Clinicopathological differences in MYC and BCL2 protein expression between primary extranodal and nodal diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) exhibits clinical, genetic, and immunohistochemical heterogeneity. However, the differences between primary extranodal or nodal DLBCL and double-expressor lymphoma (DEL), which is characterized by high MYC and BCL2 expression, remain unclear. This study aimed to elucidate the clinicopathological features, response to therapy, and clinical outcomes of primary extranodal (n=61) and nodal (n=128) DLBCL. Patients with primary nodal DLBCL had higher BCL2 expression than those with extranodal DLBCL (p=0.048), with high MYC expression and DEL as poor prognostic factors. Conversely, in patients with primary extranodal DLBCL, high BCL2 expression, low BCL6 expression, non-germinal center B-cell-like type, and DEL indicated poor prognosis. DEL was significantly associated with progression free survival and overall survival in patients with primary extranodal DLBCL (p=0.014 and p=0.021, respectively) but not in patients with primary nodal DLBCL (p=0.37 and p=0.084, respectively). Our findings highlight primary extranodal DEL as a strong adverse prognostic factor in DLBCL.

Effect of peptide-binding motif on survival of HLA-haploidentical transplantation with post-transplant cyclophosphamide.

Peptide-binding motif (PBM) model, a hierarchical clustering of HLA class I based on their binding specificity, was developed to predict immunopeptidome divergence. The effect of PBM mismatches on outcomes is unknown in HLA-haploidentical haematopoietic cell transplantation with post-transplant cyclophosphamide (PTCy-haplo). We therefore conducted a retrospective study using national registry data in PTCy-haplo. Overall, 1352 patients were included in the study. PBM-A bidirectional mismatch was associated with an increased risk of overall mortality in multivariable analysis (hazard ratio, 1.26; 95% confidence interval, 1.06 to 1.50; p = 0.010). None of relapse, non-relapse mortality (NRM) and graft-versus-host disease showed significant differences according to PBM-A bidirectional mismatch status in the entire cohort. The impact of PBM-A bidirectional mismatch on overall survival (OS) was preserved within the HLA-A genotype bidirectional mismatch population, and their lower OS stemmed from higher relapse rate in this population. The worse OS due to high NRM with PBM-A bidirectional mismatch was prominent in lymphoid malignancies receiving reduced-intensity conditioning. The PBM model may predict outcomes more accurately than HLA genotype mismatches. In conclusion, this study demonstrated that the presence of PBM-A bidirectional mismatch elevated the risk of mortality of PTCy-haplo. Avoiding PBM-A bidirectional mismatch might achieve better outcomes in PTCy-haplo.

Influence of FOXP3 single-nucleotide polymorphism after allogeneic hematopoietic stem cell transplantation.

The impact of FOXP3 single-nucleotide polymorphisms (SNP) on clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains poorly understood. We investigated the relationship between a FOXP3 SNP (rs3761548) and clinical outcomes in 91 patients with hematological malignancies after allo-HSCT. Multivariate analysis showed that risk of severe chronic graft-versus-host disease (cGVHD) was significantly higher in patients with the FOXP3-3279C/A or FOXP3-3279A/A genotype than those with the FOXP3-3279C/C genotype [hazard ratio (HR), 2.69; 95% confidence interval (CI) 1.14-6.31; p = 0.023]. Therefore, FOXP3 at SNP rs3761548 can be a useful marker for predicting the occurrence of severe cGVHD.

Blastic Plasmacytoid Dendritic Cell Neoplasm in Long-Term Complete Remission After Venetoclax Monotherapy.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy associated with a poor prognosis and limited treatment options. Although allogeneic hematopoietic stem cell transplantation or intensive chemotherapy prolongs overall survival in patients with BPDCN, intensive chemotherapy is inappropriate for older or unfit patients. Venetoclax (VEN), an oral BCL2 inhibitor, is approved for use in patients with acute myeloid leukemia (AML). BPDCN cells require BCL2 protein and are uniformly sensitive to VEN in vivo. Moreover, patients with AML who have achieved complete remission after induction therapy are reportedly considered to receive VEN monotherapy as maintenance therapy, especially older patients. However, the efficacy of VEN monotherapy as a maintenance therapy for patients with BPDCN remains controversial. Recently, BPDCN has been classified into MYC+ and MYC- subtypes, which show clinical differences. Hence, BPDCN treatment strategies based on the MYC classification may be necessary. Here, we report a case of MYC- BPDCN in an older patient in long-term complete remission after VEN monotherapy following VEN and azacitidine induction chemotherapy.

