Biomedical subjects

J Geradts

Publications and source records attributed to J Geradts.

At least 37 records · Page 2Linked to original sources

PubMed2000

Immunohistochemical p16INK4a analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene. A comparison of four commercial antibodies.

The MTS1/CDKN2/p16 gene encoding the p16INK4a tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti-p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody ZJ11 from Neo-Markers. Optimal staining conditions were established for each antibody. The pancreatic carcinomas with homozygous p16 deletions were largely devoid of nuclear staining (admixed nonneoplastic cells served as internal positive controls); only one adenocarcinoma each reacted with DCS-50 and the polyclonal antibody, and five were positive with ZJ11, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.

Antibodies↗

PubMed2000

Human breast cancer specimens: diffraction-enhanced imaging with histologic correlation--improved conspicuity of lesion detail compared with digital radiography.

Seven breast cancer specimens were examined with diffraction-enhanced imaging at 18 keV with a silicon crystal with use of the silicon 333 reflection in Bragg mode. Images were compared with digital radiographs of the specimen, and regions of increased detail were identified. Six of the seven cases (86%) showed enhanced visibility of surface spiculation that correlated with histopathologic information, including extension of tumor into surrounding tissue.

Biopsy, Needle↗

PubMed2000

Genomic FHIT analysis in RER+ and RER- adenocarcinomas of the pancreas.

Alterations of the candidate tumor suppressor gene FHIT have been reported in multiple tumor types, including pancreatic carcinoma. The mechanism of FHIT genomic inactivation is unusual, most frequently occurring by homozygous deletion, whereas only rare cases have missense mutations. Altered (shortened) transcripts and reduced protein expression are reported, but a genetic basis for these is often inapparent. We studied FHIT genomic alterations of pancreatic carcinomas. Loss of heterozygosity (LOH) was found in 41% of 93 carcinomas without microsatellite instability (RER(-)), but no mutations were found by genomic sequencing. Homozygous deletions inside the FRA3B fragile site were found in four RER(-) tumors, but only two affected the FHIT coding region. In contrast, FHIT alterations were found in the three RER(+) pancreatic carcinomas screened; two had FHIT homozygous deletions affecting exon 5 and the third had a heterozygous missense mutation (H76N). The excess occurrence of homozygous deletions at this site in RER(+) pancreatic cancers is statistically significant (P < 0.01). Since homozygous deletions have not previously been reported in RER(+) carcinomas at any genomic site, an extremely high rate of site-specific deletion must exist within the FRA3B-related FHIT gene. Consequently, the paucity of documented inactivating point mutations cannot be used to judge the presence or absence of putative FHIT-related selective pressures that act during tumorigenesis of RER(-) neoplasia. Nonetheless, the identification of a heterozygous mutation as the sole sequence abnormality might raise doubt as to whether strong selective pressures are afforded by FHIT genomic inactivation in this tumor type. Genes Chromosomes Cancer 27:239-243, 2000.

Acid Anhydride Hydrolases↗

PubMed1999

Estrogen receptor status, determined by immunohistochemistry, as a predictor of the recurrence of stage I endometrial carcinoma.

BACKGROUND: The aim of this study was to compare the concordance between immunohistochemical (IHC) and biochemical (RIA) methods for determining hormone receptor status in patients with endometrial carcinoma and to determine whether IHC expression of estrogen and progesterone receptors (ER and PR) has prognostic significance. METHODS: Paraffin blocks were obtained from patients diagnosed with endometrial carcinoma between 1987 and 1991. IHC analysis for ER and PR expression was performed and scored based on staining intensity and the percentage of tumor cells with nuclear staining. Biochemical assays were performed on frozen tissues. Concordance between the two methods was evaluated and hormone receptor status was correlated with tumor grade, stage, recurrence and survival. RESULTS: ER and PR expression, determined by IHC, correlated well with RIA levels (Spearmans correlation coefficient, P = 0.006 and 0.0005, respectively). Determination of ER and PR expression by both methods was correlated with tumor grade. Hazards ratios revealed that the absence of ER and PR expression, determined by both IHC and RIA, independently correlated with recurrence in early stage disease (P < 0.05). CONCLUSIONS: Historically, receptors have been determined by RIA. In this study, IHC and RIA were equally suitable for determination of ER and PR. This is significant clinically as IHC has several advantages over RIA, including easier processing, lower cost, greater speed, and applicability to fixed tissue samples. In addition, ER negative status was predictive of the recurrence of Stage I tumors independent of tumor grade. ER status may aid the clinician in planning treatment when adjuvant treatment is controversial.

