Cristina Jiménez

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).

Absence of platelet response after eradication of Helicobacter pylori infection in patients with chronic idiopathic thrombocytopenic purpura

Eradication of Helicobacter pylori infection has been associated with the correction of thrombocytopenia in patients with idiopathic thrombocytopenic purpura (ITP). We have analysed the response to eradication of H. pylori in a series of 56 adult patients with chronic ITP. Forty patients had H. pylori infection (71%) that was eradicated in 23 of 32 evaluable patients (72%). Platelet counts did not significantly vary according to H. pylori treatment outcome. Three of 56 patients (5%) achieved a partial response attributable to H. pylori eradication. Therefore, detection of H. pylori infection should not be routinely included in the initial work‐up of ITP.

MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström's macroglobulinemia.

We evaluated the MYD88 L265P mutation in Waldenström’s macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10−3). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23−CD27++FMC7++), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3–23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

Early infections in adult patients undergoing unrelated donor cord blood transplantation

Early transplant-related mortality after cord blood transplantation from unrelated donors (UD-CBT) is close to 50%, mainly due to infectious complications. We have studied the incidence and characteristics of early infections (before day 100) in a series of 27 adult patients (median age 30 years, range 16–46) undergoing UD-CBT at a single institution. All 27 patients experienced at least one infectious episode and 18 (66%) suffered a severe infection. Bacteremia occurred in 55% of patients (13 with Gram-positive and 11 with Gram-negative microorganisms). Eleven of 19 CMV-seropositive patients (58%) developed CMV antigenemia and one patient had CMV disease. Fungal infections were documented in three patients (11%), comprising invasive fungal infections in two cases and a localized esophagitis in one. Ten patients (37%) died before day 100 after transplantation. Infection was considered the primary cause of death in four patients (sepsis by Acinetobacter spp. bacteremia in three cases) and contributed to death in another four. The most striking findings in this series were the high incidence of, and mortality due to multiresistant Acinetobacter spp. and the low incidence of and lack of mortality due to CMV disease. This report confirms that infection is a major complication in adults undergoing UD-CBT.

C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma

The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating cell trafficking in hematopoietic stem cells and clonal B cells. We screened 418 patients with B-cell lymphoproliferative disorders and described the presence of the C1013G/CXCR4 warts, hypogammaglobulinemia, infections, and myelokathexis-associated mutation in 28.2% (37/131) of patients with lymphoplasmacytic lymphoma (Waldenström macroglobulinemia [WM]), being either absent or present in only 7% of other B-cell lymphomas. In vivo functional characterization demonstrates its activating role in WM cells, as demonstrated by significant tumor proliferation and dissemination to extramedullary organs, leading to disease progression and decreased survival. The use of a monoclonal antibody anti-CXCR4 led to significant tumor reduction in a C1013G/CXCR4 WM model, whereas drug resistance was observed in mutated WM cells exposed to Brutons tyrosine kinase, mammalian target of rapamycin, and phosphatidylinositol 3-kinase inhibitors, but not proteasome inhibitors. These findings demonstrate that C1013G/CXCR4 is an activating mutation in WM and support its role as a critical regulator of WM molecular pathogenesis and as an important therapeutic target.

Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry

We have analyzed the applicability, sensitivity and prognostic value of allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) as a method for minimal residual disease (MRD) assessment in patients with multiple myeloma (MM), comparing the results with those of multiparameter flow cytometry (MFC). A total of 170 patients enrolled in three consecutive Spanish trials achieving at least partial response after treatment were included. Lack of clonality detection (n=31), unsuccessful sequencing (n=17) and suboptimal ASO performance (n=51) limited the applicability of PCR to 42% of cases. MRD was finally investigated in 103 patients (including 32 previously studied) with persistent disease identified by PCR and MFC in 54% and 46% of cases, respectively. A significant correlation in MRD quantitation by both the techniques was noted (r=0.881, P<0.001), being reflective of treatment intensity. Patients with <10−4 residual tumor cells showed longer progression-free survival (PFS) compared with the rest (not reached (NR) vs 31 months, P=0.002), with similar results observed with MFC. Among complete responders (n=62), PCR discriminated two risk groups with different PFS (49 vs 26 months, P=0.001) and overall survival (NR vs 60 months, P=0.008). Thus, although less applicable than MFC, ASO RQ-PCR is a powerful technique to assess treatment efficacy and risk stratification in MM.

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.

