KRAS G12V Mutation Research Articles

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.

SIL-204 siRNA encapsulated in extended release microparticles for the treatment of localized cancer that harbors a KRAS G12x or G13D mutation.

745 Background: In a Phase 2 trial, treatment of non-resectable locally advanced pancreatic cancer (LAPC) patients harboring the KRAS G12D or G12V mutations, with SiG12DLoder combined with chemotherapy resulted in superior median overall survival (OS) of 9.3 months over chemotherapy (not statistically significant) and Objective Response Rate of 55% (35% over the chemotherapy control), when administered once every 3 months (Clinical trial # NCT01676259, ESMO 2023, abstract FPN: 1626). SIL-204 which is a modified version of siRNA siG12D is composed of a 21-base sense and 23-base antisense strands, both chemically modified and linked to a lipid. SIL-204 is encapsulated in biodegradable PLGA microparticles (MP), for direct release and injection into KRAS mutated solid tumors using an endoscopic ultrasound procedure. The strategy of preventing the synthesis of KRAS mutated protein may have an advantage over other approaches targeting KRAS function using small molecules. Methods: Methods are presented in results section. Results: SIL-204 has an increase stability over siG12D.To enhance stability, base modifications and phosphorothiate bonds at cleavage sites were implemented. SIL-204 exhibited a half-life of more than 48 hours in human serum. SIL-204 has robust KRAS silencing . When tested in Hepa1-6 cells transfected with individual human KRAS mutations, SIL-204 effectively silenced the various G12x mutations found in pancreatic cancer (D,V,R,C) at sub-nanomolar concentration (IC50 range=0.19-0.59) as well as G13D (IC50 =0.37). Addition of a hydrophobic tail to a prototype of SIL-204, SIL-101, increased the silencing of KRAS. Using PANC-1 cells (KRAS G12D mutation), lipid conjugation of the siRNA doubled the siRNA’s silencing efficiency in these cells. In addition, release of SIL-204 from the MP was tested in rats and indicates a prolong and sustained release over our intended treatment regimen SIL-204-MP was administered to tumors from human pancreatic tumor cell lines Capan-1 (KRAS G12V mutation and labelled with luciferase) grown in NSG mice and Panc-1 (grown in Athymic Nude mice). Bioluminescence imaging of tumor growth showed that SIL-204-MP significantly reduced Capan-1 tumor growth compared to vehicle controls, p<0.0005. Furthermore, histopathological analyses of tumor center slices from Capan-1 and Panc-1 models showed induction of significant tumor necrosis with SIL-204 compared to vehicle controls. Conclusions: The preclinical results of SIL-204-MP are promising and as the potential to further enhance the anti-tumor effect demonstrated by siG12DLoder in pancreatic patients. SIL-204 is now in toxicology studies as preparation for clinical trials in non-resectable pancreatic cancer.

Phase I study of autologous CD8+ and CD4+ transgenic T cells expressing high-affinity KRAS G12V mutation-specific T cell receptors (FH-A11KRASG12V-TCR) in patients with metastatic pancreatic, colorectal, and non-small cell lung cancers with KRAS G12V mutations.

TPS792 Background: KRAS, the most common driver oncogene in human malignancies, has predictable mutations. 85% of RAS mutations occur at the Glycine (G) 12 position with G12V being amongst the most common. Cancers driven by mutant KRAS include pancreatic (PDAC), colorectal (CRC), and non-small cell lung (NSCLC) malignancies. The prognosis of patients with activating KRAS mutations remains poor. Preliminary anti-tumor activity has been observed with T cell receptor (TCR) T cell therapies targeting KRAS mutations, but durable clinical benefit is lacking. FH-A11KRASG12V-TCR is an autologous CD8+ and CD4+ transgenic T cell product expressing (1) an HLA-A*11-restricted, high affinity KRASG12V mutation-specific T cell receptor (TCR) and (2) the wildtype CD8α/β coreceptor enabling CD4+ T cell recognition of KRAS G12V presented on an MHC class I allele mediating helper T cell activity and cytotoxicity. The TCR sequence is derived from the peripheral blood of a healthy HLA-A*11+ adult based on a strong pre-clinical dataset. Methods: This is an ongoing, investigator-initiated, phase 1, first-in-human, single-center, dose escalation study of FH-A11KRASG12V-TCR. The aims are to evaluate the safety, tolerability, maximum tolerated dose (MTD), and preliminary anti-tumor activity of FH-A11KRASG12V-TCR in patients with advanced PDAC, CRC, and NSCLC who express the HLA-A*11-restricted KRAS G12V mutation. Dose modification is guided by the Bayesian Optimal Interval design (BOIN). Patients are initially treated in cohorts of 3 at each dose level (DL). BOIN will be used to identify the recommended dose for subsequent patients. The planned target doses are 5 x 10 9 transgenic TCR T cells in DL 1 with dose escalation up to 15 x 10 9 transgenic TCR T cells in DL 3. This study enrolls patients who are 18 years or older, HLA-A*11:01-positive, with KRAS G12V mutated metastatic PDAC, CRC, or NSCLC. Patients must have progressed on or be intolerant of at least one line of prior therapies including any targeted therapies indicated for their current malignancy. Patients undergo leukapheresis prior to treatment and receive lymphodepleting chemotherapy with either cyclophosphamide intravenously (IV) and fludarabine IV on days -6, -5, and -4 or bendamustine IV on days -4 and -3. FHA11KRASG12V-TCR is administered IV on day 0. The dose-limiting toxicity observation period is 28 days. Patients may receive an additional FHA11KRASG12V-TCR IV infusion at the same or lower dose as soon as 28 days or up to 1 year after the first infusion. The study is actively recruiting. Clinical trial information: NCT06043713 .

