Efficacy and Resistance of Afatinib in Chinese Non-Small Cell Lung Cancer Patients With HER2 Alterations: A Multicenter Retrospective Study

Background Non-small cell lung cancer (NSCLC) patients with HER2 mutations and amplification may benefit from HER2-targeted therapy, including afatinib. However, the data regarding the clinical activity of afatinib in Chinese patients with NSCLC harboring HER2 alterations are limited. Patients and methods We retrospectively included metastatic NSCLC patients harboring HER2 alterations who treated with afatinib. The clinical outcomes included overall response rate (ORR), progression-free survival (PFS) and overall survival (OS). The genomic profiling data after progression on afatinib were analyzed. Results We included 54 patients harboring HER2 mutations and 12 patients harboring HER2 amplification. The ORR was 24% (95% CI, 16–36%), the median PFS was 3.3 months (95% CI, 2.2–4.4), and the median OS was 13.9 months (95% CI, 11.4–16.5). Patients with HER2 exon 20 mutations had numerically worse ORR (17% vs 42%), shorter PFS (2.6 vs 5.8 months, HR, 2.5; 95% CI, 1.2–5.5; P = 0.015) and OS (12.9 vs 33.3 months, HR, 4.4; 95% CI, 1.3–14.8; P = 0.009) than patients with other mutations. For HER2-amplified patients, the ORR was 33% (95% CI, 14–61%), the median PFS was 3.3 months (95% CI, 2.6–4.0), and the median OS was 13.4 months (95% CI, 0–27.6). The most frequently mutated genes in afatinib-resistant patients were TP53 (44%) and EGFR (33%). Three afatinib-resistant patients harbored secondary HER2 alterations. Conclusions Our results suggest that afatinib has a promising anti-tumor activity in patients with NSCLC harboring HER2 alterations. To our knowledge, this is the largest retrospective study about the clinical activity of afatinib in NSCLC patients with HER2 alterations.


INTRODUCTION
Lung cancer is one of the most common malignant tumors, causing approximate 25% of the total cancer-related deaths (1). About 85% of patients with lung cancer are histologically diagnosed as non-small cell lung cancer (NSCLC) (2). Several driver genes alterations, including EGFR (epidermal growth factor receptor) activating mutations, ALK (anaplastic lymphoma kinase) rearrangement, ROS1 (repressor of silencing 1) fusions, BRAF (B-Raf proto-oncogene, serine/ threonine kinase) mutations, MET (MET proto-oncogene, receptor tyrosine kinase) alterations, and RET (ret protooncogene) fusions, are frequently detected in the patients with NSCLC (3). Targeted therapies based on these genes have been approved by the Food and Drug Administration (FDA), changing the treatment of NSCLC (4).
Afatinib is an irreversible ERBB family inhibitor, which has been approved for EGFR-mutated lung cancer and become one of the most common therapy in NSCLC patients. In a phase II trial with 13 advanced NSCLC with HER2 exon 20 mutations, the overall response rate (ORR) of afatinib as second-line treatment was 7.7% and the median progression-free survival (PFS) was 15.9 weeks (11). Several retrospective trials revealed better activity of afatinib in patients with HER2 exon 20 mutations, with an ORR from 13 to 33% (5,(12)(13)(14)(15). However, the interpretation of the results from all these studies were limited by the small sample sizes. In addition, the efficacy of HER2-TKI in patients with HER mutations besides HER2 exon 20 mutations and HER2 amplification has been rarely studied. Seven patients with other HER2 mutations except exon 20 mutations were enrolled into the phase II trial of T-DM1, and two of these patients had a partial response, with a S310F (exon 8) mutation and a V659E (exon 17) mutation, respectively (10). Another research showed that three of four NSCLC patients with V659E or G660R (exon 17, located in transmembrane domain) achieved responses from afatinib treatment (16).
Herein, we conducted a multicenter, retrospective study to analyze the anti-tumor activity of afatinib in patients with NSCLC harboring HER2 alterations including mutations and amplification. Furthermore, we tried to explore the potential secondary resistant mechanisms of afatinib by next generation sequencing (NGS). We present the following article/case in accordance with the STROBE reporting checklist.

Data Collection and Response Assessment
Baseline clinical information were collected from electronic medical records, including age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, tumor histology, smoking status, HER2 alteration subtype, and afatinib treatment line. These clinical data were verified independently by two oncologist physicians. Tumor size measurement according to radiologic imaging was conducted by radiologists. Best response was determined according to Response Evaluation Criteria in Solid Tumors (RECIST, v1.1). The outcomes were ORR, PFS, and overall survival (OS). ORR was defined as the proportion of patients who have a partial response (PR) or complete response (CR). PFS was defined as the time interval from initial afatinib treatment to progression or death from any cause. OS was defined as the duration from the beginning of afatinib treatment to death from any cause.

