We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ⩽1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.
Anaplastic large-cell lymphoma (ALCL) accounts for 10 to 15% of pediatric and adolescent non-Hodgkin lymphomas.1 The majority of ALCL is associated with abnormalities of the anaplastic lymphoma kinase (ALK) gene and constitutes the tumor entity ALK-positive ALCL.2 Although current treatment strategies achieve an event-free survival of ∼75% in children with ALK-positive ALCL, 25% of the patients relapse.3, 4 Importantly, some children who now receive intensive multi-agent chemotherapy likely are over-treated and may be cured using less-intensive regimens.5 Risk stratification of children with ALK-positive ALCL was, until now, based on clinical parameters identified in an analysis of three different European clinical trials performed during the 1990s.6 The presence of mediastinal or visceral involvement and skin lesions were found associated with an increased risk of failure.
In addition to clinical risk factors, other important biological parameters predicting relapse have since been identified for pediatric ALK-positive ALCL.7, 8, 9, 11, 12 First, minimal disseminated disease (MDD)-positivity detected by qualitative RT-PCR for NPM-ALK in bone marrow (BM) or peripheral blood (PB) conferred a relapse risk of ∼50% in two trials.7, 9 Quantification of MDD by real-time-PCR in BM and PB allowed the identification of a poorer risk group in one study with a relapse risk of almost 70% for patients with >10 normalized copies of NPM-ALK transcript in BM or PB.9 Second, the NPM-ALK oncogenic fusion protein elicits the production of autologous anti-ALK antibodies in patients with ALK-positive ALCL.10 The strength of the ALK autoantibody response inversely correlated with stage and tumor dissemination, as well as with risk of relapse.11, 12 Finally, histological subtype is also of importance with a poorer prognosis associated with ALK-positive ALCL of a subtype other than the common type.8
However, these prognostic factors could have a higher discriminatory power when considered together and not as single factors. The measurements of more than one parameter might be needed to identify a very low-risk group amenable to therapy reduction.
The current study was performed in a large cohort of uniformly treated ALCL children from the AIEOP (Italian Association of Pediatric Hematology and Oncology) and the BFM (Berlin-Frankfurt-Muenster) study groups to investigate: (a) the prevalence of MDD and antibody response to ALK at diagnosis, and (b) whether the combination of two biological risk factors could subdivide ALK-positive ALCL patients in distinct biological risk groups (bRG). This is, to our knowledge, the first report to demonstrate that a combination of two biological variables, that is, anti-ALK antibody titer and MDD, can stratify patients in different bRGs. This raises the possibility of establishing an effective biological based tailored therapy to increase the survival of ALCL patients.
Materials and methods
Patients, samples and treatment protocol
This collaborative study was conducted on German and Italian patients with diagnosis of ALK-positive ALCL confirmed by histological review. Eligibility criteria were the confirmation of NPM-ALK positivity of the tumor by at least one of the following: NPM-ALK-specific RT-PCR, positive two color fluorescence in situ hybridization for t(2;5) or cytoplasmic and nuclear ALK-positive staining of the tumor biopsy. Additionally, BM and/or PB samples taken at the time of diagnosis had to be available for the assessment of NPM-ALK transcript and antibody titer.
The study was approved by the ethics committee or by the internal review board of each participating institution and informed consent was obtained from parents or legal guardians before patient enrollment. MDD results of patients diagnosed before 2003 in the AIEOP and 2005 in the BFM trials and ALK-antibody titers of patients before 2007 in both groups were published previously.7, 9, 11, 12
Detection of MDD by RT-PCR
Mononuclear cells from BM and PB were obtained by density gradient centrifugation. cDNA synthesis and RT-PCRs were performed according to recently described protocols.7, 9 Briefly, total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. An amount of 1 μg of total RNA was reverse transcribed using SuperScript II reverse transcriptase (Life Technologies, Milan, Italy) and random hexamers. For each sample, ABL expression was assessed as a control for the presence of amplifiable RNA and the efficiency of reverse transcription, using the primers 5′-IndexTermCGGCCAGTAGCATCTGACTTTG-3′ and 5′-IndexTermCCTTGGCCATTTTTGGTTTGG-3′. The primers specific for the chimeric transcript NPM-ALK were IndexTermTCCCTTGGGGGCTTTGAAATAACACC (NPM) and IndexTermCGAGGTGCGGAGCTTGCTCAGC (ALK). Each reaction mixture contained 10 × buffer, 1.5 mM MgCl2, 1.6 mM dNTPs, 400 nM of each primer, 0.2 IU of Taq polymerase and 5% of the RT product in a final 20 microl reaction volume. PCR reaction consisted of initial denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, 68 °C for 15 s, 72 °C for 30 s and a final extension at 72 °C for 10 min. PCR products were analyzed by 3% agarose gel electrophoresis and visualized under UV illumination after ethidium bromide staining. Ladder 50 (Invitrogen, Milan, Italy) was used as a molecular weight standard.