SMAD4 and KRAS Status Shape Cancer Cell-Stromal Crosstalk and Therapeutic Response in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) contains an extensive stroma that modulates response to therapy, contributing to the dismal prognosis associated with this cancer. Evidence suggests that PDAC stromal composition is shaped by mutations within malignant cells, but most previous work has focused on pre-clinical models driven by KrasG12D and mutant Trp53. Elucidation of the contribution of additional known oncogenic drivers, including KrasG12V mutation and Smad4 loss, is needed to increase understanding of malignant cell-stroma crosstalk in PDAC. Here, we used single-cell RNA-sequencing to analyze the cellular landscape of Trp53-mutant mouse models driven by KrasG12D or KrasG12V in which Smad4 was wild-type or deleted. KrasG12D Smad4-deleted PDAC developed a fibro-inflammatory rich stroma with increased malignant JAK/STAT cell signaling and enhanced therapeutic response to JAK/STAT inhibition. SMAD4 loss in KrasG12V PDAC differently altered the tumor microenvironment compared to KrasG12D PDAC, and the malignant compartment lacked JAK/STAT signaling dependency. Thus, malignant cell genotype impacts cancer cell and stromal cell phenotypes in PDAC, directly affecting therapeutic efficacy.

Associations of KRAS Point Mutations with Survival of Patients Who Underwent Curative-Intent Resection of Colorectal Liver Metastases.

The oncologic significance of specific KRAS point mutations for patients with colorectal liver metastases (CLM) is uncertain. This study aimed to assess the prognostic impact of KRAS point mutations on patients who underwent surgery for CLM. Patients who underwent curative-intent surgery for CLM from 2001 to 2020 were selected for the study. In the study, KRAS point mutations and other clinicopathologic variables were examined for association with survival. The study classified 798 patients into five groups by KRAS mutation status as follows: wild-type (n = 412, 51.6%), G12D (n = 123, 15.4%), G12V (n = 88, 11.0%), G13D (n = 61, 7.6%), and "Other" mutations (n = 114, 14.3%). For the patients with G12V substitutions, TP53 mutation was associated with worse overall survival (OS) (hazard ratio [HR], 2.64; 95% confidence interval [CI], 1.04-6.66; P = 0.041), but was not associated with a survival difference for the other four groups. The patients with co-occurring KRAS G12V and TP53 had a median OS of 4.4 years and a 5-year OS rate of 39.8%. In contrast, the patients with KRAS G12V mutation and wild-type TP53 had a median OS of 7.3 years and a 5-year OS rate of 75.9%, similar to the corresponding values for the patients with wild-type KRAS. Co-occurring KRAS G12V and TP53 mutations were independently associated with worse OS in the entire cohort (HR, 2.08; 95% CI, 1.15-3.76; P = 0.015). This study showed that KRAS G12V mutation is associated with worse OS for patients undergoing curative-intent CLM resection, but only those with co-occurring TP53 mutation. Prognosis after surgery for CLM should not be stratified by KRAS mutation site alone.

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.

Association of Oncogene Driver Mutations with Recurrence and Survival in Stage I Non-small Cell Lung Cancer

