Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients.

The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval [CI], 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P < .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P < .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.

Dynamic International Prognostic Scoring System scores, pre-transplant therapy and chronic graft-versus-host disease determine outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis.

BACKGROUND: Myelofibrosis is a myeloproliferative stem cell disorder curable exclusively by allogeneic hematopoietic stem cell transplantation and is associated with substantial mortality and morbidity. The aim of this study was to assess disease-specific and transplant-related risk factors that influence post-transplant outcome in patients with myelofibrosis. DESIGN AND METHODS: We retrospectively assessed 76 consecutive patients with primary (n=47) or secondary (n=29) myelofibrosis who underwent bone marrow (n=6) or peripheral blood stem cell (n=70) transplantation from sibling (n=30) or unrelated (n=46) donors between January 1994 and December 2010. The median follow-up of surviving patients was 55 ± 7.5 months. RESULTS: Primary graft failure occurred in 5% and the non-relapse mortality rate at 1 year was 28%. The relapse-free survival rate was 50% with a relapse rate of 19% at 5 years. The use of pharmacological pre-treatment and the post-transplant occurrence of chronic graft-versus-host disease were significant independent unfavourable risk factors for post-transplant survival in multivariate analysis. Using the Dynamic International Prognostic Scoring System for risk stratification, low-risk patients had significantly better overall survival (P=0.014, hazard ratio 1.4) and relapse-free survival (P=0.02, hazard ratio 1.3) compared to the other risk groups of patients. The additional inclusion of thrombocytopenia, abnormal karyotype and transfusion need (Dynamic International Prognostic Scoring System Plus) resulted in a predicted 5-year overall survival of 100%, 51%, 54% and 30% for low, intermediate-1, intermediate-2 and high-risk groups, respectively. The relapse incidence was significantly higher in the absence of chronic graft-versus-host disease (P=0.006), and pharmacological pre-treatment (n=43) was associated with reduced relapse-free survival (P=0.001). CONCLUSIONS: The data corroborate a strong correlation between alloreactivity and long-term post-transplant disease control and confirm an inverse relationship between disease stage, pharmacotherapy and outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis. The Dynamic International Prognostic Scoring System was demonstrated to be useful for risk stratification of patients with myelofibrosis who are to undergo hematopoietic stem cell transplantation.

Progressive improvement in cutaneous and extracutaneous chronic graft-versus-host disease after a 24-week course of extracorporeal photopheresis--results of a crossover randomized study.

In a prior multicenter randomized controlled trial, we found that a 12-week course of extracorporeal photopheresis (ECP) plus standard immunosuppressive therapy resulted in several beneficial outcomes in patients with corticosteroid-refractory/intolerant/dependent chronic graft-versus-host disease (GVHD). Here, we report the results of an open-label crossover ECP study in 29 eligible participants randomized initially to the standard of care non-ECP (control) arm. Eligible for the crossover ECP study were control arm patients who either (1) had progression of cutaneous chronic GVHD (cGVHD), defined as >25% worsening from baseline as measured by the percent change in the total skin score (TSS) at any time, or (2) had less than 15% improvement in the TSS, or had a ≤25% reduction in corticosteroid dose at week 12 of the initial study. ECP was administered 3 times during week 1, then twice weekly until week 12, followed by 2 treatments monthly until week 24. The median age of the study cohort was 43 (20-67) years and 90% had extensive cGVHD. The median months from onset of cGVHD to start of ECP were 26 (range: 4-79). Twenty-five of 29 patients (86%) completed the 24-week course of ECP. Complete or partial skin response at week 24 was noted in 9 patients (31%). The median percent of decrease in TSS from baseline to weeks 12 and 24 was -7.9 and -25.8, respectively. In 4 (17%) and 8 (33%) patients, a ≥50% reduction in corticosteroid dose at weeks 12 and 24 was observed. Extracutaneous cGVHD response was highest in oral mucosa with 70% complete and partial resolution after week 24. In conclusion, progressive improvement in cutaneous and extracutaneous cGVHD was observed after a 24-week course of ECP in patients who previously had no clinical improvement or exhibited worsening of cGVHD while receiving standard immunosuppressive therapy alone in a randomized study. These results confirm previous findings and support the notion that prolonged ECP appears to be necessary for optimal therapeutic effects in corticosteroid-refractory cGVHD patients.

