Introduction

Cyclin B1 is a tumor-associated antigen that is overexpressed in multiple types of cancer, including prostate cancer (Mashal et al. 1996; Kallakury et al. 1999). It is immunogenic and the patients generate both cellular and humoral immune responses to this antigen. Interestingly, highly variable levels of anticyclin B1 antibodies are also present in healthy subjects with no history of cancer. These antibodies—possibly generated by some noncancerous events (e.g., viral infections), may influence the host immunosurveillance mechanism against cyclin B1 overexpressing tumors (Vella et al. 2009a). This raises the possibility of a cyclin B1-based vaccine for subjects at risk of developing cancers characterized by the overexpression of this tumor antigen. Some studies in mice support this contention: vaccine-induced cellular and humoral immunity to cyclin B1 protected the mice from spontaneous cancer development later in life (Vella et al. 2009b). Since no animal model can fully replicate a human disease, a thorough understanding of the mechanisms responsible for natural immunity to this antigen in humans is an important prerequisite to designing an effective cyclin B1-based immunotherapy. In prostate cancer, it has been known for some time that there are inter-individual differences in naturally occurring antibody responses to cyclin B1 (Koziol et al. 2003), but no host genetic factors that might contribute to these differences have hitherto been identified.

Immunoglobulin (Ig) GM allotypes, antigenic determinants of IgG heavy chains, are encoded by three highly polymorphic IGHG loci on chromosome 14. There are currently 18 testable GM specificities—4 on γ1, 1 on γ2, and 13 on γ3 (Lefranc and Lefranc 2012). GM alleles are associated with immunity to several exogenous and endogenous antigens, including some tumor antigens (Pandey et al. 1982, 2008, 2009, 2010), but their possible role in immunity to cyclin B1 has not been investigated. A recent comprehensive analysis of human gene expression has identified Ig κ constant (IGKC) gene as a strong prognostic marker in several human solid tumors (Schmidt et al. 2012). These results provide an excellent rationale for investigating the role of KM alleles, genetic variants of IGKC on chromosome 2, in humoral immunity to tumor antigens. Particular KM alleles have been shown to epistatically interact with GM alleles and contribute to antibody responses to mucin 1 in gastric cancer (Pandey et al. 2008). All GM markers, except GM 3 and 17, are expressed in the Fc region of Ig γ1, γ2, and γ3 chains, thus making them the most likely candidates in the genome for epistatic interactions with Fcgamma receptor (FcγR) genes, which have been implicated in the immunobiology of several cancers (Stockmeyer et al. 1997; Musolino et al. 2008; Bibeau et al. 2009; Taylor et al. 2009).

In the present investigation, we evaluated whether GM, KM, and FcγR genotypes—individually or in particular epistatic combinations—are associated with antibody responsiveness to cyclin B1 in patients with prostate cancer. We found a highly significant association between an FcγRIIIa genotype and low antibody responses to cyclin B1 in AA patients with prostate cancer.

Materials and methods

Human subjects and biological specimens

Buffy coat and plasma samples were obtained from 205 patients with histologically verified adenocarcinoma of prostate diagnosed at the Hollings Cancer Center, Medical University of South Carolina. The study protocol was approved by the institutional IRB. The samples were kept frozen in the cancer center’s tissue bank until required. Age, Gleason score, and stage of disease of the patients included in the study are listed in Table 1.

Table 1 Demographic and disease characteristic of the study population
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Detection of IgG antibodies to cyclin B1

Anticyclin B1 antibodies present in plasma samples were measured by an enzyme-linked immunosorbent assay (ELISA), modified from a previously described method (Egloff et al. 2005). Briefly, 96-well microtiter plates were coated with 1 μg/ml of recombinant human cyclin B1 (GenWay Biotech Inc., San Diego, CA, USA). Wells were washed with phosphate-buffered saline (PBS; pH 7.4) containing 0.05 % tween 20 (PBS-T) and blocked with 1 % BSA in PBS-T (serum diluent buffer, SDB). Wells were further washed and incubated with 1:500 dilution of plasma in SDB. Finally, wells were washed and incubated with antihuman IgG HRP conjugate and probed with peroxidase substrate hydrogen peroxide along with 3,3′,5,5′-tetramethylbenzidine as co-substrate. Reaction was stopped by the addition of 2 N HCl and the absorbance values were measured at 450 nm in an ELISA reader. Wells incubated with buffer alone served as blank. The amount of cyclin B1 specific IgG present in three samples used as reference was measured using normal IgG as standard. Amount of cyclin B1 specific IgG present in the plasma were calculated based on mean of these references and multiplied by the dilution factor to give micrograms per milliliter of cyclin B1-specific IgG.

