Clinical, biological, and prognostic implications of SF3B1 co-occurrence mutations in very low/low- and intermediate-risk MDS patients.

SF3B1 is a highly mutated gene in myelodysplastic syndrome (MDS) patients, related to a specific subtype and parameters of good prognosis in MDS without excess blasts. More than 40% of MDS patients carry at least two myeloid-related gene mutations but little is known about the impact of concurrent mutations on the outcome of MDS patients. In applying next-generation sequencing (NGS) with a 117 myeloid gene custom panel, we analyzed the co-occurrence of SF3B1 with other mutations to reveal their clinical, biological, and prognostic implications in very low/low- and intermediate-risk MDS patients. Mutations in addition to those of SF3B1 were present in 80.4% of patients (median of 2 additional mutations/patient, range 0-5). The most frequently mutated genes were as follows: TET2 (39.2%), DNMT3A (25.5%), SRSF2 (10.8%), CDH23 (5.9%), and ASXL1, CUX1, and KMT2D (4.9% each). The presence of at least two mutations concomitant with that of SF3B1 had an adverse impact on survival compared with those with the SF3B1 mutation and fewer than two additional mutations (median of 54 vs. 87 months, respectively: p = 0.007). The co-occurrence of SF3B1 mutations with specific genes is also linked to a dismal prognosis: SRSF2 mutations were associated with shorter overall survival (OS) than SRSF2wt (median, 27 vs. 75 months, respectively; p = 0.001), concomitant IDH2 mutations (median OS, 11 [mut] vs. 75 [wt] months; p = 0.001), BCOR mutations (median OS, 11 [mut] vs. 71 [wt] months; p = 0.036), and NUP98 and STAG2 mutations (median OS, 27 and 11 vs. 71 months, respectively; p = 0.008 and p = 0.002). Mutations in CHIP genes (TET2, DNMT3A) did not significantly affect the clinical features or outcome. Our results suggest that a more comprehensive NGS study in low-risk MDS SF3B1mut patients is essential for a better prognostic evaluation.

Optimal large-scale CD34+ enrichment from a leukapheresis collection using the clinimacs prodigy platform.

Optimization of Hematology Patient's treatment: It is possible to obtain a 100% CD34+ recovery after CD34+ selection using the CliniMACS Prodigy.

Results of an Early Access Treatment Protocol of Daratumumab Monotherapy in Spanish Patients With Relapsed or Refractory Multiple Myeloma.

Daratumumab is a human CD38-targeted monoclonal antibody approved as monotherapy for heavily pretreated relapsed and refractory multiple myeloma. We report findings for the Spanish cohort of an open-label treatment protocol that provided early access to daratumumab monotherapy and collected safety and patient-reported outcomes data for patients with relapsed or refractory multiple myeloma. At 15 centers across Spain, intravenous daratumumab (16 mg/kg) was administered to 73 patients who had ≥3 prior lines of therapy, including a proteasome inhibitor and an immunomodulatory drug, or who were double refractory to both. The median duration of daratumumab treatment was 3.3 (range: 0.03-13.17) months, with a median number of 12 (range: 1-25) infusions. Grade 3/4 treatment-emergent adverse events were reported in 74% of patients and included lymphopenia (28.8%), thrombocytopenia (27.4%), neutropenia (21.9%), leukopenia (19.2%), and anemia (15.1%). Common (>5%) serious treatment-emergent adverse events included respiratory tract infection (9.6%), general physical health deterioration (6.8%), and back pain (5.5%). Infusion-related reactions occurred in 45% of patients. The median change from baseline in all domains of the EQ-5D-5L and EORTC QLQ-C30 was mostly 0. A total of 18 (24.7%) patients achieved a partial response or better, with 10 (13.7%) patients achieving a very good partial response or better. Median progression-free survival was 3.98 months. The results of this early access treatment protocol are consistent with previously reported trials of daratumumab monotherapy and confirm its safety and antitumoral efficacy in Spanish patients with heavily treated relapsed or refractory multiple myeloma. European Clinical Trials Database number: 2015-002993-19.

