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Background: Recent observations showed that systemic immune changes are detectable in case of breast cancer (BC). In this preliminary study, we investigated routinely measured peripheral blood (PB) parameters for malignant BC cases in comparison to benign breast conditions. Complete blood count, circulating lymphoid subpopulation, and serological carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3) levels were considered., Methods: A total of 127 female patients affected by malignant (n = 77, mean age = 63 years, min = 36, max = 90) BC at diagnosis (naïve patients) or benign breast conditions (n = 50, mean age = 33 years, min = 18, max = 60) were included in this study. For each patient, complete blood count and lymphoid subpopulations (T-helper, T-cytotoxic, B-, NK-, and NKT-cells) analysis on PB samples were performed. Hormonal receptor status, Ki-67 expression, and serological CEA and CA15-3 levels were assessed in the case of patients with malignant BC via statistical analysis., Results: Women with malignant BC disclosed increased circulating T-helper lymphocytes and CD4/CD8 ratio in PB when compared to those affected by benign breast conditions (2.345 vs 1.894, P < .05 Wilcoxon rank-sum test). In the case of malignant BC patients, additive logistic regression method was able to identify malignant BC cases with increased CA15-3 levels (CA15-3 >25 UI/mL) via the hematocrit and neutrophils/lymphocytes ratio values. Moreover, in the case of women with aggressive malignant BC featured by high levels of Ki-67 proliferation marker, an increasing number of correlations were found among blood count parameters and lymphocytes subpopulations by performing a Spearman's correlation analysis., Conclusions: This preliminary study confirms the ability of malignant BC to determine systemic modifications. The stratification of malignant BC cases according to the Ki-67 proliferation marker highlighted increasing detectable alterations in the periphery of women with aggressive BC. The advent of novel and more sensitive biomarkers, as well as deep immunophenotyping technologies, will provide additional insights for describing the relationship between tumor onset and peripheral alterations.

Aims: The main objective of this work was to evaluate the influence of end-stage renal disease (ESRD) on concentrations of five tumor markers (TMs): carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 (CA19-9), CA15-3, CA125, and prostate specific antigen (PSA) in a group of chronic hemodialysis patients (CHPs); and to study the influence of hemodialysis (HD) sessions on concentrations of the same TMs., Methods: We compared TMs levels in CHP before HD sessions to a control group of 50 healthy volunteers, the dosages were determined before and immediately after the HD session Comparisons were made before and after correction for dialysis-induced hemoconcentrations., Results: We enrolled 74 CHPs, all TM concentrations were higher in this group compared to control group, but this increase was significant for CEA (4.25 ± 2.89 vs 2.41 ± 1.81ng/ml; p<0.0001), CA125 (27.84 ± 92.27 vs 13.30 ± 9.85 ng/ml; p = 0.048) and CA19-9 (19.65 ± 25.02 vs 10.23 ± 11.00 U/ml; p = 0.011). Post-dialysis levels were significantly higher than those in pre-dialysis. CEA (3.35 [2,46-5.51] vs 4,06 [2.60-6.78] ng/ml; p<0.0001), CA125 (13.24 [9.66-18.63] vs 16.01 [11.33-22.53] ng/ml; p<0.0001), CA19-9 (12.29 [5.59-21.97] vs 16.29 [7.18-24.7] U/ml; p<0.0001), CA15-3 (13.06 [10.05-17.48] vs 14.58 [11.72-19.35] ng/ml; p<0.0001 and PSA (0.83 [0.5-1.24] vs 1.06 [0.62-1.43] ng/ml; p<0.0001)., Conclusions: Our work confirms that HD increases concentrations of the five TMs evaluated and suggests that the use of CA15-3 and PSA remains valid in CHPs since their concentrations were not altered by ESRD, unlike CEA, CA125, and CA19-9.

The aim of this study was to elucidate whether the IgG humoral immune response to breast cancer cells is directed to the aberrant mucin-1 (MUC1) associated to this type of cancer. To this aim, an adaptation of immunohistochemistry (IHC) was performed on samples of 45 breast cancer tissues, 12 benign disease tissues, and 31 normal tissues, incubated with matched serum samples from the same patients. Each serum sample was also incubated, with a modified immunocytochemistry (ICC), with MCF7 cells. In both techniques, serum was employed instead of the primary antibody. In the case of IHC, the reactivity with sera diminished when added after previous incubation of the tumor/tissue with an anti-MUC1 mAb; the reduction in reactivity was: from 93% to 44% in breast cancer tissues, and from 100% to 67% in benign disease tissues. The reactivity of normal samples (36%) remained unchanged. In the case of ICC, the reactivity with sera decreased after incubation with anti-MUC1 mAb from 71% to 16% in breast cancer tissues, from 83% to 0% in benign disease tissues, and from 52% to 10% in normal serum samples. These results were confirmed employing siRNA MUC1 transient gene knockdown. By Western blot analysis -
after immunoprecipitation (IP) of the circulating MUC1- and ELISA, the TF antigen was detected in circulating MUC1 in all breast cancer and benign samples while Tn was detected in 38% of the samples.
The existence of IgG autoantibodies against aberrantly glycosylated MUC1 may have a protective role and may contribute to a better prognosis in some patients. Enhancement of this natural immune response may constitute an alternative therapeutic strategy.

