Tumor Mutation Status Research Articles

KRAS Mutation Status in Colorectal Epithelial Tumors of Colorectal Cancer

Introduction: Colorectal cancer (CRC) is one of the commonest malignancies in industrialized nations with lower incidence in Asia and Africa. Majority of CRCs are non-hypermutated. KRAS is one of the commonest mutated genes in CRC patients. Given the KRAS mutated CRCs are resistant to EGFR targeted chemotherapy, expanded KRAS mutation testing is recommended before starting chemotherapy. The aim of this study was to study the genetic variation of KRAS in group of CRC cancer patients referred to a teaching hospital in Kolkata, India.Methods: Out of 15 cases of colorectal cancer studied, 2 specimens were right hemicolectomies for clinically diagnosed colon carcinomas and 13 specimens were biopsies of colonoscopy done for anaemia, changed bowel habit, occult bleeding. Formalin fixed paraffin embedded (FFPE) tissue sections, stained by routine Hematoxylin and Eosin were examined microscopically. The DNA was extracted from FFPE tissue with Qiagen kit. KRAS Mutation analysis was done by TheraScreen – KRAS Mutation Kit.Results: The age of patients ranged from 28 to 64 years. Nine were male and remaining 6 were female. histopathological examination revealed 10 adenomas and 5 adenocarcinomas. One adenoma, tubulovillous with high grade dysplasia featured positive KRAS mutation (Gly12Asp). Three out of 4 adenocarcinomas with positive KRAS mutation, one showed Gly12Ala and 2 showed Gly12Asp. Conclusion: The frequency of KRAS mutations was very low in the studied sample.

Tumor Mutational Burden in Metastatic Castration-Resistant Prostate Cancer and Response to Checkpoint Inhibition

This single-center cohort study assesses the association of tumor mutational burden status in patients with metastatic castration-resistant prostate cancer and response to immune checkpoint inhibitor therapy.

Association of RNF43 Genetic Alterations With BRAFV600E and MSIhigh in Colorectal Cancer.

Recent studies have provided evidence for a predictive value of RNF43 genetic alterations (GAs) as biomarkers for targeted therapies in microsatellite-stable (MSS) colorectal cancer (CRC). These data have the potential to prioritize treatment strategies in patients with BRAFV600E-mutant CRC and help to identify a subgroup that is more likely to derive benefit versus those patients for whom alternative treatment approaches are needed. We were therefore interested in defining the precise frequency of BRAFV600E and RNF43 GAs and their respective overlap in a large cohort of patients with CRC. To address this question, we performed a retrospective analysis that included 52,969 patients diagnosed with CRC from the FoundationCORE database. We observed a striking association of RNF43 GAs with MSI and tumor mutational burden status and BRAFV600E mutations. Overall, 23% of MSS patients with confirmed BRAFV600E mutation harbor an RNF43 GA-which accounts for 1.1% of all patients with CRC and for 15.7% of all CRC BRAFV600E cases. Ongoing phase III clinical trials, such as BREAKWATER, should aim to incorporate broader genetic profiling to further validate the superior sensitivity of patients with RNF43-mutant, MSS BRAFV600E CRC to anti-EGFR-/BRAFi-based therapies.

Triplet chemotherapy with or without bevacizumab as first line treatment for metastatic colorectal cancer: An AGEO multicenter real-world study

BackgroundPrior trials validated triplet chemotherapy (Tri-CT) with bevacizumab as first line treatment for metastatic colorectal cancer (mCRC) but real-world data are scarce and practices remain heterogeneous. AimsTo evaluate Tri-CT +/- bevacizumab efficacy and safety, and to identify factors influencing treatment decisions. MethodsThe COLOTRIP retrospective study enrolled mCRC patients treated from 2014 to 2019 in 14 French centers. ResultsOf 299 patients (81% PS 0-1, 58% RAS-mutated and 19% BRAF-mutated), 51% received Tri-CT and 49% Tri-CT + bevacizumab. Metastatic disease was classified as resectable (6.5%), potentially resectable (40%), and unresectable (54%). Bevacizumab use was associated with primary tumor location, mutational status and number of metastases. Median overall survival was 33.5 months in the Tri-CT group and 23.9 months in the Tri-CT + bevacizumab group, with median progression-free survival being 14.5 and 11.4 months. After adjusting for initial characteristics, no difference in survival was noted. Around 30% of patients experienced grade ≥3 adverse events. ConclusionsThis study highlights several factors influencing Tri-CT use +/- bevacizumab decision and confirms the real-world good oncological outcomes and tolerability of these regimens in mCRC patients. Our results suggest that Tri-CT alone may by an appropriate option for specific subgroups of patients.