Diagnostic value of bone marrow core biopsy patterns in lymphoplasmacytic lymphoma/Waldenström macroglobulinaemia and description of its mutational profiles by targeted NGS.

AIMS: The aim of this study was to describe the characteristics of the bone marrow infiltration found in a series of clinically defined lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinaemia (WM) and IgM-monoclonal gammopathy of undetermined significance (MGUS) and to perform a targeted next-generation sequencing (NGS) for the identification of additional somatic mutations to MYD88p.L265P in LPL/WM. METHODS: We have reviewed a series of 35 bone marrow biopsies from 28 patients with a clinical diagnosis of LPL/WM (24 cases) or MGUS (4 cases). Bone marrow infiltration characteristics by morphology, immunohistochemistry, flow cytometry (FCM), allele-specific real-time PCR for the detection of MYD88p.L265P mutation, targeted exonic amplicon-based NGS of 35 lymphoma-related genes and direct sequencing were analysed. RESULTS: Our findings show that bone marrow trephine biopsy evaluation is superior to FCM in the identification of significant lymphoid infiltrates. A combined paratrabecular and interstitial infiltration pattern is the most common feature in LPL/WM while a patchy interstitial pattern characterises IgM-MGUS cases. MYD88p.L265P mutation was found by allele-specific-PCR in 92% of the LPL cases (22 out of 24) and 25% of IgM-MGUS cases (1 out of 4 cases). In addition to MYD88p.L265P somatic mutations in CXCR4, KMT2D, PRDM1/Blimp1, MYC and ID3 were found by NGS and direct sequencing in 4 cases. CONCLUSIONS: In conclusion, bone marrow core biopsy evaluation is critical in the identification of unequivocal bone marrow infiltration by LPL/WM. In addition to MYD88p.L265P, somatic mutations in CXCR4, KMT2D, PRDM1/Blimp1, MYC and ID3 can appear in a fraction of LPL/WM.

Multidimensional assessment of patient condition and mutational analysis in peripheral blood, as tools to improve outcome prediction in myelodysplastic syndromes: A prospective study of the Spanish MDS group.

The International Prognostic Scoring System and its revised form (IPSS-R) are the most widely used indices for prognostic assessment of patients with myelodysplastic syndromes (MDS), but can only partially account for the observed variation in patient outcomes. This study aimed to evaluate the relative contribution of patient condition and mutational status in peripheral blood when added to the IPSS-R, for estimating overall survival and the risk of leukemic transformation in patients with MDS. A prospective cohort (2006-2015) of 200 consecutive patients with MDS were included in the study series and categorized according to the IPSS-R. Patients were further stratified according to patient condition (assessed using the multidimensional Lee index for older adults) and genetic mutations (peripheral blood samples screened using next-generation sequencing). The change in likelihood-ratio was tested in Cox models after adding individual covariates. The addition of the Lee index to the IPSS-R significantly improved prediction of overall survival [hazard ratio (HR) 3.02, 95% confidence interval (CI) 1.96-4.66, P < 0.001), and mutational analysis significantly improved prediction of leukemic evolution (HR 2.64, 1.56-4.46, P < 0.001). Non-leukemic death was strongly linked to patient condition (HR 2.71, 1.72-4.25, P < 0.001), but not to IPSS-R score (P = 0.35) or mutational status (P = 0.75). Adjustment for exposure to disease-modifying therapy, evaluated as a time-dependent covariate, had no effect on the proposed model's predictive ability. In conclusion, patient condition, assessed by the multidimensional Lee index and patient mutational status can improve the prediction of clinical outcomes of patients with MDS already stratified by IPSS-R.

Bone marrow fibrosis in myelodysplastic syndromes: a prospective evaluation including mutational analysis.

