Marcos González

Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma

We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively. Patients who were MRD(-) by sequencing had a significantly longer time to tumor progression (TTP) (median 80 vs 31 months; P < .0001) and overall survival (median not reached vs 81 months; P = .02), compared with patients who were MRD(+). When stratifying patients by different levels of MRD, the respective TTP medians were: MRD ≥10(-3) 27 months, MRD 10(-3) to 10(-5) 48 months, and MRD <10(-5) 80 months (P = .003 to .0001). Ninety-two percent of VGPR patients were MRD(+). In complete response patients, the TTP remained significantly longer for MRD(-) compared with MRD(+) patients (131 vs 35 months; P = .0009).

Deregulation of microRNA expression in the different genetic subtypes of multiple myeloma and correlation with gene expression profiling

Specific microRNA (miRNA) signatures have been associated with different cytogenetic subtypes in acute leukemias. This finding prompted us to investigate potential associations between genetic abnormalities in multiple myeloma (MM) and singular miRNA expression profiles. Moreover, global gene expression profiling was also analyzed to find correlated miRNA gene expression and select miRNA target genes that show such correlation. For this purpose, we analyzed the expression level of 365 miRNAs and the gene expression profiling in 60 newly diagnosed MM patients, selected to represent the most relevant recurrent genetic abnormalities. Supervised analysis showed significantly deregulated miRNAs in the different cytogenetic subtypes as compared with normal PC. It is interesting to note that miR-1 and miR-133a clustered on the same chromosomal loci, were specifically overexpressed in the cases with t(14;16). The analysis of the relationship between miRNA expression and their respective target genes showed a conserved inverse correlation between several miRNAs deregulated in MM cells and CCND2 expression level. These results illustrate, for the first time, that miRNA expression pattern in MM is associated with genetic abnormalities, and that the correlation of the expression profile of miRNA and their putative mRNA targets is useful to find statistically significant protein-coding genes in MM pathogenesis associated with changes in specific miRNAs.

Outcome of patients with acute promyelocytic leukemia failing to front-line treatment with all-trans retinoic acid and anthracycline-based chemotherapy (PETHEMA protocols LPA96 and LPA99): benefit of an early intervention.

To determine prognosis of acute promyelocytic leukemia (APL) failing to front-line therapy with all-trans retinoic acid (ATRA) and anthracyclines, outcome of 52 patients (32 M/20 F; age: 37, 3–72) included in PETHEMA trials LPA96 and LPA99 who presented with either molecular failure (MOLrel, n=16) or hematological relapse (HEMrel, n=36) was analyzed. Salvage therapy consisted of ATRA and high-dose ara-C-based chemotherapy (HDAC) in most cases (83%), followed by stem-cell transplantation (autologous, 18; allogeneic, 10; syngeneic, 1). Fourteen patients with MOLrel (88%) achieved second molecular complete response (molCR), whereas 81% HEMrel patients responded to second-line treatment, with 58% molCR. After median follow-up of 45 months, four MOLrel and 18 HEMrel patients, respectively, experienced a second relapse. Outcome after MOLrel compared favorably to HEMrel, with longer survival (5-year survival: 64±14 vs 24±8%, P=0.01) and lower relapse risk (5-year relapse risk: 30±13 vs 64±9%; P=0.044). Additionally, age⩽40 and male gender were favorable variables for survival, whereas molecular response predicted longer leukemia-free survival. In conclusion, early institution of salvage therapy at molecular failure, before onset of hematological relapse, is beneficial in APL. Moreover, given the poor outcome of HEMrel managed with ATRA and HDAC, use of alternative therapeutic strategies in this setting is warranted.

