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J Geradts

Publications and source records attributed to J Geradts.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular characterization of adenocarcinoma of the cervix.

In an attempt to characterize the molecular alterations of cervical adenocarcinoma, we analyzed 32 paraffin-embedded specimens for the presence of K-ras mutations, p53 overexpression, p16 and Rb protein expression, and the presence of HPV 16 and 18 DNA. Overall 25/32 (78%) of the tumors displayed an abnormality in at least one of these analyses. K-ras mutations were detected by PCR amplification and RFLP analysis in 3 tumors, including 2 at codon 12 and 1 at codon 61. p53 overexpression determined by immunohistochemistry was demonstrated with > 80% of tumor nuclei staining in 4 cases, 10-15% of nuclei staining in 3 cases, and < 1% of nuclei staining in 5 cases. The pattern of staining was diffuse in 6 cases, focal in 1 case, and scattered in 5 cases. Analysis of p16 protein expression in 23 specimens revealed 1 tumor with abnormal staining, while Rb protein expression was determined to be normal in all 25 tumors tested. HPV DNA, detected by PCR with type-specific primers, was found in 16 tumors (50%), including 7 (22%) with HPV 16 and 9 (28%) with HPV 18. There was no correlation among these abnormalities except that the presence of HPV and strong p53 overexpression (> 80% tumor nuclei staining) were mutually exclusive events. Clinical correlation demonstrated that p53 overexpression involving the majority of tumor cell nuclei is characteristic of advanced stage disease, while HPV positivity and activated ras genes are associated with early stage disease.

Adenocarcinoma↗
PubMed1997

Loss of RB and MTS1/CDKN2 (p16) expression in human sarcomas.

The product of the MTS1/CDKN2 gene (p16) and the retinoblastoma protein (pRB) inhibit cell cycle progression at the late G1 checkpoint. The absence of functional p16 or pRB has been identified in a variety of human tumors but has not been well studied in mesenchymal neoplasia. Using an immunohistochemical approach, the authors identified abnormal expression of either p16 or RB in 16 and 14 of 59 sarcomas, respectively, for an overall abnormality rate of 51%. Specific rates of abnormality varied by histological subtype, with leiomyosarcomas most commonly affected by loss of either tumor-suppressor gene product. There was no significant correlation between p16 or RB expression and overall grade, mitotic grade, or tumor progression for sarcomas. In contrast, no fibromatoses and other spindle cell neoplasms of low malignant potential displayed abnormal p16 expression, and only 4 of 23 cases showed loss of pRB expression. These data show that aberrant expression of p16/pRB is one of the most common molecular derangements in sarcomagenesis.

Carrier Proteins↗
PubMed1996

Rb and p16INK4a expression in resected non-small cell lung tumors.

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.

Adenocarcinoma↗
PubMed1996

Molecular and immunochemical analyses of RB1 and cyclin D1 in human ductal pancreatic carcinomas and cell lines.

Somatic mutations in the retinoblastoma-1 gene (RB1) and loss of RB1 protein function have been implicated in a number of human malignancies, but the role of RB1 gene and protein abnormalities in ductal pancreatic cancer (DPCA) is virtually unknown. We therefore analyzed expression of the RB1 protein immunohistochemically and/or by western blotting in a total of 54 sporadic and eight familial cases of archival and frozen DPCA and in 18 pancreatic carcinoma cell lines by using the antibodies RB-WL-1, 84-B3-1, and PMG3-245. Mutations in the RB1 promotor region and exons 13-21 of the RB1 gene were likewise examined by single-strand conformation polymorphism (SSCP) analyses and DNA sequencing of genomic DNA from 30 microdissected primary pancreatic tumors and the pancreatic carcinoma cell lines. Moreover, amplification and expression of a major regulatory component of RB1 function, cyclin D1, were assessed by southern and immunohistochemical analyses, respectively. The DPCAs were heterogeneous in both the intensity of RB1 nuclear staining and the percentage of immunoreactive cells. The tumors often had areas where RB1 staining was weak or absent adjacent to normal pancreatic tissue; however, only two of 32 archival cases and one of 30 frozen cases of DPCA completely lacked RB1 nuclear staining. Immunohistochemical and western blot analyses of 18 pancreatic carcinoma cell lines demonstrated the absence of RB1 expression in only two cell lines, Capan-1 and QGP-1. Analyses of the RB1 gene and promotor region by SSCP and DNA sequencing largely confirmed the immunochemical findings. Three of 30 primary carcinomas had abnormalities revealed by SSCP analyses. In one case a single base-pair deletion was confirmed in exon 18 and resulted in premature termination and the absence of detectable RB1 protein. A second case had TAC-->TTC missense mutation in exon 13. The third primary carcinoma could not be reliably sequenced because it had a low percentage of epithelial cells. The cyclin D1 gene was not amplified in any of the primary pancreatic tumors or cell lines examined. These immunochemical and molecular analyses of the RB1 tumor suppressor gene and cyclin D1 proto-oncogene in a large series of human pancreatic cancers and cell lines indicate that RB1 and cyclin D1 alterations occur during the development of some human DPCAs. Nevertheless, it is probable that alterations in cell-cycle regulation in DPCAs more frequently involve pathways other than those involving RB1 and cyclin D1.

