bromochloroacetic-acid and Papillomavirus-Infections

Although clinical history and morphologic appearance should be the initial considerations when evaluating small round blue cell tumors of the sinonasal tract, the final diagnosis often hinges on immunohistochemical findings. Unfortunately, interpretation of stains in these tumors is fraught with numerous pitfalls and limitations. This article presents an approach to sinonasal small round blue cell tumors based on four common immunohistochemical patterns: cytokeratin positivity, squamous marker positivity, neuroendocrine marker positivity, and cytokeratin negativity.

Topics: Biomarkers, Tumor; Carcinoma, Adenoid Cystic; Carcinoma, Squamous Cell; Diagnosis, Differential; Humans; Keratins; Nasal Cavity; Papillomavirus Infections; Paranasal Sinus Neoplasms

Squamous cell carcinoma is the most common malignant tumor of the uterine cervix with a well-documented link to infection with human papillomaviruses (HPV). According to a recent classification, there are several morphological variants of cervical squamous carcinoma, without reference to sarcomatoid squamous cell carcinoma, which is well described in other organs.. In this paper, we describe an extremely rare case of a 77-year-old woman with primary malignant cervical tumor displaying biphasic histomorphology with an epithelioid and sarcomatoid part; the latter was immunohistochemistry positive for cytokeratin and vimentin. The association with a high-grade squamous intraepithelial lesion and molecular proof of HPV33 infection in the tumor tissue supported our diagnosis of carcinoma with partial sarcomatoid differentiation.. We report a rare case of a primary cervical epithelial tumor with a partial sarcomatoid phenotype, an unequivocal HPV infection, and an associated precancerous lesion in the cervical mucosa. This is the first description of an HPV33 infection underlying a biphasic epithelioid-sarcomatous tumor of the uterine cervix. The terminology overlap between sarcomatoid carcinoma and carcinosarcoma is also discussed.

Topics: Aged; Carcinoma, Squamous Cell; Carcinosarcoma; Cell Differentiation; Cervix Uteri; Female; Humans; Keratins; Papillomavirus Infections; Sarcoma; Uterine Cervical Neoplasms

Impaired keratinocyte differentiation has recently been suggested as a key event in equine hoof canker development. Koilocytotic appearance of keratinocytes, one of the most characteristic morphological alterations in hoof canker tissue, is also a common marker for papillomavirus (PV) infection, and bovine PV-1 and/or -2 (BPV-1/2) has previously been detected in equine canker patients. Therefore, the present study aimed to correlate the frequency and severity of koilocytotic keratinocytes with BPV detection in hoof canker samples. Hoof tissue of 5/18 canker-affected horses and 2/6 control horses tested positive for BPV-1/2 DNA using polymerase chain reaction. Thus, no association between the presence of BPV-1/2 papillomaviral DNA and koilocytotic appearance was found. Proteins associated with but not specific for PV infection were also investigated. Using immunohistochemistry, specific adhesion molecules (E-cadherin and β-catenin) and intermediate filaments (keratins 6 and 14) important for intact epidermal barrier function and keratinocyte differentiation were documented in control samples (

Topics: Animals; beta Catenin; Bovine papillomavirus 1; Cadherins; Cell Differentiation; DNA, Viral; Hoof and Claw; Horse Diseases; Horses; Immunohistochemistry; Keratinocytes; Keratins; Papillomavirus Infections

Heck's disease (focal or multifocal epithelial hyperplasia) is a benign, rare condition of the skin and mucous membranes induced by human papillomavirus (HPV) infection. Other entities that can induce large papillomatous lesions that involve the mucous membranes and skin include condyloma acuminatum, which is sexually transmitted, and white sponge nevus, often due to a mutation of cytokeratin 4 or 13. Six cases diagnosed as either Heck's disease (n = 2) or white sponge nevus (n = 4) and 6 oral condyloma were compared on histologic grounds and analyzed in situ for HPV DNA, including HPVs 6,11, and 13, as well as cytokeratins 4 and 13. Each case showed marked acanthosis, and para/hyperkeratosis. More variable histologic findings included rete ridge elongation, keratinocyte degeneration, and perinuclear halos. High copy HPV 13 DNA was evident in the squamous cells towards the surface in the two cases diagnosed as Heck's disease and in two cases diagnosed as white sponge nevus on clinical grounds. HPV 6/11 was found in each of the six condyloma. Marked decrease in either cytokeratin 4 or 13 was evident in the two cases diagnosed as white sponge nevus that were HPV DNA negative. It is concluded that in situ hybridization analyses including HPVs 6, 11, and 13 as well as immunohistochemistry for cytokeratins 4 and 13 can differentiate Heck's disease from condyloma and white sponge nevus, which can be difficult to differentiate on clinical and histologic grounds.

Topics: Adult; Biomarkers; Cell Differentiation; Condylomata Acuminata; DNA, Viral; Female; Focal Epithelial Hyperplasia; Humans; Hyperplasia; In Situ Hybridization; Keratins; Leukokeratosis, Hereditary Mucosal; Male; Middle Aged; Nevus; Papilloma; Papillomaviridae; Papillomavirus Infections; Skin

Rectal squamous cell carcinoma (SCC) is a rare tumor with unresolved etiology. Human immunodeficiency virus-infected individuals and solid organ transplant recipients experience >30-fold and approximately 3-fold elevated rates of rectal SCC, respectively, suggesting immunosuppression plays a role.

