Taku Tsukamoto

8q24 amplified segments involve novel fusion genes between NSMCE2 and long noncoding RNAs in acute myelogenous leukemia

The pathogenetic roles of 8q24 amplified segments in leukemic cells with double minute chromosomes remain to be verified. Through comprehensive molecular analyses of 8q24 amplicons in leukemic cells from an acute myelogenous leukemia (AML) patient and AML-derived cell line HL60 cells, we identified two novel fusion genes between NSMCE2 and long noncoding RNAs (lncRNAs), namely, PVT1-NSMCE2 and BF104016-NSMCE2. Our study suggests that 8q24 amplicons are associated with the emergence of aberrant chimeric genes between NSMCE2 and oncogenic lncRNAs, and also implicate that the chimeric genes involving lncRNAs potentially possess as-yet-unknown oncogenic functional roles.

Epigenetic repression of miR-375 is the dominant mechanism for constitutive activation of the PDPK1/RPS6KA3 signalling axis in multiple myeloma

Cytogenetic/molecular heterogeneity is the hallmark of multiple myeloma (MM). However, we recently showed that the serine/threonine kinase PDPK1 and its substrate RPS6KA3 (also termed RSK2) are universally active in MM, and play pivotal roles in myeloma pathophysiology. In this study, we assessed involvement of aberrant miR‐375 repression in PDPK1 overexpression in MM. An analysis of plasma cells from 30 pre‐malignant monoclonal gammopathies of undetermined significance and 73 MM patients showed a significant decrease in miR‐375 expression in patient‐derived plasma cells regardless of the clinical stage, compared to normal plasma cells. Introduction of miR‐375 reduced PDPK1 expression in human myeloma cell lines (HMCLs), indicating that miR‐375 is the dominant regulator of PDPK1 expression. In addition, miR‐375 introduction also downregulated IGF1R and JAK2 in HMCLs. CpG islands in the MIR375 promoter were pathologically hypermethylated in all 8 HMCLs examined and in most of 58 patient‐derived myeloma cells. Treatment with SGI‐110, a hypomethylating agent, and/or trichostatin A, a histone deacetylase inhibitor, increased miR‐375 expression, but repressed PDPK1, IGF1R and JAK2 in HMCLs. Collectively, these results show the universal involvement of overlapping epigenetic dysregulation for abnormal miR‐375 repression in MM, which is likely to contribute to myelomagenesis and to subsequent myeloma progression by activating oncogenic signalling pathways.

Hairy Cell Leukemia with Systemic Lymphadenopathy: Detection of BRAF Mutations in Both Lymph Node and Peripheral Blood Specimens

A 47-year-old woman with pancytopenia, excessive systemic lymphadenopathy and splenomegaly was referred to our hospital. The peripheral blood (PB) smear findings indicated neutropenia with lymphoid cells exhibiting hairy projections, while the histological findings of the cervical lymph node (LN) suggested hairy cell leukemia (HCL). In addition, the BRAF V600E mutation was detected, and the immunoglobulin gene rearrangement patterns were identical in both the cervical LN and PB specimens. Based on these findings, we diagnosed the patient with systemic lymphadenopathy due to HCL. This is the first report of a BRAF mutation detected in both the PB and LN at the onset of HCL.

Transcriptional dysregulation of the deleted in colorectal carcinoma gene in multiple myeloma and monoclonal gammopathy of undetermined significance.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin‐1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma‐derived cell lines (HMCLs) expressed non‐translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co‐expressed wild type transcripts (wt.DCC), while eight co‐expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient‐derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

Chromosomal abnormality variation detected by G-banding is associated with prognosis of diffuse large B-cell lymphoma treated by R-CHOP-based therapy

Diffuse large B‐cell lymphoma (DLBCL), which is the most prevalent disease subtype of non‐Hodgkin lymphoma, is highly heterogeneous in terms of cytogenetic and molecular features. This study retrospectively investigated the clinical impact of G‐banding‐defined chromosomal abnormality on treatment outcomes of DLBCL in the era of rituximab‐containing immunochemotherapy. Of 181 patients who were diagnosed with DLBCL and treated with R‐CHOP or an R‐CHOP‐like regimen between January 2006 and April 2014, metaphase spreads were evaluable for G‐banding in 120. In these 120 patients, 40 were found to harbor a single chromosomal aberration type; 63 showed chromosomal abnormality variations (CAVs), which are defined by the presence of different types of chromosomal abnormalities in G‐banding, including 19 with two CAVs and 44 with ≥3 CAVs; and 17 had normal karyotypes. No specific chromosomal break point or numerical abnormality was associated with overall survival (OS) or progression‐free survival (PFS), but the presence of ≥3 CAVs was significantly associated with inferior OS rates (hazard ratio (HR): 2.222, 95% confidence interval (CI): 1.056–4.677, P = 0.031) and tended to be associated with shorter PFS (HR: 1.796, 95% CI: 0.965–3.344, P = 0.061). In addition, ≥3 CAVs more frequently accumulated in high‐risk patients, as defined by several conventional prognostic indices, such as the revised International Prognostic Index. In conclusion, our results suggest that the emergence of more CAVs, especially ≥3, based on chromosomal instability underlies the development of high‐risk disease features and a poor prognosis in DLBCL.

