Biological evidence for a causal role of HPV16 in a small fraction of laryngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) is a causal factor in virtually all cervical and a subset of oropharyngeal squamous cell carcinoma (OP-SCC), whereas its role in laryngeal squamous cell carcinoma (L-SCC) is unclear. METHODS: Formalin-fixed paraffin-embedded (N=154) and deep-frozen tissues (N=55) of 102 L-SCC patients were analysed for the presence of 51 mucosal HPV types. HPV DNA-positive (HPV DNA+) cases were analysed for E6*I mRNA transcripts of all high risk (HR)/probably/possibly (p)HR-HPV identified, and for HPV type 16 (HPV16) viral load. Expression of p16(INK4a), pRb, cyclin D1 and p53 was analysed by immunohistochemistry. RESULTS: Ninety-two patients were valid in DNA analysis, of which 32 (35%) had at least one HPV DNA+ sample. Among the 29 single infections, 22 (76%) were HPV16, 2 (7%) HPV56 and 1 each (4%) HPV45, HPV53, HPV70, HPV11 and HPV42. Three cases harboured HPV16 with HPV33 (twice) or HPV45. Only 32% of HPV DNA+ findings were reproducible. Among HPV16 DNA+ L-SCC, 2 out of 23 (9%) had high viral loads, 5 out of 25 (21%) expressed E6*I mRNA and 3 out of 21 (14%) showed high p16(INK4a) and low pRb expression (all three HPV16 RNA-positive), immunohistochemical marker combination not identified in any other HPV DNA+ or HPV DNA-negative (HPV DNA-) L-SCC, respectively. CONCLUSION: HPV type 16 has a causative role in a small subgroup of L-SCC (<5% in this German hospital series).

Epidemiology of human papillomavirus-related oropharyngeal cancer in a classically low-burden region of southern Europe.

The incidence of human papillomavirus (HPV)-related oropharyngeal cancer is increasing in some regions. Nevertheless, the epidemiology of this disease has not been extensively investigated in southern Europe. We conducted a retrospective cohort study of patients diagnosed with primary oropharyngeal cancer from 1991 to 2016. Cancer tissues underwent histopathological evaluation, DNA quality control, HPV-DNA detection and p16INK4a immunohistochemistry. Data were collected from medical records. Factors associated with HPV positivity and time trends were evaluated with multivariable Bayesian models. The adjusted prevalence of HPV-related cases in 864 patients with a valid HPV-DNA result was 9.7%, with HPV-DNA/p16INK4a double positivity being considered. HPV-related oropharyngeal cancer was likely to occur in non-smokers and non-drinkers, to be located in the tonsil or diagnosed at advanced stages. Time-trend analysis showed an increasing risk of HPV-related oropharyngeal cancer in the most recent periods (5-year period increase of 30%). This increase was highest and with a clear increasing trend only in the most recent years (2012-2016). The prevalence of HPV-related oropharyngeal cancer started to sharply increase in the most recent years in our setting, as occurred two decades ago in areas where most oropharyngeal cancer cases are currently HPV-related. Our results provide a comprehensive assessment of the epidemiological landscape of HPV-related oropharyngeal cancer in a region of southern Europe.

Prognostic impact of CD133 expression in Endometrial Cancer Patients.

To assess the impact of CD133 expression on the prognosis of endometrioid endometrial carcinoma (EEC). We retrospectively assessed CD133 expression in tissue microarray of 116 surgically treated FIGO I-III EEC. Tumors with ≥10% of CD133-expressing cells were considered CD133-positive (CD133+). On the basis of CD133 expression, clinical and pathological parameters, progression-free survival (PFS) and overall survival (OS) were evaluated. Of the EEC studied 85.2% showed CD133-expressing cells. Only 61% (n = 66) of EEC presented ≥10% of CD133 expressing cells and were considered CD133+. The mean OS for CD133+ tumour patients was 161 months (95% CI, 154-168) as compared with 146 months (95% CI, 123-160) for those with CD133- tumors (p = 0.012). The mean PFS for CD133+ tumour was 159 months (95% CI, 149-168) as compared with 147 months (95% CI, 132-161) in those with a CD133-tumour (p = 0.014). CD133+ tumours were less likely to have vascular invasion (p = 0.010) and more likely to be well differentiated (p = 0.034). C133+ tumours predicted favorable OS and PFS of EEC patients, with a Hazard Ratio 4.731 (95% CI, 1.251-17.89; p = 0.022). CD133+ tumor status correlates with favorable prognosis of EEC. Our findings are in agreement with studies addressing brain and colorectal tumours.

