Reversible suspension culture of human vascular smooth muscle cells using the functional biopolymer FP003.

Human vascular smooth muscle cells (SMCs) are adherent cells, and they cannot survive without scaffolds in suspension culture. Here, we aimed to establish a suspension culture of SMCs using the functional biopolymer FP003 and to investigate the proliferation status of the cells. When SMCs were suspension cultured with FP003, their proliferation was inhibited with a viability of 75% until day 15. When SMCs were re-plated on plastic plates after suspension culture with FP003 for 48 h, the SMCs proliferated as in a normal plate culture. The SMCs cultured in suspension with FP003 showed a relatively low phosphorylation of retinoblastoma protein, low expression of cyclin D1, high proportion of G0/G1 phase cells, low proportion of S phase cells, and no obvious signs of apoptosis, indicating that this culture system inhibited progression from the G1 to S phase. This growth arrest was a reversible property that showed no significant changes in the expressions of the marker proteins α-smooth muscle actin and smooth muscle myosin heavy chain. These results suggest that human SMCs can be stably cultured in suspension with FP003 without losing their characteristics when they are cultured on plastic plates again.

Slowly progressing acute lymphoblastic leukemia with prolonged leukopenia.

Acute lymphoblastic leukemia is typically characterized by leukocytosis, resulting from the uncontrolled proliferation of malignant cells. However, we report an atypical case of acute lymphoblastic leukemia that presented with leukopenia and exhibited a protracted clinical course spanning 6 months. The patient, a 45-year-old female, initially presented to our hospital with recurrent fever and was found to have lymphoblasts in a hypoplastic bone marrow. Upon further investigation, the patient was diagnosed with B-cell lymphoblastic leukemia, not otherwise specified, based on cell surface antigen expression and genetic abnormalities. Notably, the patient demonstrated persistently low white blood cell and neutrophil counts, without evidence of increasing lymphoblast infiltration in the bone marrow during the ensuing 6-month period. Subsequent chemotherapy led to normalization of hematopoiesis and disappearance of lymphoblasts, resulting in complete remission of the disease.

Therapy-related myeloid neoplasms of recipient origin after allogeneic hematopoietic stem cell transplantation for acute leukemia.

Some allogeneic stem cell transplantation (allo-SCT) recipients develop therapy-related myeloid neoplasms (t-MNs) of recipient origin with features including karyotypically abnormal hematopoiesis without cell dysplasia and myeloblast increase. However, due to their rarity their clinical course remains unclear. We report six cases of t-MN in patients with chromosomal abnormalities (CAs) after autologous recovery following allo-SCT for acute leukemia. CAs were first detected at a median interval of 422 (range 30-1941) days from allo-SCT. The fraction of CA-bearing cells of recipient origin increased with time, and cytogenetic relapse of underlying disease was not observed. Continuous emergence of identical autologous CAs was observed in one patient who did not receive total body irradiation (TBI). The other five patients received TBI, and complex karyotypes with the appearance of different types of CAs were the most dominant feature. Despite the persistence of complex abnormalities in the irradiated patients, no patient developed therapy-related acute myeloid leukemia (t-AML). TBI appears to be the major cause of t-MN of recipient origin with different types of CAs. Although t-MNs in patients receiving TBI do not initially seem to evolve to overt t-AML, they were associated with higher risk of underlying disease and greater oncogenic potential of irradiation.

PARP1 V762A polymorphism affects the prognosis of myelodysplastic syndromes.

OBJECTIVE: Myelodysplastic syndromes (MDS), caused by various genetic mutations in hematopoietic stem cells, are associated with highly variable outcomes. Poly (ADP-ribose) polymerase-1 (PARP1) plays an important role in DNA damage repair and contributes to the progression of several types of cancer. Here, we investigated the impact of PARP1 V762A polymorphism on the susceptibility to and prognosis of MDS. METHODS: Samples collected from 105 MDS patients and 202 race-matched healthy controls were subjected to polymerase chain reaction-restriction fragment length polymorphism for genotyping. RESULTS: The allele and genotype frequencies of PARP1 V762A did not differ between MDS patients and the control group. However, MDS patients with the PARP1 V762A non-AA genotype, which is associated with high gene activity, had shorter overall survival rates (P = .01) than those with the AA genotype. Multivariate analysis of overall survival also revealed PARP1 V762A non-AA genotype as a poor prognostic factor (P = .02). When patients were analyzed according to treatment history, the PARP1 V762A non-AA genotype was only associated with poor survival in patients who had received treatment (P = .02). CONCLUSION: PARP1 V762A polymorphism may be an independent prognostic factor for MDS, and a predictive biomarker for MDS treatment.

