI, TMB, and PD-L1 across all cancer types. ConclusionsThe frequency of individual biomarkers pertinent to IO therapy varies by cancer type. HPV-driven genital tract cancers have higher frequencies of PD-L1 expression, MSI-H, and TMBH. Endometrial cancers are characterized by MSI-H and TMB, whereas ovarian cancers have a low frequency of MSI-H and modest PD-L1 or TMBH. The incidence of ‘triple positive” cases was less than 2%.

Genomic characterization of p16+ compared to p16- HNSCC patients treated with immune checkpoint inhibitors and overall survival.

e18024 Background: Multiple studies investigating immune checkpoint inhibitors (ICI) in head and neck squamous cell cancer (HNSCC) patients have demonstrated prolonged survival of p16+ versus p16- tumors. However, the data has been conflicting. Methods: We queried the Caris Life Sciences CODEai database to assess survival outcomes of HNSCC patients who received ICIs comparing p16+ and p16- subgroups. A standard cut-off of 2+, &gt;70% p16 staining was used. Presence of HPV16/18 genomes was tested using whole exome sequencing. Patients were considered smokers if they had &gt;15 pack-years of tobacco use. PD-L1 expression was assessed by the 22c3 antibody, ≥1 being positive. Gene mutations were evaluated using Next-Generation Sequencing (NGS) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was assessed by somatic nonsynonymous missense mutations. Real world overall survival (rwOS) was obtained from insurance claims data and Kaplan-Meier estimates were calculated from the date of treatment start to the date of last contact. Time on treatment (TOT) was calculated from date of start to completion of ICIs. Results: 2905 patients with HNSCC were identified in the Caris database. 41% (215/525) were smokers. Among patients who were tested for p16 and/or HPV, 32% (251/791) expressed p16 and 28% (91/236) were HPV+. The majority of p16+ tumors were oropharynx (OP) in origin (68%, 171/251). 87% (970/1111) of tumors expressed PD-L1 and 16% (216/1362) had TMB ≥10/Mb. TP53 (54%, n=1115/2076) and CDKN2A (17%, n=281/1649) were the most common mutations. When compared to p16-, the p16+ group had a higher prevalence of TMB ≥10/Mb (26% vs 17%) and RB1 mutations (21% vs 4%) but lower number of smokers (15% vs 34%) and TP53 mutations (32% vs 58%). Similar to previous reports, p16+ oropharynx squamous cell carcinoma (OPSCC) patients survived longer than p16- patients, rwOS, 47 vs. 20 months (HR=0.55, p = 0.014) respectively. Among patients who were treated with ICIs, p16+ and p16- non-OP HNSCC, rwOS was not reached (NR) for both groups at follow up of 22 months while TOT for p16+ and p16- respectively, was 3.5 vs. 2.7 months, HR 0.507, p=0.039. No statistically significant difference was found in rwOS (19.3 vs. 24 months, HR 1.8, p=0.27) or TOT (3.7 vs. 4.1 months, HR 0.78, p=0.38) between p16+ and p16- OPSCC groups treated with ICIs, respectively. Conclusions: P16+ non-OP HNSCC patients receiving ICIs were found to remain on treatment longer compared to p16- patients which was not reproduced in the OPSCC subgroup. Randomized controlled trials are needed to verify p16 as a prognostic marker for ICI therapy.[Table: see text]

Molecular profiling and survival outcomes of p16+ compared to p16- oropharynx squamous cell cancer patients.