Topics: Adenocarcinoma; Anus Neoplasms; Biomarkers; Carcinoma, Squamous Cell; Case-Control Studies; DNA-Binding Proteins; DNA, Viral; Human papillomavirus 16; Humans; In Situ Hybridization; Keratins; Oncogene Proteins, Viral; Papillomavirus Infections; Rectal Neoplasms; Repressor Proteins; Transcription Factors; Tumor Suppressor Proteins; Viral Envelope Proteins

High-risk human papilloma virus (HPV) testing should be performed on all patients with newly diagnosed oropharyngeal HPV-associated squamous cell carcinoma (OPHPVSCC), and p16 immunostaining can be used as a surrogate marker. Although in surgical pathology specimens p16 staining in > 70% of the tumor cells is considered a positive result, the interpretation in fine-needle aspiration (FNA) specimens has remained controversial.. FNA of neck lymph nodes and corresponding surgical specimens from 42 patients with OPHPVSCC were reviewed.. In FNA specimens, 38 cases (90.5%) had viable tumor cells, 32 (76.2%) had keratin debris, and 36 (85.7%) had degenerated keratinized tumor cells. Twenty-seven of 27 (100%) had positive p16 staining in > 70% of viable tumor cells, while the degenerated tumor cells were negative. Twenty of 24 (83.3%) primary OPHPVSCC exhibited focal degenerated keratinized tumor cells and/or keratin debris.. This study showed that the majority of the OPHPVSCC metastases in lymph nodes had degenerated keratinized tumor cells and keratin debris. Many primary OPHPVSCC also demonstrated focal keratinization and/or degeneration. The degenerated tumor cells showed no immunoreactivity to p16. The same 70% cutoff used in histologic specimens should be applied in cytologic specimens, but only the viable tumor cells should be counted.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy, Fine-Needle; Cell Survival; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Keratins; Lymph Nodes; Male; Middle Aged; Oropharyngeal Neoplasms; Papillomavirus Infections; Predictive Value of Tests; Reproducibility of Results; Squamous Cell Carcinoma of Head and Neck

Sarcomatoid carcinoma (SC) is a variant of head and neck squamous cell carcinoma characterized by a prominent and sometimes exclusive spindle cell component. Distinction from a sarcoma or reactive stroma can be problematic, particularly in cases in which the conventional component is not obvious. The value of immunohistochemistry is limited because of the loss of cytokeratin expression in a sizable percentage of cases. Staining for p63 can enhance detection of epithelial differentiation, but its usefulness is offset by expression in various soft tissue proliferations. Staining for p40--a squamous-specific isoform of p63--could potentially improve diagnostic accuracy. Immunohistochemistry for pancytokeratin, p63, and p40 was performed on 37 head and neck SCs, 201 soft tissue neoplasms, and 40 reactive stromal proliferations. The SCs were also stained for p16 in the event that some of the tumors were human papillomavirus (HPV) related. HPV in situ hybridization was performed on p16-positive cases. Twenty-three of 37 (62%) SCs were positive for pancytokeratin, 23 of 37 (62%) were positive for p63, and 20 of 37 (54%) were positive for p40. Compared with p63, p40 staining was less likely to be observed in soft tissue tumors (5% vs. 30%) and reactive stromal proliferations (0% vs. 30%). HPV16 was detected in 3 of 10 (30%) SCs of the oropharynx but in none of the nonoropharyngeal SCs. p40 staining does not improve the sensitivity for diagnosing SC, but it does diminish the risk of misdiagnosing a sarcoma or reactive stroma as SC. The presence of a sarcomatoid variant of HPV-related oropharyngeal cancer points to HPV testing as a useful diagnostic tool for atypical spindle cell proliferations of the oropharynx.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; Diagnosis, Differential; DNA, Viral; Head and Neck Neoplasms; Human papillomavirus 16; Human Papillomavirus DNA Tests; Humans; Immunohistochemistry; In Situ Hybridization; Keratins; Oropharyngeal Neoplasms; Papillomavirus Infections; Predictive Value of Tests; Protein Isoforms; Stromal Cells; Transcription Factors; Tumor Suppressor Proteins

