KRAS Alterations Research Articles

Comparison of mutational landscape in primary and metastatic pancreatic tumors.

781 Background: Genomic profiling has brought to the forefront the clinical relevance of intratumoral heterogeneity. Oncogenic KRAS mutation is the most common driver event in pancreatic ductal adenocarcinoma (PDAC), but little is known about how KRAS alteration may cooperate with other genetic lesions to support dissemination and growth of metastatic disease. Therefore, an improved understanding of molecular events affecting the clinical course of KRAS -altered PDAC is needed to identify biomarkers of early disease and expand the repertoire of targeted therapies. Methods: We retrospectively analyzed tissue samples from PDAC patients using the OncoExTra tumor-normal whole exome and whole transcriptome test between April 2018 and May 2024. The prevalence of actionable somatic alterations was determined overall and by specimen location (primary vs. metastatic). Fisher’s exact test with FDR-based correction for multiple testing was used to identify co-alterations with KRAS and 19 DNA damage response (DDR) gene alterations. Results: PDAC samples from 434 patients (51.2% female; mean age 65.4±10.9 years) were analyzed. KRAS alterations were detected in 369 (85.0%) samples: 198 (45.6%) primary and 239 (55.1%) metastatic. KRAS G12D (32.7%) and KRAS G12V (29.5%) were the most common KRAS mutations. The frequency of KRAS alterations was 85.9% and 84.5% in primary and metastatic samples, respectively. TP53 alterations occurred in 68.2% samples overall and in 64.6% and 71.1% of primary and metastatic samples, respectively. Examination of co-alterations in KRAS -altered versus KRAS -WT samples uncovered enrichment of TP53 alterations in KRAS -altered (277 samples, 75.1%) compared to KRAS -WT samples (19 samples, 29.2%) ( p <0.001). Also, BRAF mutations and SND1:BRAF fusions exclusively occurred in metastatic KRAS -WT samples, although the number of events was small ( n =4 and n =2, respectively). We also found that MTAP was significantly co-altered in metastatic KRAS -WT samples (n=6, 16.2%) compared to KRAS -altered samples (6 samples, 3.0%) ( p =0.004). A total of 21.9% of all PDAC samples had an alteration in at least 1 DDR gene. No associations were observed between KRAS alteration status and alteration in DDR genes. However, TP53 alterations were more frequent in non-DDR-altered samples ( p <0.001), while CDKN2B alterations were enriched in DDR-altered samples ( p =0.025). Conclusions: We observed expected rates of alterations in known PDAC driver genes and uncovered higher co-occurrence of KRAS and TP53 alteration in metastatic samples. Additionally, TP53 alterations were less frequent in DDR-altered samples. Our findings suggest that a number of therapy approaches including BRAF, WEE1, PARP, and KRAS inhibitors as well as epigenetic modulators, alone or in combination, could improve clinical outcomes in PDACs with specific mutational profiles.

Relationship of circulating tumor DNA (ctDNA) tumor fraction with prognosis in patients with metastatic pancreatobiliary cancer.

763 Background: Carcinomas of the pancreas and biliary tract are among the most lethal cancers. Currently, it is very difficult to predict individual patient outcomes with only general prognostic information available. Emerging evidence shows that baseline ctDNA tumor fraction (TF) is a prognostic factor for many tumor types, including metastatic pancreatic ductal adenocarcinoma (mPDAC). With an additional year of follow-up, we refresh our prior study of the prognostic value of ctDNA TF in mPDAC and extend the analysis to biliary tract cancers (BTC). Methods: A genomic dataset comprising patients who underwent ctDNA testing using FoundationOne Liquid or FoundationOne Liquid CDx as part of routine care was used to determine the distribution of ctDNA TF across pancreatobiliary cancer subtypes. A subset of patients was also included in the nationwide (~280 US cancer clinics, ~800 sites of care) de-identified Flatiron Health-Foundation Medicine clinico-genomic database, which was used separately for all other analyses. Clinical data were derived from the electronic health record. Real-world overall survival (rwOS) was evaluated by ctDNA TF while controlling for relevant covariates. In parallel, ctDNA TF cutoffs of ≥1% and ≥10% were evaluated. Exploratory analysis of trichotomized ctDNA TF at 1% and 10% was also performed. Results: In 8817 patients with pancreatobiliary carcinoma, 46.1% of PDAC and 41.9% of extra-hepatic cholangiocarcinoma patients had detectable levels of ctDNA TF, while patients with gallbladder carcinomas had the highest levels of ctDNA TF (median 0.8%). 470 patients with mPDAC and 89 patients with BTC were included in the outcomes analysis. High ctDNA TF was associated with clinical features linked to poor prognosis in mPDAC: Stage IV at diagnosis, pretherapy opioid prescriptions, high neutrophil-to-lymphocyte ratio and CA19-9 at baseline, and alterations in KRAS , TP53 , CDKN2A , or SMAD4 . In BTC, high ctDNA TF was associated with Stage IV at diagnosis and cholangiocarcinoma histology. Higher ctDNA TF in mPDAC was associated with significantly reduced rwOS for both cutoffs (1%: HR 1.55 [1.25-1.93], P < 0.001; 10%: HR 1.85 [1.42-2.41], P < 0.001). When ctDNA TF was trichotomized, higher ctDNA TF groups had reduced rwOS stepwise. Similar trends were observed in patients with BTC, but results were not statistically significant. Conclusions: ctDNA from pancreatobiliary carcinomas can be frequently detected and characterized in liquid biopsy while also assessing key genomic biomarkers. ctDNA TF was a prognostic biomarker for mPDAC, but not for BTC in this cohort. Detection of ctDNA associated with other clinical factors linked to poor outcomes. Cohorts given uniform treatment could possibly help further evaluate the ability of ctDNA TF to identify patients with more aggressive disease and inform future studies to personalize therapeutic decision-making.

