R. Einarsson

Tumor Markers in Breast Cancer – European Group on Tumor Markers Recommendations

Recommendations are presented for the routine clinical use of serum and tissue-based markers in the diagnosis and management of patients with breast cancer. Their low sensitivity and specificity preclude the use of serum markers such as the MUC-1 mucin glycoproteins (CA 15.3, BR 27.29) and carcinoembryonic antigen in the diagnosis of early breast cancer. However, serial measurement of these markers can result in the early detection of recurrent disease as well as indicate the efficacy of therapy. Of the tissue-based markers, measurement of estrogen and progesterone receptors is mandatory in the selection of patients for treatment with hormone therapy, while HER-2 is essential in selecting patients with advanced breast cancer for treatment with Herceptin (trastuzumab). Urokinase plasminogen activator and plasminogen activator inhibitor 1 are recently validated prognostic markers for lymph node-negative breast cancer patients and thus may be of value in selecting node-negative patients that do not require adjuvant chemotherapy.

Epitope Specificity of 30 Monoclonal Antibodies against Cytokeratin Antigens: The ISOBM TD5-1 Workshop

The epitope specificities of 30 monoclonal antibodies (MAbs) against the most common human cytokeratins, i.e., Nos. 8, 18, and 19, in epithelial cells were investigated in the ISOBM TD-5 Workshop. Seven research groups from universities or companies participated independently in the evaluation of the antibody specificities. The complex assembly of cytokeratins in vivo, with obligatory heterologous dimeric combinations of different cytokeratins from each of the two major groups, comprising together more than 20 different individual cytokeratins, made analysis of the antibody reactivity patterns with isolated single cytokeratins necessary. The concordance of the evaluations was striking and independent of the technologies used. As antigens purified individual cytokeratins, chemically degraded purified cytokeratins, recombinant intact and truncated cytokeratins, as well as specific synthesized shorter peptides were used. In order to elucidate the epitope specificity, reactivity patterns in ELISA assays and immunoblots with partial enzymatic degradation of the antigens were performed. Competitive cross-inhibition experiments between antibodies using antigens and antibodies in all possible combinations were performed with radioimmunometric assays, BIAcore, and ELISA technology.All 30 antibodies could convincingly be classified with regard to target cytokeratin. One MAb (192) had to be deleted due to dual specificities in both isotype and epitope specificity against its target. Six antibodies bound selectively to cytokeratin 8, 14 to cytokeratin 18, and 10 to cytokeratin 19, as demonstrated by using native, recombinant, and synthesized antigens. The immunodominant part of the molecule for all three types of cytokeratins was located in the region of amino acid (aa) 270–400. Out of the six MAbs reactive with cytokeratin 8, four MAbs, i.e., 178, 199, 202, and 206, were reactive with a sequence in the interval aa 340–365, and MAb 191 reacted with a closely related epitope. The remaining antibody, 192, presented dual specificities. At least two closely related major immunogenic epitopes could be identified in cytokeratin 8. In cytokeratin 18 four distinct epitopes could be documented, again with the dominating sequence region 270–429 as target for 10 (181, 184, 186, 188, 189, 190, 193, 196, 198, and 200) out of 14 antibodies. Since MAb 193 is known to react with the M3 epitope, aa 322–342 in cytokeratin 18, this entire group is reactive in the region close to the charge shift, in the middle of the rod 2B region, as shown by competitive binding. The remaining four anticytokeratin 18 antibodies (180, 185, 203, and 205) displayed unique, noncompetitive binding to this filament. Cytokeratin 19, reactive with altogether ten antibodies, displayed two major epitopes, all of them also within the large immunodominant region. MAbs 179, 195, 197, and 204 were reactive with the peptides aa 311–335 also known as the KS 19.1 epitope, and MAbs 182, 183, 187, 194, and 201 bound to peptide aa 346–367, known as the BM 19.21 epitope. One antibody, 231, was selectively reactive with aa 356–370 in cytokeratin 19. A complex pattern of binding specificities comprising at least ten different, noncompetitive epitopes, mainly situated in the rod portion, 2A and 2B, situated close to the charge shift in the rod of all three cytokeratins was documented. Out of the 29 classifiable antibodies, altogether 22 were reactive in this very short region, i.e., from aa 311 to 370 in all cytokeratin filaments. The remaining seven antibodies displayed unique binding properties. The implications of the findings are of significance both for immunohistochemistry and for assaying circulating heterodimeric, partially degraded complexes in patients’ blood for tumor marker evaluation.

