Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy.

PURPOSE: HER-2 is overexpressed in 20% to 30% of human breast cancer and is associated with poor outcome. Studies suggest an association between HER-2 overexpression and resistance to alkylating agents. To further evaluate this relationship, we assessed the interaction of HER-2, measured by different methods, and outcome after dose intensification with alkylating agents in metastatic breast cancer. PATIENTS AND METHODS: From 1988 to 1995 at Duke University, 425 patients with metastatic breast cancer were enrolled in a study of high-dose alkylating agents (HDC) with autologous cellular support after doxorubicin-based therapy (AFM). HER-2 was measured in serum for shed extracellular domain (ECD) and in tissue by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). RESULTS: HER-2 ECD was positive in 29% (19 of 65) of patients pre-AFM and in 11.7% (34 of 290) pre-HDC. Higher pre-AFM and higher pre-HDC HER-2 ECD predicted worse overall survival (P =.045 and P =.0096, respectively). HER-2 overexpression by IHC and FISH showed no correlation with worse disease-free survival or overall survival. FISH and ECD were highly specific for IHC (97.3% and 97.7% respectively). However, ECD had a low sensitivity for IHC-only 22% of patients with HER-2 in the primary tumor shed ECD into the serum. CONCLUSION: These data suggest that the method of measuring HER-2 is important in predicting clinical outcome. HER2 ECD may identify a poor prognosis subgroup of HER-2-positive tumors. Lack of association of HER2 by IHC/FISH with worse outcome suggests that therapy with AFM and/or HDC therapy may be able to overcome the effect of this prognostic factor or it may not be a prognostic factor in this setting.

Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma.

Soluble MUC1 (sMUC1) levels are elevated in many MUC1(+) cancers. We and others have shown that MUC1 is expressed on multiple myeloma (MM) plasma cells and B cells. In this study, we measured sMUC1 levels in bone marrow (BM) plasma from 71 MM patients and 21 healthy donors (HDs), and in peripheral blood (PB) plasma from 42 MM patients and 13 HDs using an immunoassay that detects the CA27.29 epitope of MUC1. sMUC1 levels were found to be significantly greater (mean 31.76 U/mL, range 5.69 to 142.48 U/mL) in MM patient BM plasma versus HD BM plasma (mean 9.68 U/mL, range 0.65 to 39.83 U/mL) (P <. 001). Importantly, BM plasma sMUC1 levels were related to tumor burden because sMUC1 levels were significantly higher for MM patients with active disease (34.62 U/mL, range 5.69 to 142.48 U/mL) versus MM patients with minimal residual disease (16.16 U/mL, range 5.7 to 56.68 U/mL) (P =.0026). sMUC1 levels were also elevated in the PB plasma of MM patients (32.79 U/mL, range 4.15 to 148.84 U/mL) versus HDs (18.47 U/mL, range 8.84 to 42.49) (P =.0052). Lastly, circulating immunglobulin M (IgM) and IgG antibodies to MUC1 were measured in 114 MM patients and 31 HDs, because natural antibodies to MUC1 have been detected in patients with other MUC1-bearing malignancies. These studies demonstrated lower levels of circulating IgM (P <.001) and IgG (P =.078) antibodies to MUC1 in MM patients compared with HDs. Our data therefore show that in MM patients, sMUC1 levels are elevated and correlate with disease burden, whereas anti-MUC1 antibody levels are decreased.

Clinical use of serum c-erbB-2 in patients with ovarian masses.

The c-erbB-2 (Her-2/neu) gene product has a large extracellular domain (ECD) and part of which could be identified in the serum. We measured the serum level of c-erbB-2 ECD in 93 patients, who presented with ovarian masses, with an enzyme immunoassay test and an elevated level was found in 5.5, 16.7 and 38% of patients with benign, borderline and malignant ovarian neoplasms, respectively. This serum marker may reflect the overexpression of c-erbB-2 gene in tumor tissues, which is associated with poor prognosis. However, measurement of c-erbB-2 ECD when used alone or in combination with CA 125 is not useful in differentiating benign from malignant ovarian tumors.

The prognostic significance of c-erbB-2 serum protein in metastatic breast cancer.

The relationship between c-erbB-2 serum positivity and prognosis was evaluated in 80 patients with metastatic breast cancer. Using 120 fmol/ml as a cutoff level, elevated concentrations were found in 31 patients (38.8%) at the time of detection of metastases. Menopausal status, steroid receptor status, site of recurrence, initial tumor size, initial degree of nodal involvement as well as relapse-free interval were unrelated to c-erbB-2 serum positivity. In addition, no association could be found between adjuvant chemotherapy and positive c-erbB-2 concentrations. Patients with elevated c-erbB-2 levels showed a lower response rate (including complete remission, partial remission, no change) to first-line therapy than those with normal levels (29 vs. 59%, p < 0.01). The median survival time after relapse was 12 months (CI: 3-22 months) for the c-erbB-2-negative patients and only 6 months (CI: 3-8 months) for the c-erbB-2-positive group (p < 0.01). In the multivariate analysis, while c-erbB-2 levels at the time of primary surgery had no significant impact on survival in metastatic breast cancer, serum c-erbB-2 turned out to be the strongest factor for predicting survival after relapse.

