Serum Insulin-Like Growth Factor-I and Platelet-Derived Growth Factor as Biomarkers of Breast Cancer Prognosis

Western diet rich in saturated and partially hydrogenated fat, refined carbohydrates, and animal protein is associated with high prevalence of overweight/obesity, metabolic abnormalities, and high serum levels of hormones and growth factors that may affect both breast cancer risk (1, 2) and prognosis (1, 3-5). Overweight is associated with inflammation (6), and there is experimental and epidemiologic evidence that inflammatory mechanisms that are mediated by specific dietary components and caloric intake (7) may contribute to breast cancer etiology (8, 9).

In the present study, we followed up women enrolled into a previously published dietary intervention trial aimed at reducing sex-hormone levels in postmenopausal breast cancer patients (3, 10) to test if the baseline serum levels of serum insulin-like growth factor I (IGF-I), platelet-derived growth factor (PDGF), fructosamine, and serum markers of inflammatory response [C-reactive protein (CRP)] are associated with breast cancer relapse.

One hundred and ten women (mean age ± SD, 56.8 ± 5.6 y) operated for breast cancer at least a year previously (4.6 ± 4.4 y on average), not undergoing chemotherapy, with at least one ovary, and with no clinical evidence of disease recurrence, volunteered to participate in a dietary intervention study (Diet and Androgens 2) that required the modification of their usual diet over a year to reduce insulin and sex-hormone levels (3, 10). The patients had to be postmenopausal, defined as absence of menses for at least 2 y and by a postmenopausal gonadotropin and estrogen profile.

The women were requested to reduce their sources of refined carbohydrates and saturated fats and to increase their intake of whole grain cereals and pulses. All patients signed an informed consent, and the study was approved by the Institutional Review Board and the Ethics Committee of the Milan National Cancer Institute.

Compliance was monitored through repeated 24 h dietary diaries and anthropometric measurements. None of the participants abandoned the study. They significantly reduced body weight, serum insulin and testosterone levels, and the prevalence of metabolic syndrome (5, 10). They were followed up for 5.5 y, and recurrences and contralateral breast cancer were registered. High baseline levels of testosterone and body weight were significantly associated with breast cancer prognosis (3).

Baseline blood samples were collected at recruitment, before any dietary modification.

Blood samples were collected between 8 a.m. and 9 a.m. after overnight fasting to reduce intraindividual variation in hormone levels and were stored at −80°C. The technicians analyzing the serum samples were blinded to the outcome recurrence status of the patients. A quality control serum sample was measured at the beginning and at the end of the batch. Serum growth factor levels were measured in duplicate through commercial radioimmunoassay kits from Biosource for IGF-I and enzyme immunoassay from R&D for PDGF. We decided to measure the PDGF-AB isoform because its circulating levels are much higher than those of the other PDGF isoforms. Furthermore, PDGF-AB binds to PDGF receptors with nearly 10-fold higher affinity than does PDGF-AA or PDGF-BB (11).

Fructosamine and CRP were assayed with a Hitachi modular automatic analyzer (Roche).

The coefficients of intraassay and interassay variation, respectively, were: 2.8 and 5.3% for a mean IGF-I level of 304 ng/mL, 3.6 and 7.5% for a mean PDGF level of 25.9 ng/mL, 0.9 and 2.9% for a mean fructosamine level of 288 μmol/L, and 1.34 and 5.7% for a mean CRP level of 0.5 mg/L.

The statistical analysis focused on the risk of breast cancer recurrences (local relapse, distant metastasis, and contralateral breast cancer) in relation to the baseline levels of the variables under study. The means of the baseline serum levels of IGF-I, PDGF, CRP, and fructosamine in patients who recurred were compared with those of patients who did not. The prognostic effect of all these parameters on risk of recurrence was assessed by hazard ratios (HR) and 95% confidence intervals (95% CI) through the Cox proportional hazards model. The following covariates were considered as potential confounders: age (in tertiles), years since diagnosis, extent of disease at diagnosis, as size of the primary (T1, T2, or more) and axillary node status (N-, N+), and estrogen and progesterone receptor status (positive, negative). Because 38% of the women were under tamoxifen treatment, which decreases IGF-I levels (12), we considered current tamoxifen treatment (yes or no) as a potential confounder. T, N, and hormone receptor status proved to be significant confounders and were retained in the analytic model. Because our previous results showed that body weight and serum testosterone levels were strongly and significantly associated with recurrences (3), adjustment for these variables (in tertiles) was also done in the present analysis. We also examined the effect of each growth factor on recurrences controlling for the other one, and we tested their interaction. The proportional hazards assumption was checked for each model by scaled Shoenfeld residual analysis. The analysis was carried out with the use of the STATA 8.0. statistical package.

Out of 110 patients enrolled into the study, 32 patients developed recurrences, either local (4), distant (16), or contralateral breast cancer (12). Consistent with previous publications, we considered all these breast cancer events as recurrences (3-5). Table 1 reports the mean baseline values for all the variables under study in the patients who recurred and in the patients who did not. In a few cases, the available sera were not sufficient for analysis (see Table 1). The patients who recurred showed significantly greater baseline serum values of PDGF (11.9 ng/mL versus 9.4 ng/mL, P = 0.01). There was no difference between patients who developed local or distant recurrences and those who developed contralateral breast cancer. Serum IGF-I was higher in the recurrence group, but the differences were not significant.

