Risk factors and prognosis of hepatic acute GvHD after allogeneic hematopoietic cell transplantation.

Hepatic acute GvHD (aGvHD) is associated with high mortality owing to poor response to immunosuppressive therapy. The pathogenesis of hepatic aGvHD differs from that of other lesions, and specific risk factors related to pre-transplant liver conditions should be determined. We conducted a cohort study by using a Japanese transplant registry database (N=8378). Of these subjects, 1.5% had hepatitis C virus Ab (HCV-Ab) and 9.4% had liver dysfunction (elevated transaminase or bilirubin levels) before hematopoietic cell transplantation (HCT). After HCT, the cumulative incidence of hepatic aGvHD was 6.7%. On multivariate analyses, HCV-Ab positivity (hazard ratio (HR), 1.93; P=0.02) and pre-transplant liver dysfunction (HR, 1.85; P<0.01), as well as advanced HCT risk, unrelated donors, HLA mismatch and cyclosporine as GvHD prophylaxis, were significant risk factors for hepatic aGvHD, whereas hepatitis B virus surface Ag was not. Hepatic aGvHD was a significant risk factor for low overall survival and high transplant-related mortality in all aGvHD grades (P<0.01). This study is the first to show the relationship between pre-transplant liver conditions and hepatic aGvHD. A prospective study is awaited to validate the results of this study and establish a new strategy especially for high-risk patients.

Impact of MRD and TKI on allogeneic hematopoietic cell transplantation for Ph+ALL: a study from the adult ALL WG of the JSHCT.

To assess the impact of minimal residual disease (MRD) and tyrosine kinase inhibitor (TKI) administration on allogeneic hematopoietic cell transplantation (allo-HCT) for Ph-positive ALL (Ph+ALL), we retrospectively analyzed data from a registry database for 432 adult Ph+ALL patients in first CR (CR1) who received pre-transplant TKI administration. Negative MRD (MRD(-)) at allo-HCT was achieved in 277 patients. OS in patients transplanted in MRD(-) was significantly better than that in patients transplanted in MRD(+) (MRD(-): 67% vs MRD(+): 55% at 4 years; P=0.001). MRD(-) at allo-HCT was a significant risk factor for survival along with age at allo-HCT in multivariate analyses. Incidence of relapse in patients transplanted in MRD(-) was significantly lower than that in patients transplanted in MRD(+) (MRD(-): 19% vs MRD(+): 29% at 4 years; P=0.006). In multivariate analyses, MRD(+) at allo-HCT was a significant risk factor for relapse. A post-transplant TKI was administered to 103 patients. In subanalyses regarding the effect of post-transplant TKI administration, post-transplant TKI administration was a significant risk factor for relapse in multivariate analyses (P<0.0001). MRD status at allo-HCT is one of the most important predictive factors for Ph+ALL patients transplanted in CR1.

Impact of conditioning intensity and TBI on acute GVHD after hematopoietic cell transplantation.

The impact of the conditioning intensity and TBI on acute GVHD (aGVHD) is still a matter of debate. We analyzed 6848 adult recipients who received allogeneic hematopoietic cell transplants (HCT) between 2006 and 2011 in Japan. The subjects were divided into groups who had received myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC), either with or without TBI. There was a significant difference in the incidence of aGVHD 2-4 among the different conditioning types: 39% in TBI-MAC, 35% in TBI-RIC and 32% in both no-TBI MAC and no-TBI-RIC (P<0.001). In a multivariate analysis, TBI-MAC, but not no-TBI MAC, was significantly associated with an increased risk of aGVHD 2-4 (hazard ratio (HR) 1.33, P<0.01), whereas TBI-RIC was associated with an increased risk of GVHD 3-4 (HR 1.36, P=0.048). TBI-MAC and TBI-RIC were significantly associated with skin and gastrointestinal aGVHD. Subgroup analyses demonstrated that not only TBI-MAC, but also TBI-RIC, was significantly associated with aGVHD 2-4 in older patients. Furthermore, high-dose TBI only had an adverse impact on aGVHD 2-4 in HLA-matched HCT. Impacts of intensity and TBI on aGVHD differ by patient backgrounds, and this difference should be considered to establish a risk-adapted strategy for the prevention of aGVHD.

Continuing increased risk of oral/esophageal cancer after allogeneic hematopoietic stem cell transplantation in adults in association with chronic graft-versus-host disease.

