BRAF V600E-induced distinct DNA damage response defines the therapeutic potential of p53 activation for TP53 wild-type colorectal cancer.

BRAF V600E, one of the most frequent mutations in the MAPK pathway, confers poor prognosis to colorectal cancers (CRCs), partly because of chemotherapeutic resistance. Oncogene-induced DNA damage responses (DDRs) that primarily activate p53 are important mechanistic barriers to the malignant transformation of cells; however, the mechanism underlying this impairment in cancer remains unknown. Here, we evaluated the responses of BRAFV600E-induced DDRs in two CRC cell lines, SW48 and LIM1215, both of which harbor wild-type TP53, KRAS, and BRAF. BRAFV600E transduction exhibited distinct phenotypes in these cells: SW48 cell proliferation markedly decreased, whereas that of LIM1215 increased. BRAFV600E expression induced the activation of oncogene-induced DDR signaling in SW48 cells, but not in LIM1215 cells, whereas chemotherapeutic agents similarly activated DDRs in both cell lines. Knockdown experiments revealed that these responses in SW48 cells were mediated by p53-p21 pathway activation. Comet assay (both alkaline and neutral) revealed that BRAFV600E increased single-strand breaks to the same extent in both cell lines; however, in case of LIM1215 cells, it only facilitated double-strand breaks. Furthermore, the proliferation of LIM1215 cells, wherein no oncogene-induced DDRs occurred, was synergistically inhibited upon MDM2 inhibitor-mediated p53 activation combined with MEK inhibition. Taken together, these distinct DDR signaling responses highlight the novel characteristics of BRAFV600E-mutated CRC cells and define the therapeutic potential of p53 activation combined with MAPK inhibition against TP53 wild-type CRC harboring a BRAFV600E mutation.

Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

BACKGROUND: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. METHODS: We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). RESULTS: Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. CONCLUSIONS: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Epstein-Barr virus-negative extranodal "true" natural killer-cell lymphoma harbouring a KDM6A mutation.

Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL) is an extranodal aggressive T or NK-cell lymphoma that is characteristically associated with Epstein-Barr virus (EBV) infection and cytotoxic tissue-destructive features. Although ENKTL is described as a distinct entity according to the 2008 WHO classification, a considerable complexity is associated with the differential diagnosis of other T-cell lymphomas with respect to tumour cell origins, locations, and the presence of EBV infection, as well as molecular and cytogenetic abnormalities. Here, we report a rare case of EBV-negative ENKTL, where the absence of EBV in the true NK-lineage cells was confirmed by extensive phenotypic and genotypic analyses. Furthermore, using the next-generation sequencing approach, we identified mutations in the tumour suppressor genes KDM6A and TP53. The clinicopathological characteristics were almost similar to those of EBV-positive ENKTL, except for the absence of EBV and histologically apparent angioinvasiveness. This is the first reported ENKTL case with mutations in the KDM6A gene. KDM6A is one of the histone-modifying genes that are mutated in many human diseases including haematological cancers. Epigenetic regulation of gene expression has recently been demonstrated in ENKTL, and a similar pathway is thought to play an oncogenic role in EBV-negative ENKTL. Our report shows the extent of comprehensive examination required before making a definitive diagnosis for NK- and T-cell neoplasms and broadens the therapeutic options for potential targets.

Efficacy of chemotherapy in extremely elderly patients with diffuse large B-cell lymphoma.

The safety and effective dose of chemotherapy in treating non-Hodgkin lymphoma in elderly patients is yet to be established. In this study, we assessed the prognosis of diffuse large B-cell lymphoma (DLBCL) in elderly patients (≥75 years) treated with an optimal dose of R-CHOP. No significant differences were observed in progression-free survival between elderly patients and patients aged <74 years with DLBCL. Furthermore, no differences were observed between full-dose R-CHOP and 80% dose R-CHOP groups. Median relative dose intensity was 0.80 in elderly patients with DLBCL. Thus, our data suggested that 80% dose R-CHOP is tolerable and effective in these patients.

BCL2 expression in DLBCL: reappraisal of immunohistochemistry with new criteria for therapeutic biomarker evaluation.

