Prevalence of blood type A and risk of vascular complications following transcatheter aortic valve implantation.

OBJECTIVES: To assess the prevalence of blood type A among patients referred for transcatheter aortic valve implantation (TAVI) and whether it is related to vascular complications. BACKGROUNDS: Vascular complications following TAVI are associated with adverse outcomes. Various blood types, particularly type A, have been shown to be more prevalent in cardiovascular diseases and to be related to prognosis. METHODS: The prevalence of various blood types in a cohort of 491 consecutive patients who underwent TAVI was compared with a control group of 6500 consecutive hospitalised patients. The prevalence and predictors of vascular complications and bleeding events were evaluated in the blood type A group and were compared with non-type A patients. RESULTS: The mean age of TAVI patients was 83 ± 6 years, and 40 % were males. Patients were divided into two groups: blood type A (n = 220) and non-type A (n = 271). Type A was significantly more prevalent in the TAVI group than in the control group (45 vs. 38 %, p = 0.023). Compared with the non-type A group, patients with blood type A had more major and fatal bleeding (14.5 vs. 8.1 %, p = 0.027) and more vascular complications (any vascular complication: 24.5 vs. 15.9 % p = 0.016; major vascular complications: 12.3 vs. 7 % p = 0.047). In a multivariable analysis, blood type A emerged as a significant and independent predictor for vascular complications and bleeding events. CONCLUSIONS: Blood type A is significantly more prevalent in TAVI patients than in the general population and is related to higher rates of vascular and bleeding complications.

The ratio between carcinoma antigen-125 levels in the seminal plasma and serum may be a marker for fertilization in intracytoplasmic sperm injection.

OBJECTIVE: To assess the relationship between tumor marker carcinoma antigen-125 levels in seminal plasma and serum and fertilization rates in an IVF program, using intracytoplasmic sperm injection (ICSI). DESIGN: A prospective study. SETTING: IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. PATIENT(S): Twenty-five infertile patients with severe oligo-terato-asthenospermia syndrome and 25 fertile male donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum and seminal plasma carcinoma antigen-125 concentrations and fertilization rate per cycle. RESULT(S): In the infertile group, the seminal plasma carcinoma antigen-125 levels ranged from 22.0 to 1,284.0 U/mL (mean level +/- SD, 229.9 +/- 274.2 U/mL). In the normospermic fertile male donors, the seminal plasma carcinoma antigen-125 concentrations ranged from 12.2 to 336.7 U/mL (mean level +/- SD, 110.1 +/- 91.6 U/mL). This difference was statistically significant. The mean +/- SD ratio between the seminal plasma/serum carcinoma antigen-125 levels differed significantly between the infertile group (47.9 +/- 61.3) and the fertile male donors (5.7 +/- 3.5). In the infertile group, the ratio between the seminal plasma/serum carcinoma antigen-125 levels was found to be negatively correlated with the oocyte fertilization rate. CONCLUSION(S): The ratio between carcinoma antigen-125 levels in the seminal plasma and serum may be an indirect marker for male infertility and fertilization rate in IVF treatment using ICSI.

Tamoxifen for disease-negative but MCA-positive breast cancer patients.

Increased levels of mucin-like carcinoma-associated antigen (MCA) in breast cancer patients with no evidence of disease following the treatment of the primary disease created a dilemma of 'to treat' or 'wait and see'. One might assume that early treatment of clinically undetectable disease on the basis of an elevated serum level of a sensitive and reliable tumor marker, may improve the treatment results, and even prolong the patient's survival. 'Wait and see' on acceptance of the notion that even early metastatic disease, still manifested only by uprising MCA levels, is incurable, and treatment should be kept in reserve for palliation of symptomatic disease. Sixty-one breast cancer patients with increasing MCA levels but without evidence of metastatic disease were randomized for tamoxifen 20 mg b.i.d. or to follow-up till relapse. The results for a median follow-up period of one year were encouraging. The non-treated patients experienced a significantly higher relapse rate (24.1%) than the tamoxifen-treated subjects (0%; p=0.012). The results for a median follow-up of 5 years were disappointing. The overall relapse rate was 22.2%. The relapse rate among the control patients was 25.8% while in the treatment arm it was 17.4% (p=0.46). The event-free survival and the pattern of relapse were similar in both arms. Tamoxifen may therefore be reserved for overt metastases, and not wasted on asymptomatic subclinical disease. It seems that there is no yield in terms of event-free survival for MCA measurements in breast cancer patients during the 5-year follow-up period.

Elevated breast cancer serum markers in otherwise healthy women.

