Akihiro Abe

Injectable Bone Tissue Engineering Using Expanded Mesenchymal Stem Cells

Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue‐engineered bone (TEB) composed of mesenchymal stem cells (MSCs) and platelet‐rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out large‐scale clinical studies in patients with long‐term follow‐up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months were significantly increased over the preoperation baseline (p < .001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long‐term follow‐up. Injectable TEB restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering. STEM CELLS2013;31:572–580

Clarifying the impact of polycomb complex component disruption in human cancers.

The dysregulation of proper transcriptional control is a major cause of developmental diseases and cancers. Polycomb proteins form chromatin-modifying complexes that transcriptionally silence genome regions in higher eukaryotes. The BCL6 corepressor (BCOR) complex comprises ring finger protein 1B (RNF2/RING1B), polycomb group ring finger 1 (PCGF1), and lysine-specific demethylase 2B (KDM2B) and is uniquely recruited to nonmethylated CpG islands, where it removes histone H3K36me2 and induces repressive histone H2A monoubiquitylation. Germline BCOR mutations have been detected in patients with oculofaciocardiodental and Lenz microphthalmia syndromes, which are inherited conditions. Recently, several variants of BCOR and BCOR-like 1 (BCORL1) chimeric fusion transcripts were reported in human cancers, including acute promyelocytic leukemia, bone sarcoma, and hepatocellular carcinoma. In addition, massively parallel sequencing has identified inactivating somatic BCOR and BCORL1 mutations in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia, medulloblastoma, and retinoblastoma. More importantly, patients with AML and MDS with BCOR mutations exhibit poor prognosis. This perspective highlights the detection of BCOR mutations and fusion transcripts of BCOR and BCORL1 and discusses their importance for diagnosing cancer subtypes and estimating the treatment responses of patients. Furthermore, this perspective proposes the need for additional functional studies to clarify the oncogenic mechanism by which BCOR and BCORL1 are disrupted in cancers, and how this may lead to the development of novel therapeutics. Mol Cancer Res; 12(4); 479–84. ©2014 AACR.

A novel RUNX1-C11orf41 fusion gene in a case of acute myeloid leukemia with a t(11;21)(p14;q22)

The RUNX1 locus, which encodes a transcription factor that is essential for normal hematopoiesis, is a frequent location of chromosomal rearrangements in human hematological malignancies. We report the case of a 78-year-old man with acute myeloid leukemia (AML), M1 subtype (French-American-British classification), with a t(11;21)(p14;q22). Fluorescence in situ hybridization showed a split signal for RUNX1, which indicated that RUNX1 was involved in this translocation. Using 3-rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction analyses, we found that RUNX1 was fused to C11orf41 on 11p14 and detected two in-frame C11orf41-RUNX1 fusion transcripts. One was a fusion between exon 5 of RUNX1 and exon 13 of C11orf41, and the other was between exon 6 of RUNX1 and exon 13 of C11orf41. This suggested that the RUNX1 breakpoint was in intron 6 and had generated alternative fusion splice variants. A reciprocal C11orf41-RUNX1 fusion was not detected. Thus, we identified C11orf41 as a novel fusion partner of RUNX1 in AML.

Severe hepatitis associated with varicella zoster virus infection in a patient with diffuse large B cell lymphoma treated with rituximab-CHOP chemotherapy.

Severe disseminated varicella zoster virus (VZV) infection rarely occurs in patients who are not recipients of hematopoietic stem cell transplantation. This report concerns severe disseminated VZV infection in a diffuse large B cell lymphoma (DLBCL) patient treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP). The patient was an 82-year-old male with DLBCL who had a history of type II diabetes mellitus. He incurred VZV infection with severe hepatitis and disseminated intravascular coagulopathy after three courses of R-CHOP. When the VZV infection occurred, anti-VZV IgG was not detected and lymphopenia was observed. We initiated treatment with acyclovir, immunoglobulin, and thrombomodulin alpha, and rescued this patient. We suggest that the use of chemotherapy for immune-suppressed elderly lymphoma patients may involve the risk of severe VZV infection.

