Delineation of Molecular Lesions in Acute Myeloid Leukemia Patients at Diagnosis: Integrated Next Generation Sequencing and Cytogenomic Studies.

Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by a wide range of genetic defects. Cytogenetics, molecular and genomic technologies have proved to be helpful for deciphering the mutational landscape of AML and impacted clinical practice. Forty-eight new AML patients were investigated with an integrated approach, including classical and molecular cytogenetics, array-based comparative genomic hybridization and targeted next generation sequencing (NGS). Various genetic defects were identified in all the patients using our strategy. Targeted NGS revealed known pathogenic mutations as well as rare or unreported variants with deleterious predictions. The mutational screening of the normal karyotype (NK) group identified clinically relevant variants in 86.2% of the patients; in the abnormal cytogenetics group, the mutation detection rate was 87.5%. Overall, the highest mutation prevalence was observed for the NPM1 gene, followed by DNMT3A, FLT3 and NRAS. An unexpected co-occurrence of KMT2A translocation and DNMT3A-R882 was identified; alterations of these genes, which are involved in epigenetic regulation, are considered to be mutually exclusive. A microarray analysis detected CNVs in 25% of the NK AML patients. In patients with complex karyotypes, the microarray analysis made a significant contribution toward the accurate characterization of chromosomal defects. In summary, our results show that the integration of multiple investigative strategies increases the detection yield of genetic defects with potential clinical relevance.

Hemophagocytic lymphohistiocytosis: a rare complication of autologous stem cell transplantation.

Hemophagocytic lymphohistiocytosis (HLH) is a very severe and rare syndrome of pathologic immune activation characterized by cytopenia and clinical signs and symptoms of extreme inflammation. HLH is usually fatal without treatment so that accurate and timely diagnosis is very important. The syndrome occurs as a familial disorder (familial HLH - FLH) or as an acquired condition (secondary - sHLH) in association with a variety of pathologic states: infections, rheumatologic, malignant or metabolic diseases. Malignancy associated HLH is primarily reported in T÷NK (natural killer)-cell malignancies but also in B-cell neoplasms and other types of cancer. HLH has also been reported in rare cases as a highly fatal and difficult to diagnose complication of stem cell transplantation (SCT). In this paper, we present the case of a young male patient who underwent autologous SCT as consolidation therapy for a T÷NK-cell lymphoma, complicated with graft failure due to HLH. The patient was successfully treated with corticosteroids, Etoposide, Cyclosporine and immunoglobulins. As a particularity, he developed a second B-cell neoplasia a few months after SCT.

2016 WHO Clinical Molecular and Pathological Criteria for Classification and Staging of Myeloproliferative Neoplasms (MPN) Caused by MPN Driver Mutations in the JAK2, MPL and CALR Genes in the Context of New 2016 WHO Classification: Prognostic and Therapeutic Implications.

The 2016 WHO-CMP classification proposal defines a broad spectrum of JAK2 V617F mutated MPN phenotypes: normocellular ET, hypercellular ET due to increased erythropoiesis (prodromal PV), hypercellular ET with megakaryocytic-granulocytic myeloproliferation and splenomegaly (EMGM or masked PV), erythrocythemic PV, early and overt classical PV, advanced PV with MF and post-PV MF. ET heterozygous for the JAK2 V617F mutation is associated with low JAK2 mutation load and normal life expectance. PV patients are hetero-homozygous versus homozygous for the JAK2 V617F mutation in their early versus advanced stages with increasing JAK2 mutation load from less than 50% to 100% and increase of MPN disease burden during life long follow-up in terms of symptomatic splenomegaly, constitutional symptoms, bone marrow hypercellularity and secondary MF. Pretreatment bone marrow biopsy in prefibrotic MPNs is of diagnostic and prognostic importance. JAK2 exon 12 mutated MPN is a distinct benign early stage PV. CALR mutated hypercellular thrombocythemia show distinct PMGM bone marrow characteristics of clustered larged immature dysmorphic megakaryocytes with bulky (bulbous) hyperchromatic nuclei, which are not seen in JAK2 mutated ET and PV. MPL mutated normocellular thrombocythemia is featured by clustered giant megakaryocytes with hyperlobulated stag-horn-like nuclei without features of PV in blood and bone marrow. Myeloproliferative disease burden in each of the JAK2, CALR and MPL MPNs is best reflected by the degree of anemia, splenomegaly, mutation allele burden, bone marrow cellularity and myelofibrosis.

Erythrocyte membrane in type 2 diabetes mellitus.

Type 2 diabetes mellitus represents a major public health challenge, due to the continuously growing prevalence and the complexity of the diabetic complications. Hyperglycemia seems to be the main mechanism for the disease progression. During erythrocyte's long life span, erythrocyte membranes are affected by the chronic exposure to glucose, which triggers several biochemical modifications that lead to both structural and functional disruption, which are further involved in the physiopathology of diabetes and its complications. Non-enzymatic protein glycation of red blood cell membrane proteins occur in two phases: early glycation, characterized by Schiff bases and Amadouri compounds formation, and advanced glycation, characterized by advanced glycation end products (AGEs). These products could be valuable tools for early diagnosis or biomarkers for disease progression, depending on how advanced they are in the glycation process. Advanced glycated end products were linked with diabetic complications. Also, lipid peroxidation and decreased activity of the enzyme pumps occur in the erythrocyte membrane of the diabetic patients. The investigation of lipid rafts and erythrocyte membrane fatty acids are a valuable tool for long-term monitoring of metabolic status. Further investigation of the erythrocyte membrane could provide novel biomarkers for monitoring of diabetes and its complications.

Peculiarities in the Diagnosis Approach of MDS /MPN-U Patients.

The most recent WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues describes a set of diseases framed as the MDS / MPN (myelodysplastic / chronic myeloproliferative syndromes). There are four subtypes comprised in this category: chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia and unclassifiable MDS / MPN. They combine both myelodysplastic and myeloproliferative features. Although the unclassifiable MDS/ MPN subtype specifically associates the myelodysplastic and myeloproliferative features, it does not meet the criteria defining the first 3 subtypes. The RARS-T subtype (refractory anemia with ringed sideroblasts associated with marked thrombocytosis) is included in the MDS / MPN-U as a temporary entity. There are two cases described in this article: one diagnosed with RARS-T and one with MDS / MPN-U. Both cases evolved towards acute myeloid leukemia.

Lymphoma immunophenotyping: "borderline" lymphomas.

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.