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Inactivation of the CDKN2A Tumor-Suppressor Gene by Deletion Or Methylation Is Common at Diagnosis in Follicular Lymphoma and Associated with Poor Clinical Outcome

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Inactivation of the CDKN2A Tumor-Suppressor Gene by Deletion Or Methylation Is Common at Diagnosis in Follicular Lymphoma and Associated with Poor Clinical Outcome Published OnlineFirst January 21, 2014; DOI: 10.1158/1078-0432.CCR-13-2175 Clinical Cancer Predictive Biomarkers and Personalized Medicine Research Inactivation of the CDKN2A Tumor-Suppressor Gene by Deletion or Methylation Is Common at Diagnosis in Follicular Lymphoma and Associated with Poor Clinical Outcome Abdulmohsen Alhejaily1, Andrew G. Day2, Harriet E. Feilotter1, Tara Baetz3, and David P. LeBrun1 Abstract Purpose: Follicular lymphoma, the most common indolent lymphoma, is clinically heterogeneous. CDKN2A encodes the tumor suppressors p16INK4a and p14ARF and frequently suffers deleterious alterations in cancer. We investigated the hypothesis that deletion or hypermethylation of CDKN2A might identify follicular lymphoma cases with distinct clinical or pathologic features potentially amenable to tailored clinical management. Experimental Design: Deletion of CDKN2A was detected in pretreatment biopsy specimens using a single nucleotide polymorphism–based approach or endpoint PCR, and methylation of CpG elements in CDKN2A was quantified by methylation-specific PCR. Correlations between CDKN2A status and pathologic or clinical characteristics, including overall survival (OS), were investigated in 106 cases using standard statistical methods. Results: Deletion of CDKN2A was detected in 9 of 111 samples (8%) and methylation was detectable in 22 of 113 (19%). CDKN2A was either deleted or methylated in 29 of 106 cases (27%) and this status was associated with inferior OS especially among patients treated with rituximab (P ¼ 0.004). CDKN2A deletion or methylation was associated with more advanced age (P ¼ 0.012) and normal hemoglobin (P ¼ 0.05) but not with sex, FLIPI score, ECOG stage, LDH, performance status, number of involved nodal sites, B symptoms, histologic grade, the presence of a component of diffuse large B-cell lymphoma, proliferation index, or other pathologic factors. Conclusions: Our results show that deletion or methylation of CDKN2A is relatively common in pretreatment follicular lymphoma biopsy specimens and defines a group of cases associated with reduced survival in the rituximab era presumably on the basis of more aggressive disease biology. Clin Cancer Res; 20(6); 1–11. Ó2014 AACR. Introduction large B-cell lymphoma (DLBCL; refs. 3 and 4). The relatively Follicular lymphoma is common, accounting for approx- recent addition of rituximab, a chimeric monoclonal anti- imately 20% to 30% of non-Hodgkin lymphoma (NHL), body directed at CD20, to therapeutic regimens used to treat and its clinical course it typically indolent (1). Nonetheless, follicular lymphoma has improved survival (2). The primary patients with follicular lymphoma experience reduced life genetic event in most cases of follicular lymphoma is a expectancy with a median overall survival (OS) of approx- somatic chromosomal translocation, t(14;18)(q32;q21), imately 10 years (2). Furthermore, some cases have a poor which results in the upregulation of the proto-oncogene BCL2 prognosis because of clinically progressive disease or histo- (5). However, t(14:18) is not sufficient to induce logic transformation to aggressive lymphoma, mostly diffuse follicular lymphoma; secondary genetic alterations are required for full manifestation of the disease. The CDKN2A genetic locus at human chromosome band ARF INK4a Authors' Affiliations: 1Department of Pathology and Molecular Medicine, 9p21 encodes the proteins p14 and p16 , which are Queen's University; 2Clinical Research Centre; and 3Cancer Centre of generated through alternative exon usage (Fig. 1; ref. 6). Southeastern Ontario, Kingston General Hospital, Kingston, Ontario, ARF Canada p14 activates p53 by rescuing it from proteosome-medi- ated proteolysis whereas p16INK4a antagonizes cyclin- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). dependent kinases 4 and 6 (CDK4/6), thereby blocking phosphorylation of the retinoblastoma protein pRb and, Corresponding Author: David P. LeBrun, Division of Cancer Biology and Genetics, Queen's Cancer Research Institute, Botterell Hall, Room 330, consequently, cell-cycle progression. The p53 and pRb Queen's University, 10 Stuart Street, Kingston K7L 3N6, ON, Canada. signaling pathways are required for the induction of apo- Phone: 613-533-3209; Fax: 613-533-6830; E-mail: [email protected] ptosis or cellular senescence in response to a number of doi: 10.1158/1078-0432.CCR-13-2175 cellular stressors, including DNA damage and oncogene Ó2014 American Association for Cancer