Endometrial Neoplasms↗

PubMed1999

Loss of KAI1 expression in the progression of colorectal cancer.

The transmembrane 4 superfamily member KAI1 (CD82) has been shown to inhibit pulmonary metastases in experimental metastasis models of prostate cancer and melanoma. KAI1 expression is decreased in the progression of common solid epithelial tumors of adulthood, including lung, prostate, breast, esophageal, gastric, pancreatic, and bladder cancers. The purpose of our study was to investigate KAI1 expression in the progression of human colorectal cancer. We first analyzed 20 colorectal cancer cell lines by immunoblot techniques. KAI1 was expressed heterogeneously, with the tumor cell lines having a more complex degree of glycosylation compared with that of the normal colonic tissue. KAI1 was highly expressed in the primary SW480 colon cancer cell line but was down-regulated 15-fold in the matched metastatic SW620 cell line. We also investigated KAI1 protein expression by immunohistochemistry in tissues from 84 patients with colorectal cancer. Each tissue section was assigned a KAI1 mean score (KMS) from 0 to 300 based on the product of the percentage of cells that stained for KAI1 and the intensity of the stain (1, 2, or 3). In 84 patients with colorectal cancer, KAI1 was expressed at high levels in normal colonic mucosa (KMS 226) but was expressed at lower levels in the primary tumors (KMS 65; P < 0.0001). In a subset of 12 patients with stage IV metastatic disease, we observed a progressive down-regulation of KAI1, from the normal adjacent colonic mucosa (KMS 193) to the primary tumor (KMS 72; P = 0.0001) to the liver metastasis (KMS 25; tumor compared with metastasis, P = 0.0135). We found no correlation between loss of KAI1 expression and stage of disease. In 10 patients, we also noted loss of KAI1 expression in the transition from normal colonic mucosa (KMS 237) to adenoma (KMS 174) to carcinoma (KMS 62; P < 0.0167 for all three comparisons). We conclude that the down-regulation of KAI1 occurs early in the progression of colorectal cancer.

Adenoma↗

PubMed1999

Abnormal Fhit expression in malignant and premalignant lesions of the cervix.

Genetic analysis of cervical cancer has demonstrated frequent allelic loss in the 3p chromosomal region. The newly described gene FHIT is located at chromosome region 3p14.2, and its expression has been demonstrated previously by reverse transcription-PCR to be abnormal in a majority of cervical cancer cell lines. In this study, 98 different lesions of the cervix were examined for Fhit expression by immunohistochemical staining. Whereas normal cervical epithelium demonstrated diffuse, moderate to intense cytoplasmic staining, many pathological lesions of the cervix displayed reduced or absent Fhit expression. Sixty-one percent of squamous carcinomas and 40% of adenocarcinomas of the cervix had abnormal Fhit expression. Sixty-five preneoplastic lesions of the cervix were examined. Eleven of 33 high-grade squamous intraepithelial lesions and 1 of 12 low-grade squamous intraepithelial lesions had abnormal Fhit expression. In summary, Fhit expression is frequently abnormal in both glandular and squamous cervical cancers, with a higher frequency of Fhit alterations observed in squamous lesions. In addition, abnormal Fhit expression can be detected in some preneoplastic lesions of the ectocervix. Alterations in Fhit expression may be an important marker of early progression in the development of cancers of the cervix.

Acid Anhydride Hydrolases↗

PubMed1999

The 100-kDa proteolytic fragment of RB is retained predominantly within the nuclear compartment of apoptotic cells.

