Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease.

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR and TCR) sequences from the blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires were associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation, and counterregulation by the co-receptors BTLA, Tim-3, PD-1, TIGIT, and CD73. Tfh, Th17-like, and nonconventional (but not classical antiviral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.

Activated granulocytes and inflammatory cytokine signaling drive T-cell lymphoma progression and disease symptoms.

Peripheral T-cell lymphomas (PTCLs), especially angioimmunoblastic and follicular TCLs, have a dismal prognosis because of the lack of efficient therapies, and patients' symptoms are often dominated by an inflammatory phenotype, including fever, night sweats, weight loss, and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on T-cell follicular helper-type PTCL (TFH-PTCL) disease progression and symptoms. We showed that ITK-SYK-driven murine PTCLs and primary human TFH-PTCL xenografts both induced inflammation in mice, including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving interferon gamma (CD4+ malignant T cells) and interleukin 6 (IL-6; activated granulocytes), ultimately inducing broad JAK activation (JAK1/2/3 and TYK2) in both cell types. Inflammatory granulocyte depletion via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox), or IL-6 deletion within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration, and enhanced mouse survival. Furthermore, unselective JAK inhibitors (ruxolitinib) inhibited both TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions, and an intact JAK/STAT signaling pathway for TFH-PTCL development and also support broad JAK inhibition as an effective treatment strategy in early disease stages.

Detection of disease-specific signatures in B cell repertoires of lymphomas using machine learning.

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.

Household transmission of Omicron variant of SARS-CoV-2 under conditions of hybrid immunity-a prospective study in Germany.

PURPOSE: We investigated the protection offered by vaccinations and previous infections for the household transmission of Omicron variant of SARS-CoV-2. METHODS: 34,666 participants of the German DigiHero cohort study with two or more household members were invited to a prospective household transmission study between June and December 2022. In case of a positive SARS-CoV-2 test in a household, symptom diaries were completed for at least 14 days. Dry blood spots (DBS) were taken from all household members at the beginning and six to eight weeks later. DBS were analyzed for SARS-CoV-2 antibodies. RESULTS: 1191 individuals from 457 households participated. The risk of acquiring a SARS-CoV-2 infection decreased with higher S-titer levels at the time of exposure (from 80% at titer of 0 binding antibody units (BAU)/ml to 20% at titer of 3000 BAU/ml) and increased linearly with the time since vaccination/previous infection (20% for less than one month to 80% at one year). Transmission probability was also reduced when the symptoms of the primary case were mild and if preventive measures were implemented. CONCLUSION: Vaccinations/previous infections offer a high protection against infection with the Omicron variant for a few months only, supporting the notion of seasonal circulation of the virus.

Autoantigen-selected B cells are bystanders in spontaneous T cell-driven experimental autoimmune hepatitis.

Autoreactive B cells are considered pathogenic drivers in many autoimmune diseases; however, it is not clear whether autoimmune B cells are invariably pathogenic or whether they can also arise as bystanders of T cell-driven autoimmune pathology. Here, we studied the B cell response in an autoantigen- and CD4+ T cell-driven model of autoimmune hepatitis (AIH), the Alb-iGP_Smarta mouse in which expression of a viral model antigen (GP) in hepatocytes and its recognition by GP-specific CD4+ T cells causes spontaneous AIH-like disease. T cell-driven AIH in Alb-iGP_Smarta mice was marked by autoantibodies and hepatic infiltration of plasma cells and B cells, particularly of isotype-switched memory B cells, indicating antigen-driven selection and activation. Immunosequencing of B cell receptor repertoires confirmed B cell expansion selectively in the liver, which was most likely driven by the hepatic GP model antigen, as indicated by branched networks of connected sequences and elevated levels of IgG antibodies to GP. However, intrahepatic B cells did not produce increased levels of cytokines and their depletion with anti-CD20 antibody did not alter the CD4+ T cell response in Alb-iGP_Smarta mice. Moreover, B cell depletion did not prevent spontaneous liver inflammation and AIH-like disease in Alb-iGP_Smarta mice. In conclusion, selection and isotype-switch of liver-infiltrating B cells was dependent on the presence of CD4+ T cells recognizing liver antigen. However, recognition of hepatic antigen by CD4+ T cells and CD4+ T cell-mediated hepatitis was not dependent on B cells. Thus, autoreactive B cells can be bystanders and need not be drivers of liver inflammation in AIH.

