UV-ARTP compound mutagenesis breeding improves macrolactins production of Bacillus siamensis and reveals metabolism changes by proteomic.

Macrolactins are a type of compound with complex macrolide structure which mainly be obtained through microbiological fermentation now. They have excellent antifungal, antibacterial and antitumor activity. In order to improve macrolactins production, Bacillus siamensis YB304 was used as the research object, and a mutant Mut-K53 with stable genetic characters was selected by UV-ARTP compound mutagenesis. The yield of macrolactins was 156.46 mg/L, 3.95 times higher than original strain. The metabolic pathway changes and regulatory mechanism of macrolactins were analyzed by quantitative proteomics combined with parallel reaction monitoring. This study revealed that 1794 proteins were extracted from strain YB304 and strain Mut-K53, most of them were related to metabolism. After UV-ARTP compound mutagenesis treatment, the expression of 628 proteins were significantly changed, of which 299 proteins were significantly up-regulated. KEGG pathway analysis showed that differentially expression proteins mainly distributed in biological process, cellular component, and molecular function processing pathways. Such as utilization of carbon sources, glycolysis pathway, and amino acid metabolism pathway. Furthermore, key precursor substances such as acyl-CoA and amino acids of macrolactin biosynthesis are mostly up-regulated, which are one of the main reasons for increased production of macrolactin.This study will provide a new way to increase the yield of macrolactins through mutagenesis breeding and proteomics.

Programmed cell death ligand 1 protein levels predicted survival of non-small cell lung cancer.

Objective: To investigate the relationship between programmed death ligand 1 (PD-L1) expression using 5%, 25%, 50% cutoffs in tumor cells (TC) and postsurgical survival in non-small-cell lung cancer (NSCLC) patients. For samples with tumor infiltrating lymphocytes (TIL), correlation between PD-L1 expression in TIL using 1% cutoff and postsurgical survival was also evaluated. Methods: Primary NSCLC tumor surgical samples staging I to IIIA of 126 patients who underwent surgical procedures from September 2009 to August 2012 in Shanghai Chest Hospital, Shanghai Jiao Tong University were retrospectively included. PD-L1 protein expression was detected by immunohistochemistry (IHC) assays. A rabbit anti-human PD-L1 (E1L3N) monoclonal antibody (1:300, CST#13684, Cell Signaling Technology) was used for PD-L1 IHC staining. PD-L1 expression was evaluated both on TC and TIL. Univariate and multivariate analyses for postsurgical survival were done using Kaplan-Meier and Cox regression model, respectively. Results: The median postsurgical survival for all patients was 44.1 months [95% confidence interval (CI): 33.9-70.0 months). The median postsurgical survival for PD-L1 expression percentage 0, 1-50% and ≥50% were 51.9 months (95%CI: 33.9-70.0 months), 33.2 months (95%CI: 20.8-45.6 months) and 14.7 months (95%CI: 1.9-27.6 months), respectively (P = 0.002). Clinical stage and PD-L1 expression in TC (25% cutoff or 50% cutoff values) were found to be independent predictors for longer postsurgical survival in all cohort. Ninety (71.4%) of the 126 samples were identified to concurrent TIL. The median postsurgical survival time was 39.6 months (95% CI: 31.8-47.4 months) in patients with TIL. PD-L1 expression in TC (25% cutoff or 50% cutoff values) was found to be the independent predictor for longer postsurgical survival time in patients with TIL. Conclusion: PD-L1 negative expression in TC at 25% or 50% cutoff values was the independent predictor for longer postsurgical survival time in both NSCLC samples and NSCLC samples with TIL. For patients with PD-L1 high expression at 25% or 50% cutoff values, PD-L1 blocking may be considered.

PD-L1 expression and its relationship with oncogenic drivers in non-small cell lung cancer (NSCLC).