BACKGROUND: The number of long-term survivors after hematopoietic stem cell transplantation (HSCT) showed steady increase in the past two decades. Second malignancies after HSCT are a devastating late complication. We analyzed the incidence of, risk compared with that in the general population, and risk factors for secondary solid cancers. PATIENTS AND METHODS: Patients were 17 545 adult recipients of a first allogeneic stem cell transplantation between 1990 and 2007 in Japan. Risks of developing secondary solid tumors were compared with general population by using standard incidence ratios (SIRs). RESULTS: Two-hundred sixty-nine secondary solid cancers were identified. The cumulative incidence was 0.7% [95% confidence interval (CI), 0.6%-0.9%] at 5 years and 1.7% (95% CI, 1.4%-1.9%) at 10 years after transplant. The risk was significantly higher than that in the general population (SIR=1.8, 95% CI, 1.5-2.0). Risk was higher for oral cancer (SIR=15.7, 95% CI, 12.1-20.1), esophageal cancer (SIR=8.5, 95% CI, 6.1-11.5), colon cancer (SIR=1.9, 95% CI, 1.2-2.7), skin cancer (SIR=7.2, 95% CI, 3.9-12.4), and brain/nervous system cancer (SIR=4.1, 95% CI, 1.6-8.4). The risk of developing oral, esophageal, or skin cancer was higher at all times after 1-year post-transplant. Extensive-type chronic graft-versus-host disease (GVHD) was a significant risk factor for the development of all solid tumors (RR=1.8, P<0.001), as well as for oral (RR=2.9, P<0.001) and esophageal (RR=5.3, P<0.001) cancers. Limited-type chronic GVHD was an independent risk factor for skin cancers (RR=5.8, P=0.016). CONCLUSION: Recipients of allogeneic HSCT had a significantly higher ∼2-fold risk of developing secondary solid cancers than the general population. Lifelong screening for high-risk organ sites, especially oral or esophageal cancers, is important for recipients with active, or a history of, chronic GVHD.

Allogeneic transplantation for primary myelofibrosis with BM, peripheral blood or umbilical cord blood: an analysis of the JSHCT.

To determine whether a difference in donor source affects the outcome of transplantation for patients with primary myelofibrosis (PMF), a retrospective study was conducted using the national registry data on patients who received first allogeneic hematopoietic cell transplantation (HCT) with related BM (n=19), related PBSCs (n=25), unrelated BM (n=28) or unrelated umbilical cord blood (UCB; n=11). The 5-year OS rates after related BM, related PBSC and unrelated BM transplantation were 63%, 43% and 41%, respectively, and the 2-year OS rate after UCB transplantation was 36%. On multivariate analysis, the donor source was not a significant factor for predicting the OS rate. Instead, performance status (PS) ≥2 (vs PS 0-1) predicted a lower OS (P=0.044), and RBC transfusion ≥20 times before transplantation (vs transfusion ≤9 times) showed a trend toward a lower OS (P=0.053). No advantage of nonmyeloablative preconditioning regimens in terms of decreasing nonrelapse mortality or increasing OS was found. Allogeneic HCT, and even unrelated BM and UCB transplantation, provides a curative treatment for PMF patients.

Risk factors and organ involvement of chronic GVHD in Japan.

Few studies have evaluated the risk factors for chronic GVHD and organ involvement associated with different graft types, including unrelated cord blood (U-CB). We retrospectively studied 4818 adult patients who received their first allogeneic transplantation and survived for at least 100 days. The incidence of chronic GVHD at 2 years was 37%. The following factors were associated with the development of chronic GVHD: female donor/male recipient, CMV-Ab seropositivity, matched related peripheral blood grafts vs matched related BM grafts, no in vivo T-cell depletion and the occurrence of grade II-IV acute GVHD. Among these factors, the association with acute GVHD occurrence was consistently significant across donor subtypes. The use of U-CB was not associated with chronic GVHD, but was associated with a low incidence of extensive chronic GVHD. Chronic GVHD patients who had received U-CB transplants showed less frequent involvement of the oral cavity (28% vs 55%), eye (12% vs 26%), liver (20% vs 44%), lung (11% vs 25%) and joint (0% vs 6%) than those with matched related BM grafts. In conclusion, we found that U-CB transplants were associated with a low incidence of extensive chronic GVHD and less frequent involvement of the oral cavity, eye, liver, lung and joints.

Reduced-intensity allogeneic stem cell transplantation for patients aged 50 years or older with B-cell ALL in remission: a retrospective study by the Adult ALL Working Group of the Japan Society for Hematopoietic Cell Transplantation.

