Toshiki Iwai

Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines.

In this study, we examined a large number of patients to clarify the distribution and frequency of a recently described FLT3 tandem duplication among hematopoietic malignancies, including 112 acute myelocytic leukemia (AML), 55 acute lymphoblastic leukemia (ALL), 37 myelodysplastic syndrome (MDS), 20 chronic myelogenous leukemia (CML), 30 non-Hodgkin’s lymphoma (NHL), 14 adult T cell leukemia, 15 chronic lymphocytic leukemia (CLL) and 38 multiple myeloma (MM). We also evaluated 71 cell lines derived from 11 AML, 31 ALL, two hairy cell leukemia, three acute unclassified leukemia, 10 CML, 12 NHL including six Burkitt’s lymphoma, and two MM. Using genomic PCR of exon 11 coding for the juxtamembrane (JM) domain and first amino acids of the 5′-tyrosine kinase (TK) domain, this length mutation was found only in AML (22/112, 20%) and MDS (1/37). According to the FAB subclassification, they were 5/18 (28%) of M1, 4/29 (14%) of M2, 3/17 (18%) of M3, 6/24 (25%) of M4, 4/20 (20%) of M5 and 1/9 of refractory anemia with excess of blast in transformation. In the various cell lines examined, this abnormality was determined in only one derived from AML and never found in other hematological malignancies. The sequence analysis of the abnormal PCR products revealed that 23 of 24 showed internal tandem duplication with or without insertion of nucleotides. In one AML, insertion and deletion without duplication was determined. All 24 lengthened sequences were in-frame. Duplication takes place in the sequence coding for the JM domain and leaves the TK domain intact. In conclusion, we emphasize that the length mutation of FLT3 at JM/TK-I domains were restricted to AML and MDS. Since all these mutations resulted in in-frame, this abnormality might function for the proliferation of leukemic cells.

Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia.

We recently reported an internal tandem duplication of the human flt3 receptor gene (FLT3) as a somatic mutation in 17% of acute myelogenous leukemia (AML). The present study revealed the duplication at the juxtamembrane and the first tyrosine kinase domains of FLT3 in seven of 92 (8%) patients with myelodysplastic syndrome (MDS) and AML with trilineage myelodysplasia (AML/TMDS), the diseases which may represent neoplastic changes of pluripotent stem cells. A tandem duplication of exon 11 of FLT3 was harbored by two of 58 (3%) patients with MDS and five of 34 (15%) with overt leukemia, including MDS-derived leukemia, AML/TMDS and therapy-related leukemia. Although the duplicated regions varied within exon 11 in each case, they occurred in-frame, and altered mRNA expressions were demonstrated by reverse-transcription polymerase chain reaction. Two cases of MDS with a FLT3 duplication transformed to overt leukemia within a few months. Longitudinal analyses in two other patients with leukemia revealed that the duplication was a late genetic event during the disease course; one of whom showed two independent duplications of FLT3 at the terminal therapy-resistant phase. Of seven patients with the FLT3 duplication, six had abnormal karyotypes, and four harbored a point mutation of the N-RAS and/or TP53 genes. Patients with FLT3 mutations have poor prognoses. This study uncovered the fact that the accumulation of genetic events, including FLT3 duplication, correlates with leukemic transformation from antecedent myelodysplasia and with subsequent disease progression.

Internal tandem duplication of the FLT3 gene and clinical evaluation in childhood acute myeloid leukemia

We analyzed tandem duplication in the juxtamembrane (JM) domain of the FLT3 (FMS-like tyrosine kinase 3/FLK2, CD135) gene in 94 children with acute myeloid leukemia (AML) and evaluated its correlation with clinical features. Longer polymerase chain reaction (PCR) products were observed in five patients; 1/3 of M0, 1/9 of M1, 1/39 of M2, 1/9 of M3 and 1/12 of M5. The sequence analyses of abnormal PCR products showed that all the abnormal products were derived from tandem duplications involving the JM domain and that all the lengthened sequences were in-frame as we previously reported. Statistical analyses revealed a significantly lower incidence of the tandem duplication in childhood AML patients than in adult patients (P < 0.05), and significantly shorter disease-free survival in patients with mutant FLT3 than in patients with wild-type FLT3 (P < 0.05). our results suggest that the tandem duplication in the jm domain of the FLT3 gene is not a frequent phenomenon but might be a factor of poor prognosis in childhood patients with AML.