TP53 Y220C mutations in pancreatic ductal adenocarcinoma (PDAC): A genomic landscape study.

761 Background: A novel targeted therapy designed to restore wild-type function to the p53 Y220C mutant protein has demonstrated promising activity in an early-phase trial (NCT04585750). While the TP53 Y220C mutation is found in < 1% of all solid malignancies, its prevalence is enriched in certain cancers including PDAC. Here we report results of comprehensive genomic profiling (CGP) in a cohort of patients with advanced PDAC, with a focus on TP53 Y220C mutations. Methods: A total of 27,377 cases of clinically advanced PDAC underwent hybrid capture-based CGP to assess all classes of genomic alterations (GAs). Cases were sequenced to a mean coverage depth of 650X. Microsatellite instability (MSI) status and tumor mutation burden (TMB) were determined using sequencing data; PD-L1 expression was measured by immunohistochemistry (Dako 22C3, tumor proportion score [TPS]). Results: TP53 Y220C alterations were identified in 488 (1.8%) PDAC patients (Y220C+). In comparison to PDAC patients lacking the TP53 Y220C alteration (Y220C-), Y220C+ patients were of similar age, but were more likely to be female (52.9% vs 46.8%; p = 0.0008) and harbor KRAS mutations (96.7% vs 92.7%; p = 0.007). Meanwhile, alterations in ATM were more frequent in the Y220C- group (4.0% vs 1.4%; p = 0.0008). Frequencies of other GAs were similar between Y220C+ and Y220C- groups, including alterations in the homologous recombination defect-associated genes BRCA1/2 and RAD21 , as well as ERBB2 , BRAF , MTAP and PIK3CA . MSI-high status and elevated TMB (≥ 10 mutations/Mb) were extremely uncommon in both groups. PD-L1 expression was similar in the Y220C+ and Y220C- groups, with positive PD-L1 expression (TPS ≥ 1) observed in 31.5% and 34.9% of patients, respectively. Conclusions: The potentially targetable TP53 Y220C mutation was identified in close to 2% of clinically advanced PDAC cases. Those with TP53 Y220C mutations were more likely to be female and harbor KRAS mutations, and less likely to harbor ATM mutations. However, TP53 Y220C mutations were not associated with other distinct genomic characteristics. These findings are consistent with previous reports of interactions between TP53 and KRAS alterations in PDAC. Given the emergence of drugs aimed at restoring p53 function in cases with the Y220C mutation, further study of this alteration in PDAC is warranted. Age, number of genomic alterations (GA) per tumor, and alteration frequency of select commonly altered genes based on TP53 Y220C status. TP53 Y220C+ (n = 488) TP53 Y220C- (n = 26,889) P Value Median Age, years (range) 66 (36-89+) 66 (21-89+) NS GA/tumor 5.2 5.0 NS KRAS 96.7% 92.7% 0.007 CDKN2A 60.1% 56.6% NS CDKN2B 28.6% 29.5% NS MTAP 24.4% 23.8% NS ARID1A 7.8% 8.7% NS ATM 1.4% 4.0% 0.0008 BRCA2 2.3% 3.1% NS NS = non-significant.

MEK inhibitor-based therapy in metastatic pancreatic adenocarcinoma with KRAS G12D or KRAS G12V alteration.

726 Background: Up to 95% of pancreatic ductal adenocarcinomas (PDAC) are driven by oncogenic alterations in the KRAS gene, with KRAS G12D (40%) and KRAS G12V (30%) mutations being the most common. However, in most cases, KRAS alterations are accompanied by alterations in other tumor suppressors such as TP53 , CDKN2A/B , and SMAD4 . Therefore, targeting KRAS alone may not be sufficient for effective treatment of PDAC . As part of the MCW-Master-PREDICT (NCT05802069) observational study, we evaluated the effectiveness of MEK inhibitor (MEK-inh)-based therapy, tailored to the unique molecular alterations of each patient’s tumor, in treating metastatic PDAC (mPDAC) with KRAS G12D or KRAS G12V alterations. Methods: From April 2022 to December 2023, 19 patients ( KRAS G12D=10 (53%), KRAS G12V=9 (47%)) were treated with MEK-inh-based therapy. Comprehensive genomic profiling was performed using Tempus xT (648 genes) in 15 patients (79%), FoundationOne (324 genes) in 4 patients (21%), and STRATA (429 genes) in 1 patient. The genomic characteristics, matched therapies, and treatment outcomes are provided (Table). Results: Of the 19 patients, 11 (58%) were female. The median age at the initiation of MEK-inh-based therapy was 67 years. Six (32%) patients received MEK-inh-based therapy as first or second-line treatment, while 13 (68%) patients were treated in the third to fifth-line. Among the 19 patients, the median overall survival (mOS) and median progression-free survival (mPFS) from the initiation of MEK-inh-based therapy were 5 and 2 months, respectively. The mOS from the time of MEK-inh-based therapy was 5.1 months for KRAS G12D, and 4.7 months for KRAS G12V (P=0.87). The mPFS from the time of MEK-inh-based therapy was 2.3 months for KRAS G12D and 1.4 months for KRAS G12V (P=0.12). Among all 19 patients, 4 (21%) patients had mPFS above 4 months ( KRAS G12V=1, KRAS G12D=3; 4.1, 4.3, 4.7, 9.9 months). Conclusions: The efficacy of MEK-inh-based therapy was limited in the late-line treatment of mPDAC for patients with KRAS G12D and KRAS G12V mutations. The potential role of MEK inhibitors in earlier lines of therapy, and their combination with KRAS inhibitors, requires further prospective evaluation. Tumor alterations and matched therapies. Altered gene KRAS G12V(N=9) KRAS G12D(N=10) Patients matched(N=19) Type of matched therapy KRAS G12D/V 9 (100%) 10 (100%) 19 (100%) MEK inhibitor TP53 7 (78%) 8 (80%) 8 (42%) VEGF/VEGR inhibitor* CDKN2A/B 8 (89%) 5 (50%) 9 (47%) CDK4/6 inhibitor** SMAD4 3 (33%) 4 (40%) 4 (21%) MEK inhibitor + (EGFR or pan-HER inhibitor) *** *PMID: 27466356; **PMID: 33472910; ***PMID: 36127339, PMID: 24625091. N= number of patients.

Landscape of metastatic colorectal cancer (CRC) using comprehensive circulating tumor DNA (ctDNA) next-generation sequencing (NGS) in India: Expanding beyond RAS and RAF.

49 Background: Traditional testing in mCRC primarily focuses on RAS and RAF mutations. However, a broader range of emerging biomarkers, collectively termed the "Pressing panel" (PP), identifies patients with RAS/RAF wild-type (wt) tumors who may particularly benefit from anti-EGFR therapy. Comprehensive liquid biopsies provide a non-invasive method for identifying traditional and emerging biomarkers. Limited genomic data, including the prevalence of PP alterations (alts), is available for mCRC patients in India tested by ctDNA NGS. Methods: We reviewed the results of Guardant360 (Guardant Health, Inc.) ordered for patients with mCRC as part of routine clinical practice in India through July 2024. NGS analyzed plasma samples at a CLIA-certified, CAP-accredited central laboratory. Guardant360 analyzes ctDNA for mutations (mts), insertions and deletions, amplifications (amps), and fusions in up to 83 genes, including PP genes, i.e., KRAS, NRAS, BRAF, ERBB2, PIK3CA, PTEN, AKT1, ALK, ROS1, NTRK, RET, and MSI status. The Indian cohort's prevalence of PP mts was compared to that from other countries in Asia and Middle East (AME). Timing of testing relative to line of therapy was unknown. Results: Among 145 samples from India, ctDNA was detected in 92% (n=134). Median age was 59 years, with women comprising 40%. The median turnaround time from blood collection to result was 7 days. The mean mts count per sample was 5, and the median variant allele frequency was 1.5%. Frequent mts were in APC (53%), KRAS (56%), and SMAD4 (13%), while amps of EGFR (16%) and FGFR1 (9%) were common. Clinically relevant fusions were observed in 3% of patients. KRAS, NRAS, and/or BRAF V600E alterations were found in 63% of both India and AME samples. PP alts were identified in 24% and 27% of Indian and AME patients, respectively. Most frequent PP alts included PIK3CA (15%), PTEN (3%), and MET amp (2%). The prevalence of most of the PP alts was similar between the Indian and AME cohorts (Table), with minor differences observed in PIK3CA (15% vs 18%) and MSI-High (3% vs 1%). In patients with RAS/RAF wild-type lacking EGFR-amp, 9.5% (India) and 18% (AME) harbored at least one PP alt. Conclusions: This study highlights the role of liquid CGP for characterizing the mutational landscape and identifying PP mts for patients from India with advanced CRC. Pressing panel alterations. PP Alteration INDIA % (n=134) AME % (n=720) ERBB2 Amp 1% 2% MET Amp 2% 3% ALK Fusion 0% 0% ROS1 Fusion 0% 0% NTRK1 Fusion 1% 0% RET Fusion 0% 1% ERBB2 Mut 1% 2% PIK3CA Mut 15% 18% PTEN Mut 3% 3% AKT1 Mut 0% 1% MSI-High 3% 1%

MEK inhibitor-based therapy in metastatic pancreatic adenocarcinoma with KRAS G12R alteration.

