Akihito Fujimi

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Expansion of CD34^+ Cells on Telomerized Human Stromal Cells without Losing Erythroid-Differentiation Potential in a Serum-Free Condition

Erythropoiesis progresses from stem cell expansion on stromal cells through the formation of an erythroblastic island. Our aim was to assess the feasibility of using human stromal cells for erythroid production and differentiation. When cord blood CD34+ cells were cocultured with telomerized human stromal cells (hTERT-stromal cells) for 2 weeks, the CD34+ cells and burst-forming units-erythroid (BFU-E) significantly expanded, and a few hematopoietic cells transmigrated below the stromal layer. When nonadherent hematopoietic progenitor cells that had expanded above the hTERT-stromal cells (group B) were collected and subjected to our erythroid-differentiation protocol, they differentiated into erythroblasts with a slight hemoglobin synthesis. When the few hematopoietic cells that had transmigrated below the stromal layer were expanded for an additional 2 to 6 weeks, they exhibited a cobblestone-like appearance, and a large amount of BFU-E clambered weekly from the underside of the stromal layer to above the stromal layer (group C).When the hematopoietic progenitor cells in group C were subjected to the erythroid-differentiation protocol, large numbers of mature erythroblasts (more than 300,000 times the initial CD34+ cell number) were produced. Our hTERT-stromal expansion protocol may contribute to the construction of a system for large-scale, long-term production of erythroid cells.

Long-term survival and late-onset complications of cancer patients treated with high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation

The antitumor effect of high-dose chemotherapy (HDC) followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) is considered superior to that of conventional chemotherapy. However, the long-term benefits of this strategy in Japan remain unclear.Therefore, in this study, 109 cancer patients enrolled between 1989 and 1999 were treated with HDC and auto-PBSCT. Patients were evaluated for long-term survival and late-onset complications, including secondary malignancy. The mean number of CD34+ cells harvested per apheresis was larger in the group receiving high-dose cytosine arabinoside or high-dose etoposide plus granulocyte colony-stimulating factor (G-CSF) than in the group receiving conventional chemotherapy plus G-CSF. The 5-year overall survival rates for non-Hodgkin’s lymphoma patients in first complete remission (CR) (83.2%), second or subsequent CR (74.1%), or first partial remission (PR) (66.7%) at the time of transplantation were significantly higher than those with no remission (35.7%) at the time of transplantation (first CR,P < .05; second or subsequent CR,P < .05; first PR,P < .05). The 5-year overall survival (OS) rates for breast cancer was 40.8%, and the disease-free survival rate was extremely low (8.8%). The 5-year OS rates for chemotherapy-sensitive and chemotherapy-resistant diseases at the time of transplantation were 32.7% and 35.7%, respectively, a difference that was not considered significant. The 5-year OS for germ cell tumor was 80.0%, and the disease-free survival rate was 77.9%. The rate of therapy-related death was 8.2%. The occurrence rate of secondary malignancy was 0.9%. Late-onset complications were observed in 4 cases (glomerulonephritis, interstitial pneumonitis, ulcerative colitis, and acute myelogenous leukemia). At 3.7%, the occurrence rate was not very high, but most complications of auto-PBSCT were life threatening and interfered with patients’ quality of life. A careful follow-up is required for at least 2 years after transplantation, because the mean occurrence time of late-onset complications is 16.7 months posttransplantation.

Reversible skin and hair depigmentation during chemotherapy with dasatinib for chronic myeloid leukemia

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Focal 18F-FDG uptake in bone marrow on PET/CT in a patient with JAK2 mutation without overt myeloproliferative neoplasm.

