ABSTRACT

Objective: To investigate the histological subtypes and mutational profiles of non-small cell lung cancer in Brazil, looking for correlations among histological subtypes, expression of anaplastic lymphoma kinase (ALK), EGFR mutation status, and programmed death-ligand 1 (PD-L1) expression. Methods: We evaluated 173 specimens obtained from patients with lung adenocarcinoma in northeastern Brazil. Expression of PD-L1 and ALK was evaluated by immunohistochemistry; EGFR mutation status was evaluated by sequencing. We categorized the histological subtypes in accordance with the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Results: The most common histological subtypes of lung adenocarcinoma were solid predominant (in 46.8%), acinar predominant (in 37.0%), and lepidic predominant (in 9.8%). ALK expression was detected in 10.4% of the samples, and 22.0% of the tumors harbored EGFR mutations. The most common EGFR mutation was an exon 21 L858R point mutation (in 45.5%), followed by an exon 19 deletion (in 36.3%). The tumor proportion score for PD-L1 expression was ≥ 50% in 18.2% of the samples, 1-49% in 32.7%, and 0% in 49.5%. The solid predominant subtype was significantly associated with wild-type EGFR status (p = 0.047). Positivity for PD-L1 expression was not found to be significantly associated with ALK expression or EGFR mutation status. Conclusions: Our results suggest that the molecular profile of non-small cell lung cancer in northeastern Brazil differs from those of populations in other regions of the country, with ALK positivity being higher than the other biomarkers. Further studies including clinical and genetic information are required to confirm these differences, as well as studies focusing on populations living in different areas of the country.

Keywords: Anaplastic lymphoma kinase; ErbB receptors; B7-H1 antigen; Carcinoma, non-small-cell lung; Brazil.

RESUMO

Objetivo: Investigar os subtipos histológicos e perfis de mutação do carcinoma pulmonar de células não pequenas no Brasil, bem como as correlações entre os subtipos histológicos, a expressão do gene anaplastic lymphoma kinase (ALK, quinase do linfoma anaplásico), o estado de mutação do gene EGFR e a expressão de programmed death-ligand 1 (PD-L1, ligante de morte celular programada 1). Métodos: Avaliamos 173 espécimes provenientes de pacientes com adenocarcinoma pulmonar no Nordeste brasileiro. A expressão de PD-L1 e ALK foi avaliada por meio de imuno-histoquímica, ao passo que o estado de mutação do EGFR foi avaliado por meio de sequenciamento. Os subtipos histológicos foram classificados de acordo com a International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society. Resultados: Os subtipos histológicos mais comuns de adenocarcinoma pulmonar foram o predominantemente sólido (em 46,8%), o predominantemente acinar (em 37,0%) e o predominantemente lepídico (em 9,8%). A expressão de ALK foi detectada em 10,4% das amostras, e 22,0% dos tumores apresentavam mutações do gene EGFR. As mutações mais comuns do EGFR foram a mutação pontual L858R no éxon 21 (em 45,5%) e a deleção do éxon 19 (em 36,3%). O tumor proportion score relativo à expressão de PD-L1 foi ≥ 50% em 18,2% das amostras, = 1-49% em 32,7% e = 0% em 49,5%. O subtipo predominantemente sólido relacionou-se significativamente com EGFR selvagem (p = 0,047). A expressão positiva de PD-L1 não se relacionou significativamente com a expressão de ALK ou o estado de mutação do EGFR. Conclusões: Nossos resultados sugerem que o perfil molecular do carcinoma pulmonar de células não pequenas no Nordeste brasileiro difere do de populações em outras regiões do país: a expressão positiva de ALK é maior que os demais biomarcadores. Mais estudos com informações clínicas e genéticas são necessários para confirmar essas diferenças, além de estudos que se concentrem em populações em diferentes áreas do país.

