Efficacy of immune checkpoint inhibitors in advanced non-small cell lung cancer patients with rare KRAS mutations: a real-world retrospective study

Introduction

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, which has the highest incidence and mortality rates among all malignancies worldwide (1). Among numerous oncogenic drivers, Kirsten rat sarcoma homolog (KRAS) mutations are commonly observed in NSCLC patients (2,3) and its prognostic impact remains unclear. Since its discovery in 1982, KRAS has been considered as an undruggable driver protein for decades. However, recent studies suggest that sotorasib and adagrasib could specifically target G12C mutation, which were approved by Food and Drug Administration (FDA) in 2021 (4,5). Nevertheless, other KRAS inhibitors are undergoing trials, which indicate that NSCLC patients with non-G12C mutation require other options.

Immune checkpoint inhibitors (ICIs) have gained extraordinary outcomes among a subset of NSCLC patients in recent years and have become one of the standard treatments for advanced NSCLC. A study reported that patients with KRAS mutation can benefit from immunotherapy (6). However, these studies either included all KRAS-mutant patients or emphasized on patients with G12A, G12C, G12D and G12V mutations (7-9), which are dominant KRAS mutations accounting for around 80% of KRAS-mutant NSCLC patient population. Meanwhile, few studies specifically investigated rare KRAS mutations including G13C, G13D, Q61H, G12S and other rare mutations on exon 12, 13 and 61, hence the therapeutic effect of ICIs on these patients needs to be explored.

In this study, we presented data on the characteristics and treatment outcomes of real-world advanced KRAS-mutant NSCLC patients, and investigated the potential value of immunotherapy on advanced NSCLC patients with rare KRAS mutations. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-372/rc).

Methods

Data and patients

Over 500 patients with KRAS mutations were identified from patients who received treatment at Shanghai Chest Hospital from July 2018 to July 2021. Stage IIIC and IV NSCLC patients were enrolled, and certain patients were excluded: (I) patients who were positive for other driver mutations; (II) patients who missed follow-up or with incomplete clinical information; (III) patients with tumor of other organs; (IV) early staged patients. Two hundred and forty patients met the criteria, and clinical information such as gender, age, smoking history, histology, co-mutant genes was obtained from the database. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Shanghai Chest Hospital Ethics Committee (No. IS21117) and informed consent was taken from all the patients.

Molecular detection

Patients’ tissue samples were obtained for pathological diagnosis, next generation sequencing (NGS) and programmed death-ligand 1 (PD-L1) detection before therapeutic treatment. NGS (LungCureCDx, Burning Rock, Suzhou, China) was used to detect mutations, and PD-L1 expression was detected via the immunohistochemistry (IHC) 22C3 pharmDx assay (Agilent Technologies China, Beijing, China).

Therapeutic schedule

One hundred and ten (45.8%) patients were treated with ICIs, of which, 35 received ICI monotherapy and 75 received combined therapy with chemotherapy or anti-angiogenic therapy. Sixty-four patients received first-line immunotherapy and 40 of them received second-line immunotherapy, while the remaining 130 patients received chemotherapy regimens such as pemetrexed/paclitaxel/docetaxel plus carboplatin/cisplatin, or bevacizumab/anlotinib. Some patients received radiotherapy if appropriate.

Endpoints and follow-up

Primary endpoints were progression-free survival (PFS) and overall survival (OS). PFS is defined as the time from initiation of first-line therapy to disease progression or death, and OS is defined as the time from initiation of first-line therapy to death or last follow-up. For patients receiving immunotherapy, initiation was defined as the first day they received immunotherapy. Relevant data were obtained from the patients’ clinical records combined with verification through telephone interview. Disease progression was assessed by senior radiologists in accordance to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1), with periodic radiology tests including high resolution chest computed tomography (HRCT) scan and abdominal ultrasound scan performed every 6–8 weeks, brain magnetic resonance imaging (MRI) performed every 6 months.

Statistical analysis

All statistical analyses were performed using SPSS (ver. 27.0 IBM Corporation, Armonk, NY, USA). Chi-squared and Fisher’s exact test were used to compare categorical variables between two groups. Survival analyses of median PFS, median OS, and between-group differences were determined via the Kaplan-Meier method and log-rank test. Cox proportional hazards model was further used to perform univariate and multivariate analyses of independent survival risk factors. P values were two-sided and differences were considered statistically significant when P<0.05.

