Feasibility of the AML profiler (Skyline™ Array) for patient risk stratification in a multicentre trial: a preliminary comparison with the conventional approach.

Deoxyribonucleic acid microarrays allow researchers to measure mRNA levels of thousands of genes in a single experiment and could be useful for diagnostic purposes in patients with acute myeloid leukaemia (AML). We assessed the feasibility of the AML profiler (Skyline™ Array) in genetic stratification of patients with de novo AML and compared the results with those obtained using the standard cytogenetic and molecular approach. Diagnostic bone marrow from 31 consecutive de novo AML cases was used to test MLL-PTD, FLT3-ITD and TKD, NPM1 and CEBPAdm mutations. Purified RNA was used to assess RUNX1-RUNX1T1, PML-RARα and CBFß-MYH11 rearrangements. RNA remnants underwent gene expression profiling analysis using the AML profiler, which detects chromosomal aberrations: t(8;21), t(15;17), inv(16), mutations (CEBPAdm, ABD-NPM1) and BAALC and EVI1 expression. Thirty cases were successfully analysed with both methods. Five cases had FLT3-ITD. In one case, a t(8;21) was correctly detected by both methods. Four cases had inv(16); in one, the RNA quality was unsatisfactory and it was not hybridized, and in the other three, the AML profiler detected the genetic lesion - this being a rare type I translocation in one case. Two cases with acute promyelocytic leukaemia were diagnosed by both methods. Results for NPM1 mutations were concordant in all but two cases (2/11, non-ABD mutations). Analysis of costs and turnaround times showed that the AML profiler was no more expensive than the conventional molecular approach. These results suggest that the AML profiler could be useful in multicentre trials to rapidly identify patients with AML with a good prognosis. Copyright © 2016 John Wiley & Sons, Ltd.

Multilineage dysplasia is associated with a poorer prognosis in patients with de novo acute myeloid leukemia with intermediate-risk cytogenetics and wild-type NPM1.

Acute myeloid leukemia (AML) with myelodysplasia-related changes is characterized by the presence of multilineage dysplasia (MLD), frequently related to high-risk cytogenetics and poor outcome. However, the presence of MLD does not modify the favorable prognostic impact of NPM1 mutation. The prognosis of patients with AML presenting marked dysplasia lacking high-risk cytogenetics and NPM1 mutation is uncertain. We evaluated the prognostic impact of MLD in 177 patients with intermediate-risk cytogenetics AML (IR-AML) and wild-type NPM1. Patients were categorized as MLD-WHO (WHO myelodysplasia criteria; n = 43, 24 %), MLD-NRW (significant MLD non-reaching WHO criteria; n = 16, 9 %), absent MLD (n = 80, 45 %), or non-evaluable MLD (n = 38, 22 %). No differences concerning the main characteristics were observed between patients with or without MLD. Outcome of patients with MLD-WHO and MLD-NRW was similar, and significantly worse than patients lacking MLD. The presence of MLD (66 vs. 80 %, p = 0.03; HR, 95 % CI = 2.3, 1.08-4.08) and higher leukocyte count at diagnosis was the only variable associated with lower probability of complete remission after frontline therapy. Concerning survival, age and leukocytes showed an independent prognostic value, whereas MLD showed a trend to a negative impact (p = 0.087, HR, 95 % CI = 1.426, 0.95-2.142). Moreover, after excluding patients receiving an allogeneic stem cell transplantation in first CR, MLD was associated with a shorter survival (HR, 95 % CI = 1.599, 1.026-2.492; p = 0.038). In conclusion, MLD identifies a subgroup of patients with poorer outcome among patients with IR-AML and wild-type NPM1.

Correlation between immature platelet fraction and reticulated platelets. Usefulness in the etiology diagnosis of thrombocytopenia.

