Ruediger Klaes

Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri

Cytological screening for cervical cancer or its precursors using Papanicolaous smear test (Pap test) has been highly efficient to reduce the morbidity and mortality of cervical cancer. However, evaluation of the Pap test relies on subjective diagnostic parameters and is affected by a high rate of false‐positive and false‐negative results. More objective diagnostic parameters to identify truly dysplastic or neoplastic cells in cervical smears as well as in cervical biopsy samples would therefore avoid insecurity for many patients and the high screening costs associated with repeated testing. Cervical dysplasia is induced by persistent infections through high‐risk types of human papillomaviruses (HPVs). Outgrowth of dysplastic lesions is triggered by increasing expression of two viral oncogenes, E6 and E7, which both interact with various cell cycle‐regulating proteins. Among these is the retinoblastoma gene product pRB, which is inactivated by E7. pRB inhibits transcription of the cyclin‐dependent kinase inhibitor gene p16INK4a. Increasing expression of the viral oncogenes in dysplastic cervical cells might thus be reflected by increased expression of p16INK4a. In line with this hypothesis, we observed marked overexpression of p16INK4a in all cervical intraepithelial neoplasm (CIN) I lesions (n = 47) except those associated with low‐risk HPV types (n = 7), all CIN II lesions (n = 32), all CIN III lesions (n = 60) and 58 of 60 invasive cervical cancers. In contrast, no detectable expression of p16INK4a was observed in normal cervical epithelium (n = 42), inflammatory lesions (n = 48) and low‐grade cervical lesions (CIN I) associated with low‐risk HPV types (n = 7). Dysplastic cells could also be identified in cervical smears using a specific p16INK4a monoclonal antibody. These data demonstrate that p16INK4a is a specific biomarker to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears.

Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

Detection of integrated papillomavirus sequences by ligation-mediated pcr (DIPS-PCR) and molecular characterization in cervical cancer cells

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV‐related cancers cells. This suggests that malignant conversion of HPV‐infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral–cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation‐mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS‐PCR). DIPS‐PCR was initially used to amplify genomic viral–cellular junctions from HPV‐associated cervical cancer cell lines (C4‐I, C4‐II, SW756, and HeLa) and HPV‐immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral–cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS‐PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS‐PCR might allow efficient therapy control and prediction of relapse in patients with HPV‐associated anogenital cancers.

Characterization of viral-cellular fusion transcripts in a large series of HPV16 and 18 positive anogenital lesions.

Persistent high risk type human papillomavirus (HR–HPVs) infections induce dysplasia or cancer of the anogenital tract, most notably of the uterine cervix. The viral genome usually persists and replicates as an episomal molecule in early dysplasia, whereas in advanced dysplasia or cervical cancer HPV genomes are frequently integrated into the chromosomal DNA of the host cell. Previous studies suggested that modification of critical cellular sequences by integration of HPV genomes might significantly contribute to the neoplastic transformation of anogenital epithelia (insertional mutagenesis). This prompted us to characterize the integration loci of high risk HPV genomes in a large set of genital lesions. We amplified E6/E7 oncogene transcripts derived from integrated HPV16 and HPV18 genomes and characterized in detail the co-transcribed cellular sequences of 64 primary genital lesions and five cervical cancer cell lines. Database analyses of the cellular parts of these fusion transcripts revealed 51 different integration loci, including 26 transcribed genes (14 known genes, 12 EST sequences with unknown gene function). Seventeen sequences showed similarity to repetitive elements, and 26 sequences did not show any database match other than genomic sequence. Chromosomal integration loci were distributed over almost all human chromosomes. Although we found HPV sequences integrated into cancer related genes and close to fragile sites, no preferential site or integration motif could be identified. These data demonstrate that target directed insertional mutagenesis might occur in few HPV-induced anogenital lesions, however, it is rather the exception than the rule.

No evidence of p53 allele-specific predisposition in human papillomavirus-associated cervical cancer

The potential association of distinct polymorphisms of the tumor suppressor gene p53 with an increased susceptibility to malignant transformation has been reported for various cancer entities. Most recently, p53 protein containing an arginine residue in codon 72 was shown to be more effectively degraded by the E6 oncoprotein of human papillomavirus (HPV) than the corresponding proline isoform in cervical carcinoma cells. Additionally, a seven times higher risk of cervical cancer for Arg homozygotes was suggested. We set out to confirm this allele-specific predisposition on a larger population, comprising 87 cervical cancer and 151 normal control samples. However, there was no significant difference in the observed frequencies of homozygous Arg genotypes in cervical cancer patients (52.8%) and normal controls (55.7%). Furthermore, the prevalence of the Arg/Arg allelotype did not vary between HPV+ (n=75) and HPV– (n=12) carcinoma samples. Thus, our investigation of a larger set of clinical samples does not support the proposed association of any polymorphic status of p53 at codon 72 with an elevated risk for cervical cancer.

