Mats Ehinger

Nordic MCL2 trial update: six-year follow-up after intensive immunochemotherapy for untreated mantle cell lymphoma followed by BEAM or BEAC + autologous stem-cell support: still very long survival but late relapses do occur

Mantle cell lymphoma (MCL) is a heterogenic non‐Hodgkin lymphoma entity, with a median survival of about 5 years. In 2008 we reported the early – based on the median observation time of 4 years – results of the Nordic Lymphoma Group MCL2 study of frontline intensive induction immunochemotherapy and autologous stem cell transplantation (ASCT), with more than 60% event‐free survival at 5 years, and no subsequent relapses reported. Here we present an update after a median observation time of 6·5 years. The overall results are still excellent, with median overall survival and response duration longer than 10 years, and a median event‐free survival of 7·4 years. However, six patients have now progressed later than 5 years after end of treatment. The international MCL Prognostic Index (MIPI) and Ki‐67‐expression were the only independent prognostic factors. Subdivided by the MIPI‐Biological Index (MIPI + Ki‐67, MIPI‐B), more than 70% of patients with low‐intermediate MIPI‐B were alive at 10 years, but only 23% of the patients with high MIPI‐B. These results, although highly encouraging regarding the majority of the patients, underline the need of a risk‐adapted treatment strategy for MCL. The study was registered at www.isrctn.org as ISRCTN 87866680.

Reduced proliferative capacity of hematopoietic stem cells deficient in Hoxb3 and Hoxb4

ABSTRACT Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin− ScaI+ c-kit+ hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.

Effective cell and gene therapy in a murine model of Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9–11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5–6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD in vivo. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.

Cell differentiation in acute myeloid leukemia

Abstract: Acute myeloid leukemia (AML) is characterized by a differentiation block leading to accumulation of immature cells. Chromosomal translocations in AML affect transcription factors that are involved in regulation of myeloid differentiation. Aberrant expression of these factors interferes with differentiation events and has a role in the pathogenesis of AML through superactivation or (dominant negative) repression of genes regulating proliferation and differentiation or by interference with assembly of the transcription complex for these genes. The maturation arrest can be reversed by certain agents as judged by results from investigations of myeloid leukemic cell lines and from treatment of acute promyelocytic leukemia (APL) patients with all‐trans retinoic acid. Inactivation of the p53 and retinoblastoma (Rb) tumor suppressor genes is also associated with the pathogenesis of leukemia through effects on the cell cycle, and manipulation of these genes can affect differentiation of AML cells. With differentiation therapy, when successful as in APL, the leukemic cell mass is reduced to allow restoration of normal hematopoiesis and clinical remission, but the disease is not cured. However, initial reduction of the cell mass by maturation can increase the probability for cure with chemotherapy. Overexpression of suppressor genes may increase the probability for differentiation. Most probably, particular molecular defects of subgroups of AML have to be explored to find optimal strategies for treatment including both blocking the cell cycle, promoting terminal differentiation, and inducing apoptosis as well as strengthening the immune response.

Nordic MCL3 study: 90Y-ibritumomab-tiuxetan added to BEAM/C in non-CR patients before transplant in mantle cell lymphoma

The main objective of the MCL3 study was to improve outcome for patients not in complete remission (CR) before transplant by adding (90)Y-ibritumomab-tiuxetan (Zevalin) to the high-dose regimen. One hundred sixty untreated, stage II-IV mantle cell lymphoma patients <66 years received rituximab (R)-maxi-CHOP (cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone) alternating with R-high-dose cytarabine (6 cycles total), followed by high-dose BEAM/C (bis-chloroethylnitrosourea, etoposide, cytarabine, and melphalan or cyclophosphamide) and autologous stem cell transplantation from 2005 to 2009. Zevalin (0.4 mCi/kg) was given to responders not in CR before transplant. Overall response rate pretransplant was 97%. The outcome did not differ from that of the historic control: the MCL2 trial with similar treatment except for Zevalin. Overall survival (OS), event-free survival (EFS), and progression-free survival (PFS) at 4 years were 78%, 62%, and 71%, respectively. For responding non-CR patients who received Zevalin, duration of response was shorter than for the CR group. Inferior PFS, EFS, and OS were predicted by positron emission tomography (PET) positivity pretransplant and detectable minimal residual disease (MRD) after transplant. In conclusion, positive PET and MRD were strong predictors of outcome. Intensification with Zevalin may be too late to improve the outcome of patients not in CR before transplant. This trial was registered at www.clinicaltrials.gov as #NCT00514475.

Tissue microarray is inappropriate for analysis of BCL6 expression in diffuse large B-cell lymphoma

Objective:u2002 In this study, our aim was to investigate how different immunohistochemical techniques may influence the result of BCL6 positivity and categorization in germinal center (GC) and non‐GC derived diffuse large B‐cell lymphoma (DLBCL), as it has been proposed that classification of DLBCL according to cell‐of‐origin by immunohistochemistry may be performed as a routine procedure in the diagnostic work‐up. However, a number of technical issues need to be solved before introducing this as a standard technique.

