Hiroatsu Iida

Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells

Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.

Differential constitutive activation between STAT‐related proteins and MAP kinase in primary acute myelogenous leukaemia

Many cytokines and growth factors stimulate multiple signal transduction pathways essential for proliferation in human acute leukaemia cells, including a mitogen‐activated protein (MAP) kinase pathway and a Janus kinase (JAK)‐STAT (signal transducers and activators of transcription) pathway. We have previously shown constitutive activation of MAP kinase in approximately 50% of acute myelogenous leukaemia (AML) samples. Recently, STAT proteins have been reported to be constitutively activated in 10–20% of AML cases. STAT3 and STAT5 are the main STAT proteins activated in haemopoietic progenitors in response to cytokines such as IL‐3, GM‐CSF, erythropoietin and thrombopoietin. Although the possibility of STAT1 protein as a substrate for MAP kinase at a serine residue has been suggested, the cross‐talk between STATs and MAP kinase pathways in vivo, especially in leukaemia cells, remains unknown.

Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults

Post transplant immune disorders are problematic in cord blood transplantation (CBT) for adult patients, and optimal prophylaxis has not been established. We investigated whether intensive graft-versus-host disease (GVHD) prophylaxis using short-term methotrexate (MTX) has a prognostic impact on CBT. Post-CBT immune reactions were classified according to time course as pre-engraftment immune reaction (PIR), engraftment syndrome (ES) or acute GVHD. Between March 2001 and November 2005, a total of 77 patients underwent CBT at eight transplantation centers. Median age was 48 years (range, 18–69 years). Preparative regimens comprised myeloablative (n=31) or reduced-intensity (n=46). Acute GVHD prophylaxis included cyclosporine alone (n=23), tacrolimus alone (n=12), cyclosporine plus MTX (n=17), tacrolimus plus short-term MTX (n=23) or cyclosporine plus methylprednisolone (n=2). Cumulative incidences of PIR, ES and grade II–IV GVHD were 36, 12 and 23%, respectively. Short-term MTX exerted significant favorable effects on post-CBT immune reactions (hazard ratio, 0.55; 95% confidence interval (95% CI), 0.31–0.98; P=0.04) in multivariate analysis. Overall survival rates for patients with and without short-term MTX at day 180 were 59% (95% CI, 42–73%) and 16% (95% CI, 6.6–30%) (P=0.0001), respectively. Short-term MTX could offer one optimal regimen to reduce immune reactions and improve outcomes in CBT.

Solid tumors after hematopoietic stem cell transplantation in Japan : incidence, risk factors and prognosis

Summary:To evaluate the incidence, risk factors and prognosis for solid tumors after hematopoietic stem cell transplantation (HSCT) in Japan, 809 patients who had received HSCT between 1981 and 2000 were retrospectively analyzed. In all, 19 newly diagnosed secondary cancers were observed. The risk for cancer development was 2.8 times as high as that for expected cases. The cumulative incidence ratios at 5 and 10 years were 1.9 and 4.2%, respectively. The risk was significantly elevated for buccal cavity cancer (standard incidental ratio (SIR), 44.42: 95% confidence interval (CI) 17.86–91.51), esophageal cancer (SIR, 22.36: 95% CI 6.09–57.25), and cervical cancer (SIR, 8.58: 95% CI 1.04–31.01). Of 15 patients who developed solid cancers following allografting, 12 had chronic graft-versus-host disease (GVHD), and all 10 patients with squamous cell carcinoma of the buccal cavity or esophagus had chronic GVHD. On multivariate analysis, extensive chronic GVHD and age over 45 years at the time of transplantation were associated with a higher risk for solid cancers. In all, 17 patients received therapy for secondary cancers, nine of whom are still alive and the 5-year probability of survival from the diagnosis is 42.8%. Our data suggest that early detection of secondary cancers is very important in prolonging overall survival.

Myeloablative allogeneic hematopoietic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia in adults: significant roles of total body irradiation and chronic graft-versus-host disease.