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma has unique biology and better outcomes. p16 immunostaining is used as a surrogate marker for transcriptionally active HPV. Although diffuse staining is generally accepted as positive, the significance of partial staining has not been established, nor has the cutoff for extent of p16 staining that should be used to identify a tumor as HPV-related. From three other large studies utilizing p16 immunohistochemistry, we identified all cases with partial positive staining. The p16-stained slides were reviewed by three study pathologists for staining (nuclear and cytoplasmic) extent (in quartiles), and also for percentage that was confluent (ie, back-to-back cell staining). Tumors were histologically typed (keratinizing, non-keratinizing, or non-keratinizing with maturation) and tested for high-risk HPV by RNA in-situ hybridization and reverse-transcriptase PCR. For the 16 cases, there were two 4+(13%), five 3+(31%), six 2+(38%), and three 1+(19%) p16 staining tumors. Extent of staining ranged from 5 to 90% of cells positive with 25% or more confluent staining in 4/16 (25%). Of the 16 (31%) cases, 5 were HPV-related on the basis of RNA in-situ hybridization and reverse-transcriptase PCR. All of these cases had >50% p16 staining, 4/5 (80%) had more than 25% confluent staining, and 4/7 (57%) had non-keratinizing histological features. Only one of the p16 1+/2+ tumors was HPV RNA-positive (by reverse-transcriptase PCR only and low level). All 1+/2+ cases were keratinizing type or undifferentiated. By sensitive detection methods, most partial p16-positive squamous cell carcinoma cases with >50% staining harbor transcriptionally active HPV, and most HPV+ tumors have significant amounts of confluent staining. Cases with <50% p16 staining and lacking significant confluent staining rarely harbor HPV. These results support that greater than 75% p16 staining or, alternatively, >50% staining combined with >25% confluent areas, are suitable cutoffs for defining positivity.

Topics: Alphapapillomavirus; Biomarkers, Tumor; Carcinoma, Squamous Cell; Combined Modality Therapy; Cyclin-Dependent Kinase Inhibitor p16; DNA, Viral; Female; Humans; Immunohistochemistry; In Situ Hybridization; Keratins; Lymph Nodes; Male; Middle Aged; Neoplasm Grading; Oropharyngeal Neoplasms; Papillomavirus Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Viral

The keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1--E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1--E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1--E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1--E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1--E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1--E4 and provide an insight into the depletion of keratin co-incident with E1--E4 accumulation observed in HPV-infected epithelium.

Topics: Amino Acid Sequence; Cell Line, Transformed; Epithelium; Humans; Keratins; Molecular Sequence Data; Oncogene Proteins, Fusion; Papillomaviridae; Papillomavirus Infections; Phosphorylation; Ubiquitination; Viral Proteins

A role for cutaneous human beta-papillomavirus (HPV) types as co-factors in the development of non-melanoma skin cancer has been postulated. Here we have investigated the effects of E7 expression on keratinocyte differentiation, proliferation and cell-cycle proteins in organotypic skin cultures.. Recombinant retroviruses containing the E7 genes from cutaneous HPV types 1, 4, 5, 8, 20, 38 and RTRX7 were produced that include types associated with benign and malignant lesions. Adult human primary keratinocytes were transduced with these recombinant retroviruses and differentiated into skin-equivalents using de-epidermalised human dermis.. Expression patterns of the basal keratinocyte marker cytokeratin 14 (CK14) were not altered by any of the viral E7 types analysed. However, expression of the early and late differentiation markers CK10 and involucrin were markedly altered in HPV 1, 4 and 38 cultures. The highest proliferation rates in basal cell layers, as judged by BrdU and Ki67 staining, were observed in HPV 1, 4 and 38 cultures. Interestingly, co-expression of cyclin E and p16(INK4a) within the same cell of the suprabasal cell layers was observed only in cultures generated using E7 of HPV 5 or HPV 8.. HPV types associated with either benign or malignant lesions perturb keratinocyte proliferation and differentiation in different ways. Moreover, expression of E7 from HPV 5 or HPV 8 seem able to overcome p16(INK4a) induced cell cycle arrest in a subset of keratinocytes.

Topics: Alphapapillomavirus; Animals; Cell Cycle; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression; Humans; Keratinocytes; Keratins; Mice; NIH 3T3 Cells; Papillomavirus E7 Proteins; Papillomavirus Infections; Skin; Species Specificity; Young Adult

Epidermodysplasia verruciformis (EV) is a rare genodermatosis with susceptibility to human papillomavirus (HPV) infection, and high risk of skin cancer considered a model of viral oncogenesis.. Fifteen cases of EV plane wart (PW)-type lesions (EV) and 14 cases of PW in healthy individuals were subjected to immunohistochemical technique for cytokeratins (K) 1, 10, 14, 16, 4, involucrin, filaggrin and e-cadherin.. K1/10 showed retarded or negative expression in EV, being substituted by K14. Expression of K14 occurred in the basal and suprabasal layers in both groups, but in EV, its expression was observed up to the more superficial layers. Both groups showed positivity for K16 and K4, involucrin expression in lower levels of the spinous layer and unaltered filaggrin expression. E-cadherin expression was diminished at the koilocytotic foci of both lesions, more superficially in EV.. Infection by HPV may alter the differentiation status of the epidermis, leading to a major expression of K14, delayed or absent expression of K1/10 and earlier involucrin expression, especially in EV. It also stimulates the expression of K16 and K4. Filaggrin expression is not altered, and e-cadherin is diminished in superficial koilocytotic cells' foci in EV.

