José-Ángel Hernández

Differences in genetic changes between multiple myeloma and plasma cell leukemia demonstrated by comparative genomic hybridization

To analyze the genomic differences between multiple myeloma (MM) and plasma cell leukemia (PCL), a total of 30 cases were studied by comparative genomic hybridization (CGH). In five cases with a low proportion of plasma cells (PC) in bone marrow, an enrichment of PC was performed by using immunomagnetic beads conjugated with the monoclonal antibody B-B4. In 24 out of the 25 MM (96%) and in all five PCL (100%) patients DNA copy number changes were identified by CGH analysis; in the MM case without chromosomal imbalances, the immunomagnetic enrichment of PC had failed. The most recurrent changes in MM patients were gains at chromosomes 15q (48%), 11q (44%), 3q (40%), 9q (40%) and 1q (36%). By contrast, all PCL patients showed gains in 1q. Losses of chromosomal material were significantly more frequent in PCL than in MM patients (P = 0.03): losses on 13q in 80% of PCL vs 28% of MM; and on chromosome 16 in 80% vs 12%, respectively. In addition, PCL patients showed losses of 2q and 6p that were not present in MM. The CGH data show differences in chromosomal imbalances between MM and PCL.

Development of acute leukaemia after idiopathic myelofibrosis.

AIMS: To determine the characteristics of blastic transformation of idiopathic myelofibrosis. METHODS: The clinical and haematological features, as well as the morphological characteristics of blast cells, were analysed in nine adults with blast transformation. RESULTS: Most of the patients were male and had enlarged spleens and livers. Five of the patients had normal platelet counts, while all had pronounced anaemia and a moderate degree of leucocytosis. The duration of the acute phase was usually short: 16 (SD 8) weeks. Most myeloid cell lineages--granulocytic, monocytic, and megakaryocytic--were similarly distributed. One patient also had a hybrid (lymphoid-myeloid) phenotype. The morphological assessment of blast cells agreed with immunophenotyping in five out of the nine cases. The onset of the blastic phase was not related to previous treatment. CONCLUSIONS: A pluripotential stem cell with preferential myeloid commitment would be the target cell of blast transformation in idiopathic myelofibrosis. Our immunophenotypic data do not support the concept of a preferential association between megakaryocytic lineage and the acute transformation of idiopathic myelofibrosis. The absence of previous treatment in some cases suggests that this kind of evolution is part of the natural history of idiopathic myelofibrosis.

A high proportion of cells carrying trisomy 12 is associated with a worse outcome in patients with chronic lymphocytic leukemia

The prognosis of chronic lymphocytic leukemia (CLL) patients displaying trisomy 12 (+12) remains unclear. In this study, we analyzed the influence of the proportion of cells with +12, and other clinical and biologic factors, in time to first therapy (TTFT) and overall survival (OS), in 289 patients diagnosed with CLL carrying +12. Median OS was 129 months. One hundred seventy‐four patients (60.2%) presented +12 in <60% of cells. TTFT and OS for this subgroup were longer than for the subgroup with +12 in ≥60% of cells, with a median TTFT of 49 months (CI95%, 39–58) vs 30 months (CI95%, 22–38) (P = 0.001); and a median OS of 159 months (CI95%, 119–182), vs 96 months (CI95%, 58–134) (P = 0.015). Other factors associated with a shorter TTFT were: Binet stage, B symptoms, lymphadenopathy, splenomegaly, high lymphocyte count, 11q‐, high β2microglobulin, and high LDH. In the multivariate analysis, clinical stage, +12 in ≥60% of cells, high lymphocyte count, B symptoms, and 11q‐ in addition, resulted of significance in predicting shorter TTFT. Significant variables for OS were: Binet stage, lymphadenopathy, splenomegaly, high LDH, high β2microglobulin, 11q‐, and CD38. In the multivariate analysis, only Binet stage, 11q‐, and high β2microglobulin significantly predicted shorter OS. CLL with +12 entails a heterogeneous group with intermediate prognosis. However, a high proportion of cells carrying +12 separates a subgroup of patients with poor outcome. Copyright

Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia

BackgroundChronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.MethodsAmplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.ResultsNGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.ConclusionsOur study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.

