Wild-type KRAS Research Articles

PROMER technology: a new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity

Abstract Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wildtype KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wildtype counterpart.

Exploratory biomarker analysis for predicting treatment effects of aflibercept in the VELOUR trial.

3592 Background: Second-line treatment with aflibercept and FOLFIRI in patients with metastatic colorectal carcinoma (mCRC) previously treated with oxaliplatin improved overall survival (OS) versus placebo [Hazard ratio (HR), 0.81; 95% CI, 0.71–0.93; P=0.003]1. However, colorectal cancer (CRC) is a heterogenous disease and the new intrinsic consensus molecular (iCMS) allows for tumours to be subtyped into unique genetic and transcriptional features2. We present a post hoc analysis of VELOUR patient data, to assess the impact of KRAS or BRAF mutation status and iCMS on efficacy parameters. Methods: Patient samples were classified as either KRAS or BRAF mutant or wild-type (WT) via a targeted mutation panel1. Gene expression data was used to classify samples as either iCMS2 or iCMS3 using a modified classifier based on iCMS signatures which are specifically up-regulated in tumour samples2. Results: Of the 439 patients with available RNA data, 54.9% (241/439) were classified as iCMS2; 45.1% (198/439) as iCMS3; 48.3% (212/439) as RAS mutant and 8.4% (37/439) as RAF mutant. A favourable aflibercept treatment effect was observed for patients within the KRAS WT group (Median OS from 11 to 17.7 months; HR 0.87; 95% CI 0.79-0.96, P=0.005), particularly for patients with KRAS WT iCMS2 tumours (Median OS from 15.5 to 30.4 months; HR 0.81; 95% CI 0.70-0.94, P=0.004). This is in comparison to the KRAS WT iCMS3 patients where no treatment benefit was seen (HR 0.97; 95% CI 0.82-1.14, P=0.683). In addition, no treatment benefit was seen in the KRAS mutant group (OS HR 1.03; 95% CI 0.96-1.12, P=0.405). Progression-free survival (PFS) data followed the same trend. Aflibercept seemed to also strongly benefit the 37 patients with BRAF-mutated tumours, (PFS HR 0.46, 95% CI 0.20-1.09; OS HR 0.65, 95% CI 0.29-1.48), although differences were not statistically significant. Irrespective of treatment, improved OS (HR 0.60; 95% CI 0.48-0.75, P< 0.001) and PFS (HR 0.75; 95% CI 0.60-0.94, P=0.011) were observed in all patients with iCMS2 versus iCMS3 tumours, as expected. Treatment-by-mutation status interaction tests (OS P=0.40, PFS P=0.33) indicated that the aflibercept benefit was not statistically different among the mutation sub-groups, although the small sample size limited the analysis. Conclusions: In the VELOUR study, the benefit of the addition of aflibercept to FOLFIRI appears to be primarily in patients with KRAS WT tumours, especially those with KRAS WT iCMS2. The improved OS and PFS seen in all patients with iCMS2 tumours in comparison to iCMS3 is prognostic but not predictive of aflibercept treatment response in addition to FOLFIRI. On the contrary, the addition of aflibercept to FOLFIRI provided a numerical benefit in BRAF-mutated tumours, however the P-values were not statistically significant.

Real-world impact of KRAS mutations on clinical outcomes in metastatic colorectal cancer treated with trifluridine-tipiracil in the USA.

e15566 Background: Defining the impact of KRAS mutation status on outcomes to standard of care treatments in metastatic colorectal cancer (mCRC) is of increasing clinical relevance. We interrogated a large real-world genomic database to further explore the association between KRAS mutations and benefit to trifluridine-tipiracil (FTD-TPI) in patients with mCRC. Methods: 19,364 patients with mCRC and known KRAS mutation status were analyzed by next-generation sequencing (NextSeq, 592 Genes; NovaSeq, whole exome sequencing (WES)) at Caris Life Sciences. Real-world overall survival (rwOS) and time on treatment (ToT) was inferred from insurance claims data and calculated from time of start of treatment with FTD-TPI to last contact and time from first to last treatment with FTD-TPI with comparison done by the Kaplan-Meier test, respectively. Chi-square test was applied where appropriate, with p-value adjusted for multiple comparisons (p < 0.05). Results: 764 of 10,571 (7.2%) patients with wild-type (WT) KRAS, 768 of 6,984 (11%) with KRAS G12 mutation, and 170 of 1,809 (9.4%) with KRAS G13 mutation received FTD-TPI. Age distribution was similar among groups. Sidedness of primary site was reported in 692 (40.3%) patients; an increased proportion of right-sided tumors was observed in KRAS G12 (53.4% vs. 28.3%, p < 0.0001) and G13 (56% vs 28.3%, p < 0.0001) groups compared to KRAS WT. An increased proportion of TP53 mutations in KRAS WT was observed compared to KRAS G12 mutation (87% vs. 70%, p < 0.0001) and KRAS G13 mutation groups (87% vs. 75%, p < 0.0001). KRAS G12 mutation was associated with shorter median rwOS compared to KRAS WT (7.33 vs. 8.43 months (mo), HR 1.13 (95% CI 1.11-1.42), p = 0.041). KRAS G13 mutation was associated with similar rwOS to KRAS WT. Median ToT was shorter in patients with KRAS G12 mutations than KRAS WT (1.8 vs. 2 mo, HR of 1.22 (95% CI 1.11-1.42), p < 0.001). Median ToT was longer in patients with KRAS G13 versus KRAS G12 (2.3 vs. 1.8 mo, HR 0.82 (95% CI 0.68-0.98), p = 0.032). Conclusions: To our knowledge, this is the largest primarily US-based cohort of mCRC treated with FTD-TPI to be reported. KRAS G12 mutation, but not KRAS G13 mutation, was associated with shorter rwOS and ToT compared to KRAS WT, despite enrichment of TP53 and BRAF mutations in the KRAS WT group. This finding is consistent with previously published analyses of large randomized prospective trials of FTD-TPI in mCRC and further support KRAS mutation status as a predictive biomarker in this setting. [Table: see text]

