EGFR-mutant NSCLC may remodel TME from non-inflamed to inflamed through acquiring resistance to EGFR-TKI treatment.

BACKGROUND: EGFR-TKI represent the standard first-line therapy for advanced NSCLC harboring EGFR mutations. However, resistance to EGFR-TKI inevitably develops in nearly all patients. Previous clinical study have demonstrated that, some patients that failed EGFR-TKI therapy show a benefit outcome from immunotherapy. Our objective is to explore the immune microenviroment remodeling induced by EGFR-TKI treatment in EGFR mutant lung cancer patients and to investigate the immune cell types and potential molecular signatures involved. METHODS: A cohort of 37 EGFR mutant advanced-stage NSCLC patients, who are resistant to at least one type of TKI treatment, was retrospectively established. Both pre-treatment and TKI resistance tumor FFPE samples of each pairs were collected. Transcriptional profiling and bioinformatics analysis were employed to evaluate the change of immune associated hallmarks before and after EGFR-TKI therapy. RESULTS: Tumor samples after EGFR-TKI treatment displayed enrichment of proinflammatory signaling like interferon-γ, allograft rejection and inflammatory response. Of note, cytotoxic factor granzyme A as well as PD-L1 were found to be more expressed in EGFR-TKI resistance samples. Approximately 33.3 % (11/33) of EGFR-TKI treated samples were classified as "hot" tumor, especially for EGFR L858R mutated NSCLC patients (46.7 %,7/15). Effector cells were significantly overexpressed in 'hot' tumors feature following TKI resistance. In addition, we found that four effector genes (CD8A, CDB8, GZMB, GZMK) showed higher expression in 'hot' tumors post-TKI resistance, and its 4-gene effector cell signature was found to have a good correlation with survival benefit in external immunotherapy database. CONCLUSIONS: TKI treatment may initiate immune activation in EGFR mutant NSCLC, leading to changes in immune cell infiltration following TKI resistance. We mechanistically explored that this might be due to an increased immune response caused by the rise in effector cells post-TKI resistance.

Sputum cell-free DNA: Valued surrogate sample for the detection of EGFR exon 20 p.T790M mutation in patients with advanced lung adenocarcinoma and acquired resistance to EGFR-TKIs.

BACKGROUND: Sputum cell-free DNA (cfDNA) is a valuable surrogate sample for assessing EGFR-sensitizing mutations in patients with advanced lung adenocarcinoma. Detecting EGFR exon 20 p.T790 M (p.T790 M) is much more challenging due to its limited availability in tumor tissues. Exploring sputum cfDNA as an alternative for liquid-based sample type in detecting p.T790 M requires potential improvement in clinical practice. METHODS: A total of 34 patients with EGFR-sensitive mutation-positive lung adenocarcinoma and acquired resistance to the first generation of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were enrolled. The sputum samples, and paired tumors and/or plasma samples were tested for p.T790 M mutation and concordance of p.T790 M status among the three sample types was analyzed. RESULTS: The overall concordance rate of p.T790 M mutation between sputum cfDNA and tumor tissue samples was 85.7%, with a sensitivity of 66.7% and a specificity of 100%. The sensitivity for detecting p.T790 M in sputum cfDNA was 100%, 66.7%, and 0% in the three sputum groups of malignant, satisfactory but no malignant cells, and unsatisfactory, respectively. The combined results of plasma cfDNA testing and sputum cfDNA testing further increased the sensitivity to 100% for p.T790 M detection in satisfactory but no malignant cells sputum group. CONCLUSION: These findings revealed that cfDNA from malignant or satisfied but no malignant cells sputum is considered suitable for detecting p.T790 M mutation in patients with acquired resistance to first or second-generation EGFR-TKIs. The sputum cytological pathological evaluation-guided sputum cfDNA testing assists in significantly improving the sensitivity of p.T790 M detection, bringing significant value for the maximal application of third-generation EGFR-TKIs in second-line treatment.

Multigene PCR using both cfDNA and cfRNA in the supernatant of pleural effusion achieves accurate and rapid detection of mutations and fusions of driver genes in patients with advanced NSCLC.

