Kirsten rat sarcoma mutation in South Indians with non-small lung cancer: A cause for concern?

Gautam Balaram; Renjan Thomas; Suhas N Ghorpade; Prarthana V Kowsik; Baby Dharman; Yogesh Shivakumar; Shekar Patil; Satheesh Chiradoni Thungappa; HP Shashidhara; Somorat Bhattacharjee; Sridhar Papaiah Susheela; Radheshyam Naik; Srinivas Belagutty Jayappa; Tejaswini Bangalore Nanjaiah; Shivakumar Swamy Shivalingappa; Mithua Ghosh; BS Ajaikumar

doi:10.4103/jpo.jpo_7_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 2 | Issue : 1 | Page : 3-8

Kirsten rat sarcoma mutation in South Indians with non-small lung cancer: A cause for concern?

Gautam Balaram¹, Renjan Thomas¹, Suhas N Ghorpade¹, Prarthana V Kowsik¹, Baby Dharman¹, Yogesh Shivakumar¹, Shekar Patil², Satheesh Chiradoni Thungappa², HP Shashidhara², Somorat Bhattacharjee³, Sridhar Papaiah Susheela³, Radheshyam Naik², Srinivas Belagutty Jayappa², Tejaswini Bangalore Nanjaiah⁴, Shivakumar Swamy Shivalingappa⁵, Mithua Ghosh¹, BS Ajaikumar³
¹ Department of Molecular Pathology and Genomics, Triesta Sciences, HCG Cancer Centre, Bengaluru, Karnataka, India
² Department of Medical Oncology, HCG Cancer Centre, Bengaluru, Karnataka, India
³ Department of Radiation Oncology, HCG Cancer Centre, Bengaluru, Karnataka, India
⁴ Department of Pathology, HCG Cancer Centre, Bengaluru, Karnataka, India
⁵ Department of Radiodiagnosis, HCG Cancer Centre, Bengaluru, Karnataka, India

Date of Submission	08-Mar-2021
Date of Decision	08-Aug-2021
Date of Acceptance	11-Mar-2022
Date of Web Publication	03-May-2022

Correspondence Address:
Dr. Mithua Ghosh
Department of Molecular Pathology and Genomics, HCG Cancer Centre, KR Road, Sampangiram Nagar, Bengaluru - 560 027, Karnataka
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_7_22

Abstract

Background: Lung cancer is the poster child for advances in molecular oncology with a myriad of targeted therapies in NSCLC (non-small cell lung cancer) management. Kirsten rat sarcoma (KRAS) mutations are routinely isolated in NSCLC and account for a third of NSCLC oncogene driver tumors in Caucasian populations. The mutation is classically notorious to target with most therapies employed in management of KRASmut NSCLC being inhibitors of downstream signaling such as mitogen-activated protein kinase inhibitors (trametinib and selumetinib). There is a lacuna of information regarding prevalence and molecular epidemiology of KRAS mutations in NSCLC from an Indian context.
Materials and Methods: The following study is a retrospective analysis of the incidence of KRAS epidemiology in high concentration epidermal growth factor receptor (EGFR) samples at a tertiary care hospital in South India from 2015 to 2017. Samples were selected following histopathological assessment and were subjected to nucleic acid extraction. KRAS mutation testing was performed using real-time polymerase chain reaction to ascertain the molecular epidemiology of KRAS in NSCLC patients.
Results: KRAS mutations were observed in 15/44 NSCLC patients (34.09%) with a M:F ratio of 2:1. Majority of the mutations were single mutations, with 3 cases showing double mutations. Codon 12 mutations were observed in 6 cases followed by codon 146 mutations seen in 5 cases. Exon 3 (codon 59 and codon 61) and exon 4 (codon 117 and codon 146) were isolated in 5 and 3 cases, respectively. The current study demonstrated an elevated frequency for KRAS mutations in comparison to Asian cohorts.
Conclusion: The advent of directly targeting KRAS inhibitors such as sotorasib (KRAS G12C inhibitor) necessitates KRAS mutation testing and warrants inclusion in the initial molecular workup of NSCLC.