e18026 Background: Previous studies have consistently shown that p16+ oropharynx squamous cell cancer (OPSCC) patients have better outcomes. The significance of molecular and transcriptional signatures and its correlation with p16 expression remains unclear. Methods: We queried the Caris Life Sciences database to assess the molecular and transcriptional signatures related to p16+ and p16- head and neck squamous cell cancer (HNSCC) patients. Comprehensive molecular profiling including whole exome sequencing (WES), targeted Next-Generation Sequencing (NGS), and immunohistochemistry (IHC) 22c3 for PD-L1 was performed (CPS ≥1 considered positive) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. A standard cut-off of 2+, &gt; 70% p16 staining was used. HPV16/18 was tested using WES. Patients were considered smokers if they had &gt; 15 pack-years. Real world overall survival (rwOS) was obtained from insurance claims data and Kaplan-Meier estimates were calculated from the date of start of treatment to the date of last contact. Results: 948 cases of OPSCC were identified in the Caris database. 41% (82/199) were smokers. Where p16 and HPV data was available, 41% were p16+ (171/420) while HPV positivity rate was 52% (71/148). We noted a small number of patients with discordant p16 and HPV status (7 p16+/HPV-, 8 p16-/HPV+) in 327 patients who had HPV status available. Most common mutations were TP53 (33%), PIK3CA (17%) and KMT2D (10.6%). 87% were PD-L1 positive (342/394), with high expression in both p16+ and p16- subgroups, 90% and 85% respectively. 10% had TMB≥10/Mb (48/463). TP53 mutations were more common in p16- (49%) tumors in contrast to p16+ (10%) (p &lt; 0.0005), while no statistical difference was detected in TMB≥10/Mb between the groups. CDKN2A, TERT and NOTCH1 mutations were more prevalent in tumors that were p16- or HPV- in contrast to tumors that were p16+ or HPV+ (p &lt; 0.05). FGF3, CCND1, FGF4, FGF19 copy number alterations (CNA) were less common in p16+ OPSCC compared to p16- or HPV16- OPSCC (p &lt; 0.0005). P16+ patients had longer rwOS when compared to p16-, 47 months versus 20 months (HR = 0.55, p = 0.014) respectively. Conclusions: Higher frequency of CDKN2A, TERT and NOTCH1 mutations among p16- (versus p16+) raise the possibility of potential targets for treatment in this group with poor prognosis. However, it remains unclear if these can serve as independent predictors of survival.[Table: see text]

Association of ATM mutations in metastatic prostate cancer with differential genomic alteration profiles from homologous recombination deficient and proficient tumors.

5063 Background: ATM mutations, one of a family of DNA repair defects prevalent in prostate cancer, have been included in a list of actionable mutations for PARP inhibitor (PARPi) therapeutic trials. Despite preclinical evidence, PARPi have shown minimal clinical activity in ATM mutant prostate cancer (ATMmPCa). The present analysis explores co-occurring genomic alterations that may drive outcomes of metastatic PCa (mPCa) patients with tumors harboring ATM mutations and provide clues for understanding therapy resistance and potential targets. Methods: This study included molecular profiling analysis of 1375 cases of mPCa. Tumors were analyzed using next-generation sequencing (NGS), whole transcriptome sequencing (WTS), and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). dMMR/MSI-H status was determined by IHC, NGS, and fragment analysis and tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. We performed differential gene expression analysis of HR-associated transcripts such as ATR, PARP1-3, RAD50, RAD51A/B/C/D and RAD54. Significance was determined using the ꭓ2 test and Benjamini-Hochberg method. Results: Fifty-nine (4.2%) cases harbored pathogenic ATM mutations, 84 (6.2%) harbored BRCA2 mutations. 1018 tumors (74%) were deemed homologous recombination proficient (HRP) and 155 tumors (11.3%) were HR Deficient (HRD); harboring one or more mutation in HR-related genes excluding ATM and BRCA2. The mutation rate of TP53 was significantly lower in ATMmPCa (12.0%) compared to BRCA2mPCa (35%), HRD (35%) and HRP (46.6%) tumors. ATMmPCa showed higher rates of SMAD2 (3.7%/1%) and FLCN (5.2%/0.3%) alterations compared to HRP cases. PARP1 and RAD51D gene expression was reduced in ATMmPCa compared to HRP (p &lt; 0.05) and BRCA2mPCa ( p &lt; 0.05) tumors, respectively. No differences in gene expression levels were detected for ATR, PARP2, PARP3, RAD50, and RAD54. Chromosomal segments demonstrating differential CNA in ATMmPCa vs HRP, HRD, or BRCA2mPCa included FGF19, FGF4, PTPN11, ALDH2, DAXX, BCL7A, CCND1, BMPR1A and MEF2B (Q-value &lt; 0.05 determined by ꭓ2). The most common CNA in ATMmPCa was CCND1, present in approximately 13% (7/55) of cases. Compared to BRCA2mPCa and HRD cases, ATMmPCa cases are less likely to display markers of immunotherapy response such as dMMR/MSI-H or TMB ≥10 mutations/MB. Conclusions: ATMmPCa demonstrated several differences in co-occurring alterations compared to BRCA2mPCa, HRD and HRP mPCa. ATMmPCa tumors were less likely to harbor alterations in TP53 compared to BRCA2, HRD or HRP tumors. CNA in ATMmPCa occurred in 9 genes across distinct mPCa molecular subtypes and were enriched for those associated with the 11q13 amplicon harboring Cyclin D1. The FGF and PTPN11 related pathways are potentially targetable pathways in ATMmPC and may merit further study.