Palavras-chave: Proteínas tirosina quinases; Receptores ErbB; Antígeno B7-H1; Carcinoma pulmonar de células não pequenas; Brasil.

INTRODUCTION

Approximately 1.8 million new cases of lung cancer are diagnosed annually, accounting for 13% of all cancer cases worldwide. In the United States, there were an estimated 150,000 deaths from lung cancer in 2018. (1) For that same year, data from the Brazilian National Cancer Institute indicate that in Brazil, there were 27,200 deaths from lung cancer, as well as 31,270 new cases of the disease.(2)

Non-small cell lung cancer (NSCLC) accounts for more than 80% of all cases of lung cancer, and this broad category (NSCLC) encompasses a number of subtypes, the most prevalent of which is adenocarcinoma. According to the World Health Organization (WHO) classification, the most common histological patterns of growth in adenocarcinomas are acinar, solid, papillary, micropapillary, and mucinous.(3)

Most lung cancer patients present with metastatic disease and receive chemotherapy, targeted therapies, immunotherapies, or a combination of those modalities. The standard of care for advanced NSCLC was transformed by the identification of oncogenic drivers and the development of tyrosine kinase inhibitors targeting such drivers, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) oncogenes. The more recent development of immune checkpoint inhibitors, such as anti-programmed death-ligand 1 (anti-PD-L1) and anti-cytotoxic T lymphocyte antigen 4, has also led to major therapeutic advances in this disease as demonstrated by the results of several clinical trials documenting improvements in overall survival.(4,5)

The PD-L1 checkpoint inhibitor (also known as B7 homolog 1) is the major ligand of PD-1, and its expression on the surface of tumors cells upregulates and inhibits the immune response. Some clinical trials in patients with NSCLC have demonstrated a correlation between increased PD-L1 expression on NSCLC cells and enhanced efficacy of single-agent anti-PD-1 or anti-PD-L1 inhibitors,(6,7) as well as of combinations of those with ipilimumab.(8) The results were independent of molecular alterations in ALK or EGFR. These advances not only represent major therapeutic breakthroughs but also amplify the importance of identifying the molecular features of tumors in order to guide the therapy and maximize its benefits. In low- and middle-income countries, the enthusiasm for these novel treatments is tempered by the limited access to molecular tools to characterize tumors, as well as by the high costs of targeted therapies and immunotherapies.(9) As a result, a large proportion of patients continue to receive conventional (i.e., non-targeted) chemotherapy, which, in many circumstances, is associated with limited efficacy and significant adverse effects.

Only a few studies have documented the molecular features of NSCLC, including the prevalence of ALK rearrangement, EGFR mutation status, and PD-L1 expression, in Brazil.(10-12) There is even more limited information regarding patients in underserved regions such as the northeastern region of the country. Here, we report the findings in a sample of 173 specimens of lung adenocarcinoma evaluated at a major referral laboratory of pathology, describing the histological subtypes, EGFR status, ALK status, and PD-L1 expression.

METHODS

Sample selection

We conducted a retrospective analysis of NSCLC specimens evaluated at a regional referral laboratory for surgical pathology in Fortaleza, Brazil (the Argos Laboratory), between 2015 and 2016. All specimens were fixed in formalin, after which they were stained with hematoxylin and eosin in a routine manner. Cases were reviewed by two independent pathologists with experience in pulmonary pathology and were classified according to the WHO classification system.(3) The morphological patterns, predominant histological subtypes, and available clinical data were recorded. Only non-small cell carcinomas were included in the study, those with sarcomatoid or neuroendocrine differentiation therefore being excluded. The study was approved by the Institutional Review Board of Messejana Heart and Lung Hospital, also located in the city of Fortaleza, and was registered with the National Commission for Ethics in Research (CAAE protocol no. 65315317.0.0000.5039).