725 Background: Pancreatic ductal adenocarcinoma (PDAC) is largely driven by oncogenic alterations in the KRAS , TP53 , CDKN2A/B , and SMAD4 genes. KRAS G12R accounts for nearly 20% of KRAS alterations in PDAC, is biologically unique, and offers the potential for improved response to MEK inhibitor (MEK-inh)-based therapy. As part of the MCW-Master-PREDICT (NCT05802069) observational study, we evaluated the efficacy of MEK-inh-based therapy matched to the unique molecular alterations in the tumor of each patient as recommended by the molecular tumor board. Methods: From March 2022 to December 2023, 8 patients with metastatic PDAC were treated with MEK-inh-based therapy. Molecular profiling was performed by Tempus (648 genes) in 7 patients, and FoundationOne (324 genes) in 1 patient. Description of molecular alterations, matched therapies, and efficacy of treatment are provided. Results: The median age of patients starting MEK inhibitor-based therapy was 69 years, with 62% of them being female. The molecular characteristics and matched therapies are summarized (Table). MEK-inh-based therapy was administered to 3 (37.5%) patients as a first or second-line treatment, and to 5 (62.5%) patients as a third to fifth-line treatment for metastatic PDAC. The overall survival and progression free survival (PFS) on MEK-inh-based therapy was 8.4 and 4.9 months, respectively. Four patients had PFS beyond 4 months (4.9, 5.5, 5.6 and 6.9 months). Conclusions: Individualized MEK-inh-based therapy demonstrated efficacy in late-line treatment of metastatic PDAC with KRAS G12R alterations. Using this approach earlier in the treatment course and in combination with RAS inhibitors or cytotoxic systemic therapies may further enhance outcomes. Tumor alterations and matched therapies. Gene altered Number of patients (%) Number of patients matched (%) Drug family matched KRAS G12R 8 (100) 8/8 (100) MEK inhibitor TP53 7 (87.5) 4/7 (57) VEGF/VEGR inhibitor* CDKN2A 4 (50) 4/4 (100) CDK4/6 inhibitor** SMAD4 2 (25) 2/2 (100) MEK inhibitor + (EGFR or Pan-HER inhibitor)*** *PMID: 27466356; **PMID: 33472910; ***PMID: 36127339, PMID: 24625091.

Precision Medicine in Colorectal Cancer: Targeted Therapies and Biomarker Insights.

The current review delves into the transformative role of precision medicine in addressing Colorectal Cancer [CRC], a pressing global health challenge. It examines closely signalling pathways, genetic and epigenetic modifications, and microsatellite in-stability. The primary focus is on elucidating biomarkers revolutionizing CRC diagnosis and treatment. Genetic biomarkers encompass non-coding RNA, epigenetic markers, TP53 mutations, and KRAS, NRAS, and BRAF gene alterations. Targeted therapies, including anti-EGFR, anti-VEGF, immune checkpoint inhibitors, and HER2-targeted treatments, are explored along with their mechanisms and clinical applications. Additionally, we highlight the importance of utilizing personalized treatment strategies by employing molecular pro-filing and genetic testing. These approaches facilitate the identification of appropriate pa-tients for targeted therapies. Clinical trials supporting these treatments are presented, em-phasizing response rates and survival outcomes. Detailed exploration of resistance mecha-nisms to targeted therapies and strategies to overcome resistance is also provided, paving the way for more effective regimens. Directions for future research in precision medicine, such as biomarkers, combinations, and liquid biopsy in the oncology field, are described. However, the precise application of precision medicine in CRC comes with questions such as costs, considerations of ethical factors, and the need to sensitize patients. Nonetheless, based on a meticulous analysis of several aspects, precision medicine finds itself equipped with the ability to enhance the patients' prognosis ,thereby, lessening the global oncologic burden of CRC and provide important information to the clinician-scientist and policy-maker that might benefit from the ongoing development of precision medicine.

Alternative driver pathways in peripheral nerve sheath tumors - including DICER1 and/or KRAS alterations.

DICER1-associated sarcoma is an emerging entity, defined by either somatic or germline dicer 1, ribonuclease III (DICER1) mutations and sharing characteristic morphologic features irrespective of the site of origin. In addition to the DICER1 driver mutation, concurrent genomic alterations, including tumor protein 53 (TP53) inactivation and RAS pathway activation, are frequently detected. Tumors that morphologically resemble malignant peripheral nerve sheath tumor (MPNST) have rarely been reported among DICER1 sarcomas and often pose diagnostic challenges. This study was prompted by a case involving morphologic features of MPNST, which harbored co-existing DICER1 and hotspot KRAS mutations. Hence, we investigated the incidence of these alterations in PNST from our molecular database compared to the genomic and morphologic spectrum of DICER1-mutant sarcomas. In total, we identified three cases diagnosed as MPNST with co-existing DICER1, ATRX chromatin remodeler (ATRX), and KRAS G12V/A alterations occurring in brain, cerebellopontine angle, and intra-abdominal sites. Two additional cases each of MPNSTs and neurofibromas were identified with hotspot KRAS mutations. All five MPNSTs lacked canonical neurofibromin 1 (NF1)/neurofibromin 2 (NF2) alterations, displaying a classic morphologic appearance with fascicular monomorphic spindle cells and followed a diverse clinical behavior. Among the 38 DICER1-associated sarcomas in our database, eight (21%) had secondary KRAS hotspot mutations, all composed of monomorphic spindle and/or round cells, including three with an MPNST-like histology. In contrast, all 10 (26%) DICER1-mutant sarcomas with TP53 mutations showed a pleomorphic phenotype. The DNA-based methylation profile of our index case clustered within the group of sarcomas with DICER1 alterations. Our results highlight a small subset of MPNST associated with DICER1 and/or KRAS mutations. However, their relationship with conventional MPNST remains to be determined in larger studies. © 2025 The Pathological Society of Great Britain and Ireland.

