Ficoll-hypaque separation vs whole blood lysis: Comparison of efficiency and impact on minimal residual disease analysis.

INTRODUCTION: The high-throughput era remarkably changed molecular laboratory practice. Actually, the increasing number of processed samples requires to reduce the risk of operator biases, by automating or simplifying as much as possible both the analytical and the pre-analytical phases. Minimal residual disease (MRD) studies in hematology often require a simultaneous processing of many bone marrow and peripheral blood samples from patients enrolled in prospective, multicenter, clinical trials, monitored at several planned time points. METHODS: In this study, we demonstrate that red blood cell lysis (RBL) pre-analytical procedure can replace the time-consuming Ficoll stratification as cell recovering step. Here, we show a MRD comparison study using both total white blood cells and mononuclear cells recovered by the 2 procedures from 46 follicular lymphoma (FL), 15 multiple myeloma (MM), and 11 mantle cell lymphoma (MCL) patients enrolled in prospective clinical trials. RESULTS: The experiments were performed in the 4 laboratories of the Fondazione Italiana Linfomi (FIL) MRD Network and showed superimposable results, in terms of good correlation (R = 0.87) of the MRD data obtained by recovering blood cells by the 2 approaches. CONCLUSION: Based on these results, the FIL MRD Network suggests to optimize the pre-analytical phases introducing RBL approach for cell recovery in the clinical trials including MRD analysis.

Type 1 diabetes in Sardinia: facts and hypotheses in the context of worldwide epidemiological data.

Type 1 diabetes (T1D) results from an autoimmune destruction of insulin-producing beta cells that requires lifelong insulin treatment. While significant advances have been achieved in treatment, prevention of complications and quality of life in diabetic people, the identification of environmental triggers of the disease is far more complex. The island of Sardinia has the second highest incidence of T1D in the world (45/100,000), right after Finland (64.2/100,000). The genetic background as well as the environment of the island's inhabitants makes it an ideal region for investigating environmental, immunological and genetic factors related to the etiopathogenesis of T1D. Several epidemiological studies, conducted over the years, have shown that exposures to important known environmental risk factors have changed over time, including nutritional factors, pollution, chemicals, toxins and infectious diseases in early life. These environmental risk factors might be involved in T1D pathogenesis, as they might initiate autoimmunity or accelerate and precipitate an already ongoing beta cell destruction. In terms of environmental factors, Sardinia is also particular in terms of the incidence of infection with Mycobacterium avium paratuberculosis (MAP) that recent studies have linked to T1D in the Sardinian population. Furthermore, the unique geochemical profile of Sardinia, with its particular density of heavy metals, leads to the assumption that exposure of the Sardinian population to heavy metals could also affect T1D incidence. These factors lead us to hypothesize that T1D incidence in Sardinia may be affected by the exposure to multifactorial agents, such as MAP, common viruses and heavy metals.

Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified.

Peripheral T-cell lymphomas not otherwise specified (PTCLs/NOS) are rare and aggressive tumours whose molecular pathogenesis and diagnosis are still challenging. The microRNA (miRNA) profile of 23 PTCLs/NOS was generated and compared with that of normal T-lymphocytes (CD4+, CD8+, naive, activated). The differentially expressed miRNA signature was compared with the gene expression profile (GEP) of the same neoplasms. The obtained gene patterns were tested in an independent cohort of PTCLs/NOS. The miRNA profile of PTCLs/NOS then was compared with that of 10 angioimmunoblastic T-cell lymphomas (AITLs), 6 anaplastic large-cell lymphomas (ALCLs)/ALK+ and 6 ALCLs/ALK-. Differentially expressed miRNAs were validated in an independent set of 20 PTCLs/NOS, 20 AITLs, 19 ALCLs/ALK- and 15 ALCLs/ALK+. Two hundred and thirty-six miRNAs were found to differentiate PTCLs/NOS from activated T-lymphocytes. To assess which miRNAs impacted on GEP, a multistep analysis was performed, which identified all miRNAs inversely correlated to different potential target genes. One of the most discriminant miRNAs was selected and its expression was found to affect the global GEP of the tumours. Moreover, two sets of miRNAs were identified distinguishing PTCL/NOS from AITL and ALCL/ALK-, respectively. The diagnostic accuracy of this tool was very high (83.54%) and its prognostic value validated.

Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.

Partial nodal involvement by marginal zone lymphoma. Use of IGK gene rearrangement analysis in diagnostic work-up.

Nodal marginal zone lymphoma (NMZL) is an indolent B-cell lymphoma that originates from the marginal zone of B-cell follicles. The tumour is rather uncommon, and shares some morphologic and immunophenotypic similarities with the extranodal form of marginal zone lymphomas. However, diagnosis of NMZL implies the exclusion of lymphoplasmacytic lymphoma, follicular lymphoma, and lymph node involvement by extra nodal or splenic marginal zone B-cell lymphoma In addition, its distinction from reactive conditions, including T-zone hyperplasia, are sometimes problematic based on morphologic grounds. We describe a patient who presented with cervical and inguinal lymphadenopathies and high inflammation indexes. Bone marrow and lymph node biopsies were performed for definitive diagnosis. Bone marrow histological and immunophenotypic examinations were normal and excluded haematological disease. In contrast, lymph node evaluation showed some features compatible with a possible lymphoproliferative disorder, even though no definite diagnosis could be made based on morphologic and immunohistochemical investigation. In particular, the problem of a differential diagnosis between NMZL and a florid hyperplasia of monocytoid B-elements was posed. Thus, in order to assess the nature (neoplastic vs. reactive) of the lesion, molecular analysis of the immunoglobulin genes was performed by PCR. Notably, although no clonal rearrangements were revealed by IGHV@ analysis, further evaluation of the immunoglobulin light chain (IGKV@) confirmed the presence of a clonal B-cell population. Accordingly, a final diagnosis of NMZL was made. In conclusion, this case is a good example of the crucial role of complete molecular analysis in the diagnostic work up of lymphoproliferative disorders.