Topics: Adult; Cadherins; Epidermodysplasia Verruciformis; Female; Filaggrin Proteins; Humans; Immunohistochemistry; Intermediate Filament Proteins; Keratins; Male; Middle Aged; Papillomavirus Infections; Protein Precursors; Skin Diseases

Primary invasive squamous cell carcinoma of the vagina is rare, and the role of human papilloma virus in its pathogenesis remains unclear. The aims of our study were to determine the distribution of human papilloma virus genotypes in 21 cases of primary invasive squamous cell carcinoma of the vagina and to correlate human papilloma virus genotype with histological subtypes. Patients' clinical records were reviewed for demographic data and the stage of the disease. Tumors (n=21) were classified according to the World Health Organization criteria. Human papilloma virus genotyping (INNO-LiPA HPV Genotyping) was performed in the whole series, and statistical analysis was performed with Fisher's Exact Test and with Student's t-test. The patients' age ranged from 36 to 88 (mean 65) years. Six cases were keratinizing squamous cell carcinoma, and 15 cases were non-keratinizing squamous cell carcinoma (seven non-keratinizing not otherwise specified, three basaloid, and five warty types). The median age of patients with keratinizing squamous cell carcinoma was 73.8 years and that of non-keratinizing squamous cell carcinoma patients was 61.5 years (P=0.08). Human papilloma virus DNA was detected in 17 cases (81%): 13 non-keratinizing squamous cell carcinoma (87%) and four keratinizing squamous cell carcinoma (67%) (P=0.31). The human papilloma virus genotypes identified were: 6, 11, 16, 18, 31, 33, 35, 40, and 58, with human papilloma virus 16 DNA the most prevalent (33%). Invasive squamous cell carcinoma of the vagina is frequently associated with human papilloma virus infection, and human papilloma virus 16 is the most common genotype. Although without statistical significance, keratinizing squamous cell carcinoma is more frequent in older patients, whereas non-keratinizing squamous cell carcinoma more frequently affects younger women. All studied histological subtypes are strongly associated with human papilloma virus infection.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; DNA, Viral; Female; Genotype; Human papillomavirus 16; Humans; Keratins; Middle Aged; Neoplasm Staging; Papillomavirus Infections; Vaginal Neoplasms

Normal human epithelial cell cultures exhibit a limited (although different between tissues) lifespan in vitro. In previous studies, urothelial cell cultures were immortalized using retroviral transformation with human papillomavirus type 16 E6 and E7 genes, in undefined culture systems containing serum or bovine pituitary extract.. Due to the variability of results in such systems, we instead developed a procedure for the immortalization of urothelial cells using a defined, serum-free culture system.. Immortalization through retroviral transformation with human papillomavirus type 16 E6 and E7 was successful, and transformation of urothelial cells conferred an extended over normal lifespan and restored telomerase activity. Transformed cells retained typical morphology and exhibited a similar growth rate, cytokeratin immunoreactivity pattern, and response to growth factors as observed in untransformed cells. Karyotype analysis revealed a gradual accumulation of genetic mutations that are consistent with previously reported mutations in epithelial cells transformed with human papillomavirus type 16 E6 and E7.. The ability to extend the in vitro lifespan of cells holds the potential to reduce the continuous need for tissue samples and to enable complete investigations with one cell line.

Topics: Animals; Cell Cycle; Cell Growth Processes; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Clone Cells; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Genes, Viral; Human papillomavirus 16; Humans; Karyotyping; Keratins; Mice; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Phenotype; Quinazolines; Repressor Proteins; Telomerase; Urothelium

We have recently shown that HPV-positive tonsillar carcinoma in young patients exhibits nonkeratinizing basaloid morphology and a characteristic immunophenotype. The purpose of this study was to review a large number of cases of oropharyngeal carcinomas, in all age groups, and to identify tumors with nonkeratinizing morphology. Using polymerase chain reaction (PCR) the prevalence and type of HPV DNA was determined in representative cases and in a control group of conventional keratinizing squamous cell carcinomas. The tumors were further characterized with a panel of immunohistochemical stains. A total of 235 carcinomas were reviewed; 141 of the tonsils and 94 in the base of tongue. Ninety (36%) of the tonsillar and 30 (32%) of the base of tongue carcinomas were nonkeratinizing (NKCa) with basal cell features; the rest were classical keratinizing squamous cell carcinomas (KSCC). HPV DNA, particularly type 16, was identified in 10 (100%) of 10 of NKCA and in only 2 (20%) of 10 of KSCC (P = .0014). NKCas were strongly reactive to p16 antibodies while KSCC showed weak and focal reactivity. Higher Ki67 and lower p53 staining scores were observed in NKCa as compared to KSCC. It is concluded that NKCa of the tonsils and base of tongue is a distinct subtype of squamous cell carcinoma of the head and neck with high prevalence of HPV DNA and a characteristic immunophenotype.

