Akinao Okamoto

BACKGROUND: Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. METHODS: In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. RESULTS: Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). CONCLUSIONS: These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

症例は56歳女性で、健康診断で白血球減少が明らかになり、骨髄検査の結果、急性骨髄性白血病(AML)と診断された。白血病細胞はアズール顆粒を欠損し、t(8;21)の典型的特徴に相当しなかった。RNA-seq分析では、RUNX1-RUNX1T1に加えて、12p11でのTM7SF3は8q22でVPS13Bへ融合され、またVPS13BはRUNX1に融合されることが明らかになった。VPS13BはRUNX1T1の近くに局在し、両者は同じ染色体バンドに局在した。TM7SF3およびVPS13Bのリーディングフレームは、それぞれVPS13BおよびRUNX1のリーディングフレームとマッチしなかった。VPS13Bの破壊はCohen症候群を起こし、骨髄において左にシフトした顆粒球形成を伴う間欠性好中球減少を示した。VPS13Bの破壊はRUNX1-RUNX1T1白血病の稀な特徴を起こした。本症例は、VPS13Bの再配列が変異t(8;21)における追加の遺伝的イベントである可能性が示唆された。

AIMS: Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) not otherwise specified is defined as monoclonal EBV+ B-cell proliferation affecting patients without any known immunosuppression. Non-neoplastic EBV+ cells proliferating in or adjacent to EBV- DLBCL were reported recently, but their clinical significance is unclear. Thus, the aim of this study was to investigate the prognostic impact of EBV+ cells in DLBCL. METHODS AND RESULTS: We compared the clinicopathological characteristics of 30 EBV+ DLBCL patients and 29 and 604 EBV- DLBCL patients with and without EBV+ bystander cells (median age of onset 71, 67 and 62 years, respectively). Both EBV+ DLBCL patients and EBV- DLBCL patients with EBV+ bystander cells tended to have high and high-intermediate International Prognostic Index scores (60% and 59%, respectively), as compared with only 46% of EBV- DLBCL patients without EBV+ bystander cells. EBV- DLBCL patients with EBV+ bystander cells showed a significantly higher incidence of lung involvement than those without EBV+ bystander cells (10% versus 2%, P < 0.05). Furthermore, EBV+ DLBCL patients and EBV- DLBCL patients with EBV+ bystander cells had a poorer prognosis than patients without any detectable EBV+ cells [median overall survival (OS) of 100 months and 40 months versus not reached, P < 0.01]. Notably, EBV+ DLBCL patients and EBV- DLBCL patients with EBV+ bystander cells treated with rituximab showed overlapping survival curves (OS, P = 0.77; progression-free survival, P = 1.0). CONCLUSIONS: EBV- DLBCL with bystander EBV+ cells has similar clinical characteristics to EBV+ DLBCL. DLBCL with EBV+ bystander cells may be related to both age-related and microenvironment-related immunological deterioration.

© 2015 John Wiley & Sons, Ltd. Epstein–Barr virus (EBV)-encoded small RNA in situ hybridization (EBER-ISH) is a widely accepted method to evaluate EBV involvement in diffuse large B-cell lymphoma (DLBCL), although little is known regarding associations between EBV DNA load and the EBER status and whether EBV DNA load data provide additional clinical information. In this study, we quantified EBV DNA load in diagnostic specimens from DLBCL patients diagnosed at our hospital to evaluate clinical implications of EBV DNA load in diagnostic specimens as contrasted with EBER-ISH. Among 140 DLBCL patients without underlying immunodeficiency, 51 were evaluable for both EBER and EBV DNA load, 83 for EBER only and one for EBV DNA load only. The median EBV DNA load was 708 copies/μg. Although EBV DNA load was significantly higher for EBER-positive patients than for EBER-negative patients (p<0.001), EBV DNA was detected in up to 72% of EBERnegative patients. Progression-free survival and overall survival were significantly worse for patients with EBV DNA load above 700 copies/μg than for those with EBV DNA load below 700 copies/μg (p = 0.009 and p = 0.003); they were also significantly worse for EBER-positive patients than for EBER-negative patients (p<0.001 and p = 0.001). Even among EBER-negative patients, higher EBV DNA load conferred worse progression-free survival and overall survival (p = 0.041 and p = 0.013). These findings indicate that EBV DNA load in diagnostic specimens is not a simple surrogate for the EBER status and may be a potential biomarker associated with EBV involvement and prognosis in DLBCL.

