Yoshio Mitamura

Mutations of the epigenetics-modifying gene (DNMT3a, TET2, IDH1/2) at diagnosis may induce FLT3-ITD at relapse in de novo acute myeloid leukemia.

Gene mutations were found in acute myeloid leukemia (AML) and their importance has been noted. To clarify the importance and stability of mutations, we examined gene mutations in paired samples at diagnosis and relapse of 34 adult AML patients. Five acquired gene mutations were detected at relapse. Of the 45 gene mutations at diagnosis, 11 of them were lost at relapse. The acquired mutations at relapse were all class I mutations as Fms-like tyrosine kinase 3 (FLT3) and rat sarcoma viral oncogene homolog (RAS) mutations. The disappeared mutations at relapse were 3 of 11 internal tandem duplications of FLT3 (FLT3-ITD) (27.3%), 3 of 3 FLT3 tyrosine kinase domain (FLT3-TKD) (100%), 3 of 13 Nucleophosmin 1 (23.1%) and 2 of 5 CCAAT/enhancer-binding protein-α (40%) mutations. However, epigenetics-modifying gene (DNMT3a, TET2 and IDH1/2) mutations had no change between diagnosis and relapse samples, and may become minimal residual disease marker. The frequency of FLT3-ITD at relapse in patients with DNMT3a mutation at diagnosis is significantly higher than those in patients without them (P=0.001). Moreover, the high frequency of FLT3-ITD at relapse is also seen in AML cases that initially present with any epigenetics-modifying gene mutations (P<0.001). Our results indicate that epigenetics-modifying gene mutations may cause genetic instability and induce FLT3-ITD, leading to resistance to therapy and relapse.

Polycythemia associated with the JAK2V617F mutation emerged during treatment of chronic myelogenous leukemia

Polycythemia associated with the JAK2V617F mutation emerged during treatment of chronic myelogenous leukemia

Importance of c-kit mutation detection method sensitivity in prognostic analyses of t(8;21)(q22;q22) acute myeloid leukemia

Recently, c-kit mutations have been reported as a novel adverse prognostic factor of acute myeloid leukemia with t(8;21)(q22;q22) translocation (t(8;21) AML). However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive mutation detection method known as mutation-biased PCR (MB-PCR) and investigated the relationship between c-kit mutations and prognosis. When c-kit mutations were analyzed for 26 cases of t(8;21) AML using the direct sequence (DS) and MB-PCR, the latter had a much higher detection rate of c-kit mutations at initial presentation (DS 5/26(19.2%) vs MB-PCR 12/26(46.2%)). Interestingly for the three cases, in which c-kit mutations were observed only at relapse with the DS, c-kit mutations were detected at initial presentation using the MB-PCR. This result suggests that a minor leukemia clone with c-kit mutations have resistance to treatment and are involved in relapse. In univariate analyses, the presence of a c-kit mutation using DS was not an adverse prognostic factor (P=0.355), but was a factor when using MB-PCR (P=0.014). The presence of c-kit mutations with MB-PCR was also an independent adverse prognostic factor by multivariate analyses (P=0.006). We conclude that sensitivity of c-kit mutation detection method is important to predict prognosis for t(8;21) AML.

Identification and functional characterization of novel telomerase variant alleles in Japanese patients with bone-marrow failure syndromes.

As the incidence of bone-marrow failure syndromes (BMFS) is 2-3x higher in East Asia than in the West, we examined peripheral blood or marrow cells of 100 Japanese patients for possible pathogenic mutations in the two main components of the telomere-synthesizing enzyme telomerase (hTERC RNA and hTERT protein) that have recently been implicated in the disease pathogenesis. We analyzed samples collected from 34 patients with acquired aplastic anemia (AA), 66 patients with myelodysplastic syndromes (MDS) and 120 healthy controls. In addition to two polymorphic germ-line sequence changes (n-771A/G and n-714 C insertion) in the promoter region of hTERC and eleven hTERT polymorphisms that were identified in both patients and healthy individuals, we found a novel germ-line C323T mutation in the hTERC RNA in an MDS patient only. This heterozygous C323T mutation abolished telomerase enzymatic activity and functioned in a haploinsufficiency manner to modulate telomerase activity in cells. In summary, this study reports a novel telomerase natural variant that abolishes telomerase function, which may lead to telomere shortening and marrow hypocellularity in patients with BMFS. This study also highlights the rarity of genetic alterations in BMFS patients in Japan, which suggests that other factors may play a more prominent role in the disease pathogenesis in East Asia.

