Longitudinal minimal residual disease assessment in multiple myeloma patients in complete remission - results from the NMSG flow-MRD substudy within the EMN02/HO95 MM trial.

BACKGROUND: Multiple myeloma remains an incurable disease with multiple relapses due to residual myeloma cells in the bone marrow of patients after therapy. Presence of small number of cancer cells in the body after cancer treatment, called minimal residual disease, has been shown to be prognostic for progression-free and overall survival. However, for multiple myeloma, it is unclear whether patients attaining minimal residual disease negativity may be candidates for treatment discontinuation. We investigated, if longitudinal flow cytometry-based monitoring of minimal residual disease (flow-MRD) may predict disease progression earlier and with higher sensitivity compared to biochemical assessments. METHODS: Patients from the Nordic countries with newly diagnosed multiple myeloma enrolled in the European-Myeloma-Network-02/Hovon-95 (EMN02/HO95) trial and undergoing bone marrow aspiration confirmation of complete response, were eligible for this Nordic Myeloma Study Group (NMSG) substudy. Longitdudinal flow-MRD assessment of bone marrow samples was performed to identify and enumerate residual malignant plasma cells until observed clinical progression. RESULTS: Minimal residual disease dynamics were compared to biochemically assessed changes in serum free light chain and M-component. Among 20 patients, reaching complete response or stringent complete response during the observation period, and with ≥3 sequential flow-MRD assessments analysed over time, increasing levels of minimal residual disease in the bone marrow were observed in six cases, preceding biochemically assessed disease and clinical progression by 5.5 months and 12.6 months (mean values), respectively. Mean malignant plasma cells doubling time for the six patients was 1.8 months (95% CI, 1.4-2.3 months). Minimal malignant plasma cells detection limit was 4 × 10-5. CONCLUSIONS: Flow-MRD is a sensitive method for longitudinal monitoring of minimal residual disease dynamics in multiple myeloma patients in complete response. Increasing minimal residual disease levels precedes biochemically assessed changes and is an early indicator of subsequent clinical progression. TRIAL REGISTRATION: NCT01208766.

TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1-p38 Signaling.

Understanding the mechanisms regulating recruitment of human skeletal (stromal or mesenchymal) stem cells (hMSC) to sites of tissue injury is a prerequisite for their successful use in cell replacement therapy. Chemokine-like protein TAFA2 is a recently discovered neurokine involved in neuronal cell migration and neurite outgrowth. Here, we demonstrate a possible role for TAFA2 in regulating recruitment of hMSC to bone fracture sites. TAFA2 increased the in vitro trans-well migration and motility of hMSC in a dose-dependent fashion and induced significant morphological changes including formation of lamellipodia as revealed by high-content-image analysis at single-cell level. Mechanistic studies revealed that TAFA2 enhanced hMSC migration through activation of the Rac1-p38 pathway. In addition, TAFA2 enhanced hMSC proliferation, whereas differentiation of hMSC toward osteoblast and adipocyte lineages was not altered. in vivo studies demonstrated transient upregulation of TAFA2 gene expression during the inflammatory phase of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in serum levels of TAFA2 in patients after hip fracture. Finally, interleukin-1ß was found as an upstream regulator of TAFA2 expression. Our findings demonstrate that TAFA2 enhances hMSC migration and recruitment and thus is relevant for regenerative medicine applications. Stem Cells 2019;37:407-416.

High-Throughput Sequencing-Based Investigation of Viruses in Human Cancers by Multienrichment Approach.

BACKGROUND: Viruses and other infectious agents cause more than 15% of human cancer cases. High-throughput sequencing-based studies of virus-cancer associations have mainly focused on cancer transcriptome data. METHODS: In this study, we applied a diverse selection of presequencing enrichment methods targeting all major viral groups, to characterize the viruses present in 197 samples from 18 sample types of cancerous origin. Using high-throughput sequencing, we generated 710 datasets constituting 57 billion sequencing reads. RESULTS: Detailed in silico investigation of the viral content, including exclusion of viral artefacts, from de novo assembled contigs and individual sequencing reads yielded a map of the viruses detected. Our data reveal a virome dominated by papillomaviruses, anelloviruses, herpesviruses, and parvoviruses. More than half of the included samples contained 1 or more viruses; however, no link between specific viruses and cancer types were found. CONCLUSIONS: Our study sheds light on viral presence in cancers and provides highly relevant virome data for future reference.

