Impact of KRASG12D subtype and concurrent pathogenic mutations on advanced non-small cell lung cancer outcomes

Purpose Mutations in the Kirsten rat sarcoma viral (KRAS) oncogene constitute a significant driver of lung adenocarcinoma, present in 10–40% of patients, which exhibit heterogeneous clinical outcomes, mainly driven by concurrent genetic alterations. However, characterization of KRAS mutational subtypes and their impact on clinical outcomes in Latin America is limited. Methods A cohort study was conducted at the National Cancer Institute (INCan) of Mexico. Individuals with advance-staged of adenocarcinoma and KRAS mutations, detected by next-generation sequencing, having undergone at least one line of therapy were included for analysis. Clinical and pathological characteristics were retrieved from institutional database from June 2014 to March 2023. Results KRAS was identified in fifty-four (15.6%) of 346 patients, among which 50 cases were included for analysis. KRASG12D (n = 16, 32%) and KRASG12C (n = 16, 32%) represented the most prevalent subtypes. KRASG12D mutations were associated with female (p = 0.018), never smokers (p = 0.108), and concurrences with EGFR (25.0% vs. 17.6%, p = 0.124) and CDKN2A (18.8% vs. 14.7%, p = 0.157). KRASG12D patients showed a better ORR (66.6% vs. 30.0%; OR 4.66, 95% CI 1.23–17.60, p = 0.023) and on multivariate analysis was significantly associated with better PFS (HR 0.36, 95% CI 0.16–0.80; p = 0.012) and OS (HR 0.24, 95% CI 0.08–0.70; p = 0.009). Conclusions To our knowledge, this study represents the first effort to comprehensively characterize the molecular heterogeneity of KRAS-mutant NSCLC in Latin American patients. Our data reinforce the current view that KRAS-mutated NSCLC is not a single oncogene-driven disease and emphasizes the prognostic impact of diverse molecular profiles in this genomically defined subset of NSCLC. Further validation is warranted in larger multicenter Latin American cohorts to confirm our findings. Supplementary Information The online version contains supplementary material available at 10.1007/s12094-023-03279-2.


Introduction
Lung cancer (LC) is the leading cause of cancer-related mortality worldwide, with 1.70 million deaths and 2.2 million new cases in 2020 [1].In recent years, mutational characterization of lung cancer has improved its therapeutic outcomes.Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) represent the most frequent oncogene alterations in NSCLC, with variable incidences across ethnicities, being less prevalently in East Asian (5-11%) and Latin American countries (14%) than in Caucasian patients (25-40%) [2,3].Most KRAS alterations occur in codon 12 (80%), mainly as a substitution of glycine by cysteine (G12C) in 39-40% of cases, followed by valine (G12V) in 17-21%, aspartate (G12D) in 14-17%, or alanine (G12A) in 9-10% [4].These mutations impair GTP hydrolysis by GTPase-activating proteins (GAPs), triggering KRAS-derived signaling through MAPK and PI3K-AKT-mTOR pathways.Despite their prevalence, the prognostic impact of KRAS mutations remains uncertain owing to their highly heterogeneous clinical course and variable response to current therapies.For instance, KRAS G12D mutation has been linked to inferior clinical outcomes among patients with KRAS-mutated NSCLC Extended author information available on the last page of the article who underwent PD-L1 blockade [5].Coexisting genomic alterations may explain this prognostic significance, potentially representing predictive biomarkers in immunotherapy setting.These include mutations in tumor protein 53 (TP53), serine/threonine 11 (STK11), and Kelch-like ECH-associated protein 1 (KEAP1), alterations in Mesenchymal Epithelial Transition (MET), and loss of cyclindependent kinase 2A (CDKN2A) [6].Understanding the role of co-occurring genomic alterations in KRAS-mutated tumors is critical for developing effective personalized treatments and improving patient's outcomes; however, they have shown inconsistent effects across various studies [7].Therefore, this study aims to analyze clinicopathological and genomic characteristics of Latin American patients with KRAS-mutated advanced NSCLC, focusing on their impact on therapeutic outcomes.

