Literature Review on Genetic Profile of Lung Cancer Patients

Table of Contents                               .     Page       .

1 Student Declaration        3

2 Abstract          4

3 Introduction         5

4 Objectives         6

5 Methodology         6 – 10

6 Results          7 – 21

7 Discussion          22 – 23

8 Conclusion         24

9 Acknowledgements        25

10 References         26 – 29

11 Appendices         30

 Abstract

Introduction: Molecular targeted therapy has been gaining traction in the treatment of advanced non-small cell lung cancer (NSCLC) in place of traditional standard chemotherapy. Given the growing significance of molecular agents, it is crucial for us to have an understanding of the genetic profile of NSCLC patients and assess the efficacy of targeted agents in terms of measurable outcomes such as progression free survival (PFS), overall survival (OS), and response rate (RR). Additionally, knowledge of treatment-resistant mutations will also play a crucial role in the care of patients with advanced NSCLC.

Objectives: This review aims to analyse the current literature on (i) frequencies of genetic mutations in different populations, (ii) demographic, clinical and histological factors associated with each mutation, (iii) efficacies of the various classes of molecular agents, and (iv) establish the role of treatment-resistant mutations in NSCLC patients.

Methods: A literature search was performed on two databases, PubMed and Scopus to identify articles which addressed the research question. Inclusion and exclusion criteria were applied to the search results to select studies for the literature review. A total of 13 studies were critically appraised and summarised. The results of each study were further analysed and synthesised.

Results: 13 studies were included in the literature review. 4 studies highlighted the heterogeneity of genetic profiles across different populations with EGFR and KRAS mutations being prominent ones. 6 studies assessed the efficacy of targeted therapy. Out of these 6 studies, 3 showed that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) and anaplastic lymphoma kinase (ALK) TKIs were superior to standard chemotherapy, 2 demonstrated clinical activity in B-raf proto-oncogene (BRAF) and human epidermal growth factor receptor 2 (HER2) mutation inhibitors but were unable to comment on whether they are superior to chemotherapy, and 1 concluded that MEK1/MEK2 inhibitors were non-superior to standard chemotherapy. Finally, 3 studies sought to identify mutations that conferred resistance to targeted therapy and they include (i) EGFR T790M, (ii) echinoderm microtubule associated protein like 4–anaplastic lymphoma kinase (EML4-ALK), and (iii) P13k/Akt/mTOR pathway mutations.

Conclusions: The scene of molecular targets in advanced NSCLC is a heterogenous and evolving one. Key driver mutations in NSCLC include EGFR, ALK, and Kirsten ras oncogene (KRAS) mutations. Their frequencies vary in different patient populations. The ‘typical’ patient with mutation-associated NSCLC tend to be EGFR mutation positive, East Asian, female, light or non-smokers, with adenocarcinoma histology. EGFR TKIs and ALK TKIs are superior to standard chemotherapy in the treatment of advanced NSCLC but other classes of targeted therapy require more research to clarify their definitive role in the treatment of metastatic disease. A key treatment-resistant mutation for EGFR TKIs is the EGFR T790M mutation, which is fortunately low in frequency. More research is expected to be conducted in the area of molecular therapy and the treatment algorithm for NSCLC patients is expected to continually evolve.

Introduction

Lung cancer

According to the World Health Organisation (WHO), lung cancer is the leading cause of all cancers and the leading cause of cancer-related deaths (1), with 80-85% being non-small cell lung cancers (2, 3). Additionally, an estimated 50% of all lung cancer patients diagnosed have metastatic disease, where 5-year survival rates are as low as 4% (4, 5).

Genetic Profile of Non-Small Cell Lung Cancer (NSCLC) patients

Given that NSCLC forms the majority of lung cancers and is associated with key driver mutations crucial to the treatment of metastatic disease (6), it is vital for us to have an understanding of the genetic profile of this population. Examples of key mutations include epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS1, Kirsten ras oncogene (KRAS), and B-raf proto-oncogene (BRAF) mutations (7, 8).

Targeted Therapy

Over the past two decades, the management of NSCLC has become increasingly targeted. Today, treatment for metastatic NSCLC is driven by molecular agents specific to a patient’s mutational status (9). The use of EGFR tyrosine kinase inhibitors (TKIs) in patients with sensitising mutations have been supported by multiple landmark trials on the basis of greater response rates (RR) and progression free survival (PFS) than chemotherapy (10-15). This has been supported by two Cochrane reviews published in 2016 (16) and 2018 (17). The use of Crizotinib for ALK- and ROS1-mutant lung cancers has also been gaining ground (18, 19). It is expected that the treatment algorithm for NSCLC patients with driver mutations will continue to evolve as more studies continue to investigate the efficacy of new and existing molecular agents.

Resistance to Treatment

The use of mutational status can also be used as a predictive factor on whether a patient will be resistant to treatment (20). This allows physicians to spare patients from adverse effects of targeted therapy should benefits of response be minimal in comparison to risk of toxicity.

Objectives

This review aims to analyse the current literature on the use of molecular targets in NSCLC through the following objectives:

1. To establish the frequencies of genetic mutations in NSCLC patients
2. To ascertain factors associated with each mutation
3. To investigate the efficacy of targeted therapy
4. To identify mutations associated with resistance to targeted therapy

 

Methods

Search Strategy

An electronic search was carried out on PubMed and Scopus to identify studies which would elucidate the research topic and achieve the objectives of this literature review.