Lenalidomide-bendamustine-rituximab in patients older than 65 years with untreated mantle cell lymphoma

For elderly patients with mantle cell lymphoma (MCL), there is no defined standard therapy. In this multicenter, open-label phase 1/2 trial, we evaluated the addition of lenalidomide (LEN) to rituximab-bendamustine (R-B) as first-line treatment for elderly patients with MCL. Patients >65 years with untreated MCL, stages II-IV were eligible for inclusion. Primary end points were maximally tolerable dose (MTD) of LEN and progression-free survival (PFS). Patients received 6 cycles every four weeks of L-B-R (L D1-14, B 90 mg/m2 IV, days 1-2 and R 375 mg/m2 IV, day 1) followed by single LEN (days 1-21, every four weeks, cycles 7-13). Fifty-one patients (median age 71 years) were enrolled from 2009 to 2013. In phase 1, the MTD of LEN was defined as 10 mg in cycles 2 through 6, and omitted in cycle 1. After 6 cycles, the complete remission rate (CRR) was 64%, and 36% were MRD negative. At a median follow-up time of 31 months, median PFS was 42 months and 3-year overall survival was 73%. Infection was the most common nonhematologic grade 3 to 5 event and occurred in 21 (42%) patients. Opportunistic infections occurred in 3 patients: 2 Pneumocystis carinii pneumonia and 1 cytomegalovirus retinitis. Second primary malignancies (SPM) were observed in 8 patients (16%). LEN could safely be combined with R-B when added from the second cycle in patients with MCL, and was associated with a high rate of CR and molecular remission. However, we observed a high degree of severe infections and an unexpected high number of SPMs, which may limit its use. This trial is registered at www.Clinicaltrials.gov as #NCT00963534.

A limited number of IgH-primers binding to framework region 1 is sufficient to detect the majority of mature small B-cell non-Hodgkin lymphomas on formalin-fixed paraffin-embedded tissue by PCR

IGH gene rearrangement analysis by PCR is the widely accepted tool to determine clonality of B-cell lymphoid proliferations on formalin-fixed, paraffin-embedded tissue, but the results are often unsatisfying in terms of sensitivity. This is mainly due to poor quality DNA because of degradation and hence difficulties to amplify products of the needed length. Therefore, most previous attempts to determine clonality have depended on primers binding to framework region 3 thus producing amplification products of relatively short length. In order to improve clonality analyses, we have developed a sensitive monoplex PCR-protocol using primers binding to framework region 1 with extended cycling (42 cycles) and subsequent heteroduplex analysis. For comparison, multiplex reactions with alternative primers binding to framework region 1 according to the BIOMED-2 protocol were analyzed. By the two methods combined, we were able to detect clonality of 94% (16/17) of mature small B-cell non-Hodgkin lymphomas. The results suggest that PCR with primers binding to frame work region 1 may be the method of choice when assessing clonality of mature small B-cell non-Hodgkin lymphomas on formalin-fixed tissue.

Receptors for hematopoietic regulatory cytokines: Overview of structure and function

The production of blood cells is regulated by the action of external factors, cytokines, that can be released by many cell types. In the first place, a population of multipotent stem cells, mostly in the resting Go phase of the cell cycle, but with self-renewal capacity, gives rise to progenitor cells that are predetermined for differentiation into all kinds of blood cells. Expression of genes for cytokine receptors leads to external regulation of hematopoiesis by cytokines which bind to the receptors, resulting in modifications of proliferation and differentiation, as cytokines are not only growth factors, but are also maturation factors capable of directing hematopoiesis towards functionally competent cells. What is more, they are survival factors capable of suppressing programmed cell death (apoptosis). This is of particular importance for the viability of stem cells which must be preserved. Thus cytokines can act at all positions of the hematopoietic family tree, and the response can differ from proliferation and differentiation of progenitor cells to functional activation of mature cells. Under physiological conditions, during constitutive hematopoiesis, the regulatory cytokines are produced locally, for instance by stromal ceils of the microenvironment, and act locally in a paracrine manner [2].

Minimal residual disease assessed with deep sequencing of NPM1 mutations predicts relapse after allogeneic stem cell transplant in AML

Abstract Mutations in NPM1 can be used for minimal residual disease (MRD) analysis in acute myeloid leukemia (AML). We here applied a newly introduced method, deep sequencing, allowing for simultaneous analysis of all recurrent NPM1 insertions and thus constituting an attractive alternative to multiple PCRs for the clinical laboratory. We retrospectively used deep sequencing for measurement of MRD pre- and post-allogeneic hematopoietic stem cell transplantation (alloHCT). For 29 patients in morphological remission at the time of alloHCT, the effect of deep sequencing MRD on outcome was assessed. MRD positivity was defined as variant allele frequency ≥0.02%. Post-transplant MRD status was significantly and independently associated with clinical outcome; 3-year relapse-free survival 20% vs 85% (pu2009<u2009.001), HR 45 (95% CI 2–1260), and overall survival 20% vs 89% (pu2009<u2009.001), HR 49 (95% CI 2–1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML.