Prophylactic antiviral therapy for hepatitis B virus surface antigen-positive patients with diffuse large B-cell lymphoma treated with rituximab-containing chemotherapy.

We conducted a nationwide retrospective analysis of 116 hepatitis B virus (HBV) surface antigen (HBsAg)-positive patients with diffuse large B-cell lymphoma (DLBCL) and 278 HBsAg-negative patients with DLBCL, as a control cohort, who received rituximab-containing regimens as an induction chemotherapy at 30 Japanese medical centers between January 2004 and December 2014. Hepatitis was defined as an absolute serum alanine aminotransferase (ALT) level of ≥100 U/L. HBV reactivation-related hepatitis was defined as hepatitis with an absolute serum HBV DNA level of ≥3.3 log IU/mL or an absolute increase of ≥2 log compared with the baseline value. HBsAg-positive patients were divided into three groups based on anti-HBV prophylactic therapy: no nucleos(t)ide analogue (non-NA, n = 9), lamivudine (LAM, n = 20), and entecavir (ETV, n = 87). The 4-year cumulative incidence (CI) of hepatitis in HBsAg-positive and HBsAg-negative patients was 21.1% and 14.6% (P = .081), respectively. The 4-year CI of HBV reactivation-related hepatitis was higher in HBsAg-positive patients than in HBsAg-negative patients (8.0% vs 0.4%; P < .001). Among HBsAg-positive patients, the 4-year CI of HBV reactivation-related hepatitis was the highest in the non-NA group (33.3%), followed by the LAM (15.0%) and ETV (3.8%) groups (P < .001). Of note, 3 non-NA patients (33%) and 1 LAM patient (5%) (but no ETV patients) died due to HBV hepatitis. Based on Cox multivariate analysis, HBsAg positivity was not associated with poor overall survival. Prophylactic use of ETV would reduce the occurrence of HBV reactivation-related hepatitis and mortality in HBsAg-positive DLBCL patients receiving rituximab-containing chemotherapy.

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

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.

Safety and persistence of WT1-specific T-cell receptor gene-transduced lymphocytes in patients with AML and MDS.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

Effect of L-Leucine Therapy on Hematopoietic Function in Elderly Myelodysplastic Syndrome Patients.

Patients with myelodysplastic syndrome (MDS) often require blood transfusion and anticancer therapy; however, elderly patients are intolerant to the associated side effects of anticancer therapy. Because L-leucine can be used to treat Diamond-Blackfan anemia, which is caused by defects in ribosomal protein (RP) genes, resulting in increased in vivo hemoglobin synthesis, it is possible that some MDS patients who have aberrations in their RP genes could also be effectively treated with L-leucine. In the present study, we investigated the effects of L-leucine on hematopoietic function (reticulocyte count), red blood cell count, and hemoglobin level in MDS patients. We administered L-leucine (1.8 g, twice daily, 3 d/week) with oral vitamin B6 supplements to a final cohort of eight MDS patients for 15 (interquartile range: 11-18) weeks. We assessed the patients at 10 ± 2 weeks after therapy initiation. Only the absolute reticulocyte count was affected, improving in 6/8 (75%) patients. The median absolute reticulocyte count was 3.5 × 104 (range: 2.7-6.4 × 104) cells/µL, an increase of 0.5 × 104 (range: 0.2-0.7 × 104) cells/µL. At 10 weeks, there was only one case of an improved hemoglobin level. Non-hematological adverse events of grade 3 were observed one raised triglycerides. These data suggest that L-leucine has little effect on MDS. However, it may contribute to the recovery of hematopoietic function, futher study be desired.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma arising in patient with a history of EBV-positive mucocutaneous ulcer and EBV-positive nodal polymorphous B-lymphoproliferative disorder.

Elderly patients with Epstein-Barr virus (EBV) infection are at increased risk for developing B-cell lymphoproliferative disorder (B-LPD) due to immunosenescence. Here, we describe a case of a 75-year-old man who developed an EBV-positive (EBV+) mucocutaneous ulcer (EBVMCU) in the gingiva with spontaneous regression. Eighteen months after regression, he had a cervical lymph node enlargement that was diagnosed as EBV+ nodal polymorphous B-LPD, Ann Arbor stage IA. Clinicians decided to observe his clinical course without any treatment. Fourteen months later, the patient developed EBV-positive diffuse large B-cell lymphoma (DLBCL), Ann Arbor stage IIA, and received six courses of age-adjusted dose chemotherapy and achieved a complete remission. No evidence of a clonal relationship was found among these three lesions by standard polymerase chain reaction (PCR) analysis for immunoglobulin heavy chain. However, they all had expression of PD-L1 in the EBV+ large B-cells and Hodgkin Reed-Sternberg-like cells. This is the first case report of a PD-L1-positive (PD-L1+) EBVMCU and the development of multiple EBV-driven B-LPDs in the setting of immunosenescence within a 32-month period.