Topics: Carcinoma, Squamous Cell; Case-Control Studies; DNA, Viral; Female; Histocytochemistry; Humans; Immunophenotyping; Keratins; Ki-67 Antigen; Male; Middle Aged; Oropharyngeal Neoplasms; Palatine Tonsil; Papillomaviridae; Papillomavirus Infections; Phenotype; Polymerase Chain Reaction; Sequence Analysis, DNA; Tongue Neoplasms; Tumor Suppressor Protein p53

Adequate brushing of oral mucosa is important for accurate human papillomavirus (HPV) detection in potentially malignant (oral leukoplakia [OL], oral lichen planus [OLP]) and malignant (oral squamous cell carcinoma [OSCC]) lesions. Since various factors may limit the adequacy of oral brushing and, consequently, the accuracy of HPV detection, modified sampling procedures should be evaluated for their effect on HPV frequency and/or types detected.. To compare the HPV frequency in samples obtained by brushing the lesion site with the frequency in samples obtained by brushing an apparently normal adjacent site. The correlation between HPV frequency and keratinization of the site affected by the lesion, as well as sociodemographic variables (age, sex, smoking and drinking habits), was also examined.. HPV DNA was detected in brushing samples from 50 patients with OL, 49 with OLP, and 17 with OSCC. Polymerase chain reaction (PCR) amplification was performed by MY09/MY11 and GP05+/GP06+ primers; the HPV type was identified by DNA sequencing and a reverse hybridization (line probe) assay. Data were analyzed by the Z test, the Fisher's exact test, the chi-square test, odds ratio (OR), and a logistic regression model.. HPV DNA was detected in 22% of samples from lesion sites and in 16% of samples from adjacent sites (p = 0.22) in patients with OL, in 24.5% and 22.4% of samples from lesion and adjacent sites, respectively, in patients with OLP (p = 0.40), and in 35.3% and 41.2% of samples from lesion and adjacent sites, respectively, in patients with OSCC (p = 0.36). Lesions adjacent to HPV-positive normal sites had an increased rate of HPV detection (OR = 30; 95% CI 9.57, 94.1). HPV-18 was the most frequent genotype, followed by HPV-6, -16, -33, and -53. HPV prevalence was reduced in lesions at keratinized sites (14.5%) compared with non-keratinized sites (34.4%; p = 0.007; OR = 0.32; 95% CI 0.13, 0.81).. In patients with OL, OLP, or OSCC, a high prevalence of HPV infection was shown in apparently normal sites adjacent to lesion sites infected by HPV. The lower HPV frequency in lesions at keratinized sites suggests that HPV detection by lesion brushing is affected by keratinization. The keratinized epithelium may be less susceptible to HPV infection or, alternatively, the highly proliferative activity in non-keratinized sites may predispose to HPV infection.. Results from this study indicate that taking samples from normal sites adjacent to oral lesions may be of value in HPV detection, particularly when the lesions are located at keratinized sites. This sampling procedure may allow more accurate diagnosis of HPV infection compared with sampling only the lesion site, and may also represent a reliable method to investigate the biological characteristics of HPV infection and related oral carcinogenesis.

Topics: Alphapapillomavirus; Carcinoma, Squamous Cell; DNA, Viral; Female; Humans; Keratins; Lichen Planus, Oral; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Papillomavirus Infections

To evaluate human papillomavirus (HPV) infection in whole cervical cone specimens with cervical intraepithelial neoplasia (CIN). In addition, to evaluate the relation between the presence of CIN lesions and HPV infection and the expression of Ki-67, p53, cytokeratins, Gp230 glycoprotein, and simple mucin-type carbohydrates.. Cervical cone specimens from five patients with CIN were studied. For each specimen, serial sections encompassing the whole cone were collected (52 samples). HPV infection and HPV types were detected by the polymerase chain reaction and enzyme immunoassay. The expression of Ki-67, p53, cytokeratins, Gp230, and simple mucin-type carbohydrates was examined immunohistochemically.. All cases showed high risk HPV types, namely types 16, 33, 35, and 58. Four of the five patients were infected by multiple viral types. HPV-58 was always seen in CIN III, whereas HPV-35 was more frequent in CIN I. The expression of Ki-67 and p53 was higher in CIN III lesions. The expression of cytokeratins 8 and 17 showed complete or almost complete overlap with CIN III. Altered expression of Gp230, Tn, and sialyl-T was often seen in all grades of CIN.. When whole cervical cone specimens are evaluated the rate of multiple HPV infection is very high. The expression of cytokeratins 8 and 17 is a useful marker of CIN III.

Topics: Biomarkers, Tumor; Biopsy; Female; Humans; Immunoenzyme Techniques; Keratins; Ki-67 Antigen; Mozambique; Neoplasm Proteins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Tumor Suppressor Protein p53; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Acantholytic carcinoma is a subtype of squamous carcinoma, characterized by tubular and alveolar formations as a consequence of the acantholysis. We report a case of vulvar squamous acantholytic carcinoma (VSAC) in a 69-year-old woman, who was admitted to our institution for vulvar pruritus and the presence of a large, bilateral, exophytic, and ulcerated lesion, measuring 7 x 8 cm. The patient had never received vulvar or pelvic radiation therapy. Pathological examination with an immunohistochemical study showed features of VSAC and high p16 protein expression. Molecular study by polymerase chain reaction amplification of DNA tumor revealed a weakly positive signal for human papillomavirus. In conclusion, our case, which is the first case of VSAC with polymerase chain reaction analysis and immunohistochemical expression of p16 protein, suggested that this neoplasm could be related to human papillomavirus infection.