Base Sequence↗
PubMed1996

Wild-type and mutant retinoblastoma protein in paraffin sections.

Inactivation of the retinoblastoma susceptibility (RB) gene plays a role in the pathogenesis of a variety of human malignancies. Recently, it has become feasible to study RB expression in archival tissues, and it is expected that immunohistochemical studies on routinely processed tumors will further elucidate the biologic and clinical significance of RB mutations. Our study was designed to address two issues that are critical for the interpretation of such studies, i.e., whether mutant RB protein (pRB) can reliably be distinguished from normal pRB and whether there are significant differences in the performance characteristics of various anti-RB antibodies. We studied cell blocks of 26 mutant RB cell lines (11 lines without any RB expression, nine lines expressing truncated mRNA/pRB, six lines carrying missense mutations) with five different anti-RB monoclonal antibodies, using a recently described procedure that includes an antigen retrieval step. The specific staining pattern for pRB was nuclear. Cytoplasmic staining was found to be nonspecific and could be strong. Some truncated and all full-length mutant pRBs localized to the nucleus, creating positive nuclear staining that might be indistinguishable from the staining pattern of cells carrying wild-type RB. The five antibodies tested showed significant differences in sensitivity, specificity, and background reactivity. Our data suggest that a significant subset of mutant pRB has preserved nuclear translocation capacity, that not all anti-RB antibodies are equally suitable for immunohistochemical analysis of RB expression, and that any such analysis is bound to include a certain, albeit probably small, number of positive stains, despite the absence of functional pRB.

Humans↗
PubMed1996

High frequency of aberrant p16(INK4A) expression in human breast cancer.

The product of the CDKN2/MTS1 gene, p16(INK4A) (16), inhibits phosphorylation of the retinoblastoma protein, pRB, and thus acts as a negative cell cycle regulator. It is inactivated in a wide range of human malignancies, including breast cancer. Using an immunohistochemical approach, we studied the expression of both p16 and pRB in 104 archival breast tumors, including 63 ductal, 33 lobular, and 8 mixed carcinomas. All specimens except one were evaluable for pRB expression, but only 87 were interpretable for p16 expression, reflecting the lower abundance and greater lability of this protein. Only six tumors showed abnormal RB expression. However, 43 carcinomas (49%) were completely (35) or focally (8) negative for p16. Abnormal p16 expression did not significantly correlate with several histopathological parameters. These findings provide evidence that aberrant p16(INK4A) expression is one of the most common abnormalities in human breast cancer.

Breast Neoplasms↗
PubMed1995

Immunohistochemical analysis of the p16INK4 cyclin-dependent kinase inhibitor in malignant mesothelioma.

BACKGROUND: The identification in 1994 of the CDKN2 gene as a target for mutations in a wide range of human cancers, including malignant mesothelioma, has been controversial because subsequent studies have detected a lower frequency of CDKN2 gene mutations in primary tumors than in cultured cell lines. These reports raised the hypothesis that another gene, distinct from CDKN2, might be the target of the chromosome 9p21 deletions frequently observed in these tumors. PURPOSE: To address whether inactivation of CDKN2 function is an essential event in the etiology of malignant mesothelioma, we examined p16INK4 protein expression in primary thoracic mesotheliomas, in nonmalignant pleural tissues, and in independent mesothelioma cell lines. We also studied the growth rate of tumor cell lines following stable transfection of CDKN2 gene. METHODS: Retinoblastoma (Rb) and p16INK4 protein expression was determined by immunohistochemical analysis from archival paraffin specimens of 12 primary thoracic mesotheliomas and a nonmalignant pleural biopsy specimen. In addition, protein immunoblot analysis for Rb and p16INK4 expression was conducted on 15 independent mesothelioma cell lines, and the ability of a transfected CDKN2 gene to suppress the growth of the mesothelioma cell lines H2373 and H2461 in vitro was examined. RESULTS: We demonstrated abnormal p16INK4 expression in 12 of 12 primary mesothelioma specimens and in 15 of 15 mesothelioma cell lines. All tumor specimens and the tumor cell lines showed expression of wild-type Rb protein. In addition, we have confirmed the ability of a transfected CDKN2 gene to suppress growth of two independent mesothelioma cell lines. CONCLUSIONS: Immunohistochemical analysis of the p16INK4 gene product is feasible in archival biopsy samples. With this analysis, CDKN2 gene inactivation can be determined in tumors that are contaminated with nonmalignant cells. Furthermore, since loss of p16INK4 protein expression can result from both genetic (gene mutations) and epigenetic (abnormal DNA hypermethylation) mechanisms, as we and others have shown recently, examination of protein expression is a highly sensitive method for analyzing the CDKN2 status in large numbers of tumor samples. IMPLICATIONS: This study suggests that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas. Defining the role of the p16INK4:Rb tumor suppressor pathway and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.

