Genome-wide association and candidate gene studies implicate different genetic variants within the 6p21 chromosomal region with different non-Hodgkin lymphoma (NHL) subtypes. Complementing these efforts, we conducted human leukocyte antigen (HLA) class I and class II genotyping among 610 NHL cases and 555 controls of non-Hispanic white descent from a US multicenter study. Allele-disease associations were assessed by logistic regression for NHL and its subtypes. Statistically significant associations between HLA and NHL subtypes include HLA-DRB1*0101 for follicular lymphoma (odds ratio [OR] = 2.14, P < .001), HLA-DRB1*0401 for diffuse large B-cell lymphoma (DLBCL; OR = 0.45, P = .006), and HLA-DRB1*13 and follicular lymphoma (OR = 0.48, P = .008). We further observed significant heterozygote advantage for HLA class I alleles and NHL, and particularly DLBCL (P trend = .01 for elevated risk with increasing number of homozygous alleles). Our results support a role for HLA in the etiology of NHL and its subtypes.

There is growing evidence indicating that genetic variation in the 6p21 chromosomal region is important in non-Hodgkin lymphoma (NHL) etiology. To date, implicated single nucleotide polymorphisms (SNPs) appear to vary by NHL subtype. A genome-wide association study reported rs6457327 on 6p21.33 (located near psoriasis susceptibility region 1 [PSORS1]) to be associated with susceptibility for follicular lymphoma.1  A consortium effort reported that a tumor necrosis factor (TNF) promoter polymorphism (TNF G-308A) on chromosome 6p21.3 was associated with increased risk of diffuse large B-cell lymphoma (DLBCL) and marginal zone lymphoma.2,3 

The 6p21 chromosomal region houses the major histocompatibility complex which plays a critical role in host immune responses to viral and other pathogens. The human leukocyte antigen (HLA) genes are the most polymorphic human genes, presumably because they encode for variations that influence the specificity of the antigenic epitopes bound and presented to T cells. In general, HLA class I molecules (HLA-A, -B, and -C) present foreign antigens to CD8+ cytotoxic T lymphocytes (CTL), and HLA class II molecules (HLA-DR, -DQ, and -DP) present antigenic peptides to CD4+ T-helper cells.

Here, we present data from 610 NHL cases and 555 controls of non-Hispanic white descent who participated in a US population-based case-control study where HLA class I (A, B, C) and class II DRB1 alleles were genotyped and evaluated for their role in NHL etiology.

The multicenter National Cancer Institute–Surveillance, Epidemiology and End Results (NCI-SEER) NHL case-control study population comprised 1321 NHL cases identified in 4 SEER registries (Iowa; Detroit, MI; Los Angeles, CA; Seattle, WA) aged 20 to 74 years and newly diagnosed between July 1998 and June 2000.4,5  Cases had no evidence of HIV infection. A total of 1057 population controls were identified by random digit dialing (< 65 years) and from Medicare eligibility files (> 65 years). Written informed consent was obtained from each participant before interview in accordance with the Declaration of Helsinki, and institutional review board approval was obtained from each participating study center and from the National Institutes of Health (NIH). All study participants were asked to provide a venous blood or mouthwash buccal cell sample. The present analysis was conducted on study participants who provided blood (773 cases, 668 controls). Data from the 610 cases and 555 controls who self-reported to be non-Hispanic white and from whom sufficient DNA were available for HLA allelotyping are included in the present manuscript (population characteristics are presented in supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Pathology information was derived from abstracted reports by the local diagnosing pathologist. All cases were histologically confirmed and coded according to the International Classification of Diseases for Oncology6  (ICD, 2nd Edition) and updated to the World Health Organization (WHO)/ICD-O-3. We evaluated risk for NHL and the histologic subtypes DLBCL, follicular lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL).

DNA were extracted using Puregene Autopure DNA extraction kits (Gentra Systems). Four-digit HLA class I (A, B, C) and class II genotyping (DRB1) was conducted at NCI-Frederick (Frederick, MD) according to sequence-specific oligonucleotide probe (SSOP) hybridization and sequence-based typing protocols developed by the 13th International Histocompatibility Workshop.7  Agreement for quality control duplicates (n = 100) was more than 99%.