FOXA2 as a Candidate Gene Responsible for Congenital Panhypopituitarism: A Review of the Literature

The pituitary gland produces hormones that regulate multiple functions including growth, metabolism, reproduction, and homeostasis. Thus, congenital hypopituitarism (CH) can have profound and widespread repercussions on physiological balance. Recent research identified the forkhead box A2 transcription factor (FOXA2) as a candidate gene responsible for CH. We sought to review the literature for mouse models and case reports describing FOXA2 mutations, to shed more light on the potential role of this gene in CH. Using a pretested search strategy, we searched the PubMed database, including in the review only original research articles reporting FOXA2 gene mutations in mouse models and human case reports. A total of 10 studies reporting different Foxa2 mutations in mouse models were included. These works described the involvement of Foxa2 in the regulation of murine organogenesis. Foxa2 was found to participate in the development of ventral midline structures and endodermal-derived organs. In addition, when mutated, it was found to determine defects in node, notochord and the neural tube, precursors of the pituitary gland. Foxa2 was also found to have important effects on glucose homeostasis and its deficiency is characterized by hyperinsulinemic hypoglycaemia. Regarding human case reports, a total of 5 cases describing nonsynonymous missense mutations of FOXA2 were identified. All mutations were localized in the DNA binding domain, which might regulate the expression of tissue-specific genes important for cell differentiation. Panhypopituitarism was a prominent feature among the cases as well as hypoglycaemia in infancy with abnormal glucose homeostasis later in life. Additional 6 cases describing patients with varying deletions of 20p11.2 that encompasses FOXA2 were selected. In addition to panhypopituitarism, the patients were found to have several other dysmorphic features, affecting the face as well as the cardiac, gastrointestinal and genital systems. Authors proposed a region of approximately 1.35 Mb that covers around 17 genes, among which FOXA2, as the critical region associated with hypopituitarism. However, the other genes also deleted from this region are associated with the development of the central nervous system and the pancreas and may be responsible of the observed phenotype of these patients. Considering this evidence, FOXA2 seems to be a strong candidate for CH. However, further research is required to elucidate its involvement in pituitary development, as well as the genetic cause that drive FOXA2 haploinsufficiency in determining CH.

