Bizarre cell dysplasia of the cervix.

AIM: The aim of this study was the characterization of a new subtype of high-grade cervical squamous intraepithelial lesion (HSIL) with enlarged cells containing bizarre nuclei: so-called bizarre cell dysplasia (BCD). METHODS: A total of 29 cervical cone biopsy samples of this type of dysplasia were studied. Multi-target polymerase chain reaction and in situ hybridization human papillomavirus (HPV) detection was performed in all cases. BCD was defined as a subtype of HSIL characterized by the presence of large dysplastic cells with abnormal, large pleomorphic nuclei or multinucleation causing nucleomegaly. This results in bizarre nuclear shapes. Bizarre cells are scattered throughout the whole thickness of the dysplastic squamous epithelium. RESULTS: The BCD lesions arise within the conventional/classic high grade or "bland" type squamous dysplasia HSIL. Statistically they were significantly associated with HVP type 16. A significant association with other studied viruses (Herpes simplex virus [HSV]1, HSV2, Varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, and human polyomaviruses BK and JC) was not confirmed. CONCLUSIONS: BCD involves cytologically characteristic morphologic changes that are recognizable, but which may pose some risk of misdiagnosis as low-grade squamous intraepithelial lesion due to the enlargement of dysplastic cells and multinucleation. Based on the unique histological, cytological and biological features of BCD including strong association with HPV 16 infection, we believe that this is a specific, and so far unrecognized variant of HSIL.

[HPV-associated head and neck cancer: update and recommendations for practice]. / HPV-asociované karcinómy hlavy a krku: Aktualizácia poznatkov a odporúcania pre prax.

Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV-OSCC) represents a specific clinical and pathological entity among head and neck cancers with more favorable prognosis than corresponding HPV-negative oropharyngeal squamous cell carcinoma (SCC). HPV-OSCC most commonly displays non-keratinzing SCC histology, although number of cases presented variable morphology. Detection of transcriptionally active HPV in oropharyngeal SCCs is of critical prognostic importance, which even supersedes the importance of their formal pathological grading. This article summarizes the current state of knowledge of HPV-positive SCC of oropharynx and of other anatomical subsites of the head and neck and discusses the role of HPV detection in the diagnostics of metastatic SCC lesions of unknown origin.

Human papillomavirus infection and p16 expression in the immunocompetent patients with extragenital/extraungual Bowen's disease.

BACKGROUND: The role of human papillomaviruses (HPV) in the development of squamous cell carcinoma (SCC) has been established for anogenital lesions but still remains controversial for carcinomas in other sites. The aim of this study was to determine the α-HPV and ß-HPV prevalence and their association with p16 expression, sun exposure, and clinicopathological findings in patients with Bowen's disease (BD). METHODS: One hundred sixty nine skin biopsy specimens from 157 immunocompetent patients with extragenital/extraungual BD were examined for HPV status and p16 expression. The presence of koilocyte-like changes, solar elastosis and papillomatosis was recorded for each specimen. RESULTS: BD was diagnosed more often in potentially sun-exposed sites with prevalence 73.6 % and a remarkable predilection for the head and neck region. High risk α-HPV or ß-HPV were detected in 34.7 % of lesions and ß-HPV infections dominated over α-HPV. Higher prevalence of koilocyte-like changes and papillomatosis was found in HPV-positive specimens but it was not statistically significant. The expression of p16 was detected in 79.8 % of lesions and displayed no correlation with the HPV status. HPV-positivity tended to be detected more often in sun-protected sites. Dual infections by α-HPV/ß-HPV genera and mixed α-HPV infections were not detected, while 37.5 % of ß-HPV positive specimens were infected by two or more ß-HPV genotypes. HPV 9 was significantly associated with mixed ß-HPV infections. CONCLUSIONS: HPV may play an etiological role at least in some SCC in situ arising in extragenital sites. Sunprotected sites may be more dependent on HPV-mediated co-carcinogenesis than sun exposed areas. The presence of the p16-expression, papillomatosis or koilocyte-like change is not a reliable marker of HPV infection in SCC in situ.

Origin of cystic squamous cell carcinoma metastases in head and neck lymph nodes: Addition of EBV testing improves diagnostic accuracy.