The following search strategy was used for both databases:

1. “carcinoma, non-small-cell lung”[MeSH Terms] OR “lung neoplasms”[MeSH Terms] OR ((“lung”[All Fields] OR “bronchogenic”[All Fields] OR “pulmonary”[All Fields]) AND (“cancer”[All Fields] OR “carcinoma”[All Fields] OR “tumour”[All Fields] OR “neoplasm”[All Fields]))

AND

1. “molecular targeted therapy”[MeSH Terms] OR (targeted[All Fields] AND (“therapy”[Subheading] OR “therapeutics”[MeSH Terms]))

AND

1. “mutation”[All Fields] OR “mutations”[All Fields]

Filters Activated

Filters activated for the search strategy included:

No.	Filter	Specifications	Comments
1	Text availability	Full text
2	Publication dates	Within the past 10 years	Landmark trials relevant to the topic date back as early as 10 years ago
3	Species	Humans
4	Language	English
5	Age	Adults; >19 years old	To exclude paediatric populations

Inclusion Criteria

• Articles on a new, authentic study
• Studies that investigate mutations with molecular targets in non-small cell lung cancer
• For studies in which patients receive treatment, at least one of the treatment options should include a molecular targeted therapy
• For studies on the prevalence or incidence of mutations in non-small cell lung cancer, more than one mutation should be investigated

Exclusion Criteria

• Articles not accessible either as a free full text, or articles with restricted access for which permission to obtain a copy was not granted
• Review articles
• Case reports or case series
• Studies evaluating molecular targets in cancers apart from lung cancer
• Studies evaluating molecular targets in other types of lung cancer i.e. small cell lung cancer
• Studies investigating techniques of obtaining tissue samples for molecular testing, or methods of molecular testing
• Studies evaluating economic, administrative, or operational aspects of the topic
• Studies on the clinical presentation of lung cancer
• Studies on other lung diseases with molecular targets e.g. cystic fibrosis
• Laboratory-based, ex-vivo studies on human tissues

 

Study Selection Process

The search results elicited from PubMed and Scopus were combined and subsequently screened for the removal of duplicates. After which, titles and abstracts of the remaining articles were carefully assessed, with application of the inclusion and exclusion criteria.

Of the 22 abstracts deemed suitable for the literature review, those with full text articles which were not accessible either as free full text, or via the University College Cork (UCC) library portal were further excluded. The remaining 17 papers with full text were carefully read through to select 10 studies which best satisfied the inclusion and exclusion criteria. Lastly, the references of all 10 selected studies were carefully reviewed and another 3 highly relevant landmark trials were suggested for inclusion by the project supervisor, an expert in the field of medical oncology. These 3 studies satisfied the inclusion and exclusion criteria, and were thus included. The selection process is summarised in Figure 1 below and reasons for exclusion are found in Table 1.

 

Figure 1. Outline of Study Selection Process

 

 

 

 

 

 

 

Table 1. Overview of studies excluded

	Reason for exclusion	Number
Abstracts	Review articles	56
	Case report and case series	107
	Studies on clinical presentation of lung cancer	6
	Studies evaluating economic, administrative, or operational aspects of the topic	14
	Studies evaluating lung cancer in relation to topics apart from molecular targets	13
	Studies on small cell lung cancer	10
	Studies on other cancers with molecular targets	120
	Studies on techniques of obtaining tissue samples or on techniques of testing for mutations	43
	Studies on other lung diseases with molecular targets	4
	Laboratory-based, ex-vivo studies on human tissues	13
	Total	386
Full Text Articles	Not free full text, unable to access via UCC library portal, permission to access not granted	5
	Studies on treatment that do not have a treatment arm for molecular targeted therapy	3
	Studies focused on treatment of sites of metastases	4
	Total	12

Results

A summary of the thirteen studies selected for this review can be found in Tables 2-4. Abbreviations used in the tables are listed in Box 1.

Box 1. Abbreviations used in the results tables

OS – overall survival   PFS – progression-free survival HR – hazard ratio RR – response rate ORR – overall response rate t1/2 – half-life CI – confidence interval SC – standard chemotherapy NSCLC – non-small cell lung cancer BAC – bronchoalveolar carcinoma EGFR – epidermal growth factor receptor TKI – tyrosine kinase inhibitor EML4 – echinoderm microtubule associated protein-like 4 ALK – anaplastic lymphoma kinase EML4-ALK – EML4-ALK fusion oncogene c-MET – tyrosine-protein kinase Met WT/WT – wildtype/wildtype KRAS – Kirsten ras oncogene BRAF – B-raf proto-oncogene NRAS – neuroblastoma ras viral oncogene PIK3CA – phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha PI3K/Akt/mTOR – Phosphatidylinositol 3-kinase/Akt/Mammalian Target of Rapamycin HER2 – human epidermal growth factor receptor 2 WHO – World Health Organisation IASLC – International Association for the Study of Lung Cancer ECOG – Eastern Cooperative Oncology Group COSMIC – Sanger Institute Catalogue of Somatic Mutations in Cancer TNM – Tumour-Node-Metastases RECIST – Response Evaluation Criteria In Solid Tumours AE – adverse effect CTCAE – Common Terminology Criteria for Adverse Events QOL – quality of life QLQ-C30 – European Organisation for Research and Treatment of Cancer quality-of-life questionnaire QLQ-LC13 – European Organisation for Research and Treatment of Cancer quality-of-life questionnaire for lung cancer FISH – fluorescence in situ hybridisation

Table 2. Studies on Genetic Profile of NSCLC (Part 1)