In order to explore the potential patient population who could benefit from anti PD-1/PD-L1 mono or combination therapies, this study aimed to profile a panel of immunotherapy related biomarkers (PD-1, PD-L1, CTLA-4 and CD8) and targeted therapy biomarkers (EGFR, KRAS, ALK, ROS1 and MET) in NSCLC.Tumor samples from 297 NSCLC patients, including 156 adenocarcinomas (AD) and 129 squamous cell carcinomas (SCC), were analyzed using immunohistochemistry, immunofluorescence, sequencing and fluorescence in situ hybridization.43.1% of NSCLC patients had PD-L1 positive staining on ≥ 5% tumor cells (TC). Furthermore, dual color immunofluorescence revealed that the majority of PD-L1/CD8 dual positive tumor infiltrating lymphocytes (TIL) had infiltrated into the tumor core. Finally, combined analysis of all eight biomarkers showed that tumor PD-L1 positivity overlapped with known alterations in NSCLC oncogenic tumor drivers in 26% of SCC and 76% of AD samples.Our illustration of the eight biomarkers' overlap provides an intuitive overview of NSCLC for personalized therapeutic strategies using anti-PD-1/PD-L1 immune therapies, either as single agents, or in combination with targeted therapies. For the first time, we also report that PD-L1 and CD8 dual positive TILs are predominantly located within the tumor core.

Prevalence and Prognostic Role of BRCA1/2 Variants in Unselected Chinese Breast Cancer Patients.

BACKGROUND: The prevalence of BRCA1/2 variants in Chinese breast cancer patients varies among studies. Germline or somatic BRCA1/2 mutations are associated with sensitivity to poly(ADP-ribose) polymerase-1 inhibitors and DNA-damaging agents. We aimed to investigate the distribution of both somatic and germline BRCA1/2 variants in unselected Chinese breast cancer patients, and explore their roles in tumor phenotype and disease prognosis. METHODS: 507 breast cancer patients, unselected for family history of breast cancer or age at diagnosis, were prospectively enrolled from West China Hospital between Feb. 2008 and Feb. 2014. BRCA1/2 variants in the exons/flanking regions were detected in fresh-frozen tumors using next-generation sequencing and confirmed by independent methods. Germline/somatic status was validated by Sanger sequencing in paired blood/normal tissue. RESULTS: BRCA1/2 pathogenic or likely pathogenic (P/LP) variants were detected in 50 patients (9.9%), including 40 germline carriers (18 in BRCA1, 22 in BRCA2), 9 patients with somatic variants (3 in BRCA1, 6 in BRCA2), and 1 patient with concurrent germline/somatic variants in BRCA2. The triple-negative (21.4%) and Luminal B (9.7%) subtypes had higher rates of BRCA1/2 variants. In patients with disease stage 0~II, presence of a germline or somatic BRCA1 P/LP variant increased the risk of relapse as compared to non-carriers [univariate hazard ratio (HR): 3.70, P = 0.04]. Germline BRCA1 P/LP variants, which were associated with aggressive tumor phenotypes, predicted worse disease-free survival in the subgroup of stage 0~II (HR: 4.52, P = 0.02) and N0 (HR: 5.4, P = 0.04) compared to non-carriers. CONCLUSION: A high frequency of germline and somatic BRCA1/2 P/LP variants was detected in unselected Chinese breast cancer patients. Luminal B subtype should be considered as a high-risk population of BRCA1/2 mutation, in addition to triple-negative breast cancer. BRCA1 status was associated with aggressive tumor phenotype and worse disease progression in early stage breast cancer patients.

Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Relationships between Chromosome 7 Gain, MET Gene Copy Number Increase and MET Protein Overexpression in Chinese Papillary Renal Cell Carcinoma Patients.

To investigate the relationships between Chromosome 7 gain, mesenchymal-epithelial transition factor (MET) gene copy number increase and MET protein overexpression in Chinese patients with papillary renal cell carcinoma (PRCC), immunohistochemistry (IHC), immunofluorescence (IF) and fluorescence in situ hybridization (FISH) were performed on 98 formalin-fixed, paraffin-embedded (FFPE) PRCC samples. Correlations between MET gene copy number increase, Chromosome 7 gain and MET protein overexpression were analyzed statistically. A highly significant correlation was observed between the percentage of tumor cells with MET gene copy number ≥3 and CEP7 copy number ≥3 (R2 = 0.90, p<0.001) across two subtypes of PRCC. In addition, the percentage of tumor cells with MET gene copy number ≥3 was found to increase along with increases in MET IHC score. This correlation was further confirmed in those PRCC tumor cells with average MET gene copy number >5 using combined IF and FISH methodology. Overall, this study provides evidence that Chromosome 7 gain drives MET gene copy number increase in PRCC tumors, and appears to subsequently lead to an increase in MET protein overexpression in these tumor cells. This supports MET activation as a potential therapeutic target in sporadic PRCC.

