Arito Yamane

Prevalence and Clinical Characteristics of Acute Myeloid Leukemia Associated with Disseminated Intravascular Coagulation

Disseminated intravascular coagulation (DIC) is one of the important complications to develop in patients with acute myeloid leukemia (AML). While acute promyelocytic leukemia (APL) is a strong risk factor for DIC, other clinical features have not been fully defined. We retrospectively analyzed 161 consecutive adult patients with de novo non-APL AML. DIC was diagnosed in 52 patients (32%); 28 patients at diagnosis and 24 soon after the initiation of induction chemotherapy. Leukocyte counts, C-reactive protein, and lactate dehydrogenase were significantly higher in the DIC+ group. Negative expressions of CD13, CD19, CD34, and HLA-DR were more prevalent in the DIC+ group. On multivariate logistic-regression analysis, variables that were independently associated with the development of DIC were high C-reactive protein, high leukocyte count, negative expressions of CD13 and HLA-DR, and cytogenetics with a normal karyotype or 11q23 abnormality. Although DIC is considered to be associated with serious morbidity and occasional mortality, we did not find any significant differences in the complete remission rate, overall or disease-free survival between DIC+ and DIC- groups. This study is the first to define the clinical characteristics associated with DIC in patients with non-APL AML, but exactly how and when DIC should be treated remains to be determined.

JAK2-V617F mutation analysis of granulocytes and platelets from patients with chronic myeloproliferative disorders: advantage of studying platelets

There have been conflicting reports over the JAK2‐V617F mutation status of platelets in chronic myeloproliferative diseases (CMPDs). The aim of this study was to analyse JAK2‐V617F status, not only in granulocytes but also in platelets. The JAK2‐V617F mutation was analysed in both granulocytes and platelets in 115 patients with CMPDs using direct sequencing. JAK2‐V617F was detected in granulocytes from 71 of those patients, all 71 of whom also had platelet JAK2‐V617F expression. The remaining 44 patients showed negative JAK2‐V617F expression on granulocytes, but positive JAK2‐V617F expression was detected on the platelets from nine of the 33 essential thrombocythaemia (ET) patients, one of the eight polycythaemia vera patients, and two of the three primary myelofibrosis patients. When ET patients were divided into three groups according to granulocyte and platelet JAK2‐V617F status (both‐positive, platelets‐only positive and both‐negative), the both‐positive and platelets‐only positive groups shared the clinical features of higher white blood cell count and frequent thrombosis. These results suggest that analysis of platelets is a more sensitive approach for detecting JAK2‐V617F in CMPD patients than analysis of granulocytes. They also suggest that previous reports of the incidence of JAK2‐V617F in CMPD patients, obtained using only analysis of granulocytes, could be underestimations.

Analysis of the distribution of CAG repeats and X-chromosome inactivation status of HUMARA gene in healthy female subjects using improved fluorescence-based assay.

We investigated the polymorphic CAG-repeat distribution and the X-inactivation status of the human androgen receptor (HUMARA) gene in 58 female Japanese volunteers. Polymerase chain reaction amplification was performed using a fluorescent-dye-labeled primer under conditions specific for GC-rich targets, and fragments were analyzed. To estimate the length of these fragments, FAM-labeled (blue fluorescent) products were simultaneously compared with ROM-labeled size markers (red) that were created by sequencing various HUMARA fragments. The number of polymorphic CAG repeats of HUMARA in 116 alleles from 58 female subjects ranged from 15 to 28. Of the 58 volunteers, 51 (88.0%) were heterozygous. In 96% of the heterozygous female subjects, the allelic differences were no greater than 6 repeats. X-chromosome inactivation was calculated as the ratio of the area of the smaller peak to the sum of the areas of the smaller and larger peaks. The average ratio was 0.38 (range, 0.09-0.50). Preferential use of 1 allele, by more than 75% (ratio, <0.25), was observed in 5 volunteers (10.9%). The clonal nature of a patient with chronic myelogenous leukemia was easily identified. This method is sensitive enough to discriminate a difference of 1 triplet repeat.