BACKGROUND: Pleural effusion from patients with advanced non-small cell lung cancer (NSCLC) has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. METHODS: In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit. RESULTS: Mutations in EGFR, KRAS, and HER2 were detected in DNA and cfDNA, fusions in ALK was detected in RNA and cfRNA. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5% and 92.3%) with both 100% for fusions. CONCLUSIONS: CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR-based multigene detection kit. Pleural effusion supernatant seems a preferred material for detection of multigene alterations to guide treatment decision of advanced NSCLC.

Refined Stratification Based on Baseline Concomitant Mutations and Longitudinal Circulating Tumor DNA Monitoring in Advanced EGFR-Mutant Lung Adenocarcinoma Under Gefitinib Treatment.

INTRODUCTION: The optimal treatment for EGFR-mutant lung adenocarcinoma (LUAD) remains challenging because of intratumor heterogeneity. We aimed to explore a refined stratification model based on the integrated analysis of circulating tumor DNA (ctDNA) tracking. METHODS: ctDNA was prospectively collected at baseline and at every 8 weeks in patients with advanced treatment-naive EGFR-mutant LUAD under gefitinib treatment enrolled in a phase 2 trial and analyzed using next-generation sequencing of a 168-gene panel. RESULTS: Three subgroups categorized by baseline comutations-EGFR-sensitizing mutations (59, 32.8%), EGFR-sensitizing mutations with tumor suppressor mutations (97, 53.9%), and EGFR-sensitizing mutations with other driver mutations (24, 13.3%)-exhibited distinct progression-free survival (13.2 [11.3-15.2] versus 9.3 [7.6-10.5] versus 4.0 [2.4-9.3] months) and overall survival (32.0 [29.2-41.5] versus 21.7 [19.3-27.0] versus 15.5 [10.5-33.7] months, respectively), providing evidence for initial stratification. A total of 63.7% of the patients achieved week 8 ctDNA clearance, with significant difference noted among the three subgroups (74.5% versus 64.0% versus 29.4%, respectively, p = 0.004, Fisher's exact test). Patients without week 8 ctDNA clearance had worse progression-free survival (clearance versus nonclearance 11.2 [9.9-13.2] versus 7.4 [5.6-9.6] months, p = 0.016, Cox regression], especially in the second subgroup [5.8 (5.6-11.5) months], suggesting the necessity of adaptive stratification during treatment. During follow-up, 56.0% and 20.8% of the patients eventually harbored p.T790M and non-p.T790M mutations, respectively, with a significant difference in non-p.T790M mutations among the three subgroups (7.5% versus 15.7% versus 80.0%, respectively, p < 0.001, Fisher's exact test), giving clues to postline treatment. CONCLUSIONS: The patients with baseline comutations and ctDNA nonclearance at first visit might require combined therapy because of the limited survival benefit of EGFR tyrosine kinase inhibitor monotherapy. We proposed a refined stratification mode for the whole-course management of EGFR-mutant LUAD.

Sputum Cell-Free DNA: Valued Surrogate Sample for Detection of EGFR Mutation in Patients with Advanced Lung Adenocarcinoma.

Sputum is a common cytologic sample type, but its potential use in EGFR mutation detection in patients with lung cancer is not fully evaluated. This study established an improved sputum cell-free DNA (cfDNA) extraction method study and applied a super-amplification refractory mutation system to detect the EGFR mutation status in sputum cfDNA. The sputum sediments were used for cytology evaluation. The study included 102 lung adenocarcinoma patients; 65 patients (63.7%) were positive for EGFR mutations in tumor samples. EGFR mutation status was positive in 30 patients (29.4%) by sputum cfDNA testing, achieving an overall sensitivity and specificity of 46.2% and 100%, respectively. Comparison of EGFR mutation status in tumor samples revealed that the sensitivity of testing sputum cfDNA in 40 patients with stage I to IIIA versus 62 patients with stage IIIB to IV was 24% (6/25) versus 65.0% (26/40). Through cytology evaluation, the sputum specimens from 62 advanced patients were classified into three categories: 10 were unsatisfactory; 34 were satisfactory but had no malignant cells; and 18 had malignant cells. The sensitivities of these three categories were 0% (0/8), 71.4% (15/21), and 100% (11/11), respectively. These findings revealed that with the improved cfDNA extraction method and sputum cytology evaluation, sputum cfDNA is a valuable surrogate sample type for detecting clinical EGFR mutations in advanced lung adenocarcinoma patients.

