Mads Holten-Andersen

National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers

BACKGROUND Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS Published reports relevant to use of tumor markers for 5 cancer sites--testicular, prostate, colorectal, breast, and ovarian--were critically reviewed. RESULTS For testicular cancer, alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. alpha-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is <10 microg/L. In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, postoperative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing may be used for screening asymptomatic adults 50 years or older. For breast cancer, estrogen and progesterone receptors are mandatory for predicting response to hormone therapy, human epidermal growth factor receptor-2 measurement is mandatory for predicting response to trastuzumab, and urokinase plasminogen activator/plasminogen activator inhibitor 1 may be used for determining prognosis in lymph node-negative patients. CA15-3/BR27-29 or carcinoembryonic antigen may be used for therapy monitoring in advanced disease. CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in postmenopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer. CONCLUSIONS Implementation of these recommendations should encourage optimal use of tumor markers.

Tumor tissue levels of tissue inhibitor of metalloproteinase-1 as a prognostic marker in primary breast cancer.

Purpose: In the present study, we investigated the association between tumor tissue levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prognosis in patients with primary breast cancer and analyzed whether TIMP-1 may be useful as a prognostic marker in combination with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1). Experimental Design: In cytosolic extracts of 2984 primary breast tumors, total levels of TIMP-1 were determined using an established, validated ELISA. Levels of uPA and PAI-1 have previously been determined in the extracts. Results: Univariate survival analysis showed a significant relationship between higher levels of TIMP-1 (continuous log-transformed variable) and poor prognosis [recurrence-free survival (RFS), overall survival (OS); P < 0.001]. Performing isotonic regression analysis, we identified a cut point to classify tumors as TIMP-1-low or TIMP-1-high. Using this cut point, high levels of TIMP-1 were significantly associated with shorter survival in univariate analysis, both in the total patient group (RFS, OS; P < 0.001), in the node-negative subgroup (RFS, hazard ratio = 1.28, P = 0.006), and in the node-positive subgroup (RFS, hazard ratio = 1.43, P < 0.001). In multivariate analysis, including uPA and PAI-1, TIMP-1 was significantly associated with shorter RFS, both when included as a continuous log-transformed (P = 0.03) and as a dichotomized variable (P = 0.002). Conclusions: This study validates previous findings that tumor tissue levels of TIMP-1 are associated with prognosis in patients with primary breast cancer. It confirms that TIMP-1 may be useful as a prognostic marker in combination with uPA/PAI-1 and adds substantial positive information on the use of TIMP-1 as a prognostic marker in breast cancer.

Tumor Tissue Concentrations of the Proteinase Inhibitors Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) and Plasminogen Activator Inhibitor Type 1 (PAI-1) Are Complementary in Determining Prognosis in Primary Breast Cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29–75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3–11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II–IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.

