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Human papillomavirus (HPV), the most prevalent sexually transmitted disease (STD), continues to pose a significant public health problem especially among the adolescent population. Most precancerous and cancerous cervical changes are associated with HPV, with adolescent women being biologically at highest risk for acquiring HPV. This survey examined the type of information taught to adolescents about HPV, and specific needs for effective HPV education in middle and high schools in Vermont. The survey addressed knowledge level, behavior, attitudes, enabling factors, motivators, and barriers. Data were analyzed by descriptive statistics and supplemented with contingency table analyses. Replies (n = 108) were received from 79 schools, with 60% of responses from nurses and 40% from teachers. In five of eight questions addressing basic knowledge of HPV, less than 60% of respondents gave the correct answer. Most (73%) felt it was important to teach about HPV relative to HIV/AIDS, but spent less classroom time teaching it. Main motivations for teaching about HPV were its importance, and a desire to increase student knowledge and prevention skills. Main barriers perceived were lack of time and materials and curricula, and need for more knowledge about HPV. The most prominent needs indicated included brochures for students, an increase in the educators' knowledge base, and a high school curriculum. Health educators in Vermont schools recognize the importance of teaching adolescents about HPV, but they lack basic knowledge and resources for teaching about HPV. [ABSTRACT FROM AUTHOR]

Objectives: Ki-67 is a molecular marker of cellular proliferation that predicts prognosis of some head and neck tumors. Studies of Ki-67 in oropharyngeal cancer have yielded conflicting findings. This study was designed to test Ki-67 as a marker for poor prognosis in N0 tongue squamous cell carcinoma., Methods: We examined 29 cases in a retrospective cohort to test the hypothesis that a high rate of tumor cell proliferation (high levels of Ki-67 staining) at the invasive edge of N0 squamous cell carcinoma of the tongue correlates with increased risk of recurrence., Results: There were 14 cases of recurrence. The average age of the patients with recurrence was 58 years. The average time to recurrence was 13.1 months. A 0% to 33% uptake of Ki-67 at the tumor's leading edge was associated with a 6-times-greater risk of recurrence. The mean length of survival for the group with 0% to 33% uptake was 21 months; for the group with > 33% uptake, it was 33 months. Overall uptake of Ki-67 and histologic grade did not correlate with risk of recurrence., Conclusions: In this sample, low rates of Ki-67 staining at the invasive edge of the tumor predicted a risk of recurrence. These results need to be confirmed before Ki-67 can be used for predicting recurrence of tongue squamous cell carcinoma.

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16 immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16 immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P<0.001). p16 immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P<0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration. Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.

Background: Recent studies have shown that minichromosome maintenance (MCM) proteins (Mcm2-7) may be useful proliferation markers in dysplasia and cancer in various tissues., Aims: To investigate the use of Mcm7 as a proliferation marker in 79 lymph node negative prostate cancers and compare it with Ki-67, a commonly used cell proliferation marker., Methods: The percentage of proliferating cells (proliferation index; PI) was calculated for basal and luminal epithelial cells in benign prostate tissue, prostatic intraepithelial neoplasia (PIN), and epithelial cells in adenocarcinoma. The PI for each biomarker was correlated with the preoperative prostate specific antigen concentration, the Gleason score, surgical resection margin status, and the AJCC pT stage for each patient., Results: The mean PIs for Ki-67 and Mcm7 were: benign luminal epithelium 0.7 and 1.2 and benign basal epithelium 0.8 and 8.2; PIN non-basal epithelium 4.9 and 10.6 and PIN basal epithelium 0.7 and 3.1; adenocarcinoma 9.8 and 22.7, respectively. Mcm7 had a significantly higher mean PI (p<0.0001) than Ki-67 for all cell categories except benign luminal epithelial cells. Mcm7 was a better discriminatory marker of proliferation between benign epithelium, PIN, and invasive adenocarcinoma (p<0.0001) than Ki-67. The drop in Mcm7 mean basal cell PI from benign epithelium to PIN epithelium was significantly larger than for Ki-67 (p<0.0001). Mcm7 had a significantly higher PI than Ki-67 at each risk level., Conclusion: Mcm7 may be a useful proliferation marker in prostatic neoplasia and warrants further evaluation as a complementary tool in the diagnosis of PIN and prostate carcinoma.

Context: Patients with hyperplastic polyposis coli syndrome are thought to harbor precursor lesions of a proposed hyperplasia-carcinoma pathway in colorectal cancer, but morphologic recognition of such lesions remains difficult. Hypermethylation of the secreted Frizzled receptor protein 1 gene on chromosome 8p12 is one of the earliest molecular alterations in colorectal carcinogenesis, potentially disrupting the Wnt signaling cascade of cellular growth control., Objective: To determine if hyperplastic polyps from patients with hyperplastic polyposis coli syndrome show a distinct immunohistochemical expression pattern for mismatch repair proteins and secreted Frizzled receptor protein 1 compared to their sporadic counterparts., Design: Immunohistochemical studies (secreted Frizzled receptor protein 1, 3 mismatch repair proteins, and p53) were performed on 23 hyperplastic polyps, 6 synchronous colon cancers, and normal colonic mucosa from 6 patients with hyperplastic polyposis coli syndrome and were compared with studies of sporadic hyperplastic polyps obtained from 13 matched control subjects., Results: The staining pattern for the mismatch repair proteins MLH-1, MSH-2, and MSH-6 did not differ between sporadic and syndromic hyperplastic polyps. In contrast, 52% of syndromic hyperplastic polyps showed a reproducible and distinct staining pattern for secreted Frizzled receptor protein 1 that was not seen in control specimens and that was associated with larger polyp size (P =.002) and location in the proximal colon (P =.01)., Conclusions: Some hyperplastic polyps from patients with hyperplastic polyposis coli syndrome show a secreted Frizzled receptor protein 1 immunophenotype that could indicate alterations of cellular growth control. These findings may help identify precursor lesions in the proposed hyperplasia-carcinoma pathway of colorectal carcinogenesis.