Keywords: Epidermal growth factor receptor, Kirsten rat sarcoma, lung cancer, nonsmall cell lung cancer, real-time polymerase chain reaction

How to cite this article:
Balaram G, Thomas R, Ghorpade SN, Kowsik PV, Dharman B, Shivakumar Y, Patil S, Thungappa SC, Shashidhara H P, Bhattacharjee S, Susheela SP, Naik R, Jayappa SB, Nanjaiah TB, Shivalingappa SS, Ghosh M, Ajaikumar B S. Kirsten rat sarcoma mutation in South Indians with non-small lung cancer: A cause for concern?. J Precis Oncol 2022;2:3-8

How to cite this URL:
Balaram G, Thomas R, Ghorpade SN, Kowsik PV, Dharman B, Shivakumar Y, Patil S, Thungappa SC, Shashidhara H P, Bhattacharjee S, Susheela SP, Naik R, Jayappa SB, Nanjaiah TB, Shivalingappa SS, Ghosh M, Ajaikumar B S. Kirsten rat sarcoma mutation in South Indians with non-small lung cancer: A cause for concern?. J Precis Oncol [serial online] 2022 [cited 2023 Mar 28];2:3-8. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/1/3/344540

Introduction

Lung cancer is the leading or one of the leading causes of cancer-related mortalities in developed nations. According to the GLOBOCAN data published in 2018 by IARC, lung cancers account for approximately 6% of all new cancer cases (67,795 cases) and 8.3% (63,475 cases) of all cancer-related deaths in India.^[1],[2] Histologically, lung cancer was classified broadly on histomorphology into small cell carcinoma and nonsmall cell lung carcinoma (NSCLC), with chemotherapy being the lynchpin of therapy. NSCLC is subcategorized into numerous pathological variants, of which squamous cell carcinoma (SCC), large cell carcinoma, and adenocarcinoma (ADCA) are frequently observed [Figure 1].^[3] Prior editions of the WHO relied primarily on morphology as the diagnostic modality of choice which in turn determined patient management [Figure 2].^[4]

Figure 1: Adapted from historical perspective of lung cancer testing from Lim C et al. Improving molecular testing and personalized medicine in non-small cell lung cancer in Ontario^[3]

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Figure 2: Selected genomic alterations observed in non-small cell lung cancer and their targeted therapeutics

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Treatment options were limited in lung cancer, with chemotherapy being the lynchpin until the discovery of epidermal growth factor receptor (EGFR) and anti-EGFR tyrosine kinase inhibitors (TKIs).^[5] The aforementioned oncology therapeutics significantly improved overall patient outcomes and ushered in the era of precision oncology in NSCLC.

Genomic insights into the landscape of NSCLC have revealed the role of key oncogenic drivers with findings resulting in development of actionable targeted therapeutics such as EGFR, Kirsten rat sarcoma (KRAS), tyrosine-protein kinase Met or hepatocyte growth factor receptor (MET), anaplastic lymphocyte kinase (ALK), and ROS-proto-oncogene (ROS-1)^[6],[7] [Figure 2].

Kirsten rat sarcoma

KRAS mutations have been isolated in 30% of patients with lung ADCA in Western populace and 10% of cases among Asian cohorts.^[8],[9] Mutations alter the hydrolytic GTPase activity of KRAS leading to prolonged activation of KRAS downstream signaling.^[10] The net effect of this uncontrolled signaling is unrestrained cell proliferation leading to development of neoplastic clones. KRAS mutations have been established as predictive biomarkers in colorectal cancer (CRC). KRAS mutations act as a negative predictive marker for treatment with anti-EGFR antibodies (cetuximab and panitumumab) in CRC.^[11]

Rapid strides have been made in management of NSCLC patients, but KRAS-mutated cases have limited options, with chemotherapy being the primary modality of intervention. Epidemiological factors such as prevalence, clinical implications, and prognosis of KRAS in Indian populations are not wholly elaborated; as a result, there is a lacuna of information with studies showing varying predictive/prognostic implications. The role of KRAS in NSCLC is not well defined like in CRC with even testing guidelines relegating KRAS mutational analysis to be performed in limited scenarios. The scenarios include KRAS testing to be performed as a component of multi-gene panels in tandem with other lung mutations such as EGFR, ALK, ROS, HER2, MET, PIK3CA, and BRAF or to be if performed as a single-gene assay only after EGFR, ALK, and ROS mutational analysis. The study would be the first to address KRAS mutational analysis employing an extended KRAS panel (exon 2–4) and not just exon 2.

