Evaluation of Dot-immunobinding Assay for Carcinoembryonic Antigen Determination in Nipple Discharge as an Adjunct in the Diagnosis of Early Breast Cancer

We have previously reported carcinoembryonic antigen (CEA) measurementin nipple discharge to be a useful adjunct in the diagnosisof non-palpable breast cancer. As an extension, a dot-immunobindingassay was developed to screen a large number of patients withnipple discharge for non-palpable breast cancer. The principleis as follows. CEA bound to a solid phase monoclonal anti-CEAantibody is detected by a second monoclonal anti-CEA antibodyconjugated with horseradish peroxidase. The use of tetramethylbenzidineas a chromogen results in a stable color reaction that can besemiquantitatively analyzed by the naked eye. The CEA levelsdetermined by this dot assay correlated well with CEA levelsdetermined using the former El-motec assay. To determine whetheror not the method could also be feasible in the detection ofnon-palpable breast cancer, a collaborative study from 12 Japaneseinstitutes was organized. The CEA levels in nipple dischargesfrom 155 patients were assayed. Thirteen of 30 patients withpalpable breast cancer and 22 of 30 patients with non-palpablebreast cancer exhibited CEA values higher than 400 ng/ml, acut-off value determined using 89 benign controls. The specificity(91%) and sensitivity (73%) of this test were higher than thoseof mammography or cytology. The incidence of elevated CEA levelsin nipple discharge correlated significantly with the incidenceof intratumoral antigen expression. Thus, the system could proveuseful in screening for early breast cancer     

Evaluation of an improved dot-immunobinding assay for carcinoembryonic antigen determination in nipple discharge in early breast cancer: results of a multicenter study.

We have previously reported carcinoembryonic antigen (CEA) measurement in nipple discharge to be a useful adjunct in the diagnosis of non-palpable breast cancer. We have now developed a "microdot-immunobinding assay" using a specially constructed device to screen efficiently large numbers of patients with nipple discharge for non-palpable breast cancer. The method is as follows: a sample of nipple discharge is placed on a solid phase monoclonal anti-CEA antibody and, if CEA is present in the discharge, it will be detected by a second monoclonal anti-CEA antibody conjugated with alkaline phosphatase. The use of bromochloroindolyl phosphate as a chromogen results in a stable color reaction that can be semiquantitatively analyzed with the naked eye. CEA levels determined by this microdot assay correlated well with those determined using the earlier Elmotec assay. To determine the accuracy of the method, a collaborative study involving 11 institutes in Japan was organized. The CEA levels in nipple discharges from 77 patients undergoing surgery, 44 of whom were diagnosed as having breast cancer, were assayed. The results were that 17 of the 23 patients with palpable breast cancer, and 16 of the 21 patients with non-palpable breast cancer exhibited CEA values > 400 ng/ml, a cut-off value determined in a previous study. The overall accuracy (78%) of this test for diagnosing non-palpable breast cancer was higher than that obtained from ductography or cytology. The system may thus be of use in the screening of early breast cancer.     

肿瘤标志物测定在乳腺癌早期诊断中的应用（之二）：乳头溢液检测

对天津市肿瘤医院乳腺科１９９１年３月至１９９５年３月收治连续１６９例乳头溢液患者，采集乳头溢液检测其中肿瘤标志物癌胚抗原（ＣＥＡ），铁蛋白（ＦＴ），降钙素（ＣＴ）含量来诊断乳腺癌。三种标志物分别以１００ｎｇ／ｍｌ、５００ｎｇ／ｍｌ及１００ｐｇ／ｍｌ为阳性阈值。结果以ＣＥＡ具高度特异性（９３．７％）及敏感性（８５．７１％），ＦＴ及ＣＴ虽敏感性较低，但具较高特异性（分别为７１．４％及８８．７％）。Ｔ０癌２３例同样以ＣＥＡ具高度敏感性（８６９６％）。因此三种联合检测对增强诊断作用不大。本法诊断符合率明显优于Ｘ线诊断（Ｐ＜０．００１）。     

Tumor markers. Personal experience--screening of breast cancer by determining CEA in nipple discharge

Secondary prevention, detection and treatment at an early stage, may be the only means of controlling breast cancer. This is rational behind screening for breast cancer. We have previously reported that CEA measurement in nipple discharge is a useful adjunct in the diagnosis of nonpalpable breast cancer. As an extension, a dot-immunobinding assay was developed to screen a large number of patients with nipple discharge for nonpalpable breast cancer. This article is a review of the current status of CEA assay in nipple discharge for mass screening of breast cancer. False positive and negative cases will be also described.     

