MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC)

Objective

We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation.

Methods

Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation.

Results

Median MET H-score was 140 (range 0–400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25–50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively).

Conclusions

MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation.     

A rat model of intravesical delivery of small interfering RNA for studying urinary carcinoma

Purpose  

siRNA has been used successfully in loss-of-function studies in vitro, but neither in vivo nor in clinical applications. The aims of the present study were (1) to establish rat models for in vivo delivery of siRNA to bladder cancer, and (2) to identify potential targets for siRNA.     

Versican is differentially expressed in human melanoma and may play a role in tumor development

Undifferentiated human melanoma cell lines produce a large chondroitin sulfate proteoglycan, different from the well-known melanoma-specific proteoglycan mel-PG (Heredia and colleagues, Arch Biochem Biophys, 333: 198-206, 1996). We have identified this proteoglycan as versican and analyzed the expression of versican in several human melanoma cell lines. Versican isoforms are expressed in undifferentiated cell lines but not in differentiated cells, and the isoform expression pattern depends on the degree of cell differentiation. The V0 and V1 isoforms are found on cells with an early degree of differentiation, whereas the V1 isoform is present in cells with an intermediate degree of differentiation. We have also characterized some functional properties of versican on human melanoma cells: the purified proteoglycan stimulates cell growth and inhibits cell adhesion when cells are grown on fibronectin or collagen type I as substrates, and thus may facilitate tumor cell detachment and proliferation. Furthermore, we have analyzed the expression of versican in human melanocytic nevi and melanoma: 10 benign melanocytic nevi, 10 dysplastic nevi, 11 primary malignant melanomas, and 8 metastatic melanomas were tested. Immunoreactivity for versican was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas. Our results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis.     

Diagnostic yield and clinical utility of whole exome sequencing using an automated variant prioritization system,EVIDENCE

EVIDENCE, an automated variant prioritization system, has been developed to facilitate whole exome sequencing analyses. This study investigated the diagnostic yield of EVIDENCE in patients with suspected genetic disorders. DNA from 330 probands (age range, 0-68 years) with suspected genetic disorders were subjected to whole exome sequencing. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments. EVIDENCE reported a total 228 variants in 200 (60.6%) of the 330 probands. The average number of organs involved per patient was 4.5 ± 5.0. After clinical reassessment and/or family member testing, 167 variants were identified in 141 probands (42.7%), including 105 novel variants. These variants were confirmed as being responsible for 121 genetic disorders. A total of 103 (61.7%) of the 167 variants in 95 patients were classified as pathogenic or probably to be pathogenic before, and 161 (96.4%) variants in 137 patients (41.5%) after, clinical assessment and/or family member testing. Factor associated with a variant being regarded as causative includes similar symptom scores of a gene variant to the phenotype of the patient. This new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 42.7% diagnostic yield.     

Survival assay of transiently transfected dopaminergic neurons

Death pathways in the apoptotic neurons are mostly studied by manipulating the levels of apoptosis-related proteins and counting the survival/death of affected neurons. Such assays are, however, technically complicated. We developed a transfection-survival assay for cultured embryonic dopaminergic (DA) neurons induced to die by deprivation of glial cell line-derived neurotrophic factor (GDNF). The calcium phosphate co-precipitation technique was used to transfect DA neurons. Microisland cultures and co-transfected enhanced green fluorescent protein allowed direct counting of transfected neurons from the same cultures at the beginning and the end of GDNF deprivation, whereas post hoc subtraction of tyrosine hydroxylase-negative neurons allowed exclusion of transfected non-DA neurons. Overexpression of dominant-negative mutant of caspase-6 significantly blocked the death of GDNF-deprived DA neurons. Thus, we have found a tool not only to transfect the neurons dissociated from midbrain, but also to analyze the apoptotic proteins particularly in DA neurons.