Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

Successful treatment of an AIDS patient with prolonged Mycobacterium avium bacteremia, high HIV RNA, HBV infection, Kaposi's sarcoma and cytomegalovirus retinitis.

We report an AIDS patient with a high HIV RNA copy number in the plasma who was successfully treated for prolonged Mycobacterium avium bacteremia and other complications. An HIV-infected patient with high fever, anemia, high alkaline phosphatase, cystic lung lesions, hepatitis B virus infection and Kaposi's sarcoma was referred to our hospital. PCR of the blood revealed Mycobacterium avium bacteremia and the time to blood culture positivity was 8 days. The HIV-1 RNA copy number in the plasma was more than ten million copies/ml and the CD4-positive T cell count was 21 cells/µL. Although the high fever resolved five days after therapy for Mycobacterium avium was started, the fever recurred just before starting anti-retroviral therapy (ART) including dolutegravir. The patient experienced repeated but self-limiting bouts of severe inflammation. Mycobacteremia was intermittently detected up to 79 days, suggesting that the recurrent episodes of inflammation were due to the intermittent dissemination of mycobacteria, and that persistent treatment is needed. Five months after the beginning of ART, the HIV-1 RNA copy number in the plasma was still 28,000 copies/ml. An HIV drug-resistance test revealed sensitivity to all anti-retroviral drugs. Eleven months after the initiation of ART, the HIV RNA copy number in the plasma decreased to 45 copies/mL and the CD4-positive T cell count recovered to 205 cells/µL. Our case also suggests that dolutegravir can be effective in cases with prolonged high levels of HIV RNA. Our findings emphasize that prompt diagnosis and persistent therapy for mycobacterial infection are important for successful treatment.

Case of a patient with Philadelphia-chromosome-positive acute lymphoblastic leukemia relapsed after myeloablative allogeneic hematopoietic stem cell transplantation treated successfully with imatinib and sequential donor lymphocyte infusions.

A 23-year-old man with Philadelphia-chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) underwent myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) from his HLA-identical brother in first hematological remission following induction chemotherapy which included imatinib. He had no acute graft-versus-host disease (GVHD), and 4.5 months after HSCT, he had a molecular relapse (180,000 copies/mug RNA of minor bcr/abl transcripts (m-bcr/abl) without mutation in 22 sites including the p-loop region). Following discontinuation of cyclosporine A, imatinib (600 mg daily) was restarted and 4 days later donor lymphocyte infusion (DLI) (5 x 10(7)/kg of CD3(+) cells) was given. In 2 weeks, the marrow m-bcr/abl became undetectable. He received two further DLIs and imatinib was continued at a reduced dose of 400 mg a day. At the time of this report, he remains in complete hematological remission more than 33 months after allo-HSCT and persists in the second molecular remission for longer than 24 months. During this clinical course, he became positive for anti-nuclear antibody after second DLI, without any other manifestations of GVHD. The standard treatment for Ph(+) ALL relapsing after allo-HSCT still remains to be established. Imatinib in combination with DLI for early molecular relapse may be a promising option.

Allogeneic stem cell transplantation for refractory adult T-cell leukemia using a non-T-cell-depleted HLA-incompatible family donor graft, with reference to the grown-up child donor to parent recipient setting: report of a pilot study.

To increase the availability of alternative stem-cell donors for patients with adult T-cell leukemia (ATL), we examined the feasibility of HLA-incompatible family transplantation, especially from a grown-up child (donor) to a parent (recipient). Since January 2004, seven patients with advanced-phase ATL (three males and four females, median age 59 years), for whom a timely HLA-compatible donor was unavailable, were enrolled. All patients received allografts from their HLA-incompatible sons with reduced-intensity conditioning stem cell transplantation (RIST). Combined graft-versus-host disease (GVHD) prophylaxis involved cyclosporine A or tacrolimus, mycophenolate mofetil or corticosteroid, and short-term methotrexate. All patients achieved prompt engraftment, and there was no 100-day relapse-related mortality. Only one patient had grade-IV acute-GVHD, but this was resolved. The median follow-up period was 251 days (range 112-1,018 days), and the estimated 1-year overall and 1-year progression-free survival rates were 57.1 and 28.6%, respectively. Four patients died, with causes of death being relapse (n = 2), transplantation-associated microangiopathy (n = 1), and septicemia (n = 1). Three are currently alive: two are in complete remission and one has stable disease. Despite a high rate of relapse, RIST using an allograft from an HLA-incompatible grown-up child donor may be feasible for patients with advanced-phase ATL, and may prolong survival.

High prevalence of human T-lymphotropic virus type I carriers among patients with myelodysplastic syndrome refractory anemia with excess of blasts (RAEB), RAEB in transformation and acute promyelocytic leukemia.