The retinoblastoma tumor suppressor protein (RB) has been shown to play a role in regulating the eukaryotic cell cycle, promoting cellular differentiation, and modulating programmed cell death. Although regulation of RB tumor suppressor activity is mediated by reversible phosphorylation, an additional posttranslational modification involves the cleavage of 42 residues from the carboxy terminus of RB during the onset of drug-induced or receptor-mediated apoptosis. We now demonstrate that a recombinant p100cl RB species localizes to the nucleus where it may retain wildtype "pocket" protein binding activity. In addition, using immunocytochemistry, we show that cleavage of the endogenous RB protein occurs in vivo in human cells and that p100cl is predominantly retained within the nuclear compartment of cells during early apoptosis. We also show that the carboxy-terminal cleavage of RB is detected immediately following caspase-3 and PARP cleavage during FAS-mediated apoptosis of MCF10 cells. These findings suggest that this cleavage event may be a component of a downstream cascade during programmed cell death.

Apoptosis↗

PubMed1999

Frequent loss of KAI1 expression in squamous and lymphoid neoplasms. An immunohistochemical study of archival tissues.

The metastasis suppressor gene KAI1 was identified by its ability to inhibit the formation of pulmonary metastases in experimental models for prostatic carcinoma. Down-regulation of this gene may be correlated with the invasive phenotype in melanomas and colon and bladder carcinomas and with the metastatic phenotype in carcinomas of the lung, breast, prostate, and pancreas. The goal of our study was to establish an immunohistochemical method to detect KAI1 expression in archival tissues. Using cell lines with known KAI1 levels and paraffin-embedded KAI1 positive tissues as controls, we observed strong membrane staining in lymphoid follicular centers and squamous epithelia. We then demonstrated the utility of our assay by studying KAI1 expression in 34 lymphoid and 57 squamous lesions. All eight reactive lymph nodes were KAI1 positive. In contrast, three of 13 follicular small cleaved and five of 13 diffuse large cell lymphomas were KAI1 negative. Seventy-nine percent (37 of 47) of invasive squamous cell carcinomas from the lung (n = 15), head and neck (n = 18), and cervix (n = 14) showed extensive KAI1 down-regulation. Loss of KAI1 expression was also found in a subset of 10 high-grade cervical dysplasias. Our data show that (i) immunohistochemistry is a suitable technique for evaluating KAI1 expression in archival tissues; (ii) KAI1 was not expressed in a subset of both low-grade and high-grade lymphomas; and (iii) there was extensive down-regulation of KAI1 in squamous cell carcinomas, suggestive of an important role of the gene in the suppression of invasion in these malignancies.

Animals↗

PubMed1999

Policy guidelines for the utilization of formalin-fixed, paraffin-embedded tissue sections: the UNC SPORE experience. University of North Carolina Specialized Program of Research Excellence.

Paraffin blocks represent a valuable resource that has allowed investigators to apply today's technology to address scientific questions in a shorter period of time and in more diverse populations than would have been possible with fresh or frozen tissue. However, in addition to being an exhaustible resource, there is concern regarding the appropriate use of these tissues, both with respect to medical or legal considerations and quality control and quality assurance practices. We describe policy guidelines to address these concerns, including: safeguards to address medical/legal and patient confidentiality issues, quality control and quality assurance for tissue sectioning, processing and storage, database management for sample tracking, and scientific review for utilization of specimens. These policies and procedures have been developed and implemented by the University of North Carolina (UNC) Specialized Program of Research Excellence (SPORE) in the Breast Cancer Immunohistochemistry (IHC) Core laboratory, in collaboration with our study pathologists, participants, and research investigators. It is our hope that the information and experience described here may stimulate discussion that can ultimately lead to a uniform policy for handling formalin-fixed paraffin-embedded tissues in research.

Breast Neoplasms↗

PubMed1999

G1 checkpoint protein and p53 abnormalities occur in most invasive transitional cell carcinomas of the urinary bladder.

