INK4a in squamous cell carcinoma (SCC) of the cervix by immunohistochemistry (IHC) and determine relation with clinico-pathological parameters. This study further explored the correlation of p16INK4a immunostaining with another proliferation marker, Ki-67 and to study if human papillomavirus (HPV) IHC can be used as a marker for detection of virus in high-grade dysplasia.Cervical cancer is the fourth most common cancer in women worldwide with highest incidence reported in Eastern Africa in 2012. The primary goal of this study was to study the expression of p16INK4a, anti-Ki-67 and anti-HPV antibodies using automated immunohistochemistry platform (ASLink 48-DAKO).A total of 90 samples, diagnosed for cervical cancer, were included in the study. Fixed Paraffin Embedded (FFPE) tissue sections were stained with anti-p16INK4a positivity was found to be highest in SCC  than other HPV tumors . The majority of cases (97.4%) were p16INK4a positive in the age group 41–60 years. In addition, a statistically significant difference between p16INK4a and HPV was observed among total cervical tumor cases and SCC cases.Immunohistochemical protein expression of p16INK4a IHC results showed positive correlation in SCC and other cervical tumors.As expected staining of invasive cervical cancer with anti-HPV showed rare positivity because HPV heralds active infection in dysplastic lesions and not of frank cervical carcinoma. In contrast, anti-p16The online version of this article (doi:10.1186/s13027-017-0159-0) contains supplementary material, which is available to authorized users. Cervical cancer continues to be a major public health problem in less developed regions of the world and ranked at fourth position among various cancers in women with estimated age standardized rates over 30 per 100, 000 women . It has HPV mediated cervical carcinogenesis is a process divided into two stages: productive and transforming infections. Productive infections are short-term lesions, which regress spontaneously, however transforming infections with certain high risk-HPV (HR-HPV) progress to invasive cancer gradually (20–30 years) if not followed up. Histomorphological types of cervical cancers include squamous cell carcinoma (SCC), adenocarcinomas and neuroendocrine carcinomas. SCC represents the majority of cases diagnosed (80%) while the remainder cases are adenocarcinomas and other types , 13.A previously published study from Sudan has reported that women are diagnosed in late stages with aggressive disease emphasizing lack of efficient screening tests . To faciINK4a, which has demonstrated to be highly sensitive and specific marker of high-grade squamous and glandular neoplasia of the cervix due to its overexpression in cancerous and precancerous cervical lesions [INK4a inhibits the phosphorylation of pRb mediated through cyclin dependent kinases and regulating mitotic transition of cell cycle from G1 to S phase [INK4a in the basal and parabasal epithelial cells of HPV transformed lesions [INK4a over-expression using immunohistochemistry (IHC) is linked with increased and disease free survival hence proving to be a marker for prognosis [INK4a is over-expressed [Among the various biomarkers established to facilitate early screening is the cell cycle regulatory protein known as p16 lesions , 15. Mor S phase . Alterat lesions . A recenrognosis . Studiesxpressed .INK4a is Ki-67. HPV infected cells/lesions that overexpressed p16INK4a, as a response to viral oncogenes will concomitantly express Ki-67 [INK4a and Ki-67 in both SCC and adenocarcinoma (AC) can be a synergistic approach to quantitate each marker at a cellular level in tumor regions instead of the benign proliferative zones, thereby increasing diagnostic accuracy in both immunohistochemistry studies [In addition, cellular proliferation marker that could be used in conjunction with p16ss Ki-67 . Severalss Ki-67 , 22. Locss Ki-67 . Increasss Ki-67 . In addi studies .INK4a protein in SCC of the cervix by IHC and relate the findings with clinico-pathological characteristics, correlate p16INK4a overexpression with proliferation marker Ki-67 and validate that anti-HPV immunostaining is not suitable for detecting presence of virus in high grade invasive carcinoma.The present study aimed to determine the expression of p16The Joint Institutional Review Board (JIRB) of Weill Cornell Medicine - Qatar (WCM-Q) and Hamad Medical Corporation (HMC) Research Office, Qatar has approved the present study . This is a retrospective cohort study with a total of 90 formalin-fixed paraffin-embedded (FFPE) cervical carcinoma tumors obtained from patients diagnosed at the pathology laboratory at the National Health Laboratory (NHL), Sudan between years 2004–2008. Clinicopathologic information such as age, tumor differentiation and diagnosis was obtained when available.. H&E stained slides were examined for histopathologic diagnosis by experienced pathologist to record morphological alterations such as presence of koilocytotic cells, extent of keratinization and significant differentiation in the squamous epithelial cells (dysplasia).All cervical biopsies were fixed in 10% neutral buffered formalin, dehydrated and paraffin embedded. FFPE specimens were sectioned on microtome (4 μm thickness) for both Hematoxylin & Eosin (H&E) staining and immunostainingINK4a , anti-HPV  and anti-human Ki-67 . Briefly, after antigen retrieval the fixed tissue sections were blocked and incubated with primary antibodies for 20 min, washed, and secondary antibody coupled to HRP was added. The reaction was developed using DAB Chromogen  and counterstained with Hematoxylin stain following dehydration in ethanol. Positive and negative tissue controls such as tonsil for p16INK4a, Ki-67 and human condyloma tissue for HPV immunostaining were included in each run to validate the stain localization whereas the negative control was the same tissue section omitting primary antibody.Immunostaining of FFPE tissue sections was performed using the Envision Flex™ protocol on ASLink48 , as described previously . The antINK4a positive. Depending on the distribution of the stain in the epithelium, positivity is scored as strong diffuse or high intensity (3+), strong focal or moderate intensity (2+) and weak sporadic or mild (1+). For anti-HPV, positive staining in the nuclei of infected squamous cells and occasional staining of cytoplasm in koilocytotic & dysplastic epithelial cells was observed. All the cases were scored as positive or negative based if protein expression of HPV capsid protein (VP1) was observed. In case of anti-Ki-67 antigen, depending on the intensity and proportion of mitotic cells in the basal and parabasal cells of the squamous epithelium, scores were assigned as follows: <10- Negative, 10–30 cells- 1+, 40–60 cells- 2+, 70–100- 3+.Scoring of immunostained tumor sections was performed by an experienced pathologist. Strong diffuse nuclear as well as cytoplasmic staining in the basal layers of the squamous epithelium is considered p16INK4a and HPV immunostaining were compared using McNemar’s test and positive predictive value (PPV) and negative predictive value (NPV) were recorded. Two tailed test were used for analysis and p-value <0.05 was considered significant. The SPSS statistical package (IBM SPSS version 22.0.) was used for the statistical analysis.Immunohistochemical intensity for each of the three markers irrespective of the score was designated as positive for presence and negative if absent. Results were presented using frequencies and percentages. Comparison between different groups was performed using ANOVA test, followed by Student’s t-test. Sensitivity and specificity between p16n = 77) of women were diagnosed with squamous cell carcinoma (SCC) and 14.4% (n = 13) of other HPV associated tumors. In addition, tumor grade, which is the histopathologic differentiation of cancerous tissue, were classified into well differentiated, moderately and poorly differentiated. The age range among the studied population was 27–80 years with a mean age of 54.2 (±13.5) years. Furthermore, the individuals were categorized into 3 age groups , in which majority (52.1%) of women were aged 41–60 years. However, age was not reported for seven patients in the study (Table n = 53) subtypes, 26% (n = 20) were keratinizing carcinomas. Data