BackgroundThe majority of pancreatic ductal adenocarcinomas (PDACs) are driven by mutant (mt) KRAS. How mt KRAS and co-driver mutations affect the immune cell (IC) landscape of PDAC remains uncertain. Herein, we characterize the types of IC in the PDAC tumor microenvironment (TME) and the prevalence of immuno-oncologic (IO) biomarkers by genomic and transcriptomic analysis in the context of KRAS status.Materials and methods4142 PDAC and 3727 colorectal cancer (CRC) cases with KRAS mt were analyzed using next-generation DNA sequencing, immunohistochemistry, and whole-transcriptome RNA sequencing. Microsatellite instability and deficiency in mismatch repair (MSI-H/dMMR) and tumor mutational burden (TMB) were also assessed.ResultsWe found KRAS mt in 81% of PDAC, with the most common variant being G12D in PDAC, and fewer cases of KRAS mt were co-expressed with the predictive IO marker MSI-H/dMMR than KRAS-wild-type (wt). However, KRASG12D, KRASG12V, and KRASQ61 mutations had significantly lower TMB than KRAS wt tumors in PDAC. The IC environment of KRAS mt PDAC showed significant differences in nearly all IC types; a similar pattern was observed in CRC but was less pronounced.ConclusionsTherapeutic IO targets like programmed death-ligand 1 are enriched in pancreatic adenocarcinoma cases harboring specific targetable variants of KRAS mt PDAC. Better understanding of the TME could lead to tailored immunotherapeutic strategies to overcome these barriers in KRAS mt PDAC, possibly in combination with molecularly targeted treatment strategies.

Uveal melanoma (UM) is a rare form of melanoma having poor responses to currently available systemic agents. Our goal is to understand differences in chemokine expression in relation to tumor genetics and response to immunotherapy. UM patient tumors (N=278, 41 primary, 174 liver mets and 63 other mets) were profiled by NGS DNA/RNA at Caris Life Sciences (Phoenix, AZ). Chemokine expression, high/Low (H/L) defined as samples with >75th- or <25th-percentile of transcripts per million (TPM). Tumor microenvironment (TME) cell fraction estimated by RNA, with median fold changes (H/L) or proportion of samples with non-zero fraction reported (Table). PDL1+ (SP142) tested by IHC. Survival data obtained from insurance claims. Primary tumors had increased expression of CXCR4, CXCR1, CXCR2, CCL27 and CXCL13 compared to liver mets (FC range 1.2-4.2), while CXCL2 expression was increased in liver mets (2.3, p<0.01). In liver mets, increased infiltration of immunosuppressive cells was observed for CXCR4-H and CXCL12-H tumors(Table). Only M1 macrophages, CD8+ T cells and B cells were increased for CXCR4-H and CXCL12-H primary tumors. PDL1+ rates were increased in CXCR4-H tumors overall (H 36% vs L 13%, p<0.05). In liver mets, SF3B1 mutation was associated with lower CXCL1 and CXCL2 expression compared to WT (0.35- and 0.47-fold, respectively, p-<0.01). BAP1-mutated liver mets showed increased CXCL1 expression (2.0-fold, p-0.04), whereas CCR10 expression was increased in BAP1-mutated primary tumors (2.6-fold, p-0.02). Among immunotherapy treated patients with liver mets, there was a trend for improved survival for CXCL12 H (n=13) vs L (n=12) (HR 0.51 (0.21-1.3), p=0.14) and CXCR4 H (n=13) vs L (n=12) (HR 0.49 (0.20-1.2), p=0.12) though not significant.Table: 1132PTME (liver mets): CXCR4/CXCL12 median fold change (H/L) (where median is 0, % non-zero H vs L).Immune CellCXCR4 Median FC (H/L) or non-zero%p-valueCXCL12 Median FC (H/L) or non-zero%p-valueMonocyte13% vs 0%0.0137% vs 11%0.31Treg2.9<0.012.9<0.01T cell CD870% vs 28%<0.0167% vs 34%<0.01T cell CD432% vs 13%0.0237% vs 11%0.01NK cell1.4<0.0011.5<0.001Macrophage M21.30.0021.3<0.001Macrophage M192% vs 50%<0.0196% vs 49%<0.001B cell1.6<0.011.30.02 Open table in a new tab Our results suggest chemokines are differentially expressed in tumors harboring the common alterations associated with medium risk of distant metastases.

