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.

Normal human skin demonstrates marked site-variation of tenascin expression, not correlated to epidermal proliferation (Ki-67 binding).

In disorders of the skin characterized by epidermal hyperproliferation, it has been demonstrated that the expression of tenascin in the dermis is markedly increased. In normal dermis, however, tenascin is slightly expressed in the upper dermis beneath the basal membrane. Using an immunohistochemical approach, tenascin expression (T2H5 binding) and recruitment of cycling epidermal cells (nuclear binding to Ki-67) were studied in normal skin at various localizations of the body surface. Whereas recruitment of cycling epidermal cells did not show a significant body-site variation, tenascin expression was most pronounced at the extensor surface of the lower arm. In normal skin, no significant correlation was observed between both phenomena in striking contrast to the well-established correlation in hyperproliferative skin conditions.

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.

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.

The quality of intrapartum fetal heart rate monitoring.

OBJECTIVE: To determine the quality of fetal heart rate (FHR) recordings during the first and second stage of labor by quantifying the amount of fetal signal loss in relation to the method of monitoring: external ultrasound or directly via a scalp electrode. STUDY DESIGN: Analysis of 239 intrapartum recordings stored between 1 January 2001 and 1 July 2001 from consecutive deliveries at the Vrije Universiteit Medical Center in Amsterdam. Singletons delivered via the vaginal route were included in the study. FHR recordings had duration of at least 1h prior to birth of the infant. Subdivision in three groups took place on the basis of the recording technique which had been used; i.e. ultrasound, scalp electrode or a combination of both methods. FHR data was obtained using HP-M1350 cardiotocographs. The status (pen on, pen off, maternal signal) and the mode of the signals were acquired. The duration of pen lifts and maternal signals was divided by the total duration of the recording. Statistical analyses were performed with the Mann-Whitney U-test and the Wilcoxon signed ranks test. RESULTS: Recordings obtained via ultrasound demonstrated significantly more fetal signal loss than those obtained via the direct mode, particularly in the second stage. The FIGO criteria for fetal signal loss with external ultrasound were not fulfilled during this stage for about half the cases. CONCLUSION: Intrapartum FHR monitoring via a scalp electrode provides far better quality FHR signals than external ultrasound and deserves a more prominent position in fetal surveillance than it currently has.

Quantitation of polymorphic epithelial mucin: a challenge for biochemists and immunologists.

Polymorphic epithelial mucin (PEM) is a heavily glycosylated protein present at the apical surface of glandular epithelial cells which is shed into the lumen of epithelial tissue. In carcinomas cell polarisation is lost, PEM is overexpressed and found on the entire cell surface. High amounts of PEM are shed into the circulation of cancer patients. CA 15.3 is the first commercial assay for the detection of PEM. After roughly one decade of use in clinical practice it is considered valuable for breast cancer therapy monitoring and, in the follow up, for early detection of metastatic disease. The extreme polymorphism of this molecule, with its varying number of multiple epitopes and tremendous variation in glycosylation which can mask catcher/tracer epitopes, impairs its precise measurement. A further impediment is complex formation with autoantibodies, as revealed by a recently developed assay.

Genetic transmission of mammary tumour virus in the DBAf mouse strain.

MTV antigens were demonstrable by radioimmunoassay in milk samples from individual DBAf mice, and in samples from (male BALB/c X female DBAf) F1 mice. Although some samples collected during the first lactation periods of these mice were virus-negative, all samples of later lactation periods were virus-positive. From 75 mice of the [ male BALB/c X female (male BALB/c X female DBAf)]Bc I population, milk samples were collected during one or more lactation periods and tested for the presence of viral antigens; the samples of 42 mice were virus-positive. In the ([BALB/c X (BALB/c X DBAf)] X BALB/c)Bc II population two groups were distinguished. In the first group, the progeny of virus-positive Bc I mothers, 37 out of 62 mice had detectable levels of viral antigen in the milk. None of the 43 samples from mice of the second group, derived from MTV-negative Bc I females, were virus-positive. These data suggest that the presence of viral antigens in the milk of DBAf mice is controlled by a single dominant gene; evidence for linkage of this gene and the albino locus was obtained (recombination percentage: 20).