were not available for four SCC cases  compared to other HPV tumors, 76.9% (n = 10)  , respectively  and SCC cases alone (p = 0.049) however; no significant difference was noted in other tumor cases (p = 0.219) (Table INK4a immunostaining.Immunohistochemistry (IHC) of FFPE tissue samples was performed using anti-p16ies Fig. . Overexp49) Fig. . Howeverely Fig. . Addition = 62) cases (Table n = 55) were found to be positive for Ki-67 immunostaining  however, no significant association was found with SCC cases , however, no association with age group was seen in Ki-67 and HPV immunostaining  showed positive p16INK4a expression, which is in agreement with previous studies [INK4a overexpression was not significantly associated with tumor grade nor tumor size, however p16INK4a overexpression was highly associated with better prognosis and increased disease-free survival which, corroborates with previous studies [n = 7) of SCC cases and 23.1% (n = 3) of AC cases were p16INK4a negative which has also been reported in previous studies [INK4a expression in the cervical tumors does not indicate lack of HR- HPV or improper IHC technique but rather implicated due to silencing of the gene through mutations in the promoter regions resulting in transcription failure, epigenetic mechanisms and hyper methylation.Correlating p16 studies , 29, 30. studies , reports studies , 32. Add studies , 34, 35.INK4a and HPV positive expression was seen in age group between 41 and 60 years . The positive correlation of HPV expression with p16INK4a intensity and dispersion on cervical carcinoma cases was reported in a study in Saudi Arabia [INK4a, but negative for HPV-1 derived capsid protein (VP1). The majority of cervical cancers does not have active dysplasia and therefore are limited in protein expression of HR-HPV.Molecular assays are the most sensitive for HPV detection in FFPE samples than IHC . In the i Arabia . HoweverINK4a expression was compared with a cellular proliferation marker, Ki-67. To date, several studies have shown co-expression of p16INK4a and Ki-67 when combined concurrently improve diagnostic accuracy and that Ki-67 expression increases linearly with tumor grade in SCC [INK4a and Ki-67 positive immunostaining when assessed individually was higher in the age group >40 and 6.6% (n = 4) were both positive for p16INK4a and Ki-67. Several studies have shown that both p16INK4a and Ki-67 proteins are co-expressed in most high-grade squamous lesions, which is also observed in this study. Ki-67 is employed as an objective marker indicating aberrant cellular proliferation that facilitates measurement of advanced disease end point, which is progress of CIN to carcinoma. Hence, direct proportionality in Ki-67 expression in relation to tumor grade along with p16INK4a is involved functionally in the process of HPV induced transformation and overexpressed in the epithelium [To increase the reproducibility of diagnosis, p16e in SCC , 34, 36.ithelium , 36. Furithelium  and alsoithelium .INK4a and Ki-67 with respect to HPV immunohistochemical protein expression, which is in agreement with previous report [In the present study, no correlation was found between p16s report . HPV dets report . In conts report .INK4a is diffusely expressed in ~60% of CIN1 and mostly associated with HR-HPV genotypes, but HR-HPV presence was detected in p16INK4a negative tumors [INK4a overexpression is already indicative of advanced viral interference progressing towards invasive cancer where aside from active viral replication there is significant morphological change occurring at histology, cellular and molecular level which can be visually scored. However, in invasive cervical cancer, the HPV protein expression was not detectable by IHC and HPV genotyping in FFPE tumor samples.Based on the tumor grade, it has been shown that in low-grade lesions, p16e tumors , 39. In Currently, in Sudan precancerous cervical lesions are visually detected by VIA –10 unlikINK4a-E6H4 has been recommended specific antibody for exfoliated dysplastic cells both in histology tissue sections and cytology smears and that normal cervical epithelium, inflammatory or metaplastic lesions were not stained [The strength of the present study lies in the use of IHC technique to study immunochemical staining along with H&E stain of SCC tumors for histopathology assessment. The monoclonal antibody clone used for p16 stained .INK4a varies between pathologists, presenting a challenge as no standard parameters. Limited sample number for Ki-67 hence proper correlation with p16INK4a in SCC cases could not be made. Larger cohort with proper diagnostic, survival data can help to correlate other clinical parameters.Limitations of this study are primarily the tissue quality, preservation and fixation of FFPE samples were not suitable to carry out analysis for HPV genotyping study. In addition, cut-off threshold for immune-positivity of p16INK4a overexpression in squamous cell carcinoma was significant and differs from expression in other cervical tumors (AC). The immunostaining of p16INK4a and HPV vary in the total sample positive and negative reaction, indicating that p16INK4a is an effective way to detect invasive carcinoma than HPV. Proliferation marker, Ki-67 showed significant correlation with p16INK4a in other HPV associated tumors. Lastly, significant correlation of p16INK4a percentage frequency in age group 41–60 years where active advance transforming CIN occurs concludes that it is an appropriate surrogate marker to help in early screening of cervical neoplasia.In this preliminary study, we evaluate the clinical utility of p16INK4a as a surrogate marker using IHC technique in invasive cervical carcinoma among women in Sudan. Overall, p16


Veterans with purely physical disorders, such as ankle-foot neuromusculoskeletal disorders, are often neglected in psychological assessments because mental health evaluations are usually focused on those with a psychological disturbance or with a high percentage of injury. The purpose of this study was to evaluate the psychological condition of veterans with ankle-foot neuromusculoskeletal disorders.A cross-sectional study was performed between 2014 and 2016 on veterans with war-related ankle-foot injuries living in two provinces of Iran. An information form for demographic data and injury-related factors was used. Additionally, the previously validated Persian version of the Symptom Checklist-90-Revision (SCL-90-R) questionnaire was used for data collection.P = 0.005), had an associated injury (P = 0.002), and had a history of hospitalization within the past 12 months for reasons other than their injury (P = 0.035).The respondents were 215 male veterans with a mean age of 51.7 ± 7.5 years. The most common mental health problems were observed for the somatization (24.7%), obsessions-compulsions (14.4%), and anxiety (12.6%). Based on the Global Severity Index (GSI), 48.6% of individuals had a possible psychiatric/psychological illness. According to the multivariate regression analysis, GSI scores were significantly higher among veterans who were older than 27 years at the time of injury (Approximately half of the combat veterans with ankle-foot neuromusculoskeletal disorders likely had psychological problems. The evaluation of the patterns and predicting factors of psychological conditions may inform strategic planning efforts and decision-making, which, in turn, may provide a better quality of life for veterans. Further studies that utilize longitudinal designs are needed to evaluate and compare the psychological status of different groups of veterans and other groups in the general population. Approximately 70% of all battlefield damages are musculoskeletal injuries, which are associated with a high morbidity despite its low mortality rate , 2. AbouAfter nearly three decades of the Iran-Iraq war, there are more than 500,000 Iranian veterans registered with the Veterans and Martyrs Affairs Foundation (VMAF) –9.Studies in different countries have found high rates of mental disorders among veterans, especially posttraumatic stress disorder (PTSD) and depression . There aWe conducted a cross-sectional, descriptive survey between 2014 and 2016. The statistical population consisted of veterans with ankle-foot neuromusculoskeletal disorders