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Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in children and young adults oncology where more individualized, effective and less toxic treatments are highly desirable. In this study, we analyzed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcome in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWS by using the Agilent Human miRNA microarray v.2 and/or qRT-PCR. Statistical power of samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of 5 miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk to disease progression and survival. Validation analysis in the extended samples set indicated that both miR-34a and miR490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated to either event-free or overall survival and emerged as significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within two years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routinely evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced cells were less proliferative, less malignant and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with Nutlin-3a. Accordingly, Nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients.

Genetic alterations affecting 9p are commonly present in many cancer types and many cancer-related genes are located in this chromosomal region. We sequenced all of the genes located in a 32Mb region of 9p by targeted next generation sequencing (NGS) in 96 patients with different cancer types, including acute lymphoblastic leukemia, bone malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma, fibrosarcoma, Ewing's sarcoma, and lung carcinoma. Copy number alterations (CNA), and mutations were studied from the NGS data. We detected a deletion at the CDKN2A locus as being the most frequent genetic alteration in all cancer types. In addition to this locus, NGS also identified other small regions of copy number loss and gain. However, different cancer types did not reveal any statistically significant differences with regard to CNA frequency or type. Of the 191 genes within the target region, two novel recurrent mutations were found in the MELK and PDCD1LG2 genes. The most commonly mutated gene in sarcomas was TLN1 (8%) and PAX5 in ALL (9%). Mutations in PAX5, and RUSC2, were seen exclusively in ALL patients and those in KIAA1432, CA9, TLN1, and MELK only in sarcomas (MFH, FS, EFT). Thus using targeted NGS of the 9p region, in addition to commonly deleted CDKN2A locus, we were able to identify a number of small deletions and gains, as well as novel recurrent mutations in different cancer types. (c) 2014 Wiley Periodicals, Inc.