[Acquired thrombotic thrombocytopenic purpura during durvalumab monotherapy for non-small cell lung cancer].

Immune checkpoint inhibitor (ICI)-induced thrombocytopenias are rare immune-related adverse events (irAE), but ICI-related thrombotic thrombocytopenic purpura (TTP) is extremely rare. A 79-year-old woman with non-small cell lung cancer received maintenance therapy with the anti-human PD-L1 monoclonal antibody durvalumab. Four weeks after the last infusion, she developed overt TTP. Remission was achieved by plasma exchange and prednisolone, and the patient has now been recurrence-free for over 12 months. To our knowledge, this is the first report of TTP occurring as an irAE of durvalumab.

Impact of microsampling on toxicological evaluation in rodent safety studies.

Recently, animal welfare has been attracting worldwide attention, and implementation of 3Rs (replacement, reduction, and refinement) is prioritized in every way possible in the drug development. Microsampling, in which small amounts of blood are collected, is attracting attention in this context. ICH S3A Q&A focused on microsampling was published in November 2017 to help accelerate the application of microsampling for toxicokinetic assessment. The increased sensitivity of drug measurement apparatuses such as mass spectrometers has made it possible to measure drug concentrations with small amounts of blood samples. In this review, we summarized the reports on toxicological influence of microsampling in rodents (rats and mice) with or without drug administration or recovery period after blood collection and influences that may arise from differences in the blood sampling site or blood sampling volume. We also summarized some perspectives on further implementation of microsampling in toxicology studies. The use of microsampling in regulatory toxicology studies has gradually increased, although at a lower rate than in discovery studies. Since more animals are used in GLP toxicology studies than in discovery studies, the effect of reducing the number of animals by microsampling is expected to be greater in the toxicology studies. This report aims to promote the application of microsampling to nonclinical studies, as it is beneficial for improving animal welfare and can contribute to the 3Rs.

Chronic neutrophilic leukemia preceded by myelodysplastic syndromes.

Chronic neutrophilic leukemia (CNL) is primarily diagnosed by excluding myelodysplastic syndromes (MDS). We report the case of a patient who developed secondary CNL 3 years after hypoplastic MDS. We used droplet digital polymerase chain reaction mutation detection assay to analyze genomic alterations during the progression from MDS to CNL. At the time of MDS diagnosis, U2AF1 Q157P and SETBP1 D868N were dominant and additional mutation of ASXL1 1934_insG was observed. CSF3R T618I and SETBP1 D868N were increasing at the time of CNL diagnosis. We revealed the accumulation of multiple gene mutations during CNL development from MDS. This suggests that CNL was clonally developed from the founding clone of MDS and CSF3R mutation contributes to the development of CNL in the present case. These findings provide insights into the pathology of CNL.

The influence of serial 50 µL microsampling on rats administered azathioprine, the immunosuppressive drug.

According to the ICH S3A Q&A, microsampling is applicable to pharmaceutical drugs and toxicological analysis. Few studies have reported the effect of microsampling on the toxicity of immunotoxicological drugs. The aim of this multicenter study was to evaluate the toxicological effects of serial microsampling on rats treated with azathioprine as a model drug with immunotoxic effects. Fifty microliters of blood were collected from the jugular vein of Sprague-Dawley rats at six time points from day 1 to 2 and 7 time points from day 27 to 28. The study was performed at three organizations independently. The microsampling effect on clinical signs, body weights, food consumption, hematological parameters, biochemical parameters, urinary parameters, organ weights, and tissue pathology was evaluated. Azathioprine-induced changes were observed in certain hematological and biochemical parameters and thymus weight and pathology. Microsampling produced minimal or no effects on almost all parameters; however, at 2 organizations, azathioprine-induced changes were apparently masked for two leukocytic, one coagulation, and two biochemical parameters. In conclusion, azathioprine toxicity could be assessed appropriately as overall profiles even with blood microsampling. However, microsampling may influence azathioprine-induced changes in certain parameters, especially leukocytic parameters, and its usage should be carefully considered.