Materials and Methods

The following study is a retrospective study looking at the prevalence of KRAS epidemiology in high concentration EGFR samples for duration from 2015 to 2017 at a tertiary care hospital in South India.

Patient selection and histopathological assessment of specimen

The analysis was performed on all patients diagnosed as nonsmall cell lung cancer (NSCLC) (with or without immunohistochemistry) followed by requisition for EGFR mutational analysis. Prior to molecular analysis of EGFR, tissue samples underwent assessment by a trainee and senior histopathologist to confirm the diagnosis, if assessed at centers outside of the hospital premises (without or without immunohistochemistry) and quantification of tumor burden to ascertain adequacy for nucleic acid extraction and mutational analysis. A requisite of >15% tumor content was necessary to isolate adequate nuclear material for testing of the sample.

Inclusion criteria

Patients diagnosed with NSCLC, referred for EGFR testing and having DNA concentrations not less than 100 ng/μl were included in this study for KRAS mutational analysis. The study patients underwent histopathological assessment for confirmation of diagnosis and tumour adequacy. Following histopathological assessment, samples with high DNA concentration (>100ng/ml) were selected for KRAS mutational analysis. The tissue not adhering to the above inclusion criteria was summarily rejected from the current study.

DNA extraction

DNA extraction required formalin-fixed, paraffin-embedded tissue sections of 20 m. Following deparaffinization with xylene, DNA was extracted (QiaAmp DNA mini kit) in accordance with the manufacturer's guidelines. DNA concentration was quantified using NanoDrop photospectrometer.

Glomerular filtration rate mutational analysis

Genomic analysis of exons 18–21 of EGFR gene was performed by Scorpion amplified refractory mutation system-based real-time polymerase chain reaction (RT-PCR) on “Therascreen” EGFR RGQ Kit from Qiagen™. The kit includes probes targeting the frequently documented EGFR mutations listed in COSMIC database including T790M exon 20 point mutation which is usually associated with treatment-induced resistance. The plate with probe and extracted DNA are placed in ROCHE LightCycler II for analysis. Calculating the difference between the test and control values and comparing to cutoff values (cycle threshold) in the kit insert, a presence/absence of a mutation was noted.

Kirsten rat sarcoma mutational analysis

Genomic analysis of exon 2 (codons 12 and 13), exon 3 (codons 59 and 61), and exon 4 (codons 117 and 146) was performed by “TRUPCR^®” real-time PCR assay from 3B BlackBio Biotech India™. The kit includes probes targeting the frequently documented KRAS mutations listed in COSMIC database. The mixture with probe and extracted DNA are placed in Rotor Gene-Q or Roche LightCycler II for analysis. On comparison of the difference between the test and control values against established cutoff values (cycle threshold) in the kit insert, a presence/absence of a mutation was noted. Three known cases of KRAS-positive NSCLC (detected by NGS) were included in the study as positive controls. Data were tabulated and statistical analysis was performed and P < 0.05 was considered statistically significant.

Results

A total of 736 samples for EGFR testing were received during the study period. Of these, 44 cases met the inclusion criteria and were tested for KRAS mutations. The EGFR status was reported to be wild type for 42 samples and exon 19 deletions were seen in 2 samples.

The M: F ratio for the study cohort was 1.93:1, the mean age of the cohort was 60.22 years, and the median age was 64 years. The age ranged from 29 to 79 years. KRAS mutations were detected in 15 cases (15/44, 34.09%), with 10 males and 5 females being positive for KRAS [Table 1]. The current study did not show any significant difference in KRAS incidence on the basis of gender or age. KRAS mutations were seen predominantly in exon 2 (codons 12 and 13) followed by exon 4 (codons 177 and 146) [Figure 3]a. The majority of the cases showed single mutations, with 3 cases harboring double mutations [Figure 3]b.