Summary:Disease-free survival in Philadelphia chromosome-positive ALL (Ph+ALL) is very poor, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently considered the only procedure with curative potential. To identify factors affecting transplant outcome, we analyzed the data from 197 Ph+ALL patients aged 16 years or older who had undergone allo-HSCT. The 5-year survival rates were 34% for patients in first complete remission (CR), 21% for those in second or subsequent CR, and 9% for those with active disease (P<0.0001). Multivariate analysis showed four pre-transplant factors as significantly associated with better survival: younger age, CR at the time of transplantation, conditioning with total body irradiation, and HLA-identical sibling donor (P<0.0001, P<0.0001, P=0.0301, P=0.0412, respectively). Severe acute GVHD increased the risk of treatment-related mortality (TRM) without diminishing the risk of relapse, whereas chronic GVHD reduced the risk of relapse without increasing the risk of TRM. Thus, patients who developed extensive chronic GVHD had better survivals (P=0.0217), and those who developed grade III–IV acute GVHD had worse survivals (P=0.0023) than did the others.

Lack of constitutive activation of MAP kinase pathway in human acute myeloid leukemia cells with N-Ras mutation

Mitogen-activated protein (MAP) kinases act as transducers of extracellular signaling via tyrosine kinase-growth factor receptors and G-protein-linked receptors to transcription factors. Constitutive activation of MAP kinase has been observed in a variety of solid tumors including renal cancer and breast cancer. Recently, we have reported that constitutively activated MAP kinase was observed in 50% of human primary acute myeloid leukemia (AML) cells. Ras is one of the components of G-proteins and transduces the signal from cytokine receptors to raf-1 theoretically resulting in the activation of MAP kinase pathway. In the present study, we have examined the correlation of Ras mutations and the activation of MAP kinase pathway in patients with AML. Twenty out of 22 AML cases with activating N-Ras mutations showed no phosphorylated forms of ERK2. ERK2 phosphorylation was tightly correlated with ERK1 phosphorylation and MAP kinase activity detected by in vitro kinase assay. Three samples with N-Ras mutations were stimulated with IL-3, GM-CSF and G-CSF separately but ERK2 activation was induced in none of these samples stimulated with these cytokines. In contrast, ERK2 was constitutively activated in all of four pancreatic carcinoma cases with K-Ras mutation at codon 12. These results suggest that function of the Ras mutations may be different between solid tumors, such as pancreatic carcinoma and colorectal carcinoma, and AML. Mutated Ras does not always stimulate MAP kinase pathway constitutively and may rather inhibit classical MAP kinase cascade in AML blasts from leukemia patients.

Allogeneic stem cell transplantation for adult Philadelphia chromosome-negative acute lymphocytic leukemia: comparable survival rates but different risk factors between related and unrelated transplantation in first complete remission

To identify factors to improve the outcomes of related and unrelated allogeneic stem cell transplantations (allo-SCT) for Philadelphia chromosome-negative acute lymphocytic leukemia (Ph(-) ALL) in the first complete remission (CR1), we retrospectively analyzed 1139 Ph(-) ALL patients using the registry data, particularly the details of 641 patients transplanted in CR1. Overall survival was significantly superior among patients transplanted in CR1, but no significant difference was observed between related and unrelated allo-SCTs (related vs unrelated: 65% vs 62% at 4 years, respectively; P = .19). Among patients transplanted in CR1, relapse rates were significantly higher in related allo-SCT compared with unrelated allo-SCT, and multivariate analysis demonstrated that less than 6 months from diagnosis to allo-SCT alone was associated with relapse. On the other hand, nonrelapse mortality (NRM) was significantly higher in unrelated allo-SCT compared with related allo-SCT, and multivariate analysis demonstrated that 10 months or longer from diagnosis to allo-SCT, human leukocyte antigen mismatch, and abnormal karyotype were associated with NRM. In conclusion, our study showed comparable survival rates but different relapse rates, NRM rates, and risk factors between related and unrelated allo-SCTs. After a close consideration of these factors, the outcome of allo-SCT for adult Ph(-) ALL in CR1 could be improved.

