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To the Editor, We report a rare case of a chronic disseminated Aichi virus infection in a pediatric patient with X-linked agammaglobulinemia (XLA) that was controlled upon HLA-haploidentical hematopoietic cell transplantation (haplo-HCT). Lack of a routinely available method of AiV screening did in fact delay the proper diagnosis in our patient, and these two cases suggest that chronic AiV infection in XLA patients may be more common than anticipated. Ig replacement therapy was commenced with subcutaneous Ig infusions maintaining mean IgG trough levels at 7 g/L. The patient had no hepatosplenomegaly, growth and development were uneventful, apart from recurrent purulent conjunctivitis, intermittent asthma, and mild eczema. Immunologic Control of Disseminated Aichi Virus Infection in X-Linked Agammaglobulinemia by Transplantation of TcR -Depleted Haploidentical Hematopoietic Cells. [Extracted from the article] more...

This review summarizes – with the practicing hematologist in mind – the methods used to determine measurable residual disease (MRD) in everyday practice with some future perspectives, and the current knowledge about the prognostic impact of MRD on outcome in acute myeloid leukemia (AML), excluding acute promyelocytic leukemia. Possible implications for choice of MRD method, timing of MRD monitoring, and guidance of therapy are discussed in general and in some detail for certain types of leukemia with specific molecular markers to monitor, including core binding factor (CBF)‐leukemias and NPM1‐mutated leukemias. [ABSTRACT FROM AUTHOR] more...

In order to confirm our earlier findings of the prognostic effects of CD23 and CD40 expression in diffuse large B-cell lymphoma (DLBCL), possibly due to association with the germinal center (GC) phenotype and/or an increased autologous tumour response, tumour specimens from 125 patients with de novo DLBCL were investigated for immunohistochemical expression of CD23, CD40, BCL6, CD10, MUM1, CD4 and CD8. CD40 was positive in 64% and was associated with improved overall survival (p = 0.03). A GC phenotype was present in 47%, and was also associated with a better overall survival (p = 0.006) but did not correlate with CD40-expression. There was no correlation between amount of tumour infiltrating T-cells and CD40-positivity. CD23 was positive in 10% and expression did not correlate with prognosis. In conclusion, the prognostic effect of CD40 expression was confirmed, but did not correlate with GC-phenotype or T-cell infiltration. [ABSTRACT FROM AUTHOR] more...

Objective: 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. Methods: Tumor specimens from 122 patients with de novo stage II–IV disease, adequately treated with anthracycline-containing chemotherapy regimens were collected. Immunohistochemical expression of BCL6, CD10, and MUM-1/IRF4 was examined using a tissue microarray (TMA) technique. BCL6 and CD10 were also evaluated on whole tissue sections. Results: Due to profound tissue heterogeneity, BCL6 showed a wide range of positivity, with a high number of false negative results by TMA (25% positive), compared to 53% on whole tissue sections (WTS). CD10 was more homogeneously expressed, and TMA results corresponded better to WTS. Consequently, the results from categorization into GC and non-GC DLBCL differed considerably by use of the two methods, and resulted in very different outcome in terms of overall survival. Conclusion: Immunohistochemical GC-status determined on TMA is not reliable enough to be used for individual treatment decisions in DLBCL, mostly due to difficulties in interpreting BCL6 status. [ABSTRACT FROM AUTHOR] more...