Most cases of cystic squamous cell carcinoma (SCC) metastases in the upper neck are associated with an oropharyngeal primary, namely human papillomavirus (HPV)-associated SCC arising in the palatine or lingual tonsil. A retrospective study was performed on 22 patients who presented with cystic head and neck SCC metastases. The purpose of the study was to find out whether histological characteristics, p16 protein expression, HPV and Epstein-Barr virus (EBV) status could be useful in predicting the localization of the primary tumor. The primary site was identified in 20 of 22 patients and included the oropharynx in 14 patients (63.6%), the nasopharynx in 3 patients (13.6%), the lungs in 2 cases (9%), and the skin of the auricle in one case (4.5%). No primary was found in two patients (9%). Sixteen of 17 cases (94.1%) originating in Waldayer's ring (oropharynx and nasopharynx), and both cases with an unknown primary showed morphology of non-keratinizing SCC or non-keratinizing SCC with maturation. All tumors with oropharyngeal primary and both cases with unknown primary showed diffuse p16 staining and presence of HPV DNA. All three cystic metastases of nasopharyngeal carcinoma were EBV-positive and p16/HPV-negative. In contrast, cutaneous and pulmonary metastases showed morphology of a well differentiated keratinizing SCC and poorly differentiated keratinizing SCC, respectively, and were HPV/EBV-negative. We confirmed that cystic SCC lymph node metastases of the head and neck region are strongly associated with the occult primary localized in the oropharynx. The oropharyngeal origin should always be corroborated by p16 immunohistochemistry and HPV-specific testing because SCC arising in other sites, such as nasopharynx, skin or lungs may manifest with cystic neck metastases as well. Addition of EBV testing in p16/HPV-negative cases can disclose the nasopharyngeal origin of the cystic neck metastases in a subset of cases.

Human Papillomavirus Infection and p16 Expression in Extragenital/Extraungual Bowen Disease in Immunocompromised Patients.

An increased rate of second nonmelanoma skin cancers is found in immunocompromised patients. Epidemiological and molecular data implicate ultraviolet radiation as the major risk factor. In addition, there is increasing evidence supporting the role of human papillomavirus (HPV) in the pathogenesis of premalignant and malignant skin lesions in both immunocompetent and immunocompromised patients. In a retrospective cross-sectional study, the authors examined the expression of p16 by immunohistochemistry and the presence of mucosal (α-genus) and cutaneous/epidermodysplasia verruciformis (ß-genus) HPV DNA by polymerase chain reaction in 29 biopsy specimens of extragenital/extraungual Bowen disease (BD) from 24 Eastern European white immunocompromised patients. Furthermore, the author evaluated the association between the expression of p16 protein and the presence of HPV DNA. Among 25 specimens from 21 patients evaluable by polymerase chain reaction, HPV DNA was detected in 10 (40%) BD lesions from 9 patients. Beta-HPV predominated over alpha-HPV types. Among 29 immunohistochemically evaluable BD specimens, 22 lesions (∼76%) from 20 patients were scored as p16 positive. HPV DNA-positive and HPV DNA-negative lesions displayed the same proportion of p16 positivity (80%) and no correlation was found between the HPV DNA presence and the p16 expression status. Our pilot study demonstrated that ß-HPV infections predominate in BD cases diagnosed among immunocompromised patients, although high- and low-risk mucosal (alpha) HPV genotypes may be detected in a minority of cases. In contrast to anogenital HPV-associated lesions, positive p16 expression is not a reliable marker of high-risk α-HPV infection in BD cases, as it can be also detected in ß-HPV infected and HPV-negative cases.

Pyloric gland adenoma: a histologic, immunohistochemical and molecular genetic study of 23 cases.