Interphase Detection of t(4;14)(p16.3;q32.3) by In Situ Hybridization and FGFR3 Overexpression in Plasma Cell Malignancies

The immunoglobulin (Ig) genes are frequently involved in chromosomal rearrangements with a wide variety of partner loci in multiple myeloma (MM). However, several partner chromosomes have not been detected by conventional cytogenetic methods; for example, 4p16.3 (FGFR3), 6p25.3 (IRF4), and 16q23 (c-maf). To clarify the incidence of t(4;14)(p16.3;q32.3) in primary tumors of MM and to evaluate possible correlations with specific manifestations of the disease, G-banding, double-color fluorescence in situ hybridization (DC-FISH), and/or reverse-transcriptase polymerase chain reaction (RT-PCR) were performed on 40 patients with MM-two with plasmacytoma (PCM) and three with plasma cell leukemia (PCL). All patients were studied by DC-FISH; 40 were studied by G-banding and 36 were studied by RT-PCR. The FISH probes consisted of a cosmid pC385.12 containing the FGFR3 gene, a YAC Y6 containing VH, and a phage Iggamma1-10 containing the gamma1 constant region (Cgamma). We identified eight patients with either FGFR3/Cgamma fusion or FGFR3 overexpression: six patients with both FGFR3/Cgamma fusion and FGFR3 overexpression, one patient with FGFR3/Cgamma, and one with FGFR3 overexpression. FGFR3/Cgamma fusion was demonstrated at a frequency of 19% to 38% on interphase nuclei in seven of the 45 patients. Lytic bone lesions were found to be associated with FGFR3 overexpression. Interphase FISH with FGFR3 and Cgamma probes combined with RT-PCR proved to be an effective tool for detection of this fully cryptic translocation, thus facilitating the characterization of clinical features of MM patients with t(4;14).

Rare Alteration of Genomic Structure or Expression of the DPC4 Gene in Myelogenous Leukemias

We examined homozygous deletion, point mutation and expression of DPC4 gene, a recently isolated candidate pancreatic tumor suppressor gene, in 53 patients with myelogenous leukemias and 5 cell lines. The patients consisted of 34 cases of chronic myelogenous leukemia including 22 in the chronic phase, 3 in the accelerated phase, and 9 in blastic crisis, and 19 with acute myelogenous leukemia including 9 at the initial presentation and 10 at relapse. Polymerase chain reaction (PCR)-based deletion analysis for DPC4 exon 8 and PCR-single strand conformation polymorphism study for the entire coding region were carried out. Homozygous deletion or subtle mutation was not detected in any of the samples examined. However, 3 patients with various clinical phases showed a decrease of DPC4 expression. These results suggest that DPC4 alteration is not a crucial event in the development or the progression of myelogenous leukemias.

Clinical manifestation and prognostic factors of 32 Japanese patients with autoimmune disease-associated diffuse large B-cell lymphoma

1 Division of Hematology and Oncology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Department of Hematology, Kyoto First Red Cross Hospital, Kyoto, Japan, 3 Department of Hematology, Social Insurance Kyoto Hospital, Kyoto, Japan, 4 Department of Hematology, Kyoto Second Red Cross Hospital, Kyoto, Japan, 5 Department of Hematology, Matsushita Memorial Hospital, Moriguchi, Japan, 6 Department of Hematology, Otsu Municipal Hospital, Otsu, Japan and 7 Department of Infl ammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

Favorable Event Free-Survival of High-Dose Chemotherapy Followed by Autologous Hematopoietic Stem Cell Transplantation for Higher Risk Diffuse Large B-Cell Lymphoma in First Complete Remission

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) has been applied to patients with diffuse large Bcell lymphoma (DLBCL); it is well established that ASCT shows significant survival benefits for chemosensitive relapse. However, half of relapsed patients are resistant to salvage chemotherapy, indicating that they are not suitable for ASCT. We retrospectively analyzed the clinical records of 47 patients with DLBCL classified as high or high-intermediate (higher) risk, according to the International Prognostic Index, who underwent upfront ASCT in first complete remission (CR1). Compared with 10 patients with similar characteristics who did not receive ASCT, event free survival at 5-year was significantly superior in ASCT group. Toxicity of ASCT was acceptable and therapy-related death was not observed. We therefore propose that upfront ASCT for higher risk DLBCL in CR1 might provide survival benefit, probably because the high-dose therapy removes minimally resided tumor.