Massive perivillous fibrin deposition (MPFD) is associated with intrauterine growth retardation and first-trimester and second-trimester spontaneous abortion. Histologically, villi near the maternal interface are completely surrounded by fibrinoid material. This work compared the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in early miscarriage specimens with and without MPFD. Ten specimens with a gestational age of 7-12 weeks (mean 10 weeks) and 10 age-matched miscarriage specimens lacking MPFD were sampled. Formalin-fixed paraffin-embedded sections were stained with monoclonal antibodies against TM and EPCR using an immunoperoxidase method. The slides were independently reviewed by two pathologists using a semiquantitative grading system. Among unaffected villi, there was no difference in staining for TM or EPCR in cases of massive perivillous fibrin deposition compared with the control group. In the MPFD cases, loss of membrane positivity was noted for both TM and EPCR at the junction between normal villous epithelium and villous epithelium with deposition of fibrin. This could imply an underlying defect of trophoblastic protein C activation. Alternatively, it may represent a degenerative change secondary to impedence of oxygen and nutrient supply to the trophoblastic epithelium.

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise kappa association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, kappa = 0.848; ThinPrep, kappa = 0.918) and moderate (ThinPrep, kappa = 0.692; formalin fixation, kappa = 0.667) to poor (ethanol, kappa = 0.300) pairwise kappa agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.

Background: The objective of the current pilot project was to assess the efficacy of laser scanning cytometry (LSC) for DNA ploidy analysis of atypical urologic cytology specimens to enhance the distinction between benign and malignant changes., Methods: Forty selected urologic cytology specimens that previously had been categorized as normal, atypical, or malignant were studied. Nuclear propidium iodide and fluorescence intensity measurements were converted to pixel values, which were used to create scattergrams that excluded debris and cell clusters from ploidy analysis, creating a gated (isolated) region of predominantly single cells for LSC ploidy analysis. Integral histograms then were created to show the number of cells present in diploid, tetraploid, and aneuploid peaks; these histograms also were used to assess DNA ploidy., Results: Ten normal specimens, 10 malignant specimens, and 20 atypical specimens were examined to assess the efficacy of LSC ploidy analysis. Normal and malignant specimens generated reference histograms for comparison with the atypical specimens and exhibited 90% specificity and 100% sensitivity. Ten atypical aneuploid specimens had histogram and scattergram patterns similar to those produced by malignant specimens and, using the cytometer's relocation feature, the presence of atypical cells was confirmed in the aneuploid regions., Conclusions: The authors determined that DNA ploidy analysis of atypical urologic cytology specimens using LSC is a useful adjunct tool for identifying malignant specimens that lack sufficient cytologic criteria for diagnosis by light microscopy alone. However, LSC is time consuming and requires expensive equipment., (Copyright 2004 American Cancer Society.)

Several recent studies have demonstrated localization of donor bone marrow-derived cells in recipient lungs following transplantation from male to female mice or patients. Donor cells are identified by detection of the Y chromosome by fluorescence in situ hybridization (FISH). However, protein digestion pretreatments usually required for tissue FISH significantly limit the ability to detect cell type-specific markers by immunohistochemistry. We have used an alternative protein digest approach that entails heating the slides in 10 mM sodium citrate rather than utilizing a protease digestion. This approach preserves cell proteins following FISH, and allows lung tissue to remain intact for subsequent detection of cell-specific markers by immunohistochemistry. We have examined this technique in mouse lungs using a Y chromosome paint and three cell-specific markers, a pan-cytokeratin for epithelial cells, PECAM-1 for endothelial cells, and CD45 for leukocytes. Excellent visualization of both the Y chromosome and cell-specific surface protein markers was obtained on a single slide. This approach will significantly enhance the ability to detect and identify donor bone marrow cells in recipient mouse lungs following male to female transplantation.

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide-based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus. Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P =.025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P <.0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus-positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P <.0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas. Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide-based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.

Cytochrome P450scc catalyzes the important first step in the steroid synthesis pathway; however, it is clear that additional factors regulating the temporal and spacial specific expression of the CYP11A1 gene remain to be identified. To isolate novel transcription factors that regulate this gene, a cis-acting element of the 5'-flanking region from nucleotides -155 to -131 (-155/-131) was used to screen a human placental lambda gt11 cDNA expression library, and an interacting clone was isolated. The open reading frame of the cDNA encodes several domains that are characteristic of transcription factors including an acidic region, a region rich in prolines and three zinc-finger motifs. Expression of the cDNA by in vitro transcription/translation and by transient transfection in HeLa cells yielded a protein of 132 kDa, which concurs with the predicted size. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells, stimulate expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. This transcriptional regulating protein of 132kDa (TReP-132) when expressed in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis, and tandem copies of this region was shown to confer activation of the heterologous HSV thymidine kinase minimal promoter. Coexpression of CBP/p300 with TReP-132 further increased promoter activity, and the proteins were demonstrated to interact physically. RNA analysis demonstrated the highest levels of expression in the adrenal cortex and testis; and transcript expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines, but not in non-steroidogenic HepG2 and HK293 cells. Subsequently it has been shown that TReP-132 interacts with steroidogenic factor-1 (SF-1) through specific domains; and along with the interaction with CBP/p300 these factors are postulated to form a complex to regulate expression of the P450scc gene.

A comparison was made between labeled antibody accumulations in nude mice having either single or multiple human xenografts. The LS174T tumors were implanted subcutaneously. All animals were given 2 micrograms of labeled murine anti-carcinoembryonic antigen (CEA) monoclonal antibody 111In-mT84.66. Some animals were also given specific antibody pretreatment (SAP) of 200 micrograms of unlabeled mT84.66 to reduce liver accumulation of activity. In order to represent these multiple tumor examples, a simple initial-phase pharmacokinetic model was first fitted to each of the two groups (SAP and PBS treated) of single-tumor animals. Using the resultant six non-adjustable parameters as constants, the n = 1 uptake model was then used to represent tumor, liver and blood accumulations (%injected dose/organ) in the multiple-tumor animals. The model was found to be a good representation; in particular, it had far better agreement than single tumor predictions in the PBS mice. Differences between the single-tumor accumulations and those seen in multiple tumor examples were generally between two- and three-fold. The model also demonstrated that the result of SAP was to essentially eliminate the effect of liver targeting of tumor-secreted CEA. We conclude that an initial-phase one-tumor model can describe the decrease of accumulation of activity in the case of multiple tumors in nude mice in both untreated (PBS) and pretreated conditions. Implications for clinical imaging and therapy with monoclonal agents are discussed.