Research. activation. Consistent with its role in the regulation of both www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst January 21, 2014; DOI: 10.1158/1078-0432.CCR-13-2175 Alhejaily et al. called acquired uniparental disomy (aUPD), has uncovered Translational Relevance such alterations affecting CDKN2A in a substantial 12% to Follicular lymphoma, although generally indolent, is 20% of nontransformed follicular lymphoma samples clinically heterogeneous in that some cases behave (9, 15–17). aggressively or are refractory to standard therapy. We Methylation of cytosine residues in CpG islands in find that deletion or CpG island methylation affecting CDKN2A is associated with transcriptional silencing and the tumor-suppressor gene CDKN2A, ascertained in therefore represents the functional equivalent of gene dele- pretreatment fllicular lymphoma biopsy specimens, is tion (10, 18, 19). CDKN2A methylation is more prevalent in associated with reduced overall survival (P ¼ 0.004) aggressive- than indolent-histology lymphomas and has among patients who received rituximab as part of their been detected in up to 52% of follicular lymphoma samples therapeutic regimen, and that this association persists (8, 10, 20, 21). Therefore, CpG island methylation is more when FLIPI score and other conventional clinical or common than gene deletion as a mechanism for inactivat- pathologic factors are added to a Cox proportional ing CDKN2A in follicular lymphoma and seems to be an hazards model. In our dataset, CDKN2A deletion and important step in tumor progression and transformation. methylation are complementary in predicting adverse These considerations suggest the possibility that detecting outcome, underscoring the importance of ascertaining deleterious alterations affecting the CDKN2A locus in pre- both genetic and epigenetic mechanisms when investi- treatment follicular lymphoma biopsy specimens might gating tumor-suppressor genes as potential biomarkers. define a substantial subset of cases with aggressive under- CDKN2A is inactivated more often by methylation than lying biology and clinical behavior, including a differential deletion in follicular lymphoma, suggesting that detect- response to therapy. Indeed, a recent study reported an ing CDKN2A methylation might identify candidates for association between the presence of CDKN2A deletion or therapy with methyltransferase inhibitors. aUPD in diagnostic follicular lymphoma specimens and reduced OS (15). However, no study has yet evaluated the prognostic impact of gene methylation, the most prevalent mechanism of CDKN2A silencing in follicular lymphoma, of these pathways, deleterious alterations of CDKN2A are in a large set of follicular lymphoma cases nor has the prevalent in human cancers and play an important role in clinicopathological impact of CDKN2A silencing been eval- oncogenesis and tumor progression. Inactivation of uated in the rituximab era. CDKN2A in lymphoma can occur by gene deletion or, more In this study, we have evaluated the clinicopathological frequently, methylation of CpG islands, whereas small associations of CDKN2A silencing ascertained at diagnosis deleterious mutations are relatively rare (7–10). Although in 106 cases of follicular lymphoma. We find that deletion deletions commonly eliminate all of the exons that encode or methylation of CDKN2A is associated with reduced OS p14ARF and p16INK4a, loss of p16INK4a may be the more and that this association is most striking when the analysis is important event in human cancers (11). restricted to the 62 patients who had been treated with Deletion of CDKN2A occurs relatively commonly upon rituximab. histologic transformation of follicular lymphoma to DBLCL, but has been considered rare in follicular lympho- Materials and Methods ma samples obtained before transformation (7, 9, 12–14). Clinical samples However, the recent application of technology capable of Cases were identified by searching the pathology and detecting homozygous deletions or copy-neutral LOH, also clinical records of Kingston General Hospital, Ontario, CDKN2B CDKN2A 5 kb 12 1β 1α 23 rs4977750 rs10757261 rs10757260 rs10965197 rs2069418 rs1063192 rs3218020 rs7036656 rs3731211 rs3731239 rs2518719 rs3731257 rs7041637 rs78545330 rs67226702 CpG island Exon 2 PCR Exon 3 PCR Figure 1. Genomic configuration of the CDKN2A locus. Exons 1a, 2, and 3 of CDKN2A encode p16INK4a whereas exons 1b and 2 encode p14ARF; these different gene products are generated through differential pre-mRNA splicing and translation of exon 2 in alternative reading frames. CDKN2B encodes p15INK4b. The relative locations of 15 highly heterozygous SNPs used in this study are shown to scale, as are those of the CpG island used to ascertain methylation status and the sequences lying adjacent to exons 2 and 3 of CDKN2A that are the targets of the PCR primer sets used to ascertain homozygous
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