ORIGINAL ARTICLE. Annals of Oncology 28: , 2017 doi: /annonc/mdx183 Published online 12 April 2017

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1 Annals of Oncology 28: , 217 doi:1.193/annonc/mdx183 Published online 12 April 217 ORIGINAL ARTICLE Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T79M status after disease progression during EGFR-TKI treatment K. Haratani 1, H. Hayashi 1 *, T. Tanaka 2, H. Kaneda 3, Y. Togashi 4,5, K. Sakai 4, K. Hayashi 6, S. Tomida 7, Y. Chiba 8, K. Yonesaka 1, Y. Nonagase 1, T. Takahama 1, J. Tanizaki 1, K. Tanaka 1, T. Yoshida 1, K. Tanimura 9, M. Takeda 1, H. Yoshioka 2, T. Ishida 2, T. Mitsudomi 1, K. Nishio 4 & K. Nakagawa 1 1 Department of Medical Oncology, Kindai University Faculty of Medicine, Osaka-Sayama; 2 Department of Respiratory Medicine, Kurashiki Central Hospital, Kurashiki; 3 Department of Medical Oncology, Kishiwada City Hospital, Kishiwada; 4 Department of Genome Biology, Kindai University Faculty of Medicine, Osaka-Sayama; 5 Division of Cancer Immunology, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa; 6 Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki; 7 Department of Biobank, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama; 8 Clinical Research Center, Kindai University Hospital, Osaka-Sayama; 9 Department of Respiratory Medicine, Kishiwada City Hospital, Kishiwada; 1 Division of Thoracic Surgery, Department of Surgery, Kindai University Faculty of Medicine, Osaka-Sayama, Japan *Correspondence to: Dr Hidetoshi Hayashi, Department of Medical Oncology, Kindai University Faculty of Medicine, Ohno-higashi, Osaka-Sayama, Osaka , Japan. Tel: þ ; Fax: þ ; hayashi_h@dotd.med.kindai.ac.jp Background: The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutationpositive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T79M resistance mutation of EGFR. Patients and methods: We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumorinfiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T79M status to PD-L1 expression or TIL density was also examined in an independent cohort of 6 patients (cohort B). Results: In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T79M-negative and T79M-positive patients, respectively (P ¼.99; hazard ratio of.48 with a 95% confidence interval of ). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of 1% or <1%, respectively (P ¼.84; hazard ratio of.37, 95% confidence interval of ). PFS tended to increase as the PD-L1 expression level increased with cutoff values of 1% and 5%. The proportion of tumors with a PD-L1 level of 1% or 5% was higher among T79M-negative patients than among T79M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8 þ TIL density and nonsynonymous mutation burden. Conclusion: T79M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T79M-positive patients. Key words: non-small-cell lung cancer, epidermal growth factor receptor, T79M, nivolumab, PD-L1, tumor-infiltrating lymphocyte VC The Author 217. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For Permissions, please journals.permissions@oup.com.