We examined human T-lymphotropic virus type I (HTLV-I) infection among patients with myelodysplastic syndrome (MDS), refractory anemia with excess of blasts (RAEB)/RAEB in transformation (RAEBt) and acute myelogenous leukemia (AML). The study population consisted of 151 patients: 46 with MDS RAEB/RAEBt and 105 with AML (M1, n = 15; M2, n = 39; M3, n = 18; M4, n = 19; M5, n = 9; M6, n = 3; M7, n = 2). As a reference, we examined 92 patients with refractory anemia (RA) and 405 patients with cardiovascular diseases (CVD). Thirteen patients with RAEB/RAEBt (28.3%), 11 with AML (11.6%), 27 with RA (29.3%), and 45 with CVD (11.0%) were positive for HTLV-I. Seven AML patients with HTLV-I infection had M3 acute promyelocytic leukemia (APL). The prevalences of HTLV-I infection among patients with RAEB/RAEBt (P < 0.001), APL (P = 0.001), and RA (P < 0.001) were significantly higher than that in patients with CVD. The prevalences of HTLV-I infection were still significantly higher in patients with RAEB/RAEBt (P = 0.007), APL (P = 0.017) and RA (P < 0.001) than in those with CVD matched by sex and age. Platelet counts and survival times of RAEB/RAEBt patients with infection were significantly lower than those of patients without infection.

Cyclosporin A inhibits HTLV-I tax expression and shows anti-tumor effects in combination with VP-16.

Adult T cell leukemia (ATL) is one of the most refractory malignant hematological diseases. Our previous studies demonstrated HTLV-1Tax protein involvement in clinical manifestation of the aggressive type of ATL and suggested the potential application of agents to inhibit Tax expression for ATL treatment. In the present study, we first examined Tax involvement in the resistance to VP-16-induced apoptosis using four HTLV-1 infected T cell clones and cTax DNA-transfected cells. Next, we examined whether cyclosporin A reduced expression of Tax and its related transfer factors on Western blot and CAT assay. We further investigated whether cyclosporin A in combination with VP-16 can induce apoptosis in HTLV-1 infected T cells. Tax-producing T cells, K3T and F6T, were resistant to VP-16 induced growth inhibition compared with that of the nonproducing cells, S1T and Su9T01. Experiments using S1T and Tax-expressing cDNA-transfected S1T demonstrated Tax-induced resistance to VP-16 induction of apoptosis by DNA ladder formation. Cyclosporin A reduced Tax expression in K3T by Western blot analysis and on CAT assay, showing maximal reduction of 61% and 60% compared to control culture using LTR CAT transfected Jurkat cells and K3T cells, respectively. Cyclosporin A also reduced the nuclear expression of two Tax-related transfer factors, ATF-1 and ATF-2 on Western blot. Cyclosporin A alone did not show any cytotoxicity by itself, but sensitized cells to VP-16 when combined with VP-16. Cyclosporin A may be a useful anti-ATL agent when combined with other anti-cancer agents possibly related to Tax inhibition.

Anti-HTLV-1 tax antibody and tax-specific cytotoxic T lymphocyte are associated with a reduction in HTLV-1 proviral load in asymptomatic carriers.

Previous studies have suggested that higher anti-human T-lymphotropic virus 1 (HTLV-1) antibody titer and lower anti-HTLV-1 Tax antibody reactivity are risk factors for adult T-cell leukemia/lymphoma. In the present study, we analyzed the relationships between these factors and clarified their significance. Forty-five carriers were examined for anti-HTLV-1 and anti-Tax antibody by ELISA. In addition, 43 of the 45 carriers with HLA-A*0201 and/or A*2402 were examined for frequency of Tax-specific cytotoxic T lymphocytes (CTLs) using HTLV-1/HLA tetramers, and 44 were examined for proviral load by real-time PCR. The relationships between these factors were analyzed statistically. The frequencies of Tax11-19 and Tax301-309-specific CTLs were significantly higher in the anti-Tax antibody-positive group as compared with the antibody-negative group (P = 0.002 and 0.033, respectively). Anti-HTLV-1 antibody titer had a positive correlation with proviral load (P = 0.019), whereas anti-Tax antibody did not show a significant correlation. Higher frequencies of both Tax11-19 and Tax301-309-specific CTLs are related to a reduction in proviral load (P = 0.017 and 0.015, respectively). Synergistic interactions of humoral and cellular immunity against Tax protein were demonstrated in HTLV-1 carriers. Tax-specific CTL may reduce HTLV-1 proviral load to prevent asymptomatic carriers from developing adult T-cell leukemia/lymphoma.

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients.

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.

Immunosuppressive treatment for mixed connective tissue disease may facilitate the development of adult T cell leukemia/lymphoma in a HTLVI carrier.

A 66-year-old woman who was positive for human T-lymphotropic virus type I (HTLV-I) antibody developed mixed connective tissue disease (MCTD) with interstitial pneumonia, and was successfully treated with corticosteroid. One year later, under maintenance treatment of prednisolone (PSL), she contracted acute type adult Tcell leukemia/lymphoma (ATLL) without flaring of MCTD. MCTD is considered to be as one of the HTL-V-I-related inflammatory diseases, however the development of ATLL during the treatment of HTL-V-I-related MCTD has not been well studied. Here, we review the literature and raise the issue of the mutual interactions between MCTD-causative anti-HTLV-I immune response and anti-ATLL immune response.

