A.A. Verstraeten

Survival in Early Breast Cancer Patients Is Favorably Influenced by a Natural Humoral Immune Response to Polymorphic Epithelial Mucin

PURPOSE Polymorphic epithelial mucin (PEM or MUC1) is being studied as a vaccine substrate for the immunotherapy of patients with adenocarcinoma. The present study analyzes the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relates the presence of these antibodies in pretreatment serum to outcome of disease. MATERIALS AND METHODS We measured immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to MUC1 with an enzyme-linked immunoassay (PEM.CIg), which uses a MUC1 triple-tandem repeat peptide conjugated to bovine serum albumin, in pretreatment serum samples obtained from 154 breast cancer patients (52 with stage I disease and 102 with stage II) and 302 controls. The median disease-specific survival time of breast cancer patients was 74 months (range, 15 to 118 months). A positive test result was defined as MUC1 IgG or IgM antibody levels equal to or greater than the corresponding rounded-up median results obtained in the total breast cancer population. RESULTS A positive test result for both MUC1 IgG and IgM antibodies in pretreatment serum was associated with a significant benefit in disease-specific survival in stage I and II (P =.0116) breast cancer patients. Positive IgG and IgM MUC1 antibody levels had significant additional prognostic value to stage (P =.0437) in multivariate analysis. Disease-free survival probability did not differ significantly. However, stage II patients who tested positive for MUC1 IgG and IgM antibody and who relapsed had predominantly local recurrences or contralateral disease, as opposed to recurrences at distant sites in the patients with a negative humoral response (P =.026). CONCLUSION Early breast cancer patients with a natural humoral response to MUC1 have a higher probability of freedom from distant failure and a better disease-specific survival. MUC1 antibodies may control hematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1-expressing tumor cells. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides in an adjuvant setting may favorably influence the outcome of disease.

Humoral immune response to polymorphic epithelial mucin (MUC-1) in patients with benign and malignant breast tumours

To investigate the clinical significance of an immune response to the MUC-1 encoded polymorphic epithelial mucin (PEM) breast cancer, circulating immune complexes containing PEM (PEM.CIC) were measured in sera from 96 healthy women, in pretreatment serum samples from 40 patients with benign breast tumours and from 140 patients with breast cancer and in serum samples from 61 breast cancer patients with recurrent or progressive disease. PEM.CIC were measured using a sandwich enzyme-linked immunoassay, and PEM serum levels were measured with CA 15.3 IRMA (Centocor Inc., Malvern, Pennsylvania, U.S.A.). Cut-off levels used for PEM.CIC and CA 15.3 were 120 Optical Density Units (O.D.) x 10(3) and 30 U/ml, respectively. In benign tumours, positivity for PEM.CIC was 37.5% (15/40). 36 of the 140 patients (25.7%) in the breast cancer pretreatment group had elevated PEM.CIC values. In patients with advanced metastatic disease, positivity for PEM.CIC was 18% (11/61). PEM.CIC was elevated in 32% (24/74) of node-negative patients, but only in 20% (12/59) of node-positive patients and absolute values were higher in node-negative patients (Mann-Whitney U test, two-tailed P = 0.0168). There was an inverse correlation between positivity for PEM.CIC and extent of disease: while 3 of the 6 patients with a carcinoma in situ were positive, only 1 of the 15 patients with more than five nodes involved had elevated levels of PEM.CIC. All 7 patients with distant metastases at first diagnosis were PEM.CIC negative. 28 out of 133 patients had a recurrence during the observation period (median 55 months, range 27-84 months). 23 of these 28 patients (82%) were PEM.CIC negative at the moment of first diagnosis. None of the patients with pretreatment elevation of both PEM.CIC and CA 15.3 (n = 13) relapsed. Our preliminary clinical results suggest that a humoral immune response to PEM protects against disease progression, and further support the idea of using synthetic peptides or glycopeptides containing the immunogenic core of the mucin as cancer vaccines.

Polymorphic epithelial mucin (MUC-1)-containing circulating immune complexes in carcinoma patients

Circulating immune complexes (CICs) containing polymorphic epithelial mucin (PEM/MUC-1) were found in sera of 24.5% of 151 primary breast carcinoma patients and 18-21.4% of patients with advanced ovarian (n = 56) and breast carcinomas (n = 61), 37% of patients with benign breast tumours, but in only 2.1% of 96 healthy individuals. The incorporation of PEM into CICs affects the detection of circulating PEM in commercial immunoassays such as the CA 15-3 assay, as suggested by a negative correlation between levels of PEM-containing immune complexes (PEM-CICs) and CA 15-3 values, and confirmed by isolation of PEM from CA 15-3-negative sera containing high levels of PEM-CICs. The amounts of PEM masked by human antibodies correspond to significant values of the CA 15-3 assay when monitoring patients for carcinoma. Most antibodies in PEM-CICs were of IgG class, suggesting their specific nature to the PEM epitopes.