EGFR mutation status

For each sample, we selected a representative formalin-fixed, paraffin-embedded block containing at least 10% viable tumor. After proteinase K digestion of the samples, we extracted DNA following standard protocols. Direct DNA sequencing of exons 18 through 21 of the EGFR gene was performed as previously described.(13) To detect gene mutations, we employed multiplex polymerase chain reaction in a next-generation sequencing instrument (MiSeq; Illumina, San Diego, CA, USA), as previously described in detail.(13)

ALK expression

For the evaluation of ALK expression, all specimens were processed in accordance with the well-established standard operating procedures adopted at the pathology laboratory. In brief, sections were stained in an automated slide staining instrument (Ventana Benchmark GX; Roche Diagnostics, Basel, Switzerland) and incubated with an approved anti-ALK rabbit monoclonal primary antibody (clone: D5F3, Cat. #: 790-4796; Roche Diagnostics), after which ALK was detected with an amplification kit (OptiView Amplification Kit, Cat. #: 760-099; Roche Diagnostics) and a diaminobenzidine immunohistochemical detection kit (OptiView DAB IHC Detection Kit, Cat. #: 760-700; Roche Diagnostics). Counterstaining was performed with hematoxylin, and negative controls were assessed. Samples were considered positive for ALK expression if any cells showed cytoplasmic staining, regardless of the proportion or intensity of staining.

PD-L1 expression

For the evaluation of PD-L1 expression, all specimens were processed in accordance with standard established protocols. Immunohistochemical staining for PD-L1 protein was carried out with the Ventana PD-L1 assay (clone: SP263, Cat. #: 740-4907; Roche Diagnostics) on the Ventana Benchmark GX system, PD-L1 being detected with the kits described for ALK. Counterstaining was performed with hematoxylin, and negative controls were assessed. In the interpretation of the results, PD-L1 expression was evaluated on tumor cells. Samples were considered positive for PD-L1 expression on the basis of the proportion of cells showing staining of any intensity, in 10% increments.(14)

Statistical analysis

Correlations between categorical variables were analyzed with Fisher's exact test (when any cell in a contingency table had an expected count < 5) or Pearson's chi-square test (when none of the cells in a contingency table had an expected count < 5). All reported p-values are two-sided, and tests were conducted at a 0.05 level of significance. Statistical analysis was performed with the Statistical Analysis System, version 9.4 (SAS Institute Inc., Cary, NC, USA).

RESULTS

A total of 173 patients with lung adenocarcinoma were evaluated. The general characteristics of the patients are shown in Table 1. The median age was 67 years (range, 36-93 years), and 103 (59.5%) of the patients were > 70 years of age. Eighty-one (46.8%) of the patients were male.
 



The most common sampling sites were the lung, pleura, and lymph nodes, which respectively accounted for 125 (72.2%), 23 (13.3%), and 12 (6.9%) of the 173 specimens collected. Most of the specimens were obtained by computed tomography-guided transthoracic biopsy, followed by lobectomy and transbronchial biopsy. In accordance with the WHO classification of lung tumors,(3) we categorized the invasive adenocarcinoma growth patterns as follows (Table 1): solid predominant, in 81 (46.2%) of the specimens; acinar predominant, in 64 (37.0%); lepidic predominant, in 17 (9.8%); and papillary predominant, in 8 (4.6%).

We analyzed the EGFR mutation status in 149 patients. In 116 (77.9%), we detected no EGFR mutations (wild-type status). Thirty-three samples (22.1%) were found to harbor mutations in the EGFR kinase domain. As shown in Table 2, the main types of EGFR mutations were an L858R point mutation in exon 21, seen in 15 (45.5%) of the samples; a deletion in exon 19, seen in 12 (36.3%); and G719X point mutations in exon 18, seen in 3 (9.1%). The histological subtype mostly associated with wild-type EGFR status was the solid predominant subtype (p = 0.0475).
 