Detection of EGFR mutations in plasma circulating tumour DNA as a selection criterion for first-line gefitinib treatment in patients with advanced lung adenocarcinoma (BENEFIT): a phase 2, single-arm, multicentre clinical trial.

BACKGROUND: Detection of EGFR mutations in tumour tissue is the gold-standard approach to ascertain if a patient will benefit from treatment with an EGFR tyrosine kinase inhibitor. However, if tissue is scant, another strategy is to use circulating tumour DNA (ctDNA), but this method needs validation in clinical trials. We did a prospective clinical trial to assess ctDNA-based EGFR mutation detection as a selection criterion for patients with lung adenocarcinoma receiving gefitinib as first-line treatment. METHODS: BENEFIT is a multicentre, single-arm, phase 2 clinical trial at 15 centres in China. Patients aged 18-75 years with stage IV metastatic lung adenocarcinoma and EGFR mutations detected in ctDNA were given oral gefitinib 250 mg once daily as first-line treatment. The primary endpoint was the proportion achieving an objective response. Secondary endpoints included median progression-free survival and safety. Next-generation sequencing (NGS) of a 168-gene panel was used for genetic analysis of baseline blood samples. The primary efficacy analysis was done by intention to treat in patients who had at least one post-baseline tumour assessment. The safety analysis was done in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02282267. FINDINGS: Between Dec 25, 2014, and Jan 16, 2016, 426 patients were screened for the trial, of whom 188 with EGFR mutations in ctDNA were enrolled and received gefitinib. 183 patients had one or more post-baseline tumour assessment and were included in the primary efficacy analysis. Median follow-up was 14·5 months (IQR 12·2-16·5). At the time of data cutoff (Jan 31, 2017), 152 patients had progressive disease or had died. The proportion achieving an objective response was 72·1% (95% CI 65·0-78·5). Median progression-free survival was 9·5 months (95% CI 9·07-11·04). Of 167 patients with available blood samples, 147 (88%) showed clearance of EGFR mutations in ctDNA at week 8, and median progression-free survival was longer for these patients than for the 20 patients whose EGFR mutations persisted at week 8 (11·0 months [95% CI 9·43-12·85] vs 2·1 months [1·81-3·65]; hazard ratio [HR] 0·14, 95% CI 0·08-0·23; p<0·0001). From baseline NGS data in 179 patients, we identified three subgroups of patients: those with EGFR mutations only (n=58), those with mutations in EGFR and tumour-suppressor genes (n=97), and those with mutations in EGFR and oncogenes (n=24). Corresponding median progression-free survival in these subgroups was 13·2 months (95% CI 11·5-15·0), 9·3 months (7·6-11·0), and 4·7 months (1·9-9·3), respectively (EGFR mutations only vs mutations in EGFR and tumour-suppressor genes, HR 1·78, 95% CI 1·23-2·58; p=0·002; EGFR mutations only vs mutations in EGFR and oncogenes, 2·66, 1·58-4·49; p=0·0003). The most common grade 3 or 4 adverse events were hepatic function abnormalities (n=24). Serious adverse events were reported in 17 (9%) patients. No unexpected safety events for gefitinib were recorded. INTERPRETATION: Detection of EGFR mutations in ctDNA is an effective method to identify patients who might benefit from first-line gefitinib treatment. Further analyses of dynamic alterations of EGFR mutations and accompanying gene aberrances could predict resistance to gefitinib. FUNDING: Guangdong Association of Clinical Trials, AstraZeneca, National Natural Sciences Foundation Key Programme, and National Key Research and Development Programme of China.

Potential Resistance Mechanisms Revealed by Targeted Sequencing from Lung Adenocarcinoma Patients with Primary Resistance to Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors (TKIs).