2 Annals of Oncology Introduction Lung cancer is the most common cause of cancer deaths worldwide [1]. The advent of molecular profiling has led to the discovery of driver mutations and to targeted therapy and personalized medicine for lung cancer. Mutations of the epidermal growth factor receptor gene (EGFR) are common drivers of non-small-cell lung cancer (NSCLC) [2]. EGFR tyrosine kinase inhibitors (TKIs) are considered the best choice for first-line treatment of advanced or recurrent nonsquamous NSCLC harboring activating mutations of EGFR [2]. However, individuals treated with EGFR-TKIs eventually develop resistance to these drugs, most commonly as the result of a secondary T79M mutation of EGFR. Osimertinib, a third-generation EGFR-TKI, confers a survival benefit compared with cytotoxic chemotherapy in such T79M-positive patients [3]. The optimal treatment of T79M-negative patients who develop EGFR-TKI resistance remains unclear, however, with such individuals appearing to have a poorer prognosis compared with T79M-positive patients [4]. A more efficacious treatment strategy for T79M-negative patients is thus needed. Monoclonal antibodies to programmed death-1 (PD-1), such as nivolumab and pembrolizumab, prolong overall survival (OS) compared with docetaxel in NSCLC patients previously treated with platinum-doublet chemotherapy [5, 6]. However, subgroup analysis of clinical trials has suggested that PD-1 inhibitor therapy is less effective for patients with EGFR mutations than for those wild-type for EGFR [5 7]. In addition, a retrospective study found that EGFR mutation positivity in NSCLC was associated with a low overall response rate (ORR) to PD-1 inhibitors and that low levels of both programmed death-ligand 1 (PD-L1) and CD8 þ tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment might underlie this unfavorable clinical response [8]. Moreover, little is known of the relation between PD- 1 inhibitor efficacy and the mechanism of acquired resistance to EGFR-TKIs. We have now carried out a retrospective study to evaluate the efficacy of nivolumab according to T79M status in patients with EGFR mutation-positive NSCLC after the development of resistance to EGFR-TKIs. The expression level of PD-L1 and density of TILs (CD8 þ, CD4 þ, or FOXP3 þ cells) were also examined in tumor tissue specimens obtained after the development of EGFR- TKI resistance. In addition, we carried out whole-exome sequencing of tumor DNA. Patients and methods Patients We reviewed the medical records of all patients with advanced or recurrent EGFR mutation-positive NSCLC treated at Kindai University Hospital, Kishiwada City Hospital, or Kurashiki Central Hospital between February 212 and August 216. EGFR mutations were detected with commercial assays such as the Cobas EGFR Mutation Test (Roche Molecular Diagnostics). Analysis of plasma cell-free DNA was allowed for determination of T79M status after the development of EGFR-TKI resistance if tumor tissue specimens were unavailable. The study was carried out according to protocols approved by the Institutional Review Board of each participating hospital. From this review, we identified 25 such patients who were assessed for T79M status after disease progression during treatment with at least one EGFR-TKI and who were treated with nivolumab monotherapy thereafter (cohort A). The clinical outcome of nivolumab treatment was analyzed, and immunohistochemical analysis and whole-exome sequencing were carried out with formalin-fixed paraffin-embedded (FFPE) tumor tissue obtained after the development of EGFR-TKI resistance. In addition, we identified an independent cohort of 6 patients with EGFR mutation-positive NSCLC who had sufficient FFPE tumor tissue collected after progression on EGFR-TKIs for immunohistochemistry (cohort B). Data collection Medical records were reviewed, and data regarding clinicopathologic features and treatment history were extracted. Data were updated as of 31 December 216. Responses were assessed according to RECIST v1.1 [9]. Progression-free survival (PFS) was measured from treatment initiation to