Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

Composite mantle cell and primary cutaneous anaplastic large cell lymphoma: case report and review of the literature.

We describe the first reported occurrence of a composite cutaneous lymphoma involving a mantle cell lymphoma (MCL) and primary cutaneous anaplastic large cell lymphoma. The lesion occurred in a 76-year-old man with longstanding MCL who developed nodular skin lesions on his trunk and extremities. Biopsy revealed a CD30-positive lymphoma with pathological features characteristic of cutaneous anaplastic large cell lymphoma in the superficial dermis and a subjacent deposit of MCL in the deep dermis and subcutaneous adipose tissue. Immunophenotyping demonstrated T versus B lymphoid origin, respectively, for the 2 neoplasms, and fluorescence in situ hybridization demonstrated an 11;14 chromosomal translocation exclusively in the MCL. These results argue that the lymphomas represented clonally distinct neoplasms. Our case illustrates the extreme diversity associated with the cutaneous manifestations of lymphoid neoplasia and in particular of composite lymphomas, which present diagnostic challenges for clinicians and pathologists alike.

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1.

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.

E2A proteins enhance the histone acetyltransferase activity of the transcriptional co-activators CBP and p300.

The E2A gene encodes the E-protein transcription factors E12 and E47 that play critical roles in B-lymphopoiesis. A somatic chromosomal translocation detectable in 5% of cases of acute lymphoblastic leukemia (ALL) involves E2A and results in expression of the oncogenic transcription factor E2A-PBX1. CREB binding protein (CBP) and its close paralog p300 are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. We and others have shown that direct binding of an N-terminal transcriptional activation domain present in E12/E47 and E2A-PBX1 to the KIX domain of CBP/p300 contributes to E2A protein function. In the current work we show for the first time that the catalytic HAT activity of CBP/p300 is increased in the presence of residues 1-483 of E2A (i.e., the portion present in E2A-PBX1). The addition of purified, recombinant E2A protein to in vitro assays results in a two-fold augmentation of CBP/p300 HAT activity, whereas in vivo assays show a ten-fold augmentation of HAT-dependent transcriptional induction and a five-fold augmentation of acetylation of reporter plasmid-associated histone by CBP in response to co-transfected E2A. Our results indicate that the HAT-enhancing effect is independent of the well-documented E2A-CBP interaction involving the KIX domain and suggest a role for direct, perhaps low affinity binding of E2A to a portion of CBP that includes the HAT domain and flanking elements. Our findings add to a growing body of literature indicating that interactions between CBP/p300 and transcription factors can function in a specific manner to modulate HAT catalytic activity.

Mapping acetylation sites in E2A identifies a conserved lysine residue in activation domain 1 that promotes CBP/p300 recruitment and transcriptional activation.

E-proteins are basic helix-loop-helix transcription factors that function in cell type specification. The gene E2A encodes two E-proteins, E12 and E47, which are required in B-lymphopoiesis. E2A proteins can interact directly with the transcriptional co-activators and lysine acetyltranferases (KATs) CBP, p300 and PCAF to induce target gene transcription. Prior investigations have shown that the E2A-encoded isoform E2-5 is acetylated by CBP, p300 or PCAF in vitro or in vivo. However, E2-5 lacks the important N-terminal activation domain AD1. Furthermore, the acetylated residues in E-proteins have not been mapped, and the functional consequences of acetylation are largely unknown. Here, we use mutagenesis to show that a lysine residue at position 34 within AD1 of E12/E47 is acetylated by CBP/p300 and PCAF. Lys34 lies adjacent to a conserved helical LXXLL motif that interacts directly with the KIX domain of CBP/p300. We show that acetylation at Lys34 increases the affinity of AD1 for the KIX domain and enhances AD1-driven transcriptional induction. Our results illustrate for the first time that AD1 can both recruit, and be acetylated by, KATs and that KAT recruitment may promote transcriptional induction in part through acetylation of AD1 itself.

Expression of TCF3 target genes defines a subclass of diffuse large B-cell lymphoma characterized by up-regulation of MYC target genes and poor clinical outcome following R-CHOP therapy.