Patients and methods
An observational longitudinal cohort study was conducted on 346 patients previously diagnosed with advanced NSCLC from June 2014 to March 2023 at the Thoracic Oncology Unit of the Instituto Nacional de Cancerología (INCan).Consecutive patients with confirmed advanced NSCLC harboring a KRAS mutation detected by nextgeneration sequencing were eligible.Patients who received at least one line of anticancer therapy were included in the analysis.Response was evaluated according to RECIST v1.1 [8].Clinical and pathological data, including baseline patient characteristics, treatment regimens, therapeutic efficacies, and survival, were collected from electronic medical records.This study protocol was approved by the institutional review board (CEI/1375/19).

Samples processing
Available formalin-fixed and paraffin-embedded tissues (FFPE) were analyzed by the institutional pathology department, which performed histologic diagnosis and quantification of the percentage of neoplastic cellularity in each sample.The procedure for DNA extraction and purification was carried out using QI Amp DNA FFPE tissue kit (QIAGEN, Netherlands, USA, Cat.Number: 56404).Concentration and integrity of genetic material were measured using a 2100 bioanalyzer system (Agilent, California, EUA, #G2939BA).Three different kits were used to evaluate KRAS mutations and their concurrences: AmpliSeq Cancer HotSpot Panel v2, TruSeq Amplicon Cancer Panel, and Foundation One (FO).Gene mutations analysis included those with nonsense mutations, frameshift, and in-frame insertion-deletion mutations (indels), splice site mutations, and missense mutations defined as oncogenic in cBio Cancer Genomics Portal repository [9].

Statistical analyses
Continuous variables were reported as means and standard deviations (SD), or medians and interquartile ranges (IQR) based on data distribution assessed by Kolmogorov-Smirnov Test.According to data distribution, comparisons for continuous variables between groups were evaluated using the Student's t-test or Mann-Whitney U-test.Categorical variables were reported as frequencies and proportions, and comparisons among them were analyzed by χ2 test or Fisher exact test.Conditional odds ratios (OR) and Fisher's exact test p-values were used to assess co-occurrence and mutual exclusivity for genes among KRAS mutated and wild-type cases.Clinical and genomic characteristics associated with ORR were presented as OR estimated using logistic regression models.Kaplan Meier curves were used to evaluate median PFS and OS.The log rank test and Cox's proportional hazards model was used to test differences over time.All p-values were two-sided, with statistical significance defined as p < 0.05.All statistical analyses were conducted using Stata/MP 14.0 for Mac (StataCorp LP, 2015), and GraphPad Prism 9.0.1 for macOS (GraphPad Software, 2021) was used for plotting.