Apoptosis loss and bcl-2 expression: key determinants of lymph node metastases in T1 breast cancer.

The Bcl-2 proto-oncogene extends cell survival but does not confer any proliferative advantage to cells that express it. Thus, the loss of apoptosis may have a role in progression allowing the acquisition of additional mutations. To determine whether apoptosis loss at diagnosis is associated with the metastatic advantage of ductal breast carcinomas and to examine the relationship between Bcl-2 expression, p53, and tumor cell death status, we examined tumor samples from 116 patients diagnosed with T1 (2 cm or less) breast cancer with (n = 49) or without (n = 67) lymph node metastases. Apoptosis loss in histological sections was considered when <1% of tumor nuclei were stained with terminal deoxynucleotidyl transferase labeled with biotin. We studied the expression of Bcl-2 and p53 by immunohistochemistry and in 37 p53 mutations by single-strand conformational polymorphism analysis and cycle sequencing. Multivariate logistic regression modeling was used to estimate prevalence odds ratios (pORs) for apoptosis loss and presence of lymph node metastases. Patients with marked apoptosis loss in their tumor cells were about 5 times more likely to present lymph node metastases than those with no apoptosis loss in their tumor cells (adjusted pOR, 4.7; 95% confidence interval, 1.4-15.6; trend test, P = 0.008). Bcl-2 expression was strongly associated with both apoptosis loss (pOR, 6.9; trend test, P < 0.0001) and presence of lymph node metastases (pOR, 5.7; trend test, P = 0.002). These associations were more evident in histological grade I and II tumors than in poorly differentiated histological grade III tumors and in p53-negative tumors than in p53-positive tumors. This study demonstrates for the first time that the lymphatic progression of T1 human breast cancer is strongly related to apoptosis loss.

Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer.

To test the hypothesis of an association between HER2 and chemotherapy resistance, we performed a prospective assessment of the predictive value of the circulating HER2 extracellular domain (ECD) in patients with advanced breast carcinoma in the setting of a multicenter Phase II trial using paclitaxel and doxorubicin. Serum samples were collected from 58 patients with metastatic breast carcinoma before first-line chemotherapy for advanced disease, and the levels of circulating HER2 ECD were measured using an enzyme immunoassay. Immunohistochemistry with anti-HER2 monoclonal antibody CB11 was used to assess the overexpression of HER2 in the primary tumors. When 450 fmol/ml was used as a cutoff, 24 cases (41%) had elevated HER2 ECD levels. Elevated levels of circulating HER2 ECD were associated with the expression of HER2 in the primary tumor tissue and with the metastatic tumor burden (evaluated with the marker CA 15-3; P = 0.032 and P = 0.002, respectively) but not with variables such as menopausal status, stage at diagnosis, previous adjuvant therapy, or the number of metastatic sites. The levels of circulating HER2 ECD correlated inversely with the response to treatment. The probability of obtaining a complete response to chemotherapy was significantly lower (P = 0.021) in patients with elevated HER2 ECD levels (0%; 95% confidence interval, 0-13%) compared with patients with nonelevated HER2 (26%; 95% confidence interval, 12-45%). In addition, the duration of clinical response was significantly shorter in patients with elevated HER2 ECD, compared with the cases with nonelevated HER2 (7.5 versus 11 months; P = 0.035). In conclusion, elevated levels of circulating HER2 ECD in patients with metastatic breast cancer correlate with reduced efficacy of a paclitaxel-doxorubicin chemotherapy combination. We suggest that the poor response rate associated with HER2 expression in advanced breast cancer may not be reversed by aggressive chemotherapy alone.

Self-sampling versus physician-sampling for human papillomavirus testing.