Study	Study population	Methods	Results	Strengths and Liminations
Xia, N., et al. (2013). Analysis of EGFR, EML4-ALK, KRAS, and c-MET mutations in Chinese lung adenocarcinoma patients. Exp Lung Res 39(8): 328-335. (21)	Inclusion criteria   -Ethnic Han Chinese -Residents of Hunan Province, China -Patients seen at Xiangya Hospital -Patients who have not undergone prior therapy -Lung adenocarcinoma patients Exclusion criteria -None specified Sample size: 110	Data was collected prospectively   Mutations tested -EGFR -EML4-ALK -KRAS -c-MET Statistical methods Pearson’s Chi square or Fisher’s exact test was used to analyse association between mutations and clinical characteristics.	70% of patients harboured mutations   Frequency of mutations -EGFR = 52.7% -EML4-ALK = 10% -KRAS = 3.6% -c-MET = 5.5% Association between mutations and clinical characteristics -EGFR: higher frequency in younger (P=0.002), non-smoking (P<0.001), female lung adenocarcinoma patients (P=0.001) -EML4ALK: higher frequency in non-smokers (P=0.035)	Strengths   -All patients were of the same ethnicity, came from Hunan, China -Single institution study; variability is minimised -All patients were pathologically confirmed as lung adenocarcinoma by more than 2 pathologists using the WHO criteria and TNM staging -All patients were treatment naïve so change in mutation profile in response to treatment was eliminated -All patients were tested for mutations using the same genetic panel Limitations -Small sample size -Other histological subtypes of NSCLC were not included -Very specific study population; data may not be applicable to other patients
Lopez-Chavez, A., et al. (2015). “Molecular profiling and targeted therapy for advanced thoracic malignancies: a biomarker-derived, multiarm, multihistology phase II basket trial.” J Clin Oncol 33(9): 1000-1007. (22)	Inclusion criteria   -Patients from the United States -Patients with histologically confirmed recurrent or advanced NSCLC, SCLC, or thymic malignancies -Patients > 18 years old -Patients with disease that can be biopsied and who are willing to undergo biopsy for molecular profiling or have paraffin embedded tissue blocks available for molecular profiling analysis Exclusion criteria -Patients with disease considered to be curable with surgery or radiation therapy Sample size: 398 (NSCLC)	Data was collected prospectively   Key mutations tested (NSCLC patients) -EGFR -ALK -KRAS -ERBB2 -PIK3CA Statistical methods Kaplan-Meier analysis was performed to compare OS between different mutation groups	Frequency of mutations   -EGFR = 22.1% (a) 84.1% were Erlotinib sensitive (exon 19 deletions and L858R) (b) 20% were resistant T790M mutations -ALK = 8.7% -KRAS = 24.9% -ERBB2 = 2.8% -PIK3CA = 3.9% -EGFR, ERBB2, PIK3A, ALK mutations were found predominantly in lung adenocarcinomas -OS for all mutation groups were found to be significantly different (P<0.001) with EGFR mutations having the longest OS, followed by ALK and then KRAS -Patients without molecular mutations had the shortest survival	Strengths   -Large sample size -Although other malignancies like SCLC and thymic malignancies were included in the study, analysis of the different cancers were done separately and clearly segregated -Study included different histological subtypes of NSCLC Limitations -Patients varied in that they had a past history of different types and numbers of previous therapies prior to enrolment -Patients with any organ function were included, making variability a possibility -Not all patients were tested for all mutations of interest

Table 2. Studies on Genetic Profile of NSCLC (Part 2)

Study	Study population	Methods	Results	Strengths and Liminations
Kris, M. G., et al. (2014). “Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs.” Jama 311(19): 1998-2006. (23)	Inclusion criteria   -Patients from 14 institutions in the United States -Patients with stage IV or recurrent lung adenocarcinoma -Patients with adequate tumour tissue for genomic characterisation -Patients previously tested for oncogenic drivers Exclusion criteria -Patients with adeno-squamous lung carcinomas -Patients with tissues samples containing insufficient material to permit diagnosis of adenocarcinoma Sample size: 1007	Data was collected prospectively   Key mutations tested -EGFR (sensitising) -EGFR (others) -KRAS -ALK -ERBB2 -BRAF -PIK3CA Statistical methods Kaplan-Meier analysis was performed to assess OS for groups of interest and the log-rank test was used for comparison of survival	64% of patients harboured mutations   Frequency of mutations -EGFR (sensitising) = 17% -EGFR (others) = 4% -KRAS – 25% -ALK =8% -ERBB2 = 3% -BRAF = 2% -PIK3CA = <1% -4% of patients had 2 concurrent driver mutations -median OS in patients with mutations and who received genotype-directed therapy was significantly longer than that of patients with no mutations and who did not receive genotype directed therapy (3.5 years vs 2.4 years; propensity score-adjusted hazard ratio 0.69 [95%  CI, 0.53-0.9], P=0.006)	Strengths   -Large sample size -Patients of all ECOG performance statuses were included -Included patients from 14 different institutions Limitations -Not all patients were tested for all the mutations of interest in the study -Decision for treatment was not randomly assigned and was decided by the primary physician; potential for bias -Study design not suitable to arrive at definitive conclusions on differences in OS – a proof-of-concept rather than a definitive study on survival
Chatziandreou, I., et al. (2015). “Comprehensive Molecular Analysis of NSCLC; Clinicopathological Associations.” PLoS One 10(7): e0133859. (24)	Inclusion criteria   -Greek patients with NSCLC -Patients who had tissue sample stored in a pathology institute receiving biopsies from all over the country Exclusion criteria -None specified Sample size: 956	Data was collected retrospectively   Key mutations tested -EGFR -KRAS -ALK -c-MET -PIK3CA Statistical methods Pearson’s Chi square or Fisher’s exact test was used to analyse association between mutations and clinical characteristics (e.g. gender, histology, grade, stage)	Frequency of mutations   -EGFR = 10.6% -KRAS =26.5% -ALK = 3.7% -c-MET = 18% -PIK3CA = 3.8% -EGFR mutations correlated with adenocarcinoma histology (P<0.001), females (P<0.001), and non-smokers (P<0.001) -Other mutations had no statistically significant correlations with other clinical characteristics	Strengths   -Large sample size -Included Hellenic patients from across the country -All molecular testing was done at one pathology centre, allowing for standardised techniques -Included all histological subtypes of NSCLC Limitations -Retrospective study design -Many of the mutations had too little patients for demonstration of statistically significant correlations with clinical characteristics -Not all patients were tested for all mutations of interest