We retrospectively assessed the outcome and pretransplantation predictors of the outcome in 118 patients aged ≥ 50 years who received fludarabine-containing reduced-intensity allo-SCT (RIST) for B-cell ALL in the first or second CR. Eighty patients received transplants from unrelated donors. Seventy-eight patients were positive for the Ph chromosome. The median follow-up period was 18 months and the 2-year OS rate was 56%. The 2-year cumulative incidence of relapse and non-relapse mortality was 28% and 26%, respectively. The incidence of grades II-IV and III-IV acute GVHD was 46% and 24%, respectively. After 2 years, the incidence of chronic GVHD was 37%. Multivariate analysis of pretransplant factors showed that a higher white blood cell count (≥ 30 × 10(9)/L) at diagnosis (hazard ratio (HR)=2.19, P=0.007) and second CR (HR=2.02, P=0.036) were significantly associated with worse OS, whereas second CR (HR=3.83, P<0.001) and related donor (HR=2.34, P=0.039) were associated with a higher incidence of relapse. Fludarabine-containing RIST may be a promising strategy for older patients with B-cell ALL in their first remission.

Recent decrease in non-relapse mortality due to GVHD and infection after allogeneic hematopoietic cell transplantation in non-remission acute leukemia.

Although recent improvements have been indicated in the outcome after allogeneic hematopoietic cell transplantation (allo-HCT), little information is available on how changes in transplant modalities have affected the outcomes after allo-HCT in non-remission, based on patient age, donor source and disease type. We compared the incidence and causes of non-relapse mortality (NRM) after allo-HCT in non-remission among three consecutive four-year periods using a nationwide transplant outcome registry database. A total of 3308 patients with acute leukemia in non-remission were analyzed. The risk of NRM decreased over the three periods, and the hazard ratios (HRs) in 2001-2004 and 2005-2008 compared with 1997-2000 were 0.86 (95% CI, 0.70-1.06; P=0.16) and 0.65 (95% CI, 0.53-0.80; P<0.01), respectively. A significant decrease in the HR for overall mortality was also observed in 2005-2008 (HR 0.85; 95% CI, 0.75-0.97; P=0.02). We found that a decrease in the incidences of death due to GVHD and infection contributed to the reduction in NRM, to which high-resolution donor-recipient HLA matching and other improvements may have contributed. As none of the subgroups showed improved survival without a reduction in NRM, the effective prevention of transplant-related complications appears to be necessary for improving outcomes after allo-HCT in non-remission.

Changes in incidence and causes of non-relapse mortality after allogeneic hematopoietic cell transplantation in patients with acute leukemia/myelodysplastic syndrome: an analysis of the Japan Transplant Outcome Registry.

The outcomes for allogeneic hematopoietic cell transplantation (allo-HCT) are heavily influenced by non-relapse mortality (NRM). We retrospectively assessed the changes in the incidence and causes of NRM after allo-HCT over the past 12 years. NRM, relapse rate and OS were analyzed using the Japan transplant outcome database of 6501 adult patients with acute leukemia or myelodysplastic syndrome who received their first allo-HCT in remission from 1997 through 2008. In multivariate analysis in patients aged 16-49 years, the adjusted hazard ratios (HRs) for NRM for 2001-2004 and 2005-2008 were 0.78 (95% confidence interval, 0.65-0.93) and 0.64 (0.54-0.78), respectively, compared with 1997-2000. The HR for overall mortality in 2005-2008 was 0.81 (0.70-0.93) compared with 1997-2000. In patients aged 50-70 years, the HRs for NRM and overall mortality in 2005-2008 were 0.56 (0.46-0.68) and 0.66 (0.47-0.93), respectively, compared with those in 2001-2004. We found that causes of death that contributed to the changes in NRM varied among subgroups. In conclusion, our study indicated that the incidence of NRM after allo-HCT has significantly decreased over the past 12 years, which has led to an improvement of OS, and also showed reductions in NRM in subgroups consisting of older patients and those who received unrelated cord blood transplantation.

Unrelated cord blood transplantation vs related transplantation with HLA 1-antigen mismatch in the graft-versus-host direction.

