Tetsumi Yoshida

All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia

We reported that T cells with anti‐CD38‐chimeric antigen receptors (CAR) eliminated B‐cell lymphoma cells expressing CD38. To employ anti‐CD38‐CAR against acute myeloid leukemia (AML) blasts not expressing CD38, it is necessary to induce or increase the intensity of CD38 expression. A lactate dehydrogenase (LDH)‐releasing assay and flow cytometry showed that anti‐CD38‐CAR T cells were cytotoxic against AML lines (THP‐1 and CMK) expressing high CD38 levels (>99%), in time‐ and number of effector‐dependent manners. In other AML lines (KG1, U937 and HL60) partially expressing CD38, CD38+ AML cells were killed by CD38‐specific T cells, but CD38− AML cells remained survived. Intriguingly, 10 nM all‐trans retinoic acid (ATRA) augmented CD38 expression in KG1, U937 and HL60 cells and primary leukemic cells from AML patients. Moreover, the withdrawal of ATRA from the medium decreased CD38 expression in AML cells. Killing effects of anti‐CD38‐CAR T cells against AML lines and AML cells were limited without ATRA, whereas CD38‐specific T cells enhanced cytotoxicity on AML cells by ATRA in association with enhanced CD38 expression. These results indicate that anti‐CD38‐CAR T cells eliminate AML cells through CD38 expression induced by ATRA.

Monthly administration of rituximab is useful for patients with ocular adnexal mucosa-associated lymphoid tissue lymphoma.

Ocular adnexal mucosa-associated lymphoid tissue-type lymphoma (OAL) is a distinct category of indolent B-cell non-Hodgkins lymphoma.1 OAL is commonly found in clinical stage IE or IIE at diagnosis, and is associated with a favorable prognosis.2, 3, 4, 5 However, patients often require treatment because of cosmetic problems, discomfort or pain. Although local irradiation therapy has been frequently used as a treatment option for OAL, it is associated with high risks of complications, such as dry eye, cataract, decreased visual acuity, conjunctivitis, conjunctival ulcer, retinopathy, optic neuropathy and cancer.2,6, 7, 8, 9, 10 The repeated administration of rituximab, weekly four times, is an effective alternative associated with high response rates and lower risks of complications. However, a high rate of relapse has reportedly been a marked concern with this modality.2,11,12 Accordingly, an optimal treatment strategy with fewer adverse effects and sustainable efficacy is required to treat patients with OAL because such patients show a more favorable prognosis. Here, we retrospectively compared the efficacy and adverse effects of the monthly administration of rituximab with that of radiotherapy as the initial treatment for OAL. We showed that monthly rituximab may be more useful in view of the better event-free survival (EFS) and fewer adverse effects on treating patients with OAL, compared with radiotherapy and weekly infusion four times of rituximab. Table 1 shows the characteristics of patients diagnosed with OAL in our department between 2008 and 2013. Informed consent for each treatment was obtained from all patients. Responses were assessed by whole-body computed tomography, FDG-positron emission tomography, magnetic resonance imaging and ophthalmologic tests after the completion of eight cycles of rituximab monotherapy. Ten patients with OAL received rituximab alone at 375 mg/m2 per day intravenously every 4 weeks for up to eight cycles. Alternatively, seven OAL patients received local irradiation of a single eye or both eyes as a historical control in our hospital. As shown in Table 1, the median age was 70 (36–79 years) in the group treated with rituximab alone, and 73 (43–82 years) in the group receiving irradiation. Proportions of females and CSIIE patients were not significantly different between the two groups (P=1.0 for females; P=0.33 for CSIIE by Fishers exact test). All rituximab-treated patients (n=10) achieved and maintained complete remission during the clinical follow-up (6–29 months). There were no critical adverse events associated with the monthly infusion of rituximab. As all patients achieved complete remission and were alive during the follow-up period, EFS, defined as survival without evidence of disease progression or relapse, was calculated by Kaplan–Meier methods according to the treatment modalities. Table 1 Characteristics of patients diagnosed with OAL EFS with the monthly administration of rituximab was sustained for a prolonged period (Figure 1). Two of eight patients relapsed in 24–26 months after the completion of radiotherapy. All patients treated with irradiation complained of dacryoadenitis, conjunctivitis, visual acuity loss or cataract. EFS of the patients receiving irradiation and monthly rituximab monotherapy is shown in Figure 1. These results demonstrated that EFS rates with the monthly administration of rituximab and that of radiotherapy were comparable (P-value=0.37). Figure 1 EFS with monthly administration of rituximab and radiotherapy. EFS of patients receiving irradiation and rituximab therapy is shown. Dotted and solid lines indicate EFS in patients with radiotherapy or rituximab alone, respectively. The recent availability of rituximab has improved the EFS, progression-free survival (PFS) and overall survival of patients with B-cell non-Hodgkins lymphoma. The frequency and severity of its side effects are non-significant during mono-treatment. Thus, rituximab is currently one of the prerequisite drugs for patients with B-cell non-Hodgkins lymphoma, especially for the aged. It was reported that four weekly cycles of rituximab at doses of 375 mg/m2 as induction therapy were significantly effective in patients with OAL, but early recurrence was common.2,11,12 Ardeshna et al.13 reported that four weekly rituximab doses of 375 mg/m2 followed by maintenance therapy every couple of months for 2 years improved PFS compared with induction therapy alone for advanced and asymptomatic follicular lymphoma, which belongs to indolent lymphoma, in which mucosa-associated lymphoid tissue-type lymphoma is classified. Notably, it is of interest that the 2-year PFS in the rituximab induction group was equivalent to the 4-year PFS in the maintenance group, and furthermore, the 4-year PFS in the former was almost the same as the 6-year PFS in the latter,13 suggesting that the duration of exposure to even a low concentration of rituximab is more critical than its concentration. The alternative modality of radiotherapy is a promising tool for OAL, as the local control rate was reportedly extremely high (85–100%).2,6, 7, 8, 9, 10,14,15 However, the risk of distant relapse is high at 10–25% and there are frequent adverse effects, such as long-term toxicity causing cataract (30–50%), xerophthalmia (20–40%), ischemic retinopathy, optic atrophy, corneal ulceration and neovascular glaucoma, as well as immediate toxicity including conjunctivitis, conjunctival ulcer and dry eyes.2,6, 7, 8, 9, 10,14,15 Patients with OAL receiving local irradiation also suffer from dry eye, cataract and conjunctivitis based on our experience. As OAL is associated with quite a favorable prognosis, therapeutic options that preserve the quality-of-life of patients with less adverse effects are chosen. Thus, we decided on the monthly administration of rituximab and obtained desirable results regarding the efficacy, EFS and reduced adverse effects in patients receiving monthly rituximab treatment. Another intriguing phenomenon is that the efficacy of rituximab may vary depending on the treatment schedule. Manipulation of the increased number of effector cells, such as NK cells and monocytes expressing Fcγ receptor, which binds to the Fc of rituximab, might be useful for patients with B-cell non-Hodgkins lymphoma cells regarding the efficacy of rituximab. Taken together, our treatment modality may provide a more beneficial treatment outcome with less adverse effect.

Splenectomy followed by administration of rituximab is useful to treat a patient with hairy cell leukemia-variant.

Dear Editor, Hairy cell leukemia-variant (HCL-v) is a rare diseasecategory separated from hairy cell leukeimia (HCL) by the WHO classification [1]. The prognosis, characterized by splenomegaly, is relatively poor among splenomegalic B cell disorders including HCL and splenic marginal zone lymphoma (SMZL) [2]. Thus, a novel therapeutic modality has been explored for HCL-v patients. Here, we show that splenectomy followed by rituximab monotherapy led to molecular complete response (CR) with an undetectable level of PCR-based immunoglobulin heavy-chain gene (PCR-based IgH) rearrangement in a patient with HCL-v. A 67-year-old female presented with an abdominal mass. Computed tomography showed significant splenomegaly (Fig. 1a). Pancytepenia (WBC count, 1.88×10/μl; Hb, 10.8 g/dl; and platelet count, 121×10/μl) and atypical lymphocytes (4.0 %) were noted in peripheral blood. Hepatitis B virus antigen and hepatitis C virus antibody were negative. Soluble interleukin-2 receptor was 835 IU/ml. Bone marrow (BM) aspirate showed normocellularity, including increased mediumto large-sized atypical lymphocytes (5.0 %) with highly irregular nuclei, prominent nucleoli, and hairy projections (Fig. 1b). Moreover, monoclonal B cells with a predominant κ light chain infiltrated the BM. She was provisionally diagnosedwith splenomegalic B cell disorder. Then, her bulky spleen was resected. Involved lymphocytes were morphologically consistent with atypical cells observed in BM (Fig. 1c, d). Cells that had infiltrated the spleenwere positive for CD19, CD20, CD103, κ, and FMC7 and negative for CD5, CD25, CD10, CD38, and CD23 (Fig. 2). Karyotypic aberration including chromosome 14 and PCR-based IgH rearrangement was detected. Although WBC and platelet numbers in peripheral blood entered the normal range, lymphocytosis and the Hb level were not improved. PCR-based IgH rearrangement was detected in BM cells, and HCL-v cells still existed to the same extent as pre-splenectomy in BM. Thus, we chose additional therapy of rituximab. After four cycles of monthly rituximab (375 mg/m), the Hb level normalized. Furthermore, PCR-based IgH rearrangement became negative and there was no phenotypic evidence of BM involvement, leading to so-called molecular CR. Patients with HCL-v have a poorer 5-year survival (50– 60 %) than those with HCL (>90 %) or SMZL (80–90 %) [2]. Currently, independent prognostic factors of HCL-v include anemia, an older age, and mutation of p53 [2]. As the presence of any one of these can stratify patients with HCL-v into a high-risk group, she was consistent with this. Thus, although she underwent splenectomy, the Hb level little improved and the proportion of HCL-v cells in BM remained unchanged. Patients with HCL-v are resistant or show a limited response T. Yoshida :K. Mihara (*) : S. Sugihara : T. Mino Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan e-mail: kmmihara@hiroshima-u.ac.jp

Lung involvement at initial presentation in blastic plasmacytoid dendritic cell neoplasm lacking cutaneous lesion

Dear Editor, Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease independently categorized in association with acute myeloid leukemia or related precursor neoplasm by the WHO [1, 2]. It is symptomatically characterized by fever, skin rash, lymphadenopathy, cytopenias, and leukemic blasts. Here, we present a rare case of BPDCN with interstitial pneumonia at the initial presentation. An 82-year-old female presented with shortness of breath, fever, and neutropenia in the peripheral blood. Fine crackles were heard in all lung fields. There was no rash. Computed tomography (CT) showed interstitial shadows prominent in the lungs, notably in the lower lobes, with no lymph node swelling. The anti-nuclear antibody titer was 320 times that of a control. She was diagnosed with interstitial pneumonia associated with autoimmune disease. After oral predonisolone, all symptoms alleviated promptly. Unfortunately, the tapering of predonisolone was followed by recurrence. Then, she was hospitalized in our university hospital. Peripheral blood analysis profiled pancytopenia (WBC count, 3.5×10/l; Hb, 8.5 g/dl; and platelet count, 122×10/l). Serum lactate dehydrogenase was slightly elevated (339 IU/l). Soluble interleukin-2 receptor was 1,664U/ml. Positron-emission tomography–CT (PET–CT) demonstrated that the incorporation of F-fluorodeoxy glucose was elevated in her lungs (SUVmax, 5.6)(Fig. 1a), consistent with interstitial lesions of the bilateral lungs. Moreover, flow cytometric results of lung biopsy showed that cells infiltrating the lung specimen were positive for CD4, CD45RA, CD56, and CD38 and negative for CD3 and CD34 (Fig. 1b). Bone marrow aspirate showed hypercellularity of increased mediumto large-sized blasts (14.5 %) with irregular nuclei and nucleoli (Fig. 1c). These cells were negative for peroxidase. Flow cytometric analyses showed that blasts were phenotypically consistent with cells in the lung specimen (data not shown). Furthermore, blasts were positive for CD43, CD123 (dendritic cellassociated antigen [3]), as well as CD56 and CD4 (>90 %) in the bone marrow mononuclear cells (Fig. 1d). Karyotypic aberration and rearrangements of T cell receptor and immunoglobulin heavy-chain genes were not detected. We diagnosed the patient with BPDCN lacking cutaneous lesions with leukemic and interstitial lung presentation. Even though the patient received multiple chemotherapeutic regimens, blasts were not entirely controlled, and she died 3 months after the diagnosis. Rauh et al. reported 22 patients diagnosed with BPDCN, with skin lesions initially observed in 81 %, lymphadenopathy in 64 %, and bone marrow involvement in 96 % [1]. BPDCN patients without cutaneous lesions and with K. Endo :H. Oiwa Department of Clinical Immunology and Rheumatology, Hiroshima University, Hiroshima, Japan

Giant granulocytic sarcoma of the vagina concurrent with acute myeloid leukemia with t(8;21)(q22;q22) translocation

Granulocytic sarcoma (GS) is a localized extramedullary solid tumor consisting of immature myeloid cells [1]. Its occurrence in the female genital tract is rare and mainly in ovary or uterus, and the most frequent symptom of GS in the female genital tract is vaginal bleeding [2, 3]. Here we report an extremely rare case of vaginal GS, noticed by vaginal discharge, concurrent with acute myeloid leukemia (AML). A previously healthy and unmarried 25-year-old female presented with profuse stinking vaginal discharge. Magnetic resonance imaging (MRI) of the pelvis demonstrated a massive 7.9 cm 9 4.4 cm tumor between the posterior vaginal wall and the rectum, with propagation into the vaginal cavity (Fig. 1a). A pathological examination of the vaginal biopsy revealed normal squamous cells of the vagina and diffuse subepithelial infiltrates of medium-sized rounded cells in its stroma (Fig. 1d, e). Immunohistochemistry revealed CD45CD56CD3CD5CD15CD20CD30 CD79 (Fig. 1f). The histological and immunological findings were suggestive of NK-cell lymphoma. The patient’s blood cell count and laboratory data were normal. Fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) examination and bone marrow aspiration were performed for the clinical staging. The vaginal tumor demonstrated an increased FDG uptake with a standard uptake value (SUV) of 7.6 (Fig. 1b, c). No other region including bone marrow showed pathological FDG uptake. However, bone marrow aspiration showed hypercellular marrow comprised predominantly of myeloblasts with Auer rods (FAB classification, M2). Surface antigens of blast cells were CD34CD13CD33 CD117CD4CD56CD3CD19CD57. A cytogenetic study of the bone marrow cells revealed the chromosomal abnormality, 46,XX,del(7)(q32),t(8;21)(q22;q22), del(12)(p?). With concern for the vaginal tumor possibly being a GS, additional immunohistochemical studies were performed. The vaginal tumor cells were positive for lysozyme, CD68, and CD34 (Fig. 1g), resulting in the alteration of the diagnosis to GS following AML. Furthermore, an RTPCR analysis of the AML1-ETO fusion gene was positive in both the bone marrow cells and the vaginal tumor tissue (Fig. 1h). Immediately, the patient underwent standard induction chemotherapy with idarubicin (12 mg/m for 3 days) and cytarabine (100 mg/m for 7 days), and achieved complete hematological remission. The vaginal tumor also disappeared and the FDG uptake became negative. After five additional cycles of consolidation therapy including a cycle of high-dose cytarabine, she has been in complete cytogenetical remission for 3 years. We have shown here the first reported case of giant GS in the vagina presenting with t(8;21) AML. This case is exceptional because vaginal discharge was the first sign of an AML. Several retrospective studies have reported that GS has been found with a relatively higher incidence (9–38%) in patients with t(8;21)(q22;q22) translocation [4, 5]. The majority of these GS cases presenting with t(8;21) AML J. Imagawa T. Yoshida A. Sakai A. Kimura H. Harada (&) Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Miniami-ku, Hiroshima 734-8553, Japan e-mail: herf1@hiroshima-u.ac.jp

Concurrent administration of darbepoetin alfa, cyclosporine A, and prednisolone is useful for T cell large granular lymphocyte leukemia associated with myelodysplastic syndrome complicated by Coombs-negative hemolytic anemia

Dear Editor, T cell large granular lymphocyte leukemia associated with myelodysplastic syndrome (T-LGL/MDS) is rare [1–4]. Its clinical features are marked anemia, an increased number of large granular lymphocytes, and neutropenia in peripheral blood. Here, we report that the concurrent administration of prednisolone (PSL), cyclosporine A (CsA), and darbepoetin alpha (DA), not cytocidal drugs, was useful in a rare T-LGL/ MDS patient complicated by advanced Coombs-negative hemolytic anemia. A 67-year-old male presented with a 13-month-long general malaise caused by anemia. Lactate dehydrogenase (LDH) elevation (1,055 IU/L), hemolytic anemia showing Coombs test negativity (Hb 6.2 g/dL, haptoglobin <10mg/dL, total bilirubin 2.3 g/dL, direct bilirubin 0.5 g/dL), a subtle increase of soluble interleukin-2 receptor (599 IU/mL) in serum, and an increased number of lymphocytes (WBC count 7.22×10/L, lymphocytes 81.0 %) were noted in peripheral blood. Computed tomography showed mild splenomegaly without lymphadenopathy. Bone marrow (BM) aspirate showed hypercellularity, including increased large-sized atypical lymphocytes (1.0 % of all nucleated cells) with large azurophilic cytoplasmic granules, highly irregular nuclei, and blebs, as in peripheral blood, and fewmyeloblasts (1.0%) (Fig. 1a). Flowcytometric analysis showed that atypical lymphocytes were positive for CD3, CD5, and CD8 and negative for CD4 and CD56 (Fig. 1b). Further immunohistochemical results supported CD57 positivity (Fig. 1c). A normal karyotype and PCR-based T cell receptor gene rearrangement were detected. These findings are consistent with T cell large granular lymphocyte leukemia (T-LGL). Moreover, BM cells showed prominent atypism featuring megaloblastoid change of erythroblasts, neutrophils with pseudo-Pelger-Hüet anomalies, and micromegakaryocytes (Fig. 1a) consistent with myelodysplastic syndrome (MDS) (refractory anemia). Thus, he was diagnosed with T-LGL/MDS. Initial treatment with oral PSL (20 mg/day) improved the symptoms associated with increased Hb, reduced lymphocytes in peripheral blood, and decreased serum LDH. However, the tapering of PSL (20 to 12.5 mg/day) exacerbated lymphocytosis and hemolytic anemia (Fig. 1d). Therefore, CsA was administered in addition to oral PSL (12.5 mg/ day). Hb improved to 7.5 g/dL, and the absolute lymphocyte count normalized in 8 weeks. However, as CsA tapering owing to a deteriorated renal function led to Hb reduction, DA was injected monthly at a dose of 60 μg. This led to further improvement of Hb to 9.5 mg/dL at 54 weeks of initial intervention (Fig. 1d). We re-evaluated the BM smear and phenotype of the BM mononuclear cells (BMMNCs) after 15 months of treatment. We noticed that the large granular lymphocytes had almost disappeared and dysplasia of the erythroid cells was inconspicuous, although atypism persisted in all trilineages. CD3CD8 T cells were reduced by approximately 10 % in BMMNCs compared with the level at diagnosis. As T-LGL/MDS is rare, there is no established standard therapy. PSL was used for initial treatment because hematopoietic colony formation reportedly improves with PSL in association with elimination of the T-LGL population in the T. Yoshida :K. Mihara (*) : T. Mino : T. Ichinohe Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan e-mail: kmmihara@hiroshima-u.ac.jp