Figure 3: (a) Distribution of Kirsten rat sarcoma mutations based on exon, (b) Distribution of Kirsten rat sarcoma mutations on the basis of mutation type

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Table 1: Distribution of cases in the study cohort on the basis of gender and age

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Codon 12 accounted for six cases in the cohort with four males and two females. Four cases were above the age of 60. Four cases showed histopathology of ADCA, one case showed ADSQCC, and for a single case, definitive histopathology diagnosis was not possible [Table 2]. On KRAS mutational analysis, four cases showed single mutation and two cases showed double mutations. Mutation G12C accounted for half of the mutations detected on codon 12. A case with double mutation on the same codon (G12C G12A) was observed, with the other double mutation showing mutations on two separate codons (G12V Q61X). There were two cases of codon 13 KRAS mutations, both in the elderly (>70 years). The KRAS mutation detected was G13D, and both cases harbored single mutation. Codon 59 KRAS-positive cases were not reported in the current study cohort.

Table 2: Histopathology of Kirsten rat sarcoma-positive non-small cell lung cancer cases, the most frequent lesion was adenocarcinoma followed by poorly differentiated carcinoma

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A total of three cases harbored codon 61 mutations. Two cases were below the age of 60. On histopathology, a single case of ADCA, PDCA, and SCC was reported [Table 2]. Two cases showed single KRAS mutation, and one case harbored double mutation with G12V mutation.

K117X mutation was detected in an elderly male ADCA lung. EGFR RT-PCR revealed exon 19 deletions. KRAS analysis showed dual K117X and A146X mutations. Five cases with codon 146 mutations were detected with four males. Two cases were below the age of 60 and three cases were above the age of 60. Metastatic deposits presented in two cases (1 – abdominal wall and 1 – liver). On histopathology, four cases were diagnosed as ADCA, and one case was diagnosed as PDCA [Table 2]. On EGFR RT-PCR, two cases showed exon 19 deletion. On KRAS RT-PCR, four cases were single mutations, and one case was a double mutation. One case was reported as KRAS WT prior but on retesting showed A146X mutation.

Discussion

The current study has a KRAS positivity rate of 34.09% which is significantly higher in comparison to studies in Asian cohorts. KRAS mutations in NSCLC vary between 11% and 38% in Western populations and between 3% and 19% in Asian cohorts.^[8],[12] The finding is concordant with studies conducted in the Western populace and a single center study conducted by Shah et al [Figure 4]. The discrepancy can be addressed when it is considered that a majority of prevalent literature for KRAS in NSCLC tests only for codon 12 and 13 mutations using RT-PCR^{[5],[9],[12],[13],[14],[15],[16],[17]} with a small subset of studies also testing for exon 3 (codons 59 and 61).

Figure 4: Comparison of Kirsten rat sarcoma rates in non-small cell lung cancer for Western and Asian cohorts in selected studies

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The mutational spectrum for KRAS in NSCLC appears to be different in comparison to CRC, with higher frequencies of nonexon 2 mutations reported. In the current study, it was observed that nonexon 2 KRAS mutations accounted for approximately 50% of all cases. The finding is in concordance with a study conducted by Scheffler et al., wherein noncodon 12 and 13 mutations accounted for 11% of cases in the study population, with a majority being Q61X mutations.^[18] In the current study cohort, a case was reclassified from KRAS wild-type to KRAS A146X mutation on switching from the old KRAS kit (codons 12 and 13) to the extended kit (codons 12, 13, 59, 61, 117, and 146). The authors are of the opinion that testing with the KRAS extended kit may provide a truer insight into the KRAS mutational spectrum in Indian NSCLC cohorts.