Overexpression of the BCL2 is associated with a poor prognosis in diffuse large B-cell lymphoma (DLBCL). The assessment of MYC immunohistochemistry (IHC) is becoming optimized, whereas the criteria for BCL2 positivity are highly variable. Furthermore, data on the frequency and prognostic value of BCL2 positivity are conflicting. We aimed to evaluate BCL2 expression by IHC and assess the prognostic significance of the histopathologically scored BCL2 expression in 456 patients with DLBCL uniformly treated with standard immunochemotherapy (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone, R-CHOP). We initially designed 4-grade BCL2 scoring criteria, from 0 to 3+, and found that ∼40% of DLBCL showed strong BCL2 expression (score 3+). The scores from the pathologist's visual estimation were confirmed to be reliable using a digital image analysis. A retrospective survival analysis revealed that BCL2 score 3+ was a significant prognostic factor independent of the international prognostic index (IPI), the IHC-determined cell of origin, and the MYC protein/rearrangement status in a training set (n = 218). The adverse prognostic impact of BCL2 score 3+ was confirmed in a validation set (n = 238). We also developed a prognostic model consisting of 3 groups with a combined BCL2 score and MYC protein/rearrangement status. Patients with BCL2 score 3+ showed a higher treatment failure rate; therefore, alternative therapeutic strategies should be considered for these patients. A highly selective BCL2 inhibitor, venetoclax, was recently introduced as breakthrough therapy. Our BCL2 scoring system could readily be used by pathologists to evaluate patients with DLBCL who might benefit from BCL2-targeted therapies.

Incidence and risk factors of febrile neutropenia in patients with non-Hodgkin B-cell lymphoma receiving R-CHOP in a single center in Japan.

PURPOSE: The incidence of and risk factors for febrile neutropenia (FN) in Japanese non-Hodgkin B-cell lymphoma (B-NHL) patients receiving rituximab, cyclophosphamide, doxorubicin, vincristine, and predonisolone (R-CHOP) chemotherapy are unknown. We conducted this study to address this issue. METHODS: In this single-center, retrospective, observational study, 466 patients with B-NHL who completed an R-CHOP regimen within a 7-year period and who planned to undergo at least three cycles of this regimen were analyzed. The following FN-related factors were assessed: fever, infection, disease state, neutrophil count, and prophylactic interventions such as use of antibiotics and/or granulocyte colony-stimulating factor (G-CSF). We simulated the FN incidence and 95% confidence interval (CI) of patients without prophylaxis with G-CSF (cycle 1) using bootstrap sampling. RESULTS: The incidence of FN was 9.1% (42 of 462) in cycle 1 and 12.3% (57 of 462 patients) throughout all cycles, with 73.7% (42/57) developing FN during cycle 1. Risk factors for FN among patients with B-NHL treated with R-CHOP were albumin <35 g/L (p = 0.0047), relative dose intensity <85% (p = 0.0007), and lack of prophylaxis with G-CSF (p = 0.0006) in cycle 1. In the simulation analysis, the estimated FN incidence in cycle 1 was 16.2% (95% CI [10.9-22.2]). CONCLUSIONS: At 9.1% in cycle 1 and 12.3% throughout all cycles, the incidence of FN was lower than previously reported, possibly reflecting the appropriate use of G-CSF in this clinical setting. For patients with risk factors, the prophylaxis with G-CSF may decrease the occurrence of FN.

Treatments and Outcomes of Patients With Extranodal Natural Killer/T-Cell Lymphoma Diagnosed Between 2000 and 2013: A Cooperative Study in Japan.

Purpose To elucidate the management and outcomes of patients with extranodal natural killer/T-cell lymphoma, nasal type (ENKL), who were diagnosed between 2000 and 2013 in Japan. Patients and Methods Data from 358 patients with ENKL diagnosed between 2000 and 2013 from 31 institutes were retrospectively analyzed. Results Patients' median age was 58 years, and 257 (72%) had localized disease. The most common first-line treatment was radiotherapy with dexamethasone, etoposide, ifosfamide, and carboplatin (RT-DeVIC) (66%) for localized ENKL and L-asparaginase-containing chemotherapy (30%) for advanced ENKL. With a median follow-up of 5.8 years, overall survival (OS) rates at 5 years for localized and advanced ENKL were 68% and 24%, respectively. The prognostic index of natural killer lymphoma was validated in our study, although only 4% of patients with localized ENKL were classified as high risk. With a median follow-up of 5.6 years, OS and progression-free survival at 5 years in the 150 patients who received RT-DeVIC in clinical practice were 72% (95% CI, 63% to 78%) and 61% (95% CI, 52% to 69%), respectively. Toxicities of RT-DeVIC were comparable to those in a previous trial. Multivariate analysis in patients with localized ENKL who received RT-DeVIC identified elevated soluble interleukin-2 receptor as an independent predictive factor for worse OS and progression-free survival (adjusted hazard ratios, 2.28 and 2.46; 95% CI, 1.24 to 4.23 and 1.42 to 4.28; P = .008 and .0014, respectively). Conclusion Favorable OS in response to new treatments was demonstrated in a large number of patients. Improved treatment approaches are needed for localized ENKL exhibiting elevated pretreatment soluble interleukin-2 receptor.