Background: Currently, prostate cancer (CaP) cytogenetics is not well defined, largely because of technical difficulties in obtaining primary tumor metaphases., Methods and Results: We examined three CaP cell lines (LNCaP, DU145, PC-3) using sequential Giemsa banding and spectral karyotyping (SKY) to search for a common structural aberration or translocation breakpoint. No consistent rearrangement common to all three cell lines was detected. A clustering of centromeric translocation breakpoints was detected in chromosomes 4, 5, 6, 8, 11, 12, 14, and 15 in DU145 and PC-3. Both these lines were found to have karyotypes with a greater level of complexity than LNCaP., Conclusion: The large number of structural aberrations present in DU145 and PC-3 implicate an underlying chromosomal instability and subsequent accumulation of cytogenetic alterations that confer a selective growth advantage. The high frequency of centromeric rearrangements in these lines indicates a potential role for mitotic irregularities associated with the centromere in CaP tumorigenesis.

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome., (Copyright 2000 Academic Press.)

Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence. In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases. We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2. Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21. We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21. The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis., (Copyright 2000 S. Karger AG, Basel)

A murine Ets2 target gene isolated by differential display cloning was identified as the phospholipase A2 activating protein (PLAA) gene. A 2.7-kb human cDNA demonstrating high homology to mouse and rat Plaa genes was then isolated and characterized. Human PLAA contains six WD-40 repeat motifs and three different protein kinase consensus domains. Fluorescence in situ hybridization (FISH) mapping placed PLAA on chromosome 9p21, a region frequently deleted in various cancers. A comprehensive mapping strategy was employed to define further the chromosomal localization of PLAA relative to CDKN2A within the 9p21 locus. Radiation hybrid mapping placed the gene 7.69 cR from WI-5735 (LOD >3.0), a marker in close proximity to CDKN2A and CDKN2B. Yeast artificial chromosome (YAC) mapping localized PLAA proximal to the CDKN2A/CDKN2B genes and to a region flanked by D9S171 and INFA commonly deleted in many neoplasms. Two YACs contained both PLAA and D9S259, a marker present in a second more proximal minimal deleted region observed in cutaneous melanoma and squamous cell lung carcinoma. Double-color fiber FISH mapping confirmed the location of PLAA centromeric to D9S171 and CDKN2A/CDKN2B. The mapping data suggest a possible tumor suppressor role for this gene., (Copyright 1999 Academic Press.)

The human MEP1A gene encodes the meprin alpha subunit that consists of a protease domain conserved in the astacin family of metalloendopeptidases and several C-terminal interaction domains present in other proteins. Using the alpha subunit cDNA, we identified two clones from a human P1-derived artificial chromosome (PAC) library. Fluorescence in situ hybridization (FISH) mapped both PACs (1e12, 65a14) to chromosome 6p21, confirming the MEP1A location. FISH also mapped PAC 65a14 to chromosome 13cen, and to chromosome 9 in three different regions, 9p12-13, 9q21, and 9q22. Southern blot analysis showed that sequences of PAC 65a14 and MEP1A were similar in the 3' end but different in the 5' end, revealing for the first time that the human genome may encode multiple interaction domains highly similar to those of the meprin alpha subunit. The symbols of MEP1AL1, MEP1AL2, MEP1AL3, and MEP1AL4 have been designated for MEP1A-like sequences on 9p12-13, 9q21, 9q22, and 13cen, respectively., (Copyright 1999 Academic Press.)

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.

The mouse looptail (Lp) mutation is an established model for neural tube defects with homozygous Lp embryos showing an open neural tube from the caudal midbrain to the tip of the tail. Heterozygous Lp mice are characterized by a "looped-tail" and wobbly head movements. The Lp gene has been mapped to a 0.6-cM interval on mouse chromosome 1 delineated by two clusters of markers, Fcer1gamma/Usf1/D1Mit113/D1Wsu1 on the proximal side and Fcer1alpha/Spna1/D1Mit149 distally. In the present study, we have created a high-resolution physical map of the Lp genetic interval that is based on long-range restriction mapping by PFGE, fluorescence in situ hybridization analysis of interphase nuclei and extended chromatid fibers, and the assembly of a cloned contig. This contig consists of 25 independent and overlapping BAC clones and 3 YAC clones. The combined analysis indicates that the 0.6-cM genetic interval for Lp corresponds to a minimal physical interval of 700 kb that is delineated by D1Mit113 proximally (two crossovers) and Fcer1alpha distally (one crossover). The overall gene order and intergene distances for the region were determined to be D1Mit113-<150 kb-Nhlh1-250 kb-Atp1alpha2-280 kb-Fcer1alpha. Partial sequencing of BAC clones from the contig yielded 42 new STS markers for this region of mouse chromosome 1. Sequence analysis of the BAC clones and assignment of ESTs from the human transcript map to the cloned contig allowed the placement of four new transcription units within this region: Pc326, Kiaa0253, and Pea15 were positioned in the Nhlh1/Atp1alpha2 nonrecombinant interval, while Girk3 was located distal to Atp1alpha2., (Copyright 1999 Academic Press.)

We have cloned and characterized the Na,K-ATPase beta 3 subunit gene (ATP1B3), and a beta 3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The beta 3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the beta 3 subunit gene is identical to that of the Na,K-ATPase beta 3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse beta 3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the beta 3 subunit gene are likely to be located within this conserved region. Dot blot analysis of beta 3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of beta 3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22-->23 region of Chromosome (Chr) 3, and the beta 3 pseudogene to the p13-->15 region of Chr 2.

