Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment.

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the b subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I b-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of b-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of b-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC 50 . The Class I b-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III b-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III b-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between b-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III b-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III b-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients. ' 2007 Wiley-Liss, Inc.

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Angiogenesis leads to the formation of blood vessels from pre-existing ones, allowing tumor growth. Vascular endothelial growth factor (VEGF) and Angiopoietins (Ang-1, Ang-2) have a pivotal role in tumor angiogenesis but few data regarding their role in hereditary breast cancer are available. The aim of the present study was to analyze Ang-1, Ang-2, tyrosine-protein kinase receptor Tie2 and VEGF expression and their correlation in a cohort of familial and sporadic breast cancers in order to verify whether the presence of germline mutations in BRCA may have a role in tumor microenvironment regulation. Tumor samples from a cohort of 41 patients with a first diagnosis and a family history of breast cancer and 19 patients with sporadic breast cancers were enrolled. The expression of Tie2, Ang-1, Ang-2 and VEGF were analyzed by quantitative real-time PCR. Patients harboring BRCA mutations had higher levels of Ang-1 (P ¼ 0.05), Ang-2 (P ¼ 0.02) and VEGF (P ¼ 0.04) mRNA compared with those without BRCA mutations (BRCAX). The same was observed in triple-negative breast cancer (TNBC). Moreover, a positive correlation between Ang-2 and VEGF was found in both the familial breast cancer group (BRCA carriers: r ¼ 0.83; Po0.0001 and BRCAX: r ¼ 0.58; P ¼ 0.008) and in TNBC (r ¼ 0.62; P ¼ 0.007). The higher levels of Ang-1, Ang-2 and VEGF mRNA found in BRCA carriers and TNBCs suggest that they could be attractive angiogenic therapeutic targets in these breast cancers.