Little information is available regarding whether an unrelated cord blood (UCB) unit or a related donor with a 1-antigen mismatch at the HLA-A, HLA-B or HLA-DR locus in the graft-versus-host direction (RD/1AG-MM-GVH) should be selected as an alternative donor for patients without an HLA-matched related/unrelated donor. Therefore, we conducted a retrospective study using national registry data on patients with leukemia or myelodysplastic syndrome who received transplantation using a single UCB (n=2288) unit or an RD/1AG-MM-GVH (n=525). We found that the survival rate in the UCB group was comparable to that in the RD/1AG-MM-GVH group, although the RD/1AG-MM-GVH group with an HLA-B mismatch showed significantly higher overall and non-relapse mortality. Neutrophil and platelet engraftment were significantly faster, whereas the incidence of acute or chronic graft-versus-host disease (GVHD) was significantly higher in the RD/1AG-MM-GVH group. The incidence of acute or chronic GVHD in the RD/1AG-MM-GVH group with in vivo T-cell depletion was comparable to that in the UCB group, which translated into a trend toward better overall survival, regardless of the presence of an HLA-B mismatch. In conclusion, UCB and RD/1AG-MM-GVH are comparable for use as an alternative donor, except for RD/1AG-MM-GVH involving an HLA-B mismatch.

Long-term outcome of cord blood transplantation from unrelated donors as an initial transplantation procedure for children with AML in Japan.

To assess the outcome of unrelated umbilical cord blood transplantation (UCBT), 141 children with AML who underwent UCBT (39 in first CR (CR1), 33 in CR2, 4 in CR3 and 65 at more advanced stages (not in CR)) were analyzed in a retrospective multicenter study in Japan. Short-term MTX was used for prophylaxis of acute GVHD in 80 cases (57%). The cumulative incidences of neutrophil recovery, platelet recovery and acute GVHD (grades 2-4) were 78.7, 62.4 and 40.1%, respectively, and the 100-day transplantation-related mortality (TRM) was 10.8%. Multivariate analysis showed that an infused CD34(+) cell dose of 1.35 x 10(5) cells per kg or more was associated with favorable neutrophil and platelet recovery, and that short-term MTX was associated with a lower 100-day TRM. The 6-year relapse rate was 38.8% and was associated with disease status. Six-year overall survival was 45.8% (70.4+/-8.3% in CR1, 59.3+/-11.3% in CR2, 75.5+/-21% in CR3 and 20.6+/-6.2% for children with non-CR). We conclude that the results of UCBT are particularly promising for children with a karyotype suggesting a poor prognosis, and for those who receive transplants in CR2 and CR3 after an early relapse.

Unrelated cord blood transplantation in CML: Japan Cord Blood Bank Network analysis.

We analysed 86 patients with CML who received unrelated cord blood transplantation (UCBT), identified through a registry of the Japan Cord Blood Bank Network. At transplantation, the median patient age was 39 years (range, 1-67 years); 38 patients were in chronic phase (CP), 13 in the accelerated phase (AP) and 35 in blast crisis (BC). Median duration from diagnosis to UCBT was 1.5 years (range, 0.2-14.6 years). A nucleated cell (NC) dose of more than 3.0 x 10(7) per kg was sufficient to achieve neutrophil (91%) and platelet recovery (86%), whereas the lower dose of NC achieved only 60 and 61%, respectively. The duration and type of pre-transplant treatment did not affect neutrophil or platelet recovery. Results of multivariate analysis indicated that older patients (>50 years) had a higher incidence of transplant-related mortality. Advanced-disease stage and lower doses of NCs were significantly associated with lower leukaemia-free and event-free survival. At 2-year survival for patients in CP, AP and BC was 71, 59 and 32%, respectively (P=0.0004). A pre-transplant European Group for Blood and Marrow Transplantation scoring system was effective in predicting the outcome of UCBT. We conclude that UCBT is a reasonable alternative therapy for patients with CML.

Cardiovascular toxicity of cryopreserved cord blood cell infusion.

Although infusion of cryopreserved bone marrow or peripheral blood stem cell is associated with a variety of symptoms, there have been no reports detailing the data of infusion-related toxicities of cryopreserved cord blood (CB) units. We prospectively evaluated the incidence and significance of infusion-related toxicities in 34 adult patients undergoing unrelated CB transplantation. Cryopreserved CB units were thawed and immediately infused, unfiltered, through a central intravenous catheter without further manipulation. Heart rate, blood pressure, oxygen saturation and clinical symptoms were recorded during and after infusion. Twenty-four percent of patients experienced non-cardiovascular toxicities related to infusion. The incidence of systolic and diastolic hypertension and bradycardia was 58, 64 and 32%, respectively. Although three patients (9%) with severe systolic hypertension after the infusion required treatment with antihypertensive agents, no patients experienced life-threatening side effects or needed discontinuation of CB unit infusion. Patient or transplant characteristics had no effect on the hypertension and bradycardia related to the infusion of CB. These data suggest that infusion of cryopreserved CB without further manipulation after thawing is safe and well tolerated. However, cardiovascular toxicities including hypertension and bradycardia were frequently observed.