Small interfering RNA against BCR-ABL transcripts sensitize mutated T315I cells to nilotinib.

BACKGROUND: Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. We, therefore, evaluated specific BCR-ABL small interfering RNA silencing in BCR-ABL-positive cell lines, including those resistant to imatinib and particularly those with the T315I mutation. DESIGN AND METHODS: The factor-independent 32Dp210 BCR-ABL oligoclonal cell lines and human imatinib-resistant BCR-ABL-positive cells from patients with leukemic disorders were investigated. The effects of BCR-ABL small interfering RNA or the combination of BCR-ABL small interfering RNA with imatinib and nilotinib were compared with those of the ABL inhibitors imatinib and nilotinib. RESULTS: Co-administration of BCR-ABL small interfering RNA with imatinib or nilotinib dramatically reduced BCR-ABL expression in wild-type and mutated BCR-ABL cells and increased the lethal capacity. BCR-ABL small interfering RNA significantly induced apoptosis and inhibited proliferation in wild-type (P<0.0001) and mutated cells (H396P, T315I, P<0.0001) versus controls. Co-treatment with BCR-ABL small interfering RNA and imatinib or nilotinib resulted in increased inhibition of proliferation and induction of apoptosis in T315I cells as compared to imatinib or nilotinib alone (P<0.0001). Furthermore, the combination of BCR-ABL small interfering RNA with imatinib or nilotinib significantly (P<0.01) reversed multidrug resistance-1 gene-dependent resistance of mutated cells. In T315I cells BCR-ABL small interfering RNA with nilotinib had powerful effects on cell cycle distribution. CONCLUSIONS: Our data suggest that silencing by BCR-ABL small interfering RNA combined with imatinib or nilotinib may be associated with an additive antileukemic activity against tyrosine kinase inhibitor-sensitive and resistant BCR-ABL cells, and might be an alternative approach to overcome BCR-ABL mutations.

Therapeutic targeting of gene expression by siRNAs directed against BCR-ABL transcripts in a patient with imatinib-resistant chronic myeloid leukemia.

Within the recent years, RNA interference (RNAi) has become an almost-standard method for in vitro knockdown of any target gene of interest. Now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Here we show that in vivo application of targeted nonvirally delivered synthetic bcr-abl siRNA in a female patient with recurrent Philadelphia chromosome positive chronic myeloid leukemia (CML) resistant to imatinib (Y253F mutation) and chemotherapy after allogeneic hematopoietic stem cell transplantation can silence the expression of bcr-abl gene. We found a remarkable inhibition of the overexpressed bcr-abl oncogene resulting in increased apoptosis of CML cells. In vivo siRNA application was well tolerated without any clinically adverse events. Our findings imply that the clinical application of synthetic siRNA is feasible, safe and has real potential for genetic-based therapies using synthetic nonviral carriers.

Small-molecule inhibition of proteasome and silencing by vascular endothelial cell growth factor-specific siRNA induce additive antitumor activity in multiple myeloma.

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM), and MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional application of bortezomib directed against the 26S proteasome. After transfection with VEGF siRNA, we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji, and Karpas-299, as well as in cells of MM and lymphoma patients. VEGF siRNA significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P<0.0001), RPMI-8226 (P<0.0001), and INA-6 (P<0.01) versus controls. Cotreatment with VEGF siRNA and bortezomib in MM cells resulted in an exaggerated inhibition of proliferation and induction of apoptosis compared with VEGF siRNA or bortezomib alone (P<0.001). In addition, the combination of VEGF siRNA and bortezomib significantly (P<0.01) reversed multidrug resistance gene 1-dependent resistance of MM cells. Our data suggest that small-molecule inhibition of proteasome and silencing by VEGF-specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new, therapeutic strategies using RNA interference in MM.

Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells.

To investigate the molecular effects of growth factor independence 1B (Gfi-1B), a transcription factor essential for the development of hematopoietic cells and differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B overexpressing cell line K562 was cultured in the presence of Gfi-1B target-specific small interfering RNA (siRNA). SiRNA treatment significantly knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the siRNA silencing protocol by colony-forming units ensured that it was not cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down Gfi-1B were analyzed by oligonucleotide microarray technology and based upon rigorous statistical analysis of the data; relevant genes were chosen for confirmation by reserve transcriptase-polymerase chain reaction, including MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290, JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in Gfi-1B overexpressing cells compared to controls. Individual genes such as ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing cells. In addition, down-regulated transcripts showed cell signaling transcripts for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30, BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several essential cell signalling factors, as well as transcriptional and post-translational regulation genes were differentially expressed in cells that overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our data indicate that Gfi-1B signalling is important for commitment and maturation of hematopoietic cell populations.

Cytomegalovirus induces HLA-class-II-restricted alloreactivity in an acute myeloid leukemia cell line.

Cytomegalovirus (HCMV) reactivation is found frequently after allogeneic hematopoietic stem cell transplantation (alloSCT) and is associated with an increased treatment-related mortality. Recent reports suggest a link between HCMV and a reduced risk of cancer progression in patients with acute leukemia or lymphoma after alloSCT. Here we show that HCMV can inhibit the proliferation of the acute myeloid leukemia cell line Kasumi-1 and the promyeloid leukemia cell line NB4. HCMV induced a significant up-regulation of HLA-class-II-molecules, especially HLA-DR expression and an increase of apoptosis, granzyme B, perforin and IFN-γ secretion in Kasumi-1 cells cocultured with peripheral blood mononuclear cells (PBMCs). Indolamin-2,3-dioxygenase on the other hand led only to a significant dose-dependent effect on IFN-γ secretion without effects on proliferation. The addition of CpG-rich oligonucleotides and ganciclovir reversed those antiproliferative effects. We conclude that HCMV can enhance alloreactivity of PBMCs against Kasumi-1 and NB4 cells in vitro. To determine if this phenomenon may be clinically relevant further investigations will be required.

Use of the activating gene mutation of the tyrosine kinase (VAL617Phe) JAK2 as a minimal residual disease marker in patients with myelofibrosis and myeloid metaplasia after allogeneic stem cell transplantation.

Here we report on the use of a new real-time polymerase chain reaction (PCR) method to detect and quantify the activating gene mutation of the tyrosine kinase JAK2. We evaluated patients with myelofibrosis with myeloid metaplasia (MMM; n=25) for the gene mutation prior to allogeneic stem cell transplantation and monitored them in the long-term follow up of 125 months (median 15, range 4-125) after transplant. The results obtained were correlated to the chimerism status of these patients. The JAK2 gene mutation was detected in 15 of 25 analyzed patients prior to transplant. Three patients who were again positive for JAK2 after transplant also had mixed chimerism status. These three patients relapsed from MMM shortly after JAK2 gene mutation was detected for the first time after transplant. Our presented data shows the feasibility of the detection of JAK2 gene mutation by real-time PCR as a minimal residual disease marker after transplant.

T-cell depletion prevents from bronchiolitis obliterans and bronchiolitis obliterans with organizing pneumonia after allogeneic hematopoietic stem cell transplantation with related donors.