Allogeneic hematopoietic stem cell transplantation for aplastic anemia with pre-transplant conditioning using fludarabine, reduced-dose cyclophosphamide, and low-dose thymoglobulin: A KSGCT prospective study.

The optimal pre-transplant conditioning for aplastic anemia (AA) remains unclear. We performed a prospective study on allogeneic transplantation from a related or unrelated donor for adult patients with AA. We assessed whether reduced-dose cyclophosphamide (CY) could decrease toxicity while maintaining engraftment, and low-dose thymoglobulin could safely prevent graft-vs-host disease (GVHD). The pre-transplant conditioning regimen consisted of fludarabine 120 mg/m2 , CY 100 mg/kg, and thymoglobulin 2.5 mg/kg with or without 2 Gy of total body irradiation. Twenty-seven patients with a median age of 36 years were analyzed. Sixteen patients received graft from related donors. The stem cell source was bone marrow in 26 patients. All of the patients but one, who died early, achieved neutrophil engraftment at a median of 19 days. Mixed chimerism was observed in six and five patients at days 30 and 90, respectively. Only one patient experienced secondary engraftment failure with complete donor-type chimerism. None of the patients developed severe acute GVHD. The cumulative incidence of chronic GVHD was 37.7% at 1 year. The overall survival rate was 96.3% at 1 year and 3 years. A high EB virus-DNA load was detected in one patient at days 60. No one developed EBV-lymphoproliferative disorder within a year. The results suggest that the conditioning regimen in this study was safe and effective. However, relatively high incidence of chronic GVHD needs further improvement.

Feasibility of salvage cord blood transplantation using a fludarabine, melphalan, and low-dose anti-thymocyte globulin conditioning regimen.

Primary graft failure (PGF) is a lethal complication that occurs early after allogeneic stem cell transplantation (allo-SCT). Cord blood transplantation (CBT) is a potential re-transplantation option. Total body irradiation (TBI) is often incorporated into the pre-salvage CBT conditioning regimen following PGF; however, patients experiencing PGF are not always amenable to TBI, and non-TBI regimens for salvage CBT should be established. Here, we report five patients with hematologic malignancies who received salvage CBT for PGF following a non-TBI regimen using fludarabine (Flu), melphalan (Mel), and low-dose anti-thymocyte globulin (ATG). The median intervals between the failed allo-SCT and salvage CBT, as well as between the diagnosis of PGF and salvage CBT, were 37 days and 8 days, respectively. The median neutrophil recovery period was 21 days (range 18-21 days). Four of five patients achieved neutrophil engraftment following salvage CBT; all four exhibited sustained engraftment with complete donor chimerism. Three of the five patients were alive after a median follow-up time of 907 days (range 315-909 days) post-salvage CBT; two patients died of causes unrelated to recurrence. These data suggest that CBT following the non-TBI regimen described here is feasible in patients with PGF.

Allogeneic haematopoietic cell transplantation for adult acute myeloid leukaemia in second remission: a retrospective study of the Adult Acute Myeloid Leukaemia Working Group of the Japan Society for Haematopoietic Cell Transplantation (JSHCT).

To evaluate the outcomes and prognostic factors following allogeneic haematopoietic cell transplantation (HCT) for adult acute myeloid leukaemia (AML) in second complete remission (CR2), we retrospectively analysed the Japanese registration data of 1080 adult AML patients in CR2 who had received allogeneic HCT. The probability of overall survival and the cumulative incidence of relapse at 3 years was 66% and 19%, respectively. In multivariate analysis, older age, poor cytogenetics and shorter duration of first complete remission were significantly associated with a higher overall mortality. Our data demonstrated the significant efficacy of allogeneic HCT for adult AML in CR2.