Multistep pathogenesis of leukemia via the MLL-AF4 chimeric gene/Flt3 gene tyrosine kinase domain (TKD) mutation-related enhancement of S100A6 expression

OBJECTIVE Concerning MLL-AF4 leukemogenesis, previous mouse models suggest that the tumorigenesis capacity of MLL-AF4 alone is insufficient for causing leukemia. Based on the finding that an Fms-like tyrosine kinase 3 (Flt3) gene mutation in the tyrosine kinase domain (TKD) was observed in approximately 15% of mixed lineage leukemia (MLL), we investigated synergistic leukemogenesis effects of the two genes in vitro. MATERIALS AND METHODS In a mouse interleukin-3 (IL-3)-dependent cell line, 32Dc, expression of MLL-AF4 and mutant Flt3 was induced using a lentiviral vector. We analyzed apoptosis induction in the absence of IL-3 and the granulocyte colony-stimulating factor-related induction of differentiation, gene expression profiling, and the mechanism involved in the synergistic effects of MLL-AF4 and Flt3-TKD. RESULTS Neither Flt3-expressing 32Dc (32Dc(Flt3-TKD)) nor MLL-AF4-expressing 32Dc (32Dc(MLL-AF4)) acquired IL-3-independent proliferative capacity in semisolid/liquid media. However, Flt3-TKD+MLL-AF4-expressing 32Dc (32Dc(Flt3-TKD+MLL-AF4)) acquired a non-IL-3-dependent proliferative capacity by inhibiting apoptosis in the two media. The 32Dc(Flt3-TKD) and 32Dc(MLL-AF4) cells differentiated into granulocytes in the presence of granulocyte colony-stimulating factor. However, in the 32Dc(Flt3-TKD+MLL-AF4) cells, there was no differentiation. Subsequently, we performed gene expression profiling. The enhancement of Hox genes expression was not identified. However, expression of S100A6 was synergistically enhanced in the presence of both MLL-AF4 and Flt3-TKD genes. Moreover, anti-S100A6 small interfering RNA downregulated leukemic proliferation. CONCLUSION We conclude that their synergistic enhancement of S100A6 expression plays an important role in MLL-AF4-associated leukemogenesis.

Analysis of the exon 12 and 14 mutations of the JAK2 gene in Philadelphia chromosome-positive leukemia.

Analysis of the exon 12 and 14 mutations of the JAK 2 gene in Philadelphia chromosome-positive leukemia

Identification of TINF2 gene mutations in adult Japanese patients with acquired bone marrow failure syndromes.

Haematologica, 118, 27–29. Finnegan, D.P., Kettle, P., Drake, M., Matthews, C., Alexander, H.D., Popat, R., Cavanagh, J.D., Wachsman, W. & Morris, T. (2006) Bortezomib is effective in primary plasma cell leukemia. Leukaemia & Lymphoma, 47, 1670–1673. Garcia-Sanz, R., Orfao, A., Gonzalez, M., Tabernero, M.D., Blade, J., Moro, M.J., Fernandez-Calvo, J., Sanz, M.A., PerezSimon, J.A., Rasillo, A. & Miguel, J.F. (1999) Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood, 93, 1032–1037. Gemmel, C., Cremer, F.W., Weis, M., Witzens, M., Moldenhauer, G., Koniczek, K.H., Imbach, U., Ho, A.D., Moos, M. & Goldschmidt, H. (2002) Anti-CD20 antibody as consolidation therapy in a patient with primary plasma cell leukemia after high-dose therapy and autologous stem cell transplantation. Annals of Hematology, 81, 119–123. Oka, S., Yokote, T., Akioka, T., Hara, S., Yamano, T., Tsuji, M. & Hanafusa, T. (2006) Successful treatment of multi-agent chemotherapy with rituximab for IgM plasma cell leukemia. Leukemia Research, 30, 1581–1583. RuizArguelles, G.J. & San Miguel, J.F. (1994) Cell surface markers in multiple myeloma. Mayo Clinic Proceedings, 69, 684–690. Saccaro, S., Fonseca, R., Veillon, D.M., Cotelingam, J., Nordberg, M.L., Bredeson, C., Glass, J. & Munker, R. (2005) Primary plasma cell leukemia: report of 17 new cases treated with autologous or allogeneic stem-cell transplantation and review of the literature. American Journal of Hematology, 78, 288–294. Tanioka, F., Tamashima, S., Shimizu, S., Kobayashi, H., Kobayashi, Y. & Sugimura, H. (2003) A case of primary plasma cell leukemia with hairycell morphology and k -type Bence-Jones protein. Immunohistochemical and molecular analysis. Japanese Journal of Clinical Oncology, 33, 232–237. Walters, M., Olteanu, H., Van Tuinen, P. & Kroft, S.H. (2010) CD23 expression in plasma cell myeloma is specific for abnormalities of chromosome 11, and is associated with primary plasma cell leukaemia in this cytogenetic sub-group. British Journal of Haematology, 149, 292–293.