Abstract Background: Epigenetic code modifications by histone deacetylase inhibitors (HDACi) have been proposed as potential new therapies for lymphoid malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma for which standard first line treatment is the chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP). The HDACi valproate, which has for long been utilized in anti-convulsive therapy, has been shown to sensitize to chemotherapy in vitro. Valproate upregulates expression of CD20 in lymphoma cell lines; therefore, 48 hour pre-treatment with valproate before first line R-CHOP in DLBCL stages II-IV is evaluated in the phase I clinical trial VALFRID; Valproate as First line therapy in combination with Rituximab and CHOP in Diffuse large B-cell lymphoma. Findings: Pretreatment with valproate at oral doses comparable to anti-convulsive therapy, resulted in upregulation of CD20 mRNA and CD20 protein on the cell surface as measured by qPCR and FACS analysis in lymphoma biopsies from three evaluated patients from the VALFRID study. Valproate-treatment corresponded to increased acetylation of Histone3Lysine9 (H3K9ac) in peripheral blood mononuclear cells (PBMCs), which were employed as surrogate tissue for valproate-related epigenetic modifications. Conclusions: Valproate treatment at pharmacologically relevant doses resulted in upregulation of CD20 in vivo, and also in expected epigenetic modifications. This suggests that pre-treatment with valproate or other HDACis before anti-CD20 therapy could be advantageous in CD20-low B-cell lymphomas. Further studies are warranted to evaluate this conclusion. [ABSTRACT FROM AUTHOR] more...

Background: Epigenetic code modifications by histone deacetylase inhibitors (HDACi) have been proposed as potential new therapies for lymphoid malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma for which standard first line treatment is the chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP). The HDACi valproate, which has for long been utilized in anti-convulsive therapy, has been shown to sensitize to chemotherapy in vitro. Valproate upregulates expression of CD20 in lymphoma cell lines; therefore, 48 hour pre-treatment with valproate before first line R-CHOP in DLBCL stages II-IV is evaluated in the phase I clinical trial VALFRID; Valproate as First line therapy in combination with Rituximab and CHOP in Diffuse large B-cell lymphoma. Findings: Pretreatment with valproate at oral doses comparable to anti-convulsive therapy, resulted in upregulation of CD20 mRNA and CD20 protein on the cell surface as measured by qPCR and FACS analysis in lymphoma biopsies from three evaluated patients from the VALFRID study. Valproate-treatment corresponded to increased acetylation of Histone3Lysine9 (H3K9ac) in peripheral blood mononuclear cells (PBMCs), which were employed as surrogate tissue for valproate-related epigenetic modifications. Conclusions: Valproate treatment at pharmacologically relevant doses resulted in upregulation of CD20 in vivo, and also in expected epigenetic modifications. This suggests that pre-treatment with valproate or other HDACis before anti-CD20 therapy could be advantageous in CD20-low B-cell lymphomas. Further studies are warranted to evaluat this conclusion. [ABSTRACT FROM AUTHOR] more...

Immunohistochemical expression of CD40 is seen in 60-70% of diffuse large B-cell lymphoma (DLBCL) and is associated with a superior prognosis. By using gene expression profiling we aimed to further explore the underlying mechanisms for this effect. Ninety-eight immunohistochemically defined CD40 positive or negative DLBCL tumors, 63 and 35 respectively, were examined using spotted 55K oligonucleotide arrays. CD40 expressing tumors were characterized by up-regulated expression of genes encoding proteins involved in cell-matrix interactions: collagens, integrin αV, proteoglycans and proteolytic enzymes, and antigen presentation. Immunohistochemistry confirmed that CD40 positive tumors co-express the proinflammatory proteoglycan biglycan ( p = 0.005), which in turn correlates with the amount of infiltrating macrophages and CD4 and CD8 positive T-cells. We postulate that immunohistochemical expression of CD40 mainly reflects the inflammatory status in tumors. A high intratumoral inflammatory reaction may correlate with an increased autologous tumor response, and thereby a better prognosis. [ABSTRACT FROM AUTHOR] more...

