Proteins and carbohydrates in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy

Cancer cell invasion and metastasis requires the degradation of the extracellular matrix (ECM) and basement membrane. This process is accomplished by several proteins, including those of the plasminogen activator (PA) system. Urokinase-type PA (uPA) plays a key role in ECM degradation. In women with breast cancer, uPA appears to promote cancer invasion and metastasis [1] through degradation of the ECM, stimulation of angiogenesis, alteration in cell migration and adhesion [2], and inhibition of apoptosis [3].

Plasminogen activator activity is inhibited by plasminogen activator inhibitor-1 (PAI-1) [4]. PAI-1 promotes breast cancer invasion and metastasis. Deficient PAI-1 expression in mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When PAI-1 expression was restored, invasion and associated angiogenesis were also restored, suggesting that host-produced PAI-1 is essential for cancer cell invasion and angiogenesis [5]. PAI-1 promotes angiogenesis by directly inhibiting proteases [6], suggesting that excessive plasmin proteolysis may prevent the assembly of tumor blood vessels. Possible mechanisms by which PAI-1 promotes breast cancer include prevention of excess ECM degradation, modulation of cell adhesion, a role in angiogenesis, and the stimulation of cell proliferation [1]. The association of uPA and PAI-1 expression with breast cancer is complex.

In a pooled analysis of 8377 breast cancer patients, higher uPA and PAI-1 levels in tumor tissue were related to worse prognosis [7]. The importance of the PA system in breast cancer detection was explored by us in a sample set ranging from healthy women to those with advanced breast cancer, demonstrating that both uPA and PAI-1 were useful in predicting which women had breast cancer [8]. Specifically, high levels in breast nipple aspirate fluid (NAF) of uPA and PAI-1 significantly contributed to a model which predicted which women had breast cancer. In that study, we observed that uPA and PAI-1 are concentrated in NAF (a type of nipple discharge-ND, in addition to spontaneous ND, which can be physiologic or pathologic) compared to plasma [8]. Pathologic spontaneous ND (PND) comes forth from one but not the other breast nipple and generally the breast with PND harbors a benign or malignant tumor [9]. On the other hand, physiologic spontaneous ND is generally from both breasts and is not associated with cancer. Both NAF and PND can be obtained non-invasively and contain concentrated secreted proteins, carbohydrates and lipids from the breast ductal epithelium, the cells that give rise to cancer. This fact, as well as the fact that ND is breast specific, undiluted by the contribution from other organs, suggests that it may be a better physiologic fluid than plasma to identify breast cancer biomarkers.

In this study we demonstrate in a specific population of participants (women with a suspicious breast lesion which required biopsy to exclude cancer) that the expression of uPA and PAI-1 is altered in women with breast atypia and cancer. Since these markers were more predictive of disease in pre- than in post-menopausal women, and were not perfectly accurate in predicting the presence of disease in either menopausal group, we wondered if additional marker(s), ideally one or more that was especially predictive of postmenopausal disease, would improve the ability of uPA and PAI-1 to predict the presence of breast atypia or cancer.

We previously reported [9] that the Thomsen-Friedenreich (TF; Galactose-β-(1 → 3)-N-acetyl-D-galactosamine) antigen, which is displayed on cell-surface proteins and lipids in 70% to 90% of adenocarcinomas of the breast [10], is upregulated in post- (but not pre-menopausal) women with breast atypia and cancer, correctly classifying either cancer or abnormal vs. benign pathology 83% of the time in postmenopausal women. Because uPA and PAI-1 was also predictive in postmenopausal women, we hoped that it might increase the predictive ability of TF in these women. Because uPA was more predictive in premenopausal women, we hoped that it would increase the predictive ability in this group. We therefore evaluated all three markers in a matched subset of the 124 samples in which TF analysis was reported [9] and sufficient ND remained.

Carbohydrate biomarkers have not been investigated as extensively as proteins in NAF or other bodily fluids. Nonetheless, there have been studies dating back to at least 1989 in which the carbohydrate lactose was measured in [12]. Lactose, whose presence was felt to indicate evidence of breast secretory activity, was detected in 44.8% of the samples overall, declining from being detected in 100% of samples from women less than age 30 to being detected in 29% of samples from women ≥ 35 years old. A more recent study demonstrated that dietary intake of lactose was a strong predictor of the ability to collect NAF [13]. The measurement of TF antigen in NAF from 23 women with biopsy proven breast cancer using the galactose oxidase-Schiff (GOS) reaction was reported in 2004 [14]. The change in color with the reaction was significantly different between NAF from cancer containing vs. NAF from healthy contralateral breasts. We reported [9] levels of TF measured by direct immunoassay in 124 ND samples, including 52 from pre- and 72 from post-menopausal women with a suspicious breast lesion which required biopsy. Age alone in this cohort provided an AUC of .69. TF and age predicted the presence of atypia and cancer in the postmenopausal group with an AUC of .83. In an analysis of 79 ND samples (different than the 124) with similar enrollment criteria, uPA was highly predictive of breast cancer in premenopausal women (.83-.87), but less so in postmenopausal women. We then determined that two markers were better than one in predicting disease in all women, with TF + uPA having an AUC of .84-.92. When TF, uPA, and PAI-1 were combined, the AUC approached 1.0%.

We previously found that the expression of some ND cancer prediction markers varied based on whether or not the subject requiring surgery did or did not have PND [15]. On the other hand, TF concentration was not influenced by the presence or absence of PND [9]. We therefore determined if uPA and PAI-1 concentrations differed based on whether the sample was PND or NAF. Since we did not identify a concentration difference, all analyses included both PND and NAF samples.

We have three NAF biomarkers (TF, uPA and PAI-1) which together appear highly predictive of the presence of cancer or precancer in women requiring breast biopsy to exclude disease. The markers appear to be highly predictive of disease, regardless of whether or not PND was present. The NAF markers appear complementary, as two of the biomarkers (TF and PAI-1) are more predictive in post- than premenopausal women, whereas uPA is more predictive in premenopausal women.

The study had limitations. The first limitation of the study was the small sample size for the analysis of all three markers. Second, TF was analyzed at a different time than uPA and PAI-1. How much the age of the samples influenced marker expression is uncertain, though we did determine that uPA and PAI-1 expression was similar between the groups of 79 and 24 samples. A validation study in a larger sample size would help to determine the predictive ability of uPA, PAI-1 and TF would help to address these limitations.

In women requiring breast biopsy to exclude disease, combining ND biomarkers that were each predictive of the presence of atypia or cancer improved the prediction of both atypia and cancer, regardless of whether or not PND was present. A validation study is warranted to determine the clinical use of these noninvasive markers.