Tumor Markers From Laboratory To Clinical Utility

A very broad definition of a tumor marker is: a tool that enables the clinician to answer clinically relevant questions regarding a cancer disease (1). However, most researchers in this field would probably prefer the following more specific definition of a tumor marker: a molecule, a process, or a substance that is altered quantitatively or qualitatively in precancerous or cancerous conditions, the alteration being detectable by an assay (2). Alterations can be produced either by the tumor itself or by the surrounding normal tissue as a response to tumor cells (2). Regardless of which definition is preferred, the tumor marker itself can be DNA, mRNA, protein, or processes (apoptosis, angiogenesis, proliferation, etc.) measured quantitatively or qualitatively by an appropriate assay. In addition, the types of specimen in which the tumor marker is detected can be different; tissue, blood (plasma/ serum), saliva, urine, etc. are all used. The tumor marker assays can be of very different formats ranging from complex animal models to immunohistochemical test kits. The most commonly used format is probably the immunoassay, which is a well-characterized methodology. However, this field is progressing rapidly, and new and advanced assays such as microarrays and mass spectrometry are becoming established technologies in tumor marker research. The first known tumor marker was described in 1846, when Henry Bence-Jones reported the precipitation of a protein in acidified urine from patients with multiple myeloma. Detection of the monoclonal immunoglobulin light chain in this disease is still in use, and since then numerous potential tumor markers have been reported on in the literature (1). Examples of such markers in clinical use are: alpha-fetoprotein for tumors of the liver, testis, and other germ cell line tumors, CA125 for ovarian cancer, prostate specific antigen (PSA) for prostate cancer, and steroid hormone receptors (estrogen and progesterone receptor) used in management of breast cancer. However, as the field of tumor markers has expanded rapidly over the last two decades with a concomitant increase in published reports, it has become increasingly apparent that a strong need exists for establishment of consensus guidelines for development and use of tumor markers. Such guidelines should be internationally accepted if any of these potential new markers are ever to reach a stage where they will benefit the patients. The guidelines should define the potential specific clinical uses of tumor markers, define specific requirements for the technical development of tumor marker assays, and state specific requirements that are to be fulfilled before clinical implementation of a tumor marker. Suggestions for such guidelines have been made; in 1996, a tumor marker expert panel convened by the American Society of Clinical Oncology proposed a framework to be used for evaluation of tumor marker studies: the tumor marker utility grading system (TMUGS), which also includes a framework for rating published evidence (2). The TMUGS framework is further discussed in “Clinical Testing.” However, work in this field is still ongoing, and some important aspects to consider in the process of designing such guidelines will be covered in this review. The possible clinical uses of tumor markers are manifold, and several categories of markers can be defined. A diagnostic tumor marker is a marker that will aid in detection of malignant disease in an individual. Preferably, the marker should be tissue specific and not be influenced by benign diseases of the particular tissue/organ. Thus, a diagnostic marker should exhibit both high levels of diagnostic sensitivity and specificity (see below) to be of clinical value, especially if the marker is to be used for (mass) screening purposes. A fundamental prerequisite for development of any diagnostic (screening) tumor marker lies in the nature of the disease From the ‡Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Ridebanevej 9, DK-1870 Frederiksberg C, Copenhagen, Denmark, §Department of Chemical Endocrinology, University Medical Centre Nijmegen, P.O. Box 9101, Geert Groteplein 10, NL-6500 HB Nijmegen, The Netherlands, ¶Clinical Research Unit, Department of Obstetrics & Gynaecology, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munchen, Germany, and Rotterdam Cancer Institute (Daniel der Hoed Klinik), Josephine Nefkens Building, Nr. BE 426, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands Received, June 9, 2003 Published, MCP Papers in Press, June 17, 2003, DOI 10.1074/mcp.R300006-MCP200 1 The abbreviations used are: PSA, prostate specific antigen; TMUGS, tumor marker utility grading system; CEA, carcino-embryonic antigen; uPA, urokinase-type plasminogen activator; PAI-1, plasminogen activator inhibitor type-1; EORTC, European Organisation for Research and Treatment of Cancer; RBG, Receptor and Biomarker Group; ER, estrogen receptor; PgR, progesterone receptor; HCG, human chorionic gonadotropin; QC, quality control; LOE, level of evidence. Review

Primary Tumor Levels of Tissue Inhibitor of Metalloproteinases-1 Are Predictive of Resistance to Chemotherapy in Patients with Metastatic Breast Cancer

