Human leukocyte antigen (HLA) class I expression on Hodgkin-Reed-Sternberg cells is an EBV-independent major determinant of microenvironment composition in classic Hodgkin lymphoma.

Hodgkin-Reed-Sternberg cells (HRSCs) in classic Hodgkin Lymphoma (HL) frequently lack expression of human leukocyte antigen class I (HLA-I), considered to hamper activation of cytotoxic T cells in the tumor microenvironment (TME). Here, we demonstrate HLA-I expression on HRSCs to be a strong determinant of TME composition whereas expression of HLA-II was associated with only minor differential gene expression in the TME. In HLA-I-positive HL the HRSC content and expression of CCL17/TARC in HRSCs are low, independent of the presence of Epstein-Barr virus in HRSCs. Additionally, HLA-I-positive HL shows a high content of CD8+ cytotoxic T cells. However, an increased expression of the inhibitory immune checkpoint LAG3 on CD8+ T cells in close proximity to HRSCs is observed. Suggesting interference with cytotoxic activity, we observed an absence of clonally expanded T cells in the TME. While HLA-I-positive HL is not associated with an unfavorable clinical course in our cohorts, they share features with the recently described H2 subtype of HL. Given the major differences in TME composition, immune checkpoint inhibitors may differ in their mechanism of action in HLA-I-positive compared to HLA-I-negative HL.

Low B-cell content is associated with a CD73-low tumour microenvironment and unfavourable prognosis in classic Hodgkin lymphoma.

B-cell content in the tumour microenvironment (TME) of classic Hodgkin lymphoma (HL) is known to be associated with prognosis. Here we demonstrate that whole slide image analysis using routinely available slides predicts outcomes in patients treated with ABVD in a prospective trial with a high B-cell content being associated with a favourable prognosis. B cells in the TME did not correlate with B cells in peripheral blood. In the TME maturation, stages of B cells (naive and memory) were consistent. However, we detected down-regulation of CD73 in HL with low B cells suggestive of an antibody-independent function of B cells in the TME of HL.

LINC00892 Is an lncRNA Induced by T Cell Activation and Expressed by Follicular Lymphoma-Resident T Helper Cells.

Successful immunotherapy in both solid tumors and in hematological malignancies relies on the ability of T lymphocytes to infiltrate the cancer tissue and mount an immune response against the tumor. Biomarkers able to discern the amount and the types of T lymphocytes infiltrating a given tumor therefore have high diagnostic and prognostic value. Given that lncRNAs are known to have a highly cell-type-specific expression pattern, we searched for lncRNAs specifically expressed by activated T cells and at the same time in a kind of lymphoma, follicular lymphoma, where the microenvironment is known to play a critical role in the regulation of antitumor immunity. We focused on a non-coding transcript, annotated as LINC00892, which reaches extremely high expression levels following cell activation in Jurkat cells. Interestingly LINC00892 has an expression pattern resembling that of genes involved in T cell memory. Accordingly, LINC00892 is mostly expressed by the effector memory and helper CD4+ T cell sub-types but not by naïve T cells. In situ analyses of LINC00892 expression in normal lymph nodes and in follicular lymphoma biopsies show that its expression is limited to CD4+ PD1hi T cells, with a subcellular localization within the germinal center matching that of follicular helper T cells. Our analysis therefore suggests that the previously uncharacterized lncRNA LINC00892 could be a useful biomarker for the detection of CD4+ memory T cells in both normal and tumor tissues.

Reverted exhaustion phenotype of circulating lymphocytes as immune correlate of anti-PD1 first-line treatment in Hodgkin lymphoma.

While classical Hodgkin lymphoma (HL) is highly susceptible to anti-programmed death protein 1 (PD1) antibodies, the exact modes of action remain controversial. To elucidate the circulating lymphocyte phenotype and systemic effects during anti-PD1 1st-line HL treatment we applied multicolor flow cytometry, FluoroSpot and NanoString to sequential samples of 81 HL patients from the NIVAHL trial (NCT03004833) compared to healthy controls. HL patients showed a decreased CD4 T-cell fraction, a higher percentage of effector-memory T cells and higher expression of activation markers at baseline. Strikingly, and in contrast to solid cancers, expression for 10 out of 16 analyzed co-inhibitory molecules on T cells (e.g., PD1, LAG3, Tim3) was higher in HL. Overall, we observed a sustained decrease of the exhausted T-cell phenotype during anti-PD1 treatment. FluoroSpot of 42.3% of patients revealed T-cell responses against ≥1 of five analyzed tumor-associated antigens. Importantly, these responses were more frequently observed in samples from patients with early excellent response to anti-PD1 therapy. In summary, an initially exhausted lymphocyte phenotype rapidly reverted during anti-PD1 1st-line treatment. The frequently observed IFN-y responses against shared tumor-associated antigens indicate T-cell-mediated cytotoxicity and could represent an important resource for immune monitoring and cellular therapy of HL.