Anthropometrics and prognosis in diffuse large B-cell lymphoma: a multicentre study of 653 patients.

OBJECTIVE: The impact of body mass index (BMI) and body surface area (BSA) on survival in diffuse large B-cell lymphoma (DLBCL) is controversial. Recent studies show superior outcomes for overweight and obese patients. PATIENTS AND METHODS: A total of 653 R-CHOP(-like)-treated DLBCL patients were included in this retrospective cohort study. Patients, baseline clinicopathologic characteristics and treatment information were retrieved from the Danish Lymphoma Registry. Anthropometric measures were obtained from chemotherapy prescription charts. RESULTS: Underweight (BMI <18.5 kg/m2 ) was associated with significantly worse progression-free survival (PFS) for male patients only in sex-stratified analyses (HR 3.92, 95% CI: 1.57-9.75, P = 0.003, for males; HR 1.65, 95% CI: 0.90-3.02, P = 0.107, for females). In multivariate analyses, underweight was associated with worse PFS for both sexes (HR 5.34, 95% CI: 2.07-13.79, P = 0.001, for males; HR 2.14, 95% CI: 1.12-4.08, P = 0.021, for females). Similar results were obtained in analyses of overall survival. In crude analyses, BSA <1.8 m2 was associated with worse PFS for men and women (HR 1.65, 95% CI: 1.03-2.65, P = 0.039, for men; HR 1.62, 95% CI: 1.03-2.56, P = 0.037, for women). In multivariate analyses, however, these associations diminished. CONCLUSIONS: Our study demonstrates that underweight DLBCL patients have worse outcomes following R-CHOP as compared to normal as well as overweight patients.

International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma.

Treatment of multiple myeloma has substantially changed over the past decade with the introduction of several classes of new effective drugs that have greatly improved the rates and depth of response. Response criteria in multiple myeloma were developed to use serum and urine assessment of monoclonal proteins and bone marrow assessment (which is relatively insensitive). Given the high rates of complete response seen in patients with multiple myeloma with new treatment approaches, new response categories need to be defined that can identify responses that are deeper than those conventionally defined as complete response. Recent attempts have focused on the identification of residual tumour cells in the bone marrow using flow cytometry or gene sequencing. Furthermore, sensitive imaging techniques can be used to detect the presence of residual disease outside of the bone marrow. Combining these new methods, the International Myeloma Working Group has defined new response categories of minimal residual disease negativity, with or without imaging-based absence of extramedullary disease, to allow uniform reporting within and outside clinical trials. In this Review, we clarify several aspects of disease response assessment, along with endpoints for clinical trials, and highlight future directions for disease response assessments.

Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates.

BACKGROUND: Accurate 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 Δ Δ C q . 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 Δ Δ C q , 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. RESULTS: We suggest and benchmark different methods to obtain the standard error of the efficiency adjusted Δ Δ C q 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. CONCLUSIONS: We 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 Δ Δ C q , whilst properly accounting for the AE uncertainty and better controlling the false positive rate.

No survival benefit associated with routine surveillance imaging for Hodgkin lymphoma in first remission: a Danish-Swedish population-based observational study.