UGT2B17 is a UDP-glucuronosyltransferase enzyme expressed in several extrahepatic steroid target tissues, including the human prostate, where it glucuronidates C19 steroids such as dihydrotestosterone (DHT), androsterone (ADT), and androstane-3alpha, 17beta-diol (3alpha-diol). To determine if UGT2B17 is regulated by physiological effectors of the human prostate, DHT and epidermal growth factor (EGF) were demonstrated to specifically down-regulate the steady-state levels of UGT2B17 transcript and protein in LNCaP cells (Guillemette et al., 1997). These results implicate regulation of UGT2B17 at the level of gene transcription, therefore, a P-1-derived artificial chromosome (PAC) clone of 120 kb containing the entire UGT2B17 gene was isolated. The gene is comprised of six exons spanning approximately 30 kb, and fluorescence in situ hybridization of the UGT2B17 PAC clone to normal human lymphocyte chromosomes, mapped the gene to chromosome 4q13. To determine if the 5'-flanking DNA of the UGT2B17 gene is sufficient to confer gene expression, a 2,942-bp fragment was subcloned into a luciferase reporter plasmid and yielded an activity of 25-fold over background when transfected in LNCaP cells. However, transfection of the construct into HK-293, MCF-7, JEG-3, and HepG2 cells yielded only a moderate activity of two- to five-fold over background. Treatment of transfected LNCaP cells with 10 nM R1881, a nonmetabolizable analog of DHT, and 10 ng/ml EGF decreased the luciferase activity by 60%. This suggests that at least part, if not all, of the inhibitory effect of EGF and DHT on UGT2B17 is at the level of transcription. Progressive 5' deletions of the UGT2B17 5'-flanking region in the luciferase constructs alleviated the inhibition by R1881 and EGF, and revealed several potential responsive elements that may confer the observed regulation of the UGT2B17 gene. This study demonstrates regulation of the UGT2B17 gene by physiological effectors of the human prostate and supports the hypothesis that UGT enzymes are involved in steroid metabolism in extrahepatic tissues.

Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p12) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.

We report the cloning and characterization of a human cDNA encoding a novel homolog of rat extracellular signal-regulated kinase 3 (ERK3). The cDNA encodes a predicted protein of 721 amino acids which shares 92% amino acid identity with rat ERK3 over their shared length. Interestingly, the human protein contains a unique extension of 178 amino acids at its carboxy terminal extremity. The human ERK3 protein also displays various degrees of homology to other members of the MAP kinases family, but does not contain the typical TXY regulatory motif between subdomains VII and VIII. Northern blot analysis revealed that ERK3 mRNA is widely distributed in human tissues, with the highest expression detected in skeletal muscle. The human ERK3 gene was mapped by fluorescence in situ hybridization to chromosome 15q21, a region associated with chromosomal abnormalities in acute nonlymphoblastic leukemias. This information should prove valuable in designing studies to define the cellular function of the ERK3 protein kinase.

Cardiotrophin-1 (CT-1) is a new member of the interleukin-6 cytokine family that was identified from a mouse embryoid body cDNA library by expression cloning. Mouse CT-1 induces features of hypertrophy in neonatal rat cardiac myocytes and binds to and activates the leukaemia inhibitory factor/gp130 receptor complex. In this work we report the isolation and characterization of cDNA and genomic clones encoding human CT-1. These clones encode a 201 amino acid protein that is 80% identical to the mouse protein. Human CT-1 produced by transfection of the cDNA clones into mammalian cells induces the hypertrophy of neonatal rat cardiac myocytes. Human and mouse CT-1 bind to the leukaemia inhibitory factor receptor on both human and mouse cell lines indicating a lack of species specificity. No binding to the human oncostatin M specific receptor was detected. A 1.7 kb CT-1 mRNA is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung. The coding region of CT-1 is contained on three exons and is located on human chromosome 16p11.1-16p11.2.

Fluorescence in situ hybridization (FISH) analysis of metaphase and decondensed free chromatin fibers from Adriamycin (ADR)-sensitive and ADR-resistant murine cells demonstrated a close juxtaposition of topoisomerase II alpha (Top2a) and retinoic acid receptor alpha (Rara) genes in adjacent chromatin in the drug-resistant cells, and a close but separate genetic proximity in normal murine chromatin. This provides physical evidence that the chromosome 11 allelic rearrangement resulting in a chimeric truncated Top2a/Rara transcript in the ADR-resistant cells is due to a novel fusion of the Topo2a and Rara genes. This is the first description of a Rara gene disruption in cells selected for antineoplastic drug resistance.

The temporal relationship of radiolabeled monoclonal antibody (Mab) uptake to tumor size in a nude mouse human colon cancer xenograft model (LS174T) was evaluated as an aid to developing a method for estimation of radioimmunotherapy absorbed dose. Tumors of heterogeneous size were treated with 4.4 MBq (120 microCi) of 90Y-labeled anti-Carcinoembryonic Antigen Mab (90Y-ZCE025). Regression analysis demonstrated an inverse log-log relationship of antibody uptake (%ID/g) to tumor mass in four time intervals investigated (N > 10 points/interval):12-24 h, 2-3 d, 5-7 d, and 10-14 d. Curves of predicted radionuclide concentration vs time were then constructed for a range of constant tumor sizes. Xenograft radiation dose was obtained by temporal integration of each curve and application of appropriate dose estimation formulas. For each assumed tumor mass, an edge correction for loss of beta energy outside the target volume was applied assuming a spherical tumor shape. Estimated average absorbed doses were found to vary only from 13.8-10.3 Gy for a 20-fold change in tumor sizes (0.1-2.0 g, respectively). Such constancy of dose may explain xenograft stasis observed by our group in earlier experiments at this level of administered 90Y activity.

Background: The organs of laboratory mice used in radioimmunotherapy experiments are relatively small compared to the ranges of high-energy yttrium-90 (Y-90) beta particles. Current Medical Internal Radiation Dose (MIRD) dosimetry methods do not account for beta energy that escapes an organ. A dosimetry model was developed to provide more realistic dose estimates for organs in mice who received Y-90-labeled antibodies by accounting for physical and geometric factors, loss of beta dose due to small organ sizes, and cross-organ doses., Methods: The dimensions, masses, surface areas, and overlapping areas of different organs of 10 athymic nude mice, each weighing approximately 25 g, were measured to form a realistic geometric model. Major organs in this model include the liver, spleen, kidneys, lungs, heart, stomach, small intestine, large intestine, thyroid, pancreas, bone, marrow, and carcass. A subcutaneous tumor mass also was included in the model. By accounting for small organ absorbed fractions and cross-organ beta doses, the MIRD methodology was extended from humans to mice for beta dose calculations., Results: Absorbed fractions of beta energy were calculated using the Berger's point kernels and the electron transport code EGS4. Except for the tumor and carcass, the self-organ absorbed fractions ranged from 15% to 20% in smaller organs (the marrow and thyroid) to 65%-70% in larger organs (the liver and small intestine). Cross-organ absorbed fractions also were calculated from estimates of the overlapping surface areas between organs., Conclusion: The mathematic mouse model presented here provides more realistic organ dosimetry of radiolabeled monoclonal antibodies in the nude mouse, which should, in turn, contribute to a better understanding of the correlation of biodistribution study results and organ-tumor toxicity information.