The G1 cell cycle checkpoint regulates entry into S phase for normal cells. Components of the G1 checkpoint, including retinoblastoma (Rb) protein, cyclin D1 and p16INK4a, are commonly altered in human malignancies, abrogating cell cycle control. Using immunohistochemistry, we examined 79 invasive transitional cell carcinomas of the urinary bladder treated by cystectomy, for loss of Rb or p16INK4a protein and for cyclin D1 overexpression. As p53 is also involved in cell cycle control, its expression was studied as well. Rb protein loss occurred in 23/79 cases (29%); it was inversely correlated with loss of p16INK4a, which occurred in 15/79 cases (19%). One biphenotypic case, with Rb+p16- and Rb-p16+ areas, was identified as well. Cyclin D1 was overexpressed in 21/79 carcinomas (27%), all of which retained Rb protein. Fifty of 79 tumours (63%) showed aberrant accumulation of p53 protein; p53 staining did not correlate with Rb, p16INK4a, or cyclin D1 status. Overall, 70% of bladder carcinomas showed abnormalities in one or more of the intrinsic proteins of the G1 checkpoint (Rb, p16INK4a and cyclin D1). Only 15% of all bladder carcinomas (12/79) showed a normal phenotype for all four proteins. In a multivariate survival analysis, cyclin D1 overexpression was linked to less aggressive disease and relatively favourable outcome. In our series, Rb, p16INK4a and p53 status did not reach statistical significance as prognostic factors. In conclusion, G1 restriction point defects can be identified in the majority of bladder carcinomas. Our findings support the hypothesis that cyclin D1 and p16INK4a can cooperate to dysregulate the cell cycle, but that loss of Rb protein abolishes the G1 checkpoint completely, removing any selective advantage for cells that alter additional cell cycle proteins.

Aged↗

PubMed1999

Correlation of abnormal RB, p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormalities, and clinical features in 103 primary non-small cell lung cancers.

This study was performed to determine the frequency of inactivation and clinical correlates in non-small cell lung cancer (NSCLC) of three known tumor suppressor genes [TSGs; RB, MTS1/CDKN2 (p16), and p53] and various regions of 3p loss of heterozygosity (LOH) as other major potential TSG sites. Paraffin sections from 103 resected NSCLCs were analyzed for expression of pRB, p16, and p53 by immunohistochemistry, whereas DNA from tumor and normal tissue were tested for LOH at 3p25-26, 3p21, and 3p14. Previously published LOH data for 5q, 11p, 17q, and 18q were also available. Loss of pRB or p16 expression and overexpression of p53 were considered abnormal. The immunohistochemical and LOH data were correlated with a variety of clinical parameters including stage, age, sex, smoking history, and survival. With respect to pRB, p16, and p53, the tumors could be grouped into four categories: normal for all three proteins (21%); abnormal for pRB or p16 and normal for p53 (30%); normal for pRB and p16 and abnormal for p53 (20%); and abnormal in both pathways (28%). Aberrant expression of pRB, p16, p53, and 3p LOH, either individually or in combination, was not associated with survival differences or any other clinical parameters, with the exception that pRB/pl6 abnormalities were more common in older patients (P = 0.0005). pRB and p16 expression showed a strong inverse correlation (P = 0.002), whereas there was no correlation between expression of pRB, p16, and p53. Abnormal expression of any of the three genes inversely correlated with K-ras codon 12 mutations (P = 0.004), but not with 3p LOH or LOH at other TSG loci. We conclude that resectable NSCLCs show distinct patterns of TSG inactivation, but that no clear clinical correlates exist either alone or in combination for pRB, p16, p53, and 3p abnormalities.

Adult↗

PubMed1998

Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression.