The use of routine imaging for patients with classical Hodgkin lymphoma (HL) in complete remission (CR) is controversial. In a population-based study, we examined the post-remission survival of Danish and Swedish HL patients for whom follow-up practices were different. Follow-up in Denmark included routine imaging, usually for a minimum of 2 years, whereas clinical follow-up without routine imaging was standard in Sweden. A total of 317 Danish and 454 Swedish comparable HL patients aged 18-65 years, diagnosed in the period 2007-2012 and having achieved CR following ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine)/BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone) therapy, were included in the study. The cumulative progression rates in the first 2 years were 4% (95% confidence interval [CI] 1-7) for patients with stage I-II disease vs. 12% (95% CI 6-18) for patients with stage III-IV disease. An imaging-based follow-up practice was not associated with a better post-remission survival in general (P = 0·2) or in stage-specific subgroups (P = 0·5 for I-II and P = 0·4 for III-IV). Age ≥45 years was the only independent adverse prognostic factor for survival. In conclusion, relapse of HL patients with CR is infrequent and systematic use of routine imaging in these patients does not improve post-remission survival. The present study supports clinical follow-up without routine imaging, as encouraged by the recent Lugano classification.

Multiplex polymerase chain reaction-based prognostic models in diffuse large B-cell lymphoma patients treated with R-CHOP.

We present a multiplex analysis for genes known to have prognostic value in an attempt to design a clinically useful classification model in patients with diffuse large B-cell lymphoma (DLBCL). Real-time polymerase chain reaction was used to measure transcript levels of 28 relevant genes in 194 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). Including International Prognostic Index (IPI) as a variable in a penalized Cox regression, we investigated the association with disease progression for single genes or gene combinations in four models. The best model was validated in data from an online available R-CHOP treated cohort. With progression-free survival (PFS) as primary endpoint, the best performing IPI independent model incorporated the LMO2 and HLADQA1 as well as gene interactions for GCSAMxMIB1, GCSAMxCTGF and FOXP1xPDE4B. This model assigned 33% of patients (n = 60) to poor outcome with an estimated 3-year PFS of 40% vs. 87% for low risk (n = 61) and intermediate (n = 60) risk groups (P < 0·001). However, a simpler, IPI independent model incorporated LMO2 and BCL2 and assigned 33% of the patients with a 3-year PFS of 35% vs. 82% for low risk group (P < 0·001). We have documented the impact of a few single genes added to IPI for assignment in new drug trials.

Uterine, but not ovarian, female reproductive organ involvement at presentation by diffuse large B-cell lymphoma is associated with poor outcomes and a high frequency of secondary CNS involvement.

Involvement of the internal female reproductive organs by diffuse large B-cell lymphoma (DLBCL) is uncommon, and there are sparse data describing the outcomes of such cases. In total, 678 female patients with DLBCL staged with positron emission tomography/computed tomography and treated with rituximab-containing chemotherapy were identified from databases in Denmark, Great Britain, Australia, and Canada. Overall, 27/678 (4%) had internal reproductive organ involvement: uterus (n = 14), ovaries (n = 10) or both (n = 3). In multivariate analysis, women with uterine DLBCL experienced inferior progression-free survival and overall survival compared to those without reproductive organ involvement, whereas ovarian DLBCL was not predictive of outcome. Secondary central nervous system (CNS) involvement (SCNS) occurred in 7/17 (41%) women with uterine DLBCL (two patients with concomitant ovarian DLBCL) and 0/10 women with ovarian DLBCL without concomitant uterine involvement. In multivariate analysis adjusted for other risk factors for SCNS, uterine involvement by DLBCL remained strongly associated with SCNS (Hazard ratio 14·13, 95% confidence interval 5·09-39·25, P < 0·001). Because involvement of the uterus by DLBCL appears to be associated with a high risk of SCNS, those patients should be considered for CNS staging and prophylaxis. However, more studies are needed to determine whether the increased risk of secondary CNS involvement also applies to women with localized reproductive organ DLBCL.

Reproducible probe-level analysis of the Affymetrix Exon 1.0 ST array with R/Bioconductor.

The presence of different transcripts of a gene across samples can be analysed by whole-transcriptome microarrays. Reproducing results from published microarray data represents a challenge owing to the vast amounts of data and the large variety of preprocessing and filtering steps used before the actual analysis is carried out. To guarantee a firm basis for methodological development where results with new methods are compared with previous results, it is crucial to ensure that all analyses are completely reproducible for other researchers. We here give a detailed workflow on how to perform reproducible analysis of the GeneChip®Human Exon 1.0 ST Array at probe and probeset level solely in R/Bioconductor, choosing packages based on their simplicity of use. To exemplify the use of the proposed workflow, we analyse differential splicing and differential gene expression in a publicly available dataset using various statistical methods. We believe this study will provide other researchers with an easy way of accessing gene expression data at different annotation levels and with the sufficient details needed for developing their own tools for reproducible analysis of the GeneChip®Human Exon 1.0 ST Array.

The myeloma stem cell concept, revisited: from phenomenology to operational terms.

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19-/CD45low/-/CD38high/CD138+ malignant plasma cell, but not the CD19+/CD38low/- memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19+/CD27+/CD38- single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno- and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine.

American Society of Blood and Marrow Transplantation, European Society of Blood and Marrow Transplantation, Blood and Marrow Transplant Clinical Trials Network, and International Myeloma Working Group Consensus Conference on Salvage Hematopoietic Cell Transplantation in Patients with Relapsed Multiple Myeloma.

In contrast to the upfront setting in which the role of high-dose therapy with autologous hematopoietic cell transplantation (HCT) as consolidation of a first remission in patients with multiple myeloma (MM) is well established, the role of high-dose therapy with autologous or allogeneic HCT has not been extensively studied in MM patients relapsing after primary therapy. The International Myeloma Working Group together with the Blood and Marrow Transplant Clinical Trials Network, the American Society of Blood and Marrow Transplantation, and the European Society of Blood and Marrow Transplantation convened a meeting of MM experts to: (1) summarize current knowledge regarding the role of autologous or allogeneic HCT in MM patients progressing after primary therapy, (2) propose guidelines for the use of salvage HCT in MM, (3) identify knowledge gaps, (4) propose a research agenda, and (5) develop a collaborative initiative to move the research agenda forward. After reviewing the available data, the expert committee came to the following consensus statement for salvage autologous HCT: (1) In transplantation-eligible patients relapsing after primary therapy that did NOT include an autologous HCT, high-dose therapy with HCT as part of salvage therapy should be considered standard; (2) High-dose therapy and autologous HCT should be considered appropriate therapy for any patients relapsing after primary therapy that includes an autologous HCT with initial remission duration of more than 18 months; (3) High-dose therapy and autologous HCT can be used as a bridging strategy to allogeneic HCT; (4) The role of postsalvage HCT maintenance needs to be explored in the context of well-designed prospective trials that should include new agents, such as monoclonal antibodies, immune-modulating agents, and oral proteasome inhibitors; (5) Autologous HCT consolidation should be explored as a strategy to develop novel conditioning regimens or post-HCT strategies in patients with short (less than 18 months remissions) after primary therapy; and (6) Prospective randomized trials need to be performed to define the role of salvage autologous HCT in patients with MM relapsing after primary therapy comparing it to "best non-HCT" therapy. The expert committee also underscored the importance of collecting enough hematopoietic stem cells to perform 2 transplantations early in the course of the disease. Regarding allogeneic HCT, the expert committee agreed on the following consensus statements: (1) Allogeneic HCT should be considered appropriate therapy for any eligible patient with early relapse (less than 24 months) after primary therapy that included an autologous HCT and/or high-risk features (ie, cytogenetics, extramedullary disease, plasma cell leukemia, or high lactate dehydrogenase); (2) Allogeneic HCT should be performed in the context of a clinical trial if possible; (3) The role of postallogeneic HCT maintenance therapy needs to be explored in the context of well-designed prospective trials; and (4) Prospective randomized trials need to be performed to define the role salvage allogeneic HCT in patients with MM relapsing after primary therapy.

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.

European Myeloma Network guidelines for the management of multiple myeloma-related complications.