Non-palpable ductal carcinomain situ (DCIS) with microinvasion arising in a radial scar presenting with spiculation alone on mammograms: A case report

A case of ductal carcinoma in situ (DCIS) with microinvasion arising in a radial scar of the breast is presented. A 57-year-old woman visited our hospital with bloody discharge from her left nipple. There were no abnormal findings on cytology, carcinoembryonic antigen (CEA) level of nipple discharge was <500 ng/ml, and mammograms were normal. After 2 years of careful periodic follow-up, spiculation without a central core appeared on mammograms. The CEA level of the nipple discharge increased to 1,000 ng/ml. Ductgraphy showed a connection between the duct with the discharge and the center of the spiculation. Since these findings suggested malignancy, she underwent segmentectomy of the breast, and pathological examination showed a radial scar and DCIS with microinvasion in the ducts within the radiating bands of fibrous tissues. We discuss the characteristics of a radial scar and its relationship to breast cancer based on our experience and a review of the literature.     

Carcinoembryonic antigen and prognosis after radical surgery for lung cancer: immunocytochemical localization and serum levels.

Eighty-two per cent of tumour sections from 105 patients with lung cancer showed positive immunocytochemical localization of an anti-carcinoembryonic antigen (CEA) immunoglobulin free of antibody to normal cross-reacting antigen (NCA). The highest incidence was found in adenocarcinomas, and no association between staining and disease stage was found. There was a relationship between positive-staining tumours and preoperative and postoperative serum CEA levels of greater than or equal to 20 ng/ml, but the high incidence of CEA+, less than 20 ng/ml serum patients indicated that immunocytochemical localization was of little value in selecting patients for sequential serum monitoring. Staining for CEA was not prognostic but a preoperative serum CEA levels greater than or equal to 20 ng/ml was associated with a poor prognosis in patients undergoing radical surgery for lung cancer (P = 0.043). this prognostic effect of CEA was seen mainly in patients whose tumours showed the greatest immunocytochemical localization (P = 0.017) and in Stage III patients (P = 0.04).     

乳腺导管内乳头状病变糖类抗原153、癌胚抗原及糖类抗原125联合检测的临床价值

目的 探讨乳头溢液糖类抗原153（CA153）、癌胚抗原（CEA）及糖类抗原125（CA125）联合检测在乳腺导管内乳头状病变诊断中的价值.方法 检测154例乳腺导管内乳头状病变患者（恶性组58例,良性组96例）乳头溢液中CA153、CEA及CA125的水平,分析其诊断价值及其与临床病理因素的关系,另选30例妊娠健康妇女乳头溢液作对照.结果 恶性组乳头溢液中CA153、CEA及CA125水平[（130.11±29.62）U/ml、（89.23±28.94） ng/ml、（41.29±16,61）U/ml]显著高于良性组和健康组（P＜0.01）;与良性组和健康组比较,恶性组溢液中CA153、CEA、CA125单项检测阳性率（62.07％、46.55％、55.17％）及联合检测阳性率（82.76％）均明显增高（P＜0.05）;恶性组乳头溢液联合检测阳性率显著高于单项检查（P＜0.01）.乳头溢液及血清三项联合检测可提高敏感性（96.55％）和阴性预测值（97.30％）.溢液CA153、CEA、CA125水平在患者年龄、部位、肿瘤大小、溢液性状不同组间差异无统计学意义（P＞0.05）;溢液CA153、CEA水平在雌激素受体（ER）、孕激素受体（PR）、人类表皮生长因子受体-2（Her-2）、缺氧诱导因子-1α（HIF-1 α）及淋巴结转移不同组差异有统计学意义（P＜0.05）;CA125在HIF-1 α、Ki-67不同组间差异有统计学意义（P＜ 0.05）;CA125在ER＆ PR、Her-2不同组间,CA153水平在Ki-67不同组间差异无统计学意义（P＞0.05）.结论 乳头溢液及血清CA153、CEA及CA125联合检测有互补性,能弥补单项检测临床应用的不足,对提高乳腺导管内乳头状癌诊断的敏感性和阴性预测值有一定意义.     

