Assessing the prognostic value of KRAS mutation combined with tumor size in stage I-II non-small cell lung cancer: a retrospective analysis

Background KRAS mutation status is a well-established independent prognostic factor in advanced non-small cell lung cancer (NSCLC), yet its role in early-stage disease is unclear. Here, we investigate the prognostic value of combining survival data on KRAS mutation status and tumor size in stage I-II NSCLC. Methods We studied the combined impact of KRAS mutational status and tumor size on overall survival (OS) in patients with stage I-II NSCLC. We performed a retrospective study including 310 diagnosed patients with early (stage I-II) NSCLCs. All molecularly assessed patients diagnosed with stage I-II NSCLC between 2016–2018 in the Västra Götaland Region of western Sweden were screened in this multi-center retrospective study. The primary study outcome was overall survival. Results Out of 310 patients with stage I-II NSCLC, 37% harbored an activating mutation in the KRAS gene. Our study confirmed staging and tumor size as prognostic factors. However, KRAS mutational status was not found to impact OS and there was no difference in the risk of death when combining KRAS mutational status and primary tumor size. Conclusions In our patient cohort, KRAS mutations in combination with primary tumor size did not impact prognosis in stage I-II NSCLC.


Introduction
Non-small cell lung cancer (NSCLC) is the second most common cancer worldwide with 2.1 million new cases annually and the highest mortality rate with 1.8 million deaths (1).Staging is a crucial aspect of NSCLC management, as it is one of the most important predictors of survival.The TNM staging system describes key tumor characteristics such as size, location, and whether the disease has spread to lymph nodes and/or distant organs (2)(3)(4)(5).There are four main stages in NSCLC (stage I-IV), with stage IV having the worst prognosis.Pathological stage is considered the most important prognostic factor for resected patients, with 5-year survival rates, gradually decreasing across stages, of 83% for stage IA, 71% for IB, 57% for IIA, 49% for IIB, 36% for IIIA, and 23% for IIIB (4).
The most frequent oncogenic driver in NSCLC is the Kirsten rat sarcoma viral oncogene (KRAS), which is present in up to 40% of all cases, with the most common mutations being G12C, G12V, and G12D (6).KRAS mutations are associated with worse outcomes after chemotherapy and radiotherapy, with shorter OS in stage III and IV patients (7)(8)(9)(10)(11)(12)(13)(14).In early-stage NSCLC, however, while several studies have shown that KRAS mutations negatively influence the prognosis (15-17), others have shown no significant effect (18)(19)(20).Most recently, it was reported that KRAS G12C mutation (but not other KRAS mutations or with no mutation in KRAS) significantly increased risk of disease recurrence in stage I surgically resected lung adenocarcinomas (21).However, while the study found this in two distinct local cohorts of IRE-LUAD (Rome, Italy) and MSK-LUAD (New York, USA), data extracted from The Cancer Genome Atlas (TCGA) showed no significant difference.Another recent study reported that while STK11 mutation decreased survival probability in stage I lung adenocarcinoma, KRAS mutation showed no significant impact (22).Hence, the debate about the prognostic value of KRAS mutational status in early NSCLC is ongoing (23,24).In fact, given the lack of consensus regarding its effects on prognosis, testing for KRAS mutations for resectable stage I and II tumors is currently not recommended in clinical guidelines (25).In addition, several inhibitors that specifically bind KRAS-G12C have been investigated in clinical trials, with sotorasib becoming the first treatment to gain approval for adults with stage IV NSCLC harboring a KRAS-G12C mutation as second-line therapy (26-30).However, treatment with sotorasib is not currently recommended for patients with early-stage NSCLC due to lack of evidence showing positive outcomes of treatment in this group.
Therefore, further investigations are warranted to identify potential subgroups in Stage I-III disease who may still have to gain from effective and well-established treatments, and to add to the pool of clinical data required to study this further.One strategy is to stratify patients according to KRAS mutational status together with other key prognostic factors, such as tumor size.Primary tumor size is an established prognostic factor in NSCLC, with larger tumors being associated with poorer survival (24,(31)(32)(33)(34).The reason for this association is not yet fully understood but larger tumors may be more resistant to therapy due to having poorer blood supply, differential metabolism, and potentially a higher likelihood of micrometastatic disease compared to smaller tumors (35).Further research is needed to elucidate the underlying mechanisms.However, when considering primary tumor size, the grouping as early (I-II), advanced (III), and metastasized (IV) NSCLC can be argued to be more clinically relevant due to that stage I-II is primarily based on tumor size whereas a spread to the lymph nodes, a negative prognostic factor, is more common in stage III (3,24).
To our knowledge, no one has investigated the combined impact of primary tumor size and KRAS mutational status on OS and risk of death in stage I-II NSCLC.However, in Sweden, reflex testing for targetable alterations in NSCLC, including KRAS mutational status, has been widely implemented since 2015 for all stages.By screening all consecutive patients diagnosed with stage I-II NSCLC and molecularly assessed between 2016-2018 in Västra Götaland, the second largest county in Sweden with a population of 1.7 million, the current retrospective cohort study provides a unique real-world dataset for assessing the impact of combining KRAS mutations with primary tumor size.
To summarize, primary tumor size is a key determinant of prognosis especially in the early stages of NSCLC.At the same time, the prognostic value of KRAS mutational status in early disease stages remains unclear.Hence patients diagnosed at an early stage are not automatically tested for KRAS mutations and recommended treatment with KRAS-targeted therapy.Here, we investigate whether there is prognostic value in combining KRAS mutational status with tumor size to aid in clinical stratification of potentially treatment-responsive subgroups in early-stage NSCLC.