clinical or radiographic progression or death from any cause. OS was measured from treatment initiation to death from any cause. Patients without documented clinical or radiographic disease progression or who were still alive were censored on the date of last follow-up. Immunohistochemistry Tumor histology was classified according to WHO criteria [1]. Immunohistochemistry was carried out with monoclonal antibodies to PD-L1 (clone 28-8, Abcam), to CD8 (clone C8/144B, Agilent Technologies), to CD4 (clone 1F6, Leica Biosystems), and to FOXP3 (clone 236A/E7, Abcam) and with the use of an automated stainer (Leica Bond-III). The stained slides were scanned with an Aperio Scan Scope (Leica Biosystems) and were evaluated by board-certified pathologists who were blinded to clinical outcome. Further details are provided in supplementary methods, supplementary Figure S1, supplementary Figure S2, available at Annals of Oncology online. Whole-exome sequencing and exome analysis pipeline DNA was extracted from FFPE tumor specimens with the use of a QIAamp DNA Micro Kit (Qiagen). Whole-exome capture libraries were constructed with the use of Agilent Sure-Select Human All Exon v5.. The constructed libraries were sequenced with the use of an Illumina HiSeq 4 sequencer. Further details are described in supplementary methods, available at Annals of Oncology online. Statistical analysis See supplementary methods, available at Annals of Oncology online. Results Original article Outcome of nivolumab treatment of EGFR mutation-positive NSCLC patients according to T79M status and PD-L1 expression level (cohort A) The characteristics of the 25 patients of cohort A are listed in Table 1. Eight patients were positive for T79M, with four patients being tested for this mutation with plasma cell-free DNA. Six of the T79M-positive patients were treated with a thirdgeneration EGFR-TKI during their disease course, four of which were after disease progression on nivolumab treatment. For the entire cohort, the ORR was 2% and the disease control rate (DCR) was 36% (supplementary Table S1, available at Volume 28 Issue doi:1.193/annonc/mdx

3 Original article Table 1. Patient characteristics of cohorts A and B Characteristics Number of patients (%) a Table 1. Continued Characteristics Annals of Oncology Number of patients (%) a Cohort A (N525) Cohort B (N56) Cohort A (N525) Cohort B (N56) Median age (range), years 69 (42 78) 66 (44 8) Sex Male 12 (48) 23 (38) Female 13 (52) 37 (62) ECOG PS at initiation of nivolumab (A) or rebiopsy (B) 1 22 (88) 45 (75) 2 3 (12) 3 (5) 3 2 (3) Unknown (not recorded) 1 (17) Smoking history b Current or former 7 (28) 22 (37) Never 18 (72) 37 (62) Unknown 1 (2) Stage Recurrence 5 (2) 15 (25) IIIB IV 2 (8) 45 (75) Metastasis (at initiation of nivolumab) CNS versus no CNS 14 (56) versus 11 (44) Intrathoracic only versus extrathoracic 5 (2) versus 2 (8) Histology (initial biopsy/pre-tki) Adeno 24 (96) 56 (93) Adenosquamous 1 (4) 1 (2) Not otherwise specified 2 (3) Pleomorphic 1 (2) Histology (rebiopsy/post-tki) Adeno 18 (72) 48 (8) Squamous/adenosquamous 3 (5) Not otherwise specified 3 (12) 6 (1) Small cell 2 (3) Large cell 1 (2) Not examined 4 (16) EGFR mutation status (initial biopsy/ pre-tki) Ex19del 14 (56) 39 (65) L858R 11 (44) 16 (27) T79M () 3 (5) c Minor () 5 (8) d T79M status (rebiopsy/post-tki) Positive 8 (32) 3 (5) Negative 17 (68) e 3 (5) Treatment before nivolumab (A) or rebiopsy (B) Cytotoxic drug naïve (TKIs only) 7 (28) 26 (43) Cytotoxic drugs used 18 (72) 34 (57) Prior treatment lines before nivolumab (A) or rebiopsy (B) 1 4 (16) 24 (4) 2 6 (24) 15 (25) 3 4 (16) 5 (8) Continued 4 5 (2) 3 (5) >4 6 (24) 13 (22) PFS of first EGFR-TKI Median (range), days 332 (52 343) ( ) <365 days 13 (52) 26 (43) 365 days 12 (48) 34 (57) a Percentages may not add up to 1 because of rounding. b Current smokers were defined as individuals who had smoked a cigarette within the previous year; former smokers were defined as those who had smoked 1 cigarettes but had quit >1 year before rebiopsy; never-smokers were defined as individuals who had smoked <1 cigarettes. c One