Expression of p16INK4a and MIB-1 in relation to histopathology and HPV types in cervical adenocarcinoma.

A total of 101 primary cervical adenocarcinomas were analyzed for the presence of p16INK4a and MIB-1 expression in correlation with the presence of 'high-risk' types of human papillomavirus (HR-HPV) and clinical outcome. We found that adenocarcinoma grading showed a significant negative correlation to p16INK4a levels (p=0.001): i.e. we found less intense p16 staining in poorly differentiated tumors than in more highly differentiated tumors as well as a highly significant correlation between HPV infection and higher levels of p16INK4a staining (p=0.00), which was similar for different HPV-types. Tumor suppressor protein p16INK4a levels were higher in HPV positive than in HPV negative tumors. Higher levels of the proliferation marker MIB-1 were associated with poorer outcome. Higher MIB-1 levels were seen in tumors with a lower grade and higher stage at diagnosis. Moreover, MIB-1 levels seem to be higher in tumors due to infection with HPV 16 and 18 compared with HPV 45. MIB-1 may be a helpful marker in grading adenocarcinoma: a high level of expression of MIB-1 indicates a low grade of tumor, whereas high expression of p16INK4a indicates a highly differentiated of the tumor. Thus, immunostaining for p16INK4a appears to be a useful diagnostic tool for cervical adenocarcinoma.

p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) typically affects male smokers older than 55 years. Recently, an increase in the incidence of HNSCC in young adults has been recognized, many of them nonsmokers and females. Functional inactivation of p16 is known to be a common event in HNSCC, mainly by either deletion or methylation. A previous study by this group has shown that p16 deletions in HNSCC are significantly associated with age. The primary objective of this study was to evaluate additional molecular alterations of p16 in HNSCC, specifically in relation to age, site, and human papillomavirus (HPV) status. Patients ranging in age from 22 to 76 years with HNSCC were prospectively identified (n = 24). Methylation-specific polymerase chain reaction and immunohistochemistry were used to evaluate p16 gene inactivation and p16 protein expression, respectively. HPV 16 status was determined for each case. Overall, p16 inactivation was a frequent event detected in 46% of cases. Methylation of p16 was more often detected in females than males (P = .05). All cases showing p16 methylation were from the anterior tongue, and 75% of them were young patients. The results indicate that p16 methylation is a more common event in those younger than 40 years in contrast to p16 deletions, which are more common in those older than 40 years. Consequently, it appears that specific modes of inactivation of p16 in HNSCC are related to specific patient risk profiles. Interestingly, HPV 16 messenger RNA was detected exclusively in HNSCC from the base of tongue lesions and was only found in males. This differs from the patient profile of HNSCC in the young, which affects the anterior tongue and commonly females, thus, making it highly unlikely that this virus is a primary causative agent of HNSCC in these young adults.

A randomized trial of basing treatment on human papillomavirus and/or cytology results in low-grade cervical lesion triage.

OBJECTIVE: This study was undertaken to compare management algorithms that base treatment with loop electrosurgical excision procedure on human papillomavirus and/or repeat Papanicolaou test smear results. STUDY DESIGN: A randomized trial that referred 674 women with either atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions cytology results, detected in organized screening to treatment either (1) if they were positive in a repeat Papanicolaou test smear and/or a human papillomavirus test or (2) if they were positive in the repeat Papanicolaou test smear test only. Women who tested positive were treated, regardless of colposcopic findings. RESULTS: There were 208 of 337 (62%) women who were treated in the human papillomavirus /Papanicolaou test smear group (187/337 because of HPV positivity) and 138 of 337 (41%) in the Papanicolaou test smear only group. Histopathologically diagnosed cervical intraepithelial neoplasia grade 2 or worse was found among 112 of 337 (33.2%) women in the human papillomavirus/Papanicolaou test smear group compared with 85 of 337 (25.2%) women in the Papanicolaou test smear only group (P < .05). Twenty-one women with cervical intraepithelial neoplasia 2+ had normal colposcopy. CONCLUSION: For adequate cervical intraepithelial neoplasia 2+ sensitivity, the decision to use loop electrosurgical excision procedure needs to be based on human papillomavirus testing results and should not exclude women with normal colposcopy.

p16(INK4a) immunocytochemistry in liquid-based cervical cytology: is it feasible for clinical use?

