Relationship of circulating tumor cells and Epstein-Barr virus DNA to progression-free survival and overall survival in metastatic nasopharyngeal carcinoma patients.

We analyzed the number of circulating tumor cells (CTCs) and Epstein-Barr virus DNA (EBV DNA) for diagnosis, monitoring and prognosis of patients with metastatic nasopharyngeal carcinoma (mNPC). The levels of CTCs and EBV DNA were measured at baseline and after first-line chemotherapy in 148 mNPC patients prospectively enrolled between December 2014 and August 2016. We also collected 122 non-mNPC cases within the same time frame for examining CTCs and EBV DNA at baseline. In 270 NPC patients, we observed improved specificity (86.0% vs. 41.0%) and inferior sensitivity (42.3% vs. 81.3%) of CTCs as compared to EBV DNA for diagnosis of distant metastasis. mNPC patients were stratified into unfavorable and favorable prognostic groups, respectively, based on CTC of 12 at baseline and 1 after first-line chemotherapy and EBV DNA of 10,000 at baseline and 4,000 after first-line chemotherapy. Conversion of baseline unfavorable CTCs and EBV DNA to favorable after first-line chemotherapy was associated with significantly longer progression-free survival (PFS) and overall survival (OS) compared to patients with unfavorable CTCs and EBV DNA at both time points. Among patients with a complete/partial response as per imaging evaluation, favorable CTCs and EBV DNA levels after first-line chemotherapy were associated with significantly longer PFS and OS. In conclusion, our data demonstrated the number of CTCs and EBV DNA before, after and during first-line chemotherapy were strong predictive markers for mNPC patients. When utilized in conjunction with imaging studies, CTCs and EBV DNA could provide additional prognostic information.

Crizotinib in advanced non-small-cell lung cancer with concomitant ALK rearrangement and c-Met overexpression.

OBJECTIVE: Crizotinib can target against mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK), which has been considered as a multi-targeted tyrosine kinase inhibitor (TKI). The objective of this study was to explore the efficacy of crizotinib in advanced non-small-cell lung cancer (NSCLC) with concomitant ALK rearrangement and c-Met overexpression. METHODS: Totally, 4622 advanced NSCLC patients from two institutes (3762 patients at the Guangdong Lung Cancer Institute from January 2011 to December 2016 and 860 cases at the Perking Cancer Hospital from January 2015 to December 2016) were screened for ALK rearrangement with any method of IHC, RACE-coupled PCR or FISH. C-Met expression was performed by IHC in ALK-rearranged patients, and more than 50% of cells with high staining were defined as c-Met overexpression. The efficacy of crizotinib was explored in the ALK-rearranged patients with or without c-Met overexpression. RESULTS: Sixteen patients were identified with c-Met overexpression in 160 ALK-rearranged cases, with the incidence of 10.0% (16/160). A total of 116 ALK-rearranged patients received the treatment of crizotinib. Objective response rate (ORR) was 86.7% (13/15) in ALK-rearranged patients with c-Met overexpression and 59.4% (60/101)in those without c-Met overexpression, P = 0.041. Median PFS showed a trend of superiority in c-Met overexpression group (15.2 versus 11.0 months, P = 0.263). Median overall survival (OS) showed a significant difference for ALK-rearranged patients with c-Met overexpression group of 33.5 months with the hazard ratio (HR) of 3.2. CONCLUSIONS: C-Met overexpression co-exists with ALK rearrangement in a small population of advanced NSCLC. There may be a trend of favorable efficacy of crizotinib in such co-altered patients.

Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay.

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

Plasma EBV quantification is associated with the efficacy of immune checkpoint blockade and disease monitoring in patients with primary pulmonary lymphoepithelioma-like carcinoma.