Clinical characteristics and survival analysis in patients with isolated pulmonary metastases from colorectal cancer (CRC).

60 Background: Recent studies seem to indicate that patients with metastatic colorectal cancer (mCRC) with exclusive pulmonary metastatic disease have better outcomes than those with other metastatic sites. The aim of this study is to identify differences in clinical and survival characteristics between the two groups in a cohort of a third level hospital. Methods: We retrospectively analyzed a sample of 569 patients with mCRC in a tumor registry from 2015 to 2021, studying clinical and molecular characteristics, as well as overall survival data of patients with isolated pulmonary metastases, versus the rest of patients. Results: Of the total sample analyzed, 61 patients (10.7%) had isolated pulmonary metastases. In these patients, several clinical characteristics were more frequent: primary rectal tumor (52.5% vs 23.4%, p < 0.000), resected primary tumor (95.1% vs 78.9%, p = 0.0026), metachronous metastatic disease (62.3% vs 42%, p = 0.0025) and complete response rate to the first line of treatment (19.3% vs 9.9%, p = 0.026). Furthermore, analytically, these patients were more likely to have normal CEA (75% vs 38.3%, p < 0.000), CA 19.9 (84.3% vs 57.7%, p = 0.0002) and LDH (91.1% vs 66.5%, p = 0.0007) levels. In our cohort, we found no statistically significant differences in tumor mutational status (KRAS, NRAS, BRAF, PI3K and HER2). There were no patients with exclusive pulmonary metastatic disease and microsatellite instability (MSI). 70.3% of patients with isolated pulmonary metastases received lung metastasectomy, and up to 58.3% received a second metastases surgery. We also found a numerical benefit in terms of overall survival (OS) in patients with exclusive metastatic lung disease, although it did not reach statistical signification (median OS 77.3 vs 58.1 months, HR for death 0.70, 95% CI, 0.49 – 1.01, p = 0.083). Conclusions: In our cohort, patients with isolated pulmonary metastases were more likely to have rectal tumors, primary tumor resection, metachronous metastatic disease, higher rate of complete responses and normal CEA, CA 19.9 and LDH levels, with a possible benefit in terms of OS.

Exploratory analysis of biomarkers of response to durvalumab in advanced HER2-negative oesophago-gastric adenocarcinoma within a phase 2 clinical trial.

384 Background: Advanced oesophago-gastric cancers have showcased varied responses to immunotherapy based on biomarkers such as tumour mutational burden, microsatellite instability, and PD-L1 expression. However, these markers may not fully capture tumour heterogeneity and evolutionary dynamics. Hence, understanding the intricate relationship between immune selection, specifically immune dN/dS,1 and tumour evolution is vital for devising personalized therapeutic strategies. Methods: In our phase 2 open label, multicentre, randomised PLATFORM trial (NCT02678182), patients received maintenance durvalumab post first-line chemotherapy. A survival advantage was not seen with maintenance durvalumab compared to surveillance and exploratory analysis of PD-L1 expression was not associated with improved survival outcomes.2 However, a subset of patients treated with durvalumab experienced durable responses and some experienced incremental radiological responses. A pilot substudy on a selected cohort (n=24) of these patients was designed to explore the potential of immunogenomics evolutionary-based metrics, notably dN/dS, as predictive biomarkers. DNA and RNA from FFPE baseline tumour samples underwent analysis, examining genomic factors including tumour mutational burden, indel signatures, PDL1 status, and immune evasion mechanisms. Furthermore, the evolutionary metric, immune dN/dS, which has implications in tumour immunoediting and its response to immunotherapies, was scrutinized. Results: A dominant indel mutational signature emerged from the analysis. The majority of patients displayed a significantly higher number of indel variants, compared to non-synonymous and synonymous single-nucleotide variants. A recurring observation was dN/dS values consistently above 1, indicating positive selection for non-synonymous somatic variants. Additionally, hypermutation in driver genes, notably TP53 and XIRP2, was detected. One patient, with a unique dN/dS < 1, showcased survival beyond four years without liver metastases. Immune dN/dS > 1 and no evidence of liver metastases displayed the best prognosis after treatment in contrast with those that have immune dN/dS < 1 with or without liver metastases. Conclusions: Immune dN/dS emerges as a promising tool to decode tumour evolutionary mechanisms, providing profound insights into the dynamics of tumour evolution in advanced HER2 negative oesophago-gastric cancer patients receiving durvalumab immunotherapy. This pilot study underscores the potential of this novel immunogenomic approach in improving patient stratification and tailoring therapeutic strategies in cancer immunotherapy. 1. Zapata et al, Nature Genetics, 2023. 2. Fong et al, J Clin Oncol, 2021. Clinical trial information: NCT02678182 .

