Cervical Local Cytokine Release Syndrome Following Chimeric Antigen Receptor T-cell Therapy in Patients With Relapsed or Refractory Diffuse Large B-cell Lymphoma.

The use of chimeric antigen receptor T-cell (CAR-T) therapy for hematologic malignancies is rapidly increasing, and appropriately managing adverse events (AEs) is crucial. Cytokine release syndrome (CRS) is a common AE of CAR-T therapy, characterized by systemic symptoms such as fever and respire-circulatory failure. We present two cases with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) accompanied by a rare complication of cervical local CRS as an acute inflammatory reaction at a specific site after CAR-T infusion. Case 1: A 60-year-old gentleman with diffuse large B cell lymphoma (DLBCL) developed grade 1 CRS on day one that required three doses of tocilizumab. Then he developed remarkable cervical edema as local CRS on day five. His local CRS spontaneously improved from day seven without additional therapy. Case 2: A 70-year-old gentleman with DLBCL developed grade 1 CRS on day two that required three doses of tocilizumab. Then he developed remarkable cervical edema and muffled voice as local CRS on day three. He received dexamethasone because of concerns about airway obstruction, and his local CRS improved immediately after dexamethasone administration. Before Tisa-Cel infusion, neither patients had a lymphoma lesion in their necks. To summarize, local CRS may occur at the site without lymphoma involvement after CAR-T therapy. An appropriate diagnosis and careful observation are required to determine the need for additional treatment.

EWSR1 overexpression is a pro-oncogenic event in multiple myeloma.

Multiple myeloma (MM) is cytogenetically, genetically and molecularly heterogenous even among subclones in one patient, therefore, it is essential to identify both frequent and patient-specific drivers of molecular abnormality. Following previous molecular investigations, we in this study investigated the expression patterns and function of the Ewing sarcoma breakpoint region 1 (EWSR1) gene in MM. The EWSR1 transcriptional level in CD138-positive myeloma cells was higher in 36.4% of monoclonal gammopathy of undetermined significance, in 67.4% of MM patients compared with normal plasma cells, and significantly higher in ten human myeloma-derived cell lines (HMCLs) examined. EWSR1 gene knockdown caused growth inhibition with an increase of apoptotic cells in NCI-H929 and KMS-12-BM cells. Gene expression profiling using microarray analysis suggested EWSR1 gene knockdown caused transcriptional modulation of several genes associated with processes such as cell proliferation, cell motility, cell metabolism, and gene expression. Of particular, EWSR1 gene knockdown caused upregulation of let-7c and downregulation of its known targets K-RAS and AKT. Finally, our analysis using community database suggested that high EWSR1 expression positively associates with poor prognosis and advanced disease stage in MM. These findings suggest that EWSR1 overexpression is a pro-oncogenic molecular abnormality that may participate in MM progression.

Lenalidomide and pomalidomide potently interfere with induction of myeloid-derived suppressor cells in multiple myeloma.

An increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) is associated with disease progression and treatment resistance in multiple myeloma (MM). We investigated the mechanisms underlying MDSC induction, and sought to discover a strategy for prevention of MDSC induction in MM. Using a transwell co-culture system, four of nine examined human myeloma-derived cell lines (HMCLs) were potent in inducing monocytic (M)-MDSCs from normal peripheral blood mononuclear cells (PBMCs). As the results, we identified that secretion of C-C motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF) by myeloma cells is a prerequisite for induction of MDSCs in MM. The immunomodulatory drug (IMiD) compounds, such as lenalidomide (LEN) and pomalidomide (POM), were identified as potent inhibitors of MDSC induction through bidirectional molecular effects of cereblon (CRBN)-dependent and -independent downregulation of CCL5 and MIF in myeloma cells; and downregulation of C-C motif chemokine receptor 5, a receptor for CCL5, and induction of interferon regulatory factor 8, a critical transcription factor for monocytic differentiation, in PBMCs. In the present study of the molecular mechanisms underlying MDSC induction, we identified a novel effect of LEN and POM of inhibiting MDSC induction via overlapping regulatory effects in myeloma cells and normal PBMCs.

Tuberculosis Peritonitis During Treatment of Polycythemia Vera with Ruxolitinib.

Ruxolitinib is a selective JAK1/2 inhibitor that is widely used for the treatment of myeloproliferative neoplasms (MPNs), including myelofibrosis and polycythemia vera (PV). Despite its clinical efficacy for MPNs, ruxolitinib possesses immunosuppressive properties that potentially increase the risks for opportunistic infection, such as mycobacterium tuberculosis (MTB) infection, and reactivation of occult viral infection. Herein, we report the case of a 76-year-old male with PV who developed tuberculosis peritonitis under ruxolitinib therapy for 28 weeks. While previous studies and case reports have suggested an increased risk of MTB infection of various organs during ruxolitinib treatment of MPNs, this case is apparently the first of tuberculosis peritonitis in a patient with MPN treated with ruxolitinib. A review of previous case reports suggests the need for careful observation for MTB from the relatively early phase of ruxolitinib treatment, given that the median duration from the start of ruxolitinib treatment to the emergence of MTB was 20 weeks (range: 3-88 weeks). Clinicians should consider tuberculosis peritonitis as a differential diagnosis when patients with MPN treated with ruxolitinib develop infectious abdominal symptoms.

Tumor-specific transcript variants of cyclin D1 in mantle cell lymphoma and multiple myeloma with chromosome 11q13 abnormalities.