Reappraisal of Epstein-Barr virus (EBV) in diffuse large B-cell lymphoma (DLBCL): comparative analysis between EBV-positive and EBV-negative DLBCL with EBV-positive bystander cells.

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.

The prognostic significance of EBV DNA load and EBER status in diagnostic specimens from diffuse large B-cell lymphoma patients.

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 EBER-negative 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. Copyright © 2015 John Wiley & Sons, Ltd.

ETV6-LPXN fusion transcript generated by t(11;12)(q12.1;p13) in a patient with relapsing acute myeloid leukemia with NUP98-HOXA9.

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.

Influence of R-CHOP Therapy on Immune System Restoration in Patients with B-Cell Lymphoma.

OBJECTIVE: To assess the immunosuppressive effect of R-CHOP in patients with B-cell lymphoma at 2 years. METHODS: Parameters of humoral and cell-mediated immunity were assessed in 89 patients with diffuse large B-cell lymphoma or follicular lymphoma before and after 6-8 cycles of R-CHOP-14 or R-CHOP-21 regimen. RESULTS: Data on pre- and posttreatment serum IgG (sIgG) levels were available for all 89 patients, while the corresponding data on serum CD20+, CD3+, CD4+, and CD8+ lymphocyte counts were available in only 43. Median sIgG levels significantly decreased from 1,221 mg/dl (baseline) to 733 mg/dl (after chemotherapy) (p < 0.001). Although CD20+ and CD4+ cell counts decreased (p < 0.001), no significant effect of chemotherapy on CD3+ and CD8+ cell counts was observed. CD20+ cell counts were restored to baseline levels at the 12-month follow-up. sIgG levels and CD4+ cell counts were not completely restored at 24 months, indicating a sustained immunosuppressive effect of R-CHOP in these patients. The incidence of infections over the 2-year period was 16.3-23.6%. CONCLUSION: The immunosuppressive effect of R-CHOP in newly diagnosed cases of B-cell lymphoma tends to persist for >2 years, although sIgG levels were restored more quickly than CD4+ cell counts.

Prognostic significance of Epstein-Barr virus DNA detection in pretreatment serum in diffuse large B-cell lymphoma.

It is still a matter of debate whether detection of Epstein-Barr virus (EBV) DNA in pretreatment serum has clinical implications for diffuse large B-cell lymphoma. For this study, we measured EBV DNA load in pretreatment serum from 127 diffuse large B-cell lymphoma patients without any underlying immunodeficiency to evaluate its effects on clinical manifestations and prognosis. Anthracycline-based chemotherapy in combination with rituximab was given as initial therapy for 119 patients (94%). Epstein-Barr virus DNA was detected in 15 patients (12%), who were older (P = 0.005) and tended to be at a more advanced disease stage (P = 0.053). They showed significantly worse progression-free survival (PFS) and overall survival (OS) than other patients (P < 0.001 each). This effect remained significant (P = 0.004 and P = 0.027, respectively) after adjustment for age, lactate dehydrogenase, performance status, stage, and extranodal sites. The status of EBV-encoded small RNA in situ hybridization was known for 123 patients; 6 of 8 positive patients (75%) and 9 of 115 negative patients (8%) had detectable EBV DNA in pretreatment serum. While patients positive for EBV-encoded small RNA had significantly worse PFS and OS than negative patients (P = 0.001 and P = 0.029, respectively), EBV DNA detection in pretreatment serum was associated with poorer PFS and OS even for the 115 patients negative for EBV-encoded small RNA (P < 0.001 each). These findings suggest that EBV DNA detection in pretreatment serum may have an adverse prognostic impact for patients with diffuse large B-cell lymphoma.

NUP214-RAC1 and RAC1-COL12A1 Fusion in Complex Variant Translocations Involving Chromosomes 6, 7 and 9 in an Acute Myeloid Leukemia Case with DEK-NUP214.

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.

A varicella outbreak in B-cell lymphoma patients receiving rituximab-containing chemotherapy.