INTRODUCTION: EGFR tyrosine kinase inhibitors (TKIs) have greatly improved the prognosis of lung adenocarcinoma. However, approximately 5% to 10% of patients with lung adenocarcinoma with EGFR sensitive mutations have primary resistance to EGFR TKI treatment. The underlying mechanism is unknown. METHODS: This study used next-generation sequencing to explore the mechanisms of primary resistance by analyzing 11 patients with primary resistance and 11 patients sensitive to EGFR TKIs. Next-generation targeted sequencing was performed on the Illumina X platform for 483 cancer-related genes. EGFR mutation was initially detected using the amplification refractory mutation system. RESULTS: Potential primary resistance mechanisms were revealed by mutations unique to the EGFR TKI resistance group. Among the 11 resistant patients, 45% (five of 11) harbored a known resistance mechanism, such as MNNG HOS Transforming gene (MET) amplification de novo T790M mutation or overlapping T790M and phosphatase and tensin homolog gene (PTEN) loss and erb-b2 receptor tyrosine kinase 2 gene (ERBB2) amplification. In six of 11 resistant cases (54%), potential novel mutations that might lead to drug resistance were identified (including transforming growth factor beta receptor 1 gene [TGFBR1] mutation and/or EGFR structural rearrangement mechanistic target of rapamycin kinase gene [MTOR] mutation, transmembrane protease, serine 2 gene [TMPRSS2] fusion gene, and v-myc avian myelocytomatosis viral oncogene homolog gene [MYC] amplification). By analyzing somatic mutation patterns, the frequency of C:G→T:A transitions in the patients with primary resistance was significantly higher than that in sensitive group and occurred more frequently in the non-CpG region (Cp(A/C/T)→T). CONCLUSION: The mechanisms of primary resistance to EGFR TKIs may be highly heterogeneous. Mutations in EGFR and its downstream pathway, as well as mutations that affect tumor cell function, are related to primary resistance. Somatic single-nucleotide mutation patterns might be associated with primary resistance to EGFR TKIs.

A consensus on liquid biopsy from the 2016 Chinese Lung Cancer Summit expert panel.

The diagnosis and treatment of lung cancer have evolved into the era of precision medicine. Liquid biopsy, a minimally invasive approach, has emerged as a promising practice in genetic profiling and monitoring of lung cancer. Translating liquid biopsy from bench to bedside has encountered various challenges, including technique selection, protocol standardisation, data analysis and cost management. Regarding these challenges, the 2016 Chinese Lung Cancer Summit expert panel organised a trilateral forum involving oncologists, clinicians, clinical researchers, and industrial expertise on the 13th Chinese Lung Cancer Summit to formally discuss these controversies. Six consensuses were reached to guide the use of liquid biopsy and perform precision medicine in both clinic and research.

Droplet digital PCR improved the EGFR mutation diagnosis with pleural fluid samples in non-small-cell lung cancer patients.

BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of nucleic acid. However, its application has not been reported in pleural fluid, which is an ideal sample source for epidermal growth factor receptor (EGFR) mutation analysis in non-small-cell lung cancer (NSCLC) patients. METHODS: The extracted DNA from supernatants of pleural fluid was selected from our sample bank and re-analyzed by our previously established ddPCR assay. The results were compared with the former outcomes detected by direct sequencing or the amplification-refractory mutation system (ARMS). RESULTS: A total of 95 samples were selected, and 64 and 31 of them had been performed with direct sequencing and ARMS tests, respectively. The EGFR mutation detection rate of ddPCR was significantly elevated, compared with both direct sequencing (75.4% vs. 43.8%, P<0.0001) and ARMS (61.3% vs. 38.7%, P=0.016). Compared with ARMS, Fisher's exact test showed that EGFR-positive patients who were redefined by ddPCR had higher objective response rates (ORRs): 57.9% vs. 16.7%, P=0.032. Compared with direct sequencing results, Kaplan-Meier curves demonstrated that EGFR-positive patients who were redefined by ddPCR had longer progression-free survival (PFS): 8.0 vs. 2.0months, P=0.0001. CONCLUSION: We have demonstrated the clinical value of ddPCR in pleural fluid samples. The experience obtained from the present study is practical and favorable for the proper application of this new assay.