patient had Ex19del and T79M, and two patients had L858R and T79M. d Two patients had G719A, one patient had G719C, one patient had L861Q, and one patient had G719S and L861Q. e T79M status for four of these patients was determined by analysis of plasma cell-free DNA. ECOG PS, Eastern Cooperative Oncology Group performance status; CNS, central nervous system; TKI, tyrosine kinase inhibitor; EGFR, epidermal growth factor receptor; Ex19del, exon 19 deletion; PFS, progression-free survival. Annals of Oncology online). The ORR (24% versus 13%, P ¼ 1.) and DCR (47% versus 13%, P ¼.182) were higher in the T79M-negative patients than in the T79M-positive patients. The median PFS for the entire cohort was 1.5 months [95% confidence interval (CI), ] (Figure 1A). The median PFS was 2.1 months (95% CI, ) and 1.3 months (95% CI,.1 1.8) for T79M-negative and T79M-positive patients (P ¼.99), respectively, with the hazard ratio (HR) being.48 (95% CI, ) (Figure 1B). The outcome of nivolumab treatment thus tended to be more favorable in T79M-negative patients than in T79M-positive patients. We next evaluated nivolumab efficacy according to PD-L1 expression level and TIL density in tumor tissue, which were evaluable in 15 and 14 of the 25 patients, respectively. The ORR increased as the PD-L1 expression level increased (38%, 75%, and 1% in patients with a PD-L1 expression level of 1%, 1%, or 5%, respectively) (supplementary Table S1, available at Annals of Oncology online). The median PFS was also longer in patients with a PD-L1 expression level of 1% than in those with a level of <1% (2.1 versus 1.3 months, P ¼.84) (Figure 2A), with the HR being.37 (95% CI, ). A trend toward a better PFS was apparent as the PD-L1 expression level increased at cutoff values of 1% and 5% (supplementary Figure S3, available at Annals of Oncology online). The proportion of individuals with a PD-L1 expression level of 1% was similar for T79M-positive and T79M-negative patients, but the proportion of those with PD-L1 expression levels of 1% or 5% 1534 Haratani et al. Volume 28 Issue 7 217

4 Annals of Oncology A 1 Progression-free survival (%) Entire cohort A (N = 25) Median: 1.5 months (95% Cl, 1.3 to 2.8) Time (months) No. at risk B Progression-free survival (%) Original article T79M negative (N = 17) Median: 2.1 months (95% Cl, 1.3 to 3.4) T79M positive (N = 8) Median: 1.3 months (95% Cl,.1 to 1.8) Log-rank test: P =.99 Hazard ratio:.48 (95% Cl,.2 to 1.24) Time (months) No. at risk T79M T79M Figure 1. Kaplan Meier curves for PFS in patients with EGFR mutation-positive NSCLC treated with nivolumab after disease progression during EGFR-TKI treatment. (A) PFS curve for the entire cohort A (N ¼ 25). (B) PFS curves for T79M-positive (N ¼ 8) and T79M-negative (N ¼ 17) patients of cohort A. Plus signs denote censoring. A Profression-free survival (%) PD-L1 1% (N = 8) Median: 2.1 months (95% Cl, 1.1 to NR) PD-L1 <1% (N = 7) Median: 1.3 months (95% Cl,.1 to 1.4) Log-rank test: P =.84 Hazard ratio:.37 (95% Cl,.1 to 1.21) No. at risk Time (months) PD-L1 1% PD-L1 <1% 7 1 B Intratumoral CD8 + TILs (/mm 2 ) Wilcoxon rank sum test P =.24 C 6 Intratumoral CD4 + TILs (/mm 2 ) E F PD-L1 PD-L1 Wilcoxon rank sum test P =.69 CD8 + TILs CD8 + TILs D 4 Intratumoral FOXP3 + TILs (/mm 2 ) FOXP3 + TILs FOXP3 + TILs Wilcoxon rank sum test P =.48 Responders Nonresponders Responders Nonresponders Responders Nonresponders Figure 2. Characteristics of cohort A revealed by immunohistochemistry. (A) Kaplan Meier curves for PFS according to PD-L1 expression level (1%, N ¼ 8; <1%, N ¼ 7) for EGFR mutation-positive NSCLC patients treated with nivolumab after disease progression during EGFR-TKI treatment. Plus signs denote censoring. NR, not reached. (B D) Density of CD8 þ TILs (B), CD4 þ TILs (C), or FOXP3 þ TILs (D) for responders and nonresponders to nivolumab treatment among patients with EGFR mutation-positive NSCLC and acquired resistance to EGFR-TKIs. Arrows denote TIL counts for a T79M-negative nonresponder with a high PD-L1 expression level (1%), high CD8 þ TIL density (1727/ mm 2 ), and the highest FOXP3 þ TIL density (373/mm 2 ). Error bars indicate the mean 6 SE; horizontal lines indicate SD. (E, F) Immunohistochemical analysis of PD-L1, CD8, and FOXP3 for the T79M-negative nonresponder with the highest FOXP3 þ TIL count (373/ mm 2 ) (F) as well as for a T79M-negative responder with a high PD-L1 expression level (5%), high CD8 þ TIL density (2292/mm 2 ), and low FOXP3 þ TIL density (115/mm 2 ) (E). Scales bars, 1 mm. Volume 28 Issue doi:1.193/annonc/mdx