Elevated carcinoembryonic antigen levels in the milk of a nursing mother with inoperable breast cancer

Carcinoembryonic antigen (CEA) determinations performed in the milk of a nursing mother with an inoperable breast cancer revealed a 10-15-fold higher level compared with CEA levels in the milk of healthy nursing women. CEA was highly elevated in the milk of the tumor-bearing breast (1100 +/- 100 ng/ml) and moderately elevated in the milk of the clinically nonaffected breast (700 +/- 50 ng/ml). However, serum CEA levels were within normal range (9.8 +/- 0.5 ng/ml). The various theoretical and practical implications of this finding, including considerations for early breast cancer detection, are discussed.     

Human tissue polypeptide antigen in breast cancer.

Serum tissue polypeptide antigen (TPA) and plasma carcinoembryonic antigen (CEA) were simultaneously measured in 108 patients with breast cancer, in 40 healthy women, and in 26 women with benign breast disease. TPA levels were elevated (0.09 microgram/ml or higher) in 53% of 19 patients with primary breast cancer, and CEA levels were elevated (2.5 ng/ml) in 21%. Among 67 patients with metastatic breast cancer, TPA and CEA levels were increased in 70% and 61%, respectively. TPA was positive in 13% and CEA in 8% of the healthy women. CEA levels were not elevated in patients with benign breast disease, but levels of TPA were elevated in 27% of those studied. Elevation of TPA levels was more frequent in patients with visceral metastasis having higher values of the test results. Among 22 women with breast cancer who had no apparent cancer recurrence, TPA levels were elevated in 12 and CEA levels in 6. In another group of 39 patients with metastatic breast cancer who received palliative therapy, a limited correlation was noted between the clinical course of the disease and changes in TPA and CEA values measured in linear fashion. Thus TPA appeared to be equal to CEA as a tumor marker in most areas analyzed.     

High levels of DJ-1 protein in nipple fluid of patients with breast cancer

As we have previously demonstrated that some breast cancer cell lines secrete DJ-1 protein, we examined here whether breast cancer cells secrete DJ-1 protein in vivo. To this end, the levels of DJ-1 protein present in 136 specimens of nipple fluid was examined by enzyme-linked immunosorbent assay (ELISA). The average concentration of DJ-1 protein detected in diluted samples from 47 patients with invasive ductal carcinoma (IDC) was 22.4 ng/mL, while it was 18.6 ng/mL in 26 patients with ductal carcinoma in situ (DCIS). In contrast, the average DJ-1 concentration in samples from 63 women with benign lesions was 2.7 ng/mL, demonstrating that higher DJ-1 protein levels were detected in nipple fluid in the presence of cancer cells than in the presence of benign lesions (P < 0.0001). When a cut-off level of 3.0 ng/mL was applied, the higher level of DJ-1 was shown to be of significant clinical value for predicting the presence of breast cancer (85.9% specificity, 75% sensitivity; P < 0.0001). Multivariate logistic analysis that included established factors such as nipple discharge cytology, ductoscopic cytology, and carcinoembryonic antigen level further showed that the level of DJ-1 protein alone is of significant value for predicting the presence of breast cancer. Immunohistochemistry and in situ hybridization also showed that the low expression of DJ-1 protein, despite high mRNA expression, was significantly correlated with high DJ-1 protein levels in the nipple fluid. These data indicate that breast cancer cells secrete DJ-1 protein in vivo, and that its level is a potential indicator of breast cancer in patients with nipple discharge.     

