Circulating Beta-2 Microglobulin and Risk of Cancer: The Atherosclerosis Risk in Communities Study (ARIC)

This article is featured in Highlights of This Issue, p. 567

Beta-2 microglobulin (B2M) is a subunit of the major histocompatibility complex (MHC) class I molecule; it is found on the surface of all nucleated cells and is especially abundant in lymphocytes and monocytes (1). Under normal physiological conditions, some amount of B2M may be secreted into circulation from cell surfaces or as a result of intracellular release (2–4) and is removed from blood predominantly by the kidneys (5). Thus, the net concentration of serum B2M is determined by its generation and secretion into serum, as well as its elimination by the kidneys, so that serum B2M concentration increases with elevated turnover of cells and/or reduced kidney function (6). Therefore, in people without kidney disease, elevated B2M serves as a marker of altered cell proliferation. Elevated serum B2M concentrations have been observed in many pathological conditions, including renal disease, immunodeficiency, and autoimmune diseases (1, 7–9). Further, B2M concentrations have been reported as elevated at the time of diagnosis in many solid (3, 10–14) and hematological cancers (15, 16).

There is also evidence to suggest that elevated serum B2M concentrations are associated with risk of all-cause death. In a prospective cohort study of nondisabled people 65 years and older, B2M concentrations better predicted death than high sensitivity C-reactive protein (CRP), an established inflammatory marker (1). In addition, several cohort studies, including the Atherosclerosis Risk in Communities Study (ARIC; ref. 7), have reported positive associations between B2M and aging, various cardiovascular conditions, and mortality from diabetes, CVD and all causes, suggesting that serum B2M may also serve as an inflammatory biomarker (7, 17, 18).

However, despite the evidence showing increased B2M concentrations in cancer patients, there have been no published studies that examined serum B2M in relation to the risk of subsequent cancer in the general population. Using ARIC data, we examined the risk of all cancers combined, the four most common solid cancers (colorectal, lung, prostate, and breast cancers) individually, and hematological cancers combined. We hypothesized that the strongest associations would be observed for colorectal and hematological cancers, but the mechanisms underlying these associations would be different. Given that colorectal cancer has been associated with immune response and inflammation (19–22), we reasoned that higher serum B2M would be an early biomarker associated with increased risk for future colorectal cancer. While this hypothesis might also be relevant for breast and prostate cancers (23, 24), associations between circulating immune and inflammatory markers and these cancers have been less consistently reported (23, 25) than those for colorectal cancer. In contrast, we hypothesized that B2M may serve as a marker of early detection for hematological cancers, because elevated B2M concentrations are consistently observed in multiple myeloma, malignant lymphomas, and leukemia and associated with progression and worse prognosis (4, 5, 15, 16). Similarly, B2M could be a potential early detection biomarker for lung cancer, which is often diagnosed at an advanced stage, given that B2M was reported to be higher and occur more frequently in lung cancer cases compared with unaffected controls (26).

ARIC, a multicenter population-based prospective cohort study of atherosclerosis, was initiated in 1987–1989 when 15,792 volunteers ages 45 to 64 years were recruited from 4 communities: suburban Minneapolis (MN), Forsyth County (NC), Jackson (MS), and Washington County (MD; refs. 27, 28). Baseline and four follow-up visits in 1990–1992, 1993–1995, 1996–1998, and 2011–2013 included interviews, laboratory measurements, and clinic examinations (27, 28). At each visit, participants were asked about their demographic characteristics, education, lifestyle behaviors, and reproductive and medical histories. Trained personnel collected anthropometric measures, blood (serum and plasma), and urine samples.

B2M was measured in serum samples collected at Visit 2 (93% of the living cohort was reexamined); therefore, most of the covariates shown in Table 1 and tested in the multivariable regression models were also collected at Visit 2, i.e., body mass index (BMI), waist-to-hip ratio (WHR), smoking status, pack-years, alcohol intake (g/week), use of hormone replacement therapy (HRT), and aspirin use during the 2 weeks before the examination. Education, consumption of red meat, and dietary fiber were collected at Visit 1. The analytical cohort was followed from Visit 2 until the end of 2006, for a median of 14.8 years of follow-up (maximum was 16.9 years).

Local institutional review boards approved the ARIC protocol and all participants provided their informed consent, which included permission for follow-up for the occurrence of non-cardiovascular diseases.