Mutated KRASas a promising target in pancreas cancer: PURPLE registry data to inform real-world incidence and prognostic significance and to aid trial recruitment.

11150 Background: With around 90% KRASmutation (mt) frequency, pancreatic ductal adenocarcinoma (PDAC) is considered the most RAS-addicted cancer. Despite guideline recommendations, genomic testing is not routine in all PDAC patients (pts) in Australia, due to unproven clinical impact. As more novel KRAS-direct therapeutics enter clinical trials, understanding the real-world frequency of individual mutations and prognostic significance, and facilitating trial recruitment are important goals, all of which can be supported by registry data. Methods: Data extracted from the PURPLE pancreatic cancer registry from 9 participating Australian cancer centres, between 2016-2022, was analysed to compare clinicopathological features, survival based on KRASmt status, and assess feasibility of the platform to identify and molecularly stratify patients for future clinical trials. Survival estimates were calculated using Kaplan-Meier curves and log-rank testing on SPSS (Macintosh v.29). Results: Of 721 PDAC routine care pts identified, next generation sequencing (NGS) was undertaken in 378/721 (52%). Median patient age was 68 years (range 52-83); 152 (40%) had resectable, 111 (29%) locally advanced and 115 (30%) metastatic disease. 57/378 (15%) were KRAS wildtype. Of 321 pts with a KRAS mt, codons 12,13, and 61 were the most common sites of mt, including G12D (45%), and G12V (30%), with lower frequencies of G12R (13%), Q61H (6%), G12A (3%), G12C (1%), and G13D (1%). Comparing KRAS wildtype to KRASmt pts, there was no difference in median age (68 vs 67, p=0.63), gender (male: 49% vs 60%, p=0.23), Charlson comorbidity index (p=0.30), or stage at first presentation (p=0.99). Overall KRAS wildtype pts were more likely to be ECOG PS 0 at diagnosis (p=0.01) and to receive at least 1 modality of treatment (p=0.004). For all pts, median overall survival (OS) in KRASwildtype versus KRAS mt pts was 29.0 months versus 19.7 months (p=0.007), and for the 115 metastatic pts 15.1 versus 10.4 months (p=0.28). Further analysis of the impact by disease stage and by individual RAS mt is underway. Conclusions: Registry based analysis informs understanding of KRAS mt status of PDAC in a community setting. Here, KRAS mt status was associated with worse OS outcomes, likely in part due to the association with ECOG PS and receiving less active treatment. With newer promising KRAS-targeted therapies becoming available in clinical trials, known RAS status will aid identification of trial candidates. The clinical utility of NGS and rationale for reflex testing in PDAC is increasing. Clinical Registry information (ACTRN12617001474347).

Development of an arenavirus-based immunotherapy for treatment of KRAS mutant cancer.