Discussion
This study provides valuable outcome information from a real-world cohort of Latin America patients with NSCLC harboring KRAS mutations and emphasizes the prognostic impact of diverse molecular profiles in this genomicallydefined subset of lung cancer.Prevalence of KRAS mutations in our cohort significantly differs from studies conducted in Caucasian patients [10,11], but aligns with that reported in Asian [12] and Latin American populations [2,3].This may be explained by a low tobacco smoke exposure; since we identified a higher proportion of never-smokers (40%) than in Caucasian populations (6.4-7.1%)[13], along with lower consumed pack per years (median 9.6) reported by smoker patients than previous studies (median 30.0) [5].We found a higher proportion of KRAS G12D cases compared with other cohorts [14], which agrees with available evidence not associating KRAS G12D with smoking-related mutational signatures [13].According to each mutational subtype, different carcinogenic patterns are activated, since KRAS G12C triggers RalA/B signaling, while KRAS G12D activates MEK and PI3K pathway [15].KRAS G12D exhibited a strong and independent association with favorable outcomes, conversely to previous evidence [5], likely explained by its infrequent concurrence with smoking-induced alterations, such as STK11 [16,17], widely known to predict reduced survival rates and diminished clinical responses to systemic treatments [18].In agreement, our observations suggested a deleterious prognostic effect of KRAS/STK11 comutation, also consistent with previous evidence in KRAS-mutated NSCLC [19].Biological comprehension of this prognostic role has revealed that loss of STK11 impairs the activation of AMP-activated protein kinase (AMPK), consequently allowing activity of the mammalian target of rapamycin (mTOR) [20], ultimately inhibiting cell proliferation, cancer-associated metabolism, and differentiation towards metastatic phenotype [21].These findings highlight the need for identification of agents capable of reactivating to improve patient outcomes.Regarding this, metformin restores AMPK-dependent signaling, leading to inhibition of tumor cell proliferation [22], but further prospective studies exploring its role in STK11-mutant NSCLC are warranted.
Differential survival outcomes among KRAS G12D and KRAS G12C cases may be driven by limited access to immunotherapy in our cohort.Consequently, deleterious responses and worse survival outcomes were noted among KRAS G12C cases after treatment regimens without immunotherapy, which is consistent with previous findings [23].Consequently, immunotherapy alone, or in combination, conferred a greater benefit in cases with KRAS G12C mutation, as it is linked to a greater TMB in NSCLC, commonly associated to tobacco-related carcinogenesis [24], as well as more efficient tumor neoantigen presentation to T cells, higher infiltration of CD8 + T cells, and increased PD-L1 expression [5].Meanwhile, KRAS G12D subtype is associated with low PD-L1 expression and TMB, lack of pro-inflammatory IL-18 production, induction of CD3 + T cell apoptosis, and impairment of CD8 + T cell activation [25].As well, the consistent benefit of immunotherapy in terms of overall survival along KRAS G12D or KRAS G12C groups may be derived from the impact of subsequent lines of treatment in KRAS G12D cases and concomitant employment of chemotherapy in almost all patients undergoing ICI-based regimens.Nevertheless, insufficient statistical power avoided comparing first-line monotherapy with PD-L1 blocking and chemoimmunotherapy in this population.
Moreover, KRAS G12D -mutated NSCLC may harbor exceptional oncogenic biology and treatment response.Regarding the coalterations, we found a higher incidence of uncommon EGFR comutations (14%) in almost all KRAS G12D cases, contrasting with available literature in Western individuals with KRAS mutations (1.3-4.0%)[14,26].As well, other comutations constituted predictive biomarkers of response to PD-L1 blockade.Specifically, STK11 was related to shorter PFS and OS, in line with previous reports [18,27], but limited sample size prevented statistical significance.Biological reasoning underlining these findings describes a lack of PD-L1 expression and lower densities of infiltrating CD8 + T cells in STK11-altered tumors [18].Consequently, STK11/LKB1 co-alteration is widely known as an independent predictor of unfavorable outcomes after PD-L1 blockade in lung adenocarcinoma [28].Thereby, it has been theorized that a triple regimen comprised of chemotherapy plus PD-L1 and CTLA-4 blockade may improve clinical response of this hard-to-treat subgroup [29].Differently, is consistent with Fig. 3 A, Type of responses to all treatments according to KRAS G12D mutation.B, progression-free survival of individuals with KRAS G12D or KRAS non−G12D mutations after all treatments.C, overall survival of patients having KRAS G12D or non-G12D mutations after all treatments.D, therapeutic responses to immunotherapy according to KRAS G12D mutation.E, progression-free survival of individuals having in KRAS G12D and KRAS non−G12D mutations undergoing immunotherapy or chemotherapy.F, overall survival of individuals having in KRAS G12D and KRAS non−G12D mutations undergoing immunotherapy or chemotherapy.G, therapeutic responses to immunotherapy in individuals harboring or not TP53 comutation.H, progression-free survival of patients having or not comutation with TP53 after immunotherapy or chemotherapy.I, overall survival of patients having or not comutation with TP53 after immunotherapy or chemotherapy.J, therapeutic responses to immunotherapy of individuals having or not STK11 comutation.K, progression-free survival of patients having or not comutation with STK11 after immunotherapy or chemotherapy.L, overall survival of patients having or not comutation with STK11 after immunotherapy or chemotherapy.IO, immunotherapy.ICI, immune checkpoint inhibitors.CT, chemotherapy.PFS, progression-free survival.OS, overall survival.KRAS, Kirsten rat sarcoma viral oncogene homolog.G12D, missense substitution of glycine for aspartate.TP53, tumor protein p53.STK11, Serine/Threonine Kinase 11.PFS was calculated from diagnosis to progression to first-line treatment.OS was determined by the period between diagnosis and death for any cause.Log-rank test was performed to determine statistical differences between Kaplan-Meyer curves.p<0.05 were considered as significative ◂ available literature describing that TP53 comutations show a remarkable benefit of PD-L1 blockade, likely derived from a TP53-related increase in PD-L1 expression and a greater infiltration of CD8 + T-cells in lung adenocarcinomas [30].
Limitations of the present study need to be considered when interpreting these results.Firstly, limited sample size in our cohort may have reduced the statistical power to detect significant differences among subgroups harboring distinct co-occurring genomic alterations.Secondly, information regarding PD-L1 expression was unavailable for all patients; therefore, we were not able correlate TMB and PD-L1 expression with distinct biological subgroups in the cohort.Thirdly, a low availability of immunotherapy-based regimens conditioned that only a minority of patients were treated with this therapeutic modality, hindering performance of a multivariate analysis evaluating factors associated with ICI-related clinical outcomes.