The European Myeloma Network provides recommendations for the management of the most common complications of multiple myeloma. Whole body low-dose computed tomography is more sensitive than conventional radiography in depicting osteolytic disease and thus we recommend it as the novel standard for the detection of lytic lesions in myeloma (grade 1A). Myeloma patients with adequate renal function and bone disease at diagnosis should be treated with zoledronic acid or pamidronate (grade 1A). Symptomatic patients without lytic lesions on conventional radiography can be treated with zoledronic acid (grade 1B), but its advantage is not clear for patients with no bone involvement on computed tomography or magnetic resonance imaging. In asymptomatic myeloma, bisphosphonates are not recommended (grade 1A). Zoledronic acid should be given continuously, but it is not clear if patients who achieve at least a very good partial response benefit from its continuous use (grade 1B). Treatment with erythropoietic-stimulating agents may be initiated in patients with persistent symptomatic anemia (hemoglobin <10g/dL) in whom other causes of anemia have been excluded (grade 1B). Erythropoietic agents should be stopped after 6-8 weeks if no adequate hemoglobin response is achieved. For renal impairment, bortezomib-based regimens are the current standard of care (grade 1A). For the management of treatment-induced peripheral neuropathy, drug modification is needed (grade 1C). Vaccination against influenza is recommended; vaccination against streptococcus pneumonia and hemophilus influenza is appropriate, but efficacy is not guaranteed due to suboptimal immune response (grade 1C). Prophylactic aciclovir (or valacyclovir) is recommended for patients receiving proteasome inhibitors, autologous or allogeneic transplantation (grade 1A).

Predicting response to multidrug regimens in cancer patients using cell line experiments and regularised regression models.

BACKGROUND: Patients suffering from cancer are often treated with a range of chemotherapeutic agents, but the treatment efficacy varies greatly between patients. Based on recent popularisation of regularised regression models the goal of this study was to establish workflows for pharmacogenomic predictors of response to standard multidrug regimens using baseline gene expression data and origin specific cell lines. The proposed workflows are tested on diffuse large B-cell lymphoma treated with R-CHOP first-line therapy. METHODS: First, B-cell cancer cell lines were tested successively for resistance towards the chemotherapeutic components of R-CHOP: cyclophosphamide (C), doxorubicin (H), and vincristine (O). Second, baseline gene expression data were obtained for each cell line before treatment. Third, regularised multivariate regression models with cross-validated tuning parameters were used to generate classifier and predictor based resistance gene signatures (REGS) for the combination and individual chemotherapeutic drugs C, H, and O. Fourth, each developed REGS was used to assign resistance levels to individual patients in three clinical cohorts. RESULTS: Both classifier and predictor based REGS, for the combination CHO, were of prognostic value. For patients classified as resistant towards CHO the risk of progression was 2.33 (95% CI: 1.6, 3.3) times greater than for those classified as sensitive. Similarly, an increase in the predicted CHO resistance index of 10 was related to a 22% (9%, 36%) increased risk of progression. Furthermore, the REGS classifier performed significantly better than the REGS predictor. CONCLUSIONS: The regularised multivariate regression models provide a flexible workflow for drug resistance studies with promising potential. However, the gene expressions defining the REGSs should be functionally validated and correlated to known biomarkers to improve understanding of molecular mechanisms of drug resistance.

Outcome prediction by extranodal involvement, IPI, R-IPI, and NCCN-IPI in the PET/CT and rituximab era: A Danish-Canadian study of 443 patients with diffuse-large B-cell lymphoma.