Guideline Concordance of Treatment and Outcomes Among Adult Non-Hodgkin Lymphoma Patients in Sub-Saharan Africa: A Multinational, Population-Based Cohort.

BACKGROUND: Although non-Hodgkin lymphoma (NHL) is the 6th most common malignancy in Sub-Saharan Africa (SSA), little is known about its management and outcome. Herein, we examined treatment patterns and survival among NHL patients. METHODS: We obtained a random sample of adult patients diagnosed between 2011 and 2015 from 11 population-based cancer registries in 10 SSA countries. Descriptive statistics for lymphoma-directed therapy (LDT) and degree of concordance with National Comprehensive Cancer Network (NCCN) guidelines were calculated, and survival rates were estimated. FINDINGS: Of 516 patients included in the study, sub-classification was available for 42.1% (121 high-grade and 64 low-grade B-cell lymphoma, 15 T-cell lymphoma and 17 otherwise sub-classified NHL), whilst the remaining 57.9% were unclassified. Any LDT was identified for 195 of all patients (37.8%). NCCN guideline-recommended treatment was initiated in 21 patients. This corresponds to 4.1% of all 516 patients, and to 11.7% of 180 patients with sub-classified B-cell lymphoma and NCCN guidelines available. Deviations from guideline-recommended treatment were initiated in another 49 (9.5% of 516, 27.2% of 180). By registry, the proportion of all patients receiving guideline-concordant LDT ranged from 30.8% in Namibia to 0% in Maputo and Bamako. Concordance with treatment recommendations was not assessable in 75.1% of patients (records not traced (43.2%), traced but no sub-classification identified (27.8%), traced but no guidelines available (4.1%)). By registry, diagnostic work-up was in part importantly limited, thus impeding guideline evaluation significantly. Overall 1-year survival was 61.2% (95%CI 55.3%-67.1%). Poor ECOG performance status, advanced stage, less than 5 cycles and absence of chemo (immuno-) therapy were associated with unfavorable survival, while HIV status, age, and gender did not impact survival. In diffuse large B-cell lymphoma, initiation of guideline-concordant treatment was associated with favorable survival. INTERPRETATION: This study shows that a majority of NHL patients in SSA are untreated or undertreated, resulting in unfavorable survival. Investments in enhanced diagnostic services, provision of chemo(immuno-)therapy and supportive care will likely improve outcomes in the region.

Liquid biomarkers of macrophage dysregulation and circulating spike protein illustrate the biological heterogeneity in patients with post-acute sequelae of COVID-19.

Post-acute sequelae of COVID-19 (PASC) are long-term consequences of SARS-CoV-2 infection that can substantially impair the quality of life. Underlying mechanisms ranging from persistent viruses to innate and adaptive immune dysregulation have been discussed. Here, we profiled the plasma of 181 individuals from the cohort study for digital health research in Germany (DigiHero), including individuals after mild to moderate COVID-19 with or without PASC and uninfected controls. We focused on soluble factors related to monocyte/macrophage biology and on circulating SARS-CoV-2 spike (S1) protein as a potential biomarker for persistent viral reservoirs. At a median time of 8 months after infection, we found pronounced dysregulation in almost all tested soluble factors, including both pro-inflammatory and pro-fibrotic cytokines. These immunological perturbations were remarkably independent of ongoing PASC symptoms per se, but further correlation and regression analyses suggested PASC-specific patterns involving CCL2/MCP-1 and IL-8 that either correlated with sCD162, sCD206/MMR, IFN-α2, IL-17A and IL-33, or IL-18 and IL-23. None of the analyzed factors correlated with the detectability or levels of circulating S1, indicating that this represents an independent subset of patients with PASC. These data confirm prior evidence of immune dysregulation and persistence of viral protein in PASC and illustrate its biological heterogeneity that still awaits correlation with clinically defined PASC subtypes.

Superantigenic character of an insert unique to SARS-CoV-2 spike supported by skewed TCR repertoire in patients with hyperinflammation.

Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

Characterization of the T cell receptor repertoire and melanoma tumor microenvironment upon combined treatment with ipilimumab and hTERT vaccination.

BACKGROUND: This clinical trial evaluated a novel telomerase-targeting therapeutic cancer vaccine, UV1, in combination with ipilimumab, in patients with metastatic melanoma. Translational research was conducted on patient-derived blood and tissue samples with the goal of elucidating the effects of treatment on the T cell receptor repertoire and tumor microenvironment. METHODS: The trial was an open-label, single-center phase I/IIa study. Eligible patients had unresectable metastatic melanoma. Patients received up to 9 UV1 vaccinations and four ipilimumab infusions. Clinical responses were assessed according to RECIST 1.1. Patients were followed up for progression-free survival (PFS) and overall survival (OS). Whole-exome and RNA sequencing, and multiplex immunofluorescence were performed on the biopsies. T cell receptor (TCR) sequencing was performed on the peripheral blood and tumor tissues. RESULTS: Twelve patients were enrolled in the study. Vaccine-specific immune responses were detected in 91% of evaluable patients. Clinical responses were observed in four patients. The mPFS was 6.7 months, and the mOS was 66.3 months. There was no association between baseline tumor mutational burden, neoantigen load, IFN-γ gene signature, tumor-infiltrating lymphocytes, and response to therapy. Tumor telomerase expression was confirmed in all available biopsies. Vaccine-enriched TCR clones were detected in blood and biopsy, and an increase in the tumor IFN-γ gene signature was detected in clinically responding patients. CONCLUSION: Clinical responses were observed irrespective of established predictive biomarkers for checkpoint inhibitor efficacy, indicating an added benefit of the vaccine-induced T cells. The clinical and immunological read-out warrants further investigation of UV1 in combination with checkpoint inhibitors. Trial registration Clinicaltrials.gov identifier: NCT02275416. Registered October 27, 2014. https://clinicaltrials.gov/ct2/show/NCT02275416?term=uv1&draw=2&rank=6.

Next-Generation Immunosequencing Reveals Pathological T-Cell Architecture in Autoimmune Hepatitis.

BACKGROUND AND AIMS: Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T-cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH. APPROACH AND RESULTS: T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next-generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV-J genes. More importantly, we found strong AIH-specific TRBV-J skewing not attributable to the HLA-DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies. CONCLUSIONS: Patients with AIH show profound and persisting T-cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T-cell clusters shared between patients may mediate liver damage and warrant further study.

Correlation of nutrition-associated parameters with non-relapse mortality in allogeneic hematopoietic stem cell transplantation.

Outcome of allogeneic stem cell transplantation (alloSCT) is hampered by substantial non-relapse mortality (NRM). Given its impact on organ function and immune response, the nutritional status has been suggested as relevant for NRM. We aimed to evaluate the association of NRM with nutritional status prior to alloSCT and in the post-SCT course. In a retrospective single-center study, we analyzed 128 alloSCTs. Besides standard characteristics, nutrition-associated parameters BMI, serum total protein, and serum albumin were recorded before conditioning and at various time points after alloSCT. Association with NRM was evaluated by univariate and multivariate survival analysis. The cohort comprised patients with a median BMI of 26 kg/m2 (16.7-46.9 kg/m2), median serum total protein of 59 g/l (41-77 g/l), and serum albumin of 36 g/l (22-46 g/l) before SCT. NRM at d+100 was 14.8% and at 1 year 26.6%. Prior to SCT, only serum albumin deficiency was associated with increased NRM (p = .010) in multivariate analysis. After SCT (d+30 and d+100), all nutrition-associated parameters decreased (p < .002), but no association of deteriorating nutritional status with NRM was found. In multivariate analysis, serum albumin (p = .03) and severe albumin deficiency (p = .02) correlated with NRM at d+30 and d+100, while BMI and serum total protein did not. In our study, albumin deficiency, particularly prior to alloSCT, shows a strong correlation with NRM. This finding may add to monitoring, risk evaluation, and counseling of patients and serve as a rational for interventions to improve the nutritional status in patients undergoing SCT.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Evolutionary clonal trajectories in nodular lymphocyte-predominant Hodgkin lymphoma with high risk of transformation.