The biological and molecular events that underlie bone marrow fibrosis in patients with myelodysplastic syndromes are poorly understood, and its prognostic role in the era of the Revised International Prognostic Scoring System (IPSS-R) is not yet fully determined. We have evaluated the clinical and biological events that underlie bone marrow fibrotic changes, as well as its prognostic role, in a well-characterized prospective patient cohort (n=77) of primary MDS patients. The degree of marrow fibrosis was linked to parameters of erythropoietic failure, marrow cellularity, p53 protein accumulation, WT1 gene expression, and serum levels of CXCL9 and CXCL10, but not to other covariates including the IPSS-R score. The presence of bone marrow fibrosis grade 2 or higher was associated with the presence of mutations in cohesin complex genes (31.5% vs. 5.4%, p=0.006). By contrast, mutations in CALR, JAK2, PDGFRA, PDGFRB,and TP53 were very rare. Survival analysis showed that marrow fibrosis grade 2 or higher was a relevant significant predictor for of overall survival, and independent of age, performance status, and IPSS-R score in multivariate analysis.

Prognostic impact of chromosomal translocations in myelodysplastic syndromes and chronic myelomonocytic leukemia patients. A study by the spanish group of myelodysplastic syndromes.

Chromosomal translocations are rare in the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). With the exception of t(3q), translocations are not explicitly considered in the cytogenetic classification of the IPSS-R and their impact on disease progression and patient survival is unknown. The present study was aimed at determining the prognostic impact of translocations in the context of the cytogenetic classification of the IPSS-R. We evaluated 1,653 patients from the Spanish Registry of MDS diagnosed with MDS or CMML and an abnormal karyotype by conventional cytogenetic analysis. Translocations were identified in 168 patients (T group). Compared with the 1,485 patients with abnormal karyotype without translocations (non-T group), the T group had a larger proportion of patients with refractory anemia with excess of blasts and higher scores in both the cytogenetic and global IPSS-R. Translocations were associated with a significantly shorter survival and higher incidence of transformation into AML at univariate analysis but both features disappeared after multivariate adjustment for the IPSS-R cytogenetic category. Patients with single or double translocations other than t(3q) had an outcome similar to those in the non-T group in the intermediate cytogenetic risk category of the IPSS-R. In conclusion, the presence of translocations identifies a subgroup of MDS/CMML patients with a more aggressive clinical presentation that can be explained by a higher incidence of complex karyotypes. Single or double translocations other than t(3q) should be explicitly considered into the intermediate risk category of cytogenetic IPSS-R classification.

CD 30-positive transformed follicular lymphoma: two case reports and literature review.

AIMS: Here we report two cases of follicular lymphoma that transformed to CD30 positive diffuse large B cell lymphoma and review the literature on this topic. RESULTS: The first case represents an example of early transformation of conventional low-grade follicular lymphoma to CD30-positive large B cell lymphoma. Immunoglobulin (Ig)H and cytogenetic identity was demonstrated between both components. High-dose and auto-stem cell transplant (SCT) was applied and complete response was achieved. The second case represents an example of d'emblee transformation of intrafollicular neoplasia to CD30-positive large B cell lymphoma. Immunoglobulin K deleting element (IgKde) and cytogenetic identity between both phases was demonstrated. The patient was in partial response after four cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). CONCLUSIONS: CD30 expression was found to be associated in these cases to the transformation event and could be considered a therapeutic target to add to conventional immunochemotherapeutic regimens, even in combination with auto-SCT. We suggest looking for CD30 expression in transformed follicular lymphoma cases.

Lymph node tuberculosis after allogeneic haematopoietic stem cell transplantation: an atypical presentation of an uncommon complication.

Mycobacterium tuberculosis infections are uncommon complications in the haematopoietic stem cell post-transplant period. Most cases are reactivations of latent infections affecting the lung. We present an atypical case of isolated lymph node tuberculosis after an allogeneic haematopoietic stem cell transplantation, which highlights the importance of having a high suspicion index, even in non-endemic countries.

Reciprocal translocations in myelodysplastic syndromes and chronic myelomonocytic leukemias: review of 5,654 patients with an evaluable karyotype.