Human MUC1 mucin: a multifaceted glycoprotein.

Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells. MUC1 is overexpressed and aberrantly glycosylated in carcinoma cells. The role MUC1 plays in cancer progression represents two sides of one coin: on the one hand, loss of polarity and overexpression of MUC1 in cancer cells interferes with cell adhesion and shields the tumor cell from immune recognition by the cellular arm of the immune system, thus favoring metastases; on the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses. Tumor-associated MUC1 has short carbohydrate sidechains and exposed epitopes on its peptide core; it gains access to the circulation and comes into contact with the immune system provoking humoral and cellular immune responses. Natural antibodies to MUC1 present in the circulation of cancer patients may be beneficial to the patient by restricting tumor growth and dissemination: early stage breast cancer patients with a humoral response to MUC1 have a better disease-specific survival. Several MUC1 peptide vaccines, differing in vectors, carrier proteins and adjuvants, have been tested in phase I clinical trials. They are capable of inducing predominantly humoral responses to the antigen, but evidence that these immune responses may be effective against the tumor in humans is still scarce.

Use of Serum Tumor Markers in the Differential Diagnosis between Ovarian and Colorectal Adenocarcinomas

In the search for a method to facilitate the preoperative discrimination of ovarian carcinomas from colorectal carcinomas serum levels of 6 tumor markers were measured in 47 patients presenting with ovarian cancer and compared to levels found in 24 female patients with advanced, untreated colorectal cancer. The markers studied were CA 125, CA 15.3, CA 19.9, CEA and two recently developed mucin markers, CA M29 and CA M26. Levels of CA 125, CA 15.3, CEA and CA M29 showed significant differences between both groups. In predicting ovarian cancer, sensitivity was highest for CA 125 at 94% (35 U/ml cutoff level). However, the specificity of CA 125 was at 71% low. Specificity increased significantly by using a combination of a CA 125-positive score (greater than 35 U/ml) and a simultaneous negative CEA score (less than or equal to 5 ng/ml) (specificity 100%, sensitivity 81%). A CA 125/CEA serum ratio greater than 25 resulted in the highest discriminative power with a specificity of 100% and a sensitivity of 91% resulting in an overall test accuracy of 94%. It is concluded that the serum tumor markers used, especially a combination of CA 125 and CEA, are helpful in the preoperative differential diagnosis between adenocarcinomas of ovarian and colorectal origin.

Maternal Serum CA125 and CA15-3 Antigen Levels in Normal and Pathological Pregnancy

Objective: To evaluate the value of maternal serum CA125 and CA15-3 concentrations for discriminating pathological from normal pregnancies. Methods: Serum samples from 120 women, in whom pregnancy outcome was pathological, i.e. spontaneous abortion, fetal death, intrauterine growth retardation, chromosomal and structural abnormalities, and (pre)eclampsia, were assessed for CA125 and CA15-3 and compared with levels found in 350 women with a normal pregnancy outcome matched for age and duration of pregnancy. Results: Maternal CA125 serum values were significantly higher in the first and the third trimester of pregnancy (median 23.0 and 21.0 U/ml; p < 0.00001 and p < 0.001, respectively), compared to those in the second trimester (median 14.0 U/ml), but not significantly different from those obtained in pathological pregnancies. Maternal serum CA15-3 values were significantly higher during the third trimester (median 26.0 U/ml) compared to the first and second trimester of pregnancy (median 14.0 and 15.0 U/ml; p < 0.0001); CA15-3 serum levels in normal and pathological pregnancies showed no significant difference. Conclusion: Maternal serum levels of CA125 are higher during the first and third trimester of pregnancy. CA15-3 maternal serum levels are higher during the third trimester compared to the first and second trimester. Maternal CA125 and CA15-3 serum levels showed no relation with a pathological outcome of pregnancy.

Cellular and humoral immune responses to MUC1 mucin and tandem-repeat peptides in ovarian cancer patients and controls.