Incident cancers in 1990–2006 were identified by linkage to the state cancer registries of MD, MN, MS, and NC and were supplemented by active surveillance of the cohort, which included recording of hospital discharges and hospitalizations that were self-reported during annual follow-up telephone calls (29, 30). Additional cancer cases (not reported by cancer registries) were included after hospital records were obtained, reviewed, and verified for cancer. Primary site, date of cancer diagnosis, and source of diagnostic information (e.g., pathology report) were recorded (29, 30). Data on cancer stage were not uniformly collected.

Deaths from cancer as the underlying cause were obtained from death certificates in 1990–2011. The follow-up for death was five years longer than for incidence (1990–2006), which explains the higher number of deaths from lung cancer (n = 352) compared with the number of incident lung cancers (n = 298). Also, although the inclusion of only first primary incident cancers did not affect cancer deaths, it resulted in a decrease of incident cancer cases.

WBC count was measured in local centers at the time of the Visit 2 exams (1990–1992). Serum concentrations of B2M, high-sensitivity CRP (hs-CRP) as well as cystatin (a marker of kidney function), were measured in 2012–2013 using stored serum from Visit 2. B2M and CRP were measured using the Roche B2M reagent and Roche latex-particle enhanced immunoturbidimetric assay kits, respectively, and read on the Roche Modular P Chemistry analyzer (Roche Diagnostics). Cystatin C was measured using the Gentian immunoassay (Gentian Technology AS). Another marker of kidney function, creatinine, was measured by the modified kinetic Jaffé method (31) in 1990–1992. Estimated glomerular filtration rate (eGFR), an index of kidney function, was calculated in ARIC using standardized cystatin C and standardized creatinine (32).

The analytical sample for this analysis (n = 12,300) excluded the participants who did not consent to participate in studies of non-cardiovascular diseases (n = 187); were not black or white (n = 48); were blacks from Minneapolis and Washington County (n = 55); did not participate in Visit 2 (n = 1,323), self-reported history of cancer at baseline (n = 882); had cancer diagnosed before Visit 2 (n = 214), or had missing information about B2M (n = 786; some individuals were excluded for more than one reason).

Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for incident cancers in relation to B2M modeled as quartiles. Tests for trends were calculated by fitting quartile numbers as an ordinal variable into the Cox regression model.

The proportional hazards assumption was tested by adding an interaction term between follow-up time and B2M concentration, and the assumption was not violated for any individual cancer or total cancer.

For each cancer, we analyzed two multivariable-adjusted models. Model 1 was adjusted for age (continuous), sex, and a 5-level variable combining race and ARIC field center. Model 2 was additionally adjusted for education (less than high school, high school, more than high school), BMI (<25, 25–29.9, ≥30 kg/m²), smoking status (yes, no) and pack-years of smoking (continuous), use of aspirin (yes, no), sex-HRT variable (men, women never taking HRT, and women who were former or current HRT users), and eGFR (continuous). We adjusted for eGFR for two reasons: (i) serum B2M concentration depends on its elimination by the kidneys (Spearman correlation coefficient between B2M and eGFR ρ = −0.72) and (ii) previous studies found that reduced kidney function and chronic kidney diseases were associated with poor prognosis for several cancers, including cancers of the lung (33), bladder (33), kidney (34, 35), and several others (36–39). Therefore, we aimed to understand whether the B2M–cancer associations were independent of reduced kidney function. Additional covariates that were tested for confounding but were not included in final models included consumption of alcohol, red meat, and dietary fiber.

The fully adjusted breast cancer models also included age at menarche (continuous), number of live births (0, 1–2, 3–4, and ≥5), and menopausal status (yes, no). The colorectal cancer analysis was also run separately by subsite (colon, rectum). In addition, we conducted several supplementary analyses for all cancer types. We further adjusted for WHR, in addition to BMI, to decrease the likelihood of residual confounding by body fatness (the Spearman correlations were: between B2M and BMI ρ = 0.15; B2M and WHR ρ = 0.18; BMI and WHR ρ = 0.46). We also assessed the independence of the B2M and colorectal cancer association from hs-CRP, which we previously found to be associated with colorectal cancer in ARIC (30). To do so, we first determined the Spearman correlation between B2M and hs-CRP (ρ = 0.27). In addition, we conducted several sensitivity analyses: we excluded participants (∼10% of participants) with evidence of acute inflammation (hs-CRP >10 mg/L) and those with reduced kidney function measured as eGFR ≤ 90 mL/min/1.73 m² (∼33% of participants), i.e., the cutoff point recommended by the National Kidney Foundation (40). To minimize the impact of undiagnosed disease, we excluded people with less than 3 years of follow-up (4% of participants). Although we were not able to study associations across cancer stage, we examined associations between B2M and risk of death from cancer.