We first calculated HLA-A, -B, -C, and -DRB1 allele frequencies and evaluated the statistical differences between cases and controls using the χ2 test for significance or by the Fisher exact test when a cell contained fewer than 5 subjects. Only for alleles found to be significantly different between case and control groups were odds ratios (ORs) and 95% confidence intervals (CIs) subsequently calculated to determine the magnitude and precision of associations. To test whether there was heterozygote advantage, we determined whether an increase in disease risk was observed for those who were homozygous at HLA loci. We assessed zygosity by calculating ORs and 95% CIs for individual HLA loci (eg, HLA-A, -B, -C, or -DRB1) and for combined zygosity across the 3 class I HLA loci. All risk estimates are adjusted for the study design variables: sex, age (< 45, 45-64, 65+ years), education (< 12, 12-15, > 15 years), and study center (Detroit, Iowa, Los Angeles, Seattle). All logistic regression models were unconditional and conducted using SAS 9.1.3 (SAS Institute). All tests of statistical significance were 2-sided.

HLA class II alleles

HLA-DRB1*13 alleles were inversely associated with overall NHL risk (OR = 0.71, 95% CI = 0.52-0.97) and most notably for follicular lymphoma (OR = 0.48, 95% CI = 0.28-0.82; Table 1). Of the HLA-DRB1*13 alleles, the association was most pronounced for HLA-DRB1*1301 and follicular lymphoma (OR = 0.35, 95% CI = 0.15-0.83). HLA-DRB1*0401 was significantly associated with DLBCL (OR = 0.45, 95% CI = 0.26-0.79), while HLA-DRB1*0101 was significantly associated with follicular lymphoma (OR = 2.14, 95% CI = 1.40-3.26). No significant associations were observed for any HLA-DRB1 alleles and CLL/SLL.

HLA class I alleles

Presence of HLA-A*2601 and HLA-Cw*0701 alleles was associated with increased NHL risk and HLA-B*3503 was associated with decreased NHL risk. The HLA class I alleles were largely consistent in the direction of association by subtype. Similarly, although statistically significant associations were observed and most pronounced for HLA-B*5101 and HLA-Cw*1502 with DLBCL, the direction of the associations was also consistent for follicular lymphoma and for NHL overall though not statistically significant.

Allele frequencies for HLA class I A, B, C and class II DRB1 alleles are shown in supplemental Table 2.

Zygosity (heterozygote advantage)

No significant increases in risk for NHL, DLBCL, or follicular lymphoma were observed for homozygosity at individual HLA loci (Table 2). However, increasing risks for NHL and DLBCL with increasing numbers of homozygous class I loci were observed. Compared with persons heterozygous in 2 or more HLA class I loci, persons homozygous at 2 HLA class I loci had a 1.81-fold risk (95% CI = 0.78-4.17) for DLBCL, and persons homozygous at all 3 HLA class I loci had a 3.66-fold risk (95% CI = 1.15-11.7) for DLBCL (P trend = .01). Results were more pronounced (P trend = .007) in analyses not restricted to non-Hispanic white (supplemental Table 3).

Overall, associations observed for HLA class II alleles were more striking for NHL subtypes than for NHL overall. Most notable were the increased risks observed with HLA-DRB1*0101 and follicular lymphoma and HLA-DRB1*0401 with DLBCL, and the risk decrease observed between HLA-DRB1*13 alleles and follicular lymphoma. Our results add further evidence of a role for HLA in NHL risk and suggest that these associations may be specific to certain NHL subtypes.

Our results are consistent with our knowledge of NHL; HLA class II molecules present antigens to CD4+ T cells and are fundamental to immune responses to external pathogens and autoimmunity, 2 well-recognized risk factors for NHL.8  Specifically, the implicated alleles have been previously linked with autoimmune conditions, many of which are also established or hypothesized risk factors for NHL, including systemic lupus erythematosus (HLA-DRB1*0301, HLA-B*0801), Sjogren syndrome (HLA-DRB1*0301, HLA-B*0801), celiac disease (HLA-DRB1*0301), rheumatoid arthritis (HLA-DRB1*0101 and *0401), and type 1 diabetes (HLA-DRB1*0401 and *0301).9  Previous risk reductions have also been reported for several other diseases in association with HLA-DRB1*13 alleles, including autoimmune conditions (eg, rheumatoid arthritis10  and multiple sclerosis11 ) and infectious diseases (eg, hepatitis B infection,12-14  hepatitis C infection,15,16  and human papillomavirus17 ). Ours is the first such report for NHL.