registered in the local branch of the VMAF in two provinces of Iran including Zanjan and Markazi [The study protocol was approved by the ethics committees of Janbazan Medical and Engineering Research Center (JMERC) and performed in accordance with the Declaration of Helsinki and its subsequent revisions. All participants were informed about the voluntary nature of their participation and the aims of the survey. Verbal informed consent was obtained from each participant.The first part of data collection was accomplished using an information form developed for the study that assessed demographic data such as age, gender, marital status (and number of children), education level, occupation, economic level, percentage of disability, and history of hospitalization within the past 12 months. These data were obtained through 10 min-interviews with the participants.The second part of data collection assessed information on mental health status, obtained using the Persian version of SCL-90-R, which is a multi-dimensional self-reported symptom inventory designed to measure current psychological symptom status. This assessment requires approximately 12–20 min to administer. It consists of 90 items on nine primary symptom dimensions, including somatization, obsessions-compulsions, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. Each of the dimensions is assessed by 6–13 items scored on a Likert-type scale, with response options for each item ranging from zero (“never”) to four (“very often”). The overall score for each dimension is the mean score of all items on the subscale and directly reflects the severity of the mental health problem. According to previous studies, subscale scores ≥2 were suggestive of potential mental health issues , 16. TheP < 0.05 was considered significant. The analyses were conducted using SPSS software, version 20.0 .Quantitative data with normal distributions are presented as the means ± standard deviations (SDs), and categorical variables are shown as frequencies and proportions. A two-tailed Student’s t-test or ANOVA was used after establishing the normal distribution of data and homogeneity of variances for continuous values. Post hoc analysis was used to compare the means across the study groups. A multiple linear stepwise regression analysis was performed to evaluate the independent contribution of demographic characteristics and injury-related factors to GSI scores. A two-tailed alpha with In total, 215 veterans participated and provided an acceptable and reliable response to the questionnaire; thus the response rate was 54.7% in our study. Since all Iranian participants in the battlefields of the Iran-Iraq war were male, all veterans were men with a mean age of 51.7 ± 7.5 (range: 29–92) years. The average percentage of injury (disability rating) was 32.8 ± 13.7% (range: 5%–70%) among participating veterans. The mean age at the time of injury was 27.9 ± 3.3 (range: 15–35) years. In total, 156 (72.6%) had an associated injury other than ankle-foot neuromusculoskeletal problems. The associated injuries included psychological problems, chemical injuries, and spinal cord damage in 49 (22.8%), 19 (8.8%), and 6 (2.8%) individuals, respectively. Additionally, other associated physical injuries  were seen in 125 (58.6%) veterans. More detailed demographic characteristics of our study population have been published elsewhere .P = 0.045; Table P = 0.038), interpersonal sensitivity (P = 0.043), and depression (P = 0.045), with higher scores seen among participants in the “less than diploma” group. Veterans who had more than four children had a higher degree of somatization (P = 0.041), interpersonal sensitivity (P = 0.027), depression (P = 0.041), and anxiety (P = 0.008). Post hoc analysis indicated a significant increase in the somatization subscale among retired individuals compared with those who were government employees (P = 0.013).Tables P = 0.017), “presence of an associated injury” (P = 0.022), “presence of abdominal injury” (P = 0.033), “presence of a limb disability” (P < 0.001), “hospitalization for the reason of injury” (P = 0.016), and “hospitalization for other reasons” (P = 0.039). Additionally, head injury was significantly associated with lower GSI scores (P = 0.039). Among the damage-related factors, disability percentage of less than 30% was significantly associated with higher scores on the subscales for obsessions-compulsions (P = 0.018), interpersonal sensitivity (P = 0.027), and phobic anxiety (P = 0.005). Age at the time of injury >27 years was related to higher scores on the subscales for interpersonal sensitivity (P = 0.007), depression (P = 0.002), anxiety (P = 0.043), and paranoid ideation (P = 0.006). All subscale scores were significantly higher in the presence of limb disability (P < 0.05). Having an associated injury in addition to ankle-foot neuromusculoskeletal problems was related to higher somatization (P = 0.006), anxiety (P = 0.007), and hostility (P = 0.003) scores. Injuries to different parts of the body were also associated with either higher or lower scores on the psychological subscales  individuals with possible psychiatric/psychological illness within the study sample. Table P = 0.005), “presence of an associated injury” (P = 0.002), and “hospitalization due to reasons other than the injury” (P = 0.035).A multivariate regression analysis showed that significant injury-related factors for higher GSI scores included “age at the time of injury”  among veterans may lead to depression , 28. MorAdditionally, veterans with associated injuries (other than ankle-foot problems) represented a large category, with several factors related to the severity of psychological distress. Although psychological problems  were a prevalent associated injury in our study population and may directly influence SCL-90-R scores or lead to hospitalizations, veterans with chemical injuries and other associated physical damages should receive more attention in mental health services, and interventions for prevention or treatment of affected veterans should be implemented.One of the limitations of this study was its cross-sectional design, which should be enhanced by following these veterans in a longitudinal study to evaluate changes in their mental health over time and the effects of interventions . Another limitation of our study was the response rate of 54.7%. While this rate may be excellent for other descriptive studies, our research evaluated mental health status. Thus, the current response rate may lead to an under estimation of prevalence by masking the veterans whose illness and its severity  did not permit them to participate in the survey. The third limitation was that the study included veterans in only two provinces of Iran. Veterans living in the other 29 provinces, especially in Iran-Iraq border provinces, who may have more severe problems due to their proximity to conflict zones and suffer from injuries in events such as land mine accidents, were not included in the study.Combat veterans with ankle-foot neuromusculoskeletal problems may be a neglected group in mental health assessments. Psychological problems may lead to a lower quality of life in this population. However, evaluation of the patterns and predicting factors of these psychological problems may support the health services and other relevant entities in strategic planning and decision-making, which may eventually provide a better quality of life for them. It is recommended that veterans with higher scores on the SCL-90-R subscales undergo complementary assessments and current diagnostic systems for mental health, such as the Diagnostic and Statistical Manual of Mental Disorders (DSM-V); using this approach, we can better detect the veterans who may need treatment or intervention. Furthermore, mental health screening using the SCL-90-R can be recommended as part of the routine evaluation of all medical and health-related affairs involving veterans to better predict and detect potential psychological problems in this population and to implement strategies to prevent the development of psychological disorders. Further studies that utilize longitudinal designs are needed to evaluate and compare the psychological status of different groups of veterans as well as groups in the general population.