Samples were positive for protein expression of ALK in 18 (10.4%) of the 173 cases analyzed. Among the ALK-positive cases, the histological subtype was acinar predominant in 10 (55.6%), solid in 6 (33.3%), lepidic in 1 (5.6%), and papillary in 1 (5.6%). Cases in which the subtype was mucinous predominant did not display ALK rearrangements (Table 3). Figure 1 shows two lung adenocarcinoma samples of the acinar predominant subtype, of which one was negative for ALK expression (Figure 1A) and one showed strong, diffuse positivity for ALK (Figure 1B).
 

 




PD-L1 expression was analyzed in 55 of the tumor samples. Of those, 27 (49.1%) were negative for PD-L1 expression and 28 (50.9%) showed some degree of PD-L1 expression. Using the tumor proportion score (TPS) cut-off values employed in clinical trials of atezolizumab,(6) we stratified PD-L1 expression by TPS, which was 0% in 27 (49.1%) of the 55 samples, 1-4% in 2 (3.6%), 5-49% in 16 (29.1%), and ≥ 50% in 10 (18.2%), as shown in Table 4. Figure 2 shows representative images of different extents of (i.e., TPS for) PD-L1 expression in lung adenocarcinoma: 0% (Figure 2A); 10% (Figure 2B); 50% (Figure 2C); and 100% (Figure 2D).
 

 




Table 3 shows the associations that gender, age, and histological subtype showed with PD-L1 expression, ALK expression, and EGFR mutation status. Neither PD-L1 positivity, EGFR mutation status, nor ALK expression was found to be significantly associated with gender or age. Fisher's exact test showed no significant relationship between positive PD-L1 expression and EGFR mutation status (p = 0.407) or between positive PD-L1 expression and ALK expression (p = 0.408).

DISCUSSION

Here, we have attempted to detect associations among PD-L1 expression, ALK expression, and EGFR mutation status in cases of NSCLC evaluated at a regional referral laboratory for surgical pathology in Brazil. Our findings show that the frequency of PD-L1 expression in patients with nonsquamous NSCLC was 50.9%, higher than the 37.9% reported in another study conducted in Brazil, in which the protocols were similar but different antibodies were used.(15) Previous studies have shown that approximately 30% of patients with advanced NSCLC have a high level of PD-L1 expression (defined as a TPS ≥ 50%).(16,17) In the present study, approximately half of the patients showed some degree of PD-L1 positivity, although only 18.2% had a TPS ≥ 50%. The relatively low proportion of patients with a high level of PD-L1 expression in our study sample could reflect variations in the antibodies, staining platforms, and assay methodologies across studies, as well as a certain degree of arbitrariness in the definition of PD-L1 positivity. There is therefore an urgent need for standardization of PD-L1 testing, which has yet to be addressed. In addition, given that the antibody used in the present study (clone SP263) exhibits staining characteristics for PD-L1 similar to those reported for other anti-PD-L1 antibodies, such as 22C3 and 28-8,(5) we can postulate that our findings are attributable to the unique molecular features of the population studied.

Knowing the proportional PD-L1 expression in lung tumors in any given population might be important not only for predicting responses to therapy but also for determining the overall prognosis. A recent clinical trial, known as the KEYNOTE-024 trial,(17) showed improved progression-free survival and overall survival in NSCLC patients whose tumors had a PD-L1 TPS ≥ 50%. A recent meta-analysis involving 47 studies and more than 11,000 patients showed a positive correlation between PD-L1 expression and a poor prognosis in lung cancer.(18) It is of note that the association with a poor prognosis was observed only in Asian populations. Given the lack of data for the population of Brazil, the present study might represent a first step toward identifying a specific prevalence.