9p24.1 alterations and programmed cell death 1 ligand 1 expression in early stage unfavourable classical Hodgkin lymphoma: an analysis from the German Hodgkin Study Group NIVAHL trial.

High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed-Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.

In-depth cell-free DNA sequencing reveals genomic landscape of Hodgkin's lymphoma and facilitates ultrasensitive residual disease detection.

BACKGROUND: Individualization of treatment in Hodgkin's lymphoma is necessary to improve cure rates and reduce treatment side effects. Currently, it is hindered by a lack of genomic characterization and sensitive molecular response assessment. Sequencing of cell-free DNA is a powerful strategy to understand the cancer genome and can be used for extremely sensitive disease monitoring. In Hodgkin's lymphoma, a high proportion of cell-free DNA is tumor-derived, whereas traditional tumor biopsies only contain a little tumor-derived DNA. METHODS: We comprehensively genotype and assess minimal residual disease in 121 patients with baseline plasma as well as 77 follow-up samples from a subset of patients with our targeted cell-free DNA sequencing platform. FINDINGS: We present an integrated landscape of mutations and copy number variations in Hodgkin's lymphoma. In addition, we perform a deep analysis of mutational processes driving Hodgkin's lymphoma, investigate the clonal structure of Hodgkin's lymphoma, and link several genotypes to Hodgkin's lymphoma phenotypes and outcome. Finally, we show that minimal residual disease assessment by repeat cell-free DNA sequencing, as early as a week after treatment initiation, predicts treatment response and progression-free survival, allowing highly improved treatment guidance and relapse prediction. CONCLUSIONS: Our targeted cell-free DNA sequencing platform reveals the genomic landscape of Hodgkin's lymphoma and facilitates ultrasensitive detection of minimal residual disease. FUNDING: Mildred Scheel School of Oncology Aachen-Bonn-Cologne-Düsseldorf MD Research Stipend, Next Generation Sequencing Competence Network grant 423957469, Deutsche Krebshilfe grant 70112502, Deutsche Forschungsgemeinschaft (DFG) grant EN 179/13-1, the HL MRD consortium, and the Frau-Weiskam und Christel Ruranski-Stiftung.

Cross-Sectional and Longitudinal Construct Validity of the Generic KINDL-A(dult)B(rief) Questionnaire in Adults with Thrombophilia or with Hereditary and Acquired Bleeding Disorders.

BACKGROUND/AIMS: The newly adapted generic KINDL-A(dult)B(rief) questionnaire showed satisfactory cross-sectional psychometric properties in adults with bleeding disorders or thrombophilia. This investigation aimed to evaluate its cross-sectional and longitudinal construct validity. METHODS: After ethical committee approval and written informed consent, 335 patients (mean age 51.8 ± 16.6 years, 60% women) with either predominant thrombophilia (n = 260) or predominant bleeding disorders (n = 75) participated. At baseline, patients answered the KINDL-AB, the MOS 36-item Short-Form Health Survey (SF-36), and the EQ-5D-3L. A subgroup of 117 patients repeated the questionnaire after a median follow-up of 2.6 years (range: 0.4-3.5). A priori hypotheses were evaluated regarding convergent correlations between KINDL-AB overall well-being and specific subscales, EQ-5D-3L index values (EQ-IV), EQ-5D visual analog scale (EQ-VAS), and SF-36 subscales. RESULTS: Contrary to hypothesis, baseline correlations between the KINDL-AB and EQ-IV/EQ-VAS were all moderate while, as hypothesized, several KINDL-AB subscales and SF-36 subscales correlated strongly. At follow-up, no significant changes in all three instruments occurred. Correlations between instruments over the follow-up were mostly moderate and partially strong. Contrary to hypothesis but consistent with no significant changes in health-related quality of life, convergent correlations between changes in KINDL-AB overall well-being, physical and psychological well-being, and EQ-IV/EQ-VAS were all weak. CONCLUSIONS: While repeated measures of KINDL-AB showed moderate to strong correlations, changes in KINDL-AB overall well-being and subscales correlated more weakly than expected with changes involving two established instruments of generic health status.