Pyloric gland adenoma is a rare neoplasm with a gastric epithelial differentiation. We report 23 cases of pyloric gland adenoma in older persons, with a mean age of 74 years (range 52 - 87 years). They occurred in the esophagus (3 cases), corporal gastric mucosa (7 cases), duodenum (10 cases), gallbladder (2 cases), and choledochus (one case). Histologically, they were characterized by closely packed pyloric gland-type tubules with a monolayer of cuboidal to low columnar epithelial cells containing basally located round nuclei, and a superficial layer of tall, columnar, foveolar-type epithelium. Immunohistochemically, most tumor glands expressed pyloric gland mucin MUC6, whereas MUC5AC was positive in superficial gastric foveolar epithelium, and in a minority of glands. In addition, scattered neuroendocrine cells positive for chromogranin A and/or synaptophysin were seen in all cases. In 3 cases (two cases in the gallbladder and one case in the esophagus), areas of intestinal metaplasia with CK20, CDX2, and MUC2 positivity were found. Focal low-grade dysplasia was found in five cases (21.7%), and diffuse high-grade dysplasia was seen in one adenoma (4.4%), i.e., 6 of 23 PGAs (26.1%) showed dysplastic features. In one esophageal case, an invasive adenocarcinoma was diagnosed. Scattered p53 positive cells were found in all cases. Their number was higher in lesions with low-grade dysplasia and it was substantially increased in adenoma with high-grade dysplasia and in adenocarcinoma. Our molecular genetic results indicate that pyloric gland adenomas neoplastic nature is associated with p53 accumulation, mutations in oncogenes GNAS, KRAS, CTTNB1 and tumor suppressor genes SMAD4, and TP53. Pyloric gland adenoma can evolve into dysplasia and adenocarcinoma.

Epidermolytic hyperkeratosis of the vulva associated with basal cell carcinoma in a patient with vaginal condyloma acuminatum and vaginal intraepithelial neoplasia harboring HPV, type 42.

The occurrence of basal cell carcinoma (BCC) of the vulva is rare. We report the case of a 79-year-old woman with a medical history of intravaginal condyloma acuminatum and vaginal intraepithelial neoplasia 3 (VaIN 3) who presented with a solitary whitish lesion sized 8x5 mm with a central desquamation located on the right labium majus. Histopathologic examination revealed a typical superficial and nodular BCC. Additionally, there were multiple remarkable foci of epidermolytic hyperkeratosis (EH). These foci both merged with superficial BCC or were sharply demarcated from the tumor. Retrospective molecular-biological examination of all the available material revealed HPV type 42 in both condyloma acuminatum and VaIN 3 specimen but not in the BCC associated with EH. To our best knowledge, involvement of the lower female genitalia by EH is a rare finding with six cases published to date. Awareness of EH in this location and its distinction is important because it may be potentially misinterpreted as a viral condyloma.

Pigmented squamous intraepithelial neoplasia of the anogenital area: a histopathological and immunohistochemical study of 64 specimens from 45 patients exploring the mechanisms of pigmentation.

Pigmented lesions in the anogenital area encompass a wide variety of disorders including squamous intraepithelial neoplasia. The authors sought to explore the mechanism(s) underlying clinically pigmented squamous intraepithelial neoplasia in the anogenital area. A light-microscopic and immunohistochemical study of 64 lesional specimens from 45 patients (32 women, 13 men; age range, 23-73 years) with pigmented lesions in the anogenital area was performed. Histopathologically, 63 (98%) specimens showed melanin incontinence into the superficial dermis beneath the dysplastic epithelium. A focal or total loss of basilar hyperpigmentation was detected in 30 (48%) and 13 (20%) of lesions, respectively. In 17 (27%) cases, absence of basal layer hyperpigmentation was accompanied by a subepithelial lichenoid infiltrate. Melanin within the upper part of dysplastic areas were seen in 63 cases (98%), whereas dendritic melanocytes colonization, mild in all but 1 specimen case, was observed in 53 (83%) cases. All cases proved to be the usual type of squamous intraepithelial neoplasia; no single case of the simplex (differentiated) variant was present. The main mechanisms of pigmented squamous intraepithelial neoplasia of the anogenital area include melanin incontinence and occurrence of melanin in dysplastic keratinocytes. Colonization of the dysplastic epithelium by dendritic melanocytes seems to contribute, but it is rarely a prominent feature.

HPV typing of high-grade dysplasia (CIN III) in cone biopsies of 38 HPV-vaccinated women.

HPV-vaccinated women develop CIN III very rarely. We have identified a study group of 38 such patients and showed that a specific HPV genotype prevalence in those cases equals the prevalence of HPV genotypes in CIN III present in the general Czech population. In all cases, CIN III was diagnosed within 3 years after having completed the HPV vaccination. We conclude that dysplasia was present before the vaccination in those women. A history of abnormal pre-vaccination PAP smear result (present in 78 % of women in the study group) and age of over 17 by the time of vaccination completion (97 % of women in the study group) are identified as probable factors increasing the risk of CIN III development after HPV vaccination.

Eosinophilic dysplasia of the cervix associated with HPV 6 infection - case report and review of the literature.