Pancreatic adenocarcinoma develops from histologically identifiable intraductal lesions that undergo a series of architectural, cytological, and genetic changes. Limited genetic evidence recently suggested that the p16 gene plays a role in the progression of these "duct lesions." Duct lesions were identified in pancreata from 33 pancreaticoduodenectomies performed for infiltrating adenocarcinoma. All of these infiltrating adenocarcinomas were previously shown to contain alterations in the p16 gene or its promoter. Monoclonal and polyclonal anti-p16 antibodies were used for histological immunodetection. One hundred twenty-six duct lesions were identified. Nine (30%) of 30 flat, 4 (27%) of 15 papillary, 37 (55%) of 67 papillary with atypia, and 10 (71%) of 14 carcinoma in situ duct lesions showed loss of p16 expression. These included 30% of the flat lesions versus 53% of the nonflat lesions and 29% of the nonatypical lesions versus 58% of the atypical lesions. For both comparisons, the differences were statistically significant (P = 0.036 and P = 0.003, respectively). Loss of p16 expression occurs more frequently, but not exclusively, in higher-grade duct lesions. These data support the hypothesis that pancreatic duct lesions are neoplastic and that they represent the precursors of infiltrating adenocarcinoma. Immunohistochemical detection of p16 provides a new technology to study the genetic alterations in and stages of progression of large numbers of morphologically defined pancreatic duct lesions.

Adenocarcinoma↗

PubMed1998

RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer.

Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb(+/-) p53(+/-) mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.

Animals↗

PubMed1998

Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression.

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.

Animals↗

PubMed1998

Protein expression and functional analysis of the FHIT gene in human tumor cells.

BACKGROUND: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. METHODS: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal anti-pFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. RESULTS: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. CONCLUSIONS: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the "classic" tumor suppressor paradigm.

Carcinoma, Small Cell↗

PubMed1998

Loss of tumor suppressor gene expression in high-grade but not low-grade non-Hodgkin's lymphomas.

The products of the MTS1/CDKN2 and retinoblastoma (RB) tumor suppressor genes, p16 and pRB, act as agonists in controlling the late G1 cell cycle checkpoint. Inactivation of either gene occurs in a wide range of human malignant neoplasms. Data on the expression of both genes in the same set of malignant lymphoid neoplasms are scarce. We studied the p16/pRB pathway in low-grade and high-grade non-Hodgkin's lymphomas, using immunohistochemical techniques. Paraffin sections of 9 reactive lymph nodes and 43 low-grade and 60 high-grade malignant lymphomas were reacted with antibodies against pRB and p16. All benign lymph nodes showed a normal pattern of RB and MTS1/CDKN2 expression. Of 101 evaluable lymphomas, only a single high-grade tumor displayed loss of RB reactivity. Loss of p16 was identified in 14 of 55 evaluable high-grade lymphomas but not in any of the low-grade lesions. All but 3 of the RB- and p16-negative cases were diffuse large cell lymphomas, for an abnormality rate of 55% in this category. While loss of RB function was a rare event in human lymphomagenesis, p16 was absent in some 25% of high-grade non-Hodgkin's lymphomas; diffuse large cell lymphomas were the primary target of tumor suppressor gene inactivation.

Cell Cycle↗

PubMed1998

Nasopharyngeal carcinomas frequently lack the p16/MTS1 tumor suppressor protein but consistently express the retinoblastoma gene product.

The p16/MTS1 gene is altered by deletion, mutation, or hypermethylation in a wide variety of human cancers. As a result of deficient p16 protein, these cancers lack a critical mechanism for halting G1/S cell cycle progression. In the current study, 59 cases of nasopharyngeal carcinoma were evaluated for expression of the p16 tumor suppressor protein by immunohistochemical analysis of paraffin-embedded tissue. There was no detectable p16 in 38/59 cases (64%), which implies a very high rate of p16 inactivation in this type of cancer. On the other hand, the retinoblastoma gene product, which also regulates the G1 to S phase transition of the cell cycle, was consistently expressed in nasopharyngeal carcinomas by immunohistochemical analysis. These results implicate p16 inactivation but not Rb alteration in the stepwise progression of nasopharyngeal carcinogenesis.

Carcinoma, Squamous Cell↗

PubMed1997

Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.

The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.

Carrier Proteins↗