Ingo Nindl

Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling

BackgroundCarcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC).ResultsThree different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes.ConclusionThe majority of identified differentially expressed genes in cutaneous SCC were previously not described.

Human Papillomavirus distribution in cervical tissues of different morphology as determined by hybrid capture assay and PCR

Distribution of various types of genital human Papillomavirus (HPV) in smears from histologically classified cervical lesions was determined by hybrid capture assay (HCA) and was compared with a polymerase chain reaction (PCR) system using general primers (GP) in first and type specific primers (TS) in a second step. The overall agreement of high-risk HPV by HCA and the more sensitive GP/TS PCR was 80.6% (204 of 253, kappa value 0.6). Human Papillomavirus frequency by GP/TS PCR was 14–20% higher compared with HCA (p = 0.02–0.004) independent of morphology. Only one sample was positive by HCA and negative by GP/TS PCR. A significantly higher frequency was found using HCA and GP/TS PCR in smears from histologically proven cervical intraepithelial lesions (CIN) II/III compared with CIN I, tissues with minimal changes (metaplasia, cervicitis, or lack of glycogenization), or normal morphology (61% and 81% vs 8–15% and 24–34%, p ≤ 0.001). Semi-quantitative estimate of HPV DNA copies by GP-PCR coincided with estimated virus load by quantitative HCA and was significantly higher in patients with CIN II/III compared with CIN I (p < 0.001). Thus, the GP-PCR may be used to monitor the amount of HPV DNA copies in clinical samples. A direct correlation between morphologic changes and HPV detection as well as virus load was found by HCA and the more sensitive GP/TS PCR.

Human papillomavirus detection in cervical intraepithelial neoplasia by the second-generation hybrid capture microplate test, comparing two different cervical specimen collection methods

BACKGROUNDnThe second generation Hybrid Capture microplate-based human papillomavirus (HPV) test (HC II) was examined to determine its sensitivity for identification of cervical intraepithelial neoplasia (CIN) by two different cervical specimen collection methods.nnnOBJECTIVESnA cohort of 115 women with a mean age of 34.6 years (SD 9.1), referred to colposcopy with a history of abnormal cytology, was studied to compare HPV prevalence and viral load in low grade CIN vs. high grade CIN.nnnSTUDY DESIGNnPrior to the application of acetic acid, cervical specimens were obtained by either method 1 or 2, as follows: method 1: A cotton-tipped swab was applied to the ectocervix and endocervix for a Papanicolaou (Pap) smear. Next, a special cone-shaped cervical brush was applied to the endocervix, the ectocervix, and to the posterior vaginal vault and suspended in 1.0 ml of transport medium for HPV testing. Method 2: a Pap smear was taken with a cyto standard cylindrical cytology brush from the endocervix, and ectocervix, and the remaining cells were suspended in 3 ml phosphate-buffered saline (PBS) for HPV testing. Next, a Dacron-tipped swab was used to take a specimen from the ectocervix and posterior fornix and suspended in the same PBS solution.