Given that each biomarker is measured with some degree of error, we sought to determine whether combining B2M and hs-CRP would better capture the association with colorectal cancer than either biomarker measurement alone; we restricted this analysis to colorectal cancer due its stronger association with B2M. To do so, we created a combined score, by summing hs-CRP and B2M as categorical variables (where the quartiles for each variable were coded 0–3). The resulting combined score, which ranged from 0 to 6, was categorized into 4 groups (0–1, 2–3, 4–5, and 6). In addition, we tested the risk of colorectal cancer in relation to two other combined scores based on hs-CRP and B2M that were created as (i) a sum of the normalized log-transformed continuous biomarkers; (ii) a linear combination of normalized log-transformed continuous biomarkers with weights equal to beta coefficients derived from Cox regression, when hs-CRP and B2M were included simultaneously.

We also tested whether the multivariable models that included both B2M and hs-CRP (normalized log-transformed) provided better fit than the model with B2M alone; we tested for differences in model fit using the likelihood ratio χ² test. Analyses were performed using SAS v9.3 (SAS Institute Inc.); all statistical tests were two sided.

The analytical cohort (n = 12,300; 55% women, 26% black and 74% white) was followed for incident cancer from 1990–1992 through 2006. During follow-up, 2,436 incident cancers were ascertained, including 255 colorectal, 298 lung, 424 breast, 524 prostate, and 176 hematological cancers. Serum B2M concentrations at the start of follow-up ranged from 0.9 to 57.8 mg/L, with a mean (SD) of 2.0 (1.5) mg/L. B2M was unrelated to sex, but higher B2M quartiles were associated with older age, higher proportions of whites (versus blacks), less than high school education, higher BMI, greater pack-years of smoking, recent aspirin use, and less ever use of hormone therapy (Table 1). These findings were mostly consistent with previous studies (41–43).

In the minimally adjusted Model 1, for the highest versus lowest B2M quartile, there were significant positive associations of B2M with the risk of total (HR, 1.20; 95% CI, 1.07–1.36; P_trend = 0.0003) and colorectal cancers (HR, 1.55; 95% CI, 1.04–2.30; P_trend = 0.02) and a possible association with hematological cancers (HR, 1.42; 95% CI, 0.92–2.21; P_trend = 0.07; Table 2). In adjusted Model 2, compared with the lowest quartile, people in the highest quartile were more likely to develop total (HR, 1.25; 95% CI, 1.06–1.47; P_trend = 0.002) and colorectal cancers (HR, 2.21; 95% CI, 1.32–3.70; P_trend = 0.001). When the analysis for colorectal cancer was stratified by subsite, HRs were similar for colon (n = 206) and rectal (n = 67) cancers: 1.72 and 1.98 for the highest versus lowest B2M quartile (not presented in the tables). After multivariable adjustment (Model 2), the HRs for hematological cancers did not change substantively. For lung cancer, there was a statistically significant association in the fourth versus first quartile only: HR = 1.64 (95% CI, 1.03–2.61) in Model 2 (Table 2). B2M was not associated with risk of breast or prostate cancer in either Model 1 or 2.

Additional adjustment of B2M–cancer associations for WHR or log-transformed hs-CRP did not markedly change any of the observed associations (Supplementary Table S1, Models 3 and 4, respectively). After excluding participants with a possible acute inflammatory response (CRP >10 mg/L), associations for colorectal and total cancer remained statistically significant (Supplementary Table S1, Model 5). Likewise, the associations for total and colorectal cancer also did not appreciably change after excluding those with eGFR ≤ 90 mL/min/1.73 m²: for the highest versus lowest B2M quartile, the HRs were 1.21 (95% CI, 0.96–1.51; P_trend = 0.004) for total cancer and 2.18 (95% CI, 1.18–4.04; P_trend = 0.004) for colorectal cancer (Supplementary Table S1, Model 6). After excluding people with less than 3 years of follow-up, the positive associations with total or colorectal cancer did not change, whereas the hazard ratios for lung and hematological cancers for the fourth quartiles (versus the first quartiles) diminished (Supplementary Table S1, Model 7). Further, there was no evidence of multiplicative interactions of serum B2M with age, BMI, smoking, HRT or aspirin use in relation to any cancer (P values for all interactions were > 0.1). Stratified associations for colorectal and lung cancers are presented in Supplementary Tables S2a and 2b, respectively.