APOBEC3B high expression status is associated with aggressive phenotype in Japanese breast cancers

BackgroundThe members of AID/APOBEC protein family possess cytidine deaminase activity that converts cytidine residue to uridine on DNA and RNA. Recent studies have shown the possible influence of APOBEC3B (A3B) as DNA mutators of breast cancer genome. However, the clinical significance of A3B expression in Japanese breast cancer has not been studied in detail.MethodsNinety-three primary breast cancer tissues (74 estrogen-receptor (ER) positive, 3 ER and HER2 positive, 6 HER2 positive, and 10 triple negative) including 37 tumor-normal pairs were assessed for A3B mRNA expression using quantitative real-time RT-PCR. We analyzed the relation between A3B expression, mutation analysis of TP53 and PIK3CA by direct sequencing, polymorphic A3B deletion allele and human papillomavirus (HPV) infection in tumors.ResultsA3B mRNA was overexpressed in tumors compared with normal tissue. Patients with high A3B expression were associated with subtype and progression of lymph node metastasis and pathological nuclear grade. However, the expression was not related to any other clinicopathological factors, including mutation of TP53 and PIK3CA, polymorphic A3B deletion allele, HPV infection and survival time.ConclusionThe expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

Sacroiliitis as an initial manifestation of acute myelogenous leukemia

Sacroiliitis is the most pathognomonic and earliest manifestation of ankylosing spondylitis.We herein report a 28-year-old female patient who presented with sacroiliitis as an initial manifestation of acute myelogenous leukemia (AML). She had a 3-month history of anemia and walking difficulty. Bone marrow findings revealed an increase of blasts with trilineage dysplasia. Although she was initially diagnosed with myelodysplastic syndrome (MDS), blasts rapidly increased and AML developed 1 month after the diagnosis of MDS with sacroiliitis. Induction chemotherapy failed to induce a complete remission of AML, but it did effectively treat the sacroiliitis. However, the sacroiliitis relapsed when the leukemia cells progressed thereafter. Oral corticosteroids helped ameliorate the sacroiliitis. She underwent bone marrow transplantation (BMT) from an HLA-identical sister during a nonremission period; however, the leukemic cells began to rapidly increase from day 30 after BMT. The close relationship between the occurrence of sacroiliitis and AML suggested that autoimmune sacroiliitis was a paraneoplastic phenomenon of AML in this patient. Although autoimmune disorders develop in a substantial number of MDS patients, they are rarely observed in de novo AML. No previous report has described sacroiliitis as the initial manifestation of de novo AML.

Autologous Peripheral Blood Stem Cell Transplantation to Treat CHOP-Refractory Aggressive Subcutaneous Panniculitis-Like T Cell Lymphoma

his right femur that had swelled for 3 months. A physical examination revealed cutaneous indurations on his lower abdomen, right buttock and right femur. Multiple nontender subcutaneous nodules (1–2 cm in diameter) were evident on his face, back and upper extremities. A nontender right inguinal lymph node (1.5 cm) was palpable. Blood chemistry demonstrated lactate dehydrogenase, 832 IU/l (normal: ! 229 IU/l), and soluble interleukin-2 receptor, 1,909 IU/ml (normal: 220–530 IU/ml). A bone marrow examination revealed normocellular marrow without any lymphoma cell involvement and hemophagocytosis. Soft tissue enlargement of the lower abdomen and right femur ( fig. 1 a) were evident on computed tomographic images and 18 F-fluorodeoxyglucose positron emission tomography images showed that these regions were hypermetabolic. The maximum standardized uptake value was 3.4 in the right inguinal lymph node. A skin biopsy from the right femur showed infiltration of the subcutaneous fatty tissue by atypical medium to large lymphocytes with irregular nuclei rimming around fat vacuoles ( fig. 2 a, b). Immunohistochemical studies revealed that the phenotype of these cells was CD3+, CD4–, CD8+ ( fig. 2 c), CD20–, CD30–, CD45RO+ and CD56–. Cytotoxic molecules, namely granzyme B and T Subcutaneous panniculitis-like T cell lymphoma (SPTCL) is a rare form of postthymic cytotoxic T cell lymphoma that represents ! 1% of all non-Hodgkin lymphomas [1, 2] . The clinical course of SPTCL ranges from mild to progressive and fatal despite aggressive chemotherapy. Treatment strategies for SPTCL remain controversial because of its rarity. Anthracycline-based regimens, cyclophosphamide (CPM), doxorubicin, vincristine and prednisone (CHOP) or CHOP-like combinations have been used most frequently for patients with aggressive disease, but the overall outcome remains poor. The presence of hemophagocytic syndrome (HPS) and CD56 expression by tumor cells are associated with inferior overall survival [2–5] . We describe here a patient with CHOP-refractory aggressive SPTCL despite being HPS and CD56 negative. He achieved complete remission after undergoing an acute lymphoblastic leukemia (ALL) regimen followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) with a conditioning regimen comprising total body irradiation and melphalan. Three years later, he remained in complete remission without treatment. The patient, a 22-year-old Japanese male, presented with persistent high fever and subcutaneous nodules on Received: March 3, 2009 Accepted after revision: March 18, 2009 Published online: June 29, 2009