Measurement of a monoclonal-antibody-defined antigen (CA19-9) in the sera of patients with malignant and nonmalignant diseases. Comparison with carcinoembryonic antigen

Immunoradiometric assay (IRMA) using monoclonal antibody for colon cancer cell surface antigen (CA19-9) was compared with carcinoembryonic antigen (CEA) with regard to sensitivity and specificity in 730 patients. In the 341 patients who had no evidence of malignant disease, CA19-9 levels ranged between less than 1.5 to 49 U/ml. Specificity of CA19-9 at a cutoff of 20 U/ml was similar to that of CEA at a cutoff of 5.0 ng/ml; CA19-9 was more sensitive than CEA in pancreatic cancer, whereas CEA was more sensitive than CA19-9 in breast, colon, and gastric cancer. Of 17 patients with pancreatic cancer, 13 had elevated levels of CA19-9 (sensitivity, 76%), whereas only 8 had elevated levels of CEA (sensitivity, 47%) and 15 had elevated levels of either CEA or CA19-9 (sensitivity, 88%). These findings suggest that, like CEA, CA19-9 is detectable in nonmalignant diseases and is not specific for gastrointestinal tumors, and has higher sensitivity than CEA only in pancreatic cancer. However, further prospective studies are required to verify its value in the diagnosis and management of pancreatic cancer.     

The value of the tumor marker CA 15-3 in diagnosing and monitoring breast cancer. A comparative study with carcinoembryonic antigen

To estimate the utility of the tumor-associated antigen CA 15-3 in the diagnosis of patients with breast cancer, this tumor marker was measured preoperatively in 1342 patients. This group included 509 patients with malignant disease (134 breast cancer patients and 375 patients with other malignancies not involving the breast) and 833 patients with benign surgical diseases (95 patients with fibroadenoma of the breast and 738 patients with other benign diseases). The results were compared with those obtained for carcinoembryonic antigen (CEA) in the diagnosis of breast cancer. The CA 15-3 level was above normal (25 U/ml) in 31% of the patients with breast cancer, in 22% of patients with other malignancies, and in 9% of patients with benign diseases. The CEA level was elevated in 26% of patients with breast cancer (more than 3 ng/ml). There was a good correlation of CA 15-3 levels with the tumor stage of breast cancer. Both CA 15-3 and CEA also were determined in 671 patients who had received initial curative surgery of breast cancer and who regularly attended our follow-up clinic. The CA 15-3 was found to be more sensitive than CEA in detecting recurrences of breast cancer. In the postcare period, carcinoma recurred in 205 patients. Of these, 73% had CA 15-3 concentrations above 25 U/ml; only 50% had CEA values above 3 ng/ml (P less than 0.0001). Although neither CA 15-3 nor CEA were sensitive enough for the screening and diagnosis of early breast cancer, CA 15-3 was significantly better than CEA in the detection of breast cancer metastases.     

Detection of microsatellite alterations in nipple discharge accompanied by breast cancer

Nipple discharge in breast cancer cases was examined loss of heterozygosity (LOH). DNA samples were extracted from both supernatant and cell pellet components of the discharge, and examined for LOH at microsatellite markers, D11S1818, D11S2000, D16S402, D16S504, D16S518, D17S520, and D17S786. At least one LOH was found in either the supernatant or cell pellet in seven out of 10 patients (70%). Five of seven samples, which were cytologically negative, were LOH positive, and only one case, which was cytologically positive, showed no LOH on the markers examined. All three samples, which were judged negative by CEA measurement (<400 ng/ml), were LOH positive. This method could be a useful novel diagnostic modality for nonpalpable breast cancer with nipple discharge.     

A case of serum CEA disappearance curve after resection of breast carcinoma

Carcinoembryonic antigen (CEA) elimination kinetics after tumor resection were measured in a case of breast cancer. A 45-year-old woman with a left breast carcinoma underwent surgery after neoadjuvant chemotherapy. The serum CEA level before surgery was 34.3 ng/ml. After sequential monitoring of serum CEA levels, postoperative serum CEA elimination kinetics were calculated using non-linear least square analysis with the fitting equation C(t)=(C0-Cp)exp(-kt)+Cp, where C(t) was the postoperative CEA level, t was the number days after surgery, C0 was the CEA level at postoperative time zero, Cp was the CEA at plateau, and k was the rate constant of elimination. Cp was calculated as 6.9 ng/ml, which was above the cut-off level and indicated residual malignancy. After adjuvant chemotherapy, CEA normalized to 1.8 ng/ml. In breast cancer patients with high preoperative serum CEA levels, our analytical method for CEA elimination might be useful for the detection of residual malignancies.     