TCF3 is a lymphopoietic transcription factor that acquires somatic driver mutations in diffuse large B-cell lymphoma (DLBCL). Hypothesizing that expression patterns of TCF3-regulated genes can inform clinical management, we found that unsupervised clustering analysis with 15 TCF3-regulated genes and eight additional ones resolved local DLBCL cases into two main clusters, denoted Groups A and B, of which Group A manifested inferior overall survival (OS, p = 0.0005). We trained a machine learning model to classify samples into the Groups based on expression of the 23 transcripts in an independent validation cohort of 569 R-CHOP-treated DLBCL cases. Group A overlapped with the ABC cell-of-origin subgroup but its prognostic power was superior. GSEA analysis demonstrated asymmetric expression of 30 gene sets between the Groups, pointing to biological differences. We present, validate and make available a novel method to assign DLBCL cases into biologically-distinct groups with divergent OS following R-CHOP therapy.

Transplacental benzene exposure increases tumor incidence in mouse offspring: possible role of fetal benzene metabolism.

Childhood cancer is the leading cause of disease-related death in children aged 1-14 years in Canada and the USA and it has been hypothesized that transplacental exposure to environmental carcinogens such as benzene may contribute to the etiology of these cancers. Our objectives were to determine if transplacental benzene exposure increased tumor incidence in mouse offspring and assess fetal benzene metabolism capability. Pregnant CD-1 and C57Bl/6N mice were given intraperitoneal injections of corn oil, 200 mg/kg, or 400 mg/kg benzene on gestational days 8, 10, 12 and 14. A significant increase in tumor incidence was observed in CD-1, but not C57BL/6N, 1-year-old offspring exposed transplacentally to 200 mg/kg benzene. Hepatic and hematopoietic tumors were predominantly observed in male and female CD-1 offspring, respectively. Female CD-1 offspring exposed transplacentally to 200 mg/kg benzene had significantly suppressed bone marrow CD11b(+) cells 1 year after birth, correlating with reduced colony-forming unit granulocyte/macrophage numbers in 2-day-old pups. CD-1 and C57Bl/6N maternal blood benzene levels and fetal liver benzene, t, t-muconic acid, hydroquinone and catechol levels were analyzed by gas chromatography/mass spectrometry. Significant strain-, gender- and dose-related differences were observed. Male CD-1 fetuses had high hydroquinone levels, whereas females had high catechol levels after maternal exposure to 200 mg/kg benzene. This is the first demonstration that transplacental benzene exposure can induce hepatic and hematopoietic tumors in mice, which may be dependent on fetal benzene metabolism capability.

Objective quantification of BCL2 protein by multiplex immunofluorescence in routine biopsy samples of diffuse large B-cell lymphoma demonstrates associations with survival and BCL2 gene alterations.

Up-regulation of BCL2 in cases of diffuse large B-cell lymphoma (DLBCL) can confer treatment resistance. Quantitative immunofluorescence (QIF) histology allows objective quantification of protein-based biomarkers. We investigated the utility of QIF for evaluating BCL2 as a biomarker in DLBCL by quantifying BCL2 selectively in CD20-expressing lymphoma cells in biopsy samples from 116 cases of DLBCL in two cohorts one of which consisted of relapsed/refractory cases from a clinical trial. BCL2 protein by QIF correlated with BCL2 mRNA abundance and was associated with both translocation and copy number gain of the BCL2 gene. Elevated BCL2 protein expression by QIF, but not immunohistochemistry or mRNA quantification, was associated with inferior overall and relapse-free survival in the relapsed/refractory cohort. QIF is an effective means of quantifying BCL2 protein objectively in routine cancer biopsy specimens and shows promise for identifying relapsed/refractory DLBCL patients at risk of inferior outcomes after salvage therapy.

Critical role for a single leucine residue in leukemia induction by E2A-PBX1.

In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

High-grade B-cell lymphoma with MYC and BCL2 rearrangements arising in a composite lymphoma.

BACKGROUND: We report the first case of composite lymphoma consisting of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL) and high-grade B-cell lymphoma with MYC and BCL2 rearrangements within the same needle biopsy in which a clonal relationship between the FL and high-grade B-cell lymphoma components was demonstrated by molecular cytogenetics. CASE PRESENTATION: An 85-year-old man presented with masses in his neck and right groin. Cutting needle biopsy of the inguinal mass revealed the three lymphoma types which were morphologically, immunophenotypically and topographically distinct. Fluorescence in situ hybridization (FISH) identified an IGH-BCL2 rearrangement in both the FL and high-grade B-cell components while a MYC rearrangement was detected in the high-grade B-cell component alone. CONCLUSIONS: Our findings suggest that the high-grade lymphoma with MYC and BCL2 translocations evolved through transformation of the FL by a process that entailed acquisition of the MYC translocation. No clonal relationship between the FL and CLL/SLL components was evident since the IGH-BCL2 rearrangement was present in in the former but not the latter. This unique case of co-localized FL, CLL/SLL, and high-grade B-cell lymphoma contributes to our understanding of the clonal relationships that may exist between the components of composite lymphomas.

