Martin Spacek

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Plasma EBV‐DNA monitoring in Epstein–Barr virus‐positive Hodgkin lymphoma patients

Spacek M, Hubacek P, Markova J, Zajac M, Vernerova Z, Kamaradova K, Stuchly J, Kozak T. Plasma EBV‐DNA monitoring in Epstein–Barr virus‐positive Hodgkin lymphoma patients. APMIS 2010.

Heat-shock protein expression in leukemia.

Heat-shock proteins (Hsps) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, Hsp27, Hsp60, Hsp90α, and HspBP1 gene expression was investigated in human leukemia cell lines as well as in leukemia cells derived from patients with the onset of the disease. Hsp70 membrane expression and expression of Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 genes were also tested in samples from leukemia patients. Relative Hsps gene expression was examined in human leukemia cell lines and also in patients, using real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Hsp70 cell surface expression was studied in patients with leukemia onset using flow cytometry. All tested cell lines showed significantly increased expression of Hsp60, Hsp90α, and HspBP1 genes compared with a cohort of healthy controls; additionally there was increased Hsp27 expression except for Jurkat and CCRF cells. Significantly higher gene expression of Hsp27, Hsp60, Hsp90α, and HspBP1 was observed in the peripheral blood of patients compared with bone marrow and healthy control samples, while Hsp70 expression was without any significant difference among cohorts. Hsp70 cell surface expression was found on defrosted and cultured leukemia cells but not on unprocessed biological samples from patients. Leukemia cells showed a heterogeneous pattern of Hsp gene expression among leukemia cell lines as well as in peripheral blood and bone marrow of patients.

Ofatumumab retreatment and maintenance in fludarabine‐refractory chronic lymphocytic leukaemia patients

There are limited data on retreatment with monoclonal antibodies (mAb) in patients with chronic lymphocytic leukaemia (CLL). In a pivotal study, ofatumumab (human anti‐CD20 mAb) monotherapy demonstrated a 47% objective response rate (ORR) in fludarabine refractory CLL patients. From this study, a subset of 29 patients who had at least stable disease and then progressed were retreated with eight weekly ofatumumab infusions (induction treatment period), followed by monthly infusions for up to 2 years (maintenance treatment period). The ORR after 8 weeks of induction retreatment was 45% and 24% had continued disease control after maintenance at 52 weeks. Efficacy and safety of the retreated patients were compared with their initial results in the pivotal study. Response duration was 24·1 months vs. 6·8 months; time to next therapy was 14·8 months vs. 12·3 months; and progression‐free survival was 7·4 months vs. 7·9 months (medians). Upon retreatment, 72% had infusion reactions, mostly Grade 1–2. Three patients had fatal infections. In summary, ofatumumab retreatment and maintenance therapy was feasible in patients with heavily pretreated CLL and appeared to result in more durable disease control than initial ofatumumab treatment in this subset of patients who may have a more favourable disease profile.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as “required” or “recommended” for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate “required” markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5–20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for “required” diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. “Recommended” markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as “required” for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus “recommended” panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated.

Expression of heat shock protein 70 and NKG2D ligands in acute myeloid leukemia cell lines

Membrane-bound heat shock protein 70 (HSP70) was found to be tumor-specific and was proposed as a target for immunotherapy. In the present study, we analyzed cell surface and relative gene expression of HSP70 in cell lines established from patients with different acute myeloid leukemia (AML) subtypes, together with the expression of natural killer (NK) cell activation/inhibitory ligands. Materials and methods: Six AML cell lines were included in this study. The relative gene expression of HSP70 was analyzed using the real-time reverse-transcriptase polymerase chain reaction. Surface expression of HSP70 and NK cell ligands was analyzed using flow cytometry. Results: All cell lines overexpressed HSP70; however, its mRNA levels were not elevated. The expression of NKG2D activation ligands was heterogeneous. Conclusion: Our study is the first to describe long-term stationary cell surface expression of HSP70 in different subtypes of AML. Combined with the results of the gene expression experiments these data provide more evidence to the idea of a self-limiting mechanism for HSP70 expression.

Prevalence of chromosomally integrated HHV-6 in patients with malignant disease and healthy donors in the Czech Republic

Chromosomal integration of human herpesvirus 6 (Ci-HHV6) was described first in 1993 by Luppi et al. (1993). Despite increasing number of studies concerning the clinical impact of HHV-6 A and HHV-6 B viral species (former known as HHV6 variants; Ablashi et al. 2012) or directly Ci-HHV-6, biological consequence of Ci-HHV-6 is yet not fully understood (Pellett et al. 2012). Classical horizontal infection of HHV-6 is transmittable from one person to another leading to subse quent latent viral infection of the host in a form of episomic viral DNA in the cells and the possibility of viral reactivation into the infection with production of infectious viral capsids (Flamand et al. 2010). Such type of HHV-6 primary infection and latency is similar to other human herpesviruses (Ward 2005). In the situation of Ci-HHV-6, viral DNA is integrated directly into the human chromosomes and is inherited through the germ line from parents to child vertically (Pellett et al. 2012). It is the reason for presence of HHV-6 DNA is in every cell of the body which leads to the ratio of human and viral DNA 1:1 and to the possibility of Ci-HHV-6 confirmation by detection of HHV-6 DNA in the nails or hair roots (Pellett et al. 2012). Despite both HHV-6 species can be chromosomally integrated, inheritance of viral DNA in vivo is unique among the human herpesviruses. So far published data locate the integrated HHV-6 DNA into the telomeric part of the chromosomes suggesting possible association of Ci-HHV-6 with development of the malignant proliferation due to the impact of the telomerase and shortening of the telomers into the regulation of cell proliferation (Pellett et al. 2012; Morissette and Flamand 2010; Martinez and Blasco 2011). Published studies with the patients having primary malignant disease show rather wide range of Ci-HHV-6 prevalence from 0.74 to 12.73 % (Pellett et al. 2012). In our previous study among 339 children treated for acute leukaemia, we detected Ci-HHV6 prevalence of 1.47 % (Hubacek et al. 2009). Hence we were interested in the more detailed calculation of the CiHHV-6 prevalence in Czech patients with malignant disease and in general population.

No association between the TP53 codon 72 polymorphism and risk or prognosis of Hodgkin and non-Hodgkin lymphoma

The role of the TP53 genes R72P polymorphism in non-Hodgkin lymphoma (NHL) has been analyzed in several studies but it has not been studied in Hodgkin lymphoma (HL). We have evaluated the role of R72P in 340 NHL and 298 HL patients. There was no difference in the R72P frequency between analyzed lymphoma cases and 749 controls. We found no association of R72P with the risk of NHL and HL development [OR(ArgPro/ProPro)=0.9 (95% CI 0.7-1.2) and 1.2 (95% CI 0.9-1.5), respectively] or with survival. Our results support the evidence that R72P is not a prognostic factor in Caucasian NHL patients, and they indicate its irrelevance for HL development or prognosis.