GM, KM, and FcγR genotyping

DNA from buffy coat was isolated using a standard protocol (Qiagen-Kit method).

IgG1 allelic markers GM3 and GM17 (arginine to lysine substitution, a G → A transition in the CH1 region of the γ1 gene) were determined by direct DNA sequencing. Polymerase chain reaction (PCR) was used to amplify the CH1 region of the γ1 gene using the following primers: 5′-CCCCTGGCACCCTCCTCCAA-3′ and 5′-GCCCTGGACTGGGGCTGCAT-3′ (Balbín et al. 1991). The double-stranded 364 bp DNA product was then purified and sequenced on an ABI Prism 377.

IgG2 allelic marker GM23 (valine-to-methionine substitution, a G → A transition in the CH2 region of the γ2 gene) was determined using a nested PCR-restriction fragment length polymorphism (RFLP) method. A 915-bp fragment that includes the polymorphic site was amplified using the following primers: 5′-AAATGTTGTGTCGAGTGCCC-3′ and 5′-GGCTTGCCGGCCGTGGCAC-3′ (Brusco et al. 1995). A 197-bp fragment was then amplified from the 915-bp fragment using the following primers: 5′-GCACCACCTGTGGCAGGACC-3′ and 5′-TTGAACTGCTCCTCCCGTGG-3′. The 197-bp product was digested by the restriction enzyme NIaIII. This resulted in the following product sizes for each genotype: GM23 (+/+), 90, 63, and 44 bp; GM23(−/−), 134 and 63 bp; and GM23(+/−), 134, 90, 63, and 44 bp.

IgG3 hetero-allelic markers GM5 (asparagine-to-serine substitution, an A → G transition in the CH3 region of the γ3 gene) and GM21 (proline-to-leucine substitution, a C → T transition in the CH2 region of the γ3 gene) were determined by a previously described PCR-RFLP method (Balbín et al. 1994).

The κ chain is triallelic—KM1, KM1,2, and KM3 alleles. KM1 allele is rare; 98 % of the individuals positive for KM1 are also positive for KM2. Thus, positivity for KM1 includes both KM1 and KM1, 2 alleles. The KM alleles were determined by a previously described PCR-RFLP method (Moxley and Gibbs 1992).

A change in the nucleotide at position 497 of FcγRIIa gene from A to G results in change of amino acid histidine to arginine (H/R131). A change in the nucleotide at position 559 of the FcγRIIIa gene from T to G results in phenylalanine to valine substitution (F/V158) in the membrane proximal IgG-like domain of FcγRIIIa. The FcγRIIa alleles were determined by a previously described PCR-RFLP method (Jiang et al. 1996). The FcγRIIIa alleles were determined by the TaqMan® SNP Genotyping Assays supplied by Applied Biosystems following manufacturer’s protocols. Due to technical reasons, certain samples could not be typed for certain alleles, causing slight variations in the sample number for each genotype. Genotype determinations were made blinded with respect to the anticyclin B1 antibody status, and vice versa. The results were provided to a genetic epidemiologist (EKG) who conducted the analyses.

Statistical analysis

Linear regression models were constructed within population groups (Caucasian American (CA) and African American (AA)). Tests of genotype models—2df tests with no assumptions about inheritance models—as well as 1df tests of additive, dominant, and recessive effects of the minor allele were constructed. The phenotype of interest, anticyclin B1 antibody level (micrograms per milliliter), was log transformed to avoid violating model assumptions. In all models, statistical significance was defined as p < 0.05.

Results

The distribution of GM, KM, and FcγR genotypes among CA study subjects in relation to the mean levels of IgG antibodies (micrograms per milliliter) to cyclin B1 is given in Table 2. None of the genotypes—either individually or epistatically—was associated with antibody responsiveness to cyclinB1 in this group of patients. The distribution of GM, KM, and FcγR genotypes among AA subjects in relation to the mean levels of IgG antibodies (micrograms per milliliter) to cyclinB1 is given in Table 3. None of the GM, KM, and FcγRIIa genotypes was associated with antibody responsiveness to cyclin B1. However, a highly significant association at the FcγRIIIa locus was observed in this group of patients. The association was significant for the genotype, additive, and recessive models, but not for the dominant model of inheritance. The FcγRIIIa V/V homozygotes had significantly lower anticyclin B1 antibody levels than F/F homozygotes and F/V heterozygotes (22.3 vs. 41.3 and 49.4 μg/mL; p = 0.0007). This association would remain significant even after the most conservative (Bonferroni’s method) correction for multiple testing. In this setting, however, given the absolute linkage disequilibrium between certain GM alleles within a race and significant linkage disequilibrium between particular FcγRIIa and FcγRIIIa alleles, such an adjustment would likely be overly punitive. No significant interactive effects of the genotypes on antibody responsiveness to cyclin B1 were observed; the p values for various interactions ranged from 0.1325 for KM and FcγRIIa alleles to 0.9741 for KM and FcγRIIIa alleles (data not shown). This study probably was underpowered to detect epistatic interactions. Anticyclin B1 antibody responses were not significantly associated with age, clinical T stage, or Gleason scores (data not shown).