Our results also suggest that heterozygote advantage may exist at HLA class I loci for NHL and DLBCL, as previously demonstrated for some autoimmune conditions (eg, psoriatic arthritis18 ) and infectious diseases (eg, hepatitis B and HIV19 ). Because HLA class I heterozygotes would in principle present a broader range of peptides for antigen presentation to cytotoxic T lymphocytes or CD8+ T cells than homozygotes, our results support the hypothesis that such advantages afforded from a broad and more productive immune response for a more diverse array for pathogens and infectious diseases20  may also decrease NHL risk. As infectious diseases are a well-known etiologic cause of NHL, our results warrant further investigation. No heterozygote advantage was observed found for HLA-DRB1 alleles and NHL (data not shown).

In summary, our results support a role for HLA in NHL etiology, and are consistent with the mounting evidence that calls for a targeted and comprehensive evaluation of the variation in the 6p21 chromosomal region in NHL etiology.1-3,5  Our results also emphasize the importance of pursuing parallel efforts of a priori regions of interest such as HLA to complement ongoing agnostic approaches in genetic discoveries.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

We thank Peter Hui of Information Management Services Inc for programming support. We also gratefully acknowledge the contributions of the staff and scientists at the SEER centers of Iowa, Los Angeles, Detroit, and Seattle for the conduct of the study's field effort.

The NCI-SEER study was supported by the Intramural Research Program of the NIH (National Cancer Institute [NCI]), and by Public Health Service (PHS) contracts N01-PC-65064, N01-PC-67008, N01-PC-67009, N01-PC-67010, and N02-PC-71105. HLA typing for the study was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E and in part by the Intramural Research Program of the NIH, NCI, Center for Cancer Research.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US government.

National Institutes of Health

Contribution: S.S.W. and M.C. designed the research; L.M.M., J.R.C., W.C., N.R., S.D., R.K.S., L.B., and P.H. designed and conducted the parent NHL study from which this analysis was based; A.M.A., R.T., X.G., and M.C. performed the research/HLA genotyping; S.S.W. and M.C. conducted data analysis; and all authors participated in manuscript preparation.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Sophia S. Wang, PhD, Division of Cancer Etiology, Department of Population Sciences, Beckman Research Institute and the City of Hope, Duarte, CA 91010; e-mail: [email protected].

1
Skibola
 
CF
Bracci
 
PM
Halperin
 
E
et al. 
Genetic variants at 6p21.33 are associated with susceptibility to follicular lymphoma.
Nat Genet
2009
, vol. 
41
 
8
(pg. 
873
-
875
)
2
Rothman
 
N
Skibola
 
CF
Wang
 
SS
et al. 
Genetic variation in TNF and IL10 and risk of non-Hodgkin lymphoma: a report from the InterLymph Consortium.
Lancet Oncol
2006
, vol. 
7
 
1
(pg. 
27
-
38
)
3
Skibola
 
CF
Bracci
 
PM
Nieters
 
A
et al. 
Tumor necrosis factor (TNF) and lymphotoxin-{alpha} (LTA) polymorphisms and risk of non-Hodgkin lymphoma in the InterLymph Consortium.
Am J Epidemiol
2010
, vol. 
171
 
3
(pg. 
267
-
276
)
4
Wang
 
SS
Cerhan
 
JR
Hartge
 
P
et al. 
Common genetic variants in proinflammatory and other immunoregulatory genes and risk for non-Hodgkin lymphoma.
Cancer Res
2006
, vol. 
66
 
19
(pg. 
9771
-
9780
)
5
Abdou
 
AM
Gao
 
X
Cozen
 
W
et al. 
Human leukocyte antigen (HLA) A1-B8-DR3 (8.1) haplotype, tumor necrosis factor (TNF) G-308A, and risk of non-Hodgkin lymphoma.
Leukemia
2010
, vol. 
24
 