The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 mu M with IC50 value of 0.13 mu M at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

次世代シーケンシングを用い、急性骨髄性白血病(AML)高齢患者におけるTP53変異を分析し、導入化学療法(IC)後の治療成果に及ぼす影響について検討した。対象は1997年〜2014年までに当院でAMLと診断され、ICが施行された(男性48例、女性29例、年齢60〜89歳)とした。その結果、12例(16%)に14のTP53変異が認められ、変異とモノソーム核型間に有意な関連性が認められた。なお、生存患者の経過観察期間中央値は4年であり、ICでの完全寛解(CR)に関しては、変異を有する患者(変異群)では42%(5/12)、変異を有さない患者(非変異群)では63%(41/65)に認められたが、変異群では全例、非変異群では41例中30例に再発が認められた。また、変異群では全例、非変異群では48例が死亡し、変異群は非変異群後に比し、有意に予後不良であった。さらに、多変量解析において、TP53変異が生存に対する最も有意な予後因子であることも確認された。なお、同一時期にICを受けなかった高齢患者は59例であったが、それら患者の生存中央値は1.7ヵ月であり、変異群のICを受けた患者の方が良好な予後が示された。

© 2015 Wiley Periodicals, Inc. ETV6, which encodes an ETS family transcription factor, is frequently rearranged in human leukemias. We show here that a patient with acute myeloid leukemia with t(7;11)(p15;p15) gained, at the time of relapse, t(11;12)(q12.1;p13) with a split ETV6 FISH signal. Using 3′-RACE PCR analysis, we found that ETV6 was fused to LPXN at 11q12.1, which encodes leupaxin. ETV6-LPXN, an in-frame fusion between exon 4 of ETV6 and exon 2 of LPXN, did not transform the interleukin-3-dependent 32D myeloid cell line to cytokine independence; however, an enhanced proliferative response was observed when these cells were treated with G-CSF without inhibition of granulocytic differentiation. The 32D and human leukemia cell lines each transduced with ETV6-LPXN showed enhanced migration towards the chemokine CXCL12. We show here for the first time that LPXN is a fusion partner of ETV6 and present evidence indicating that ETV6-LPXN plays a crucial role in leukemia progression through enhancing the response to G-CSF and CXCL12.

A novel series of Mannich derivatives of 3a-g and 3a'-g' were designed and synthesized from pseudophenylpropanolamine (Psi-PPA). The stereo chemical aspects of the synthesized compounds were studied and all compounds were well characterized with respect to spectral techniques. All Mannich derivatives, 3a-g and 3a'-g' were evaluated for their anti-proliferative activity against A549 and HepG-2 cells. Among the tested compounds, 3a showed significant anti-proliferative activity against HepG-2 cells at 25 mu M when compared to other compounds. The treatment of 3a exhibited morphological changes, nuclear condensation, colony formatting ability, apoptosis and cell cycle arrest at G2/M phase in HepG-2 cells. Besides, 3a triggered mitochondrial mediated apoptotic pathway as indicated by down regulation of Bcl-2, up-regulation of Bax, and release of cytochrome c and caspases-3. Furthermore, 3a effectively suppressed the cell proliferation and cell growth via JAK2/STAT3 signaling pathway in a time and dose dependent manner. In vivo administration of 3a inhibited tumor growth without significant change in body weight in HepG-2 xenograft mice model. Molecular docking studies revealed that good binding energies of compound 3a against JAK2 (-6.10 kcal mol(-1)) and Bcl-2 (-6.04 kcal mol(-1)) receptors. Taken together, 3a possessed potent antitumor activity; it could be a promising lead candidate for the potential treatment of human hepatocellular carcinoma.

© 2015 Elsevier Ireland Ltd. All rights reserved. The present study was undertaken to investigate the anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit and to explore the molecular mechanisms of action in MCF-7 cells. Cytotoxic properties of hexane, ethyl acetate and methanol extracts were carried out against MCF-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Ethyl acetate extract showed good cytototoxic activities compared to hexane and methanol extracts. Methyl caffeate was isolated from the ethyl acetate extract using column chromatography. Cytotoxic properties of methyl caffeate was investigated against MCF-7, A549, COLO320, HepG-2 and Vero cells. The compound showed potent cytotoxic properties against MCF-7 cells compared to A549, COLO320 and HepG-2 cells. Methyl caffeate significantly reduced cell proliferation and increased formation of fragmented DNA and apoptotic body in MCF-7 cells. Bcl-2, Bax, Bid, p53, caspase-3, PARP and cytochrome c release were detected by western blot analysis. The activities of caspases-3 and PARP gradually increased after the addition of isolated compound. Bcl-2 protein was down regulated; Bid and Bax were up regulated after the treatment with methyl caffeate. Molecular docking studies showed that the compound bound stably to the active sites of poly (ADP-ribose) polymerase-1 (PARP1), B cell CLL/lymphoma-2 (BCL-2), E3 ubiquitin-protein ligase (MDM2) and tubulin. The results strongly suggested that methyl caffeate induced apoptosis in MCF-7 cells via caspase activation through cytochrome c release from mitochondria.