Carrier Proteins↗
PubMed1995

Immunohistochemical detection of the cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) product p16INK4A in archival human solid tumors: correlation with retinoblastoma protein expression.

The retinoblastoma (RB) and cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) tumor suppressor genes play important roles in the regulation of the cell cycle. The protein products of these two genes, pRB and p16INK4A ("p16"), respectively, inhibit progression from G1 to S phase. Moreover, p16 has been shown to exert its function through inhibition of CDK4-mediated phosphorylation of pRB. Both genes have been found to be mutated or deleted in a wide range of primary human tumors and tumor cell lines. However, the presence of CDKN2/MTS1 containing nonneoplastic elements in every tumor specimen may contribute to the apparent lower deletion detection rate in resected neoplasms compared to cell lines. We have developed an immunohistochemical assay that allows us to assess p16 expression in formalin-fixed, paraffin-embedded tissues. As controls, we used paraffin-embedded pellets of cell lines with well-defined p16 status (four positive and four negative lines), as well as routinely processed nude mouse xenografts of two p16-positive cell lines. p16-negative cells were characterized by the absence of nuclear staining, whereas cytoplasmic staining was variable. In neoplastic and normal tissues, the level of p16 generally appeared to be low. We tested 75 random human malignancies from 4 different anatomic sites for p16 expression and correlated the findings with the immunohistochemical presence or absence of pRB. Twenty % of tumors selectively lacked pRB, while 37% of neoplasms had undetectable p16. In 43% of all carcinomas, both pRB and p16 could be detected. Significant differences existed in the expression of both tumor suppressor genes between carcinomas from different sites. Breast cancers had the highest rate of p16 negativity (13 of 20). Our data show that: (a) immunohistochemistry may be a suitable modality to screen for RB and CDKN2/MTS1 abnormalities in paraffin-embedded tissues; (b) undetectable levels of p16 expression occur at a relatively high frequency; (c) p16 and pRB expression in common human malignancies is not mutually exclusive; (d) loss of function of both tumor suppressor genes appears to be a distinctly uncommon phenomenon; and (e) different types of carcinomas have variable rates of disturbance in the p16/pRB pathway.

Carrier Proteins↗
PubMed1995

The Carolina Breast Cancer Study: integrating population-based epidemiology and molecular biology.

The integration of epidemiology and molecular biology provides a new strategy to identify additional risk factors for breast cancer and to better understand the role played by traditionally recognized risk factors. The Carolina Breast Cancer Study (CBCS) is a population-based, case-control study designed to identify causes of breast cancer among Caucasian and African-American women who are residents of a 24-county area of central and eastern North Carolina. Information on established and potential breast cancer risk factors is obtained by personal interviews. Blood samples are collected from all consenting participants. Medical record documentation and paraffin-embedded tumor specimens are obtained for all breast cancer patients. DNA from tumor tissue is tested for a variety of molecular alterations characteristic of breast cancer. Germline DNA from blood lymphocytes is evaluated for presence of alleles increasing susceptibility to breast cancer. Statistical analyses evaluate gene-environment interaction by exploring the associations between environmental/behavioral factors and breast cancer in relation to specific molecular alterations (germline and tumor). Results will help identify high-risk women, clarify causal pathways, and hopefully contribute to the prevention of breast cancer.

Adult↗
PubMed1995

The need for epidemiologic studies of in-situ carcinoma of the breast.