e14672 Background: Oncogenic mutations in KRAS are prevalent in more than 80% of pancreatic ductal adenocarcinomas (PDAC), approximately 30% of colorectal cancer (CRC) and 15-20% of lung adenocarcinoma (LUAD). It has been demonstrated previously that CD8+ T cell responses specific for KRAS mutations can lead to tumor control and regression of metastasis. Replicating arenavirus vectors are currently evaluated in multiple clinical trials in infectious diseases and oncology and have demonstrated potent induction of target antigen specific CD8+ T cell responses of up to almost 50% of the total CD8+ T cell compartment in peripheral blood. To augment KRAS mutation specific CD8+ T cell responses in patients, we are using replicating arenavirus vectors targeting five prevalent KRAS mutations which are found in approximately 95% (PDAC), 74% (CRC), and 83% (LUAD) of the KRAS mutated tumors, representing a promising off-the-shelf immunotherapy for these indications. Here, we systematically investigate immune responses induced by mutant KRAS targeted arenavirus-vector-based cancer vaccines in HLA transgenic mice and characterize the efficiency of target cell killing of activated KRAS mutation specific CD8+ T cells. Methods: A transgene cassette consisting of peptide stretches including KRAS mutations G12D, G12V, G12C, G12R and G13D was generated by in silico aided antigen design. Vectors encoding peptide stretches of single KRAS mutations as well as the corresponding wildtype KRAS sequence served as controls in preclinical experiments. KRAS mutation specific CD8+ T cell expansion was evaluated in HLA transgenic mice and functionality of induced CD8+ T cells was evaluated by assessing CD8+ T cell mediated killing of mutant KRAS peptide loaded target cells in vivo. Results: All treatment regimens were well tolerated, and no mortalities or major adverse events were observed. Following vector administration, HLA I restricted antigen-specific CD8+ T cell responses were detected against 4 encoded mutant KRAS epitopes in HLA-A*11:01 (KRAS G12D and G12V), or HLA-B*07:02 (KRAS G12C and G12R) transgenic mice. These cells did not cross-react with wildtype KRAS epitopes presented in the same HLA I molecules. Specific cytotoxicity against KRAS G12D/V or KRAS G12C/R pulsed target cells was observed in vivo in HLA transgenic animals, indicating functionality of the induced KRAS mutation specific T cells. No specific cytotoxicity towards target cells pulsed with KRAS WT peptides was observed in any of the groups. Conclusions: This study indicates efficient induction of KRAS mutation specific T cell responses in HLA transgenic mice using an arenavirus vector based vaccine. Expanded CD8+ T cells were capable of killing cells loaded with KRAS mutation specific epitopes in vivo indicating functionality of the induced T cell responses. Based on these results, initiation of a Phase I study for the treatment of KRAS mutated cancers is planned.

A phase 1 trial evaluating the safety, tolerability, PK, and preliminary efficacy of QTX3034, an oral G12D-preferring multi-KRAS inhibitor, in patients with solid tumors with KRASG12D mutation.

TPS3172 Background: KRAS is one of the most mutated driver oncogenes across solid tumors, and KRAS G12D missense mutations have high prevalence in pancreatic, colorectal (CRC), and endometrial cancers. QTX3034 is a highly-selective, non-covalent, orally bioavailable, multi-KRAS inhibitor with potent activity across several KRAS variants, particularly G12D. QTX3034 binds to GDP-bound forms of mutant and wild-type KRAS with (sub)nanomolar affinities, allosterically inhibiting its conversion to the active state. QTX3034 inhibits KRAS signaling in in vitro studies and displays synergy with EGFR inhibitors. In vivo evaluation of QTX3034 resulted in significant tumor growth inhibition across various tumor models, with tumor regressions observed in 100% of animals in both HPAC pancreatic and GP2D colorectal KRAS G12D xenograft models. QTX3034 exhibits brain penetration in preclinical studies, including an orthotopic model of intracranial efficacy. This clinical trial is now enrolling patients with advanced solid tumors harboring a KRASG12D mutation. Methods: This is a first-in-human, multicenter, open-label phase 1 study in adult patients with advanced solid tumors with KRASG12D mutations, without prior direct KRAS inhibitor treatment (NCT06227377). The primary objectives are to evaluate the safety and tolerability and to determine maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) of QTX3034 alone, and in combination with cetuximab. Secondary objectives are characterization of the PK profile and preliminary antitumor activity. Part 1 consists of two dose-escalation arms: QTX3034 monotherapy (advanced solid tumors) and QTX3034 plus cetuximab (CRC) and will enroll 3-4 subjects per cohort in a Bayesian Logistic Regression Model (BLRM) design. QTX3034 will be administered orally in 21-day cycles until disease progression, intolerable toxicity, or withdrawal from treatment. Exploratory paired biopsies will be collected in Cycle 2 in a group of patients in the dose-escalation phase. Serial ctDNA will be collected in all patients. Following identification of a RP2D, QTX3034 will be evaluated in dose-expansion cohorts of up to 30 patients with various KRASG12D mutated tumor types to determine preliminary activity: pancreatic cancer, CRC, non-small cell lung cancer and endometrial cancer. To further optimize the recommended dose, patients will be randomized between two dose levels in the pancreatic cancer cohort. QTX3034 plus cetuximab will be explored following identification of a recommended combination dose in patients with CRC. Clinical trial information: NCT06227377 .

Molecular and clinical features of pancreatic acinar cell carcinoma: A single institutional experience.