Frequent BCOR aberrations in extranodal NK/T-Cell lymphoma, nasal type.

Extranodal natural killer/T cell lymphoma (ENKTL) is a rare subtype of lymphoma. Recurrent mutations in the JAK-STAT pathway, recently reported in ENKTL cases, are interesting in terms of both pathogenesis and inhibitor therapy. However, the frequencies of these mutations are low and variable among reports, and other pathognomonic mutations in ENKTL remain to be elucidated. In the present study, targeted capture sequencing of 602 cancer-related genes from 25 frozen ENKTL samples was performed, 11 of which were matched to normal samples. Several recurrent somatic mutations involving BCOR (32%), TP53 (16%), DDX3X (12%), FAT4 (8%), NRAS (8%), MLL3 (12%), and MIR17HG (8%) were identified. The pattern of BCOR aberrations (1 nonsense and 5 frame-shift mutations, a mutation leading to a splicing error, and gene loss) suggested that loss of function of BCOR was the functionally important outcome of such changes. The literature was reviewed and the public data on BCOR aberrations was reanalyzed and it was found that the aberrations were frequently found in myeloid neoplasms, but, interestingly, were highly specific to ENKTL among lymphoid malignancies. Given the high frequency and pattern of aberration, BCOR is likely to play an important role in ENKTL pathogenesis as a tumor suppressor gene.

Absolute peripheral monocyte count at diagnosis predicts central nervous system relapse in diffuse large B-cell lymphoma.

Recently, elevated peripheral blood monocyte counts at diagnosis have been shown to be an independent marker associated with poor prognosis in patients with both non-Hodgkin and Hodgkin lymphoma. In this study, we retrospectively analyzed the data from a total of 550 patients with diffuse large B-cell lymphoma and evaluated the relationship between central nervous system relapse and absolute monocyte counts at diagnosis. Twenty-six patients developed central nervous system relapse. The central nervous system relapse-free survival rate was significantly lower in patients with the absolute monocyte counts ≥ 0.51 × 10(9)/L (87.8% versus 96.4%; P<0.001). This association was independently significant after adjusting for other significant factors, including systemic relapse as a time-dependent covariate by multivariate analysis (hazard ratio 2.46; 95% confidence intervals 1.05-5.75; P=0.039). These results suggest that the absolute monocyte count at diagnosis is an independent significant risk factor for central nervous system relapse in patients with diffuse large B-cell lymphoma.

Rapid progression of anemia related to tumor-lysis syndrome associated with bortezomib treatment in myeloma patients.

OBJECTIVE: Tumor-lysis syndrome is a rare complication in patients with multiple myeloma. However, bortezomib treatment for myeloma is often associated with tumor-lysis syndrome. METHODS: We developed an index called the rapid anemia progression index, which represents the duration and progression of anemia, to evaluate risk factors for tumor-lysis syndrome. We retrospectively reviewed 35 relapsed or refractory myeloma patients treated with bortezomib-containing treatment in our institution. We analyzed various parameters, including albumin, lactase dehydrogenase, ß2-microglobulin and creatinine, similar to the rapid anemia progression index, and evaluated the risk factors for tumor-lysis syndrome associated with bortezomib by the Cairo-Bishop definition. RESULTS: Clinical tumor-lysis syndrome occurred in six patients (17.1%). Tumor-lysis syndrome occurred during the first course of bortezomib-containing treatment among all the patients. The result of the area under the receiver operating characteristic curve for the rapid anemia progression index was 0.759 (P = 0.049). The rapid anemia progression index was more accurate than the index of lactate dehydrogenase, ß2-microglobulin, albumin and creatinine according to the receiver operating characteristic curve. For a cut-off point of -1.12 for the rapid anemia progression index, the sensitivity and specificity were 66.7 and 82.8%, respectively. CONCLUSIONS: The rapid anemia progression index is related to clinical tumor-lysis syndrome associated with bortezomib treatment for multiple myeloma patients with a cut-off point of -1.12 g/dl/month.