A 58-year-old female diagnosed with early stage esophageal carcinoma in our hospital underwent endoscopic resection by endoscopic submucosal dissection (ESD) in April 2013. F-FDG PET/CT performed immediately prior to the ESD showed focal F-FDG accumulations in the vertebral body of Th8, and vertebral body and arch of L4 with SUV max of 3.98–4.42 (Fig. 1a–c). No masses or osteoclastic lesions were observed. MRI findings of the lesions showed low intensity on T1WI and high intensity on STIR image (Fig. 2a–d). To clarify the cause of the FFDG accumulation in the bone, we performed bone biopsy from the vertebral arch of L4. Histopathological findings revealed hypercellular marrow (80 % cellularity) and increases in number and size of megakaryocytes, most of which were in maturated form with hyperlobulated nuclei, which are usually found in cases with myeloproliferative neoplasm (MPN) (Fig. 3a, b). Reticulin fibrosis of marrow was observed minimally by Gitter staining (Fig. 3c), and collagen fibrosis was not observed. In contrast, laboratory workup for peripheral blood showed no abnormality: WBC 6,920/lL (stab 0.0 %, seg 61.0 %, lymph 30.0 %, mono 6.0 %, eosino 1.5 %, and baso 1.5 %), RBC 452 9 10/ lL, Hb 14.1 g/dL, Ht 40.6 %, PLT 33.3 9 10/lL, reticulocytes 1.2 %, LDH 229 U/L, VB12 351 mg/dL, and NAP score 223. Bone marrow biopsy subsequently performed from the left iliac bone showed normocellular marrow, but the number of megakaryocytes was also increased (Fig. 3d). Furthermore, JAK2 V617F mutation was detected in the bone marrow sample by real-time qualitative PCR with a sensitivity of more than 2 % of all alleles. G-banding showed normal diploid karyotype, and BCR–ABL translocation was not detected by FISH analysis. 5 months after the first visit, her laboratory data of peripheral blood was within the normal range, and FFDG PET/CT also showed similar F-FDG accumulation in the bone, without new lesions. We will continue to follow her progress carefully. The JAK2 V617F mutation is present in patients with Philadelphia-negative MPN, including over 90 % of polycythemia vera cases and about half of essential thrombocythemia and primary myelofibrosis cases [1]. The JAK2 V617F mutation may also be detected in healthy individuals without overt MPN [2]. Nielsen et al. [2] reported that the JAK2 V617F mutation was detected in 18 of 10,507 participants (0.2 %) in the general population, and three of these 18 individuals with the JAK2 V617F mutation developed overt myeloproliferative disorder during up to 17.6 years of follow-up. In the present case, focal F-FDG accumulation in bone marrow and histopathological findings, other than the finding of the left iliac bone marrow as positive for JAK2 V617F mutation, suggest that the patient is more likely to develop some form of overt MPN in the A. Fujimi (&) Y. Kanisawa Department of Hematology and Oncology, Oji General Hospital, 3-4-8 Wakakusa-cho, Tomakomai 053-8506, Japan e-mail: Akihito.fujimi@ojihosp.or.jp

Anti-erythropoietin receptor antibody-associated pure red cell aplasia accompanied by Coombs-negative autoimmune hemolytic anemia in a patient with T cell/histiocyte-rich large B cell lymphoma

A 79-year-old female diagnosed with T cell/histiocyte-rich large B cell lymphoma in complete remission after six cycles of rituximab-combined chemotherapy developed severe anemia, reticulocytopenia, and bone marrow erythroid hypoplasia. She was diagnosed with pure red cell aplasia (PRCA) accompanied by Coombs-negative autoimmune hemolytic anemia evidenced by a lack of glycophorin-A-positive cells in the bone marrow, haptoglobin under the detection level, and a high titer of RBC-bound IgG. Anti-erythropoietin receptor (EPOR) antibody was detected in the serum, and oligoclonal α/β and γ/δ T cells were also detected in her peripheral blood by Southern blotting analysis. Parvovirus B19 DNA was not detected by PCR. Although the treatment with rituximab had limited efficacy (specifically, only for hemolysis), subsequent cyclosporine therapy led to prompt recovery of erythropoiesis with the disappearance of anti-EPOR antibody and oligoclonal T cells. This is the first case report of anti-EPOR antibody-associated PRCA in a patient with malignant lymphoma treated successfully with cyclosporine.