A consecutive series of 118 samples from patients referred to colposcopy assessment and follow-up with cytology and biopsies were analysed with immunocytochemical staining to determinate the expression of p16(INK4a). Accumulation of p16(INK4a) antigen has been proposed as a biomarker helpful for the identification of dysplastic cervical cells. In our study all benign cases were negative for p16(INK4a), while more than half of the high grade lesions showed moderate or strong reactivity. There was a correlation between CIN grade and p16(INK4a) expression levels with more advanced lesions showing stronger reactivity. The correlation between p16(INK4a) immunoreactivity and the severity of cytological abnormality was stronger, when the diagnosis was based on simultaneous routine cytology (p<0.001, chi(2) exact test for trend). There was no or weak reactivity in benign cases, as well as almost all low-grade lesions, while two thirds of high-grade lesions showed moderate or strong staining for p16(INK4a) antigen. Thus p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and is a simple way of emphasizing the presence of premalignant cell reactive atypias. This staining can be applied to cytological samples, and might be a complement prognostic procedure in order to find women at risk for cervical cancer.

A comparison of liquid-based cytology and Pap smear as a screening method for cervical cancer.

The implementation of population-based screening for cervical cancer with Pap smear in the early sixties was set to detect and treat precancerous lesions, hopefully preventing a subsequent invasive cervical cancer. Epidemiological data indicate that organized screening has a major impact on morbidity and mortality from cervical cancer. The limited sensitivity of a single smear necessitates repeated smears in organized program. It is suggested that liquid-based cytology improves the sensitivity. The aim of this split-sample study was to compare ThinPrep liquid-based cytology with conventional Pap smear, relying on a laboratory with long-term experience of the latter. In total, 137 women with atypical Pap smear in population-based cervical screening were enrolled for the split-sample study. The performance of both techniques (ThinPrep liquid-based cytology and conventional Pap smear) were compared and validated by a histological follow-up. Women without representative histological biopsy were excluded from the study. Pap smear had sensitivity for detection of CIN2-3 of 47% compared to 66% for liquid-based material. The concordance of the two sampling techniques with the histological diagnosis was 37% and 53%, respectively, this difference being statistically significant. The proportion of reports on atypical squamous cells of undetermined significance (ASCUS) was significantly less in the liquid-based material, 4.3% compared to 8% of the conventional smears. This improved sensitivity in combination with the possibility to perform reflex testing such as HPV DNA or p16 immunocytochemistry without renewed sampling gives ThinPrep a substantial advantage and makes the liquid-based technique interesting.

[Are analyses of cytological cervix smears from young women more harmful than beneficial?]. / Er cervixcytologisk undersøkelse av unge kvinner mer til skade enn til gagn?

BACKGROUND: The Norwegian coordinated cervical cancer screening program invites women from 25 to 69 years of age to have a cytological smear from the cervix uteri analysed every third year. The aim of this study was to identify the volume of smears collected and to evaluate the consequences of having or not having this done for 24-year-old and younger women not included in the screening program and to compare the results with those for other age groups. MATERIAL AND METHODS: Data were obtained from the following Norwegian Cancer Registry sources: the Cancer Registry for incident cases of cancer cervix uteri (1995-2004), the Cytology Registry for volume of cervical specimen collection (1992-2005) and outcome of smears (2004), the CIN-treatment database for the number of incident cases of cervical intraepithelial neoplasia grade 2 or 3 (2004) and the Cervix Histology Registry for histological outcome of cervical biopsies (2004). Data were displayed by age (< or = 24, 25-29, 30-69 and 70 years or older). No statistical testing was done. RESULTS: Cancer in the cervix uteri is extremely rare among 24 year-old and younger women (0-3 cases per year). The volume of cervix smears taken from young women has decreased over the past 10 years; in 2005 approximately 35,000 out of 440,000 smears came from young women. More smears from young women were inconclusive and more lesions were of low grade than in older women; repeat testing was therefore required more frequently in young women. More young than older women were diagnosed with grade 1 or grade 2 lesions. INTERPRETATION: Most lesions in young women regress spontaneously. By not collecting smears from young women, many lesions that would anyway regress spontaneously, would not be discovered and follow-up could be avoided. Prospective follow-up studies of viral processes and oncogeneses indicate that it takes many years for normal cervix cells in the uterus to develop into cancer. For most young women it makes no difference whether low grade lesions or lesions that require treatment are diagnosed at age 23, 24 or shortly after 25. With reference to data from the Norwegian Cancer Registry and international literature, we conclude that analysis of cytological cervix smears from young women is more harmful than beneficial.