Results

Patient characteristics

Two hundred and forty patients were enrolled (Figure 1A). The cohort included 211 males and 29 females, and the majority (n=234, 97.5%) had clinical stage IV disease at the time of diagnosis. One hundred and forty-nine (62.0%) of patients had a smoking history. The median age was 66 years (range, 32−86 years) and the median follow-up time was 36.5 months (range, 30.8−42.1 months) by December 15th, 2022. Tumor progression occurred in 211 patients and 180 of patients passed away. One hundred and forty (58.3%) patients had distant metastasis, 101 (42.1%) had bone metastasis and 41 (17.1%) had brain metastasis. Genetic sequencing revealed the distribution of KRAS mutational subtypes, including G12A (n=25, 10.4%), G12C (n=68, 28.3%), G12D (n=50, 20.8%), G12V (n=36, 15.0%) and rare mutations (n=61, 25.4%) (Figure 1B,1C). All variables were balanced between patients with conventional mutations (G12A, G12C, G12D and G12V) and rare mutations and results did not differ statistically (P>0.05). Other baseline characteristics of all patients are shown in Table 1.

Figure 1 The enrolling process (A), distribution of KRAS mutation subtypes (B), and specific composition of rare KRAS mutations (C), the others included G12L (n=1), G12F (n=3), A146T (n=1), A59G (n=1), Q61K (n=1), G13Y (n=1), A59T (n=1) and G12I (n=1). KRAS, Kirsten rat sarcoma homolog; ICI, immune checkpoint inhibitor.

Survival analysis

Survival analysis was conducted to reveal whether survival differences exist among KRAS subtypes. Of the 240 advanced NSCLC patients with KRAS mutations, 36 patients with G12V and 68 patients with G12C had more PFS (G12C P=0.39, G12V P=0.08) and OS (G12C P=0.88, G12V P=0.29) benefit after standard therapy, however neither was statistically significant. As for the 25 G12A patients, 50 G12D patients and 61 rare mutation patients, potential differences in survival outcomes were not observed (Figure 2A,2B, Figure S1A,S1B).

Figure 2 Comparison of progression-free survival (A) and overall survival (B) between all conventional mutation patients (G12ACDV) and other rare mutation patients; and comparison of progression-free survival (C) and overall survival (D) between conventional mutation patients and other rare mutation patients who were treated with chemotherapy.

Moreover, we analyzed patients who received standard treatments exclusive of immunotherapy (n=130) and patients who received ICIs (n=110). In patients who received standard treatments, patients harboring rare KRAS mutations (including G13C, G13D, Q61H, etc.) have shorter PFS (3.4 vs. 4.1 months, P=0.047) and OS (5.2 vs. 7.1 months, P=0.02) (Figure 2C,2D) than other subtypes. As for patients who received immunotherapy, differences in survival outcomes were not observed among different KRAS subtypes (Figure S1C,S1D).

The 110 patients who received ICIs therapy had significantly longer PFS and OS (median PFS 7.6 vs. 3.8 months, P<0.001; median OS, 18.5 vs. 6.1 months, P<0.001, Figure 3A,3B), compared with the 130 patients who received other standard therapies. Comparisons were further conducted in subgroups and significant differences were observed among patients who did or did not receive ICIs in the G12D group (median PFS 13.4 vs. 3.8 months, P=0.002; median OS, 30.1 vs. 8.6 months, P=0.002, Figure S2A,S2B) and rare mutation patients (median PFS 7.3 vs. 3.4 months, P<0.001; median OS, 13.3 vs. 5.2 months, P<0.001, Figure 3C,3D). Similarly, a trend towards longer OS appeared in G12C and G12V patients (Figure S2C,S2D).

Figure 3 Comparison of progression-free survival (A) and overall survival (B) between all immunotherapy and non-immune patients; and comparison of progression-free survival (C) and overall survival (D) between rare mutation patients who received ICIs or not. ICI, immune checkpoint inhibitor.