A patient was considered MDD-positive, if at least one of the two samples (BM or PB) was RT-PCR positive. Several comparative analyses for quality control performed between AIEOP and BFM central laboratories demonstrated identical results in serial diluted NPM-ALK-positive cell lines and blinded patient samples.
Detection of antibody response to ALK
Cytocentrifuge preparations of COS-1 monkey epithelial cells transiently transfected with a pcDNA3-based vector encoding NPM-ALK or with empty vector only were prepared and stained with patient serum or plasma in an indirect immunoperoxidase technique as previously described.10 The cytocentrifuge preparations were incubated with patient’s serum and/or plasma diluted from 1:50 to 1:60 750. The cutoff for a positive result was defined as the highest dilution that gave a positive reaction with NPM-ALK transfectants.
All patients included in the study had at least 24 months follow-up from diagnosis. A receiver–operator characteristic curve was calculated to define the cutoff value of circulating anti-NPM-ALK antibodies that may predict relapse with the highest sensitivity and specificity. The sensitivity of the assay for each antibody titer group was defined based on the percentage of relapses in each group and specificity was calculated using the percentage of non-relapse in all groups except the test one. Survival analysis was performed according to Kaplan–Meier method;15 differences were compared by the log-rank test. The 5-years overall survival (OS) was calculated from the date of diagnosis to the date of death from any cause or to the date of last follow-up. The 5-years progression-free survival (PFS) was calculated from the date of diagnosis to the date of the first event (tumor progression or relapse) or to the date of last follow-up. Cox’s regression analysis was used16 in which variables with a P-value <0.2 in univariate analysis were considered to determine prognostic factors affecting PFS of patient subgroups. The association of MDD and antibody titer with specific clinical characteristics (that is stage, mediastinal or visceral involvement) was analyzed by the χ2-test or the Fisher exact test when the frequency of cases in a given subgroup was <5.
All P-values are two-sided, with a type I error rate fixed at 0.05. Statistical analysis was performed by using the SAS statistical program (SAS-PC, version 9.2; SAS Institute Inc., Cary, NC, USA).
Between April 1996 and December 2008, 128 ALCL patients, 85 males and 43 females, median age at diagnosis 11 years (range, 3 months to 17.7 years), were included in the study. The main clinical characteristics and treatment results of the study population are listed in Table 1. Histological diagnosis demonstrated that most cases were common type ALCL (59%) and were characterized by the presence of abundant hallmark cells. Tumor cells in all cases were CD30-, ALK- and EMA-positive. Based on St Jude classification, 81% of them were stage III–IV. The median follow-up of the patients alive was 4.7 years (range, 2.0–11.7). Forty patients suffered progression of disease or relapse after a median time from diagnosis of 7.7 months (range, 0.24–24.1 months). Eighteen patients died, 11 due to disease progression/relapse, 5 due to treatment-related toxicity (4 after relapse) and the remaining 2 from complications arising following blood stem cell transplantation as second line treatment. The 5-years PFS and OS of the whole study group were 68% (s.e., 4%) and 86% (s.e., 3%), respectively.
No statistically significant difference in either OS or PFS was observed between the study population and all other patients treated concomitantly in the same protocols (OS: 86% (s.e., 3%) and 89% (s.e., 2%), P=0.39; PFS: 68% (s.e., 4%) and 73%(s.e., 3%), P=0.27, respectively).
Prognostic impact of MDD and antibody titer against ALK
The BM/PB sample obtained at diagnosis was MDD-positive by RT-PCR in 59% of patients (75/128) studied (BM+/PB+ 38 patients; BM+/PB− 3 patients; BM−/PB+11 patients, for the remaining cases only BM was available and 23 were positive). Importantly, only 11 of these 75 patients were positive by standard morphological analysis of BM smears.
Circulating antibodies recognizing the NPM-ALK protein were detected at diagnosis in 96% (123/128) of the cases. A receiver–operator characteristic curve was calculated to define the cutoff value that may predict relapse. The antibody titer cutoff value of 1:750 dilution had the best sensitivity and specificity compared with other antibody levels (Supplementary Figure 1). Thirty percent of patients had antibody titers below the cutoff (that is, 1:750 dilution). These patients had a significantly higher relapse risk (HR: 3.8, 95% confidence interval 2.0–7.1) than those children with higher antibody titers.