Topics: Acantholysis; Aged; Carcinoma, Squamous Cell; Cyclin-Dependent Kinase Inhibitor p16; DNA, Viral; Female; Humans; Immunohistochemistry; Keratin-14; Keratins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Tumor Virus Infections; Vimentin; Vulvar Neoplasms

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1=E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1=E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1=E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1=E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1=E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1=E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1=E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1=E4Delta87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1=E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1=E4 to sequester its cellular targets in the cytoplasm.

Topics: Culture Techniques; Cytoskeleton; Female; Humans; Intermediate Filaments; Keratins; Oncogene Proteins, Fusion; Papillomaviridae; Papillomavirus Infections; Tumor Cells, Cultured; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Viral Proteins

In the K14-HPV16 transgenic mouse model of human papillomavirus (HPV)-associated squamous cell cancers, HPV16 E6 and E7 oncogenes and E1 and E2 regulatory genes are driven by the K14 keratinocyte-specific promoter. HPV transcription varies within the different layers of the epithelium. The correlation between HPV transcription patterns and disease pathogenesis is not well understood. Understanding these patterns is critical to designing and testing new HPV-specific therapeutic strategies. We examined HPV gene expression in homogenous populations of cells microdissected from the stratum basale, stratum spinosum, and stratum corneum of lesions from the transgenic mice using PALM microlaser technology. RNA extracted from each cell layer was subjected to two-step gene-specific RT-PCR and real-time quantitative nested PCR. To ensure specific amplification of spliced transcripts, the primers used for real-time nested PCR spanned the splice sites. High levels of E2 were detected in the basal and supra-basal layers of hyperplastic and dysplastic lesions. E7 and E6* levels increased significantly over time in stratum basale and stratum spinosum. E6** was expressed at much lower levels. We showed that the transgenic mice express correctly spliced E2 transcripts and are suitable as a preclinical model to test a therapeutic strategy using transcriptional regulation by the E2 protein.

Topics: Animals; Carcinoma, Squamous Cell; Computer Systems; Disease Models, Animal; Dissection; DNA-Binding Proteins; Epidermis; Genes, Viral; Humans; Keratin-14; Keratins; Lasers; Mice; Mice, Transgenic; Micromanipulation; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Papillomavirus Infections; Polymerase Chain Reaction; Promoter Regions, Genetic; Repressor Proteins; Skin Neoplasms; Transcription, Genetic; Transgenes

During normal keratinocyte differentiation, a coordinated expression of many cytoskeletal and regulatory proteins occurs. Several studies suggest that expression of some of these proteins is altered in epithelium infected by the human papillomavirus (HPV). To examine protein expression, human foreskin tissue was infected with either the low-risk HPV type 11 or with HPV 83, a high-risk type. The foreskin tissue was implanted and grown in the athymic mouse xenograft system. Immunohistochemistry and immunoblot analysis of human foreskin xenografts were performed to detect cytokeratin 16 (K16), a protein previously identified in proliferative disorders of the skin. K16 was abundant in HPV 11-infected xenograft tissue, but was not detected in uninfected or HPV 83-infected tissue. Analysis of protein extracted from human biopsy tissue demonstrated the same expression patterns in natural infection by HPV 11. Reverse transcriptase PCR detected mRNA transcripts for K16 in both experimental and natural HPV 11-infected tissues, but not in uninfected tissue. These studies suggest that K16 overexpression during HPV 11-infection is regulated at the level of transcription. The marked epithelial proliferation that occurs in HPV 11 infection may involve alterations in expression of cytoskeletal proteins, including K16. Determining the mechanisms of K16 transcriptional induction could lead to therapies with the ability to reduce cell proliferation within infected tissue.

Topics: Animals; Culture Techniques; Epithelium; Genitalia, Male; Humans; Keratins; Male; Mice; Mice, Nude; Papillomaviridae; Papillomavirus Infections; Skin; Skin Transplantation; Transplantation, Heterologous; Tumor Virus Infections

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; DNA, Neoplasm; DNA, Viral; Female; Humans; In Situ Hybridization; Keratins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The purpose of this study is to report an unusual variant of cervical squamous cell carcinoma, not associated with either human papillomavirus infection or antecedent squamous intraepithelial lesions. Five women had a diagnosis of invasive cervical cancer discovered at hysterectomy performed for prolapse (two cases), leiomyoma (one case), or a vaginal fistula (two cases). The women ranged in age from 47 to 78 years (mean 59 years). Four of the five had a history of normal Papanicolaou (Pap) smears; the other had a Pap smear diagnosis of atypical squamous cells of undetermined significance (ASCUS). All had large cervical tumors (two with parametrial involvement and one with vaginal involvement) that showed extensive keratin formation, an inverted pattern of growth, and, except for one case, minimal cytologic atypia. There was extensive hyperkeratosis and parakeratosis adjacent to each tumor; none had evidence of squamous intraepithelial lesion. Human papillomavirus testing by polymerase chain reaction in situ hybridization and reverse-transcribed polymerase chain reaction in situ was negative in each case, compared with a detection rate of 107 of 108 (99%) for squamous intraepithelial lesion-associated cervical squamous cell and adenocarcinomas. Two of the women died of extensive local recurrence; two other women were recently diagnosed. We conclude that highly differentiated keratinizing squamous cell carcinoma of the cervix is a rare entity not associated with human papillomavirus infection or squamous intraepithelial lesion and thus difficult to detect on routine cervical cancer screening.