Stevens-Johnson syndrome induced by mizoribine in a patient with systemic lupus erythematosus.

A 32-year-old Japanese woman, who had a treatment history of systemic lupus erythematosus (SLE) with lupus nephritis World Health Organization class IV for 11 months, visited our hospital due to fever, facial erythema, and erosion of the oral cavity on November 10, 2003. Her mucosal erosion and facial skin erythema progressed over the following week, and Stevens-Johnson syndrome was diagnosed due to pathological findings of the skin. Among the administrated drugs, only mizoribine, started 6 months earlier, produced a positive reaction in the drug lymphocyte stimulation test. Increased prednisolone and high dose intravenous gamma-globulin were given successfully. Cyclosporine at 50 mg was administered to control the SLE, followed by an increase to 100 mg on January 7, 2004. She suffered from abdominal pain, blindness, and convulsion on January 9. The magnetic resonance image of her brain prompted a diagnosis of reversible posterior leukoencephalopathy syndrome. After withdrawal of cyclosporine and control of hypertension, symptoms disappeared rapidly. Cyclophosphamide pulse therapy was successfully administrated to control lupus nephritis. This is the first report describing the relationship between Stevens-Johnson syndrome and mizoribine. Although the use of mizoribine is thought to be safe, careful observation is necessary.

Case of a patient with progressive adult T-cell leukemia/lymphoma treated successfully by reduced-intensity conditioning stem cell transplantation from an HLA-incompatible related donor.

A 61-year-old man with progressive adult T-cell leukemia/lymphoma (ATLL) successfully received reduced-intensity conditioning stem cell transplantation (RIST) without T-cell depletion (TCD) from his HLA-incompatible son, who had negative results for human T-lymphotropic virus type 1 (HTLV-1) (1-locus, 1-allele mismatch in the graft-versus-host [GVH] direction; 2-loci, 1-allele mismatch in the host-versus-graft direction). The preparatory regimen consisted of fludarabine, busulfan, and rabbit antithymocyte globulin. GVH disease (GVHD) prophylaxis consisted of short-term administration of methotrexate, tacrolimus, and methylprednisolone. The patient achieved complete donor chimerism on day 30 after transplantation. On approximately day 50 the patient started to experience steroid-refractory skin GVHD (grade IV), which was successfully managed with basiliximab (anti-CD25 monoclonal antibody) and mycophenolate mofetil (MMF). Serial analysis of HTLV-1 proviral load by quantitative polymerase chain reaction analysis using whole peripheral blood demonstrated undetectable levels from day 90. At the time of this writing the patient had been in complete remission for more than 16 months. The results in this case suggest the potential of non-TCD RIST from an HLA-incompatible relative donor as an alternative source of hematopoietic stem cells even for an elderly patient with advanced ATLL. In addition, basiliximab combined with MMF may be effective for the treatment of steroid-refractory skin GVHD without deteriorating the graft-versus-ATL effect.

Matrix metalloproteinase inhibitor reduces apoptosis induction of bone marrow cells in MDS-RA.

BACKGROUND AND OBJECTIVES: We examined the involvement of apoptosis with myelodysplastic syndrome (MDS) accompanied by peripheral cytopenias despite normo-hypercellular bone marrow. MATERIALS AND METHODS: Bone marrow smears from 31 patients with MDS-refractory anemia (RA) and five normal controls were stained using the in situ end labeling (ISEL) method. Next, the inhibitory effects of a caspase-3 inhibitor, matrix metalloproteinase inhibitor (MMPI), anti-tumor necrosis factor (TNF)-alpha or anti-Fas antibody upon the apoptosis induction in overnight cultures of bone marrow cells from the patients were examined. Further, TNF-alpha, transforming growth factor (TGF)-beta and soluble Fas ligand (sFasL) concentrations in culture supernatants of the cells were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The incidence of ISEL-positive cells among MDS patients was significantly higher than in normal controls (50.8 +/- 14.0% vs. 11.3 +/- 2.4%; P < 0.0001). A caspase-3 inhibitor reduced significantly the ISEL-positive rates (32.6 +/- 15.2% vs. 50.2 +/- 16.5%; P < 0.0001). Anti-TNF-alpha or anti-Fas antibody reduced the ISEL-positive rates significantly (28.2 +/- 6.0%, 29.2 +/- 5.8%, vs. 44.2 +/- 3.4%, P < 0.001, P = 0.001, respectively). KB-R7785 also significantly decreased the ISEL-positive rates (18.0 +/- 9.3% vs. 43.6 +/- 14.0%; P < 0.0001). The concentration of TNF-alpha was significantly reduced by KB-R7785 (P < 0.05), whereas that of TGF-beta was not. Concentration of sFasL was under detectable level in the present assay system. The derivatives of KB-R7785 that can be administrated orally showed inhibitory effect on apoptosis induction as well. CONCLUSIONS: These findings suggest that MMPIs inhibits the apoptosis induction of MDS bone marrow cells via the inhibition of TNF-alpha and probably sFasL secretion, and that MMPIs can be used to control the abnormal induction of apoptosis in MDS.