Presentation of extramedullary Philadelphia chromosome-positive biphenotypic acute leukemia as testicular mass: Response to imatinib-combined chemotherapy

Leukemias of ambiguous lineage are uncommon, representing *4% of all leukemias [1] and have been categorized into three sub-types; undifferentiated acute leukemia, bilineal acute leukemia and biphenotypic acute leukemia. Biphenotypic acute leukemia is characterized by blasts which co-express myeloid and T or B lineage specific antigens or concurrent B and T lineage antigens [1,2]. About a third of cases have Philadelphia (Ph) chromosome [1]. The prognosis of biphenotypic acute leukemia is generally unfavorable, especially accompanied with the occurrence of the t(4;11) or Ph chromosome abnormalities [1,3]. We here describe successful treatment for a patient with Ph-positive biphenotypic acute leukemia with testicular involvement who had received allogenic stem cell transplantation after imatinib combined chemotherapy. A 54-year-old Japanese male without a significant past medical history was admitted to the hospital in January 2005 because of enlargement of left testis, leukocytosis, anemia and thrombocytopenia. The blood count was hemoglobin 8.7 g dL (87 g L), platelets 3.5610 mL (35610 L) and WBC 94.3610 mL (94.3610 L) with a differential of 93% blasts, 0% neutrophils, 6% lymphocytes and 1% monocytes. Lactate dehydrogenase (LDH) was 439 IU L (119 – 229 IU L). Bone marrow showed marked hypercellularity with 99% blasts. Flow cytometric analysis revealed that the blast cells were positive for CD13, CD33, CD117, CD79a, CD19 and TdT (Figure 1). Cytogenetic study of the bone marrow cells revealed 46, XY, t(9;22)(q34;q11) in all metaphases. Real-time RT-PCR analysis revealed 950 000 copies per 1 mg RNA of major BCR/ABL. Furthermore, Southern blot analysis revealed IgH rearrangement. Scrotal ultrasound examination revealed a well-circumscribed and hypoechoic mass measuring 6 cm65 cm64 cm. He was diagnosed as having Ph-positive biphenotypic acute leukemia with testicular involvement. At first, the patient was treated with acute myeloid leukemia (AML)-directed therapy [idarubicin (12 mg m per day intravenously) for 3 days (days 1 – 3) and cytarabine (100 mg m per day) by continuous infusion for 7 days (days 1 – 7)], however the response was poor and more than 80% of blasts persisted in the bone marrow cells as well as enlargement of left testis. We started to treat with 600 mg of imatinib orally daily (days 1 – 40) and acute lymphoblastic leukemia (ALL)-directed therapy [cyclophosphamide (1200 mg m per day i.v.) on day 1, daunorubicin (60 mg m per day i.v.) for 3 days (days 1 – 3), vincristine (1.3 mg m per day i.v.) on days 1, 8, 15, 22, prednisolone (60 mg m orally daily on days 1 – 21)]. After this chemotherapy, he achieved hematological complete remission (CR) without testicular mass and we continued 600 mg of