Topics: Adult; Aged; Carcinoma, Squamous Cell; DNA, Neoplasm; DNA, Viral; Female; Humans; In Situ Hybridization; Keratins; Middle Aged; Papillomaviridae; Papillomavirus Infections; Reverse Transcriptase Polymerase Chain Reaction; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.

Topics: DNA, Viral; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization; Keratin-14; Keratins; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Protein Precursors; Transcription Factor AP-1; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Human Papillomavirus type 16 (HPV-16) is the cause of both benign lesions and ano-genital cancers. In HPV-associated cancers the transforming properties of the expressed viral E6 and E7 proteins have been revealed by a number of different assays. We have generated transgenic mice expressing HPV-16 E6/E7 genes under the control of the murine keratin 5 gene promoter, which should confer cell-type specific expression in the basal cells of squamous stratified epithelia. Transgenic mice developed thymic hyperplasia and lung neoplasia with 100% frequency, the thymus showing a size increase at 2 months and reaching the maximum dimension at 6 months, when lung carcinomas appeared. After this time the size of hyperplastic thymi decreased, while malignant formations invaded the mediastinal area. Hepatic metastasis could be also observed in some of the animals at the autopsy and death invariably occurred around 10-11 months of age.

Topics: Animals; Carcinoma; Keratin-15; Keratin-5; Keratins; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Transgenic; Oncogene Proteins, Viral; Organ Size; Papillomavirus E7 Proteins; Papillomavirus Infections; Promoter Regions, Genetic; Recombinant Fusion Proteins; Repressor Proteins; Thymus Gland; Thymus Hyperplasia; Tumor Virus Infections

Topics: Antibodies; Eccrine Glands; Epidermal Cyst; Foot Dermatoses; Humans; Immunohistochemistry; Keratins; Papillomaviridae; Papillomavirus Infections; Tumor Virus Infections

Two cell lines derived from vaginal intraepithelial neoplasias (VAINs) expressing human papillomavirus (HPV) 33 (VAIN I, UT-DEC-1) and 16 (VAIN II, UT-DEC-2) E6-E7 mRNA were studied in organotypic culture for their keratins and cell cycle regulatory proteins in relation to replicative aging. Early-passage UT-DEC-1 and UT-DEC-2 cells reproduced epithelial patterns consistent with VAIN. Cells from later passages resembled full-thickness intraepithelial neoplasia (UT-DEC-1) and microinvasive cancer (UT-DEC-2). The morphological changes were compatible with these cell lines' ability for anchorage-independent growth at later passages. Simple epithelial keratins were aberrantly expressed in both cell lines. K18 (absent in normal vaginal keratinocytes) and K17 expression increased in UT-DEC-1 and UT-DEC-2 cells at late passages. No marked differences in expression of p53 (wild type in both cell lines), mdm-2 or PCNA were detected in parallel with progression. The expression of p21WAF1/cip1 localized mostly to the upper half of the epithelium at early passage and was more intense in the HPV 16-positive UT-DEC-2 cell line expressing K10. In Northern blot analyses, the transcription pattern of the HPV 33 E6-E7 of the UT-DEC-1 cell line changed during later passages, whereas that of the HPV 16 E6-E7 of the UT-DEC-2 cell line remained unaltered. The present characterization of the phenotype of these cell lines derived from natural squamous intraepithelial lesions shows an association between simple epithelial-type keratin expression and progressive changes in growth and morphology, but fails to demonstrate consistent changes in the expression of cell cycle regulatory proteins studied in parallel with progression.

Topics: Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Adhesion; Cell Culture Techniques; Cell Cycle Proteins; Cellular Senescence; Disease Progression; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Keratins; Neoplasm Proteins; Papillomaviridae; Papillomavirus Infections; Phenotype; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Virus Infections; Vagina