Populations living in low- and middle-income countries face many challenges in order to gain access to new therapies. Not only are the prices of immune checkpoint inhibitors higher in Brazil but the implementation of biomarker selection also represents a barrier to access to the best immunotherapies.(19) In this context, health care systems are also penalized; in one study, a decision-analytic model showed that the use of PD-L1 expression as a biomarker increases the cost-effectiveness of immunotherapy.(20) Some studies have demonstrated that PD-L1 expression tends to be associated with smoking status, high pathologic grade, positive lymph nodes, and tumor size.(16,21)

The echinoderm microtubule-associated protein-like 4-ALK fusion gene was first identified in 2007 by Soda et al.,(22) who estimated its frequency to be 6.7% in patients with NSCLC. Since then, other studies, using immunohistochemistry, have estimated the frequency of ALK expression to be 3-7% among such patients. (23,24) The frequency of ALK expression in the present study (10.4%) was higher than the 3.2-4.8% previously reported for patients with NSCLC in Brazil.(10,12) To our knowledge, ours is the first study focusing on a population of patients in northeastern Brazil. The few previous studies reporting the prevalence of ALK and other biomarkers of NSCLC in Brazil have all focused on the population living in the southeastern region of the country. Socioeconomic disparities between the more developed southeastern region and the northeastern region might have contributed to the higher frequency of ALK positivity found in our study. In fact, clinical variables and genetic information should also be considered in order to explain such differences, and further studies certainly will be required.

We found that 22.1% of patients with lung adenocarcinoma harbored EGFR mutations, which is similar to the 21.6% reported in another study conducted in Brazil,(12) albeit lower than the 26-33% reported for Latin America at large(25,26) and the 30-50% reported for Asia,(27) whereas it is higher than the 11-17% reported for White patients in the United States and the 8-13% reported for patients in Europe. (28) The most common EGFR mutations found in the present study were an exon 19 deletion and an exon 21 L858R point mutation, as has been reported for other populations.(12,21,29)

The associations between PD-L1 expression and EGFR mutations vary across studies. Some authors have shown a direct association between high PD-L1 expression and a positive EGFR mutation status.(30,31) However, Takada et al.(21) found that PD-L1 expression was significantly associated with a wild-type EGFR status. In the present study, we found no association between EGFR mutation status and PD-L1 expression. Our results are more akin to those described in a recent meta-analysis conducted by Yang et al.,(32) in which the authors concluded that the relationship between PD-L1 expression and EGFR mutation status was variable and not significant.

An association between ALK positivity and PD-L1 expression has been demonstrated in some clinical studies,(33,34) although not in others.(35,36) Although we identified a discrete trend toward such an association in the present study, it did not reach statistical significance. The unclear relationship between PD-L1 expression and the activation of oncogenic drivers (EGFR and ALK) in NSCLC, together with the discrepancies among studies, might be attributable to differences across studies in terms of the baseline clinical characteristics of the patients, heterogeneity among study populations, and the lack of standardization in the definition of PD-L1 positivity.

In attempts to determine whether molecular alterations are able to alter morphology, there have been several studies investigating the associations between EGFR mutations and the major histological patterns. A study conducted in Japan showed that EGFR mutations were significantly associated with the papillary predominant and lepidic predominant subtypes.(37) In a study conducted in China, Song et al.(38) found that the micropapillary predominant and lepidic predominant subtypes were associated with EGFR mutations. In a population of patients in the United States, the lepidic predominant subtype was found to be the only histological subtype associated with EGFR mutations,(39) whereas the acinar predominant subtype was the only subtype found to be associated with EGFR mutations in a population of patients in Brazil. (12) Our finding that the solid predominant subtype was most strongly associated with wild-type EGFR status underscores the fact that the relationship between EGFR mutation status and the histological subtype of lung adenocarcinoma remains unclear.

Our study has several limitations. First, it was a single-center retrospective study, which makes it impossible to rule out the possibility of bias. Second, because we focused mainly on pathological findings, there is a lack of clinical data, which could have improved the study. Finally, the immunohistochemical analysis of PD-L1 involved the use of only one antibody, which might have been inappropriate if there was heterogeneity in the PD-L1 expression within a tumor sample.