e16335 Background: Acinar Cell Carcinoma (ACC) accounts for around 1% of pancreatic cancer. The molecular and clinical features of acinar cell carcinoma are less characterized. We sought to evaluate the clinical and molecular features of patients with ACC at MD Anderson Cancer Center. Methods: Patients with ACC who had germline and/or somatic molecular testing at MD Anderson Cancer Center from 2008 to 2022 were identified retrospectively. After obtaining approval of approval of the institutional review board genomic alterations including germline or somatic BRCA mutations, KRAS mutation, demographic, clinical, and pathological information were extracted from institutional databases using the Palantir Foundry software platform (Syntropy); this included natural language processing with subsequent validation by manual chart review. Overall Survival (OS) was calculated from the date of diagnosis to death or last follow-up. The Kaplan-Meier method was used for survival analysis. Results: 16 patients were identified with molecular testing results available. There were 13 males and 3 females. The median age of diagnosis was 62.5 years (range 49 -72). 14/16 patients (87.5%) had metastatic disease, one (6.25%) patient had borderline resectable disease and one (6.25%) had localized resectable disease at initial diagnosis. Median OS was for 24 months for patients with metastatic disease. There were 2 patients with mixed pathology, 1 with acinar cell carcinoma with neuroendocrine features and the 2nd had mixed acinar cell-ductal carcinoma. 15/16 (93.75%) patients were tested for KRAS and all 15 were KRAS wildtype. Somatic mutations in DNA damage repair genes (BRCA2, PALB2) were present in 3 patients (25%) and 2 patients had germline mutation (BRCA and ATM) (16%) out of 12 patients tested for those genes. 6/16 (37.5%) patients were treated with FOLFIRINOX at first line. 3/6 (50%) of these patients had partial response, 2/6 (33%) had stable disease and only 1/6 (16%) had progression. 5 patients were treated with FOLFOX based regimen and 1/5 (20%) had response, 2/5 (40%) had stable disease and 2/5 (40%) had progression of disease. 5 patients were treated with gemcitabine-based regimen and only 1 had response which received gemcitabine/nab-paclitaxel combined with cisplatin. Conclusions: ACC were KRAS wild type and had higher rates of germline/somatic mutations in DNA damage repair genes. Better treatment outcome of FOLFIRNIOX/ platinum-based regimens was suggested based on this limited patient dataset. Larger studies are needed to further understand the molecular features of ACC and guide the treatment of this rare entity of pancreatic cancer.

Clinical and molecular characterization of AXL in colorectal cancer: CALGB (Alliance)/SWOG 80405 and real-world data.

3145 Background: Dysregulation of AXL signaling is linked with chemotherapy and targeted therapy resistance. However, it remains unclear which patients may benefit from therapies targeting AXL, given its role in both tumor cell intrinsic resistance and immune microenvironmental reprogramming. Here, we present a clinical and molecular characterization of AXL in colorectal cancer (CRC). Methods: 24,257 CRC samples tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq), NextGen DNA sequencing (NextSeq, 592 genes and NovaSEQ, WES), and PD-L1 expression (22C3 clone; TPS ≥ 1%) were analyzed. RNA deconvolution analysis with QuantiSEQ estimated cell infiltration in the tumor microenvironment (TME). In this cohort, AXL expression was categorized as high (H) or low (L) using median cutoff: overall survival (OS) was evaluated from treatment initiation using insurance claims data. Data from the phase III CALGB/SWOG 80405 trial on 433 metastatic CRC (mCRC) patients treated with bevacizumab (Bev, n = 226) or cetuximab (Cet, n = 207) in combination with first-line chemotherapy were also evaluated. RNA isolated from FFPE tumor samples were sequenced with HiSeq 2500 (Illumina). OS and progression-free survival (PFS) were compared using categorical gene expression tertiles (high (T3), medium (T2), and low (T1)) adjusting for age, demographics, ECOG, and tumor/treatment characteristics. Results: BRAF and RNF43 mutations were more prevalent in AXL-H, but APC, NRAS, KRAS, and PIK3CA correlated with AXL-L (all q< .001). AXL-H had increased B cells, M1/M2 macrophages, T-regs, and NK cells in the TME while increased neutrophils and dendritic cells were associated with AXL-L (all q< .001). AXL-H had increased PD-L1 positivity (5.1 vs 2.6%) and pathway activation of epithelial-mesenchymal transition (EMT), inflammatory/IFN-G response, and TNF-a signaling (all q< .005). AXL-H demonstrated worse OS in FOLFOX/FOLFIRI treated CRC (H: 35.7 vs L: 38.7 months [mo], P = .003; HR 1.08, 95% CI [1.03-1.14]). The effect persisted within KRAS wildtype (wt) CRC (45.4 vs 40.1 mo, P = .005; HR 1.12 [1.04-1.22]) but not KRASmutant (mt). In 80405, higher AXLexpression was associated with worse PFS and OS ( Plinear = 2E-04 and 2.2E-05, respectively). AXL-T3 showed significantly shorter PFS (T3: 9.2 vs T2: 11.7 vs T1: 12.9 mo, T3 vs T1 (reference) adjusted HR 1.53 [1.19-1.97]) and OS (24.2 vs 34.0 vs 34.7 mo, adjusted HR 1.73 [1.33-2.25]). No significant interaction between AXL expression and treatment was observed for PFS and OS (likelihood ratio test). Conclusions: Our results indicate increased AXL expression is associated with immune cell infiltration, EMT, and inflammatory signaling. AXL-H confers worse OS/PFS on first-line chemotherapy, with a more pronounced effect in KRASwt CRC. This data supports evaluation of AXL as a prognostic marker and potential therapeutic target in CRC. Trial Identifier: NCT00265850.