Low burden of a JAK2-V617F mutated clone in monoclonal haematopoiesis in a Japanese woman with Budd-Chiari syndrome

Approximately one-half of the cases of Budd-Chiari syndrome (BCS) are caused by bcr/abl negative chronic myeloproliferative disorders (CMPDs). Furthermore, a mutation in the Janus kinase protein (JAK2-V617F) is detected in half of the patients with BCS. However, whether the JAK2 mutation is the primary event leading to CMPDs and BCS is controversial. We present a report concerning a young woman who suffered from BCS prior to the onset of CMPDs. Analysis of X-chromosome inactivation patterns in this patient, using the human androgen receptor gene demonstrated monoclonal haematopoiesis in her granulocytes. In contrast, she had a low burden of a JAK2-V617F mutation positive clone among granulocyte populations. These results suggest that the JAK2-V617F mutation occurs after the onset of monoclonal haematopoiesis; thus the V617F mutation of JAK2 may not be the primary event in the induction of BCS.

Expression of CD55 and CD59 on peripheral blood cells in patients with lymphoproliferative disease of granular lymphocytes.

Lymphoproliferative disease of granular lymphocytes (LDGL) is a disorder characterized by the clonal expansion of granular lymphocytes. It has recently been shown that the clonal expansion of granular lymphocytes occurs in patients with paroxysmal nocturnal hemoglobinuria (PNH) in a subclinical fashion. To test the possibility that LDGL patients share a PNH phenotype, we obtained peripheral blood cells from 20 patients with LDGL and examined the expression of the glycosylphosphatidyl inositol (GPI)‐anchored proteins, CD55 and CD59. Compared with normal controls, however, a defective expression of CD55/59 was not observed on either granulocytes or erythrocytes from LDGL patients. An unexpected finding was the significantly lower CD55/59 expression on granular lymphocytes from patients with CD16+CD56− phenotype LDGL than from patients with CD16+CD56+ phenotype LDGL, or natural killer (NK) and NK/T lymphocytes from healthy individuals. The positive correlation between the expression of CD56 and CD55/59 might have some relevance to the functional properties of the CD56+ subset of large granular lymphocytes.

Overexpression of B-cell lymphoma 6 alters gene expression profile in a myeloma cell line and is associated with decreased DNA damage response

B‐cell lymphoma 6 (BCL6) attenuates DNA damage response (DDR) through gene repression and facilitates tolerance to genomic instability during immunoglobulin affinity maturation in germinal center (GC) B cells. Although BCL6 expression is repressed through normal differentiation of GC B cells into plasma cells, a recent study showed the ectopic expression of BCL6 in primary multiple myeloma (MM) cells. However, the functional roles of BCL6 in MM cells are largely unknown. Here, we report that overexpression of BCL6 in a MM cell line, KMS12PE, induced transcriptional repression of ataxia telangiectasia mutated (ATM), a DDR signaling kinase, which was associated with a reduction in γH2AX formation after DNA damage. In contrast, transcription of known targets of BCL6 in GC B cells was not affected, suggesting a cell type‐specific function of BCL6. To further investigate the effects of BCL6 overexpression on the MM cell line, we undertook mRNA sequence analysis and found an upregulation in the genomic mutator activation‐induced cytidine deaminase (AID) with alteration in the gene expression profile, which is suggestive of de‐differentiation from plasma cells. Moreover, interleukin‐6 exposure to KMS12PE led to upregulation of BCL6 and AID, downregulation of ATM, and attenuation of DDR, which were consistent with the effects of BCL6 overexpression in this MM cell line. Taken together, these results indicated that overexpression of BCL6 alters gene expression profile and confers decreased DDR in MM cells. This phenotypic change could be reproduced by interleukin‐6 stimulation, suggesting an important role of external stimuli in inducing genomic instability, which is a hallmark of MM cells.