 

Table 3. Studies on Targeted Therapy (Part 1)

Study	Study population	Methods	Results	Strengths and Liminations
Rosell, R., et al. (2012). “Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.” Lancet Oncol 13(3): 239-246. (25)	Inclusion criteria   -Histological diagnosis of stage IIIB or stage IV NSCLC -Measurable or evaluable disease -Presence of activating EGFR mutations (i.e. exon 19 deletion, L858R mutation in exon 21) -Age >18 years -No history of chemotherapy for metastatic disease in 6 months prior to enrolment -Patients with asymptomatic brain metastases Exclusion criteria -Non-specified Sample size: 174	-Randomised prospective trial at 42 hospitals in France, Italy and Spain   -Patients randomly assigned to receive erlotinib or SC (1:1) Primary endpoint: PFS Secondary endpoints: OS, RR Statistical methods -Kaplan-Meier curves for OS, and PFS analysis  and comparisons made using the log-rank test -Calculated HRs (95% CI) with a Cox proportional hazards analysis	PFS   -Significantly longer in gefitinib group than SC group -Erlotinib: 9.7 months (95% CI 8.4-12.3) -SC: 5.2 months (95% CI 4.5-5.8) -HR: 0.37 (95% CI 0.25-0.54; p<0.0001) OS -No significant difference -Erlotinib: 19.3 months (95% CI 14.7-26.8) -SC: 19.5 months (16.1 – not accessible) -HR: 1.04, 95% CI 0.65-1.68; p=0.87) RR -Erlotinib: 64% -SC: 18%	Strengths   -Centralised randomisation for patients across the 3 countries was done to avoid bias -PFS and response as defined by the RECIST criteria were confirmed externally by a board of reviewers to avoid bias -Tumour specimens were obtained and tested before any treatment -Baseline characteristics were balanced between the erlotinib and standard chemotherapy group Limitations -Small sample size -Protocol included completion of a lung cancer symptom scale questionnaire by patients but analysis of results was excluded due to poor response -Results only applicable to European populations
Maemondo, M., et al. (2010). “Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.” N Engl J Med 362(25): 2380-2388 (26)	Inclusion criteria   -Advanced NSCLC -Sensitising EGFR mutation present -No previous chemotherapy -Age < 75 years Exclusion criteria -Presence of resistant T790M EGFR mutation Sample size: 230	-Multi-center, randomised prospective trial conducted in Japan   -Patients randomly assigned to receive gefitinib or SC Primary endpoint: PFS Secondary endpoints: OS, RR, AE Statistical methods -Kaplan-Meier curves for OS, and PFS analysis  and comparisons made using the log-rank test -Calculated HRs (95% CI) with a Cox proportional hazards analysis -RR compared using Fisher’s exact test -Toxic effects compared using Wilcoxon test	PFS   -Significantly longer in gefitinib group -Gefitinib: 10.8 months -SC: 5.4 months -HR: 0.30 (95% CI 0.22-0.41; P<0.001) OS -No significant difference -Gefitinib: 30.5 months -SC: 23.6 months RR -Significantly higher in gefitinib group -Gefitinib: 73.7% -SC: 30.7% -P<0.001 AE -Severe, grade III toxic effects significantly lower in Gefitinib group (P<0.001) -SC: 71.7% -Gefitinib: 41.2%	Strengths   -Prospective, randomised trial -Patients were all treatment naïve – allows assessment of suitability of treatment as first line therapy -All histological subtypes of NSCLC included Limitations -Does not include all EGFR mutations -Results only applicable to the Japanese population

Table 3. Studies on Targeted Therapy (Part 2)