© 2015 S. Karger AG, Basel. DEK-NUP214 gene fusion in acute myeloid leukemia (AML) is associated with poor prognosis. It is most often a sole translocation and more rarely observed as complex chromosomal forms. We describe an AML case with complex karyotype abnormalities involving chromosome bands 6p23, 6q13, 7p22, and 9q34. RNA sequencing analysis revealed that exon 17 of NUP214 (9q34) was fused to exon 2 of RAC1 (7p22). We also detected that the 5′-end of intron 1 of RAC1 was fused with the antisense strand of intron 5 of COL12A1 (6q13). RT-PCR analysis confirmed the expression of DEK-NUP214, NUP214-RAC1, RAC1-COL12A1, NUP214, and RAC1. These results suggest that the 5′- and 3′-ends of NUP214 from the breakpoint in the same locus were fused to RAC1 and DEK, respectively, and the 5′-end of RAC1 was fused to COL12A1. The reading frame of NUP214 was not matched with RAC1; however, high expression of the RAC1 protein was detected by Western blotting. This study identifies the variant complex fusion genesNUP214-RAC1 and RAC1- COL12A1 in a case of AML.

リンパ腫、HIV感染症、関節リウマチの病歴のない18歳以上のび漫性大細胞型B細胞性リンパ腫(DLBCL)127例を対象に、治療前の血清中のEpstein-Barrウイルス(HBV)DNA量を測定し、臨床症状および予後への影響を評価した。119例(94%)に初回治療としてアントラサイクリンベース化学療法とリツキシマブの併用療法を行っていた。127例のうち、EBV DNA陰性患者は112例(男性65例、年齢33〜89歳)、EBV DNA陽性患者は15例(男性8例、年齢51〜91歳)で、EBV DNA陽性患者の方が高齢で、病期が進行しており、国際予後スコアが高いことが示された。また、EBV DNA陽性患者は無増悪生存期間(PFS)と全生存期間(OS)が有意に不良であった。EBV-encoded small RNAを標的としたin situハイブリダイゼーション法では、EBV-encoded small RNAが陽性であった8例中6例、陰性であった115例中9例で治療前の血清からEBV DNAが検出された。EBV-encoded small RNA陽性患者では陰性患者に比べてPFSとOSが有意に不良であったのに対し、EBV-encoded small RNA陰性の115例においても治療前血清からのEBV DNA検出はPFSおよびOSの不良と関連していた。以上から、DLBCL患者における治療前血清からのEBV DNA検出は、予後に有害な影響を及ぼす可能性があると考えられた。

最近の支持療法によって、導入化学療法を受けていない患者の生存率が上昇するか検討し、長期生存患者の特徴について検討した。当院で2004〜2012年の間に急性骨髄性白血病(AML)と診断された患者158名を遡及的に調査した。158名中43名(男24名、女19名、平均78歳)が導入化学療法を受けていなかった。生存期間中央値は1.5ヵ月で、3ヵ月、6ヵ月および12ヵ月以上生存は11名、6名、4名であった。これら患者の生存率は強度の、または低強度の導入化学療法を施行された患者よりも悪かった。AML診断時の白血球数低値と骨髄異形成症候群の既往が、長期生存期間と有意に関連していた。これらの所見から、現在の支持療法は導入化学療法を受けていないAML患者を延命させないが、一部の患者は比較的長く生存することが示唆された。

帯状疱疹を発症した発端者に続いて4名の入院患者が水痘を発症した。発症者と同じ病室に滞在し、水痘を発症しなかった18名を対照として、集団発生の要因を調べた。発症者は全てリツキシマブを含む化学療法を受けたび漫性大細胞型B細胞性リンパ腫患者であった。非発症者でリツキシマブを含む化学療法を受けた患者は18名中3名のみであった。全ての発症者において化学療法前から抗水痘・帯状疱疹ウイルスIgG型抗体が検出されていた。ウイルスに対する中和抗体が存在しても、リツキシマブを含む化学療法を受けたリンパ腫患者は水痘に再感染する可能性がある。帯状疱疹患者は血液科病室や腫瘍科病室から隔離する必要がある。