Association between OGG1 S326C CC genotype and elevated relapse risk in acute myeloid leukemia.

Recent studies have shown that tumors of relapsed acute myeloid leukemia (AML) present additional genetic mutations compared to the primary tumors. The base excision repair (BER) pathway corrects oxidatively damaged mutagenic bases and plays an important role in maintaining genetic stability. The purpose of the present study was to investigate the relationship between BER functional polymorphisms and AML relapse. We focused on five major polymorphisms: OGG1 S326C, MUTYH Q324H, APE1 D148E, XRCC1 R194W, and XRCC1 R399Q. Ninety-four adults with AML who achieved first complete remission were recruited. Genotyping was performed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The OGG1 S326C CC genotype (associated with lower OGG1 activity) was observed more frequently in patients with AML relapse [28.9 vs. 8.9%, odds ratio (OR) = 4.10, 95% confidence interval (CI) = 1.35-12.70, P = 0.01]. Patients with the CC genotype exhibited shorter relapse-free survival (RFS). Moreover, the TCGA database suggested that low OGG1 expression in AML cells is associated with a higher frequency of mutations. The present findings suggest that the OGG1 S326C polymorphism increased the probability of AML relapse and may be useful as a prognostic factor for AML relapse risk.

Increased non-relapse mortality due to high-dose cytarabine plus CY/TBI in BMT/PBSCT for acute lymphoblastic leukaemia in adults.

The efficacy of high-dose cytarabine (HDCA) plus cyclophosphamide/total-body irradiation (CY/TBI) has been proved in cord blood transplantation (CBT) for acute lymphoblastic leukaemia (ALL), but not in bone marrow or peripheral blood stem cell transplantation (BMT/PBSCT). In this cohort study, we compared the prognosis of CY/TBI (N = 1244) and HDCA/CY/TBI (N = 316) regimens in BMT/PBSCT for ALL. The addition of HDCA decreased post-transplant relapse, while significantly increasing non-relapse mortality (risk ratio, 1·33), and overall survival was not improved. The positive effects of HDCA reported in CBT cannot be extrapolated to BMT/PBSCT, and HDCA may not be recommended in these procedures.

Influence of Differently Licensed KIR2DL1-Positive Natural Killer Cells in Transplant Recipients with Acute Leukemia: A Japanese National Registry Study.

Licensing by self MHC class I ligands is required for proper natural killer (NK) cell response. NK cells with inhibitory killer cell immunoglobulin-like receptors for nonself MHC exhibit transient alloreactivity after hematopoietic stem cell transplantation (HSCT). We analyzed 3866 recipients in the Japan national registry who underwent their first allogeneic HSCT for acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) from HLA-A, -B, and -DRB1 allele-genomatched unrelated donors. By classifying them into 5 independent groups based on HLA-C group matching and assumed donor NK cell status, we found that for HLA-C-matched HSCT for AML in HLA-C1/C1 recipients, in whom transient alloreactivity against HLA-C2-negative leukemic cells was expected, the relapse rate was significantly lower than it was in HLA-C-matched HSCT for AML in HLA-C1/C2 recipients (hazard ratio [HR], .72; P = .011). This difference was not observed in HLA-C-matched HSCT for ALL. Compared with HLA-C-matched HSCT, significantly higher mortality was observed in HLA-C1/C1 AML patients who received transplants from HLA-C-mismatched HLA-C1/C1 donors (HR, 1.37; P = .001) and in HLA-C1/C1 ALL patients who received transplants from HLA-C2-positive donors (HR, 2.13; P = .005). In conclusion, donor selection based on leukemic subtype and donor HLA-C group matching improves transplantation outcome after HLA-C-mismatched HSCT.

[Relapsed primary intraocular lymphoma treated with intravitreal methotrexate and high-dose chemotherapy followed by autologous stem cell rescue].

A 58-year-old woman with primary intraocular lymphoma (PIOL) of her right eye was treated with combination chemotherapy (methotrexate, procarbazine and vincristine) followed by irradiation to her brain and right eye. However, the disease recurred in the right eye four months later. She was treated with intravitreal injection of methotrexate and high-dose chemotherapy in combination with autologous stem cell transplantation after salvage therapy consisting of cytarabine, etoposide and rituximab. With this treatment strategy, she has been in remission for more than one year with no deterioration of either leukoencephalopathy or cognitive function. Intravitreal injection of methotrexate and high-dose chemotherapy may now be regarded as one of the treatment choices for relapsed PIOL.