Patient population
We conducted a multi-center retrospective study screening all consecutive NSCLC patients diagnosed with stage I-II NSCLC and molecular assessment performed between 2016-2018 in Västra Götaland, Sweden (n = 354).Further inclusion criteria included the availability of tumor size from CT scanning or a pathology report as well as follow-up data.

Mutational status
Patients were assessed with next-generation sequencing (NGS) for mutational status on DNA from formalin-fixed paraffinembedded (FFPE) blocks or cytological smears using the Ion AmpliSeq ™ Colon and Lung Cancer Panel v2 from Thermo Fisher Scientific as part of the diagnostic workup process at the Department of Clinical Pathology at Sahlgrenska University Hospital, assessing hotspot mutations in EGFR, BRAF, KRAS, and NRAS.Until June 2017, ALK-fusions were assessed with immunohistochemistry (IHC), and with fluorescence in situ hybridization (FISH) if positive or inconclusive IHC.ROS1 was analyzed upon request with FISH.Thereafter, ALK, ROS1, and RET fusions were assessed on RNA using the Oncomine Solid Tumor Fusion Panel from Thermo Fisher Scientific.

Tumor size
To obtain the most recent and accurate untreated primary tumor size, measurements were collected from the radiology report of computed tomography (CT) performed before a final diagnosis of NSCLC was established; this is referred as clinical staging.In patients who underwent surgical resection, the actual primary tumor size was also collected from the pathology report, also referred as pathological staging (PAD).The largest tumor diameter was collected and reported in millimeters.

Study objectives
The primary outcome of this study was OS, defined as the interval between the date of first treatment and the date of death from any cause.Patients alive or lost to follow-up at the cut-off date were censored at last contact.Median follow-up time was estimated using the reverse Kaplan-Meier method.We compared OS and risk of death stratified by KRAS mutational status, i.e., with no mutation in KRAS (wildtype, KRAS WT ), all KRAS mutations (KRAS MUT ), KRAS G12C mutations (KRAS MUT G12C ) and all KRAS mutations other than G12C (KRAS MUT not G12C ).

