Frequency Of KRAS Mutations Research Articles

Innovative PNA-LB mediated allele-specific LAMP for KRAS mutation profiling on a compact lab-on-a-disc device

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (Tf) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence Tf values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies.

CtDNA whole exome sequencing in pancreatic ductal adenocarcinoma unveils organ-dependent metastatic mechanisms and identifies actionable alterations in fast progressing patients

Understanding progression mechanisms and developing new targeted therapies is imperative in pancreatic ductal adenocarcinoma (PDAC). In this study, 80 metastatic PDAC patients were prospectively recruited and divided into discovery (n=37) and validation (n=43) cohorts. Tumor and plasma samples taken at diagnosis were pair analyzed using whole exome sequencing (WES) in patients belonging to the discovery cohort alone. The variant allele frequency (VAF) of KRAS mutations was measured by ddPCR in plasma at baseline and response assessment in all patients. Plasma WES identified at least one pathogenic variant across the cohort, uncovering oncogenic mechanisms, DNA repair, microsatellite instability, and alterations in the TGFb pathway. Interestingly, actionable mutations were mostly found in plasma rather than tissue. Patients with shorter survival showed enrichment in cellular organization regulatory pathways. Through WES we could identify a specific molecular profile of patients with liver metastasis, which exhibited exclusive mutations in genes related to the adaptive immune response pathway, highlighting the importance of the immune system in liver metastasis development. Moreover, KRAS mutations in plasma (both at diagnosis and persistent at follow-up) correlated with shorter progression free survival (PFS). Patients presenting a reduction of over 84.75 % in KRAS VAF at response assessment had similar PFS to KRAS-negative patients. Overall, plasma WES reveals molecular profiles indicative of rapid progression, potentially actionable targets, and associations between adaptive immune response pathway alterations and liver tropism.

Identification and validation of a lactate metabolism-related six-gene prognostic signature in intrahepatic cholangiocarcinoma

PurposeIntrahepatic cholangiocarcinoma (iCCA) is a highly malignant and fatal liver tumor with increasing incidence worldwide. Lactate metabolism has been recently reported as a crucial contributor to tumor progression and immune regulation in the tumor microenvironment. However, it remains poorly identified about the biological functions of lactate metabolism in iCCA, which hinders the development of prognostic tools and therapeutic interventions.MethodsThe univariate Cox regression analysis and Boruta algorithm were utilized to identify key lactate metabolism-related genes (LMRGs), and a prognostic signature was constructed based on LMRG scores. Genomic variations and immune cell infiltration were evaluated in the high and low LMRG score groups. Finally, the biological functions of key LMRGs were verified with in vitro and in vivo experiments.ResultsPatients in the high LMRG score group exhibit a poor prognosis compared to those in the low LMRG score group, with a high frequency of TP53 and KRAS mutations. Moreover, the infiltration and function of NK cells were compromised in the high LMRG score group, consistent with the results from two independent single-cell RNA sequencing datasets and immunohistochemistry of tissue microarrays. Experimental data revealed that lactate dehydrogenase A (LDHA) knockdown inhibited proliferation and migration in iCCA cell lines and tumor growth in immunocompetent mice.ConclusionOur study revealed the biological roles of LDHA in iCCA and developed a reliable lactate metabolism-related prognostic signature for iCCA, offering promising therapeutic targets for iCCA in the clinic.

Temporal trends and regional variability in BRAF and KRAS genetic testing in Denmark (2010-2022): Implications for precision medicine.

This study aims to evaluate the developments in the testing of Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations across different cancer types and regions in Denmark from 2010 to 2022. Using comprehensive data from the Danish health registries, we linked molecular test results from the Danish Pathology Registry with cancer diagnoses from the Danish National Patient Registry between 2010 and 2022. We assessed the frequency and distribution of KRAS and BRAF mutations across all cancer types, years of testing, and the five Danish regions. The study included records of KRAS testing for 30 671 patients and BRAF testing for 30 860 patients. Most KRAS testing was performed in colorectal (78%) and lung cancer (18%), and BRAF testing in malignant melanoma (13%), colorectal cancer (67%), and lung cancer (12%). Testing rates and documentation mutational subtypes increased over time. Reporting of wildtype results varied between lung and colorectal cancer, with underreporting in lung cancer. Regional variations in testing and reporting were observed. Our study highlights substantial progress in KRAS and BRAF testing in Denmark from 2010 to 2022, evidenced by increased and more specific reporting of mutational test results, thereby improving the precision of cancer diagnosis and treatment. However, persistent regional variations and limited testing for cancer types beyond melanoma, colorectal, and lung cancer highlight the necessity for a nationwide assessment of the optimal testing approach.