In order to identify genes associated with primary chemotherapy-resistance, gene expression profiles (GEP) in tumour tissue from 37 patients with de novo diffuse large B-cell lymphoma (DLBCL), stage II–IV, either in continuous complete remission ( n = 24) or with progressive disease during primary treatment ( n = 13), were examined using spotted 55K oligonucleotide arrays. Immunohistochemistry was used for confirmation at the protein level. The top 86 genes that best discriminated between the two cohorts were chosen for further analysis. Only seven of 86 genes were overexpressed in the refractory cohort, e.g. RABGGTB and POLE, both potential targets for drug intervention. Seventy-nine of 86 genes were overexpressed in the cured cohort and mainly coded for proteins expressed in the tumour microenvironment, many of them involved in proteolytic activity and remodelling of extra cellular matrix. Furthermore, major histocompatibility complex class I molecules, CD3D and ICAM1 were overexpressed, indicating an enhanced immunological reaction. Immunohistochemistry confirmed the GEP results. The frequency of tumour infiltrating lymphocytes, macrophages, and reactive cells expressing ICAM-1, lysozyme, cathepsin D, urokinase plasminogen activator receptor, signal transducer and activator of transcription 1, and galectin-3 was higher in the cured cohort. These findings indicate that a reactive microenvironment has an impact on the outcome of chemotherapy in DLBCL. [ABSTRACT FROM AUTHOR] more...

Objective: Infantile malignant osteopetrosis (IMO) is a rare autosomal recessive disorder affecting osteoclast function. Fifty percent of the patients have a mutation in the TCIRG1 gene coding for one subunit of an osteoclast proton pump. The only curative treatment is hematopoietic stem cell transplantation (SCT). The oc/oc mouse has a mutation in the gene homologous to TCIRG1 and its expected lifespan is only 3 to 4 weeks. Previous attempts to cure these mice with SCT have been unsuccessful. We wanted to determine if early hematopoietic SCT using enriched and MHC-matched stem cells can cure oc/oc mice from osteopetrosis. Methods: One- and 8-day-old oc/oc and control mice were radiated with 200, 400, or 600 cGy and transplanted intraperitoneally with 1 or 5 × 106 normal lineage-depleted bone marrow cells. Blood, x-ray, and pathology analyses were performed on transplanted mice. Results: All 1-day-old mice irradiated with 400 cGy and transplanted with 5 × 106 cells survived long term. An engraftment level of 20% is sufficient to correct most features of the disease. X-ray and histopathology examination of transplanted animals showed normalization of bone structure. However, although a correction of bone structure occurred, the transplanted oc/oc mice were smaller in size than their littermates. In contrast to untreated animals, oc/oc mice developed teeth after transplantation, but with abnormal structure and shape making them unusable. Conclusion: We have shown that this murine form of IMO is curable with neonatal SCT using enriched stem cells. [Copyright &y& Elsevier] more...

AML with mutated NPM1 occurs in all age groups. Yet, the mutational pattern is not extensively studied in the very old, which may hamper appropriate risk assessment. Herein we examined 22 cases of NPM1-mutated de novo AML in patients older than 75, with a median age of 84. All diagnostic samples were sequenced aiming for coverage of the most relevant AML-associated mutations. For comparison with younger patients, we used already published data on several cohorts. A total of 76 mutations including 50 different variants were identified in 16 recurrently mutated AML genes. Compared with younger patients, a significant enrichment of TET2 and SRSF2 was observed, together with a reduced frequency of DNMT3A mutations. Our results indicate that the mutational pattern may be different in the very old as compared to younger patients with NPM1-mutated AML. The mutational spectrum of NPM1-mutated AML in patients above 75 years displays distinct features. A significant enrichment of TET2 and SRSF2 mutations together with a reduced frequency of DNMT3A mutations was observed in the elderly. NPM1 mutation is a secondary event in the development of AML in the very old. [ABSTRACT FROM AUTHOR] more...

The Swedish national guidelines for treatment of acute myeloid leukemia (AML) recommend analysis of measurable residual disease (MRD) by multiparameter flow cytometry (MFC) in bone marrow in the routine clinical setting. The Swedish AML registry contains such MRD data in AML patients diagnosed 2011–2019. Of 327 patients with AML (non-APL) with MRD-results reported in complete remission after two courses of intensive chemotherapy 229 were MRD-negative (70%), as defined by <0.1% cells with leukemia-associated immunophenotype in the bone marrow. MRD-results were reported to clinicians in real time. Multivariate statistical analysis adjusted for known established risk factors did not indicate an association between MFC-MRD and overall survival (HR: 1.00 [95% CI 0.61, 1.63]) with a median follow-up of 2.7 years. Knowledge of the importance of MRD status by clinicians and individualized decisions could have ameliorated the effects of MRD as an independent prognostic factor of overall survival. [ABSTRACT FROM AUTHOR] more...