Stage I nonsmall cell lung cancer (NSCLC) is primarily treated with surgical resection and has a favorable prognosis with an expected recurrence rate of 30%. New methods to risk stratify patients with stage I NSCLC are needed to help select those that might benefit from more active surveillance or adjuvant therapy. We analyzed clinical data from 1330 patients (1469 tumors) with NSCLC and correlated it with next-generation sequencing (NGS). To reduce the potential confounding variables of stage and treatment, this analysis only included patients with stage I NSCLC in whom surgical resection was the primary treatment. In 570 patients (600 tumors), 75 (12.5%) developed recurrence. Recurrence occurred in 37.5% of patients with KRAS G12V mutation versus 11.1% of patients without this mutation (P < .001). A lower chance of recurrence was associated with "any EGFR" mutation (6.74% vs. 14.9%, P = .006). A history of coronary artery disease (CAD) increased the chance of recurrence: OR 2.7 (1.57-4.89, P < .001). Shorter survival was predicted by KRAS G12V (P = .009) and "other TP53" mutation (P = .025). KRAS G12V, KRAS G13D, MET E168D, PTEN, and "other TP53" were oncogene mutations associated with reduced survival in stage I NSCLC. CAD, type 2 diabetes (DM2), and "other cancer" were medical comorbidities associated with reduced survival in stage I NSCLC. Oncogene mutations such as KRAS G12V and EGFR may have implications for cancer surveillance strategies and inform future treatment trials of stage I NSCLC.

Abstract A020: Enhanced cell-free RNA (cfRNA) recovery for liquid biopsy applications: Comparative analysis using controlled RNA samples for early and advanced cancer detection

Abstract Introduction: Extracting cfRNA from biological samples is crucial in liquid biopsy applications, particularly for detecting low-frequency mutations like KRAS G12V, essential for early cancer diagnosis and minimal residual disease (MRD) monitoring. Traditional cfRNA extraction methods involve multiple transfer steps, aliquoting, and pooling, leading to cfRNA loss and affecting mutation detection sensitivity and accuracy. The nRichDX Revolution cfTNA Max 20 kit, with its innovative nRicher cartridge, simplifies the extraction process by eliminating these extra steps, potentially enhancing yield and efficiency. This study evaluates the cfRNA recovery efficiency of the nRichDX kit compared to the Qiagen Circulating Total Nucleic Acid kit. Methods: Plasma samples (5 mL each) were spiked with RNA containing KRAS G12V mutation at concentrations ranging from 100 to 2 million copies, mimicking early-stage cancer detection and MRD monitoring, where low copy numbers represent early disease and high copy numbers reflect advanced cancer stages. cfRNA was extracted using the nRichDX and Qiagen kits, with each condition tested in triplicate. cfRNA recovery efficiency was assessed using quantitative reverse transcription PCR (qRT-PCR) to quantify KRAS G12V RNA copies recovered. High-sensitivity RNA ScreenTape Analysis was also used to evaluate cfRNA profile quality. Results: The nRichDX kit recovered significantly higher cfRNA yields than Qiagen across all concentrations. Recovery rates were consistent and reproducible, with over 70% of input RNA recovered, even at lower inputs (100 to 100,000 copies), where recovery rates were approximately 70-85%. High Sensitivity RNA ScreenTape Analysis showed a linear increase in 18S and 28S ribosomal peaks and high RNA Integrity Number (RIN) scores in nRichDX profiles. The ability to robustly recover low copy numbers is significant for early cancer detection and MRD monitoring. In contrast, the kit’s effectiveness in high copy number samples reflects its ability to monitor advanced cancer stages accurately. Conclusion: The nRichDX cfTNA kit demonstrates exceptional efficiency in cfRNA extraction from plasma samples across a broad dynamic range, from as few as 100 copies to as many as 2 million copies of the KRAS G12V mutation. Its versatility makes it suitable for various cancer detection and monitoring applications. The streamlined process offered by the nRicher cartridge minimizes sample handling and reduces cfRNA loss, enhancing performance. This translates to more accurate and sensitive detection of early-stage and high-burden mutations associated with advanced cancer. The high-quality profiles indicated by RNA ScreenTape Analysis further validate the reliability of the nRichDX kit, making it a valuable tool for liquid biopsy applications, with significant advantages in early cancer detection and monitoring disease progression. Citation Format: Nafiseh Jafari, Mayer Saidian, Jason Saenz, Andrew Dunnigan, Carlos Hernandez, Leila Aghili. Enhanced cell-free RNA (cfRNA) recovery for liquid biopsy applications: Comparative analysis using controlled RNA samples for early and advanced cancer detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A020.

More T cell receptors to the RAScue in cancer?

Treatment with T cells genetically engineered to express tumor-reactive T cell receptors (TCRs), known as TCR-gene therapy (TCR-T), is a promising immunotherapeutic approach for patients with cancer. The identification of optimal TCRs to use and tumor antigens to target are key considerations for TCR-T. In this issue of the JCI, Bear and colleagues report on their use of in vitro assays to characterize four HLA-A*03:01- or HLA-A*11:01-restricted TCRs targeting the oncogenic KRAS G12V mutation. The TCRs were derived from healthy donors or patients with pancreatic cancer who had received a vaccine against mutant KRAS. The most promising TCRs warrant testing in patients with KRAS G12V-positive cancers.

Mutant KRAS in Circulating Tumor DNA as a Biomarker in Localized Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy.

The primary objective was to determine the prognostic significance of circulating tumor DNA (ctDNA) in patients receiving neoadjuvant chemotherapy (NAC) for localized pancreatic ductal adenocarcinoma (PDAC) using digital droplet polymerase chain reaction (ddPCR). Increasingly, ctDNA is being used for clinical decision-making in a variety of solid malignancies. However, the detection and prognostic value of KRAS ctDNA as assessed by ddPCR during NAC has yet to be characterized. Patients with localized PDAC eligible to receive NAC were prospectively enrolled. Peripheral blood samples were obtained at diagnosis, after NAC, and after resection and analyzed for ctDNA using ddPCR. Log-rank tests and Cox proportional hazards model were used to assess for association with OS. 84 patients were included in the analysis. Mutant KRAS ctDNA was detected in 49.3% of patients at diagnosis, 69.6% of patients after NAC, and 69.7% of patients after resection, respectively. There were 15 (17.9%) patients that cleared mutational ctDNA over the course of treatment. Clearance of ctDNA during NAC was associated with improved overall survival (OS) (18.4 mo. vs NR, P<0.05). Detection of mutant KRAS G12V after NAC and resection was associated with shorter OS (18.0 versus NR months, P<0.031). Detection of the KRAS G12V mutation after resection was associated with reduced OS (aHR 36.75, 95% CI 2.93-461.38). Throughout treatment, KRAS ctDNA is detectable by ddPCR in patients with localized PDAC treated with NAC. Detection of mutant KRAS G12V after resection was associated with reduced OS.

100 (PB088): KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications.

100 (PB088): KRASmulti inhibitor BI 3706674 shows efficacy in KRAS-driven preclinical models of cancer that supports clinical testing in patients with tumors harboring KRASG12V mutations and KRAS wild-type amplifications.

Clinical characteristics of KRAS mutation subtypes in non-small cell lung cancer population in Xinjiang, China, and their impact on the prognosis of immunotherapy

PurposeNon-small cell lung cancer (NSCLC) is a highly fatal malignancy. The Kirsten rat sarcoma viral oncogene (KRAS) gene profoundly impacts patient prognosis. This study aims to explore the correlation between KRAS mutation subtypes, clinical data, and the impact of these subtypes on immunotherapy.Materials and methodsTumor samples from 269 NSCLC patients at the Affiliated Cancer Hospital of Xinjiang Medical University were analyzed. Patients received first- or second-line therapy without targeted therapy. Molecular and clinical data were used to analysis KRAS mutation subtypes and treatment outcomes.ResultsKRAS mutations predominantly included G12C, G12D, and G12V subtypes. TP53 had the highest mutation frequency among KRAS mutations, followed by MST1, STK11, and KMT2C. Gender differences were noted among KRAS mutation subtypes, with G12C and G12V mutations prevalent in males, while G12D mutations were less common among males. Smokers exhibited varied KRAS mutation subtypes, with G12C and G12V prevalent in smokers and G12D in nonsmokers. KRAS mutations were mainly in lung adenocarcinoma. TTF-1 and PD-L1 expression differed significantly among KRAS mutations. Patients with G12C and G12V mutations showed higher TMB levels and better immunotherapy outcomes compared to those without KRAS mutations. Conversely, patients with G12D mutations had poorer immunotherapy responses.ConclusionsKRAS mutation subtypes exhibit distinct clinical and molecular characteristics and varying responses to immunotherapy. G12C and G12V mutations correlate with better immunotherapy outcomes, while G12D mutations are associated with poorer responses.

Identification and structural characterization of a mutant KRAS-G12V specific TCR restricted by HLA-A3.

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3β and CDR1β formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.

Molecular and clinical characteristics of patients with non-small-cell lung cancer (NSCLC) harboring KRAS G12V mutations.