Background: Radioimmunotherapy of nude mice bearing human tumor xenografts using 90Y-labeled monoclonal antibodies has resulted in slower tumor growth, decreased tumor burden, and increased survival times. Dosimetry estimates in the murine model usually were based on biodistribution data and standard Medical Internal Radiation Dose approaches. A new dosimetric model for the mouse that takes into consideration the small dimensions, mass, and proximity of murine organs has been developed based on self-organ absorbed and cross-organ doses., Methods: Nude mice bearing carcinoembryonic antigen-expressing WiDr human colon cancer xenografts were injected with 240 microCi 90Y-anti-carcinoembryonic-antigen monoclonal antibodies and then killed at 12, 24, 72, 120, and 168 hours. Tumors and major organs were removed, weighed, and counted on a gamma counter. Using the resulting biodistribution data, the radiation doses to tumor and normal organs were calculated using the new dosimetric model for the mouse., Results: Three organs (the liver, kidneys, and large bowel) directly received > 50% of the total absorbed beta dose from radioactivity. Lungs, stomach, and marrow received the highest percentage (70-75%) of the total absorbed dose from adjacent organs. Tumor absorbed dose, estimated with the new dosimetric model, was three times less than that obtained with a MIRD-style calculation without correction for self-absorbed and cross-organ doses., Conclusions: The new dosimetric model, which accounts more accurately for self-organ absorbed and cross-organ beta dose fraction, allows the calculation of tumor and organ doses in the murine model. Accurate estimation of radiation doses to tumor and critical organs, such as the marrow, spleen and kidneys, is important in determining the efficacy and toxicity of radioimmunotherapy regimens in animals and in subsequent human applications.

An anticarcinoembryonic antigen (CEA) monoclonal antibody, T84.66, has been conjugated to a metallocarborane complex (Venus flytrap cluster, VFC) containing 57Co. This radioimmunoconjugate, 57Co-VFC-T84.66, retained > 90% immunoreactivity, was stable in serum (7 days) and demonstrated good localization in LS174T tumor xenografts. Pharmacokinetics of 57Co-VFC-T84.66 in tumor-bearing mice were compared to T84.66 Mab conjugated with either DTPA or its benzylisothiocyanate derivative (BzDTPA) labeled with 111In. Whole-body half-life for VFC-T84.66 was less (t1/2 = 62 hr) than that for either DTPA-T84.66 (t1/2 = 157 hr) or BzDTPA-T84.66 (t1/2 = 167 hr). Blood clearance was similar for all three radioimmunoconjugates (t1/2 = 22 hr). Hepatic uptake of the radiolabel was rapid and remained constant for 7 days for both DTPA radioimmunoconjugates (DTPA radioimmunoconjugate = 13.7 +/- 1.5 %ID/g; BzDTPA radioimmunoconjugate = 10.4 +/- 1.7%ID/g). For VFC, however, liver radioactivity decreased from 19.1 +/- 0.6%ID/g at 1 hr to 0.9 +/- 0.1 %ID/g 7 days postinjection, suggesting a possible role for VFC radioimmunoconjugate in the imaging and therapy of liver metastases.

Radioimmunotherapy and external beam radiotherapy were compared in a nude mouse human colon cancer model. Radioimmunotherapy was delivered by intraperitoneal injection of 90Y-labeled anticarcinoembryonic antigen monoclonal antibody (anti-CEA MAB). Single fraction external beam radiotherapy was delivered using a 60Co teletherapy unit. Control groups received saline, unlabeled anti-CEA monoclonal antibody and labeled nonspecific monoclonal antibody. Subcutaneous CEA-expressing LS174T human colon carcinoma tumors were measured over time. Tumor growth suppression was expressed as delay to reach 2g compared to saline controls. Unlabeled anti-CEA monoclonal antibody and labeled nonspecific monoclonal antibody had no effect. External beam radiotherapy of 300, 600, 1000 and 2000 cGy produced growth delays of 3, 12, 17, and 22 days, respectively. Radioimmunotherapy with 120 microCi, 175 microCi, and 225 microCi resulted in growth delays of 20, 34, and 36 days. Estimated absorbed tumor dose was 1750 cGy in the 120 microCi group. Similar comparisons were done with the more radioresistant WiDr human colon carcinoma cell line. External beam radiotherapy doses of 400, 800, 1200, and 1600 cGy resulted in growth delays of 6, 21, 36 and 48 days, respectively. Radioimmunotherapy of 120 microCi and 175 microCi resulted in growth delays of 9 and 19 days, respectively. The 120 microCi dose delivered an estimated absorbed tumor dose of 1080 cGy to WiDr tumors. In summary, for the radiosensitive LS174T line, radioimmunotherapy produced biologic effects that were comparable to a similar dose of single fraction external beam radiotherapy. For the more radioresistant WiDr tumor, radioimmunotherapy produced a biologic effect which was less than a similar dose of single fraction external beam radiotherapy. These studies suggest that a tumor's response to radioimmunotherapy relative to that of external beam radiotherapy is, in part, dependent on tumor radiosensitivity and repair capacity.

Heterophilic antibodies interfere in two-site immunoassays. Our purpose was to screen for the samples containing heterophilic antibodies that react with mouse immunoglobulins and to use them to determine how to eliminate interference in various two-site immunoassays being developed in our laboratory. Of approximately 2600 samples screened, 81 had heterophilic antibodies. When creatine kinase MB (CKMB) concentration was measured with intact antibody conjugate in these 81 samples, 18 (22%) samples had apparent CKMB values significantly greater than values measured with Hybritech's "Tandem -E CKMB immunoenzymetric" assay and Corning agarose-gel electrophoresis. Adding up to 133 mg/L of polymerized IgG or up to 1666 mg/L polyclonal mouse IgG in the assay did not eliminate the interference in all the samples. However, adding F(ab')2 conjugate plus 40 mg/L of polymerized IgG or 83 mg/L of polyclonal mouse IgG eliminated the interference in all the samples. This approach was also effective in eliminating the interference in 15 samples containing 4.7-165.2 mg/L of human antimouse antibody (HAMA). Combined use of F(ab')2 conjugate and polyclonal mouse IgG is recommended to eliminate interference from heterophilic antibodies that react with murine immunoglobulins or HAMA in two-site murine-antibody-based assays.

