Barry L. Dowell

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.

Free, Complexed and Total Serum Prostate Specific Antigen: The Establishment of Appropriate Reference Ranges for their Concentrations and Ratios

PURPOSE Prostate specific antigen (PSA) exists in the serum in several molecular forms that can be measured by immunodetectable assays: free PSA, PSA complexed to alpha 1-antichymotrypsin (complexed PSA) and total PSA, which represents the sum of the free and complexed forms. We determined the normal distribution of values and established the appropriate reference ranges for these 3 molecular forms of PSA and their ratios (free-to-total, complexed-to-total and free-to-complexed PSA). Knowing the amount and ratio of these molecular forms appears to be useful in enhancing the ability of PSA to distinguish potentially curable prostate cancer from benign prostatic hyperplasia and in decreasing the number of unnecessary prostate biopsies. MATERIALS AND METHODS A total of 422 healthy men 40 to 79 years old was randomly chosen from the male population of Olmsted County, Minnesota and underwent a detailed clinical examination that included digital rectal examination, serum PSA determination and transrectal ultrasound to exclude the presence of prostate cancer. Using newly developed, monoclonal-monoclonal immunofluorometric assays for each molecular form, the free, complexed and total PSA, and the ratios of these 3 forms were determined for each study participant. RESULTS All 3 molecular forms correlated directly with patient age (r = 0.45, r = 0.43 and r = 0.45, respectively). Using the 95th percentile, the recommended age-specific reference ranges for the free, complexed and total PSA forms, respectively, are 0.5, 1.0 and 2.0 ng./ml. for men 40 to 49 years old; 0.7, 1.5 and 3.0 ng./ml. for men 50 to 59 years old; 1.0, 2.0 and 4.0 ng./ml. for men 60 to 69 years old, and 1.2, 3.0 and 5.5 ng./ml. for men 70 to 79 years old. With regard to each of the ratios (free-to-total, complexed-to-total and free-to-complexed PSA) none correlated with patient age. As a result, the appropriate upper limit of normal (95th percentile) for all 3 ratios is constant for men of all ages. These reference ranges are greater than 0.15 for free-to-total PSA ratio, less than 0.70 for complexed-to-total PSA ratio and greater than 0.25 for free-to-complexed PSA ratio. The free-to-total PSA ratio will have its greatest value for men with a serum PSA value between 2 and 10 ng./ml. CONCLUSIONS The establishment of appropriate reference ranges for free, complexed and total PSA as well as the ratios will allow the practicing urologist to incorporate these new parameters into the diagnostic evaluation of men at risk for early, potentially curable prostate cancer.

Emerging Biomarkers for the Diagnosis and Prognosis of Prostate Cancer

BACKGROUND Early detection of prostate cancer (CaP), the most prevalent cancer and the second-leading cause of death in men, has proved difficult, and current detection methods are inadequate. Prostate-specific antigen (PSA) testing is a significant advance for early diagnosis of patients with CaP. CONTENT PSA is produced almost exclusively in the prostate, and abnormalities of this organ are frequently associated with increased serum concentrations. Because of PSAs lack of specificity for CaP, however, many patients undergo unnecessary biopsies or treatments for benign or latent tumors, respectively. Thus, a more specific method of CaP detection is required to augment or replace screening with PSA. The focus recently has been on creating cost-effective assays for circulating protein biomarkers in the blood, but because of the heterogeneity of CaP, it has become clear that this effort will be a formidable challenge. Each marker will require proper validation to ensure clinical utility. Although much work has been done on variations of the PSA test (i.e., velocity, density, free vs bound, proisoforms) with limited usefulness, there are many emerging markers at various stages of development that show some promise for CaP diagnosis. These markers include kallikrein-related peptidase 2 (KLK2), early prostate cancer antigen (EPCA), PCA3, hepsin, prostate stem cell antigen, and alpha-methylacyl-CoA racemase (AMACR). We review biomarkers under investigation for the early diagnosis and management of prostate cancer. SUMMARY It is hoped that the use of panels of markers can improve CaP diagnosis and prognosis and help predict the therapeutic response in CaP patients.