8618 Background: KRAS G12V is one of the most common drivers of non-small cell lung cancer (NSCLC), accounting for around 3 percent of cases. Recently, there have been promising approaches in early clinical research to therapeutically target this mutation. However, this subset of patients is poorly characterized both molecularly and clinically, and data on therapy-dependent outcome are lacking. We performed this real world analysis to gain insight into the genomic and clinical characteristics of a large cohort of NSCLC patients with KRAS G12V mutations. Methods: Molecular data of 662 UICC stage I-IV lung cancer patients diagnosed between 2018 and 2023 and harbouring a KRAS G12V mutation in their tumor tissue were linked to the medical record data in the Network Genomic Medicine (NGM) database. The mutational status was determined by next-generation sequencing (NGS) and further immunohistochemistry (IHC) for PD-L1 status was performed. Results: 662 patients with a KRAS G12V mutation were diagnosed between 01/2018 and 03/2023 (stage I: 33 (6.0%), II: 29 (5.3%), III: 89 (16.3%), IV: 396 (72.4%)), which is a proportion of 15.3% among all registered KRAS-mutated lung cancer patients in this period (n=4332). Among the KRASG12V positive primary lung carcinomas, 605 (94.3%) showed an adenocarcinoma and 17 (2.6%) a squamous cell carcinoma. The median age at diagnosis was 67 years (range: 33-91), of which 338 were men (52.6%) and 304 women (47.4%). The majority of the cohort were active or former smokers (active: 289 (54.9%); former: 208 (39.2%)). In the group of never smokers (n=33, 6.2%), there were 5 men (15.2% of never smokers) and 28 women (84.8% of never smokers). The most common mutation site at initial diagnosis in patients with metastatic disease was brain and bone metastases with 41.5% and 37.4%, respectively. The most common co-mutations with a frequency of ≥ 5% were TP53 (40.2%), STK11 (28.3%), KEAP1 (22.6%) and NTRK1-3 (8.7%). Preliminary median overall survival at UICC stage IV was 525 (95% CI: 427 – 579) days. The first PD-L1-independent analysis showed a response rate of 40.9% (18/44) to initial chemotherapy (CTX)/immune-checkpoint blockade (ICB) combination, 66.7% (8/13) to ICB alone and 18.8% (6/32) to CTX alone. Conclusions: The cohort is characterized by heavy tobacco consumption. Among the never-smokers, the high proportion of women is noteworthy and will be analyzed further. Metastases at first diagnosis and concurrent mutations show clear differences to other KRAS subtypes, which highlights the heterogeneity of KRAS subtypes. We will continue to analyze the overall prognosis as well as the first indications of a PD-L1-independent benefit of immunotherapy compared to chemotherapy alone.

Synergistic inhibition of CXCL8 following MRTX1133 treatment with ONC212 or 5-FU in KRAS G12D and G12V mutant CRC and PDAC cell lines.

e15196 Background: CXCL8, serves as a crucial multifunctional cytokine that plays a significant role in regulating tumor growth, invasion, and migration. Although in some cancers the critical role of CXCL8 in patient prognosis and disease progression is stablished but its role in CRC &amp; PDAC patients is under investigation. High levels of CXCL8 in tumor microenvironment (TME) and association with cancer stem cell survival, immune surveillance escape &amp; metastasis make it an interesting therapeutic target. Both CRC &amp; PDAC are among the deadliest cancers with dismal prognosis. The need for novel therapeutics with a focus on TME is emerging. MRTX1133 was identified as an inhibitor of KRASG12D. ONC212 is a fluorinated imipridone with strong anti-cancer activity in nM range and preclinical efficacy against pancreatic and other malignancies. Here we report the synergistic inhibition of CXCL8 in both KRAS G12D &amp; KRAS G12V cell lines with use of KRAS G12D inhibitor in combination with 5-FU or ONC212. Methods: Different CRC &amp; PDAC cells were treated with MRTX1133 with 5-FU or ONC212 cytokine profiling at 48h time point was done. Human protein atlas was used for protein and RNA data &amp;TCGA data for survival was used. Results: We previously have shown the synergistic inhibition of pERK and cytokine alteration between MRTX1133 KRAS G12D inhibitor with 5FU or ONC212 in both cell lines with KRAS G12D &amp; KRAS G12V mutation (Tajiknia V et al. AACR 2023 annual meeting). In LS513 KRAS G12D CRC cell line, treatment of ONC212, MRTX1133 and combination of both showed a decrease of 52.36%,58.83% and 71.65% respectively in CXCL8 levels compared to control at 48h time point. In HPAF-II KRAS G12V PDAC cell line, treatment of ONC212, MRTX1133 and combination of both resulted in 38.69%, 64.44%, 57% inhibition of CXCL8 levels respectively compared to control. In SW480 KRAS G12V CRC cell line, treatment of ONC212, MRTX1133 and combination of both demonstrated 25.7%,47.3% and 66.5% inhibition in CXCL8 level respectively compared to control at 48h time point. Single treatment of MRTX1133 caused a decline in CXCL8/IL-8 levels by 30% while single treatment of 5-FU showed a 36% decrease, the combination of both resulted in more than 55% decrease compared to control in LS513 KRAS G12D cell line. In SW480 KRAS G12V cell line, combination of 5-FU &amp; MRTX1133 showed 30% inhibition while in Capan-2 PDAC KRAS G12V cells the same combination showed a 48.2% reduction in CXCL8 compared to control at 48h time point. TCGA data for RNA shows low specificity of CXCL8 for cancer type but it is significantly high in CRC and PDAC. Protein concentration in the Pan-cancer cohort shows significant high level of CXCL8 in CRC. Conclusions: Considering the important role of KRAS mutations and CXCL8 in pathogenesis of CRC &amp; PDAC, the effective inhibition of CXCL8 with the use of targeted therapy in combination could enhance treatment response and patient survival.

