Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p = 0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

A Case of the nephrotic syndrome in bone marrow transplantation recipient, histologically showing overlapped glomerular lesions of thrombotic microangiopathy and membranous nephropathy.

Nephrotic syndrome (NS) rarely occurs in post-hematopoietic stem cell transplantation (HSCT) recipients but represents the renal manifestation of graft-versus-host disease (GVHD). Membranous nephropathy (MN) accounts for almost two thirds of post-HSCT NS and is caused by immune complex deposition. Renal thrombotic microangiopathy (TMA) without fulfillment of clinical criteria for TMA has been underreported because of reduced opportunity for histological examination. However, renal TMA has recently been reported in association with GVHD and humoral immunological reactions. Although both MN and TMA after HSCT are associated with GVHD and immunological abnormalities, these diseases are exceptionally coexistent in renal biopsy specimens. We herein describe a case of post-HSCT NS, histologically showing overlapped lesions of TMA and MN. Renal biopsy specimen after presentation of NS revealed early stage MN and TMA with evidence of chronicity. TMA was thought to have preceded MN, and renal biopsy at the phase of pre-nephrotic proteinuria might reveal earlier histological changes of isolated renal TMA. Detection of subclinical renal TMA earlier by spontaneous renal biopsy can help prevent progression of renal injury or overlapping of other renal pathologies. We also demonstrated Th2 predominant intraglomerular infiltration of lymphocytes by immunohistochemistry.

Unique clonal relationship between T-cell acute lymphoblastic leukemia and subsequent Langerhans cell histiocytosis with TCR rearrangement and NOTCH1 mutation.

Acute lymphoblastic leukemia (ALL) occasionally develops before or after the onset of Langerhans cell histiocytosis (LCH). The mechanism of LCH developing after ALL remains unclear; thus the clonality of LCH developing during maintenance chemotherapy for T-cell ALL (T-ALL) was investigated. The T-ALL and LCH cells tested had the same T-cell receptor (TCR) gamma rearrangement. Mutation analysis of the NOTCH1 gene revealed 7213C>T (Q2405X) in exon 34 in T-ALL and LCH cells, but 5156T>C (I1719T) in exon 27 only in T-ALL. Polymerase chain reaction-restriction fragment length polymorphism analysis revealed three patterns of NOTCH1 mutations in T-ALL cells. The results suggest that the T-ALL and LCH cells were derived from a common precursor with TCR rearrangement and a single NOTCH1 mutation, rather than LCH cells developing from a minor clone of T-ALL with single NOTCH1 mutation.

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.

Galectin-3 (Gal-3) induced by leukemia microenvironment promotes drug resistance and bone marrow lodgment in chronic myelogenous leukemia.

Bone marrow (BM) microenvironment (BMME) constitutes the sanctuary for leukemic cells. In this study, we investigated the molecular mechanisms for BMME-mediated drug resistance and BM lodgment in chronic myelogenous leukemia (CML). Gene-expression profile as well as signal pathway and protein analyses revealed that galectin-3 (Gal-3), a member of the ß-gal-binding galectin family of proteins, was specifically induced by coculture with HS-5 cells, a BM stroma cell-derived cell line, in all five CML cell lines examined. It was also found that primary CML cells expressed high levels of Gal-3 in BM. Enforced expression of Gal-3 activated Akt and Erk, induced accumulation of Mcl-1, and promoted in vitro cell proliferation, multidrug resistance to tyrosine kinase inhibitors for Bcr-Abl and genotoxic agents as a result of impaired apoptosis induction, and chemotactic cell migration to HS-5-derived soluble factors in CML cell lines independently of Bcr-Abl tyrosine kinase. The conditioned medium from Gal-3-overexpressing CML cells promoted in vitro cell proliferation of CML cells and HS-5 cells more than did the conditioned medium from parental cells. Moreover, the in vivo study in a mice transplantation model showed that Gal-3 overexpression promoted the long-term BM lodgment of CML cells. These results demonstrate that leukemia microenvironment-specific Gal-3 expression supports molecular signaling pathways for disease maintenance in BM and resistance to therapy in CML. They also suggest that Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance.

