Oncogenic pathways in hereditary and sporadic breast cancer.

Cancer is a genetic disease. Breast cancer tumorigenesis can be described as a multi-step process in which each step is thought to correlate with one or more distinct mutations in major regulatory genes. The question addressed is how far a multi-step progression model for sporadic breast cancer would differ from that for hereditary breast cancer. Hereditary breast cancer is characterized by an inherited susceptibility to breast cancer on basis of an identified germline mutation in one allele of a high penetrance susceptibility gene (such as BRCA1, BRCA2, CHEK 2, TP53 or PTEN). Inactivation of the second allele of these tumour suppressor genes would be an early event in this oncogenic pathway (Knudson's "two-hit" model). Sporadic breast cancers result from a serial stepwise accumulation of acquired and uncorrected mutations in somatic genes, without any germline mutation playing a role. Mutational activation of oncogenes, often coupled with non-mutational inactivation of tumour suppressor genes, is probably an early event in sporadic tumours, followed by more, independent mutations in at least four or five other genes, the chronological order of which is likely less important. Oncogenes that have been reported to play an early role in sporadic breast cancer are MYC, CCND1 (Cyclin D1) and ERBB2 (HER2/neu). In sporadic breast cancer, mutational inactivation of BRCA1/2 is rare, as inactivation requires both gene copies to be mutated or totally deleted. However, non-mutational functional suppression could result from various mechanisms, such as hypermethylation of the BRCA1 promoter or binding of BRCA2 by EMSY. In sporadic breast tumorigenesis, at least three different pathway-specific mechanisms of tumour progression are recognizable, with breast carcinogenesis being different in ductal versus lobular carcinoma, and in well differentiated versus poorly differentiated ductal cancers. Thus, different breast cancer pathways emerge early in the process of carcinogenesis, ultimately leading to clinically different tumour types. As mutations acquired early during tumorigenesis will be present in all later stages, large-scale gene expression profiling using DNA microarray analysis techniques can help to classify breast cancers into clinically relevant subtypes.

Oncogenic pathways in hereditary and sporadic breast cancer.

Cancer is a genetic disease. Breast cancer tumorigenesis can be described as a multi-step process in which each step is thought to correlate with one or more distinct mutations in major regulatory genes. The question addressed is how far a multi-step progression model for sporadic breast cancer would differ from that for hereditary breast cancer. Hereditary breast cancer is characterized by an inherited susceptibility to breast cancer on basis of an identified germline mutation in one allele of a high penetrance susceptibility gene (such as BRCA1, BRCA2, CHEK 2, TP53 or PTEN). Inactivation of the second allele of these tumour suppressor genes would be an early event in this oncogenic pathway (Knudson's "two-hit" model). Sporadic breast cancers result from a serial stepwise accumulation of acquired and uncorrected mutations in somatic genes, without any germline mutation playing a role. Mutational activation of oncogenes, often coupled with non-mutational inactivation of tumour suppressor genes, is probably an early event in sporadic tumours, followed by more, independent mutations in at least four or five other genes, the chronological order of which is likely less important. Oncogenes that have been reported to play an early role in sporadic breast cancer are MYC, CCND1 (Cyclin D1) and ERBB2 (HER2/neu). In sporadic breast cancer, mutational inactivation of BRCA1/2 is rare, as inactivation requires both gene copies to be mutated or totally deleted. However, non-mutational functional suppression could result from various mechanisms, such as hypermethylation of the BRCA1 promoter or binding of BRCA2 by EMSY. In sporadic breast tumorigenesis, at least three different pathway-specific mechanisms of tumour progression are recognizable, with breast carcinogenesis being different in ductal versus lobular carcinoma, and in well differentiated versus poorly differentiated ductal cancers. Thus, different breast cancer pathways emerge early in the process of carcinogenesis, ultimately leading to clinically different tumour types. As mutations acquired early during tumorigenesis will be present in all later stages, large-scale gene expression profiling using DNA microarray analysis techniques can help to classify breast cancers into clinically relevant subtypes.

T cell infiltration and MHC I and II expression in the presence of tumor antigens: An immunohistochemical study in patients with serous epithelial ovarian cancer.