Even if vulvar cancer is not common, over one hundred women are affected in Austria per year. There is strong evidence that basaloid and warty variants are associated with types of human papillomavirus (HPV).INK4a was analyzed by immunohistochemistry . A tumor was considered HPV-driven if an overexpression of p16INK4a was detected.The aim of this study is to analyze the types of HPV in vulvar cancer in Austria. This cross-sectional period-prevalence international collaborative study on archival specimens was performed in cooperation with the Institut Catalan di Oncologia in Barcelona, Spain. A total of 177 consecutive samples of Austrian women were analyzed to detect the presence of various HPV types using the SPF10 PCR/DEIA/LiPA25 system. Furthermore, the expression of the tumor suppressor protein p16p = 0.021). In addition, 77.4% of all cases suffering from warty or basaloid squamous cell cancer tested positive for HPV, compared to 9.5% of the keratinizing squamous cell cancer cases (p < 0.001). The most commonly detected HPV strain was type 16, followed by 31 and 33.In all, 41 cases of vulvar cancer tested positive for HPV DNA (23%) and 32 (18%) were p16 positive. Patients with warty and basaloid squamous cell cancer were significantly younger than those with keratinizing squamous cell cancer (63.3 years vs. 71.0 years, Infection with HPV type 16 appears to be strongly correlated to the development of warty or basaloid squamous cell cancer. Vaccination against HPV can be expected to prevent this type of vulvar cancer. In Europe, the incidence of vulvar cancer has ranged between 2.9 and 4.1 cases per 100,000 women over the last few decades. Due to the fact that this disease mostly affects women above the age of 60, this rate is age-adjusted. In Austria, the number of newly diagnosed cases per year has been between 114 and 163 in the period from 1990 to 2010. Vulvar cancer is thus a rare disease [Over the past few decades, the incidence rates of vulvar intraepithelial neoplasia (VIN) and invasive vulvar cancer (IVC) have both been reported to have increased in Austria, in other European countries and worldwide, particularly among younger women –6. SquamINK4a in cancers of the vulva.We present the Austrian data of an international collaborative study to evaluate the HPV contribution and genotype distribution in vulvar lesions from pathological archives in 39 countries from five continents . The priINK4A) that is reported to be overexpressed in at least 90% of VIN- and HPV-related IVC cases [The cross-sectional period-prevalence study on archival specimens included a common standard protocol and a sensitive assay was used for HPV-DNA detection (SPF10/DEIA/LiPA25 system). IVC cases were further tested for the cyclin-dependent kinase-4 inhibitor (p16VC cases .INK4A was performed following manufacturer’s instructions (CINtec® PLUS Cytology Kit by Roche) in the invasive vulvar cancer cases. A case was considered to be positive if more than 25% of invasive cancer cells showed a diffuse overexpression. External audit from the reference laboratory and from the Steering Committee of the Study was done periodically. A committee of pathologists monitored the study periodically. The protocol was approved by local and the ICO ethics committees [A total of 204 consecutive samples was sent to the study center at the Institut Catalan di Oncologia (ICO) at Barcelona, Spain. Of those, 16 were submitted as control specimens according to the protocol. The remaining 188 cases of invasive vulvar cancers were conserved in paraffin blocks at the Medical University of Vienna between 1994 and 2010. The tissue material was re-embedded at the ICO if the paraffin block did not satisfy the requirements of the study. Microtome sectioning of the specimens under noncontamination conditions and sandwich technique were carried out to confirm an optimal number of sections to be used for DNA extraction, HPV-DNA detection, and HPV genotyping. First and last sections were hematoxylin–eosin stained for pathological review and the in-between sections were used for the HPV-DNA detection. All cases were reviewed by a trained pathologist at the ICO to assess the diagnosis and quality of the specimen before HPV testing. A case was considered suitable for HPV-DNA testing when tumoral cells were found in the two hematoxylin–eosin slides. Cases difficult to classify, cases with a dissenting diagnosis compared to the original one and all the rare histological types were further reviewed by two senior expert pathologists at the ICO. DNA was extracted under noncontamination protocols and aliquoted. HPV testing was performed on each specimen using the SPF-10 broad spectrum primers PCR followed by DNA enzyme immunoassay (DEIA). HPV-DNA-positive samples were subsequently analyzed by SPF10 PCR/DEIA/LIPA25 , a reverse hybridization technique that detects 25 high-risk (HR) and low-risk (LR) HPV types . p16Of the 188 analyzed cases, 7 were excluded according to protocol and 4 cases were not verified as invasive disease. For this analysis, 177 cases of IVC remained. These can be divided into 31 cases of warty or basaloid squamous cell cancer (SCC) and 126 cases of keratinizing SCC, as well as 18 mixed and 2 other cases (one adenocarcinoma not otherwise specified and one basocellular carcinoma) (Table p = 0.021) (Table p = 0.002) . In addition, 38 cases of the 41 HPV-positive cases exhibited a single infection. The most common type was HPV 16 (31/38), followed by HPV 31, 33, and 44 (2/38 each). This distribution can also be observed when focusing only on cases of warty or basaloid SCC. Cases of keratinizing SCC could be linked to HPV 16 (8/10) and HPV 33 and 74 (1/10 each) (Table INK4a positive (Table INK4a. Among HPV-positive cases, it seems that keratinizing tumors are less p16INK4a positive than warty or basaloid ones. Finally, 88% of the p16INK4a-positive cases exhibited HPV 16 (Table Overall, 77% of all cases suffering from warty or basaloid SCC were HPV positive, compared to 10% of the keratinizing SCC cases (INK4a positivity was included in the criteria to consider a tumor to be HPV driven. p16INK4a is reported to be overexpressed in at least 90% of VIN- and HPV-related IVC cases [This analysis confirms the predominant contribution of HPV 16 to the etiology of HPV-positive vulvar cancer and suggests that other HPV types, such as HPV 33 and HPV 31, which are common in cervical cancer and precancer, also play a role in vulvar carcinogenesis although to a much lesser extent. In contrast to the global results, there was no case related to HPV 18 or 45 observed in the Austrian series. A major contribution of this study is that p16VC cases .INK4A could be a reflection of a transient infection with no role in carcinogenesis. The details of the methods have been published previously [HPV-DNA detected without over-expression of p16eviously .The presented data show an important inverse association of age with HPV prevalence in vulvar cancer tissue. HPV-positive cases with keratinizing vulvar carcinomas were significantly younger, whereas no significant age difference was observed in the other histological subtypes.INK4a positivity excluding a possible transient infection. This is in accordance with the report of vulvar intraepithelial and invasive neoplasms in Austria [INK4A positive [INK4a positive. Vaginal neoplasms are also dominated by HPV 16 [In all, 23% of our cases were HPV positive, 18% due to p16positive . In a rey HPV 16 . We alsoy HPV 16 , 16.To conclude, in Austria HPV contributed to approximately a quarter of invasive vulvar cancers. HPV 16 was present in about three-quarters of all HPV positive cases. HPV vaccines may reduce a quarter of IVC based on the reported efficacy of the trial , 18. The


