W. Jeffrey Allard

Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases

Purpose: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. Experimental Design: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. Results: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of ≥85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had ≥2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 ± 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≥2 CTCs. Detection of ≥2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. Conclusions: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

A novel multiple marker bioassay utilizing HE4 and CA125 for the prediction of ovarian cancer in patients with a pelvic mass

INTRODUCTION Patients diagnosed with epithelial ovarian cancer (EOC) have improved outcomes when cared for at centers experienced in the management of EOC. The objective of this trial was to validate a predictive model to assess the risk for EOC in women with a pelvic mass. METHODS Women diagnosed with a pelvic mass and scheduled to have surgery were enrolled on a multicenter prospective study. Preoperative serum levels of HE4 and CA125 were measured. Separate logistic regression algorithms for premenopausal and postmenopausal women were utilized to categorize patients into low and high risk groups for EOC. RESULTS Twelve sites enrolled 531 evaluable patients with 352 benign tumors, 129 EOC, 22 LMP tumors, 6 non EOC and 22 non ovarian cancers. The postmenopausal group contained 150 benign cases of which 112 were classified as low risk giving a specificity of 75.0% (95% CI 66.9-81.4), and 111 EOC and 6 LMP tumors of which 108 were classified as high risk giving a sensitivity of 92.3% (95% CI=85.9-96.4). The premenopausal group had 202 benign cases of which 151 were classified as low risk providing a specificity of 74.8% (95% CI=68.2-80.6), and 18 EOC and 16 LMP tumors of which 26 were classified as high risk, providing a sensitivity of 76.5% (95% CI=58.8-89.3). CONCLUSION An algorithm utilizing HE4 and CA125 successfully classified patients into high and low risk groups with 93.8% of EOC correctly classified as high risk. This model can be used to effectively triage patients to centers of excellence.

MEASUREMENT OF COMPLEXED PSA IMPROVES SPECIFICITY FOR EARLY DETECTION OF PROSTATE CANCER

OBJECTIVES Prostate-specific antigen (PSA) is the most useful of all tumor markers. Although the sensitivity is impressive, low specificity results in a lack of cancer detection in a significant proportion of patients undergoing prostate biopsy. Several recent studies have addressed the need for improved specificity. Of all these approaches, the free/total PSA ratio appears to be the most promising. Given that most circulating PSA is complexed to alpha1-antichymotrypsin, and that this moiety represents a greater proportion of the total PSA in those men with carcinoma, we set out to determine whether complexed PSA would improve specificity in the detection of men with prostate cancer. METHODS Archival sera were obtained from 300 men, 75 of whom had biopsy-proved prostate cancer. All sera had been previously stored at -70 degrees C for variable periods. An investigative assay for complexed PSA (Bayer) was used. The Tandem-R free and total PSA assays (Hybritech) were used according to the manufacturers recommendations. RESULTS Among all patients, specificities for the total PSA, free/total PSA, and complexed PSA alone were 21.8%, 15.6%, and 26.7%, respectively, at cutoffs yielding 95% sensitivity. Similar equivalence or superior performance, in terms of specificity relative to the free/total PSA ratio, was seen at other sensitivity thresholds and other total PSA ranges. CONCLUSIONS Complexed PSA alone performs better than total PSA or the free/total PSA ratio and obviates the need for a second analyte determination. We believe this marker may offer significant enhancement in PSA testing with significant economic advantages.

Utility of a novel serum tumor biomarker HE4 in patients with endometrioid adenocarcinoma of the uterus

OBJECTIVE Tumor markers with increased sensitivity and specificity for endometrial cancer are needed to help monitor response to therapy and to detect recurrent disease. Currently, the tumor maker CA125 is utilized in this role with limited value. The objectives of this study were to examine the levels of several novel tumor markers HE4, SMRP, CA72.4 and CA125 as potential markers in patients diagnosed with endometrioid adenocarcinoma of the uterus. METHODS Pre-operative serum samples from surgically staged patients with endometrioid adenocarcinoma of the uterus were analyzed for levels of HE4, SMRP, CA72-4 and CA125. Control samples were obtained from healthy postmenopausal women. Logistic regression models and receiver operating characteristic (ROC) curves were constructed for each tumor marker and for all combinations, with cross-validation analyses to obtain average sensitivities at set specificities of 90%, 95%, and 98%. RESULTS Serum samples from 156 healthy subjects and 171 patients with endometrial cancer (122 stage I, 17 stage II, 26 stage III, and 6 stage IV) were analyzed. At a 95% specificity, the sensitivities for differentiating between healthy subjects and all stages of cancer were 45.5% for HE4 and 24.6% for CA125. For stage I disease, HE4 yielded a 17.1% improvement in sensitivity compared with CA125. CONCLUSION HE4 is elevated in all stages of endometrial can100cer and is more sensitive in early-stage endometrial cancer compared to CA125. Further investigation of HE4 as a marker for early detection of recurrent endometrial cancer and monitoring response to therapy is warranted.