Molecular insights from comprehensive genomic profiling data in advanced metastatic colorectal cancer in South Asian population: A retrospective observational study

ABSTRACT Background: An increase in colorectal cancer incidence has been reported in India, often presenting in advanced stages and resulting in poor survival. However, the genomic and therapeutic landscape is not well understood. Objective: The primary objective of the study was to understand the mutational profile of metastatic colorectal cancer in the Southeast Asian cohort, and the secondary objective was to define the proportion of patients with therapeutically significant variants. Materials and Methods: This retrospective study was conducted between January 2021 and September 2023, at 4baseCare Onco Solutions Pvt. Ltd., Bengaluru, Karnataka, India. Comprehensive genomic profiling (CGP) and biomarker testing for MSI, TMB, and PD-L1 was carried out in 477 metastatic advanced (Stage III/IV) colorectal cancer patients, for the current retrospective-observational study. Results: With CGP, we identified drivers/clinically actionable variants in 78.6% of the cohort (375 patients). Although 30.8% of our cohort (147 patients) was eligible to available targeted therapy, 29.5% (141 patients) were found to harbor variants imparting therapeutic resistance. The combined mutation frequency of APC, TP53, and KRAS was >50%, while KRAS constituted >90% of all RAS mutations. The mismatch repair (MMR) genes including MLH1, MLH3, MSH3, and POLE were exclusively found in colon cancers. Genomic alterations in several genes of prognostic/therapeutic significance were seen (mutations in PIK3CA, SMAD4, BRAF, and amplifications in KRAS, EGFR, and ERBB2). Of those tested, 15.8% (41 patients) of the cohort had high tumor mutation burden (TMB-H), 14% had high microsatellite instability (MSI-H) (46 patients), and 26.8% were programmed death-ligand 1 (PD-L1) positive (30 patients). Conclusion: Our study shows that CGP is an advantageous option for identifying subsets of patients eligible for various targeted therapies, thus, improving patient outcomes.

Primary Rosai-Dorfman disease of the central nervous system: A clinical, histological, and molecular appraisal.

Rosai-Dorfman disease (RDD) is characterized by clonal proliferation of S-100 positive histiocytes and variable emperipolesis. It commonly affects cervical lymph nodes. Central nervous system (CNS) involvement is extremely rare. We attempted to evaluate the Cyclin D1 expression and frequency of KRAS and BRAF mutations in the RDD involving the CNS. All patients with histopathologically diagnosed RDD involving CNS were recruited from 2011 to 2022. All cases were subjected to immunohistochemistry for CD68, CD163, S100, CD1a, GFAP, CD207, EMA, ALK, BRAFV600E, IgG4, IgG, and CyclinD1. The real-time polymerase chain reaction (RT-PCR) for hotspot mutation analysis of KRAS (exons 2, 3, and 4) and BRAF (V600E) was conducted on formalin-fixed paraffin-embedded tissue using a commercial kit (EntroGen). A total of seven cases were included. The median age was 31 years, with six men and one woman. It showed spinal cord (n = 4) and intracranial (n = 3) involvement. Histologically, all cases showed histiocyte-rich inflammation with evidence of emperipolesis. These histiocytes were positive for S100, CD68, CD163, and Cyclin D1, whereas negative for CD1a, CD207, and EMA. BRAF V600E was expressed in a single case. None of the control cases (demyelination and infarction) with histiocytic infiltrate showed Cyclin D1 expression. Four RDD cases showed increased IgG4-positive plasma cells (>10/HPF) and IgG4/IgG ratio (>40%). BRAF V600E mutation was detected in one case (14.28%), while none showed KRAS mutation. RDD involving CNS is extremely rare and diagnostically challenging. Nuclear Cyclin D1 expression along with S-100 positivity in the tumor cells is a strong diagnostic clue. BRAF and KRAS mutations are rare in CNS RDD.