Introduction: Reverse transcriptase quantitative PCR (RT‐qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1‐mutated acute myeloid leukemia (AML). MRD can also be determined with DNA‐based methods offering certain advantages. We here compared the DNA‐based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT‐qPCR. Methods: Of 110 follow‐up samples from 30 patients with NPM1‐mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT‐qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT‐qPCR cutoffs. Results: The DNA‐based methods showed strong intermethod correlation, but were less sensitive than RT‐qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT‐qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. Conclusion: DNA‐based MRD techniques may complement RT‐qPCR for assessment of residual leukemia. DNA‐based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA‐based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA. [ABSTRACT FROM AUTHOR] more...

We studied mutation kinetics in ten relapsing and four non-relapsing patients with acute myeloid leukemia by whole exome sequencing at diagnosis to identify leukemia-specific mutations and monitored selected mutations at multiple time-points using IBSAFE droplet digital PCR. Five to nine selected mutations could identify and track leukemic clones prior to clinical relapse in 10/10 patients at the time-points where measurable residual disease was negative by multicolor flow cytometry. In the non-relapsing patients, the load of mutations gradually declined in response to different therapeutic strategies. Three distinct patterns of relapse were observed: (1) one or more different clones with all monitored mutations reappearing at relapse; (2) one or more separate clones of which one prevailed at relapse; and (3) persistent clonal hematopoiesis with high variant allele frequency and most mutations present at relapse. These pilot results demonstrate that IBSAFE analyses detect leukemic clones missed by flow cytometry with possible clinical implications. The IBSAFE ddPCR MRD method seems applicable on virtually all newly diagnosed AML patients and was more sensitive than flow cytometry. Monitoring a few mutations captured the kinetics of the evolving recurrent leukemia. NPM1-mutation alone may not be a reliable MRD-marker. [ABSTRACT FROM AUTHOR] more...

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% (p < .001), HR 45 (95% CI 2-1260), and overall survival 20% vs 89% (p < .001), HR 49 (95% CI 2-1253). Thus, the new methodology deep sequencing is an applicable and predictive tool for MRD assessment in AML. [ABSTRACT FROM AUTHOR] more...

The main objectives of the present study were to monitor minimal residual disease (MRD) in the bone marrow of patients with mantle cell lymphoma (MCL) to predict clinical relapse and guide preemptive treatment with rituximab. Among the patients enrolled in 2 prospective trials by the Nordic Lymphoma Group, 183 who had completed autologous stem cell transplantation (ASCT) and in whom an MRD marker had been obtained were included in our analysis. Fresh samples of bone marrow were analyzed for MRD by a combined standard nested and quantitative real-time PCR assay for Bcl-1/immunoglobulin heavy chain gene ( IgH ) and clonal IgH rearrangements. Significantly shorter progression-free survival (PFS) and overall survival (OS) was demonstrated for patients who were MRD positive pre-ASCT (54 patients) or in the first analysis post-ASCT (23 patients). The median PFS was only 20 months in those who were MRD-positive in the first sample post-ASCT, compared with 142 months in the MRD-negative group ( P  < .0001). OS was 75% at 10 years and median not reached in the MRD-negative group, compared with only 35 months in the MRD-positive group ( P  < .0001). Of the 86 patients (47%) who remained in continuous molecular remission, 73% were still in clinical remission after 10 years. For all patients, the median time from ASCT to first molecular relapse was 55 months, with a continuous occurrence of late molecular relapses. Fifty-eight patients who experienced MRD relapse received rituximab as preemptive treatment on 1 or more occasions, and in this group, the median time from first molecular relapse to clinical relapse was 55 months. In most cases, rituximab converted patients to MRD negativity (87%), but many patients became MRD-positive again later during follow-up (69%). By multivariate analysis, high-risk Mantle Cell Lymphoma International Prognostic Index score and positive MRD status pre-ASCT predicted early molecular relapse. In conclusion, preemptive rituximab treatment converts patients to MRD negativity and likely postpones clinical relapse. Molecular monitoring offers an opportunity to select some patients for therapeutic intervention and to avoid unnecessary treatment in others. MRD-positive patients in the first analysis post-ASCT have a dismal prognosis and thus are in need of novel strategies. [ABSTRACT FROM AUTHOR] more...