Eosinophilic dysplasia of the cervix is recently described unusual and somewhat obscure dysplastic lesion of squamous epithelium. We present histological features of a lesion in 41 years old woman. It was composed of cells with brightly eosinophilic cytoplasm contoured by a sharp and slightly broader cytoplasmic membrane, lacking maturation, with mild increase in nuclear-cytoplasmic ratio, slight chromatin clumping and uneven mild nuclear clearing. Electronmicroscopic study showed mild crevices of the nuclear membrane in some dysplastic cells. Tissue in situ hybridization study confirmed the presence of HPV 6 in the form of patchy dotted pattern of integrated type. Immunohistochemistry revealed diffuse positive expression of antigen p16, extraordinarily in this case focally sparing basal part of the epithelium. Underestimation of this lesion can be avoided by paying attention to strong eosinophilia of the cytoplasm and sharp cellular contouring of the examined epithelium in routine hematoxylin-eosin staining.

Human papillomavirus type-specific prevalence in the cervical cancer screening population of Czech women.

BACKGROUND: Infection with high-risk human papillomavirus (HPV)types has been recognized as a causal factor for the development of cervical cancer and a number of other malignancies. Today, vaccines against HPV, highly effective in the prevention of persistent infection and precancerous lesions, are available for the routine clinical practice. OBJECTIVES: The data on the prevalence and type-specific HPV distribution in the population of each country are crucial for the surveillance of HPV type-specific prevalence at the onset of vaccination against HPV. METHODS: Women attending a preventive gynecological examination who had no history of abnormal cytological finding and/or surgery for cervical lesions were enrolled. All samples were tested for the presence of HPV by High-Risk Hybrid Capture 2 (HR HC2) and by a modified PCR-reverse line blot assay with broad spectrum primers (BS-RLB). RESULTS: Cervical smears of 1393 women were analyzed. In 6.5% of women, atypical cytological findings were detected. Altogether, 28.3% (394/1393) of women were positive for any HPV type by BS-RLB, 18.2% (254/1393) by HR HC2, and 22.3% (310/1393) by BS-RLB for HR HPV types. In women with atypical findings the prevalence for HR and any HPV types were significantly higher than in women with normal cytological findings. Overall, 36 different HPV types were detected, with HPV 16 being the most prevalent (4.8%). HPV positivity decreased with age; the highest prevalence was 31.5% in the age group 21-25 years. CONCLUSIONS: Our study subjects represent the real screening population. HPV prevalence in this population in the Czech Republic is higher than in other countries of Eastern Europe. Also the spectrum of the most prevalent HPV types differs from those reported by others but HPV 16 is, concordantly, the most prevalent type. Country-specific HPV type-specific prevalences provide baseline information which will enable to measure the impact of HPV vaccination in the future.

[Sequencing - classical method]. / Sekvenování - klasická metodika.

In this article the basic methods of reading nucleotide sequences in DNA molecules are summarized. Sanger sequencing is described most thoroughly as it is the most frequent routine method currently being utilized. The article describes in detail the principle of sequence determination through the production of fragments with a known end base using chain termination synthesis of DNA and ways of separation and detection of the fragments. Some alternative methods of sequencing are mentioned in short. Basic approaches of analyzing sequence data are explained as well as different outcomes, obstacles and challenges.

Human papillomaviruses are not involved in the etiopathogenesis of salivary gland tumors.

BACKGROUND: Tumor-related high risk human papillomaviruses (HPV) 16 and 18 have been repeatedly detected in head and neck cancers, particularly, they are well known risk factors in squamous cell carcinoma of the oropharyngeal and tonsillar region. Little is known, however, about the possible role of HPV in salivary gland tumors. METHODS: Fifty-five cases of benign and malignant salivary gland tumors were tested using p16 immunohistochemistry followed by HPV DNA polymerase chain reaction (PCR) using SPF, CPSGB, GP5+/GP6+ primers, and type specific primers for HPV 16, 18, 31, 33, 35, 45 in the cases with strong immunohistochemical expression for p16 protein (score 3+). RESULTS: Only 5 tumors of 55 (9 %) were completely devoid of any p16 staining, and in 10 cases (18 %), less than 25 % of tumor cells stained (score 1+). In the majority of cases (35 of 55; 64 %) there was a patchy nuclear and cytoplasmic strong staining in 26 to 50 % of tumor cells (score 2+). In five cases (9 %), strong nuclear and cytoplasmic staining in more than 51% of tumor cells was detected (score 3+). However, none of the p16-positive cases showed any evidence of high-risk HPV by PCR. CONCLUSIONS: The results of the study indicate that HPV, in particular oncogenic types 16 and 18, are not involved in the etiology of benign and malignant epithelial tumors of salivary glands. Therefore, it is likely that salivary gland tumors belong to the category of tissues in which the p16 positive immunohistochemistry is not biologically relevant to the oncogenic role of HPV infection.