Abstract 3318: GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy

Abstract Background: KRAS is the best-known oncogene but had long been considered ‘undruggable’ until the approval of the first KRAS G12C inhibitor sotorasib in 2021. However, most current clinical-stage KRAS inhibitors target the KRAS G12C mutation. Inhibitors of other KRAS mutants such as KRAS G12D, the most frequent KRAS mutation in human cancer, are needed for patients. We have developed GFH375 (VS-7375), an oral, selective KRAS G12D inhibitor targeting both “ON” (GTP-bound) and “OFF” (GDP-bound) states of KRAS proteins. The in vitro potency, selectivity, and in vivo efficacy of monotherapy and combination therapy with avutometinib, a unique RAF/MEK clamp, were evaluated in preclinical studies. Methods: Biochemical and cellular assays were used to investigate inhibition to KRAS cycling and signaling. CellTiter-Glo assay was performed to determine the effects on proliferation of tumor cell lines. Several KRAS G12D CDX tumor models were employed to study the in vivo pharmacodynamic and anti-tumor effects. Results: GFH375 inhibited both nucleotide exchange on GDP-bound KRAS G12D and interaction between GMPPNP-bound KRAS G12D with RAF1 with single-digit nanomolar IC50 values. GFH375 suppressed phosho-ERK1/2 (p-ERK) level with sub-nanomolar IC50 values and potently inhibited cell proliferation across a panel of KRAS G12D tumor cell lines. GFH375 showed high selectivity for KRAS G12D relative to non-G12D KRAS variants, KRAS wild type cells, and NRAS, HRAS, or BRAF mutated cells. Following a single oral dose, GFH375 produced deep and durable inhibition of p-ERK in KRAS G12D CDX tumors. GFH375 demonstrated dose-dependent anti-tumor activity with tumor regressions at 10 or 30 mg/kg given orally twice daily in multiple KRAS G12D PDAC and CRC CDX tumor models. GFH375 also showed strong anti-tumor efficacy in an intracranial KRAS G12D tumor model starting as low as 10 mg/kg orally twice daily. Strong synergy between GFH375 and avutometinib was observed in vitro and was validated in vivo as anti-tumor activity of GFH375 was further enhanced by avutometinib. Conclusions: GFH375 is a highly potent and selective orally active inhibitor of KRAS G12D (ON/OFF) and demonstrated promising anti-tumor activity in multiple KRAS G12D tumor models. GFH375 was also effective in an intracranial tumor model. GFH375 showed strong synergy with avutometinib in vitro and in vivo. GFH375 is currently in IND-enabling development in preparation for clinical studies of monotherapy and potentially in combination with other agents for patients with KRAS G12D mutant tumors. Citation Format: Feng Yan, Tao Jiang, Tao Liang, Lijian Cai, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaoling Lan, Xiaohui Zhang, Meng Liu, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Lili Tang, Silvia Coma, Yaofeng Cheng, Nathan Sanburn, Jonathan A. Pachter, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH375 (VS-7375): An oral, selective KRAS G12D (ON/OFF) inhibitor with potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3318.

Abstract 786: Genomic distinctions of HIV- and EBV-associated DLBCL in a diverse African cohort

Abstract Lymphoma, a leading cancer cause of death among people living with HIV (PLHIV) globally, poses a pressing public health challenge in Sub-Saharan Africa (SSA), the epicentre of the HIV epidemic. Despite its prevalence, the genetic underpinnings of lymphoma in Black Africans and PLHIV remain poorly defined, contributing to regional disparities. This study aims to elucidate the impact of HIV status and EBV on the genetic landscape, molecular subtypes and survival outcomes of diffuse large B-cell lymphoma (DLBCL) in ethnically diverse SSA patients. We established a cohort of 117 newly diagnosed DBLCL cases (55 HIV+, 21 EBV+, 62 Black Africans), with histopathologic and clinical annotation from a public teaching hospital in Cape Town, South Africa (2010-22). Tumor specimens underwent histologic review, whole exome sequencing (WES) and bulk whole transcriptome sequencing. Germline variants were filtered by paired normal WES (when available) and normal references augmented by supervised machine learning in tumor-only specimens. Aberrant somatic hypermutation (ASHM) was assessed in curated genes within paired samples. Microsatellite instability (MSI) was assessed via MSI-sensor. Canonical genetic aberrations reproduced in this cohort included recurrent SNVs in TP53, CARD11, TET2, SOCS1, MYD88, and KMT2D with differences in relative proportion compared to the NCI consortium cohort being more attributable to HIV or EBV status than ethnicity. HIV+ cases harbored frequent mutations in TET2, BRAF, and KRAS, with fewer in NFKB pathway genes including CARD11, IKBK, and BTK. ABC-type HIV+ cases manifested higher expression of genes in the JAK-STAT pathway. EBV+ cases exhibited distinct genomic features including amplifications in NOTCH1, CARD11, NFKBIE, MYC, CD274, and JAK2, as well as frequent gain-of-function (GOF) mutations in the NOTCH1 PEST domain (40% vs 3.8% q=0.04). Tumor mutation burden was consistent across ethnic and virological strata (median mutations 139-167, p>0.05). Notably, ASHM was not identified as a predominant mutational signature in either HIV-positive or negative cases, and 4 HIV+ DLBCL cases exhibited MSI, corroborated by protein-affecting mutations in mismatch repair genes. The 2-year overall survival (OS) was less than 40%, markedly inferior to published Western cohorts, even when controlled for clinical prognostic factors and treatment regimen. This study highlights genetic and biological distinctions in DLBCL due to HIV and EBV. Key findings include the absence of ASHM correlates of pathogenic driver mutations, challenging a prevailing hypothesis of HIV-associated lymphomagenesis, and the identification of MSI in HIV+DLBCL. The dismal survival of this cohort and distinct genomic features, especially in EBV+DLBCL, highlight the urgency of further biological interrogation and inclusive clinical trials for these demonstrably underserved populations. Citation Format: Katherine Antel, Aleksander Nesmelov, Meng-Xuan Wu, Charlotte Slome, Dharshnee Chetty, Raymond Kriel, Ekaterina Postovalova, Robert Redd, Martin Aryee, Delphine Armand, Lauren McGrath, Nina Xiong, Jenna Oosthuizen, Karryn Brown, Andrey Tyshevich, Svetlana Podsvirova, Felix Frenkel, Natalia Kuzkina, Jessica Scher, Cagdas Tazearslan, Ksenia Zornikova, Nikita Kotlov, Aleksander Bagaev, Nathan Fowler, Arnold Freedman, Scott Rodig, Estelle Verburgh, Mark A. Murakami. Genomic distinctions of HIV- and EBV-associated DLBCL in a diverse African cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 786.