[CD5- and CD10-positive MYC and BCL2 double-expressor diffuse large B-cell lymphoma with MYD88L265P mutation: a case report and literature review].

A 75-year-old man who had lymphadenopathy was admitted to our hospital. Histopathological examination of cervical lymph node biopsy specimens showed diffuse proliferation of lymphoma cells with large nuclei. In immunohistochemistry, the lymphoma cells were positive for CD5, CD10, CD20, BCL2, BCL6, and MYC. The patient was diagnosed with CD5- and CD10-positive diffuse large B-cell lymphoma (DLBCL). MYD88L265P mutations have been detected in DLBCL. Partial response was achieved after six courses of R-THP-COP therapy. However, the patient died because of disease progression 18 months after the diagnosis. On autopsy, lymphoma cells were found in the lymph nodes throughout the body, central nervous system, adrenals, and skin. CD5- and CD10-positive DLBCL account for 0.5-1% of DLBCL cases and have a very poor disease prognosis. This is a rare case of CD5- and CD10-positive DLBCL with MYC and BCL2 expressions harboring MYD88L265P mutation.

Effect of microsampling (50 µL) on toxicological evaluation of methapyrilene, a hepatotoxic substance, in a collaborative 28-day study in female rats.

Although microsampling of blood is recommended to promote the 3Rs in toxicokinetic (TK) evaluation, there are few reports applying microsampling in actual toxicity evaluation. Here, we assessed the effects of microsampling on toxicological evaluation of methapyrilene hydrochloride, a hepatotoxic substance. Female SD rats received methapyrilene hydrochloride orally at dose levels of 0 (vehicle), 10, and 30 mg/kg BW, once daily for 4 weeks. Each dose level included a microsampling group and a non-microsampling group (n = 5). In the microsampling groups, blood sampling (50 µL/time point) was performed at 6 time points on day 1 of administration and 7 time points on day 27-28; all the animals underwent necropsy on day 29. Toxicity studies and TK analysis were performed, and through these studies in 2 organizations, cross-organization validation of the effect on toxicity evaluation was conducted. In one organization, microsampling obscured changes in some parameters in hematology due to the administration of methapyrilene hydrochloride. In the other organization, although the relationship between the developing pattern of histopathological findings in the liver and the blood sampling was suspected, it was associated with poor reproducibility; this was considered as a change within a variation range of biological reactions. Each of these phenomena was observed in only one organization without consistency. In both organizations, no effect of blood microsampling was observed in other endpoints. In conclusion, microsampling is considered to be a technique applicable to safety studies of drugs showing hepatotoxicity, as it did not show a marked influence on the toxicological evaluation of methapyrilene hydrochloride.

A prospective, multicenter, open-label phase III study of emicizumab prophylaxis in patients with acquired hemophilia A.

BACKGROUND: Emicizumab is a bispecific antibody that mimics the cofactor function of activated factor (F) VIII. It prevents bleeds in patients with congenital hemophilia A regardless of the inhibitor status; however, no prospective clinical studies have been conducted for emicizumab in patients with acquired hemophilia A (PwAHA). OBJECTIVES: To describe the primary analysis results from a prospective, multicenter, open-label phase III study evaluating the efficacy, safety, and pharmacokinetics of emicizumab in PwAHA (AGEHA; JapicCTI-205151). METHODS: Emicizumab was administered subcutaneously at 6 mg/kg on day 1 and 3 mg/kg on day 2, followed by 1.5 mg/kg once weekly from day 8 onward. Predefined criteria for the completion of dosing included FVIII activity of >50 IU/dL. RESULTS: By the cutoff date (April 23, 2021), 12 patients on immunosuppressive therapy were enrolled, and 11 of them (91.7%) completed emicizumab treatment. The mean trough plasma emicizumab concentration rapidly reached a steady state (1 week), achieving the efficacious level that was established in patients with congenital hemophilia A (>30 µg/mL). Before first emicizumab administration, 7 patients (58.3%) experienced 77 major bleeds. During emicizumab treatment, no major bleeds occurred in any patient. Neither death due to bleeding or infection nor any study treatment-related serious adverse event was reported. One asymptomatic, nonserious deep vein thrombosis was discovered with no laboratory findings indicating any trend toward hypercoagulation. CONCLUSION: These results suggest that emicizumab prophylaxis with the tested dosing regimen and completion criteria may have a favorable benefit-risk profile in PwAHA.