First-line (1L) nivolumab (NIVO) + ipilimumab (IPI) in patients (pts) with microsatellite instability-high/mismatch repair deficient (MSI-H/dMMR) metastatic colorectal cancer (mCRC): 64-month (mo) follow-up from CheckMate 142.

97 Background: NIVO + IPI demonstrated robust, durable clinical benefit and was well tolerated as a 1L therapy in pts with MSI-H/dMMR mCRC in the phase 2 CheckMate 142 study (NCT02060188), leading to the inclusion of NIVO + IPI in the NCCN guidelines as an initial therapy option for these pts. At 52-mo median follow-up, 1L NIVO + IPI continued to demonstrate durable clinical benefit, and no new safety signals were identified. Here we report longer follow-up results. Methods: Pts with MSI-H/dMMR mCRC and no prior treatment for metastatic disease received NIVO 3 mg/kg Q2W + IPI 1 mg/kg Q6W until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) by investigator assessment (INV) per RECIST v1.1. Other key endpoints were disease control rate (DCR), duration of response (DOR), progression-free survival (PFS), all by INV; overall survival (OS); and safety. Results: In total, 45 pts received 1L NIVO + IPI. With median follow-up of 64.2 mo (range, 59.4–68.9 mo), ORR by INV was 71% (95% CI, 56–84%). The proportion of pts with a best overall response of complete response (CR) was 20%, partial response (PR) was 51%, stable disease (SD) was 13%, and progressive disease was 16%. Median DOR (mDOR) was not reached, and the 60-mo DOR rate was 72%. Median PFS (mPFS) by INV and median OS (mOS) were not reached, with 60-mo PFS and OS rates of 55% and 67%, respectively (Table). Among pts alive at the data cutoff (n = 31), 30 remained treatment-free after initial study treatment without receiving any subsequent systemic therapy, with a median treatment-free interval of 34.7 mo (range, 1.6–61.4 mo). Exploratory analysis by tumor mutational burden status will be presented. Safety data are shown in the Table. Conclusions: At 64-mo follow-up, NIVO + IPI continued to demonstrate clinically meaningful survival and durable responses, with mPFS, mOS, and mDOR still not reached, suggesting the potential for long-term clinical benefit. Safety remained consistent with previous data. These findings further support current recommendations for NIVO + IPI as a 1L treatment for pts with MSI-H/dMMR mCRC. Clinical trial information: NCT02060188 . [Table: see text]

Systematic analysis of the prognostic value and immunological function of LTBR in human cancer.

