Shin-ichiro Fujiwara

Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4–ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability.

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44 000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI− CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5′-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or ‘proteome,’ of malignant cells in addition to the ‘transcriptome.’ To identify proteins specifically expressed in leukemias, here we isolated AC133+ hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G2–M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Rituximab plus 70% cyclophosphamide, doxorubicin, vincristine and prednisone for Japanese patients with diffuse large B-cell lymphoma aged 70 years and older

Abstract In the rituximab era, several large studies have suggested that full-dose rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) might be the best treatment for patients with diffuse large B-cell lymphoma (DLBCL) aged 60 years and older. However, it remains unclear whether this is also the case for those aged 70 years and older. Previously untreated patients with DLBCL aged 70 years and older (elderly) were treated with R-70%CHOP, and patients younger than 70 years (younger) were treated with full-dose R-CHOP every 3 weeks, for a total of 6–8 cycles. Complete remission (CR) rates in elderly versus younger patients were 75 vs. 78% (p = 0.7), respectively. The 3-year overall survival, event-free survival and progression-free survival of elderly versus younger patients were 58 vs. 78% (p < 0.05), 45 vs. 70% (p < 0.05) and 64 vs. 72% (p = 0.43), respectively. Severe adverse events were more frequent in the elderly, even with the dose reduction in that age group. Three-year PFS with R-70%CHOP for patients aged 70 years and older was not significantly worse than that with full-dose R-CHOP for younger patients, suggesting that R-70% CHOP might be a reasonable choice for patients with DLBCL aged 70 years and older, especially for those with comorbidities.

High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide polymorphism-typing microarrays

Angioimmunoblastic T-cell lymphoma (AILT) and peripheral T-cell lymphoma, unspecified (PTCL-u) are relatively frequent subtypes of T- or natural killer cell lymphoma. To characterize the structural anomalies of chromosomes associated with these disorders, we here determined chromosome copy number alterations (CNAs) and loss of heterozygosity (LOH) at >55 000 single nucleotide polymorphism loci for clinical specimens of AILT (n=40) or PTCL-u (n=33). Recurrent copy number gain common to both conditions was detected on chromosomes 8, 9 and 19, whereas common LOH was most frequent for a region of chromosome 2. AILT- or PTCL-u-specific CNAs or LOH were also identified at 21 regions, some spanning only a few hundred base pairs. We also identified prognosis-related CNAs or LOH by several approaches, including Coxs proportional hazard analysis. Among the genes that mapped to such loci, a poor prognosis was linked to overexpression of CARMA1 at 7p22 and of MYCBP2 at 13q22, with both genes being localized within regions of frequent copy number gain. For a frequent LOH region at 2q34, we also identified IKAROS family zinc-finger 2 cDNAs encoding truncated proteins. Our data indicate that AILT and PTCL-u consist of heterogeneous subgroups with distinct transforming genetic alterations.

MicroRNA expression profiles of human leukemias.

1 Taksin AL, Legrand O, Raffoux E, de Revel T, Thomas X, Contentin N et al. High efficacy and safety profile of fractionated doses of Mylotarg as induction therapy in patients with relapsed acute myeloblastic leukemia: a prospective study of the alfa group. Leukemia 2007; 21: 66–71. 2 Mrozek K, Bloomfield CD. Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia. Hematology 2006 Education program book. American Society of Hematology: Washington, DC, 2006, 169–177. 3 Garrido SM, Bryant E, Appelbaum FR. Allogeneic stem cell transplantation for relapsed and refractory acute myeloid leukemia patients with 11q23 abnormalities. Leuk Res 2000; 24: 481–486. 4 Larson RA, Sivers EL, Stadtmauer EA, Lowenberg B, Estey EH, Dombret H et al. Final report on the efficacy and safety of gemtuzumab ozogamicin (Mylotarg) in patients with CD33 positive acute myeloid leukemia in first recurrence. Cancer 2001; 104: 1442–1452. 5 Muñoz L, Nomdedéu JF, Villamor N, Guardia R, Colomer D, Ribera JM et al. Acute myeloid leukemia with MLL rearrangements: clinicobiological features, prognostic impact and value of flow cytometry in the detection of residual leukemic cells. Leukemia 2003; 17: 76–82.

Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.

Biphenotypic acute leukemia (BAL) is a relatively rare subtype of acute leukemia characterized by the presence of both myeloid and lymphoid cell surface antigens. We have now screened for transforming genes in BAL blasts with the use of the focus formation assay with a retroviral cDNA expression library constructed from malignant blasts isolated from a BAL patient. Some of the retroviral inserts recovered from transformed foci were found to encode wild-type purinergic receptor P2Y, G protein coupled, 8 (P2RY8). The oncogenic potential of P2RY8 was confirmed with the in vitro focus formation assay as well as with an in vivo tumorigenicity assay in nude mice. A variety of luciferase-based reporter assays revealed that P2RY8 increased both the trans-activation activities of CREB and Elk-1 as well as the transcriptional activities of the serum response element and enhancer-promoter fragments of the c-Fos and c-Myc genes. Quantitation of P2RY8 mRNA in CD34+ cells of bone marrow showed that P2RY8 expression is frequently increased in leukemia patients, especially in those with refractory disease. Our data thus reveal an abundant expression of P2RY8 in leukemic cells and its unexpected role in the pathogenesis of acute leukemia.

Incidental carcinomas detected by PET/CT scans in patients with malignant lymphoma

According to the international working group response criteria for malignant lymphoma revised in 2007, 18F-fluorodeoxyglucose positron emission tomography (18FDG-PET) combined with or without computed tomography (CT) is recommended for pre-treatment staging and response assessment among patients with diffuse large B-cell lymphoma and Hodgkin lymphoma. Recently, along with the widespread use of PET/CT, unexpected uptake and accumulation of 18FDG has been reported. Discussed in the present report are patients with malignant lymphoma and second primary carcinomas that were incidentally found by PET/CT. A total of 497 consecutive PET/CT were performed on 290 patients with malignant lymphoma in our institution from April 2008 through March 2010. Eight patients (2.8%) had pathologically confirmed second primary carcinomas consisting of 4 colon cancers, 3 lung cancers, and 1 pancreatic cancer. Two cases were diagnosed at the initial staging, and the others were detected after treatment for lymphoma. It is noteworthy that PET revealed high accumulations of 18FDG in 5 (62.5%) of the 8 patients without corresponding tumors in conventional CT. All of the 4 patients with colon carcinoma underwent curative surgery. The present study suggests that incidental findings by PET in malignant lymphoma can lead to early detection and successful treatment of second malignancies.

Prospective randomized and crossover comparison of two apheresis machines for peripheral blood stem cell collection: a multicenter study

Improving apheresis technology may lead to an efficient and safe peripheral blood stem cell (PBSC) collection. Recently, the Spectra Optia (Optia, Terumo BCT) was introduced as an automated apheresis instrument, but comparisons with other instruments have been few. This is the first randomized multicenter and crossover comparison of the Optia with the automated program of the established apheresis instrument, the Spectra (Spectra‐Auto, Terumo BCT).

Clinical features of de novo CD25-positive follicular lymphoma

Abstract CD25 expression in follicular lymphoma (FL) has not yet been investigated. Eighty-five patients with newly diagnosed FL were retrospectively evaluated. On two-color flow cytometric analysis, CD25 was detected on CD19 + and CD20 + lymphoma cells. CD25 expression in FL tended to be higher than in reactive lymphadenopathy, but was lower than in diffuse large B-cell lymphoma. Patients with CD25 + FL (n = 12) showed clinical features of elevated soluble interleukin-2 receptor (IL-2R) levels, B symptoms and an advanced age compared with CD25 − FL (n = 73). The overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) in patients with CD25 + FL were significantly inferior to those with CD25 − FL (ORR, 60 vs. 93%; 2-year PFS, 32 vs. 80.3%; 6-year OS, 47.4 vs. 85.9%, respectively). Multivariate analysis demonstrated that CD25 positivity is an independent prognostic factor for PFS and OS in FL. CD25 + FL may constitute a distinct subgroup associated with aggressiveness and an inferior prognosis.