Spontaneous cholesterol crystal embolism to lymph node

A 65-year-old male diagnosed with hypertension and hypertrophic cardiomyopathy in April 2010 at a different hospital was administered angiotensin II receptor blocker and low-dose aspirin. Although laboratory data at that time showed eosinophilia (2,860/lL), further examination was not performed. He had a history of smoking 1.5 packs of cigarettes a day for 45 years, but no history of diabetes mellitus. He developed cerebral infarction in January 2012, but recovered uneventfully with conservative treatment, including statins for dyslipidemia. He was subsequently referred to our hospital to investigate the eosinophilia. On physical examination, he had several swollen lymph nodes in bilateral inguinal regions, but no cutaneous lesion was observed. Laboratory data were as follows: WBC 10,600/ lL, eosinophil 840/lL, Hb 11.2 g/dL, Plt 8.8 9 10/lL, FDP 12.0 lg/mL, LDH 352 U/L, BUN 17.5 mg/dL, Cr 1.00 mg/dL, IgE 8,600 IU/mL, ACTH 15.3 pg/mL and cortisol 9.4 lg/dL, as well as negative test results for ANA and MPO-ANCA. The urinalysis showed proteinuria and microhematuria. Parasite eggs were not detected in the feces. Bone marrow examination showed 9.1 % eosinophils among all nucleated cells without dysplasia, and FIP1L1-PDGFRa and BCR-ABL chromosomal aberrations were not detected by FISH analysis. Chest and abdominal CT showed several enlarged inguinal lymph nodes up to 18 mm in the minor axis. Although he stated that he had recognized these inguinal masses about 10 years previously and that they had not changed markedly in size, we performed biopsy from the right inguinal lymph node. Histopathological findings revealed needle-shaped clefts in the lumen of arterioles with multinucleated giant cell infiltration surrounded by normal lymphoid follicles (Fig. 1a–c). Perivascular inflammatory cell infiltration, mainly of eosinophils, was also observed. Flow cytometric analysis of lymph node showed no abnormality. The diagnosis of cholesterol crystal embolism (CCE) to lymph node was made. As he presented no other clinical manifestations of CCE, no further therapeutic intervention was performed. CCE is a rare systemic disease caused by occlusion of small arteries by cholesterol crystals released from atheromatous plaques of the aorta or major branches. Chest CT in this patient also showed calcification and wall thickness of the thoracic aorta, which can be a source of cholesterol crystals (Fig. 2). The common manifestations of CCE are characteristic skin lesions, such as livedo reticularis, cyanosis or ulceration, renal impairment, and gastrointestinal disorder. CCE involvement of lymph node is extremely rare. Only a few preand postmortem cases of CCE to lymph node have been reported to date [1, 2]. CCE usually occurs following an invasive vascular procedure, or anticoagulant or thrombolytic therapy, but it can also occur spontaneously. We surmised that the CCE in this patient was spontaneous, as he had not undergone any such intervention during this clinical course. The exact time at which the CCE developed was unclear, but pathological findings of lymph nodes showing CCE with giant cell infiltration and no signs of fibrosis suggested that it had been a relatively recent event. Hence, we suspect that the A. Fujimi (&) A. Hashimoto Y. Kanisawa Department of Hematology and Oncology, Oji General Hospital, 3-4-8 Wakakusa-cho, Tomakomai 053-8506, Japan e-mail: Akihito.fujimi@ojihosp.or.jp

Thrombocytopenia and Anemia with Anti-c-Mpl antibodies Effectively Treated with Cyclosporine in a Patient with Rheumatoid Arthritis and Chronic Renal Failure.