Unrelated donor cord blood transplantation in adults with chronic myelogenous leukemia: results in nine patients from a single institution

The potential role of unrelated donor cord blood transplantation (UD-CBT) in adults is not well established. We report the results of UD-CBT in nine adult patients with chronic myeloid leukemia (CML). The median age was 27 years (range, 19–41 years), and the median weight was 62 kg (range, 45–78 kg). At transplant, six patients were in chronic phase (five in first, and one in second), two in blast crisis, and one in accelerated phase. Eight had received intensive chemotherapy, and three had undergone autologous peripheral blood hematopoietic stem cell transplantation. Four had received interferon with no cytogenetic response, and only three underwent UD-CBT within 1 year of diagnosis. After serological typing for class I antigens, and high-resolution DNA typing for DRB1, the degree of HLA match between patients and cord blood (CB) units was 4/6 in six cases and 5/6 in three cases. The median number of nucleated cells infused was 1.7 × 107/kg (range, 1.2 to 4.9 × 107/kg), and was above 2 × 107/kg in only two cases. All patients received thiotepa, busulfan, cyclophosphamide and anti-thymocyte globulin as conditioning; cyclosporine and prednisone for graft-versus-host disease (GVHD) prophylaxis; and G-CSF from day +7 until engraftment. All seven evaluable cases engrafted. The median time to reach an absolute neutrophil count ⩾0.5 × 109/l and ⩾1 × 109/l was 22 days (range, 19–52 days) and 28 days (range, 23–64 days), respectively. In the four patients evaluable for platelet recovery time to levels of ⩾20 × 109 platelets/l, ⩾50 × 109 platelets/l, and ⩾100 × 109 platelets/l, these ranged from 50 to 128 days, 60 to 139 days, and 105 to 167 days, respectively. Three patients developed acute GVHD above grade II, and three of the five patients at risk developed extensive chronic GVHD. Four patients, all transplanted in chronic phase, remain alive in molecular remission more than 18, 19, 24 and 42 months after transplantation. These preliminary results suggest that UD-CBT may be considered a reasonable alternative in adults with CML who lack an appropriate bone marrow donor. Bone Marrow Transplantation (2001) 27, 693–701.

Marked reduction in the incidence of hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation with CD34(+) positive selection.

Veno-occlusive disease of the liver (VOD) is a common and severe complication of allogeneic hematopoietic stem cell transplantation (HSCT). To determine the incidence of, and the risk factors for the development of VOD, we performed a retrospective analysis of a series of 178 patients, who underwent allogeneic HSCT at our institution between 1990 and 1999. Busulfan and cyclophosphamide constituted the conditioning regimen most frequently administered. Bone marrow was the source of stem cells in 129 patients (73%), and peripheral blood (PBSC) in 49 patients (27%). Thirty-one patients of the PBSC group received CD34+ positively selected grafts. Most patients were given cyclosporin A and methotrexate (MTX) as graft-versus-host disease (GVHD) prophylaxis. Overall, 30 patients (17%) developed VOD. In univariate analyses, the incidence of VOD was significantly higher in recipients of unmanipulated grafts (20% vs 0%; P = 0.01), in patients with active malignant disease at transplantation (24% vs 9%; P = 0.03), in recipients of marrow from unrelated donors (33% vs 15%; P = 0.03), in patients grafted with bone marrow (21% vs 6%; P = 0.03), and in those receiving MTX as GVHD prophylaxis (21% vs 6%; P = 0.05). Under multivariate analysis, only CD34+ positive selection (P = 0.0004) and the status of the disease at transplant (P = 0.03) were statistically significant variables for the development of VOD. We conclude that CD34+ positively selected PBSC transplantation could result in a marked reduction in the incidence of VOD after allogeneic HSCT. Bone Marrow Transplantation (2001) 27, 983–988.

Prolonged molecular remission after PML/RARα-positive autologous peripheral blood stem cell transplantation in acute promyelocytic leukemia: is relevant pretransplant minimal residual disease in the graft?

The contribution of residual malignant cells contaminating the autologous graft with the occurrence of post-transplant relapse in acute myeloid leukemia (AML) is still unclear. The presence of a specific molecular marker (the PML/RARα rearrangement) in acute promyelocytic leukemia (APL) offers the opportunity to investigate better the pathogenesis of disease recurrence after transplant. We report an APL patient who received high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft in second hematologic remission. Two leukaphereses that tested PML/RARα positive by RT-PCR were obtained during the post-reinduction hematopoietic recovery, while the patient also tested PCR positive in the BM, and was reinfused after myeloablative chemotherapy (BUCY4), when the patient had spontaneously converted to PCR negative in the marrow. At present, he remains in continuous molecular and hematologic remission 22 months after PBSC transplantation. This is the second report of an APL patient who was transplanted in molecular remission with a PML/RARα-positive PBSC autograft. As in the previous report, the prolonged clinical and molecular remission experienced post-transplant suggests that autologous PBSC infusion is still worthy of consideration for patients with APL in spite of the detection of PML/RARα-positive cells in the PBSC collections. Possible underlying mechanisms and the potential role of molecular monitoring of the graft, as well as the host, before and after transplant, in patients with APL undergoing autologous HSCT are also discussed.