Downregulated C-type lectin domain family 3 member B (CLEC3B) is observed in NSCLC and is linked with immune cell infiltration. We aimed to analyse the impact of CLEC3B mRNA expression on survival and its molecular link to NSCLC subtypes and to immune cell infiltration. 19,892 samples were tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes/WES), RNA (WTS) and IHC. Top quartile transcripts per million (TPM) were defined as high (Q4, ≥4.03 TPM) and bottom quartile as low (Q1, ≤0.89 TPM). Cell infiltration was estimated by QuantiSEQ. X2/Fisher-Exact were used and significance was determined as p-value adjusted for multiple comparison (q<0.05). Real-world overall survival (OS) information was obtained from insurance claims data. Median age was 69 years. High CLEC3B mRNA expression was associated with female sex (55.2% women vs 44.8% men, p<0.001). Adenocarcinomas vs squamous-cell carcinomas had higher CLEC3B expression (2.28 vs 1.51, q<0.001). CLEC3B correlated negatively with mutations in TP53 (74.7% in Q1 vs 52.4% in Q4), KEAP1 (15.9% vs 11.2%), RB1 (13.5 vs 9.2%), CDKN2A (13.0% vs 8.3%), NF1 (10.3% vs 6.9%) (all q<0.001). KRAS (31.8% in Q4 vs 25.6% in Q1), EGFR (17.2% vs 8.1%), STK11 (15.9% vs 11.3%) mutations were more frequently observed in CLEC3B high expressors (all q<0.001). High CLEC3B mRNA expression was negatively associated with high TMB (43.0% vs 29.0%) and high PD-L1 expression (62.5% vs 47.7%) (all q<0.05). Transcriptomics revealed an upregulation of various immunological markers (i.e. LAG3, INF-G, PDCD1) and a higher abundance of several immune cells (i.e. B cells, macrophages, T cells) in the CLEC3B high subgroup (all q<0.001). Patients with high CLEC3B mRNA expression showed an improved OS when compared to CLEC3B low expression (p<0.001, HR: 1.35); a similar observation was made in patients treated with checkpoint inhibitors (p<0.001, HR: 1.29). Our study represents the largest analysis of CLEC3B mRNA expression in NSCLC. High CLEC3B expression levels are linked to a distinct molecular/immunological profile and to improved survival. Further experiments are now ongoing to unravel functional aspects of CLEC3B biology in NSCLC.

Two ROS1 tyrosine kinase inhibitors have been approved for ROS1 fusion-positive (ROS1+) non-small cell lung cancer (NSCLC). We performed a pan-tumor analysis of the incidence and characteristics of ROS1 fusions across all solid tumors. A retrospective analysis was performed on ROS1-positive solid malignancies identified by targeted RNA sequencing and whole transcriptome sequencing of clinical tumor samples through Caris Life Science (Phoenix, AZ). Real-world overall survival (rwOS) was obtained from insurance claims data and calculated from either tissue collection to last contact or time on treatment (TOT) from start to finish of ICI. Comparison of survival was performed by Kaplan-Meier analysis. A total of 259 ROS1+ samples across 17 distinct solid malignancies were identified from approximately 175,350 tumors that underwent sequencing (88% from whole transcriptome; 12% from targeted RNA; (Table). ROS1+ NSCLC constituted 78.8% of the ROS1+ solid malignancies, followed by glioblastoma (6.9%) and breast cancer (2.7%). The frequency of ROS1 fusion was approximately 0.47% among NSCLC, 0.29% for glioblastoma and 0.04% for breast cancer. The mean tumor mutation burden for all ROS1+ tumors was 4.8 mutations/Mb (SD 2.8). The distribution of PD-L1 (22C3) expression among all ROS1+ malignancies were 0% (18.6%), 1%-49% (29.4%), and > 50% (52.0%). The mean junctional read among all tumor types was 54.7 with NSCLC at 64.0 (SD 107.77), glioblastoma at 32.2 (SD 55.12) and breast cancer at 6.7 (SD 5.20). The most common genetic co-alterations of ROS1+ NSCLC were TP53 (29.1%) and SETD2 (7.3%). Although the analysis was limited in sample size, ROS1+ in glioblastoma may represent a potential poor prognostic factor (HR 0.56 95% CI: 0.23-1.35, p=0.19). Also, treatment with immunotherapy in ROS1+ fusions demonstrated shorter survival than those treated with ROS1 TKIs (HR 2.77 95% CI: 0.85-9.03, p=0.08).Table: 105PAllNSCLCGlioblastomaBreastN259204187Age median(range)63 (18-89)65 (27-89)63 (41-89)60 (40-77)Male11386110Female14611877Sequencing methodsTargeted RNA (Archer)554941WTS204155146Mean junction read54.764.032.26.7TMB (median)4435 Open table in a new tab ROS1 fusions occurred at a low frequency among a diverse range of solid tumors.