Abstract PS17-27: Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer and accounts for 10-15% of all cases. Though ILC has distinct clinical, prognostic and molecular features, studies are limited and include few patients. ILCs show a decreased response to neoadjuvant chemotherapy and an increased resistance to endocrine therapy. Thus, there is a great need to identify alternative therapies, such as immunotherapy, that could improve overall survival. Success of immunotherapy largely depends on tumor immunogenicity which varies with histologic type. Determination of predictive and prognostic biomarkers for ILC will help determine who can benefit the most. Our study investigates canonical markers of immunogenicity - PD-L1 expression and Tumor Mutational Burden (TMB) - in patients with ILC compared to invasive ductal carcinoma (IDC). We further correlate these markers in different subtypes i.e. hormone receptor positive (HR+), HER2 positive (HER2+), and in triple negative breast cancers (TNBC). We also analyze differences in immune cell profiles constituting the tumor microenvironment (TME) and differences in genomic alterations between ILC and IDC. Methods: A retrospective data analysis was performed to identify breast cancer tumors with ILC or IDC histology profiled at Caris Life Sciences (Phoenix, AZ) that were tested for PD-L1 by SP142 assay and had whole transcriptome sequencing data available. ILC and IDC cases were further subtyped as HR+ and HER2+ and TNBC. PD-L1 expression in immune cell was assessed using Ventana PD-L1 (SP142) histochemical assay and PD-L1 expression in tumor cell was assessed by laboratory developed test using SP142 clone with staining higher than 2+ considered positive. TMB was measured by counting somatic non-synonymous missense mutations on the 592 gene panel (Nextseq) next generation sequencing (NGS) assay, and ≥ 10 mutations/megabase (mut/Mb) was considered high. Using the whole transcriptome RNA sequencing (NovaSeq) data we analyzed the difference in immune cell profiles constituting the TME using a computational RNA deconvolution approach. NGS was used to identify significant differences in genomic alterations between ILC and IDC. Results: We identified 1,359 breast cancer patients (ILC: 194 vs IDC: 1,165). Among ILC, 79% were HR +, 11% TNBC, &amp;lt;2% HER2+ and the rest were of unclear subtype, compared to 55% HR +, 31% TNBC, 10% HER2+ in the IDC group. PD-L1 expression in immune cells was lower in ILC (PD-L1≥ 1% ILC: 13% vs. IDC: 35% p &amp;lt;0.0001; PD-L1&amp;gt;10% ILC: 1% vs. IDC: 5% p=0.017). PD-L1 expression in tumor cells was also lower in ILC (ILC 1% vs IDC 5%; p =0.0136). All subtypes of ILC had lower PD-L1 expression in immune and tumor cells when compared to IDC. TMB was similar and comparable in IDC and ILC (median TMB 7 mut/Mb). Assessment of TME showed a significantly increased abundance of cytotoxic lymphocytes and monocytic cells in IDC, and stromal endothelial cells in ILC. Androgen receptor (AR) expression, and mutations in CDH1 (ILC 76% vs IDC 2%) and PIK3CA (ILC 44% vs IDC 30%) were more common in ILC. CDH1 mutation was significantly higher in both TN ILC (ILC 60% vs IDC 1%; p&amp;lt;0.05) and HR+ ILC (77% vs IDC 2%; p&amp;lt;0.05) subtypes of ILC. TP53 mutation was lower in ILC (ILC 26% vs IDC 65%) irrespective of subtype. Conclusion: In summary, PD-L1 expression in immune cells and tumor cells was lower in ILC, however TMB was comparable between ILC and IDC. Immune cell profiling supports a cold or less immunogenic TME for ILC. A composite immune biomarker may be able to better characterize immunogenicity of ILC. Citation Format: Upama Giri, Joanne Xiu, Tatiana Karadimitriou, Sofi Castanon, Foluso Ademuyiwa, Paula Pohlmann, Elias Obeid, Jasgit Sachdev, Elisa Krill-Jackson, Michael Simon, Antoinette Tan, Neelima Denduluri, Lee Schwartzberg, Michael Korn, Evanthia T Roussos Torres. Molecular evaluation of immunogenicity and genomic alterations in invasive lobular breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-27.

Patient characteristics and survival outcomes in gastrointestinal cancers harboring RNF43 mutations or RSPO3 fusions: a retrospective analysis.

473 Background: The growth of intestinal epithelium is dependent on the Wnt/β-catenin signaling pathway. The R-Spondin (RSPO) family of proteins form complexes with Wnt ligand and RNF43 protein, which promote ubiquitination and degradation of Wnt receptors. Patients with gastrointestinal cancers harboring RSPO3 gene fusions or RNF43 gene mutations may benefit from Wnt pathway inhibitors. Methods: We retrospectively studied 51 patients at our institution diagnosed with gastrointestinal cancers with RSPO3 gene fusions or RNF43 gene mutations identified using Caris Life Sciences next-generation sequencing (NGS). Microsatellite-instability (MSI) was evaluated using NGS. Tumor mutational burden (TMB) was estimated from 592 genes by counting all nonsynonymous missense mutations not previously defined as germline mutations, expressed as mutations per megabase. First-line median progression-free survival (PFS) and median overall survival (mOS) were estimated using the Kaplan-Meier method. Results: Of the 51 patients in our study, 44 (86.3%) had RNF43 mutations and 7 (13.7%) had RSPO3 fusions, 6 of whom had colorectal cancer (CRC) and had PTPRK as the fusion partner, 1 case of small bowel cancer had a RSPO3: IFNGR1 fusion. Median age at diagnosis was 64.0 ± 12.0 years. The most common primary tumors were CRC in 23 patients (45.1%) and pancreas in 12 patients (23.5%). Co-occurring mutations in CRC patients with RSPO3 fusions included KRAS (50%, Q61H, G12C, and G12V), BRAF V600E (50%), and TP53 (100%), none were MSI-H, median TMB (mTMB) was 9.5 (range 7-11). Sixty-seven percent of RNF43-mutated CRC patients had the G659fs mutation. Co-occurring findings in RNF43-mutated CRC patients included 67% MSI-H, median TMB 27 (range 6-123), KRAS (28%), BRAF V600E (28%), and TP53 mutations (33%). Twenty-two patients (43.1%) had stage IV disease, 18 (35.3%) had stage III disease and 11 (21.6%) had stage II disease. Thirty RNF43-mutated patients (58.8%) underwent definitive surgical resection, with a mean time to recurrence of 22.2 months and mOS of 43.0 months (95% CI, 15.6-70.4). Among RNF43-mutated stage IV CRC (mCRC) patients, median PFS was 8.0 months (95% CI, 5.9-10.1). Stratified by stage, mOS was 111.0 months for stage II disease, 72.0 months for stage III disease, and 37.0 months for stage IV disease (95% CI, 14.7-59.3). Conclusions: RNF43 correlates with overall favorable mOS in patients with mCRC. mOS in RNF43-mutated mCRC is similar to those of RAS and BRAF wild-type subgroups as reported in the TRIBE study (Cremolini et al. 2015). Therefore, RNF43 mutational status appears to be useful in predicting clinical outcomes for patients with stage IV CRC and overlaps significantly with MSI-H. RSPO3 fusions are rare events and the clinical outcomes of this subset of patients with tumors with RSPO3 fusions remains largely unknown.