Introduction: Cancer antigen 15-3 (CA 15-3) is the most widely used serum marker in diagnosing and monitoring breast cancer. The aim of this work was to analyze preoperative CA 15-3 serum levels in patients with ductal breast carcinoma in relation to 1) clinicopathological parameters, 2) hormone receptors, and 3) tissue-based tumor markers., Methods: A group of 340 women with infiltrating ductal carcinoma of the breast who had undergone no prior treatment was studied. These women ranged in age between 27 and 83 years (mean age 61.5±9.9 years). Preoperative CA 15-3 serum levels were determined by an immunoradiometric method. Hormone receptors (estrogen, progesterone and androgen receptors), p53, bcl-2 and Ki67 were determined by different immunohistochemical methods. Epidermal growth factor receptor (EGFR) was analyzed in the cell membranes by radioligand assay whereas cathepsin D and pS2 were determined by immunoradiometric analysis. Tumor ploidy and S-phase fraction were studied by flow cytometry., Results: CA 15-3 serum levels were higher in postmenopausal women (p=0.032), in patients with tumors exceeding 2 cm (p=0.003), lymph node involvement (p=0.026), distant metastases (M1) (p<0.0001), S-phase fraction <7% (p=0.015), EGFR <6 fmol/mg protein (p=0.025), and cathepsin D <50 pmol/mg protein (p=0.023)., Conclusions: Preoperative CA 15-3 serum levels were associated with cell proliferation determined by the S-phase fraction, the concentration of cathepsin D in the cytosol, and the EGFR concentration in the cell membrane.

Up to 80% of breast cancer patients developing metastases have high levels of CA 15.3. We studied the prognostic implications of CA 15.3 kinetics in 119 patients before and at first metastasis by univariate and multivariate statistics. At first metastasis, CA 15.3 was elevated in 82.4% of patients, with a lead time (median 162 days) in 42.0% of them. Kaplan-Meier analysis showed overall survival (median 1477 days) to be significantly related to estrogen receptor (ER) and progesterone receptor (PgR) status (p=0.0001) and tumor size (p=0.025). The interval between diagnosis and first abnormal CA 15.3 (p=0.0001), the CA 15.3 concentration (p=0.013), and the presence or absence of a lead time (p=0.001) also had prognostic value. ER and PgR status (p=0.0005 and p=0.0103, respectively), metastasis-free interval (p=0.0003), existence of a CA 15.3 lead time (p=0.0028), and days from diagnosis to first abnormal CA 15.3 (p=0.0055) entered in the Cox model. After first metastasis (median survival 573 days), ER and PgR status (p=0.0001 and p=0.0004, respectively), existence of a lead time for CA 15.3 (p=0.0138), and the concentration of first abnormal CA 15.3 (p=0.0145) had individual prognostic value. In the Cox model ER status (p=0.0001), nodal status (p=0.0191), existence of a lead time for CA 15.3 (p=0.0033), days from diagnosis to first abnormal CA 15.3 (p=0.0132), and concentration of first abnormal CA 15.3 (p=0.0320) were found to be independent prognostic variables. Compared to a matched historical control group that was not monitored by CA 15.3 assaying (n=140), the study group had a significantly longer survival after the first metastasis (p=0.0005). In conclusion, the kinetics of CA 15.3 before the first metastasis is of prognostic value. When associated with 18-fluorodeoxyglucose imaging, serial CA 15.3 assays may help to implement early treatment of metastases.