The 5-year PFS was 54% (s.e., 6%) for MDD-positive and 87% (s.e., 5%) for MDD-negative patients, respectively (P<0.0001) (Figure 1a). Based on antibody titer levels, the 5-year PFS was 42% (s.e., 8%) for patients with low antibody titer (⩽1/750) and 79% (s.e., 4%) for patients with high antibody titer (>1/750) (P<0.0001) (Figure 1b). In addition, MDD and antibody titer appear to be independent biological variables (χ2-test for association, P=0.22).
Combination of MDD and antibody titers for risk stratification
When the MDD and antibody titers were considered in combination, the following three subgroups of patients with different prognosis could be identified: (1) a biological high risk (bHR) group defined by MDD-positivity and antibody titer ⩽1/750, 26/128 (20%); (2) a biological low risk (bLR) group defined by MDD-negativity and an antibody titer >1/750, 40/128 (31%); (3) a biological intermediate risk (bIR) group including all other patients (MDD-negative/antibody titer ⩽1/750 or MDD-positive/antibody titer >1/750), 62/128 (48%). PFS was 28% (s.e., 9%) for bHR patients and 93% (s.e. 4%) for bLR patients (P<0.0001) (Figure 2a). The bIR group showed a 5-years PFS of 68% (s.e., 6%) and was predominantly composed of patients with high antibody titer/MDD-positivity (79%). The 5-years OS was 72% (s.e., 9%) for bHR patients, 84% (s.e., 5%) for bIR cases and 98% (s.e., 2%) for bLR patients (P=0.02) (Figure 2b).
Univariate and multivariate risk factor analysis
Results from a univariate analysis that accounted for clinical variables and for the new biologically based groups bHR vs bLR and bIR are included in Table 1. Among the variables entered in the Cox regression analysis (stage of disease, involvement of peripheral lymph nodes, mediastinum, visceral organs, morphological BM involvement, histology, MDD, titer and bRG) it was only the bHR group and a histology other than common type that were negative prognostic factors with a relative risk of relapse of 4.89 and 2.70, respectively (Table 1). Of note, patients with bHR had a significantly lower PFS independently of the histological subgroup (common vs non-common histology). Within the non-common type histology, patients belonging to the bHR category had a PFS of 23 vs 57% for the bLR+bIR (P 0.003); within the histological common type group PFS was also significantly lower for patients with bHR features compared with bLR+bIR patients: 33 vs 88%, respectively (P<0.0001). Significant association was found between the bRG and selected clinical parameters, such as B symptoms, stage, mediastinal involvement, visceral involvement, BM morphology and histological subtype (Table 2).
Patient stratification according to risk factors is a prerequisite for optimized individualized treatment in pediatric oncology. In lymphoma, treatment intensity has, until now, been largely based on clinical parameters. In particular, high-risk ALK-positive ALCL patients treated according to the international ALCL-99 protocol were defined by the presence of mediastinal, visceral and/or skin involvement. We recently demonstrated that the submicroscopic involvement of BM and/or PB is an important prognostic factor in ALCL.10, 11, 12 This study represents the largest international cohort of ALCL pediatric patients analyzed for MDD and antibody titer. As previously reported in two separate studies,11, 12 we observed a high prevalence of MDD and anti-ALK antibody at diagnosis. Previously, we also demonstrated the existence of an inverse correlation between the anti-ALK antibody titers and the risk of relapse.11, 12 The present study showed that by combining MDD and antibody titer, patients can be stratified into three different groups with significantly different PFS and OS probabilities. This permits, for the first time, the identification of high-risk patients on the basis of easily measurable biological parameters early after diagnosis, thereby allowing a more effective first-line therapy to be designed that may prevent relapse and improve survival. In this context, the bHR patients could be considered for early-phase clinical trials with newly available and promising targeted therapies, including brentuximab-vedotin and ALK-inhibitors. In fact, based on clinical risk stratification 3 years PFS was 58% (5% s.e.) for patients with clinical high-risk features vs 88% (5% s.e.) for standard risk patients (P=0.0007), whereas bHR patients had a PFS of 28% (9% s.e.) vs 93% (4% s.e.) for bLR patients, Figure 2a. Only three patients who were clinically SR were bHR, two of them relapsed. The patients who were clinically HR but bIR or bLR had a PFS of 74% (6% s.e.), bHR patients had a PFS of 24%. Of interest, relapses in bHR group occurred mostly within the first 12–18 months from diagnosis and this was associated with a significantly lower OS of bHR patients compared with the others. Using multivariate analysis the histological subtype also retained a prognostic value in addition to the bRG. However, bRG had a stronger prognostic impact, with an HR of 4.89 for bHRG, compared with 2.70 of non-common histology (Table 1). The association of common histological subtype with bLR is in accordance with our earlier studies hinting to a possible correlation between MDD and histological subtype.9 Furthermore, this correlation supports a study in which ALCL patients with the common type and small cell/lymphohistiocytic variants were associated with distinct gene signatures.17 Among the 32 patients with common-type ALCL there was a statistically significant overexpression of genes encoding proteins involved in responses to external stimuli and stress and, importantly, in inflammatory and immune responses. In contrast, genes-encoding proteins involved in the regulation of the cell cycle and in cell proliferation were overexpressed in the variant subgroup.