Abstract The objective of this study was to demonstrate the presence of proliferative T cell responses to human polymorphic epithelial mucin (MUC1) and its tandem-repeat peptides in peripheral blood mononuclear cells (PBMC) from ovarian cancer patients and from controls and to correlate these cellular responses to a humoral response to MUC1. PBMC were obtained from 6 healthy women, from 13 women in the third trimester of pregnancy and from 21 ovarian cancer patients. Only 1 of the 6 healthy women showed a weak primary proliferative response (stimulation index, SI<2) to a 20-mer MUC1 tandem-repeat peptide in the presence of interleukin-2 (IL-2). In PBMC from 5/13 pregnant women (38%) a weak response could be induced by the 20-mer and/or 60-mer tandem-repeat peptides (SI ≤ 3.0) and in PBMC from 8/15 ovarian cancer patients (53%) 20-mer and/or 60-mer MUC1 tandem-repeat peptides induced primary responses (SI ≤ 5.4). MUC1 mucin purified from a breast tumor cell line and/or from urine of a healthy donor had a relatively strong stimulating effect (SI ≤ 19) on PBMC from 4 of 16 ovarian cancer patients (25%). In contrast, in PBMC of 9 ovarian cancer patients stimulated by the addition of a Candida albicans extract, MUC1 mucin strongly inhibited proliferation. This inhibition could partially be abrogated by the addition of IL-2. MUC1 (CA 15.3 assay) and free circulating MUC1 IgG and IgM antibodies (PEM.CIg assay) were determined in the plasma of all subjects. The MUC1 and the free circulating MUC1 IgG antibody plasma levels were significantly higher in the ovarian cancer patients than in the healthy women. Although no significant correlations were found between MUC1 mucin, MUC1 Ab plasma levels and the individual proliferative responses to the MUC1 antigens, an association may exist between them, since all three are significantly higher in the ovarian cancer patients than in the healthy women.

The Second Generation CA125 Assays

Optimal management of ovarian cancer patients can only be provided using the CA 125 serum test for treatment monitoring, early prediction of outcome and early detection of recurrence. The newly introduced second generation CA 125 assays, the Centocor CA 125 II IRMA, the Boehringer Mannheim Enzymun CA 125 II and the BYK Liamat CA 125 II are one-step heterologous double-determinant solid phase assays that utilize the M11 as capture antibody and the original OC 125 as tracer. The CA 125 II assays will probably replace the original CA 125 assays within a short period of time. For comparison reasons the Abbott IMx CA 125 assay was also included in this study.Highly similar CA 125 distribution patterns were obtained with these new CA 125 II assays. Linear regression analysis in ovarian cancer patients showed the following: Centocor CA 125 II = 0.98×CA 125 IRMA+ 10.7 (r = 0.8717, P<0.0001), Sy|x = 89.9; Enzymun CA 125 II = 1.03×CA 125 IRMA + 9.0 (r=0.8988, P<0.0001), Sy|x = 81.8; BYK Liamat CA 125 II = 1.17×CA 1...

Mucin-Like Carcinoma-Associated Antigen Serum Levels in Patients with Adenocarcinomas Originating from Ovary, Breast and Colon

The mucin-like carcinoma-associated antigen (MCA) enzyme immunoassay was tested in 962 healthy controls. MCA levels were compared with CA 125 in 70 patients with benign and 76 with malignant ovarian tumors. In addition MCA was compared with CA 15.3 in 58 patients with breast cancer and with CEA in 50 patients with colon carcinoma. In healthy controls the 95th percentile cutoff of 19.2 U/ml appeared to be higher than generally used. With the common cutoff value of 14 U/ml, a 38% sensitivity and 100% specificity was reached in malignant versus benign ovarian tumors. In colorectal cancer only 4% of patients had elevated MCA serum levels (CEA: 50%). In breast cancer patients the MCA assay performed better than CA 15.3 although only 17.2% showed elevated levels (CA 15.3: 7.4%). Thus MCA seems to be of limited value in the diagnosis and follow-up of adenocarcinomas of breast, ovary or colon.

Discordant serum CA 125 values in commercial immunoassays

The CA 125 assay is from a clinical point of view very well suited to monitor ovarian cancer patients. Although most commercial assay systems include as standards CA 125 preparations derived from the OVCA 433 cell line, the various assay configurations do not produce identical results. The so called cut-off value of 35 U/ml obtained in the original Centocor CA 125 assay appears to be equivalent to CA 125 values ranging as low as 18 U/ml and as high as 53 U/ml in other commercial CA 125 assays now available. Because of these dissimilar results, obtained with different immunoassays, switching from one assay to another during follow-up, should not be tolerated. The newly introduced second generation Centocor CA 125 II IRMA was shown to retain the cut-off values of 35 U/ml and 65 U/ml as defined with the original CA 125 IRMA.