Acute promyelocytic leukemia following aleukemic leukemia cutis harboring NPM/RARA fusion gene.

To the Editor: Leukemia cutis is occasionally documented in patients with infant leukemia, and it is observed more frequently in acute myeloid leukemia with monocytic or myelomonocytic differentiation [1]. Leukemia cutis can occur before bone marrow involvement and systemic manifestations, and spontaneous resolution has been described [2]. We previously described 6-month-old male with aleukemic leukemia cutis harboringNPM/RARA fusion gene,whohad spontaneous regression of leukemia associated with cutaneous mastocytosis at the age of 19 months [3]. At 4 years and 4 months of age, the patient presented with fever and leg pain. Physical examination disclosed swelling of the right cheek and hepatosplenomegaly. Laboratory studies showed a hemoglobin level of 9.2 g/dl, a platelet count of 15.2 10/ml, elevated white blood cell count of 25.56 10/ml with 15.5% promyelocytes, 26.5% myelocytes, and 8.5% metamyelocytes, but no obvious blastic cells, and an elevated level of lactate dehydrogenase of 1,456 IU/L. Coagulopathy was associated with fibrinogen of 142mg/dl, FDP of 47.1 mg/ml (normal: <4.9), and D-Dimer of 21.5 mg/ml (normal: <1.0). Bone marrow specimens showed pathologic promyelocytic cells, which contained cytoplasmic azurophilic granules, but no Auer rods (Fig. 1A). The leukemic cells were strongly positive for myeloperoxidase staining (Fig. 1B). Karyotype analysis of the bone marrow cells showed 46,XY,t (5;17)(q35;q12),i(21)(q10) [16/20], as previously described [3]. Fluorescence in situ hybridization analysis with PML and RARA probes revealed two split RARA signals in 91.0% cells, but no fusion signal, and reverse transcriptase polymerase chain reaction showed a transcript of NPM/RARA fusion gene. The patient was finally diagnosed as having overt acute promyelocytic leukemia (APL) with NPM/RARA fusion gene as the relapse of aleukemic leukemia cutis. Therefore, he was treated with systemic chemotherapy followed by all-trans retinoic acid (ATRA) administration and achieved complete remission with the lesion in the head disappearing. Among eight patients with spontaneous resolution following aleukemic leukemia cutis, four patients were reported to have monocytic or myelomonocytic leukemia and one patient had acute myeloid leukemia (AML) M2, though AML subtypes of three patients were not described [2]. Cytogenetic and molecular studies suggested that APL clone was the same as that of the first presentation in our case.

A varicella outbreak in B-cell lymphoma patients receiving rituximab-containing chemotherapy

Varicella, characterized by a vesicular rash, occurs primarily in young children. Although older individuals can also be affected or vaccinated, outbreaks among adults are rare. We investigated a small outbreak of varicella in B-cell lymphoma patients for elucidation of risk factor of the disease. We experienced four cases of varicella after an index herpes zoster case. All varicella cases were confirmed varicella zoster virus (VZV) infection by PCR. All varicella cases occurred in diffuse large B-cell lymphoma patients receiving rituximab-containing chemotherapy. On the other hand, only three of the 18 non-varicella patients in the same room were receiving rituximab-containing chemotherapy (P = 0.005). All varicella patients had detectable serum anti-varicella zoster virus IgG antibodies before chemotherapy. Even in the presence of neutralizing antibodies to the virus, lymphoma patients treated with rituximab-containing chemotherapy can possibly become re-infected with varicella. These findings suggest that zoster patients should be strictly isolated in hematology and oncology ward, and prophylactic acyclovir should be considered for such patients when exposed to zoster/varicella.

Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice

Patients in the chronic phase (CP) of chronic myelogenous leukemia (CML) have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC) phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt.) AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.