Objectives: Primary pulmonary lymphoepithelioma-like carcinoma (PLELC) is a subtype of lung carcinoma associated with the Epstein-Barr virus (EBV). The clinical predictive biomarkers of immune checkpoint blockade (ICB) in PLELC require further investigation. Methods: We prospectively analysed EBV levels in the blood and immune tumor biomarkers of 31 patients with ICB-treated PLELC. Viral EBNA-1 and BamHI-W DNA fragments in the plasma were quantified in parallel using quantitative polymerase chain reaction. Results: Progression-free survival (PFS) was significantly longer in EBNA-1 high or BamHI-W high groups. A longer PFS was also observed in patients with both high plasma EBNA-1 or BamHI-W and PD-L1 ≥ 1%. Intriguingly, the tumor mutational burden was inversely correlated with EBNA-1 and BamHI-W. Plasma EBV load was negatively associated with intratumoral CD8+ immune cell infiltration. Dynamic changes in plasma EBV DNA level were in accordance with the changes in tumor volume. An increase in EBV DNA levels during treatment indicated molecular progression that preceded the imaging progression by several months. Conclusions: Plasma EBV DNA could be a useful and easy-to-use biomarker for predicting the clinical activity of ICB in PLELC and could serve to monitor disease progression earlier than computed tomography imaging.

Circulating tumor cells dynamics during chemotherapy predict survival and response in advanced non-small-cell lung cancer patients.

Background: Circulating tumor cells (CTCs) are prognostic biomarker in non-small-cell lung cancer (NSCLC). CTCs could also be used as predictor of efficacy of systemic treatments in advanced NSCLC. Objectives: We described the dynamic changes of CTCs during first-line platinum-based chemotherapy in advanced NSCLC and clarified the correlation between CTC counts and efficacy of chemotherapy. Design: Chemotherapy is administered and blood specimens are collected at four time points from baseline to disease progression for CTC detection. Methods: This multicenter prospective study enrolled patients with previously untreated stage III or IV NSCLC fit for standard platinum-based chemotherapy. Bloods were sampled as per standard operating procedures at baseline, cycle 1 and cycle 4 of chemotherapy, and at disease progression for CTC analysis using the CellSearch system. Results: Among 150 patients enrolled, median overall survival (OS) was 13.8, 8.4, and 7.9 months in patients with CTC-, KIT-CTC, and KIT+CTC at baseline (p = 0.002). Patients with persistent negative CTC (46.0%) had longer progression-free survival [5.7 months, 95% confidence interval (CI): 5.0-6.5 versus 3.0 months, 0.6-5.4; hazard ratio (HR): 0.34, 95% CI: 0.18-0.67) and OS (13.1 months, 10.9-15.3 versus 5.6 months, 4.1-7.1; HR: 0.17, 0.08-0.36) compared with patients with persistent positive CTC (10.7%), which was not impacted by chemotherapy. Chemotherapy decreased CTC from 36.0% (54/150) to 13.7% (13/95). Conclusions: CTC persistent presence during treatment represents poor prognosis and resistance to chemotherapy in advanced NSCLC. Chemotherapy could effectively eliminate CTCs. Molecular characterization and the functionalization of CTC will be warranted for further intensive investigation. Trial registration: NCT01740804.

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.

Clinical Utility of Cerebrospinal Fluid Cell-Free DNA as Liquid Biopsy for Leptomeningeal Metastases in ALK-Rearranged NSCLC.

INTRODUCTION: Leptomeningeal metastases (LMs) indicated a poor prognosis in NSCLC. LMs were more frequent in driver gene-mutated patients, and cerebrospinal fluid (CSF) cell-free DNA has shown unique genetic profiles of LM in EGFR-mutated LM. However, studies in patients with ALK receptor tyrosine kinase gene (ALK)-rearranged NSCLC with LMs are scarce. METHODS: Patients with lung cancer with ALK rearrangement were screened from September 2011 to February 2018 at our institute. CSF and paired plasma were tested by next-generation sequencing. RESULTS: LMs were diagnosed in 30 (10.3%) of 291 patients with ALK-rearranged lung cancer. A total of 11 paired CSF and plasma samples tested by next-generation sequencing were analyzed. Driver genes were detected in 81.8% of the CSF samples (9 of 11) and 45.5% of the plasma samples (5 of 11) (p = 0.183). The maximum allelic fractions were all higher in CSF than in plasma (p = 0.009). ALK and tumor protein p53 gene (TP53) were the two most frequently mutated genes in CSF. Gatekeeper gene ALK G1202R and C1156F mutations were identified in CSF after resistance to alectinib. Multiple copy number variants were mainly found in CSF, including in EGFR, cyclin D1 gene (CCND1), fibroblast growth factor 3 gene (FGF3), and fibroblast growth factor 4 gene (FGF4). Also found were v-myc avian myelocytomatosis viral oncogene homolog gene (MYC) copy number gains and TP53 and cyclin dependent kinase inhibitor 2A gene (CDKN2A) copy number deletions. Brigatinib seemed to be effective in controlling LM. One case showed that CSF could be used to monitor disease development of LM and longitudinally monitor tumor response. CONCLUSION: Liquid biopsy of CSF is more sensitive than liquid biopsy of plasma to detect targetable alterations, characterizing resistance mechanisms on progression and monitoring tumor response in patients with ALK-rearranged NSCLC with LM. Thus, CSF might be promising as a medium of liquid biopsy in LM.

