Anders Ellern Bilgrau

Diffuse Large B-Cell Lymphoma Classification System That Associates Normal B-Cell Subset Phenotypes With Prognosis

PURPOSE Current diagnostic tests for diffuse large B-cell lymphoma use the updated WHO criteria based on biologic, morphologic, and clinical heterogeneity. We propose a refined classification system based on subset-specific B-cell-associated gene signatures (BAGS) in the normal B-cell hierarchy, hypothesizing that it can provide new biologic insight and diagnostic and prognostic value. PATIENTS AND METHODS We combined fluorescence-activated cell sorting, gene expression profiling, and statistical modeling to generate BAGS for naive, centrocyte, centroblast, memory, and plasmablast B cells from normal human tonsils. The impact of BAGS-assigned subtyping was analyzed using five clinical cohorts (treated with cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP], n = 270; treated with rituximab plus CHOP [R-CHOP], n = 869) gathered across geographic regions, time eras, and sampling methods. The analysis estimated subtype frequencies and drug-specific resistance and included a prognostic meta-analysis of patients treated with first-line R-CHOP therapy. RESULTS Similar BAGS subtype frequencies were assigned across 1,139 samples from five different cohorts. Among R-CHOP-treated patients, BAGS assignment was significantly associated with overall survival and progression-free survival within the germinal center B-cell-like subclass; the centrocyte subtype had a superior prognosis compared with the centroblast subtype. In agreement with the observed therapeutic outcome, centrocyte subtypes were estimated as being less resistant than the centroblast subtype to doxorubicin and vincristine. The centroblast subtype had a complex genotype, whereas the centrocyte subtype had high TP53 mutation and insertion/deletion frequencies and expressed LMO2, CD58, and stromal-1-signature and major histocompatibility complex class II-signature genes, which are known to have a positive impact on prognosis. CONCLUSION Further development of a diagnostic platform using BAGS-assigned subtypes may allow pathogenetic studies to improve disease management.

High miR-34a expression improves response to doxorubicin in diffuse large B-cell lymphoma

The standard treatment for patients with diffuse large B-cell lymphoma (DLBCL) is the immunochemotherapy-based R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone). Resistance to treatment, intrinsic or acquired, is observed in approximately 40% of patients with DLBCL, who thus require novel interventions to survive. To identify biomarkers for cytotoxic response assessment, microRNAs (miRNAs) associated with doxorubicin sensitivity were determined by combining global miRNA expression profiling with systematic dose-response screens in 15 human DLBCL cell lines. One candidate, miR-34a, was tested in functional in vitro studies and in vivo in a retrospective clinical cohort. High expression of miR-34a was observed in cell lines sensitive to doxorubicin, and upregulation of miR-34a is documented here to increase doxorubicin sensitivity in in vitro lentiviral transduction assays. High expression of miR-34a had a prognostic impact using overall survival as outcome. With risk stratification of DLBCL samples based on resistance gene signatures (REGS), doxorubicin-responsive samples had statistically significant upregulated miR-34a expression. Classification of the DLBCL samples into subset-specific B cell-associated gene signatures (BAGS) revealed differentiation-specific expression of miR-34a. Our data further support FOXP1 as a target of miR-34a, suggesting that downregulation of FOXP1 may sensitize DLBCL cells to doxorubicin. We conclude that miRNAs, in particular miR-34a, may have clinical utility in DLBCL patients as both predictive and prognostic biomarkers.

Stable Phenotype Of B‐Cell Subsets Following Cryopreservation and Thawing of Normal Human Lymphocytes Stored in a Tissue Biobank

Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping.