Cox proportional-hazards models were used to analyze other factors that may influence PFS and OS. P<0.1 was considered significant to increase sensitivity. After univariate and multivariate analyses, we found that TNM stage (P=0.04), histology (P=0.03), immunotherapy (P<0.001), G12C (P=0.04) and other rare KRAS subtypes (P=0.04) were significant factors affecting PFS (Table 2), and receiving immunotherapy was the independent risk factor for OS (P<0.001, P<0.001, Table 3).

PD-L1 expression and concurrent mutations

Among patients who received immunotherapy, PD-L1 expression was observed in 72 patients. Compared to PD-L1 negative tumors, patients with PD-L1 positive tumors, especially those with higher rate of PD-L1 positive tumor cells (≥50%), had elevated PFS and OS (Figure 4A-4D).

Figure 4 For all immunotherapy patients (PD-L1 expressions were available in 72 of them), their PFS (A) and OS (B) elevated with their expression of PD-L1 rate (P=0.01, P<0.001). For rare mutation patients receiving ICIs (17 with available PD-L1 expressions), their PFS (C) and OS (D) also elevated with their expression of PD-L1 rate (P=0.07, P=0.02). PD-L1, programmed death-ligand 1; PFS, progression-free survival; OS, overall survival; ICI, immune checkpoint inhibitor.

STK11 and KEAP1 have been considered a negative prognosis factor for KRAS patients (10,11). Regrettably, due to insufficiency of the NGS test kit, only STK11 mutational conditions of patients were detected. Fifty-two (21.6%) patients had positive STK11 mutations and they had significant less survival benefit than other patients, regardless of whether they accepted immunotherapy or not (Figure 5A-5D). On the other hand, the most common co-mutation, TP53 (n=131, 54.5%) had minimal effect over patients’ OS (P=0.93).

Figure 5 STK11/KRAS co-mutation have brought patients worse PFS (4.7 vs. 5.5 months, P=0.06, A) and OS (6.6 vs. 10.6 months, P=0.03, B), such differences were also observed in patients received immunotherapy (PFS: 5.8 vs. 8.8 months, P=0.002, C; OS: 13.3 vs. 20.9 months, P=0.02, D). KRAS, Kirsten rat sarcoma homolog; PFS, progression-free survival; OS, overall survival.

Discussion

KRAS mutations are highly prevalent in solid tumors like lung, colorectal and pancreatic cancers, found in about 30% of lung cancer patients and less in Asian patients (12), and may cause worse prognosis in advanced and metastatic NSCLC (13). KRAS mutations are involved in many vital oncogenic processes like proliferation, differentiation and survival through different intracellular downstream pathways such as rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway and phosphatidylinositol 3-kinase (PI3K)-AKT pathway (14). However, owing to its intrinsic characteristics such as its picomolar affinity for GDP and GTP, KRAS was considered as an “undruggable” target (15), which resulted in the delayed discovery of targeted inhibitors two decades later than EGFR. Nevertheless, after the approval of sotorasib in 2021, which targets the G12C subtype, standard first-line treatment for KRAS mutation patients continues to follow the principles of driver-gene-negative NSCLC treatment, which is based on cytotoxic chemotherapy or immunotherapy. In addition, a meta-analysis suggested that immunotherapy brought KRAS-mutant patients greater OS benefit than wild type (WT) patients (16), indicating that with the introduction of immunotherapy, the efficacy of immunotherapy on KRAS-mutant patients is still worth exploring.

Since the discovery of new targetable allosteric regulatory sites on G12C in 2013 (15), which suggested that KRAS signaling process could be interfered through irreversible binding of covalent inhibitors seemed to be the most effective way to overcome KRAS, and correlative research gained increasing attention. As the most common KRAS pathogenic variant, KRAS G12C occurs in around 30% of all cases (2,17). With the discovery of enhanced G12C inhibitors, research about KRAS mutation lung cancers mainly emphasized on the G12C subtype, while others focused on major subtypes like G12A, G12D and G12V. With the approval of sotorasib and adagrasib by the FDA and research conducted on other G12C, G12D, or pan-KRAS inhibitors (18,19), rare subtype inhibitors are being evaluated with novel compounds including RMC-6236.