DETERMINATION OF THE "REFLEX RANGE" AND APPROPRIATE CUTPOINTS FOR PERCENT FREE PROSTATE- SPECIFIC ANTIGEN IN 413 MEN REFERRED FOR PROSTATIC EVALUATION USING THE AxSYM SYSTEM

OBJECTIVES Prostate-specific antigen (PSA) exists in the serum in two clinically important molecular forms: free PSA and PSA complexed to alpha 1-antichymotrypsin. Total PSA approximates the sum of the free and complexed forms. Preliminary investigations have illustrated the potential benefits of using percent free PSA to enhance the clinical utility of PSA in distinguishing benign prostate disease from prostate cancer. The current study defines the optimal range of total PSA for measuring percent free PSA (reflex range) and generates appropriate cutpoints for percent free PSA within this range. METHODS A total of 413 patients, 225 (54%) with benign prostate disease (mean age, 67 years) and 188 (46%) with prostate cancer (mean age, 66 years), who had PSA values between 2.0 and 20.0 ng/mL participated in the investigation. All patients underwent a sextant biopsy to establish the diagnosis. The serum specimens were assayed with the AxSYM PSA assay (total PSA) and AxSYM Free PSA assay (Abbott Laboratories; Abbott Park, IL). Percent free PSA was calculated for all patients. Receiver operating characteristic (ROC) curves were generated for various ranges of total PSA to determine the reflex range that maximized the increase in sensitivity and specificity of percent free PSA over total PSA. Within the optimal range, the ROC curves were utilized to generate cutpoints for percent free PSA to be used in clinical practice. RESULTS The appropriate reflex range for the utility of percent free PSA was 3.0 to 10.0 ng/mL. The appropriate cutpoint for percent free PSA when the total PSA value was 3.0 to 4.0 ng/mL to achieve 90% sensitivity for the detection of prostate cancer was 0.19. This approach resulted in a biopsy rate of 73% and a cancer detection rate of 44% in men with a total PSA value between 3.0 and 4.0 ng/mL. The appropriate cutpoint for percent free PSA when the total PSA value was 4.1 to 10.0 ng/mL to ensure 95% sensitivity for detection of prostate cancer was 0.24. Within the range of 4.1 to 10.0 ng/mL, this approach resulted in 13% fewer negative biopsies and failure to detect 5% of the cancers. CONCLUSIONS Percent free PSA should be utilized in patients with a total serum PSA value between 3.0 and 10.0 ng/mL. In patients with a total PSA value between 3.0 and 4.0 ng/mL, percent free PSA enhanced the detection of prostate cancer (improving sensitivity). In patients with a total PSA concentration ranging from 4.1 to 10.0 ng/mL, negative biopsies were eliminated (improving specificity).

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Phase 2 trial of linifanib (ABT-869) in patients with unresectable or metastatic hepatocellular carcinoma

The efficacy and safety of linifanib (ABT‐869), a selective inhibitor of vascular endothelial growth factor and platelet‐derived growth factor receptor tyrosine kinases, were assessed in this phase 2, single‐arm, open‐label, multicenter trial.

Plasma TIMP-1 and CEA in detection of primary colorectal cancer: a prospective, population based study of 4509 high-risk individuals

Abstract Objective. The combination of plasma tissue inhibitor of metalloproteinases-1 (TIMP-1) and carcinoembryonic antigen (CEA) may be valuable biomarkers for early detection of colorectal cancer (CRC). A prospective, population based study was performed to validate this hypothesis. Material and methods. Individuals (n = 4509) referred for large bowel endoscopy due to symptoms of CRC were prospectively included. Baseline data and concurrent diseases were recorded. The primary endpoint was detection of CRC and findings at examinations were recorded using International Classification of Diseases-10 codes. Plasma was obtained before endoscopy and TIMP-1 and CEA levels were determined after the inclusion of all individuals. Results. Findings were based on sigmoidoscopy in 1766 and colonoscopy in 2743 individuals. Colon cancer (CC) was detected in 184 and rectal cancer in 110 individuals. Ten individuals with other cancers, 856 with adenomas and 1176 with non-neoplastic findings were also detected. The biomarker levels were increased in a variety of diseases including CRC compared to individuals without any findings at endoscopy. A multivariable analysis demonstrated that both markers were significant and independent detectors of CRC. Combining both biomarkers, independent contributions from each (TIMP-1, odds ratio (OR) = 1.8 (95% confidence interval (CI): 1.4–2.2), p < 0.0001; CEA < 5 ng/ml, OR = 1.6, 1.3–1.9, or ≥5 ng/ml, OR = 2.3, 95% CI: 1.9–2.7 (p < 0.0001)) were obtained. Subgroup analysis of individuals examined by colonoscopy with CC as the endpoint showed that combining both biomarkers, independent contributions from each (TIMP-1, OR = 2.5, 95% CI: 1.8–3.4, p < 0.0001; CEA < 5 ng/ml, OR = 1.4, 95% CI: 1.1–1.8, and CEA ≥ 5 ng/ml, OR = 2.3, 95% CI: 1.8–3.0 (p < 0.0001)) were obtained. Conclusions. This prospective validation study supports the use of the combination of plasma TIMP-1 and CEA protein measurements as a potential aid in early detection of CRC and specifically of CC.