The majority of patients diagnosed with oropharyngeal squamous cell cancer (OPSCC) are due to HPV infection. At present, there are no reliable tests for screening HPV in patients with OPSCC. The objective of this study was to assess the Cobas® HPV Test on oral rinse specimens as an early, non-invasive tool for HPV-related OPSCC.Oral rinse specimens were collected from 187 patients  and 81 control patients who had benign or malignant thyroid nodules) treated at MSKCC. The Cobas® HPV Test was used to detect 14 high-risk HPV types in these samples. Performance of the HPV Test was correlated with p16 tumor immunohistochemistry as gold standard.91.1% of the oropharynx cancer patients had p16 positive tumors compared to 3.3% of oral cavity cancer. Of the 81 control patients, 79 (97.5%) had no HPV in their oral rinse giving a specificity of the HPV test of 98%. For the combined oral cavity oropharynx cancer cohort, the sensitivity, specificity, positive predictive value and negative predictive value of the HPV Test were 79.1%, 90.5%, 85.0% and 86.4% respectively when p16 immunohistochemistry was used as the reference.The Cobas® HPV Test on oral rinse is a highly specific and potentially sensitive test for oropharyngeal cancer and may be a potentially useful screening test for early oropharyngeal cancer.We describe an oral rinse test for the detection of HPV related oropharyngeal cancer. The link between human papillomavirus (HPV) and oropharyngeal squamous cell carcinoma (OPSCC) has been well-documented over the past two decades –4. CurreExpectations are that the rate of HPV oropharynx cancer will increase in incidence over the next decade. The efficacy of current HPV vaccination practices at reducing the disease burden of oral HPV remains to be established but the rates of HPV-related OPSCC are eventually predicted to decline mirroring the decline of cervical cancer . Until tPrevious studies in Europe have suggested that an oral rinse test might be highly specific , 12 and Our study included 187 patients separated into three cohorts based on tumor site: there were 45 patients with OPSCC tumors, 61 with OCSCC tumors, and 81 patients in our normal cohort. Table Our normal cohort consisted of 81 patients who attended the head and neck clinic with either thyroid cancer or benign thyroid nodules. All patients had a comprehensive head and neck examination. This involved an examination of the oral cavity and oropharynx and either flexible or mirror laryngoscopy to examine the larynx, hypopharynx and base of tongue. All control patients had no clinical evidence for oral cavity cancer or oropharyngeal cancer. Of the 81 patients, 79 (97.5%) had no HPV in their oral rinse on PCR. Two patients were positive for HPV 16 but clinical examination showed no clinical evidence for tonsil, base of tongue cancer or oral cavity cancer. These results indicate the very high specificity (97.5%) of the Cobas® HPV Test for the detection of HPV with only 2 false positive results from 81 control patients studied.Our oral cavity cancer cohort consisted of 61 patients with the majority of patients with cancer of the oral tongue (n=40). 6 patients (9.8%) had a positive HPV oral rinse test and this was for HPV16. Of these 6 patients, 2 had oral tongue, 1 lower gum, 1 floor of mouth, 1 retromolar trigone and 1 buccal mucosa cancer. Two of the 6 patients with a positive oral rinse test were confirmed to have p16 positive tumors . The other 4 patients who had a positive HPV oral rinse test had negative p16 immunohistochemistry in the oral cavity tumors; these 4 patients are currently under follow up but none have developed an oropharyngeal cancer to date.The oropharynx cancer cohort consisted of 45 patients. Of these, 34 of 45 (76%) patients were positive for HPV16 or 18 in their oral rinse (33 HPV16 and 1 HPV18). Of the 34 patients, 32 had p16 positive tumors and 2 patients had p16 negative tumors. Of the 33 pts that were HPV16 positive in their oral rinse, 18 were also positive for another HPV virus serotype. In addition, of the 11 patients that were negative for HPV16/18 on oral rinse, 2 were positive for another HPV serotype.Table We also present these data with the oral cavity cohort removed, and then broken down into oropharynx subsite for comparison Table . The posThe patients selected for our oropharynx and oral cavity cohorts were not chosen based on any known information regarding their HPV status. However, p16 testing confirmed that the OPSCC population was 91.1% (41/45) positive for HPV, while our OCSCC population was only 3.3% (2/61) for HPV. Analysis showed these groups to be significantly different in the p16 status (p<0.001) which is consistent with the observed demographic differences in these two diseases over the past 2 decades, where HPV has become a predominant cause of OPSCC . AdditioThe purpose of this project was to evaluate the potential for an oral rinse test as an early detection tool in head and neck squamous cell carcinoma. Our analysis proved this test to be fairly sensitive (79.1%) and highly specific (90.5%) with a PPV of 85.0% and NPV of 86.4%. This suggests that the Cobas® HPV Test has value in characterizing lesions of the oropharynx or oral cavity. The test was particularly sensitive to HPV-related cancers of the tonsil (86.4%) with a PPV of 100%. However, the results were less consistent for tumors of the base of tongue . The fact that these HPV markers were more likely to be detected in tonsil cancer than base of tongue cancer may be a result of the anatomy of the oropharynx. Portions of the tonsils are more likely to have contact with an oral rinse collection when a patient is asked to gargle and expectorate saline solution as in this study. Additionally, the number of viral copies of HPV has been shown to be greater in cancers of the tonsil than other sites . PreviouThis study and others like it , 12, sugA recent study has also suggested that the oral rinse test could be used as a post treatment prognostic indicator . HoweverOur study has several limitations. Although we had larger study cohorts than other studies of the oral rinse test, larger studies are still required to further validate these findings. An expansion of this study to a greater population through multi-institutional collaboration is warranted. In this study, we used an off-label process to extract nucleic acids before the nucleic acid amplification step run on the cobas z 480, which may have contributed to reduced sensitivity. Although HPV positivity confers a more favorable prognosis for head and neck cancer  there isWe collected oral rinse specimens from patients presenting to the Head and Neck service at Memorial Sloan Kettering Cancer Center (MSKCC). This study was approved by the MSKCC Institutional Research Board (IRB 15-256) and patients gave informed consent to use their oral rinse specimens and tumors for HPV analysis. Study participants were selected from three separate cohorts: (1) patients with biopsy proven OPSCC, (2) patients with biopsy proven SCC of the oral cavity (OCSCC) and (3) a non-SCC cohort of patients comprised of patients with benign or malignant thyroid nodules. The patients from our third cohort were deemed to be a representative “normal” population because complete head and neck examination including flexible laryngoscopy or mirror laryngoscopy of the laryngopharynx did not show any evidence for oral cavity or oropharyngeal pathology.Patients were instructed to swish and gargle 10 mL of 0.9% NaCl solution for 30 seconds before expectorating into a sterile 50 mL vial. The vials were then stored on ice for transport to our institution’s microbiology lab.g and 4°C on a Sorvall Legend RT. The supernatant was then separated from the pellet and both samples were stored at -80°C. DNA was extracted from the pellet using the PowerLyzer PowerSoil DNA Isolation Kit  per the manufacturer’s guidelines [Mouthwash samples were initially processed by spinning for 20 minutes at 2916 x idelines . Extractidelines .Cobas® HPV Test , which was approved by the US FDA for diagnostic and screening use in cervical specimens, allows HPV16 and 18 genotyping concurrently with the detection of 12 other high-risk HPV types . The test reports the genotypes of the other 12 high risk HPV types as “other”. The HPV Test performs PCR amplification and real-time detection in an automated fashion (the oche cobas c4800 System). Detection of the human β-globin gene is used as an internal control to monitor specimen cellularity . In thisin situ hybridization or by PCR. Ang et al. has reported that the expression of p16INK4a by immunohistochemistry correlated well  with the presence of HPV DNA in tumors [The gold standard test for the detection of HPV in tumor specimens is an invasive test by carrying out a biopsy of the tumor and then detecting HPV DNA or RNA in the specimen by n tumors . This isn tumors . In prosn tumors  but unfoIn our study, all pathology specimens were examined by a single pathologist specialized in head and neck pathology (NK) for p16 status. p16 immunohistochemistry was performed as follows: Four-micrometer tumor sections were deparaffinized, and after heat-induced epitope retrieval, immunohistochemistry for p16INK4a was performed with the primary antibody dilution of 1:7 as per manufacturer’s protocol . Cases with nuclear and cytoplasmic immunolabeling in at least 70% of the tumor cells were considered positive for p16.Statistical analysis was carried out using SPSS . Pearson’s chi-squared test was used to compare variables between groups. Confidence intervals were calculated with the R package epiR as described by Collett (1999) .