Increased MYC expression without MYC gene translocation in patients with the diffuse large B-cell-lymphoma subtype of iatrogenic immunodeficiency-associated lymphoproliferative disorders.

Post-transplant lymphoproliferative disorder (PTLD) and other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) are iatrogenic lymphoproliferative disorders (LPD) that develop in association with immunosuppressive treatment in the setting of organ transplantation and autoimmune disease, respectively. Each has a spectrum of pathologies ranging from lymphoid hyperplasia to lymphoma. To clarify the characteristics of the diffuse large B-cell lymphoma (DLBCL) subtype in a cohort of 25 patients with PTLD or OIIA-LPD from our institute, we selected 13 with a histological subtype of DLBCL, including 2 cases of PTLD and 11 of OIIA-LPD. The median patient age at diagnosis was 70 years, with a female predominance. Both PTLD cases developed after kidney transplant. Of the patients with OIIA-LPD, 10 had rheumatoid arthritis, 1 had mixed connective tissue disease, and 8 were treated using methotrexate. Both of the PTLD patients and 6 of the OIIA-LPD patients had extranodal manifestations. All patients except for one were classified as having the non-germinal center B-cell (non-GCB) subtype according to the Hans algorithm. Tissue samples from 8 patients were positive for CD30 and 8 were positive for Epstein-Barr virus (EBV)-encoded small RNA. Seven patients had MYC-positive tissue samples, but none had MYC translocation. Our study suggests that extranodal manifestations and the non-GCB subtype are common, that EBV is associated with the DLBCL subtype of PTLD and OIIA-LPD, and that anti-CD30 therapy is applicable. In addition, our patients with the DLBCL subtype of PTLD and OIIA-LPD exhibited MYC overexpression without MYC translocation, suggesting an alternative mechanism of MYC upregulation.

Increased serum C-reactive protein is an adverse prognostic factor in low-risk myelodysplastic syndromes.

Inflammatory cytokines play a role in hematopoiesis and development of myelodysplastic syndromes (MDS). Although increased serum levels of inflammatory cytokines are associated with poor survival in MDS patients, clinical management does not include assessment of inflammation. We investigated the significance of inflammation in MDS using serum C-reactive protein (CRP) levels, an indicator of the degree of systemic inflammation that can be used in routine practice. We hypothesized that serum CRP levels can be used to further classify low-risk MDS. We conducted a retrospective analysis of 90 patients with low-risk MDS, defined by the international prognostic scoring system (IPSS). We examined the prognostic relevance of CRP and known prognostic factors at diagnosis. Increased serum CRP (≥ 0.58 mg/dL) was associated with poor survival (hazard ratio [HR]: 17.63, 95% confidence interval [CI] 5.83-53.28, P < 0.001) both overall and among the 73 patients with low-risk MDS as defined by the revised IPSS (HR: 28.05, 95% CI 6.15-128.04, P < 0.001). Increased CRP might predict poor prognosis and serum CRP levels can indicate clonal hematopoiesis and non-hematological comorbidity in patients with low-risk MDS.

Single cord blood transplantation for acute myeloid leukemia patients aged 60 years or older: a retrospective study in Japan.