A high value of mucin-like carcinoma associated antigen (MCA), CA-15.3 or H23, in a woman known to have a diagnosis of breast cancer, may reflect presence of disease. A low level in a breast cancer patient may be accepted for remission, but a false negative result cannot be excluded. On the other hand, a low level of serum tumor marker in the general population actually lacks any significance. However, what is the meaning of an elevated level of marker, known to have a relatively high sensitivity and specificity, in an otherwise healthy woman? Does it mean an occult breast cancer or a false positive? Sera samples were obtained from 155 consecutive, otherwise healthy women, who were referred for mammography, and assayed for tumor markers. MCA was elevated in 15-24% of patients with normal mammogram, depending on their ages. Lack of elevation of a second marker in most of the cases supported the assumption that the elevation of the MCA was insignificant. Elevation of H23 occurred more frequently in younger women than in the elders, but was not associated with elevation of a second marker. In the cases with abnormal mammogram due to histologically proven benign disorders, serum tumor markers were generally within the normal ranges. Our results pointed to the lack of diagnostic significance of an elevated level of serum tumor marker, as far as the mammogram was normal or benign, there was no history of cancer nor any other systemic disease (including malignancy), and a second tumor marker was within the normal range. The women with presently false positive marker level may, however, be followed, because of the possible risk for future development of breast cancer.

Effect of elective prolonged urethral catheterization on serum prostate-specific antigen concentration.

OBJECTIVES: To determine the effect of an indwelling catheter on prostate-specific antigen (PSA) levels. PSA is an organ (prostate)-specific marker, and its level can be elevated in various pathologies as well as following urologic manipulations. An elevated marker may indicate the presence of prostate cancer. In the presence of an indwelling catheter, our inability to decide whether an elevated PSA value represents genuine pathology or is related to the catheter itself is often of great clinical importance. METHODS: A prospective study was conducted on 21 men with an indwelling catheter inserted electively for major nonurologic abdominal surgery to determine its influence on PSA concentration. Sera were collected before catheter insertion, 2 hours after, and then every day (average, 16 days). Catheters were left in place for an average of 5.5 days. RESULTS: Follow-up data compared to baseline and to the previous day's PSA concentrations revealed no significant change in any of the subjects. In 2 men with elevated preinsertion PSA levels (more than 10.0 ng/mL), the change over time did not differ in magnitude from changes in the other 19 men with normal pretreatment values. CONCLUSIONS: Inserting a urethral catheter and maintaining it for several days does not result in any clinically or statistically significant change in PSA levels. PSA values obtained in patients with an indwelling catheter are reliable and independent of its presence. An elevated level mandates prompt evaluation to exclude prostate cancer.

Treatment of disease-negative but mucin-like carcinoma-associated antigen-positive breast cancer patients with tamoxifen: preliminary results of a prospective controlled randomized trial.

Increasing levels of tumor markers such as carcinoembryonic antigen, mucin-like carcinoma-associated antigen (MCA), CA 15.3, and monoclonal antibody H23 in breast cancer patients following the treatment of the primary disease and adjuvant radiation and chemotherapy reflect subclinical development of metastatic disease. Overt metastatic disease is usually incurable and prolongation of life at this stage is impossible, and the treatment is only palliative. The efficacy of tamoxifen, a least-toxic agent, in the treatment of early and minimal metastatic disease detected only by increasing serum levels of MCA was studied prospectively in a randomized study. Our preliminary, albeit encouraging, results showed that the rate of relapse within a median follow-up period of 11 months was 24.1% in the control arm as compared with 0% in the tamoxifen arm (Fisher's exact test, P = 0.012). None of the patients with a relapse had positive progesterone receptors (PR). We may carefully conclude that early treatment may be warranted in young patients with negative PR and continuously increasing serum levels of the marker.

Vaccinia virus MUC1 immunization of mice: immune response and protection against the growth of murine tumors bearing the MUC1 antigen.