MiRNA expression profile of chronic lymphocytic leukemia patients with 13q deletion

Deletion 13q (13q-) is the most common cytogenetic aberration in chronic lymphocytic leukemia (CLL) and is associated with the most favorable prognosis as the sole cytogenetic abnormality. However, it is heterogeneous whereby CLL patients with higher percentages of 13q- cells (13q-H) have a more aggressive clinical course and a distinct gene expression profile. The microRNA (miRNA) expression profile of CLL gives additional biological and prognostic information, but its expression in 13q- CLL has not been examined in detail. The miRNA expression of clonal B cell lymphocytes (CD19+ cells) of 38 CLL patients and normal B cells of six healthy donors was analyzed. CLL patients with higher percentages of 13q- cells (≥80%) showed a different level of miRNA expression from patients with lower percentages (<80%). Interestingly, miR-143 was downregulated and miR-155 was overexpressed in 13q-H. This deregulation affected important validated target genes involved in apoptosis (BCL2, MDM2, TP53INP1) and proliferation (KRAS, PI3K-AKT signaling), that could lead to decreased apoptosis and increased proliferation in 13q-H patients. This study provides new evidence about the heterogeneity of the 13q deletion in CLL patients, showing that miRNA regulation could be involved in several significant pathways deregulated in CLL patients with a high number of losses in 13q.

The International Prognostic Index for Patients with Chronic Lymphocytic Leukemia Has the Higher Value in Predicting Overall Outcome Compared with the Barcelona-Brno Biomarkers Only Prognostic Model and the MD Anderson Cancer Center Prognostic Index

In recent years, new prognostic indexes (PIs) for chronic lymphocytic leukemia (CLL), which include clinical, biological, and genetic variables, have been validated, highlighting the MD Anderson Cancer Center prognostic index (MDACC PI), the CLL-international prognostic index (CLL-IPI), and the Barcelona-Brno biomarkers only prognostic model. The aim of this study is to compare the utility of these PIs in a cohort of Spanish patients. A retrospective analysis of 696 unselected CLL patients newly diagnosed and previously untreated from different Spanish institutions was performed. The MDACC PI, the CLL-IPI, and the biomarkers only PI were applied to these patients, and a comparison of the three PIs was performed. With a median follow-up time of 46 months, 394 patients were alive and 187 had received treatment. The median overall survival (OS) was 173 months and the median time to first therapy (TTFT) was 32 months. Significant differences were obtained in OS and TTFT for all subgroups when applying these PIs, with the CLL-IPI being the one with the higher c-index (0.676 for OS and 0.757 for TTFT). The three PIs were able to discriminate patients in different prognostic subgroups. In our cohort, the CLL-IPI showed higher power in predicting TTFT and OS.

Hyperdiploidy as a rare event that accompanies poor prognosis markers in CLL

The presence of chromosomal gains other than trisomy 12 in chronic lymphocytic leukaemia (CLL) is unusual. However, some patients may show gains on several chromosomes simultaneously suggesting a hyperdiploid karyotype.

Prognostic Factors in Chronic Lymphoid Leukemia and Identification of New Clinically Relevant Molecular Markers

Chronic lymphocytic leukemia (CLL) is a hematological malignancy with significant clinical heterogeneity, due in part to the genetic alterations that leukemic cells present in each patient (Chiorazzi et al, 2005). CLL has a highly variable clinical course. Traditionally, it has been considered that about one-third of patients will never require treatment, as they will have prolonged survival and they will die from causes unrelated to the disease. In another third of cases, after an indolent phase disease progression occurs. In the remaining third of patients early treatment is required because of the aggressiveness of the disorder. However, due to the routine performance of blood counts in the population, the number of asymptomatic patients is increasing and, conversely, those who require initial treatment account for fewer than 15% of cases (Hernandez et al, 2010). Since the first descriptions of the disease, researchers have attempted to establish prognostic factors with which to make a risk assessment of disease progression and probability of death. The ultimate aim is to try and apply a targeted and early treatment that increases overall survival and quality of life in patients with more aggressive forms, and to determine reliably the cases who do not need further treatment. (Dighiero & Hamblin, 2008).