Further, we analyzed serum B2M and risk of death from cancer during follow-up through 2011 and showed that the patterns of associations were similar to the associations for incident total cancer and each cancer type, respectively (Table 3). The association for death from hematological cancers was stronger than that for incidence and was statistically significant, while death from colorectal cancer was not significantly associated with B2M (with only 85 deaths observed). The association for death from lung cancer was of the same magnitude as for incidence and was significant.

Finally, we examined whether the combined score based on hs-CRP and B2M was more strongly associated with colorectal cancer incidence than was B2M alone. As shown in Table 4, the associations for combined score 1, which included categorical B2M and hs-CRP, were very similar in magnitude and behaved in a parallel fashion to the B2M–colorectal cancer association: the HR for highest versus lowest quartile was 2.49 (95% CI, 1.42–4.38; P_trend = 0.0002). The associations with two other combined scores based on continuous log-transformed hs-CRP and B2M were also similar to those with B2M and to each other (Supplementary Table S3). However, the multivariable model with continuous hs-CRP and B2M included simultaneously in the model showed improvement in model fit compared with the multivariable model that included B2M only (P = 0.02).

In this population-based cohort study of adults followed for a maximum of 17 years, participants with a serum B2M concentration in the highest versus lowest quartiles had a 25% higher risk of total cancer incidence and 121% higher risk of colorectal cancer incidence. These findings support our a priori hypothesis that B2M, a biomarker of increased cell turnover, is associated with colorectal cancer risk.

The robust association between B2M and colorectal cancer risk was independent of the inflammatory marker hs-CRP and persisted in all sensitivity analyses: it remained after excluding those with eGFR ≤90 mL/min/1.73 m² (potentially abnormal kidney function) or those with CRP <10 mg/L (acute inflammation). It also did not markedly change after excluding the first three years of follow-up, which implies that higher B2M concentrations do not reflect the subclinical presence of malignancy. The magnitude of the B2M–colorectal associations was similar to the strength of the association between hs-CRP and colorectal cancer in this study: for the highest versus lowest hs-CRP quartile, the HR was 2.17 (95% CI, 1.39–3.37; P_trend = 0.0002). This association is unlikely to be explained by residual confounding by obesity, because B2M concentration was only weakly correlated with BMI or WHR (Spearman ρ = 0.15 and ρ = 0.18, respectively). This characteristic distinguishes B2M from CRP, because CRP concentrations are moderately associated with obesity, both in our study (Spearman r = 0.39) and other studies (44, 45). Also, we showed that linear combinations of two biomarkers, B2M and CRP, were positively associated with the risk of developing colorectal cancer. Importantly, the linear combinations of B2M and CRP did not yield a higher HR than either biomarker put into the model alone. However, the inclusion of CRP and B2M simultaneously in the model improved the fit of the model, suggesting that the combined score may be a better marker for the development of colorectal cancer than B2M alone.

The associations for incidence and death associated with B2M behaved in parallel fashion for total cancer and each cancer type. The trends for deaths were significant for total, lung, and hematological cancers. For the lung cancer death, the association was driven by the increased hazard ratio in the highest versus lowest B2M quartile. For death from hematological cancers, the HRs in all quartiles were higher than the corresponding HRs for risk of hematological cancers, which is consistent with a strong correlation between B2M concentration and the progression of hematological cancer (4, 5). Additional studies are needed to better understand the link between B2M and risk of hematological and lung cancers.

The observed associations of elevated B2M with the total cancer incidence and death, colorectal cancer incidence and the death from lung and hematological cancers are consistent with experimental evidence that links B2M expression with carcinogenesis. In mouse studies, Josson and colleagues reported that B2M overexpression in cancer cells promoted the growth and progression of human prostate, breast, lung, and renal cancer cells and led to metastases and lethality (46). In addition, several human studies have shown that serum B2M is elevated at the time of cancer diagnosis or thereafter and increases with progression in many solid cancers, including those of the prostate (11, 12), breast (10, 13, 14), lung (26, 47), and kidneys (48), and, to a greater extent, in hematological cancers, such as lymphomas and multiple myeloma (15, 16, 49). Further, serum B2M was reported to be a strong indicator of poor prognosis and reduced survival of patients with solid and hematological cancers, including kidney, prostate and breast tumors, multiple myeloma, lymphomas and leukemia (11, 14, 16, 50–52).