The purpose of this paper is to present background information on carcinoma in situ (CIS) of the breast and to provide a theoretical framework for planning epidemiologic studies which may further our understanding of breast cancer. Two types of epidemiologic studies are needed which incorporate CIS of the breast: (i) case-control studies, in which in-situ lesions serve as disease outcomes (endpoints), and (ii) cohort studies and clinical trials, in which diagnosis of in-situ carcinoma serves as a starting point for patient treatment and follow-up. Case-control studies focusing on the causes of CIS have distinct advantages: if risk factors for cancer contribute to pathways involving some intermediate stages but not others (e.g. comedo-type but not non-comedo-type DCIS; LCIS versus DCIS), the use of precursor lesions may more clearly reveal risk factor associations than studies of invasive breast cancer alone; epidemiologic studies of precursor lesions are conducted closer in time to the exposures suspected to be causes and may reduce recall bias or other forms of misclassification; genetic alterations in early lesions are more likely to represent causal events in development of the malignant phenotype. Population-based case-control studies of CIS may thus prove useful in understanding breast cancer etiology and designing preventive strategies. CIS patients identified for case-control studies may be followed up over time as a cohort. Cohort studies (and clinical trials) of CIS aim to elucidate mechanisms influencing progression of CIS to invasive cancer as well as to evaluate effectiveness of specific treatment modalities. Although the majority of CIS lesions of the breast are ductal carcinoma in situ (DCIS), epidemiologic studies which also include patients with lobular carcinoma in situ (LCIS) address potential differences between DCIS and LCIS with respect to both etiology and progression.

Breast Neoplasms↗
PubMed1994

Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti-RB antibodies.

The retinoblastoma (RB) susceptibility gene encodes a nuclear phosphoprotein which is likely involved in cell cycle control and cell differentiation. The RB protein is mutated or absent in a variety of human malignancies. Its role as a molecular marker for clinical tumor behavior is under extensive investigation. However, studies on the status of the RB protein in primary or metastatic tumors and their precursor lesions have been slowed by the lack of availability of a sensitive, reliable assay which allows examination of RB expression in selected cell populations within archival tissues. Thus far, meaningful immunohistochemical analysis of RB protein in formalin-fixed, paraffin-embedded specimens has been achieved only with the polyclonal antibody RB-WL-I. We now describe a method which produced excellent staining results in formalin-fixed, routinely processed tissues, using commercially available monoclonal antibodies (MAbs) in conjunction with an antigen-retrieval step. The resulting stains were superior to those on frozen sections and comparable to those obtained with RB-WL-I. Twelve of 51 random invasive bladder cancers (24%) had abnormal expression of the RB gene, as determined by immunohistochemistry. Smaller cohorts of breast, prostate and lung carcinomas had incidences of aberrant RB gene expression ranging from 9% to 24%. Since the staining method was widely applicable to essentially all formalin-fixed, archival tissues, it may expedite studies on the biological and clinical significance of altered RB expression in human neoplasia.

Antibodies, Monoclonal↗
PubMed1994

Pathology and laboratory medicine.

Image analysis and flow cytometry are complementary methods in assessing the DNA content and proliferative capacity of a tumor. In prostatic adenocarcinomas, DNA aneuploidy is associated with poor outcome. The current 21 days for identification of mycobacteria and 28 days for susceptibility testing should be reduced in the coming years.

Clinical Laboratory Techniques↗
PubMed1994

Retinoblastoma protein monoclonal antibodies with novel characteristics.

We have developed a family of monoclonal antibodies directed against the retinoblastoma gene product (p110RB). One of these monoclonal antibodies, 3C8, binds p110RB near the C-terminal end of the protein (aa886-aa905). It was characterized by immunoblotting, ELISA, fluorescence-activated flow cytometry and immunohistostaining. It was shown to be useful for the detection of p110RB in formalin-fixed and paraffin-embedded tissue sections. Because 3C8 binds outside of regions shown to be involved in p110RB interactions with other cellular proteins, it may be an especially useful reagent for the reliable detection of p110RB in tumor cells, and for the isolation by affinity chromatography of p110RB complexes with other cellular proteins.

Animals↗
PubMed1994

Partial inactivation of the RB product in a family with incomplete penetrance of familial retinoblastoma and benign retinal tumors.

