|Year : 2015 | Volume
| Issue : 2 | Page : 243-248
Genomic profiling in a homogeneous molecular subtype of non-small cell lung cancer: An effort to explore new drug targets
Vidya H Veldore1, S Patil2, CT Satheesh2, HP Shashidhara2, R Tejaswi1, Shilpa A Prabhudesai1, N Krishnamoorthy1, D Hazarika1, R Naik2, Raghavendra M Rao3, BS Ajai Kumar4
1 Department of Molecular Pathology, Triesta Reference Laboratory, Triesta Sciences, A Unit of Health Care Global Enterprises Ltd., Bengaluru, Karnataka, India
2 Department of Medical Oncology, HCG Cancer Hospitals, Bengaluru, Karnataka, India
3 Department of Medical Oncology, HCG Cancer Hospitals; Centre for Academics and Research, HCG Foundation, Bengaluru, Karnataka, India
4 Health Care Global Enterprises Ltd., Bengaluru, Karnataka, India
|Date of Web Publication||5-Feb-2016|
Vidya H Veldore
Department of Molecular Pathology, Triesta Reference Laboratory, Triesta Sciences, A Unit of Health Care Global Enterprises Ltd., Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Patients' who are positive for kinase domain activating mutations in epidermal growth factor receptor (EGFR) gene, constitute 30–40% of non-small cell lung cancer (NSCLC), and are suitable candidates for Tyrosine Kinase Inhibitor based targeted/personalized therapy. In EGFR non-mutated subset, 8–10% that show molecular abnormalities such as EML4-ALK, ROS1-ALK, KIP4-ALK, may also derive the benefit of targeted therapy. However, 40% of NSCLC belong to a grey zone of tumours that are negative for the clinically approved biomarkers for personalized therapy. This pilot study aims to identify and classify molecular subtypes of this group to address the un-met need for new drug targets in this category. Here we screened for known/novel oncogenic driver mutations using a 46 gene Ampliseq Panel V1.0 that includes Ser/Thr/Tyr kinases, transcription factors and tumor suppressors. Methods: NSCLC with tumor burden of at least 40% on histopathology were screened for 29 somatic mutations in the EGFR kinase domain by real-time polymerase chain reaction methods. 20 cases which were EGFR non-mutated for TK domain mutations were included in this study. DNA Quality was verified from each of the 20 cases by fluorimeter, pooled and subjected to targeted re-sequencing in the Ion Torrent platform. Torrent Suite software was used for next generation sequencing raw data processing and variant calling. Results: The clinical relevance and pathological role of all the mutations/variants that include SNPs and Indels was assessed using polyphen-2/SIFT/PROVEAN/mutation assessor structure function prediction programs. There were 10 pathogenic mutations in six different oncogenes for which annotation was available in the COSMIC database; C420R mutation in PIK3CA, Q472H mutation in vascular endothelial growth factor receptor 2 (VEGFR2) (KDR), C630W and C634R in RET, K367M mutation in fibroblast growth factor receptor 2 (FGFR2), G12C in KRAS and 4 pathogenic mutations in TP53 in the DNA binding domain (E285K, R213L, R175H, V173G). Conclusion: Results suggest, a potential role for PIK3CA, VEGFR2, RET and FGFR2 as therapeutic targets in EGFR non-mutated NSCLC that requires further clinical validation.
Keywords: Epidermal growth factor receptor mutation, genomic profiling, ion torrent, next generation sequencing, non-small cell lung cancer, targeted re-sequencing
|How to cite this article:|
Veldore VH, Patil S, Satheesh C T, Shashidhara H P, Tejaswi R, Prabhudesai SA, Krishnamoorthy N, Hazarika D, Naik R, Rao RM, Ajai Kumar B S. Genomic profiling in a homogeneous molecular subtype of non-small cell lung cancer: An effort to explore new drug targets. Indian J Cancer 2015;52:243-8
|How to cite this URL:|
Veldore VH, Patil S, Satheesh C T, Shashidhara H P, Tejaswi R, Prabhudesai SA, Krishnamoorthy N, Hazarika D, Naik R, Rao RM, Ajai Kumar B S. Genomic profiling in a homogeneous molecular subtype of non-small cell lung cancer: An effort to explore new drug targets. Indian J Cancer [serial online] 2015 [cited 2021 Aug 5];52:243-8. Available from: https://www.indianjcancer.com/text.asp?2015/52/2/243/175843
| » Introduction|| |
There is an increasing incidence of lung cancer in India and across the world, with mortality in the range of 25–30% which is far more than breast and colorectal cancer., One of the major challenges we face today with lung cancer is late diagnosis and advanced disease at presentation, as it is asymptomatic. This is reflected in the poor clinical outcome with an overall 5 years survival of 16–20%.