Study	Study population	Methods	Results	Strengths and Liminations
Shaw, A. T., et al. (2013). “Crizotinib versus chemotherapy in advanced ALK-positive lung cancer.” N Engl J Med 368(25): 2385-2394 (27)	Inclusion criteria   -Locally advanced or metastatic NSCLC -Positive for ALK rearrangement mutations -Age > 18 years -Progressive disease after a previous platinum-based chemotherapy -Measurable disease as defined by RECIST version 1.1 criteria -ECOG status of 0-2 -Stable brain metastases that have been previously treated, or are untreated but asymptomatic Exclusion criteria -None specified Sample size: 347	-Randomised prospective trial   -Patients randomly assigned to receive crizotinib or SC (1:1) Primary endpoint: PFS Secondary endpoints: OS. RR, CTCAE-defined AE, QOL assessed by QLQ-C30 and QLQ-LC13 Statistical methods -Kaplan-Meier curves for OS, and PFS analysis  and comparison made using the log-rank test -Calculated HRs (95% CI) with a stratified Cox proportional hazards analysis -RR compared using two-sided stratified Cochran-Mentel-Haeszel test	PFS   -Significantly longer in crizotinib group -Crizotinib: 7.7 months (95% CI 6.0-8.8) -SC: 3.0 months (95% CI 2.6-4.3) -HR: 0.49 (95% CI 0.37-0.64; P<0.001) OS -No significant difference -Crizotinib: 20.3 months -SC: 22.8 months RR -Significantly higher in crizotinib group -Crizotinib: 65% (95% CI 58-72) -SC: 20% (95% CI 14-26) -P<0.001 CTCAE AE -Incidence of severe grade 3-4 AE similar QOL -Significantly greater global improvement of QLQ-C30 and QLQ-LC13 determined quality of life in crizotinib group (P<0.001)	Strengths   -Baseline characteristics of patients from both groups were well balanced -Use of evidenced-based measures of AE i.e. the CTCAE -Use of evidence-based measures of quality of life in cancer patients i.e. QLQ-C30 and QLQLC13 Limitations -Analysis of OS confounded by high crossover rate in SC group patients -Factors causing imbalance of analysis of incidence of AEs: (a)significantly longer duration of treatment in crizotinib group than SC group (b)significantly greater number of patients in crizotinib group continued treatment beyond disease progression
Planchard, D., et al. (2016). “Dabrafenib in patients with BRAF(V600E)-positive advanced non-small-cell lung cancer: a single-arm, multicentre, open-label, phase 2 trial.” Lancet Oncol 17(5): 642-650. (28)	Inclusion criteria   -Age >18 years -Histologically confirmed stage IV NSCLC – –Progressed after one systemic therapy for metastatic disease -BRAFV600E mutation positive -ECOG status 2 -Tumour sample adequate for BRAFV600E mutation testing Exclusion criteria -Previous BRAF or MEK inhibitor therapy -Symptomatic or unstable brain metastases -Anti-cancer therapy within 14 days prior to start of study treatment Sample size: 84	Multicenter, non-randomised study in 34 centres across 10 countries within Asia, North America, and Europe.   Primary endpoint: ORR Secondary endpoints: PFS, OS Statistical methods -ORR: 2-stage Green-Dahlberg design for phase 2 cancer trials -ORR CI: Clopper-Pearson method -PFS, OS: Kaplan-meier curves, CI calculated using Brookmeyer-Browley method	Conclusion   -Results show clinical activity of Dabrafenib against BRAF-mutant lung cancer ORR 33% (95% CI 23-45) PFS 5.5 months (95% CI 3.4-7.3) OS 12.7 months (95% CI 7.3-16.9)	Strengths   -Demonstrates anticancer activity of Dabrafenib in patietns with BRAFV600E mutation -1st trial on BRAF mutation focusing on V600E mutation, good focus on one mutation -Comparison made to other trials in discussion Limitations -Small sample size -Lack of comparison group -Multicenter study may lead to inter-institution variability

Table 3. Studies on Targeted Therapy (Part 3)

Study	Study population	Methods	Results	Strengths and Liminations
Mazieres, J., et al. (2016). “Lung cancer patients with HER2 mutations treated with chemotherapy and HER2-targeted drugs: results from the European EUHER2 cohort.” Ann Oncol 27(2): 281-286. (29)	Inclusion criteria   -Advanced NSCLC -Exon 20 HER2 mutation/insertion -Undergone at least 1 systemic anti-cancer therapy -Documented follow-up of tumour Exclusion criteria -None specified Sample size: 101	Retrospective cohort study conducted in France, Switzerland, Spain, Italy, Poland, Portugal and Netherlands.   Patients received one of the following: (A)HER2 targeting agent i.e. Trastuzumab (B)1st line without HER2 targeting agent (C)2nd line without HER2 targeting agent (D)EGFR-TKI (E)Neratinib, lapatinib and afatinib Primary endpoint: ORR Secondary endpoint: PFS, OS Statistical methods – Kaplan-Meier curves used for PFS, OS rates	Conclusion   -Results show sensitivity of HER2 mutant lung cancer to Trastuzumab ORR (A)50.9% (B)43.5% (C)10% (D)7.6% (E)7.4% PFS (95% CI) (A)4.8 months (3.4-6.5) (B)6 months (5-7.1) (C)4.3 months (3.1-5) (D)2.99 months (1.87-4.47) (E)3.4 months (2.4-4) OS (95% CI) (A)13.3 (8.1-15) (B)24(19.1-36.4) (C)19.4 (9.6-24.7) (D)13.3 (8.1-15) (E)6.5 (4.7-30.6)	Strengths   -Largest study of date of patients with advanced HER2 mutant NSCLC Limitation -Small sample size -Retrospective study -Lack of comparison group -All patients had adenocarcinoma histology, limiting variety of NSCLC histological subtypes -Non-randomised trial -No clear indication of which treatment should be first line
Blumenschein, G. R., Jr., et al. (2015). “A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)dagger.” Ann Oncol 26(5): 894-901. (30)	Inclusion criteria   -Histologically confirmed KRAS/NRAS/BRAF/MEK1-mutant lung adenocarcinoma previously treated with 1 platinum-based chemotherapy -ECOG status 0 or 1 -Measurable disease defined by RECIST v1.1 -Adequate bone marrow, cardiac, hepatic, functions Exclusion criteria -Previous BRAF or MEK inhibitor treatment -Risk of brain metastasis Sample size: 129	Prospective, randomised trial where patients are assigned to trametinib or docetaxel (2:1)   Primary endpoint: PFS Secondary endpoints: RR Statistical methods -Kaplan-Meier curve used for PFS, compared using stratified log-rank test (stratified for sex) -Fisher’s exact test used to compare RR	Trametinib exhibited similar PFS and RR as docetaxel in KRAS mutant NSCLC patients who received prior treatment i.e. trametinib did not demonstrate superiority to docetaxel   PFS -Trametinib: 12 weeks -Docetaxel: 11 weeks -HR: 1.14 (95% CI 0.75-1.75) RR -Trametinib: 12% -Docetaxel: 12% -No significant difference	Strengths   -Prospective, randomised study -Randomisation was stratified by gender and mutational status (KRAS vs NRAS/BRAF/MEK1) -Multicenter study conducted in European countries Limitations -Small study sample -Population was mainly of European origin with only a small representation from Asians and no representation from other countries

 

