Shouhei Yokota

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.

Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product.

An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To eludicate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gain-of-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.

Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia

FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high ldh level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.

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.

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.

Role of human interleukin-9 as a megakaryocyte potentiator in culture

OBJECTIVE This study investigated the effect of interleukin-9 (IL-9) on the proliferation and differentiation of human colony-forming unit megakaryocytic progenitor cells (CFU-Meg). MATERIALS AND METHODS Peripheral blood-derived CD34(+)IL-6R(-) cells were sorted and cultured in the presence of IL-9, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) alone or in combination. The number of pure and mixed megakaryocyte colonies, the size of pure megakaryocyte colonies, the ploidy distribution of megakaryocytes, and proplatelet formation were investigated. RESULTS Apart from TPO, no single factor could support CFU-Meg-derived colony formation, but each two-factor combination among IL-9, Epo, and SCF supported a few CFU-Meg colonies. Interestingly, the combination of Epo+SCF+IL-9 induced four to six times as many CFU-Meg colonies as any of the two-factor combinations. Neutralizing monoclonal antibodies (mAbs) for IL-9 receptor and c-kit completely abolished this synergistic effect. In contrast, addition of neutralizing anti-c-Mpl or anti-CXCR4 Abs did not influence colony formation, indicating that this synergistic effect was independent of TPO or SDF-1. Moreover, the endogenous production of TPO by cultured CD34(+)IL-6R(-) cells in the presence of Epo+SCF+IL-9 was ruled out by reverse transcriptase polymerase chain reaction for TPO mRNA. Interestingly, the combination of TPO, Epo, SCF, and IL-9 supported the largest number of pure and mixed megakaryocyte colonies, suggesting that this combination of cytokines might recruit primitive megakaryocytic as well as multipotential progenitors. This combination also potently enhanced proplatelet formation compared with TPO alone or a combination of Epo, SCF, and IL-9. CONCLUSION This study demonstrated for the first time that human IL-9 can potentiate human megakaryocytopoiesis in the presence of Epo and/or SCF.

Detection of karyotypic abnormalities in most patients with acute nonlymphocytic leukemia by adding ethidium bromide to short-term cultures

A modified short-term culture method, in which cultured bone marrow cells were treated with ethidium bromide to prevent chromosome condensation was used to study the chromosomes of 70 patients with acute nonlymphocytic leukemia. Clonal karyotypic abnormalities were detected in 60 patients. Among these, 35 patients showed one of recurrent type specific alterations. A close relationship between karyotypes and clinical outcome was shown: thus, t(8;21) or a single miscellaneous chromosomal defect associated with a favourable prognosis whereas t(9;11) or a complex karyotype related to a poor prognosis. The ten cytogenetically normal patients did not appear to have a favourable prognosis.

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein

Abstract:  We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34+ blood progenitors which express different levels of c‐kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte‐colony stimulating factor (G–CSF) by peripheral blood (PB)‐derived CD34+c‐kit− cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)‐3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst‐promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB‐derived CD34+c‐kithigh cells in which erythroid‐burst (BFU‐E) was highly enriched. However, FL could synergize with IL‐3 or GM–CSF in support of erythrocyte‐containing mixed (E‐Mix) colony by PB‐derived CD34+c‐kithigh or low cells in the presence of Epo. Replating of E‐Mix colonies derived from CD34+c‐kithigh cells supported by IL‐3+Epo+SCF yielded more secondary colonies than those supported by IL‐3+Epo or IL‐3+Epo+FL. When PB‐derived CD34+c‐kitlow cells which represent a more immature population than CD34+c‐kithigh cells were used as the target, number of secondary colonies supported by IL‐3+Epo, IL‐3+Epo+SCF or IL‐3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL‐3+Epo+FL than in that containing IL‐3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.

Monitoring of minimal residual disease (MRD) is useful to predict prognosis of adult patients with Ph-negative ALL: results of a prospective study (ALL MRD2002 Study)

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) for patients with Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) in first complete remission (CR1) is much more intensive than multi-agent combined chemotherapy, although allogeneic HSCT is associated with increased morbidity and mortality when compared with such chemotherapy. Minimal residual disease (MRD) status has been proven to be a strong prognostic factor for adult patients with Ph-negative ALL.MethodsWe investigated whether MRD status in adult patients with ALL is useful to decide clinical indications for allogeneic HSCT. We prospectively monitored MRD after induction and consolidation therapy in adult patients with Ph-negative ALL.ResultsOf 110 adult ALL patients enrolled between July 2002 and August 2008, 101 were eligible, including 59 Ph-negative patients. MRD status was assessed in 43 patients by the detection of major rearrangements in TCR and Ig and the presence of chimeric mRNA. Thirty-nine patients achieved CR1, and their probabilities of 3-year overall survival and disease-free survival (DFS) were 74% and 56%, respectively. Patients who were MRD-negative after induction therapy (n = 26) had a significantly better 3-year DFS compared with those who were MRD-positive (n = 13; 69% vs. 31%, p = 0.004). All of 3 patients who were MRD-positive following consolidation chemotherapy and did not undergo allogeneic HSCT, relapsed and died within 3 years after CR.ConclusionsThese results indicate that MRD monitoring is useful for determining the clinical indications for allogeneic HSCT in the treatment of ALL in CR1.

Haematopoietic action of flt3 ligand on cord blood‐derived CD34‐positive cells expressing different levels of flt3 or c‐kit tyrosine kinase receptor: comparison with stem cell factor

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)‐derived CD34+ cells expressing different levels of flt3 or c‐kit tyrosine kinase (TK) receptor in clonal cell culture. The c‐kit receptor was expressed by 58.5±16.7% of CB CD34+ cells (n = 19), in which c‐kithigh, c‐kitlow and c‐kit‐ cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6±8.3% (n = 9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU–E) formation by any subpopulation of CD34+ cells. However, SCF+Epo supported BFU–E and erythrocyte‐containing mixed (CFU–mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU–mix colony formation supported by interleukin (IL)‐ 3+Epo when CD34+c‐kitlow or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU‐mix colony formation supported by IL‐3+Epo when CD34+c‐kithigh or low and CD34+FR+ cells were used. The replating potential of CFU–mix supported by IL‐3 + Epo + FL was greater when CD34+c‐kitlow or CD34+FR+ cells were used. When the CD34+c‐kitlow cells were used, the number of lineages expressed in secondary cultures of CFU–mix colonies derived from primary cultures containing IL‐3 + Epo+FL or SCF was significantly larger than when the primary cultures contained IL‐3+Epo. Furthermore, the number of long‐term culture‐initiating cells found in CD34+FR+ cells was larger than that in FR‐ cells. CB‐derived CD34+c‐kitlow cells represent a less mature population than c‐kithigh cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB‐derived CD34+FR+ cells are less mature than CD34+FR‐ cells.