EGFR mutation status of paired cerebrospinal fluid and plasma samples in EGFR mutant non-small cell lung cancer with leptomeningeal metastases.

PURPOSE: Central nervous system (CNS) is the prevalent site for metastases in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-relapsed NSCLC patients. To understand the EGFR mutation status in paired cerebrospinal fluid (CSF) and plasma samples after EGFR-TKI treatment failure might be useful to guide the treatment of intra- and extracranial tumors in those patients. METHODS: Paired CSF and plasma samples were collected from seven NSCLC patients with CNS metastases after EGFR-TKI failure. EGFR mutations were tested by amplification refractory mutation system (ARMS) and droplet digital PCR (ddPCR) methods. Gefitinib concentrations were evaluated by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). RESULTS: EGFR mutations were detected in all seven CSF samples, including three of E19-Del, three of L858R and one of E19-Del&T790M by both methods. On the other hand, majority of the matched plasma samples (5/7) were negative for EGFR mutations by both methods. The other two plasma samples were positive for E19-Del&T790M by ddPCR, and one of them had undetectable T790M by ARMS. Gefitinib concentration in CSF was much lower than that in plasma (mean CSF/plasma ratio: 1.8 %). CONCLUSIONS: After EGFR-TKI failure, majority of the NSCLC patients with CNS metastases remained positive detection of EGFR sensitive mutations in CSF, but much less detection in the matched plasma. Significantly low exposure of gefitinib in CSF might explain the intracranial protection of the EGFR sensitive mutation positive tumor cells.

Recurrent amplification of MYC and TNFRSF11B in 8q24 is associated with poor survival in patients with gastric cancer.

BACKGROUND: Gastric cancer (GC) is an aggressive malignancy whose mechanisms of development and progression are poorly understood. The identification of prognosis-related genomic loci and genes may suffer from the relatively small case numbers and a lack of systematic validation in previous studies. METHODS: Array-based comparative genomic hybridization (aCGH) coupled with patient clinical information was applied to identify prognosis-related loci and genes with high-frequency recurrent gains in 129 GC patients. The candidate loci and genes were then validated using an independent cohort of 384 patients through branched DNA signal amplification analysis (QuantiGene assays). RESULTS: In the 129 patients, a copy number gain of three chromosome regions-namely, 8q22 (including ESRP1 and CCNE2), 8q24 (including MYC and TNFRSF11B), and 20q11-q13 (including SRC, MMP9, and CSE1L)--conferred poor survival for patients. In addition, the correlation between the branched DNA signal amplification analysis results and the aCGH results was analyzed in 73 of these 129 patients, and MYC, TNFRSF11B, ESRP1, CSE1L, and MMP9 were found to be well correlated. Further validation using an independent cohort (n = 384) verified that only MYC and TNFRSF11B within 8q24 are related to survival. Patients with gains in both MYC and TNFRSF11B had poorer survival than those with no gains, particularly those with noncardia GC. Gains in both of these genes were also a significant independent prognostic indicator. CONCLUSIONS: Our results revealed that copy number gains in MYC and TNFRSF11B located at 8q24 are associated with survival in GC, particularly noncardia GC.

Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma.

BACKGROUND: MAPK7/ERK5 (extracellular-signal-regulated kinase 5) functions within a canonical three-tiered MAPK (mitogen activated protein kinase) signaling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself. Despite being the least well studied of the MAPK-modules, evidence supports a role for MAPK7-signaling in the pathology of several cancer types. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) analysis identified MAPK7 gene amplification in 4% (3/74) of non-small cell lung cancers (NSCLC) (enriched to 6% (3/49) in squamous cell carcinoma) and 2% (2/95) of squamous esophageal cancers (sqEC). Immunohistochemical (IHC) analysis revealed a good correlation between MAPK7 gene amplification and protein expression. MAPK7 was validated as a proliferative oncogenic driver by performing in vitro siRNA knockdown of MAPK7 in tumor cell lines. Finally, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. Phosphorylation antibody microarray analysis also identified novel downstream pharmacodynamic (PD) biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2D). CONCLUSIONS: Together, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade.