OBJECTIVES: Ovarian cancer is a frequent cause of death among women with gynaecologic malignancies despite the introduction of combination chemotherapy. There is therefore a need for new therapeutic strategies for patients with ovarian cancer, such as cellular immunotherapy. In this immunohistochemical study we analysed the expression of three tumor antigens, p53, HER-2/neu and MUC-1 in relation to the expression of major histocompatibility complex (MHC) class I and II on tumor cells, and we searched for the presence of (activated) immune effector cells at the tumor site. STUDY DESIGN: The study was carried out retrospectively in tumor tissue from 29 patients with serous ovarian cancer. Material used had been formalin fixed and paraffin embedded. Material had been obtained from 15 patients at staging laparotomy and from 14 patients during second look or intervention laparotomy. RESULTS: A positive staining for p53 was found in 19/29 (66%) of the tumors, with a high positivity in 13/29 (45%). HER-2/neu and MUC-1 staining was positive in 8/29 (28%) and 21/28 (75%), respectively. Downregulation of MHC class I on tumor cells was found in a minority of the patients, beta-2-microglobin (beta2m) was expressed on tumor cells in all patients. High staining for CD45RO correlated with a high positive staining for granzyme-B (R=0.40, P=0.04) and TIA-1 (R=0.39, P=0.04). A statistically significant better survival in the group with lower stage of disease was found. CONCLUSIONS: As only three out of 29 patients were negative for the tumor antigens p53, HER-2/neu and MUC-1, immunotherapy aiming at all three could serve almost all patients with ovarian cancer. We found that granzyme-B, TIA-1 and CD45RO+ T cells are present in the tumor biopsies, increasing this number by immunotherapy may be beneficial. The immune escape mechanism by MHC class I and beta2m downregulation seems to be of minor importance. Our data support the view that immunotherapy may offer new possibilities with high specificity in ovarian cancer.

Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1.

A monoclonal antibody (MAb), VU-2-G7, was generated against a synthetic 60-mer MUC1 triple tandem repeat peptide with N-acetyl-galactosamine (GalNAc) O-linked to the threonine in the PDTR region of each repeat (3M GalNAc). VU-2-G7 and 8 MUC1 MAbs (VU-3-C6, VU-4-H5, 139H2, A76-A/C7, VU-12-E1, BCP9, MF11 and BW835) were tested against various glycosylated and nonglycosylated MUC1 tandem repeat peptides. VU-2-G7 showed strong reactivity with its immunogen, 3M GalNAc, and much lower reactivity with the nonglycosylated 60-mer MUC1 triple tandem repeat peptide. VU-2-G7 showed no reactivity with a 60-mer MUC1 triple tandem repeat peptide modified at the PDTR region or with a 60-mer MUC1 triple tandem repeat peptide with 3 GalNAc per repeat outside the PDTR region (9M GalNAc). In ELISA and flow cytometry, VU-2-G7 ubiquitously reacted with 4 MUC1-expressing breast cancer and 2 ovarian cancer cell lines and with a MUC1-gene-transfected Chinese hamster ovary cell line. The reactivity of VU-2-G7 was always higher than that of VU-4-H5, raised against a nonglycosylated 60-mer MUC1 triple tandem repeat peptide. Immunohistochemical staining of paraffin sections of breast and ovarian tumor tissues showed strong binding of VU-2-G7 predominantly at the cell membrane. The dominant epitope of VU-2-G7 is in the glycosylated PDTR motif of the MUC1 tandem repeat, and this epitope is abundantly present on the surface of tumor cell lines and breast and ovarian tumor tissues. Given the ubiquitously aberrant glycosylation of MUC1 in malignant cells, the production of MAbs against highly purified glycosylated MUC1 tandem repeat peptides may yield MAbs better suited for the immunotherapy of carcinomas than those available at the moment.

Influence of chemotherapy on the expression of p53, HER-2/neu and proliferation markers in ovarian cancer.

OBJECTIVE: Mutated p53 and HER-2/neu play a role in the etiology of ovarian cancer. It is important to know whether the expression of these proteins is affected by platinum-containing chemotherapy. STUDY DESIGN: Together with the cell proliferation markers Ki-67 and PCNA, the expression of p53 and HER-2/neu was assessed before and after chemotherapy. Paraffin-embedded tumor sections from 20 patients with ovarian cancer and four patients with benign disorders of the ovaries (controls) were analyzed. The expression of p53 was determined by the antibodies DO-1 and BP53-12. In addition to HER-2/neu and PCNA specific antibodies, MIB-1 was used to detect Ki-67. RESULTS: The expression of all markers was higher in ovarian cancer patients than in non-malignant controls. MIB-1 showed a significant increase of expression after chemotherapy (P=0.002). HER-2/neu, p53 and PCNA also showed a clear increase after treatment, but this was not statistically significant. HER-2/neu is of prognostic relevance with respect to the response to chemotherapy (P=0.005) and survival (P=0.0002). CONCLUSION: The different markers tested all increase after chemotherapy, but the differences are not statistically significant. Low HER-2/neu expression correlates with good outcome at second look.