Influence of circulating c-erbB-2 serum protein on response to adjuvant chemotherapy in node-positive breast cancer patients.

This retrospective case control study investigated the therametric value of the circulating c-erbB-2 gene product (Her-2, NEU) as (1) an eligibility criterion for high doses of chemotherapy and (2) response to standard adjuvant chemotherapy in node-positive breast cancer patients. Preoperative c-erbB-2 levels were measured in 211 locally advanced (> 3 nodes positive), pre- and perimenopausal breast cancer patients to determine if circulating levels of the gene product can assist in the determination of appropriate therapeutic options. 152 of 211 breast cancer patients received post-operatively a combination chemotherapy including the anthracycline analog mitoxantrone, while 59 patients were treated with conventional CMF therapy. Using 120 fmol/ml as a cut-off level, elevated c-erbB-2 values were found in 26 (12.3%) patients with locally advanced breast cancer. In univariate analysis significant survival differences were detected when c-erbB-2 'positive' patients were compared with c-erbB-2 'negative' patients. However, no significant survival differences were detected, when c-erbB-2 'positive' patients were compared according to regimen of adjuvant treatment. In multivariate analysis c-erbB-2 was an independent prognostic factor for predicting disease-free survival, but not for overall survival. High levels of c-erbB-2 were associated with low estrogen and progesterone receptor concentrations of the tumor cytosol. There was no correlation between elevated c-erbB-2 values and age, tumor size or degree of nodal involvement. c-erbB-2 was a better predictor of risk of recurrence than extent of nodal involvement or hormone receptor status.

Mucins bearing the cancer-associated sialosyl-Tn antigen mediate inhibition of natural killer cell cytotoxicity.

The sialosyl-Tn (STn) antigen is a mucin-associated carbohydrate antigen expressed by a variety of adenocarcinomas. In the colon, expression of this antigen has been associated with a poor prognosis, independent of tumor stage or histology. The present study was performed to determine whether this adverse clinical outcome might be due to an interaction between STn-positive mucin and natural killer (NK) cell cytotoxicity. Ovine submaxillary mucin (OSM), a mucin highly rich in STn antigen, partially inhibited NK cell cytotoxicity against K562 target cells, but only at high concentrations. Low concentrations of OSM were not inhibitory but became markedly inhibitory in the presence of ammonium ions. Two other STn-positive submaxillary mucins also markedly inhibited NK cytotoxicity when combined with ammonium ions. Removal of sialic acid from OSM reversed the OSM/ammonium-mediated inhibition of NK cell activity. Unlike the submaxillary mucins, two mucins derived from human breast and lung cancer cells which lack the STn antigen, did not inhibit NK cell activity in this system. Likewise, four other non-mucin glycoproteins which lack STn expression did not inhibit NK cells despite having levels of sialic acid that were, in some cases, comparable to submaxillary mucin. These results indicate that mucins bearing the cancer-associated STn antigen can effectively inhibit NK cell cytotoxicity in the presence of ammonium ions. While this NK cell inhibition is likely to be caused by ammonium, mucin markedly enhances this effect, thereby implicating a novel immunomodulatory property of mucin.

Tumor selective reactivity of a monoclonal antibody prepared against a recombinant peptide derived from the DF3 human breast carcinoma-associated antigen.

The DF3 antigen is a member of a family of high molecular weight glycoproteins aberrantly expressed in malignant mammary epithelium. We have generated a monoclonal antibody (MAb), designated DF3-P, against a recombinant DF3/beta-galactosidase fusion protein. Characterization of this MAb has demonstrated reactivity with immature precursors of DF3 antigen and not with the secreted form. These findings are in contrast to those obtained with MAb DF3, a previously described antibody with predominant reactivity against the mature glycoprotein. The finding that deglycosylation of secreted DF3 antigen with neuraminidase and endo-alpha-N-acetylgalactosaminidase is associated with increased MAb DF3-P reactivity provided additional support for the selectivity of this antibody against the protein core. Epitope mapping studies demonstrate that both the DF3-P and DF3 epitopes are located at a TRPAPGS domain in the 20-amino acid tandem repeat. The results of competition studies with synthetic peptides indicate that the proline in this domain is involved in both epitopes, while the potential glycosylation sites at threonine and serine may contribute to the differential reactivity of MAbs DF3 and DF3-P. Taken together, these findings suggest that both antibodies react with a similar epitope that is modified by the presence of carbohydrate moieties. The results of immunoperoxidase staining studies further demonstrate that while MAb DF3-P reacts with formalin-fixed sections of breast carcinomas, this antibody exhibits little if any reactivity with normal mammary epithelium. Selective expression of the DF3-P epitope in malignant breast cells may be useful in identifying this transformed phenotype.

A new radioimmunoassay detecting early stages of colon cancer: a comparison with CEA, AFP, and Ca 19-9.