Subclonal TP53 and KRAS variants combined with poor treatment response identify ultra-high-risk pediatric T-ALL patients.

Variations in the TP53 and KRAS genes indicate a particularly adverse prognosis in relapsed pediatric T-ALL. We hypothesized that these variations might be subclonally present at disease onset and contribute to relapse risk. To test this, we examined two cohorts of children diagnosed with T-ALL: one with 81 patients who relapsed and 79 matched non-relapsing controls, and another with 226 consecutive patients, 30 of whom relapsed. In cohort 1, targeted sequencing revealed TP53 clonal and subclonal variants in 6 of 81 relapsing patients but none in the non-relapsing group (p=0.014). KRAS alterations were found in 9 of 81 relapsing patients compared to 2 of 79 non-relapsing patients (p=0.032). Survival analysis showed that none of the relapsed patients with TP53 and/or KRAS alterations survived, whereas 19 of 67 relapsed patients without such variants did with a minimum follow-up time of 3 years (p=0.023). In cohort 2, none of the relapsing patients but 10 of 196 non-relapsing patients carried TP53 or KRAS variants, indicating that mutation status alone does not predict poor prognosis. All ten non-relapsing patients with mutations had a favorable early treatment response. Among the total cohort of 386 patients, 188 showed poor treatment response of whom 69 relapsed. Nine of these poor responders harbored TP53 or KRAS variants. In conclusion, subclonal TP53 and KRAS alterations identified at the time of initial diagnosis, along with a poor treatment response, characterize a subset of children with T-ALL who face a dismal prognosis and may benefit from alternative treatment approaches.

KRAS Mutation Status and Treatment Outcomes in Patients With Metastatic Pancreatic Adenocarcinoma

Despite the high prevalence of KRAS alterations in pancreatic ductal adenocarcinoma (PDAC), the clinical impact of common KRAS mutations with different cytotoxic regimens is unknown. This evidence is important to inform current treatment and provide a benchmark for emergent targeted KRAS therapies in metastatic PDAC. To assess the clinical implications of common KRAS G12 mutations in PDAC and to compare outcomes of standard-of-care multiagent therapies across these common mutations. This retrospective cohort study obtained deidentified clinical data for 5382 patients from a nationwide (US-based) clinicogenomic database. The deidentified data originated from approximately 280 US cancer clinics (approximately 800 sites of care). Patients diagnosed with metastatic PDAC from February 9, 2010, to September 20, 2022, and with sufficient follow-up and treatment data were included. Median overall survival (OS) and time to next treatment (TTNT) were calculated for each KRAS mutation group. Hazard ratios (HRs) were generated using multivariate Cox proportional hazards models for KRAS mutations and mutation-treatment combinations. A total of 2433 patients with PDAC were included in the analysis (mean age at first treatment, 67.0 [range, 66.0-68.0] years; 1340 male [55.1%]). Among 2023 patients with KRAS mutations, those with G12R had the longest median TTNT (6.0 [95% CI, 5.2-6.6] months) and the longest median OS (13.2 [95% CI, 10.6-15.2] months). Patients with KRAS G12D and G12V mutations had a significantly higher risk of disease progression (HRs, 1.15; [95% CI, 1.04-1.29; P = .009] and 1.16 [95% CI, 1.04-1.30; P = .01], respectively) and death (HRs, 1.29 [95% CI, 1.15-1.45; P < .001] and 1.23 [95% CI, 1.09-1.39; P < .001], respectively) compared with KRAS wild type. The FOLFIRINOX regimen (fluorouracil, irinotecan, oxaliplatin, and leucovorin) had a significantly lower risk of treatment progression and death than gemcitabine with (HRs, 1.19 [95% CI, [1.09-1.29; P < .001] and 1.18 [95% CI, 1.07-1.29; P < .001], respectively) or without (HRs, 1.37 [95% CI, 1.11-1.69; P = .003] and 1.41 [95% CI 1.13-1.75; P = .002], respectively) nab-paclitaxel across all patients. In this cohort study of 2433 patients with PDAC, KRAS G12D and G12V mutations were associated with worse patient outcomes compared with KRAS wild type. KRAS G12R was associated with more favorable patient outcomes, and FOLFIRINOX was associated with better patient outcomes than gemcitabine-based therapies. These findings highlight the need for more effective systemic therapies for these groups of patients.

Pan-KRAS inhibitors BI-2493 and BI-2865 display potent anti-tumor activity in tumors with KRAS wild-type allele amplification.