Varicella, characterized by a vesicular rash, occurs primarily in young children. Although older individuals can also be affected or vaccinated, outbreaks among adults are rare. We investigated a small outbreak of varicella in B-cell lymphoma patients for elucidation of risk factor of the disease. We experienced four cases of varicella after an index herpes zoster case. All varicella cases were confirmed varicella zoster virus (VZV) infection by PCR. All varicella cases occurred in diffuse large B-cell lymphoma patients receiving rituximab-containing chemotherapy. On the other hand, only three of the 18 non-varicella patients in the same room were receiving rituximab-containing chemotherapy (P = 0.005). All varicella patients had detectable serum anti-varicella zoster virus IgG antibodies before chemotherapy. Even in the presence of neutralizing antibodies to the virus, lymphoma patients treated with rituximab-containing chemotherapy can possibly become re-infected with varicella. These findings suggest that zoster patients should be strictly isolated in hematology and oncology ward, and prophylactic acyclovir should be considered for such patients when exposed to zoster/varicella.

BCOR as a novel fusion partner of retinoic acid receptor alpha in a t(X;17)(p11;q12) variant of acute promyelocytic leukemia.

The majority of acute promyelocytic leukemia (APL) cases are characterized by the presence of a promyelocytic leukemia-retinoic acid receptor alpha(RARA) fusion gene. In a small subset, RARA is fused to a different partner, usually involved in regulating cell growth and differentiation. Here, we identified a novel RARA fusion transcript, BCOR-RARA, in a t(X;17)(p11;q12) variant of APL with unique morphologic features, including rectangular and round cytoplasmic inclusion bodies. Although the patient was clinically responsive to all-trans retinoic acid, several relapses occurred with standard chemotherapy and all-trans retinoic acid. BCOR is a transcriptional corepressor through the proto-oncoprotein, BCL6, recruiting histone deacetylases and polycomb repressive complex 1 components. BCOR-RARA was found to possess common features with other RARA fusion proteins. These included: (1) the same break point in RARA cDNA; (2) self-association; (3) retinoid X receptor alpha is necessary for BCOR-RARA to associate with the RARA responsive element; (4) action in a dominant-negative manner on RARA transcriptional activation; and (5) aberrant subcellular relocalization. It should be noted that there was no intact BCOR found in the 45,-Y,t(X;17)(p11;q12) APL cells because they featured only a rearranged X chromosome. These results highlight essential features of pathogenesis in APL in more detail. BCOR appears to be involved not only in human congenital diseases, but also in a human cancer.

[Therapeutic choice for the chronic myeloid leukemia patients in chronic phase showing late suboptimal response to imatinib].

The response criteria proposed by European Leukemia Net are useful to predict the prognosis of de novo chronic myeloid leukemia (CML) patients in the chronic phase (CP) treated with imatinib. However, the clinical significance of late suboptimal response, which is defined as the achievement of CCgR without MMR after 18 months, is controversial. In this study, we retrospectively analyzed the clinical courses of 16 CML-CP patients, who satisfied the criteria for late suboptimal response. The median duration of imatinib treatment was 62 (25∼87) months. The median starting dose of imatinib was 400 mg/day. Imatinib dose was escalated to 600∼800 mg/day in 10 patients for various reasons. Among 4 patients who continued high-dose imatinib for late suboptimal response, 2 patients subsequently achieved MMR, and BCR-ABL mRNA transcript levels were decreasing in 2 patients. However, imatinib was kept at 300 or 400 mg/day in 6 patients. Among these six patients, 4 patients achieved MMR, while 2 failed to achieve MMR. None of 16 patients progressed to the acute phase or blast phase. Imatinib dose escalation was effective for late suboptimal response. Furthermore, a second tyrosine kinase inhibitor such as nilotinib may be more potent to reduce the risk of disease progression by achieving earlier MMR.

Rearrangement of VPS13B, a causative gene of Cohen syndrome, in a case of RUNX1-RUNX1T1 leukemia with t(8;12;21).

Variant chromosomal translocations associated with t(8;21) are observed in 3-4% of acute myeloid leukemia (AML) cases with a RUNX1-RUNX1T1 fusion gene. However, the molecular events that occur in variants of t(8;21) are not well characterized. In the present study, we report genetic features of a variant three-way translocation of t(8;12;21)(q22;p11;q22) in a patient with AML. In this patient, leukemia cells lacked azurophilic granules, which does not correspond with the classic features of t(8;21). RNA-seq analysis revealed that TM7SF3 at 12p11 was fused to VPS13B at 8q22 and VPS13B to RUNX1, in addition to RUNX1-RUNX1T1. VPS13B was located near RUNX1T1 and both were localized at the same chromosomal bands. The reading frames of TM7SF3 and VPS13B did not match to those of VPS13B and RUNX1, respectively. Disruption of VPS13B causes Cohen syndrome, which presents intermittent neutropenia with a left-shifted granulopoiesis in the bone marrow. Disruption of VPS13B may thus cause the unusual features of RUNX1-RUNX1T1 leukemia. Our case indicates that rearrangement of VPS13B may be additional genetic events in variant t(8;21).