Molecular Testing
The baseline HER2 gene alterations were tested by NGS in an accredited local laboratory (for example as shown in Figure S1). Genomic profiling when progression on afatinib treatment was tested in a CLIA-accredited/CAP-certified laboratory (3D Medicines Inc., Shanghai, China). The NGS panel targeted cancer-related genes was performed on the NextSeq500 platform (Illumina, CA, USA) (17). DNA extracts (30-200 ng) were sheared to 250 bp fragments using an S220 focusedultrasonicator (Covaris). Libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) following the manufacturer's protocol. The captured libraries were loaded onto a NextSeq500 platform for 100 bp paired-end sequencing with a mean sequencing depth of 500×.
Raw data of paired samples (an FFPE sample and its normal tissue control) were mapped to the reference human genome hg19 using the Burrows-Wheeler Aligner (v0.7.12). PCR duplicate reads were removed and sequence metrics were collected using Picard (v1.130) and SAMtools (v1.1.19), respectively. Variant calling was performed only in the targeted regions. Somatic single nucleotide variants (SNVs) were detected using an in-house developed R package to execute a variant detection model based on binomial test. Local realignment was performed to detect indels. Variants were then filtered by their unique supporting read depth, strand bias, base quality as previously described. All variants were then filtered using an automated false positive filtering pipeline to ensure sensitivity and specificity at an allele frequency (AF) of ≥1%. Singlenucleotide polymorphism (SNPs) and indels were annotated by ANNOVAR against the following databases: dbSNP (v138), 1000Genome and ESP6500 (population frequency >0.015). Only missense, stopgain, frameshift and non-frameshift indel mutations were kept. Copy number variations (CNVs) and gene rearrangements were detected. The interpretation of variants were based on American College of Medical Genetics and Genomics (ACMG) standards and guidelines.

Statistical Analyses
All statistical analyses were conducted using the SPSS statistical package, version 20.0 (SPSS Inc ® , Chicago, Illinois, USA) and GraphPad prism v6 (GraphPad, La Jolla, CA, USA). The PFS and OS were estimated by Kaplan-Meier curves, with P value determined by a log-rank test. And we calculated hazard ratio (HR) and its 95% confidence intervals (CIs) by Cox regression. Univariate and multivariate analyses were performed by Cox proportional hazard model. A two-sided P <.05 was considered statistically significant.
Since HER2 exon 20 mutation is the most common mutation for HER2 in patients with NSCLC, we further compared the outcomes of patients with exon 20 mutation and other mutations. As for HER2 exon 20 mutations, the total ORR was 17%, and the ORRs of the patients with Y772_A775dup mutation and G778_P780dup were 33 and 10%, respectively, while the ORR was 0% in patients with other exon 20 mutations including G776delinsVC/LC, A775_G776insSVMA, A775_G776insVVMA, and V777L (  Figure S2).
We also performed subgroup analysis according afatinib treatment lines. The ORR was 42% in patients who received afatinib as first-line treatment compared with 14% in those who received afatinib as secondary-line or beyond treatment. Patients who received afatinib as secondary-line or beyond treatment had shorter PFS and OS compared with patients who received afatinib as first-line treatment (mPFS = 2.7 vs 4.7 months; OS = 11.2 vs 15.6 months; Figure S3). Multivariate analysis showed that afatinib treatment line and brain metastasis were associated with PFS (P = 0.026 and 0.017, respectively), and ECOG performance status was associated with OS (P = 0.046) (Tables S1 and S2).

Potential Biomarkers for Resistance to Afatinib
To reveal potential biomarkers of resistance to afatinib, NGS was performed from blood or tissue samples of nine patients after progression on afatinib treatment. Pathogenic and likely pathogenic mutations were analyzed. We observed most patients (78%, 7/9) still harbored HER2 alterations after afatinib treatment (Table S3) Table S3). The most frequently mutated genes in afatinib-resistant patients were TP53 (44%) and EGFR (33%). Besides, one patient carried a NRAS mutation and another patient had no HER2 alteration nor other pathogenic mutation when progression on afatinib (Table S3).