AFNT-211: A phase 1 study of autologous CD4+ and CD8+ T cells engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR, a CD8α/β coreceptor, and a FAS41BB switch receptor in patients with advanced/metastatic solid tumors.

TPS8650 Background: Activating mutations in KRAS (including KRAS G12V) are well-described oncogenic drivers in solid tumors. Patients with KRAS driver mutations have a poor prognosis, and most KRAS-mutated cancers lack effective therapies. T cell receptor (TCR)-T cell therapies targeting mutant KRAS have demonstrated proof of concept in the clinic, but duration of response remains a challenge. AFNT-211 represents a novel strategy to address the immunosuppressive tumor microenvironment and improve response rate and duration in solid tumors. AFNT-211 autologous CD4+ and CD8+ T cells are engineered to express a high avidity HLA-A*11:01-restricted, KRAS G12V-specific, transgenic TCR; the wildtype CD8α/β coreceptor; and a FAS-41BB switch receptor. The intended mechanism of action is recognition and elimination of KRAS G12V-mutated tumor cells by AFNT-211. AFNT-211 is designed to enhance anti-tumor activity via the following mechanisms of action as shown in preclinical models: (1) the CD8α/β coreceptor enables MHC-I– restricted TCR recognition of the KRAS G12V target by CD4+ T cells; (2) engaging CD4+ helper T cell responses drives enhanced CD8+ T cell cytotoxic activity and prevents T cell exhaustion; (3) the FAS-41BB chimeric switch receptor drives increased T cell activity, circumventing native FAS apoptotic signaling. Methods: This ongoing Phase 1, first-in-human, multicenter, open-label study of AFNT-211 (1) evaluates safety/tolerability, (2) determines the optimal biological dose (OBD) and recommended Phase 2 dose (RP2D), and (3) assesses preliminary antitumor activity. The study consists of 2 parts: dose escalation and dose expansion. Dose escalation is guided by the Bayesian optimal interval Phase 1/2 (BOIN12) design to determine the OBD. The BOIN12 design quantifies the desirability of a dose in terms of toxicity-efficacy tradeoff and adaptively allocates patients to the dose with the highest estimated desirability. The RP2D will be selected based on the BOIN12 design and the totality of benefit/risk assessment. Patients enrolling in dose expansion will be treated at the OBD/RP2D in indication-specific cohorts. This study is conducted in patients aged ≥ 18 years who are HLA-A*11:01-positive with advanced/metastatic solid tumors harboring a KRAS G12V mutation, and intolerance of/progression on at least 1 prior systemic therapy. Patients undergo leukapheresis to collect T cells for the manufacturing of AFNT-211 and receive lymphodepleting chemotherapy prior to AFNT-211 infusion. This is followed by a 28-day dose-limiting toxicity observation period (dose escalation only) and a post-treatment follow-up period for 24 months/ until disease progression. The study is open for recruitment in the United States. Clinical trial information: NCT06105021 .

Results of a first-in-human, dose-escalation phase 1 study of the ERK1/2 inhibitor ATG-017 in patients with advanced solid tumors.

e15114 Background: This open-label, first-in-human, phase 1 study evaluates the safety, pharmacokinetics, and maximum tolerated dose (MTD) of ATG-017, an oral, potent, and highly selective inhibitor of ERK1/2, in patients with refractory advanced solid tumors. Methods: Twenty-one patients with solid tumors harboring activating alterations in the RAS-MAPK pathway were enrolled in the 3+3 design dose-escalation phase for continuous dosing: 5 mg QD (n=1), 5 mg BID (3), 10 mg BID (3), 20 mg BID (7), 30 mg BID (4), and 40 mg BID (3). Results: Safety: All patients reported at least 1 treatment-emergent adverse event (TEAE). Fourteen patients (66.7%) experienced serious adverse events. At 40mg BID, 2 of 3 patients experienced a dose-limiting toxicity (DLT) (grade 3 diarrhea and grade 3 retinopathy). At 30mg BID, 2 of 4 patients experienced a DLT (grade 3 acneiform dermatitis and grade 2 blurred vision). All DLTs resolved with drug interruption. At 20mg BID, a potentially biologically active dose, 7 patients were treated with no DLTs observed. At all dose levels, the most commonly observed TEAEs were consistent with previously reported toxicities with other ERK pathway inhibitors (gastrointestinal, skin, and ocular adverse events). Pharmacokinetics: Between 5mg BID and 30mg BID, exposure increased proportionally. At doses 20mg BID or higher, the concentration in the whole dosing interval (≥ 328ng/mL, geometric mean) was higher than the target concentration (231 ng/mL) for effective ERK inhibition derived from preclinical PK/PD models. Efficacy: Of the 21 treated patients, one patient (4.8%) with mesonephric-like ovarian adenocarcinoma (KRAS G12V mutation) in the 30 mg BID cohort achieved a best response of partial response (sustained for six cycles of treatment), and 8 (38%) patients were assessed as having stable disease (sustained over an average of five cycles), based on RECIST v1.1. Conclusions: In patients with solid tumors, 20mg BID continuous dosing of ATG-017 was identified as the MTD and delivers exposure that is likely to be active in inhibiting ERK. This dose is currently being explored in a dose expansion phase. A combination regimen with nivolumab is also being investigated. Clinical trial information: NCT04305249 .