The B-cell architecture of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is complex since it is composed of malignant lymphocyte-predominant cells along with a rich B-cell bystander environment. To gain insight into molecular determinants of disease transformation, we studied B-cell evolutionary trajectories in lymphoma tissue from diagnosis to relapse or transformation to non- Hodgkin lymphoma by next-generation sequencing of immunoglobulin heavy chains. Patients with NLPHL that later transformed were older and showed IgD negativity, absence of the characteristic IGHV3/IGHD3/IGHJ6 lymphocyte-predominant rearrangement and high repertoire clonality. We constructed phylogenetic trees within the compartment of the malignant clone to investigate clonal evolution. In all relapsing cases, the lymphocyte-predominant rearrangement was identical at diagnosis and relapse. NLPHL cases with transformation showed more complex trajectories with strong intraclonal diversification. The dominant founder clone in transformations showed clonal evolution if derived from the same cell of origin, or arose from a different cell of origin. Together, our data point to a significant role of antigenic drive in the transformation of NLHPL and identify high B-cell repertoire clonality with dominant intraclonal lymphocyte-predominant cell diversification as a hallmark of transformation. Sequencing of initial paraffin-embedded tissue may therefore be applied diagnostically to identify NLPHL cases with high risk of transformation.

Clinical presentation and diagnosis of adult patients with non-Hodgkin lymphoma in Sub-Saharan Africa.

Non-Hodgkin lymphoma (NHL) is the sixth most common cancer in Sub-Saharan Africa (SSA). Comprehensive diagnostics of NHL are essential for effective treatment. Our objective was to assess the frequency of NHL subtypes, disease stage and further diagnostic aspects. Eleven population-based cancer registries in 10 countries participated in our observational study. A random sample of 516 patients was included. Histological confirmation of NHL was available for 76.2% and cytological confirmation for another 17.3%. NHL subclassification was determined in 42.1%. Of these, diffuse large B cell lymphoma, chronic lymphocytic leukaemia and Burkitt lymphoma were the most common subtypes identified (48.8%, 18.4% and 6.0%, respectively). We traced 293 patients, for whom recorded data were amended using clinical records. For these, information on stage, human immunodeficiency virus (HIV) status and Eastern Cooperative Oncology Group Performance Status (ECOG PS) was available for 60.8%, 52.6% and 45.1%, respectively. Stage at diagnosis was advanced for 130 of 178 (73.0%) patients, HIV status was positive for 97 of 154 (63.0%) and ECOG PS was ≥2 for 81 of 132 (61.4%). Knowledge about NHL subclassification and baseline clinical characteristics is crucial for guideline-recommended treatment. Hence, regionally adapted investments in pathological capacity, as well as standardised clinical diagnostics, will significantly improve the therapeutic precision for NHL in SSA.

Ipilimumab or FOLFOX with Nivolumab and Trastuzumab in previously untreated HER2-positive locally advanced or metastatic EsophagoGastric Adenocarcinoma - the randomized phase 2 INTEGA trial (AIO STO 0217).

BACKGROUND: Esophagogastric adenocarcinoma (EGA) currently represents a main cause of cancer related death. Despite an intensified treatment for locally advanced or metastatic EGA with a doublet chemotherapy consisting of a platinum compound and a fluoropyrimidine in combination with trastuzumab for HER2-positive disease or in selected cases with docetaxel, survival remains poor. Recently, immune-oncology based strategies relevantly improved the treatment of different solid tumors and showed some promise in late or later stage trials in EGA. Notably, the combination of immunotherapy with trastuzumab to enhance anti-tumor immunity through activation of innate and adaptive immunity was beneficial in preclinical studies or clinical studies in breast cancer. METHODS: The INTEGA study is an open-label, randomized, multicenter, exploratory phase II trial designed to assess clinical performance, safety and tolerability of ipilimumab or 5-FU/folinic acid and oxaliplatin (FOLFOX) in combination with nivolumab and trastuzumab in patients with previously untreated HER2-positive, locally advanced or metastatic EGA. The primary objective is to determine the clinical performance of ipilimumab or FOLFOX in combination with nivolumab and trastuzumab in terms of overall survival. Secondary objectives are safety and tolerability, efficacy in terms of progression-free survival and objective response rate and blood-based signatures (e.g. immune response or suppression of anti-HER2 resistance) that may correlate with treatment response. DISCUSSION: Recent evidence from the phase II NCT02954536 study (oxaliplatin, capecitabine, trastuzumab and pembrolizumab) suggests the clinical feasibility of combining chemotherapy, trastuzumab and checkpoint inhibition in EGA. However, evidence for a chemotherapy-free regimen is also mounting in HER2-positive disease (NCT02689284) i.e. margetuximab and Pembrolizumab. Both studies excelled with high overall response rates and manageable toxicities. The INTEGA study aims to comparatively assess these results and select a promising new 1st line regimen which then needs to be confirmed in a randomized phase III trial. Further, the translational part of the study might help to better stratify patients and tailor treatment of either arm. TRIAL REGISTRATION: NCT03409848 24.01.2018.