Abstract 250: Sex differences in cancer stemness among individuals with early onset colorectal cancer

Abstract Since 1990, the overall incidence and mortality rates of late onset colorectal cancer (LOCRC ≥50 years) have been decreasing due to improvements in access to screening and available treatment options. However, the rates for early onset CRC (EOCRC ≤ 49 years) have steadily increased in men and women. Less than 30% of LOCRC patients present with late-stage tumors (clinical stages III or IV) at the time of diagnosis compared to 70% of EOCRC patients. Despite this, the cellular and molecular mechanisms underlying the incidence and stage are unclear, as are appropriate strategies for prevention of recurrence in men and women with EOCRC. Effective prognostic markers are therefore urgently needed. To address this issue, we performed a detailed bioinformatics analysis based on Gene Expression Omnibus database (GSE39582; n=585 patients), wherein we identified 7 prognostic biomarkers for EOCRC: KRAS, LGR5, CTNNB1, MYC, BMI1, CD44 and ASNS. Importantly, Kaplan Meier analysis revealed tumor onset at a younger age was significantly (p = 0.0082) associated with poorer 5-year survival in men with EOCRC only, after adjusting for KRAS status. CTNBB1 and CD44 transcript levels were higher in men with EOCRC versus men with LOCRC. Conversely, female EOCRC had a higher frequency of KRAS mutations (52.6%) compared to their LOCRC counterpart (38.8%), and an increased expression of markers of tumor stem cell and metabolic reprogramming including LGR5, MYC, BMI, and ASNS. A single CRISPR-Cas9 deletion of ASNS in HCT116 cell lines, a pre-clinical model of EOCRC, caused an inhibition of 3D spheroid formation compared to HCT116 ASNS wild type cells. In vivo study using a cell line derived R2G2 mouse xenograft model showed that ASNS disruption caused a reduction in tumor burden that was accompanied by low Lgr5 and Myc transcripts compared to the ASNS wild type group, indicating that ASNS may regulate cancer stemness. Together, this data suggests a unique molecular fingerprint for EOCRC and highlights sex-specific influences on cellular metabolism during EOCRC progression. Citation Format: Oladimeji Aladelokun, Kaelyn Sumigray, Sajid A. Khan, Caroline Johnson. Sex differences in cancer stemness among individuals with early onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 250.

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.

Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D.

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

Genetic landscape of 482 thyroid carcinomas: analysis with the national datacenter for cancer genomic medicine in Japan.