KRASG12C selective inhibitors, such as sotorasib and adagrasib, have raised hopes of targeting other KRAS mutant alleles in cancer patients. We report that KRAS wild-type amplified tumor models are sensitive to treatment with the small molecule KRAS inhibitors BI-2493 and BI-2865. These pan-KRAS inhibitors directly target the "OFF" state of KRAS and result in potent anti-tumor activity in pre-clinical models of cancers driven by KRAS mutant proteins. Here, we used the high-throughput cellular viability PRISM assay to assess the anti-proliferative activity of BI-2493 in a 900+ cancer cell line panel, expanding on our previous work. KRAS wild-type amplified cancer cell lines, with a copy number >7, were identified as the most sensitive, across cell lines with any KRAS alterations, to our pan-KRAS inhibitors. Importantly, our data suggest that a KRAS "OFF" inhibitor is better suited to treat KRAS wild-type amplified tumors than a KRAS "ON" inhibitor. KRAS wild-type amplification is common in patients with gastroesophageal cancers where it has been shown to act as a unique cancer driver with little overlap to other actionable mutations. The pan-KRAS inhibitors BI-2493 and BI-2865 show potent anti-tumor activity in vitro and in vivo in KRAS wild-type amplified cell lines from this and other tumor types. In conclusion, this is the first study to demonstrate that direct pharmacological inhibition of KRAS shows anti-tumor activity in preclinical models of cancer with KRAS wild-type amplification, suggesting a novel therapeutic concept for patients with cancers bearing this KRAS alteration.

PATH-11. TECTAL GLIOMA: CLINICAL, PATHOLOGICAL, AND MOLECULAR FEATURES

Abstract Tectal glioma (TG) is a rare lower-grade glioma (LrGG) that occurs in the tectum, mainly affecting children. The clinical course is usually indolent, with common symptoms including headache, gait disturbance, and ataxia resulting from obstructive hydrocephalus. TG shares pathological similarities with pilocytic astrocytoma (PA), but recent genetic analyses have revealed distinct features, such as alterations in KRAS and BRAF. Here, we report the clinical, radiological, pathological, and molecular characteristics of TG cases in our hospital. We conducted a retrospective review of cases clinically diagnosed as TG and surgically treated at our institute between January 2005 and March 2023. Molecular analysis, including genome-wide DNA methylation profiling, was performed. Six cases were identified and the median age at diagnosis was 30.5 years (range: 6–45 years). The mean tumor diameter was 22.8 mm (range: 14.7–33.2 mm), with obstructive hydrocephalus observed in all cases. Contrast enhancement of the tumor was noted in four cases and cyst formation occurred in two cases. Four patients underwent biopsy or biopsy with endoscopic third ventriculostomy, and two patients underwent tumor resection. Postoperatively, three patients received radiotherapy and two were treated with chemotherapy. The median overall survival was 6.2 years (range: 1.4–7.3 years), and the median progression-free survival was 3.4 years (range: 1.4–7.3 years). Histological diagnoses included three cases of PA, one case of astrocytoma, and two cases of high-grade glioma. Among the three patients who underwent molecular evaluation, including genome-wide methylation analysis, two had KRAS mutation and one had H3-3A K27M mutation. Our results demonstrate the diverse histological and molecular characteristics of TG distinct from other LrGGs. Given the heterogeneous pathological background and the risk of pathological progression in TG, we emphasize the importance of comprehensive diagnosis, including molecular evaluation.

PATH-51. CENTRAL NERVOUS SYSTEM METASTASES ARE GENOMICALLY DIVERGENT FROM THEIR RESPECTIVE EXTRACRANIAL SITES

Abstract INTRODUCTION Next-generation sequencing (NGS) has become standard of care in establishing the diagnosis and guiding treatment of cancers. Our understanding of genomic evolution of brain metastases (BM) and leptomeningeal disease (LMD) from solid tumor malignancies remains limited. Here we present updated data from our pilot study. METHODS We prospectively collected clinical data and NGS of seventeen patients undergoing craniotomy for symptomatic BM from solid tumor malignancies or cerebrospinal fluid (CSF) sampling for LMD. Matched systemic and CNS analyses were performed using a research-based NGS assay. When extracranial disease tissue was unavailable, liquid NGS served as a surrogate. RESULTS CNS metastases were present at initial diagnosis in 10/17 and 11/17 patients had not received systemic therapy prior to CNS tissue sampling. The predominant cancer type was NSCLC (n=11), followed by breast cancer (n=3), ovarian high grade serous carcinoma (n=1), GEJ adenocarcinoma (n=1), and basosquamous carcinoma of skin (n=1). The mean tumor mutational burden in extracranial samples was 3.65 (SD 3.34; SEM 0.97), in contrast to CNS samples 21.63 (SD 26.47; SEM 7.64). Concordant mutations in matched sampling had an increase in CNS variant allele frequency (VAF) (27.8% vs 17.3%; arithmetic difference = 10.5, p=0.054) and CNS gene copy number alterations (CNA) (2.4 copies vs 0.8 copies; arithmetic difference = 1.6, p=0.171). Known driver alterations in EGFR, KRAS and PIK3CA were retained. LMD was present in 4/17 patients, with two present at enrollment and two developing during the study. CONCLUSIONS Matched sequencing reveals CNS metastases to harbor a significantly higher TMB, retention of primary driver alterations with a trend towards higher VAF and CNA of concordant mutations. While limited by small sample size, these data indicate genetic evolution in CNS metastases regardless of prior treatment status.

Tectal glioma: clinical, radiological, and pathological features, and the importance of molecular analysis.