While increasing evidence points to a role of the immune response in tumor development, it is becoming evident that an individual immune response to a tumor relies on multiple factors. Although both cytotoxic T-cells and CD4-T-helper cell responses (both being of primary importance in tumor immunity) have been identified in ALK-positive ALCL,18 other workers have reported the expression of immunosuppressive molecules by ALK-positive ALCL cells can contribute to downregulation of the immune response (for example, CD274).19 Furthermore, tumor cells may escape immune recognition by downregulating major histocompatibility complex class II expression as shown for diffuse large B-cell lymphoma.20 Abnormalities linked with genes such as PERFORIN (PRF1) involved in the immune response have also been reported: Cannella et al.21 demonstrated that patients with ALCL have a higher prevalence of PRF1 mutations compared with healthy controls. This is important as PRF1 has a key role in the cytotoxicity of natural killer cells and cytotoxic T-cells. Thus, although the anti-ALK antibody titer appears to be associated with the anti-tumor immune response as suggested by its association with stage and relapse risk, it may not completely reflect the tumor immune response of individual patients. Indeed, the multiplicity of immunological mechanisms involved in ALK-positive ALCL may explain why we failed to demonstrate an association between the anti-ALK antibody titer and MDD when using the cutoff defined by the receiver–operator characteristic curve-calculation.
We were, nevertheless, to identify three distinct groups of patients each exhibiting a different prognosis using measurements of MDD and anti-ALK titers. This biological risk stratification has some important advantages compared with clinical stratification. These include the ease and reproducibility of the assays, reasonable cost and a clear prognostic cutoff is clear, which allows a very good separation of both high-risk and low-risk patients. As noted above, the histological subtype was also an important prognostic factor but, as demonstrated recently by Lamant et al.,8 the concordance between national and international reviews is not easy to achieve. Highly reproducible stratification criteria are needed for future clinical trials and this is especially true for ALCL due to the necessity of worldwide international studies in this rare disease. Although quantitative real-time PCR allowed the identification of a poor risk group in one study,9 it was expensive when compared with qualitative RT-PCR, and good risk patients could not be separated. Furthermore, the method is difficult to transfer to other laboratories and an international cutoff value would need to be defined. International quality control is difficult to achieve because this assay requires set-up of several parameters with reliable measurement at the lowest end of the standard curve. As part of an international biological NHL Study Group, establishment of a quality controlled quantitative real-time PCR for NPM-ALK is one of our major efforts at present, but in terms of clinical feasibility, qualitative RT-PCR is now the ‘gold standard’ because it is easily reproducible, inexpensive, highly sensitive and, as demonstrated, has relevant prognostic impact. Stratifying patients according to the biological risk factors MDD and antibody titers against ALK identifies not only the high-risk patients, but also a very low-risk patient group. Risk stratification in future ALCL clinical trials based on these newly identified risk categories should open up improved therapies enabling improved survival for high-risk patients while decrease toxicity for the low-risk group.
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The study was supported by Fondazione Città Della Speranza, Associazione Italiana contro le Leucemie (AIL) and by Camera di Commercio di Venezia. The study was also supported by a grant from the Deutsche Jose Carreras Leukämie-Stiftung (DJCLS08/09) to WW. CDW and WW are additionally supported by the Forschungshilfe peiper. KP was supported by Leukemia and Lymphoma research, the Sam Foye Fund, Julian Starmer-Smith Lymphoma fund and the National Institute for Health Research Biomedical Research Center Program. We are grateful to Dr Elisa Carraro for data management. We thank the Colleagues from various AIEOP and BFM centers for contributing biological samples and patient clinical information.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Mussolin, L., Damm-Welk, C., Pillon, M. et al. Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis. Leukemia 27, 416–422 (2013). https://doi.org/10.1038/leu.2012.205
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