5 Original article Annals of Oncology Table 2. PD-L1 expression level and TIL density in cohorts A and B according to T79M status after EGFR-TKI treatment Cohort A P value a Cohort B P value a T79M positive (N58) T79M negative (N517) T79M positive (N53) T79M negative (N53) PD-L1 expression level N¼6 N¼9 N¼28 N¼26 1% 3/6 (5%) 5/9 (56%) 1. 1/28 (36%) 1/26 (38%) 1. 1% 1/6 (17%) 3/9 (33%).6 3/28 (11%) 7/26 (27%).167 5% /6 (%) 2/9 (22%).489 1/28 (4%) 3/26 (12%).34 CD8 þ TIL density (/mm 2 ) N¼4 N¼1.572 N¼24 N¼ Median (range) (44 586) ( ) (4 1162) (32 968) FOXP3 þ TIL density (/mm 2 ) N¼4 N¼1.157 N¼23 N¼29.13 Median (range) (39 91) (57 373) ( 25) ( 226) a Fisher s exact test or Wilcoxon rank sum test for PD-L1 expression level and TIL density, respectively. PD-L1, programmed death-ligand 1; TIL, tumor-infiltrating lymphocyte; EGFR, epidermal growth factor receptor; TKI, tyrosine kinase inhibitor. was higher for T79M-negative patients than for T79M-positive patients (Table 2). The Wilcoxon rank sum test revealed that the CD8 þ TIL count was significantly (P ¼.24) higher in nivolumab responders than in nonresponders (Figure 2B). In contrast, the numbers of CD4 þ TILs and FOXP3 þ TILs were not associated with nivolumab efficacy (Figure 2C and D). The FOXP3 þ TIL count was highest (373/mm 2 ) for a T79M-negative patient who had a PD- L1 expression level of 1% and a high CD8 þ TIL count (1727/ mm 2 ) but who did not respond to nivolumab (arrows in Figure 2B and D). Representative immunohistochemistry images for a T79M-negative patient who showed a response to nivolumab and had a high PD-L1 expression level (5%), high CD8 þ TIL count (2292/mm 2 ), and low FOXP3 þ TIL count (115/mm 2 ) are shown in Figure 2E, whereas those for the nonresponder with the highest FOXP3 þ TIL count are shown in Figure 2F. The numbers of CD8 þ TILs and FOXP3 þ TILs were similar for T79Mpositive and T79M-negative patients in this cohort (Table 2). Although the median OS was not achieved at a median followup of 7.3 months for all patients in cohort A, the HR for OS was.38 (95% CI, ) for the T79M-negative group versus the T79M-positive group (supplementary Figure S4, available at Annals of Oncology online), suggestive of a survival benefit of nivolumab treatment in T79M-negative patients. PD-L1 expression level and TIL density according to T79M status after EGFR-TKI treatment (cohort B) To validate our finding that the PD-L1 expression level was higher in T79M-negative patients of cohort A, we evaluated such expression in an independent cohort (cohort B) (Table 1). We also determined CD8 þ TIL density and FOXP3 þ TIL density in this cohort. All tumor specimens were obtained by biopsy after disease progression during EGFR-TKI treatment. Six, seven, and eight patients could not be evaluated for PD-L1 expression, CD8 þ TIL density, or FOXP3 þ TIL density, respectively. The proportion of individuals with a PD-L1 expression level of 1% was similar for both T79M-positive and T79Mnegative patients, whereas the proportion of those with a PD-L1 expression level of 1% or 5% was higher for T79Mnegative patients than for T79M-positive patients (Table 2). The number of CD8 þ TILs was similar for T79M-positive and T79M-negative patients, although there was a more frequent coexistence of a high PD-L1 expression level (1% or 5%) and a high CD8 þ TIL count (more than or equal to the median) in the T79M-negative group than in the T79M-positive group [5/25 (2%) versus 1/24 (4%) and 3/25 (12%) versus 1/24 (4%), respectively]. The FOXP3 þ TIL count was significantly (P ¼.13) lower in T79M-negative than in T79M-positive patients of cohort B. Whole-exome sequencing analysis