The infrequency of translocations in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemias (CMML) makes their identification and reporting interesting for the recognition of the recurrent ones and the genes involved in these neoplasias. The aims of this study were to identify new translocations associated with MDS and CMML and to establish their frequency in a cohort of 8,016 patients from the Spanish Group of MDS database. The karyotype was evaluable in 5,654 (70%) patients. Among those, 2,014 (36%) had chromosomal abnormalities, including 213 (10%) translocations identified in 195 patients. The translocations were balanced in 183 (86%) cases and unbalanced in 30 (14%) cases. All chromosomes were found to be involved in translocations, with the single exception of the Y chromosome. The chromosomes most frequently involved were in decreasing frequency: 3, 1, 7, 2, 11, 5, 12, 6, and 17. Translocations were found in karyotypes as the unique chromosomal abnormality (33%), associated with another chromosomal abnormality (11%), as a part of a complex karyotype (17%), and as a part of a monosomal karyotype (38%). There were 155 translocations not previously described in MDS or CMML and nine of them appeared to be recurrent.

Simultaneous analysis of the expression of 14 genes with individual prognostic value in myelodysplastic syndrome patients at diagnosis: WT1 detection in peripheral blood adversely affects survival.

Several studies have evaluated the prognostic value of the individual expression of certain genes in patients with myelodysplastic syndromes (MDS). However, none of them includes their simultaneous analysis by quantitative polymerase chain reaction (PCR). We evaluated relative expression levels of 14 molecular markers in 193 peripheral blood samples from untreated MDS patients using real-time PCR. Detectable WT1 expression levels, low TET2, and low IER3 gene expression were the only markers showing in univariate analysis a poor prognostic value for all treatment-free (TFS), progression-free (PFS), and overall survival (OS). In multivariate analysis, molecular parameters associated with a shorter TFS were: WT1 detection (p = 0.014), low TET2 (p = 0.002), and low IER3 expression (p = 0.025). WT1 detection (p = 0.006) and low TET2 (p = 0.006) expression were associated with a shorter PFS when multivariate analysis was carried out by including only molecular markers. Molecular values with an independent value in OS were: WT1 detection (p = 0.003), high EVI1 expression (p = 0.001), and undetectatable p15-CDKN2B (p = 0.037). WT1 expressers were associated with adverse clinical-biological features, high IPSS and WPSS scoring, and unfavorable molecular expression profile. In summary, detectable WT1 expression levels, and low TET2 and low IER3 expression in peripheral blood showed a strong association with adverse prognosis in MDS patients at diagnosis. However, WT1 was the only molecular marker displaying an independent prognostic value in both OS and TFS.

FLAGIDA-lite is an effective regimen for patients between 70 and 80 years with acute myeloid leukemia or refractory anemia with excess blasts-2 and is feasible as outpatient treatment.

We investigated a FLAGIDA-lite protocol (fludarabine 40 mg/m(2)/d orally days 1-5, cytarabine 20 mg/m(2)/d subcutaneously days 1-5, G-CSF 300 µg/d subcutaneously days 1-5, and idarrubicin 15 mg/m(2)/d orally days 1-3) in 38 consecutive patients older than 70 years of age with acute myeloid leukemia (32 patients) or refractory anemia with excess blasts-2 (six patients) and no prior therapy. Seventy-nine percent had intermediate/unfavorable karyotype and 79% had a high comorbidity. Overall response was 55% [complete response (CR) 47%] and 37% were refractory. CR rate was 52% in patients between 71 and 79 years of age and 38% in patients 80 years or older. The 4-week induction mortality was 16% (8% in patients between 70 and 79 years of age and 32% in patients 80 years or older). Overall survival (OS) at 3 years was 22% (31.3% in patients between 70 and 79 years and 15.4% in patients 80 years or older). Relapse-free survival (RFS) at 3 years was 15%. A total of 65 cycles (47 as induction and 18 as consolidation) were administered, 46 of them (70%) in an outpatient setting. In summary, this FLAGIDA-lite protocol is an effective and well-tolerated option for patients between the ages of 70 and 79 years with acute myeloid leukemia or refractory anemia with excess blasts-2 and is usually feasible as outpatient treatment, but is not beneficial for most patients 80 years or older.