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors.

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.

Hematopoietic progenitor cell mobilization using low-dose cyclophosphamide and granulocyte colony-stimulating factor for multiple myeloma.

High-dose chemotherapy (HDT) supported by autologous stem cell transplantation (ASCT) has long been one of the standards of care for younger patients with multiple myeloma (MM). Cyclophosphamide (CY) plus granulocyte colony-stimulating factor (G-CSF) has been the conventional preparation for hematopoietic progenitor cell (HPC) mobilization, although the optimal dosage of CY in this setting has not yet been clearly defined. This study investigated the efficacy and safety of low-dose (LD-)CY (1.5 g/m(2)) plus G-CSF for conditioning for HPC apheresis harvest (HPC-A) in 18 MM patients, and compared it with a regimen consisting of intermediate-dose (ID)-CY (4 g/m(2)) plus G-CSF for 13 MM patients. Eleven patients in the former and six in the latter were treated with bortezomib (BTZ) during the induction therapy. Both regimens were comparably effective in terms of CD34(+) cell yields, while adverse events, such as leukopenia, thrombocytopenia, and febrile neutropenia, occurred significantly less frequently in the LD-CY cohort. All patients in LD-CY cohort started and completed their apheresis on day 7 or 8, whereas for the ID-CY cohort the day of first apheresis varied widely from day 8 to 15. These findings indicate that the LD-CY regimen is as effective as ID-CY for HPC mobilization, while the former is clearly more practicable and convenient than the ID-CY regimen for patients with MM.

Establishment of a novel human myeloid leukemia cell line, AMU-AML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

Double-hit lymphomas constitute a highly aggressive subgroup in diffuse large B-cell lymphomas in the era of rituximab.

OBJECTIVE: The incorporation of rituximab in immunochemotherapy has improved treatment outcomes for diffuse large B-cell lymphoma, but the prognosis for some diffuse large B-cell lymphomas remains dismal. Identification of adverse prognostic subgroups is essential for the choice of appropriate therapeutic strategy. METHODS: We retrospectively investigated the impact of so-called 'double-hit' cytogenetic abnormalities, i.e. cytogenetic abnormalities involving c-MYC co-existing with other poor prognostic cytogenetic abnormalities involving BCL2, BCL6 or BACH2, on treatment outcomes for 93 consecutive diffuse large B-cell lymphoma patients. RESULTS: According to the revised international prognostic index, no patients were cytogenetically diagnosed with double-hit lymphomas in the 'very good' risk group or in the 'good' risk group, while 5 of 33 patients had double-hit lymphomas in the 'poor' risk group. All the double-hit lymphoma patients possessed both nodal and extranodal involvement. The overall complete response rate was 89.3%, overall survival 87.1% and progression-free survival 75.8% over 2 years (median observation period: 644 days). The complete response rates were 93.2% for the non-double-hit lymphoma patients and 40.0% for the double-hit lymphoma patients. Significantly longer progression-free survival and overall survival were observed for the 'very good' and the 'good' risk patients than for the 'poor' risk patients. Moreover, the progression-free survival of double-hit lymphoma was significantly shorter than that of the non-double-hit lymphoma 'poor' risk patients (P = 0.016). In addition, the overall survival of the double-hit lymphoma patients also tended to be shorter than that of the non-double-hit lymphoma 'poor' risk group. CONCLUSIONS: The diagnosis of double-hit lymphoma can help discriminate a subgroup of highly aggressive diffuse large B-cell lymphomas and indicate the need for the development of novel therapeutic strategies for double-hit lymphoma.

Periodontal inflamed surface area in oral cavity associated with febrile neutropenia in patients with hematologic malignancy undergoing chemotherapy.