African Americans had lower HPV vaccine awareness (p = .006) but not lower intentions to vaccinate (p = .086). Parental willingness was positively associated with the following variables: Child’s age (p < .039), Perceived Advantages of HPV Vaccination for Adolescents (p = .002), Parental Trust in Medical Researchers (p < .001), and Level of Ease in Understanding Clinical Trial Information (p = .010). Educating parents about the advantages of HPV vaccines for younger adolescents using low-literacy educational materials and building trust between parents and researchers may increase parental willingness to consent to adolescent participation in HPV vaccine clinical trials.Approximately one-quarter of human papillomavirus (HPV) infections are acquired by adolescents, with a higher burden among racial/ethnic minorities. However, racial/ethnic minorities have been underrepresented in previous HPV vaccine trials. Ongoing and future HPV vaccine optimization trials would benefit from racially- and ethnically-diverse sample of adolescent trial participants. This study examined factors influencing parental willingness to consent to their adolescents’ participation in HPV vaccine clinical trials and tested for possible racial differences. A convenience sample of parents of adolescents (N = 256) completed a cross-sectional survey. Chi square analyses were used to assess racial differences in parental HPV vaccine awareness and intentions and willingness to consent to their child participating in an HPV vaccine clinical trial. Ordinal logistic regression was used to identify factors associated with willingness. Approximately 47% of parents were willing to allow their adolescent to participate in HPV vaccine clinical trials (30.7% African American and 48.3% Caucasian,  Cancer incidence associated with the human papillomavirus (HPV) is on the rise . ProlifeThe clinical trials that evaluated efficacy of three HPV vaccines  for primary prevention against HPV infections were conducted in adolescents and young adults aged 16–26 years –11. AlthWhile findings of these international studies demonstrated that the vaccine was efficacious and safe and induced higher immunogenicity under age 15 , 14–16, Parents or legal guardians must give consent for an adolescent child to participate in vaccine clinical trials . The pedThe purpose of this study was to assess, among parents of African American and Caucasian adolescents ages 9–15 in Tennessee, U.S., factors influencing their willingness to allow their adolescent children to participate in HPV vaccine clinical trials. Using the Health Belief Model (HBM) as an overarching conceptual framework, this study’s objectives were to: (1) determine whether racial differences exist in parental HPV vaccine awareness, HPV vaccine intentions, and their willingness to provide consent for their adolescent’s participation in HPV vaccine clinical trials, and (2) assess the independent association of the various HBM-related factors on parental willingness while controlling for race. Findings from this study will inform strategies to recruit adolescents for upcoming immunogenicity studies and the development of interventions aimed to educate parents about HPV vaccine efficacy studies to inform their decision-making regarding their child’s participation in vaccine clinical trials such as HPV vaccines.We applied the HBM  to examiFrom November 2014 to May 2015, we conducted a cross sectional study among a convenience sample of 256 parents across the state of Tennessee. This study (Protocol#: 14-11-306) was approved by the Meharry Medical College Institutional Review Board. Inclusion criteria were: (1) female or male, (2) English-speaking, (3) have, care for, or make medical decisions for an adolescent child aged 9–15 years. If parents/guardians had more than one child in the targeted age range, they were asked to complete the survey in reference to their youngest child in the age range.Consistent with principles of community-engaged research, we solicited input on recruitment strategies and sources from parents, community members . As a result, parents were recruited in person in a variety of community-based settings  in Nashville, Tennessee, U.S. We also identified participants in other parts of Tennessee using an online recruitment tool called ResearchMatch.org. ResearchMatch is a national health volunteer registry that allows researchers to identify individuals searching for research studies, and allows individuals to identify research studies of interest . Other oBoth online and in-person respondents first completed informed consent by reviewing the background/purpose of the survey and checking a box online or on paper to indicate their consent to participate in the survey. Participants then completed the anonymous online or paper-based survey. Paper surveys were completed individually or in groups ranging from 2 to 30 participants. Upon completion, per recommendation from the community stakeholders, participants completing the survey in person received a $10 gift card for their time. Online survey respondents filled out a separate compensation form, and a $10 gift card was mailed to them. All study procedures took approximately 15–30 min.HPV Clinical Trial Survey for Parents with Children Aged 9–15 (CTSP-HPV). Steps in the survey development process have been described in detail previously [We combined community-engaged research principles with traditional research study methods to assist in the development of the eviously . In brieeviously . In addieviously . The meaeviously  for a fuParental willingness to consent to adolescent participation in a HPV vaccine clinical trial was measured with one item asking, “How willing would you be to allow your child to participate in a HPV vaccine clinical trial to test the HPV vaccine?” using a 5-point response scale ranging from very unwilling to very willing.Perceived Benefits (Advantages of HPV Vaccination for Adolescents) and Perceived Barriers (Disadvantages of HPV Vaccination for Adolescents). Perceived Benefits was a 4-item measure, and Perceived Barriers was a 5-item measure. For each measure, parents indicated their level of agreement using a 5-point Likert scale. Items related to advantages were represented by statements such as “The HPV vaccine is a good way to protect my child’s health.” “I lack information about the HPV vaccine to decide whether my child should receive it” is an example of a disadvantage-related item. Mean scores were computed for each scale. Higher scores were reflective of more perceived advantages and perceived disadvantages on the respective scales.HPV Vaccine Awareness Parents were asked, “Have you heard of the Human Papillomavirus (HPV) Vaccine?” with the response options Yes, No, and I don’t know. Response options No and I don’t know were combined for the analysis.Race This single item asked parents “How would you describe yourself?” The categorical response options were: (1) Black, African American, African, or Afro-Caribbean, (2) Native American Indian or Alaskan Native, (3) Hispanic, Latino, or Spanish origin, (4) White, (5) Native Hawaiian/Other Pacific Islander, (6) Middle Eastern/North African, (7) Asian, (8) Prefer not to answer, and (9) Other. The race variable was transformed to the response options African American, White, and Other.Child’s Age This single item asked parents “Please select the YOUNGEST of your children in this age range. Answer ALL the questions in this survey specifically about this child”. Responses option were 9, 10, 11, 12, 13, 14, and 15.HPV Vaccine Intention Parents were asked, “Which of the following describes how you feel about getting your child the HPV vaccine?” The four ordinal response options from lowest to highest intentions were I do not plan to get my child the HPV vaccine, I am unsure about getting my child the HPV vaccine, I plan to get my child the HPV vaccine, and My child is already vaccinated against HPV.Parental Trust in Medical Researchers This 12-item measure included statements such as “Researchers who make mistakes try to cover them up.” Response options ranged from 1 (strongly disagree) to 5 (strongly agree). Negatively worded questions were reverse coded so that higher scores indicated more trust in medical researchers. The mean score was computed for the scale.Level of Ease in Understanding Clinical Trial Information A single item was used as a proxy measure for health literacy, “Has the information you heard about clinical trials for your child been easy to understand?” The response options included not at all, a little, some, a great deal, completely, and I haven’t received anything. For the analysis, the responses of not at all, a little, and some were combined into one category, and the responses of a great deal and completely were combined together. The response option I haven’t received anything was used as the reference category.Data were analyzed using Version 23 of the IBM Statistical Package for the Social Sciences (SPSS). Due to a small sample (N = 17) of the participants reporting Hispanic or other ethnic origins, these individuals were excluded from the current analysis and comparisons were conducted between Caucasians and African Americans, resulting in an analytic sample size of N = 239 participants. Chi square analyses were used to test the bivariate association between race and HPV vaccine awareness, HPV vaccine intentions, and the degree of willingness to allow their adolescent children’s participation in HPV vaccine clinical trials. Multivariate ordinal logistic regression was used to estimate the independent association of each of the HBM-based CTSP-HPV measures on parental willingness while controlling for socio-demographics (race and child’s age), and the other independent variables. We employed