Histologically, lung cancer is classified into two broad categories; non-small cell lung cancer (NSCLC) which constitutes 85% of all the lung cancers and small cell lung cancer (SCLC) which constitutes 15%. This histological distinction at the time of diagnosis has significant impact on the clinical management as the responsiveness to various chemotherapy drugs is different for SCLC and NSCLC. While the evolution of NSCLC roots to epithelial origin, the SCLC is derived from neuro-endocrine origin, and hence the difference in the disease manifestation and response.,,,
Evolution of technologies and impact on clinical management of lung cancer
In the last three decades several clinical trials from various research groups across the world had focused on trying different combinations of treatment modalities to improve the survival outcomes in NSCLC, however, there was very minimal success in terms of improving the efficacy of treatment. Later, during a retrospective sub-analysis from these results, it was realized that, the histo-morphological heterogeneity within the subtypes of NSCLC, namely the adenocarcinoma, squamous cell carcinoma, large cell carcinoma contributes significantly to survival outcomes. The histological subtyping in NSCLC is confirmed based on the protein expression using immunohistochemistry for basic lineage specific markers such as TTF1, CK, NP40 and TP63. This opened challenges on further classification of NSCLC depending on the molecular alterations resulting in a particular disease phenotype.
The past decade has seen major advancements in terms of personalized therapy options for NSCLC, as an add on to the existing treatment modalities, which has resulted in increased median overall survival from 2 months to more than 12 months., Hence, there exists a need for understanding of the disease in terms of molecular subtype/classification to achieve improved outcomes with reduced treatment toxicities in this era of personalized therapy and genomics.
Existing molecular targets in non-small cell lung cancer
Today, molecular testing for epidermal growth factor receptor (EGFR) and ALK has become the standard of care in frontline management of NSCLC for treatment selection.
Our own experience, and as well as reports from different groups across Asia and south east Asia; mutations in EGFR gene, constitutes ~30–50% of NSCLC, which makes these patients as suitable candidates for Tyrosine Kinase Inhibitor (TKI) based targeted therapy.,,, Also, little is known in terms of co-existing activating mechanisms in EGFR mutated population, wherein there is an add-on therapeutic benefit by other biologics to improve their quality of life and clinical outcome. Gefitinib, Erlotinib, more recently Afatinib remains the main standard of care drugs incorporated in the routine clinical management of EGFR mutated lung cancer patients.
Several groups have reported unique cohorts of NSCLC wherein attempts were made to do molecular subtyping using latest DNA sequencing technologies. A comprehensive understanding of all these results reveals a high level of molecular heterogeneity in EGFR non-mutated NSCLC patients. Molecular abnormalities such as EML4-ALK, ROS1-ALK, KIP4-ALK together constitutes 8–10% in small proportions, who may derive the benefit of targeted therapy as per the guidelines [Figure 1].
|Figure 1: Frequency/distribution of driver mutation subsets in Non-Small Cell Lung Cancer (Gou and Wu 2014, Lung Cancer: Targets and Therapy, Vol 2014: 5: Pg 1-9)|
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Beyond EGFR and other smaller subsets, there exists another subgroup, which is a grey zone/orphan group of tumours which constitutes 40% of NSCLC [Figure 1] and is negative for the clinically approved biomarkers for personalized therapy. These patients undergo conventional chemotherapy regimen, and hence do not get the benefit of targeted therapy which has reduced toxicities and improved quality of life. Here in this pilot study, we aim to understand the underlying molecular subtypes in terms of oncogenic activation in EGFR non-mutated tumours, which includes the grey zone/orphan group that could provide leads to address the un-met medical need for new drug targets in this subcategory of NSCLC. We screened for known/novel oncogenic activating mutations in a panel of 46 cancer related genes (Ion Ampliseq Panel V1.0) [Figure 2] that includes serine/threonine kinases, tyrosine kinases, transcription factors and tumor suppressors, in 20 cases of NSCLC which were EGFR negative for kinase domain activating mutations.
|Figure 2: Ion Ampliseq Cancer Panel version 1.0 (Courtesy: Life technologies Inc.)|
Click here to view
| » Methods and Sample Selection|| |
Twenty cases of NSCLC which had a tumor burden of at least 40% on histological evaluation were chosen for this study. Another important criterion of the choice of subjects is based on the EGFR mutation status of the tumor. Those tumours which were non-mutated for the 29 somatic mutations spanning the Exons 18, 19, 20 and 21 in the EGFR kinase domain were selected for this study.