Quantification of MUC1 in breast cancer patients. A method comparison study.

OBJECTIVE: To compare the performance of four serum assays for the quantification of MUC1 in breast cancer patients. STUDY DESIGN: A total of 282 serum samples were evaluated with two automated (Boehringer Mannheim Enzymun-Test CA 15-3 and Chiron ACS BR) and two manual assays (Centocor CA 15-3 radioimmunoassay [RIA] and Biomira Truquant BR RIA). Sera were obtained from healthy controls (n=50), patients with benign (n=25) and malignant breast disease (n=77) and patients with other malignancies (n=69). In addition, sera from pregnant women (n=56) and patients with liver cirrhosis (n=5) were included. RESULTS: Intraassay coefficients of variation (C.V.s) were highest for the manual Centocor CA 15-3 assay (7.4% for values below 50 kU/l and 8.1% for values above 180 kU/l). Interassay C.Vs were highest for the manual Truquant BR assay (11.7% for the lower concentration values and 18.6% for the higher concentration values). False positive rates ranged between 0% for the Centocor CA 15-3 RIA and 14% for the ACS BR assay (cut-off: 30 kU/l). In monitoring breast cancer patients all four assays show similar patterns, although absolute MUC1 values found may differ up to 50%. CONCLUSION: For monitoring purposes all assays perform equally well, however, automated assays show lower inter- and intraassay variability, especially in the higher value range. Therefore we recommend the use of the same, automated, assay for quantification of MUC1 during the follow-up of breast cancer patients.

Fluctuations in CA 125 and CA 15-3 serum concentrations during spontaneous ovulatory cycles.

The aim of this study was to investigate cycle dependent changes of serum CA 125 and CA 15-3 concentrations during spontaneous ovulatory cycles. Twenty apparently healthy women with spontaneous menstrual cycles attending our infertility clinic were included. Of these women, 18 had occluded tubes as a result of sterilization. Ovulation was confirmed by luteinizing hormone test and ultrasonography and, to exclude endometriosis, a laparoscopy was performed. Serum samples for CA 125, CA 15-3, 17 beta-oestradiol and progesterone determinations were taken every second day starting on the 2nd day of the cycle until the 7th day of the next cycle. After correction for inter-individual variation in serum concentrations, highest CA 125 concentrations were found during the menstruation. During the follicular and peri-ovulatory phase CA 125 serum concentrations were lowest. For CA 15-3, serum concentrations were not statistically different throughout the cycle. CA 125 and oestradiol concentrations were negatively correlated, CA 15-3 and oestradiol concentrations were positively correlated. Absolute serum concentrations of both CA 125 and CA 15-3 vary among females. Within the female, fluctuations of CA 125 are phase related. In the population studied most of the patients had tubal obstruction and high CA 125 serum concentrations during menstruation, which revokes the theory that the menstrual rise of CA 125 is due only to retrograde menstruation.

Comparison of seven immunoassays for the quantification of CA 125 antigen in serum.

Seven CA 125 immunoassays were compared for their clinical performance. CA 125 concentrations were determined in 289 serum samples obtained from women with benign pelvic tumors (samples from 98 patients) and patients with various cancers (samples from 111 patients). In the range of 0-1000 kilounits/L, all assays tested were linearly correlated, with correlation coefficients ranging from 0.89 to 0.99. In relation to the original Centocor CA 125 assay, there was an overall tendency to measure higher absolute values in the lower CA 125 value range. This was not seen in relation to the Centocor CA 125 II assay. ROC curves (benign vs pretreatment ovarian cancer patients) were nearly identical for all assays, and the areas under the ROC curves were not markedly different. We conclude that the CA 125 assays tested are strongly related to each other and are clinically reliable for the quantification of serum CA 125 and that none of the assays offers higher diagnostic accuracy or better discrimination between patient groups, especially not in the lower ranges.

Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin. San Diego, Calif., November 17-23, 1996.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.

'Epitope fingerprinting' using overlapping 20-mer peptides of the MUC1 tandem repeat sequence.

The ISOBM TD-4 Workshop antibodies 122-177 were tested for reactivity with 20 overlapping MUC1 tandem repeat 20-mer peptides by an ELISA, in order to determine the complete amino acid sequences of the epitopes. Of the 56 antibodies studied, 30 showed specific binding and thus the epitopes were characterized. The epitopes appear to be 'broader' when compared to those deduced from studies using smaller peptides. Interassay variation is remarkably small, allowing for precise grouping of clusters with very similar epitope patterns. Five groups of antibodies show remarkable similarity: BC3 and VU-4-H5; BC4W154, C595 and Mc5; MF06 and B27.29; VU-11-D1 and VU-11-E2; Ma552, VU-3-C6, 7540MR and BC4E549. We have used the term 'epitope fingerprinting' to refer to the 'fine structure' of the epitope with all its essential and flanking amino acids. We believe this method is more precise than the usual epitope mapping with short peptides.