MUC1 is a mucin found on the apical surfaces of some normal mammalian mucin-secreting cells. It is characterized by heavy glycosylation and a 20-amino-acid tandem repeat segment. In most cases of human breast adenocarcinoma, this antigen is overexpressed. Moreover, abnormal glycosylation exposes a novel peptide epitope within the tandem repeat, such that antibodies to this epitope can distinguish normal from malignant adenocarcinomatous breast tissue. We have constructed a vaccinia virus (VV) that carries the cDNA for the MUC1 antigen. Murine and human cells infected with this virus express the MUC1 molecule, with three to four tandem repeats per molecule and with the tumor-associated epitopes exposed. Mice immunized with this virus produce antibodies that recognize MUC1 outside the tandem repeat, within the tandem repeat, and within the tumor-associated protein core epitope. Tumorigenic P815 (DBA) and 3T3 (BALB/c) cells have been transfected with MUC1. Thirty percent of DBA mice immunized with VV-MUC1 are protected from growth of P815-MUC1 tumors when implanted with 10(5) cells. Immunized BALB/c mice show a late development of transfected 3T3 tumor cells. Immunized mice show a moderate MUC1-specific IgG titer, but it cannot be correlated with subsequent tumor rejection. No evidence for a MUC1-specific cytotoxic T lymphocyte response has been found after immunization with VV-MUC1.

Vaccinia recombinants expressing secreted and transmembrane forms of breast cancer-associated epithelial tumour antigen (ETA).

Monoclonal antibody H23 identifies a polymorphic epithelial tumour antigen (ETA) that is aberrantly expressed in breast cancer and which may afford a target for active immunotherapy. We recently reported the cloning of H23-ETA genomic and cDNA clones. H23-ETA contains a multiple internal tandem repetition of a 20 amino acid motif and sequence analysis predicted two mRNA species encoding different ETA proteins, one harbouring a C-terminal potentially transmembrane hydrophobic zone (T) and a second form (S) that lacks this zone. We report that both RNA species can be detected in breast cancer cells. To further characterize the encoded proteins we have constructed vaccinia virus recombinants, VV-ETA-S and VV-ETA-T, separately expressing the alternative forms. Despite selective loss of internal tandem repeat elements during propagation of recombinant vaccinia, the encoded polypeptides were efficiently recognized by H23 monoclonal antibody. Immunoprecipitation revealed that ETA encoded by the S recombinant was secreted into the culture medium whereas the T form remained tethered at the cell surface. Both forms were readily detected in infected cells by immunofluorescence. Abnormal mobility of the T polypeptide indicated post-translational cleavage that may permit the extracellular domain of the T-polypeptide to be shed from the cell surface. Further, fluorescence-activated cell sorting analysis shows that the S form of the polypeptide is also partly present at the cell surface. Vaccinia recombinants expressing ETA may be of utility in the active immunotherapy of breast cancer.

Serial serum MCA measurements in the follow-up of breast cancer patients.

Mucin-like carcinoma-associated antigen (MCA) was serially assayed in 58 women with histologically proven breast cancer after their treatment for primary disease. MCA sensitivity and specificity were 87.5% and 76.9%, respectively, and the positive predictive value 82.4%. 10 patients had elevated MCA and no evidence of disease (NED) during their follow-up, of whom 4 finally developed overt metastases. The 4 had a mean (S.D.) MCA value of 46.48 (18.26) U/ml during the lead time, versus 18.76 (2.69) U/ml in the other 6, who are still at high risk for developing overt metastases. Raised levels of MCA in patients with NED create a dilemma of "treat" versus "wait and see". Early treatment of patients with serially uprising MCA levels should be evaluated in a prospective randomised study to assess its ability to prevent or delay the development of overt metastatic disease and influence survival.

Vaccination against tumor cells expressing breast cancer epithelial tumor antigen.

Ninety-one percent of breast tumors aberrantly express an epithelial tumor antigen (ETA) identified by monoclonal antibody H23. Vaccinia virus recombinants expressing tumor antigens have considerable promise in the active immunotherapy of cancer, and we have evaluated the potential of vaccinia recombinants expressing the secreted (S) and cell-associated (transmembrane, T) forms of H23 ETA to elicit immunity to tumor cells expressing ETA. Tumorigenic ras-transformed Fischer rat fibroblast lines FR-S and FR-T, expressing the S or T form of H23 ETA, respectively, were constructed for use in challenge experiments. Expression of H23 ETA in these lines was confirmed by Western blotting and immunofluorescence. When challenged by subcutaneous seeding of tumor cells, 97% (FR-S) and 91% (FR-T) of syngeneic Fischer rats rapidly developed tumors that failed to regress. Vaccination with recombinant vaccinia virus expressing ETA-T prior to challenge prevented tumor development in 82% of animals seeded with FR-T cells but in only 61% of animals seeded with FR-S. The vaccinia recombinant expressing the S form was a less effective immunogen, and vaccination protected only 29-30% of animals from developing tumors upon challenge with either FR-S or -T cells. The increased immunogenicity of the recombinant expressing ETA-T was reflected in elevated levels of ETA-reactive antibody in vaccinated animals, confirming that secreted antigens expressed from vaccinia virus are less effective immunogens than their membrane-associated counterparts.

Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen.

A human gene and cDNA coding for a breast-cancer-associated antigen (H23Ag) were isolated and characterized. The gene contains two exons and one intron. Part of the second exon is a tandem repeat array (TRA) consisting of multiple 60-bp G + C-rich units. We report here the characterization of unique sequences that are found in the H23Ag gene and cDNA, in addition to the 60-bp repeats. Analysis of the cDNA sequences revealed a putative ATG start codon preceded by two overlapping initiation consensus sequences (CCACC). The open reading frame determines an amino acid (aa) sequence consisting of three regions. The first region contains an initiating methionine and a highly hydrophobic putative signal peptide. This is followed by a variable number of highly conserved 20-aa repeat units (TRA). The last region, C-terminal to TRA, contains four potential N-linked glycosylation sites. The genomic nucleotide sequences demonstrate a putative promoter region that includes a 'TATA' box. A putative estrogen regulatory element is located 5' to the promoter region. The characterization of the gene and cDNA coding for the H23Ag presented here, may help to elucidate its possible function in human breast cancer.

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen. A comparative analysis in breast cancer.

Expression of the gene coding for a new breast tumor-associated antigen, H23, was compared to expression of genes coding for pS2, c-erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non-breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c-erbB2, ER and pS2, respectively. Non-malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.

Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms.

The isolation and characterization of complementary DNAs (cDNAs) which code for an epithelial antigen aberrantly expressed in human breast tumor tissue are described here. The only information regarding the primary structure of this potentially important antigen has been a 20-amino-acid repeat motif. We now report the complete amino acid sequences of different forms of the human epithelial tumor antigen as deduced from the nucleotide sequence of isolated non-repeat cDNAs. The diversity of protein forms is generated by a series of alternative splicing events that occur in the regions located upstream and downstream to a central tandem repeat array. Isolated cDNAs coding for the upstream region show that differential usage of alternative splice acceptor sites may generate two protein forms containing putative signal peptides of varying hydrophobicities. The complexity of possible antigen forms is further compounded by alternative splicing events occurring in the region 3' to the repeat array. The isolated cDNAs 3' to the tandem repeats indicate that whereas one mRNA transcript is colinear with the gene, and defines an open reading frame (ORF) containing 160 amino acids downstream to the repeat array, a second cDNA correlates with a mRNA that is generated by a series of splicing events. The deduced amino acid sequence of the spliced cDNA contains an ORF that is identical for 149 amino acids downstream to the repeat array with the amino acid sequence of the unspliced cDNA. At this point it diverges and continues for an additional 179 amino acids. The sequence contains a highly hydrophobic 28-amino-acid peptide, located towards the carboxyl terminus, that may correspond to a transmembrane region. The cDNAs and deduced amino acid sequences, presented here, define the complete amino acid sequences of the epithelial tumor antigen and demonstrate the existence of multiple protein forms that probably localize to different cellular and extracellular compartments.

A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen. cDNA cloning, expression of the transfected gene and over-expression in breast cancer tissue.

A monoclonal antibody, H23, that specifically recognizes a breast-tumor-associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850-bp cDNA insert, shows that they are composed of 60-bp (G + C)-rich tandem repeating units. The coding strand was determined and codes for a proline-rich 20-amino-acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60-bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60-bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragment produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over-expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.

hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase.

Vaccination against polyoma virus (PyV) tumors using vaccinia-PyV recombinants: a major tumor-specific transplantation antigen (TSTA) epitope resides within the C-terminal segment of middle-T protein.

We previously reported that inoculation of rats with live vaccinia virus (VV) recombinants VVpyMT, VVpyLT expressing either the middle-T (MT) or large-T (LT) proteins of polyomavirus (PyV) can elicit immunity to challenge with syngeneic PyV-tumor cells. We now report the results of cross-vaccination studies. VVpyMT was ineffective against cells expressing LT protein but prevented development of MT-expressing cells. Conversely, the VVpyLT was ineffective against MT-expressing cells. In the two experiments performed, tumor growth enhancement rather than retardation was observed in VVpyLT-vaccinated animals receiving PyV-LT (FRLTI) challenge tumor cells. To determine the location of the major TSTA within MT, a further VV recombinant (VVpyMT/Cfr) was constructed that expresses only the unique C-terminal segment of MT. VVpyMT-Cfr and VVpyMT were equally effective in eliciting tumor immunity, indicating the presence of a major TSTA epitope within the unique C-terminal region of MT.

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.