Bronchiolitis obliterans (BO) and bronchiolitis obliterans organizing pneumonia (BOOP) are late-onset non-infectious pulmonary complications (LONIPCs) following allogeneic hematopoietic stem cell transplantation (HSCT). In the present study 10 of 197 conventionally prepared stem cell recipients developed BOOP after 365 days and 6 patients developed BO 333 days post-transplant. No BOOP or BO was diagnosed following T-cell depletion (p<0.05). Chronic GVHD was ascertained in all BOOP patients and appeared significantly (p<0,001) more frequent in the conventional transplant group. The data confirm a strong association between T-cell activity, chronic GVHD, BO and BOOP and point out the impact of T lymphocytes in the pathomechanism of BOOP.

Molecular methods used for detection of minimal residual disease following hematopoietic stem cell transplantation in myeloid disorders.

Monitoring of minimal residual disease (MRD) in patients with acute or chronic myeloid disorders is routinely performed after allogeneic or autologous transplantation. The detection of MRD helps to identify patients who are at high risk for leukemic relapse after transplantation. The most commonly used techniques for MRD detection are qualitative and quantitative PCR methods, fluorescence in situ hybridization (FISH), fluorescence-activated cell sorting (FACS), and cytogenetic analysis, which are often performed complementary in order to assess more precisely MRD. Here, we describe the most used sensitive real-time reverse-transcription (RT)-PCR methods for chronic and acute myeloid disorders. Besides protocols for real-time RT-PCR and multiplex RT-PCR procedures for the most common fusion-gene transcripts in acute and chronic myeloid disorders, methods for detection of disease-specific genetic mutated alterations as FLT3 gene-length mutations, and aberrantly expressed genes as WT1 gene transcripts, are described in detail for daily use.

Donor cell reaction to OKT3 as predictor of chronic graft-vs-host disease in hematopoietic stem cell recipients.

OBJECTIVE: In the hematopoietic stem cell transplantation setting, granulocyte colony-stimulating factor (G-CSF) administration can reduce donor cell reactivity in vitro, but the clinical significance of this phenomenon was only sparsely defined. METHODS: We performed lymphocyte transformation tests in 28 related stem cell donors pre and 5 days post G-CSF treatment, respectively, and correlated proliferative responses of donor peripheral blood mononuclear cells with clinical parameters in the corresponding recipients. RESULTS: In vitro reactions towards 4 mitogens and 12 recall antigens at day 5 post G-CSF administration were predictive for the occurrence of chronic graft-vs-host disease (cGVHD). Here, proliferative responses towards the mitogen anti-CD3 monoclonal antibody (OKT3) above median were most informative; this threshold could be determined by discrimination and receiver operating curve (ROC) analyses. In the whole cohort (18 human leukocyte antigen [HLA]-identical and 10 partially mismatched donor-recipient pairs), OKT3 responses predicted cGVHD with an odds ratio of 33.0, a sensitivity of 79%, and a specificity of 90%. A subgroup analysis of HLA-identical pairs even yielded an odds ratio of 85.0. Furthermore, bivariate analysis defined HLA compatibility and responses towards OKT3 as independent risk factors for cGVHD (p = 0.02 and p = 0.0007, respectively). CONCLUSION: The proliferative capacity of G-CSF-mobilized donor cells appears as a graft factor that determines the future incidence of cGVHD in the corresponding recipient.

Early CMV-replication after allogeneic stem cell transplantation is associated with a reduced relapse risk in lymphoma.

A preventive effect of early human cytomegalovirus (HCMV) replication was evaluated in 136 non-Hodgkin lymphoma (NHL) patients with mature B-cell NHLs (n = 94), and mature T- and NK-cell NHLs (n = 42) after allogeneic stem cell transplantation (alloSCT). Most study-patients (85%) had received at least 2 cycles of chemotherapy and 60% had also received an autograft prior to alloSCT. First detection of CMV-replication by HCMV antigenemia/viremia was found at a median of day +33 after alloSCT. The cumulative incidence of relapse at 5 years after alloSCT was 38% (95% confidence interval [95%CI]: 26-49) in 82 patients without compared to 22% (95%CI: 8-37) in 54 patients with HCMV antigenemia/viremia (p = .013). A decreased relapse risk of HCMV replication was confirmed by multivariate analysis for HCMV antigenemia/viremia (Hazard ratio [HR]: 0.29, 95%CI: 0.11-0.76, p < .014). This report demonstrated a possible improvement of relapse incidence after replicative HCMV infection in patients with NHL after alloSCT.