Comprehensive genomic profiling is useful for patients with Thyroid carcinoma (TC) for whom standard treatment has become refractory. We analyzed the clinical and genomic characteristics of patients with TC using the Japanese nationwide Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database. This retrospective observational study used the data obtained from the C-CAT database. Genomic information has been accumulated on representative gene mutations associated with TC. Among the 482 patients, 212 (44%) were male and 270 (56%) were female. According to histological type, 259 (54%), 46 (10%), 16 (3%), 51 (11%), and 110 (23%) patients had papillary TC (PTC), follicular TC, medullary TC, poorly differentiated TC, and anaplastic TC (ATC), respectively. Among the genomic profiling tests, FoundationOne CDx (n = 388; 80%) was the most frequently performed. The frequencies of BRAF, NRAS, HRAS, KRAS, and RET mutations were 259 (54%), 62 (13%), 13 (3%), 16 (3%), and 12 (2%), respectively. The BRAF V600E mutation (n = 257) was the predominant BRAF mutation. TERT promoter mutations, which are associated with tumor aggressiveness, were detected in 308 patients (64%). PTC was the most common histologic type of TC for which genetic profiling was performed in Japan, followed by ATC. Since the most common targetable mutation is the BRAF mutation, practical application of BRAF-targeted therapy can be an important treatment option for Japanese patients with TC.

Outcomes in patients treated with frontline immune checkpoint inhibition (ICI) for advanced NSCLC with KRAS mutations and STK11/KEAP1 comutations across PD-L1 levels

IntroductionIn patients with advanced NSCLC (aNSCLC), the impact of KRAS mutations (m) and comutations with STK11 and KEAP1 on outcomes across different PD-L1 levels remains incompletely understood. We aimed to investigate the frequency of KRAS mutations and comutations across PD-L1 levels, and the association between these mutations and survival, stratified by PD-L1 expression. MethodsWe conducted a nationwide cohort study of patients diagnosed with aNSCLC between 2016 and 2021 treated with frontline (chemo)immunotherapy, who underwent molecular genotyping including KRAS, STK11, and KEAP1. Real-world overall survival (OS) and progression-free survival (rwPFS) were estimated using Kaplan-Meier methodology. Cox multivariable regressions were used to evaluate the association between KRASm and survival across different PD-L1 strata, and to assess whether the association between KRASm and survival differed by PD-L1 level. Finally, within subgroups defined by PD-L1 expression, we used interaction terms to assess whether co-mutations with STK11 and KEAP1 moderated the association between KRAS mutation and survival. ResultsOf our 2593-patient cohort, 982 (37.9 %) were KRASm and 1611 (62.1 %) KRASwt. KRASm were enriched in the PD-L1 ≥50 % cohort (334/743, 45 %), but within patients with KRASm, co-mutations with STK11 and KEAP1 were enriched in the PD-L1 0 % cohort. KRASm was associated with significantly worse OS in the PD-L1 0 % cohort compared to the PD-L1 ≥50 % cohort (P for interaction = 0.008). On adjusted analyses stratified by PD-L1, KRASm was associated with worse survival only in the PD-L1 0 % group (OS HR 1.46, p = 0.001). KEAP1 and STK11 comutations were most strongly associated with worse OS in the PD-L1 0 % subgroup; patients with triple KRASm/KEAPm/STK11m PD-L1 0 % NSCLC experienced the worst outcomes. ConclusionsKRASm are associated with worse overall survival in PD-L1 negative NSCLC; however, this association is largely driven by comutations with STK11 and KEAP1, which are enriched in PD-L1 negative tumors.

KRAS, NRAS and BRAF Mutational Profile of Colorectal Cancer in a Series of Moroccan Patients.

The present study aimed to evaluate the frequencies of KRAS, NRAS, and BRAF mutations and their possible associations with clinicopathological features in 249 Moroccan patients with colorectal cancer (CRC). A retrospective investigation of a cohort of formalin-fixed paraffin-embedded tissues of 249 patients with CRC was screened for KRAS/NRAS/BRAF mutations using Idylla™ technology and pyrosequencing. KRAS, NRAS, and BRAF mutations were revealed in 46.6% (116/249), 5.6% (14/249), and 2.4% (6/249) of patients. KRAS exon 2 mutations were identified in 87.9% of patients (102/116). KRAS G12D and G12C were the most frequent, at 32.8% and 12.93%, respectively. Among the patients with KRAS exon 2 wild-type (wt), 27.6% (32/116) harbored additional KRAS mutations. Concurrent KRAS mutations were identified in 9.5% (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12A and Q61H), and one in codon 13 (G13D and Q61L). Among the NRAS exon 2wt patients, 64.3% (9/14) harbored additional NRAS mutations. Concurrent NRAS mutations were identified in 28.6% (4/14) of NRAS-mutant patients. Since 3.2% wt KRAS were identified with NRAS mutations, concomitant KRAS and NRAS mutations were identified in 2.4% (6/249) of patients. KRAS mutations were higher in the >50-year-old age-group (P = .031), and the tumor location was revealed to be significantly associated with KRAS mutations (P = .028) predominantly in left colon (27.5%) and colon (42.2%) locations. NRAS mutations were most prevalent in the left colon (42.8%) and in well-differentiated tumors (64.2%). Detection of KRAS mutations, particularly the G12C subtype, may be significant for patients with CRC and has possible therapeutic implications. However, rare KRAS concomitant mutations in CRC patients suggest that each individual may present distinct therapeutic responses. KRAS testing alongside the identification of other affected genes in the same patient will make the treatments even more personalized by contributing more accurately to the clinical decision process. Overall, early diagnosis using novel molecular techniques may improve the management of CRC by providing the most efficient therapies for Moroccan patients.