Whole-slide image analysis of the tumor microenvironment identifies low B-cell content as a predictor of adverse outcome in patients with advanced-stage classical Hodgkin lymphoma treated with BEACOPP.

A subset of patients with advanced-stage classical Hodgkin Lymphoma (cHL) relapse or progress following standard treatment. Given their dismal prognosis, identifying this group of patients upfront represents an important medical need. While prior research has identified characteristics of the tumor microenvironment, which are associated with cHL outcomes, biomarkers that are developed and validated in this high-risk group are still missing. Here, we applied whole-slide image analysis (WSI), a quantitative, large-scale assessment of tumor composition that utilizes conventional histopathology slides. We conducted WSI on a study cohort with pre-treatment biopsies of 340 advanced-stage cHL patients enrolled in the HD12 and HD15 trials of the German Hodgkin Study Group (GHSG), and tested our results in in a validation cohort of 147 advanced-stage cHL patients within the GHSG HD18 trial. All patients were treated with BEACOPP-based regimens. By quantifying T cells, B cells, Hodgkin-Reed-Sternberg-cells and macrophages with WSI, 80% of all cells in the tumor tissue were identified. Crucially, low B cell count was associated with significantly reduced progression-free survival (PFS) and overall survival (OS), while T cell-, macrophage- and Hodgkin-Reed-Sternberg-cell content was not associated with the risk of progression or relapse in the study cohort. We further validated low B cell content as a prognostic factor of PFS and OS in the validation cohort and demonstrate good inter-observer agreement of WSI. WSI may represent a key tool for risk stratification of advanced-stage cHL that can easily be added to the standard diagnostic histopathology work-up.

Tumor and microenvironment response but no cytotoxic T-cell activation in classic Hodgkin lymphoma treated with anti-PD1.

Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

An image dataset related to automated macrophage detection in immunostained lymphoma tissue samples.

BACKGROUND: We present an image dataset related to automated segmentation and counting of macrophages in diffuse large B-cell lymphoma (DLBCL) tissue sections. For the classification of DLBCL subtypes, as well as for providing a prognosis of the clinical outcome, the analysis of the tumor microenvironment and, particularly, of the different types and functions of tumor-associated macrophages is indispensable. Until now, however, most information about macrophages has been obtained either in a completely indirect way by gene expression profiling or by manual counts in immunohistochemically (IHC) fluorescence-stained tissue samples while automated recognition of single IHC stained macrophages remains a difficult task. In an accompanying publication, a reliable approach to this problem has been established, and a large set of related images has been generated and analyzed. RESULTS: Provided image data comprise (i) fluorescence microscopy images of 44 multiple immunohistostained DLBCL tumor subregions, captured at 4 channels corresponding to CD14, CD163, Pax5, and DAPI; (ii) "cartoon-like" total variation-filtered versions of these images, generated by Rudin-Osher-Fatemi denoising; (iii) an automatically generated mask of the evaluation subregion, based on information from the DAPI channel; and (iv) automatically generated segmentation masks for macrophages (using information from CD14 and CD163 channels), B-cells (using information from Pax5 channel), and all cell nuclei (using information from DAPI channel). CONCLUSIONS: A large set of IHC stained DLBCL specimens is provided together with segmentation masks for different cell populations generated by a reference method for automated image analysis, thus featuring considerable reuse potential.

Metabolic tumor volume, cancer cell fraction, and prognosis - the case of T-cell/histiocyte-rich large B-cell lymphoma.

T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a rare aggressive lymphoma characterized by a paucity of neoplastic B-cells and a majority of reactive T-cells with or without histiocytes. In the 'Positron Emission Tomography-Guided Therapy of Aggressive Non-Hodgkin Lymphomas' trial (clinicaltrials.gov NCT00554164; EudraCT 2006-001641-33), its frequency was less than 3%. While cancer cell content by quantitative histology was 10-times lower, baseline total metabolic tumor volume (TMTV) by 18-fluorodesoxyglucose positron emission tomography was 10-times higher in THRLBCL than in diffuse large B-cell lymphoma (DLBCL). When THRLBCL and DLBCL populations were matched for TMTV, the survival curves were superimposable. However, when the populations were matched for cancer cell volume by multiplying TMTV by cancer cell fraction, outcome in THRLBCL was worse than in DLBCL. Whether genetic differences between cancer cells, tumor-promoting properties of non-neoplastic cells, or both are responsible for inferior cancer cell volume-related outcome in THRLBCL, remains to be elucidated.