Tectal glioma (TG) is a rare lower grade glioma (LrGG) that occurs in the tectum, mainly affecting children. TG shares pathological similarities with pilocytic astrocytoma (PA), but recent genetic analyses have revealed distinct features, such as alterations in KRAS and BRAF. We conducted a retrospective review of cases clinically diagnosed as TG and treated at our institute between January 2005 and March 2023. Six cases were identified and the median age was 30.5years. Four patients underwent biopsy and two patients underwent tumor resection. Histological diagnoses included three cases of PA, one case of astrocytoma, and two cases of high-grade glioma. The integrated diagnosis, according to the fifth edition of the World Health Organization Classification of Tumours of the central nervous system, included two cases of PA and one case each of diffuse high-grade glioma; diffuse midline glioma H3 K27-altered; glioblastoma; and circumscribed astrocytic glioma. Among the three patients who underwent molecular evaluation, two had KRAS mutation and one had H3-3A K27M mutation. Our results demonstrate the diverse histological and molecular characteristics of TG distinct from other LrGGs. Given the heterogeneous pathological background and the risk of pathological progression in TG, we emphasize the importance of comprehensive diagnosis, including molecular evaluation.

Therapeutic vulnerabilities and pan-cancer landscape of BRAF class III mutations in epithelial solid tumors

BackgroundKinase-impaired class III BRAF mutations have recently received attention as a possible prognostic factor and therapeutic target. Class III BRAF variants differ from class I and class II mutations in terms of mechanism of pathway activation and therapeutic vulnerabilities. Genomic landscape analyses of tumors in large real-world cohorts represent a great opportunity to further characterize tumor-related molecular events and treatment vulnerabilities, however, such data is not yet available for tumors with BRAF class III mutations.MethodsWe investigated the pan-cancer genomic landscape of BRAF class III mutations in 376,302 patients. Patients had comprehensive genomic profiling either by FoundationOne® or FoundationOne®CDx from formalin-fixed, paraffin embedded tissue biopsies. 2 patient cases that harbored BRAF class III mutations who demonstrated dramatic response to anti-EGFR treatment were presented.ResultsBRAF class III mutations are likely to co-occur with RAF1, NRAS and HRAS alterations, while concomitant KRAS alterations were rare. Moreover, we found that alterations that predict resistance to anti-EGFR agents were significantly less common in tumors harboring BRAF class III mutations, which is of great importance as anti-EGFR therapies are a potential targeted treatment option in these tumors.DiscussionOur findings suggest a heterogenous interplay of oncogenic alterations in BRAF class III mutated tumors and have important implications for the molecular mechanisms of carcinogenesis while revealing potential therapeutic vulnerabilities.HighlightsTumors harboring BRAF class III (BRAF vIII) mutations comprise a novel subset with distinct genomic heterogeneity.BRAF vIII mutations may sensitize tumors to anti-EGFR treatments.BRAF vIII alterations show significantly less co-occurrence with alterations that predict resistance to anti-EGFR agents.Rare tumors with limited therapy options should be screened for BRAF vIII mutations as they may benefit from anti-EGFR agents.

MO50-6 Characterization of patients with advanced biliary tract cancer by KRAS alteration subtypes

MO50-6 Characterization of patients with advanced biliary tract cancer by KRAS alteration subtypes

Cost-effectiveness of next-generation sequencing for advanced EGFR/ALK-negative non-small cell lung cancer

ObjectivesThis study aimed to evaluate the cost-effectiveness of next-generation sequencing (NGS) versus sequential single-gene testing (SGT), including the long-term costs and survival outcomes of relevant treatments for advanced EGFR/ALK-negative non-small cell lung cancer (NSCLC). Materials and MethodsWe developed a decision tree linked to a partitioned survival model to estimate the clinical outcomes and costs over the five-year analysis period. The decision tree consisted of treatment types based on molecular biomarker (ROS1, BRAF, NTRK, MET, RET, and KRAS alterations) test results. The probability of receiving each targeted therapy was estimated based on 1) the testing rate, 2) the proportion of alterations detected, and 3) the proportion of patients receiving treatment consistent with the testing results. We estimated the long-term overall survival and progression-free survival for each treatment using parametric estimation by reconstructing patient-level data from clinical trials. The costs of testing, drugs, administration, physician visits, monitoring, adverse events, post-progression, and end-of-life care were included. The utility values were obtained from a previous study. The incremental cost-effectiveness ratio (ICER) was used to evaluate the cost-effectiveness of NGS within a threshold of $38,701 (50,000,000 KRW) per quality-adjusted life year (QALY). ResultsThe incremental life-years (LYs) and QALYs for the NGS group versus the SGT group were 0.028 and 0.023, respectively. The total medical cost for the NGS group was $8,375 higher than that for the SGT group. The difference in drug costs accounted for most of the differences in total medical costs. NGS was not cost-effective compared to sequential SGT, with an ICER of $300,233/LY and $359,405/QALY, respectively. ConclusionsNGS is not cost-effective for advanced EGFR/ALK-negative NSCLC, but has a survival benefit over sequential SGT. Our findings provide a basis for decision-making regarding the coverage and clinical utilization of NGS in regions where EGFR alterations are prevalent.

A Novel AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53 NGS Assay: A Non-Invasive Tool to Monitor Resistance Mechanisms to Hormonal Therapy and CDK4/6 Inhibitors.