Studies of genetic transmission of mammary tumour virus by C3Hf mice.

By radioimmunoassay (RIA) mammary tumour virus (MTV) antigens were detected in individual milk samples of C3Hf mice, (female BALB/c X male C3Hf)F1 mice and (female C3Hf X male BALB/c)F1 mice; milk samples of BALB/c mice were negative. In the segregating backcross I population, female BALB/c X male (female BALB/c X male C3Hf) viral antigens were found in the milk of 93 out of 169 mice (55%). In the Bc II population (daughters of Bc I mothers and BALB/c fathers) two groups were distinguished. In the first group, derived from positive Bc I mothers, 55 out of 110 mice (50%) had detectable levels of viral antigens in the milk. In the second group, progeny of negative Bc I mothers, 1 mouse out of 47 was positive. These data are consistent with the assumption that one dominant gene is responsible for the presence of viral antigens in the milk of C3Hf mice. This gene (Mtv-1) seems to be linked with the albino locus situated on chromosome 7; the recombination percentage was about 29. In the first experiment with Bc I mice a significant difference was found between the tumour ages of the mice with virus-positive milk and of the mice with virus-negative milk: all mice (18) with viral antigens in the milk developed mammary tumours at an age ranging from 7 to 18 months, whereas in only 7 out of 16 mice with virus-negative milk were mammary tumours found before the age of 21 months. Viral antigens were detectable (by RIA) in the tumours of mice of both subgroups; however, the amounts (mU/mg tumour) were significantly lower in the tumours derived from mice with virus-negative milk. Although MTV-L of C3Hf mothers could be transmitted to BALB/c mice by foster-nursing, viral antigens could not be detected in milk samples collected prior to the third lactation period; thus an influence on the data of extrachromosomally transmitted MTV-L is unlikely.

Genetic transmission of mammary tumour virus by GR mice.

By immunodiffusion assay (ID-test) milk samples of mice of several strains and of F1-hybrids of the GR strain were tested for the presence of mammary tumour virus (MTV) antigens. The results clearly demonstrated that the presence of viral antigens in the milk of the first lactation period is restricted to mice harbouring endogenous MTV-GR. Viral antigens were detectable in about 50% of the milk samples collected during the first (occasionally the second) lactation periods of mice of the segregating backcross I (Bc I) populations: DBAfX(DBAfXGR), AKRX(AKRXGR), BALB/cX(BALB/cXGR) and C57BLX(C57BLXGR), indicating that one dominant gene is responsible for the presence of viral antigens in the milk of GR mice. The proposed gene symbol is Mtv-2. Milk samples from female mice of three different Bc II populations were tested for the occurrence of viral antigens. In the first Bc II: [BALB/cX(BALB/cXGR)]XBALB/c 33 out of 51 mice, descending from ID-positive mothers, had ID-positive milk and only one out of 71 mice, which were the progeny of ID-NEGATIVE Bc I mothers, was ID-positive. These results may be influenced by the MTV transmitted extrachromosomally via the milk of the mother. The two other Bx II populations were derived from Bc I fathers, either [BALB/cX(BALB/cXGR)] or [(BALB/cXGR)XBALB/c] f and BALB/c females. The results obtained with these Bc II populations suggested that 6 Bc I fathers were heterozygous for Mtv-2. Since the segregation ratio (60:29) in the Bc II population (progeny of these 6 Bc I male) deviates significantly from the expected 1:1 ratio, one may assume extrachromosomal transmission of MTV via the seminal fluid of the father to the progeny. A close correlation was found between the presence of MTV antigens in the milk and the occurrence of both early mammary tumours after hormone treatment and spontaneous mammary tumours before the age of 13 months. These results suggest that the early appearance of mammary tumours in the GR strain and the early expression of MTV antigens in the milk appear to be controlled by the same genetic factors.