[Emergence of donor-derived anti-HLA antibody and subsequent transfusion-refractory thrombocytopenia after allogeneic hematopoietic stem cell transplantation from an HLA-matched sibling donor in a patient with acute myeloid leukemia].

A 45-year-old woman with acute myelogenous leukemia developed platelet transfusion refractoriness (PTR) after the engraftment of an allogeneic peripheral blood stem cell transplantation (PBSCT) from her multiparous sister, which was attributed to HLA antibodies that could not be detected in the patient's serum before transplantation. She achieved neutrophil engraftment by day 18 and megakaryocytopoiesis and complete donor chimerism was confirmed in the bone marrow on day 21. IgG-class HLA antibodies were detected in her serum on day 24 after PBSCT; however, on day 15, no HLA antibodies were detected. The specificity of the antibodies that emerged in the patient closely resembled that of the antibodies found in the donor. The donor had probably been immunized during pregnancy by their partner's HLA-antigens expressed by the fetus. Consequently, transplanted donor-derived cells provoked HLA antibodies in the recipient early after PBSCT, and those HLA antibodies induced PTR. The presence of HLA antibodies should be examined at least in pregnant female donors whose recipients developed PTR attributable to HLA antibodies after SCT.

Persistence of anti-HLA antibody after cord blood transplantation engraftment in acute myelogenous leukemia: a potential marker of minimal residual disease, but not a significant factor in secondary humoral engraftment failure.

The presence of pre-transplant anti-HLA antibodies in recipients of cord blood transplantation (CBT) is associated with failed engraftment. However, only a small number of studies have reported that recipient-derived anti-HLA antibodies persist after CBT and have potential impact on the outcome. Of 61 patients who underwent HLA-mismatched CBT at Saiseikai Maebashi Hospital, three patients were identified as having anti-HLA antibodies not corresponding to HLA antigens in the transplanted CB. All patients achieved successful engraftment. However, the three patients with the pre-transplant anti-HLA antibodies not corresponding to HLA antigens in the transplanted CB continued to produce these antibodies even after engraftment; the persistence of these antibodies served as a sensitive minimal residual disease (MRD) marker. In contrast, donor HLA-specific and newly produced third party antibodies were not detectable even after relapse. The persistence of anti-HLA antibodies even after engraftment may be a potential marker for MRD, but is not a significant factor in secondary humoral engraftment failure.

[Toxoplasmic encephalitis in a case of acute myelogenous leukemia after conventional consolidation chemotherapy].

Toxoplasmic encephalitis is a rare complication in patients with hematological malignancies. It is infrequently reported in patients undergoing allogeneic hematopoietic stem cell transplantation, much less in patients after conventional chemotherapy. A 75-year old female with acute myelogenous leukemia had an episode of aphasia, right homonymous hemianopsia, and consciousness disturbance 122 days after consolidation chemotherapy. Multiple enhancing space occupying lesions involving the left occipital lobe were seen on magnetic resonance imaging (MRI). HIV antibody was negative. Based on radiological findings, clinical presentation and positive Toxoplasma gondii serostatus, sulfadoxine and pyrimethamine were added. A rapid improvement was observed thereafter. Heightened awareness of the occurrence of toxoplasmic encephalitis, even in non-HIV patients, is needed for a better outcome.

Long-term follow-up of EBV-positive lymphoproliferative disorders in a patient with systemic lupus erythematosus.