QIA Amp DNA Mini kit (QIAGEN Inc.) was used for the isolation of tumor genomic DNA and the procedure was followed as per the manufacturer's recommendation. A minimum of 2 × 20 µ curls of FFPE was used for isolation of DNA. The concentration and the quality of the DNA were first assessed by Nano drop spectrophotometer. Following which, 10 ng of DNA from each of the 20 tumor tissues was pooled and the quality was verified by fluorometric methods (Cubit platform) and used as template for next generation sequencing (NGS) on Ion Torrent Personalized Genome Machine (PGM).
Assuring molecular homogeneity in the sample selection
Intrinsic molecular heterogeneity is hallmark of most cancers and also NSCLC. To assure the selected tumor subtypes were all EGFR non-mutated, we used most sensitive technologies for screening of somatic mutations in the kinase domain of EGFR. The choice of the technology assures a limit of detection 5–10 copies for mutation positivity: THERASCREEN EGFR mutation detection kit (QIAGEN Inc.) based on Scorpion amplified refractory mutation system (ARMS) real time polymerase chain reaction (PCR). All the samples in the study subset passed the criteria for being EGFR wild type otherwise called non-mutated for kinase domain activating mutations.
Polymerase chain reaction sequencing/targeted re-sequencing
The genotyping of the PCR products was performed as previously described. Ion Ampliseq Cancer Panel (V1.0) was used which is designed to identify 739 somatic/germ line mutations in 46 cancer related genes. The panel is designed based on the mutational spectrum in various cancers documented in public domain cancer databases such as COSMIC, TCGA, dbSNP, HUMVAR and individual findings reported in the PubMed literature database. A minimum of 10 ng of DNA was used as input for generation of genomic libraries. DNA libraries were then purified (Life Technologies Inc.) and then subjected to target enrichment using emulsion PCR technologies. The Emulsion PCR product was further purified to clear any nonamplified libraries and other PCR impurities and then loaded on the Ion 316 Semiconductor chip for sequencing. The raw data output from the PGM sequencer was analysed used the Torrent Suite Software version 2.0 (Thermofisher Inc., Waltham, Massachusetts, USA).
Assembly and analysis of the raw data using Torrent Suite software
The raw data of sequencing run from the Ion torrent platform was subjected to iterative cycles of sequence alignment based on the raw data profiles with quality control filters and those regions where the Q20 value was satisfactory was considered for further assembly of the sequenced fragments and analysis., The variants were analysed by variant caller software which is integral part of the TSS, and the functional role of variants was predicted using SIFT/PROVEAN , Mutation Assessor , and Polyphen-2 suite of programs.
| » Results|| |
Analysis of the raw data with mutation assessor revealed the presence of 27 different locations on the human DNA wherein nucleotide variations were observed. 97 missense and 38 coding-synonymous mutations were observed with 13 mutations in introns, 1 in the utr-3' and 2 in the utr-5' regions. A total of 151 alleles could be annotated in 15 different genes with varying transcripts.
Mutations and their clinical/physiological relevance
A missense mutation W515R was identified in MPL gene. This mutation, although poorly represented, is predicted to be deleterious and damaging, thus warrants further screening on the larger cohort. This mutation is commonly observed in as one of the driver mutations in oncogenesis in hematolymphoid malignancies.
Three different mutations were noted in PIK3CA of which one is a missense mutation, one in the utr-3 and the third one is a coding synonymous. The coding synonymous mutation, T1025T is with 30% mutation burden and is well documented in solid tumours in COSMIC database. The mutation in the 3-utr in PIK3CA is strongly represented hence requires further experimental validation to demonstrate its clinical significance. The C420R is less frequent, nevertheless predicted to be deleterious and damaging and hence it's clinical importance.