18F-fluorodeoxyglucose PET/CT (PET/CT) is the current state-of-the-art in the staging of diffuse large B-cell lymphoma (DLBCL) and has a high sensitivity for extranodal involvement. Therefore, reassessment of extranodal involvement and the current prognostic indices in the PET/CT era is warranted. We screened patients with newly diagnosed DLBCL seen at the academic centers of Aalborg, Copenhagen, and British Columbia for eligibility. Patients that had been staged with PET/CT and treated with R-CHOP(-like) 1(st) line treatment were retrospectively included. In total 443 patients met the inclusion criteria. With a median follow-up of 2.4 years, the 3-year overall (OS) and progression-free survival (PFS) were 73% and 69%, respectively. The Ann Arbor classification had no prognostic impact in itself with the exception of stage IV disease (HR 2.14 for PFS, P<0.01). Extranodal involvement was associated with a worse outcome in general, and in particular for patients with involvement of >2 extranodal sites, including HR 7.81 (P < 0.001) for PFS for >3 sites. Bone/bone marrow involvement was the most commonly involved extranodal site identified by PET/CT (29%) and was associated with an inferior PFS and OS. The IPI, R-IPI, and NCCN-IPI were predictive of PFS and OS, and the two latter could identify a very good prognostic subgroup with 3-year PFS and OS of 100%. PET/CT-ascertained extranodal involvement in DLBCL is common and involvement of >2 extranodal sites is associated with a dismal outcome. The IPI, R-IPI, and NCCN-IPI predict outcome with high accuracy.

International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma.

This International Myeloma Working Group consensus updates the disease definition of multiple myeloma to include validated biomarkers in addition to existing requirements of attributable CRAB features (hypercalcaemia, renal failure, anaemia, and bone lesions). These changes are based on the identification of biomarkers associated with near inevitable development of CRAB features in patients who would otherwise be regarded as having smouldering multiple myeloma. A delay in application of the label of multiple myeloma and postponement of therapy could be detrimental to these patients. In addition to this change, we clarify and update the underlying laboratory and radiographic variables that fulfil the criteria for the presence of myeloma-defining CRAB features, and the histological and monoclonal protein requirements for the disease diagnosis. Finally, we provide specific metrics that new biomarkers should meet for inclusion in the disease definition. The International Myeloma Working Group recommends the implementation of these criteria in routine practice and in future clinical trials, and recommends that future studies analyse any differences in outcome that might occur as a result of the new disease definition.

Exposure time independent summary statistics for assessment of drug dependent cell line growth inhibition.

BACKGROUND: In vitro generated dose-response curves of human cancer cell lines are widely used to develop new therapeutics. The curves are summarised by simplified statistics that ignore the conventionally used dose-response curves' dependency on drug exposure time and growth kinetics. This may lead to suboptimal exploitation of data and biased conclusions on the potential of the drug in question. Therefore we set out to improve the dose-response assessments by eliminating the impact of time dependency. RESULTS: First, a mathematical model for drug induced cell growth inhibition was formulated and used to derive novel dose-response curves and improved summary statistics that are independent of time under the proposed model. Next, a statistical analysis workflow for estimating the improved statistics was suggested consisting of 1) nonlinear regression models for estimation of cell counts and doubling times, 2) isotonic regression for modelling the suggested dose-response curves, and 3) resampling based method for assessing variation of the novel summary statistics. We document that conventionally used summary statistics for dose-response experiments depend on time so that fast growing cell lines compared to slowly growing ones are considered overly sensitive. The adequacy of the mathematical model is tested for doxorubicin and found to fit real data to an acceptable degree. Dose-response data from the NCI60 drug screen were used to illustrate the time dependency and demonstrate an adjustment correcting for it. The applicability of the workflow was illustrated by simulation and application on a doxorubicin growth inhibition screen. The simulations show that under the proposed mathematical model the suggested statistical workflow results in unbiased estimates of the time independent summary statistics. Variance estimates of the novel summary statistics are used to conclude that the doxorubicin screen covers a significant diverse range of responses ensuring it is useful for biological interpretations. CONCLUSION: Time independent summary statistics may aid the understanding of drugs' action mechanism on tumour cells and potentially renew previous drug sensitivity evaluation studies.

Validation and implementation of a method for microarray gene expression profiling of minor B-cell subpopulations in man.

BACKGROUND: This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. METHODS: Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. RESULTS: Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r ≥ 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r ≥ 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas. CONCLUSION: A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.