Cytomegalovirus antigenemia and outcome of patients treated with pre-emptive ganciclovir: Retrospective analysis of 241 consecutive patients undergoing allogeneic hematopoietic stem cell transplantation

Summary:CMV disease remains a major infectious complication after allogeneic hematopoietic stem cell transplantation (HSCT). To investigate the relationship between CMV antigenemia, treatment with ganciclovir (GCV), and outcome, we retrospectively analyzed 241 consecutive patients at risk for CMV infection who underwent allogeneic HSCT. Antigenemia-guided pre-emptive strategy with GCV was used for all patients. CMV antigenemia developed in 169 patients (70.1%), and CMV disease in 18 patients (7.5%). Multivariate analysis showed that acute GVHD (grades II–IV) was the only risk factor for developing antigenemia, and acute GVHD and advanced age for CMV disease. GCV use, as well as acute GVHD and advanced age, significantly increased the risk for bacterial and fungal infection after engraftment. Those who developed CMV antigenemia had a poorer outcome than those who did not (log-rank, P=0.0269), although the development of CMV disease worsened the outcome with only borderline significance (log-rank, P=0.0526). In conclusion, detection of antigenemia proved to be a poor prognostic factor for HSCT patients, which may be attributed to a combination of factors, including CMV disease itself, the effect of treatment, and a host status that allows for reactivation of CMV. Optimal pre-emptive strategy needs to be determined.

Trough plasma concentration of imatinib reflects BCR-ABL kinase inhibitory activity and clinical response in chronic-phase chronic myeloid leukemia : A report from the BINGO study

Pharmacokinetic (PK) factors have been suggested to be involved in the unfavorable clinical responses of chronic myeloid leukemia (CML) patients treated with imatinib. The purpose of this study was to clarify prognostic implications of PK factors in CML patients treated with imatinib. The plasma trough (Cmin) level of imatinib and serum α1‐acid glycoprotein (AGP) level were measured on two different days in 65 CML patients treated with imatinib for more than 12 months. We further examined whether the Cmin level of imatinib actually reflects inhibitory activity against BCR–ABL kinase using the plasma inhibitory activity (PIA) assay. Since the differences of five patients were statistically rejected by the Smirnov–Grubbs’ test, we excluded them for further analysis. The Cmin level was strongly associated with the achievement of MMR at the 12th month, and ROC analysis demonstrated Cmin levels and their discrimination potential for major molecular response (MMR) with the best sensitivity (63.2%) and specificity (68.2%) at a Cmin threshold of 974 ng/mL. The α1‐Acid glycoprotein (AGP) level was within the normal range in 57 of 60 patients, indicating little impact of AGP on our study. There was a weak correlation between PIA against phospho (P)‐BCR–ABL and the Cmin level of imatinib (r2 = 0.2501, P = 0.0007), and patient plasma containing >974 ng/mL imatinib sufficiently inhibited P‐BCR‐ABL. These results collectively indicated that maintaining ∼1000 ng/mL of Cmin was clinically and biologically important for the optimal response in CML patients treated with imatinib. A prospective intervention study is required to establish PK‐based management in CML patients treated with imatinib. (Cancer Sci 2010)

Clinicopathological manifestations and treatment of intestinal transplant-associated microangiopathy

Intestinal transplant-associated microangiopathy (i-TAM) is an important complication after allogeneic hematopoietic SCT. From 1997 to 2006, 87 of 886 patients with diarrhea after transplantation received colonoscopic biopsy. i-TAM, GVHD and CMV colitis were diagnosed histopathologically. The median duration from transplantation to the onset of diarrhea was 32 days (range: 9–130 days) and that from the onset of diarrhea to biopsy was 12 days (range: 0–74 days). The median maximal amount of diarrhea was 2 l/day (range: 130–5600 ml/day). Histopathological diagnosis included i-TAM (n=80), GVHD (n=26), CMV colitis (n=17) and nonspecific findings (n=2) with overlapping. Among 80 patients with i-TAM, abdominal pain was a major symptom, and only 11 patients fulfilled the proposed criteria for systemic TAM. Non-relapse mortality (NRM) among patients without resolution of diarrhea was 72% and i-TAM comprised 57% of NRM. NRM was 25% among patients without intensified immunosuppression, but was 52, 79 and 100% among those with intensified immunosuppression before diarrhea, after diarrhea, and before and after diarrhea, respectively. In conclusion, i-TAM is a major complication presenting massive refractory diarrhea and abdominal pain, which causes NRM. Avoiding intensified immunosuppression that damages vascular endothelium until the resolution of i-TAM may improve transplant outcome.