Enhancing the specificity of the COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae by retesting specimens with equivocal results.

The COBAS AMPLICOR CT/NG test for Neisseria gonorrhoeae cross-reacts with certain strains of nonpathogenic Neisseria species. In some strains, the target sequence is identical to that of N. gonorrhoeae, whereas other strains have a small number of mismatches within the regions recognized by the primers or probe used in the COBAS AMPLICOR NG test. These cross-reactive strains are occasionally present in urogenital specimens, causing false-positive results in the COBAS AMPLICOR NG test. Analysis of the data generated in a large multicenter clinical trial showed that 2.9% of the specimens gave signals between A(660)s of 0.2 and 3.5 but that one-half of these equivocal specimens did not contain N. gonorrhoeae. Most of these equivocal specimens were correctly classified as true positive or true negative by retesting in duplicate and defining a PCR-positive result as two of three results with an A(660) of > or =2.0. If specimens had been classified as positive or negative based on a single test result using a cutoff of an A(660) of 0.2, specificity would have ranged from 96.2 to 98.9% depending on specimen type, sex, and presence of symptoms. By employing the equivocal zone-retesting algorithm, specificity increased to 98.6 to 99.9% with little effect (0.1 to 4.9% decrease) on sensitivity in most specimen types, enabling the test to achieve a positive predictive value of at least 90% in populations with a prevalence of 4% or higher. In lower-prevalence populations, the test could be used to screen for presumptive infections that would have to be confirmed by an independent test.

Performance characteristics of the COBAS AmpliScreen HIV-1 test, version 1.5, an assay designed for screening plasma mini-pools.

BACKGROUND: The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma. A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA. Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes. STUDY DESIGN AND METHODS: Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm. Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma. Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing. RESULTS: The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma. Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL. The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors. Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test. HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen. The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit. CONCLUSION: The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.

Ultrasensitive Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma by the AMPLICOR and COBAS AMPLICOR HIV-1 MONITOR™ Tests.

The measurement of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels has become an important tool for identifying individuals likely to benefit from antiretroviral therapy (1-7) as well as monitoring patients on therapy (8-14), and is now regarded as standard medical practice for managing the treatment of HIV-1-infected individuals (15-21). Three commercially available assays for measuring HIV-1 RNA levels are available. The firstgeneration AMPLICOR HIV-1 MONITOR™ Test, which uses reverse transcriptase-polymerase chain reaction (RT-PCR) technology, has a lower limit of quantitation of 400 copies/mL of plasma (22,23). The NucliSens HIV-1 QT Test (Organon Teknika, Boxtel, Netherlands), a second-generation assay based on the nucleic acid sequence-based amplification technique, has a lower limit of quantitation of 100 HIV-1 RNA copies/mL of plasma (24). The Quantiplex HIV-1 Version 2.0 Test (Chiron, Emeryville, CA), which uses the branched-chain DNA signal amplification technique, has a lower limit of quantitation of 500 HIV-1 RNA copies/mL of plasma (25,26).

Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics.

Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2. 0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.

Performance characteristics of a quantitative, homogeneous TaqMan RT-PCR test for HCV RNA.

We developed a homogeneous format reverse transcription-polymerase chain reaction assay for quantitating hepatitis C virus (HCV) RNA based on the TaqMan principle, in which signal is generated by cleaving a target-specific probe during amplification. The test uses two probes, one specific for HCV and one specific for an internal control, containing fluorophores with different emission spectra. Titers are calculated in international units (IU)/ml by comparing the HCV signal generated by test samples to that generated by a set of external standards. Endpoint titration experiments demonstrated that samples containing 28 IU/ml give positive results 95% of the time. Based on these data, the limit of detection was set conservatively at 40 IU/ml. All HCV genotypes were amplified with equal efficiency and accurately quantitated: when equal quantities of RNA were tested, each genotype produced virtually identical fluorescent signals. The test exhibited a linear range extending from 64 to 4,180,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 21.6 to 30.4%, which implies that titers that differ by a factor of twofold (0.3 log10) are statistically significant (P = 0.005). The test did not react with other organisms likely to co-infect patients with hepatitis C and exhibited a specificity of 99% when evaluated on a set of samples from HCV seronegative blood donors. In interferon-treated patients, the patterns of viral load changes revealed by the TaqMan HCV quantitative test distinguished responders from nonresponders and responder-relapsers. These data indicate that the TaqMan quantitative HCV test provides an attractive alternative for measuring HCV viral load and should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.

Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood.

BACKGROUND: Testing for viral nucleic acids should reduce the residual risk of transmitting viral infections by transfusion of blood components. The AmpliScreen Hepatitis C (HCV) Test, Version 2.0, was designed for screening pools composed of samples from individual units of blood or plasma. STUDY DESIGN AND METHODS: An ultracentrifugation step during sample processing simultaneously extracts and concentrates HCV, HIV type 1, and hepatitis B virus particles from plasma. An HCV internal control RNA serves as an extraction and amplification control for each processed sample. Processed samples are amplified by reverse transcriptase polymerase chain reaction and detected by hybridization of the amplified products to HCV- and internal-control-specific oligonucleotide probes. Plasma samples containing known quantities of HCV were used to evaluate analytical sensitivity and precision. RESULTS: The analytical sensitivity of the test was 25 IU of HCV per mL of pooled plasma; all HCV genotypes were detected with similar efficiency. The test did not react with other blood-borne viruses. The within-run and total coefficients of variation were 1.3-13.0 percent and 1.7-22.0 percent, respectively, with low copy samples producing the more variable results. The test performed well using plasma collected in either EDTA, ACD, or PPT Vacutainer tubes. Plasma samples containing elevated levels of hemoglobin, albumin, triglycerides, or bilirubin did not interfere with the test. The test detected HCV RNA 23 to 32 days prior to seroconversion for four of the five seroconversion panels tested. CONCLUSION: The AmpliScreen HCV Test, Version 2.0 provides a reproducible and specific method for screening pooled blood units for HCV. Theoretically, this test has sufficient sensitivity to detect a single infected unit containing 2.4 x 10(3) IU of HCV per mL in a pool with 95 uninfected units and should reduce the window period by at least 20 to 30 days.

Multicenter evaluation of the fully automated COBAS AMPLICOR PCR test for detection of Chlamydia trachomatis in urogenital specimens.

The fully automated COBAS AMPLICOR CT/NG test for the detection of Chlamydia trachomatis was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the C. trachomatis major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98. 5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99. 1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of C. trachomatis exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.

The AMPLICOR® HCV Tests for the Detection and Quantitation of Serum or Plasma HCV RNA.

Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.

Quality control of the polymerase chain reaction.

The polymerase chain reaction (PCR) has revolutionized both the basic and the applied aspects of the biomedical field with more than 40,000 papers having been published employing this technique. PCR has become an indispensable tool for basic research applications, such as cloning (1), sequencing (2), mRNA analysis (3), DNA and RNA quantitation (4-6), mutagenesis (7), and gene detection (8). Because of its ability to amplify minute quantities of nucleic acid specifically, PCR has also been applied with great success in clinical diagnostics (9-11), genetic testing (12-15), forensics (16,17), and environmental testing (18,19).

Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma.

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This "ultrasensitive" method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.

An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance.

We constructed internal controls (ICs) to provide assurance that clinical specimens are successfully amplified and detected. The IC nucleic acids contain primer binding regions identical to those of the target sequence and contain a unique probe binding region that differentiates the IC from amplified target nucleic acid. Because only 20 copies of the IC are introduced into each test sample, a positive IC signal indicates that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity. The COBAS AMPLICOR Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and human hepatitis C virus tests exhibited inhibition rates ranging from 5 to 9%. Approximately 64% of these inhibitory specimens were not inhibitory when a second aliquot was tested. Because repeatedly inhibitory specimens were not reported as false negative and because additional infected specimens were detected during retesting, test sensitivities were 1 to 6% greater than they would have been if the IC had not been used.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.

COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR.