Two model peptides rich in boron and prepared by Merrifield syntheses, dansyl.(nido-CB)2, (1) and dansyl.(nido-CB)10.Lys.Ac (2), where nido-CB represents the alpha-amino acid [nido-7-CH3-8-(CH2)3CH-(NH2)COOH-7,8-C2B9H10]-, were conjugated with the anti-CEA mAb T84.66 using peptide active ester reagents. The dansyl groups provided a means of fluorimetric analysis of mAb conjugates which was augmented by conventional amino acid analyses for nido-CB. The conjugate of 1 contained an average of 63 B atoms per mAb molecule. The mAb conjugate of 2 was chromatographically separated into a strongly fluorescent high molecular weight aggregated fraction (HMW) and a less intensely fluorescent monomeric fraction. Both fractions retained immunoreactivity. The HMW species contained an average of ca. 490 B atoms/mAb molecule, as determined by amino acid analysis. Biodistribution data were collected using nude mice bearing LS174T xenografts and 125I-labeled mAb conjugates. While the lightly B-loaded dipeptide conjugate gave biodistribution results which resembled those of native T84.66 mAb, the undecapeptide conjugate displayed greatly enhanced liver uptake and decreased tumor accretion. These results suggest that as the boron-containing burden on the supporting immunoprotein is greatly increased, as in the case of the T84.66-2 conjugate, loss of circulating conjugate to liver effectively competes with the desired tumor localization. Means which might be taken to circumvent this difficulty have been described elsewhere (ref 15).

Athymic nu/nu mice bearing a subcutaneous human colon cancer xenograft (WiDr, low CEA expression) were treated with gamma-interferon (gamma IFN) at varying doses, frequencies, and periods of duration. CEA content (micrograms/g) and uptake of radiolabeled anti-CEA monoclonal antibody (MAB) (percent injected dose per gram, % ID/g) were measured at 48 h following administration of the MAB, The optimal enhancement of tumor CEA content and tumor localization of [111In] anti-CEA monoclonal antibody (MAB) was seen at gamma IFN doses of 100,000 U i.p. every 8 h for 4 days (4.7 micrograms/g; 29% ID/g) compared to control animals (0.9 micrograms/g; 10% ID/g). The effects of gamma IFN on CEA content and MAB localization were less pronounced when administered (a) at lower doses: 5,000 to 50,000 U i.p. every 8 h, (b) at varying frequencies: 300,000 U/day delivered in divided doses every 4 or 24 h, or (c) for varying periods: 2 or 6 days of therapy. In each case, the biologic effects on tumor CEA content and uptake of [111In]MAB correlated closely with the serum gamma IFN level. Therefore, we conclude that enhancement of in vivo CEA expression by gamma IFN may have clinical relevance for tumor imaging and therapy using radiolabeled monoclonal antibodies.

The effects of human recombinant gamma-interferon (gamma-IFN) on the levels of carcinoembryonic antigen (CEA) expression were investigated in vitro in three human colon adenocarcinoma cell lines (WiDr, HT29, and SW403). Subconfluent cultures were exposed continuously to IFN at concentrations of 1-1,000 antiviral units/ml for up to 6 consecutive days. IFN resulted in a significant increase in CEA levels when assayed by cellular enzyme-linked immunosorbent assay (ELISA), with higher concentrations and longer exposure times resulting in greater CEA enhancement. A three to five-fold enhancement of CEA was observed after 5-6 days of continuous exposures at concentrations of 100-1,000 antiviral units/ml. CEA levels returned to baseline over a 4-day period after discontinuation of IFN. Levels of IFN that resulted in CEA enhancement also resulted in cell growth inhibition, with a direct correlation observed. Flow cytometric studies, which evaluated changes in CEA membrane expression of only the viable cells remaining after IFN exposure, gave similar results to cellular ELISA. Quantitative CEA ELISA, which quantitated changes in total cellular CEA content, demonstrated greater increase in CEA than predicted by cellular ELISA. Continuous IFN exposures for 5-6 days at 1,000 U/ml led to a 96-, 26-, and 5-fold increase in total CEA for the WiDr, HT29, and SW403 cell lines, respectively. WiDr cells exposed to daily 6-h IFN pulses demonstrated intermediate increases in CEA compared with cells exposed continuously to IFN.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human gamma-interferon (IFN-gamma) has recently been shown to enhance localization of radiolabeled monoclonal antibodies (MAb) to human colon carcinoma xenografts in athymic mice. The present study investigates the ability of gamma-interferon to enhance radioimmunotherapy of a low carcinoembryonic antigen-expressing human colon cancer (WiDr) in athymic mice. Growth curve analysis, antibody localization, and dose estimation studies were performed. A significant tumor growth delay, measured as the time to reach 1.0 g, was noted for animals receiving specific anti-carcinoembryonic antigen 90Y-MAb (ZCE025, 120 microCi) plus IFN-gamma (61.8 days) as compared to animals that received specific 90Y-MAb with phosphate-buffered saline (34.9 days; P less than 0.005). IFN-gamma (100,000 units) was given i.p. every 8 h for 2 days before and 4 days after 90Y-MAb therapy. The time required to reach 1.0 g for animals treated with nonspecific 90Y-MAb (ZME018) was significantly less either with (38.3 days) or without (34.4 days) IFN-gamma. The difference was more apparent when compared to animals receiving IFN-gamma alone (30.0 days) or phosphate-buffered saline alone (28.9 days; P less than 0.001). Increased antibody localization in the tumors of animals treated with IFN-gamma plus specific 90Y-MAb (43.2% injected dose/g) was seen in comparison to animals treated with specific 90Y-MAb without IFN-gamma (18.2% injected dose/g). The estimate of radiation dose delivered to the tumors, based on biodistribution data over time, revealed significantly higher levels in animals treated with specific 90Y-MAb with IFN-gamma (2477 cGy) compared to animals treated without IFN-gamma (1217 cGy). These results provide support for the use of gamma-interferon as an immunomodulating agent prior to radioimmunotherapy.

A novel transition metal complex, Venus flytrap cluster (VFC), is described for the preparation of radio-labeled antibodies. VFC contained 57Co, which was held tightly between the faces of two covalently bridged carborane ligands by cluster bonding of the metal with appropriate ligand orbitals. Anti-carcinoembryonic antigen monoclonal antibody T84.66 was conjugated to 57Co-VFC with full retention of immunological activity. Biodistribution studies in nude mice bearing carcinoembryonic antigen-producing tumors showed excellent tumor localization of 57Co-VFC-T84.66. The accumulation of radionuclide in normal liver was low and independent of dose, which may reflect the stability of the radionuclide complex. These results presage the use of VFC systems for binding transition metals that are clinically useful for radioimmunodiagnosis and radioimmunotherapy.