Febrile neutropenia (FN) is an infectious complication that develops during chemotherapy. Although the oral cavity can be an important infection route, it is unknown whether the oral environment is associated with FN. The present study examined the relationship between the oral environment using periodontal inflamed surface area (PISA), a new periodontal disease parameter, and FN in hematologic cancer patients undergoing chemotherapy. In this retrospective cohort study, 157 patients were divided into FN onset during chemotherapy (n = 75) and the FN negative groups (n = 82). The associations of risk factors related to the intraoral environment were assessed. Logistic regression analysis showed that types of blood cancer (odds ratio 1.98; P < 0.01), use of a high-risk regimen (odds ratio 4.44; P < 0.05), prophylaxis treatment with human granulocyte colony-stimulating factor (G-CSF) (odds ratio 4.15; P < 0.01) and PISA (odds ratio 1.02; P < 0.01) were independent factors associated with FN onset. Finally, propensity score matching was performed between two groups; 37 matched pairs were generated. PISA was significantly higher in the FN group than the FN negative group. There was a significant relationship between PISA and FN onset (P = 0.035). The present findings indicate that periodontitis treatment before starting cancer treatment is recommended as supportive care for preventing FN onset during chemotherapy.

Prognostic impact of CEBPA bZIP domain mutation in acute myeloid leukemia.

Mutations of CCAAT/enhancer-binding protein alpha (CEBPAmu) are found in 10% to 15% of de novo acute myeloid leukemia (AML) cases. Double-mutated CEBPA (CEBPAdm) is associated with a favorable prognosis; however, single-mutated CEBPA (CEBPAsm) does not seem to improve prognosis. We investigated CEBPAmu for prognosis in 1028 patients with AML, registered in the Multi-center Collaborative Program for Gene Sequencing of Japanese AML. It was found that CEBPAmu in the basic leucine zipper domain (bZIP) was strongly associated with a favorable prognosis, but CEBPAmu out of the bZIP domain was not. The presence of CEBPAmu in bZIP was a strong indicator of a higher chance of achieving complete remission (P < .001), better overall survival (OS; P < .001) and a lower risk of relapse (P < .001). The prognostic significance of CEBPAmu in bZIP was also observed in the subgroup with CEBPAsm (all patients: OS, P = .008; the cumulative incidence of relapse, P = .063; patients aged ≤70 years and with intermediate-risk karyotype: OS, P = .008; cumulative incidence of relapse, P = .026). Multivariate analysis of 744 patients aged ≤70 years showed that CEBPAmu in bZIP was the most potent predictor of OS (hazard ratio, 0.3287; P < .001). CEBPAdm was validated as a cofounding factor, which was overlapping with CEBPAmu in bZIP. In summary, these findings indicate that CEBPAmu in bZIP is a potent marker for AML prognosis. It holds potential in the refinement of treatment stratification and the development of targeted therapeutic approaches in CEBPA-mutated AML.

Detection of novel and recurrent conjoined genes in non-Hodgkin B-cell lymphoma.

For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.

Allogeneic hematopoietic cell transplantation using fludarabine plus myeloablative busulfan and melphalan confers promising survival in high-risk hematopoietic neoplasms: a single-center retrospective analysis.

OBJECTIVES: Optimal selection of pretransplant conditioning is crucially vital for improving survival and quality-of-life of patients who receive allogeneic hematopoietic cell transplantation (allo-HCT), particularly in those with high-risk diseases. In this study, we evaluated the efficacy and safety of recently-developed reduced-toxicity myeloablative regimen that combines fludarabine, intravenous busulfan, and melphalan (FBM). METHODS: We conducted a single-center retrospective analysis of 39 patients (23 with myeloid neoplasms and 16 with lymphoid neoplasms), with a median age of 50 (range, 17-68) years, who underwent their first allo-HCT using the FBM regimen. Graft types were bone marrow in 11, peripheral blood in 11, and cord blood in 17 patients. Cyclosporine- or tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was administered. The primary end point of the study was the overall survival rate at 2-year after transplantation. RESULTS: After a median follow-up of 910 days for the surviving patients, 2-year overall survival was 62% for the entire cohort; 73% in the low-to-intermediate-risk group and 44% in the high-to-very high-risk group classified by the refined CIBMTR Disease Risk Index. Cumulative incidences of engraftment, grade II-IV acute GVHD, chronic GVHD, relapse, and non-relapse mortality were 95%, 56%, 56%, 31%, and 17%, respectively. CONCLUSION: These results suggest that our FBM regimen can be applied to allo-HCT using various graft types and yields acceptable outcomes with relatively low non-relapse mortality in both myeloid and lymphoid neoplasms. Also, we observed a promising survival in the group of patients with high-risk diseases, warranting more accumulation of patients and longer follow-up.