To investigate the relation of nonsynonymous mutation burden to nivolumab efficacy in EGFR mutation-positive NSCLC, we carried out whole-exome sequencing for nine patients (three responders and six nonresponders) of cohort A (Table 3). The median nonsynonymous mutation burden per tumor was 11. The burden was significantly (P ¼.38) higher in responders than in nonresponders, but it did not differ (P ¼.71) between T79Mnegative and T79M-positive patients. Two of the five T79Mnegative patients were positive for KRAS(G13D) or PIK3CA (E545K) mutations, both of which are thought to confer resistance to EGFR-TKIs. In addition, comparative copy number analysis revealed a significant copy number gain for MET in another two of the T79M-negative patients who also expressed PD-L1 at a high level (5%) and responded to nivolumab treatment. Discussion Our results have revealed that nivolumab efficacy after disease progression during EGFR-TKI treatment tended to be greater for EGFR mutation-positive NSCLC patients negative for T79M 1536 Haratani et al. Volume 28 Issue 7 217

6 Annals of Oncology Table 3. Nonsynonymous mutation burden for nine patients of cohort A Patient Age (years) Smoking status Sensitive EGFR mutation T79M status (post-tki) Previous treatment lines before biopsy PFS on first EGFR-TKI (months) Response to nivolumab PD-L1 expression level (%) Original article NMB/tumor NMB/Mb Other gene alterations 1 52 Never L858R Negative Yes MET amp 2 7 Former Ex19del Positive Yes Never L858R Negative Yes MET amp 4 66 Never Ex19del Positive 5 1,7 No < Never L858R Negative No KRAS(G13D) 6 42 Never Ex19del Positive No Never L858R Positive No < PTEN(R13Q) 8 74 Never L858R Negative No PIK3CA(E545K) 9 75 Never L858R Negative No < EGFR, epidermal growth factor receptor; Ex19del, exon 19 deletion; TKI, tyrosine kinase inhibitor; PFS, progression-free survival; PD-L1, programmed death-ligand 1; NMB, nonsynonymous mutation burden; amp, amplification. Nonsynonymous mutation burden differed significantly between responders to nivolumab treatment and nonresponders (P ¼.38, Wilcoxon rank sum test), but not between T79M-negative and T79M-positive patients (P ¼.71). than for those positive for this resistance mutation, likely as a result of a higher PD-L1 expression level in the T79M-negative group. Analysis of an independent cohort confirmed a higher level of PD-L1 expression in T79M-negative patients compared with those positive for T79M. Our study is the first to examine the relation of the tumor immune microenvironment to the efficacy of a PD-1 inhibitor in EGFR mutation-positive NSCLC according to T79M status after disease progression during EGFR-TKI treatment. Previous studies have indicated that PD-1 inhibition is a promising treatment strategy for tumors that overexpress c-met or have undergone epithelial-to-mesenchymal transition, both of which constitute mechanisms of resistance to EGFR-TKIs [11 13]. Consistent with these previous findings, our wholeexome sequencing analysis revealed that two nivolumab responders with a high level of PD-L1 expression manifested a copy number gain for MET. The specific mechanisms that underlie the relation between EGFR-TKI resistance in T79M-negative tumors and PD-L1 expression warrant further study. CD8 þ TIL density is a predictive marker for the efficacy of PD-1 inhibitors [8]. We found that the CD8 þ TIL count was significantly higher in nivolumab responders with EGFR mutationpositive NSCLC. Although the CD8 þ TIL density did not differ between T79M-positive and T79M-negative patients, there was a more frequent coexistence of a high PD-L1 expression level and a high CD8 þ TIL count in T79M-negative patients, consistent with the notion that such patients are potential candidates for PD-1 inhibitor therapy. Nonsynonymous mutation burden is emerging as a predictive marker for the efficacy of PD-1 blockade [14]. EGFR mutationpositive NSCLC including that positive for T79M has previously been shown to have a low mutation burden by whole-exome sequencing [15], suggesting that PD-1 inhibitors might not be appropriate for such tumors. On the