Abundant expression of BMI1 in follicular lymphoma is associated with reduced overall survival.

Although generally indolent, follicular lymphoma (FL) sometimes pursues a more aggressive course leading to early death. B-cell-specific Mo-MLV insertion site-1 (BMI1) is a member of the polycomb group (PcG) proteins that confer stem cell properties through gene silencing. We used multi-channel immunofluorescence and automated image analysis to quantify BMI1 selectively in the nuclei of FL-derived B-cells in routine biopsy specimens. Applying this assay to 109 pretreatment FL biopsy samples demonstrates a significant association between abundant BMI1 and reduced overall survival (p = .001); the statistically significant association with mortality persists in a Cox proportional hazards model that includes Follicular Lymphoma International Prognostic Index (FLIPI) score, histological grade, and the presence of a component of diffuse large B-cell lymphoma in the biopsy sample. Ascertaining BMI1 over-expression may be useful in identifying patients who might benefit from novel therapies directed at reversing the chromatin-modifying functions of BMI1.

The reliability of lymphoma diagnosis in small tissue samples is heavily influenced by lymphoma subtype.

A specific pathologic diagnosis is important in malignant lymphoma because the diverse disease subtypes require tailored approaches to clinical management. Reliance on small samples obtained with cutting needles has been advocated as a less invasive alternative to using larger, excised samples. Although published studies have demonstrated the safety and apparent sufficiency of this approach in informing clinical care, none have systematically determined the accuracy of pathologic lymphoma subtyping based on very small samples. We used a tissue microarray representing 67 cases of malignant lymphoma and 17 samples of nonneoplastic lymphoid tissue to model lymphoma diagnosis in small samples. Overall, 73.8% of the cases were diagnosed with a level of confidence deemed sufficient for directing clinical management; 85.9% of these diagnoses were accurate. Small cell lymphomas with highly distinctive immunophenotypes, including small lymphocytic, mantle cell, and T-lymphoblastic lymphoma, were recognized most consistently and accurately in the small samples. In contrast, follicular lymphoma and marginal zone lymphoma were especially difficult. Our results indicate that the reliability of lymphoma diagnoses based on small samples is heavily influenced by lymphoma subtype.

Tumor suppressors in follicular lymphoma.

Follicular lymphoma (FL) is the most common indolent lymphoma. The vast majority of cases are associated with the chromosome translocation t(14;18), a somatic rearrangement that leads to constitutive expression of the anti-apoptotic BCL2 protein. Although t(14;18) clearly represents an important early event in FL pathogenesis, abundant evidence indicates that it is not sufficient. In particular, the recent application of next-generation DNA sequencing technology has uncovered numerous recurrent somatic genomic alterations associated with FL, most of which affect tumor suppressor genes (TSGs). In this article we review the existing literature on TSGs involved in the development and progression of FL. We consider the genes that are most frequently targeted by deleterious mutation, deletion or epigenetic silencing, along with strategies for developing new treatments that exploit the susceptibilities that may be conferred on lymphoma cells by the loss of particular TSGs.

Interleukin-4 Expressed By Neoplastic Cells Provokes an Anti-Metastatic Myeloid Immune Response.