The availability of alternative donor sources could allow elderly patients to receive allogeneic hematopoietic cell transplantation (HCT). We retrospectively evaluated the outcomes of single-unit cord blood transplantation (CBT) in 1577 patients aged ≥60 years with acute myeloid leukemia (AML) in Japan between 2002 and 2017. In total, 990 (63%) patients were not in complete remission (CR) at the time of CBT. A myeloablative conditioning regimen (52%) and calcineurin inhibitor (CI) + mycophenolate mofetil (MMF)-based graft-versus-host disease (GVHD) prophylaxis (45%) were more commonly used. With a median follow-up for survivors of 31 months, the probability of overall survival and the cumulative incidence of leukemia-related mortality at 3 years was 31% and 29%, respectively. The cumulative incidence of non-relapse mortality (NRM) at 100 days and 3 years were 24% and 41%, respectively. The cumulative incidences of grade II-IV and grade III-IV acute GVHD at 100 days and extensive chronic GVHD at 2 years were 44%, 16%, and 14%, respectively. The cumulative incidence of neutrophil engraftment was 80% at 42 days. Results of multivariate analysis indicated that the following factors were significantly associated with higher overall mortality: performance status ≥1, hematopoietic cell transplantation-specific comorbidity index ≥3, adverse cytogenetics, extramedullary disease at diagnosis, and non-CR status at CBT. By contrast, female sex, HLA disparities ≥2, mycophenolate mofetil-based GVHD prophylaxis, and recent CBT were significantly associated with lower overall mortality. In conclusion, single CBT offers a curative option for AML patients aged ≥60 years with careful patient selection.

Characteristics and predictors of post-transplant-associated hemophagocytic lymphohistiocytosis in adults.

Hemophagocytic lymphohistiocytosis (HLH) is an uncontrolled hyperinflammatory disorder driven by an overactive immune system that results in high mortality. Post-transplant-associated hemophagocytic lymphohistiocytosis (PT-HLH) is a type of secondary HLH that occurs following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical features of PT-HLH remain unclear and diagnostic and prognostic tools have not yet been established. Here, we retrospectively evaluated the clinical manifestations and outcomes of PT-HLH in 94 patients who underwent allo-HSCT. According to our PT-HLH criteria (hyperferritinemia and increased macrophage count in bone marrow), PT-HLH occurred in 12 patients (12.8%). The PT-HLH patients showed splenomegaly (P = .001), a higher risk of engraftment failure (P = .013), and an increased percentage of macrophages and hemophagocytes in bone marrow aspirates (P = .0009 and P = .0006, respectively). Moreover, univariate and multivariate analyses revealed that the survival rate was lower in PT-HLH patients than non-PT-HLH patients (P = .0017 and P = .034, respectively). This study defines the clinical features of PT-HLH and PT-HLH criteria that could be useful tools for diagnosing PT-HLH.

Effects of serial cervical or tail blood sampling on toxicity and toxicokinetic evaluation in rats.

To assess the influences of blood sampling volumes or sites on toxicological and toxicokinetic (TK) evaluations, 4-week duration animal studies and a single-dose TK study of imipramine were conducted. In the toxicological evaluation, six-week-old Sprague-Dawley rats were divided into no blood and blood sampling groups. Fifty microliters (microsampling) or 100 µL (larger sampling) of blood/time point was collected from the jugular vein (50 µL of data was reported previously as Yokoyama et al., 2020) or the tail vein 6 to 7 times on days 1/2 and in week 4. Although no parameters were affected by the 100 µL sample from the tail vein, the 100 µL jugular vein sampling decreased the red blood cell parameters in females, possibly due to hemorrhage at the sampling site. Regarding the TK assessment, 50 µL of blood/site/time point was collected at 6 time points from the tail and jugular vein of the same male rats after single oral administration of 10 or 100 mg/kg imipramine, which was selected as a representative drug with high distribution volume. Although there were no differences in the AUC0-24hr and Cmax values between the sites, the plasma concentrations at the early time points were significantly lower from the tail vein than the jugular vein. From our studies, 50 µL of jugular and tail vein microsampling did not affect the toxicity parameters or AUC/Cmax. However, appropriate toxicity considerations and/or selection of the blood sampling site may be important in the case of larger sampling volumes or blood concentration assessment.

