Mitsushige Nakao

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.

International prognostic scoring system and TP53 mutations are independent prognostic indicators for patients with myelodysplastic syndrome

We applied the International Prognostic Scoring System (IPSS) to our series of 118 patients with myelodysplastic syndrome (MDS) to determine its validity, and also used univariate and multivariate analyses to evaluate the prognostic significance of TP53 configurations. Sixteen patients with the mutation had a strikingly worse prognosis and the multivariate analysis demonstrated that this alteration was the most significant factor. The prognostic comparison between patients with and without the mutation within each IPSS subgroup showed a significant difference in the intermediate subgroups. A combination of clinical manifestations and genetic configurations provided us with more accurate prognostic information in MDS patients.

RUNX1/AML1: A Central Player in Hematopoiesis

It has been well established that a number of transcription factors play critical roles in regulating the fate of hematopoietic stem cell populations. One of them is the leukemia-associated transcription factor acute myeloid leukemia 1 (AML1; also known as runt-related transcription factor 1, or RUNX1). This gene was originally cloned from the breakpoint of the t(8;21) reciprocal chromosome translocation and was later recognized as one of the most frequent targets of leukemia-associated gene aberrations. Gene-targeting experiments revealed that transcriptionally active AML1 is essential for the establishment of definitive hematopoiesis. More specifically, this gene functions in the emergence of the hematopoietic progenitor cells from the hemogenic endothelium by budding in the aorta-gonad-mesonephros region, and its expression points to the sites with strong potential for the emergence of hematopoietic stem cells. This review discusses aspects of the biologic properties of AML1 in early hematopoietic development.

Successful treatment with Erwinia L-asparaginase for recurrent natural killer/T cell lymphoma.

We describe a patient with natural killer (NK)/T cell lymphoma who relapsed after autologous peripheral blood stem cell transplantation (auto-PBSCT) and was successfully treated with Escherichia coli (E. coli ) and Erwinia l -asparaginase. A 38-year-old male patient with ulcerated tumor at the left thigh was diagnosed as having nasal type NK/T cell lymphoma on the basis of histopathological and flowcytometric findings of tumor, revealing diffuse infiltration of atypical lymphoid cells into blood vessels and expression of CD7 and CD56 antigens, but not CD3. He had tumor infiltration in the bone marrow and at the right lower lung field. After five cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) therapy, the patient achieved complete remission and received high-dose chemotherapy with auto-PBSCT, although the tumor recurred in the right leg 10 months later. Despite salvage chemotherapy, followed by local irradiation and surgical amputation, a tumor recurred at the left upper gingiva 10 days after. Using E. coli l -asparaginase (6000 U/m 2 /day), the tumor regressed, fever was alleviated and the serum lactate dehydrogenase decreased to normal range after several days. The asparagine synthetase expression in tumor cells was immunohistochemically negative on paraffin-embedded tissues. Because of the anaphylactoid reaction developing after E. coli l -asparaginase, alternative Erwinia l -asparaginase (6000 U/m 2 /day) was administered, resulting in regression of tumor and fever lysis. l -asparaginase is a promising agent for the treatment of NK/T cell lymphoma.

Configuration of the TP53 Gene as an Independent Prognostic Parameter of Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) consists of a heterogeneous group of acquired hematopoietic stem cell disorders, characterized by bone marrow failure and leukemic transformation. Since hematological manifestations and clinical outcomes vary widely among MDS patients, a considerable number of studies have tried to identify the prognostic parameters for the stratification of patients into different risk groups. Based on reported risk-based studies, the International Prognostic Scoring System (IPSS) was proposed as a reliable risk assessment method for primary MDS patients, and several validating studies have clarified its usefulness. Critical prognostic parameters of the IPSS consist of chromosome findings, the percentage of marrow blasts, and the number of peripheral blood cytopenias. Although other laboratory findings, including several molecular alterations, have been identified as convincing prognostic factors in MDS, these molecular configurations were not selected as prognostic parameters in the IPSS, because analysis for these alterations were not routinely available for the management of patients with MDS. Because recent advances in molecular genetics may make it available as a routine work-up of MDS in the future, we discuss potential improvement of the IPSS by the addition of molecular analysis to the system, with particular reference to the configuration of the TP53 gene.

Novel loss-of-function mutations of the haematopoiesis-related transcription factor, acute myeloid leukaemia 1/runt-related transcription factor 1, detected in acute myeloblastic leukaemia and myelodysplastic syndrome.

AML1/RUNX1, which encodes a transcription factor essential for definitive haematopoiesis, is a frequent target of leukaemia‐associated chromosome translocations. Point mutations of this gene have also recently been associated with leukaemia and myelodysplastic syndrome (MDS). To further define the frequency and biological characteristics of AML1 mutations, we have examined 170 cases of such diseases. Mutations within the runt‐domain were identified in five cases: one of de novo acute myeloid leukaemia (AML) and four of MDS. Where multiple time point samples were available, mutations were detected in the earliest samples, which persisted throughout the disease course. Of the five mutations, one was a silent mutation, two were apparent loss‐of‐function mutations caused by N‐terminal truncation, and two were insertions, I150ins and K168ins, which preserved most of the AML1 DNA‐binding domain. Both AML1 molecules with insertion mutations were non‐functional in that they were unable to rescue haematological defects in AML1‐deficient mouse embryonic stem cells. In addition, activating mutations of N‐ras, deletion of chromosome 12p, or inactivation of TP53 accompanied some of the AML1 mutations. Together, these observations strongly suggest that one‐allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations.

Differentiation of follicular from mucosa-associated lymphoid tissue lymphoma by detection of t(14;18) on single-cell preparations and paraffin-embedded sections.

A 57‐year‐old woman was referred to the Kyoto Prefectural University of Medicine because of multiple polypoid lesions in the duodenum. On the basis of the histopathologic findings, mucosa‐associated lymphoid tissue lymphoma had been diagnosed. The polypoid lesions did not show any improvement in spite of antimicrobial therapy for elimination of Helicobacter pylori. Because the disease remained stable during the clinical course, no other specific treatment was administered. We performed fluorescence in situ hybridization (FISH) analysis on a single‐cell preparation obtained from the duodenal lesions, to assess the specific chromosome translocation. We identified BCL2/IGH fusion at a frequency of 83%. Two‐color FISH was also performed on paraffin‐embedded tissue sections, which demonstrated BCL2/IGH fusion–positive cells in neoplastic follicles. These findings, together with the CD10+ immunophenotyping of tumor cells, led to a diagnosis of primary follicular lymphoma of the duodenum. Interphase FISH with the IGH gene and oncogene probes is a rapid and powerful tool for assessing genomic changes in gastrointestinal lymphoma on single‐cell preparations and tissue sections. This technique is particularly useful in view of the increasingly small core biopsy samples and needle aspirations obtained for diagnostic purposes.

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