Intra-Tumoral Heterogeneity of HER2, FGFR2, cMET and ATM in Gastric Cancer: Optimizing Personalized Healthcare through Innovative Pathological and Statistical Analysis.

Current drug development efforts on gastric cancer are directed against several molecular targets driving the growth of this neoplasm. Intra-tumoral biomarker heterogeneity however, commonly observed in gastric cancer, could lead to biased selection of patients. MET, ATM, FGFR2, and HER2 were profiled on gastric cancer biopsy samples. An innovative pathological assessment was performed through scoring of individual biopsies against whole biopsies from a single patient to enable heterogeneity evaluation. Following this, false negative risks for each biomarker were estimated in silico. 166 gastric cancer cases with multiple biopsies from single patients were collected from Shanghai Renji Hospital. Following pre-set criteria, 56 ~ 78% cases showed low, 15 ~ 35% showed medium and 0 ~ 11% showed high heterogeneity within the biomarkers profiled. If 3 biopsies were collected from a single patient, the false negative risk for detection of the biomarkers was close to 5% (exception for FGFR2: 12.2%). When 6 biopsies were collected, the false negative risk approached 0%. Our study demonstrates the benefit of multiple biopsy sampling when considering personalized healthcare biomarker strategy, and provides an example to address the challenge of intra-tumoral biomarker heterogeneity using alternative pathological assessment and statistical methods.

Patient-Derived Gastric Carcinoma Xenograft Mouse Models Faithfully Represent Human Tumor Molecular Diversity.

Patient-derived cancer xenografts (PDCX) generally represent more reliable models of human disease in which to evaluate a potential drugs preclinical efficacy. However to date, only a few patient-derived gastric cancer xenograft (PDGCX) models have been reported. In this study, we aimed to establish additional PDGCX models and to evaluate whether these models accurately reflected the histological and genetic diversities of the corresponding patient tumors. By engrafting fresh patient gastric cancer (GC) tissues into immune-compromised mice (SCID and/or nude mice), thirty two PDGCX models were established. Histological features were assessed by a qualified pathologist based on H&E staining. Genomic comparison was performed for several biomarkers including ERBB1, ERBB2, ERBB3, FGFR2, MET and PTEN. These biomarkers were profiled to assess gene copy number by fluorescent in situ hybridization (FISH) and/or protein expression by immunohistochemistry (IHC). All 32 PDGCX models retained the histological features of the corresponding human tumors. Furthermore, among the 32 models, 78% (25/32) highly expressed ERBB1 (EGFR), 22% (7/32) were ERBB2 (HER2) positive, 78% (25/32) showed ERBB3 (HER3) high expression, 66% (21/32) lost PTEN expression, 3% (1/32) harbored FGFR2 amplification, 41% (13/32) were positive for MET expression and 16% (5/32) were MET gene amplified. Between the PDGCX models and their parental tumors, a high degree of similarity was observed for FGFR2 and MET gene amplification, and also for ERBB2 status (agreement rate = 94~100%; kappa value = 0.81~1). Protein expression of PTEN and MET also showed moderate agreement (agreement rate = 78%; kappa value = 0.46~0.56), while ERBB1 and ERBB3 expression showed slight agreement (agreement rate = 59~75%; kappa value = 0.18~0.19). ERBB2 positivity, FGFR2 or MET gene amplification was all maintained until passage 12 in mice. The stability of the molecular profiles observed across subsequent passages within the individual models provides confidence in the utility and translational significance of these models for in vivo testing of personalized therapies.

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.

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.

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies.

BACKGROUND: To determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment. METHODS: Archival tumor samples from the ZODIAC ( NCT00312377 , vandetanib ± docetaxel), ZEAL ( NCT00418886 , vandetanib ± pemetrexed), ZEPHYR ( NCT00404924 , vandetanib vs placebo) and ZEST ( NCT00364351 , vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients. RESULTS: The prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3-1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4-6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression. CONCLUSIONS: We have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation. TRIAL REGISTRATION: Randomized Phase III clinical trials ( NCT00312377 , ZODIAC; NCT00418886 , ZEAL; NCT00364351 , ZEST; NCT00404924 , ZEPHYR).

