Post-influenzal triazole-resistant aspergillosis following allogeneic stem cell transplantation.

Influenza virus infection is now recognised as a risk factor for invasive pulmonary aspergillosis (IPA). Delays in diagnosis contribute to delayed commencement of antifungal therapy. In addition, the emergence of resistance to first-line triazole antifungal agents puts emphasis on early detection to prevent adverse outcomes. We present 2 allogeneic stem cell transplant patients who developed IPA due to triazole-resistant Aspergillus fumigatus following influenza infection. We underline the challenges faced in the management of these cases, the importance of early diagnosis and need for surveillance given the emergence of triazole resistance.

A multi-centre retrospective study of rituximab use in the treatment of relapsed or resistant warm autoimmune haemolytic anaemia.

This retrospective analysis assessed the response, safety and duration of response to standard dose rituximab 375 mg/m(2) weekly for four weeks as therapy for patients with primary or secondary warm autoimmune haemolytic anaemia (WAIHA), who had failed initial treatment. Thirty-four patients received rituximab for WAIHA in seven centres in the Republic of Ireland. The overall response rate was 70·6% (24/34) with 26·5% (9/34) achieving a complete response (CR). The time to response was 1 month post-initiation of rituximab in 87·5% (21/24) and 3 months in 12·5% (3/24) of patients. The median duration of follow-up was 36 months (range 6-90 months). Of the patients who responded, 50% (12/24) relapsed during follow up with a median time to next treatment of 16·5 months (range 6-60 months). Three patients were re-treated with rituximab 375 mg/m2 weekly for four weeks at relapse and responded. There was a single episode of neutropenic sepsis. Rituximab is an effective and safe treatment for WAIHA but a significant number of patients will relapse in the first two years post treatment. Re-treatment was effective in a small number of patients, suggesting that intermittent pulse treatment or maintenance treatment may improve long-term response.

Epstein-Barr virus-positive mucocutaneous ulcer: A unique case occurring in association with cholelithiasis in a gallbladder.

Epstein-Barr virus (EBV)-positive mucocutaneous ulcer is a lymphoproliferative disorder occurring in patients due to iatrogenic or age-related immunosuppression confined to the oropharynx, skin, and gastrointestinal tract. Here, we report the first case to our knowledge of EBV-positive mucocutaneous ulcer occurring in a gallbladder.

Molecular Subtyping of Diffuse Large B-Cell Lymphoma Using a Novel Quantitative RT-PCR Assay.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Cell-of-origin classification in DLBCL has identified activated B cell (ABC) and germinal center B cell (GCB) as two major subtypes. Patients with the ABC subtype show reduced overall survival with standard therapies. Development of a quantitative RT-PCR-based lymphoma cell-of-origin (LCOO) assay to determine ABC, GCB, and unclassifiable subtypes in formalin-fixed, paraffin-embedded tissue (FFPET) DLBCL samples is reported. The LCOO classifier was trained on two DLBCL cohorts with validation performed by using an analytical grade assay in an independent cohort of 60 FFPET DLBCL samples. In the validation cohort, LCOO classification was 88.1%, 84.7%, and 84.7% concordant with microarray, immunohistochemistry (Hans classification), and Lymphoma Subtyping Test, respectively. Importantly, LCOO and Lymphoma Subtyping Test assays commonly assigned subtypes in 17 (94.4%) of 18 ABC samples and 34 (89.5%) of 38 GCB DLBCL samples from this cohort. Progression-free survival and overall survival of ABC and GCB subtypes, as classified by all platforms, were not significantly different in the validation cohort. LCOO classification using publicly available microarray gene expression from two independent data sets (414 fresh frozen and 474 FFPET DLBCL biopsies) revealed a significantly worse outcome for the ABC subtype compared with that of the GCB subtype. Thus, a sensitive, reproducible, LCOO assay developed on an easy to standardize quantitative RT-PCR platform may be an important clinical tool for DLBCL cell-of-origin classification.

A Phase 1b Study to Evaluate the Safety and Efficacy of Durvalumab in Combination With Tremelimumab or Danvatirsen in Patients With Relapsed or Refractory Diffuse Large B-Cell Lymphoma.