Statistical analysis
Clinical characteristics were summarized using descriptive statistics and analysis of associations between KRAS mutational status and clinicopathological parameters was performed using Pearson´s X 2 test or T-test.Survival was estimated using the Kaplan-Meier method, visualized at 5-year follow up.The logrank test was used to assess significant differences in OS between KRAS WT and KRAS MUT groups.To evaluate if there was a significant difference in primary tumor size between KRAS MUT and KRAS WT , the Mann-Whitney U test was used.We defined an interaction term between tumor size (largest diameter in mm) and KRAS mutational status to assess the combined impact on the risk of death (HR).First, the mean size of all primary tumors was calculated.Thereafter a dummy variable was calculated by subtracting the mean size from all individual measurements following multiplication with 1 if KRAS was mutated and 0 if KRAS was WT.The interaction term was included in Multivariable Cox regression analysis, also correcting for sex, age, tumor size in mm and KRAS mutational status.Statistical significance was set at p < 0.05 and no adjustments were made for multiple comparisons.Data analysis was conducted using IBM SPSS Statistics version 27 and GraphPad Prism version 9.

Patients and tumor characteristics
A total of 310 patients, who were diagnosed with stage I-II NSCLC during 2016-2018 in Västra Götaland, Sweden and for whom genetic data was available, were included in this retrospective cohort study (Figure 1).In the total population, majority of patients were female (187, 60.3%), with a median age of 70 years, and most were current or former smokers (267, 86%) (Table 1).Most patients had good PS with ECOG 0-1 at diagnosis (285, 92%) and the proportion of N1 was low (18, 5.8%).NSCLC was predominantly adenocarcinoma of the lung (281, 90.6%), while squamous cell carcinoma incidence was relatively low (11, 3.5%), which was expected due to the selection of histological type for NGS assessment.Of included patients, over a third (115, 37%) had a KRAS mutation (Table 1).This percentage matches what has been previously reported (9), showing good representativeness of the patient group studied here.When comparing the baseline characteristics of KRAS WT with KRAS MUT patients, a greater proportion of those with KRAS MUT were female and current or former smokers.There were no cases of squamous cell carcinoma in the KRAS MUT group.The most common KRAS mutation was G12C (47%).In the total population, majority of patients underwent surgical resection (273, 88%; Table 2).Three patients did not receive any treatment and were excluded from further survival analyses.Median follow-up time was 63 months (95% CI, 59.7-68.3)and the data cut-off date was 31 October 2022.

No significant difference in survival for all patients stratified by KRAS mutations
When comparing OS for all (stage I-II) patients stratified by KRAS mutational status, no significant difference was detected with a mean OS (median not reached) of 74 months for KRAS WT vs 63 months for KRAS MUT (p = 0.847; Figure 2A).Further stratification of the KRAS mutated group by the G12C mutation also did not significantly change survival: 74 months for KRAS WT , 61 months for KRAS MUT not G12C and 63 months for KRAS MUT G12C (p = 0.834; Figure 2B).

No significant difference in survival for patients in stage I or stage II disease combined with KRAS mutations
There were also no significant differences according to KRAS mutational status in stage I (Figures 2C, D) or Stage II (Figures 2E,  F).Similarly in resected patients, no significant difference was observed with a mean OS (median not reached) of 78 months for KRAS WT vs 65 months for KRAS MUT (p = 0.856; Supplementary Figure 1A), or between the subgroups of KRAS MUT (p = 0.471; Supplementary Figure 1B).
Next, we stratified by stage and found mean OS (median not reached) of 79 months for stage I vs 50 months for stage II (Supplementary Figure 2A).We then conducted the analysis separately according to KRAS mutational status.For KRAS WT , the mean OS (median not reached) was 78 months for stage I vs 46 months for stage II (Supplementary Figure 2B), and for KRAS MUT , 65 months for stage I vs 53 months for stage II (Supplementary Figure 2C).
No significant difference in survival for patients with TNM-stage T1, T2 or T3 disease combined with KRAS mutations Next, we stratified patients using T-staging and studied OS according to KRAS mutational status.Among those with T1 disease, KRAS WT had 83 months while KRAS MUT had 66 months OS (p = 0.751; Figure 3A).Further, KRAS MUT not G12C patients had survival of 70 months and KRAS MUT G12C had 61 months (p = 0.344; Figure 3B).In the T2 group, KRAS WT had 53 months while KRAS MUT had 59 months OS (p = 0.495; Figure 3C).KRAS MUT not G12C patients  had survival of 51 months and KRAS MUT G12C had 66 months (p = 0.389; Figure 3D).Similarly, in the T3 group, KRAS WT had 47 months while KRAS MUT had 50 months OS (p = 0.966; Figure 3E).KRAS MUT not G12C patients had survival of 50 months and KRAS MUT G12C had 53 months (p = 0.984; Figure 3F).We further analyzed the impact of T stage on survival and found that it correlated as expected with mean OS of 82 months for T1, 55 months for T2, and 46 months for T3 (p < 0.001; Supplementary Figure 3A).The same trend was observed when separately analyzing KRAS WT with a mean OS (median not reached) of 83 months for T1, 53 months for T2, and 45 months for T2 (p < 0.001; Supplementary Figure 3B), and KRAS MUT with a mean OS of 65 months for T1, 58 months for T2, and 48 months for T3 (p < 0.023; Supplementary Figure 3C).