Evaluation of Trastuzumab Anti-Tumor Efficacy and its Correlation with HER-2 Status in Patient-Derived Gastric Adenocarcinoma Xenograft Models.

The aim of the study was to investigate trastuzumab anti-tumor efficacy and its correlation with HER-2 status in primary xenograft models derived from Chinese patients with gastric adenocarcinoma. Patient-derived gastric adenocarcinoma xenograft (PDGAX) mouse models were firstly generated by implanting gastric adenocarcinoma tissues from patients into immune deficient mice. A high degree of histological and molecular similarity between the PDGAX mouse models and their corresponding patients' gastric adenocarcinoma tissues was shown by pathological observation, HER-2 expression, HER-2 gene copy number, and mutation detection. Based on Hoffmann's criteria in gastric cancer, three models (PDGAX001, PDGAX003 and PDGAX005) were defined as HER-2 positive with fluorescence in situ hybridization (FISH) amplification or immunohistochemistry (IHC) 2+/ 3+, while two models (PDGAX002, PDGAX004) were defined as HER-2 negative. Upon trastuzumab treatment, significant tumor regression (105 % TGI) was observed in model PDGAX005 (TP53 wt), while moderate sensitivity (26 % TGI) was observed in PDGAX003, and resistance was observed in PDGAX001, 002 and 004. A significant increase in HER-2 gene copy number was only observed in PDGAX005 (TP53 wt). Interestingly, trastuzumab showed no efficacy in PDGAX001 (HER2 IHC 3+ and FISH amplification, but with mutant TP53). Consistent with this finding, phosphor-HER2 modulation by trastuzumab was observed in model PDGAX005, but not in PDGAX001.

Assessment and prognostic analysis of EGFR, HER2, and HER3 protein expression in surgically resected gastric adenocarcinomas.

AIM: To investigate the significance of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)2, and HER3 expression on survival outcomes in Chinese gastric cancer patients. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded specimens from 121 patients who underwent gastrectomy at Shanghai Renji Hospital from 2007-2010 were retrospectively examined. Fluorescence in situ hybridization and immunohistochemistry techniques were used to identify gene amplification and protein overexpression. Correlations between the expression or amplification of HER family genes and clinicopathological parameters were then determined using statistical analysis. RESULTS: EGFR protein overexpression, an increase in HER2 copy number and gene amplification, and HER3 protein overexpression were identified in 33.1%, 17.4%, and 62.0% of samples, respectively. Statistical analysis showed a significant association between EGFR expression and tumor invasion depth or tumor stage. HER2 was also shown to be significantly associated with the tumor grade. In addition, EGFR protein overexpression was found to be significantly associated with worse overall survival (P=0.03). CONCLUSION: The HER family members showed a high expression in gastric cancer. EGFR protein expression was associated with overall survival.

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.

Volitinib, a potent and highly selective c-Met inhibitor, effectively blocks c-Met signaling and growth in c-MET amplified gastric cancer patient-derived tumor xenograft models.

PURPOSE: To investigate the incidence of cMET gene copy number changes and protein overexpression in Chinese gastric cancer (GC) and to preclinically test the hypothesis that the novel, potent and selective cMET small-molecule inhibitor volitinib, will deliver potent anti-tumor activity in cMET-dysregulated GC patient-derived tumor xenograft (PDX) models. EXPERIMENTAL DESIGN: A range of assays were used and included; in vitro cell line panel screening and pharmacodynamic (PD) analysis, cMET fluorescence in-situ hybridization (FISH) and immunohistochemical (IHC) tissue microarray (TMA) analysis of Chinese GC (n = 170), and anti-tumor efficacy testing and PD analysis of gastric PDX models using volitinib. RESULTS: The incidence of cMET gene amplification and protein overexpression within Chinese patient GC tumors was 6% and 13%, respectively. Volitinib displayed a highly selective profile across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L-12.5 nM/L). Volitinib treatment led to pharmacodynamic modulation of cMET signaling and potent tumor stasis in 3/3 cMET-dysregulated GC PDX models, but had negligible activity in a GC control model. CONCLUSIONS: This study provides an assessment of tumor cMET gene copy number changes and protein overexpression incidence in a cohort of Chinese GC patients. To our knowledge, this is the first study to demonstrate anti-tumor efficacy in a panel of cMET-dysregulated gastric cancer PDX models, using a novel selective cMET-inhibitor (volitinib). Thus, the translational science presented here provides strong rationale for the investigation of volitinib as a therapeutic option for patients with GC tumors harboring amplified cMET.