other hand, the mechanism of EGFR-TKI resistance in T79M-negative tumors is molecularly heterogeneous [16], leaving open the possibility that mutation burden may be higher in some of these tumors. Our findings suggest that a subset of EGFR mutation-positive NSCLC patients with acquired resistance to EGFR-TKIs may have a relatively high nonsynonymous mutation burden and therefore benefit from PD-1 inhibitor treatment, although there was no clear difference in mutation burden based on T79M status, possibly as a result of our small sample size. The role of FOXP3 þ TILs in PD-1 blockade therapy has been unclear. Although we did not detect a clear relation between nivolumab efficacy and FOXP3 þ TIL density, our immunohistochemical findings in individual patients hinted that FOXP3 þ TILs might contribute to resistance to PD-1 inhibitors (Figure 2F). Inhibitors of regulatory T cells such as FOXP3 þ TILs, including CTLA-4 inhibitors, are considered candidates for combination therapy with PD-1 inhibitors in PD-L1-positive NSCLC patients with EGFR mutations [17]. Indeed, the CheckMate 12 trial showed that combination therapy with nivolumab and ipilimumab was effective (ORR of 5%) in a small cohort of patients with EGFR mutation-positive NSCLC [18]. There are several limitations to our study. The study was thus retrospective in nature and the number of patients was small, making it difficult to draw definitive conclusions. Multivariate analysis could not be carried out because of the small number of patients, leaving open the possibility for bias in the clinical data. Nevertheless, given that our study is the first to report that T79M-negative status tended to be associated with a more favorable efficacy of a PD-1 inhibitor, likely as a result, at least in part, of a higher PD-L1 expression level, our findings may prove helpful for clinical decision-making regarding the use of PD-1 inhibitors in patients with EGFR mutation-positive NSCLC. PD-1 inhibitors may thus be more promising therapeutic agents for T79M-negative patients, especially those positive for PD-L1, after disease progression during EGFR-TKI treatment, whereas osimertinib has been recognized as the first treatment choice for T79M-positive patients. Prospective clinical trials are required to confirm the efficacy of PD-1 inhibitors in T79M-negative Volume 28 Issue doi:1.193/annonc/mdx

7 Original article patients with EGFR mutation-positive NSCLC. The WJOG8515L study, a randomized phase II trial to compare nivolumab with platinum-doublet therapy in patients with EGFR mutation-positive nonsquamous NSCLC who have developed resistance to EGFR-TKIs due to mechanisms other than T79M is currently in progress (UMIN-CTR ID: UMIN21133). In conclusion, our findings suggest that EGFR mutation-positive NSCLC patients with acquired resistance to EGFR-TKIs not due to T79M are more likely to receive benefit from PD-1 inhibitors than are those positive for this resistance mutation, possibly as a result of a higher PD-L1 expression level in the T79Mnegative patients. Prospective clinical trials are warranted to confirm these results. Acknowledgements We thank Junya Fukuoka and Yuki Imaoka of Nagasaki University as well as Haruka Sakamoto, Yume Shinkai, Michiko Kitano, and Mami Kitano of Kindai University for technical support. Funding This work was supported by Japan Society for the Promotion of Science (KAKENHI grant number 16K2156 to HH). Disclosure KH has received honoraria from Ono Pharmaceutical Co. Ltd. and lecture fees from Pfizer Japan Inc. HH has received honoraria from AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Eli Lilly Japan K.K., Ono Pharmaceutical Co. Ltd., and Taiho Pharmaceutical Co. Ltd.; lecture fees from Chugai Pharmaceutical Co. Ltd., Kyowa Hakko Kirin Co. Ltd., MSD K.K., and Pfizer Japan Inc.; and research funding from Ono Pharmaceutical Co. Ltd. HK has received lecture fees from AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Chugai Pharmaceutical Co. Ltd., Eli Lilly Japan K.K., and Pfizer Japan Inc. YT has received honoraria from Boehringer Ingelheim Japan Inc.; Bristol-Myers