CA 15-3 has been most widely used as a serum tumor marker in follow-up and detection of breast cancer recurrence. In this study we have specifically focused upon the prognostic implications and utility of preoperative CA 15-3 levels. We have identified on our database 414 patients with breast cancer in whom serial levels of the serum tumor marker CA 15-3 had been determined at diagnosis and follow-up. We have analyzed the follow-up and clinical outcomes in these patients and from this data we have assessed the potential of CA 15-3 as a predictor of five-year overall and disease-free survival. Our results show that an initially elevated CA 15-3 level is associated with a very poor prognosis in both early and late stage disease. Elevated pre-biopsy CA 15-3 levels are associated with 14% five-year disease-free survival rates and 17% overall survival rates at five years. In contrast, normal CA 15-3 levels are associated with 47% five-year disease-free survival rates and 54% overall survival rates at five years (p<0.01). Comparison of five-year survival rates between patients with elevated and normal CA 15-3 levels in early breast cancer (stage I and II) also showed significant differences, with survival being 41% and 75%, respectively (p<0.01).

CA 15-3 (also known as MUC1) is the most widely used serum marker in breast cancer. MUC1 is a large transmembrane glycoprotein which is frequently overexpressed and aberrantly glycosylated in cancer. Physiologically, MUC1 appears to play a role in cell adhesion and the high levels present in cancer may be causally involved in metastasis. At present the main uses of CA 15-3 are in preclinically detecting recurrent breast cancer and monitoring the treatment of patients with advanced breast cancer. In a prospective study of 368 patients we show that patients with high preoperative levels of CA 15-3 (>30.4 U/mL) had a worse outcome than patients with low levels of the marker. In multivariate analysis CA 15-3 as a prognostic marker was independent of both tumor size and nodal status. Furthermore, in multivariate analysis the prognostic impact of CA 15-3 was stronger than that of tumor size and at least as strong as nodal status. CA 15-3 may thus be the first independent prognostic serum marker in breast cancer.

Background: Angiogenesis is a prerequisite for tumor growth and metastasis. Vascular cell adhesion molecule1 (VCAM-1) is expressed on endothelial cells as a result of vascular endothelial growth factor (VEGF) stimulation., Purpose: To determine if measurement in serum of VEGF or VCAM-1 provides an accurate measure of tumor angiogenesis., Methods: VCAM-1 and VEGF were measured in the serum of women with early and advanced breast cancer by ELISA. Levels were compared to levels of VCAM-1 and VEGF in women with normal breasts and levels of the endothelial glycoprotein von Willebrand factor. Levels of VEGF and VCAM-1 in women with early breast cancer were correlated with established clinicopathological prognostic markers and intratumoral microvessel density (IMD)., Results: In early breast cancer serum VCAM-1 correlated closely with the microvessel density in tumors (r=0.61, p<0.001). Women with lymph node-positive and high-grade tumors had higher levels of serum VCAM-1 than women with lymph node-negative and low-grade tumors. Serum VEGF demonstrated no correlation with established prognostic features or IMD. Levels of VCAM-1 and VEGF were raised in women with advanced breast cancer., Conclusion: Serum VCAM-1 is a surrogate marker of angiogenesis in breast cancer and its measurement may help in the assessment of antiangiogenic drugs currently in phase II trials.

In this prospective study the correlation of pathological with biological prognostic factors and serum tumor markers has been investigated in 574 patients with primary invasive breast cancer. The p53 protein and Bax level correlated positively with tumor size, lymph node status and histological grade. The serum levels of CEA, CA 15.3, TPA-M and TK correlated with tumor extent. There was a significant difference between pre- and postmenopausal breast cancer patients in serum levels of TPA-M and cytosol levels of Bax. Whether these correlations can help in predicting the prognosis of breast cancer by providing additional information with respect to the conventional factors, will have to be investigated by several years of careful clinical follow-up.

TPA and CA 15.3 concentrations were routinely determined in serum of patients treated for breast cancer during a 15-month period. ROC curves did not show differences in the ability to differentiate between NED and PD on the basis of matching tumor marker values. During monitoring of patients with NED, TPA levels showed fluctuations of more than 25% that were not disease related. We concluded that CA 15.3 is a more slowly reacting marker of tumor burden than TPA, which is an immediate indicator of cell turnover.

TPS, CA 15-3 and CEA were determined in metastatic breast cancer patients during treatment. After six months of follow-up the patients were divided into four groups according to the UICC criteria for treatment response. Forty-six patients with a more favorable prognosis (complete remission, partial remission or stable disease) were followed for an extended period. In 30 of the 46 patients at least one marker had increased at the end of the six-month period by at least 25% (TPS in 54%, CA 15-3 in 20%, CEA in 20%). All these 30 patients subsequently developed progression. The prognostic sensitivity was 83%, 30% and 30%, respectively, for TPS, CA 15-3 and CEA. The combination of TPS and CA 15-3 showed a sensitivity of 96%. The median lead time was about 8 months for TPS and CA 15-3, but less than 50% of the patients showed a lead time for CA 15-3 as compared to TPS. We conclude that TPS and CA 15-3 determinations are helpful for the prediction of progression during the follow-up of breast cancer patients.