5
(pg. 
1055
-
1058
)
6
Percy
 
C
Van Hollen
 
V
Muir
 
C
International Classification of Diseases for Oncology. 2nd Edition
1990
Geneva, Switzerland
World Health Organization
7
Tilanu
 
MG
Hansen
 
JA
Hurley
 
CK
IHWG Technical Manual: Genomic Analysis of the Human MHC
2002
Seattle, WA
IHWG
8
Wang
 
SS
Hartge
 
P
Mauch
 
PM
Armitage
 
JO
Harris
 
NL
Coiffier
 
B
Dalla-Favera
 
R
Etiology and epidemiology of non-Hodgkin lymphoma.
Non-Hodgkin Lymphoma
2010
Baltimore, MD
Lippincott, Williams and Wilkens
9
De Bakker
 
PI
McVean
 
G
Sabeti
 
PC
et al. 
A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC.
Nat Genet
2006
, vol. 
38
 
10
(pg. 
1166
-
1172
)
10
van der Woude
 
D
Lie
 
BA
Lundström
 
E
et al. 
Protection against anti-citrullinated protein antibody-positive rheumatoid arthritis is predominantly associated with HLA-DRB1*1301: a meta-analysis of HLA-DRB1 associations with anti-citrullinated protein antibody-positive and anti-citrullinated protein antibody-negative rheumatoid arthritis in four European populations.
Arthritis Rheum
2010
, vol. 
62
 
5
(pg. 
1236
-
1245
)
11
Laaksonen
 
M
Pastinen
 
T
Sjoroos
 
M
et al. 
HLA class II associated risk and protection against multiple sclerosis–a Finnish family study.
J Neuroimmunol
2002
, vol. 
122
 
1–2
(pg. 
140
-
145
)
12
Ramezani
 
A
Hasanjani Roshan
 
MR
Kalantar
 
E
et al. 
Association of human leukocyte antigen polymorphism with outcomes of hepatitis B virus infection.
J Gastroenterol Hepatol
2008
, vol. 
23
 
11
(pg. 
1716
-
1721
)
13
Kummee
 
P
Tangkijvanich
 
P
Poovorawan
 
Y
Hirankarn
 
N
Association of HLA-DRB1*13 and TNF-alpha gene polymorphisms with clearance of chronic hepatitis B infection and risk of hepatocellular carcinoma in Thai population.
J Viral Hepat
2007
, vol. 
14
 
12
(pg. 
841
-
848
)
14
Hohler
 
T
Gerken
 
G
Notghi
 
A
et al. 
HLA-DRB1*1301 and *1302 protect against chronic hepatitis B.
J Hepatol
1997
, vol. 
26
 
3
(pg. 
503
-
507
)
15
Hohler
 
T
Gerken
 
G
Notghi
 
A
et al. 
MHC class II genes influence the susceptibility to chronic active hepatitis C.
J Hepatol
1997
, vol. 
27
 
2
(pg. 
259
-
264
)
16
Kuzushita
 
N
Hayashi
 
N
Moribe
 
T
et al. 
Influence of HLA haplotypes on the clinical courses of individuals infected with hepatitis C virus.
Hepatology
1998
, vol. 
27
 
1
(pg. 
240
-
244
)
17
Wang
 
SS
Hildesheim
 
A
Chapter 5: viral and host factors in human papillomavirus persistence and progression.
J Natl Cancer Inst Monogr
2003
31
(pg. 
35
-
40
)
18
Nelson
 
GW
Martin
 
MP
Gladman
 
D
Wade
 
J
Trowsdale
 
J
Carrington
 
M
Cutting edge: heterozygote advantage in autoimmune disease: hierarchy of protection/susceptibility conferred by HLA and killer Ig-like receptor combinations in psoriatic arthritis.
J Immunol
2004
, vol. 
173
 
7
(pg. 
4273
-
4276
)
19
Carrington
 
M
Nelson
 
GW
Martin
 
MP
et al. 
HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage.
Science
1999
, vol. 
283
 
5408
(pg. 
1748
-
1752
)
20
Doherty
 
PC
Zinkernagel
 
RM
Enhanced immunological surveillance in mice heterozygous at the H-2 gene complex.
Nature
1975
, vol. 
256
 
5512
(pg. 
50
-
52
)
Sign in via your Institution