an alpha level of 0.05, while tests with marginal significance  were also noted for exploration in future research.Demographic characteristics for the 239 parents in the analytic sample are shown in Table 2(1) = 7.613, p = .006). There was not a significant racial difference in parental intentions to obtain the HPV vaccine (χ2(2) = .767, p = .086), with 36.7% of African American and 40.4% of Caucasian reporting that they planned to do so, and 39.3% of African American and 34.8% of Caucasian parents reporting they were unsure of their plans.Table 2(3) = 8.293, p = .081).Only 30.7% of African American parents and 48.3% of Caucasian parents stated they would be willing to allow their child to participate in a HPV vaccine clinical trial. While African American parents appeared to be less willing to allow their children to participate in a HPV vaccine clinical trial in comparison to Caucasian parents, the racial difference for this variable was only marginally significant . Regarding child’s age, for each additional year in age, the odds of a one unit increase in parental willingness were 19% greater . For each one unit increase on the Perceived Advantages of HPV Vaccination for Adolescents and the Parental Trust in Medical Researchers scales , the odds of each unit increase in parental willingness were 25% and 335% greater . In addition, Level of Ease Understanding Clinical Trial Information was positively associated with willingness; specifically, parents with low levels of understanding of clinical trial information prior to the current study  were 69% less willing to allow a child to participate in an HPV vaccine clinical trial compared to parents who had high levels of understanding information . HPV Vaccine Intentions and Perceived Disadvantages of HPV Vaccination for Adolescents were not found to be significantly associated with willingness while controlling for the other variables in the model.Table The HPV vaccine demonstrates great promise to reduce the prevalence of HPV infection in adolescents and future HPV-related cancer incidence among men and women. Adolescents, especially those of populations disproportionately affected by HPV-related cancers , could greatly benefit from future versions of vaccine that cover a wider range of high-risk HPV types and from further changes in immunization guidelines to reduce the number of doses, given the low rates of compliance observed under the three-dose schedule , 36. To In this study, most of the respondents were aware of the HPV vaccine, and many had intentions to obtain the vaccination. Yet, similar to past studies , AfricanPerceived Advantages of HPV vaccination of Adolescents, Parental Trust in Medical Researchers, Child’s Age, and Level of Ease Understanding Clinical Trial Information) were identified as positive correlates. The total number of perceived advantages, not surprisingly, along with greater degree of trust in medical researchers, were associated with greater willingness. This finding reflects a need for further research to determine strategies to increase awareness of benefits associated with HPV clinical trial participation among parents of adolescents. The finding that trust was positively associated with willingness suggests a need to reduce misconceptions or skepticism some may perceive about research [This study identified several factors influencing willingness of parents to allow their adolescent to participate in a HPV vaccine clinical trial. Similar to past studies reporting factors influencing adolescent participation in clinical trials in general , (Perceiresearch . Hence, research . This coresearch .This study was not without limitations. Survey participants were recruited using a convenience sample. Therefore, findings may not be generalizable to a specific population, and selection bias may have been introduced in the sampling. Because the data were self-reported, there was potential for social desirability bias as participants may have provided answers they perceived favorable to the researchers. There was also potential for recall-bias as participants attempted to recall information related to their awareness of the HPV vaccine and clinical trials. Since this was a cross-sectional study, the determination of temporal sequence and causation was not possible. Last, there may be other factors that influence parental willingness among African Americans and Caucasians differently that were not included in the current model. Despite these limitations, there are some strengths. Community engagement was applied at all phases of the research project which was vital to the success of this project. Furthermore, this study clearly documents the procedures so that the validity of the findings can be assessed by other researchers. Last, this study provides implications for future studies as it relates to recruitment of adolescents in clinical trials involving vaccines.Perceived benefits of adolescent clinical trial participation, ease of understanding clinical trials, and trust in medical researchers were positively associated with greater parental willingness of adolescent participation. Since this is among the first study of its kind, further studies should be conducted to assess potential differences between racial groups on willingness to allow a child to participate in an HPV vaccine clinical trial. Future research should also examine the information parents need in order to make an informed decision along with the optimal sources to distribute this information. Based on this study’s findings, one area to advise parents on is the benefits of adolescent clinical trials with an emphasis on the HPV vaccine. The incorporation of community-engaged research approaches may also be beneficial for future studies to develop interventions that address factors to increase parental willingness to participate in HPV vaccine clinical trials.


Small cell carcinoma of the uterine cervix is a rare and highly malignant tumor, and its etiopathogenesis is strongly related to high-risk HPV infections.The clinicopathological data of 30 cases of cervical primary small cell carcinoma were retrospectively analyzed. In situ hybridization, polymerase chain reaction and reverse dot-blot hybridization were employed to detect HPV DNA in both small cell carcinoma and other coexisting epithelial tumors. Immunohistochemistry was used to detect the protein expression of p16 and p53.P = 0.002). However, similar infection rates of HPV 16 were detected in both tumors (P = 0.383). Both small cell carcinoma and other types of epithelial tumors exhibited strong nuclear and cytoplasmic staining for p16 in all cases. Three cases of small cell carcinoma revealed completely negative p53 immunohistochemical expression in 15 cases of composite tumors, which suggested TP53 nonsense mutation pattern. The pure small cell carcinoma of uterine cervix had similar mutation or wild type pattern for TP53 compared with composite tumor (P = 0.224).Amongst 30 patients with cervical primary small cell carcinoma, 15 patients simultaneously exhibited other types of epithelial tumors, including squamous cell carcinoma, adenocarcinoma, squamous cell carcinoma in situ, and adenocarcinoma in situ. Most tumor cells infected with HPV presented integrated patterns in the nuclei by in situ hybridization. HPV DNA was detected in every small cell carcinoma case (100%) by polymerase chain reaction and reverse dot blot hybridization. 27 cases (90%) harbored type 18, and 15 (50%) displayed multiple HPV18 and 16 infections. The prevalence of HPV 18 infection in small cell carcinoma was higher than in cervical squamous and glandular epithelial neoplasms (Cervical small cell carcinomas are often associated with squamous or glandular epithelial tumors, which might result from multiple HPV infections, especially HPV 16 infection. Multiple HPV infections were not correlated with tumor stage, size, lymphovascular invasion, lymph node metastasis, or prognosis. Furthermore, careful observation of specimens is very important in finding little proportion of small cell carcinoma in the composite lesions, specifically in cervical biopsy specimens, in order to avoid the missed diagnosis of small cell carcinoma. Numerous clinical and experimental studies have demonstrated a closed etiopathogenetic relationship between the development of cervical cancers  and high-risk human papillomavirus (HPV) infection . It has In this report, the clinicopathological features of 30 patients were analyzed, including 15 patients with pure cervical small cell carcinoma, and 15 cases with composite cervical tumors composed of small cell carcinoma and other types of epithelial tumors. The HPV infection in these tumors was detected by ISH and PCR-RDDH, and the expression of p16 and p53 proteins were examined using immunohistochemistry. Our results demonstrate that cervical small cell carcinoma is often associated with squamous or glandular epithelial tumors, and these tumor cells usually exhibit overexpression of p16. Small cell carcinomas and adenocarcinomas or squamous cell carcinoma in situ showed all completely negative p53 immunohistochemical expression in three of 15 composite tumors of uterine cervix. To the best of our knowledge, this is the first study to report that multiple infections of HPV 18 and 16 developed in half of cervical small cell carcinomas. Furthermore, this is the first report to make the distinction that HPV 18 infection is closely correlated to the occurrence of cervical small cell carcinoma while HPV 16 infection is involved in cervical squamous cell carcinoma or adenocarcinoma in the co-existing tumor cases. Multiple infections of HPV subtypes were not related to tumor stage, size, lymphovascular invasion, lymph node metastasis, or prognosis. In addition, our