Exclusion of BRAFV599E as a melanoma susceptibility mutation

Very recently, BRAF mutations were found in about 2/3 of malignant melanomas and at lower frequencies in other human cancers. The BRAF gene codes for a protein in the mitogen‐activated protein kinase (MAPK) pathway. All mutations identified to date are within the kinase domain, with a single missense mutation (V599E) accounting for 80%. We investigated the hypothesis that this common somatic BRAF mutation (V599E) would contribute to melanoma predisposition in familial and polygenic malignant melanoma if occuring as a germ‐line mutation. We performed comprehensive mutational screening of exon 15 of BRAF using DHPLC (denaturing high‐performance liquid chromatography) and DNA sequencing techniques. No V599E mutation could be detected in 172 melanoma patients comprising 46 familial cases, 21 multiple melanoma patients and 106 cases with at least one first‐degree relative suffering from other cancers. We therefore conclude that the common somatic BRAF mutation V599E does not contribute to polygenic and familial melanoma predisposition.

Interlaboratory agreement of different human papillomavirus DNA detection and typing assays in cervical scrapes

More than 90% of high-grade cervical intraepithelial neoplasia (CIN 2/3) and cervical cancers are associated with high-risk (HR) human papillomavirus (HPV) types. HPV tests applicable for population screening have to detect all HR HPV types in a simple manner. It is likely that HPV testing will augment cytological investigations in screening programs (Cuzick et al., 1995; Meijeret al., 1992; Reidet al., 1991; Schneider et al., 1996). In addition, women with the cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) can be triaged on the basis of HPV positivity (Cox et al., 1995; Hatchet al., 1995). Persistent HR HPV infections are a marker for progressive CIN disease (Remmink et al., 1995), indicating the importance of HPV detection methods. In view of such broad applications, it is essential that HPV detection assays are standardized with respect to sensitivity and specificity. We designed a study to assess inter-method variations according to HR HPV detection and HR HPV typing in cervical scrapes. Three different polymerase chain reaction (PCR)-based methods using 2 different PCR primer pairs to generate either a 150-bp or a 450-bp PCR fragment were used in 5 laboratories. Cervical scrapes were obtained from women attending the outpatient clinics of the University Hospital Vrije Universiteit (Amsterdam, The Netherlands). The cervical scrapes were collected in 5 ml phosphate-buffered saline (PBS), centrifuged and the cell pellets were resuspended in 1 ml 10 mM Tris-HCl, pH 7.5, and frozen at 270°C. One hundred microliters were taken, boiled for 10 min at 100 °C, cooled on ice and centrifuged for 1 min at 3,000 g. Ten microliters of these crude cell suspensions were used for PCR. In the reference laboratory (laboratory 1) crude cell suspensions were analyzed by general primers GP5 1 and bioGP61 PCR (de Roda Husman et al., 1995b; Jacobset al., 1997). Fifteen HPV-positive and 5 HPV-negative cervical scrapes were encoded and distributed to the 4 participating laboratories (laboratories 2 –5) for HPV testing. All 20 cervical scrapes were positive by b-globin PCR according to de Roda Husman et al. (1995a) and thus contained DNA of at least 509 bp that could be amplified. In addition, laboratories 2–5 received 10μl of 5 aliquots representing a range of 10-fold DNA dilutions of the cervical carcinoma cell line SiHa (10 ng/ μl, 1 ng/μl, 100 pg/μl, 10 pg/μl and 1 pg/μl). Laboratories 1 and 2: For HPV analysis, the general primers GP51 and bioGP61, which span a region of 140–150 bp from the L1 open reading frame of a broad spectrum of HPV genotypes, were used in general primer (GP)-PCR (de Roda Husman et al., 1995b), with the exception that the GP6 1 primer was biotinylated (bioGP6 1). The GP-PCR amplicons were analyzed by an enzyme immunoassay (EIA) with 14 different HPV type-specific oligonucleotides to detect HR HPV types individually (Jacobs et al., 1997). Laboratory 3: General primers GP5 1/GP61 were used and PCR amplicons were separated electrophoretically. Positive samples were further analyzed by a type specific (TS)-PCR. The TS-PCR was performed using combinations of HPV 6/11, 16, and 18 specific cloning site-flanking primers as described by van den Bruleet al. (1990). The PCR amplicons of samples positive by GP-PCR and negative by TS-PCR were sequenced for HPV typing. Laboratories 4 and 5: The presence of HPV DNA was analyzed by PCR using L1 degenerate primer MY09/MY11 for a broad spectrum of mucosal HPV types. For HPV typing, PCR fragments were analyzed by a restriction fragment length polymorphism (RFLP) and hybridization analysis (Meyer et al., 1995). Hybridization signals were detected using a chemiluminescence-based system (ECL, Amersham, Ayelsbury, UK). All samples were tested in a blinded manner. Of 20 patients, 5 cervical scrapes were HPV negative, 6 contained 1 HPV type, 8 contained 2 and 1 contained 3 different HPV types. All 5 HPV negative scrapes were found to be HPV negative by all laboratories. Thus, false-positive HPV results due to contamination did not occur. Laboratories 1 and 2 detected 4 dilutions of SiHa DNA (100 ng to 100 pg), laboratories 3 and 5 detected 3 dilutions (100 ng to 1 ng SiHa DNA). For laboratory 4, data concerning this dilution series were not available. The different sensitivity for HPV 16 DNA detection in SiHa cells may be explained by the higher sensitivity of the EIA used in laboratories 1 and 2 and appears not to be due to the different primer pairs GP51/GP61 and MY09/MY11. The overall agreement rate for the detection of HR HPVs was between 75% and 100% (k values 0.50 to 1.0) (Table I). Lower agreement rates, between 54% and 92%, were found for HPV typing, especially in cervical scrapes with multiple infections (Table I). Direct sequencing used by laboratory 3 appears to detect only the overrepresented HPV type in cervical scrapes, because not more than 1 HPV type was detected in 9 multiple infections. By the MY09/MY11 PCR RFLP detection method in 2 cervical scrapes (samples 13 and 29, see Table I) a different HPV type