Pediatric Oncology Group Utilization of Immunologic Markers in the Designation of Acute Lymphocytic Leukemia Subgroups: Influence on Treatment Responsea

The clinical application of blast cell immunophenotype testing is important in childhood ALL for the following reasons. (1) Knowledge of the immunologic group is important in predicting prognosis. Prognostic grouping may prove to be accomplished best by using a combination of traditional risk factors and immunologic phenotyping. However, definition of traditional risk factors may vary within the immunologic groups of ALL. (2) In assessing the relative effectiveness of different treatment regimens for children with ALL it is important to make comparisons among patients within the same major immunologic groups of ALL. (3) Identification of specific immunologic groups of patients within ALL may help in designing therapy for each group. The POG has already made preliminary attempts in this direction for T-ALL and B-ALL. However, leukemia species-specific therapy is still only a long-range goal. Laboratory research must endeavor to identify additional biologic characteristics peculiar to each major immunologic group of ALL. These characteristics may dictate therapeutic maneuvers in the future.

Biology and potential clinical implications of tissue inhibitor of metalloproteinases-1 in colorectal cancer treatment

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the industrialized world. About half of “curatively” resected patients develop recurrent disease within the next 3–5 years despite the lack of clinical, histological and biochemical evidence of remaining overt disease after resection of the primary tumour. Availability of validated biological markers for early detection, selection for adjuvant therapy, prediction of treatment efficacy and monitoring of treatment efficacy would most probably increase survival. Tissue inhibitor of metalloproteinases-1 (TIMP-1) may be such a marker. TIMP-1 inhibits the proteolytic activity of metalloproteinases, which are centrally involved in tumour invasion and metastases. However, in clinical investigations high tumour tissue or plasma levels of TIMP-1 have shown a strong and independent association with a shorter survival time in CRC patients, suggesting that TIMP-1 could have a tumour-promoting function. Furthermore, measurement of plasma TIMP-1 has been shown to be useful for disease detection, with a high sensitivity and high specificity for early-stage colon cancer. This review describes some basic information on the current knowledge of the biology of TIMP-1 as well as the potential use of TIMP-1 as a biological marker in the management of CRC patients.

A comparative evaluation of Golgi protein-73, fucosylated hemopexin, α-fetoprotein, and PIVKA-II in the serum of patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Abstract Background: Golgi protein-73 (GP73) and fucosylated proteins have been proposed as potential serum markers for liver disease and/or hepatocellular carcinoma (HCC). The purpose of this study was to compare the sensitivity and specificity of serum GP73 and fucosylated hemopexin (Fuc-HPX) with α-fetoprotein (AFP) and with protein induced by the absence of vitamin K or antagonist-II (PIVKA-II) for diagnosing chronic hepatitis, cirrhosis, and HCC. Methods: The concentration of GP73 in human sera was determined using an enzyme-linked immunosorbent assay employing mouse monoclonal and rabbit polyclonal GP73 antibodies. Fuc-HPX was detected using a lectin chemiluminescence-linked immunosorbent assay using a mouse monoclonal anti-hemopexin antibody and Aleuria aurantia lectin. A total of 229 serum samples from patients with chronic hepatitis, cirrhosis, and HCC, as well as from normal individuals were evaluated using these four markers. Results: GP73 and Fuc-HPX showed significantly higher values in samples from patients with cirrhosis and HCC than in samples from patients with hepatitis and from normal individuals. The areas under the curves (AUCs) for GP73, Fuc-HPX, AFP, and PIVKA-II were 0.90, 0.77, 0.74, and 0.88, respectively, for liver cirrhosis and HCC samples vs. hepatitis and normal samples. The AUCs of GP73, Fuc-HPX, AFP, and PIVKA-II were 0.78, 0.72, 0.81, and 0.90, respectively, for HCC samples vs. all other samples. Conclusions: PIVKA-II showed superior sensitivity and specificity for HCC compared with the other three markers. GP73 may be useful for detecting cirrhosis as a risk factor for HCC. Fuc-HPX showed inferior sensitivity and specificity compared to the other markers.