OBJECTIVE: Interleukin-4 (IL-4) can induce macrophages to undergo alternative activation and polarize toward an M2-like or wound healing phenotype. Tumor associated macrophages (TAMs) are thought to assume M2-like properties, and it has been suggested they promote tumor growth and metastasis through effects on the tumor stroma, including extracelluar matrix remodeling and angiogenesis. IL-4 also promotes macrophage survival and formation of multinucleated giant cells, which have enhanced phagocytic behavior. This study was designed to explore the effect of cancer cell derived IL-4 on the tumor immune stroma and metastasis. METHODS: The metastatic mouse mammary carcinoma cell line AC2M2 was transduced with control or IL-4 encoding retroviruses and employed in orthotopic engraftment models. Tumor growth and metastasis were assessed. The cellular composition and biomarker expression of tumors were examined by immunohistochemical staining and flow cytometry; the transcriptome of the immune stroma was analyzed by nanoString based transcript quantitation; and in vivo and in vitro interactions between cancer cells and macrophages were assessed by flow cytometry and co-culture with video-time lapse microscopy, respectively. RESULTS: Unexpectedly, tumors from IL-4 expressing AC2M2 engrafted cells grew at reduced rates, and most surprising, they lost all metastatic potential relative to tumors from control AC2M2 cells. Myeloid cell numbers were not increased in IL-4 expressing tumors, but their expression of the M2 marker arginase I was elevated. Transcriptome analysis revealed an immune signature consistent with IL-4 induced M2 polarization of the tumor microenvironment and a generalized increase in myeloid involvement in the tumor stroma. Flow cytometry analysis indicated enhanced cancer cell phagocytosis by TAMs from IL-4 expressing tumors, and co-culture studies showed that IL-4 expressing cancer cells supported the survival and promoted the in vitro phagocytic behavior of macrophages. CONCLUSIONS: Although M2-like TAMs have been linked to enhanced tumorigenesis, this study shows that IL-4 production by cancer cells is associated with suppressed tumor growth and loss of metastatic potential as well as enhanced phagocytic behavior of TAMs.

Retrovirus-Mediated Expression of E2A-PBX1 Blocks Lymphoid Fate but Permits Retention of Myeloid Potential in Early Hematopoietic Progenitors.

The oncogenic transcription factor E2A-PBX1 is expressed consequent to chromosomal translocation 1;19 and is an important oncogenic driver in cases of pre-B-cell acute lymphoblastic leukemia (ALL). Elucidating the mechanism by which E2A-PBX1 induces lymphoid leukemia would be expedited by the availability of a tractable experimental model in which enforced expression of E2A-PBX1 in hematopoietic progenitors induces pre-B-cell ALL. However, hematopoietic reconstitution of irradiated mice with bone marrow infected with E2A-PBX1-expressing retroviruses consistently gives rise to myeloid, not lymphoid, leukemia. Here, we elucidate the hematopoietic consequences of forced E2A-PBX1 expression in primary murine hematopoietic progenitors. We show that introducing E2A-PBX1 into multipotent progenitors permits the retention of myeloid potential but imposes a dense barrier to lymphoid development prior to the common lymphoid progenitor stage, thus helping to explain the eventual development of myeloid, and not lymphoid, leukemia in transplanted mice. Our findings also indicate that E2A-PBX1 enforces the aberrant, persistent expression of some genes that would normally have been down-regulated in the subsequent course of hematopoietic maturation. We show that enforced expression of one such gene, Hoxa9, a proto-oncogene associated with myeloid leukemia, partially reproduces the phenotype produced by E2A-PBX1 itself. Existing evidence suggests that the 1;19 translocation event takes place in committed B-lymphoid progenitors. However, we find that retrovirus-enforced expression of E2A-PBX1 in committed pro-B-cells results in cell cycle arrest and apoptosis. Our findings indicate that the neoplastic phenotype induced by E2A-PBX1 is determined by the developmental stage of the cell into which the oncoprotein is introduced.

E2A basic helix-loop-helix transcription factors in human leukemia.

The gene E2A on chromosome 19 is involved in recurrent chromosomal rearrangements associated with pediatric acute lymphoblastic leukemia. The resulting fusion of 5' E2A sequences with 3' portions of other genes leads to the expression of two well-characterized fusion proteins: E2A-PBX1 and E2A-HLF. Since the E2A, PBX1 and HLF proteins all appear to function as transcription factors, it appears likely that the oncogenic fusion proteins contribute to leukemia development by causing abnormal transcriptional regulation of key target genes. Furthermore, since the E2A portion of the fusion proteins contains transcriptional activation domains, and the PBX1 and HLF portions contain DNA binding domains, leukemogenesis may be due, at least in part, to excessive transcriptional induction of target genes defined by PBX1 or HLF. However, recent findings suggest that this model is simplistic and possibly incorrect. In this article, I review the evidence pertaining to leukemogenesis by the well-characterized E2A-fusion proteins and consider its mechanistic implications.

Inactivation of the CDKN2A tumor-suppressor gene by deletion or methylation is common at diagnosis in follicular lymphoma and associated with poor clinical outcome.

PURPOSE: Follicular lymphoma, the most common indolent lymphoma, is clinically heterogeneous. CDKN2A encodes the tumor suppressors p16(INK4a) and p14(ARF) 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 = 0.004). CDKN2A deletion or methylation was associated with more advanced age (P = 0.012) and normal hemoglobin (P = 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.