High and differential expression of HER-2/neu extracellular domain in bilateral ductal fluids from women with unilateral invasive breast cancer.

PURPOSE: Overexpression of HER-2/neu is associated with aggressive diseaseand perhaps with increased risk of breast cancer when presentin benign breast tissue. Breast ductal fluid can be obtained from women by simple nipple aspiration and may be useful for analyzing the microenvironment of the breast. EXPERIMENTAL DESIGN: After obtaining informed consent, we prospectively compared the volume of fluid collected, protein concentration, and level of HER-2/neu expression in nipple aspiration fluid (NAF) samples from both breasts and serum samples in 65 patients with unilateral primary invasive breast cancer (median age, 54 years). HER-2/neu concentrations were determined by immunoassay, with a sensitivity of 0.1 ng/ml. RESULTS: The mean NAF volume obtained and the mean NAF protein concentration were no different in the normal versus the affected breast (62.4 versus 60.4 micro l and 140.9 versus 107.8 mg/ml, respectively). Mean serum HER-2/neu level was 4.36 ng/ml (range, 0-16.8 ng/ml), approximately 50 times less than the mean NAF HER-2/neu level from all patients and all breasts (209.2 ng/ml; range, 1.0-3480.0). NAF HER-2/neu levels were significantly correlated between breasts for each individual patient (r = 0.302; P = 0.038). HER-2/neu-overexpressing tumors produced significantly more HER-2/neu in the affected breast (653.6 ng/ml) than in the unaffected breast (101.7 ng/ml) or serum (3.46 ng/ml; P = 0.016). CONCLUSIONS: Nipple aspiration is a noninvasive method for detecting tumor-specific relevant molecular changes from ductal fluid. The presence of high HER-2/neu levels in the ductal systems of breast cancer patients may have clinical implications for monoclonal antibody directed therapy.     

Carcinoembryonic antigen in gastric cancer patients

Carcinoembryonic antigen (CEA) levels were determined in 252 gastric cancer patients. In patients with resectable cancer, the preoperative CEA values and CEA positivity rates were 2.4 +/- 1.5 ng/ml and 7.7% for stage I, 24.9 +/- 72.0 ng/ml and 10.0% for stage II, 21.6 +/- 84.1 ng/ml and 17.9% for stage III, and 6.3 +/- 8.4 ng/ml and 27.1% for stage IV cancers, respectively. In patients with nonresectable cancers, the CEA value was 83.0 +/- 235.5 ng/ml, the CEA positivity rate was 47.8%. Overall, of 252 patients with primary gastric cancer, 47(18.7%) were positive for CEA. In patients with cancer recurrence, the CEA value averaged 41.8 +/- 101.8 ng/ml, the positivity rate was 63%. This rate increased as the cancer stage increased; it was highest in gastric cancer patients with liver metastasis. In 4 of 13 patients with recurrence, an elevation in CEA was observed about 4.8 months before the clinical detection of cancer recurrence. Our results suggest that in gastric cancer patients, the preoperative and periodic postoperative assay of CEA levels has predictive value in determining cancer stage, progression and recurrence.     