Identification of HBV-MLL4 Integration and Its Molecular Basis in Chinese Hepatocellular Carcinoma.

To gain molecular insights of HBV integration that may contribute to HCC tumorigenesis, we performed whole transcriptome sequencing and whole genome copy number profiling of hepatocellular carcinoma (HCC) samples from 50 Chinese patients. We identified a total of 33 HBV-human integration sites in 16 of 44 HBV-positive HCC tissues, which were enriched in HBV genotype C-infected patients. In addition, significantly recurrent HBV-MLL4 integration (18%; 8/44) was found in this cohort of patients. Using long-range PCR and Sanger sequencing, we comprehensively characterized gDNA and cDNA sequences that encode for the HBV-MLL4 transcripts, and we revealed that HBV integration into MLL4 exons led to much higher mRNA expression of MLL4 than the integration into MLL4 introns due to an alternative splicing mechanism. Moreover, the HBV-MLL4 integration occurred almost exclusively in CTNNB1 and TP53 wild-type patients. The integration was also associated with a distinct gene expression profile. In conclusion, this is the first report on the molecular basis of the MLL4 integration driving MLL4 over-expression. HBV-MLL4 integration occurred frequently in Chinese HCC patients, representing a unique molecular segment for HCC with HBV infection.

Oncogenic HER2 fusions in gastric cancer.

BACKGROUND: Genetic amplification of HER2 drives tumorigenesis and cancer progression in a subset of patients with gastric cancer (GC), and treatment with trastuzumab, a humanized HER2-neutralizing antibody, improves the overall survival rate of HER2-positive patients. However, a considerable portion of the patients does not respond to trastuzumab and the molecular mechanisms underlying the intrinsic resistance to anti-HER2 therapy in GC is not fully understood. METHODS: We performed whole-transcriptome sequencing on 21 HER2-positive tumor specimens from Chinese GC patients. Whole genome sequencing was performed on the three samples with HER2 fusion to discover the DNA integration structure. A multicolor FISH assay for HER2 split screening was conducted to confirm HER2 fusion and IHC (HercepTest™) was used to detect the membranous expression of HER2. Fusion cDNA were transfected into NIH/3T3 cells and generate stable cell line by lentivirus. The expression of exogenous HER2 fusion proteins and pHER2 were examined by western blot analysis. In vitro efficacy studies were also conducted by PD assay and softagar assay in cell line expression wild type and fusion HER2. T-DM1 was used to assess its binding to NIH/3T3 cells ectopically expressing wild-type and fusion HER2. Finally, the anti-tumor efficacy of trastuzumab was tested in NIH/3 T3 xenografts expressing the HER2 fusion variants. RESULTS: We identified three new HER2 fusions with ZNF207, MDK, or NOS2 in 21 HER2-amplified GC samples (14%; 3/21). Two of the fusions, ZNF207-HER2, and MDK-HER2, which are oncogenic, lead to aberrant activation of HER2 kinase. Treatment with trastuzumab inhibited tumor growth significantly in xenografts expressing MDK-HER2 fusion. In contrast, trastuzumab had no effect on the growth of xenografts expressing ZNF207-HER2 fusion, due to its inability to bind to trastuzumab. CONCLUSIONS: Our results provide the molecular basis of a novel resistance mechanism to trastuzumab-based anti-HER2 therapy, supporting additional molecule stratification within HER2-positive GC patients for more effective therapy options.

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell-Free DNA from Patients with Advanced Non-Small Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing.

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma.

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break-apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break-apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break-apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.

Whole genome gene copy number profiling of gastric cancer identifies PAK1 and KRAS gene amplification as therapy targets.