Retrospective analysis of change in frequency of STK11 mutation in lung adenocarcinomas over a 10-year period.

8032 Background: Previous studies have linked the presence of inactivating mutations in the tumor suppressor gene STK11 with reduced efficacy of immune checkpoint inhibitor (ICI) treatments in lung adenocarcinoma (LA). We queried whether there has been a change in the STK11 mutation frequency over the last 10-year period that could potentially reflect the emergence of additional acquired resistance mutations. Methods: Over a 10-year period from 2013 through 2022, 109,763 clinically advanced LA underwent hybrid capture based comprehensive genomic profiling (CGP) to assess all classes of genomic alterations (GA) at a single reference laboratory (Foundation Medicine). MSI-high status and tumor mutational burden (TMB) were determined from the sequencing data. PD-L1 status was measured by immunohistochemistry (IHC) using the Dako 22C3 kit with TPS scoring. Statistical comparisons utilized the 2-tailed Chi Square method with the Yates’ correction. Results: The frequency of STK11 inactivating GA in all cases of LA significantly increased during the study period from 2013 to 2022 (16.1% vs 20.1%; p=0.0005). This increase appeared to be predominantly in KRAS wild type LA (10.1% vs 15.5%; p<0.0001). The STK11 GA frequency slightly declined in the KRAS mutated LA over the 10-year period (29.9% vs o 27.0%, P=0.25). During the same time period, the KEAP1 GA frequency increased from 12.6% to 14.9% (p= 0.076) and the frequency of combined STK11 and KEAP1 GA increased from 4.8% to 8.0% (p=.0006). The frequencies of MSI-high status, TMB and PD-L1 expression did not significantly change over the observation period. Conclusions: The continuously increasing approved indications for ICI-based treatments for both early stage and clinically advanced LA appears to be accompanied by a significant increase in the frequency of inactivating STK11 GA predominantly in the KRAS mutation negative LA population. This inactivation is associated with an attenuated response rate and progression-free survival in this population. Given the development of novel therapies focused on potential restoration of STK11 function by HDAC/CoREST inhibition, further study of the STK11 GA distribution in LA appears warranted. [Table: see text]

Stress-induced Rab11a-exosomes induce amphiregulin-mediated cetuximab resistance in colorectal cancer.

Exosomes are secreted vesicles made intracellularly in the endosomal system. We have previously shown that exosomes are not only made in late endosomes, but also in recycling endosomes marked by the monomeric G-protein Rab11a. These vesicles, termed Rab11a-exosomes, are preferentially secreted under nutrient stress from several cancer cell types, including HCT116 colorectal cancer (CRC) cells. HCT116 Rab11a-exosomes have particularly potent signalling activities, some mediated by the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). Mutant activating forms of KRAS, a downstream target of EGFR, are often found in advanced CRC. When absent, monoclonal antibodies, such as cetuximab, which target the EGFR and block the effects of EGFR ligands, such as AREG, can be administered. Patients, however, inevitably develop resistance to cetuximab, either by acquiring KRAS mutations or via non-genetic microenvironmental changes. Here we show that nutrient stress in several CRC cell lines causes the release of AREG-carrying Rab11a-exosomes. We demonstrate that while soluble AREG has no effect, much lower levels of AREG bound to Rab11a-exosomes from cetuximab-resistant KRAS-mutant HCT116 cells, can suppress the effects of cetuximab on KRAS-wild type Caco-2 CRC cells. Using neutralising anti-AREG antibodies and an intracellular EGFR kinase inhibitor, we show that this effect is mediated via AREG activation of EGFR, and not transfer of activated KRAS. Therefore, presentation of AREG on Rab11a-exosomes affects its ability to compete with cetuximab. We propose that this Rab11a-exosome-mediated mechanism contributes to the establishment of resistance in cetuximab-sensitive cells and may explain why in cetuximab-resistant tumours only some cells carry mutant KRAS.

Correlation of allele-specific HLA loss of heterozygosity in patients with cancer with driver mutations.