Epidermal growth factor receptor
In EGFR there were two mutations one coding synonymous and another a missense mutation. The coding synonymous Q787Q, is very strongly represented with 25% mutation burden with unknown clinical significance. The missense mutation R803Q is predicted to be deleterious and damaging and it is represented with low burden. It implies that the panel of 29 mutations might miss out certain other rare oncogenic drivers in EGFR, which further confirms the probable role of TKIs being sensitive to a small group of lung cancer patients who are EGFR Wild type for the clinically validated subset of mutations in Exons 18 to 21.
Vascular endothelial growth factor receptor 2/KDR
There was a single mutation, Q472H, with approx. 40% mutation burden with demonstrated pre-clinical significance.
In C-MET, we identified two different mutations one is coding synonymous and one is a missense mutation, with latter being reported in other cancers in the cosmic database. They are S178S and N375S both at close to 30% burden. The latter mutation has demonstrated resistance to MET specific inhibitor.
Three mutations were identified in RET, one of them is coding synonymous and other two is are missense mutations. The coding synonymous is L769 L. The two missense mutations are C630W and C634R. Although these two mutations are poorly represented in our study subset, they are worth screening due to their deleterious and damaging potential on the function of the protein.
Fibroblast growth factor receptor 2
Two different mutations at the same functional site were noted for fibroblast growth factor receptor 2 (FGFR2) at position 368: K368M and K368E. These mutations are strongly represented however they are predicted to be benign. They are often observed as a germline mutation in craniofacial disorders; nevertheless, their relevance in the context of lung cancer needs to be explored.
10% of the tumours presented with G12C mutation in KRAS which is commonly observed when EGFR is non-mutated.
Four missense mutations were identified in TP53 and were poorly represented in terms of mutation burden since most of them were non-smokers. Of the four mutations, three of them: E285K, R213 L and V173G, predicted to be deleterious and damaging and fourth mutation, R175H is predicted to be benign. The V173G is represented with 15% burden, although not reported in COSMIC database.
Mutations were also observed in FGFR3, platelet-derived growth factor receptor-α (COSM22413) and APC (COSM19349) and all of them were coding synonymous. Two mutations were noted in the intronic segments in FGFR3 and ATM genes wherein the clinical significance is not known. Two utr-5 alleles in FGFR2 with different transcript lengths were observed. Variable transcript length is a biological event in FGFR family in different medical conditions. A single mutation: N181S in ERBB4 (COSM48369), predicted benign with unknown clinical significance.
In summary, there were 10 pathogenic mutations in six different oncogenes for which annotation was available in the COSMIC database; C420R mutation in PIK3CA, Q472H mutation in vascular endothelial growth factor receptor 2 (VEGFR2) (KDR), C630W and C634R in RET, K367M mutation in FGFR2, G12C in KRAS and 4 pathogenic mutations in TP53 in the DNA binding domain (E285K, R213 L, R175H, V173G) [Table 1].
|Table 1: Summary of nonsynonymous mutations observed and their clinical relevance based on structure-function prediction algorithmns|
Click here to view
| » Discussion|| |
The central hypothesis of personalized cancer therapy is that treatment is tailored based on the individuals' tumour morphology, phenotype and genomic profile, thus, improving clinical outcomes, as measured by response rate, survival, and safety. Along similar lines, our focus in this pilot study was to identify a set of novel/clinically validated oncogenic driver mutations, in a unique molecular subtype of NSCLC wherein the EGFR is non-mutated. In order, to arrive at this subset, we screened for 29 somatic oncogeneic driver mutations of EGFR kinase domain using highly sensitive methods.
One of our first key findings, were 100% concordance in the results from NGS for the kinase domain mutations in EGFR with that of the Scorpion ARMS real time PCR, thus validating both the technologies for EGFR mutation testing. Beyond EGFR, we explored the tumor genome to identify potential oncogenic driver mutations in NSCLC using NGS or second generation sequencing technologies.
Role of multi-gene profiling
The underlying genetic modifications that effect oncogenesis is most often reflected with unique histological and molecular sub-categories. Therefore, understanding the diversity in the molecular subtypes particularly with the variety and frequency of mutations could answer some of the potential challenges in addressing novel biomarker driven combinatorial anticancer therapy. Hence, the need for high throughput multi-gene profiling.
