Heterogeneity of EGFR-mutant clones and PD-L1 highly expressing clones affects treatment efficacy of EGFR-TKI and PD-1 inhibitor
Authors: K. Kunimasa, H. Nakamura, K. Sakai, M. Kimura, T. Inoue, M. Tamiya, K. Nishino, T. Kumagai, S. Nakatsuka, H. Endo, M. Inoue, K. Nishio, F. Imamura
Journal: Annals of Oncology (2018)

Current clinical trials have suggested poor efficacies of programmed death-1 (PD-1) blockade therapies for non-small-cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations [1.Lee C.K. Man J. Lord S. et al.Clinical and molecular characteristics associated with survival among patients treated with checkpoint inhibitors for advanced non-small cell lung carcinoma: a systematic review and meta-analysis.JAMA Oncol. 2017; Google Scholar]. However, there is controversy about the use of PD-L1 expression as a surrogate marker for response to PD-1 blockade therapy as a biomarker [2.Hata A. Katakami N. Nanjo S. et al.Programmed death-ligand 1 expression according to epidermal growth factor receptor mutation status in pretreated non-small cell lung cancer.Oncotarget. 2017; 8: 113807-113816Crossref PubMed Scopus (6) Google Scholar]. Here, we show an NSCLC case with PD-L1 highly expressing EGFR-mutant NSCLC who responded dramatically to PD-1 blockade therapy but not to an EGFR-TKI due to intratumor heterogeneity. A 62-year-old Asian woman presented at our hospital complaining of dry cough and gait disturbance due to right hip pain. She was a current smoker with 62 pack-years and had a history of hypertension and osteoarthritis of the right hip. She had no previous malignancies in the past and no family history of cancer. A computed tomography (CT) scan revealed a 65 mm mass in the right ilium and a 16 mm nodule in the lung. CT-guided biopsy showed the mass of the right ilium was metastasis from lung adenocarcinoma with EGFR exon 19 deletion (Ex.19 del) detected by the PNA-LNA PCR clamp method. PD-L1 tumor proportion score (TPS) was 90%. Erlotinib and radiotherapy to the right ilium were started. However, follow-up CT carried out 2 months later detected multiple new metastases. CT-guided biopsy of the right chest wall metastasis was carried out. Biomarker screening detected no EGFR mutations in the tumor and PD-L1 staining showed TPS 95%. Pembrolizumab was administered as the second-line therapy. During the first course, her right hip pain dramatically improved and CT after three cycles of pembrolizumab showed complete disappearance of primary lesion and multiple metastases. Immunofluorescent analysis of both right ilium and chest wall lesions with an anti-EGFR antibody specific to Ex.19 del and an anti-PD-L1 antibody revealed the presence of distinct heterogeneity of EGFR-mutant clones and PD-L1 highly expressing clones (Figure 1). We analyzed both of the right ilium (pre-EGFR-TKI) and chest wall (post-EGFR-TKI) lesions with a QIAseq comprehensive cancer panel (Qiagen, Hilden, Germany) (supplementary Table S1, available at Annals of Oncology online). More than 90% mutations including KRAS and TP53 were shared by the both specimens. The ratio of EGFR Ex.19 del allele to wild-type allele was evaluated by droplet digital PCR (ddPCR) showing EGFR Ex.19 del mutation index decrease from 0.18% to 0.06% (supplementary Figure S1, available at Annals of Oncology online). The present case suggests that appreciable clonal heterogeneity exists in EGFR-mutant NSCLC. Dominant PD-L1 highly expressing clones correlated to her clinical course. Immunofluorescent analysis could successfully distinguish EGFR-mutant and PD-L1 highly expressing clones in this case. TRACERx study [3.Jamal-Hanjani M. Wilson G.A. McGranahan N. et al.Tracking the evolution of non-small-cell lung cancer.N Engl J Med. 2017; 376: 2109-2121Crossref PubMed Scopus (1326) Google Scholar] has recently demonstrated that most driver mutations in EGFR, MET, BRAF and TP53 are clonal and likely represents early events involved in initial lung cancer development. In contrast, we here report that EGFR mutation may also be a subclonal alteration, and play a role in late-stage lung cancer development. The reported sensitivity and the specificity of the PNA-LNA PCR clamp method for EGFR mutations of 97% and 100% [4.Tanaka T. Nagai Y. Miyazawa H. et al.Reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers.Cancer Sci. 2007; 98: 246-252Crossref PubMed Scopus (86) Google Scholar]. Our cancer panel sequencing (QIAseq comprehensive cancer panel) with median read depth value of 3154 and variant calling threshold of 29 in this analysis had a limit of detection of 1% variant frequency for hot spot mutations. This is far above the EGFR Ex.19 mutation allele frequencies of those detected by ddPCR (supplementary Figure S1, available at Annals of Oncology online). This discrepancy highlights intratumor heterogeneity and sparse representation of EGFR Ex.19 del mutated clones that on NGS-based analyses would not have been easily identified. This result presents the caveat of using ultra-sensitive approaches to actionable variant detection to inform clinical management. For EGFR-mutant NSCLC patients, we should use EGFR-TKIs as first-line therapy [5.Reck M. Popat S. Reinmuth N. et al.Metastatic non-small-cell lung cancer (NSCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.Ann Oncol. 2014; 25: iii27-iii39Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar]. However, the present case suggested that shorter PFS in EGFR-TKI treatment of EGFR-mutant NSCLC patients with high PD-L1 TPS and without acquired T790M mutation could encourage us to try PD-1 blockade therapy as second-line therapy. There are PD-1 blockade therapy good responders among EGFR-mutant NSCLC patients. To refine the selection, comprehensive consideration of clinical profiles including PFS in EGFR-TKI treatment, further targetable resistant mutations including T790M mutation, tumor mutation burden and PD-L1 TPS is necessary. Written informed consent about publication was obtained from the patient in this report. We thank the patient who contribute to this report and Yachiyo Kumamoto for her contribution to immunofluorescent experiment. None declared.