e14658 Background: While immunotherapies have revolutionized cancer treatment, the limited predictability of response and resistance mechanisms present challenges to the development of novel therapeutics for solid tumors. Many of these use a sensitive and specific T cell receptor (TCR)-HLA:peptide interaction as a mechanism of action and can be impacted by HLA loss of heterozygosity (LOH). Allele-agnostic HLA LOH analyses assessing loss of any MHC class I allele are relevant for HLA-agnostic approaches such as immune checkpoint inhibitors; however, context-specific studies remain limited. For example, adoptive TCR T cell therapies and bi-specific TCR T cell engagers target neoantigen peptides derived from oncogenic driver mutations, such as those of KRAS and TP53, presented by a specific HLA allele. Thus, allele-agnostic analyses may overestimate the frequency and relevance of HLA LOH for allele-specific modalities. Methods: A real-world comprehensive genomic profiling dataset consisting of 78,418 cases (1), germline heterozygous for one or more HLA class I locus, was analyzed for HLA LOH in the context of frequent driver mutations (KRAS G12C, G12D, G12V and TP53 R175H), the corresponding HLA alleles (A*02:01, A*03:01, and A*11:01), and within tumor subtypes. Results: Allele-agnostic HLA LOH was observed in 16.6% of all samples and varied between indications. Notably, allele-specific loss of HLAs A*02:01, A*03:01, and A*11:01 across all samples was much lower than allele-agnostic loss (6.3%, 6.6%, and 6.3% cases, respectively). Although, a trend for increased allele-specific HLA LOH was observed when analyzed by driver mutation, the frequency remained low. Specifically, across all cases, A*02:01, A*03:01, and A*11:01 allele-specific LOH ranged between 5.9% - 9.7% in relation to KRAS G12C, G12D, and G12V compared to 5.9% - 6.2% for wild-type (WT). Moreover, it did not achieve statistical significance for any HLA-KRAS mutation pairs in colorectal (CRC), non-small cell lung (NSCLC), and pancreatic (PDAC) cancers, except for A*11:01 and KRAS G12C in NSCLC, which was lower in mutated cases (5.3% for G12C vs 10.1% for WT KRAS, p=0.045). Both allele-agnostic and A*02:01-specific HLA LOH in TP53 WT samples were lower than average. The TP53 R175H mutation was associated with limited increase in HLA-A*02:01 LOH (from 4.4% for WT to 8.1% for TP53 R175H) and varied between indications, e.g. no significant increase was observed in R175H-positive breast cancer and NSCLC cases contrary to CRC and PDAC. Conclusions: Based on this real-world dataset, over 90% of cases retain the TCR-targeted allele and, therefore, preserve the molecular complex necessary for HLA/driver mutation-based therapies. Notably, the database is enriched for advanced cancer patients. Further studies should test correlations with treatments and whether tumors in earlier lines of treatment have an even lower rate of HLA LOH. 1. Montesion et al., PMID: 33127846.

Prognostic significance of EGFR, AREG and EREG amplification and gene expression in muscle invasive bladder cancer.

Muscle invasive bladder cancer (MIBC) remains a prevalent cancer with limited therapeutic options, obviating the need for innovative therapies. The epidermal growth factor receptor (EGFR) is a linchpin in tumor progression and presents a potential therapeutic target in MIBC. Additionally, the EGFR ligands AREG and EREG have shown associations with response to anti-EGFR therapy and improved progression-free survival in colorectal carcinoma. We investigated the prognostic significance of EGFR, AREG, and EREG in MIBC. Gene expression and copy number analyses were performed via qRT-PCR on tissue samples from 100 patients with MIBC who underwent radical cystectomy at the University Hospital Mannheim (MA; median age 72, interquartile range [IQR] 64-78 years, 25% female). Results were validated in 361 patients from the 2017 TCGA MIBC cohort (median age 69, IQR 60-77 years, 27% female), in the Chungbuk and MDACC cohort. Gene expressions were correlated with clinicopathologic parameters using the Mann-Whitney test, Kruskal-Wallis- test and Spearman correlation. For overall survival (OS), cancer-specific survival (CSS) and disease-free survival (DFS) gene expression was analyzed with Kaplan-Meier and Cox-proportional hazard models. Significant gene expression differences in EGFR, AREG, and EREG could be detected in all cohorts. In the TCGA cohort, EGFR expression was significantly elevated in patients with EGFR amplification and KRAS wildtype. High AREG expression independently predicted longer OS (HR = 0.35, CI 0.19 - 0.63, p = 0.0004) and CSS (HR = 0.42, CI 0.18 - 0.95, p = 0.0378) in the MA cohort. In the TCGA cohort, high EGFR, AREG, and EREG expression correlated with shorter OS (AREG: HR = 1.57, CI 1.12 - 2.20, p = 0.0090) and DFS (EGFR: HR = 1.91, CI 1.31 - 2.8, p = 0.0008). EGFR amplification was also associated with reduced DFS. High EGFR and EREG indicate worse survival in patients with MIBC. The prognostic role of AREG should further be investigated in large, prospective series. Divergent survival outcomes between the four cohorts should be interpreted cautiously, considering differences in analysis methods and demographics. Further in vitro investigations are necessary to elucidate the functional mechanisms underlying the associations observed in this study.

Combining antivascular endothelial growth factor and anti-epidermal growth factor receptor antibodies: randomized phase II study of irinotecan and cetuximab with/without ramucirumab in second-line colorectal cancer (ECOG-ACRIN E7208).

