Hiroto Kaneko

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.

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.

Distinct genetic involvement of the TP53 gene in therapy-related leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype

We examined chromosomes and molecular aberrations in 21 patients with therapy-related leukemias (t-AML) or myelodysplastic syndromes (t-MDS). All patients showed abnormal karyotypes, and chromosomal losses of No. 5 and/or No. 7 (−5/5q− and/or −7/7q−) were identified in 12 patients. Among these 12, six patients (50%) harbored a TP53 mutation, and two of five examined showed microsatellite instability, suggesting replication error (RER+) phenotype. Meanwhile, among the other nine patients without −5/5q− and/or −7/7q−, none harbored a TP53 mutation, and none of five examined showed RER+ phenotype. Thus, TP53 mutations and RER+phenotype were preferentially associated with specific chromosomal losses in t-AML/MDS. We then screened for mutational events in representative DNA mismatch repair genes; exons 5–7 and 12–15 of the hMSH2 gene and exon 9 of hMLH1. Notably, two unrelated patients showing RER+ phenotype had an identical missense alteration at codon 419 of hMSH2 in their marrow cells and fibroblasts, which were not found in 120 DNA samples from healthy volunteers or patients with other hematological disorders. Consequently, this study revealed a possible relationship of RER+ phenotype accompanying an hMSH2 alteration to the development of therapy-related AML/MDS in association with TP53 mutations and specific chromosomal losses, and suggests that some patients may be predisposed to myelodysplasia after chemotherapy for their primary tumor.

Human cord blood-derived primitive progenitors are enriched in CD34+c-kit- cells: correlation between long-term culture-initiating cells and telomerase expression.

We studied the functional characteristics of subpopulations of cord blood-derived CD34+ cells expressing different levels of CD38 and c-kit antigens, using clonal cell culture and long-term culture with allogeneic bone marrow stromal cells or the MS-5 murine stromal cell line to assay long-term culture-initiating cells (LTC-IC) in each subpopulation. To investigate the capacity for replication, proliferation, and differentiation of each subpopulation of CD34+ cells, we also studied the correlation between LTC-IC and telomerase activity. After 5 weeks of coculture, LTC-IC accounted for one out of 32 CD34+CD38− cells and one out of 33 CD34+c-kit− cells. In contrast, the frequency of LTC-IC was low in their antigen-positive counterparts (one per 84 CD34+CD38+ cells, one per 90 CD34+c-kitlow cells, and very low among CD34+c-kithigh cells). It was noteworthy that some LTC-IC derived from CD34+CD38− as well as CD34+c-kit− cells generated colony-forming cells (CFCs) after up to 9 weeks of coculture. Telomerase activity was consistently low in CD34+CD38− and CD34+c-kit− cells compared to CD38+ or c-kithigh or low cells, suggesting that CD34+CD38− or c-kit− cells are likely to be more quiescent. These results suggest that the CD34+CD38− and CD34+c-kit− cell populations are primitive stem/progenitor cells, and that the telomerase activity of these cells correlates with their proliferative capacity as well as their stage of differentiation.

Genotype of glutathione S-transferase and other genetic configurations in myelodysplasia

We examined polymorphisms of glutathione S-transferase (GST) genes in 159 Japanese patients with myelodysplasia and compared the incidence with that in 43 normal individuals to clarify their pathogenetic significance in myelodysplasia. In individuals with the GSTT1 null genotype, the odds ratios for disease risk were elevated to 2.65 (95%CI; 1.27-5.52) in de novo MDS, 4.62 (1.48-14.4) in therapy-related AML, and 2.94 (1.07-8.07) in AML with triliniage dysplasia. Other representative polymorphisms of GSTs had a similar incidence among patients with myelodysplasia, and those of the controls and other hematological disorders. To further investigate the genetic pathway of myelodysplasia, the association between GST genotype and karyotype or configurations of TP53 and NRAS was evaluated, but no relationship was noted. These results suggest that the GSTT1 null genotype may play a role in an increased risk of myelodysplasia unrelated to other mechanisms of myelodysplasia, such as chromosomal alterations or mutation of TP53 or NRAS.

Helicobacter pylori-negative gastric and duodenal ulcers.

Abstract: It is unclear whether Helicobacter pylori infection is essential to the development of peptic ulcers. In this study, we examined the rates of H. pylori-negativity among patients with peptic ulcers. We also attempted to clarify the characteristics of H. pylori-negative peptic ulcers to throw light on the pathogenesis of peptic ulcers. The study included 215 consecutive patients with gastric ulcers (GUs) and 120 consecutive patients with duodenal ulcers (DUs). After routine endoscopic examination and phenol red dye endoscopy, forceps biopsies were performed for culture, histology, and the rapid urease test. A patient was considered H. pylori-negative when the serum anti-H. pylori IgG and the three tests on biopsied specimens were all negative. H. pylori-negative rates were 3.2% in the patients with GUs and 1.7% in the patients with DUs. Lack of atrophy of the gastric mucosa was significantly more common in the H. pylori-negative patients with GUs. A history of ulcer disease was less common and antral ulcers were more common in H. pylori-negative GU patients, but not significantly so. As the urea breath test had not been performed, the possibility of a false-negative result cannot be completely ruled out, but we believe that the H. pylori-negative rate in our study is more reliable than these rates in previous reports, because we visualized H. pylori distribution by phenol red dye endoscopy to avoid false-negative results in biopsies, and we used both biopsy and serum anti-H. pylori IgG findings to establish an H. pylori-negative diagnosis. Since H. pylori-negative peptic ulcers certainly exist, H. pylori infection is thought not to be essential to the development of peptic ulcers. There were few differences between the characteristics of H. pylori-negative and H. pylori-positive peptic ulcers in our study. A large-scale study is required to clarify the characteristics of H. pylori-negative peptic ulcers.