Cyclin D1 (CCND1) overexpression is an early and unifying oncogenic event in mantle cell lymphoma (MCL) and multiple myeloma (MM) with chromosome 11q13 abnormalities. Herein, we report newly discovered transcript variants of the CCND1 gene in MCL and MM cells with chromosome 11q13 abnormalities. These transcript variants, designated CCND1.tv., covered the full-length coding region of CCND1 with longer 5'-untranslated regions (5'-UTRs) of CCND1 and occasionally contained a novel exon. CCND1.tv. was specifically detectable in patient-derived primary MCL or MM cells with chromosomal translocation t(11;14)(q13;q32), but not in t(11;14)-negative cells. The lengths of the 5'-UTR sequences of CCND1.tv. differed among patients and cell lines. Introduction of CCND1.tv. led to increased expression of normal-sized CCND1 protein in HEK293 cells. Furthermore, mTOR inhibition by rapamycin or serum starvation reduced ectopic expression of CCND1.tv.-derived CCND1 protein, but not 5'-UTR less CCND1-derived CCND1 protein in HEK293 cells, suggesting that the protein expression of CCND1.tv. is regulated by the mTOR pathway. Our results suggest that the aberrant expression of CCND1.tv. may contribute to the understanding of the pathogenesis of MCL and MM with 11q13 abnormalities.

BRD4-Regulated Molecular Targets in Mantle Cell Lymphoma: Insights into Targeted Therapeutic Approach.

BACKGROUND: Since bromodomain-containing protein 4 (BRD4) facilitates the transcription of genes important for neoplastic cells in a cancer-type specific manner, BRD4-regulated molecules may also include therapeutic targets for mantle cell lymphoma (MCL), a treatment-refractory subtype of malignant lymphoma. MATERIALS AND METHODS: In order to uncover direct BRD4-regulated targets in MCL, we performed integrated analysis using the pathway database and the results of both gene-expression profiling and chromatin immunoprecipitation with parallel sequencing for BRD4. RESULTS: Treatment with BRD4 inhibitor I-BET151 exerted a dose-dependent inhibitory effect on cell proliferation in MCL cell lines. BRD4 was found to directly regulate series of genes involved in the B-cell receptor (BCR) signaling pathway, including B-cell linker (BLNK), paired box 5 (PAX5), and IKAROS family zinc finger 3 (IKZF3), and several oncogenes, such as MYB. Indeed, the combinatory inhibition of BCR pathway and IKZF showed an additive antitumor effect. CONCLUSION: Concomitant targeting multiple BRD4-regulated molecules may constitute a rational therapeutic strategy for MCL.

Sequential therapy of four cycles of bortezomib, melphalan, and prednisolone followed by continuous lenalidomide and dexamethasone for transplant-ineligible newly diagnosed multiple myeloma.

The combinations of melphalan, bortezomib, and prednisolone (VMP) and of lenalidomide and dexamethasone (Rd) are standard treatment strategies for transplant-ineligible newly diagnosed multiple myeloma (NDMM). To make the most of these two strategies, we investigated the efficacy and feasibility of first-line treatment with 4 cycles of VMP followed by continuous Rd therapy in a multi-institutional phase 2 study in Japanese patients with transplant-ineligible NDMM. Thirty-six patients of median age 74 years old with NDMM initially received 35-day cycles of VMP: oral melphalan (6 mg/m2) and prednisolone (60 mg/m2) on days 1 to 4 and bortezomib (1.3 mg/m2) on days 1, 8, 15, and 22. After 4 cycles of VMP, treatment was switched to 28-day cycles of Rd, which was continued until disease progression or emergence of an unacceptable adverse event (AE) in 33 patients, while one patient who achieved CR after VMP continued VMP at the physician's discretion. The overall response rates after VMP and after Rd were 66.7% and 86.1%, including CR rates of 5.6% and 36.1%, respectively. In a median follow-up period of 34.3 months, the progression-free survival and overall survival rates at 3 years were 43.2% and 81.3%, respectively. Grade 3-4 hematological AEs included neutropenia (39% with VMP and 24% with Rd) and thrombocytopenia (11% with VMP and 3% with Rd). There was no death due to an AE. In conclusion, sequential therapy with VMP followed by Rd is effective and mostly feasible for transplant-ineligible NDMM. The study is registered as UMIN000034815.

Prediction of delayed platelet engraftment after autologous stem cell transplantation for B-cell non-Hodgkin lymphoma.

Delayed platelet engraftment (DPE) is occasionally observed despite prompt neutrophil engraftment after autologous peripheral blood stem cell transplantation (auto-PBSCT). To identify risk factors for DPE and to develop a simple and clinically applicable system for predicting the time required for platelet recovery, we conducted a multi-institutional retrospective study in 144 patients with B-cell non-Hodgkin lymphoma who underwent auto-PBSCT. In a median observation period of 930 days (range: 25-5272 days), 139 patients successfully achieved platelet engraftment (≥50.0 × 109/L). The median duration for platelet engraftment was 19 days, and 130 patients had platelet engraftment within 40 days after auto-PBSCT; however, the other 14 patients failed to achieve platelet engraftment within 60 days. These 14 patients with DPE required a significantly greater number of apheresis procedures and had a lower pre-apheresis absolute lymphocyte count (PA-ALC) compared to those without DPE. Importantly, multivariate analysis revealed that the number of transplanted CD34+ cells (≤2.0 × 106/kg), number of required apheresis procedures (≥3 days), and PA-ALC (≤1.0 × 109/L) were independently associated with a longer time for platelet engraftment after auto-PBSCT. By incorporating these three independent factors as variables, we generated a new scoring system for prediction of the time and probability for platelet engraftment after auto-PBSCT.

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.

A novel method of amplified fluorescent in situ hybridization for detection of chromosomal microdeletions in B cell lymphoma.

Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.

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

Despite 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.

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.