Research paperPerforin gene variation influences survival in childhood acute lymphoblastic leukemia
Introduction
Perforin is a pore-forming protein stored in the acidic secretory granules of cytotoxic lymphocytes [1]. Perforin-mediated cytotoxicity is crucial in killing transformed cells and cells harboring intracellular pathogens; it is also involved in downmodulation of the immune response by inducing apoptosis of effector lymphocytes and antigen-presenting cells [[2], [3], [4]].
The belief that perforin is critical for immune homeostasis and tumor immune surveillance is supported by the spectrum of pathologies associated with perforin deficiency [5]. Perforin-depleted (Prf1−/−) mice are highly immunosuppressed and susceptible to diverse intracellular pathogens [6], as well as to transplanted [7], virus-induced [8] and spontaneous malignancies, primarily to aggressive B-cell lymphomas [9]. Perforin has also been shown to play an important role in NK-cell mediated suppression of tumor metastasis [10] and in controlling the growth of carcinogen-induced sarcoma [11]. In humans, biallelic PRF1 gene mutations are well recognized as the cause of 20–50% of familial heamophagocytic lymphohistiocytosis (FHL2) [[12], [13], [14], [15]], a severe hyperinflammatory syndrome with uncontrolled activation of antigen-presenting cells, CD8+ T cells and massive cytokinaemia [16]. Recent studies also describe the presence of perforin-dependent immune surveillance in humans. Carriers of mutant PRF1 were shown to suffer more frequently from relatively early-onset melanoma – a malignancy in which CD8+ T cells may play a role in the prevention of disease progression and metastatis [17]. Both mono- and biallelic PRF1 gene mutations have also been found in a proportion of patients with Hodgkin and non-Hodgkin lymphomas [[18], [19], [20]]. A limited number of studies on acute lymphoblastic leukemia (ALL), the single most common pediatric malignancy [21], have provided contradictory results: the A91V polymorphism was identified as an ALL-predisposing factor in a relatively small group of children [22], but was not confirmed on a larger cohort [23]. It was also suggested that perforin mutation may be more prevalent in ALL with BCR-ABL1 translocation [24].
As perforin plays a pivotal role in maintaining immune homeostasis, not only regarding tumor immune surveillance, but also pathogen defense and immune regulation, the aim of the present study is to examine the impact of perforin gene variation on the clinical outcome in pediatric ALL patients. As such, this is the only study so far to address the role of perforin in the clinical course of ALL.
Section snippets
The study population
The study enrolled 312 children aged 1–18 years, treated for ALL in reference centers of the Polish Pediatric Leukemia/Lymphoma Study Group between April 20 and September 2015. The diagnosis, risk group assignment and treatment were performed according to the guidelines of BFM ALL-IC 2002 and BFM ALL-IC 2009 Protocols, as described before [25]. Clinical variables are presented in Table 1. The Ethics Committee approved the research protocol and informed consent was obtained from all participants
Distribution of PRF1 allele frequencies in ALL patients and in controls
The sequencing of coding exons and exon-intron boundaries of PRF1 gene revealed many genetic variants (see Supplementary Table S1).
Out of these, three polymorphic sites with MAF (minor allele frequency) >0.05: rs885822, rs885821 and rs35947132 were found in the study group. All three SNPs were in the Hardy-Weinberg equilibrium in the control group (p = 0.15, p = 0.29 and p = 1.0, respectively). Moreover, rs885822 and rs35947132 SNPs were in complete linkage disequilibrium (D’ = 1, in that A
Discussion and conclusions
After identification of loss-of-function perforin mutation as the cause of FHL2 in 1999 [15], it was observed that FHL can sometimes be greatly delayed in carriers of biallelic PRF1 gene mutations [28]. It was later shown that a large proportion of these patients develop either leukemia or lymphoma [29]. Since then, perforin mutations have been linked with a spectrum of human malignancies [30]. All previous studies on the topic, however, have so far focused on rare genetic variants and their
Contributions
AJ performed genetic studies in leukemia patients and wrote the draft of the manuscript, AP and JT collected clinical data for survival analysis, KW and RP performed genetic studies in the control group, MB performed genetic studies in leukemia patients, WF performed statistical analysis, LS and TS provided clinical data in leukemia patients, WM designed the study and prepared the final version of the manuscript. Wojciech Mlynarski is the guarantor of the study and, as such, had full access to
Acknowledgments
We thank the patients and their families for agreeing to participate in this study. This study was supported by National Science Center grant No 2011/03/N/NZ5/04363 and Medical University of Lodz grant No 502-03/1-090-01/502-14-161.
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