A 61-year-old woman with rheumatoid arthritis who was undergoing hemodialysis for end-stage renal failure was transferred to our hospital due to severe thrombocytopenia and anemia. A bone marrow biopsy showed the complete absence of megakaryocytes and erythroblasts. Cyclosporine treatment resulted in the improvement of her megakaryocyte and erythroblast levels, and a decrease in her serum level of anti-c-Mpl (thrombopoietin receptor) antibodies. After this initial improvement, her anemia progressively worsened, despite the continuous administration of immunosuppressive therapy with cyclosporine. Her platelet and leukocyte counts remained stable. This is the first report of a probable case of anti-c-Mpl antibody-associated pure red cell aplasia and acquired amegakaryocytic thrombocytopenic purpura.

Extramedullary involvement of the stomach presenting as multiple white elevations in the initial diagnosis of chronic myeloid leukemia treated with dasatinib

Dear Editor, A 68-year-old man, who had been treated for diabetes mellitus and hypertension for 15 years, was admitted to our hospital because of an elevated white blood cell (WBC) count. Approximately 1 year ago, his complete blood count was within the reference range. Laboratory data on admission were as follows: WBC count, 185.4 × 10/L (myeloblasts, 2.0%; promyelocytes, 3.0%; basophils, 0.0%); hemoglobin level, 11.2 g/dL; and platelet count, 132 × 10/L. The bone marrow was hypercellular without excess blasts (myeloblasts, 3.0%; promyelocytes, 10.0%), stained strongly positive for myeloperoxidase (MPO) and CD68, and stained negative for CD34 and p53. CD31 and glycophorin A staining were positive on megakaryocytes and erythroblasts, respectively. Gitter staining revealed no reticulin fibrosis. Chromosomal analysis by G-banding in the bone marrow showed 46,XY,t(9;22)(q34;q11.2) in all 24 analyzed cells. He was diagnosed with chronic myeloid leukemia (CML). The prognostic scores for Sokal, Hasford, and EUTOS were intermediate (0.92 points), intermediate (783 points), and low (0 points), respectively. Notably, esophagogastroduodenoscopy performed as gastrointestinal screening showed multiple, white, flat elevations up to 2 cm in diameter, mainly in the corpus of the stomach (Fig. 1a–d). Histopathological findings of the biopsy specimen revealed diffuse infiltration of small round cells in the lamina propria of the gastric mucosa (Fig. 1e, f), and immunohistochemical staining was positive for MPO and CD68 and negative for CD20, CD45RO, CD56, CD34, and p53—characteristics similar to those in bone marrow leukemic cells. Although most infiltrated cells were immature myeloid cells, more differentiated myeloid cells, such as stab cells and segmented cells, were also observed (Fig. 1f). CD31-positive megakaryocytic cells were also evident; however, extramedullary hematopoiesis was excluded because glycophorin A staining was only positive for mature erythrocytes. Fluorescence in situ hybridization analysis of BCR-ABL fusion genes using paraffin-embedded gastric tissue revealed 80% positive results of the analyzed cells. Accordingly, the patient was diagnosed with blast-phase CML by extramedullary blast proliferation into the stomach. Low-dose dasatinib (50 mg qd) was administered considering the emergence of adverse effects; however, it was discontinued on day 6 before increasing the dose because of persistent bleeding from the biopsy region of the gastric mucosa. Nilotinib was then administered, but was also discontinued owing to marked hyperglycemia. Therefore, dasatinib was readministered at a lower dose of 20 mg qd considering the bleeding tendency, then gradually increased to 100 mg qd, which resulted in no further bleeding from the gastric mucosa. A month after dasatinib re-administration, complete hematological response and disappearance of all elevated lesions of the stomach were achieved. Complete cytogenetic response was obtained at 3 months; however, the major BCR-ABL1 mRNA transcript level on the international scale at 12 months was 0.2680%. Currently, the patient receives dasatinib at a dose of 70 mg bid without gastric lesion recurrence. Extramedullary blast proliferation of CML has been reported to develop in 16% of blast crisis cases in the pre-tyrosine kinase inhibitor (TKI) era [1], whereas the diagnosis of extramedullary disease at initial presentation * Akihito Fujimi akihito.fujimi@gmail.com