Colposcopic appearance of cervical intraepithelial neoplasia is age dependent

Objective: We investigated to determine whether colposcopic, histologic, and virologic parameters of cervical intraepithelial neoplasia are influenced by a patient’s age. Study Design: A cohort of 967 women with a mean age of 37.1 years underwent screening for detection of cervical intraepithelial neoplasia by colposcopy, cytologic examination, and testing for high-risk human papillomaviruses with the Hybrid Capture System (Digene, Silver Springs, Md) and a general primer and type-specific primer polymerase chain reaction system. Cervicography was used for documentation and reproducible evaluation of the colposcopic appearance of the cervix. In 86% of patients with trivial colposcopic changes of doubtful significance (100/116) and 89% of patients with colposcopic changes consistent with cervical intraepithelial neoplasia (89/99), punch biopsy specimens were taken for histologic evaluation. Results: In patients with trivial colposcopic changes of doubtful significance, histologically confirmed cervical intraepithelial neoplasia was almost as frequent (32%, 37/116) as in patients with colposcopic changes consistent with cervical intraepithelial neoplasia (43%, 43/99, difference not significant). The ratio between colposcopic evidence of cervical intraepithelial neoplasia and trivial colposcopic changes was 1.9 in patients <35 years old with cervical intraepithelial neoplasia, versus 0.5 in patients ≥35 years old with cervical intra-epithelial neoplasia (P = .005). Patients with trivial colposcopic changes of doubtful significance were older (median age 36 years) than were patients with colposcopic changes consistent with cervical intraepithelial neoplasia (median age 29 years, P = .008). In patients with cervical intraepithelial neoplasia who had no or trivial colposcopic changes, the thickness of neoplastic epithelium was smaller (P = .008) and the number of cellular layers was lower (P = .01) than in patients with cervical intraepithelial neoplasia who had colposcopic changes consistent with cervical intraepithelial neoplasia. In patients <35 years old the rate of positive results for a high-risk human papillomavirus (P < .005) and the viral load (difference not significant) were higher than in women ≥35 years old. The rate of positive results for high-risk human papillomaviruses differed independently of age among patients with normal colposcopic findings, patients with trivial colposcopic changes of doubtful significance, and patients with colposcopic changes consistent with cervical intraepithelial neoplasia (P < .005). Conclusions: In women ≥35 years old cervical lesions associated with intraepithelial neoplasia are thinner and thus less colposcopically conspicuous than those in women <35 years old. Patients ≥35 years old with acetowhite cervical lesions consistent with trivial changes of doubtful significance should therefore undergo punch biopsy for histologic evaluation. (Am J Obstet Gynecol 1998;179:1298-304.)

Distribution of 14 high risk HPV types in cervical intraepithelial neoplasia detected by a non-radioactive general primer PCR mediated enzyme immunoassay.

AIM: To evaluate the presence of high risk human papillomaviruses (HPV) in cervical smears showing intraepithelial neoplasia (CIN). METHODS: The presence of 14 high risk HPV was evaluated in 114 cervical smears with CIN of different degrees, by comparing a non-radioactive polymerase chain reaction (PCR) enzyme immunoassay (EIA) with conventional PCR followed by radioactive Southern blot hybridisation. General primer PCR amplicons detecting low risk and high risk HPV were typed for 14 different high risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) by a non-radioactive PCR-EIA. Virus load of HPV 16 positive CIN was assessed using the semiquantitative PCR-EIA. RESULTS: Histological evaluation confirmed CIN I in 49 cases (mean age 29.0 years, range 17 to 52), CIN II in 31 cases (mean age 30.8 years, 18 to 54), and CIN III in 34 cases (mean age 31.1 years, 16 to 57). The non-radioactive PCR-EIA showed an overall agreement rate of 90% (kappa value 0.75) when compared with conventional general primer PCR followed by radioactive Southern blot hybridisation. High risk HPVs were detected in 47% of CIN I, 77% of CIN II, and 97% of CIN III (p < or = 0.02). HPV types 39, 51, 56, and 58 were found in CIN I exclusively (between 2% and 8%). HPV 16 and HPV 31 were detected in 12% and 2% of CIN I, 35% and 21% of CIN II, and 74% and 13% of CIN III, respectively (p < or = 0.03 and p < or = 0.04). The virus load estimated by the semiquantitative PCR-EIA of HPV 16 was similar in CIN I, CIN II, and CIN III. CONCLUSIONS: The PCR-EIA has high clinical sensitivity for detecting CIN II/III (90%). There was a significantly higher prevalence rate of HPV 16 and 31 in CIN III than in CIN I and II.