The molecular diversity in NSCLC is well studied globally, and approx. 50–60% of these patients derive the benefit of personalized therapy [Figure 1]. Nevertheless, due to complexity in the inherent genetic makeup among different ethnic groups, the disease onset at the molecular level is known to vary significantly across the world. Therefore, in Indian context, discovery of novel driver mutations which could form unique molecular subtypes, in absence of EGFR being an oncogenic driver would always add value in improving the clinical outcome and personalized management of lung cancers.
As described earlier, in 20 cases of NSCLC, we found 10 pathogenic mutations in six different cancer related genes with potential role for novel therapeutic options and prognostication.
The C320R mutation in PIK3CA is predicted to have a pathogenic role as it disturbs the disulphide bridges in the functional domain of the protein. This activating mutation is commonly found in breast, endometrial, cervical and ovarian cancers  with a potential role for AKT1/mTOR inhibitors such as Everolimus.
Vascular endothelial growth factor receptor 2
Q472H mutation in VEGFR2 (KDR) is associated with upregulation of KDR expression or increase in Micro Vessel Density in NSCLC , with potential therapeutic options as Sorafenib and Bevacizumab.
C630W and C634R mutations on RET result in a significant change in the conformation of active site pocket in the RET kinase. These mutations were often reported in hereditary cancers such as multiple endocrine neoplasia (MEN IIA). They also occur as somatic mutations in the kinase domain, often reported in sporadic MTCs and rarely in other solid tumours. Activated RET feeds RAS/RAF/MAPK, PIK3/AKT and JNK signalling  and hence probable therapeutic options include pan-kinase inhibitor Sorafenib and several other inhibitors such as Vandetanib, specific to RET in clinical trials for lung cancer.
Fibroblast growth factor receptor 2
The K367M/E mutation in FGFR2 receptor does not predict a functional pathogenic role of this kinase being one of the drivers in oncogenesis, however its passive role in combination with other somatic oncogenic driver events needs to be ruled out.
Codon 12 mutations in KRAS gene is observed in 13% of the lung cancers which are EGFR wild type and our observation here in a minor subset of individuals as reflected by low percentage of mutations is in concordance. Mutant KRAS confers resistance to Anti-EGFR targeted therapy. Also, patients with co-existing KRAS and PIK3CA confer resistance to MEK1 inhibitors.,
The four pathogenic mutations in TP53 (E285K, R213L, R175H, V173G) have been shown to negatively impact its functional role as tumor suppressor, thus resulting in poor prognosis in terms of chemo and radio resistance. Nevertheless, there are several upcoming studies which have demonstrated the potential role of mutant TP53 as a targeted therapy marker.
The finding of germline FGFR2 mutations in cancer also provides an interesting convergence with genetic cancer risk: genome wide association studies have implicated germ-line polymorphisms of FGFR2 in susceptibility to breast cancer. Similarly, somatic mutations in FGFR2 are indicated in endometrial, cervix and lung squamous carcinoma.
As per the data reported from TCGA group, whole tumour genomic profiling of lung squamous cell carcinoma revealed the presence of 8% mutations in FGFR receptors, with most abundant mutations in FGFR2 and FGFR3 which accounts to 6%. However, these mutations were not uniformly distributed across all the samples. FGFR2 mutant tumours are sensitive to Pazopanib. Nintedanib is an investigational triple angio-kinase inhibitor that targets three receptor tyrosine kinases involved in the regulation of angiogenesis: VEGFR, FGFR, and platelet-derived growth factor receptor in the LUME-Lung 2 clinical trial. Also, FGFR mutations were found to repeatedly co-occur with TP53 mutations but rarely with any other gene mutations.
In conclusion, results from this pilot study suggests, a potential role for PIK3CA, VEGFR2, RET and FGFR as therapeutic targets in EGFR non-mutated lung cancer patients which requires further clinical validation. Such studies aid in understanding the diversity of molecular drivers in lung cancer and its clinical management for these patients, which is continuously evolving. Considering the heterogeneity in the gene pool, ethnic variations play a significant role in genetic predisposition to NSCLC. In future, we plan to validate our findings from this report on a larger subset of NSCLC in this population to tailor novel alternate strategies for lung cancer screening, diagnosis and prognosis.
| » Acknowledgments|| |
The team also acknowledges Invitrogen Bioservices India Pvt Ltd for the technical support on the NGS analysis.
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[Figure 1], [Figure 2]
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