Usually classified as ‘others’, the remaining portion of rare KRAS mutations manifests in 10% to 20% (and potentially higher in smokers) of the KRAS mutation lung cancer patient population (20,21). These rare mutations include more than 10 subtypes and are located on codon 12, 13 and 61 of the KRAS gene, which indicate that they do not share a common molecular structure which as a result, researchers may deem developing specific targeting inhibitors to be uneconomical and less important. Furthermore, rare KRAS mutations were often reported as impact factor for inferior survival, despite slight difference in grouping from our study’s definition, like codon 12 vs. codon 13 according to Yu et al. (22). In this situation, the efficacy of ICIs in patients with KRAS-mutated lung cancer may provide another option for targeted inhibitors. A study suggested that immunotherapy confers significant survival benefits over chemotherapy in all KRAS mutational status (16). Our research found that rare KRAS mutation patients had worse PFS and OS than major KRAS-subtypes when their therapeutic schedules were exclusive of ICIs, with a median PFS of 3.4 months and median OS of 5.2 months. However, the same result was not observed in the whole population inclusive of immunotherapy patients. Hence, we analyzed the difference in survival outcome between patients receiving ICIs or not, and found that the effect of ICIs on rare KRAS mutation patients were the most statistically significant (P<0.001). This suggests that advanced NSCLC patients with rare KRAS mutation with poor prognosis can benefit more from immunotherapy. As we mentioned above, in previous studies these rare KRAS mutation patients were to a certain degree neglected, this study may provide reference for future clinical practice.

We also investigated the survival benefit of immunotherapy treatment lines on patients, however significant difference between first-line and second-line treatment were not observed (Figure S3A,S3B). On the other hand, high PD-L1 expression could elevate the efficacy of ICIs on rare KRAS-mutant patients. This indicates that PD-L1 expression may have greater impact than other factors on these patients.

STK11/KRAS co-mutations are associated with poor prognoses and may predict primary resistance to ICI (10). We found that 21.6% (n=52) patients are KRAS/STK11 co-mutated in our study. The co-mutations predict worse OS and PFS than KRAS-mutated STK11 WT. While receiving immunotherapy could bring better outcomes than standard chemotherapy, the difference in survival outcomes between STK11 mutation type (MT) and WT still exists. While analyzing survival outcomes of immunotherapy, we found that ICI had a statistically significant effect on G12D patients, and these patients may see greater improvement than rare mutation patients. Our results, however, contradict some other studies (9,23), and this may be due to selection bias present in our study. Literature has suggested that ICIs combined with chemotherapy seems to achieve longer OS than immunotherapy alone for KRAS-mutant advanced NSCLCs (16). We found no difference between the two regimens (Figure S3C,S3D). Notably, up to 87.9% (n=211) of our patients were male, though KRAS mutations were more frequently found in male and east-Asians (21,24).

There are several limitations in our study. First, it is a retrospective single-center study with a sample size of 240, which selection bias may exist, like our population was consisted mainly of patients from Shanghai and surrounding areas, as well as the higher proportion of male patients. Second, due to practical reasons, information gathered was incomplete, such as the lack of PD-L1 expression status of partial patients and KEAP1 mutation situations of all patients.

Conclusions

In conclusion, our study suggests that among KRAS mutations, rare mutations (other than G12A, C, D, V) presented worse survival outcomes in advanced NSCLC patients. Though specific inhibitors are unavailable, ICI-based treatment could provide significant survival improvement than chemotherapy, which correlates with patients’ PD-L1 expression.

Acknowledgments

Funding: This work was supported by the foundation of National Natural Science Foundation of China grants (No. 82272913); National Multi-disciplinary Treatment Project for Major Disease (No. 2020NMDTP); the foundation of Shanghai Jiao Tong University (No. YG2021QN121); the foundation of Chinese Society of Clinical Oncology (Nos. Y-2019AZZD-0355 & Y-QL2019-0125).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-372/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-372/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-372/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-372/coif). H.H. received grants or contracts from BMS, Lilly Oncology, Roche Diagnostics, BillionToOne; consulting fees from Mirati, Janssen, Astrazeneca, Regeneron, BMS, Guardant, Foundation Medicine, Merck, Regeneron; payment or honoraria from Amgen, Mirati, Foundation Medicine, Janssen, Astrazeneca, EMD Serono, Merck, Regeneron. F.F. serves as the Advisory board for MSD laboratory. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Shanghai Chest Hospital Ethics Committee (No. IS21117) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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