Expression of p16(INK4a) in relation to histopathology and viral load of 'high-risk' HPV types in cervical neoplastic lesions.

A total of 91 cervical archival biopsy series were analysed for the presence and viral load of 'high-risk' types of human papillomavirus (HR-HPV), and p16(INK4a) expression. The women had various degrees of CIN (cervical intraepithelial neoplasia). HPV 16 was the most prevalent type found, at 47% frequency. The frequency of HPV 16 increased with increasing immunoreactivity to p16(INK4a), from 39% to 44% at cases scored low to medium, to 65% at high reactivity. Thirty (33%) of the samples had negative p16(INK4a) analysis results, but were positive for HR-HPV. There was no significant correlation between viral load and the level of p16(INK4a) expression, while the grade of CIN correlated to such expressions. Thus, p16(INK4a) expression analysis yielded information which is consistent with results from the histopathology and might complement the HPV analysis in a clinical prognostic procedure in order to find women at risk for cervical cancer.

Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.

The purpose of this study was to compare the detection of human papillomavirus (HPV) DNA with detection of mRNA. The study included 4,136 women >30 years of age. E6/E7 mRNA expression from the carcinogenic HPV types 16, 18, 31, 33, and 45 was detected by the PreTect HPV-Proofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by type-specific PCR. A total of 4.0% had an abnormal cytologic diagnosis, 3.0% were positive by PreTect HPV-Proofer, 4.4% by type-specific PCR, and 10.4% by consensus PCR. For detection of HPV in high-grade squamous intraepithelial lesion (HSIL), no significant difference was observed between PreTect HPV-Proofer and consensus PCR. For women with a cytologic normal, atypical squamous cell of uncertain significance, and low-grade SIL diagnosis, the detection rate of HPV was significantly higher by Gp5+/6+ consensus PCR (P < 0.005) than by PreTect HPV-Proofer. Histology confirmed 14 of 23 cytologic HSIL as cervical intraepithelial neoplasia grade >2. Of these women, PreTect HPV-Proofer and type-specific PCR detected 12, whereas consensus PCR detected 13. In conclusion, for HSIL, detection of E6/E7 transcripts from HPV types 16, 18, 31, 33, and 45 are present to the same degree as DNA detected by consensus PCR. Equally important, only a small proportion of the HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or low-grade SIL diagnosis had a detectable mRNA expression. HPV E6/E7 mRNA detection by PreTect HPV-Proofer represents a new promising test as an adjunct to cytology.

Detection and quantitation of HER-2 gene amplification and protein expression in breast carcinoma.

We compared fluorescence in situ hybridization (FISH), immunohistochemical analysis, immunocytochemical analysis, and relative quantification assays using polymerase chain reaction (PCR) as methods for estimating HER-2 gene amplification and protein overexpression infine-needle aspirate (FNA) specimens and paraffin-embedded tissue samples from 49 cases of breast cancer: FISH can be performed successfully on FNA smears. Immunohistochemical and immunocytochemical staining intensity of 3+ corresponds to a FISH ratio of more than 2.5. Immunohistochemical and immunocytochemical staining of 2+ and 1+ are not necessarily associated with gene amplification. Increased DNA PCR ratios might be seen without amplification, reflecting polysomy. HER-2 messenger RNA relative quantitation scores correlate well with HER-2 gene amplification. Owing to the ease with which it can be performed and interpreted, we conclude that FISH is the test of choice for HER-2 estimation and, when possible, should be performed on whole nuclei, which are readily available in FNA smears or imprint cytology. FISH may be used primarily or to confirm immunohistochemical, immunocytochemical, and PCR results.

Cytological criteria for the diagnosis of intraductal hyperplasia, ductal carcinoma in situ, and invasive carcinoma of the breast.

The advent of mammography screening presents a diagnostic challenge to the cytopathologist as an increasing proportion of breast lesions requiring investigation will be nonpalpable and up to 40% will be accounted for by atypical intraductal hyperplasia and ductal carcinoma in situ, as opposed to previously, when these lesions represented less than 10% of palpable tumors. We studied 133 fine-needle aspirates from breast tumors and found that nuclear morphology, myoepithelial cells, signs of invasion, and degree of cellular dissociation are among the most potent factors discriminating between benign epithelial proliferations, atypical intraductal hyperplasia, ductal carcinoma in situ, and invasive carcinoma.

Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening.

BACKGROUND: Primary cervical screening with both human papillomavirus (HPV) DNA testing and cytological examination of cervical cells with a Pap test (cytology) has been evaluated in randomized clinical trials. Because the vast majority of women with positive cytology are also HPV DNA positive, screening strategies that use HPV DNA testing as the primary screening test may be more effective. METHODS: We used the database from the intervention arm (n = 6,257 women) of a population-based randomized trial of double screening with cytology and HPV DNA testing to evaluate the efficacy of 11 possible cervical screening strategies that are based on HPV DNA testing alone, cytology alone, and HPV DNA testing combined with cytology among women aged 32-38 years. The main outcome measures were sensitivity for detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) within 6 months of enrollment or at colposcopy for women with a persistent type-specific HPV infection and the number of screening tests and positive predictive value (PPV) for each screening strategy. All statistical tests were two-sided. RESULTS: Compared with screening by cytology alone, double testing with cytology and for type-specific HPV persistence resulted in a 35% (95% confidence interval [CI] = 15% to 60%) increase in sensitivity to detect CIN3+, without a statistically significant reduction in the PPV (relative PPV = 0.76, 95% CI = 0.52 to 1.10), but with more than twice as many screening tests needed. Several strategies that incorporated screening for high-risk HPV subtypes were explored, but they resulted in reduced PPV compared with cytology. Compared with cytology, primary screening with HPV DNA testing followed by cytological triage and repeat HPV DNA testing of HPV DNA-positive women with normal cytology increased the CIN3+ sensitivity by 30% (95% CI = 9% to 54%), maintained a high PPV (relative PPV = 0.87, 95% CI = 0.60 to 1.26), and resulted in a mere 12% increase in the number of screening tests (from 6,257 to 7,019 tests). CONCLUSIONS: Primary HPV DNA-based screening with cytology triage and repeat HPV DNA testing of cytology-negative women appears to be the most feasible cervical screening strategy.

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear.

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.

Ki-67 antigen detection in unstained and destained cytologic samples.

OBJECTIVE: To evaluate the effect of different tissue preservation, fixation and staining procedures on the expression of proliferation-associated Ki-67 antigen in cytologic samples to establish an easy and uniform way to handle cytologic specimens with an automated staining technique. STUDY DESIGN: Multiple touch imprints were made from eight breast tumors. The specimens were treated according to different protocols, and Ki-67 nuclear expression was compared to that in the corresponding histologic sections. RESULTS: In the unstained specimens, air drying at room temperature for up to four months or ethanol spray fixation preserved the material and offered excellent results. Processing effectively removed previous stain without additional chemical destaining. Antigen retrieval was not achieved in the previously Giemsa stained imprints and was suboptimal in those stained according to Papanicolaou. CONCLUSION: Immunocytochemical detection of Ki-67 is recommended for previously unstained cytologic specimens.

Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia.

DNA methylation contributes to the chromatin conformation that represses transcription of human papillomavirus type16 (HPV-16), which is prevalent in the etiology of cervical carcinoma. In an effort to clarify the role of this phenomenon in the regulation and carcinogenicity of HPV-16, 115 clinical samples were studied to establish the methylation patterns of the 19 CpG dinucleotides within the long control region and part of the L1 gene by bisulfite modification, PCR amplification, DNA cloning, and sequencing. We observed major heterogeneities between clones from different samples as well as between clones from individual samples. The methylation frequency of CpGs was measured at 14.5%. In addition, 0.21 and 0.23%, respectively, of the CpA and CpT sites, indicators of de novo methylation, were methylated. Methylation frequencies exceeded 30% in the CpGs overlapping with the L1 gene and were about 10% for most other positions. A CpG site located in the linker between two nucleosomes positioned over the enhancer and promoter of HPV-16 had minimal methylation. This region forms part of the HPV replication origin and is close to binding sites of master-regulators of transcription during epithelial differentiation. Methylation of most sites was highest in carcinomas, possibly due to tandem repetition and chromosomal integration of HPV-16 DNA. Methylation was lowest in dysplasia, likely reflecting the transcriptional activity in these infections. Our data document the efficient targeting of HPV genomes by the epithelial methylation machinery, possibly as a cellular defense mechanism, and suggest involvement of methylation in HPV oncogene expression and the early-late switch.