The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

Immunohistochemical and pharmacokinetic characterization of site-specific immunoconjugate 15A8-glycyl-tyrosyl-(N-epsilon-diethylenetriamine pentaacetic acid)-lysine derived from anti-breast carcinoma monoclonal antibody 15A8.

In this study, the breast carcinoma-reactive monoclonal antibody 15A8 and a site-specific immunoconjugate of the antibody, 15A8-glycyl-tyrosyl-(N-epsilon-diethylenetriamine pentaacetic acid)-lysine (15A8-GYK-DTPA), were characterized by immunohistological methods for reactivity with normal and neoplastic human tissues and normal cynomolgus monkey tissues. In addition, 15A8-GYK-DTPA labeled with 111In was assessed by in vivo imaging and pharmacokinetic studies for localization to human tumor xenografts in nude mice. The native antibody and the site-specific immunoconjugate exhibited similar limited reactivity with normal human tissues. Specifically, epithelial structures, including normal breast epithelium, lung alveoli, bronchial epithelium and glands, liver bile ducts, pancreatic ducts, kidney distal and collecting tubules, epidermal and esophageal epithelium, endometrial glands, and thymic Hassall's corpuscles, were reactive. Normal monkey tissues stained with 15A8 exhibited a similar pattern of reactivities. Antibody 15A8 reacted broadly with epithelium-derived tumors; more than 60% of the cells in all of the breast, colon, non-small cell lung, ovarian, prostate, bladder, and renal carcinomas tested expressed the antigen. In contrast, a variety of nonepithelial neoplasms, including lymphomas, melanomas, sarcomas, and small cell lung carcinomas, were nonreactive. 15A8-GYK-DTPA-111In administered i.v. rapidly localized to and imaged both MX-1 and MCF-7 human breast carcinoma xenografts in nude mice, reaching maximal levels of about 20% of injected dose/g of tumor within 4 days. No unusual localization to any nontumor tissue or organ was seen; the level of radioactivity in the normal tissues and organs was at or below that seen in the blood. Furthermore, the immunoconjugate did not accumulate in xenografts of the antigen-negative breast carcinoma ZR-75-1, which indicates that tumor localization was antigen specific. Pharmacokinetic studies in cynomolgus monkeys suggested that significant amounts of 15A8-GYK-DTPA-111In did not localize to normal epithelia and demonstrated that the immunoconjugate was not toxic. These findings suggest that antibody 15A8 may be useful in the diagnosis and therapy of breast cancer and possibly other carcinomas.

Immunohistochemical and pharmacokinetic characterization of the site-specific immunoconjugate CYT-356 derived from antiprostate monoclonal antibody 7E11-C5.

In this study, a site-specific immunoconjugate, designated CYT-356, of the prostate-reactive monoclonal antibody 7E11-C5 was characterized by immunohistological methods for reactivity with normal and neoplastic human tissues. In addition, CYT-356 labeled with 111In was assessed by in vivo imaging and pharmacokinetic studies for localization to human tumor xenografts in nude mice. The native antibody and the site-specific immunoconjugate exhibited similar patterns of reactivity with normal human tissues. Although the majority of tissues tested were negative, weak reactivity with cardiac muscle, proximal kidney tubules, and sweat glands was observed. Positive staining of normal prostate epithelial cells and glandular lumina and strong reactivity with a subset of skeletal muscle cells were also observed. CYT-356 reacted with 100% of prostate tumors examined but was negative on a variety of other neoplasms. Following i.v. administration, CYT-356-111In rapidly localized to and imaged LNCaP human prostate adenocarcinoma xenografts in nude mice, reaching maximal levels of about 30% of injected dose/g of tumor within 3 days. No unusual localization was seen to any nontumor tissue or organ; the level of radioactivity in the normal tissues and organs was at or below that seen in the blood. The localization to xenografts was antigen specific and the accessible binding sites in 100-200-mg tumors appeared to be saturated at an antibody dose between 10 and 100 micrograms. These findings suggest that the CYT-356 immunoconjugate may be useful in the diagnosis and therapy of prostate cancer.

