Expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed small cell lung cancer.

Background: The transformation of epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) into small cell lung cancer (SCLC) accounts for 3-14% of the resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs). At present, there is no relevant research to explore the dynamic expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed SCLC/neuroendocrine carcinoma (NEC). Methods: Genetic analysis and protein level analysis by next-generation sequencing (NGS), Whole-exome sequencing (WES) and immunohistochemistry were performed to explore expression of EGFR-mutant proteins and genomic evolution in EGFR-mutant transformed SCLC. The research used three patient-derived organoids (PDOs) to explore the efficacy of combo [chemotherapy (chemo) plus TKI or bevacizumab] treatment. According to the subsequent treatment regimens after SCLC/NEC transformation, 35 patients were divided into chemo (n=21) and combo (n=14) groups. Results: EGFR L858R and EGFR E746-750 del protein expression by immunohistochemistry was 80.0% (4/5) and 100% (6/6), respectively (P=0.455) in initially-transformed tissues. Meanwhile, EGFR-mutant proteins were expressed in 85.7% (6/7) of dynamic rebiopsy tissues or effusion samples after the first transformation. Then, by the pathway enrichment analysis of tissue and plasma NGS, the EGFR-related pathways were still activated after SCLC/NEC transformation. Moreover, WES analysis revealed that transformed SCLC shared a common clonal origin from the baseline LUAD. The drug sensitivity of three PDOs demonstrated potent anti-cancer activity of EGFR-TKIs plus chemo, compared with chemo or TKI alone. There were significant differences in objective response rate (ORR) between the combo and chemo groups [42.9 % vs. 4.8%, P=0.010, 95% confidence interval (CI): 1.5-145.2]. Furthermore, the median post-transformation progression-free survival (pPFS) was significantly prolonged in the combo group, with 5.4 (95% CI: 3.4-7.4) versus 3.5 (95% CI: 2.7-4.3, P=0.012) months. Conclusions: EGFR 19del or L858R-mutant proteins could be constantly expressed, and EGFR pathway still existed in EGFR-mutant transformed SCLC/NEC with a common clonal origin from the baseline LUAD. Taking together, these molecular characteristics potentially favored clinical efficacy in transformed SCLC/NEC treated with the combo regimen.

Prognostic features and comprehensive genomic analysis of NF1 mutations in EGFR mutant lung cancer patients.

OBJECTIVE: NF1 is a tumor suppressor gene that encodes the neurofibromin protein and negatively regulates Ras signaling. This study was aimed to investigate the molecular, clinical characteristics, and prognostic features of NF1 gene in EGFR mutant lung cancer patients. METHOD: The next-generation sequencing (NGS) was used to analyze the data from lung cancer patients in the Guangdong Lung Cancer Institute (GLCI) from June 2016 to December 2020. RESULTS: Somatic NF1 mutations were present in 4.2% (135/3220) of Chinese lung cancer patients. NF1 mutations where clearly enriched in older (p < 0.001), male (p < 0.001), and smoking (p < 0.001) patients. Patients with NF1 mutations were more likely to have TP53 (p = 0.003), BRAF (p = 0.001) and RASA1 (p = 0.026) mutations and mutually exclusive with EGFR mutations (p = 0.006). TP53 mutation had worsen prognosis in cases of NF1 mutant (p = 0.026) or EGFR/NF1 co-mutant (p = 0.031) lung adenocarcinomas (LUAD) patients. There was no effect on overall survival (OS) in LUAD patients with and without NF1 mutations, even in LUAD driver-gene negative patients. NF1/EGFR co-mutation patients had a longer OS than a single mutation of either the EGFR gene (median OS: 47.7 m vs. 30.2 m, hazard ratio [95% CI], 0.47 [0.30-0.74], p = 0.004) or NF1 gene (47.7 m vs. 19.0 m, 0.44 [0.27-0.73], p = 0.003). Furthermore, NF1 mutations significantly prolonged OS in EGFR mutant/TP53 wild-type LUAD patients (106.5 m vs. 25.5 m, 0.28 [0.13-0.59], p = 0.003) but not in patients with EGFR/TP53 co-mutations (36.8 m vs. 30.2 m, 0.70 [0.39-1.26], p = 0.280). CONCLUSION: Our results indicated NF1 mutations served as a good prognostic factor in EGFR mutant/TP53 wild-type lung cancer patients in this single-center study. TP53 mutation was obviously enriched in NF1 mutant patients and had shorter OS.

PD-1/PD-L1 combined with LAG3 is associated with clinical activity of immune checkpoint inhibitors in metastatic primary pulmonary lymphoepithelioma-like carcinoma.

Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is an Epstein-Barr virus (EBV)-related, rare subtype of non-small-cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICI) show durable responses in advanced NSCLC. However, their effects and predictive biomarkers in PLELC remain poorly understood. We retrospectively analyzed the data of 48 metastatic PLELC patients treated with ICI. Pretreated paraffin-embedded specimens (n = 19) were stained for PD-1, PD-L1, LAG3, TIM3, CD3, CD4, CD8, CD68, FOXP3, and cytokeratin (CK) by multiple immunohistochemistry (mIHC). Next-generation sequencing was performed for 33 PLELC samples. Among patients treated with ICI monotherapy (n = 30), the objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), and overall survival (mOS) were 13.3%, 80.0%, 7.7 months, and 24.9 months, respectively. Patients with PD-L1 ≥1% showed a longer PFS (8.4 vs. 2.1 months, p = 0.015) relative to those with PD-L1 <1%. Among patients treated with ICI combination therapy (n = 18), ORR, DCR, mPFS, and mOS were 27.8%, 100.0%, 10.1 months, and 19.7 months, respectively. Patients with PD-L1 ≥1% showed a significantly superior OS than those with PD-L1 <1% (NA versus 11.7 months, p = 0.001). Among the 19 mIHC patients, those with high PD-1/PD-L1 and LAG3 expression showed a longer PFS (19.0 vs. 3.9 months, p = 0.003). ICI also showed promising efficacy for treating metastatic PLELC. PD-L1 may be both predictive of ICI treatment efficacy and prognostic for survival in PLELC. PD-1/PD-L1 combined with LAG3 may serve as a predictor of ICI treatment effectiveness in PLELC. Larger and prospective trials are warranted to validate both ICI activity and predictive biomarkers in PLELC. This study was partly presented as a poster at the IASLC 20th World Conference on Lung Cancer 2019, 7-10 September 2019, Barcelona, Spain.

An integrated biomarker of PD-L1 expression and intraepithelial CD8+ T cell infiltration was associated with the prognosis of lung cancer patients after intracranial resection of brain metastases.

BACKGROUND: Brain metastases (BM) are common in lung cancer. However, data on the status of immune biomarkers in BM lesions remain limited. METHODS: We retrospectively analyzed PD-L1 expression and infiltration levels of CD3+ , CD4+ , CD8+ T cells as biomarkers by immunohistochemistry in both BM lesions and primary lung cancer (PL) lesions of 29 lung cancer (LC) patients. In addition, the correlations between these biomarkers and the clinical outcome were analyzed using log-rank test. RESULTS: Intratumoral heterogeneous expression of PD-L1 was observed on tumor cells (TCs) in 11 cases and on immune cells (ICs) in 10 cases with BM samples from multiple regions. There was a disagreement in PD-L1 expression on TCs between paired BM and PL lesions in 15 cases and on ICs in seven cases. Intraepithelial CD3+ and CD8+ T cell infiltration levels in BM samples were lower than those in the paired PL samples. PD-L1 positivity on both TCs and ICs was associated with a better post-BM-surgery prognosis (p = 0.010; p = 0.041). Notably, PD-L1 positivity on TCs and a high level of intraepithelial CD8+ T cell infiltration could serve as an integrated biomarker that indicates longer survival time (p = 0.004) in LC patients. CONCLUSION: The heterogeneity in PD-L1 expression was common in both stromal and intraepithelial regions in BM lesions of LC patients, suggesting the need for multiregional PD-L1 testing in clinical practice. More importantly, a combination of PD-L1 expression on TCs with intraepithelial CD8+ T cell infiltration might predict better post-BM-surgery outcomes.

High SHP2 expression determines the efficacy of PD-1/PD-L1 inhibitors in advanced KRAS mutant non-small cell lung cancer.

BACKGROUND: Src homology region 2 domain-containing phosphatase 2 (SHP2) is a novel target for Kirsten rat sarcoma oncogene (KRAS) mutant cancer. We retrospectively studied the significance of SHP2 in KRAS mutant non-small cell lung cancer (NSCLC) treated with immunotherapy and its relationship with tumor microenvironment (TME). METHODS: Sixty-one advanced KRAS mutant NSCLC patients who underwent immunotherapy were enrolled. Next-generation sequencing (NGS) was used to profile mutation status. The expression of SHP2, phospho-SHP2 (pSHP2), and programmed death ligand 1 (PD-L1) were analyzed by immunohistochemistry (IHC). Quantitative multiplexed immunofluorescence cytochemistry (mIFC) analysis was conducted to describe the TME. RESULTS: SHP2 was heterogeneously expressed in 32 samples in both tumor cells and immune cells and highly expressed (H-score >10) in 25 (78.1%) samples. The expression levels of SHP2 and pSHP2 were positively correlated. Stromal SHP2 (s-SHP2) was higher in tumors with PD-L1 ≥50% versus PD-L1 <50% (p = 0.039). By quantitative mIFC analysis, the expression of s-SHP2 had positive correlation with CD8, CD4, CD68, and PD-L1 levels in stromal area. Patients with high SHP2 expression made up 100.0% of the partial respond (PR) and 80.0% of the stable disease (SD), whereas 50.0% of the progress disease (PD). High SHP2 expression was associated with longer progression-free survival (PFS) and overall survival (OS) (p < 0.001, p = 0.013). Patients with high expression of both SHP2 and PD-L1 had longer PFS (p < 0.001). CONCLUSION: High SHP2 expression could predict the efficacy of immunotherapy and better survival in advanced KRAS mutant NSCLC. SHP2 may function in both tumor cells and immune cells, warranting further study on the potential diverse effects of SHP2 inhibition in TME.