While familial retinoblastoma has served as the paradigm for the two-hit theory of tumorigenesis and for the concept of the tumor suppressor gene, the etiology of incomplete penetrance of familial retinoblastoma is poorly understood. To address the molecular basis for this phenotype we have studied the functional properties of a mutant Rb gene identified in a kindred with incomplete penetrance of familial retinoblastoma and evidence for regressed retinal lesions (retinomas). In contrast to all previously isolated RB mutant proteins, we demonstrated that the mutant product from this kindred retained the wildtype properties of nuclear localization, the ability to undergo hyperphosphorylation in vivo, and the capacity to suppress growth of RB(-) cells. Protein binding ('pocket') activity, however, was defective defining a new class of RB mutant with partial inactivation. The presence of this unique RB mutant in the germline of obligate carriers with incomplete penetrance and regressed retinal lesions suggests a molecular basis for this phenotype and supports the hypothesis that a minimum 'RB threshold' level of protein binding activity is required to suppress tumorigenesis.

Alleles↗
PubMed1993

Human lung cancer cell lines exhibit resistance to retinoic acid treatment.

Retinoic acid (RA) and nuclear retinoic acid receptors (RARs) have been implicated in a variety of human malignancies including lung cancer, and RA has been proposed as a chemopreventive agent for bronchogenic carcinoma. Normal human tracheobronchial epithelial cells show dramatic induction of RAR-beta mRNA and significant growth inhibition after RA treatment. In contrast, 17 of 22 small cell lung cancer (SCLC) and 9 of 15 non-SCLC lines treated with 1 microM RA showed no significant growth inhibition. Of interest, 5 SCLC lines with high levels of myc gene family expression related to c-, N-, or L-myc gene amplification exhibited growth inhibition (28-87%), whereas 2 non-SCLC lines actually showed growth stimulation after treatment with 1 microM RA. The lines varied greatly in their constitutive expression of RAR-beta mRNA, and 15 of 20 SCLC and 8 of 15 non-SCLC lines failed to show RAR-beta mRNA induction after RA treatment. Six cell lines showed possible alterations in the coding region of RAR-beta by complementary DNA (cDNA)/polymerase chain reaction (PCR) analysis using primers common to the RAR-beta1,2,3 isoforms, since other regions would undergo cDNA/PCR amplification whereas the DNA binding domain would not. Nonetheless, no abnormal band shift patterns in cDNA amplified by PCR were found by single strand conformation polymorphism analysis covering all 1344 base pairs of the RAR-beta open reading frame. Finally, no abnormalities in RAR-alpha gene structure or expression were identified by Southern and Northern blot analysis, including lines with cytogenetic abnormalities of 17q21. We conclude that abnormalities of the RAR-beta system are common in human lung cancer cell lines.

Base Sequence↗
PubMed1993

RB-mediated tumor suppression of a lung cancer cell line is abrogated by an extract enriched in extracellular matrix.

To examine whether the expression of retinoblastoma (RB) protein could mediate tumor suppression in a lung carcinoma cell line carrying multiple genetic defects, we transfected the Rb gene into a non-small cell lung cancer cell line with absent RB protein. We observed that the stable expression of a functional RB product was associated with a decreased efficiency of soft agar cloning and partial suppression of tumorigenicity in nude mice. The suppression of tumorigenicity was marked when 10(6) cells were injected into the flanks of nude mice but was less prominent when 10(7) cells were injected. RB-mediated tumor suppression, however, was consistently blocked by preincubation of the transfected cells with an extract enriched with an extracellular matrix (Matrigel). Analysis of tumors harvested from nude mice showed that they continued to express detectable levels of human RB protein which retained functional E1A binding activity and nuclear localization. RB-mediated inhibition of soft agar cloning was also blocked in a dose-dependent manner by the addition of Matrigel. These observations demonstrate that the expression of wild-type RB may suppress certain parameters of tumorigenicity in lung carcinoma cells that contain multiple genetic alterations. In addition, the reversal of tumor suppression by an extract enriched in basement membrane components may offer clues for further investigations into the mechanism of RB-mediated tumor suppression.

Animals↗
PubMed1990

Use of the polymerase chain reaction in the diagnosis of unsuspected herpes simplex viral pneumonia: report of a case.

A 30-year-old apparently immunocompetent woman presented with acute respiratory failure (acute respiratory distress syndrome). No etiologic agent was found, and she died 2 weeks later despite antibiotic therapy. Postmortem examination of the lungs showed diffuse organizing alveolar damage with superimposed focal necrotizing peribronchiolar pneumonia. Cultures obtained from lung tissue were negative for virus, fungi, and bacteria. Histopathologic and electron microscopic studies showed that the necrotizing changes were consistent with herpesvirus infection. With the use of a new diagnostic tool, the polymerase chain reaction, a specific diagnosis of herpes simplex virus pneumonia was made, and other viral agents were excluded. The polymerase chain reaction is a sensitive, specific, and rapid technique that may greatly facilitate establishing an infectious etiology in cases of pneumonia.

Adult↗