DISCUSSION
In the present study, afatinib showed promising anti-tumor activity in patients with NSCLC harboring HER2 alterations including HER2 exon 20 mutations, other mutations and HER2 amplification. To our knowledge, this is the largest retrospective study on clinical activity of afatinib in NSCLC patients with HER2 alterations. Most previous studies focused on HER2 exon 20 insertions. Recent studies reported that NSCLC patients with HER2 exon 20 insertions had an ORR of 13-19% from afatinib treatment (14,15,18). The sole to date prospective study (11) on afatinib in NSCLC patients with HER2 exon 20 insertions only enrolled 13 patients, with a modest clinical outcomes (ORR = 7.7%). In the largest cohort of NSCLC patients with HER2 exon 20 insertions, Mazières et al. (12) reported clinical activity of chemotherapy and HER2-targeted drugs. The ORR of the patients (N = 29) treated with TKIs (neratinib, afatinib, and lapatinib) was 7.4%. Among the patients (N = 11) who were treated with afatinib, the ORR was 18.2%. In the present study, most HER2 mutations were exon 20 insertions (61%), which was similar with previous studies. We observed an ORR of 17% in these patients, which was comparable with previous retrospective studies. Of the patents with most common Y772_A775dup mutation, ORR was 33%, which suggests that these patients might have better clinical outcome from afatinib.
Moreover, we found HER2 other mutations except exon 20 mutations were also sensitive to afatinib. Five (42%) of these patients achieved response from afatinib treatment. Among these patients who were response to afatinib, one patient with a L655V (exon 17) mutation had a PFS of 8.1 months. L655V (exon 17) is located in transmembrane domain (TMD) that is important to   stabilize the active HER2 homodimer (19). And L655V is close to V659/G660, which were demonstrated to be sensitive to afatinib (16). One lung squamous cell carcinoma patient with a HER2 R896G (exon 22) mutation had a long PFS of 14.5 months, which was recently reported as a case report (20). Another patient with a M960V (exon 24) mutation received afatinib as third-line therapy, and achieved a PR and a PFS of 7.1 months. The other two patients respectively harbored H878Y (exon 21) and L1173V (exon 27), and the PFS were 22.7 and 25.0 months, respectively. These results suggest that the patients with HER2 other mutations except exon 20 mutations could also benefit from HER2-targeted inhibitors. So far, the standard care for NSCLC patients with HER2 amplification is chemotherapy. Although T-DM1 is recommended by NCCN Guidelines for HER2-mutated NSCLC patients, no HER2-targerd inhibitors are approved for NSCLC patients with HER2 mutations or amplification. In a phase II trial of dacomitinib in lung cancer patients with HER2 alterations, none of four patients with HER2-amplified tumors responded (21). Recently, two studies on HER-mutated NSCLC patients treated with pyrotinib, a pan-HER inhibitor, showed that ORRs were 53.3 and 30%, and mPFSs were 6.4 months and 6.9 months, respectively (22,23). An in vitro study and phase II trial demonstrated another pan-HER inhibitor poziotinib had potent clinical activity against HER2 mutations (24,25). In breast cancer, gastric Cancer, and colorectal cancer, HER2 amplification was demonstrated to be associated with the clinical outcomes of HER2-targeted treatment (26,27). In this study, we presented an ORR of 33% in the NSCLC patients with HER2 amplification, and this is the first time that clinical activity of afatinib in HER2amplified NSCLC patients has been reported. These results indicate HER2-targeted treatment might be one of the choices for these patients.
Primary and acquired resistance is the main reason for progression disease when patients received TKIs treatment. Currently, we know much about the mechanisms for resistance of EGFR-targeted treatment, but researches about resistance to HER2-targerted inhibitors in NSCLC patients are lacking. Chuang et al. (13) suggested PIK3CA mutation and HER2 gene amplification may be the potential mechanisms for resistance during HER2-targeted treatment. However, the results were analyzed from four cases, which is hard to reach statistical significance. Herein, we performed NGS for nine patients when progression on afatinib treatment. Of three patients harbored secondary HER2 alterations, two carried a HER2 exon 20 insertion and another carried HER2 amplification as secondary alteration. Previous studies demonstrated that secondary ALK mutations could induce resistance of ALK inhibitors (28,29). Whether HER2 secondary alterations resistance mechanism to afatinib need to be determined in further studies. In addition, we found TP53 was recurrently mutated (44%) in afatinib-resistant patients. Several studies reported that TP53 mutations were associated with inferior clinical effect of EGFR-targeted inhibitors (30)(31)(32). One patient harbored TP53 and RB1 co-mutations, which were associated with an increasing risk for small cell transformation and resistance to TKIs treatment (33)(34)(35)(36).
This study still has several limitations. Firstly, we cannot completely avoid the reporting bias because of this work's retrospective nature. Secondly, due to a lack of control arm, comparison with other therapies was not feasible. Thirdly, only nine patients were performed NGS when progression, so these data cannot fully reflect the whole cohort and no statistical significance can be reached about resistance of afatinib. Despite these limitations, this study provides deep insights into clinical activity of afatinib in NSCLC with HER2 alterations.

CONCLUSION
Our results suggest that afatinib has a potential efficacy in these patients, especially in the patients with HER2 amplification or other pathologic mutations in exons except exon 20. Further studies, especially prospective studies, are warranted to investigate the clinical activity of afatinib and the mechanism of resistance to HER2-targeted therapy.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Zhejiang Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.