Next-generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow-up samples. The aim of this study was to identify leukemia-specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence-activated cell sorting. Exome sequencing of these cells identified 240 leukemia-specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow-up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia-specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high-sensitivity quantification of minimal residual disease in a patient-tailored manner. [ABSTRACT FROM AUTHOR] more...

In recent decades, the prognosis of Mantle Cell Lymphoma ( MCL) has been significantly improved by intensified first-line regimens containing cytarabine, rituximab and consolidation with high-dose-therapy and autologous stem cell transplantation. One such strategy is the Nordic MCL2 regimen, developed by the Nordic Lymphoma Group. We here present the 15-year updated results of the Nordic MCL2 study after a median follow-up of 11·4 years: For all patients on an intent-to-treat basis, the median overall and progression-free survival was 12·7 and 8·5 years, respectively. The MCL International Prognostic Index ( MIPI), biological MIPI, including Ki67 expression ( MIPI-B) and the MIPI-B including mIR-18b expression ( MIPI-B-miR), in particular, significantly divided patients into distinct risk groups. Despite very long response durations of the low and intermediate risk groups, we observed a continuous pattern of relapse and the survival curves never reached a plateau. In conclusion, despite half of the patients being still alive and 40% in first remission after more than 12 years, we still see an excess disease-related mortality, even among patients experiencing long remissions. Even though we consider the Nordic regimen as a very good choice of regimen, we recommend inclusion in prospective studies to explore the benefit of novel agents in the frontline treatment of MCL. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

Recent studies have shown that a large proportion of patients classified as essential thrombocythemia (ET) actually have early primary prefibrotic myelofibrosis (prePMF), which implies an inferior prognosis as compared to patients being diagnosed with so-called genuine or true ET. According to the World Health Organization (WHO) 2008 classification, bone marrow histology is a major component in the distinction between these disease entities. However, the differential diagnosis between them may be challenging and several studies have not been able to distinguish between them. Most lately, it has been argued that simple blood tests, including the leukocyte count and plasma lactate dehydrogenase (LDH) may be useful tools to separate genuine ET from prePMF, the latter disease entity more often being featured by anemia, leukocytosis and elevated LDH. Whole blood gene expression profiling was performed in 17 and 9 patients diagnosed with ET and PMF, respectively. Using elevated LDH obtained at the time of diagnosis as a marker of prePMF, a 7-gene signature was identified which correctly predicted the prePMF group with a sensitivity of 100% and a specificity of 89%. The 7 genes included MPO, CEACAM8, CRISP3, MS4A3, CEACAM6, HEMGN, and MMP8, which are genes known to be involved in inflammation, cell adhesion, differentiation and proliferation. Evaluation of bone marrow biopsies and the 7-gene signature showed a concordance rate of 71%, 79%, 62%, and 38%. Our 7-gene signature may be a useful tool to differentiate between genuine ET and prePMF but needs to be validated in a larger cohort of “ET” patients. [ABSTRACT FROM AUTHOR] more...

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease. [ABSTRACT FROM AUTHOR] more...

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progressionfree survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance. [ABSTRACT FROM AUTHOR] more...

Background: Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease that arises in a multistep fashion through acquisition of several genetic aberrations, subsequently giving rise to a malignant, clonal expansion of T-lymphoblasts. The aim of the present study was to identify additional as well as cooperative genetic events in T-ALL. Methods: A population-based pediatric T-ALL series comprising 47 cases was investigated by SNP array and deep sequencing analyses of 75 genes, in order to ascertain pathogenetically pertinent aberrations and to identify cooperative events. Results: The majority (92%) of cases harbored copy number aberrations/uniparental isodisomies (UPIDs), with a median of three changes (range 0-11) per case. The genes recurrently deleted comprised CDKN2A, CDKN2B, LEF1, PTEN, RBI, and STIL. No case had a whole chromosome UPID; in fact, literature data show that this is a rare phenomenon in T-ALL. However, segmental UPIDs (sUPIDs) were seen in 42% of our cases, with most being sUPID9p that always were associated with homozygous CDKN2A deletions, with a heterozygous deletion occurring prior to the sUPID9p in all instances. Among the 75 genes sequenced, 14 (19%) were mutated in 28 (72%) of 39 analyzed cases. The genes targeted are involved in signaling transduction, epigenetic regulation, and transcription. In some cases, NOTCH1 mutations were seen in minor subclones and lost at relapse; thus, such mutations can be secondary events. Conclusions: Deep sequencing and SNP array analyses of T-ALL revealed lack of wUPIDs, a high proportion of sUPID9p targeting CDKN2A, NOTCH1 mutations in subclones, and recurrent mutations of genes involved in signaling transduction, epigenetic regulation, and transcription. [ABSTRACT FROM AUTHOR] more...

Mantle cell lymphoma ( MCL) is an aggressive B cell lymphoma, where survival has been remarkably improved by use of protocols including high dose cytarabine, rituximab and autologous stem cell transplantation, such as the Nordic MCL2/3 protocols. In 2008, a MCL international prognostic index ( MIPI) was created to enable stratification of the clinical diverse MCL patients into three risk groups. So far, use of the MIPI in clinical routine has been limited, as it has been shown that it inadequately separates low and intermediate risk group patients. To improve outcome and minimize treatment-related morbidity, additional parameters need to be evaluated to enable risk-adapted treatment selection. We have investigated the individual prognostic role of the MIPI and molecular markers including SOX11, TP53 (p53), MKI67 (Ki-67) and CCND1 (cyclin D1). Furthermore, we explored the possibility of creating an improved prognostic tool by combining the MIPI with information on molecular markers. SOX11 was shown to significantly add prognostic information to the MIPI, but in multivariate analysis TP53 was the only significant independent molecular marker. Based on these findings, we propose that TP53 and SOX11 should routinely be assessed and that a combined TP53/ MIPI score may be used to guide treatment decisions. [ABSTRACT FROM AUTHOR] more...

The main objective of the MCL3 study was to improve outcome for patients not in complete remission (CR) before transplant by adding 90Y-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. [ABSTRACT FROM AUTHOR] more...

Combined core needle biopsy and fine-needle aspiration with ancillary studies correlate highly with traditional techniques in the diagnosis of nodal-based lymphoma. We read with interest the letter to the Editor by Pizzi et al: "Lymph node core-needle biopsy for the diagnosis of lymphoproliferative disorders: a word of caution"1 which is a comment to an article by Cohen et al2, Pizzi et al1 maintain that lymph node core-needle biopsy (LN-CB)3-5 should not be adopted routinely and that incisional/excisional (surgical) biopsy should be performed in all suitable cases. Letter to the Editor: Fine-needle aspiration cytology and core-needle biopsy in the diagnosis of lymphadenopathies: Words of endorsement. [Extracted from the article] more...

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 as ISRCTN 87866680. [ABSTRACT FROM AUTHOR] more...

Diamond-Blackfan anemia (DBA) isa congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) Is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 downregulation. We report the generation of mouse models for RPS19 deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19 deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene. [ABSTRACT FROM AUTHOR] more...