Frequency and oncologic outcomes of KRAS mutations in circulating tumor DNA of patients with pancreatic ductal adenocarcinoma.

636 Background: KRAS genetic alterations have been reported in more than 90% of pancreatic ductal adenocarcinoma (PDAC). The association of KRAS mutation subtypes detected in circulating tumor DNA (ct-DNA) with survival remains unclear. We investigated the rate of liquid biopsy detected KRAS mutation subtypes, and their association with overall survival (OS) in patients with PDAC. Methods: The Foundry software platform was used to retrospectively query a single-institution Electronic Health Records to identify patients with PDAC who underwent ct-DNA liquid biopsy from 2018 to 2023 and extract their oncologic outcomes. Results: Liquid biopsies were performed for 313 patients with PDAC; 58% (n=181) were diagnosed with stage IV disease. The median follow-up was 25 months, with median OS 24 months (95%CI=21-29). Overall, 52% (n=162) tested positive for KRAS mutations. Among patients with metastatic disease at diagnosis (n=181), 66.7% (n= 120) tested positive for KRAS mutations. In contrast, only 31.8% (n= 42) of patients with stage I to III disease at diagnosis (n=132) tested positive. Among patients with metastatic disease, those with KRAS positive disease had significantly shorter OS (median OS 13 vs 27 months, HR=2.7, 95%CI=1.7-4.3, P<0.0001). In patients with non-metastatic disease, the median OS was 28 vs 43 months, respectively (HR=1.7, 95%CI=0.97-2.8, P=0.064). The most frequent KRAS mutation was G12D (n=66, 40.7% of KRAS mutations) followed by G12V (n=58, 35.8%), G12R (n=15, 9.3%), and Q61H (n=10, 6.2%). Among KRAS mutations allelic subtypes, KRAS G12D and Q61 mutations had significantly worse OS with median OS 13 and 10 months respectively (P <0.001). The most frequently co-occurring mutations detected in KRAS positive patients were TP53 (n=126, 77.8%), CDKN2A (n=34, 21%), SMAD4 (n=33, 20.4%), and ARID1A (n=12, 7.4%). Examining the prognostic significance of co-occurring mutations among patients with metastatic disease, only detection of co-occurring CDKN2A mutation was associated with worse OS as compared to patients with KRAS mutations without co-occurring CDKN2A mutations (median OS 8 vs 14 months, HR=2, 95%CI=1.1-3.5, P value=0.015). Conclusions: Detection of KRAS mutation in liquid biopsies of patients with metastatic PDAC is associated with worse OS. KRAS G12D/Q61 alterations detection is associated with worse OS compared to other KRAS mutation subtypes. Ct-DNA detection of co-occurring CDKN2A mutation is associated with worse OS in patients with KRAS positive metastatic disease.

Comparison of colorectal cancer (CRC) characteristics across genetic ancestries: Implications for early cancer detection (ECD).