Purpose: Only about 50% of metastatic breast cancer patients benefit from cytotoxic chemotherapy. Today, no validated markers exist for prediction of chemotherapy sensitivity/resistance in this patient group. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to protect against apoptosis, and the purpose of the present study was to test the hypothesis that tumors expressing high levels of TIMP-1 are protected against apoptosis-inducing agents and thus less sensitive to apoptosis-inducing chemotherapeutic drugs. Experimental Design: We investigated the association between primary tumor expression levels of TIMP-1 protein and objective response to first-line chemotherapy in 173 patients with metastatic breast cancer. Results: When analyzed as a continuous log-transformed variable, increasing TIMP-1 levels were significantly associated with lack of response to cyclophosphamide/methotrexate/5-fluorouracil and anthracycline-based chemotherapy (P = 0.01; odds ratio, 2.0; 95% confidence interval, 1.1-3.3). In a multivariate model, including lymph node status, steroid hormone receptor status, menopausal status, dominant metastases site, type of chemotherapy, and disease-free interval, TIMP-1 was significantly associated with resistance to treatment (P = 0.03; odds ratio, 1.7; 95% confidence interval, 1.1-3.3). Conclusions: In the present exploratory study, we showed that elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to chemotherapy. By using TIMP-1, we identified a group of patients with metastatic breast cancer, which hardly respond to the most frequently used chemotherapy regimes (i.e., cyclophosphamide/methotrexate/5-fluorouracil and anthracyclines).

Evaluation of sample handling in relation to levels of tissue inhibitor of metalloproteinases-1 measured in blood by immunoassay

BACKGROUND The possible effect of preanalytical conditions such as blood sample preparation and handling on TIMP-1 levels in blood needs thorough investigation. MATERIALS AND METHODS Blood was collected in dry tubes and tubes containing EDTA and kept at 4 degrees C or 20 degrees C for 1, 3, 8, 24 or 72 hours before processing into serum or EDTA plasma. In addition, serum and EDTA plasma samples were frozen and thawed 1-8 times. TIMP-1 was measured by ELISA. RESULTS Time to processing for up to 72 hours did not significantly affect TIMP-1 levels in serum. In EDTA plasma, TIMP-1 levels were stable for up to eight hours; however, if samples were kept for 24 hours or longer the TIMP-1 levels increased (p < 0.0001). Repeated freezing and thawing had a significant effect on TIMP-1 levels in serum (p = 0.04). In plasma, repeated freezing and thawing for up to six times did not influence TIMP-1. However, in plasma samples exposed to seven or eight freeze/thaw cycles TIMP-1 levels decreased, although not significantly (p = 0.23). CONCLUSIONS Handling and processing of blood samples is crucial for TIMP-1 measurement by immunoassay. In serum, TIMP-1 levels are unaffected by time to processing. Plasma samples should be processed within eight hours to avoid a TIMP-1 increase. For the measurement of TIMP-1 in archival material, serum should not be used because TIMP-1 levels are significantly affected by repeated freezing and thawing; archival plasma can readily be used provided that samples have not been frozen and thawed more than six times.

Levels of tissue inhibitor of metalloproteinases 1 in plasma and urine from patients with bladder cancer

AIM To assess the potential use of plasma and urine levels of tissue inhibitor of metalloproteinases 1 (TIMP-1) in urothelial cancer. METHODS TIMP-1 levels were determined in urine and plasma from healthy donors (n=26), patients with bacterial bladder infection (n=24), urothelial bladder adenoma (n=3) or adenocarcinoma (n=7). RESULTS Free and total TIMP-1 in plasma were weakly but significantly correlated with age; urinary TIMP-1 was not. A strong correlation between free and total TIMP-1 in plasma was observed, with an average ratio of 0.85. No correlation between total TIMP-1 in urine and plasma was found (p=0.55). No significant differences in free or total TIMP-1 in plasma were found between healthy individuals, patients with cystitis or bladder cancer (p=0.4). Urinary TIMP-1 levels were significantly increased in patients with cystitis (p=0.001). No apparent differences in TIMP-1 levels were found in patients with bladder cancer at different stages. CONCLUSION Our previous observation of a weak but significant correlation between plasma TIMP-1 and age was confirmed. Likewise, an association between free and total TIMP-1 in plasma with a ratio of 0.85 was established. No correlation between plasma and urine TIMP-1 was found. Measurement of TIMP-1 in plasma and/or urine is apparently not useful for the identification of bladder cancer.

New Tumor Biomarkers

This chapter describes the key elements in tumor biomarker development toward clinical use. In particular, it focuses on the analytical aspects of assay development and how to implement new tumor biomarkers in the clinical setting.