TCRγ-Chain Gene Rearrangement by PCR-Based GeneScan: Diagnostic Accuracy Improvement and Clonal Heterogeneity Analysis in Multiple Cutaneous T-Cell Lymphoma Samples

Cutaneous T-cell lymphomas are a heterogeneous group of lymphomas where the tumor population emerges within a multiple subclone pattern (“clonal heterogeneity”). PCR analysis has been shown to be useful in the diagnosis of mycosis fungoides (MF) and Sézary Syndrome (SS). Focusing the attention on clonal heterogeneity, the efficacy of the multiplex/heteroduplex (HD) PCR and the GeneScan (GS) capillary electrophoresis analysis was compared in the early diagnosis of MF/SS, using a multiple sample approach. Indeed, GS demonstrated TCRγ gene rearrangement (GR) in all the 57 SS (100%) and in 123/146 (84%) of the MF samples, whereas the multiplex/HD PCR was less sensitive. An increase in clonality was observed in connection with both a worsening of the cutaneous disease (79% T1/T2; 100% T3/T4) and an increase in the histopathological score (HS<5, 76%; HS≥5, 94%). Clonal heterogeneity with adjunctive reproducible skin TCRγ-GRs was also observed. “Clonal instability,” with different GRs, was present in a small percentage of patients. Therefore, it can be concluded that GS analysis in TCRγ-GR is able to improve diagnosis in MF/SS patients and the multiple sample approach is helpful for a correct interpretation of clonal patterns in skin lesions, especially in early-stage MF and in SS skin/blood samples

An Italian Multicenter Perspective Harmonization Trial for the Assessment of MET Exon 14 Skipping Mutations in Standard Reference Samples

Lung cancer remains the leading cause of cancer deaths worldwide. International societies have promoted the molecular analysis of MET proto-oncogene, receptor tyrosine kinase (MET) exon 14 skipping for the clinical stratification of non-small cell lung cancer (NSCLC) patients. Different technical approaches are available to detect MET exon 14 skipping in routine practice. Here, the technical performance and reproducibility of testing strategies for MET exon 14 skipping carried out in various centers were evaluated. In this retrospective study, each institution received a set (n = 10) of a customized artificial formalin-fixed paraffin-embedded (FFPE) cell line (Custom METex14 skipping FFPE block) that harbored the MET exon 14 skipping mutation (Seracare Life Sciences, Milford, MA, USA), which was previously validated by the Predictive Molecular Pathology Laboratory at the University of Naples Federico II. Each participating institution managed the reference slides according to their internal routine workflow. MET exon 14 skipping was successfully detected by all participating institutions. Molecular analysis highlighted a median Cq cut off of 29.3 (ranging from 27.1 to 30.7) and 2514 (ranging from 160 to 7526) read counts for real-time polymerase chain reaction (RT-PCR) and NGS-based analyses, respectively. Artificial reference slides were a valid tool to harmonize technical workflows in the evaluation of MET exon 14 skipping molecular alterations in routine practice

NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, Anaplastic Large Cell Lymphoma (ALCL) is a T cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor (TCR) and other T-cell specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation and migration of transformed T cells. Here we show that loss of T cell specific molecules in ALCL cases is broader than previously reported and involves most TCR-related signalling molecules, including CD3ε, ZAP70, LAT and SLP76. We further demonstrate that NPM-ALK, but not the kinase dead NPM-ALK(K210R), down-regulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing since this down-regulation was reverted by treating ALCL cells with 5-aza-2′-deoxycytidine or by knocking-down STAT3 through sh-RNA. Finally, NPM-ALK increased the methylation of ZAP-70 intron1-exon2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 (DNMT1) in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T cell identity and signalling