BACKGROUND: Reticulated platelets (RP) are a surrogate marker for megakaryocytic activity, but the limitation of this determination is the lack of standardization of methodology. The determination of the immature platelet fraction (IPF) is performed in a simple, automated, and reproducible way between laboratories. We analyzed the correlation between IPF and RP, and usefulness of IPF in patients with thrombocytopenia. METHODS: RP were determined by flow cytometry using double staining with thiazole orange and CD61 PerCP. IPF was performed with Sysmex XE2100 analyzer. We used a control group with normal platelets, and thrombocytopenic patients were classified into three groups: Group 1. Central thrombocytopenia, Group 2. Thrombocytopenia as a result of enhanced peripheral platelet destruction, and Group 3. Peripheral non-immune thrombocytopenia by abnormal distribution. RESULTS: Fourteen controls and 66 patients were analyzed. Group 1: 25 patients, they had mean and confidence interval 95% (95% CI) for IPF 8.67% (6.49-10.46%) and RP 4.08% (2.86-5.30%). Group 2: 20 patients, they had mean and 95%CI for IPF 16.80% (12.20-21.39%) and RP 16.14% (9.89-22.40%). Group 3: 21 patients, they had mean and 95% CI for IPF 9.04% (6.95-11.14%) and RP 5.23% (3.41-7.05%). The overall Pearson linear correlation between IPF and RP was r: 0.65. There were statistically significant differences in values of IPF and RP between Group 2 and the other two groups (P < 0.01). CONCLUSION: There is a good correlation between IPF and RP mainly in thrombocytopenia by peripheral destruction. Determination of IPF is an easy technique in their implementation, standardized and reproducible, so it could be a useful screening technique in patients with thrombocytopenia.

Gamma heavy-chain disease accompanied with follicular lymphoma: a case report.

Heavy chain diseases (HCD) are B-cell lymphoprolipherative disorders characterized by the production of monoclonal heavy chains without associated light chains. Some cases of gamma-HCD (γ-HCD) are concurrent with other lymphoid neoplasm. The monoclonal component is not always detectable by serum electrophoresis, and often an immunofixation procedure is necessary to detect this component. Prognosis is variable, and no established guidelines for follow-up are available. We describe a case of a challenging diagnosis of γ-HCD due to the absence of clinical signs frequently reported in the disease (anaemia and palatal oedema among others). Haematological malignancy was the first suspicion but bone marrow examination was negative. In addition, the presence of an autoimmune bicytopenia and a Klinefelter syndrome complicated the clinical context of the patient. A thoracoabdominal computed tomography reported many small adenopathies whose pathological and immunohystochemical study revealed a follicular lymphoma. Shortly after, serum inmunofixation secondary to an abnormal electrophoretic pattern revealed a gamma paraprotein without light chains. Eventually, γ-HCD in association with follicular lymphoma was the final diagnosis. This is the first case reporting this association.

Tromboc@t Working Group recommendations for management in patients receiving direct oral anticoagulants. / Recomendaciones del Grupo Catalán de Trombosis (Tromboc@t Working Group) para el tratamiento de los pacientes que reciben anticoagulantes orales directos.

BACKGROUND AND OBJECTIVES: In recent years, direct oral anticoagulants (DOACs) have become an alternative to vitamin K antagonists (VKA) for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF) as well as for prevention and treatment of deep venous thrombosis. Pivotal trials have demonstrated non-inferiority and potential superiority compared to warfarin, which increases the options of anticoagulant treatment. In our setting, the Anticoagulant Treatment Units (ATUs) and Primary Care Centres (PCCs) play an important role in the education, follow-up, adherence control and management in special situations of anticoagulated patients. These considerations have motivated us to elaborate the present consensus document that aims to establish clear recommendations that incorporate the findings of scientific research into clinical practice to improve the quality of care in the field of anticoagulation. MATERIAL AND METHODS: A group of experts from the Catalan Thrombosis Group (TROMBOC@T) reviewed all published literature from 2009 to 2016, in order to provide recommendations based on clinical evidence. RESULTS: As a result of the project, a set of practical recommendations have been established that will facilitate treatment, education, follow-up and management in special situations of anticoagulated patients with ACODs. CONCLUSIONS: Progressive increase in the use of DOACs calls for measures to establish and homogenise clinical management guidelines for patients anticoagulated with DOACs in ATUs and PCCs.

A new D816 c-KIT gene mutation in refractory AML1-ETO leukemia.

One of the most common genetic events in acute myeloid leukemia (AML) is the t(8;21) (q22;q22) translocation, which contributes to leukemic transformation. However, different lines of evidence suggest that the AML1-ETO rearrangement is not sufficient to cause the full leukemic phenotype. Secondary genetic alterations such as mutations in receptor tyrosine kinases are thus required to induce overt AML. The incidence of c-KIT mutations in exon 17 was evaluated in 37 Spanish patients with AML1-ETO+ leukemias. c-KIT mutations were present in only two cases (6.6%) and were shown to be associated with an adverse outcome. The frequency of c-KIT mutations described here is much lower than in other reports.

Low frequency of exon 3 PTPN11 mutations in adult de novo acute myeloid leukemia. Analysis of a consecutive series of 173 patients.

A total of 173 samples obtained from adult patients with de novo acute myeloid leukemia (AML) were assayed for exon 3 PTPN11 mutations by single strand conformation polymorphism (SSCP) analysis and direct sequencing. Only three monocytic leukemias had point mutations (1.73%).