A treatment refractory CD30-positive diffuse large B cell lymphoma in the ileal neobladder

Dear Editor, We here report a rare case of a CD30-positive diffuse large B cell lymphoma (DLBCL) in the ileal neobladder. A 78-yearold male was admitted to our hospital complaining of abdominal distension, hematuria, decreased urine volume, and edema for 5 months. The patient had a history of invasive bladder cancer that was cured by cystectomy with bladder reconstruction using an ileal conduit 15 years ago. On admission, a large tumor of 20 cm in diameter was palpable on the right side of his abdomen, and F-fluorodeoxy glucose (FDG)-positron emission (PET)-computed tomography (CT) revealed the presence of an enlarged FDG acid neobladder with a thickened wall and bilateral hydronephrosis (Fig. 1a, b). The ECOG performance status was 1. Serological tests revealed increased levels of lactate dehydrogenase (253 IU/l, normal range 114–243), soluble interleukin-2 receptor (4260 U/ml, normal range 145–519), creatinine (1.18 mg/dl, normal range 0.30–1.10), and C-reactive protein (2.73 mg/dl, normal range 0.00–0.20). Testing showed that the patient was negative for hepatitis B, hepatitis C, human immunodeficiency virus II, Epstein-Barr virus (EBV) DNA, and HHV-8 DNA. A transurethrally biopsied specimen showed a diffuse infiltration of abnormal, large lymphoid cells (Fig. 1c) that were positive for CD20 (Fig. 1d), CD30 (Fig. 1e), CD79a, and MUM1, but were negative for CD3, CD5, and CD10.MIB-1-positive cells comprised more than 90% of the large lymphoid neoplastic cells, while no EBV latent membrane protein 1 was detected (data not shown). Accordingly, the patient was diagnosed as having DLBCL, non-germinal center type, in the ileal neobladder. The disease stage according to the Ann Arbor staging system and the disease risk by the international prognostic index were estimated as IAE and low-intermediate risk, respectively. Although systemic immunochemotherapies, containing rituximab with cyclophosphamide, pirarubicin, vincristine, and prednisolone (R-THP-COP), induced a transient partial remission, the disease relapsed after the sixth cycle of R-THP-COP and was refractory to salvage chemotherapy, containing rituximab, gemcitabine, carboplatin, and dexamethasone, and irradiation. The patient eventually died of disease progression 8 months after the diagnosis. The development of malignant lymphoma in a reconstructed organ, such as the stoma, ileal conduit, or ileal neobladder, has been extremely rare with only seven previous cases reported [1]. All reported patients were male, and most patients with lymphoma diagnosed at ileostomy had inflammatory bowel diseases that were treated by immunosuppressive therapy [1–4]. Based on our search in the English language literature, this case was the first report of the development of malignant lymphoma in an ileal neobladder. While our patient had no history of inflammatory bowel disease or immunosuppressive therapy, it is conceivable that the focal chronic inflammation due to chronic infection may have associated with tumorigenesis in our case [2]. It is possible that CD30 positivity associated with the disease development in our case. CD30 expression associates with prominent cytokine and stromal signatures in gene expression profiling [5]. Although the prognostic impact of CD30 expression has been controversial in DLBCL [5–7], it was associated * Junya Kuroda junkuro@koto.kpu-m.ac.jp

Long-Term Maintenance of Hematological and Cytogenetic Remission in 5q- Syndrome After Short-Term Administration of Lenalidomide