Single copies of HIV proviral DNA detected by fluorescent in situ hybridization.

A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency of detection of individual loci of proviral DNA was 57% to 78%. Only 1% of uninfected control cells exhibited a false-positive signal. HIV proviral DNA could be accurately identified in mixed populations comprised of only 5% infected cells. Thus, this assay could be used to identify cells that harbor HIV proviral DNA and to monitor the status of proviral DNA throughout the course of HIV infection.

Carbohydrate-derivatized immunoconjugate of the anti-(carcinoembryonic antigen) monoclonal antibody C46: immunohistological reactivity and pharmacokinetic comparison with a randomly derivatized C46 immunoconjugate.

In this study, a site-specific glycyl-tyrosyl-(N-epsilon-diethylenetriaminepentaacetic acid)-lysine (GYK-DTPA) immunoconjugate of the anti-carcinoembryonic antigen monoclonal antibody C46 (C46-GYK-DTPA) was characterized by immunohistological and immunofluorescence methods for reactivity with normal and neoplastic human tissues. In addition, pharmacokinetic studies assessed the ability of C46-GYK-DTPA labeled with 111In to localize to and image human tumor xenografts in nude mice. The native antibody and the site-specific immunoconjugate exhibited similar patterns of reactivity with normal human tissues. C46 did not bind to the surface of normal human granulocytes, which indicates lack of reactivity with normal cross-reacting antigen. C46-GYK-DTPA reacted with 100% of the colon, breast and renal carcinomas examined and with two of three lung carcinomas, but did not react with any sarcomas, melanomas or lymphomas examined. Intravenously administered C46-GYK-DTPA-111In rapidly localized to and imaged LS174T human colon adenocarcinoma xenografts in nude mice, reaching maximal levels of about 25% of injected dose/g tumor within 1 day. No unusual localization to any non-tumor tissue or organ was seen; the level of radioactivity in the normal tissues and organs was at or below that in the blood. The accessible binding sites in 1 g tumors appeared to be saturated at an antibody dose between 100 micrograms and 1000 micrograms/mouse. Further, in a direct in vivo comparison, the site-specific conjugate C46-GYK-DTPA had more favorable pharmacokinetics and better tumor localization than a randomly derivatized C46 immunoconjugate (C46-DTPA). These findings suggest that the site-specific immunoconjugate C46-GYK-DTPA may be useful in the diagnosis and therapy of colon cancer and other adenocarcinomas expressing carcinoembryonic antigen.

Variant embryonal carcinoma cells lacking SSEA-1 and Forsmann antigens remain developmentally pluripotent.

Six embryonal carcinoma (EC) cell lines that are resistant to the cytotoxic, galactose-specific lectin abrin were isolated from mutagenized populations of either PSA-1 or F9 cells. The LD10 for each of the variant lines was at least 150-fold greater than that for parental cells. Indirect cytotoxicity tests demonstrated that all of the variant cell lines lacked both Stage Specific Embryonic Antigen-1 (SSEA-1, less than 1% of wild-type levels) and Forsmann antigen (less than 5% of wild-type levels). When abrin-resistant cells were fused to previously isolated SSEA-1-negative cells (M. J. Rosenstraus (1983), Dev. Biol. 99, 318-323) that express Forsmann antigen, the resulting hybrids expressed SSEA-1. This implies the mutation conferring abrin resistance is in a different gene than that defined by the previously isolated mutation. Thus, we have identified two genes that are required for SSEA-1 expression, one of which also appears to be required for Forsmann antigen expression. The F9-derived variants differentiated into visceral-like or parietal-like endoderm when treated with retinoic acid in the absence or presence of 8-bromo-cAMP, respectively. PSA-1-derived variants formed differentiated teratocarcinomas containing derivatives of all three germ layers. Thus the SSEA-1 and Forsmann haptenic determinants are not required for EC cells to differentiate into a broad spectrum of cell types; nor do they appear to be involved in the cell-cell interactions that are postulated to regulate visceral versus parietal endoderm differentiation.

Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal.

The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.