We evaluated the detection of human papillomavirus (HPV) infection using two sampling methods of cervical exfoliated cells, consisting of self-sampling of vaginal cells and cervical sampling performed by the physician. Women included were 379 patients of the general population attending outpatient clinics in Northern Greece for routine cytological cervical dysplasia screening. HPV DNA detection was similar with both sampling techniques. The HPV prevalences in self-collected samples were 4.7% and 3.7% in the physician-collected samples (P>0.05). The Kappa statistic for HPV DNA agreement between the two methods was 0.54 (95% Confidence interval = 0.33-0.75). Self-sampling of cervico-vaginal exfoliated cells could be used as an alternative option to test for HPV infection.

Evaluation of in vitro bromodeoxyuridine labeling of breast carcinomas with the use of a commercial kit.

The prognostic significance of S-phase fraction analyses of breast carcinomas has been reported by several investigators. The Cell Proliferation Kit (Amersham Corporation, Arlington Heights, IL), which uses in vitro bromodeoxyuridine (BRDU) labeling to evaluate cell cycle kinetics without a flow cytometer or radioisotopes, simplifies this assay for the clinical-based laboratory by providing standardized reagents and recommended methods. This study was performed to determine whether data derived from its use were comparable to published S-phase data from the use of thymidine labeling, BRDU, or other methods on breast carcinomas. Primary infiltrating ductal carcinomas (n = 142) and primary lobular carcinomas (n = 6) showed mean and median BRDU labeling of 4.63% and 3%, 1.3% and 1%, respectively, with a range of 0-28%. Benign lesions always had less than 3% BRDU uptake (n = 21). Estrogen receptor (ER) and progesterone receptor (PR) status correlated with BRDU labeling (P less than 0.05), with the highest S-phase fractions in ER- and PR-negative tumors. Correlations between BRDU uptake and histologic tumor type or size were observed. Significant correlations between BRDU uptake and lymph node status, patient age, or histologic tumor grade were not observed. S-phase studies of breast carcinomas using other techniques have shown similar data, therefore, the Cell Proliferation Kit appears to be a practical and useful method for in vitro S-phase analysis that allows concomitant histologic examination of the same tumor tissue sample.

In vitro bromodeoxyuridine labeling of malignant neoplasms. A comparative study with flow cytometry cell-cycle analysis.

Proliferative fraction has been defined as an independent prognostic marker for some malignant neoplasms. An estimate of the proliferative fraction can be obtained by cell-cycle analysis of flow cytometric DNA measurements. However, overlapping DNA distributions of aneuploid neoplasms are difficult to analyze, even with sophisticated computer programs, and results are not always reproducible. Bromodeoxyuridine (BrdUrd) labeling followed by immunohistochemical detection has been proposed as an alternative, simple, and accurate method for identifying and counting DNA synthesizing cells. In vitro BrdUrd labeling was performed on 87 tumors, including 35 lung cancers, 25 breast carcinomas, and 27 tumors of other origin. Results were compared with flow cytometric S-phase estimates in 46 cases. Mean BrdUrd labeling of lung tumors was 8.5% +/- 5.2%, compared with a mean flow cytometric S-phase fraction of 20% +/- 18.4%. Mean BrdUrd labeling of breast carcinomas was 6.8% +/- 3.8%, compared with a mean flow cytometric S-phase fraction of 12.5% +/- 9.9%. In both tumor types, BrdUrd labeling correlated well with histologic grade. Correlation between BrdUrd labeling and flow cytometric S-phase estimates were generally poor, particularly with aneuploid tumors.

Cervical human papillomavirus infection in women attending gynaecological outpatient clinics in northern Greece.

Human papillomavirus (HPV) is the necessary cause for the development of invasive cervical cancer. Identification of HPV determinants may contribute to the targeting of high-risk groups for cervical cancer. The study was aimed at estimating HPV prevalence and its determinants among 1296 women attending six gynaecological outpatient clinics in northern Greece. Information was available through personal interview and the study of cervical exfoliated cells. HPV DNA was detected by reverse line-blot polymerase chain reaction using the L1 primers PGMY09/11. The overall HPV prevalence was 2.5%. After controlling for potential confounders, the two independent risk factors associated with an increased prevalence were young age and parity. The prevalence odds ratio (POR) for those younger than 27 years against those older than 42 years was 5.31 (95% confidence interval (CI)=1.53-18.44) and the POR for nulliparous women compared with women with two or more children was 4.15 (95% CI=1.35-12.76). HPV was present in 10 of 12 women with low-grade cervical intraepithelial lesions (CIN) (83.3%) and in 3 of 4 with high-grade CIN (75%). The prevalence of genital HPV infections in the study population was among the lowest ever reported internationally.