A population-based study of prognosis in advanced stage follicular lymphoma managed by watch and wait

Watch and wait (WAW) is a common approach for asymptomatic, advanced stage follicular lymphoma (FL), but single‐agent rituximab is an alternative for these patients. In this nationwide study we describe the outcome of patients selected for WAW. A cohort of 286 out of 849 (34%) stage III‐IVA FL patients seen between 2000 and 2011, were managed expectantly and included. The 5‐year progression‐free survival (PFS) was 35% [95% confidence interval (CI) 29–42]. The 10‐year overall survival (OS) was 65% (95%CI 54–78), and the cumulative risk of dying from lymphoma within 10 years of diagnosis was 13% (95%CI 7–20). Elevated lactate dehydrogenase and > four nodal regions involved were associated with a higher risk of lymphoma treatment and death from lymphoma. The WAW patients and a matched background population had similar OS during the first 50 months after diagnosis (P = 0·7), but WAW patients had increased risk of death after 50 months (P < 0·001). The estimated loss of residual life after 10 years was 6·8 months. The 10‐year cumulative risk of histological transformation was 22% (95%CI 15–29) and the 3‐year OS after transformation was 71% (95%CI 58–87%). In conclusion, advanced stage FL managed by WAW had a favourable outcome and abandoning this strategy could lead to overtreatment in some patients.

Proof of the concept to use a malignant B cell line drug screen strategy for identification and weight of melphalan resistance genes in multiple myeloma.

In a conceptual study of drug resistance we have used a preclinical model of malignant B-cell lines by combining drug induced growth inhibition and gene expression profiling. In the current report a melphalan resistance profile of 19 genes were weighted by microarray data from the MRC Myeloma IX trial and time to progression following high dose melphalan, to generate an individual melphalan resistance index. The resistance index was subsequently validated in the HOVON65/GMMG-HD4 trial data set to prove the concept. Biologically, the assigned resistance indices were differentially distributed among translocations and cyclin D expression classes. Clinically, the 25% most melphalan resistant, the intermediate 50% and the 25% most sensitive patients had a median progression free survival of 18, 32 and 28 months, respectively (log-rank P-value  = 0.05). Furthermore, the median overall survival was 45 months for the resistant group and not reached for the intermediate and sensitive groups (log-rank P-value  = 0.003) following 38 months median observation. In a multivariate analysis, correcting for age, sex and ISS-staging, we found a high resistance index to be an independent variable associated with inferior progression free survival and overall survival. This study provides clinical proof of concept to use in vitro drug screen for identification of melphalan resistance gene signatures for future functional analysis.

Interrater Reliability of Diagnostic Methods in Traditional Indian Ayurvedic Medicine

This study assesses the interrater reliability of Ayurvedic pulse (nadi), tongue (jivha), and body constitution (prakriti) assessments. Fifteen registered Ayurvedic doctors with 3–15 years of experience independently examined twenty healthy subjects. Subjects completed self-assessment questionnaires and software analyses for prakriti assessment. Weighted kappa statistics for all 105 pairs of doctors were computed for the pulse, tongue, and prakriti data sets. According to the Landis-Koch scale, the pairwise kappas ranged from poor to slight, slight to fair, and fair to moderate for pulse, tongue, and prakriti assessments, respectively. The average pairwise kappa for pulse, tongue, and prakriti was 0.07, 0.17, and 0.28, respectively. For each data set and pair of doctors, the null hypothesis of random rating was rejected for just twelve pairs of doctors for prakriti, one pair of doctors for pulse examination, and no pairs of doctors for tongue assessment. Thus, the results demonstrate a low level of reliability for all types of assessment made by doctors. There was significant evidence against random rating by software and questionnaire use and by the diagnosis preferred by the majority of doctors. Prakriti assessment appears reliable when questionnaire and software assessment are used, while other diagnostic methods have room for improvement.

HemaClass.org: Online one-by-one microarray normalization and classification of hematological cancers for precision medicine

Background Dozens of omics based cancer classification systems have been introduced with prognostic, diagnostic, and predictive capabilities. However, they often employ complex algorithms and are only applicable on whole cohorts of patients, making them difficult to apply in a personalized clinical setting. Results This prompted us to create hemaClass.org, an online web application providing an easy interface to one-by-one RMA normalization of microarrays and subsequent risk classifications of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin and chemotherapeutic sensitivity classes. Classification results for one-by-one array pre-processing with and without a laboratory specific RMA reference dataset were compared to cohort based classifiers in 4 publicly available datasets. Classifications showed high agreement between one-by-one and whole cohort pre-processsed data when a laboratory specific reference set was supplied. The website is essentially the R-package hemaClass accompanied by a Shiny web application. The well-documented package can be used to run the website locally or to use the developed methods programmatically. Conclusions The website and R-package is relevant for biological and clinical lymphoma researchers using affymetrix U-133 Plus 2 arrays, as it provides reliable and swift methods for calculation of disease subclasses. The proposed one-by-one pre-processing method is relevant for all researchers using microarrays.