Table 4. Studies on Mutations Resistant to Targeted Therapy

Study	Study population	Methods	Results	Strengths and Liminations
Shaw, A. T., et al. (2009). “Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.” J Clin Oncol 27(26): 4247-4253. (31)	Inclusion criteria   -NSCLC patients with 2 or more of the following characteristics: female sex, Asian ethnicity, never/light smoking history, adenocarcinoma histology Exclusion criteria -Patients with insufficient tissue sample for genetic testing -Patients with EML4-ALK FISH inconclusive -Multifocal bronchoalveolar carcinoma (BAC) Sample size: 141	Retrospective study conducted in United States.   Genetic profiling performed for patients. Primary endpoint: RR Statistical methods -Fishers’ exact test used to compare RR	RR   -Small difference between EML4-ALK and WT/WT patients on erlotinib -Not statistically significant (P=0.536) -Significantly higher RR in EGFR positive patients versus EML4-ALK positive patients (P<0.001)	Strengths   -Includes all histological subtypes of NSCLC -Excluded patients of BAC histology which was not relevant to the study Limitations -Small study sample -Study population was selected based on specific features associated with EGFR mutations and so most were female, light/non-smokers. This excludes all other EML4-ALK patients and limits the applicability of study results to other patients
Lim, S. M., et al. (2016). “Targeted sequencing identifies genetic alterations that confer primary resistance to EGFR tyrosine kinase inhibitor (Korean Lung Cancer Consortium).” Oncotarget 7(24): 36311-36320. (32)	Inclusion criteria   -NSCLC patients with activating EGFR mutations Exclusion criteria -None specified Sample size: 152	Prospective study with patients from Korean population treated with gefitinib to assess resistance.   Genomic testing was done to determine genetic profile of each patient Primary endpoints: PFS, OS Statistical methods -Kaplan-Meier curves for PFS and OS -Differences compared by log-rank test, 2-sided P-values < 0.05 were considered significant	PFS   -Significantly shorter in patients with P13K/Akt/mTOR mutation than those without the mutation -P13K/Akt/mTOR positive: 2.1 months -P13K/Akt/mTOR negative: 12.8 months -P=0.04 OS -Significantly shorter in patients with P13K/Akt/mTOR mutation than those without the mutation -P13K/Akt/mTOR positive: 15.7 months -P13K/Akt/mTOR negative: not reached -P=<0.001	Strengths   -Comprehensive genetic profiling of patients allows detection of treatment-resistant genes -Evidence-based definition of response to treatment using RECIST criteria Limitations -Small study sample -Limited to the Korean population -Only mutations reported in COSMIC were reported and intronic or silent mutations were not reported
Yu, H. A., et al. (2014). “Poor response to erlotinib in patients with tumors containing baseline EGFR T790M mutations found by routine clinical molecular testing.” Ann Oncol 25(2): 423-428. (33)	Inclusion criteria   -Patients with lung cancer who underwent molecular testing -Metastatic or recurrent disease Exclusion criteria -None specified Sample size: 2774	Retrospective study conducted in United States   Frequency of EGFR T790M mutations established Primary endpoints: RR, OS, PFS Statistical methods -Kaplan-Meier curves for OS and PFS	EGFR T790M mutation:   -Low incidence – 0.5% of all lung cancers, 2% in EGFR mutant lung cancers -Predicts lack of response to EGFR TKI in the form of lower RR to Erlotinib, and shorter PFS and shorter OS when compared to the literature RR: 8% PFS: 1.5 months OS: 16 months	Strengths   -Mutation status determined from pre-treatment tumour samples – eliminates possible acquired mutations due to prior treatment -Large sample size Limitations -No specification of histological subtype of lung cancer -Lack of comparison group

 

Results Cont’d

Genetic profile of NSCLC patients

Four studies focused on the frequencies of specific mutations in different populations and demographic, clinical, or histological factors associated with each mutation (Table 2.) (21-24).

(A) Frequency of mutations

The frequency of EGFR mutations varied from 10.6% to 52.7%, and tended to be higher in the East Asian Chinese population than the Caucasian populations (21-24). In contrast, KRAS mutations tended to be higher in Caucasian populations, at about 25% compared to 3.6% in the Asian population (21-24). ALK mutations had the highest frequencies in the Chinese population, followed by United States, and then Greece (21-24). Lastly, the c-MET mutation was higher in the Greek population than the Chinese population (21, 24). ERBB2 and PIK3CA mutations were similar in studies that reported it, being at low levels of 3.9% or less (22-24). Only one study from the United States reported BRAF mutation frequency and it was 2% (23). One study reported that 4% of patients were doubletons i.e. had two concurrent mutations (23). However, no information was given on which mutations were concurrent (23).

Table 5. Summary of frequencies of mutations

Study	Xia et al,   2013	Lopez-Chavez et al, 2015	Kris et al,   2014	Chatziandreou et al, 2015
Population	Chinese	United States	United States	Greek
EGFR	52.7%	22.1%	21%	10.6%
KRAS	3.6%	24.9%	25%	26.5%
ALK	10%	8.7%	8%	3.7%
ERBB2	–	2.8%	3%
PIK3CA	–	3.9%	<1%	3.8%
c-MET	5.5%	–	–	18%
BRAF	–	–	2%	–

(B) Factors associated with mutations

EGFR mutation frequency was significantly higher in Chinese, younger, female, non-smoking, adenocarcinoma patients (21, 24). ALK mutations were also significantly associated with non-smokers. Lastly, most mutations were predominant in patients with adenocarcinoma (22), and were associated with longer OS compared to wildtype patients (22, 23).

Efficacy of Targeted Therapy

Six studies assessed the use of targeted therapy in NSCLC with a focus on one mutation per study (Table 3.) (25-30). Three randomised controlled trials concluded that targeted therapy was superior to standard chemotherapy on the basis of significantly longer PFS set as the primary endpoint (25-27). However, one randomised trial concluded that targeted therapy was non-superior to chemotherapy by exhibiting similar PFS in both treatment groups as the primary endpoint (30). Two non-randomised studies demonstrated clinical activity of targeted therapy to sensitising mutations with RR defined as a primary endpoint, but no comparison was made to chemotherapy group (28, 29).