Fifty-eight plasma specimens from 30 patients who had undergone presurgical radioimmunoscintigraphy with 111In-labeled anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (Mab) and who had no clinical evidence of disease after surgical resection showed increased concentrations of CEA (greater than or equal to 5 micrograms/L) in plasma when studied with the previously available commercial CEA enzyme immunoassay (EIA) from Roche. The possible role of anti-murine antibody (HAMA) interference was addressed by adding mouse IgG (mIgG) to the plasma (2 g/L) before assay. Fifteen specimens (26%) showed no change in CEA (reflecting a true increase as shown by the original results), 22 (38%) showed a decrease in CEA of greater than 15% but remained positive (reflecting an artefactual increase), and 21 (36%) became CEA-negative (less than or equal to 5 micrograms/L; reflecting a false increase). Subsequently, we assayed the same samples with a modified version of this CEA EIA kit and 47 specimens remained CEA positive (greater than 5 micrograms/L): 25 (53%) were truly increased, 12 (26%) remained artefactually increased, and 10 (21%) continued to show a false increase. The degree of interference in the original EIA kit correlated with the plasma concentration of HAMA (P less than 0.005). All artefactually and falsely increased CEA values observed in both kits were corrected by addition of polyclonal mIgG or of a mixture of IgG1, IgG2a, and IgG2b Mabs before assay. This correction is important in the follow-up of patients who receive murine Mabs for treatment or diagnosis.

An intraperitoneal xenograft tumor model was developed in the nude mouse to represent advanced colorectal disease in the human patient. Intraperitoneal (ip) and subcutaneous (sc) tumor xenografts were successfully localized by indium 111-labeled anti-carcinoembryonic antigen monoclonal antibodies (Indacea) administered intravenously (IV) or intraperitoneally (IP). In the sc model, tumor uptake (% injected dose per g of tumor tissue--%ID/g) was significantly improved at 72 hours postinjection when Indacea was administered IP (44.01 +/- 1.94 vs. 33.74 +/- 0.87, P less than 0.005). In the ip model, tumor uptake at 72 hours was improved with IP Indacea (28.23 +/- 5.42 vs. 19.36 +/- 5.50, P less than 0.01) and the tumor to blood ratio was significantly improved for IP Indacea (17.83 +/- 2.95 vs. 8.45 +/- 1.38, P less than 0.01). Tumor mass was not a contributing factor in these differences. This study demonstrates that ip xenografts can be successfully imaged using radiolabeled antibodies and that the IP route of administration results in better tumor uptake of the antibody in ip lesions.

Nude mice bearing subcutaneous human colon cancer xenografts (LS174T) were treated with 120 microCi of yttrium 90-labeled anti-carcinoembryonic antigen monoclonal antibodies (specific therapy), 120 microCi of 90Y-labeled anti-melanoma monoclonal antibodies (nonspecific therapy), or phosphate-buffered saline solution (no treatment control). Mean (+/- SD) tumor growth rates (percent increase per day) over the first 30 days of the study were as follows: 0.6% +/- 0.2% per day (specific therapy); 17.7% +/- 5.7% per day (nonspecific therapy); and 30.5% +/- 4.2% per day (control). In all three groups, tumors over 1 g had similar doubling times (5.74 +/- 0.71 d). Specific therapy caused a lag in tumor growth corresponding to a 3-logarithm cell kill. Estimated tumor dose of radiation obtained by tissue analysis was 34 and 14 Gy for specific and nonspecific therapy, respectively. In conclusion, 120 microCi of 90Y-labeled anti-carcinoembryonic antigen monoclonal antibodies was effective in suppressing growth of human colon cancer xenografts. Clinical studies with this preparation are recommended.

Monoclonal antibody ZCE025 recognizes an epitope of the carcinoembryonic molecule (CEA). We have shown that when linked to 90Y, its localization in the tumor was sufficient to result in a significant tumoricidal effect in human colon carcinomatosis grown in the peritoneum of athymic mice. Intraperitoneal tumors were present 7 days after inoculation of the CEA-producing human colon carcinoma cell line LS174T, when the mice received i.p. injections with 40 to 160 microCi of 90Y-labeled ZCE025 or 96.5c (nonspecific monoclonal antibody). The animals that were autopsied 12 days after treatment displayed a significant (P less than 0.001) inhibition of tumor growth when compared to the control animals that received no treatment or similar doses of nonspecific monoclonal antibody. Microscopically, the treated tumors showed extensive radiation effect and they became progressively necrotic until only a rim of viable tissue remained in the periphery of the nodules. CEA expression was practically absent on the newly grown nodules that began to appear 3 weeks after therapy, and remained so 6 weeks thereafter. In contrast, over 80% of the tumor cells from the untreated animals expressed CEA. There was no mortality due to treatment; however, the hematopoietic organs were markedly depleted at the higher doses. The marrow and the spleen recovery began 2 weeks after treatment, and it was completed by the 4th week. No evidence of toxicity was present in any of the other organs examined. These studies suggest that 90Y-ZCE025 therapy results in clonal selection of cells lacking or minimally expressing CEA. The inherent implications of these findings are discussed.

The purpose of this study was to examine the mechanism of specific antibody pretreatment for reduction of liver uptake of 111In-labeled monoclonal antibody (MAB). Previous work with an anti-carcinoembryonic antigen (CEA) MAB (T84.66) and LS174T human colon cancer xenografts in nude mice has shown that giving a high dose (0.2 mg) of unlabeled T84.66 in conjunction with the same MAB (T84.66) labeled with 111In (Indacea) significantly lowered the liver uptake of 111In. High performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to assess the radiolabeled components in serum and liver at different times following administration of Indacea in normal and tumor bearing mice. In serum the 111In remained associated with the IgG in both tumor bearing and non-tumor bearing mice. Liver uptake of 111In in mice without tumor was low (8-12% injected dose/g) and both IgG and a low molecular weight metabolite were found in the liver homogenates. Liver uptake in tumor bearing mice increased dramatically (15-40% injected dose/g) with size of tumor and in addition to the IgG and low molecular weight components, a high molecular weight compound was identified. Administration of CEA: Indacea complexes to non-tumor bearing mice produced the same high pressure liquid chromatography and gel patterns as those seen in mice with large (greater than 1 g) tumors. Liver homogenates from tumor bearing mice given specific antibody pretreatment showed the same patterns seen with non-tumor bearing mice (no high molecular weight peak). In conclusion, CEA:Indacea complexes are formed in tumor bearing mice and rapidly cleared by the liver. Specific antibody pretreatment results in the production of unlabeled CEA:MAB complexes causing a reduction in the formation of CEA:Indacea complexes and a lower liver uptake of 111In.