correspondence: Identification of TINF2 gene mutations in adult Japanese patients with acquired bone marrow failure syndromes

Haematologica, 118, 27–29. Finnegan, D.P., Kettle, P., Drake, M., Matthews, C., Alexander, H.D., Popat, R., Cavanagh, J.D., Wachsman, W. & Morris, T. (2006) Bortezomib is effective in primary plasma cell leukemia. Leukaemia & Lymphoma, 47, 1670–1673. Garcia-Sanz, R., Orfao, A., Gonzalez, M., Tabernero, M.D., Blade, J., Moro, M.J., Fernandez-Calvo, J., Sanz, M.A., PerezSimon, J.A., Rasillo, A. & Miguel, J.F. (1999) Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood, 93, 1032–1037. Gemmel, C., Cremer, F.W., Weis, M., Witzens, M., Moldenhauer, G., Koniczek, K.H., Imbach, U., Ho, A.D., Moos, M. & Goldschmidt, H. (2002) Anti-CD20 antibody as consolidation therapy in a patient with primary plasma cell leukemia after high-dose therapy and autologous stem cell transplantation. Annals of Hematology, 81, 119–123. Oka, S., Yokote, T., Akioka, T., Hara, S., Yamano, T., Tsuji, M. & Hanafusa, T. (2006) Successful treatment of multi-agent chemotherapy with rituximab for IgM plasma cell leukemia. Leukemia Research, 30, 1581–1583. RuizArguelles, G.J. & San Miguel, J.F. (1994) Cell surface markers in multiple myeloma. Mayo Clinic Proceedings, 69, 684–690. Saccaro, S., Fonseca, R., Veillon, D.M., Cotelingam, J., Nordberg, M.L., Bredeson, C., Glass, J. & Munker, R. (2005) Primary plasma cell leukemia: report of 17 new cases treated with autologous or allogeneic stem-cell transplantation and review of the literature. American Journal of Hematology, 78, 288–294. Tanioka, F., Tamashima, S., Shimizu, S., Kobayashi, H., Kobayashi, Y. & Sugimura, H. (2003) A case of primary plasma cell leukemia with hairycell morphology and k -type Bence-Jones protein. Immunohistochemical and molecular analysis. Japanese Journal of Clinical Oncology, 33, 232–237. Walters, M., Olteanu, H., Van Tuinen, P. & Kroft, S.H. (2010) CD23 expression in plasma cell myeloma is specific for abnormalities of chromosome 11, and is associated with primary plasma cell leukaemia in this cytogenetic sub-group. British Journal of Haematology, 149, 292–293.

No racial difference in allele frequencies of FCGR3A gene F158V polymorphisms in diffuse large B‐cell lymphoma