Tyrosine kinase inhibitors (TKIs) are effective in EGFR mutant non-small cell lung cancer (NSCLC), but acquired resistance is virtually inevitable. Known resistance mechanisms include acquired EGFR mutations (e.g, 718V, c797x, 724s, 721s or T790M); copy number amplifications in MET, ERBB2, and PIK3CA; gene fusion events; and histological transformation. We herein present the prevalence of resistance mutations in the largest reported cohort of EGFR mutant NSCLC.

Authors: Julia Judd, Nagla Abdel Karim, Hina Khan, Abdul R. Naqash, Yasmine Baca, Joanne Xiu, Ari M. VanderWalde, Hirva Mamdani, Luis E. Raez, Misako Nagasaka, Sachin Gopalkrishna Pai, Mark A. Socinski, Jorge J. Nieva, Chul Kim, Antoinette J. Wozniak, Chukwuemeka Ikpeazu, Gilberto de Lima Lopes, Alexander I. Spira, W. Michael Korn, Edward S. Kim, Stephen V. Liu, Hossein Borghaei. 1 Department of Hematology-Oncology, Fox Chase Cancer Center, Temple University Health System, Philadelphia, PA, USA

NANOG is a pluripotency transcription factor that serves as a signaling hub in cancer stemness pathways while also mediating immune evasion. This study aimed to clarify molecular characters relating to gene expression levels of NANOG and NANOGP8 (P8), both of which encode full-length NANOG protein, in patients with colorectal cancer (CRC).

Objective. Immuno-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. Methods. A 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. Results. Of 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. Conclusions. The 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 TMB\\H. Endometrial cancers are characterized by MSI-H and TMB, whereas ovarian cancers have a low frequency of MSI-H and modest PD-L1 or TMB\\H. The incidence of 'triple positive" cases was less than 2%. (c) 2021 Elsevier Inc. All rights reserved.

Background: The impact of molecular alterations on programmed death-ligand 1 (PD-L1) combined positive score (CPS) is not well studied in gastroesophageal adenocarcinomas (GEAs). We aimed to characterize genomic features of tumors with different CPSs in GEAs. Patients and methods: Genomic alterations of 2518 GEAs were compared in three groups (PD-11 CPS >= 10, high; CPS 1-9, intermediate; CPS < 1, low) using next-generation sequencing. We assessed the impact of gene mutations on the efficacy of immune checkpoint inhibitors (ICIs) and tumor immune environment based on the Memorial Sloan Kettering Cancer Center and The Cancer Genome Atlas databases. Results: High, intermediate, and low CPSs were seen in 18%, 54% and 28% of GEAs, respectively. PD-L1 positivity was less prevalent in women and in tissues derived from metastatic sites. PD-L1 CPS was positively associated with mismatch repair deficiency/microsatellite instability-high, but independent of tumor mutation burden distribution. Tumors with mutations in KRAS, TP53, and RAS-mitogen-activated protein kinase (MAPK) pathway were associated with higher PD-L1 CPSs in the mismatch repair proficiency and microsatellite stability (pMMR&MSS) subgroup. Patients with RAS-MAPK pathway alterations had longer overall survival (OS) from ICIs compared to wildtype (WT) patients [27 versus 13 months, hazard ratio (HR) = 0.36, 95% confidence interval (a): 0.19-0.7, P = 0.016] and a similar trend was observed in the MSS subgroup (P 0.11). In contrast, patients with TP53 mutations had worse OS from ICIs compared to TP53-WT patients in the MSS subgroup (5 versus 21 months, HR = 2.39, 95% CI: 1.24- 4.61, P 0.016). Conclusions: This is the largest study to investigate the distinct genomic landscapes of GEAs with different PD-L1 CPSs. Our data may provide novel insights for patient selection using mutations in TP53 and RAS-MAPK pathway and for the development of rational combination immunotherapies in GEAs.

Roughly 10% of breast cancers have an identifiable germline pathogenic variant, the most commonly being in the BRCA1/2 genes. Alterations in other genes, such as PALB2, ATM, CHK2, and RAD51C/D are also associated with increased risk of breast cancer when mutated. Pathogenic variants in these genes are also associated with other cancers. An increasing number of somatic mutations in synthetic lethal genes have been noted in “non-syndromic tumor” types; however, the clinical significance is unknown.