The efficacy of bone marrow transplantation (BMT) for the prevention of 90Y toxicity and extension of survival in nude mice with i.p. LS174T carcinomatosis was evaluated. 90Y-labeled monoclonal antibody (MAB) directed against carcinoembryonic antigen (90Y-anti-CEA MAB) at a dose of 120 microCi caused no deaths due to treatment toxicity and increased the duration of animal survival. No long term cures were obtained in these mice. At doses of 160 microCi or more 90Y-anti-CEA MAB led to hematological deaths. Nude mice were given i.p. injections of 10(6) LS174T tumor cells on day 0. On day 7 the mice received 90Y-anti-CEA MAB i.p. at doses of 120-225 microCi. Syngeneic bone marrow cells (10(7) cells) were then injected i.v. into the mice at 1, 3, 5, 7, 10, or 14 days following 90Y treatment. In the absence of BMT, toxic deaths for animals given 175 microCi 90Y were 11 of 24 (46%) with a median survival of 17 days and 13 of 20 (65%) for animals given 225 microCi 90Y with a median survival of 14 days. Animals receiving the same two doses of 90Y and given BMT 5 days following the 90Y treatment showed 0 of 24 (0%) and 0 of 54 (0%) toxicity deaths, respectively. The optimal time of BMT in relation to 90Y therapy was dependent upon the dose of 90Y-anti-CEA MAB (225 microCi, 3-5 days; 175 microCi, 5-14 days). The mean survival in tumor bearing animals was extended from 31.7 +/- 1.2 (SE) to 45.3 +/- 2.0 days by treatment with 120 microCi of 90Y-anti-CEA MAB. By increasing the dose of 90Y-anti-CEA MAB to 225 microCi and undertaking BMT 5 days later the mean survival was further extended to 63.2 +/- 3.6 days (P less than 0.005). BMT administered at the optimal times can prevent toxic deaths and facilitates higher, more effective doses of tumor specific 90Y-MAB.

In these measurements, we quantitated, via surgical samples, human primary tumor uptake of the anti-carcinoembryonic antigen monoclonal antibody T84.66. Uptake was measured in units of percentage of injected dose/kg with 111In as the radiolabel. All 11 colorectal lesions were nonnecrotic and were visualized upon scanning. Tumor volume was calculated using the three orthogonal dimensions as described by pathology. Uptake mean +/- SD was 6.55 +/- 3.55% injected dose/kg with a range of 1.2 to 10.4% injected dose/kg. Lesion mean volume was 36 cm3 with a range of 1.5 to 304 cm3. Using mean values, assuming no biological clearance and that the biodistribution of the monoclonal is independent of its radiolabel, the predicted human tumor 90Y beta dose was 0.12 Gy/mCi. Therefore a 10-Gy tumor dose would require 83 mCi of i.v. activity. Using least and highest uptake results, requisite activity values were 3-fold larger and smaller respectively. Thus, there was approximately an order of magnitude variation in the amount of 90Y predicted to achieve a given tumor dose in colorectal cancer patients. Murine and human uptake values were consistent if lesion mass and carcinoembryonic antigen content were taken into account.

Over a 4-year period, 108 patients with known or suspected colorectal cancer were studied by radioimmunoconjugate scintigraphy prior to operative procedures. Study subjects received 0.2 to 40 mg i.v. of murine anti-carcinoembryonic antigen monoclonal antibody labeled with 2-5 mCi of 111In (Indacea). Resected tissues were analyzed for 111In and carcinoembryonic antigen content. Tumor, liver, and draining lymph nodes had over 10% injected dose/kg compared to less than 2.5% injected dose/kg for other normal tissues. Primary tumors that were successfully imaged were significantly larger and had higher 111In and carcinoembryonic antigen content. In 54 patients, primary tumors were visualized with a sensitivity of 78%. Hepatic metastases (58 patients) were visualized as negative filling defects (sensitivity, 45%). Extrahepatic (intraabdominal) metastases (25 patients) were visualized (sensitivity, 48%) as areas of increased uptake. Extraabdominal metastases were uncommon (10 patients; sensitivity, 80%). Of 56 patients with known or suspected hepatic metastases who presented with no evidence of extrahepatic disease by conventional tests (X-ray, computerized tomographic scan), 20 (36%) were documented to have extrahepatic metastases at exploratory surgery and 10 of these (50%) had the extrahepatic disease localized by the Indacea scan. The management of these 10 patients was, or could have been, modified by the scan findings and unnecessary surgery eliminated.

Scintigraphic studies in animals and in humans have demonstrated uptake of radiolabeled antibody by both normal and tumor tissue. Normal tissues most commonly visualized are blood, liver, spleen, kidneys, lymph nodes, bone, and thyroid. A number of factors have been demonstrated to affect the uptake by normal and tumor tissue, including radioisotope properties, immunoglobulin characteristics, antibody specificity, tumor size, vascular permeability, and antigen expression. Clarification of the mechanisms of tumor and normal tissue uptake depends upon comparison of scintigraphic findings with analysis of tissue for such factors as radioactivity, antigen content, and tumor size. One of the major limitations of 111In labeled monoclonal antibody imaging has been extensive 111In uptake by histologically normal liver, especially in a host bearing a large tumor mass. By high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis analysis of liver and blood it can be demonstrated that much of the liver uptake is related to the formation of antigen:antibody complexes. The normal liver intensity can be decreased by inhibition of radiolabeled complex formation. Understanding of the mechanisms of tissue uptake, both normal and tumor, and of radiolabeled antibody metabolism is crucial to the rational planning and use of radioimmunoconjugates for tumor imaging and treatment. Animal and human studies complement one another in examination of these mechanisms.