ETV6‐LPXN fusion transcript generated by t(11;12)(q12.1;p13) in a patient with relapsing acute myeloid leukemia with NUP98‐HOXA9

ETV6, which encodes an ETS family transcription factor, is frequently rearranged in human leukemias. We show here that a patient with acute myeloid leukemia with t(7;11)(p15;p15) gained, at the time of relapse, t(11;12)(q12.1;p13) with a split ETV6 FISH signal. Using 3′‐RACE PCR analysis, we found that ETV6 was fused to LPXN at 11q12.1, which encodes leupaxin. ETV6‐LPXN, an in‐frame fusion between exon 4 of ETV6 and exon 2 of LPXN, did not transform the interleukin‐3‐dependent 32D myeloid cell line to cytokine independence; however, an enhanced proliferative response was observed when these cells were treated with G‐CSF without inhibition of granulocytic differentiation. The 32D and human leukemia cell lines each transduced with ETV6‐LPXN showed enhanced migration towards the chemokine CXCL12. We show here for the first time that LPXN is a fusion partner of ETV6 and present evidence indicating that ETV6‐LPXN plays a crucial role in leukemia progression through enhancing the response to G‐CSF and CXCL12.

NUP214-RAC1 and RAC1-COL12A1 Fusion in Complex Variant Translocations Involving Chromosomes 6, 7 and 9 in an Acute Myeloid Leukemia Case with DEK-NUP214

DEK-NUP214 gene fusion in acute myeloid leukemia (AML) is associated with poor prognosis. It is most often a sole translocation and more rarely observed as complex chromosomal forms. We describe an AML case with complex karyotype abnormalities involving chromosome bands 6p23, 6q13, 7p22, and 9q34. RNA sequencing analysis revealed that exon 17 of NUP214 (9q34) was fused to exon 2 of RAC1 (7p22). We also detected that the 5′-end of intron 1 of RAC1 was fused with the antisense strand of intron 5 of COL12A1 (6q13). RT-PCR analysis confirmed the expression of DEK-NUP214, NUP214-RAC1, RAC1-COL12A1, NUP214, and RAC1. These results suggest that the 5′- and 3′-ends of NUP214 from the breakpoint in the same locus were fused to RAC1 and DEK, respectively, and the 5′-end of RAC1 was fused to COL12A1. The reading frame of NUP214 was not matched with RAC1; however, high expression of the RAC1 protein was detected by Western blotting. This study identifies the variant complex fusion genesNUP214-RAC1 and RAC1- COL12A1 in a case of AML.

Isolation of human mAbs that directly modulate FMS-related tyrosine kinase 3 signaling.

FMS‐related tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase that plays important roles in hematopoiesis, including early progenitors and dendritic cell development. FLT3 is expressed at high levels in 70–100% of cases of AML and in virtually all cases of B‐lineage acute lymphoblastic leukemia. FLT3 is regarded as a molecular target in the development of novel therapies for acute leukemia patients. Currently, many small‐molecule FLT3 inhibitors have been developed, but clinical trials have resulted in limited antileukemia effects because of off‐target toxicities and drug resistance. The development of anti‐FLT3 Abs might overcome these difficulties and enhance the antileukemia efficacy of FLT3 inhibitors. In the present study, we demonstrate the isolation of novel human mAbs against FLT3 with antagonistic or agonistic activities. An antagonistic Ab, designated A2, continuously inhibits FLT3 ligand (FL)‐induced phosphorylation of FLT3 and MAPK. A2 cooperatively induces apoptosis with daunorubicin, even in the presence of FL. An agonistic Ab, designated 3E6, surprisingly induces the phosphorylation of FLT3 and MAPK, and supports the growth of a factor‐dependent cell line independently of FL addition. In addition, A2 showed complement‐dependent cytotoxicity activity, but was devoid of Ab‐dependent cell mediated cytotoxicity. Finally, we evaluated Ab internalization in a cell line. Immunofluorescence and flow cytometry analyses revealed that A2 is efficiently internalized. Collectively, these data demonstrate that A2 is a potent human Ab that might be capable of delivering cytotoxic reagents and that has antagonistic effects on FLT3 signaling. In addition, 3E6 might be a potential scaffold for novel dendritic cell‐based immunotherapies. (Cancer Sci 2012; 103: 350–359)