Single-unit umbilical cord blood transplantation from unrelated donors in patients with hematological malignancy using busulfan, thiotepa, fludarabine and ATG as myeloablative conditioning regimen

Attempts to optimize outcomes in cord blood transplantation (CBT) by using new conditioning regimens and standardization of cord blood unit selection are warranted. In all, 88 patients (18 children and 70 adults) with hematological malignancy from nine Spanish institutions underwent a single-unit CBT after an i.v. BU-based myeloablative conditioning regimen. All evaluable patients except one engrafted. The overall cumulative incidence (CI) of myeloid engraftment was 94% at a median time of 19 days. In multivariate analysis, nonadvanced disease stage was the only factor with a favorable impact on myeloid engraftment. The CI of acute GVHD grades II–IV and chronic extensive GVHD were 24% each. The CI of nonrelapse mortality at 100 days, 180 days and 5 years was 14, 23 and 44%, respectively. The 5-year CI of relapse was 18%, whereas disease-free survival (DFS) was 46%, 39% and 11% for patients transplanted in early, intermediate and advanced stages of the disease, respectively. Our study shows high rates of engraftment with fast neutrophil recovery in patients undergoing single-unit CBT using a BU-based conditioning regimen. Long-term DFS can be achieved in a substantial number of patients with high-risk hematological malignancies, particularly when transplanted in an early stage of the disease.

Bisphosphonate‐related osteonecrosis: genetic and acquired risk factors

The objectives of this study were to review epidemiological, clinical and biological aspects associated with the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ) in multiple myeloma (MM) patients, with special emphasis on the genetic aspects. A detailed review of previously described risk factors as well as recent genetic findings mostly comprises this work. The most recent meeting abstracts and relevant articles published in journals covered by the Science Citation Index and Medline are also examined. The review pays special attention to the genetic component of BRONJ. A total of 15 series and 14 guidelines or revisions were selected to fit the aims of the review. Gene variability was reviewed in depth to give a clinical illustration on the genetic aspects of BRONJ. Crude prevalence and 5-year cumulative incidence were considered as the most important end points for predictive purposes. Several acquired factors were recognized as predictors for BRONJ in MM, especially intravenous bisphosphonates, dental trauma and advanced age. Among genetic factors, polymorphisms on CYP2C8 gene arise as a promising risk factor. Bisphosphonate-related osteonecrosis of the jaw can be predicted with a conjunction of genetic and environmental risk factors.

Pretreatment characteristics and clinical outcome of acute promyelocytic leukaemia patients according to the PML‐RARα isoforms: a study of the PETHEMA group

Of 167 newly diagnosed acute promyelocytic leukaemia patients, 83 patients were long (L)‐form (50%), eight variable (V)‐form (5%) and 76 short (S)‐form (45%). The V‐form and S‐form groups presented a significantly higher percentage of patients with white blood cell counts >u200a10u2003×u2003109/l (Pu2003<u20030·05). The S‐form cases displayed a significantly higher number of cases with M3v microgranular features (Pu2003=u20030·005) and CD34 expression (Pu2003<u20030·0001). There were no differences between the three isoforms in complete remission (CR) rate (overall CR 90%), but the 3‐year disease‐free survival was lower for V‐form cases than it was for L‐ and S‐form cases (62% vs. 94% and 89%, Pu2003=u20030·056). We conclude that the V‐form and S‐form types are associated with some negative prognostic features at diagnosis. However, our data were only able to demonstrate an association with adverse prognosis in the V‐form type and, moreover, as the number of cases was limited, needs to be confirmed in large, uniformly treated series.