We have previously shown that expression of CD40 has a favorable prognostic impact in diffuse large B-cell lymphoma (DLBCL) after anthracycline-based chemotherapy. Here we examined the prognostic value of immunohistochemically defined CD40 expression in 95 patients with DLBCL treated with both anthracycline-based chemotherapy and rituximab. Using a 10% cut-off level, 77% of the patients had CD40-positive tumors and showed a superior overall survival ( p = 0.02 log-rank, hazard ratio 0.35, 95% CI 0.14–0.88, p = 0.03 Cox regression). When adjusted for International Prognostic Index in multivariate analysis, CD40 was not an independent prognostic factor (hazard ratio 0.39, 95% CI 0.15–1.04, p = 0.06 Cox regression). However, even after the introduction of immunochemotherapy, CD40 has a potential prognostic impact in DLBCL. Additional and larger studies are necessary, regarding the immunohistochemical robustness of CD40 and the biological mechanisms that contribute to the superior prognosis in CD40-expressing DLBCL. [ABSTRACT FROM AUTHOR] more...

Objective: Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. Materials and Methods: Pregnant dams were conditioned with busulphan and their litters transplanted with 1 × 106 normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. Results: Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. Conclusion: Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels. [Copyright &y& Elsevier] more...

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucosidase, beta, acid (GBA) gene that encodes the lysosomal enzyme glucosylceramidase (GCase). GCase deficiency leads to characteristic visceral pathology and, in some patients, lethal neurological manifestations. Here, we report the generation of mouse models with the severe neuronopathic form of GD. To circumvent the lethal skin phenotype observed in several of the previous Gcase-deficient animals, we genetically engineered a mouse model with strong reduction in GCase activity in all tissues except the skin. These mice exhibit rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death within the brain, reminiscent of neuronopathic GD. In addition, we have created a second mouse model, in which GCase deficiency is restricted to neural and glial cell progenitors and progeny. These mice develop similar pathology as the first mouse model, but with a delayed onset and slower disease progression, which indicates that GCase deficiency within micro- glial cells that are of hematopoietic origin is not the primary determinant of the CNS pathology. These findings also demonstrate that normal microglial cells cannot rescue this neurodegenerative disease. These mouse models have significant implications for the development of therapy for patients with neuronopathic GD. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

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 WI 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. [ABSTRACT FROM AUTHOR] more...

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[sup -] ScaI[sup +] c-kit[sup +] 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. [ABSTRACT FROM AUTHOR] more...

c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32°C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer. Oncogene (2000) 19, 2194–2204 [ABSTRACT FROM AUTHOR] more...

Transforming growth factor-β (TGFβ) is a member of a large family of polypeptide growth factors. TGFβ signals mainly through the intracellular proteins Smad2 and Smad3, which are highly similar in amino acid sequence identity. A number of studies have shown that these proteins, dependent on context, have distinct roles in the TGFβ signaling pathway. TGFβ is one of the most potent inhibitors of hematopoietic stem and progenitor cell proliferation in vitro, but its role in hematopoiesis in vivo is still being determined. To circumvent possible redundancies at the receptor level and to address specifically the role of the Smad circuitry downstream of TGFβ and activin in hematopoiesis, we studied the effect of genetically deleting both Smad2 and Smad3 in adult murine hematopoietic cells. Indeed, TGFβ signaling is impaired in vitro in primitive bone marrow (BM) cells of Smad2 and Smad3 single knockout models. However, blood parameters appear normal under steady state and in the transplantation setting. Interestingly, upon deletion of both Smad2 and Smad3 in vivo, mice quickly develop a lethal inflammatory disease, suggesting that activin/TGFβ signaling is crucial for immune cell homeostasis in the adult context. Furthermore, concurrent deletion of Smad2 and Smad3 in BM cells in immune-deficient nude mice did not result in any significant alterations of the hematopoietic system. Our findings suggest that Smad2 and Smad3 function to mediate crucial aspects of the immunoregulatory properties of TGFβ, but are dispensable for any effect that TGFβ has on primitive hematopoietic cells in vivo. [ABSTRACT FROM AUTHOR] more...

A correction to the article "Nordic MCL2 Trial Update: 6-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" published in previous issue is presented. more...