Mutations in innate immune system NOD2/CARD 15 and TLR-4 (Thr399Ile) genes influence the risk for severe acute graft-versus-host disease in patients who underwent an allogeneic transplantation.

BACKGROUND: NOD2 and TLR-4 genes belong to the innate immune system that detects invading pathogens through several pattern-recognition receptors. Here we analyzed 403 patients for NOD2 gene mutations and 307 patients for TLR-4 gene mutations (Thr399Ile) with their respective donors and correlated the results with the incidence of acute graft-versus-host disease (aGVHD), severe acute GVHD (saGVHD), the risk for transplant-related mortality (TRM), overall survival (OS) and incidence of infectious complications. METHODS: We performed a retrospective single-center study. Genotyping of TLR-4 and NOD2 were evaluated by real-time polymerase chain reaction. RESULTS: Surprisingly, we found a significant reduced incidence of aGVHD, saGVHD, and intestinal GVHD for patients with NOD2 gene mutations on the donor side with 50%, 0% and 2% compared to patients with the wild-type NOD2 gene with 65%, 17%, and 26%, respectively (P<0.02). However, the incidence of saGVHD increased in patients with NOD2 mutations on the patient and donor (P/D) side with 44% versus 17% compared to patients with the wild-type gene (P<0.03). TLR-4 gene mutations at P/D side had an increased risk for saGVHD with 42% versus 15% of patients with wild-type gene (P<0.04). OS, TRM, and incidence of infectious complications were not influenced by the mutated genes. Multivariate analysis confirmed that NOD2 gene mutations on the donor side had a reduced risk for saGVHD (P<0.001), whereas mutations of the NOD2 gene on P/D side had an increased risk for saGVHD (P<0.01) in our analysis. CONCLUSIONS: These results suggest that NOD2 mutations have influence on the occurrence of acute GVHD after transplantation.

Reduced risk for molecular disease in patients with chronic myeloid leukemia after transplantation from a KIR-mismatched donor.

BACKGROUND: To examine how killer-cell immunoglobulin-like receptor (KIR) ligand incompatibilities effect molecular relapse (MR), we compared the occurrence of bcr-abl-positive reverse-transcriptase polymerase chain reaction (RT-PCR) results in 236 CML patients (pts) after human leukocyte antigen (HLA)-identical (n=158) (group 1), HLA class I antigen mismatched and KIR-ligand compatible (n=49) (group 2), and HLA class I antigen mismatched and KIR-ligand incompatible (n=29) (group 3) hematopoietic stem-cell transplantation. METHODS: We performed a retrospective single-center study. MR was evaluated using the real-time RT-PCR method for the detection of bcr-abl transcripts. RESULTS: In the first group, 133 of 158 (84%) pts were in the first chronic phase of CML, and the corresponding figures were 33 of 49 (67%) pts in group 2 and 19 of 29 (64%) in group 3 (P<0.05). MR occurred in 1 of 29 (3%) pts in group 3 compared with 62 of 158 (39%) pts in group 1 and in 11 of 49 (22%) pts in group 2 (P<0.001). A hematologic relapse developed in 20 of 158 (13%) pts in group 1, 2 of 49 (4%) pts in group 2, and in 0 of 29 (0%) pts in group 3 (P<0.05). Multivariate analysis confirmed that KIR mismatches are a strong independent predictor for the occurrence of MR after transplantation (P<0.02). The 5-year overall survival rate did not vary greatly between the three groups (67% in group 1, 52% in group 2, and 66% in group 3). CONCLUSIONS: These results suggest that KIR-ligand incompatibility is an important prognostic factor in the occurrence of MR after transplantation for CML.