164 Background: Blood-based ECD has the potential to reduce mortality rates by improving strategies for, adoption of, and adherence to screening. A deeper understanding of the cancer genomic landscape across genetic ancestries will aid comparable detection performance across a broad population. We evaluated the mutational landscape in patients with CRC across genetic ancestries. Methods: 16,080 de-identified patients with stage I-IV CRC who had personalized, tumor-informed commercial ctDNA testing (Signatera) based on whole exome sequencing (WES) were included. Genetic ancestry determined via supervised local ancestry inference (pmid36042219) included African (AFR), East Asian (EAS), European (EUR), Latino (LAT), and South Asian (SAS). Patients assigned to a single ancestry were included (except SAS due to small cohort size). Somatic single nucleotide variants called from WES were used for mutational signature inference. EUR was the reference group for standard statistical tests with corrections for multiple hypothesis testing. Results: A single genomic ancestry was assigned for 16,257 (99.2%) patients (AFR n=1697, 10%; EAS n=2247, 14%; EUR n=10,526, 64%; LAT n=1291, 1%; SAS n=184, 1%). Across AFR, EAS, EUR, and LAT, approximately half of patients were male (range, 47-54%) and most were aged >50 years. Microsatellite instability (MSI) rates were significantly higher in EUR (14.5%) v AFR (11%, p<0.001), EAS (8%, p<0.001), and LAT (11%, p=0.002). Compared to EUR (range 0.027-875.1 mut/Mb), the distribution of TMB was significantly higher in EAS (0.304-906.2 mut/Mb, p<0.001) and lower in LAT (0.053-476.7 mut/Mb, p<0.001). The POLE hyper-mutator signature was seen in 1.2% of all patients and in 7.6% of all hyper-mutated cases. Patients with the POLE signature were less common in EUR (1.1%) v AFR (2%, p=0.001). There were significant differences in the frequency of driver mutations in APC, BRAF, KRAS, TP53, and PIK3CA between EUR and the other ancestry groups in both MSI and MSS tumors (Table). Median exposure levels to alkylation were higher in EAS v EUR. Conclusions: These data confirm previous results in AFR populations, and reveal novel differences in the EAS and LAT populations. Findings from this study may provide information for developing risk stratification and prevention strategies for the early detection of cancer and provide rationale for precision treatment based on ancestry. [Table: see text]

Abstract A044: Tumor-infiltrating lymphocyte density correlates with survival in pancreatic adenosquamous carcinoma

Abstract Background: Pancreatic adenosquamous carcinoma (PASC) is a rare and aggressive form of pancreatic cancer, representing only 1-5% of cases. Histologically, it is defined by the presence of at least 30% squamous differentiation mixed with ductal adenocarcinoma. No standard therapies or prognostic biomarkers have been validated to date in this understudied disease. Methods: All patients diagnosed with pancreatic cancer in the Johns Hopkins Cancer Registry from 2013 to 2018 were queried for adenosquamous histology. Archived slides of available tumor specimens were examined to quantify the percentage of squamous differentiation and tumor-infiltrating lymphocyte (TIL) density. Descriptive statistics were used to summarize clinical, pathologic, and molecular characteristics. Kaplan-Meier estimates and log-rank tests were used for survival analyses. Results: Among a total of 8,908 patients queried, 155 patients with adenosquamous histology were identified. Median overall survival (OS) was 14.2 months and 7.5 months for resectable and unresectable/metastatic disease, respectively. Molecular tumor testing revealed frequent somatic mutations in KRAS, TP53, and SMAD4 in addition to multiple DNA damage repair genes. The percentage of squamous differentiation and TIL density was correlated with survival outcomes in a subset of 28 patients with evaluable tumor specimens. No association between the percentage of squamous differentiation and OS was identified. Of note, compared to those with low TIL density (0-10%), patients with an intermediate/high TIL density (>10%) exhibited significantly improved survival (median OS 36.3 vs 14.2 months, p = 0.011). Conclusions: For the first time, intermediate/high TIL density is associated with prolonged OS in a single-institution cohort of PASC patients. Prospective validation in larger cohorts is needed to establish prognostic biomarkers that can guide therapeutic decision-making in this lethal and under-examined disease subtype. Citation Format: Saurav D. Haldar, Christopher D. Jakubowski, Su Jin Lim, Hao Wang, Richard A. Burkhart, Kelly Lafaro, William R. Burns III, Jin He, Daniel A. Laheru, Elizabeth D. Thompson, Nilofer S. Azad. Tumor-infiltrating lymphocyte density correlates with survival in pancreatic adenosquamous carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A044.