Elevated C-reactive protein level is associated with poor prognosis in follicular lymphoma patients undergoing rituximab-containing chemotherapy.

Although follicular lymphoma (FL) is a pathological entity characterized by relatively uniform histological and molecular findings, its clinical course is highly variable. Establishment of therapeutic strategies based on a simple and practical prognostic model is important. C-reactive protein (CRP) is an adverse prognostic marker for various tumors and aggressive lymphomas. However, the significance of serum CRP levels as a prognostic index in low-grade lymphomas, such as FL, has not been thoroughly investigated. We retrospectively analyzed the relationship between serum CRP levels at diagnosis and the prognosis in patients with FL (n = 61) undergoing rituximab-containing chemotherapy. Elevated CRP levels showed a significant association with elevated fibrinogen (P = 0.002) in univariate analysis. Patients with higher CRP levels (> 5 mg/L) had a significantly shorter progression-free survival in multivariate analysis (P = 0.044). We concluded that serum CRP levels are important in prognostic stratification of patients with FL.

Lack of toxicological influences by microsampling (50 µL) from jugular vein of rats in a collaborative 28-day study.

Due to finalization of the ICH S3A Q&A focusing on microsampling, application of microsampling technique to regular non-clinical animal studies is expected for non-clinical safety assessment of pharmaceuticals. In Europe, microsampling from the tail vein or saphenous vein has often been used, whereas sampling from the jugular vein is thought to be more common for non-clinical studies in Japan. Therefore, we assessed the toxicological effects of serial microsampling from the jugular vein of SD rats in a common 28-day study at 4 independent organizations. Fifty microliter sampling was performed at 6 timepoints on day 1 to 2 and 7 timepoints on day 27 to 28 and its toxicological influences on body weight, food consumption, hematological and clinical chemistry parameters, and organ weights (on day 29 for 3 and day 28 for 1 organizations) were evaluated. The serial microsampling was shown to have no or minimal influences on the assessed parameters. The observed statistical differences for the 18 parameters were sporadic and did not appear to be systemically associated with microsampling. However, the sporadic changes were more often observed in females (14/18 parameters) than in males (6/18), suggesting the possibility that female rats were more susceptible to treatment-based influences. The current results indicate that serial 50 µL sampling from the jugular vein of SD rats had no or very slight toxicological effects, suggesting that this microsampling condition is applicable for toxicokinetic evaluation of non-clinical rat toxicity studies.

Comparative Study of Tacrolimus and Short-Term Methotrexate: 2-Day versus 3-Day Methotrexate as Graft-versus-Host-Disease Prophylaxis after Umbilical Cord Blood Transplantation in Adults.

Methotrexate (MTX) in combination with a calcineurin inhibitor has been commonly used for prophylaxis of graft-versus-host disease (GVHD) following umbilical cord blood transplantation (UCBT) in Japan. However, the appropriate prophylactic MTX dosage in UCBT has not been established to date. To determine the preferential GVHD prophylaxis in UCBT, this study retrospectively investigated the administration of short-term MTX for 2 days versus 3 days. Of 103 adult patients submitted to UCBT enrolled in the study, 73 received tacrolimus (TAC) with 2 days of MTX given at 10 mg/m2 on day 1 and 7 mg/m2 on day 3 (very short-term [vs] MTX), whereas 30 patients received TAC with 3 days of MTX given at 10 mg/m2 on day 1, 7 mg/m2 on day 3, and 7 mg/m2 on day 6 (short-term [s] MTX). In univariate analysis, neutrophil engraftment was shown to be significantly better (P = .039) in the vsMTX/TAC group. Among high-risk patients, the vsMTX/TAC group also exhibited earlier neutrophil engraftment (P = .042); however, the incidence of acute GVHD was higher in the vsMTX/TAC group (P = .035) on univariate analysis. In multivariate analysis, compared with sMTX/TAC, vsMTX/TAC was associated with lower risk of relapse (hazard ratio, .27; 95% confidence interval, .11 to .64; P = .003) . These results suggest that vsMTX/TAC can be appropriate GVHD prophylaxis after UCBT, especially in higher-risk patients.