Simultaneous cytomorphologic and multiparametric flow cytometric analysis on lymph node samples is faster than and as valid as histopathologic study to diagnose most non-Hodgkin lymphomas.

We evaluated the validity and accuracy of cytomorphology and multiparametric flow cytometry (C-FCM) in diagnosing oncohematologic disease in 223 consecutive lymph node biopsy specimens from patients with lymphadenopathy, from 2004 to 2007. C-FCM and histopathologic studies were interpreted independently by hematologists and pathologists, respectively. C-FCM detected neoplastic disorders in 133 samples (59.6%): 92 non-Hodgkin lymphomas (NHLs; 41.3%), 21 Hodgkin lymphomas (HLs; 9.4%), 19 malignant nonhematologic neoplasms (8.5%), and 1 multiple myeloma (0.4%). Sensitivity and specificity were 87.25% and 95.95%, respectively. Positive predictive value and negative predictive value (NPV) were 97.74% and 78.89%, respectively. Sensitivity and NPV were 94.79% and 96.81% upon excluding HL and malignant nonhematologic neoplasms from the analysis. Of the 92 NHLs, 89 (97%) were categorized according to the 2001 World Health Organization classification of hematolymphoid neoplasms with a concordance of 87%. The C-FCM study was significantly faster than the histopathologic study. C-FCM has high sensitivity and specificity, allowing for a valid and reliable diagnosis, especially in NHLs and enabling their subclassification. C-FCM is faster than the histopathologic examination, allowing for therapeutic decisions to be made quickly. However, in the samples in which C-FCM cannot establish a diagnosis, histopathologic results are needed.

Fluorescence in situ hybridization improves the detection of 5q31 deletion in myelodysplastic syndromes without cytogenetic evidence of 5q-.

BACKGROUND: More than 50% of patients with myelodysplastic syndromes present cytogenetic aberrations at diagnosis. Partial or complete deletion of the long arm of chromosome 5 is the most frequent abnormality. The aim of this study was to apply fluorescence in situ hybridization of 5q31 in patients diagnosed with de novo myelodysplastic syndromes in whom conventional banding cytogenetics study had shown a normal karyotype, absence of metaphases or an abnormal karyotype without evidence of del(5q). DESIGN AND METHODS: We performed fluorescence in situ hybridization of 5q31 in 716 patients, divided into two groups: group A patients (n=637) in whom the 5q deletion had not been detected at diagnosis by conventional banding cytogenetics and group B patients (n=79), in whom cytogenetic analysis had revealed the 5q deletion (positive control group). RESULTS: In group A (n=637), the 5q deletion was detected by fluorescence in situ hybridization in 38 cases (5.96%). The majority of positive cases were diagnosed as having the 5q- syndrome. The deletion was mainly observed in cases in which the cytogenetics study had shown no metaphases or an aberrant karyotype with chromosome 5 involved. In group B (n=79), the 5q deletion had been observed by cytogenetics and was confirmed to be present in all cases by fluorescence in situ hybridization of 5q31. CONCLUSIONS: Fluorescence in situ hybridization of 5q31 detected the 5q deletion in 6% of cases without clear evidence of del(5q) by conventional banding cytogenetics. We suggest that fluorescence in situ hybridization of 5q31 should be performed in cases of a suspected '5q- syndrome' and/or if the cytogenetic study shows no metaphases or an aberrant karyotype with chromosome 5 involved (no 5q- chromosome).

Follow-up of healthy donors receiving granulocyte colony-stimulating factor for peripheral blood progenitor cell mobilization and collection. Results of the Spanish Donor Registry.