Infusion Rate Escalation Study of Rituximab in Patients with CD20+ B-Cell Lymphomas: A Single Institution Analysis in Japan.

Background. To determine the maximum tolerable infusion rate of rituximab, and investigate the safety and feasibility of rapid infusion of rituximab for patients with CD20 positive B-cell lymphomas (CD20+NHL). Patients and Methods. 18 patients with CD20+NHL were registered. This study had six cohorts of administration rate of rituximab. The median age was 56 years (range, 38-79), and five of 18 patients were male. Two patients (11%) with diffuse large B-cell lymphoma were receiving R-CHOP therapy, two (11%) with indolent lymphoma were receiving R-CVP therapy, and 14 (78%) with indolent lymphoma were receiving rituximab as maintenance therapy. Results. A total of 88 cycles of rituximab was administered. Rapid infusion of rituximab was well tolerated, with only one grade 3 leukocytepenia and one grade 4 neutropenia. Four patients (22%) developed grade 1 infusion-related toxicities at the first administration of rituximab. No patient with severe drug-related events was observed. Conclusions. We determined that the maximum tolerable infusion rate of rituximab is 300 mL/h (under 700 mg/h), and confirmed that administration of over 60 minutes is safe and feasible. We recommend rapid administration of rituximab for practice setting in patients with CD20+NHL being treated with rituximab or rituximab-containing chemotherapy. (Clinical trial no. JFCR2009-1027).

Prognostic value of high thymidine kinase activity in patients with previously untreated diffuse large B-cell lymphoma treated by rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone.

The purpose of this study was to investigate prognostic factors for overall survival (OS) among patients with previously untreated diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP). We evaluated four biological parameters, including thymidine kinase (TK) activity. This study included 183 patients. The median level of TK was 14.0 IU/L, which we chose as the cut-off. After a median follow-up time of 50.0 months, the OS rate at 4 years in the high and low TK arm were 46.7% and 66.7%, respectively (p = 0.001). By multivariate analysis, OS was significantly inferior in the high TK arm (hazard ratio 2.705; p = 0.045). The complete response (CR) rate in the high TK arm was significantly worse than in the low TK arm. OS was significantly better in patients who had achieved CR than in those with partial response or less. In conclusion, high TK activity was a strong predictor for short OS and poor response among patients with previously untreated DLBCL treated with R-CHOP.

Inactivation of pathogenic viruses by plant-derived tannins: strong effects of extracts from persimmon (Diospyros kaki) on a broad range of viruses.

Tannins, plant-derived polyphenols and other related compounds, have been utilized for a long time in many fields such as the food industry and manufacturing. In this study, we investigated the anti-viral effects of tannins on 12 different viruses including both enveloped viruses (influenza virus H3N2, H5N3, herpes simplex virus-1, vesicular stomatitis virus, Sendai virus and Newcastle disease virus) and non-enveloped viruses (poliovirus, coxsachievirus, adenovirus, rotavirus, feline calicivirus and mouse norovirus). We found that extracts from persimmon (Diospyros kaki), which contains ca. 22% of persimmon tannin, reduced viral infectivity in more than 4-log scale against all of the viruses tested, showing strong anti-viral effects against a broad range of viruses. Other tannins derived from green tea, acacia and gallnuts were effective for some of the viruses, while the coffee extracts were not effective for any of the virus. We then investigated the mechanism of the anti-viral effects of persimmon extracts by using mainly influenza virus. Persimmon extracts were effective within 30 seconds at a concentration of 0.25% and inhibited attachment of the virus to cells. Pretreatment of cells with the persimmon extracts before virus infection or post-treatment after virus infection did not inhibit virus replication. Protein aggregation seems to be a fundamental mechanism underlying the anti-viral effect of persimmon tannin, since viral proteins formed aggregates when purified virions were treated with the persimmon extracts and since the anti-viral effect was competitively inhibited by a non-specific protein, bovine serum albumin. Considering that persimmon tannin is a food supplement, it has a potential to be utilized as a safe and highly effective anti-viral reagent against pathogenic viruses.