Autoantibodies to p53 in ovarian cancer patients and healthy women: a comparison between whole p53 protein and 18-mer peptides for screening purposes.

Autoantibodies against complete p53 protein and 18-mer peptides of p53 in ovarian cancer patients and healthy women were examined. Sera from 9% (4/46) of ovarian cancer patients but none (0/51) of healthy women recognized complete p53 protein. The antibodies were mainly of the IgG1 isotype. Two patients had also IgG2 antibodies. Sera from 28% (13/46) of cancer patients and 21% (11/52) of healthy women contained either IgM, or IgM plus IgG2 antibodies against 18-mer p53 peptides. Screening against complete p53 protein instead of peptides seems necessary for identifying patients with tumor-related antibodies. IgG2 antibodies against p53 suggest p53-specific CD4+ T helper 1 cell activity in some of the ovarian cancer patients.

Clinical and technical evaluation of ACS BR serum assay of MUC1 gene-derived glycoprotein in breast cancer, and comparison with CA 15-3 assays.

The mucin glycoprotein-detecting assay CA 15-3 is a valuable tool for monitoring the course of disease in breast cancer patients. Assays of CA 15-3 are based on the use of two MAbs to polymorphic epithelial mucin (PEM). We evaluated the technical and clinical performance of the Chiron ACS BR, an automated competitive chemiluminescence assay using a single MAb, B27.29, and compared the assay's results with those of the Centocor CA 15-3 RIA, the Abbott IMx CA 15-3, and the Boehringer Mannheim Enzymun-Test CA 15-3. The study population consisted of 253 healthy women, 66 patients with benign breast disease, 168 breast cancer patients, and 76 patients with other carcinomas. In the technical evaluation, we assessed the precision and linearity on dilution of the ACS BR assay. Cutoff values (upper limits of values seen in healthy subjects) were determined for all four assays. Agreement between the assays was studied by linear regression analysis. The ACS BR assay gave within- and between-assay CVs of 2.2% and 3.9%, respectively. Three samples from healthy women gave discordant values by ACS BR and were not included in the calculations. All four assays exhibit a highly similar pattern when monitoring breast cancer disease; the closest agreement of values was obtained between ACS BR and Centocor CA 15-3. We conclude that the ACS BR assay is a fast and reliable immunoassay for measuring PEM in serum. Although it detects a slightly different epitope on the PEM molecule than is targeted in other assays, for cancer serum samples it agreed better with the original Centocor CA 15-3 assay than did the other two CA 15-3 assays tested.

Clinical evaluation of the Byk LIA-mat CA125 II assay: discussion of a reference value.

The Byk LIA-mat CA125 II assay was compared with the Centocor IRMA CA125 II. Serum samples studied (n = 1012) were obtained from 652 apparently healthy females, 61 pregnant women, and 299 patients with benign and malignant gynecological tumors. The CA125 II assay value at the 95th percentile of the total healthy group was 29 kU/L for the LIA-mat and 32 kU/L for the Centocor assay. For the LIA-mat assay the 95th percentile was 31 kU/L (Centocor 36 kU/L) for the group < 45 years and 21 kU/L (Centocor 25 kU/L) for women > 55 years of age. By using ROC curves we found the optimal pretreatment Byk LIA-mat CA125 II value differentiating between benign and malignant ovarian tumors to be 95 kU/L. Pretreatment CA125 values > 1000 kU/L were detected in serum samples of patients with advanced epithelial ovarian cancer.

CA 125: a valid marker in ovarian carcinoma patients treated with paclitaxel?