Lymphotoxin beta receptor (LTBR) is a positive T cell proliferation regulator gene. It is closely associated with the tumor immune microenvironment. However, its role in cancer and immunotherapy is unclear. Firstly, the expression level and prognostic value of LTBR were analyzed. Secondly, the expression of LTBR in clinical stages, immune subtypes, and molecular subtypes was analyzed. The correlation between LTBR and immune regulatory genes, immune checkpoint genes, and RNA modification genes was then analyzed. Correlations between LTBR and immune cells, scores, cancer-related functional status, tumor stemness index, mismatch repair (MMR) genes, and DNA methyltransferase were also analyzed. In addition, we analyzed the role of LTBR in DNA methylation, mutational status, tumor mutation burden (TMB), and microsatellite instability (MSI). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were used to explore the role of LTBR in pan-cancer. Finally, the drugs associated with LTBR were analyzed. The expression of LTBR was confirmed using quantitative real-time PCR and Western blot. LTBR is significantly overexpressed in most cancers and is associated with low patient survival. In addition, LTBR expression was strongly correlated with immune cells, score, cancer-related functional status, tumor stemness index, MMR genes, DNA methyltransferase, DNA methylation, mutational status, TMB, and MSI. Enrichment analysis revealed that LTBR was associated with apoptosis, necroptosis, and immune-related pathways. Finally, multiple drugs targeting LTBR were identified. LTBR is overexpressed in several tumors and is associated with a poor prognosis. It is related to immune-related genes and immune cell infiltration.

Abstract LB_A03: Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples

Abstract Activating KRAS mutations, homologous recombination deficiency (HRD), deficient mismatch repair (dMMR), and other prognostic or predictive biomarkers are seen in fewer than half of pancreatic ductal adenocarcinoma (PDAC) cases. Structural variants (SV), such as gene fusions, translocations, deletions, duplications, etc, are known to play a role in oncogenesis, metastasis, and/or treatment resistance. In solid tumors, SV discovery and validation has been limited, often due to a fresh or live tissue requirement in cytogenomic analyses. Recent innovations in processing formalin-fixed paraffin-embedded (FFPE) samples for “next-generation” chromatin capture and long-range sequencing platforms (i.e. Hi-C) have overcome this barrier. Hi-C is a genome-wide technology which surveys rearrangements, generating genomic contiguity data from highly fragmented chromatin. Unlike targeted (e.g. hybrid capture) assays, Hi-C can survey the landscape of genomic aberrations in an unbiased manner. Archived FFPE has previously been an obstacle to Hi-C analysis of solid tumors. We present here, to our knowledge, the first Hi-C analysis of FFPE PDAC samples using a previously described, proprietary Hi-C platform. Thirty-one samples from 20 unique patients diagnosed and treated at the University of Colorado Comprehensive Cancer Center between 2014-2022 were processed and analyzed. Twenty-two samples were surgical resections paired with later biopsies of metastatic recurrences. Nine were unpaired surgical resections or metastatic biopsies. Seven of these cases had somatic KRAS mutations, five had germline mutations in DNA repair genes, and none had evidence of dMMR or MSI-high. Fifty percent of samples had sufficient tumor content for processing and successful library generation. Notably, two paired samples from 2014 were successfully processed and sequenced demonstrating the applicability of this technology to archival samples. In successfully analyzed samples, the vast majority of SVs observed in metastases were also seen in paired primary tumors. Several duplications and deletions recurred across two or more patients. Furthermore, at least three SVs from this cohort were observed in other tumor types from a separate cohort analysis using this technology. Cases with relatively more SVs demonstrated a longer time to metastatic recurrence than cases with fewer SVs. Cases with germline-mutated cancer-predisposing genes demonstrated a similar SV signature and time to metastatic recurrence as some cases with or without unknown germline and somatic tumor mutation status. Overall, our findings show that this novel platform can successfully profile archived FFPE PDAC samples. Some paired samples show a shared SV signature suggesting the presence of metastatic clones at the time of initial diagnosis. Following these results, we plan to process and analyze a larger cohort of PDAC cases enriched for wild-type KRAS and HRD carriers. We plan to compare our findings to Hi-C results from other tumors and further focus on any SVs recurrent across tumor types. Citation Format: Abhishek Pandey, Stephen Eacker, Adrie Van Bokhoven, Kathleen Torko, Christopher H Lieu. Proximity ligation sequencing reveals novel and recurrent structural genomic variants in FFPE pancreatic ductal adenocarcinoma samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A03.