[The testing strategy for detection of biologically relevant infection of human papillomavirus in head and neck tumors for routine pathological analysis]. / Moznosti prukazu biologicky relevantní papilomavirové infekce u maligních nádoru hlavy a krku v diagnostické patologii.

There is a subgroup among head and neck squamous cell carcinomas, which is etiologically linked to the infection of high-risk human papillomavirus (HPV). In recent studies, HPV related squamous cell carcinomas have been placed in a separate group because of their different epidemiology, distinctive histopathological characteristics, therapeutic response and clinical outcome. The reported prevalence of high-risk HPV in head and neck tumors varies in different studies. This fact occurs mainly due to the absence of a widely accepted consensus for HPV detection in head and neck malignancies. We present a methodological algorithm for detection of biologically relevant HPV infection: a combination of an immunohistochemical staining of the p16 protein - a surrogate marker for a transforming HPV infection, and a molecular genetic identification of HPV DNA by three different polymerase chain reactions (PCR). The study group consisted of 41 patients with a tumor in head and neck region. A verification of detection of biologically relevant HPV infection has been performed in 10 available samples using an alternative approach, which comprised the detection of RNA transcript of HPV by reverse transcription followed by PCR (RT-PCR), and further in situ hybridization (ISH) with a commercial high-risk HPV probe. We have found a high correlation between HPV DNA detection using triple-PCR approach and strong diffuse positivity of the p16 protein (correlation coefficient 0.94) and have confirmed the validity of this algorithm. In 94 % of HPV related squamous cell carcinomas HPV type 16 was detected. In one case HPV type 33 was identified. That is in agreement with earlier published data. A more appropriate alternative method for the detection of biologically relevant- transforming HPV infection seems to be RT-PCR, which proved 100 % agreement with the original methodological approach of p16 determination and PCR status. Interpretation of the ISH has been complicated by frequent nonspecific staining of the sample and its routine usage in the diagnostic algorithm of our laboratory is currently not feasible.

Cribriform adenocarcinoma of minor salivary glands may express galectin-3, cytokeratin 19, and HBME-1 and contains polymorphisms of RET and H-RAS proto-oncogenes.

The aim of the study was to further elucidate the immunohistochemical and genetic characteristics of cribriform adenocarcinoma of minor salivary glands (CAMSG). The study comprised five CAMSG from two males and three females, aged 21-72 years. Four tumors were localized at the base of tongue and one in the floor of mouth. At the time of diagnosis, four tumors had metastasised to regional lymph nodes. After tumor resection, two patients were treated by radiotherapy and one by chemoradiotherapy. During the follow-up (median 14 months), two patients developed lymph node metastasis. Microscopically, all tumors showed cribriform, papillary, follicular, and microcystic growth patterns. The tumor cells displayed vesicular nuclei with intranuclear grooves. Immunohistochemically, all tumors showed expression of cytokeratin (CK) 7, CK8, CK18, vimentin, smooth muscle actin, calponin, S-100 protein, and p16 protein. In addition, we observed expression of galectin-3, CK19, and HBME-1, but not of thyroglobulin and TTF-1. No mutations of RET, BRAF, K-RAS, H-RAS, and N-RAS proto-oncogenes were detected. However, in RET proto-oncogene, we found polymorphisms Gly691Ser (exon 11) and Ser904Ser (exon 15) in one case, p.Leu769Leu (exon 13) in one case, and variant p.IVS14-24 G/A of intron 14 in two cases, and in H-RAS proto-oncogene we found polymorphism 81 T-C (exon 1) in three cases. Thyroglobulin and TTF-1 are the only useful markers in the differential diagnosis between CAMSG and papillary thyroid carcinoma as both tumors may express galectin-3, CK19, and HBME-1. The RET, H-RAS, and N-RAS proto-oncoogenes are not mutated in CAMSG.