BACKGROUND: Despite recent advances, outcomes in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) remain poor. Immune checkpoint inhibitors have shown limited efficacy in this setting, but combinations with novel agents may enhance benefit. Combination therapy with durvalumab, an anti-programmed death ligand 1 (PD-L1) antibody, and danvatirsen (AZD9150; an antisense oligonucleotide inhibiting signal transducer and activator of transcription 3 [STAT3]) or tremelimumab (an anti-cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4] antibody) may augment endogenous antitumor activity. PATIENTS AND METHODS: In this phase 1b dose escalation and dose expansion study, we evaluated durvalumab 20 mg/kg every 4 weeks plus either tremelimumab 1 mg/kg every 4 weeks or danvatirsen 2 or 3 mg/kg (administered on days 1, 3, 5, 8, 15, and 22, then every week). Treatment continued until disease progression. The primary endpoint was safety; secondary endpoints included efficacy, pharmacokinetics, and immunogenicity. RESULTS: As of April 4, 2019, 32 patients were enrolled and treated, receiving a median of 2 prior lines of systemic therapy. Treatment-related adverse events occurred in 21 patients (65.6%), most commonly alanine aminotransferase/aspartate aminotransferase increased (grade 1-3), anemia (grade 1-3), and fatigue (grade 1). The overall objective response rate was 6.3%, with 2 partial responses. Median time to response was 11.0 weeks (range, 7.7-14.3 weeks). Median progression-free survival was 7.4 weeks (range, 0.1-31.4 weeks), and median overall survival was 28.0 weeks (range, 1.9-115.4 weeks). CONCLUSION: The primary endpoint was met, with durvalumab plus tremelimumab/danvatirsen generally well tolerated in patients with relapsed/refractory DLBCL; however, antitumor activity was limited.

Impact and importance of a centralised review panel for lymphoma diagnostics in the WHO era: a single-centre experience.

Lymphoma diagnosis is complex, requiring a wide array of adjunctive tests to reach accurate diagnoses. We retrospectively examined the rates of concordance between referral and review lymphoma diagnoses on cases referred to St James's Hospital, Dublin for multidisciplinary team review between 2013 and 2016. Frequency and cost of adjunctive diagnostic tests performed were also analysed. The overall discordance rate was 7.8% (14/179), compared with rates of 6%-48% in the published literature. 13 discordant cases required a change in clinical management following review of the referred diagnosis. Of all referred cases, 33.5% (60/179) required extra analyses to reach a final diagnosis, costing the reference laboratory €35463.40. We conclude that establishment of centralised haematopathology diagnostic networks would help reduce the rate of revision made to lymphoma diagnoses by providing specialist haematopathologist input and access to ancillary testing.

Development of an endogenous virus-free line of chickens susceptible to all subgroups of avian leukosis virus.

Primary chicken embryo fibroblasts (CEF) from special specific pathogen-free chicken lines are used for detection of contamination of adult or embryonic tissues, meconium, or tissue culture fluids with avian leukosis viruses (ALV). The suitability and efficiency of such tests depend on the susceptibility of CEF to the various subgroups of exogenous as well as endogenous ALV. The ideal CEF for such tests should be not only susceptible to all retroviruses, but also free of endogenous viruses so that such tests are immune to any interference that may occur between the endogenous and the tested (exogenous) viruses. CEF and/or chickens free of endogenous viruses are also desirable for gene transfer studies using retroviral vectors, such as RNA interference (RNAi) experiments and transgenic work. The absence of ev genes in CEF or chickens can empower clean detection of successful RNAi construct delivery or gene transfer. CEF free of ev genes are also essential reagents routinely used in growing and detecting unknown retroviruses in varied viral assays. This report documents the development of a new line of chickens, 0.TVB*S1, that is free of endogenous viruses and susceptible to all subgroups of ALV identified in chickens.

Moxetumomab pasudotox in relapsed/refractory hairy cell leukemia.

This is a pivotal, multicenter, open-label study of moxetumomab pasudotox, a recombinant CD22-targeting immunotoxin, in hairy cell leukemia (HCL), a rare B cell malignancy with high CD22 expression. The study enrolled patients with relapsed/refractory HCL who had ≥2 prior systemic therapies, including ≥1 purine nucleoside analog. Patients received moxetumomab pasudotox 40 µg/kg intravenously on days 1, 3, and 5 every 28 days for ≤6 cycles. Blinded independent central review determined disease response and minimal residual disease (MRD) status. Among 80 patients (79% males; median age, 60.0 years), durable complete response (CR) rate was 30%, CR rate was 41%, and objective response rate (CR and partial response) was 75%; 64 patients (80%) achieved hematologic remission. Among complete responders, 27 (85%) achieved MRD negativity by immunohistochemistry. The most frequent adverse events (AEs) were peripheral edema (39%), nausea (35%), fatigue (34%), and headache (33%). Treatment-related serious AEs of hemolytic uremic syndrome (7.5%) and capillary leak syndrome (5%) were reversible and generally manageable with supportive care and treatment discontinuation (6 patients; 7.5%). Moxetumomab pasudotox treatment achieved a high rate of independently assessed durable response and MRD eradication in heavily pretreated patients with HCL, with acceptable tolerability.