KRAS mutations are associated with smaller tumor size measured from CT scans, but not resection specimens
To evaluate differences between primary tumor size from CT scans at diagnosis stratified by KRAS mutational status, we used the Mann-Whitney U test.The test revealed that KRAS MUT primary tumors were significantly smaller at diagnosis, with a median size of 20 mm (n = 115) vs KRAS WT primary tumors with a median size of 25 mm (n = 190) (p = 0.043; Figure 4A).However, when looking at tumor size as assessed in resected specimens, there were no differences; KRAS WT median size 22 mm (n = 171) vs KRAS MUT median size 21 mm (n = 102) (p = 0.16; Figure 4B).
Larger tumor size measured from resection specimens, but not CT scans, is associated with a higher risk of death We found that increase in primary tumor size determined from CT scans did not have a significant effect on risk of death (HR, 1.006; 95% CI, 0.922-1.021;p > 0.5) (Figure 4C).However, when testing the correlation between primary tumor size as assessed in No treatment 3 (1.0) 3 (1.5)0 (0) Data are presented as n (%).
resection specimens, we found a significantly increased risk of death (HR, 1.029; 95% CI, 1.012-1.046;p < 0.001) (Figure 4D).The risk of death increases with 2.9% for every mm increase of size.When analyzing stage I and stage II patients separately we found that the primary tumor size, as assessed in resection specimens, correlated to a significantly increased risk of death (HR, 1.051; 95% CI, 1.026-1.077;p < 0.001) (Supplementary Table 1) for stage I patients, with 5.1% for every mm increase in tumor size.However, for stage II patients, no correlation was found between tumor size and risk of death.Furthermore, the primary tumor size determined by CT did not impact the risk of death in either stage.Along these lines, we analyzed the risk of death separately for T1, T2 and T3 groups regarding tumor size from CT and resected specimens and no correlation was found (Supplementary Table 1).

The combination of KRAS mutational status and tumor size does not impact the risk of death
To test if the combination of tumor size and KRAS mutational status impacts the risk of death, we defined an interaction term including both variables.For primary tumor size from CT scans and KRAS mutational status, no significant difference in the risk of death was detected (HR, 1.008; 95% CI, 0.988-1.030;p > 0.5) (Figure 4C).Similarly, there were no significant differences for primary tumor size and KRAS mutational status when measured in resection specimens (HR, 1.002; 95% CI, 0.978-1.027;p = 0.807) (Figure 4D).Along these lines, we analyzed the risk of death separately for stage I and II, as well as for T1, T2 and T3 groups regarding the interaction term combining tumor size and KRAS mutational status and no correlation was found (Supplementary Table 1).