Serum CA 15.3, CEA and ESR were longitudinally determined in 298 patients with breast cancer during postsurgical follow-up and/or therapy. Observation lasted until the death of the patient or at least for three years. With regards to longitudinal serum markers and ESR curves, four different patterns have been identified: pattern I: the markers and ESR stayed at normal levels; pattern II: the markers and ESR decreased from a peak level; pattern III: the markers and ESR fluctuated widely; pattern IV: the markers and ESR increased steadily. We have looked at over all survival (OS) and relapse-free survival ( RFS) versus longitudinal CA 15.3, CEA and ESR patterns. Univariate Cox regression analysis showed that OS and RFS were significantly associated with all four patterns.

By means of the retrospective study of the clinical records of 158 women followed for breast cancer, we aimed to evaluate the consequences of a non-systematic indication for bone scan (BS) based either on CA 15-3 levels alone or a combination of tumor marker levels and clinical criteria. With the first option, the negative predictive value was 95% and 82% of the BS would have been avoided. With the second option, the negative predictive value was 97% and 59% of the BS would have been avoided. Furthermore, the preliminary results of a longitudinal study showed that those patients with normal CA 15-3 levels and positive bone scans showed a subsequent rise in CA 15-3 levels which frequently became elevated with a average delay of 15 months. Omission of systematic bone scans in the follow-up of breast cancer patients is likely to lead to a delay in the diagnosis of bone metastasis in 3% to 5%, the consequences of which have to be examined carefully.

The aim of this study was to investigate whether immunoassays for circulating MUC1 antigen in breast cancer could be compressed in time so that serum level results would be made available during the time of the patient's visit to clinic. Two assays were used: -The EMCA (Euro DPC) is a liquid phase immunoassay and the ELSA CA15-3 (CIS) is a double determinant solid phase immunoradiometric assay. The effects of shortened incubation times were investigated by assaying standards and unknown samples and comparing the results with those using the standard kit protocols. The binding kinetics of the monoclonal antibodies employed in the assays were analysed separately. We conclude that the EMCA assay can be shortened to 35 min and we have attributed this to the fast binding kinetics inherent in a liquid phase assay. This shortened assay may produce the basis for a useful "near patient" assay. By comparison, the solid phase ELSA CA15-3 assay cannot be compressed without loss in assay performance.

The Prostate-Specific Antigen (PSA) and the Cancer-Associated Serum Antigen (CASA) assay for the MUC1 mucin were compared in the serum of 303 patients with malignant or benign prostatic disease. Using cutpoints of 4, 10, and 20 micrograms/l, PSA was elevated in 93%, 81%, and 64% of patients with prostate cancer (n = 113), with corresponding specificities of 55%, 84%, and 96% in benign prostate disease (prostatic hyperplasia or prostatitis, n = 190). Using the recommended cutpoint of 4 Units/ml, CASA was elevated in 38% of patients with prostate cancer, with a specificity of 91% in benign disease. PSA and CASA showed a poor correlation in prostate cancer (r = 0.367) and benign disease (r = 0.158), and CASA was elevated in some PSA negative samples. Used together, PSA > or = 20 micrograms/l and CASA > or = 4 kU/l gave perfect specificity in benign disease, with a corresponding sensitivity of 29% (positive and negative predictive values of 100% and 70%, respectively). However, this combination gave no improvement over the use of PSA alone, with sensitivity 47% when the cutpoint was raised to give perfect specificity. These data suggest that CASA is of little use as an adjunct to PSA in the differentiation of benign and malignant prostate disease.

Immunoradiometric determination of the bone isoenzyme of alkaline phosphatase with a method provided by Hybritech Inc., San Diego CA (USA) was carried out in 145 female patients, 97 of whom with radically operated breast cancer and 48 with benign mammary cysts, in order to evaluate the correlation of serum levels with the metabolic process of bone rearrangement in patients with bone metastases. This study shows that skeletal ALP, having high specificity (86.48%) and sensitivity (78.6%) for early progression (the average anticipation time compared to scintigraphic detection was 101 days) could represent a valid marker for bone metastases in association with mucinous markers in the follow-up of patients operated for breast cancer. In addition, dynamic serum determination of skeletal ALP could be a valid help in monitoring the efficacy of therapy in patients with bone progression.