Response of white leghorn chickens of various B haplotypes to infection at hatch with subgroup J avian leukosis virus.

White leghorn chickens from seven 15.B congenic lines (genetically similar except for genes linked to the major histocompatibility complex [MHC] B haplotype) and two Line 0.B semicongenic lines were infected at hatch with strain ADOL Hc-1 of subgroup J avian leukosis virus (ALV-J). At 5, 8, 16, and 36 wk of age, chickens were tested for viremia, serum-neutralizing antibody, and cloacal shedding. Chickens were also monitored for development of neoplasia. In the 15.B congenic lines (B*2, B*5, B*12, B*13, B*15, B*19, and B*21) there were no significant differences in the incidence of viremia between B haplotypes. In fact, infection at hatch in all of the 15.B congenic lines induced tolerance to ALV-J because 100% of these chickens were viremic and transient circulating serum-neutralizing antibody was detected in only a few chickens throughout the 36 wk experiment. However, at 16 wk of age more B*15 chickens had antibody and fewer B*15 chickens shed virus than did the 16-wk-old B*2, B*5, or B*13 chickens. Moreover, compared with B*15 chickens, a higher percentage of B*13 chickens consistently shed virus from 8 wk postinfection to termination at 36 wk postinfection. The B haplotype had a transient effect on viral clearance in Line 0.B semicongenics, as more B*13 than B*21 chickens remained viremic through 5 wk of age. Very few (0%-18%) of the Line 0.B semicongenic chickens shed virus. By 36 wk of age, all Line 0 B*13 and B*21 chickens produced serum-neutralizing antibodies and cleared the virus. These results show that following ALV-J infection at hatch the immune response is influenced transiently by the B haplotype and strongly by the line of chicken. Although this study was not designed to study the effect of endogenous virus on ALV-J infection, the data suggest that endogenous virus expression reduced immunity to ALV-J in Line 15I5, compared with Line 0, a line known to lack endogenous virus genes.

Response of white leghorn chickens of various genetic lines to infection with avian leukosis virus subgroup J.

In Experiment 1, chickens from various white leghorn experimental lines were inoculated with strain ADOL-Hcl of subgroup J avian leukosis virus (ALV-J) either as embryos or at 1 day of age. At various ages, chickens were tested for ALV-J induced viremia, antibody, and packed cell volume (PCV). Also, at 4 and 10 wk of age, bursal tissues were examined for avian leukosis virus (ALV)-induced preneoplastic lesions with the methyl green-pyronine (MGP) stain. In Experiment 2, chickens harboring or lacking endogenous virus 21 (EV21) were inoculated with strain ADOL-Hcl of ALV-J at hatch. All embryo-inoculated chickens in Experiment 1 tested positive for ALV-J and lacked antibody throughout the experimental period of 30 wk and were considered viremic tolerant, regardless of line of chickens. By 10 wk of age, the incidence of ALV-J viremia in chickens inoculated with virus at hatch varied from 0 (line 0 chickens) to 97% (line 1515); no influence of ALV-J infection was noted on PCV. Results from microscopic examination of MGP-stained bursal tissues indicate that ALV-J can induce typical ALV-induced transformation in bursal follicles of white leghorn chickens. Lymphoid leukosis and hemangiomas were the most common ALV-J-induced tumors noted in chickens in Experiment 1. At termination of Experiment 2 (31 wk of age), 54% of chickens harboring EV21 were viremic tolerant compared with 5% of chickens lacking EV21 after inoculation with ALV-J at hatch. The data indicate that genetic differences among lines of white leghorn chickens, including the presence or absence of EV21, can influence response of chickens to infection with ALV-J.

Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines.

Brain-derived neurotrophic factor (BDNF) has multiple alternative splicing variants and plays diverse biological functions in mammals, including neuronal survival, cholesterol metabolism, cell differentiation and tumor development. However, genomic structures of chicken BDNF (cBDNF) variants and its potential functions are still undefined. Here, we characterized two novel alternative splicing variants of cBDNF, cBDNF1 and cBDNF2, via combining comparative genomics methods and molecular techniques in inbred chicken line 6(3) and line 7(2), which have been developed to be resistant and susceptible, respectively, to Marek's disease tumor since 1939. Both cBDNFs consist of a bipartite transcript, with different 5' exons, exon I (298 bp) in cBDNF1 and exon II (286 bp) in cBDNF2, each of which is spliced to the common 3' exon IV. Exon I and IV are highly conserved between chicken and mammals, whereas exon II is unique for chicken. The amino acid sequence of cBDNF1 contains 8 additional amino acids in the N terminal compared to cBDNF2. cBDNF1 and cBDNF2 were only expressed in the hypothalamus among eight tissues, and cBDNF2 showed lower expression than that of cBDNF1 in both lines. The expression level of cBDNF1 was significantly higher in line 7(2) than in line 6(3) (P<0.01). Notably, the DNA methylation levels on the cis-regulatory region of cBDNF1 was negatively correlated with its expression level, which suggests that the mRNA expression level of cBDNF1 may be related to the DNA methylation status in the chickens. We also discussed a potential role of variant cBDNF1 in MD tumor resistance and susceptibility.