MDM2 is a key negative regulator of tumor suppressor p53. MDM2 gene amplification has been reported as a potential marker for hyperprogression under checkpoint inhibitors (ICI). We aimed to characterize the molecular and gene expression profile of MDM2 amplified (a-MDM2) GI cancers. 23632 samples were included: 11692 colorectal, 3830 gastric/esophageal, 3960 pancreatic, 1860 biliary cancers (BC), 2330 other GI. Samples were analyzed using NextGen DNA seq for gene mutation (mut) and amplification (copy number > 6), in situ hybridization, whole transcriptome RNA seq and immunohistochemistry (Caris Life Sciences). EBseq was used to identify differentially expressed genes based on MDM2 expression levels (above vs below median) with control for FDR (Q < 0.2). Pathway and functional enrichment analysis was performed using Reactome. A-MDM2 frequency was highest in small bowel cancers (4.5%, 28/627), gastric/esophageal (3.3%, 125/3830), and BC (3.4%, 64/1860). a-MDM2 was more common in males (P = .01). Compared to MDM2 not amplified (na), a-MDM2 GI tumors showed lower mut rates in TP53 (23 vs 69%), KRAS (15 vs 44%), APC (5 vs 41%), and PIK3CA (4 vs 12%), whereas ATM mut were higher (9.5 vs 4%) (Q < .001). Copy number alterations (CNA) were significantly higher in a-MDM2 vs na, including CDK4, ERBB3, HMGA2, LGR5, NACA and WIF1 (Q < .001). Main results according to tumor type are summarized in the table. MDM2 amplification had an inverse relationship with TMB (Q = .04) and MSI-H/dMMR (Q = .03). No association was found with PDL1 levels and CPS score. A total of 785 genes were significantly differentially expressed based on MDM2 levels, with an increase in FGF signaling related pathways in MDM2 overexpressing tumors (Q < .05).Table: 1952PBCEsophagealGastricSmall Bowela-MDM2 %na %Q-valuea-MDM2 %na %Q-valuea-MDM2 %na %Q-valuea-MDM2 %na %Q-valueNGS TP5316440.0024189< 0.0001957< 0.0001860< 0.0001CNA CDK4190.2< 0.0001210.2< 0.000160.10.02110.30.02CNA ERBB3300.0560.10.01200.440.20.8CNA GNAS60.20.00580.70.0300.31370.5< 0.0001CNA HMGA2420.3< 0.0001350.5< 0.0001270.2< 0.0001520.3< 0.0001CNA LGR5310.10.01250.1< 0.000160.10.01180< 0.0001CNA NACA50.1< 0.0001600.002200.400.21CNA WIF1360.1< 0.0001270.4< 0.0001180.1< 0.0001380.2< 0.0001 Open table in a new tab This is the most extensive profiling study to investigate a-MDM2 GI tumors. Our data show distinct molecular patterns of a-MDM2 GI cancers involving WNT pathway genes, upregulation of FGF signaling and inverse association with TMB and MSI which may explain the resistance mechanisms to ICI.

Objective: While the cause of uterine cancer disparities in African-American women is multifactorial, there is significant evidence suggesting a genetic basis of disparity. Racial differences in the molecular landscape of uterine cancer have yet to be fully characterized. We aim to examine uterine cancer tumors stratified by race and to identify potential therapeutic targets.