Reproducibility of histologic prognostic parameters for mantle cell lymphoma: cytology, Ki67, p53 and SOX11.

Mantle cell lymphoma (MCL) shows a clinical aggressiveness that varies from patient to patient. Despite major advances in outcomes with current immunochemotherapy, the future development of therapies requires risk stratification to tailor therapy intensity. Within the group of reference pathologists for the ongoing trials of the European MCL Network, we performed a round robin test on a tissue microarray to evaluate the reproducibility in assessing the biomarkers of outcome in MCL. Cytological subtype, Ki67-index and expression of p53 and SOX11 were evaluated on 20 diagnostic tumour samples by eight participating labs independently. We demonstrate that the assessment of the proliferation index by counting the Ki67 positive cells as well as assessment of SOX11 and p53 expression status is reproducible between labs. For the most established prognostic biomarker, Ki67, the intra-class correlation coefficient was very good when assessed as a continuous parameter (0.87). The agreement was lower when the values were analysed in a dichotomized way applying the commonly used cutoff of 30% (kappa = 0.65, complete concordance of all labs in 13/20 (65%)). Cases with discrepant results between labs in the dichotomized analysis showed mean values close to the cutoff of 30%. Centralised scoring and digital image analysis revealed results in line with the scores from individual labs. All cases in our cohort were additionally assessed for gene expression signatures and of TP53 gene alterations. Given the good reproducibility when guidelines of assessment are applied, the biomarker studied in this inter-laboratory test presents potential candidates to be enhanced for risk-stratification in the future clinical trials.

High CD206 levels in Hodgkin lymphoma-educated macrophages are linked to matrix-remodeling and lymphoma dissemination.

Macrophages (Mφ) are abundantly present in the tumor microenvironment and may predict outcome in solid tumors and defined lymphoma subtypes. Mφ heterogeneity, the mechanisms of their recruitment, and their differentiation into lymphoma-promoting, alternatively activated M2-like phenotypes are still not fully understood. Therefore, further functional studies are required to understand biological mechanisms associated with human tumor-associated Mφ (TAM). Here, we show that the global mRNA expression and protein abundance of human Mφ differentiated in Hodgkin lymphoma (HL)-conditioned medium (CM) differ from those of Mφ educated by conditioned media from diffuse large B-cell lymphoma (DLBCL) cells or, classically, by macrophage colony-stimulating factor (M-CSF). Conditioned media from HL cells support TAM differentiation through upregulation of surface antigens such as CD40, CD163, CD206, and PD-L1. In particular, RNA and cell surface protein expression of mannose receptor 1 (MRC1)/CD206 significantly exceed the levels induced by classical M-CSF stimulation in M2-like Mφ; this is regulated by interleukin 13 to a large extent. Functionally, high CD206 enhances mannose-dependent endocytosis and uptake of type I collagen. Together with high matrix metalloprotease9 secretion, HL-TAMs appear to be active modulators of the tumor matrix. Preclinical in ovo models show that co-cultures of HL cells with monocytes or Mφ support dissemination of lymphoma cells via lymphatic vessels, while tumor size and vessel destruction are decreased in comparison with lymphoma-only tumors. Immunohistology of human HL tissues reveals a fraction of cases feature large numbers of CD206-positive cells, with high MRC1 expression being characteristic of HL-stage IV. In summary, the lymphoma-TAM interaction contributes to matrix-remodeling and lymphoma cell dissemination.

Automated macrophage counting in DLBCL tissue samples: a ROF filter based approach.

