Page 118 - Haematologica3

F. Knörr et al.
found to be elevated in seven ALK-positive ALCL patients.12 sCD30 levels correlated with an inferior survival in a series of adult ALCL patients with unknown ALK-status.13,14 IL-9 has been described as part of an autocrine signaling path- way including JAK3 with a possible role in the pathogenesis of ALK-positive ALCL.15 IL-22 expressed in three ALCL cell lines contributes to STAT3 activation and tumorigenicity of ALK-positive ALCL.16
While above-mentioned reports highlight ALCL as a cytokine-active lymphoma with hints towards an ALCL- type cytokine signature, the type and pattern of cytokine expression among ALK-positive ALCL patients has not been analyzed systematically. Pretreatment serum cytokine levels may reflect the biological activity of the tumor as well as host immune characteristics. Correlations of initial cytokine concentrations and patterns with patients’ charac- teristics, antibody titers against ALK as a surrogate measure of an autologous immune response against ALK, as well as outcomes might allow the definition of cytokine profiles that are associated with disease activity, tumor burden and the patients’ specific immune response.
We, therefore, investigated whether pretreatment cytokine concentrations in patients with ALK-positive ALCL correlate with biological and clinical characteristics and ALK-antibody titers in a uniformly treated, large cohort of 119 children and adolescents with ALK-positive ALCL.
Methods
Eligibility
NPM-ALK-positive ALCL patients treated in the Berlin- Frankfurt-Muenster group study NHL-BFM95 or German patients enrolled in the European intergroup trial ALCL99 between August 1998 and December 2008 were potentially eligible for inclusion in this study after giving written informed consent to participation. Both studies were approved by the institutional ethics committee of the primary investigator of the NHL-BFM study group (AR). Patients with completely resected stage I disease were excluded because their treatment was different.
Patients were included if there were pretreatment serum or plas- ma samples available and had detectable anti-ALK antibody titer levels. Eligibility was confirmed by demonstration of NPM-ALK positivity of the tumor either by NPM-ALK polymerase chain reaction, two-color fluorescence in situ hybridization for t(2;5) or nuclear and cytoplasmic staining for ALK.
Patients
The inclusion criteria were fulfilled by 119 patients. Staging pro- cedures included bone marrow aspiration cytology and a spinal tap. Bone marrow involvement was defined by cytologically detectable ALCL cells, irrespective of their number. The patients’ treatment consisted of a cytoreductive prephase followed by six chemotherapy courses, as described elsewhere.17
As control, serum or plasma samples from 15 of those patients in remission without concurrent infection taken before the start of the sixth chemotherapy course were analyzed.
In addition, serum samples taken at the time of diagnosis from 11 age-matched patients with Burkitt lymphoma from risk groups R1 and R2 (stage I – III, lactate dehydrogenase below 500 U/L) included in the B-NHL BFM 04 study served as a second control group.
Methods and the patients’ results regarding ALK-antibody titers and minimal disseminated disease at diagnosis were described and published previously.18-20
Measurement of cytokine levels
Blood samples were centrifuged and supernatants were immediately frozen and stored at -80°C until analysis. Samples were assessed for the levels of following soluble mediators: IL- 1β, IL-2, sIL-2R, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL- 13, IL-17a, IL-22, IL-23, tumor necrosis factor-α (TNF-α), IFN- γ, monokine induced by γ-interferon (MIG), interferon γ- induced protein 10 (IP-10), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, granulocyte colony-stimulating fac- tor (G-CSF) and soluble CD30 (sCD30). The measurements were performed using FlowCytomix kits (eBioscience, Frankfurt, Germany) according to the manufacturer’s instruc- tions. Samples were processed on a FACS Calibur (BD, Heidelberg, Germany) and data were analyzed using the FlowCytomix Software (version 2.4, eBioscience, Frankfurt, Germany).
Statistical methods
Statistical calculations were performed using the R statistical package (R Foundation for Statistical Computing, Vienna, Austria).
Cytokine levels are reported as median values and were com- pared between different groups according to diagnosis, clinical and biological characteristics using Mann-Whitney U and Kruskal-Wallis tests. The level of statistical significance was 0.05. Event-free survival was defined as the time from diagnosis to relapse, secondary tumor or death from any cause. Estimates of overall and event-free survival were performed using the Kaplan-Meier method. Differences between groups were com- pared by log-rank test. A multivariate analysis was performed using the proportional hazards method described by Cox on cytokines showing significant differences in univariate analysis and known risk factors20 with forward selection keeping only significant variables (P<0.05) in the model.
Results
Patients’ characteristics
The median age of the 119 ALCL patients at diagnosis was 12.0 years (range, 0.3 – 17.8) and 58% (69 patients) were male. The median follow-up was 6.6 years. The 3- year event-free survival rate of the 119 ALCL patients was 66.4 ± 4.3% and their 3-year overall survival rate was 86.5 ± 3.1%. Detailed clinical data of the patients and controls are shown in Online Supplementary Table S1.
Pretreatment cytokine levels in patients with anaplastic lymphoma kinase-positive anaplastic large cell lymphoma
Concentrations of IL-9, IL-10, IL-17a, HGF, sIL-2R, and sCD30 were significantly higher in ALCL patients at the time of diagnosis than in the patients in either control group (patients with B-cell non-Hodgkin lymphoma and ALCL patients in remission) (Figure 1).
The median concentrations of all cytokines are shown in Online Supplementary Table S2. In 28 ALCL patients, measurements of sIL-2R were above the upper detection limit of 221 869.99 pg/mL. For the analyses, these sam- ples were attributed a value of 221 869.99 pg/mL. IP-10 could not be measured in one patient.
Correlations between cytokine concentrations are shown in Online Supplementary Table S3.
478
haematologica | 2018; 103(3)