Protein profiling of pleural effusions to identify malignant pleural mesothelioma using SELDI-TOF MS.

Diagnosis of malignant pleural mesothelioma (MM) is limited. Novel proteomic technologies can be utilized to discover changes in expression of pleural proteins that might have diagnostic value. The objective of this study was to detect protein profiles that could be used to identify malignant pleural mesothelioma with surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Pleural effusions were collected from patients with confirmed mesothelioma (n = 41) and from patients with effusions due to other causes ([n = 48] cancerous and non-cancerous). Samples were fractionated using anion exchange chromatography and bound to different types of ProteinChip array surfaces. All samples were also subjected to other commercially available immunoassays (human epididymes protein 4 [HE4], osteopontin [OPN], soluble mesothelin-related proteins [SMRP], and the cytokeratin 19 fragment [CYFRA 21–1]). Peak intensity data obtained by SELDI-TOF were subjected to classification algorithms in order to identify potential classifier peaks. A protein peak at m/z 6614 was characterized as apolipoprotein (Apo) CI. In this setting, the sensitivity and specificity of this potential biomarker was 76 % and 69 %, respectively. The area under the receiver operating characteristic curve (AUC) for Apo CI was 0.755, thereby outperforming OPN, HE4, and CYFRA 21–1. SMRP performed best with an AUC of 0.860 with a sensitivity of 83% and specificity of 74%. Our study validates the use of SMRP as a diagnostic marker for pleural mesothelioma and furthermore suggests that Apo CI levels could be used in the future to discriminate pleural mesothelioma from other causes of exudates.

Measurement of four tumor marker antigens in the sera of pregnant women

We sought to determine the maternal serum levels of four tumor‐associated antigens during the three trimesters of pregnancy in healthy women. CEA, CA 228, CA 15‐3, and Her2/neu oncogene product p105 assay values were determined for 90 healthy pregnant women during the three trimesters of pregnancy at five participating evaluation sites. Results were compared to means and cut‐off values determined for healthy nonpregnant women. Differences in assay values in the 1st and 3rd trimester were analyzed for statistical significance (Students t‐test). CEA, CA 228 and CA 15‐3 assay values in general were found to be within the normal range. CA 15‐3 and Her2/neu p105 serum assay values were above the cut‐off (3.3% and 8.2%, respectively) and were significantly elevated in the 3rd trimester as compared to the 1st trimester of pregnancy (P < 0.05 and P < 0.001, respectively). CEA and CA 228 may be of potential value in monitoring pregnant women with malignant disease. Normal elevations in 3rd trimester serum Her2/neu p105 and CA 15‐3 assay values should be considered when monitoring a pregnant patient with malignant disease. J. Clin. Lab. Anal. 13:35–39, 1999.

Technicon Immuno 1 PSA assay measures both free and alpha-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis.

In this study, we investigated the immunoreactivity of the Technicon Immuno 1® PSA assay, a monoclonal‐polyclonal sandwich immunoassay, with free and ACT‐complexed PSA. Assay calibrators prepared with free PSA (standard immuno 1 calibrators) and calibrators consisting of 90% ACT‐complexed and 10% free PSA (90:10 calibrators) yielded virtually identical PSA recoveries at all concentrations tested. Concentrations of total PSA at ∼4 and 10 ng/mL, prepared in varying ratios of free PSA to PSA‐ACT complex, recovered from 92–104% at 4 ng/mL and from 98–102% at 10 ng/mL. Additionally, an excellent correlation of serum total PSA values from a panel of 40 prostatic cancer patient samples was obtained using calibration curves generated with the standard Immuno 1 calibrators or the 90:10 calibrators. These results demonstrate that the Technicon Immuno 1® PSA assay measures free and ACT‐complexed PSA on an equal molar basis.

BAYER IMMUNO 1 PSA ASSAY : AN AUTOMATED, ULTRASENSITIVE METHOD TO QUANTITATE TOTAL PSA IN SERUM

The Bayer Immuno 1™ PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV ≤ 3.4% and a biological detection limit of 0.03 μg/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 μg/L for males 50–59 years and 3.3 μg/L for males 60–69 years, to 4.6 μg/L for males ≥ 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5–100%. Specificity of 60–70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx® PSA Assay over the range 0.0–6,238 μg/L (Y = 1.10 × + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum. J. Clin. Lab. Anal. 12:65–74, 1998.

MESOMARK® in vitro diagnostic test for mesothelioma

Mesothelioma is a highly progressive tumor with a poor prognosis. In this article, the authors give a thorough introduction into and evaluation of the MESOMARK(®) in vitro test - the only blood test for the management of patients with mesothelioma approved by the US FDA. In Europe, Australia, New Zealand and Canada, the test is approved or licensed for the measurement of the soluble mesothelin, also termed as soluble mesothelin-related peptides. In the US, it is approved for the monitoring of patients with epithelioid and biphasic malignant mesothelioma.