In stratified squamous epithelia, altered expression of keratins (Ks) is one possible marker of malignant potential. In the epithelium of the uterine cervix, presence of human papillomaviruses (HPVs) is increasingly regarded as a marker of risk for cervical cancer. However, a similar role in oral cancer and precancer remains controversial. To address these questions, formalin-fixed, paraffin-embedded oral carcinomas from Sudanese snuff dippers (n=14) and oral carcinomas from Sudanese (n=14), Swedish (n=19) and Norwegian (n=41) non-snuff dippers were examined by immunohistochemistry for expression of K types 13, 14 and 19 using monoclonal antibodies. HPV infection was searched for in all the carcinomas by in situ hybridization (ISH) using the cocktail HPV OmniProbe and the ViraType probe. Carcinomas from Sudanese (snuff dippers/non-snuff dippers) were also examined for HPV infection by polymerase chain reaction (PCR) using the general HPV primers GP5+/GP6+. For the oral carcinomas from snuff dippers, moderate to intense expression of K13 (71%; 10/14), K14 (86%; 12/14) and K19 (93%; 13/14) was found. For the oral carcinomas from non-snuff dippers, weak to moderate expression of K13 (64%; 47/74), K14 (43%; 32/74) and K19 (45%; 33/74) was found. HPV DNA was not detected in any of the carcinomas from three countries when examined by ISH. The Sudanese (from snuff dippers/non-snuff dippers) oral carcinomas were also negative for HPV DNA with the PCR. The present study shows that (i) there is a high level of expression of K13, K14 and K19 in oral carcinomas from snuff dippers compared to those from non-snuff dippers, (ii) this high level of expression may arise from dysregulation of keratinocyte proliferation and maturation caused by damaging effects of snuff, (iii) the HPV genome is not found in Sudanese (snuff dippers/non-snuff dippers), Swedish or Norwegian oral carcinomas, and (iv) this may suggest that these viruses do not play a prominent role in the aetiology of oral carcinomas from these countries.

Topics: Carcinoma, Squamous Cell; Female; Humans; In Situ Hybridization; Keratin-14; Keratins; Male; Mouth Neoplasms; Norway; Papillomaviridae; Papillomavirus Infections; Plants, Toxic; Polymerase Chain Reaction; Sudan; Sweden; Tobacco, Smokeless; Tumor Virus Infections

The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1-E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers.

Topics: Base Sequence; Cell Line; DNA Primers; Humans; Keratins; Molecular Sequence Data; Molecular Weight; Oncogene Proteins, Fusion; Papillomaviridae; Papillomavirus Infections; Protein Binding; Tumor Virus Infections; Viral Proteins

Given the prevalence of human papilloma virus (HPV) infection, an attempt was made to determine whether certain factors such as keratinization and/or squamous atypia are associated with its development. Review of our gynecologic cytology files from 1989 yielded 1,615 specimens showing parakeratosis and/or hyperkeratosis, without cytologic evidence of HPV. Concomitant diagnoses included no atypia [keratinization only (KO)], inflammatory squamous atypia (ISA), and squamous atypia (SA). Morphologic follow-up including repeat cytology or biopsy was available for 916 cases, 92 (10.0%) of which possessed changes of HPV. For any case with both cytologic and biopsy evidence of HPV, only the biopsy result was tabulated. HPV on follow-up examination was detected in 52 (6.7%) of the 764 cases with KO; in 20 (20.8%) of the 96 cases with keratinization and ISA (KISA); and in 20 (35.7%) of the 56 cases with keratinization and SA (KSA). The definitive diagnosis of HPV was based on previously described features (Gupta, In: Comprehensive Cytopathology, Philadelphia: WB Saunders, 1991:133-140) including nuclear enlargement with nuclear membrane irregularities in combination with sharply demarcated paranuclear cytoplasmic clearing. Affected cells have rounded borders. Binucleated cells are not uncommon. The increasing percentage of HPV from KO to KISA to KSA is not necessarily surprising. However, mathematical analysis revealed statistically significant differences in the development of HPV in each of the 3 groups: KISA vs. KO (P < 0.001), KSA vs. KO (P < 0.001), and KSA vs. KISA (P < 0.05). Therefore, a cytologic diagnosis of keratinization with ISA or especially SA should warrant closer follow-up than that of KO.

Topics: Biopsy; Female; Follow-Up Studies; Humans; Incidence; Keratins; Papillomaviridae; Papillomavirus Infections; Parakeratosis; Predictive Value of Tests; Prevalence; Retrospective Studies; Tumor Virus Infections; Vaginal Smears

Human papillomaviruses (HPVs) are important human pathogens associated with a range of epithelial neoplasia. The rising incidence of HPV infection and association of HPV with malignancy has led to increased interest in appropriate management of these infections. Development of new therapies for viral warts has been frustrated by the lack of availability of models permissive for viral replication. Here we describe the development of HPV-severe combined immunodeficient mouse model which reproduces mature HPV-infected epithelia. Grafting of anogenital and laryngeal papillomas harbouring either HPV-6 or HPV-11 resulted in the formation of a differentiated neo-epithelium exhibiting the hallmark features of HPV infection including basal hyperplasia, acanthosis and koilocytosis. The reformed warty epithelium contained amplified HPV DNA and expressed capsid protein in the differentiated layers. A striking feature is the production of macroscopic papillomata in an anatomically relevant and accessible site, providing a system of particular relevance for the temporal evaluation of developing lesions and selection of antiviral agents.

Topics: Animals; Capsid; Condylomata Acuminata; Disease Models, Animal; DNA Replication; Epithelium; Gene Expression; Humans; Keratins; Laryngeal Neoplasms; Mice; Mice, SCID; Neoplasm Transplantation; Papilloma; Papillomaviridae; Papillomavirus Infections; Proliferating Cell Nuclear Antigen; Protein Precursors; Skin; Skin Transplantation; Transplantation, Heterologous; Tumor Virus Infections; Virus Replication