Stromal PD-L1-Positive Regulatory T cells and PD-1-Positive CD8-Positive T cells Define the Response of Different Subsets of Non-Small Cell Lung Cancer to PD-1/PD-L1 Blockade Immunotherapy.

INTRODUCTION: Inhibition of programmed cell death-1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) by using an immune checkpoint inhibitor has emerged as a promising immunotherapy for NSCLC. The correlation of PD-L1 expression in tumor cells with treatment outcomes has been reported in many pivotal trials; however, the relationship remains unclear. Here, we demonstrate that those patients with both high density of PD-1-positive CD8 and PD-L1-positive CD4-positive CD25-positive (PD-1hi PD-L1hi) regulatory T cells (Tregs) have a better response to PD1/PD-L1 blockade. METHODS: In our study between April 1, 2014, and May 30, 2017, a total of 73 NSCLC peripheral blood samples and fresh tumor specimens were collected for study. Of these, 42 large (10-mm3) fresh tumor specimens were obtained from surgical procedures and checked for expression of immunology biomarkers, including PD-L1, PD-1, CD8, CD4, and CD25, in tumor cells and tumor-infiltrating lymphocytes (TILs) by flow cytometry, immunohistochemistry, and immunofluorescence (IF). Moreover, 31 small biopsy specimens from patients who received immunotherapy (pembrolizumab or nivolumab) were analyzed by immunohistochemistry and IF. The correlation between flow cytometry and IF detected for TILs' density was evaluated by Spearman's rank correlation test; the primary end point was progression-free survival. For the PD-1/PD-L1 blockade assay, the TILs and peripheral blood mononuclear CD8 T cells were cultured (1×105 per well) with anti-PD-1 (clone MIH4), anti-PD-L1 (clone MIH1). The cytotoxic activity of TILs in killing NSCLC cells after stimulation by anti-PD-1 and anti-PD-L1 was measured by a conventional 51Cr release assay. RESULTS: We first identified a population of high-PD-L1-expressing CD25-positive CD4-positive T cells (PD-L1hi Tregs) in the tumor microenvironment. The frequency of PD-L1hi Tregs was higher in tumor tissue (mean 48.6 ± 14.3% in CD25-positive CD3-positive CD4-positive T cells) than in blood (mean 35.4 ± 10.2% in CD25-positive CD3-positive CD4-positive T cells) and normal tissue (mean 38.6 ± 9.7% in CD25-positive CD3-positive CD4-positive T cells) (p < 0.05), as determined by flow cytometry. The frequency of PD-L1hi Tregs was positively correlated with PD-1-positive CD8 in Tregs. In addition, the TILs from these patients (PD-1hi PD-L1hi) showed PD-1/PD-L1 pathway dependence and could induce a greater killing effect of TILs by PD-1/PD-L1 blockade treatment. The patients with PD-L1-positive NSCLC with PD-1hi PD-L1hi TILs showed a better clinical outcome than those with a low frequency of PD-1hi CD8 or PD-L1hi Tregs (median progression-free survival not reached versus 2 months). CONCLUSIONS: Our findings suggested that the density of PD-L1-positive CD4-positive CD25-positive Tregs in the tumor microenvironment can serve as a diagnostic factor to supplement PD-L1 expression in tumor cells and predict the response to PD-1/PD-L1 blockade immunotherapy in NSCLC.

Clinical characteristics and sequence complexity of anaplastic lymphoma kinase gene fusions in Chinese lung cancer patients.