Phase III study of nivolumab alone or combined with ipilimumab as immunotherapy versus standard of care in resectable head and neck squamous cell carcinoma.

Locally advanced head and neck squamous cell carcinoma (LA-HNSCC) often requires postoperative chemoradiation with high risk of toxicity. Disease-free survival (DFS) after 2 years is approximately 70%. Combining nivolumab (N), a PD-1-inhibitor and ipilimumab (I), a CTLA4- inhibitor, may improve DFS due to antitumor effects of immunotherapy. The IMSTAR-HN study compares neoadjuvant N and N ± I 6 months after adjuvant therapy versus standard therapy as first-line treatment for LA-HNSCC. Eligible patients have treatment-naive LA-HNSCC, Eastern cooperative oncology group performance score (PS) ≤1 and no distant metastasis. 276 patients will be randomized into two arms. Primary endpoint is DFS and secondary endpoint includes locoregional control (LRC) and overall survival (OS). This study is one of the first in HNSCCs implementing immunotherapy in first-line treatment in a curative setting. Clinical Trial Registration: NCT03700905 (ClinicalTrials.gov).

The burden of hepatitis E among patients with haematological malignancies: A retrospective European cohort study.

BACKGROUND & AIMS: The burden of hepatitis E virus (HEV) infection among patients with haematological malignancy has only been scarcely reported. Therefore, we aimed to describe this burden in patients with haematological malignancies, including those receiving allogeneic haematopoietic stem cell transplantation. METHODS: We conducted a retrospective, multicentre cohort study across 11 European centres and collected clinical characteristics of 50 patients with haematological malignancy and RNA-positive, clinically overt hepatitis E between April 2014 and March 2017. The primary endpoint was HEV-associated mortality; the secondary endpoint was HEV-associated liver-related morbidity. RESULTS: The most frequent underlying haematological malignancies were aggressive non-Hodgkin lymphoma (NHL) (34%), indolent NHL (iNHL) (24%), and acute leukaemia (36%). Twenty-one (42%) patients had received allogeneic haematopoietic stem cell transplantation (alloHSCT). Death with ongoing hepatitis E occurred in 8 (16%) patients, including 1 patient with iNHL and 1 patient >100 days after alloHSCT in complete remission, and was associated with male sex (p = 0.040), cirrhosis (p = 0.006) and alloHSCT (p = 0.056). Blood-borne transmission of hepatitis E was demonstrated in 5 (10%) patients, and associated with liver-related mortality in 2 patients. Hepatitis E progressed to chronic hepatitis in 17 (34%) patients overall, and in 10 (47.6%) and 6 (50%) alloHSCT and iNHL patients, respectively. Hepatitis E was associated with acute or acute-on-chronic liver failure in 4 (8%) patients with 75% mortality. Ribavirin was administered to 24 (48%) patients, with an HEV clearance rate of 79.2%. Ribavirin treatment was associated with lower mortality (p = 0.037) and by trend with lower rates of chronicity (p = 0.407) when initiated <24 and <12 weeks after diagnosis of hepatitis E, respectively. Immunosuppressive treatment reductions were associated with mortality in 2 patients (28.6%). CONCLUSION: Hepatitis E is associated with mortality and liver-related morbidity in patients with haematological malignancy. Blood-borne transmission contributes to the burden. Ribavirin should be initiated early, whereas reduction of immunosuppressive treatment requires caution. LAY SUMMARY: Little is known about the burden of hepatitis E among patients with haematological malignancy. We conducted a retrospective European cohort study among 50 patients with haematological malignancy, including haematopoietic stem cell transplant recipients, with clinically significant HEV infection and found that hepatitis E is associated with hepatic and extrahepatic mortality, including among patients with indolent disease or among stem cell transplant recipients in complete remission. Hepatitis E virus infection evolved to chronic hepatitis in 5 (45.5%) patients exposed to a rituximab-containing regimen and 10 (47.6%) stem cell transplant recipients. Reducing immunosuppressive therapy because of hepatitis E was associated with mortality, while early ribavirin treatment was safe and effective.

Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking.

The CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19- relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19- ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19- escape variant first detected after only 2 treatment courses. In 1 patient, the CD19- clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19- progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post-endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

Deep sequencing of bone marrow microenvironments of patients with del(5q) myelodysplastic syndrome reveals imprints of antigenic selection as well as generation of novel T-cell clusters as a response pattern to lenalidomide.

In myelodysplastic syndromes with a partial deletion of the long arm of chromosome 5, del(5q), lenalidomide is believed to reverse anergic T-cell immunity in the bone marrow resulting in suppression of the del(5q) clone. In this study we used next-generation sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements in bone marrow-residing and peripheral blood-circulating lymphocytes of patients with del(5q) myelodysplastic syndromes to assess the immune architecture and track adaptive immune responses during treatment with lenalidomide. The baseline bone marrow B-cell space in patients was comparable to that of age-matched healthy controls in terms of gene usage and IGH clonality, but showed a higher percentage of hypermutated IGH sequences, indicating an expanded number of antigen-experienced B lineage cells. Bone marrow B lineage clonality decreased significantly and hypermutated IGH clones normalized upon lenalidomide treatment, well in line with the proliferative effect on healthy antigen-inexperienced B-cell precursors previously described for this drug. The T-cell space in bone marrow of patients with del(5q) myelodysplastic syndromes showed higher TRB clonality compared to that of healthy controls. Upon lenalidomide treatment, myelodysplastic syndrome-specific T-cell clusters with low to medium spontaneous generation probabilities emerged; these clusters were shared across patients, indicating a common antigen-driven T-cell response pattern. Hence, we observed B lineage diversification and generation of new, antigen-dependent T-cell clusters, compatible with a model of adaptive immunity induced against the del(5q) clone by lenalidomide. Overall, this supports the concept that lenalidomide not only alters the functional T-cell state, but also the composition of the T- and B-cell repertoires in del(5q) myelodysplastic syndromes.

A transplant "immunome" screening platform defines a targetable epitope fingerprint of multiple myeloma.

Multiple myeloma (MM) is a hematological cancer for which immune-based treatments are currently in development. Many of these rely on the identification of highly disease-specific, strongly and stably expressed antigens. Here, we profiled the myeloma B-cell immunome both to explore its predictive role in the context of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and to identify novel immunotherapeutic targets. We used random peptide phage display, reverse immunization, and next-generation sequencing-assisted antibody phage display to establish a highly myeloma-specific epitope fingerprint targeted by B-cell responses of 18 patients in clinical remission. We found that allogeneic HSCT more efficiently allowed production of myeloma-specific antibodies compared with autologous HSCT and that a highly reactive epitope recognition signature correlated with superior response to treatment. Next, we performed myeloma cell surface screenings of phage-displayed patient transplant immunomes. Although some of the screenings yielded clear-cut surface binders, the majority of screenings did not, suggesting that many of the targeted antigens may in fact not be accessible to the B-cell immune system in untreated myeloma cells. This fit well with the identification of heat-shock proteins as a class of antigens that showed overall the broadest reactivity with myeloma patient sera after allogeneic HSCT and that may be significantly translocated to the cell surface upon treatment as a result of immunogenic cell death. Our data reveal a disease-specific epitope signature of MM that is predictive for response to treatment. Mining of transplant immunomes for strong myeloma surface binders may open up avenues for myeloma immunotherapy.