A previous study reported the results of a radioimmunoassay that analysed immune complexes (IC) with a specific labeled polyclonal antibody for the detection of the early stages of colon cancer. In order to investigate further the possible clinical use of this assay, a blind study that screened 505 patients referred for colonoscopy, compared their pathology reports with the results of four assays that measure levels of circulating tumor markers. These were CEA, AFP, Ca 19-9, and our previously described radioimmunoassay (RIA). Of the patients with no malignancies, the results that were in the normal range were as follows: CEA-473/495 (95.6 per cent), Ca 19-9-486/495 (98.2 per cent) and our RIA-488/495 (98.6 per cent). AFP levels were in the normal range for all patients in the study. The only assay to identify any Dukes' C and D patients was Ca 19-9, which detected 2/3 (67 per cent). Of the patients with Dukes' A and B colon cancer, CEA only identified 1/7 (14 per cent), AFP and Ca 19-9 0/7 (0 per cent), and our own RIA 5/7 (71 per cent). The positive results of our assay were significantly different from those of the other three assays with p values all less than 0.05. These preliminary results suggest that this RIA, because of its ability to detect the early stages of colon cancer, may be an effective complement to the currently available assays. The combination may provide a more comprehensive evaluation in monitoring colon cancer.

Lung cancer-associated protein: development and characterization of a new assay that detects a circulating lung cancer marker.

A panel of murine monoclonal antibodies was generated against a high-molecular-weight glycoprotein produced by human lung cancer cells. This lung cancer-associated protein (LCAP) has been shown to circulate in the plasma of patients with lung cancer. Various combinations of MAbs were used in solid-phase enzyme-linked sandwich immunoassays to optimize the detection of LCAP in the plasma of these patients. One of these monoclonal antibodies, designated DF-L1, used both in the solid phase as well as the tracer, was selected to evaluate circulating levels of LCAP in normal subjects and in patients with lung cancer. In 341 normal subjects, the mean LCAP level was 7 units/ml, with 47 (13.8%) and 18 (5.3%) subjects having levels greater than or equal to 15 units/ml and 23 units/ml, respectively. In contrast, 27 of 35 (77.1%) patients with lung cancer had LCAP levels greater than or equal to 23 units/ml. A total of 16 of 19 (84.2%) patients with adenocarcinoma, four of seven (57.1%) patients with squamous cell carcinoma, and four of six (66.7%) patients with small cell carcinoma had levels greater than or equal to 23 units/ml. Moreover, in a small group of patients, serial LCAP levels correlated with clinical course during therapy. The LCAP assay is technically reproducible and unaffected by interfering substances in the blood or by variations in the handling of samples. These results indicate that LCAP is a new and potentially useful marker for the evaluation of patients with lung cancer.

Detection and characterization of a high molecular weight human lung carcinoma-associated glycoprotein.

We have defined a human lung carcinoma antigen using murine monoclonal antibodies (DF-L1 and DF-L2) prepared against a primary adenocarcinoma of the lung. This antigen is expressed on the surface of human lung carcinoma cell lines and has an apparent Mr of 350,000-420,000. Immunoperoxidase staining has demonstrated expression of the antigen in the cytoplasm and membranes of adenocarcinomas and squamous cell carcinomas but not small cell tumors of the lung. Immunoprecipitation of the antigen following radiolabeling has demonstrated the presence of both protein and carbohydrate. Antigen purified by immunoaffinity was used to study the epitopes defined by monoclonal antibodies DF-L1 and DF-L2. The results indicate that the DF-L1 epitope primarily involves a peptide structure, while the DF-L2 epitope is comprised in part by peptide and O-linked carbohydrate. In contrast, there was no detectable evidence for the presence of N-linked glycosylation. The results also demonstrate that this antigen circulates at elevated levels in patients with carcinoma of the lung. These findings are similar to previous reports of high molecular weight glycoproteins in breast and ovarian carcinomas. Indeed, the present results in lung cancer identify another member of this heterogeneous family of human carcinoma-associated glycoproteins.

An analysis of immunocomplexes for the detection of the early stages of colon cancer.

A radioimmunoassay was developed for the detection of the early stages of colon cancer by analysis of immune complexes (IC) with a specific polyclonal antibody. Human colon cancer cells were grown in a capillary culture system to provide unaltered antigens for the development of a specific antibody. The antibody was labeled with iodine 125 (125I) and used to analyze the antigen component of IC removed from whole serum. The assay was positive in 50% and 88% of known Dukes' A and Dukes' B colon cancer patients, respectively. It was also positive for only 25% of Dukes' C and 14% of Dukes' D patients, possibly because of the decreased quantity of specific IC found in the late stages of colon cancer. A blind study of patients referred for colonoscopy compared pathology diagnosis with the test results. The assay was positive for one patient with a polypoid adenocarcinoma (Dukes' B) and one with a villous adenoma and negative for 38 patients with benign polyps and 43 with no polyps. The assay was negative for all patients with stomach cancer and inflammatory bowel diseases and positive for about 10% of the patients with pancreas or breast cancer. The results of this preliminary investigation suggest that this radioimmunoassay may be useful for the detection of the early stages of colon cancer.