Discussion
In this study, we assessed the prognostic value of combining KRAS mutational status with tumor size in early-stage NSCLC.We found that combining these variables had no significant effect on overall survival or the risk of death.
In alignment with previous findings, we found in our patient cohort that later disease stage and larger primary tumor size is associated with worse survival.Interestingly, we found that these correlations are sustained independent of KRAS mutational status.Importantly, the established literature on how KRAS mutations affect outcomes in early-stage NSCLC is varying between worse survival and no significant difference.We find that KRAS mutational status alone does not significantly impact OS or risk of death in patients with stage I-II NSCLC.Taken together, these findings show good representativeness of this well-defined patient cohort.
Our study included only patients with stage I-II disease due to the focus on primary tumor size and to limit the prognostic impact of local invasion and regional lymph node involvement.Only 5.8% of the patients had N1 disease that could affect the prognosis.The major portion of the patients had tumor resection and more than 90% of tumors were adenocarcinoma.During this period, patients diagnosed with squamous cell carcinoma were molecularly assessed to a lesser extent, thus our study is more representative of adenocarcinoma.Even though most tumors were classified according the TNM staging guidelines 7 th edition, changes included in the 8 th edition, mainly covering substages that were not analyzed in this study, do not alter our findings (4).No significant differences were observed when comparing OS for all stage I-II patients stratified by KRAS mutational status.However, the mean OS was 11 months shorter for KRAS MUT patients.The same trend was observed when looking at resected patients with a 13month shorter mean survival for KRAS MUT patients.Although trends toward a poorer prognosis were present, the KRAS MUT subgroup consisted of a relatively small number of patients, potentially necessitating larger cohorts to achieve statistical significance.This observation aligns with recent findings in a similar cohort of stage I LUAD with relatively small sample size in the KRAS MUT group (22).Within the KRAS mutational subgroups in our cohort, contrary to prior reports (36,37), KRAS G12C mutation in stage I disease did not indicate a worse prognosis.However, as noted in another study (38), there was a tendency toward improved survival among KRAS G12C patients with stage II disease and T2/T3 tumors, although these differences did not reach statistical significance.
Outcome variables other than survival such as recurrence rates and progression-free survival were not examined here.In addition, there remain confounders that were not include in the analyses such as the effect of different treatment methods on survival.Further, we use the T descriptor of the TNM staging system for tumor size but the descriptor also includes invasion status and or intrapulmonary metastasis.In addition, we found that larger tumor size measured from resection specimens, but not CT scans, is associated with a higher risk of death.However, one confounder here is that nonresected patients are included in the CT group but not in the PAD group, which biases toward worse prognosis.
Going forward, much remains to be explored on the role of KRAS mutation in early NSCLC.In the age of precision medicine, our study contributes toward the detailed level clinical data that is required for future pooled analysis of prognosis assessments that can help guide clinical decisions.
In conclusion, we confirm the importance of primary tumor size and stage as a prognostic factor for survival in stage I-II NSCLC.KRAS mutations were not found to impact OS and no difference in the risk of death was observed when combining KRAS mutations and primary tumor size.
Gothenburg, for a critical review of the manuscript.We would also like to thank "Akademistatistik -consulting services in statistics and health economics" at the University of Gothenburg for their support with defining the interaction term.In addition, we thank members of the Swedish Lung Cancer Registry and the continuous reporting by Swedish healthcare employees.

FIGURE 1
FIGURE 1 Patient selection.Flow chart showing patient selection for the study.

2
FIGURE 2 Impact of KRAS mutational status on overall survival in Stage I and II NSCLC.Kaplan-Meier estimates comparing overall survival between (A, B) all patients, (C, D) Stage I and (E, F) Stage II patients with no mutation in KRAS (wildtype, KRAS WT ), with all KRAS mutations (KRAS MUT ), only KRAS-G12C mutations (KRAS MUT G12C ) and KRAS mutations other than G12C (KRAS MUT not G12C ).
Swedish guidelines in 2018, and full implementation was reached in 2019.Ethical approval was obtained from the Swedish Ethical Review Authority prior to study commencement (Dnr 2019-04771 and 2021-04987).No informed consent was required due to all data presented in a de-identified form according to the Swedish Ethical Review Authority.

TABLE 1
Characteristics of the total cohort as well as stratified by KRAS WT and KRAS MUT .

TABLE 1 Continued
TotalKRAS WT KRAS MUT P-value

TABLE 2
Summary of first-line treatments in the total cohort as well as stratified by KRAS WT and KRAS MUT .