Bone Marrow Aspiration: A Randomized Controlled Trial Assessing the Quality of Bone Marrow Specimens Using Slow and Rapid Aspiration Techniques and Evaluating Pain Intensity

Background/Aims: Bone marrow aspiration (BMA) is an essential procedure in the examination of hematological disorders, but there is limited evidence as to whether the aspiration rate affects specimen quality. We aimed to assess the specimen quality and pain intensity using slow (S-technique) or rapid (R-technique) aspiration. Methods: This was a single-center, prospective, randomized patient- and assessor-blinded study of 482 patients scheduled for BMA. Specimen quality was evaluated by grading bone marrow (BM) cellularity and counting the number of marrow particles. Pain was assessed using a visual analog scale (VAS). Results: We found a significant difference between the 2 groups with regard to the quality of specimens. For cellularity, the odds ratio (OR) for having a poor quality aspirate using the S-technique versus the R-technique was 3.05 [confidence interval (CI) 1.79-5.31]. For BM particles, the quality of specimens with the S-technique proved to be poor compared with the R-technique (OR 2.52; CI 1.51-4.28). We found a statistically significant difference of 1 VAS point (p < 0.001) of the median pain intensity in favor of the S-technique. Conclusion: Even though the pain intensity is significantly higher with the R-technique, the median difference is relatively small. We propose that the R-technique is preferable to the S-technique due to better specimen quality.

Circulating tumor necrosis factor-α and YKL-40 level is associated with remission status following salvage therapy in relapsed non-Hodgkin lymphoma

1 Department of Hematology, 4 Department of Clinical Immunology and 5 Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark, 2 Department of Hematology and 7 Department of Medicine and Oncology, Herlev University Hospital, Herlev, Denmark, 3 Department of Hematology, Roskilde University Hospital, Roskilde, Denmark and 6 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark

Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates

BackgroundAccurate adjustment for the amplification efficiency (AE) is an important part of real-time quantitative polymerase chain reaction (qPCR) experiments. The most commonly used correction strategy is to estimate the AE by dilution experiments and use this as a plug-in when efficiency correcting the ΔΔCq. Currently, it is recommended to determine the AE with high precision as this plug-in approach does not account for the AE uncertainty, implicitly assuming an infinitely precise AE estimate. Determining the AE with such precision, however, requires tedious laboratory work and vast amounts of biological material. Violation of the assumption leads to overly optimistic standard errors of the ΔΔCq, confidence intervals, and p-values which ultimately increase the type I error rate beyond the expected significance level. As qPCR is often used for validation it should be a high priority to account for the uncertainty of the AE estimate and thereby properly bounding the type I error rate and achieve the desired significance level.ResultsWe suggest and benchmark different methods to obtain the standard error of the efficiency adjusted ΔΔCq using the statistical delta method, Monte Carlo integration, or bootstrapping. Our suggested methods are founded in a linear mixed effects model (LMM) framework, but the problem and ideas apply in all qPCR experiments. The methods and impact of the AE uncertainty are illustrated in three qPCR applications and a simulation study. In addition, we validate findings suggesting that MGST1 is differentially expressed between high and low abundance culture initiating cells in multiple myeloma and that microRNA-127 is differentially expressed between testicular and nodal lymphomas.ConclusionsWe conclude, that the commonly used efficiency corrected quantities disregard the uncertainty of the AE, which can drastically impact the standard error and lead to increased false positive rates. Our suggestions show that it is possible to easily perform statistical inference of ΔΔCq, whilst properly accounting for the AE uncertainty and better controlling the false positive rate.