In order to verify the efficiency of the tumor markers CA 15.3 and CA 549 in the follow-up of breast cancer patients, it was necessary first to check the cutoff levels of each tumor marker in women with an increased age-related risk, but with no evidence of disease. From 132 serum samples in this age group, we confirmed the CA 549 cutoff level of 12.1 U/ml. However, the cutoff of CA 15.3 was 34 U/ml, which is higher than previously reported in the literature. Fifty-two breast cancer patients with or without metastases at the time of entry into the study were followed for 2 to 3 years with both tumor markers. The sensitivity, specificity and the test efficiency for the presence of metastases were analyzed with each tumor marker. Taking into account the different cutoff levels, we concluded that both tumor markers can be used independently to follow the clinical situation of patients. In several cases an increase in both tumor markers was observed before a clinical diagnosis of metastases could be made. Combination of these two tumor markers gave no more significant information about the patient's clinical situation than each tumor marker alone.

The value of tumour-associated antigens CEA and CA 15-3 was studied in patients with breast cancer over a 4-year period. A total of 252 patients with primary or recurrent disease had available and corresponding CEA and CA 15-3 values at diagnosis and during follow-up and were studied in detail. Preoperative and three-monthly serial postoperative levels were measured in each patient. Ten of 11 patients presenting with primary and concurrent metastatic disease had elevated CA 15-3 levels (> 25 I.U./ml) as compared to 6 with CEA (> 5 ng/ml). Fourty-seven patients developed locoregional recurrence of which 15 had concurrent metastatic disease. CA 15-3 was elevated in 14 cases while CEA in 11. Of 32 patients with locoregional recurrence alone, 18 later developed metastatic disease at a mean follow-up time of 17.5 months. There was a significant correlation between CA 15-3 value at locoregional recurrence and time to subsequent metastasis (r = 0.-0.57, P = 0.0133). CEA was elevated in 64%, CA 15-3 in 87% and either marker in 94% of 87 patients diagnosed with metastatic disease. Of 53 patients with serial markers and metastatic disease, 72% (38/53) had rising CA 15-3 levels prior to diagnosis with a mean lead time of 9.9 months. Use of CEA in conjunction improved lead time detection to 83%. This study demonstrates that CA 15-3 is superior to CEA at detecting metastatic disease at initial presentation and during follow-up. Use of CEA in conjunction with CA 15-3 improves the detection of systemic disease.

Serum levels of CEA, CA 15.3 and CA 27.29 were measured during the follow-up of 499 breast cancer patients. Studies included three different groups of women: 82 blood donors free of disease, 42 patients with non-malignant breast diseases and 499 breast cancer patients. After the determination of cut-off values, serum levels of tumor markers did not show significant elevations in benign breast diseases. On the basis of our results CA 15.3 (sensitivity = 57%; accuracy = 87%) was the most effective marker, CA 27.29 (sensitivity = 62%; accuracy = 83%) was the most sensitive and CEA (sensitivity = 45%; accuracy = 81%) was the least sensitive and effective marker. The combined use of markers was evaluated by step-wise logistic regression analysis. The regression coefficients showed that CA 15.3 (coeff. = 2.97) and CA 27.29 (coeff. = 1.46) were suitable for the detection of possible metastases during follow-up. Finally, we studied the relationship between pT, pN, pM and circulating levels of CA 15.3 and CA 27.29.

In the present investigation we evaluated the variability of tumor marker levels in the follow-up of patients without evidence of disease after resection of primary breast cancer. CEA and CA15.3 were measured using commercially available methods in serial blood samples collected from 170 patients. The coefficient of variation among all samples from each patient, which accounts for the total variability (analytical variability+biological variability), was widely scattered (from 4 to 99% for CEA; from 4 to 52% for CA15.3). The critical difference was calculated using the formula designed by Fraser [CD = 2.77. (CVa2 + CVb2)1/2]. It ranged from 11 to 276 for CEA and from 11 to 144 for CA15.3. From the present findings we conclude that: 1) it is possible to identify individually tailored decision criteria to evaluate tumor marker variations in the follow-up of breast cancer patients; 2) in a considerable number of cases the non-tumor-related variability is too high to allow the early identification of minor tumor marker variations that are of clinical relevance.

Two specific monoclonal antibodies for breast tissue (DF3 and MCAb-12) and the corresponding tumor markers CA15-3 and MCA in serum have been evaluated in 50 patients with breast cancer and in 15 controls. The expression of these antigens in tissue was poorly correlated with the common prognostic parameters. Their presence in serum was associated with an altered distribution of the antigens in the cell. The expression of these antigens in tissue enables us to select patients for serological follow-up and to evaluate tumor differentiation from a functional point of view.