OS was measured in the three randomised controlled trials and no significant difference was found between targeted therapy and standard chemotherapy (25-27).

One study reported a significantly greater improvement of global as well as and lung cancer-associated quality of life based on evidence-based questionnaires in patients on targeted therapy versus chemotherapy (27).

Lastly, two studies took into account adverse effects in their conclusion of results – one reported that severe grade III toxic effects were significantly lower in the targeted therapy group (26), while the other reported similar incidence of severe grade III-IV toxicities in targeted therapy and chemotherapy groups (27).

Resistance to Treatment

Three studies examined mutations exhibiting resistance to targeted therapy particularly of the EGFR TKI class (Table 4.) (31-33). Patients with EML4-ALK, P13K/Akt/mTOR, and EGFR T790M mutations had PFS and OS that were either significantly shorter than patients with sensitising mutations or similar to wildtype patients (31-33). They also had RR that were lower than patients with sensitising mutations or similar to wildtype patients (31, 33).

 

Table 6. EBL Critical Appraisal Validity Scores for Each Study (Full Details in Appendix 1)

Article	Population Validity Score (%)	Data Collection Validity Score (%)	Study Design Validity Score (%)	Results Validity Score (%)	Overall   Validity Score (%)
Xia et al, 2013 (21)	80	80	100	67	81
Lopez-Chavez et al, 2015 (22)	71	85	100	67	80
Kris et al, 2014 (23)	83	85	100	83	91
Chatziandreou et al, 2015 (24)	83	85	100	67	82
Rosell et al, 2012 (25)	100	85	100	67	88
Maemondo et al, 2010 (26)	87	85	100	83	88
Shaw et al, 2015 (27)	87	85	100	83	88
Planchard et al, 2016 (28)	79	85	100	67	74
Mazières et al, 2015 (29)	66	85	100	67	75
Blumenschein et al, 2015 (30)	75	85	100	67	80
Shaw et al, 2009 (31)	60	85	100	67	78
Lim et al, 2016 (32)	66	85	80	67	75
Yu et al, 2014 (33)	83	85	100	67	83

A score of >75% implies validity.

Quality of Studies

The validity scores generated for each study using the EBL critical appraisal checklist is summarised in Table 6, and the full details can be found in Appendix 1.

Only one study (28) was deemed to be invalid with a score of 74%. Nine studies (21-23, 25-28, 30, 32) were prospective in nature which allowed for collection of data best suited to achieve the objectives of the studies.

Additionally, four (25-27, 30) out of six studies investigating the efficacy of targeted therapy were randomised controlled trials which allowed effective comparison of targeted therapy against standard chemotherapy. However, the heterogeneity of study designs resulted in difficulties in comparison of results with the remaining two studies.

Three studies (21, 23, 30) focused only on adenocarcinomas, which only forms 50% of all NSCLCs (2). While adenocarcinomas have been shown to be associated with most mutations relevant to lung cancer (6, 22), for a more complete analysis, other histological subtypes should be included.

In order to expand numbers for the study population, many studies involved multiple institutions (22, 23, 25-29, 31-33). While this helps to increase the sample size in situations where mutations are rare, studies without a clear protocol are at risk of inter-institution variability in patient diagnosis and management. For example, in three of the four studies examining frequencies of mutations (22-24), some patients were tested for all mutations while others were not, making the methodology inconsistent. Additionally, none of the studies performed sample size calculations, making it impossible to comment on whether the sample size is sufficient to generate accurate estimates.

Of the studies examining efficacy and resistance to treatment, six used PFS as the primary endpoint (25-27, 30, 32, 33), while three used RR as the primary endpoint (28, 29, 31). OS was hardly used as an endpoint as it often was not found to be significantly different between targeted therapy and chemotherapy patients (25-27). Given the variations in primary endpoints of the study, comparison of studies was more challenging. Lastly, primary endpoints could have been deliberately chosen by authors to reinforce their conclusions, and any endpoint that might contradict their conclusions were deemed as secondary endpoints.

Discussion

Genetic Profile of NSCLC Patients

This literature review has demonstrated the heterogeneity of genetic profiles across different populations with frequency of EGFR mutations highest in East Asian populations (21), and frequency of KRAS mutations highest in Caucasian populations (22-24). Frequency of treatment-resistant EGFR T790M mutations remain low (22, 33), which bodes well for patient outcomes. An interesting observation was the possibility of concurrent mutations highlighted by Kris et al (23). In future analyses, it would be worthwhile further examining this subset of patients by looking at which mutations tend to coexist, the possibility of gene interactions, and whether these patients require a different management algorithm.

It is also evident that the ‘typical’ NSCLC patient with mutation-associated features were EGFR-positive, East Asian, female, younger, light or non-smokers, with adenocarcinomas (21, 22, 24). This will be useful to clinicians in proactively considering genetic testing for such patients. Additionally, factors associated with most of the other mutations have not be well established, and this could be a potentially promising area to explore in future lung cancer research. Lastly, patients with mutations tended to have longer OS than wildtype patients (22, 23), and more can be done in future studies to analyse the role of mutational status as a prognostic factor for survival.

The Use of Targeted Therapy

It is clear from this review that targeted therapy is superior to standard chemotherapy for advanced NSCLC patients with sensitising mutations to EGFR TKIs, like erlotinib (25) and gefitinib (26), as well as the ALK TKI, crizotinib (27). PFS and RR were significantly greater in the groups receiving targeted therapy versus chemotherapy (25-27). This has practice-changing implications given that platinum-based doublet chemotherapy has traditionally been the gold standard for the management of advanced NSCLC (34). In fact, targeted therapy may not only be used singly in place of chemotherapy, but also concomitantly with chemotherapy to exert a synergistic effect (35).