Myelodysplastic syndrome (MDS) is a disease of the hematopoietic stem cell, characterized by peripheral blood pancytopenia and morphological dysplasia of hematopoietic cells. Deletions of the long arm of chromosome 5 (5q-) are commonly found in MDS as a part of complex chromosomal abnormalities. Patients with 5qas a sole abnormality are classified as 5qsyndrome, and are well known to present severe anemia and transfusion dependence [1]. Remarkably, lenalidomide is sensitive to 5qsyndrome by inhibiting proliferation of the 5qclone and promoting effective erythropoiesis in non-5qMDS progenitors [2]. The participation of various molecules has been suggested in the pathogenesis of this disease, such as SPARC, and RPS14, which are encoded on 5q31-33, the commonly deleted segment [3]. In recent years, the reports on discontinuing lenalidomide treatment for 5qsyndrome have been increasing, and we present a patient who maintains hematological complete remission over 3 years after the discontinuation of this drug. A 67-year-old man presented to our hospital with dizziness lasting for 4 months in September 2011. His previous medical past history included a total gastrectomy for duodenal ulcer. Physical examination revealed anemia of the skin and conjunctiva. He did not have enlarged superficial lymph nodes or hepatosplenomegaly. Vital signs were unremarkable. His blood cell count showed normocytic anemia with hemoglobin (Hb) at 8.6 g/dl. White blood cell and platelet levels remained within normal limits. Serum lactate dehydrogenase was not elevated. Serum levels of vitamin B12, folic acid, and ferritin were normal. Examination of an aspirated specimen of the bone marrow revealed hypoplasticity. The nucleated cell count was 50,000/ll, with 2.4% myeloblasts. His M/E ratio was elevated at 11.77. The blood cells exhibited abnormal morphological features; the pseudo-Pelger-Huet anomaly, and small megakaryocytes. Chromosomal analysis (Giemsa-banding stain) showed a 5qas the sole abnormality in 12 of 20 (60%) metaphases (Fig. 1). The precise chromosomal breakpoint was unclear. He was diagnosed with refractory anemia according to the French-AmericanBritish criteria and was also classified as MDS with isolated del(5q) by the World Health Organization criteria [4]. Administration of lenalidomide at a dose of 10 mg/day was started. This treatment cycle consisted of dosing for 3 weeks, followed by 1 week of rest. Although the patient initially needed transfusion of red blood cell products once every 2 weeks, within a month after beginning the therapy, he no longer required transfusions. Adverse events included grade 2 neutropenia (according to Common Terminology Criteria for Adverse Events Ver. 4.0) and grade 3 thrombocytopenia that spontaneously improved. A grade 2 itchy skin rash also appeared on his back, which was treated with an anti-histaminergic agent and ointment. We administered the second and third courses of lenalidomide and his Hb level increased gradually. In January 2012, after 12 weeks of treatment, lenalidomide was & Yuka Kawaji yuka0914@koto.kpu-m.ac.jp

Inversion of chromosome 7q22 and q36 as a sole abnormality presenting in myelodysplastic syndrome: a case report.

IntroductionDeletions of chromosome 7 are often detected in myelodysplastic syndrome. The most commonly deleted segments are clustered at band 7q22. A critical gene is therefore suggested to be located in this region. We report a patient with myelodysplastic syndrome whose marrow cells carried an inversion of 7q22 and q36 as a sole karyotypic abnormality. How this extremely rare chromosomal aberration contributes to the pathogenesis of myelodysplastic syndrome should be clarified by accumulating clinical data of such cases.Case presentationA 74-year-old Japanese man presented with pancytopenia incidentally detected by routine medical check-up. His complete blood cell counts revealed that his white blood cells had decreased to 2100/mm3, neutrophils 940/mm3, red blood cells 320×104/mm3, hemoglobin 11.1g/dL, hematocrit 33.1%, and platelets 12.6×104/mm3. Bone marrow examination showed normal cellularity with nucleated cells of 9.4×104/mm3. The proportion of blasts was 4%. A morphological examination showed only basophilic stippling of erythroblasts which was seen as dysplasia. According to World Health Organization classification, the diagnosis was myelodysplastic syndrome-u. Karyotypic analysis showed 46,XY,inv(7)(q22q36) in all of 20 metaphases examined. Additional analysis revealed the karyotype of his lymphocytes was 46,XY. He is asymptomatic and cytopenia has slowly progressed.ConclusionsTo the best of our knowledge, this karyotype from a clinical sample of de novo malignancies has never been documented although the identical karyotype from secondary myelodysplastic syndrome was reported. Despite the extremely low frequency, inversion of 7q22 appears to play a crucial role for myelodysplastic syndrome in this patient.