In summary, we have reported the frequency of clinical biomarkers of NSCLC, together with the corresponding pathological findings, in a population of 173 patients in northeastern Brazil. We found no significant associations among those biomarkers. Despite the fact that the frequency of PD-L1 expression and EGFR mutation status were consistent with the few data available for Brazil, the frequency of ALK expression was higher than that previously reported for populations in Brazil. Further studies are encouraged in order to understand how such biomarkers are distributed throughout this heterogeneous population and, more importantly, how to translate that knowledge into better routine clinical practice.

REFERENCES

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30. https://doi.org/10.3322/caac.21442
2. Brasil. Ministério da Saúde. Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA) [homepage on the Internet]. Rio de Janeiro: INCA; c2018 [cited 2018 Apr 1]. Estimativa 2017: Incidência de Câncer no Brasil. Available from: http://www.inca.gov.br/estimativa/2018/casos-brasil-consolidado.asp
3. Travis WD, Brambilla E, Burke A, Marx A, Nicholson AG. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 4th ed. Lyon: International Agency for Research on Cancer; 2015.
4. Rittmeyer A, Barlesi F, Waterkamp D, Park K, Ciardiello F, von Pawel J, et al. Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial. Lancet. 2017;389(10066):255-265. https://doi.org/10.1016/S0140-6736(16)32517-X
5. Herbst RS. Can you Hear Music. Proceedings of the AACR Annual Meeting 2018; 2018 Apr 14-18; Chicago, USA. Philadelphia: AACR; 2018.
6. Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372(21):2018-28. https://doi.org/10.1056/NEJMoa1501824
7. Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Csőszi T, Fülöp A, et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med. 2016;375(19):1823-1833. https://doi.org/10.1056/NEJMoa1606774
8. Antonia SJ, López-Martin JA, Bendell J, Ott PA, Taylor M, Eder JP, et al. Nivolumab alone and nivolumab plus ipilimumab in recurrent small-cell lung cancer (CheckMate 032): a multicentre, open-label, phase 1/2 trial. Lancet Oncol. 2016;17(7):883-895. https://doi.org/10.1016/S1470-2045(16)30098-5
9. Araujo LH, Baldotto C, Castro G Jr, Katz A, Ferreira CG, Mathias C, et al. Lung cancer in Brazil. J Bras Pneumol. 2018;44(1):55-64. https://doi.org/10.1590/s1806-37562017000000135
10. Lopes LF, Bacchi CE. Anaplastic lymphoma kinase gene rearrangement in non-small-cell lung cancer in a Brazilian population. Clinics (Sao Paulo). 2012;67(7):845-7. https://doi.org/10.6061/clinics/2012(07)23
11. Pontes LDB, Bacchi CE, Queiroga EM, Piha T, Miranda PA, Freire S, et al. EGFR mutation screening in non-small cell lung cancer: Results from an access program in Brazil. J Clin Oncol. 2014;32(15 suppl):1526. https://doi.org/10.1200/jco.2014.32.15_suppl.1526
12. de Melo AC, Karen de Sá V, Sternberg C, Olivieri ER, Werneck da Cunha I, Fabro AT, et al. Mutational Profile and New IASLC/ATS/ERS Classification Provide Additional Prognostic Information about Lung Adenocarcinoma: A Study of 125 Patients from Brazil. Oncology. 2015;89(3):175-86. https://doi.org/10.1159/000376552
13. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129-39. https://doi.org/10.1056/NEJMoa040938