The second generation CA 125 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 heterlogous 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 Abbot 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 x CA 125 IRMA + 10.7 (r = 0.8717, P < 0.0001), Syx = 89.9; Enzymun CA 125 II = 1.03 x CA 125 + 9.0 (r = 0.8988, P < 0.0001) Syx = 81.8; BYK Liamat CA 125 II = 1.17 x CA 125 IRMA + 0.6 (r = 0.8930, P < 0.0001), Syx = 96.8. Our first technical and clinical evaluation of these three new CA 125 II assays shows their superior analytical performance, in addition to a high qualitative and quantitative correlation with the original CA 125 IRMA.

Quantitative estimation of mouse mammary tumor virus (MTV) antigens by radioimmunoassay;.

A radioimmunoassay for the antigens of the mouse mammary tumour virus has been developed. -125Iodine-labelling of intact virus [derived from mammary tumours of (C3H TIMES 020)F1 mice] followed by ether extraction and separation of the ether and water layers resulted in a separation of the labelled material into two main groups of antigenic components. The intact labelled material from each group was separated from viral debris and other possible contaminants by affinity chromatography. The antigens of the ether phase were proved to belong mainly to the viral nucleoid whereas the water phase contained mainly envelope antigens. No type-specific antigens could be demonstrated in either of the phases. Radioimmunological assessment of plasma revealed that in the plasma of MTV-S- and MTV-P-positive animals viral antigens were only measurable when palpable mammary tumours were present, whereas in MTV-L-positive tumour-bearing animals some negative samples were found. In milk of individual nontumour-bearing mice a close correlation was found between the expression of viral antigens and the generally accepted presence of virus in the strain of mice. In milk, viral antigen levels tend to increase with parity with a possible decrease after a finite number of pregnancies. In a pilot study the presence of MTV antigens could also be demonstrated in epididymis extracts of male GRS/A mice. Genetical analysis of the low-virulent MTV-L by radioimmunoassay of milk is in progress.

Expression of tenascin, biglycan and decorin in disorders of keratinization.

The distribution of three (recently discovered) extracellular matrix components (tenascin, biglycan and decorin) was studied in normal adult human skin and in a number of monogenic disorders of keratinization, using immunohistology. The expression of tenascin, which is sparsely distributed in normal human dermis, was found to be grossly increased in epidermolytic hyperkeratoses and in Darier's disease. Tenascin expression in three types of ichthyosis (X-linked recessive ichthyosis, autosomal dominant ichthyosis vulgaris, non-erythrodermic lamellar ichthyosis) was similar to that of normal skin. The presence of biglycan and decorin did not show a marked variation between the different disorders studied, suggesting that their expression is subject to regulatory mechanisms distinct from those of tenascin. The increased expression of tenascin in two disorders of keratinization with a hyperproliferative phenotype, lends further support to the hypothesis that dermal tenascin expression is increased as a result of epidermal hyperproliferation.

Tenascin expression in human dermis is related to epidermal proliferation.

The extracellular matrix glycoprotein tenascin is sparsely distributed in normal human dermis. The authors have shown that in a number of skin diseases (psoriasis, skin tumors), tenascin expression is strongly increased. In this immunohistochemical study, using polyclonal and monoclonal antisera, we have tested the hypothesis that tenascin expression in vivo is linked to epidermal proliferation. Using the sellotape stripping model in normal human skin, which causes a rapid recruitment of keratinocytes into the cell cycle, induction of tenascin expression was found in the upper dermis within 24 hours after stripping. In contrast, in normoproliferative monogenic disorders of keratinization (X-linked recessive ichthyosis, autosomal dominant ichthyosis vulgaris, non-erythrodermic lamellar ichthyosis), no increase in tenascin expression was found compared with normal skin. These findings demonstrate a relationship between epidermal proliferation and metabolic alterations in the dermal compartment.

Quality of intrapartum cardiotocography in twin deliveries.