OBJECTIVES: To investigate the clinical characteristics of anaplastic lymphoma kinase (ALK) rearrangements and sequence complexity of the ALK fusion gene in Chinese lung cancer patients. METHODS: We prospectively screened ALK rearrangements in 1474 lung cancer specimens, including 1387 cases of non-small cell lung cancer (NSCLC), 54 cases of small cell lung cancer (SCLC), and 33 cases of cancer with lung metastasis from other organs by both standard polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE)-coupled PCR. Fifteen cases of ALK-positive RACE-coupled PCR products were transformed into Escherichia coli for molecular cloning and sequenced for complexity analysis. RESULTS: The overall frequency of ALK rearrangements was 5.1% (71/1387) in NSCLC. In 71 positive cases, the coexistence of epidermal growth factor receptor (EGFR) and ALK variations was found in 6 cases (8.5%), and the coexistence of different ALK variants was found in 2 cases (2.8%) (1 case with variants 1 and 9; the other case with variants 3 and 2) by PCR analysis. Furthermore, through sequence cloning analysis of 15 cases of non-selective ALK-positive samples, two cases with variants 1 and 3 harbored the coexistence of three subtypes (variant 1 subtypes: E13; A20, E13del63; A20 and E7E12E13; A20 and variant 3 subtypes: E6; A20, E6ins33; A20 and E3E6; A20). Variant 3a and 3b subtypes were always coexistent and had the same proportion of ALK variant 3 rearrangements. ALK rearrangement was associated with young age, female gender, never-smokers, those with adenocarcinoma, advanced stage, and EGFR mutations. No ALK fusion was detected in 54 cases of SCLC or 33 cases of cancer with lung metastasis from other organs. CONCLUSIONS: The identification of novel ALK variants, the coexistence of EGFR mutations and ALK fusions, the coexistence of ALK variants, and the coexistence of subtypes reveal the diversity and sequence complexity of ALK fusions.

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

BACKGROUND: Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform. METHODS: A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility. RESULTS: Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform. CONCLUSIONS: We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

Detection of Driver and Resistance Mutations in Leptomeningeal Metastases of NSCLC by Next-Generation Sequencing of Cerebrospinal Fluid Circulating Tumor Cells.

Purpose: Leptomeningeal metastases are more common in non-small cell lung cancer (NSCLC) with EGFR mutations. The diagnosis is difficult by traditional imaging only, and leads to poor understanding of resistance mechanisms of leptomeningeal metastases.Experimental Design: We compared the CellSearch Assay, the Thinprep cytologic test (TCT), and brain magnetic resonance imaging (MRI) in 21 NSCLC patients with suspected leptomeningeal metastases. Next-generation sequencing that included 416 cancer-associated genes was also performed on cerebrospinal fluid circulating tumor cells (CSFCTC) of 19 patients.Results: Twenty-one patients were diagnosed with leptomeningeal metastases, and CSFCTCs were captured by CellSearch in 20 patients (median, 969 CSFCTCs/7.5 mL; range, 27-14,888). CellSearch had a sensitivity of 95.2% for leptomeningeal metastases diagnosis, which was higher than that of TCT (12/21, 57.1%), MRI (10/21, 47.6%), and MRI plus TCT (19/21, 90.5%), respectively. CTCs were found only in 5 of 14 patients (median, 2 CTCs/7.5 mL; range, 2-4), which was a much lower ratio than CSFCTCs. Genetic profiles of CSFCTCs were highly concordant with molecular mutations identified in the primary tumor (17/19, 89.5%). The resistance gene EGFR T790M was detected in 7 of 9 patients with extracranial lesions, but was detected in only 1 of 14 CSFCTC samples. Other potential resistant mutations, such as MET amplification and ERBB2 mutation, were also identified in CSFCTCs.Conclusions: CSFCTCs captured by CellSearch may be a more sensitive and effective way to diagnose leptomeningeal metastases, and may serve as a liquid biopsy medium for gene profiles in NSCLC patients with leptomeningeal metastases. Clin Cancer Res; 23(18); 5480-8. ©2017 AACR.

Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform.