BACKGROUND: Changes in serum CA 125 from baseline do not reflect response to paclitaxel in relapsed ovarian carcinoma patients. Our study aimed to determine whether CA 125 changes relate to tumor response and overall survival during paclitaxel salvage treatment. METHODS: Response data and CA 125 values of 77 platinum pretreated ovarian carcinoma patients were included in the study. Patients received 496 courses of paclitaxel in total (median 6; range: 2-18 courses). RESULTS: Response group numbers on the basis of World Health Organization (WHO) criteria were: 7 partial response, 22 stable disease, and 48 progressive disease. CA 125 values at the moment of clinical response allocation, the median survival duration, and the 3-year survival rate did not differ among WHO defined response groups. For both the stable disease group and the responders, the slopes of the exponential CA 125 regression curves during paclitaxel treatment were negative. Response groups, as defined by CA 125 changes, i.e., halving or doubling of baseline values, after 4 courses were concordant with WHO defined response groups in only 27%, but predicted survival far better. CONCLUSIONS: This study confirms that CA 125 changes in patients receiving paclitaxel treatment do not correlate with response allocations according to WHO criteria. In particular, patients with clinically and radiologically defined progression will often not show an increase in CA 125 concentrations from baseline. Those patients who do show doubling of CA 125 values, however, have a very poor prognosis. The CA 125 ratio, as determined after 4 courses of paclitaxel treatment, may be a better indicator of response than WHO defined response status.

Multicenter evaluation of the Abbott IMx CA 15-3 assay.

We describe a new fully automated procedure for the quantitative measurement of CA 15-3: the microparticle enzyme immunoassay (MEIA) technology developed by Abbott Labs. for the IMx automated immunoassay analyzer. The new IMx CA 15-3 test uses two mouse monoclonal antibodies, 115D8 and DF3. The test has a dynamic range to 250 kilounits/L and a minimal detectable dose of CA 15-3 < 0.2 kilounits/L. On dilution, linearity is excellent, with recoveries ranging from 94% to 101%. Studies were conducted at four sites to evaluate the performance characteristics of this assay. The intra- and interassay CVs were < 4.7% and < 5.6%, respectively, and showed a between-laboratory CV < 5.9%. Test results of the Abbott IMx CA 15-3 (y) were correlated with those obtained with the Centocor CA 15-3 RIA (x), a solid-phase heterologous RIA. Linear regression analysis on results for 1973 samples yielded: y = 0.97x - 2.09 (r = 0.9899, Sy/x = 22.2, range 1-4089 kilounits/L).

Heterologous double-determinant immunoradiometric assay CA 125 II: reliable second-generation immunoassay for determining CA 125 in serum.

The new CA 125 II (Centocor) serum assay utilizing the M11 mouse monoclonal antibody as capture antibody and OC125 as tracer antibody, was investigated for its technical and clinical performance against the original CA 125 assay. The CA 125 II test revealed a quadruple increase in signal-to-noise ratio, good intra- and interassay precision (with CVs < 5% and 7%, respectively), improved dilution linearity, and a minimal detectable dose of 0.38 units/mL. Sera were obtained from healthy females (n = 192), women with benign conditions (n = 208), and patients with various cancers (n = 379). Both assays measured highly similar CA 125 distributions with equal reference ranges and nearly identical positivity (> 35 units/mL) rates, resulting in similar receiver-operating characteristic curves and monitoring graphs. Linear regression analysis of results by the two assays (CA 125 = x, CA 125 II = y) in ovarian cancer patients showed, for CA 125 assay values between 0 and 1000 units/mL, a slope of 1.00 and a y-intercept of 12.6 (n = 254, r = 0.8617, P < 0.0001). The heterologous CA 125 II assay appeared to be more accurate in patients who had human anti-mouse antibodies after immunoscintigraphy. The CA 125 II immunoradiometric assay is sensitive and reliable for measuring serum CA 125, and fully retains the cutoff values of 35 and 65 units/mL that were defined with the original CA 125 immunoradiometric assay.

Effect of hormones on the expression of proviral genes Mtv-2 and Mtv-3 in mouse mammary gland.

We have investigated the expression of the Mtv-2 and Mtv-3 proviral genes in mouse mammary glands by examining the effect of hormones on levels of mammary tumor virus (MTV) proteins p27 (gag) and gp52 (env) in mouse mammary explants. We also investigated the effect of the hormones on DNA synthesis in the explants. The mammary glands were derived from inbred GR and 020 mice, and from the respective congenic mouse strains GR/Mtv-2- and 020/Mtv-2+. The addition of insulin to the culture medium caused increases in p27 and gp52 levels in GR and 020/Mtv-2+ glands; a further increase in the viral proteins was obtained by also adding dexamethasone. Prolactin in combination with progesterone enhanced p27 and gp52 levels, but to a lesser extent than did dexamethasone. Dexamethasone caused a slight but significant increase in p27 protein in mammary explants from GR/Mtv-2- mice. Our data indicate that the Mtv-2 locus and the Mtv-3 locus in mouse mammary gland are under separate glucocorticoid control, and that Mtv-2 expression is also stimulated by the prolactin and progesterone combination. Whereas dexamethasone enhances MTV protein levels in mouse mammary explants, it inhibits DNA synthesis in the explants.