Successful Treatment of Chronic Myeloid Leukemia With Dasatinib After Kidney Transplantation: A Case Report.

OBJECTIVE: Chronic myeloid leukemia (CML) is a rare malignancy in kidney transplant (KT) recipients. Although dasatinib is the first-line treatment for CML, it has inhibitory activity against CYP3A4; this might increase the blood concentration of tacrolimus (administered to KT patients for immune suppression). Furthermore, tacrolimus can also increase blood concentrations of dasatinib through P-glycoprotein inhibition. METHODS: Here, we report a case of sustained molecular remission of CML with prolonged first-line dasatinib therapy in a KT recipient being treated with tacrolimus. A 61-year-old woman developed CML-chronic phase (CML-CP) 38 months post KT. Her maintenance immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil, and methylprednisolone. Considering the potential drug interaction with tacrolimus, dasatinib was administered at a low dose of 50 mg/day. Her immune status was evaluated regularly by assessing the mixed lymphocyte reaction (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique; immunosuppressive therapy was adjusted accordingly. RESULTS: The patient achieved complete hematologic remission (CHR) after 1 month of dasatinib treatment. Six months after dasatinib treatment, she achieved a major molecular response. During the observation period, neither antibody-mediated nor acute cellular rejection were encountered in the patient. She remained in CHR with a major molecular response 12 months after the diagnosis of CML-CP. CONCLUSION: Data obtained from immune monitoring assays using CFSE-MLR helped us to successfully manage a KT recipient with CML-CP being treated with dasatinib. Drug-drug interactions are a key consideration while designing treatment regimens; such strategies would ensure that drug-drug interactions do not negatively affect the treatment outcomes.

Recurrent intragenic exon rearrangements of SOBP and AUTS2 in non-Hodgkin B-cell lymphoma.

Expression of intragenic exon rearrangements (IERs) has reportedly been detected in both normal and cancer cells. However, there have been few reports of occurrence of these rearrangements specific to neoplasms including malignant lymphoma. In this study, we detected IERs of ten genes (NBPF8, SOBP, AUTS2, RAB21, SPATA13, ABCC4, WDR7, PHLPP1, NFATC1 and MAGED1) in non-Hodgkin B cell lymphoma (B-NHL) cell line KPUM-UH1 using a high-resolution single nucleotide polymorphism array and reverse transcription polymerase chain reaction using reversely directed divergent primers within exons involved in genomic intragenic gains followed by sequencing analysis. Among them, the IERs involved in SOBP (6q21) exon 2 and 3 and AUTS2 (7q11.22) exon 2-4 were the molecular lesions specific to tumors and were frequently detected in B-NHL samples. These IERs constitute novel genetic alterations of B-NHL, which might be associated with tumorigenesis and be useful as genetic biological markers.

Combined rituximab, bendamustine, and dexamethasone chemotherapy for relapsed or refractory indolent B-cell non-Hodgkin lymphoma and mantle cell lymphoma: a multicenter phase II study.