Non-gastric advanced mucosa-associated lymphoid tissue (MALT) lymphoma has worse prognosis than gastric MALT lymphoma even when treated with rituximab-containing chemotherapy.

The aim of this study was to assess the clinical characteristics, treatment results, and analyze the prognostic factors among patients with extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). We retrospectively reviewed 98 patients with MALT lymphoma consecutively diagnosed at the Cancer Institute Hospital. Eighty-one patients (82%) had localized disease and 17 patients (17%) had advanced disease. The primary site was gastric in 52, and extra-gastric in 46. With a median follow-up of 40 months, the estimated 3-year overall survival (OS) and progression free survival (PFS) of the entire group were 100% and 89%, respectively. Three-year PFS was significantly better in patients with gastric lymphoma than in those with non-gastric lymphoma (95% vs. 82%, p = 0.043). Patients with localized disease had significantly better 3-year PFS than those with advanced disease (94% vs. 73%, p = 0.026). Upon multivariate analysis, non-gastric lymphoma retained prognostic significance for PFS.

R-CHOP with dose-attenuated radiation therapy could induce good prognosis in gastric diffuse large B cell lymphoma.

BACKGROUND: The treatment strategy for gastric diffuse large cell lymphoma (DLBCL) has not been standardized in such as to the cycles of chemotherapy, dose of radiation, or necessity for the surgery. Although the results of CHOP or R-CHOP treatments have demonstrated the good prognosis, the treatments have been controversial in many cases. METHODS: We retrospectively analyzed 40 gastric DLBCL patients receiving chemotherapy with or without radiation in our institute. Those in stages II-IV were treated with six cycles of R-CHOP without radiation; for those in stage I, we administered three cycles of R-CHOP with radiation. RESULTS: The three-year overall survival (OS) and progression-free survival (PFS) rates were 95.2 and 91.8%, respectively. Those in stage I obtained 100% of OS. The radiation dose prescribed was 30.6 Gy for CR cases and 39.6 to 40 Gy for PR after chemotherapy. Although survival rates tended to correlate with staging groups or age-adjusted IPI classifications, multivariate statistical analysis did not show clear differences. All 14 patients with initial bleeding were successfully managed without surgery during treatment. CONCLUSION: R-CHOP therapy was very effective for gastric DLBCL. It may be not necessary to use more than 30.6 Gy of radiotherapy in the highly chemo-sensitive cases. Less toxic treatments should be made available to gastric DLBCL patients.

Prospective evaluation of incidence and severity of oral mucositis induced by conventional chemotherapy in solid tumors and malignant lymphomas.

PURPOSE: Oral mucositis (OM), a complication frequently associated with cancer chemotherapy, may decrease treatment efficacy due to dose reduction or impair the patient's quality of life. The purpose was to determine the incidence and severity of OM and its sequelae in patients receiving conventional chemotherapy for various malignancies. METHODS: Two hundred twenty-seven patients (male, 33%; female, 66%) who received chemotherapy for head and neck cancer, esophageal cancer, colorectal cancer, breast cancer, and malignant lymphomas at the Cancer Institute Hospital between January 2007 and December 2008 were examined with questionnaires, prospectively. RESULTS: The incidence of OM was highest in patients with breast cancer (76.5%), then head and neck cancer (67.7%), colorectal cancer (63%), esophageal cancer (57.8%), and malignant lymphoma (42.9%). However, patients who experienced severe OM (≥grade 3) were rare: at most 4.8%. The high-risk regimens for OM were TPF (85.7%), FOLFIRI (80%), CAF (78.8%), AC (70.6%), and FOLFOX (60%). OM was associated with gastrointestinal adverse events, anorexia, diarrhea, and dysphagia, which aggravated quality of life. There was no correlation between incidence of OM and prior therapy, PS, oral care, or laboratory data. There was no statistically significant correlation between OM and overall survival. The predictive factor was history of OM in previous chemotherapy. CONCLUSION: OM frequently occurs in patients with various tumors receiving conventional chemotherapy. Despite low-grade OM, they might cause gastrointestinal adverse events. Adequate preventive treatment for OM is required depending on each chemotherapy regimen and each patient's OM history.