Quantitative evaluation of DNA methylation patterns for ALVE and TVB genes in a neoplastic disease susceptible and resistant chicken model.

Chicken endogenous viruses, ALVE (Avian Leukosis Virus subgroup E), are inherited as LTR (long terminal repeat) retrotransposons, which are negatively correlated with disease resistance, and any changes in DNA methylation may contribute to the susceptibility to neoplastic disease. The relationship between ALVE methylation status and neoplastic disease in the chicken is undefined. White Leghorn inbred lines 7(2) and 6(3) at the ADOL have been respectively selected for resistance and susceptibility to tumors that are induced by avian viruses. In this study, the DNA methylation patterns of 3 approximately 6 CpG sites of four conserved regions in ALVE, including one unique region in ALVE1, the promoter region in the TVB (tumor virus receptor of ALV subgroup B, D and E) locus, were analyzed in the two lines using pyrosequencing methods in four tissues, i.e., liver, spleen, blood and hypothalamus. A significant CpG hypermethylation level was seen in line 7(2) in all four tissues, e.g., 91.86 +/- 1.63% for ALVE region2 in blood, whereas the same region was hemimethylated (46.16 +/- 2.56%) in line 6(3). CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver. RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01). The methylation levels of six recombinant congenic strains (RCSs) closely resembled to the background line 6(3) in ALVE-region 2, which imply the methylation pattern of ALVE-region 2 may be a biomarker in resistant disease breeding. The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively. Our data disclosed a hypermethylation pattern of ALVE that may be relevant for resistance against ALV induced tumors in chickens.

At least one YMHCI molecule in the chicken is alloimmunogenic and dynamically expressed on spleen cells during development.

Transcriptionally active, MHC class I (MHCI) loci are located in two separate polymorphic genomic regions in the chicken called B and Y. The YMHCI gene sequences encode molecules with uncommon substitutions in the antigen-binding region indicating that YMHCI molecules are likely unique and may bind a specialized form of antigen distinct from that of other antigen-binding MHCI molecules. To learn whether YMHCI gene expression results in the production of alloantigens at the cell surface, we immunized 15I(5) x 7(2) : chickens using syngeneic RP9 cells expressing transduced YF1w*7.1, a potentially alloimmunogenic YMHCI allele from the Y7 haplotype present in line C. The resulting antisera show that YF1w*7.1 MHCI molecules are immunogenic and expressed on the surfaces of cells in blood and spleen of line C chickens. Virtually all CD3+, CD4+, and CD8+ cells circulating in line C blood are positive, as are BU1+ cells. The YF1w*7.1 MHCI allele is dynamically expressed at levels comparable to but transcriptionally independent of classical BMHCI on erythrocytes, lymphocytes, granulocytes, monocytes, and thrombocytes within the spleen pre- and post-hatching. The antisera react with cells from two among four haplotypes segregating in closed populations of lines N and P. YMHCI shares features associated with both classical and non-classical MHCI. It is becoming increasingly likely that YMHCI has a fundamental role in avian immunity and thereby needs to be included in the growing spectrum of functionally active, diverse MHCI molecules no longer adequately described by the classical/non-classical dichotomy.

2004 Nomenclature for the chicken major histocompatibility (B and Y) complex.

The first standard nomenclature for the chicken (Gallus gallus) major histocompatibility (B) complex published in 1982 describing chicken major histocompatibility complex (MHC) variability is being revised to include subsequent findings. Considerable progress has been made in identifying the genes that define this polymorphic region. Allelic sequences for MHC genes are accumulating at an increasing rate without a standard system of nomenclature in place. The recommendations presented here were derived in workshops held during International Society of Animal Genetics and Avian Immunology Research Group meetings. A nomenclature for B and Y (Rfp-Y) loci and alleles has been developed that can be applied to existing and newly defined haplotypes including recombinants. A list of the current standard B haplotypes is provided with reference stock, allele designations, and GenBank numbers for corresponding MHC class I and class IIbeta sequences. An updated list of proposed names for B recombinant haplotypes is included, as well as a list of over 17 Y haplotypes designated to date.