Role of MTHFR (677, 1298) haplotype in the risk of developing secondary leukemia after treatment of breast cancer and hematological malignancies

Therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) is a malignancy occurring after exposure to chemotherapy and/or radiotherapy. Polymorphisms involved in chemotherapy/radiotherapy response genes could be related to an increased risk of developing this neoplasia. We have studied 11 polymorphisms in genes of drug detoxification pathways (NQO1, glutathione S-transferase pi) and DNA repair xeroderma pigmentosum, complementation group (3) (XPC(3), X-ray repair cross complementing protein (1)), Nijmegen breakage syndrome (1), excision repair cross-complementing rodent repair deficiency, complementation group (5) and X-ray repair cross complementing protein (3) and in the methylene tetrahydrofolate reductase gene (MTHFR(2), 677C>T, 1298A>C), involved in DNA synthesis. The analyzed groups were a t-MDS/AML patients group (n=81) and a matched control group (n=64) treated similarly, and they did not develop t-MDS/AML. We found no significant differences when the groups were compared globally. However, when analysis was carried out according to the primary neoplasia involved, a significant association was observed between the MTHFR haplotype (single nucleotide polymorphisms 677 and 1298) and the risk of developing t-MDS/AML in the breast cancer patients group (P=0.016) and cyclophosphamide-treated hematological disease group (P=0.005). Risk haplotype was different for each case, corresponding to the 677T1298A haplotype after breast cancer treatment and the 677C1298C haplotype after hematological malignancy treatment. We postulate that such differences are related to variations in chemotherapy schemes between hematological and breast cancers and their differential interaction with the MTHFR route.

Molecular characterization of immunoglobulin gene rearrangements in diffuse large B-cell lymphoma: antigen-driven origin and IGHV4-34 as a particular subgroup of the non-GCB subtype.

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.

Observational prospective study of viral infections in children undergoing allogeneic hematopoietic cell transplantation: a 3-year GETMON experience

We studied surveillance, incidence and outcome of viral infections in children undergoing allogeneic hematopoietic cell transplantation (HCT) in the main pediatric transplant units in Spain. We prospectively collected data from first year post-HCT in every consecutive allogeneic HCT performed during 3 years (N=215): first HCT=188 and second HCT=27; median age=6.6 years (0.1–20.7). Most patients had acute leukemia (N=137) and 135 recipients (63%) were CMV seropositive. A total of 46 patients underwent cord blood transplant, 133 patients underwent HCT from alternative donors (62%) and 101 patients received anti-thymocyte globulin. Observational time was completed in 137 patients, whereas the remaining 78 died after a median survival time of 99 days (3–352). CMV was monitored in all patients; adenovirus (ADV) and human herpesvirus 6 (HHV-6) were monitored in 101 and 33 patients, respectively. We found 145 viral infections in 103 patients: CMV (n=42), ADV (n=32), HHV-6 (n=7), polyomavirus (n=20), EBV (n=6), VZV (n=17) and others (n=8). CMV infection was significantly higher in seropositive patients (25 vs 7%) (P=0.02). Extensive chronic GVHD (cGVHD) was significantly associated with an increased rate of viral infections (12 of 16 patients with cGVHD had infections vs 91 of 199 without GVHD) (P=0.035). In total, 10 patients (4.6%) died of viral infections (CMV=5, ADV=3, respiratory=2). We found a high incidence of viral infection, but mortality was low.

BAALC is an important predictor of refractoriness to chemotherapy and poor survival in intermediate-risk acute myeloid leukemia (AML)

We have analyzed brain and acute leukemia, cytoplasmic (BAALC) gene expression and other genetic markers (ERG, EVI1, MN1, PRAME, WT1, FLT3, and NPM1 mutations) in 127 intermediate-risk acute myeloid leukemia (AML) patients: 98 cytogenetically normal and 29 with intermediate-risk cytogenetic alterations. High versus low BAALC expressers showed a higher refractoriness to induction treatment (31% vs 10%; pu2009=u2009.005), lower complete remission rate after salvage therapy (82% vs 97%; pu2009=u2009.010), and lower 3-year overall (23% vs 58%, pu2009<u2009.001) and relapse-free survival (26% vs 52%, pu2009=u2009.006). Similar results were found when cytogenetic subgroups were analyzed separately. Multivariate models confirmed the unfavorable prognosis of this marker. In conclusion, BAALC is a relevant prognostic marker in intermediate-risk AML.