Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.

Significance: microRNA profiles of anaplastic lymphoma kinase ( ALK )-driven non-small cell lung cancers (NSCLCs) are currently not available in publically accessible databases. Identifying translocated ALK , mutant EGF receptor, and mutant V-Ki-ras2 Kirsten rat sarcoma cases in NSCLC is of value for determining which patients are more likely to benefit from a targeted therapy, to explicate mechanisms underlying chemotherapy survival, and ultimately in new drug development. microRNA-based classifiers are newly developed prognostic and diagnostic tools that can improve and complement the current gold-standard techniques. These classifiers also potentially represent an engine for boosting research on the role of these microRNAs in response to commonly used chemotherapy regimens in NSCLC to maximize patient outcomes.

Aim: We have studied the antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3 and CLM29) in primary papillary dedifferentiated thyroid cancer (DePTC) cells. Methods: The antiproliferative effect was tested in DePTC cells obtained at reoperation from patients with recurrence of the tumor. The concentrations of CLM3 and CLM29 used in the in vitro experiments were 1, 10, 30, and 50 μm. Results: Proliferation assays in DePTC cells showed a significant reduction of proliferation by CLM3 and CLM29, which was by 12% with CLM3 (the most potent compound) 10 μm, 43% with CLM3 30 μm, and 60% with CLM3 50 μm. CLM3 and CLM29 increased the percentage of apoptotic cells in DePTC cells dose dependently (P < 0.001) and inhibited migration (P < 0.001). A DePTC cell line (AL) was injected sc in CD nu/nu mice, and tumor masses became detectable 10 d after xenotransplantation. CLM3 (40 mg/kg · die) significantly inhibited tumor growth and weight, and the therapeutic effect was significant starting on the 19th day after cell implantation (4 d after the beginning of treatment). The CLM3-treated group of animals did not show any appreciable toxicity. CLM3 and CLM29 increased thrombospondin-1 expression in the AL cell line. A significant reduction of microvessels and in the percentage of antivascular endothelial growth factor antibody immunoreactivity was observed in the CLM3 treated tumors, with a simultaneous increase of the percentage of necrosis. Conclusion: The antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3, CLM29) in vitro and CLM3 in vivo in DePTC has been shown, opening the way to a future clinical evaluation.

MicroRNAs (miRNAs) play a key role in cancer pathogenesis and are involved in several human cancers, including non-small cell lung cancer (NSCLC). This study evaluated Let-7g and miR-21 expression by quantitative real-time PCR in 80 NSCLC patients and correlated the results with their main clinicopathological and molecular features. MiR-21 expression was significantly higher in NSCLC tissues compared to non-cancer lung tissues (p<0.0001), while no significant changes in Let-7g expression were observed between the tumor and normal lung tissues. Target prediction analysis led to the identification of 26 miR-21 and 24 Let-7g putative target genes that play important roles in cancer pathogenesis and progression. No significant association was observed between the analysed miRNAs and the main clinicopathological or molecular characteristics of the NSCLC patients, although both miRNAs were downregulated in squamous cell carcinomas compared to adenocarcinomas. Noteworthy, we observed a significant association between low Let-7g expression and metastatic lymph nodes at diagnosis (p=0.046), as well as between high miR-21 expression and K-Ras mutations (p=0.0003). Survival analysis did not show any significant correlation between prognosis and the analysed miRNAs, although the patients with a high Let-7g and miR-21 expression showed a significantly lower short-term progression-free survival (p=0.01 and p=0.0003, respectively) and overall survival (p=0.023 and p=0.0045, respectively). In conclusion, we showed that Let-7g and miR-21 expression was deregulated in NSCLC and we demonstrated a strong relationship between miR-21 overexpression and K-Ras mutations. Our data indicate that Let-7g and miR-21 profiling combined with the determination of K-Ras mutational status may be considered a useful biomarker for a more effective molecular characterization and clinical management of NSCLC patients.

Context and Objective: We have studied the antitumor activity of a novel cyclic amide, CLM94, with anti-vascular endothelial growth factor (VEGF) receptor-2 and antiangiogenic activity in primary anaplastic thyroid cancer (ATC) cells in vitro and in vivo. Design and Main Outcome Measures: CLM94 was tested: 1) in two human cell lines (HMVEC-d, dermal microvascular endothelial cells; and 8305C, undifferentiated thyroid cancer) at 0.001–100 μm; 2) in ATC cells at the concentrations of 10, 30, and 50 μm; and 3) in an ATC cell line (AF) in CD nu/nu mice. Results: CLM94 significantly inhibited VEGF receptor-2 and epidermal growth factor receptor phosphorylation in HMVEC-d and proliferation in HMVEC-d and 8305C cells. A significant reduction of proliferation with CLM94 in ATC cells (P < 0.01, ANOVA) and a slight but significant reduction of proliferation with CLM94 30 and 50 μm in normal thyroid follicular cells (P < 0.01, ANOVA) were shown. CLM94 increased the percentage of apoptotic ATC cells dose-dependently (P < 0.001, ANOVA) and inhibited migration (P < 0.01) and invasion (P < 0.001). AF cell line was injected sc in CD nu/nu mice, and tumor masses became detectable 25 d afterward. CLM94 (40 mg/kg · d) significantly inhibited tumor growth (starting 10 d after the beginning of treatment). CLM94 significantly decreased the VEGF-A gene expression in the AF cell line and the VEGF-A protein and microvessel density in AF tumor tissues. Conclusions: The antitumor and antiangiogenic activity of a new “cyclic amide” compound, CLM94, is very promising in ATC, opening the way to a future clinical evaluation.

We report the first case of a heterozygous T78M mutation in the caveolin-3 gene (CAV3) associated with rippling muscle disease and proximal myopathy. The patient displayed also bilateral winged scapula with limited abduction of upper arms and marked asymmetric atrophy of leg muscles shown by magnetic resonance imaging. Immunohistochemistry on the patient's muscle biopsy demonstrated a reduction of caveolin-3 staining, compatible with the diagnosis of caveolinopathy. Interestingly, consistent with the possible diagnosis of FSHD, the patient carried a 35 kb D4Z4 allele on chromosome 4q35. We discuss the hypothesis that the two genetic mutations may exert a synergistic effect in determining the phenotype observed in this patient.