Comparative analysis of CA125, tissue polypeptide specific antigen, and soluble interleukin-2 receptor α levels in sera, cyst, and ascitic fluids from patients with ovarian carcinoma

The serum markers CA125, tissue polypeptide specific antigen (TPS), and soluble interleukin‐2 receptor alpha (sIL‐2Rα) concentrations were determined in sera, cyst, and ascitic fluids from patients with malignant and benign ovarian neoplasms.

Tissue polypeptide specific antigen (TPS), a marker for differentiation between pancreatic carcinoma and chronic pancreatitis

The value of serum tissue polypeptide specific antigen (TPS) as a complement to CA 19‐9 in the detection of pancreatic carcinoma was determined prospectively. TPS and CA 19‐9 levels obtained at the time of diagnosis in patients suspected of having chronic pancreatitis or pancreatic carcinoma were evaluated in receiver operating characteristic (ROC) curve analysis.

Treatment response in metastatic breast cancer. A multicentre study comparing UICC criteria and tumour marker changes

Abstract One hundred and twenty-nine patients with dissiminated breast cancer were selected to study the clinical applicability of changes in CA 15-3, tissue polypeptide specific antigen (TPS) and carcinoembryonic antigen (CEA) during therapy and how these compare with responses as assessed by UICC criteria in a multicentre study. The alterations in tumour marker levels followed the course of disease but indicated changes in the disease state earlier than clinical assessment (UICC criteria). Patients were grouped according to whether they had progressive disease (PD in groups III and IV), stable disease (SD in group II) or partial remission/complete remission (PR/CR in group I) during a treatment period of 6 months. The number of patients with a 50% decrease of the marker level, and the time at which this reduction was reached, was analysed in patient groups I–III. TPS showed this 50% decrease more frequently (69%) and also earlier (1.1 months) than either CA 15-3 (46% and 2.5 months) or CEA (47% and 1.7 months). The number of patients for whom marker levels increased by 25% or more was analysed in patient groups III and IV versus groups I and II. Such rises were seen more often in patients with progressive disease (TPS 82%, CA 15-3 62% and CEA 61%) than in patients with a more favourable prognosis (TPS 43%, CA 15-3 22% and CEA 24%). The dynamics of changes in tumour marker levels were different during treatment in patients with SD, PR or CR. However, the markers showed similar behaviour in patients developing PD during the study period. TPS appeared to indicate outcome somewhat better than did either CA 15-3 or CEA. Combined determination of CA 15-3 and TPS during therapy for metastatic breast cancer is recommended.

The prognostic significance of increasing marker levels in metastatic breast cancer patients with clinically complete remission, partial remission or stable disease

TPS, CA 15-3 and CEA were determined in metastatic breast cancer patients during treatment. After six months of follow-up the patients were divided into four groups according to the UICC criteria for treatment response. Forty-six patients with a more favorable prognosis (complete remission, partial remission or stable disease) were followed for an extended period. In 30 of the 46 patients at least one marker had increased at the end of the six-month period by at least 25% (TPS in 54%, CA 15-3 in 20%, CEA in 20%). All these 30 patients subsequently developed progression. The prognostic sensitivity was 83%, 30% and 30%, respectively, for TPS, CA 15-3 and CEA. The combination of TPS and CA 15-3 showed a sensitivity of 96%. The median lead time was about 8 months for TPS and CA 15-3, but less than 50% of the patients showed a lead time for CA 15-3 as compared to TPS. We conclude that TPS and CA 15-3 determinations are helpful for the prediction of progression during the follow-up of breast cancer patients.

TPS and CA 19-9 measurements in the follow-up of patients with pancreatic cancer and chronic pancreatitis

The aim of this study was to assess the value of TPS and CA 19–9 in a long-term follow-up analysis of 11 patients with chronic pancreatitis (CP) and 15 patients with pancreatic cancer (PC). In all monitored patients with chronic pancreatitis the initial TPS level was below 200 U/L, whereas CA 19–9 was elevated in two of them. In one patient a dramatic increase in the TPS concentration (820 U/L) was measured at the last follow-up visit (after 8.6 months), which led to the detection of PC. In all patients with PC the preoperative TPS level exceeded 200 U/L, whereas CA 19–9 was elevated in only nine patients. After the Kausch-Whipple operation 11 patients showed no evidence of disease and in eight of these patients both TPS and CA 19–9 were within the reference range; however, in three patients liver metastases were detected after 8–24 months from the last tumor marker measurement. In four of the 15 patients both markers were elevated at the end of the follow-up period and distant metastases were clinically confirmed. Our results indicate that in patients with CP and PC undergoing long-term follow-up, TPS reflects the clinical status of patients more accurately than CA 19–9.

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 μg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340–365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311–335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.