Early studies showed promise of combined anti-epidermal growth factor receptor (EGFR) plus anti-vascular endothelial growth factor (VEGF) antibodies for advanced colorectal cancer (CRC), yet this was later rejected as toxic and ineffective in studies not selected for RAS status. We studied advanced KRAS wild-type CRC, as second-line treatment, using irinotecan-cetuximab with or without the anti-VEGF receptor antibody ramucirumab. Patients with 1 prior regimen including fluoropyrimidine, oxaliplatin, and bevacizumab, with KRAS wild-type tumors were stratified by Eastern Cooperative Oncology Group Performance Score, time since last chemotherapy, and progression on oxaliplatin to irinotecan-cetuximab (IC) (180 mg/m2 and 500 mg/m2 every 2 weeks) vs modified ICR (irinotecan-cetuximab with ramucirumab 150 mg/m2 and 400 mg/m2 plus 6 mg/kg, respectively). A total of 102 patients were compared for progression-free survival (PFS) as primary endpoint (85% power for 70% improvement in median PFS from 4.5 to 7.65 months). Of the 102 enrolled, 44 treated with irinotecan-cetuximab and 45 with modified ramucirumab were evaluable. Median PFS was 6.0 months vs 9.2 months, respectively (hazard ratio = 0.75, P = .07; statistically significant by study design for P < .128). Response rate was 23% vs 36% (P = .27), and disease-control rate was 52% vs 73% (P = .05). Grade 3 or higher toxicity was equivalent. Overall survival was not significantly different at approximately 19 months. Previous phase 3 trials without RAS genotyping rejected combining anti-epidermal growth factor receptor and anti-VEGF drugs. In this randomized multicenter phase 2 study for KRAS wild-type CRC (all previously bevacizumab treated), the addition of ramucirumab to irinotecan and cetuximab improved PFS and disease control rate, showing the combination is feasible and effective. Further, phase 3 trials with appropriate patient-selection are required. (NCT01079780).

Molecular Mechanism of Phosphorylation-Mediated Impacts on the Conformation Dynamics of GTP-Bound KRAS Probed by GaMD Trajectory-Based Deep Learning.

The phosphorylation of different sites produces a significant effect on the conformational dynamics of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations were combined with deep learning (DL) to explore the molecular mechanism of the phosphorylation-mediated effect on conformational dynamics of the GTP-bound KRAS. The DL finds that the switch domains are involved in obvious differences in conformation contacts and suggests that the switch domains play a key role in the function of KRAS. The analyses of free energy landscapes (FELs) reveal that the phosphorylation of pY32, pY64, and pY137 leads to more disordered states of the switch domains than the wild-type (WT) KRAS and induces conformational transformations between the closed and open states. The results from principal component analysis (PCA) indicate that principal motions PC1 and PC2 are responsible for the closed and open states of the phosphorylated KRAS. Interaction networks were analyzed and the results verify that the phosphorylation alters interactions of GTP and magnesium ion Mg2+ with the switch domains. It is concluded that the phosphorylation pY32, pY64, and pY137 tune the activity of KRAS through changing conformational dynamics and interactions of the switch domains. We anticipated that this work could provide theoretical aids for deeply understanding the function of KRAS.

Molecular Characterization and Therapeutic Opportunities in KRAS Wildtype Pancreatic Ductal Adenocarcinoma.

To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

KRAS as a Prognostic and Predictive Marker in Metastatic Non-Small Cell Lung Carcinoma: A Systematic Review.

Metastatic non-small cell lung cancer (NSCLC) poses a significant clinical challenge, prompting a focused investigation into the role of KRAS mutations in prognosis and treatment response. Targeted therapies offer promising avenues for intervention, motivating a comprehensive analysis of existing evidence. Conducted in June 2023, our review delved into MEDLINE (Medical Literature Analysis and Retrieval System Online), Embase, Scopus, and the Cochrane Register of Controlled Trials. Rigorous inclusion and exclusion criteria guided the selection of 12 articles, comprising two randomized controlled trials (RCTs) and 10 observational studies. Multiple investigators independently executed data extraction, evaluating prognostic factors (overall and progression-free survival) and predictive outcomes (treatment and objective response). The Newcastle-Ottawa Scale (NOS) and modified Jadad scores were used for study quality assessment of observational studies and RCTs, respectively. From an initial pool of 120 articles, the 12 selected studies, spanning 2013 to 2022, encompassed 2,845 metastatic NSCLC patients. KRAS mutations, particularly the G12C variant, emerged as a pivotal factor influencing treatment response. Notably, KRAS wild type patients displayed enhanced responses to platinum-based chemotherapy, while those with KRAS mutations exhibited favourable outcomes with immune checkpoint inhibitors (ICIs). The role of KRAS mutations as prognostic indicators in metastatic NSCLC is underscored by this systematic review, with implications for both survival and treatment response. The discernment between KRAS wild type and mutant patients offers insights into tailored therapeutic strategies, with platinum-based chemotherapy and immune checkpoint inhibitors emerging as context-dependent options. Nevertheless, more research is required to solidify the predictive role of KRAS and explore the efficacy of KRAS inhibitors and other targeted therapies, paving the way for refined and personalized interventions in the management of metastatic NSCLC.

Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selectiveinhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selectiveinhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selectiveinhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Abstract LB031: Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies

Abstract Besides marketed inhibitors targeting KRAS G12C, novel therapies are needed to effectively treat cancer patients harboring other KRAS mutations. SOS1, a guanine nucleotide-exchange factors (GEFs), plays a critical role in catalyzing the conversion of KRAS from its GDP- to GTP-bound form, regardless of KRAS mutation status and represents a promising therapeutic target for all KRAS-driven tumors. Up to date, several SOS1 inhibitors have entered early phase clinical trials. But a more potent SOS1 inhibitor with improved drug-like properties is still needed to prove efficacy as monotherapy or in combination with other drugs targeting the KRAS/MAPK pathway.We discovered a potent SOS1 inhibitor, HH100937 that effectively disrupted the interaction between SOS1 and wild-type KRAS or with G12C, G12D or G12V mutations. HH100937 exhibited lower IC50s than BI-3406 and MTRX0902 in blocking ERK phosphorylation and cancer cell proliferation, leading to better anti-tumor efficacies in xenografts than reference compounds at same doses. HH100937 also showed synergistic effect with AMG510 in cancer cells with KRAS G12C mutation but refractory to AMG510. The combination of HH100937 and AMG510 in xenografts led to tumor regression in all mice, while some tumors did not response well to AMG510 monotherapy. Our SOS1 inhibitor also exhibited synergistic effect with HH2710, a clinical-stage ERK inhibitor, in a broad panel of cancer cells with KRAS mutations.The pharmacological potency of HH100937 was highly selective over SOS2, other kinases tested or other targets in a safety panel of 47 targets. HH100937 was well tolerated in 28-day DRF study in rats and dogs up to 250 mg/kg QD and 30 mg/kg QD, respectively. The AUC of drug exposure in DRF studies was approximately three to six times higher than that at the efficacious dose. HH100937 also showed favorable DMPK properties in mice, rats and dogs, and acceptable safety profiles in vitro.Taken together, HH100937 is a highly potent and selective SOS1 inhibitor with better anti-tumor activities than some SOS1 inhibitors at clinical stage. It can be developed as mono therapy or in combination with other KRAS/MAPK targeting therapies. Citation Format: Qing Xie, Dongsheng Li, Shujuan Jiang, Jie Zhu, Songda Yu, Maozhi Yang, Yalei Cai, Lei Liu, Bing Yu, Wangyang Tu, Yixiang Zhang, Leping Li. Discovery and Characterization of HH100937, a potent and selective SOS1 inhibitor demonstrates synergistic efficacy in combination with KRAS/MAPK therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB031.

Abstract LB266: AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer

Abstract Introduction: Studies have shown that molecular targeted therapy can mediate Gasdermin-E (GSDME) dependent pyroptosis. However, GSDME expression is suppressed in many mutated patients, including those with KRAS-G12C-mutated non-small-cell lung cancer (NSCLC). The efficacy of AMG510 against KRAS-G12C inhibition (KRAS-G12Ci) is constrained by tumor recurrence. Pyroptosis is widely involved in the occurrence and development of tumors. However, the role and molecular mechanism of AMG510 in pyroptosis have not been well identified. Here, we demonstrate that inactivation of anti-apoptotic Bcl-2 family members during KRAS-G12Ci treatment enhances pyroptosis, which then promotes the sensitivity of AMG510 in NSCLC. Methods: We assessed pyroptosis using live cells imaging, LDH release assays, ELISA, Western blotting, and flow cytometry. In vitro assays were used to investigate the function of GSDME. Co-culture between peripheral blood mononuclear cells (PBMCs) and KRAS-G12C mutant lung cancer cell lines explored the anti-tumor immunity of cytotoxic T lymphocytes affected by knockdown of GSDME. The molecular mechanism was determined using bulk RNA sequencing, molecular cloning technology, and Western blotting. Results: We demonstrated that KRAS G12Ci treatment promotes cleavage of GSDME, release of high mobility group protein B1 (HMGB1), and LDH, markers of pyroptotic cell death. Additionally, GSDME was significantly downregulated in KRAS-mutated NSCLC compared with patients of KRAS wild type in TCGA cohort, while significantly upregulated after AMG510 treatment and downregulated after AMG510 resistance in vitro assay. We identified that KRAS-G12Ci resulted in the inactivation of BCL-xL, leading to caspase-3-dependent cleavage of gasdermin E. Conversely, amplification of BCL-xL downregulated the expression of GSDME, therefore suppressing the process of pyroptosis. GSDME knockdown in KRAS-G12C mutant lung cancer cell lines showed defective HMGB1 release, reduced caspase-3 viability, and decreased granzyme B and IFN-γ secretion by CD8+ T cells in response to KRAS-G12Ci, and more frequent tumor cell growth. Conclusion: Our findings indicate that GSDME-dependent pyroptosis is a critical determinant of KRAS-G12C-targeted inhibitor AMG510, and through the modulation exerted by the Bcl-2 family protein BCL-xL upon GSDME. These findings suggest that activation of GSDME may provide an effective strategy to sensitize cancer cells to KRAS-G12Ci treatment. Citation Format: Linfeng Luo, Anlin Li, Wei Du, Maojian Chen, JIng Li, Yixin Zhou, Li Zhang, Shaodong Hong. AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB266.