This multicenter phase II study (UMIN000008145) aims to investigate the efficacy and safety of six cycles of combination therapy (RBD) comprising rituximab, bendamustine, and dexamethasone (DEX) for relapsed or refractory (RR) indolent B-cell non-Hodgkin lymphoma (B-NHL) and mantle cell lymphoma (MCL). Although the initial study protocol comprised 20 mg/body DEX on days 1 and 2, and 10 mg/body on days 3-5 [high-dose (HD-) DEX group], the dose of DEX was later decreased to 8 mg/body on days 1 and 2 [low-dose (LD-) DEX group] due to frequent cytomegalovirus (CMV) antigenemia and recurrent retinitis. We enrolled 33 patients, and LD-DEX and HD-DEX were administered in 15 and 18 patients, respectively. The overall response and the 3-year progression-free survival rates were 88% and 75.5%, respectively. The leading adverse event was myelosuppression. Incidence of grade 3-4 leukocytopenia, neutropenia, and lymphocytopenia was 55%, 67%, and 91%, respectively. The most frequent nonhematological adverse events were CMV antigenemia and rash (33% and 30%, respectively). Incidence of CMV antigenemia over 10/100,000 white blood cells was significantly lower with LD-DEX than that with HD-DEX (P = 0.0127). In conclusion, RBD showed significant effectiveness for RR indolent B-NHL and MCL.

Establishment and Characteristics of a Novel Mantle Cell Lymphoma-derived Cell Line and a Bendamustine-resistant Subline.

BACKGROUND/AIM: Bendamustine hydrochloride (BH) is a key therapeutic agent for mantle cell lymphoma (MCL), while the mechanism underlying BH-resistance has not been verified. MATERIALS AND METHODS: We compared molecular/biological characteristics of a newly-generated MCL-derived cell line KPUM-YY1 and its BH-resistant subline KPUM-YY1R. RESULTS: The growth-inhibitory IC50 for BH was 20 µM in KPUM-YY1 cells, while cell proliferation was not inhibited by up to 60 µM BH in KPUM-YY1R cells. Compared to KPUM-YY1 cells, gene expression profiling in KPUM-YY1R cells revealed up-regulation of 312 genes, including ABCB1 encoding P-glycoprotein (P-gp), and microsomal glutathione S-transferase 1 (MGST1). Addition of either a P-gp inhibitor or a GST inhibitor, at least partly, restored sensitivity to BH in KPUM-YY1R cells. In addition, KPUM-YY1R cells showed cross-resistance against various anti-MCL chemotherapeutics. CONCLUSION: BH resistance is mediated by overlapping mechanisms with overexpression of ABCB1 and MGST1, and is potentially accompanied by multidrug resistance in MCL.

Molecular heterogeneity in the novel fusion gene APIP-FGFR2: Diversity of genomic breakpoints in gastric cancer with high-level amplifications at 11p13 and 10q26.

Several novel fusion transcripts were identified by next-generation sequencing in gastric cancer; however, the breakpoint junctions have yet to be characterized. The present study characterized a plethora of APIP-FGFR2 genomic breakpoints in the SNU-16 gastric cancer cell line, which harbored homogeneously staining regions (hsrs) and double minute chromosomes. Oligonucleotide microarrays revealed high-level amplifications at chromosomes 8q24.1 (0.8 Mb region), 10q26 (1.1 Mb) and 11p13 (1.1 Mb). These amplicons contained MYC and PVT1 at chromosome 8q24.1, BRWD2, FGFR2 and ATE1 at chromosome 10q26, and 24 genes, including APIP, CD44, RAG1 and RAG2, at chromosome 11p13. Based on these findings, reverse transcription-polymerase chain reaction (PCR) was performed using various candidate gene primers to detect possible fusion transcripts, and several products using primer sets for the APIP and FGFR2 genes were detected. Eventually, three in-frame and two out-of-frame fusion transcripts were detected. Notably, PCR analysis of the entire genomic DNA detected three distinct genomic junctions. The breakpoints were within intron 5 of APIP, which contained three distinct breakpoints, and introns 5, 7 and 9 of FGFR2. Fluorescence in situ hybridization showed several fusion signals within hsrs using two short probes (~10-kb segments of a bacterial artificial chromosome clone) containing exons 2-5 of APIP or exons 11-13 of FGFR2. Although, for any given fusion, a multiplicity of transcripts is thought to be created by alternative splicing of one rearranged allele, the results of the present study suggested that genomic fusions of APIP and FGFR2 are generated in hsrs with a diversity of breakpoints that are then faithfully transcribed.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.