Novel specific TCRs targeting KRASG12V-related cancers.

e14528 Background: KRAS mutations act as oncogenic drivers in a variety of tumors, such as pancreatic, colorectal and lung cancer. Among these, the KRASG12V mutation, being one of the most common mutations, is associated with adverse outcomes in patients. KRAS has been regarded as an “undruggable” target, and no clinical trials targeting tumors carrying shared KRASG12V mutation have been initiated. Notably, KRASG12D specific TCR-T has shown high effectiveness, including the regression of metastases in patients with pancreatic cancer. Therefore, TCR-engineered T cells could be a promising strategy for the treatment of solid tumors harboring G12V mutation. Methods: We have developed an innovative approach to rapidly and sensitively identify natural TCRs from single T cell derived from patients using CorreGene’s Single T cell TCR Cloning platform, thus obviating the requirement for NGS sequencing and TCR gene synthesis. Additionally, natural TCRs can be refined with SMART-TCR system, utilizing T cell reporter cell lines to construct a high-throughput T-cell Display Platform for functional screening of TCRs. This system is meticulously designed to enrich TCRs with superior potency and specificity from a natural TCR mutation library. We performed a thorough assessment of each TCR identified, evaluating them across both in vitro and in vivo settings. Results: In the present study, we identified three natural TCRs from patients' TILs, designated as KVA11-N01, KVA11-N02 and KVA11-N04 respectively, that specifically recognized the HLA-A*11:01-restricted KRAS G12V epitope. KVA11-N01 exhibited lower affinity and potency, prompting us to develop an affinity-optimized variant, KVA11-M01-231, using the SMART-TCR system. For comparison, we used the murine TCR TRAV3-301/BV401, identified by NCI from HLA-A11:01 transgenic mice immunized with KRAS G12V7-16, as a benchmark. Three of our TCRs, including KVA11-N02, KVA11-N04 and the engineered KVA11-M01-231, outperformed TRAV3-301/BV401 in both in vitro and in vivo assays. These three TCRs exhibited excellent expression intensity and stability on T cells, and notably, their IFN-γ release potency surpassed that of TRAV3-3*01/BV4*01. Furthermore, in vivo experiments confirmed the significant tumor suppression efficacy of our TCRs in a tumor-bearing mouse model, showing enhanced tumor growth inhibition compared to TRAV3-301/BV401. TCRs from Corregene’s platform exhibited exceptional specificity, with no allo-reactivity, no reaction to KRASG12V-negative cell lines, and no off-target effects. Additionally, all our TCRs demonstrates favorable tolerance in mice during in vivo studies. Conclusions: TCRs via Corregene’s Single T cell TCR Cloning platform and SMART-TCR system showed significantly excellent anti-tumor activities with great specificity and offers a promising solution for the treatment of KRASG12V-related cancers.

Association of KRAS G12D vs. G12V circulating tumor DNA variant allele fraction and real-world overall survival in metastatic non-small cell lung and colorectal cancers.

3041 Background: We previously reported KRAS G12D but not G12V variant allele fraction (VAF), as measured in circulating tumor DNA (ctDNA), was associated with overall survival (OS) in metastatic treatment-naïve pancreatic cancer (mPDAC). However, this has not been examined in other RAS-driven cancers. Here we assessed association of real-world OS (rwOS) with KRAS G12D and G12V VAF in metastatic colorectal cancer (mCRC) and non-small cell lung cancer (mNSCLC). Methods: We queriedGuardantINFORM, a real-world evidence database of aggregated commercial payer health claims and de-identified results from Guardant360 (G360) ctDNA testing from 2014 to 2023, to identify mNSCLC/mCRC patients who were treatment-naive and had KRAS G12D or G12V mutations identified &lt; 30 days prior to first-line therapy initiation. Those with co-occurring KRAS alterations, KRAS amplifications, and/or MSI-H were excluded. Outcomes were assessed as above vs. below median VAF and stratified by treatment type. The Cox regression model was run for continuous VAF, adjusting for age, gender, summary comorbidity score [Elixhauser Comorbidity Index (ECI)], and year of G360. Results: For mCRC, KRAS VAF above median was significantly associated with shorter rwOS for both G12D and G12V (Table 1); subgroup analysis of the predominant standard-of-care chemotherapy, FOLFOX, showed similar findings. For mNSCLC, KRAS VAF above median was associated with shorter rwOS for G12V, but not G12D (Table). Subgroup analysis by first-line treatment type revealed this association to be similar for chemotherapy-containing regimens but not immunotherapy (IO) alone. Conclusions: While a variant-specific association of KRAS VAF with rwOS was observed in mNSCLC overall (G12V associated, G12D not), above vs below median VAF was not associated with rwOS for the subset of patients receiving IO only. In contrast, for mCRC both G12D and G12V VAF were associated with rwOS, including for the subset of patients who received chemotherapy. These data suggest a complex interplay between KRAS variant, ctDNA shedding, first-line treatment, and OS that will be important in the interpretation of the prognostic implications of ctDNA-identified KRAS alterations. [Table: see text]