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.

The oncogenic role of PKCiota gene amplification and overexpression in Chinese non-small cell lung cancer.

BACKGROUND: The atypical protein kinase C isozyme iota (PKCiota) has been proposed as an oncogene based on its transformation property and amplification identified in Caucasian non-small cell lung cancer (NSCLC) patients. Because the geography difference of some genetic aberrance such as EGFR mutations between Caucasian and Asian NSCLC patients has been identified previously, it is important to know whether the PKCiota amplification also occurs in Asian NSCLC patients. METHODS: The PKCiota gene copy number changes and protein expression in Chinese patients samples were detected by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Logistic regression was used to assess the association of PKCiota expression with clinicopathological parameters. siRNA-mediated gene silencing was applied to demonstrate the role of PKCiota in promoting cell growth in PKCiota gene amplified and protein overexpressed cancer cells. RESULTS: The result showed that PKCiota gene was amplified in 20.2% (24/119) of the tested primary tumor samples from Chinese NSCLC patients. Interestingly this gene amplification was highly enriched in squamous NSCLC patients (37.1%, 23/62). Further IHC analysis indicated that PKCiota protein was highly expressed (IHC score 2+ and 3+) in 91.6% (109/119) of Chinese NSCLC tumors. Moreover, the PKCiota gene amplification was also correlated with gender, subtype and distant metastasis. Knockdown of PKCiota gene in the PKCiota gene amplified and protein overexpressed cells led to significant growth inhibition. CONCLUSION: Taken together, our data demonstrate that PKCiota is a potential oncogene and therapeutic target in Chinese NSCLC.

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target.

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.

Identification of a subset of human non-small cell lung cancer patients with high PI3Kß and low PTEN expression, more prevalent in squamous cell carcinoma.

PURPOSE: The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kß and its relation to PTEN in non-small cell lung cancer NSCLC remain unclear. EXPERIMENTAL DESIGN: Antibodies directed against PI3Kß and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kß and PTEN were explored and colocalization of these markers in individual tumor cores were correlated. RESULTS: PI3Kß expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kß and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2. CONCLUSIONS: We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kß accompanied by a reduction/loss of PTEN, for whom selective PI3Kß inhibitors may be predicted to achieve greater clinical benefit.

Low frequency KRAS mutations in colorectal cancer patients and the presence of multiple mutations in oncogenic drivers in non-small cell lung cancer patients.

Intratumor heterogeneity can confound the results of mutation analyses in oncodriver genes using traditional methods thereby challenging the application of targeted cancer therapy strategies for patients Ultradeep sequencing can detect low frequency and expanded clonal mutations in primary tumors to better inform treatment decisions. KRAS coding exons in 61 treatment-naive colorectal cancer (CRC) tumors and KRAS, EGFR, ALK, and MET in lung tumors from three Chinese non-small cell lung cancer (NSCLC) patients were sequenced using ultradeep sequencing methods. Forty-one percent of CRC patients (25/61) harbored mutations in the KRAS active domain, eight of which (13%) were not detected by Sanger sequencing. Three (of eight) had frequencies less than 10% and one patient harbored more than one mutation. Low frequency KRAS active (G12R) and EGFR kinase domain mutations (G719A) were identified in one NSCLC patient. A second NSCLC patient showed an EML4-ALK fusion with ALK, EGFR, and MET mutations. A third NSCLC patient harbored multiple low frequency mutations in KRAS, EGFR, and MET as well as ALK gene copy number increases. Within the same patient, multiple low frequency mutations occurred within a gene. A complex pattern of intrinsic low frequency driver mutations in well-known tumor oncogenes may exist prior to treatment, resulting in resistance to targeted therapies. Ultradeep sequencing can characterize intratumor heterogeneity and identify such mutations to ultimately affect treatment decisions.