Table 2 Tests of associations between genetic variants (GM, KM, FcγRIIa, FcγRIIIa) and anticyclin B1 antibody levels (micrograms per milliliters) in Caucasian American patients with prostate cancer
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Table 3 Tests of associations between genetic variants (GM, KM, FcγRIIa, FcγRIIIa) and anticyclin B1 antibody levels (micrograms per milliliter) in African American patients prostate cancer
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Discussion

The results presented here show that AA patients with prostate cancer who were homozygous for the V allele of FcγRIIIa generated significantly lower antibody responses to cyclin B1 than F/F homozygotes and F/V heterozygotes. Either the V allele itself, or another gene in linkage disequilibrium with it, somehow contributes to the low anticyclin B1 antibody responsiveness when in homozygous state. The V allele is associated with effector functions, such as antibody-dependent cellular cytotoxicity, mediated by some monoclonal antitumor antibodies (Musolino et al. 2008; Taylor et al. 2009; Namboodiri and Pandey 2011), but the exact mechanism(s) underlying its association with endogenous anticyclin B1 antibody responses is not clear. One could speculate on a mechanism involving the antigen processing/presenting pathway. It is known that FcγR-mediated uptake of antigen–antibody complexes can enhance antigen presentation. Perhaps FcγRIIIa-F/F and F/V expressing antigen-presenting cells are more efficient than those expressing FcγRIIIa-V/V in the uptake of opsonized cyclin B1 and presenting it to collaborating helper T cells, thus resulting in higher B-cell activation.

Irrespective of the mechanism(s) involved, these findings could aid in dividing the population into “naturally” high and low responders to cyclin B1, the former (F carriers) being more likely to benefit from cyclin B1-based vaccines. For carriers of the low-responder V/V genotype, cyclin B1 could be fused with appropriate adjuvants, such as heat shock proteins or flagellin, to circumvent the possible FcγR restriction in immune responsiveness.

The results presented here also could shed some light on the racial disparity in prostate cancer. AA men have higher incidence of prostate cancer and low survival rate from the disease compared to CA men. As mentioned before, antibodies to cyclin B1 appear to protect from cancer development (Vella et al. 2009b). Association of V/V genotype with low anticyclin B1 antibody responsiveness could result in a weaker immunosurveillance mechanism against cyclin B1-overexpressing tumors in AA subjects possessing this genotype. This would suggest a higher frequency of this low-responder genotype in AA men with prostate cancer than in healthy people of African descent. This appears to be the case: compared to the frequency in a historic AA healthy population (Lehrnbecher et al. 1999), the frequency of the V/V genotype in AA patients in the present study was higher (8 vs. 13 %). However, a formal case–control study needs to be done to conclusively determine whether or not the frequency of FcγRIIIa-V/V genotype is higher in AA men with prostate cancer than in ethnically matched controls.

If the FcγRIIIa polymorphism (rs396991) is relevant to the etiopathogenesis of prostate cancer in people of African descent, why has it not been detected by the genome-wide association studies (GWAS) of this malignancy? One reason could be that the majority of the GWAS have involved people of European descent only. Another likely reason might be the absence of this single nucleotide polymorphism in the commonly used genotyping platforms. DNA segments harboring FcγR genes on chromosome 1 are highly homologous and may not be amenable to the high throughput genotyping technology; this attribute may have contributed to their exclusion from many genotyping panels.

Despite a strong rationale for the involvement of GM alleles in antitumor immunity, we did not find a statistically significant association between these alleles and anticyclin B1 antibody responses in the present investigation. It is possible that these alleles do contribute to immunity to this tumor antigen but their effect is smaller than that of FcγRIIIa alleles and, therefore, would require a much larger study population for its detection.

Conclusions

We found a highly significant association between the FcγRIIIa-V/V genotype and low antibody responses to cyclin B1 in AA patients with prostate cancer. This is the first report, to our knowledge, implicating the FcγRIIIa locus in immunity to this tumor antigen. The results suggest that inherent variability in immune responsiveness to a tumor antigen could contribute to the racial disparity in mortality from prostate cancer. Although the observed association is very strong, it nonetheless needs to be replicated in a larger and independent study population.