Abstract C009: Biomarker analyses for predicting the benefit from immune checkpoint inhibitors in EGFR-mutated non-small cell lung cancer

Abstract Background: Although immune checkpoint inhibitors (ICIs) are effective for non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR)-mutated NSCLC patients are unlikely to benefit from ICIs treatment. It has been reported, however, a small subset of patients harboring EGFR-mutations respond to the treatment, although no biomarkers have been identified. In this study, we retrospectively explored predictive biomarkers to identify the responders by addressing tumor mutational status and tumor microenvironment (TME). Methods: NSCLC patients harboring EGFR-sensitizing mutations who were treated with EGFR-TKIs followed by ICIs alone or ICIs combined with chemotherapy between October 1, 2014 and September 31, 2019 were enrolled into the study. Next-generation sequencing (NGS) was performed on Ion S5 system using DNAs extracted from tissue samples at diagnosis for evaluating genetic mutations and tumor mutational burden (TMB) using Oncomine Tumor Mutation Load Assay. We also evaluated the expression of PD-L1, CD8 and FoxP3 on tumor tissues by immunohistochemical staining. Gene enrichment analysis was performed using gene symbol of somatic mutations by KOBAS in database of KEGG pathway. Mutational signature was identified by SigProfilerAssignment with reference mutational signatures of COSMIC v3.3 using exported vcf file from Ion torrent FileExporter plugin. Fisher's exact test was performed by using EZR software version 1.61. Results: Thirty-four patients were enrolled and the tissue DNA for NGS was successfully collected from 27 patients. Patient demographics were as follows: male/female, 17/17; nonsmoker/smoker, 17/17; 32/2 adenocarcinoma/non-adenocarcinoma, 32/2; EGFR mutation in exon19del/L858R/other, 21/11/2; overall response (ORR) to the past EGFR-TKI treatment CR-PR/SD/PD, 27/2/5; type of ICI pembrolizumab/nivolumab/atezolizumab/other, 17/11/5/1; response to ICI treatment CR-PR/SD/PD, 10/1/22. There was no correlation between the duration of initial EGFR-TKI treatment and the duration of ICI therapy. Patients who had immune-related adverse event (irAE) had significantly longer progression free survival (PFS) with ICIs than those without irAE (p = 0.049). Significantly more CR and PR were observed in patients who do not harbor TP53 mutations (p = 0.033). No correlation was found between TMB and the response to ICI treatment. With regard to PD-L1 expression, tissue tumor proportion score or combined positive score did not correlate with the response to ICI treatment. Patients with high expression level of CD8 showed significantly longer PFS and overall survival (p = 0.039 and p = 0.006). Gene enrichment analysis in CR and PR group indicated that most enriched KEGG pathway was platinum drug resistance and one CR case had a mutational signature of SBS31, relevant to platinum drug treatment, suggesting the involvement of platinum drug treatment in the response to ICI treatment. Conclusions: TME defined by CD8 and TP53 mutational status may have predictive impact on ICIs treatment in EGFR-mutated NSCLC. Citation Format: Mitsuo Osuga, Koichi Azuma, Shoichi Itoh, Yoshitaka Kawa, Daijiro Harada, Yukihiro Toi, Kenta Murotani, Akito Hata, Toshiyuki Kozuki, Shunichi Sugawara, Miyako Satouchi, Nobuyuki Yamamoto, Yasuhiro Koh. Biomarker analyses for predicting the benefit from immune checkpoint inhibitors in EGFR-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C009.

Nivolumab plus chemotherapy in first-line metastatic non-small-cell lung cancer: results of the phase III CheckMate 227 Part 2 trial