OBJECTIVE: Intrapartum fetal heart rate (FHR) recordings in twins were compared for fetal signal loss during both stages of labor to assess the quality of these recordings by the method that had been used: external ultrasound or directly via a scalp electrode. STUDY DESIGN: Analysis of recordings collected between January 1, 1994, and January 1, 2002, from consecutive twin deliveries at the Vrije Universiteit Medical Center in Amsterdam. One hundred seventy-two twins that delivered via the vaginal route were included in the study. FHR recordings had a duration of at least 1 hour before the birth of the second twin. Subdivision took place on the basis of the recording technique, ie, ultrasound or scalp electrode. FHR data was obtained with HP-M1350 cardiotocographs. The status (pen on, pen off, maternal signal) and the mode of the signals were acquired. The duration of pen lifts and maternal signals was divided by the total duration of the recording. Statistical analyses were performed with the Mann-Whitney U test and the Wilcoxon signed ranks test. RESULTS: Recordings obtained via ultrasound demonstrated significantly more fetal signal loss than those obtained via the direct mode, particularly in the second stage. Approximately 26% to 33% of first stage and 41% to 63% of second stage ultrasound intrapartum FHR recordings in twins exceeded the International Federation of Gynecology and Obstetrics (FIGO) criteria for fetal signal loss. CONCLUSION: Intrapartum FHR monitoring via ultrasound provides far poorer quality FHR signals than the direct mode. The direct mode deserves a more prominent position in fetal surveillance than it currently has.

Multicenter technical and clinical evaluation of a fully automated enzyme immunoassay for CA 125.

The technical performance and clinical usefulness of the newly developed Enzymun-Test CA 125 (Boehringer Mannheim) was evaluated in a multicenter study. Sera tested were obtained from healthy control subjects (n = 1003) and from patients with benign conditions (379), ovarian cancer (518), or other malignancies (479). Intra- and interassay variability was low at CA 125 concentrations greater than 100 units/mL. Intra- and interassay CVs were respectively less than or equal to 36% and 23% for the low CA 125 concentrations (less than or equal to 35 units/mL) and less than or equal to 15% and 14% for the medium CA 125 concentrations (36- less than or equal to 100 units/mL). Interlaboratory reproducibility of the Enzymun-Test CA 125 was excellent. A strong linear correlation was observed between Enzymun-Test CA 125 and three of four other commercially available assays of CA 125 in serum. Cutoff values of 35 units/mL in three of these other tests corresponded to Enzymun-Test CA 125 values ranging from 27.0 to 42.1 units/mL. In the fourth test, an enzyme immunoassay, a cutoff of 35 units/mL corresponded to Enzymun-Test values ranging from 64.1 to 77.0 units/mL. Discordant, as yet unexplained, results in which Enzymun-Test values were greater than 65 units/mL and Centocor CA 125 immunoradiometric assay results were less than 35 units/mL were found in 15 of 1003 samples (1.5%) of apparently healthy control subjects. Sensitivity and performance of the Enzymun-Test were very similar to those of the Centocor assay for sera from the patients with various benign disorders or malignant diseases. Given its excellent automated technical performance, this new CA 125 serum assay is feasible for monitoring ovarian cancer patients. Test results are interchangeable with those from other laboratories and, in general, with those obtained by most other CA 125 tests.

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.

An abnormal early evening peak of plasma prolactin in nulliparous and obese post-menopausal women.

Plasma specimens were obtained in the afternoon (13.30 to 17.29 h) from 668 post-menopausal women and in the evening (17.30 to 20.30 h) from 219 further women. The mean evening plasma prolactin level was significantly higher than that found in the afternoon (p less than 0.0007). Mean afternoon prolactin levels were not related to nulliparity and body weight. In nulliparous obese women the mean of the evening peak prolactin concentrations was double that of comparable women studied in the afternoon (p less than 0.003).

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.

Tenascin expression in basal cell carcinoma.

Tenascin (hexabrachion, cytotactin) is an extracellular matrix glycoprotein whose expression is strongly increased in hyperproliferative skin diseases, as shown by immunohistochemistry with polyclonal sera. In this study we describe a new monoclonal antibody (T2H5) against human tenascin. The specificity of T2H5 was validated by sequential immunoprecipitation of tenascin with polyclonal sera. T2H5 was used to analyse the presence of tenascin in basal cell carcinoma. Using Western blotting, at least two forms of tenascin were found, with approximate molecular weights of 210 and 300 kDa. In cultured human skin fibroblasts only the high molecular weight form was found.