Gastric cancer is the second leading cause of death from cancer worldwide, with an approximately 20% 5-year survival rate. To identify molecular subtypes associated with the clinical prognosis, in addition to genetic aberrations for potential targeted therapeutics, we conducted a comprehensive whole-genome analysis of 131 Chinese gastric cancer tissue specimens using whole-genome array comparative genomic hybridization. The analyses revealed gene focal amplifications, including CTSB, PRKCI, PAK1, STARD13, KRAS, and ABCC4, in addition to ERBB2, FGFR2, and MET. The growth of PAK1-amplified gastric cancer cells in vitro and in vivo was inhibited when the corresponding mRNA was knocked down. Furthermore, both KRAS amplification and KRAS mutation were identified in the gastric cancer specimens. KRAS amplification was associated with worse clinical outcomes, and the KRAS gene mutation predicted sensitivity to the MEK1/2 inhibitor AZD6244 in gastric cancer cell lines. In summary, amplified PAK1, as well as KRAS amplification/mutation, may represent unique opportunities for developing targeted therapeutics for the treatment of gastric cancer.

Malignant pleural effusion supernatants are substitutes for metastatic pleural tumor tissues in EGFR mutation test in patients with advanced lung adenocarcinoma.

BACKGROUND: Though the possibility of using malignant pleural effusions (MPEs) as alternatives for metastatic pleural tumor tissues (MPTTs) in epidermal growth factor receptor (EGFR) mutation test has been examined, due to the lack of studies comparing the results in matching MPEs and MPTTs, the clinical value of MPEs for advanced adenocarcinoma patients with pleural effusions is not confirmed. METHODS: EGFR mutation statuses in matching MPTTs, MPE supernatants and cell blocks, of 41 patients with advanced lung adenocarcinoma as diagnosed by thoracoscopy were analyzed using amplification refractory mutation system (ARMS). RESULTS: EGFR mutations were detected in 46.3% (19/41) of MPTTs, 43.9% (18/41) of MPE supernatants and 56.3% (18/32) of MPE cell blocks by ARMS analysis. Generally, the same EGFR statuses were identified in both MPTTs and matching MPE cell blocks of 81.3% patients (26/32), whereas MPTTs and matching MPE supernatants of 87.8% (36/41) patients shared the same EGFR status. Compared with EGFR mutation detection in MPTTs, the sensitivity of EGFR mutation detection in MPE-cell blocks was 87.5% (14/16), specificity was 75.0% (12/16), while the sensitivity of EGFR mutation detection in MPE-supernatants was 84.2% (16/19), specificity was 90.9% (20/22). CONCLUSIONS: The high concordance of EGFR mutation statuses between MPEs and MPTTs in lung adenocarcinoma patients with pleural metastasis as determined by ARMS analysis suggests that MPEs, particularly MPE supernatants, may be substitutes for MPTTs in EGFR mutation test.

The use of mutation-specific antibodies in predicting the effect of EGFR-TKIs in patients with non-small-cell lung cancer.

PURPOSE: We aimed to quantify the epidermal growth factor receptor (EGFR) mutation in tumors and to analyze its prediction of EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment efficacy in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients. METHODS: We examined EGFR mutation status in 124 lung cancer samples by direct sequencing and amplification refractory mutation system. Among them, 41 were appropriate to quantify the expression of mutant EGFR proteins using immunohistochemistry (IHC) with mutation-specific antibodies. The quantification was determined by both the staining intensity and the proportion of stained tumor cells. RESULTS: The median progression-free survival (PFS) in patients with a high score for mutant EGFR expression was 18.0 months (95 % CI 16.0-20.0), which was significantly longer than that in patients with a low score (8.0 months; 95 % CI 2.6-13.4; P = 0.048). Such significant association with patients' PFS was also apparent in the proportion of stained tumor cells (median, 19.0 vs. 8.0 months; P = 0.019), but not in the staining intensity (P = 0.787). Among the 41 specimens, 32 were detected EGFR mutation positive by both direct sequencing and ARMS, referring to a relatively high abundance of mutation, and 26 (81.3 %) of them gained a high expression score of mutant proteins as well. Six samples with mutation negative by direct sequencing but positive by ARMS, which showed a low abundance, and 5 (83.3 %) of them also revealed a low expression score. The EGFR mutation quantitative analysis using mutation-specific IHC was moderately consistent with that by molecular-based assays (P = 0.001, kappa value 0.50). CONCLUSIONS: Our results suggest that immunohistochemical analysis with mutation-specific antibodies is a promising approach for quantifying EGFR mutations, and may predict the effect of EGFR-TKI treatment for EGFR mutation-positive NSCLC.