Establishment and application of a method of next generation sequencing of 285 genes in lung cancer based on Ion-Proton platform.

BACKGROUND: The development of "precision medicine" needs a novel genetic screening and diagnostic technique for clinical detection. This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through testing 285 genes by customized next generation sequencing (NGS) on Ion-Proton platform. METHODS: We reviewed the related literature and collected data of genomic alteration that occurred in lung cancer. We identified 285 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. Targeted hybridization probes were designed using SureDesign software. The detection method was established by analyzing four cell lines and 13 lung cancer specimens which had been validated through Sanger sequencing. The sensitivity and specificity of the proposed method were preliminarily evaluated by comparisons with the Sanger sequencing and a LungCarta mutation-detection method. RESULTS: The proposed method was able to detect mutations of 285 genes in lung cancer cell lines and clinical lung cancer specimens. The reads, mapped reads, on target, mean depth and uniformity were 14.90±4.37 (×106), 98.68%±0.61%, 60.49%±10.72%, 714.42±264.13 and 90.51%±6.91%, respectively. The detected mutation result of cell lines was consistent with the observations on previously reported mutations, and the congruence rate was 100%. The proposed method can detect single nucleotide polymorphism (SNP), InDel, Fusion and copy number variation (CNV). The complete congruence rate of detected result of specimens between the proposed method and Sanger sequencing, LungCarta mutation-detection method, immunohistochemistry (IHC), real-time polymerase chain reaction (RT-PCR) method were all 100% regarding mutations in common genes like EGFR, KRAS, or fusions of ALK, RET, etc. In addition, NFE2L3_p.Ser511_Pro513del, ERBB2_E770delinsEAYVM, MET_S701N, PDGFRA_T674I, TP53_G245V, TP53_V274A, TP53_A276F, TP53_G334L, TP53_R337L and TP53_Y220C mutations were detected only through the proposed method. The proposed method can detect mutations from blood, this detection result was consistent with the cancer tissues of the same clinical lung cancer patient. CONCLUSIONS: The proposed Ion-Proton technology-based NGS method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method could be used to detect mutations in other cancer tissues and plasma, which needs further validation.

Low frequency of mutation of epidermal growth factor receptor (EGFR) and arrangement of anaplastic lymphoma kinase (ALK) in primary pulmonary lymphoepithelioma-like carcinoma.

BACKGROUND: Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare and unique subtype of lung cancer. However, the prevalence of driver alterations, such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements, and the response of tyrosine kinase inhibitor (TKIs) in PLELC has not been thoroughly investigated. METHOD: We retrospectively reviewed the genetic profiles and treatment course of 330 PLELC patients at the Guangdong Lung Cancer Institute (GLCI) from 1st January, 2008 to 30th December, 2018. We searched and analyzed related literature published in PubMed and Web of Science from 1st January, 2000 and 31th August, 2019 based on their mention of "driver mutations" and "the response of TKIs to mutant PLELC". RESULTS: Genetic alterations of EGFR/ALK were tested in 203 patients (203/330, 61.5%). Five patients (5/175, 2.9%) had EGFR mutation and three patients (3/140, 2.1%) had ALK alteration. From the total of 15 articles identified from electronic searches, 1071 PLELC cases mentioned the driver mutations. EGFR mutation and ALK rearrangement were detected in 15 patients and one patient, respectively. In total, there were four EGFR/ALK mutant PLELC patients who received targeted therapy as palliative treatment at the GLCI and in the literature. However, there was disease progression in all cases one month after use of TKIs. CONCLUSION: The mutation rates of EGFR and ALK were low in PLELC. EGFR and ALK TKIs showed limited response in EGFR/ALK mutant PLELC. Further studies are needed to explore other molecular targets to optimize the therapeutic strategy for PLELC.