Therapeutic implications

Presence of concomitant estimated glomerular filtration rate and Kirsten rat sarcoma mutations

The consensus is that driver mutations such as EGFR and KRAS are mutually exclusive and this finding has been replicated in the various cohorts.^[8],[19] Recent studies of KRAS among Asian cohorts have revealed the presence of dual-driver EGFR and KRAS mutations in a small percentage of cases.^[20],[21] The finding was also observed in the current study with two cases demonstrating the presence of EGFR exon 19 deletions in tandem with KRAS. The importance of this finding has been studied in a meta-analysis which demonstrated lower response rates of EGFR TKIs in KRAS-mutated NSCLC.^[22] G12C-mutated patients had shorter PFS and OS in comparison to non-G12C patients treated with EGFR-TKIs in a study conducted by Fiala et al.^[16] Two cases in the current cohort received therapy with afatinib with OS of 2.6 and 19.3 months, respectively, and had G12C and A146X mutations, respectively. Considering the above findings, oncologists are advised to include KRAS mutation testing in the initial molecular workup of recurrent NSCLC patients or EGFR-positive NSCLC patients prior to initiation of therapy with EGFR TKIs.

Kirsten rat sarcoma mutations and immune-checkpoint inhibitors

NSCLC associated with smoking/tobacco consumption has shown to have significantly elevated greater tumor mutation burden (TMB) and programmed death-ligand 1 (PD-L1) expression.^{[23],[24],[25]} Scheel et al. demonstrated significantly elevated PD-L1 expression in KRASmut in comparison to KRASWT.^[18] KEYNOTE-189 trial revealed elevated PD-L1 expression and TMB in KRASmut patients.^[26] A meta-analysis in 2017 demonstrated that immune-checkpoint inhibitors prolonged the OS of the KRASmut subgroup in comparison to KRASWT cohorts.^[27] KEYNOTE-42 trial revealed pembrolizumab reduced risk of death by 58% in the KRASmut subgroup and by 72% in individuals with KRAS G12C mutation in comparison to chemotherapy. ORR and PFS in the pembrolizumab arm elevated in the KRAS mutated cohort when compared to the KRAS wildtype cohort (56.7% vs. 29.1% and 12 vs. 6 months, respectively).^[28] The findings suggest that KRAS mutations could be employed as a potential biomarker for immune-checkpoint inhibitors.

Directly acting Kirsten rat sarcoma inhibitors

KRAS has traditionally been a difficult gene to target and poses a significant unmet need in precision oncology. KRAS-mutated NSCLC has traditionally been treated with chemotherapy or inhibitors of downstream signaling such as mitogen-activated protein kinase with poor outcomes. KRAS has recently re-emerged as a druggable targeted with the development of sotorasib (AMG510), a KRAS G12C-targeted inhibitor.^[29] A phase 2 clinical trial for sotorasib composed of 126 previously treated NSCLC cases with KRAS G12C mutation demonstrated partial response in 42 cases and complete response in 4 cases.^[30] The drug has received FDA approval for use in KRAS G12C-mutated NSCLC who have received at least one line of prior treatment with either immune-checkpoint inhibitors or chemotherapy.

Nassar et al.^[31] demonstrated a prevalence of KRAS G12C to be 13.8% in NSCLC cases wherein the current study demonstrated a prevalence rate of 6.8%.^[31] Further, KRAS mutations and particularly KRAS G12C are observed at lower frequencies in Asian populations compared to Caucasian cohorts and may explain for the low incidence rate of KRAS mutations in the current study. The supplementary data from the abovementioned correspondence show that Asian males in the cohort had a KRAS G12C incidence rate of around 6.47% which is concordant with the current study. Preclinical studies have also demonstrated durable responses of novel molecules such as MRTX 1133 which targets KRAS G12D. The KRAS novel inhibitors could be deployed in the management for almost a quarter of KRASmut NSCLC in the current study cohort.

Conclusion

In the current NSCLC cohort, KRAS mutations are found in a significant proportion of cases. The study's findings suggest that KRAS mutation analysis should be included in the initial workup of NSCLC testing in order to identify patient subsets who might respond to KRAS targeted therapies or benefit from the addition of immune-checkpoint inhibitors to their treatment.

Ethical approval

The study has been approved by the Human Research Ethics Committee of the institute (Ethics Committee Number EC/509/20/09).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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