[Human papillomavirus and human immunodeficiency virus infections as risk factors for cervix cancer in women prisoners]. / Infección por los virus del papiloma humano y de la inmunodeficiencia humana como factores de riesgo para el cáncer de cuello uterino en mujeres reclusas.

BACKGROUND: The identification of high risk groups for genital human papillomavirus (HPV) infection may contribute to cervical cancer prevention. The study was designed to estimate the prevalence of HPV infection and the related risk of cervical cancer among imprisoned women. PATIENTS AND METHODS: 157 women were visited at the Medical Office of a prison in Barcelona, Spain. Women underwent a structured interview, determination of HIV serostatus and detection of HPV cervical infection by means of PCR. RESULTS: The prevalence of HPV infection was 46%. Prostitution was reported by 38.2% and intravenous drug use by 64.3%. HIV infection was detected in 56.1%. Cervical cytology revealed 19 women with ASCUS and 28 with squamous intraepithelial lesions (SIL) (all grades). HPV infection was associated with an increased risk of intravenous drug use for more than 10 years (prevalence odds ratio [POR] = 2.9) and seropositivity to HIV (POR = 4.7). The increase in risk for SIL related to HIV was explained by the presence of HPV. HIV positive women with low CD4 counts may increase the risk for SIL independently of HPV. CONCLUSION: HIV positive women are at high risk for HPV infection and as a consequence, for developing SIL. HIV positive women should be closely monitored for cervical cancer.

[Laryngeal reinnervation in the dog]. / Reinervación laríngea en el perro.

In a group of 7 operated dogs, atrophy of the laryngeal musculature begins 3-4 weeks after recurrent nerve division. The atrophy increases progressively as time runs out, so that at the ninth week (last control done) the degree of atrophy of the posterior cricoarytenoid muscle is well marked as compared with that of the initial stage (4-5 weeks). In another set of 8 dogs reinnervation of this muscle was successful in 5 animals. In some of them up to 36 months elapsed after the cutting of the recurrent nerve and 14 weeks after application of a neuromuscular graft (composed of the hypoglossal ansa and a sheet of sternothyroid muscle). Histologic recuperation was good in all cases, but in only 2 of them the motility of the vocal cord at stimulation of the branch of the nerve was effective. The conclusion drawn is that histologic regeneration does now always parallel the functional one. Nevertheless, it seems possible that the elapsing of more time should be necessary in order to restore motility. This point demands new and broader experience before a positive answer can be given.

Human beta papillomavirus DNA study in primary cutaneous squamous cell carcinomas and their corresponding metastases.

The association between beta human papillomavirus (HPV) types and cutaneous squamous cell carcinomas (cSCCs) is controversial. Several studies have found such an association, especially at early stages of carcinogenesis, but the presence of beta HPV types in aggressive cSCCs has only been reported in three patients previously. We aimed to search for beta HPV DNA in primary cSCCs and their corresponding lymph node metastases in a series of patients. The presence of DNA from 25 beta HPV types was determined using a multiplex PCR protocol in 35 primary cSCCs from 35 patients and their corresponding lymph node metastases. DNA from beta HPV types was detected in 9 % of primary cSCCs and in 13 % of metastases. No primary cutaneous SCC or lymphatic metastases were found to share the same HPV DNA. These data suggest that beta HPV types do not play an etiopathogenic role in advanced stages of squamous cell carcinogenesis.

Urine human papillomavirus prevalence in women with high-grade cervical lesions.

OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) in urine samples from women with high-grade cervical lesions. Secondary objectives are to identify the influence of socio-demographic factors and the different genotypes with urinary HPV positivity. STUDY DESIGN: 75 women with a positive biopsy for CIN2+ were included in the study from October 2010 to July 2011. A sample of urine was collected immediately before conization at the outpatient clinic. We analyzed the presence of HPV using a PCR technique. RESULTS: The mean age of the patients was 34.8 years (range 24 to 61). All patients had histological CIN2+, of whom 54.67% had CIN3. The prevalence of HPV in urine test was 58.82% in CIN2 population versus 78.05% in CIN3 patients (p 0.072). 31 different genotypes were found. The most frequent HPV genotype was 16-HPV, which was identified in 58% of women with positive HPV-DNA in urine samples. No demographic characteristics were significantly associated to urinary HPV prevalence. CONCLUSION: Most of the patients with CIN2+ showed positive results for urine HPV test. The prevalence of positive urinary HPV test was higher for patients with CIN3. HPV urine detection could be considered as an acceptable option for high-risk population who skip regular screening programs.

Human papillomavirus testing for primary screening in women at low risk of developing cervical cancer. The Greek experience.

OBJECTIVE: To compare the performance of human papillomavirus (HPV) DNA detection against routine Papanicolaou smear for the detection of low- and high-grade cervical intraepithelial neoplasia in a low-risk population. MATERIALS AND METHODS: A cross-sectional study was performed involving 1296 women attending six outpatient clinics in Northern Greece (Thessaloniki, Thermi, Mihaniona, Corfu, Veria, and Serres). Women underwent a gynecological examination, including collection of exfoliated cervical cells for Papanicolaou cytology and HPV DNA detection. Cytology was processed according the conventional routine manner, and HPV DNA was determined using the polymerase chain reaction technique. In positive cases of either method, a complete colposcopic evaluation was performed with directed biopsies. Tests (HPV DNA, cytology, and colposcopy) performance characteristics were determined using the histopathologic diagnosis as the reference standard. RESULTS: HPV DNA testing showed a significantly better sensitivity than the Papanicolaou smear in detecting cervical intraepithelial neoplasia (75% versus 50% for high-grade lesions and 81.2% versus 50% for lesions of any grade, respectively). Specificity, and positive and negative predictive values did not significantly differ. Even after dividing women in younger or older than 30 years, the sensitivity of the HPV DNA test was greater than cytology (100% and 70% versus 50% for cytology in both groups, respectively), with a 6.3% loss in specificity when performed in women younger than 30 years. CONCLUSION: HPV testing could be useful in screening women at low risk for cervical cancer, either as an adjunct tool to augment existing cytology programs or as a unique test of its own.

Estrogen receptors in skin appendage tumors and extramammary Paget's disease.

Determination of estrogen receptors (ER) in breast carcinoma is valuable in the management of patients. However, little is known about the presence of these receptors in other tumors. Normal skin appendages and their neoplasms, including extramammary Paget's disease (EPD), might be expected to express ER since the breast is histogenetically related to sweat glands. In this study, 41 cases of skin appendage tumors (SAT) and 11 cases of EPD were stained using the ER-ICA monoclonal kit (Abbott, Chicago, IL) with a modified technique for paraffin-embedded sections. Controls included 10 biopsies of primary breast carcinoma and 4 cases of metastatic breast carcinoma to skin, all positive for ER. None of the samples of SAT or EPD showed staining for ER. Normal skin appendages were also negative. Normal vaginal epithelium in one case of EPD showed positive nuclear staining for ER. ER determination using immunohistochemical technique in paraffin-embedded sections may be useful in the differential diagnosis between malignant SAT and metastatic breast carcinoma in the skin. The absence of ER in normal skin appendages suggests that its apparition is a feature of specialized differentiation of breast epithelium.

Apoptosis in ductal carcinoma in situ of the breast.

Programmed cell death (apoptosis) may play a role in tumor development and progression. The importance of apoptosis dysregulations in ductal carcinoma in situ (DCIS) and its relationships with p53 mutations and expression of bcl-2 has received little attention in the literature. In a series of 58 DCIS patients, we evaluated the number of apoptotic cells by the TUNEL technique in subgroups of DCIS and correlated it with immunohistochemical expression of hormone receptors, c-erbB-2, p53, bcl-2, and Ki-67 (MIB-1) and DNA content measured by image cytometry. High apoptotic index (greater than 3%) was related to high tumor grade, negative hormone receptors, c-erbB-2 overexpression, aneuploidy and lack of bcl-2 immunohistochemical stain. Apoptosis was not related to p53 or proliferative index. The findings are similar to those found in infiltrating breast cancer.