Detection of KRAS Mutations in Triple-negative Breast Cancers by Polymerase Chain Reaction

Abstract Background: Recently, it has been proven that KRAS gene mutation analysis is an additional in vitro diagnostic method for determining colorectal cancer patients who will not respond to anti-epidermal growth factor receptor (EGFR) therapy. KRAS mutation status assessment may be important in other EGFR-overexpressed cancers, such as breast cancer tumors. Only 5% of breast tumors are KRAS-mutated, yet 60% of triple-negative breast tumors express EGFR, making them potential targets for EGFR inhibitors. To provide a biological foundation for assessing anti-EGFR therapy, we aimed to examine the frequency of KRAS mutations in triple-negative breast cancer (TNBC). Materials and Methods: Forty breast cancer cases were evaluated. These 40 cases have undergone immunohistochemistry for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 markers which exhibited a negative reaction and were ascertained as triple negative. The mutation status of KRAS was determined by real-time polymerase chain reaction (RT-PCR) to detect wild or mutant types of KRAS. Results: In our study, we observed that, out of the total 40 cases of TNBCs, 14 (35%) cases were of wild type and 26 (65%) cases were mutant type detected by RT-PCR. Conclusion: According to our study, KRAS mutations were detected in TNBCs, and EGFR inhibitors may be effective in the treatment of these tumors, which overexpress EGFR in around 65% of cases.

Genomic Landscape of Endometrial, Ovarian, and Cervical Cancers in Japan from the Database in the Center for Cancer Genomics and Advanced Therapeutics.

This study aimed to comprehensively clarify the genomic landscape and its association with tumor mutational burden-high (TMB-H, ≥10 mut/Mb) and microsatellite instability-high (MSI-H) in endometrial, cervical, and ovarian cancers. We obtained genomic datasets of a comprehensive genomic profiling test, FoundationOne® CDx, with clinical information using the "Center for Cancer Genomics and Advanced Therapeutics" (C-CAT) database in Japan. Patients can undergo the tests only after standardized treatments under universal health insurance coverage. Endometrial cancers were characterized by a high frequency of TMB-H and MSI-H, especially in endometrioid carcinomas. The lower ratio of POLE exonuclease mutations and the higher ratio of TP53 mutations compared to previous reports suggested the prognostic effects of the molecular subtypes. Among the 839 cervical cancer samples, frequent mutations of KRAS, TP53, PIK3CA, STK11, CDKN2A, and ERBB2 were observed in adenocarcinomas, whereas the ratio of TMB-H was significantly higher in squamous cell carcinomas. Among the 1606 ovarian cancer samples, genomic profiling of serous, clear cell, endometrioid, and mucinous carcinomas was characterized. Pathogenic mutations in the POLE exonuclease domain were associated with high TMB, and the mutation ratio was low in both cervical and ovarian cancers. The C-CAT database is useful for determining the mutational landscape of each cancer type and histological subtype. As the dataset is exclusively collected from patients after the standardized treatments, the information on "druggable" alterations highlights the unmet needs for drug development in major gynecological cancers.

The landscape of actionable genomic alterations in lung adenocarcinomas in India.

Lung adenocarcinoma (LUAD), the most prevalent form of non-small cell lung cancer (NSCLC), remains a leading cause of cancer-related death globally, including in India, with a 5-year survival rate below 10%. Despite these grim statistics, recent advances in the use of next-generation sequencing (NGS) for identifying genetic alterations and the emergence of targeted therapies have opened new possibilities for personalized treatment based on distinct molecular signatures. To understand the molecular pattern of NSCLC, a retrospective study was conducted with 53 Indian LUAD patient samples, using a targeted NGS panel of 46 cancer-relevant oncogenes to identify clinically relevant variants. Pathogenic or likely pathogenic variants were detected in 94% of the 53 cases. Non-synonymous mutations, rearrangements, copy number alterations, insertions, and deletions of functional relevance were observed in 31 out of 46 genes. The most frequently mutated genes included TP53 (52.8%) and EGFR (50.9%), followed by RET, PIK3CA and ERBB2; some patients had multiple alterations in the same gene. Gender-based enrichment analysis indicated that ALK and IDH2 alterations were more prevalent in females, while TP53 and PTEN were more common in males. No significant correlation was found between mutations and other clinicopathological attributes, such as age, stage, and subtype. A higher prevalence of EGFR, RET, PIK3CA, ERBB2 and ALK mutations were observed compared to previous LUAD genetic studies coupled with a lower frequency of KRAS mutations. Clinically actionable variants were annotated using OncoKB and categorized into the four therapeutic levels based on their clinical evidence. Seventy-nine percent of cases had at least one clinically actionable alteration. Most patients (39.6%) had the highest level of actionability (Level 1) wherein an FDA-approved drug is available specifically for the observed mutation in lung cancer patients. EGFR Exon19 in-frame deletions and EGFR L858R were the most frequent among targetable variants (20.7%). These findings emphasize the importance of a selective NGS panel in enabling personalized medicine approaches by identifying actionable molecular alterations and informing the choice of targeted therapy for more effective treatment options in Indian NSCLC patients.