Novel papillomavirus isolates from Erinaceus europaeus (Erinaceidae, Insectivora) and the Cervidae (Artiodactyla), Cervus timorensis and Pudu puda, and phylogenetic analysis of partial sequence data

The diversity of papillomaviruses (PVes) infecting stratified squamous epithelia of warm-blooded animals, such as birds and mammals, is only fragmentarily documented. The PV types are sequenced from 9 of 18 placental taxa at the order level to date. Current phylogenetic analyses of PV sequences frequently do not consider evolutionary polarity and statistical evaluation of internal nodes, that are required for robust evolutionary conclusions. In this study, we isolated and characterized three putatively novel animal PV types from hair follicles comprising the first known insectivoran PV and two cervid PVes. With the help of the primer pair FAP59/FAP64, we amplified L1 gene fragments consisting of approximately 470 base pairs. Phylogenetic analyses were performed with a representative set of 73xa0PV sequences that included the three novel PVes using Maximum Likelihood, Bayesian inference, Maximum Parsimony, and distance-based methods on amino acid alignments. The three novel PVes appear to be components of the β+γ+π+ξ-PV supertaxon, within which the insectivoran PV has an isolated phylogenetic position. The two cervid PVes constitute a distinct group that is only distantly related to the core cervid PVes of the δ-PVes. The molecular data supports a complex evolutionary scenario for PVes which is driven by multiple mechanisms comprising host-linked evolution, adaptive radiation establishing different ecological niches, and multiple infections across species borders.

Cervical cancer, HPV 16 E6, variant genotypes, and serology

1 Hill MD, Hachinski V. Stroke treatment: time is brain. Lancet 1998; 352 (suppl III): 10–14. 2 Chinese Acute Stroke Trial collaborative group. CAST: a randomised placebocontrolled trial of early aspirin use in 20 000 patients with acute ischaemic stroke. Lancet 1997; 349: 1641–49. 3 International Stroke Trial Collaborative Group. The International Stroke Trial (IST): a randomised trial of aspirin, subcutaneous heparin, both, or neither among 19 435 patients with acute ischaemic stroke. Lancet 1997; 349: 1569–81. 4 Antiplatelet Trialists’ Collaboration. Collaborative overview of randomised trials of antiplatelet therapy. I: Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. BMJ 1994; 308: 81–106. multivariants V-131/350 (9%), and V345/350 (3%). Seroreactivity with the prototype antigen was observed in 79%. Of the seven E6 seronegative patients, the prevalence rate of the prototype was 29%, of the variant V-350 43%, and of the variant V-131/350 29%. All serum samples were reanalysed with five different variant antigens.* All the 27 seropositive serum samples remained seropositive independent of the antigen. No HPV 16 E6 antibodies were detectable in the seven seronegative cases, irrespective of the antigen used, and all seronegative sera were positive for IgG antibodies against Epstein-Barr virus, cytomegalovirus antigens, or both. Age of the seronegative patients (mean 46 years) and stages of disease (71% FIGO stage I, 29% stage II) did not differ from the HPV-seropositive group. Do different HPV 16 serotypes exist? By contrast to extensive genotype analyses, only serological studies using virus-like particles of L1 from different HPV 16 variants have been done and no different serotypes were found. In our study, the same seroreactivity was seen for six different 16 E6 variant antigens, suggesting that different HPV 16 E6 serotypes do not exist. Moreover, HPV 16 E6 negative serum samples cannot be explained on the basis of variant proteins. The presence of HPV 16 E6 variants will not, therefore, alter the outcome of past or current screening tests for cervical cancer that are all based on the prototype E6 antigen.

High sensitivity and reproducibility of immunohistochemistry with microagitation

We examined the practicability, reproducibility and analytical sensitivity of classical immunohistochemistry (IHC) and IHC with microagitation. Two monoclonal antibodies, Ki-67 (proliferation marker) and p53 (tumor suppressor marker), were used. Consecutive paraffin sections of biopsies of suspicious lesions of patients with non-melanoma skin cancer were used in the study. Reproducibility was examined using specimens from four patients in three independent experiments with antibodies against Ki-67 and p53. Analytical sensitivity of the two methods was determined using serial dilutions in two independent experiments. IHC with microagitation could be carried out without destroying the tissue. The new technique was consistent and reproducible, and no background staining was observed. The primary antibodies Ki-67 and p53 could be used at higher dilutions (four to ten times) with microagitation compared with classical IHC. Microagitation can be used for immunohistochemistry; it was reproducible, highly sensitive, and antibodies could be used at higher dilutions. Further analyses with other antibodies using this technique are warranted.