Identification and characterization of UEV3, a human cDNA with similarities to inactive E2 ubiquitin-conjugating enzymes

Recent studies have shown that ubiquitination is an essential factor in endosomal sorting and virus assembly. The human TSG101 gene has been demonstrated to belong to a group of genes coding for apparently inactive E2 ubiquitin-conjugating enzymes, which exert regulatory effects on E2 activity in cellular ubiquitination processes. In this study, a novel human cDNA (UEV3) encoding a putative protein of 379 amino acids was isolated from a human placenta library that may represent a partial paralogue of human TSG101. The predicted protein contains an N-terminal domain homologous to the catalytic domain of ubiquitin-conjugating enzymes (Ubc), which is fused to a sequence showing significant homology to members of the lactate dehydrogenase protein family. The UEV3 gene is located on chromosome 11 closely adjacent to TSG101 and LDH-C. Northern blot and UEV3-specific reverse transcription/polymerase chain reaction (RT/PCR) analyses of various colon carcinoma cell lines as well as both normal and tumor samples from colon revealed an expression of the UEV3 cDNA in all tested samples.

State of human papilloma virus DNA in cervical carcinomas

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.

Trisomy 8q and partial trisomy 22 in a 43-year-old man with moderate intellectual disability, epilepsy and large cell non-Hodgkin lymphoma

Partial trisomies are chromosome abnormalities resulting in a broad range of malformations depending on the size and location of the chromosomal rearrangement. Whereas diagnosis of these syndromes is usually made in early childhood, few descriptions exist about the clinical picture in adulthood. We report on a patient diagnosed at the age of 43 years with a 47,XY,+der(22)t(8;22)(q24.13;q11.21) karyotype and predominant clinical features of trisomy 8q. To our knowledge, this is the oldest patient described with a partial trisomy 8. The patient presented with moderate intellectual disability, a past history of epilepsy and facial anomalies. In addition, a large cell non‐Hodgkin lymphoma was diagnosed in adulthood. Detailed breakpoint mapping by single nucleotide polymorphism (SNP) arrays showed that the derivative chromosome contains a full‐length copy of the C‐MYC oncogene. Given that trisomy 8q is the most frequent secondary chromosomal abnormality in hematological diseases, the possibility of a genetic predisposition for these disorders in patients with 8q duplication is raised.