Conclusions
To our knowledge, this study represents the first effort to comprehensively characterize the molecular heterogeneity of KRAS-mutant NSCLC in Latin American patients.Our data reinforce the current view that KRAS-mutated NSCLC is not a single oncogene-driven disease and emphasizes the prognostic impact of diverse molecular profiles in this genomically defined subset of NSCLC.Further validation is warranted in larger multicenter Latin American cohorts to confirm our findings.

Table 1
KRAS, Kirsten rat sarcoma viral oncogene homolog, G12D missense substitution of glycine for aspartate, ECOG eastern cooperative oncology group performance status, WSE wood smoke exposure, LEP lepidic, CAN acinar, PAP papillary, MCP micropapillary, SOL solid, TPS tumor proportion score, PD-L1 TPS programmed death ligand 1 tumor proportion score, TMB tumor mutational burden, EGFR epidermal growth factor receptor, TKI tyrosine kinase inhibitor, CNS central nervous system, Mts mutations, MB megabase.Comparisons were made using: a t-test or b Mann-Whitney test according to normal distribution determined by the Kolmogorov-Smirnov test.Nominal variables were analyzed by c Pearson Chi-Square test, except when small size of sample (n < 5) required using d Fisher's exact test.Significance was set at p < 0.05 (two-sided), and shown as bold values in tables

Table 2
Bivariate and multivariate analysis of progression-free survival

Table 3
Bivariate and multivariate analysis of overall survival according to diverse clinical characteristics mOS median progression-free survival, CI confidence interval, HR hazard ratio, ECOG PS Eastern Cooperative Oncology Group Performance Status, LEP lepidic, CAN acinar, PAP papillary, MCP micropapillary, SOL solid, PD-L1 TPS programmed death ligand 1 tumor proportion score, IO immunotherapy, KRAS Kirsten rat sarcoma viral oncogene homolo, G12C missense substitution of glycine for cysteine, G12D missense substitution of glycine for aspartate, TP53 tumor protein p53, STK11 Serine/Threonine Kinase 11, GNAS guanine nucleotide binding protein, alpha stimulating.HER2 human epidermal growth factor receptor 2. CDKN2A Cyclin-dependent kinase inhibitor 2A MET mesenchymal epithelial transition.RB1 Retinoblastoma 1, PI3KCA phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.Comparisons were performed using *Log-rank test.Statistically significant p values were determined as p ≤ 0.05 and shown as bold values in tables