Additionally, molecular agents targeting BRAF mutations, such as dabrafenib (28), and HER2 mutations, such as trastuzumab (29), have been proven to be clinically active in patients with sensitising mutations primarily on the basis of RR analysis. However, much has to be done to ascertain whether these agents should be considered for first-line treatment in place of chemotherapy.

Lastly, not all targeted agents show promise in improving patient outcomes. For example, the MEK1/MEK2 inhibitor, trametinib, was shown to have similar PFS and RR as chemotherapy in KRAS-mutant patients (30).

Overall, the scene for molecular targets in advanced NSCLC is a heterogeneous and evolving one. Future developments in this area is expected to encourage greater use of these agents given favourable outcomes on survival, response, and quality of life (27), with side effect profiles similar to or better than standard chemotherapy (26, 27).

Treatment-resistant Mutations

Equally important in this literature review are the insights gained on treatment-resistant mutations. An awareness of mutations such as EML4-ALK (31), P13K/Akt/mTOR (32), and EGFR T790M (33) allows physicians to spare patients from having to experience unnecessary adverse effects should the drawbacks of toxicities outweigh the minimal response from targeted therapy. This brings into consideration of genetic testing not just to seek out sensitising mutations but also to tease out resistant ones. In future reviews, it would be useful to examine how genetic profiles of treatment-resistant patients change with disease progression or recurrence –  through re-biopsies (36) –  in order to generate a treatment algorithm best suited for this subset of patients.

Limitations

The exclusion of studies that were not in English or were inaccessible was a limitation of this review. The 2000 word limit is also a major constraint. Given that this is generated by a single-author, there could also be potential for bias and error. Additionally, although programme death-ligand 1 (PD-L1) immune checkpoint inhibitors like pembrolizumab and nivolumab are also being explored for the management of advanced NSCLC (37, 38), articles on this topic did not satisfy the inclusion and exclusion criteria, and were thus excluded. This serves as a potential area for future analysis. Lastly, this review was done at a single time-point, not allowing for literature updates.

Conclusion

The results of this literature review have demonstrated the heterogeneity of the efficacy of targeted agents in advanced NSCLC patients with sensitising mutations. EGFR TKIs and ALK TKIs are clearly superior to standard chemotherapy, while other agents require more to be done to clarify their definitive role in the care of NSCLC patients. The genetic profiles of NSCLC patients also have significant variations across different populations and it is important to keep in mind a patient’s demographic, clinical and histological background when performing genetic tests. Lastly, treatment-resistant mutations remain at low frequencies and should be taken into consideration when managing patients with NSCLC. Overall, molecular therapy has a promising future in the management of advanced NSCLC given its potential for favourable treatment outcomes, side effect profiles and impact on patients’ quality of life. It is expected that future developments in the field will bring about new changes to the treatment algorithm for this group of patients.

 

Acknowledgements

I would like to express my sincere gratitude to my supervisor Dr Brian Healy Bird and co-supervisor Dr Richard Bambury for their patient guidance, and for pointing out the crucial areas for analysis in this literature review.

 

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Appendices

Appendix 1 – EBL Critical Approisal Checklist

EBL Critical Appraisal Checklist

Xia   (2013)

Lopez-Chavez   (2015)

Kris   (2014)

Chatziandreou   (2015)

Rosell   (2012)

Maemondo   (2010)

Shaw   (2015)

Planchard   (2016)

Mazières   (2015)

Blumenschein   (2015)

Shaw   (2009)

Lim   (2016)

Yu   (2014)

Section A: Population

Is the study population representative of all users, actual and eligible who might be included in the study?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Are inclusion and exclusion criteria definitively outlined?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is the sample size large enough for sufficient precise estimates?

N

Y

Y

Y

Y

Y

Y

N

N

N

U

U

Y

Is the response rate large enough for sufficiently precise estimates?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is the choice of population bias-free?

Y

N

N

N

Y

N

N

N

N

N

N

N

N

If a comparative study:   Were participants randomised into groups? Were the groups comparable at baseline? If groups were not comparable at baseline, was incompatibility addressed by the authors in the analysis?

N/A

N   Y N/A

N/A

N/A

Y   Y N/A

Y   Y N/A

Y Y   N/A

N/A

N/A

Y   Y N/A

N/A

N/A

N/A

Was informed consent obtained?

N/A

N/A

Y

N/A

Y

Y

Y

Y

U

Y

N/A

Y

Y

Section B: Data Collection

Are data collection methods clearly described?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

If a face-to-face survey, were inter-observer and intra-observer bias reduced?

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Is the data collection instrument validated?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

If based on regularly collected statistics, are the statistics free from subjectivity?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Does the study measure the outcome at a time appropriate for capturing the intervention’s effect?

N/A

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is the instrument included in the publication?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Are questions posed clearly enough to be able to elicit precise answers?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Were those involved in the data collection not involved in delivering a service to the target population?

N

N

N

N

N

U

N

N

N

N

N

N

N

Section C: Study Design

Is the study type/methodology utilised appropriate?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is there face validity?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is the research methodology clearly stated at a level of detail that would allow its replication?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Was ethics approval obtained?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Are the outcomes clearly stated and discussed in relation to the data collection?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Section D: Results

Are all the results clearly outlined?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Are confounding variables accounted for?

N

N

Y

N

N

N

Y

N

N

N

N

N

N

Do the conclusions accurately reflect the analysis?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is subset analysis a minor, rather than a major focus of the article?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Are suggestions provided for further areas to research?

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Is there external validity?

N

N

N

N

N

N

N

N

N

N

N

N

N