Background Although rare, the incidence of BM is increasing due to the improvement in metastatic CRC treatment and longer survival. Genomic analyses of small series revealed that BM can harbor potentially unique driver mutations. We aimed to comprehensively characterize the molecular profile of BM and explore the differences between BM vs other distant metastases (OM) and primary tumors (PT) in CRC. Methods Tumor samples from BM (n = 81), PT (n = 6898) and OM (n = 5918) were analyzed using NGS (TruSEQ on 45 genes or NextSEQ on 592 genes), in situ hybridization and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. Results The most frequently mutated genes in BM were TP53 (80%), APC (73%), KRAS (68%), ARID1A (18%), PIK3CA (13%) and FBXW7 (9%). The most prevalent copy number increase was seen in CDX2 (20%), CCND2 (7%), FLT1 (7%), FLT3 (7%) and FOXO1 (7%). When compared to OM and PT, mutations in KRAS (BM: 68%, OM: 49%, PT: 48%), CDKN2A (5%, 1%, 1%), ERCC2 (2%, 0, 0) and HRAS (1%, 0, 0) were significantly higher in BM (P 17mut/MB) and MSI-high were seen in BM vs PT (2 vs 9% and 1 vs 8%, P=.049 and .031, respectively) but not compared to OM. No RSPO3 nor NTRK1 fusions were seen in BM (n = 5). Female gender was associated with younger age in BM (53.5 vs 62 yr, P=.0014) and OM (58.8 vs 60.2 yr, P Conclusions This is the largest and most extensive profiling study to investigate the molecular makeup of BM and the differences with PT and OM in CRC. Our data show distinct mutations and CNA characterizing BM and lower expression of immune related markers, supporting the rationale to develop tailored approaches to the treatment of this metastatic site. Legal entity responsible for the study The authors. Funding National Cancer Institute grant number P30CA014089, Gloria Borges WunderGlo Foundation-The Wunder Project, Dhont Family Foundation, San Pedro Peninsula Cancer Guild, Daniel Butler Research Fund, Call to Cure Research Fund and Fong Research Project. Disclosure J. Xiu: Full / Part-time employment: Caris Life Sciences. Y. Baca: Full / Part-time employment: Caris Life Sciences. R.M. Goldberg: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Grothey: Travel / Accommodation / Expenses: Caris Life Sciences. A.F. Shields: Research grant / Funding (self), Travel / Accommodation / Expenses: Caris Life Sciences. A. Seeber: Advisory / Consultancy: Caris Life Sciences. M.E. Salem: Travel / Accommodation / Expenses: Caris Life Sciences. P.A. Philip: Travel / Accommodation / Expenses: Caris Life Sciences. J.L. Marshall: Advisory / Consultancy: Caris Life Sciences. W..M. Korn: Full / Part-time employment: Caris Life Sciences. H.J. Lenz: Travel / Accommodation / Expenses: Caris Life Sciences. All other authors have declared no conflicts of interest.

Background Werner syndrome gene (WRN) encodes a DNA helicase with an exonuclease activity that contributes to DNA repair. In cancer, WRN mutations lead to genomic instability. It is known that WRN is necessary to sustain in-vivo growth of cancers cells with microsatellite instability (MSI), including CRC. WRN is a very promising new target especially in cancers with MSI. There is still a lack of knowledge about the frequency of WRN alterations and their association with immunological and molecular phenotypes. Methods Tumour samples from 6854 CRC patients were analyzed using NGS (NextSEQ on 592 genes), in-situ hybridization and immunohistochemistry (Caris Life Sciences, Phoenix, AZ, USA). Tumour mutational burden (TMB) was calculated based on somatic non-synonymous missense mutations, and MSI was evaluated by NGS of known MSI loci. Results WRN mutations (WRN-mut) were observed in 80 of 6854 samples (1.2%). A higher prevalence of WRN-mut was detected in right- compared to left-sided CRC (2.4% vs 0.7%, p<.0001). In WRN-mut (MT) CRC, TMB (43 vs. 8.6 mutations/megabase [mut/MB], p<.0001) and PD-L1 expression (13% vs 4%, p<.0001) were higher compared to WRN wild-type (WT). A higher frequency of MSI-H was seen in cancers harboring WRN-mut (56% vs 7%, p<.0001). Also, WRN-mut was associated with a higher TMB in both MSI-H subgroup of tumors (54 vs 40 mut/MB, p=.03) and MSS subgroup (43 vs 8.6 mut/MB, p<.0001). Several differences between WRN-mut and WRN-WT CRC was observed, including TP53 (47% vs 73%), KRAS (34% vs 49%), APC (56% vs 73%), BRAF (26% vs 9%), ASXL1 (25% vs 4%), ERBB2 (9% vs 2%), BRCA1 (8% vs 1%), BRCA2 (15% vs 2%), CDK12 (10% vs 1%), (p<.01 for all). Copy number alterations (CNA) of CDX2 were seen only in WRN-WT tumours (6.4% vs 1%, p=.026) and CNAs seen more frequently in WRN-mut tumours included CD274, CALR, CRTC1, ELL, JAK3, KEAP1, LYL1, MEF2B (p<.01). Conclusions This is the largest profiling study to investigate the molecular and immunological landscape of WRN-mut CRCs. We show the high prevalence of MSI in WRN-mut tumours and their association with higher TMB and PD-L1 expression. Furthermore, it revealed that WRN-mut CRC is characterized by a distinct genetic profile. Our data might serve to tailor treatment in WRN-mut CRC. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.