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.

Transcription factors that regulate growth and differentiation of myeloid cells.

Recently much progress has been made in our understanding of how myeloid progenitor cells undergo commitment and become mature granulocytes or monocytes/macrophages. Studies of normal and leukemic myeloid cells as well as those of cells derived from mice with targeted disruption showed that a series of transcription factors play a major role in both commitment and maturation of myeloid cells. This is primarily because these transcription factors direct an ordered pattern of gene expression according to a well-defined developmental program. PU.1, an Ets family member, is one of the master transcription factors identified to regulate development of both granulocytes and monocytes/macrophages. Further, C/EBPalpha and C/EBPvarepsilon of the bZip family have important roles in directing granulocytic maturation. A number of additional transcription factors such as AML1, RARalpha, MZF-1, Hox and STAT families of transcription factors, Egr-1 and c-myb etc are shown to play roles in myeloid cell differentiation. Our laboratory has recently obtained evidence that ICSBP, a member of the IRF family, is involved in lineage commitment during myeloid cell differentiation and stimulates maturation of functional macrophages. Future elucidation of pathways and networks through which these transcription factors act in various stages of development would provide a more definitive picture of myeloid cell commitment and maturation.

[Autologous bone marrow transplantation for CML].

The first line therapy for CML is IFN-alpha and/or allogeneic hematopoietic stem cell transplantation(HSCT). The indication of autologous HSCT is limited for patients with no HLA-matched donor, and is recognized as an experimental therapy. Major problems of autologous HSCT for CML are contamination of Ph1-positive cells to graft and no GVL effects. To concur these problems, many attempts have been made such as the collection of peripheral blood stem cells after high dose chemotherapy, ex vivo purging using antisense, administration cyclosporin to induce GVL effect, and post-HSCT therapy by IFN-alpha +/- Interleukin-2. Autologous HSCT for CML is still to be a hopeful candidate for pursuing cure.

ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages.

During hematopoiesis, myeloid progenitor cells give rise to granulocytes and macrophages. To study the role for ICSBP, a hematopoietic cell-specific transcription factor in myeloid cell development, the gene was introduced into myeloid progenitor cells established from ICSBP-/- mice. ICSBP retrovirus-transduced cells differentiated into mature macrophages with phagocytic activity, which coincided with the induction of specific target DNA binding activity. Similar to macrophages in vivo, ICSBP-transduced cells were growth arrested, expressed many macrophage-specific genes, and responded to macrophage activation signals. Contrary to this, ICSBP transducion led to repression of granulocyte-specific genes and inhibited G-CSF-mediated granulocytic differentiation in these and other myeloid progenitor cells. Together, ICSBP has a key role in the myeloid cell lineage selection and macrophage maturation.

Hyperfunction of neutrophils in a patient with BCR/ABL negative chronic myeloid leukemia: a case report with in vitro studies.

BACKGROUND: Among patients diagnosed with chronic myeloid leukemia (CML), a small percentage lack a BCR/ABL fusion gene, a landmark of CML. Their clinical features are distinct from patients with BCR/ABL positive CML, although to the authors' knowledge the pathogenesis to date has been unknown. METHODS: A 50-year-old female patient with BCR/ABL negative CML and multiple complications of Graves disease, Sweet syndrome, and a fatal pulmonary alveolar proteinosis (PAP) is described in the current study. To show a clonal origin of her myeloid cells, hypoxanthine phosphoribosyltransferase (HPRT) assay was applied. Because the patient developed a progressive and fatal neutrophilia, a screening of cell functions in neutrophilic lineage, including in vitro colony assay of her bone marrow cells and production of superoxide and interleukin-8 (IL-8) by blood neutrophils was performed. RESULTS: Southern blot analysis based on the polymorphism of the HPRT gene was compatible with monoclonality of her neutrophils. The patient had an increased amount of bone marrow granulocyte-macrophage progenitor cells, which formed colonies in response to a very low dose (0.1 ng/mL) of granulocyte-colony stimulating factor. In vitro production of superoxide and IL-8, which is an inducer of positive chemotaxis of neutrophils, by her peripheral neutrophils was markedly augmented. Her bronchoalveolar lavage fluid also contained a significant amount of IL-8 as well as an unusual infiltration of neutrophils. CONCLUSIONS: In the patient in the current study, hyperfunction of the neutrophils might have contributed to the onset of PAP as well as Sweet syndrome and to the pathogenesis of BCR/ABL negative CML.