Masahiko Sumi

Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells : Involvement of ATM-dependent DNA damage response pathways

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. In order to determine whether G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), might have effects on telomere dynamics and to evaluate the clinical utility, we assessed the effects of telomestatin on BCR-ABL-positive human leukemia cells. We found that treatment with telomestatin reproducibly inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines OM9;22 and K562, resulting in telomere shortening. Inhibition of telomerase activity by telomestatin disrupts telomere maintenance and ultimately results in telomere dysfunction. Telomestatin completely suppressed the plating efficiency of K562 cells at 1 μM; however, telomestatin had less effects on BFU-Es and CFU-GMs colony formation from normal bone marrow CD34-positive cells. Enhanced chemosensitivity toward imatinib and chemotherapeutic agents was also observed in telomestatin-treated K562 cells. Further, the combination of telomestatin plus imatinib more effectively inhibited hematopoietic colony formation by primary human chronic myelogenous leukemia cells. Last, telomestatin induced the activation of ATM and Chk2, and subsequently increased the expression of p21CIP1 and p27KIP1. These results demonstrate that telomere dysfunction induced by telomestatin activates the ATM-dependent DNA damage response. We conclude that telomerase inhibitors combined with the use of imatinib and other chemotherapeutic agents may be very useful for the treatment of human leukemia.

Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines. In the present study, we investigated the mechanisms of apoptosis induced by telomerase inhibition in acute leukemia. We have found the activation of caspase-3 and poly-(ADP-ribose) polymerase in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25). Activation of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these cells. To examine the effect of p38 MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580. Dominant-negative-hTERT-expressing U937 cells stopped proliferation on PD25; however, a significant increase in growth rate was observed in the presence of SB203580. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38 MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperitoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.

Telomerase inhibition enhances apoptosis in human acute leukemia cells : possibility of antitelomerase therapy

Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. We examined the impact of telomerase inhibition by the dominant-negative human catalytic subunit of telomerase (DN-hTERT) on the biological features of acute leukemia. We introduced vectors encoding dominant- negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistant marker into a telomerase-positive human acute lymphoblastic leukemia cell line, HAL-01. Expression of DN-hTERT dramatically inhibited telomerase activity, leading to apoptotic cell death. Mutant telomerase expression also enhanced daunorubicin-induced apoptosis. Nude mice (n=5 per group) received subcutanous implants of HAL-01 cells expressing the control vector or DN-hTERT or WT-hTERT. Implantation of HAL-01 cells expressing control vector (n=5) rapidly produced tumors, whereas implantation of those expressing DN-hTERT (n=5) did not. Thus, telomerase inhibition both growth of HAL-01 cells in vitro and tumorigenic capacity in vivo. Furthermore, the G-quadruplex-interactive telomerase-specific inhibitor, telomestatin, shortened the telomere length and induced apoptosis in freshly isolated primary acute leukemia cells. These results suggest that antitelomerase therapy may be useful in some acute leukemias in combination with antileukemic agents such as daunorubicin.

Lymphadenopathy of IgG4-related sclerosing disease: three case reports and review of literature

Immunoglobulin (Ig) G4-related sclerosing disease is a recently described syndrome characterized by lymphoplasmacytic infiltration of exocrine glands or extranodal tissues and elevated serum IgG4. We report three cases of lymphadenopathy secondary to IgG4-related sclerosing disease. Histologic features of involved lymph nodes included interfollicular immunoblasts and plasma cells, similar to Castleman’s disease. The percentage of IgG4-/IgG-positive plasma cells in the three patients was markedly elevated (30, 50, and 60%). Administration of prednisolone led to remission in every case. One of three cases was consulted to our hospital due to suspected diagnosis of angioimmunoblastic T cell lymphoma (AITL). The case demonstrates many clinical and pathologic similarities between IgG4-related sclerosing disease and AITL. Pathological similarities between AITL and the lymphoplasmacytic subtype of IgG4-sclerosing disease have recently been reported. It is important to accurately diagnose IgG4-related lymphadenopathy given its ready response to steroid therapy and the potential for misdiagnosing lymphoma on clinical grounds.

Peripheral T-cell lymphoma together with myelofibrosis with elevated plasma transforming growth factor-β1

Myelofibrosis is usually observed in myeloproliferative disorders, such as chronic myeloid leukemia. However, there are only a few reports showing an association between T-cell lymphoma and myelofibrosis. We report a case of peripheral T-cell lymphoma, unspecified (diffuse large cell) type, involving the bone marrow that was associated with severe myelofibrosis. In the present case, the plasma concentration of transforming growth factor-beta1 (TGF-beta1) was increased to 8.95 ng/ml (normal range: 1.56-3.24 ng/ml). No lymphadenopathy or skin lesions were observed during the entire clinical course. Although the mechanism of secondary myelofibrosis is still unclear, elevated plasma TGF-beta1 might be involved in the pathogenesis of bone marrow fibrosis in the present case.

Successful engraftment of cord blood following a one-day reduced-intensity conditioning regimen in two patients suffering primary graft failure and sepsis

Successful engraftment of cord blood following a one-day reduced-intensity conditioning regimen in two patients suffering primary graft failure and sepsis

Use of percutaneous image‐guided coaxial core‐needle biopsy for diagnosis of intraabdominal lymphoma

Although pathological diagnosis is essential for managing malignant lymphoma, intraabdominal lesions are generally difficult to approach due to the invasiveness of abdominal surgery. Here, we report the use of percutaneous image‐guided coaxial core‐needle biopsy (CNB) to obtain intraabdominal specimens for diagnosing intraabdominal lymphomas, which typically requires histopathological and immunohistochemical evaluation. We retrospectively reviewed consecutive cases involving computed tomography (CT)‐ or ultrasonography (US)‐guided CNB to obtain pathological specimens for intraabdominal lesions from 1999 to 2011. Liver, spleen, kidney, and inguinal node biopsies were excluded. We compared CNBs with laparotomic biopsies. A total of 66 CNBs were performed for 59 patients (32 males, 27 females; median age, 63.5), including second or third repeat procedures. Overall diagnostic rate was 88.5%. None of the patients required additional surgical biopsies. Notably, the median interval between recognition of an intraabdominal mass and biopsy was only 1 day. Forty‐five procedures were performed for hematological malignancies. Adequate specimens were obtained for histopathological diagnosis in 86% of cases. Flow cytometry detected lymphoma cells in 79.5% of cases. Twelve patients (nine males, three females; median age, 60) were eligible for surgical biopsy. While every postoperative course was satisfactory, median duration from lesion recognition to therapy initiation for lymphoma cases was significantly shorter for CNB than for surgical biopsy (14 vs. 35 days). While one‐fourth of the patients were not eligible for the procedures, CNB is safe and highly effective for diagnosis of intraabdominal lymphomas. This method significantly improves sampling and potentially helps attain immunohistological distinction, allowing for more timely therapy initiation.

Reduced-intensity cord blood transplantation without prior remission induction therapy induces durable remission in adult patients with relapsed acute leukemia after the first allogeneic transplantation

To the Editor: Leukemia relapse following allogeneic stem cell transplantation (allo-SCT) remains a major cause of treatment failure (1, 2). Among treatment options, second allo-SCT appears to increase disease-free survival compared with other interventions (3). The most important factors influencing outcomes of second allo-SCT may be the length of remission after the first transplantation and age (2). While disease status may also be an important factor, the probability of remission by re-induction therapy is generally low for relapsed acute leukemia after allo-SCT (4, 5). Limited data exist on other factors, such as conditioning regimens or use of different donors (2). Reducedintensity conditioning (RIC) regimens are now widely used in umbilical cord blood transplantation (CBT) (6). However, few reports exist on the use of RIC regimens in second allo-SCT with CB to treat relapsed patients after first allo-SCT (7). We report the outcomes of RIC CBT without prior reinduction chemotherapy for 11 consecutive adult patients from 2007 to 2009 at our institution (Table 1). Seven had acute myeloid leukemia (AML) and four had acute lymphoblastic leukemia (ALL). Median duration between first SCT and relapse was 6 months (3–24). All but one underwent conditioning with fludarabine (125 mg ⁄m), L-PAM (80 mg ⁄m), and total-body irradiation (TBI; 4 Gy). To achieve the maximum graft-versus-leukemia effect, a relatively low concentration of continuous tacrolimus was used as graft-versus-host disease (GVHD) prophylaxis (mean concentrations for each 10-d period spanning days 1 to 40 were 8.9, 8.9, 10.2, and 9.8 ng ⁄mL, respectively). Six patients received minimal cytoreductive chemotherapy prior to or during conditioning to control the rapid increase in leukemic cells in peripheral blood. Median duration between relapse to second SCT was 37 d (22–60). Four were in complete remission (CR) at the last follow-up (15–37 months). Except for three patients who died early after the second SCT, eight achieved longer CR after the second SCT than after the first. According to the definitions for pre-engraftment immune reaction (PIR) (8) and hemophagocytic syndrome (HPS) following SCT (9), PIR was observed in all cases and HPS in seven. In 10 cases, prednisolone (median dose, 1 mg ⁄kg, ranging 0.2–6.9) was introduced for a short period immediately following the diagnosis of PIR to avoid HPS, which may correlate with graft failure of CB. Graft failure was observed in three patients, two of whom underwent further CBTs as salvage treatment. Complications included severe infections (bacterial, n = 4; viral, n = 5). Other toxicities greater than grade 2 included mucositis (n = 3), sinusoidal obstruction syndrome (n = 2), and generalized skin pain (n = 2). While high grade acute GVHD (III or IV) was not observed in the four patients maintaining CR, all had chronic GVHD. To the best of our knowledge, the largest studies of second allo-SCTs using CB included seven relapsed adult leukemia patients (10) and 22 pediatric (11) leukemia patients; none of the adults and 10 of the pediatric patients received RIC regimens. Compared with these two studies, the interval from first SCT to relapse in the surviving four patients maintaining CR was extremely short in our cohort; in three of four patients, it was 5 months. Compared with the first SCT, an extremely high incidence of PIR was observed with the second SCT (one case vs. all cases), whereas no difference was seen in the incidence of acute GVHD (grade II–IV) and chronic GVHD. Although the biological mechanisms are unclear, PIR is assumed to reflect alloimmune reaction and is observed frequently in CBTs. (8). While the use of CB and a relatively low tacrolimus concentration may have caused the high incidence of PIR and HPS, PIR may have contributed to the suppression of leukemic cells that remained in the early phase after the RIC regimen. Graft failure was a devastating complication that has recently been reported to be closely correlated with HPS following CBT (9). As we previously reported, two of three patients who suffered from graft failure and sepsis underwent successful salvage therapy with a one-day regimen using CB (12). However, further studies are needed to clarify how PIR and HPS should be controlled. LETTER TO THE EDITOR doi:10.1111/j.1600-0609.2010.01555.x European Journal of Haematology 86 (268–271)

Gemtuzumab ozogamicin-induced long-term remission in a woman with acute myelomonocytic leukemia and bone marrow relapse following allogeneic transplantation

A 56-year-old woman with acute myelomonocytic leukemia underwent myeloablative allogeneic hematopoietic stem cell transplantation (allo-SCT) from a matched unrelated donor in her first complete remission (CR). Veno-occlusive disease (VOD) prophylaxis consisted of low-dose heparin and ursodeoxycholic acid. Graft-versus-host disease (GVHD) prophylaxis comprised tacrolimus and short-term methotrexate. On day 14, VOD developed, but gradually resolved with supportive therapy. On day 58, she showed grade II acute GVHD, but this resolved spontaneously. On day 140, she developed hematological relapse with 40.2% marrow infiltration of CD33-positive blasts. Following the discontinuation of tacrolimus, gemtuzumab ozogamicin (GO) was administered. After GO administration, the patient exhibited mild VOD and severe pancytopenia with a sustained high fever for 6 weeks without evident infection. Bone marrow examination revealed severe hypoplastic marrow with 1.3% blasts 4 weeks after GO administration. Although transfusion-dependent pancytopenia persisted for 8 months after GO administration, bone marrow examination revealed the recovery of normal hematopoietic cells with 0.8% blasts. The patient has remained in CR with incomplete blood count recovery for 7 years following GO administration. Although the standard treatment for acute myeloid leukemia relapse after allo-SCT still remains to be established, GO may be a promising option.

Hypereosinophilia in a patient with invasive thymoma with clonal T-lymphocyte expansion expressing CD4, CD8, and CD25 antigens

We report the case of a patient with hypereosinophilia and invasive thymoma harboring probable clonal proliferation of CD4+, CD8+, and CD25+ T-lymphocytes. A 64-year-old woman had eosinophilia (14.1 × 109/L) and an anterior mediastinal tumor with elevated levels of serum immunoglobulin E (609.8 mg/dL) and interleukin 5 (239 pg/mL). Bone marrow aspirate showed marked infiltration by morphologically normal eosinophils with a normal karyotype but no FIP1L1-PDGFRA fusion gene. Flow cytometric analysis revealed an increasing number of CD3+/CD25+ lymphocytes in the peripheral blood, and the resected thymoma had infiltrated lymphocytes with CD4/CD8/CD25 antigens. Moreover, the thymoma had T-cell receptor rearrangements with a cytogenetically clonal nature, ie, t(2;4)(p22;q26). Although the number of patients with thymoma showing hypereosinophilia is small, this case suggests that a subset of patients with thymoma may have clonal expansion of T-lymphocytes with abnormal phenotypes that affect clinical manifestations, including hypereosinophilia.