BACKGROUND: Information about the long-term follow-up and safety of granulocyte colony-stimulating factor administration to healthy donors is limited. The aims of this study were to analyze the side effects of granulocyte colony-stimulating factor administration in donors included in a Spanish Registry of hematopoietic stem cell donors and to determine the long-term outcome of these donors. DESIGN AND METHODS: The Spanish National Donor Registry was developed to record the short- and long-term results of granulocyte colony-stimulating factor administration to mobilize peripheral blood progenitor cells in normal donors. To date, 1436 donors (771 males, 665 females) with a median age of 37 years (range, 1 to 74 years) have been registered. Granulocyte colony-stimulating factor was the only cytokine administered. A baseline investigation was performed in every donor before granulocyte colony-stimulating factor administration and follow-up investigations (controls) were planned at 4 weeks and annually thereafter for up to 5 years after the mobilization. RESULTS: At least one of the scheduled controls was performed in 736 donors, while 320 donors have been followed for 2 years or more. The peripheral white blood cell count decreased significantly from 6.8 x 10(9)/L at baseline to 5.9 x 10(9)/L at 4 weeks after leukapheresis (p<0.0001) and remained at values lower than those observed premobilization until 2 years after mobilization. In contrast, hemoglobin concentration and platelet count returned to normal values within 1 year after mobilization. Bone pain (90%) and headache (33%) were the most frequently reported granulocyte colony-stimulating factor-related side effects. Five patients (0.68%) were diagnosed as having solid tumors (lung cancer in two patients and thyroid carcinoma, choroid melanoma, and colon carcinoma in one patient each) between 10 and 64 months after administration of granulocyte colony-stimulating factor. No hematologic malignancies have been reported. CONCLUSIONS: The clinical side effects of granulocyte colony-stimulating factor administration in healthy donors are generally mild. Changes in blood counts were minimal and mainly affected white blood cell counts, which returned to normal values within 2 years after granulocyte-colony stimulating factor administration. No patient developed a hematologic malignancy. A larger number of donors and longer follow-up are needed to determine the safety of granulocyte colony-stimulating factor administration definitively.

Basal CD34(+) cell count predicts peripheral blood progenitor cell mobilization and collection in healthy donors after administration of granulocyte colony-stimulating factor.

We analyzed factors predicting CD34(+) cell mobilization and collection after granulocyte colony-stimulating factor (G-CSF) administration in 47 healthy donors. Basal CD34(+) cell count and sex were the two variables that significantly predicted a better CD34(+) cell mobilization, and greater age was the only variable associated with lower CD34+ cell yields.

Tandem transplants with different high-dose regimens improve the complete remission rates in multiple myeloma. Results of a Grupo Español de Síndromes Linfoproliferativos/Trasplante Autólogo de Médula Osea phase II trial.

Between 1994 and 1999, 88 multiple myeloma (MM) patients were included in a phase II study to evaluate a tandem autologous stem cell transplantation (ASCT) programme. The first was conditioned with melphalan 200 mg/m2 (MEL200-ASCT1), and the second with cyclophosphamide, etoposide and BCNU (CBV-ASCT2). All patients were in response after MEL200-ASCT1. A control group of MM patients with response to a single ASCT was selected to compare outcomes. After MEL200-ASCT1, 26 patients (30%) achieved complete remission (CR). Of the remaining 48 evaluable patients, 16 (33%) achieved CR with CBV-ASCT2. The final CR rate was 48%. The 5-year survival (OS) was 55%[95% confidence interval (CI) 43-67%] while the event-free survival (EFS) was 28% (95% CI 15-39%). CR status after CBV-ASCT2 was the most important prognostic factor for OS and EFS (P = 0.00001), although no differences in outcomes were detected when the patients in CR after MEL200-ASCT1 were compared with those who obtained CR after CBV-ASCT2. Univariate and multivariate analyses showed improved OS and EFS for the tandem series as compared with the control series treated with a single MEL200-ASCT. However, in a stratified comparison by response, there were no prognostic differences between tandem patients and control patients treated with a single ASCT. In summary, our study suggests that the benefit of a second high-dose therapy course depends on its capacity to result in CR for MM patients who have not attained CR after ASCT1.