In CheckMate 227 Part 1, first-line nivolumab plus ipilimumab prolonged overall survival (OS) in patients with metastatic non-small-cell lung cancer (NSCLC) and tumor programmed death-ligand 1 (PD-L1) expression ≥1% versus chemotherapy. We report results from CheckMate 227 Part 2, which evaluated nivolumab plus chemotherapy versus chemotherapy in patients with metastatic NSCLC regardless of tumor PD-L1 expression. Seven hundred and fifty-five patients with systemic therapy-naive, stage IV/recurrent NSCLC without EGFR mutations or ALK alterations were randomized 1 : 1 to nivolumab 360 mg every 3 weeks plus chemotherapy or chemotherapy. Primary endpoint was OS with nivolumab plus chemotherapy versus chemotherapy in patients with nonsquamous NSCLC. OS in all randomized patients was a hierarchically tested secondary endpoint. At 19.5 months' minimum follow-up, no significant improvement in OS was seen with nivolumab plus chemotherapy versus chemotherapy in patients with nonsquamous NSCLC [median OS 18.8 versus 15.6 months, hazard ratio (HR) 0.86, 95.62% confidence interval (CI) 0.69-1.08, P= 0.1859]. Descriptive analyses showed OS improvement with nivolumab plus chemotherapy versus chemotherapy in all randomized patients (median OS 18.3 versus 14.7 months, HR 0.81, 95.62% CI 0.67-0.97) and in an exploratory analysis in squamous NSCLC (median OS 18.3 versus 12.0 months, HR 0.69, 95% CI 0.50-0.97). A trend toward improved OS was seen with nivolumab plus chemotherapy versus chemotherapy, regardless of the tumor mutation status of STK11 or TP53, regardless of tumor mutational burden, and in patients with intermediate/poor Lung Immune Prognostic Index scores. Safety with nivolumab plus chemotherapy was consistent with previous reports of first-line settings. CheckMate 227 Part 2 did not meet the primary endpoint of OS with nivolumab plus chemotherapy versus chemotherapy in patients with metastatic nonsquamous NSCLC. Descriptive analyses showed prolonged OS with nivolumab plus chemotherapy in all-randomized and squamous NSCLC populations, suggesting that this combination may benefit patients with untreated metastatic NSCLC.

TMIC-22. IDENTIFICATION OF EXOSOME MIRNA-MRNA INTERACTIONS IN THE GLIOMA TUMOR IMMUNE MICROENVIRONMENT

Abstract INTRODUCTION Exosomes have emerged as key local and systemic immune modulators in glioma, particularly in tumors with an IDH mutation. However, the exact mechanism by which these vehicles communicate with the tumor immune microenvironment warrants further exploration. We sought to identify exosomal miRNA differences in discrete genomic subsets in glioma and predict potential mRNA targets in tumor immune cell populations. METHODS Exosomes were isolated from IDH wild-type (IDHwt) and IDH mutant (IDHm) glioma organoids using ultracentrifugation. Nanoparticle Tracking Analysis and western blot were used to validate exosome capture. Exosomal miRNA was sequenced using the Illumina Novaseq platform and targets were predicted using TargetScan. Peripheral blood mononuclear cells (PBMCs) were isolated from glioma patients and processed for scRNA-seq. RESULTS The characteristics of isolated exosomes were consistent with previously published data. Six miRNAs were over-expressed in IDHm exosomes compared to IDHwt. Interestingly, none of these miRNAs were significantly up-regulated in IDHm tumor tissue. Comparison between predicted targets of IDHm exosome miRNA and scRNA-seq of circulating tumor PBMCs identified a set of expressed genes involved in the FOXO pathway, which is implicated in microglia activation and polarization. Furthermore, scRNA-seq analysis showed that the FOXO3 pathway is activated in CD14+AREG+ monocytes and CD14+RNA-lo monocytes, which are two cell populations that are fractionally down-regulated in IDHm glioma. CONCLUSION Our group and others have defined mutation-dependent mechanisms of immunosuppression in glioma. Here, we characterized the effects of tumor mutational status on exosome miRNA cargo and conducted a miRNA-mRNA network analysis to identify potential targets in tumor-associated myeloid cells. Our results suggest that exosomes may play a role in suppressing specific populations of monocytes via the FOXO pathway in an IDH-mutation dependent manner. This data sets the stage for mechanistic exploration of glioma exosomes on peripheral myeloid tumor immunity.

Simultaneous medullary and papillary thyroid carcinoma associated with a primary renal carcinoma: Case report and review of literature

Background: The association of primary two thyroid cancer (TC) and papillary renal carcinoma is an uncommon situation and only few cases have been reported. Case presentation: We report the case of a 49-year-old man with a papillary, medullary thyroid cancer in whom a papillary renal cancer was subsequently discovered. The patient was treated with total thyroidectomy and dissection of lymph nodes followed by radiotherapy and radioactive iodine. Conclusions: The simultaneous thyroid and renal carcinomas remains a subject of research, further investigations including tumor mutational status and histopathologic evaluation for each primary tumor are needed.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

1472P Radiotherapy affects immunotherapy efficacy based on tumor mutation status in patients with metastatic NSCLC