OBJECTIVE: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. METHODS: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCarta(TM) kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. RESULTS: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCarta(TM) kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. CONCLUSIONS: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues.

Leptomeningeal Metastases in Patients with NSCLC with EGFR Mutations.

INTRODUCTION: Leptomeningeal metastases (LM) have increased in patients with NSCLC, and prognostic factors and outcomes for LM with EGFR gene mutations have not been well studied. METHODS: We retrospectively analyzed patients with lung cancer from January 2011 to June 2015 at our institute. Treatments and clinical outcomes of LM were reviewed. RESULTS: LM were diagnosed in 184 (3.4%) of 5387 patients with lung cancer. Patients with LM harboring EGFR mutations (9.4%) were significantly more frequent than those with wild-type EGFR (1.7% [p < 0.001]). The median overall survival (OS) after LM was 8.7 months (95% confidence interval [CI]: 7.3-10.1). Among the 109 patients with common EGFR mutations, the 88 patients who received tyrosine kinase inhibitor (TKI) therapy demonstrated longer OS than those who did not (10.0 months versus 3.3 months [p < 0.001]), but 42 patients who underwent whole brain radiotherapy (WBRT) did not show longer OS than those without WBRT, and a combination of WBRT and TKIs did not add any survival benefit beyond that in patients receiving only TKIs. A multivariate analysis indicated that TKI therapy (p < 0.001, hazard ratio = 0.218) was an independent predictor of favorable survival, whereas poor Eastern Cooperative Oncology Group performance status (p < 0.001, hazard ratio = 3.657) was a predictor of poor survival. CONCLUSIONS: LM were much more frequent in patients with NSCLC harboring EGFR mutations. EGFR TKIs were the optimal treatment for LM, and active treatment with WBRT did not prolong OS for EGFR-mutated patients.

Plasma dynamic monitoring of soluble c-Met level for EGFR-TKI treatment in advanced non-small cell lung cancer.

BACKGROUND: The activation of c-Met has been associated with both primary and acquired resistance to EGFR-TKI therapy in NSCLC patients. Thus, c-Met status during EGFR-TKI therapy should receive much attention. RESULTS: Forty-nine patients were selected as training cohort and 52 cases as validation cohort. With disease progression, IHC results showed that 37 (75.5%) of the patients were tissue c-Met-negative, and 12 (24.5%) were tissue c-Met-positive. There was a statistically significant difference in the dynamic change in soluble c-Met between the tissue c-Met-negative and c-Met-positive groups (P = 0.002). Patients with a baseline soluble c-Met level >766 ng/ml showed inferior median progression-free survival (PFS; 10.2 vs. 14.0 months; P = 0.003) after EGFR-TKI treatment. Multivariate Cox proportional hazards model analyses demonstrated that the soluble c-Met level was an independent prognostic factor for PFS after EGFR-TKI treatment (P = 0.009; hazard ratio: 3.583; 95% confidence interval: 1.379-9.312). In the validation cohort, patients with soluble c-Met levels >766 ng/ml were also determined to have significant short median PFS after EGFR-TKI treatment (6.8 vs. 14.5 months, P < 0.001). PATIENTS AND METHODS: We retrospectively investigated the dynamic change in the soluble c-Met level in plasma and its relationship with clinical outcomes of EGFR-TKI therapy in advanced NSCLC. Immunohistochemistry (IHC) was used to assess the expression of c-Met in the resistant tissue. Plasma c-Met levels were assayed in duplicate using a human soluble c-Met quantitative enzyme-linked immunosorbent assay (ELISA) kit. CONCLUSIONS: Quantitatively determining the soluble c-Met level in plasma by ELISA might provide a non-invasive and sensitive method to predict EGFR-TKI prognosis.

Anaplastic Lymphoma Kinase Variants and the Percentage of ALK-Positive Tumor Cells and the Efficacy of Crizotinib in Advanced NSCLC.