Background: Patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (mBC) generally receive hormonal therapy (HT) combined with CDK4/6 inhibitors (CDK4/6i). Despite this treatment, resistance mechanisms to CDK4/6i emerge and the majority of these patients experience disease progression (PD). This highlight the necessity to uncover the resistance mechanism to CDK4/6i through the identification of specific biomarkers. The primary objective is to assess the accuracy and feasibility of a novel multi-gene target panel NGS assay on circulating tumor DNA (ctDNA) to detect molecular alterations of AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53 genes in women with BC undergoing HT plus CDK4/6i treatment. Secondarily, the study aims to explore the relationship between genomic profiling and clinical outcomes. Materials and Methods: Plasma samples were collected from 16 patients diagnosed with advanced/locally advanced HR+/HER2- BC at 2 time points: T0 (baseline) and at T1 (3 months after CDK4/6i treatment). Starting from 2 mL of plasma, ctDNA was isolated and libraries were set up using the Plasma-SeqSensei (PQS)® Breast Cancer IVD Kit, sequenced on Nextseq 550 and analyzed using the Plasma-SeqSensei™ IVD Software®. Results: Among the five patients who presented PD, three had PIK3CA mutations and, of these, two showed a higher mutant allele frequency (MAF) at T1. In three patients with stable disease and in eight patients with partial response, the MAF of the detected alterations decreased dramatically or disappeared during CDK4/6i treatment. Conclusions: Based on our findings, the liquid biopsy analysis using the PQS panel seems to be both feasible and accurate, demonstrating a strong sensitivity in detecting mutations. This exploratory analysis of the clinical outcome associated to the mutational status of patients highlights the potential of molecular analysis on liquid biopsy for disease monitoring, although further validation with a larger patient cohort is necessary to confirm these preliminary observations.

Abstract C066: Identifying Transcriptional Drivers of Plasticity in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease with few effective treatments. Even with the advent of mutationally-directed therapies targeting the KRAS oncogene, PDAC transcriptional plasticity remains a major mechanism of therapeutic resistance. As a result, there is a desperate need to develop additional strategies to treat this deadly disease. In addition to the canonical genomic alterations in KRAS, TP53, CDKN2A, and SMAD4, PDAC tumors exhibit diverse transcriptional programs, or "cell states," with prognostic implications: PDAC tumors in the “classical state” are more responsive to upfront chemotherapy and have better outcomes, while those in the “basal state” are more aggressive with poorer prognoses. Currently, PDAC cell states are not used to direct therapy. Cell state is an integrative property shaped by both cell-intrinsic (e.g., mutations, epigenetics) and cell-extrinsic (e.g., tumor microenvironment) factors. In our prior work, single-cell analyses of clinical PDAC samples demonstrated notable cell state heterogeneity and plasticity. In addition, longitudinal monitoring of patient-derived organoids showed that cells can shift from classical to basal or coexpressor states as the environment changes, and that these cell states can drastically influence drug response. However, it remains unknown which signaling and transcriptional pathways induce basal or classical states, and whether these gene regulatory networks can be targeted therapeutically. Thus, there is a pressing need to devise scalable methods to determine essential drivers for prognostically relevant cancer cell states.Here, we aim to systematically and unbiasedly identify transcription factors (TFs) that drive basal and classical cell states in PDAC. We have successfully established a high-throughput screening pipeline to 1) over-express all human TF isoforms in a pooled format within a cohort of PDAC models; 2) sort cells using antibodies against state-specific cell surface markers; and 3) perform single-cell RNA sequencing on sorted cells to nominate individual TFs or combinations that induce specific cell state transitions. Preliminary results identify both previously reported (e.g., GATA4) and new TFs that can induce the classical state. By overexpressing TFs individually or in combination, we are able to engineer PDAC cell line models that more faithfully reflect the transcriptional phenotypes observed in patient tumors. Ultimately, we aim to use this approach to construct high-fidelity isogenic but state-variant PDAC models for use both in therapeutic screening and as a substrate for investigating mechanisms governing cancer cell state plasticity. Citation Format: Yuzhou Evelyn Tong, Aswanth Mahalingam, Walaa E Katan, Julia Joung, Alex K Shalek, Peter S Winter, Srivatsan Raghavan. Identifying Transcriptional Drivers of Plasticity in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C066.

Clinical and Biological Impact of KRAS Overexpression in Stomach Adenocarcinoma

This study analyzes Kirsten rat sarcoma viral oncogene (KRAS) expression heterogeneity, and biological and clinical relevance in stomach adenocarcinoma (STAD). The study utilized various tools including UALCAN, GEPIA2, Kaplan-Meier (KM) plotter, cBioPortal, STRING, DAVID, and TIMER 2.0 to conduct this analysis. The results illustrated overexpression of KRAS in STAD and the analysis based on various clinicopathological parameters also verified overexpression of KRAS in STAD. Eventually, this overexpression was linked to poor overall survival (OS) of STAD patients. These results suggested the role of KRAS is involved in the development and progression of STAD. The study also assessed several significant correlations of KRAS expression with promoter methylation tumor purity and immune cell infiltration. Genetic alteration of KRAS revealed to have a strong role in STAD initiation. Gene enrichment analysis highlighted the enrichment of KRAS with various pathways. In conclusion, the findings illustrated the potential of KRAS as a diagnostic, prognostic, and therapeutic biomarker in STAD.