Carcinoembryonic Antigen (CEA) Levels in Pleural Effusions and Sera of Lung Cancer Patients

For determining the value of carcinoembryonic antigen (CEA)levels in diagnosis of malignant tumors of the lung, the CEAlevels in 187 specimens of pleural fluid and sera obtained simultaneouslyfrom patients with pleural fluid were measured. In all 70 patientswith benign diseases, the CEA levels in the effusions were lessthan the cut-off value of 5 ng/ml (mean±SD: 1.44±1.01ng/ml). In contrast, in 88 of 117 patients (75.2%) with malignantdiseases, the CEA levels in the effusions were over 5 ng/ml(25.3±24.5 ng/ml) and in 58 of the 117 patients (50.4%),the CEA levels in the serum were values of 5 ng/ml or more (11.9±18.4ng/ml). There was a significant correlation between the CEAlevels in the effusions and in the sera. The CEA levels in effusionsin patients with malignant lung tumors were usually much higherthan those in their sera. The incidence of CEA levels of 5 ng/mlor more in both the serum and effusion was highest in the patientswith adenocarcinoma. These data indicate that determination of the CEA level in effusions,when done in combination with cytological examinations, mayhave additional value in diagnosis of lung cancer.     

Circulating levels of epithelial membrane antigen in patients with breast or colorectal-cancer

Epithelial membrane antigen (EMA) is expressed by adenocarcinomas of the breast, ovary and colon and has been suggested as a circulating tumour marker. Serum EMA levels were measured in 126 patients, 31 with colorectal cancer, 52 with breast cancer and 43 age matched controls using a competitive binding radioimmunoassay and the rat anti-EMA monoclonal antibody (MAb) ICR2. The EMA levels in the control group varied widely from 90-3240ng/ml with a median value of 570ng/ml. This was not significantly different from the levels in patients with colorectal cancer (60-8530, median 580ng/ml) or those with breast cancer (210-13300, median 655ng/ml). However, the highest EMA levels (>5000ng/ml) were found in patients with cancer. The wide range of EMA levels in the control group prohibit its use for screening.     

Carcinoembryonic antigen and phosphohexose isomerase, gammaglutamyl transpeptidase and lactate dehydorgenase levels in patients with and without liver metastases.

Plasma carcinoembryonic antigen (CEA) and serum enzyme levels of phosphohexose isomerase (PHI), gamma-glutamyl transpeptidase (psi-GTP), and lactate dehydrogenase (LDH) were measured in 147 patients with malignancy. Levels were higher in patients (particularly with G.I., breast and lung cancers) than in normals or in patients with cancer in clinical remission. Elevations of CEA and of all three enzymes in blood were most frequent in patients with hepatic metastases. CEA elevations correlated directly with PHI levels. Seventy-eight percent of patients with metastatic G.I. cancer could be identified by CEA (greater than 5 ng/ml) alone, as well as 38% with breast cancer and 85% with lung cancer; but only 17% of other cancers could be identified by CEA alone. CEA or one or more enzymes was elevated in 64% of metastatic breast cancer patients, 92% of lung cancer and 41% of other cancers, but enzyme measurement did not increase identification of G.I. cancer over that achieved by CEA alone. These findings suggest that circulating levels of CEA, PHI, psi-GTP and LDH may reflect a direct contribution from the malignant tissue and/or liver malfunction secondary to liver replacement.     

TPS in breast cancer--a comparative study with carcinoembryonic antigen and CA 15-3.

The serum levels of tissue polypeptide antigen were determined using the M3 monoclonal antibody (TPS) and compared with the serum levels of carcinoembryonic antigen (CEA) and breast carcinoma antigen 15-3 (CA 15-3) in 96 patients with benign breast tumors, in 25 breast cancer patients with no evidence of disease, in 139 preoperative breast cancer patients and in 298 samples of 25 breast cancer patients during therapy monitoring (4-22 samples per patient). The 95th percentile of TPS in 89 apparently healthy females was 51 U/l. The 95th percentile of TPS in patients with benign breast tumors was 55 U/l. The maximum TPS level in breast cancer patients with no evidence of disease was 56 U/l. In preoperative breast cancer the number of patients with TPS levels above the 95th percentile of TPS in benign breast tumors was significantly higher in stage III breast cancer as compared with stage I+II. This was not established for CEA and CA 15-3. During therapy monitoring TPS followed the course of the disease faster than CEA and CA 15-3 in patients with bone metastases, liver metastases, lung metastases and pleural effusion, with one exception. TPS levels could be correlated with progression of disease in patients with normal and steady levels of CEA and/or CA 15-3.