TTF-1 negativity as a biomarker of outcomes to immunotherapy, chemoimmunotherapy, and KRAS G12C inhibitors in lung adenocarcinoma.

8608 Background: Thyroid transcription factor 1 (TTF-1) expression is routinely assessed in lung adenocarcinomas (LUAD) and is negative (TTF-1Neg) in 15-20% of cases. Whether these tumors differ from LUAD expressing TTF-1 (TTF-1Pos) in terms of biologic features and outcomes is unclear. Methods: Patients (pts) with LUAD and available TTF-1 expression by IHC at five institutions were included. Clinicopathologic, genomic, and outcomes data were compared according to TTF-1 expression. Results: Among 3,315 pts with LUAD, TTF-1 was negative in 15% (N=499). Compared to TTF-1Pos (N=2,816), pts with TTF-1Neg LUAD were more likely male (44% vs 38%, P=0.01), smokers (82% vs 74%, P&lt;0.001), with stage IV disease at diagnosis (76% vs 71%, P&lt;0.001), and lower median PD-L1 tumor proportion score (TPS, 1% vs 10%, P&lt;0.001). At stage IV diagnosis, TTF-1Neg cases less commonly had brain metastasis than TTF-1Pos cases (35% vs 44%, P=0.04), but more frequently had bone metastasis (53% vs 45%, P=0.03). TTF-1Neg LUAD was enriched for KRAS, STK11, KEAP1, SMARCA4, and CDKN2A mutations (q&lt;0.05), while TTF-1Pos LUAD was enriched for EGFR and MET mutations (q&lt;0.05). Compared to TTF-1Pos LUAD, TTF-1Neg LUAD had higher prevalence of KRAS G12D (12% vs 4%, P&lt;0.001) and G12V mutations (11% vs 7%, P=0.001), but similar frequency of G12C mutations (14% vs 15%, P=0.82). The association between TTF-1 and treatment outcomes was next investigated. Pts with stage III unresectable TTF-1Neg LUAD who received durvalumab after chemo-radiation (N=20), compared to TTF-1Pos cases (N=174), had shorter median progression-free survival (mPFS, 7.6 vs 24.7 months, P=0.01) and overall survival (mOS, 22.7 months vs not reached, P=0.01). Among pts with stage IV LUAD, TTF-1Neg cases treated with immune checkpoint inhibitors (N=237), compared to TTF-1Pos (N=1,161), had worse objective response rate (ORR, 17% vs 28%, P&lt;0.001), mPFS (2.5 vs 4.7 months, P&lt;0.001) and mOS (9.8 vs 20.9 months, P&lt;0.001). Similarly, pts with TTF-1Neg LUAD (N=297) treated with chemoimmunotherapy, compared to TTF-1Pos cases (N=920), had worse ORR (27% vs 42%, P&lt;0.001), mPFS (4.6 vs 8.1 months, P&lt;0.001) and mOS (11.2 vs 23.4 months, P&lt;0.001). TTF-1 negativity retained its association with worse outcomes after adjusting for treatment line, ECOG performance status, smoking status, PD-L1 TPS, STK11/ KEAP1 mutation status in multivariable cox regression models. Lastly, pts with KRAS G12C mutated TTF-1Neg LUAD treated with KRAS inhibitors (N=25), compared to TTF-1Pos (N=138), had worse ORR (12% vs 35%, P=0.03), mPFS (2.7 vs 5.9 months, P&lt;0.001), and mOS (4.4 vs 12.1 months, P&lt;0.001). After excluding mucinous LUAD cases, which are more commonly TTF-1Neg, similar results were observed. Conclusions: TTF-1 negativity identifies a unique clinicopathologic and genomic subset of LUAD with worse outcomes to diverse systemic therapies.