Signal through gp130 Activated by Soluble Interleukin (IL)‐6 Receptor (R) and IL‐6 or IL‐6R/IL‐6 Fusion Protein Enhances Ex Vivo Expansion of Human Peripheral Blood‐Derived Hematopoietic Progenitors

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)‐6 receptor (R) and IL‐6 on the ex vivo expansion of human peripheral blood (PB)‐derived hematopoietic progenitor cells in a short‐term serum‐free liquid suspension culture system, using PB‐derived CD34+IL‐6R+/– cells as a target. In combination with stem cell factor (SCF), IL‐3, and sIL‐6R/IL‐6, the expansion efficiency (EE) for granulocyte/macrophage colony‐forming unit (CFU‐GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU‐E) and mixed colony‐forming unit (CFU‐Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL‐3 + sIL‐6R/IL‐6 + flt3 ligand (FL) most effectively expanded CFU‐GM and CFU‐Mix. The maximum EEs for CFU‐GM and CFU‐Mix were 208‐fold and 42‐fold, respectively. While the EE for BFU‐E was 70‐90‐fold in the presence of SCF + IL‐3 + sIL‐6R/IL‐6, FL significantly augmented the EE for CFU‐GM and CFU‐Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU‐Mix. Interestingly, in combination with IL‐3 and SCF, newly generated IL‐6R/IL‐6 fusion protein (FP) expanded PB‐derived BFU‐E and CFU‐Mix twice more effectively than a combination of sIL‐6R and IL‐6. These results demonstrated that human PB‐derived committed progenitors were effectively expanded in vitro using sIL‐6R/IL‐6 or FP, in combination with IL‐3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL‐6R and IL‐6.

Interleukin 6 receptor expression by human cord blood- or peripheral blood-derived primitive haematopoietic progenitors implies acquisition of different functional properties

The significance of interleukin 6 receptor (IL‐6R) expression by cord blood (CB)‐ and peripheral blood (PB)‐derived primitive haematopoietic progenitors was investigated. IL‐6R was preferentially expressed by PB‐derived myeloid progenitors. Most PB‐derived erythroid bursts (BFU‐E) and mixed colony‐forming cells (CFU‐Mix) did not express this receptor. However, CB‐derived primitive progenitor cells possessed multipotentiality, irrespective of IL‐6R expression. Interestingly, the long‐term culture‐initiating cell (LTC‐IC) population was enriched in PB‐derived CD34+ IL‐6R+ cells, but the extended LTC‐IC (ELTC‐IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL‐6R+ and IL‐6R− cell populations. In contrast, the number of LTC‐ICs and ELTC‐ICs was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. It is noteworthy that the number of LTC‐ICs and ELTC‐ICs in CB‐derived CD34+ cells was markedly higher than that in PB‐derived CD34+ cells regardless of IL‐6R expression. Telomerase activity was consistently lower in PB‐derived CD34+ IL‐6R− cells than in CD34+ IL‐6R+ cells. In contrast, telomerase activity was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. The pattern of telomerase induction upon cytokine stimulation differed between CB‐ and PB‐derived CD34+ IL‐6R+ or IL‐6R− cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB‐derived primitive progenitors are less mature than PB‐derived progenitors and that the expression of IL‐6R by primitive haematopoietic progenitors may have different implications for PB‐ and CB‐derived CD34+ cells.

Significance of chromosomal alterations and mutations of the N-RAS and TP53 genes in relation to leukemogenesis of acute myeloid leukemia

We examined chromosomes and mutations of the N-RAS and TP53 genes in 73 patients with acute myeloid leukemia (AML). Twenty-six patients showed a reciprocal chromosomal translocation or an inversion, and 34 patients showed only unbalanced aberrations. Balanced aberrations were predominantly detected in the AML patients who did not have myelodysplasia, preceding myelodysplastic syndrome, and a history of chemotherapy or radiation therapy. In contrast, unbalanced aberrations were more frequently seen in the patients with AML with trilineage myelodysplasia, AML transformed from MDS, and therapy-related AML. Twenty-two mutations of the N-RAS and TP53 genes were detected, and these mutations were frequently associated with unbalanced chromosomal aberrations. Furthermore, the spectrum of mutations was suggestive of an exposure to alkylating chemicals.

Effectiveness and Limitation of Gamma Knife Radiosurgery for Relapsed Central Nervous System Lymphoma: A Retrospective Analysis in One Institution

We describe 6 patients with relapsed central nervous system lymphoma (CNSL) treated with Gamma Knife radiosurgery (GKR). The histologic diagnosis in all 6 patients was diffuse large B-cell lymphoma without human immunodeficiency virus infection. Two patients had intracranial relapse of primary CNSL, and the remaining 4 had CNS relapse of systemic lymphoma. All patients were treated with GKR without severe adverse effects, and all but 1 patient received subsequent chemotherapy shortly after GKR. Four patients showed a complete response, and the remaining 2 patients had a partial response or stable disease. Although the neurologic symptoms disappeared or improved markedly in all patients, all of the diseases recurred or progressed 3 to 13 months after the first GKR. A second GKR was eventually performed in 4 patients. The median overall survival and progression-free survival times after the first GKR were 17 and 11 months, respectively. In our experience, GKR seems to be a useful procedure for the treatment of relapsed CNSL, because it facilitates excellent local control in a short-term treatment period without severe complications, although the efficacy period is not long enough.