14. Buttner R, Gosney JR, Skov BG, Adam J, Motoi N, Bloom KJ, et al. Programmed Death-Ligand 1 Immunohistochemistry Testing: A Review of Analytical Assays and Clinical Implementation in Non-Small-Cell Lung Cancer. J Clin Oncol. 2017;35(34):3867-3876. https://doi.org/10.1200/JCO.2017.74.7642
15. Dix Junqueira Pinto G, de Souza Viana L, Scapulatempo Neto C, Vicente Serrano S. Evaluation of PD-L1 Expression in Tumor Tissue of Patients with Lung Carcinoma and Correlation with Clinical and Demographic Data. J Immunol Res. 2016;2016:9839685. https://doi.org/10.1155/2016/9839685
16. Rangachari D, VanderLaan PA, Shea M, Le X, Huberman MS, Kobayashi SS, et al. Correlation between Classic Driver Oncogene Mutations in EGFR, ALK, or ROS1 and 22C3-PD-L1 ≥50% Expression in Lung Adenocarcinoma. J Thorac Oncol. 2017;12(5):878-883. https://doi.org/10.1016/j.jtho.2016.12.026
17. Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fülöp A, et al. KEYNOTE-024: Pembrolizumab (pembro) vs platinum-based chemotherapy (chemo) as first-line therapy for advanced NSCLC with a PD-L1 tumor proportion score (TPS) ≥50%. Ann Oncol. Ann Oncology. 2016;27(6 suppl): LBA8_PR. https://doi.org/10.1093/annonc/mdw435.40
18. Zhang M, Li G, Wang Y, Wang Y, Zhao S, Haihong P, et al. PD-L1 expression in lung cancer and its correlation with driver mutations: a meta-analysis. Sci Rep. 2017;7(1):10255. https://doi.org/10.1038/s41598-017-10925-7
19. Aguiar PN, De Mello RA, Tadokoro H, Lopes G. Economic impact of immune checkpoint inhibitor therapy in Brazil and strategies to improve access. J Clin Oncol. 2017;35:6612. https://doi.org/10.1200/JCO.2017.35.15_suppl.6612
20. Aguiar PN, Perry LA, Penny-Dimri J, Babiker H, Tadokoro H, de Mello RA, et al. The effect of PD-L1 testing on the cost-effectiveness and economic impact of immune checkpoint inhibitors for the second-line treatment of NSCLC. Ann Oncol. 2018;29(4):1078 https://doi.org/10.1093/annonc/mdx478
21. Takada K, Toyokawa G, Tagawa T, Kohashi K, Shimokawa M, Akamine T, et al. PD-L1 expression according to the EGFR status in primary lung adenocarcinoma. Lung Cancer 2018;116:1-6. https://doi.org/10.1016/j.lungcan.2017.12.003
22. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153):561-6. https://doi.org/10.1038/nature05945
23. Camidge DR, Kono SA, Flacco A, Tan AC, Doebele RC, Zhou Q, et al. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res. 2010;16(22):5581-90. https://doi.org/10.1158/1078-0432.CCR-10-0851
24. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med. 2010;363(18):1693-703. https://doi.org/10.1056/NEJMoa1006448
25. Arrieta O, Cardona AF, Martín C, Más-López L, Corrales-Rodríguez L, Bramuglia G, et al. Updated Frequency of EGFR and KRAS Mutations in NonSmall-Cell Lung Cancer in Latin America: The Latin-American Consortium for the Investigation of Lung Cancer (CLICaP). J Thorac Oncol. 2015;10(5):838-43. https://doi.org/10.1097/JTO.0000000000000481
26. Arrieta O, Cardona AF, Federico Bramuglia G, Gallo A, Campos-Parra AD, Serrano S, et al. Genotyping non-small cell lung cancer (NSCLC) in Latin America. J Thorac Oncol. 2011;6(11):1955-9. https://doi.org/10.1097/JTO.0b013e31822f655f
27. Shi Y, Au JS, Thongprasert S, Srinivasan S, Tsai CM, Khoa MT, et al. A prospective, molecular epidemiology study of EGFR mutations in Asian patients with advanced non-small-cell lung cancer of adenocarcinoma histology (PIONEER). J Thorac Oncol. 2014;9(2):154-62 https://doi.org/10.1097/JTO.0000000000000033
28. Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst. 2005;97(5):339-46. https://doi.org/10.1093/jnci/dji055
29. Inoue A, Yoshida K, Morita S, Imamura F, Seto T, Okamoto I, et al. Characteristics and overall survival of EGFR mutation-positive non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors: a retrospective analysis for 1660 Japanese patients. Jpn J Clin Oncol. 2016;46(5):462-7. https://doi.org/10.1093/jjco/hyw014
30. D'Incecco A, Andreozzi M, Ludovini V, Rossi E, Capodanno A, Landi L, et al. PD-1 and PD-L1 expression in molecularly selected non-small-cell lung cancer patients. Br J Cancer. 2015;112(1):95-102. https://doi.org/10.1038/bjc.2014.555
31. Bylicki O, Paleiron N, Margery J, Guisier F, Vergnenegre A, Robinet G, et al. Targeting the PD-1/PD-L1 Immune Checkpoint in EGFR-Mutated or ALK-Translocated Non-Small-Cell Lung Cancer. Target Oncol. 2017;12(5):563-569. https://doi.org/10.1007/s11523-017-0510-9
32. Yang H, Chen H, Luo S, Li L, Zhou S, Shen R, et al. The correlation between programmed death-ligand 1 expression and driver gene mutations in NSCLC. Oncotarget. 2017;8(14):23517-23528. https://doi.org/10.18632/oncotarget.15627
33. Huynh TG, Morales-Oyarvide V, Campo MJ, Gainor JF, Bozkurtlar E, Uruga H, et al. Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment. J Thorac Oncol. 2016;11(11):1869-1878. https://doi.org/10.1016/j.jtho.2016.08.134
34. Ota K, Azuma K, Kawahara A, Hattori S, Iwama E, Tanizaki J, et al. Induction of PD-L1 Expression by the EML4-ALK Oncoprotein and Downstream Signaling Pathways in Non-Small Cell Lung Cancer. Clin Cancer Res. 2015;21(17):4014-21. https://doi.org/10.1158/1078-0432.CCR-15-0016
35. Yang CY, Lin MW, Chang YL, Wu CT, Yang PC. Programmed cell death-ligand 1 expression in surgically resected stage I pulmonary adenocarcinoma and its correlation with driver mutations and clinical outcomes. Eur J Cancer. 2014;50(7):1361-9. https://doi.org/10.1016/j.ejca.2014.01.018
36. Zhang Y, Wang L, Li Y, Pan Y, Wang R, Hu H, et al. Protein expression of programmed death 1 ligand 1 and ligand 2 independently predict poor prognosis in surgically resected lung adenocarcinoma. Onco Targets Ther. 2014;7:567-73. https://doi.org/10.2147/OTT.S59959
37. Yoshizawa A, Sumiyoshi S, Sonobe M, Kobayashi M, Fujimoto M, Kawakami F, et al. Validation of the IASLC/ATS/ERS lung adenocarcinoma classification for prognosis and association with EGFR and KRAS gene mutations: analysis of 440 Japanese patients. J Thorac Oncol. 2013;8(1):52-61. https://doi.org/10.1097/JTO.0b013e3182769aa8
38. Song Z, Zhu H, Guo Z, Wu W, Sun W, Zhang Y. Correlation of EGFR mutation and predominant histologic subtype according to the new lung adenocarcinoma classification in Chinese patients. Med Oncol. 2013;30(3):645. https://doi.org/10.1007/s12032-013-0645-1
39. Villa C, Cagle PT, Johnson M, Patel JD, Yeldandi AV, Raj R, et al. Correlation of EGFR mutation status with predominant histologic subtype of adenocarcinoma according to the new lung adenocarcinoma classification of the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society. Arch Pathol Lab Med. 2014;138(10):1353-7. https://doi.org/10.5858/arpa.2013-0376-OA