Intravascular lymphoma in a rheumatoid arthritis patient following short-term methotrexate treatment

SIR, It has been reported that patients with RA have an increased risk of lymphoproliferative disorders (LPDs) compared with the general population [1, 2]. The abnormal immune state found in RA itself and the immunosuppressive effect of anti-rheumatic drugs such as MTX may contribute to development of LPDs. Several studies have reported that the median duration of MTX treatment until lymphoma diagnosis is 48 56 months, and extranodal locations are frequent [3 5]. Intravascular lymphoma (IVL) is a rare form of extranodal lymphoma that is characterized by the proliferation of lymphoma cells within the vascular lumen without an obvious extravascular tumour mass. Because IVL has no specific symptoms, random biopsies from healthy-appearing skin have been reported to be useful in its diagnosis [6]. Herein, we report a case of IVL in an RA patient with only 2 months’ MTX treatment. According to the extremely few reports concerning IVL patients with RA, IVL normally develops after prolonged RA duration or historic MTX therapy [7, 8]. To our knowledge, there are no reports of IVL concomitant with early RA. A 75-year-old Japanese woman was admitted following a week-long history of fever and malaise. Four months previously, she had been diagnosed with anti-CCP antibodypositive RA, and SSZ had been initiated. The patient started MTX (6 mg/week) 2 months before the present admission and her disease activity was improving. She also had a 30-year history of diabetes mellitus, which was well controlled by intensive insulin therapy. On physical examination, the patient’s temperature was 38.4 C, blood pressure was 152/84 mmHg, pulse rate was 90/min and respiration rate was 18/min. There were no crackles, heart murmurs, pain spots, lymph node swellings or skin rashes. Her laboratory examination showed elevated levels of lactate dehydrogenase (1164 IU/l) and soluble IL-2 receptor (1210 U/ml). After hospitalization, despite the discontinuation of MTX, fever with nausea persisted for 1 week and her general condition progressively deteriorated. She also presented with haemolytic anaemia and positive cold agglutinin with a titre of 1:131 072; however, there were no clinical findings of the underlying disease, such as infection, macroglobulinaemia or lymphadenopathy. We therefore performed a random skin biopsy on the eighth hospital day, and intravascular large B cell lymphoma (IVLBCL) was revealed (Fig. 1). Tumour cell invasions were also detected in the bone marrow aspirate and the cerebrospinal fluid. 18-fluorodeoxyglucose PET/CT showed neither lymph node enlargement nor specific fluorodeoxyglucose uptake in lymphoid or extra-lymphoid organs. Consequently, we diagnosed this case as IVLBCL with paraneoplastic autoimmune haemolytic anaemia and started R-hyper-CVAD/R-MA (rituximab plus cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with rituximab plus high-dose MTX and cytarabine) chemotherapy. After the 6 months’ treatment, she achieved complete remission of IVLBCL, and her cold agglutinin had disappeared. One year after that, she restarted DMARDs other than MTX for RA relapse, while there is no recurrence of IVLBCL. It is not clear whether MTX caused the patient’s IVL. MTXassociated LPD is often related to EBV and regress with the cessation of MTX. In this case, however, EBV was not determined and her IVL did not regress after the discontinuation of MTX. Since there were no clinical findings that would suggest LPD such as fever or elevation of lactate dehydrogenase until the start of MTX use, we considered her IVL may have developed after the initiation of treatment for RA. As in the present case, diagnosis of IVL developing in recent-onset RA patients can be challenging. Clinicians should not rule out the possibility of LPD, even if the disease duration is short or there are no obvious mass lesions. In this case, the clinical findings of unknown cause, such as fever, malaise and a newly developed autoimmune disorder (haemolytic anaemia), provided clues for the successful diagnosis of this rare condition.

Quadruple Cancers of Non-producing Multiple Myeloma, Cholangiocellular Carcinoma, and Two Different Thyroid Cancers

We report the case of a 72-year-old man who presented with non-producing multiple myeloma (MM) with three additional concomitant solid tumors that were identified by postmortem autopsy. The disease was refractory to anti-MM therapy including bortezomib and lenalidomide, and he finally died of bacterial pneumonia with diffuse alveolar damage 8 months after the diagnosis. An autopsy revealed that he was also affected by three other solid cancers, cholangiocellular carcinoma, medullary thyroid cancer and papillary thyroid cancer that were clinically asymptomatic and remained undiagnosed before death. A review of the literature suggests that primary quadruple cancers including MM are extremely rare.