International and Italian prognostic indices in follicular lymphoma.

BACKGROUND AND OBJECTIVES: The International Prognostic Index (IPI), initially designed for aggressive lymphomas, has been successfully used in patients with follicular lymphoma (FL). The Italian Lymphoma Intergroup (ILI) created a new prognostic index specific for FL. The aim of this study was to compare which of these two indices is more useful when applied to a large group of FL patients. DESIGN AND METHODS: Both indices, IPI (age >60 years, extranodal involvement >=2 sites, elevated lactate dehydrogenase, ECOG >=2, stage >=3) and ILI (age >60 years, extranodal involvement >=2 sites, elevated lactate dehydrogenase, male sex, B symptoms, erythrocyte sedimentation rate >=30 mm 1(st) hour) were calculated in a group of 398 FL patients. Overall survival (OS) and progression-free survival (PFS) associated with each prognostic group were calculated according to the Kaplan-Meier method. RESULTS: The overall concordance between both indices was 73%. According to the IPI 122 patients (31%) were in the higher risk group, whereas according to the ILI 132 (33%) were; concordance between the high risk groups was 66%. The 10-years OS and PFS rates after applying the IPI system were 73% and 37%, respectively, in the low risk groups; 47% and 26%, in the intermediate risk groups and 25% and 2%, in the high risk groups (log-rank=69.2 and 41.3, respectively; p<0.0001). According to ILI index the 10-year OS and PFS were 60% and 34%, respectively, in the low risk groups; 59% and 30%, in the intermediate risk groups and 17% and 0%, in the high risk groups (log-rank=86.6 and 58.5, respectively; p<0.0001). INTERPRETATION AND CONCLUSIONS: Both the IPI and ILI index, are useful for classifying FL patients into different risk groups. Although it seems that the ILI index has a higher discriminating power among groups, significant differences were not observed in identifying FL patients with a poor outcome.

Clonal heterogeneity assessed by flow cytometry in B-cell lymphomas arising from germinal centers.

Patients with mature follicular B-cell lymphomas develop aggressive non-Hodgkin lymphomas (NHLs) during disease progression. It is controversial whether most diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) emerge as de novo lymphomas or from an original follicular lymphoma. To distinguish clonally related populations in aggressive NHL, we studied the immunophenotypic features of 18 consecutive samples from 16 patients. Three flow cytometric patterns were distinguished: (1) a homogeneous neoplastic population of large B cells with phenotypic features of follicular center cells; (2) 2 atypical populations of B cells, small monoclonal B cells, and large B cells with loss of some surface antigens; and (3) 2 clonal populations of small and large B cells sharing the same light-chain isotype. The 3 flow cytometric patterns were observed, respectively, in de novo DLBCL and BL, transformation into BL, and transformation into DLBCL. Flow cytometric data can provide valuable information about the natural history of NHL.

Primary bone marrow lymphoma: an uncommon extranodal presentation of aggressive non-hodgkin lymphomas.

Bone marrow involvement by lymphoma is considered a systemic dissemination of the disease arising elsewhere, although some tumors may arise primarily in the bone marrow microenvironment. Primary bone marrow lymphoma (PBML) is a rare entity whose real boundaries and clinicobiological significance are not well defined. Criteria to diagnose PBML encompass isolated bone marrow infiltration, with no evidence of nodal or extranodal involvement, including the bone, and the exclusion of leukemia/lymphomas that are considered to primarily involve the bone marrow. Twenty-one out of 40 lymphomas retrospectively reviewed by the International Extranodal Lymphoma Study Group from 12 institutions in 7 different countries over a 25-year period fulfilled the inclusion criteria. These cases comprised 4 follicular lymphomas (FLs), 15 diffuse large B-cell lymphomas (DLBCLs), and 2 peripheral T-cell lymphomas, not otherwise specified. The FL cases showed paratrabecular infiltration, BCL2 protein and CD10 expression, and BCL2 gene rearrangement. DLBCL showed nodular infiltration in 6 cases and was diffuse in 9 cases; it also showed positivity for BCL2 protein (9/10) and IRF4 (6/8). Median age was 65 years with male predominance. All but 3 FL patients were symptomatic. Most cases presented with cytopenias and high lactate dehydrogenase. Four patients (3 FL cases and 1 DLBCL case) had leukemic involvement. Most DLBCL patients received CHOP-like or R-CHOP-like regimens. The outcome was unfavorable, with a median overall survival of 1.8 years. In conclusion, PBML is a very uncommon lymphoma with particular clinical features and heterogenous histology. Its recognition is important to establish accurate diagnosis and adequate therapy.