BACKGROUND: For analysis of the tumor microenvironment in diffuse large B-cell lymphoma (DLBCL) tissue samples, it is desirable to obtain information about counts and distribution of different macrophage subtypes. Until now, macrophage counts are mostly inferred from gene expression analysis of whole tissue sections, providing only indirect information. Direct analysis of immunohistochemically (IHC) fluorescence stained tissue samples is confronted with several difficulties, e.g. high variability of shape and size of target macrophages and strongly inhomogeneous intensity of staining. Consequently, application of commercial software is largely restricted to very rough analysis modes, and most macrophage counts are still obtained by manual counting in microarrays or high power fields, thus failing to represent the heterogeneity of tumor microenvironment adequately. METHODS: We describe a Rudin-Osher-Fatemi (ROF) filter based segmentation approach for whole tissue samples, combining floating intensity thresholding and rule-based feature detection. Method is validated against manual counts and compared with two commercial software kits (Tissue Studio 64, Definiens AG, and Halo, Indica Labs) and a straightforward machine-learning approach in a set of 50 test images. Further, the novel method and both commercial packages are applied to a set of 44 whole tissue sections. Outputs are compared with gene expression data available for the same tissue samples. Finally, the ROF based method is applied to 44 expert-specified tumor subregions for testing selection and subsampling strategies. RESULTS: Among all tested methods, the novel approach is best correlated with manual count (0.9297). Automated detection of evaluation subregions proved to be fully reliable. Comparison with gene expression data obtained for the same tissue samples reveals only moderate to low correlation levels. Subsampling within tumor subregions is possible with results almost identical to full sampling. Mean macrophage size in tumor subregions is 152.5±111.3 µm2. CONCLUSIONS: ROF based approach is successfully applied to detection of IHC stained macrophages in DLBCL tissue samples. The method competes well with existing commercial software kits. In difference to them, it is fully automated, externally repeatable, independent on training data and completely documented. Comparison with gene expression data indicates that image morphometry constitutes an independent source of information about antibody-polarized macrophage occurence and distribution.

The translational science of hodgkin lymphoma.

Hodgkin Lymphoma (HL) is an unusual B-cell lymphoma because the malignant cells exist as a minority population in a densely cellular microenvironment. The microenvironment is comprised predominately of inflammatory and immune cells with fibrosis in some cases. There are multiple dysregulated signalling pathways that sustain HL within this microenvironment, such as the Nuclear factor-κB and Janus kinase/signal transducers and activators of transcription pathways. Advances in genomic medicine have enabled a better characterisation of the rare tumour cells and improved our understanding of the signalling mechanisms that exist between the malignant cell and its microenvironment. Current therapy for HL produces excellent clinical outcomes in most younger patients. However, problems with current treatment approaches include poorer outcomes in the elderly, toxicity of highly-effective combination chemotherapy regimens and relapse in high-risk patients. Better understanding of disease biology aids in upfront prognostication of patients, defines new methods for treatment monitoring and assists in the recognition of novel targets for therapy. Biology-driven therapies, including anti-CD30 antibody conjugates, cellular immunotherapies and immune modulation, particularly with checkpoint inhibitors, have changed treatment algorithms for relapsed/refractory patients. Future challenges exist in incorporating immune-based therapies earlier in treatment algorithms to reduce toxicity and prevent relapse for patients with HL.

T-Cell Clustering in Neoplastic Follicles of Follicular Lymphoma.

The nonneoplastic microenvironment is abundant in follicular lymphoma. Its composition has been reported to be associated with the course of the disease. Lack of animal models hampers studies of interaction between lymphoma and bystander cells. We aimed to identify indicators of cellular interaction exemplified by nonrandom distribution of cell types within neoplastic follicles. Physiological germinal centers and follicles in follicular lymphoma were stained to identify macrophages, all T, follicular T-helper, dendritic and B cells. Density of cell types and cell distribution (spatial point pattern) were analyzed by digital image analysis. The density of all T, follicular T-helper and dendritic cells was higher in the dark zone than in the light zone of physiological germinal centers. Densities of cell types in follicular lymphoma were intermediate between the light and the dark zone. All cell types analyzed showed a completely random spatial distribution pattern within the dark and the light zone, respectively. In follicular lymphoma B cells and macrophages displayed complete spatial randomness. In contrast, all T cells, follicular T-helper cells and dendritic cells showed clustering of each individual cell type within a radius of 6-10 µm in the lymphoma. We conclude that the distribution of nonneoplastic cells within follicles of follicular lymphoma is not random. T cells and dendritic cells form clusters within the follicles, suggestive of sites of interaction between microenvironment and lymphoma cells. These clusters might help to understand the interaction of lymphoma cells with the microenvironment and might provide a structure for therapeutic intervention.

Psychometric Properties of a Modified KINDL-R Questionnaire for Adolescents and Adults, and Construction of a Brief Version, the KINDL-A(dult)B(rief) Questionnaire, KINDL-AB.