Squibb Co. Ltd.; Becton, Dickinson and Company; Chugai Pharmaceutical Co. Ltd.; Kyowa Hakko Kirin Co. Ltd.; MSD K.K.; and Ono Pharmaceutical Co. Ltd. YN has received lecture fees from Taiho Pharmaceutical Co. Ltd. TT has received honoraria from AstraZeneca K.K. and lecture fees from Eli Lilly Japan K.K., Ono Pharmaceutical Co. Ltd., and Takeda Pharmaceutical Co. Ltd. KTana has received lecture fees from AstraZeneca K.K. and Merck Serono Co. Ltd. KTani has received lecture fees from Boehringer Ingelheim Japan Inc., Meiji Seika Pharma Co. Ltd., and Novartis Pharma K.K. H.Y has received honoraria from AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Chugai Pharmaceutical Co. Ltd., Eli Lilly Japan K.K., Ono Pharmaceutical Co. Ltd., Pfizer Japan Inc., Taiho Pharmaceutical Co. Ltd., and Takeda Pharmaceutical Co. Ltd. T.M has received honoraria from AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Eli Lilly Japan K.K., Kyowa Hakko Kirin Co. Ltd., MSD K.K., Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd., Pfizer Japan Inc., and Taiho Pharmaceutical Co. Ltd. as well as research funding from Boehringer Ingelheim Japan Inc., Chugai Pharmaceutical Co. Ltd., Ono Pharmaceutical Co. Ltd., Pfizer Japan Inc., and Taiho Pharmaceutical Co. Ltd. KN has received honoraria from Astellas Pharma Inc., AstraZeneca K.K., AYUMI Pharmaceutical Corporation, Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co. Ltd., Eli Lilly Japan K.K., Kissei Pharmaceutical Co. Ltd., Kyowa Hakko Kirin Co. Ltd., MSD K.K., Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd., Pfizer Japan Inc., Showa Yakuhin Kako Co. Ltd., Sym Bio Pharmaceuticals Ltd., and Taiho Pharmaceutical Co. Ltd.; research funding from AbbVie Inc., Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim Japan Inc., Bristol-Myers Squibb Co. Ltd., Daiichi Sankyo Co. Ltd., Eisai Co. Ltd., Eli Lilly Japan K.K., GlaxoSmithKline K.K., Kyowa Hakko Kirin Co. Ltd., Merck Serono Co. Ltd., MSD K.K., Novartis Pharma K.K., Ono Pharmaceutical Co. Ltd., Otsuka Pharmaceutical Co. Ltd., Pfizer Japan Inc., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Co. Ltd., and Yakult Honsha Co. Ltd.; and consulting or advisory fees from Astellas Pharma Inc., Eli Lilly Japan K.K., and Ono Pharmaceutical Co. Ltd. All remaining authors have declared no conflicts of interest. References Annals of Oncology 1. Ferlay J, Soerjomataram I, Dikshit R et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 212. Int J Cancer 215; 136: E359 E National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology: Non-Small Cell Lung Cancer (Version 5) 217; 3. Mok TS, Wu YL, Ahn MJ et al. Osimertinib or platinumpemetrexed in EGFR T79M-positive lung cancer. N Engl J Med 217; 376: Oxnard GR, Arcila ME, Sima CS et al. 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8 Annals of Oncology 11. Chen L, Gibbons DL, Goswami S et al. Metastasis is regulated via microrna-2/zeb1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun 214; 5: Balan M, Mier y Teran E, Waaga-Gasser AM et al. Novel roles of c-met in the survival of renal cancer cells through the regulation of HO-1 and PD-L1 expression. J Biol Chem 215; 29: Han JJ, Kim DW, Koh J et al. Change in PD-L1 expression after acquiring resistance to gefitinib in EGFR-mutant non-small-cell lung cancer. Clin Lung Cancer 216; 17: , e Rizvi NA, Hellmann MD, Snyder A et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in nonsmall cell lung cancer. Science 215; 348: Original article 15. Spigel DR, Schrock AB, Fabrizio D et al. Total mutation burden (TMB) in lung cancer (LC) and relationship with response to PD-1/PD-L1 targeted therapies. J Clin Oncol 216; 34 (suppl): abstr Stewart EL, Tan SZ, Liu G et al. mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations a review. Transl Lung Cancer Res 215; 4: Melero I, Berman DM, Aznar MA et al. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer 215; 15: Hellmann MD, Rizvi NA, Goldman JW et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 12): results of an open-label, phase 1, multicohort study. Lancet Oncol 217; 18: Volume 28 Issue doi:1.193/annonc/mdx