[Persistent polyclonal B-cell lymphocytosis: study of 35 cases]. / Linfocitosis B policlonal persistente: estudio de 35 casos.

BACKGROUND AND OBJECTIVES: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare entity, presenting especially in adult smoker women. It is characterized by an increase of serum IgM, DR7-HLA haplotype, cytogenetic abnormalities and multiple IgH/BCL-2 rearrangements. To date, it has not been elucidated whether this is a benign or premalignant disorder. We analyzed the PPBL characteristics with especial attention to its evolution. PATIENTS AND METHODS: Thirty-five PPBL patients from 5 hospitals in Catalonia were retrospectively analyzed. A simultaneous morphologic review of the blood smears was performed by members of the GCCH in a 16 multiple-observer optic microscope. Clinical and biological data were also analyzed. RESULTS: PPBL presents in the majority of cases with persistent polyclonal B-cell lymphocytosis and affects primarily smoker women. The morphologic hallmark, in absence of viral infections, is the presence of activated lymphocytes with bilobulated and/or cleaved nuclei, and nuclear pockets in the ultrastructural study. Increased serum IgM, HLA-DR7 haplotype, chromosomal abnormalities such as i(3)(q10) and multiple IgH/BCL-2 rearrangements were detected. Thirty-four out of 35 patients are alive after a median follow up of 70.7 months. One patient died because of lung adenocarcinoma and another developed a follicular lymphoma without relation to PPBL. CONCLUSIONS: PPBL has an asymptomatic and stable evolution, although it frequently presents genetic abnormalities. It remains unknown whether it is a premalignant entity, similar to monoclonal gammopathies of unknown significance. Hence, accurate cytologic diagnosis and follow-up are essential.

Acute myeloid leukemia subgroups identified by pathway-restricted gene expression signatures.

Acute myeloid leukemia (AML) is a heterogeneous group of disorders characterized by abnormal proliferation of myeloid precursors and a maturation block. Underlying genetic lesions determine an altered expression program (transcriptosome) that can be studied in depth by massive technologies. Alternatively, we selected a pathway profiling strategy based on the current knowledge in order to stratify de novo AML patients and identify those cases which would potentially benefit from the use of new chemotherapeutic agents. One hundred and thirty-two RNA samples obtained from de novo adult AML patients were tested for FLT3, FLT3-LG, NDST1, HDAC2, ATRX, FOS, DNMT1, DNMT3A, DNMT3B, NBS1, RAD50, MRE11A, Meis1 and Meis2 expression using quantitative PCR (qPCR) assays. Clinical and biologic findings were correlated with expression results. Cluster analysis was also performed. FLT3 expression defined three subgroups of patients. The best outcome was found in the group with the lowest FLT3 expression. Intermediate levels of FLT3 were associated with the worst outcome. Patients with low levels of ATRX more frequently presented an adverse karyotype whereas cases with preserved ATRX levels showed an excellent outcome. In accordance with previous results, Meis1 downregulation is a useful surrogate marker indicating a good prognosis in AML patients. Simple qPCR platforms may help to identify different biologic subgroups in AML.

Genomic polymorphisms provide prognostic information in intermediate-risk acute myeloblastic leukemia.

Current prognostic factors for acute myeloblastic leukemia (AML) are not sufficient to accurately predict the group of patients in the intermediate-risk category who will successfully respond to treatment. Distinct patterns of inherited functional genomic polymorphisms might explain part of these heterogeneous prognoses. We used the allelic discrimination method to identify polymorphisms in GSTT1, SULT1C2, CDA, SXR (drug metabolic pathways), XPD, XPA, XPG, ERCC1, TOP2A (DNA repair), VEGF (angiogenesis), and MDR1 (multidrug resistance) genes in 110 adult patients with intermediate-risk AML, enrolled in the CETLAM-99 prospective trial. A multivariate prognostic model adjusted for age, white blood cell (WBC) count, French-American-British group, cytogenetics, MLL rearrangement, internal tandem duplication of FLT3 (FLT3-ITD), induction courses to achieve complete remission, and germline polymorphisms, was used to detect independent risk factors associated with clinical outcome. This analysis showed an increased risk of refractoriness to chemotherapy in the group of patients with XPA variant alleles (RR = 14; P = .02). In the same model, increased relapse risk was associated with SULT1C2 heterozygosity (RR = 4.1; P = .004), FLT3-ITD (RR 3.3; P = .003), and MDR1 variant alleles (RR = 2.4; P = .02). Adverse prognostic variables for overall survival were XPA (RR = 3.4; P = .02) and MDR1 (RR = 2.1; P = .02) variant alleles, and WBC count (RR = 2.1; P = .02). These findings might be useful in selecting risk-adapted treatment strategies in intermediate-risk AML.