Genomic diversity of human papillomavirus-16, 18, 31, and 35 isolates in a Mexican population and relationship to European, African, and Native American variants.

Cervical cancer, mainly caused by infection with human papillomaviruses (HPVs), is a major public health problem in Mexico. During a study of the prevalence of HPV types in northeastern Mexico, we identified, as expected from worldwide comparisons, HPV-16, 18, 31, and 35 as highly prevalent. It is well known that the genomes of HPV types differ geographically because of evolution linked to ethnic groups separated in prehistoric times. As HPV intra-type variation results in pathogenic differences, we analyzed genomic sequences of Mexican variants of these four HPV types. Among 112 HPV-16 samples, 14 contained European and 98 American Indian (AA) variants. This ratio is unexpected as people of European ethnicity predominate in this part of Mexico. Among 15 HPV-18 samples, 13 contained European and 2 African variants, the latter possibly due to migration of Africans to the Caribbean coast of Mexico. We constructed phylogenetic trees of HPV-31 and 35 variants, which have never been studied. Forty-six HPV-31 isolates from Mexico, Europe, Africa, and the United States (US) contained a total of 35 nucleotide exchanges in a 428-bp segment, with maximal distances between any two variants of 16 bp (3.7%), similar to those between HPV-16 variants. The HPV-31 variants formed two branches, one apparently the European, the other one an African branch. The European branch contained 13 of 29 Mexican isolates, the African branch 16 Mexican isolates. These may represent the HPV-31 variants of American Indians, as a 55% prevalence of African variants in Mexico seems incomprehensible. Twenty-seven HPV-35 samples from Mexico, Europe, Africa, and the US contained 11 mutations in a 893-bp segment with maximal distances between any two variants of only 5 mutations (0.6%), including a characteristic 16-bp insertion/deletion. These HPV-35 variants formed several phylogenetic clusters rather than two- or three-branched trees as HPV-16, 18, and 31. An HPV-35 variant typical for American Indians was not identifiable. Our research suggests type specific patterns of evolution and spread of HPV-16, 18, 31, and 35 both before and after the worldwide migrations of the last four centuries. The high prevalence of highly carcinogenic HPV-16 AA variants, and the extensive diversity of HPV-18, 31, and 35 variants with unknown pathogenic properties raise the possibility that HPV intra-type variation contributes to the high cervical cancer burden in Mexico.

Use of human papillomavirus DNA testing to compare equivocal cervical cytologic interpretations in the United States, Scandinavia, and the United Kingdom.

BACKGROUND: Human papillomavirus (HPV) DNA testing may be useful in clarifying equivocal cervical cytologic interpretations. One application might be to standardize the meaning of equivocal interpretations from laboratories in various regions. Because international differences may be particularly marked, international comparisons of emerging data will require clear translations of "equivocal" and similar terms. METHODS: To perform a three-country comparison, the authors selected a morphologically diverse set of 188 conventional Papanicolaou tests initially classified as "squamous atypia" from a study of more than 20,000 women in Portland, Oregon (1989-1990). Previously, five U.S. expert cytopathologists independently interpreted the slides with screening cytotechnologists' marks in place. For this comparison, one British and two Scandinavian reviewers involved in HPV research reviewed the slides after original marks had been removed. The authors compared all eight reviewers' classifications of negative, equivocal, or abnormal in a series of pairwise comparisons using the kappa statistic. They then compared cytologic interpretations with HPV DNA testing. RESULTS: Oncogenic HPV DNA detection was significantly associated with increasingly abnormal interpretations for each reader. The British reader tended to rate tests as more abnormal than the American pathologists did, whereas the Scandinavians tended to rate tests as more normal. Reference to the HPV DNA standard clarified the tendency of readers to render systematically more or less severe interpretations. For example, the Scandinavian cytologists discounted subtle (often HPV-associated) changes in favor of cytologic certainty, making HPV triage of equivocal tests less applicable there. CONCLUSIONS: International research on cytopathology, particularly on the possible uses of HPV DNA testing, will require calibration of local cytologic definitions.