1472P Radiotherapy affects immunotherapy efficacy based on tumor mutation status in patients with metastatic NSCLC

Molecular Subtypes and Outcomes in a Multi-Institutional Review of Rectal Cancer Patients

Colorectal cancer (CRC) is a heterogeneous malignancy associated with a variety of genetic mutations. More recent data indicates that CRC can be broken down into unique biologically distinct consensus molecular subtypes (CMS) based on different pathological and genetic signatures. The purpose of this study is to evaluate the outcomes of rectal cancer patients in a multi-institutional network based on tumor mutational assessment and CMS group. Patient exome and transcriptome sequencing data and clinical outcomes were collected under the Total Cancer Care Protocol and Avatar® project within the Oncology Research Information Exchange Network (ORIEN). A total of 101 patients with demographic and outcomes information had data for microsatellite instability (MSI), tumor mutation burden (TMB), transcriptome, and whole exome sequencing (WES). Molecular subclasses (CMS1, CMS2, CMS3, CMS4, CMS-Mixed) were assigned based on transcriptional signatures by the R package "CMS Caller." Survival analysis was performed with the R packages "Survival" and "Survminer." A total of 101 rectal cancer patients, with a median age of 56.8, had a median follow up of 3.5 years (range 0.26-23.5). 78 patients were treated with curative intent for clinically localized disease and 35% of these patients developed metastatic disease. The remaining 23 patients had synchronous metastatic disease at presentation. There were 5 (5%) CMS1, 29 (29%) CMS2, 13 (13%) CMS 3, 49 (49%) CMS 4, and 5 (5%) CMS-Mixed patients in our cohort respectively. The cohort included 5 (5%) BRAF, 51 (50%) KRAS, and 63 (62%) TP53 mutated patients and 5 (5%) MSI high patients. Median survival was 18.8, 117.2, 125.7 and 119 months for CMS1, CMS2, CMS3, and CMS4 patients respectively, with insufficient events in CMS-mixed for calculation (p = 0.15). CMS1 patients had a significantly shorter survival compared to the other cohorts (p = 0.02), with 2 of 5 of these patients having received immunotherapy. 40% (2,0) CMS1, 52% (7,8) of CMS2, 15% (1,1) of CMS3, 59% (13,15) of CMS4, and 40% (1,1) of CMS-Mixed presented with or developed metastatic disease respectively. When divided into mutation groups, median survival was 43 versus 119, 119 versus 117, and 126 versus 119 months for BRAF, KRAS, and TP53 mutated and wild type patients respectively (p = 0.18, p = 0.48, p = 0.93). Evaluation of TMB and MSI status did not reveal significant differences in outcomes (p = 0.54, p = 0.7), with median survival of 126 months versus 117 in TMB high versus low patients and unreached versus 119 months in MSI versus MSS patients respectively. Of note, 3 of the 5 MSI patients were also CMS1, with the other two coming from CMS4 and CMS-mixed cohorts. CMS classification and tumor mutation status are associated with differential outcomes for rectal cancer patients, with some groups having a large likelihood of developing metastatic disease. Further work on optimizing and personalizing treatments for these high-risk populations is necessary.

Association between Contrast-Enhanced Computed Tomography Radiomic Features, Genomic Alterations and Prognosis in Advanced Lung Adenocarcinoma Patients.

Non-invasive methods to assess mutational status, as well as novel prognostic biomarkers, are warranted to foster therapy personalization of patients with advanced non-small cell lung cancer (NSCLC). This study investigated the association of contrast-enhanced Computed Tomography (CT) radiomic features of lung adenocarcinoma lesions, alone or integrated with clinical parameters, with tumor mutational status (EGFR, KRAS, ALK alterations) and Overall Survival (OS). In total, 261 retrospective and 48 prospective patients were enrolled. A Radiomic Score (RS) was created with LASSO-Logistic regression models to predict mutational status. Radiomic, clinical and clinical-radiomic models were trained on retrospective data and tested (Area Under the Curve, AUC) on prospective data. OS prediction models were trained and tested on retrospective data with internal cross-validation (C-index). RS significantly predicted each alteration at training (radiomic and clinical-radiomic AUC 0.95-0.98); validation performance was good for EGFR (AUC 0.86), moderate for KRAS and ALK (AUC 0.61-0.65). RS was also associated with OS at univariate and multivariable analysis, in the latter with stage and type of treatment. The validation C-index was 0.63, 0.79, and 0.80 for clinical, radiomic, and clinical-radiomic models. The study supports the potential role of CT radiomics for non-invasive identification of gene alterations and prognosis prediction in patients with advanced lung adenocarcinoma, to be confirmed with independent studies.