For CRP and fructosamine, the average concentration was similar in the two patient groups.

Table 2 reports the HRs of recurrence for quartiles of the baseline serum levels of PDGF and IGF-I. The HR of recurrence for the upper quartile of PDGF with reference to the lower quartile was 3.3 (95% CI, 1.1-9.9) but decreased to 2.3 (95% CI, 0.7-7.1) after adjustment for body weight and serum testosterone. As for IGF-I, the risk of recurrence was higher above the first quartile, but the differences were not statistically significant and there was no concentration effect trend. When we examined the effect of each growth factor on recurrences controlling for the other, the results did not substantially change. In this analysis, the fully adjusted HR of recurrence for the upper quartile of PDGF with reference to the lower quartile was 2.8 (95% CI, 0.9-9.0); the fully adjusted HR of recurrence for the upper quartile of IGF-I versus the lower one was 3.7 (95% CI, 0.9-15.1). The interaction test between PDGF and IGF-I was not significant.

By combining PDGF and IGF-I, the fully adjusted HR of recurrence among the women with PDGF and IGF-I levels > median values (respectively, 9.3 and 174.4 ng/mL) was 6.4 (95% CI, 1.5-26.7) compared with the women with PDGF and IGF-I levels ≤ their median values (Table 2). High IGF-I levels did not show any association with recurrence risk (HR, 0.7; 95% CI, 0.1-3.0) in the patients with PDGF levels under the median value. In the presence of high serum levels of PDGF, the risk associated with low IGF-I levels was 1.7 (95% CI, 0.4-7.0).

There was no association of fructosamine and CRP with breast cancer events (data not shown).

The data from the present study on the risk of breast cancer recurrences during 5.5 years of follow-up suggest that baseline serum levels of PDGF are associated with breast cancer prognosis. Furthermore, these data suggest that high IGF-I levels may increase the risk of breast cancer recurrences only in the presence of high PDGF levels. Contrary to our a priori hypothesis, we did not find any relationship with the baseline serum levels of fructosamine, a marker of insulin resistance and abnormal glucose metabolism, or with the baseline serum levels of CRP, a marker of inflammation.

IGF-I and PDGF are two essential growth factors that synergistically promote cell proliferation and the transformation of several types of cells. Several in vitro studies have pointed to interactions between PDGF and IGF-I receptor (13-15). The results from our study suggest that in vivo IGF-I and PDGF may also interact to increase the risk of breast cancer progression. We found that high IGF-I levels were associated with recurrence risk only when PDGF levels were also high above the median value.

The mechanism(s) responsible for the higher breast cancer recurrence rate in the women with high serum levels of PDGF is not known, but a PDGF “priming” effect on IGF-I receptors may be involved. In vitro studies have shown that PDGF stimulated IGF-I receptor mRNA expression and increased the number of IGF-I binding sites at the cell surface through the activation of the IGF-I receptor gene promoter, thus enhancing IGF-I responsiveness and cell proliferation (13-15).

The present study deals with small numbers of patients, and its results require confirmation by larger studies; however, they were not unexpected because two recent prospective cohort studies reported a positive association of serum IGF-I and breast cancer risk in postmenopausal women (16, 17). Previous smaller studies, however, did not find any association (18). A few small studies suggested that serum PDGF may be associated with breast cancer progression (19, 20). We previously showed that metabolic syndrome, especially if associated with high testosterone, an anabolic hormone, is a major predictor of breast cancer recurrence (3, 5). High levels of PDGF and IGF-I may belong to the same pattern of metabolic risk factors associated with western lifestyle that increase breast cancer risk and relapse.

A limitation of the present study is the lack of information about adjuvant chemotherapy. No patient, however, was under chemotherapy at the time of recruitment. Because chemotherapy regimen is strongly associated with the stage of disease defined by T and N status and with the hormonal receptor status, we do not expect that controlling for chemotherapy regimen would have modified the stage and hormonal adjusted odds ratio estimates.

The Diet and Androgens intervention trials showed that a sustainable dietary modification aimed at lowering circulating insulin levels reduces serum insulin and testosterone levels; increases serum sex hormones binding globulin, IGF binding protein 1, and IGF binding protein 2 levels, thus reducing the bioavailability of both IGF-I and sex hormones; and favorably modifies anthropometric and metabolic parameters (3, 10, 21, 22). Studies by Fontana et al. on individuals who voluntarily restricted calorie and protein intake also showed decreased levels of circulating PDGF and IGF-I (23, 24).

Our previous analysis suggested that the reduction of body weight and serum testosterone induced by such diet may favorably influence breast cancer prognosis (3). Unfortunately, for the present study the available sera were not sufficient to analyze the prognostic effect of the change in PDGF levels.

If the predictive value of serum PDGF and IGF-I will be confirmed in larger studies, dietary or other medical intervention to reduce their serum levels should be considered.

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