We report a woman in her early thirties with a long-term history of systemic lupus erythematosus (SLE) and prednisolone administration, who progressed to Epstein-Barr virus (EBV)-positive lymphoproliferative disorder (LPD). Treatment for SLE consisted of 1 mg/kg/ day prednisolone followed by 5 mg/day of maintenance therapy. Lymph node biopsies were performed when the patient was in her early thirties, mid-forties, and late fifties. Histologically, the initial lymph node lesion was characterized by numerous enlarged, coalescing lymphoid follicles. The second biopsy showed effacement of the follicles and expansion of the paracortical area. A polymorphous population of small- to medium-sized lymphocytes, plasma cells, and immunoblasts had diffusely infiltrated the paracortical area. In the third lymph node biopsy, fibrous collagen bands divided the epithelioid cell granulomas into nodules. There were numerous Hodgkin and Reed-Sternberg cells in the epithelioid cell granuloma. In situ hybridization demonstrated there were no EBV-infected lymphocytes in the first biopsy; however, EBER(+) cells were detected in the second and third biopsy specimens. The current findings illustrate the natural progression in a patient with a long-term history of EBV(+) B-cell LPD in which the immunodeficiency was caused by SLE and probably her aging, which together resulted in histological change.

Discrepancy in EBV-DNA load between peripheral blood and cerebrospinal fluid in a patient with isolated CNS post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a fatal complication of allogeneic hematopoietic stem cell transplantation (HSCT) that is caused by reactivation of Epstein-Barr virus (EBV). A successful approach, monitoring EBV-DNA load in peripheral blood (PB) accompanied by preemptive rituximab therapy, has recently been reported. Here, we describe a 29-year-old woman who developed isolated central nervous system (CNS) PTLD. She received HSCT against acute myelogenous leukemia from a related human leukocyte antigen-haploidentical donor, following a conditioning regimen that included antithymocyte globulin. Tacrolimus and methylprednisolone were given as prophylaxis for graft-versus-host disease. On day +172, the patient's consciousness deteriorated. Magnetic resonance imaging showed six ring-enhanced lesions in the cerebral hemispheres. These tumors were diagnosed, via a craniotomy and tumorectomy, as PTLD. EBV-DNA load was elevated in the cerebrospinal fluid (CSF) but not detected in PB. She was treated with whole-brain irradiation and rituximab, and achieved partial remission of the tumors. This case serves as a reminder that vigilance is required regarding the development of isolated CNS PTLD; it is worth examining EBV-DNA replication in CSF for diagnosis even when the EBV-DNA load is negative in PB.

[Glossitis by mixed infection of cytomegalovirus and herpes simplex virus during therapy for pure red cell aplasia complicated with Good syndrome].

A 64-year-old man with a 10-year history of Good syndrome had been treated with periodic replacement of γ-globulin. He also had a 6-year history of lichen planus of the tongue. In 2009, the patient was diagnosed as having pure red cell aplasia (PRCA) based on bone marrow aspiration. Thymectomy was not effective. Then, immunosuppressive therapy with PSL and cyclosporine was initiated. Twenty days after treatment painful ulcer appeared on the left side of the tongue. Biopsy specimen of the ulcer demonstrated cells infected with cytomegalovirus and herpes simplex virus. Cytomegalovirus antigenemia was also positive. The tongue ulcer promptly improved after gancyclovir administration for a few weeks. Viral glossitis should be considered as part of the differential diagnoses of oral lesions not only in patients with HIV infection but also in those under immunosuppressive therapy.

[Clinical profiles of 7 patients with chronic myelogenous leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia treated with dasatinib].

We retrospectively analyzed the clinical outcome of dasatinib in 7 patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) resistant or intolerant to imatinib. Three patients with chronic phase CML and two patients with Ph+ALL achieved major molecular response, however, two CML patients in accelerated phase (AP)/blast crisis (BC), did not. Grade> or =3 pancytopenia was seen in four patients. Among these, two AP/BC-CML patients required interruption/or dose reduction of dasatinib. As for nonhematologic adverse events, pleural effusion was seen in one patient and cytomegalovirus (CMV) colitis was observed in two patients. No patients who had been intolerant to imatinib experienced the same nonhematologic toxicity following treatment with dasatinib. We identified three patients who developed peripheral lymphocytosis, identified as natural killer cells or cytotoxic T-cells based on their large granular lymphocyte (LGL) morphologies and immunophenotypic profiles, out of six patients receiving dasatinib therapy. All three cases that developed LGL lymphocytosis achieved optimal molecular response, two of the patients, however, had pleural effusion and CMV colitis, respectively. Dasatinib inhibits off-target kinases, which may result in unexpected drug responses.