To the Editor: In a frequency analysis of FCGR3A gene polymorphism F158V (rs396991 T ⁄G) for diffuse large B-cell lymphoma (DLBCL), Kim et al. (1) found that in contrast to Caucasian patients, who have a higher frequency of the F allele, the V allele was more common in Korean patients (VV 47%, VF 48%, FF 5%). They also found that those with a homozygous pair of V alleles (VV) had significantly higher remission rates under Rituximab plus CHOP therapy compared to those with non-VV pairs (1). NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) associated with Rituximab is more severe with the V allele than with the F allele (2, 3), and some have reported that this influences the effectiveness of Rituximab therapy (1, 4, 5). If the frequencies of FCGR3A gene polymorphisms differ significantly by race, then treatment results for lymphoma using Rituximab in Asian patients may also differ significantly from that in Caucasian patients. Therefore, this study examined Japanese subjects to investigate whether Asian subjects differ significantly from Caucasian subjects with regard to FCGR3A gene polymorphism frequency. Our study subjects included 73 Japanese patients with DLBCL and 141 normal control subjects. As FCGR3B is a pseudogene of FCGR3A, and the base in FCGR3B corresponding to the FCGR3A gene polymorphism F158V (rs396991 T ⁄G) is G, we selected a primer sequence that would not amplify FCGR3B. We used the Q-probe (quenching probe system) to analyze gene polymorphisms on i-densy, a fully automated SNP analyzer developed by ARKRAY, Inc. (6). Frequency analyses of the F158V alleles for FCGR3A revealed that FF was the most common and VV the least common for both DLBCL and normal control groups [DLBCL: VV n = 7 (9.6%), VF n = 30 (41.1%), FF n = 36 (49.3%); normal control (n = 141): VV n = 8 (5.7%), VF n = 47 (33.3%), FF n = 86 (61.0%)] (Table 1). These two groups did not differ significantly in terms of allele frequencies for FCGR3A gene polymorphisms. In addition, we observed no significant correlations between the F158V alleles for FCGR3A and patient age or other clinical background factors. While 74% of all symptomatic patients went into remission, remission rates were not significantly different between VV, VF, and FF groups (complete remission rate: VV 71%, VF 69.4%, FF 78.4%, P = 0.809). Table 1 shows the frequency of FCGR3A gene F158V polymorphisms in lymphoid malignancies. Frequency analysis of F158V polymorphisms in Japan revealed significantly higher frequency of cases with the F allele rather than the V allele when compared to Korean cases (P < 0.001), and a tendency for more cases with the F allele compared to Western cases (P = 0.052). Similar to Western cases, few cases in Japan had homozygous V alleles (VV cases) with strong ADCC. Similar results have been reported concerning IgA nephropathy and rheumatoid arthritis in Japanese patients, as well as anti-glomerular basement membrane disease in Chinese patients (11–13). We therefore

correspondence: Identification of TINF2 gene mutations in adult Japanese patients with acquired bone marrow failure syndromes: Correspondence

Haematologica, 118, 27–29. Finnegan, D.P., Kettle, P., Drake, M., Matthews, C., Alexander, H.D., Popat, R., Cavanagh, J.D., Wachsman, W. & Morris, T. (2006) Bortezomib is effective in primary plasma cell leukemia. Leukaemia & Lymphoma, 47, 1670–1673. Garcia-Sanz, R., Orfao, A., Gonzalez, M., Tabernero, M.D., Blade, J., Moro, M.J., Fernandez-Calvo, J., Sanz, M.A., PerezSimon, J.A., Rasillo, A. & Miguel, J.F. (1999) Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics. Blood, 93, 1032–1037. Gemmel, C., Cremer, F.W., Weis, M., Witzens, M., Moldenhauer, G., Koniczek, K.H., Imbach, U., Ho, A.D., Moos, M. & Goldschmidt, H. (2002) Anti-CD20 antibody as consolidation therapy in a patient with primary plasma cell leukemia after high-dose therapy and autologous stem cell transplantation. Annals of Hematology, 81, 119–123. Oka, S., Yokote, T., Akioka, T., Hara, S., Yamano, T., Tsuji, M. & Hanafusa, T. (2006) Successful treatment of multi-agent chemotherapy with rituximab for IgM plasma cell leukemia. Leukemia Research, 30, 1581–1583. RuizArguelles, G.J. & San Miguel, J.F. (1994) Cell surface markers in multiple myeloma. Mayo Clinic Proceedings, 69, 684–690. Saccaro, S., Fonseca, R., Veillon, D.M., Cotelingam, J., Nordberg, M.L., Bredeson, C., Glass, J. & Munker, R. (2005) Primary plasma cell leukemia: report of 17 new cases treated with autologous or allogeneic stem-cell transplantation and review of the literature. American Journal of Hematology, 78, 288–294. Tanioka, F., Tamashima, S., Shimizu, S., Kobayashi, H., Kobayashi, Y. & Sugimura, H. (2003) A case of primary plasma cell leukemia with hairycell morphology and k -type Bence-Jones protein. Immunohistochemical and molecular analysis. Japanese Journal of Clinical Oncology, 33, 232–237. Walters, M., Olteanu, H., Van Tuinen, P. & Kroft, S.H. (2010) CD23 expression in plasma cell myeloma is specific for abnormalities of chromosome 11, and is associated with primary plasma cell leukaemia in this cytogenetic sub-group. British Journal of Haematology, 149, 292–293.