Burkitt Lymphoma Preceded by Autoimmune Hemolytic Anemia due to Anti-D Antibody

We herein report a rare case of Burkitt lymphoma (BL) preceded by autoimmune hemolytic anemia (AIHA) caused by autoantibodies against D antigen. After a partial response to AIHA with prednisolone (PSL) treatment for 7 months, the patient developed BL with a t(8;22)(q24;q11.2) chromosomal translocation. Intensive immunochemotherapy, including rituximab, led to a complete response (CR) of BL; however, anti-D antibody remained detectable in the plasma and antibody-dissociated solution from erythrocytes, thus continuous therapy with PSL was necessary even after achievement of the CR. BL with AIHA is extremely rare, with only one previously reported case in the literature.

Dual targeting of bromodomain-containing 4 by AZD5153 and BCL2 by AZD4320 against B-cell lymphomas concomitantly overexpressing c-MYC and BCL2

SummaryDespite the recent therapeutic progress, the prognoses of diffuse large B-cell lymphomas (DLBCLs) that concomitantly overexpress c-MYC and BCL2, i.e., double hit lymphoma (DHL) and double expressing lymphoma (DEL), remain poor. This study examined triple targeting of c-MYC, BCL2 and the B-cell receptor (BCR) signaling pathway for DHL and DEL. We first used AZD5153, a novel bivalent inhibitor for bromodomain-containing 4 (BRD4), in DHL- and DEL-derived cell lines, because BRD4 regulates disease type-oriented key molecules for oncogenesis. AZD5153 was more effective than conventional monovalent BRD4 inhibitors, JQ1 and I-BET151, in inhibiting cell proliferation of a DHL-derived cell line and two DEL-derived cell lines, with at least 10-fold lower half growth inhibitory concentrations. AZD5153 caused G1/S cell cycle blockade, while the apoptosis-inducing effect was relatively modest. At the molecular level, AZD5153 was potent in downregulating various molecules for oncogenesis, such as c-MYC, AKT2 and MAP3K; those involved in the BCR signaling pathway, such as CD19, BLNK and CD79B; and those associated with B-cell development, such as IKZF1, IKZF3, PAX5, POU2AF1 and EBF1. In contrast, AZD5153 did not decrease anti-apoptotic BCL2 proteins, and did not activate pro-apoptotic BH3-only proteins, except BAD. To augment cell death induction, we added a novel BH3-mimicking BCL2 inhibitor AZD4320 to AZD5153, and found that these two agents had a mostly synergistic antitumor effect by increasing cells undergoing apoptosis in all three cell lines. These results provide a rationale for dual targeting of BRD4 and BCL2 using AZD5153 and AZD4320 as a therapeutic strategy against DHL and DEL.

Human herpesvirus-6 pneumonitis in a patient with follicular lymphoma following immunochemotherapy with rituximab

Primary infection with human herpesvirus-6 (HHV-6) commonly occurs at an early age in children, most often at 3 years of age, and is associated with childhood diseases, such as exanthema subitum, hepatitis, febrile convulsions, or encephalitis. However, the virus occasionally reactivates from its latent state in immunosuppressed adults, especially post-transplant, resulting in serious disseminated, sometimes life-threatening end-organ complications. Herein, we report a case of a 68-year-old man with relapsed follicular lymphoma who developed HHV-6 pneumonitis. Eighteen months after achieving second complete remission by salvage immunochemotherapy with rituximab, the patient was complicated by pneumonia, with chest computed tomography finding showing disseminated nodular shadows with ground-glass opacity in both lungs. While empiric antibiotic and antifungal therapies did not improve the pneumonia, polymerase chain reaction–based viral screening tests on his bronchoalveolar lavage fluid detected the presence of HHV-6 DNA, and ganciclovir treatment quickly resolved the pneumonia, indicating that he suffered from HHV-6 pneumonitis. He had no other HHV-6–related end-organ damage, such as encephalitis. This case suggests that, although extremely rare, HHV-6 reactivation should be considered as one of the candidate pathogens for pulmonary complications of uncertain etiology in patients who have been treated with intensive immunosuppressive chemotherapy, even without hematopoietic stem cell transplantation. Furthermore, polymerase chain reaction–based viral screening testing on bronchoalveolar lavage fluid is a powerful diagnostic tool for pneumonitis due to viral reactivation, including HHV-6 reactivation.