Comprehensive Genomic Profiling of Gastroenteropancreatic Neuroendocrine Neoplasms (GEP-NENs).

GEP-NENs are rare malignancies with increasing incidence. Their molecular characteristics are still undefined. We explored the underlying biology of GEP-NENs and the differences between gastrointestinal (GI) and pancreatic (PNEN), high-grade (HG), and low-grade (LG) tumors. GEP-NENs were analyzed using next-generation sequencing (NGS; MiSeq on 47 genes, NextSeq on 592 genes), IHC, and in situ hybridization. Tumor mutational burden (TMB) was calculated on the basis of somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. In total, 724 GEP-NENs were examined: GI (N = 469), PNEN (N = 255), HG (N = 135), and LG (N = 335). Forty-nine percent were female, and median age was 59. Among LG tumors, the most frequently mutated genes were ATRX (13%), ARID1A (10%), and MEN1 (10%). HG tumors showed TP53 (51%), KRAS (30%), APC (27%), and ARID1A (23%). Immune-related biomarkers yielded a lower prevalence in LG tumors compared with HG [MSI-H 0% vs. 4% (P = 0.04), PD-L1 overexpression 1% vs. 6% (P = 0.03), TMB-high 1% vs. 7% (P = 0.05)]. Compared with LG, HG NENs showed a higher mutation rate in BRAF (5.4% vs. 0%, P < 0.0001), KRAS (29.4% vs. 2.6%, P < 0.0001), and PI3KCA (7% vs. 0.3%, P < 0.0001). When compared with GI, PNEN carried higher frequency of MEN1 (25.9% vs. 0.0%, P < 0.0001), FOXO3 (8.6% vs. 0.8%, P = 0.005), ATRX (20.6% vs. 2.0%, P = 0.007), and TSC2 (6.3% vs. 0.0%, P = 0.007), but lower frequency of mutations in APC (1.0% vs. 13.8%, P < 0.0001). Significant molecular differences were observed in GEP-NENs by tumor location and grade, indicating differences in carcinogenic pathways and biology.

Clinically and biologically relevant subgroups of Wilms tumour defined by genomic and epigenomic analyses

BackgroundAlthough cure rates for Wilms tumours (WT) are high, many patients receive therapy with attendant long-term complications. Our goal was to stratify WT using genome-wide analyses to identify candidate molecular features for patients who would benefit from a reduction in therapy.MethodsWe generated DNA methylation and exome sequencing data on WT–kidney pairs (n = 57) and unpaired tumours (n = 27) collected either at our centre or by the Children’s Oncology Group. Samples were divided into a discovery set (n = 32) and validation set (n = 52).ResultsAnalysis of DNA methylation revealed two subgroups of WT with distinct features. Subgroup A has a similar DNA methylation profile to mature kidney, while Subgroup B has genome-wide dysregulation of DNA methylation. The rate of non-synonymous missense mutations and segmental chromosomal aberrations was higher in Subgroup B tumours, suggesting that this group has genome instability related to its epigenetic state. Subgroup A had a higher proportion of cases of bilateral disease. Tumours with high-risk histology or from patients who relapsed were only found in Subgroup B.ConclusionWe have identified subgroup-specific molecular events that could inform future work supporting more targeted therapeutic approaches and patient stratification. We propose a novel developmental tumour model based on these findings.