Circulating Tumor Cell Subpopulations Predict Treatment Outcome in Pancreatic Ductal Adenocarcinoma (PDAC) Patients.

There is a high clinical unmet need to improve outcomes for pancreatic ductal adenocarcinoma (PDAC) patients, either with the discovery of new therapies or biomarkers that can track response to treatment more efficiently than imaging. We report an innovative approach that will generate renewed interest in using circulating tumor cells (CTCs) to monitor treatment efficacy, which, in this case, used PDAC patients receiving an exploratory new therapy, poly ADP-ribose polymerase inhibitor (PARPi)-niraparib-as a case study. CTCs were enumerated from whole blood using a microfluidic approach that affinity captures epithelial and mesenchymal CTCs using anti-EpCAM and anti-FAPα monoclonal antibodies, respectively. These antibodies were poised on the surface of two separate microfluidic devices to discretely capture each subpopulation for interrogation. The isolated CTCs were enumerated using immunophenotyping to produce a numerical ratio consisting of the number of mesenchymal to epithelial CTCs (denoted "Φ"), which was used as an indicator of response to therapy, as determined using computed tomography (CT). A decreasing value of Φ during treatment was indicative of tumor response to the PARPi and was observed in 88% of the enrolled patients (n = 31). Changes in Φ during longitudinal testing were a better predictor of treatment response than the current standard CA19-9. We were able to differentiate between responders and non-responders using ΔΦ (p = 0.0093) with higher confidence than CA19-9 (p = 0.033). For CA19-9 non-producers, ΔΦ correctly predicted the outcome in 72% of the PDAC patients. Sequencing of the gDNA extracted from affinity-selected CTC subpopulations provided information that could be used for patient enrollment into the clinical trial based on their tumor mutational status in DNA repair genes.

Investigating genomic, proteomic, and post-transcriptional regulation profiles in colorectal cancer: a comparative study between primary tumors and associated metastases

IntroductionApproximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases.MethodsA total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients’ tumour pairs.ResultsAmong the primary and metastatic tumours the mutation rates for the individual genes are as follows:TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci (z-score = 6.246, difference = 0.3793, P < 0.0001). Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14–80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed.ConclusionThe molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients’ prognoses and response to therapy.

Histopathology and levels of proteins in plasma associate with survival after colorectal cancer diagnosis

BackgroundThe TNM system is used to assess prognosis after colorectal cancer (CRC) diagnosis. Other prognostic factors reported include histopathological assessments of the tumour, tumour mutations and proteins in the blood. As some of these factors are strongly correlated, it is important to evaluate the independent effects they may have on survival.MethodsTumour samples from 2162 CRC patients were visually assessed for amount of tumour stroma, severity of lymphocytic infiltrate at the tumour margins and the presence of lymphoid follicles. Somatic mutations in the tumour were assessed for 2134 individuals. Pre-surgical levels of 4963 plasma proteins were measured in 128 individuals. The associations between these features and prognosis were inspected by a Cox Proportional Hazards Model (CPH).ResultsLevels of stroma, lymphocytic infiltration and presence of lymphoid follicles all associate with prognosis, along with high tumour mutation burden, high microsatellite instability and TP53 and BRAF mutations. The somatic mutations are correlated with the histopathology and none of the somatic mutations associate with survival in a multivariate analysis. Amount of stroma and lymphocytic infiltration associate with local invasion of tumours. Elevated levels of two plasma proteins, CA-125 and PPP1R1A, associate with a worse prognosis.ConclusionsTumour stroma and lymphocytic infiltration variables are strongly associated with prognosis of CRC and capture the prognostic effects of tumour mutation status. CA-125 and PPP1R1A may be useful prognostic biomarkers in CRC.