Mechanisms explaining the positive associations between serum B2M concentrations and carcinogenesis have not been precisely established, but, most likely, B2M plays several roles: (i) proangiogenic; (ii) protumorigenic; (iii) it may drive innate proinflammatory cytokines (e.g., TNFα, IL1, IL6, and IL8) and growth-promoting factors (e.g., vascular endothelial growth factor, epidermal growth factor receptor, fatty acid synthase, insulin and insulin-like growth factor 1 receptor) in both cancer patients and unaffected people (3, 4, 12, 53); and (iv) may drive epithelial–mesenchymal transition (46).

Thus, B2M may have a direct pathogenic role in immune response and inflammation or may serve as an early biomarker of increased cell turnover. Its role as an inflammation marker is corroborated by studies reporting positive associations of B2M with aging, CVD, diabetes, and mortality from those diseases and all causes (1, 7–9). In a prospective cohort study of 1,034 healthy people 65 years and older followed for 8 years, the risk of all-cause death was significantly increased by 102% in the second and by 184% in the third tertile versus the first B2M tertile, independent of lifestyle factors, renal dysfunction, and preexisting comorbidities (1). Of note, B2M was a better predictor of mortality than CRP or cystatin.

The function of B2M as a marker of increased cell turnover may be particularly relevant to colorectal cancer, because the tissues in colon and rectum are characterized by rapidly dividing epithelia cells and immune response elicited by gut microbiota (22, 54). However, we do not know exactly whether B2M is secreted by immune or tumor cells, i.e., whether the elevated B2M concentrations reflect local or systemic response. Finally, B2M could be a marker of undiagnosed cancer, especially for hematological malignancies, because B2M is an established marker of progression and worse prognosis in those cancers (4, 5, 16).

A limitation of our study is that B2M was measured only once at Visit 2 (1990–92). However, a recent study using Third National Health and Nutrition Examination Survey (NHANES III) data on 795 people examined two B2M measurements taken 18 days apart and concluded that B2M has a low short-term variability (coefficient of variation was 8.4%; ref. 55). Another limitation of our study is the low number of cases ascertained in each cancer group, especially hematological cancers, which precluded both the study of leukemia and lymphoma separately and the conduct of subgroup analyses. The main strength of this study is that, to our knowledge, it is the first to examine serum B2M concentrations in relation to future cancer risk. We studied this association in a large prospective population-based study that has rigorously collected data on multiple risk factors related to B2M and cancer, including information on other markers of kidney function and inflammation. This allowed us to evaluate the association of B2M with cancer development independently of other risk factors.

In conclusion, our study provides the first evidence that higher serum B2M in people without a prior cancer diagnosis is associated with increased colorectal cancer risk. Significant associations were also observed for mortality from total, lung, and hematological cancers. Further research is needed to determine whether for these associations, circulating B2M is marking increased turnover of tumor or immune cells.

E. Selvin is a consultant/advisory board member for Roche Diagnostics. No potential conflicts of interest were disclosed by the other authors.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conception and design: A.E. Prizment, P.L. Lutsey

Development of methodology: A.E. Prizment, T.R. Church

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E. Selvin, A.R. Folsom

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.E. Prizment, A.M. Linabery, P.L. Lutsey, H.H. Nelson, T.R. Church, E.A. Platz, C. Joshu

Writing, review, and/or revision of the manuscript: A.E. Prizment, A.M. Linabery, P.L. Lutsey, E. Selvin, H.H. Nelson, A.R. Folsom, T.R. Church, C.G. Drake, E.A. Platz, C. Joshu

Study supervision: T.R. Church

Cancer incidence data have been provided by Maryland Cancer Registry, Center of Cancer Surveillance and Control, Department of Health and Mental Hygiene, 201 West Preston Street, Room 400, Baltimore, MD 21201. The authors acknowledge the State of Maryland, the Maryland Cigarette Restitution Fund, and the National Program of Cancer Registries of the CDC for the funds that helped support the availability of the cancer registry data. Reagents for the β-2 microglobulin and C-reactive protein assays were donated by Roche Diagnostics.

The Atherosclerosis Risk in Communities (ARIC) study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C, HHSN268201100008C, HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and HHSN268201100012C), and UL1RR025005, a component of the NIH and NIH Roadmap for Medical Research. Studies on cancer in ARIC are also supported by the National Cancer Institute U01 CA164975 (PI – Elizabeth Platz, Co-Is: Aaron Folsom, Corrine Joshu, Heather Nelson, Anna Prizment, Elizabeth Selvin). This research was supported by NIH/NIDDK grant R01DK089174 to Dr. Elizabeth Selvin. A.E. Prizment was supported by the National Center for Advancing Translational Sciences of the NIH Award Number UL1 TR000114.