BACKGROUND: Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer patients exhibited heterogeneous magnitude of response and duration time to criztotinib treatment. This study explored ALK variants and the percentage of ALK-positive cells using fluorescent in situ hybridization (FISH) on clinical efficacy of crizotinib. PATIENTS AND METHODS: A total of 120 patients with ALK rearrangement who were treated with criztotinib were enrolled. ALK variants were clarified in 61 patients, and ALK percentages were evaluated using FISH in 114 ALK-positive patients. Retrospectively, objective response rate, and progression-free survival (PFS) were evaluated. RESULTS: A total of 61 patients with specific ALK variants were divided into 3 subgroups, echinoderm microtubule-associated protein like 4 (EML4)-ALK variant 1 (n = 22), EML4-ALK variant 3a/b (n = 18), and other ALK variants (n = 21). Median PFS in the 3 subgroups was 11.0 months (95% confidence interval [CI], 5.5-16.5), 10.9 months (95% CI, 5.9-15.8), 7.4 months (95% CI, 3.2-11.6), respectively, and no significant difference (P = .795) existed among them. The percentage of ALK-positive cells in FISH analysis was weakly correlated with PFS (rs = 0.235; P = .015). Additionally, it was also weakly correlated with best response to crizotinib (rs = 0.288; P = .003). Overall, there were 45, 49, and 26 patients receiving first, second, and third or further-line crizotinib, respectively. Median PFS in the first-line setting (10.5 months; 95% CI, 8.6-12.4) was significantly longer than that in the second-line setting (8.3 months; 95% CI, 4.7-12.0; P = .020). CONCLUSION: Anaplastic lymphoma kinase variants might have no correlation with clinical response to crizotinib. The percentage of ALK-positive cells might correlate with the extent of benefit from crizotinib treatment.

Polymorphism of rs9387478 correlates with overall survival in female nonsmoking patients with lung cancer.

BACKGROUND: Our previous study identified rs9387478 as a new susceptibility locus associated with lung cancer in never-smoking women in Asia; however, the clinical and prognostic significance of this finding is not known. METHODS: We analyzed the relationship between the rs9387478 single nucleotide polymorphism and i) clinical parameters and ii) overall survival time in 505 female nonsmoking lung cancer patients, using the chi-square test and Kaplan-Meier analysis with the log-rank test, respectively. We further established the epidermal growth factor receptor (EGFR) mutation status and assessed its association with rs9387478 genotypes as well as the efficacy of EGFR tyrosine kinase inhibitors. RESULTS: The frequency of the AA genotype was significantly higher in the EGFR-mutation-negative group than in the EGFR-mutation-positive group (32% vs. 16%, χ2 = 13.025, p = 0.011). Patients with the CC genotype had a better overall survival time than patients with the AA/AC genotype (median survival time: 54.2 vs. 32.9 months, χ2 = 4.593, p = 0.032). The distribution of rs9387478 genotypes differed according to the clinical disease stage. CONCLUSIONS: This study indicates that the rs9387478 genotype was associated with overall survival in nonsmoking female patients with lung cancer, although it was not significant after adjusting for multiple testing. The identification of the location of the rs9387478 single nucleotide polymorphism in the genomic interval containing the DCBLD1 and ROS1 genes, together with the finding that the rs9387478 polymorphism correlates with EGFR mutation status, may have important implications for therapeutic approaches targeting EGFR or ROS1 in patients with lung cancer.

Integrative Analyses of Lung Squamous Cell Carcinoma in Ten Chinese Patients with Transcriptome Sequencing.

Few effective therapies have been developed for the treatment of lung squamous cell carcinoma (SQCC), in part due to a lack of understanding regarding the mechanisms underlying the initiation and development of this disease. Whole transcriptome sequencing not only provides insight into the expression of all transcribed genes, but offers an efficient approach for identifying genetic variations, including gene fusions, mutations and alternative splicing. In this study, we performed whole transcriptome sequencing of 10 patients with stage IIIA lung SQCC, and discovered a large number of single nucleotide variants (SNVs; mean of 12.2 SNVs/Mb), with C>T/G>A and A>G/T>C transitions being the most frequently observed. Additionally, a total of 132 gene fusions were identified based upon TopHat alignments, 70.5% (93/132) of which occurred as a result of intra-chromosomal rearrangements. Based on the number of supporting reads for each fusion, we further validated 20 of the 26 top gene fusions by RT-PCR and Sanger sequencing. Taken together, these data provide an in-depth view of transcriptional alterations in lung SQCC patients, and may be useful for identification of new therapeutic targets.