Histologic and cytologic review of biopsies confirmed the presence of breast cancer in 147 Senegalese patients. The mean age of subjects was 47.7 years, with a range of 13 to 77. Approximately half (47%) were menopausal, and only ten currently used any form of hormonal contraception. Mean gravidity was 5.8, with a range of zero to 19, and mean age of first pregnancy was 19.4 years. Few (3%) had any history of hormone replacement therapy, and less than 1% reported tobacco or alcohol use. Only eleven women (7%) had a history of obesity. Most of these women had advanced disease and large tumors, with only 33% of tumors being 5 cm or less in size, while 43% were 10 cm or greater (). Evidence of nodal involvement was present in 92% of cases, and a vast majority (80%) of the cancers were stage III and higher.
Clinical Characteristics of Tumors in Women with Confirmed Breast Cancer in Senegal (n=147). N (% with characteristic)
Expression of mammaglobin, GABAπ, B726P, and B305D in tissue and in blood
We first examined the expression frequency of the four genes in breast cancer biopsies. Using single gene real-time RT-PCR, mammaglobin expression was detected in 85% (39 of 46) cases (). GABAπ was detected in 52% of the tumors, but only when mammaglobin was also present. In two other cases, biopsy samples were positive for B726P, but not mammaglobin, resulting in 89% of tissues examined expressing at least one gene. Gene expression analysis of cells collected from blood of 84 women with breast cancer and 50 healthy female volunteers was then performed using the multigene RT-PCR assay. Mammaglobin expression was detected in 51 (61%), GABAπ was found in 18%, B726P in 5% and B305D in 8% of the blood samples. Overall, expression of one or more of these genes was present in 77% of samples from women with proven breast cancer, but none of the 50 samples from women without cancer.
The detection of Mammaglobin protein in sera and Gene expression in Tissue and Blood in Women with Confirmed Breast Cancer in Senegal (n=147). N (% with detection)
Associations of detection of gene transcripts in peripheral blood with subject and tumor characteristics
We next examined the associations between detection of gene transcripts in peripheral blood and tumor characteristics. Overall, among women with biopsy confirmed breast cancer, detection in peripheral blood of any of the four genes included in the multigene assay was not significantly associated with increasing tumor size (p=0.4) Likewise, detection of mammaglobin transcripts, alone, were not associated with tumor size (p=0.4). However, peripheral blood based detection of GABAπ was found to be associated with tumor size (p<0.001, test for trend) with no expression in the 30 small (2–5 cm) tumors, expression in 14% of medium (6–9 cm) tumors and in 40% of largest tumors 10 cm or greater. Mammaglobin (p=0.07) and GABAπ (p=0.05) were each marginally associated with increased nodal involvement; only 20% of those with N0 disease were positive for mammaglobin, compared to 60–70% of those with N1 or N2 disease. Similarly, 10% of women with N0 or N1, as compared to 29% of N2 had GABAπ detected in blood samples. In contrast, B726P detection was inversely associated with nodal involvement (p=0.01) as 20% of those who were N0 were B726P positive, compared to only 2% of those who were N1 and none of those who were N2 or N3. Interestingly, in the ten women with N0 cancer, six were positive in the multigene assay, including two positive for mammaglobin, two for B726P, one for B305D and one for GABAπ.
Lastly, detection of mammaglobin transcripts in the blood did not vary consistently with increasing tumor stage (p=0.4). In contrast, GABAπ was not detected in stage I, II, or IIIA cancers, but was expressed in blood samples from 29% of those with stage IIIB or IV disease (p=0.004, test for trend). Overall, the detection of the four gene transcripts was associated with stage of disease (p=0.03), being present in 53% of those with stage II, 82% of those with stage III, and 87% of those with stage IV breast cancer.
Demographic and behavioral characteristics were also examined in association with detection of gene transcripts in peripheral blood. Detection of mammaglobin or transcripts did not differ significantly by the age or gravidity of the patient, however, detection of transcripts of GABAπ was inversely associated with menopausal status, as 28% of premenopausal compared to 9% of postmenopausal women with breast cancer had transcripts of GABAπ detected in peripheral blood (p=0.02).
Detection of mammaglobin protein and/or transcripts of mammaglobin, GABAπ, B726P, and/or B305D in peripheral blood samples
Blood samples from 79 women with breast cancer could be analyzed for both the mammaglobin protein as well as detection of the transcripts from the four genes of interest. In these women, 66 (83.5%) had either elevated mammaglobin protein, or transcripts of one of the four genes detected in a single peripheral blood sample. Over half (59.5%) of the blood samples had mammaglobin transcripts detected, fourteen more had gene transcripts from one or more of B305D, GABAπ and B726P, and an additional five had elevated mammaglobin protein noted.
Associations of elevated mammaglobin serum protein with subject and tumor characteristics in women with breast cancer
Among the 142 women with confirmed breast cancer mammaglobin serum protein >1.706 ng/ml was marginally associated with increasing tumor size (p=0.09, test for trend), but not lymph node involvement (p=0.5). In addition, mammaglobin protein >1.706 ng/ml was marginally associated with increasing clinical stage of disease (p=0.10, test for trend), with only 29% of samples from women with stage I or II cancer being positive for mammaglobin protein, compared to 31% of those with stage IIIA, 34% of those with stage IIIB, and 48% of those with stage IV disease having elevated serum levels. However, among those with mammaglobin serum protein above the level selected as the upper limit of normal (>1.706 ng/ml, n=54), mammaglobin ELISA dilution values were predictive of disease severity. Mean natural log transformed ELISA dilution values in clinical stages I to IIIB were all between 0.9 – 1.4, but increased to 2.3 ng/ml in stage IV disease (p=0.02 in ANOVA, ). Similarly, mammaglobin ELISA dilution values were strongly association with increasing tumor size (p=0.001 in ANOVA), as the mean natural log transformed dilution values in tumors <10 cm was 1.2 compared to 2.3 in tumors 10 cm or larger.
Serum Mammaglobin concentration in control and breast cancer specimens.
Mammaglobin serum protein concentration was also associated with the detection of mammaglobin transcript in the blood by RT-PCR. In mammaglobin PCR-negative blood samples the mean ELISA value for circulating protein was 1.8 ng/ml compared to the mean ELISA value of 18.5 ng/ml in mammaglobin PCR-positive samples (p=0.01 in t-test). Mammaglobin ELISA values were also correlated to the log(copy numbers) of the multigene RT-PCR assay (p=0.02). In the patient population (n=40) with mammaglobin expressing tumors, mammaglobin ELISA values were associated with mammaglobin transcript detection in blood (p=0.008 in t-test). Mean ELISA values are 2.2 ng/ml in those without mammaglobin expression in blood, compared to 26.6 ng/ml in those MG RT-PCR positive.