BACKGROUND/AIMS: The generic quality of life KINDL-R -questionnaire is validated for use in children/adolescents ≤16 years. The aim of this cross-sectional investigation was to modify the KINDL-R questionnaire for use in adults and to validate its psychometric properties. METHODS: Five items of the KINDL-R questionnaire were adapted and the newly developed KINDL-A(dult) questionnaire administered to 255 patients with hereditary and acquired bleeding disorders (mean age 53 years). Its internal consistency and convergent and divergent construct validity were investigated and confirmatory factor analysis was used to evaluate the latent factor structure. RESULTS: The KINDL-A questionnaire showed satisfactory reliability, varying construct validity, but inconclusive factor structure. The KINDL-AB(rief) was developed by removing half of the items and combining 2 sub-axes. This led to factor loadings between 0.62 and 0.91 and increased overall fit (Goodness of fit > 0.8 and Root Mean Square Error of Approximation, RMSEA, < 0.08). Results were validated in 966 healthy blood donors (mean age 38 years). In this group, the KINDL-AB questionnaire showed factor loadings between 0.43 and 0.77, Goodness of fit > 0.95 and RMSEA < 0.05. CONCLUSIONS: The new KINDL-AB suggests sufficient to good psychometric properties in adult patients with hereditary and acquired bleeding disorders.

Round-robin test for the cell-of-origin classification of diffuse large B-cell lymphoma-a feasibility study using full slide staining.

Diffuse large B-cell lymphoma (DLBCL) is subdivided by gene expression analysis (GEP) into two molecular subtypes named germinal center B-cell-like (GCB) and activated B-cell-like (ABC) after their putative cell-of-origin (COO). Determination of the COO is considered mandatory in any new-diagnosed DLBCL, not otherwise specified according to the updated WHO classification. Despite the fact that pathologists are free to choose the method for COO classification, immunohistochemical (IHC) assays are most widely used. However, to the best of our knowledge, no round-robin test to evaluate the interlaboratory variability has been published so far. Eight hematopathology laboratories participated in an interlaboratory test for COO classification of 10 DLBCL tumors using the IHC classifier comprising the expression of CD10, BCL6, and MUM1 (so-called Hans classifier). The results were compared with GEP for COO signature and, in a subset, with results obtained by image analysis. In 7/10 cases (70%), at least seven laboratories assigned a given case to the same COO subtype (one center assessed one sample as not analyzable), which was in agreement with the COO subtype determined by GEP. The results in 3/10 cases (30%) revealed discrepancies between centers and/or between IHC and GEP subtype. Whereas the CD10 staining results were highly reproducible, staining for MUM1 was inconsistent in 50% and for BCL6 in 40% of cases. Image analysis of 16 slides stained for BCL6 (N = 8) and MUM1 (N = 8) of the two cases with the highest disagreement in COO classification were in line with the score of the pathologists in 14/16 stainings analyzed (87.5%). This study describes the first round-robin test for COO subtyping in DLBCL using IHC and demonstrates that COO classification using the Hans classifier yields consistent results among experienced hematopathologists, even when variable staining protocols are used. Data from this small feasibility study need to be validated in larger cohorts.

Health-related quality of life in children, adolescents and adults with hereditary and acquired bleeding disorders.

BACKGROUND: To better understand self-reported health-related quality-of-life (HrQoL) in children and adults with chronic hemostatic conditions compared with healthy controls. METHODS/PATIENTS/RESULTS: Group 1 consisted of 74 children/adolescents aged 8-18years with hereditary bleeding disorders (H-BD), 12 siblings and 34 peers. Group 2 consisted of 82 adult patients with hereditary/acquired bleeding disorders (H/A-BD), and group 3 of 198 patients with deep venous thrombosis (DVT) on anticoagulant therapy. Adult patients were compared to 1011 healthy blood donors. HrQoL was assessed with a 'revised KINDer Lebensqualitaetsfragebogen' (KINDL-R)-questionnaire adapted to adolescents and adults. No differences were found in multivariate analyses of self-reported HrQoL in children with H-BD. In contrast, apart from family and school-/work-related wellbeing in female patients with DVT the adult patients showed significantly lower HrQoL sub-dimensions compared to heathy control subjects. Furthermore, adults with H/A-BD disorders reported better friend-related HrQoL compared to patients with DVT, mainly due to a decreased HrQoL subscale in women on anticoagulation. CONCLUSION: In children with H-BD, HrQoL was comparable to siblings and peers. In adults with H/A-BD HrQoL was comparable to patients with DVT while healthy blood donors showed better HrQoL. The friend-related HrQoL subscale was significantly reduced in female compared to male patients.