Microsatellite instability is not an uncommon finding in adult de novo acute myeloid leukemia.

To investigate the biologic relevance of microsatellite instability (MSI) in de novo acute myeloid leukemia (AML), 102 consecutive adult patients were analyzed by using a panel of seven microsatellites (BAT25, BAT26, D13S1267, D13S174, D2S123, D5S346 and Mdf15). Frame-shift mutations in the repetitive sequences in the coding region of MSH3, MSH6, BAX, TGFBRII and IGFRII were also investigated by using a fluorescent PCR-based assay. Methylation-specific PCR was used to determine the methylation status of hMLH1 in MSI+ cases. MSH3, MSH6 and MLH1 expression was also analyzed in 68 cases by means of real-time quantitative PCR. MSI was detected in 20 cases: 14 cases had MSI-high (instability of at least two microsatellite markers) and 6 cases corresponded to MSI-low (a single polymorphic marker with instability). Six MSI+ cases showed an associated MLL rearrangement (p=0.002). No single case showed a mutation in the repetitive sequences of the MSH3, MSH6, BAX, TGFBRII and IGFRII genes. Most samples displayed low mRNA levels of the repair genes. hMLH1 promoter was hypermethylated in five MSI+ cases. Overall survival analysis revealed no adverse effect of MSI positivity. These results suggest that MSI may be a common biologic finding in de novo AML.

High-dose infusional ifosfamide, etoposide plus methylprednisolone followed by dexamethasone, high-dose ara-C and cisplatinum and autologous stem cell transplantation for refractory or relapsed aggressive non-Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVES: A salvage program including infusional high-dose ifosfamide plus etoposide (IFOVM) was evaluated in patients with refractory or relapsed aggressive non-Hodgkin's lymphoma. DESIGN AND METHODS: Forty-six patients were included. IFOVM consisted of ifosfamide (10 g/m2 as a 72-hour continuous infusion), etoposide (900 mg/m2) and methylprednisolone; responding patients underwent two cycles of DHAP and subsequently an autologous peripheral blood stem cell transplantation (APBSCT) with BEAM as the conditioning regimen. RESULTS: All but one patient showed tumor regression following IFOVM. Myelosuppression was brief but 26 patients developed neutropenic fever. All but two patients proceeded to DHAP. Overall response rate to IFOVM/DHAP was 59% (29% CR and 30% PR). Refractory patients had a significantly lower response rate than relapsed patients (39% vs. 85% p=0.002). All refractory patients with intermediate-high or high IPI progressed during IFOVM/DHAP. Twenty-seven patients proceeded to APBSCT. Two-year overall survival of patients with low or low-intermediate IPI was 47% [95% CI 25-69%], which was significantly better than that obtained in patients with intermediate-high or high IPI (11% [95% CI 0-22%] p=0.0001). INTERPRETATION AND CONCLUSIONS: This sequential regimen of IFOVM, followed by DHAP and consolidated with BEAM is active in relapsed or refractory patients with low or low-intermediate IPI aggressive lymphoma. However, it has little activity in those patients with intermediate or high IPI, especially in refractory lymphomas.

Cyclophosphamide, pegylated liposomal doxorubicin (Caelyx), vincristine and prednisone (CCOP) in elderly patients with diffuse large B-cell lymphoma: results from a prospective phase II study.

BACKGROUND AND OBJECTIVES: Anthracycline-based combination chemotherapy regimens are the standard therapy for patients with diffuse large B-cell lymphoma (DLBCL), but such regimens may be poorly tolerated in elderly patients. DESIGN AND METHODS: In a prospective phase II study we analyzed the feasibility of a regimen (CCOP) that includes pegylated liposomal doxorubicin (Caelyx ) plus vincristine, cyclophosphamide and prednisone in patients with DLBCL above the age of 60 years. RESULTS: Thirty-three patients, with a median age of 74 years, were enrolled in the study. The overall response rate was 64% (49% complete remissions and 15% partial remissions). The estimated one-year overall and event-free survivals were 55% (95% CI, 38-72) and 45% (95%CI, 28-62), respectively. The only relevant toxicity was neutropenia, which reached grades 3-4 in 21 cases (64%). INTERPRETATION AND CONCLUSIONS: These results suggest that CCOP appears to be an acceptable alternative for elderly patients with DLBCL, and randomized trials against a conventional doxorubicin-containing regimen are justified.