study also confirmed for the first time that patients with composite tumors had similar HPV infection subtypes, clinicopathological features, and prognosis compared to patients with pure small cell carcinoma.Thirty cases of cervical primary small cell carcinoma were retrieved from the 2009–2017 surgical pathology archives of the Xijing Hospital. These included 23 hysterectomies, 1 conization, and 6 biopsies. To rule out the possibility of metastatic small cell carcinoma from the lung or other organs, the clinical data were carefully reviewed. Hematoxylin and eosin-stained slides of primary cervical neoplasm were re-examined to confirm the original diagnosis. Immunohistochemical staining for neuroendocrine markers, including Leu-19, synaptophysin, chromogranin, and neuron-specific enolase, was performed, and more than half of small cell carcinoma cells showed positive expression for two or more markers. The clinical records and follow-up of these patients were examined. This study received approval from the Ethics Committee of the Xijing Hospital.Three-micron sections containing small cell carcinoma and other types of epithelial tumors were examined for HPV DNA by ISH staining with INFORM® HPV III Family 16 and 6 Probe  for high-risk HPV types  and low-risk HPV types (6 and 11), respectively. The ISH assay was performed according to the manufacturer’s protocol using the Ventana BenchMark XT system . Labeling was detected with the ISH iVIEW™ Blue Plus Detection Kit . The positive and negative controls were carried out using HPV quality control slides provided by Ventana Medical Systems, Inc. The HPV signals were detected in the nuclei of tumor cells, and the signal patterns were categorized as either episomal staining pattern or punctuate staining pattern. The former presents as a homogeneous globular navy-blue to black signal in the entire nuclei of tumor cells, and the latter shows single or multiple sparsely distributed and dot-like navy-blue punctae in the nuclei of the tumor cells.2O2) for 60 min in the dark. HPV subtypes were determined by the positive point on the HPV genotype profile on the membrane. HPV-positive and negative controls were also included in every experiment.Eight-micron sections were prepared from formalin-fixed, paraffin-embedded (FFPE) tissues of cervical tumor samples. Tumor tissues were dissected using sterile blades from slides and were collected into 1.5 ml Eppendorf tubes. Amongst 15 specimens with composite carcinomas, small cell carcinoma and other types of epithelial tumors were successfully isolated in seven specimens. 37 samples of tumor cells were collected, including 22 cases of small cell carcinoma, 8 cases of mixed tumors, 2 cases of squamous cell carcinoma, 2 cases of adenocarcinoma, 2 cases of squamous cell carcinoma in situ, and 1 case of adenocarcinoma in situ. DNA extraction was carried out using QIAamp® DNA FFPE Tissue Kit  following the manufacturer’s guidelines. The samples were digested with proteinase K in a volume of 200 μL at 56 °C overnight, and 20 μL of DNA aliquot was obtained finally. The quality of the genomic DNA extracted was tested by agarose gel electrophoresis with ethidium bromide staining. For the PCR, 2 μL of DNA aliquot was used, and broad-spectrum HPV DNA amplification was performed using primers for GP5+/6+  with a total reaction volume of 50 μL. PCR amplification was performed in the following conditions: 50 °C for 15 min, 95 °C for 10 min, 94 °C for 30 s, 46 °C for 90 s, 72 °C for 30 s, 40 cycles, 72 °C for 5 min. DNA RDDH was performed with the HPV Genotyping Detection Kit  to simultaneously identify 23 HPV subtypes, including HPV 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 81, 82, and 83. PCR amplification products were boiled 10 min to obtain single-stranded DNA. Samples were then added in low density gene patches with a total of 23 gene probes of different HPV subtypes and hybridization was performed at 51 °C for 3 h. The gene patches were incubated in peroxidase solution for 30 min, and then developed in color reagent . P16 (clone: 6H12) and p53 (clone: DO-7) antibodies were purchased from Maxin Corp.  and DAKO Corp. , respectively. Immunohistochemical staining was conducted employing the Roche Ultraview DAB Detection Kit  following the manufacturer’s instructions. The positive and negative control slices were also run simultaneously. Nuclear/cytoplasmic staining was considered positive for p16, and p53 protein showed nuclear expression. A tumor was recorded positive for p16 if more than 50% of the tumor cells showed immunoreactivity. IHC for p53 includes mutation pattern and wild type pattern. Strong and diffuse nuclear staining or complete negative with internal control is considered to be mutation pattern, and focal and weak staining correlate with a wild type pattern of p53.2 test or Fisher’s exact test was adopted for correlation analysis of enumeration data. Wilcoxon rank sum test was used for comparison of patients’ age and tumor size. P<0.05 was considered as a statistically significant.Statistical software SPSS17.0  was employed in this report. Pearson ΧAmong 30 patients with cervical primary small cell carcinomas included in this study, 15 patients were diagnosed with pure cervical small cell carcinoma. In the remaining 15 cases of composite tumors, 7 cases also exhibited invasive cervical squamous cell carcinoma, 4 cases also showed cervical squamous cell carcinoma in situ, 3 cases also had cervical adenocarcinoma, and one patient also displayed cervical adenocarcinoma in situ. The age of all 30 patients at diagnosis ranged from 31 to 74 years . The mean and median age of the 15 patients with cervical composite tumors was 46.4 and 45 years, respectively. 22 patients presented with abnormal vaginal bleeding or contact bleeding. Two patients showed no clinical symptoms, and the tumors were found following physical examination. Most of the tumors displayed exophytic growth with or without cervical erosion. The tumor sizes in the 24 hysterectomies and cervical conization specimens ranged from 1 to 6 cm . 16 tumors (66.7%) were FIGO stage IB, 3 stage IIA, 2 stage IA, 1 stage IIB, 1 stage IIIA, and 1 stage IVB. Accurate staging information was not acquired for 6 cases of cervical biopsy specimens. 21 patients underwent radical hysterectomy with bilateral or partial adnexectomy and pelvic lymph node dissection. Two patients received radical hysterectomy with pelvic lymph node dissection, and one case underwent cervical conization owing to the superficial invasion depth of small cell carcinoma. Accurate surgical methods in 6 out-patients were not obtained in this study. 15 patients underwent postoperative chemotherapy combined with radiotherapy, and 6 patients received postoperative chemotherapy. Postoperative treatment strategies were not obtained in 9 other patients. Follow-up data of 16 cases was obtained: 7 patients died with a survival time ranging from 5 to 24 months . Survival time was more than two years in 4 out of 10 confirmed surviving patients, and two patients free of disease have undergone a follow-up at 67 and 88 months.P = 0.651). There was no statistical difference in patients’ age, FIGO stage, lymph node metastasis, lymphovascular invasion, or prognosis between pure small cell carcinoma and composite tumors (Table P = 0.716). The prognosis of these patients was determined by the composition of small cell carcinoma. Six cases (26.1%) had regional nodal metastasis, including 5 cases of metastatic small cell carcinoma and one case of metastatic squamous cell carcinoma. One patient displayed small cell carcinoma involving vaginal stump, and ovarian metastatic small cell carcinoma was found in another patient. In the 24 patients who underwent hysterectomies or cervical conization, lymphovascular invasion of small cell carcinoma was detected inside or within the tumor in 21 cases, of which two patients had simultaneous lymphovascular invasion of squamous cell carcinoma. Only one case presented with lymphovascular invasion of small cell carcinoma in 6 cervical biopsy specimens. Lymphovascular invasion was more likely to be found in hysterectomy or conization specimens than in biopsy specimens (P = 0.002).Histologically, cervical primary small cell carcinomas showed morphological features similar to those seen in the pulmonary counterpart. Densely packed small tumor cells often formed a sheet-like diffuse growth pattern. Neuroendocrine growth patterns, such as orderly tubular, trabecular, organoid, and nuclear palisading patterns, were less illustrated. Tumor cells showed round, ovoid, or spindled nuclei and scant cytoplasm. Nuclear chromatin is finely granular, and nucleoli were absent or inconspicuous. Numerous mitotic figures and extensive necrosis were commonly observed. Furthermore, squamous and glandular epithelial neoplasms were observed in 15 cases. Most squamous cell carcinomas developed in the superficial parts of small cell carcinomas Fig. . Howeverrs Table . PatientISH of tissue sections revealed that most specimens were positive for the INFORM HPV III Family 16 Probe in both small cell carcinomas and other types of epithelial tumors. 29 out of 30 cases of small cell carcinomas showed positive staining, including 21 cases with only one navy-blue to black puncta Fig. , 6 casesP = 0.136). The infection prevalence of HPV 16 and 18 was not significantly different between the composite tumor group and the pure tumor group . The HPV DNA subtypes in different tumor components were detected in seven cases of composite tumors, and the consistent HPV type was detected in two cases with single infection. Four cases showed that tumor cells of small cell carcinoma were positive for HPV both 16 and 18, while other types of epithelial tumors, including adenocarcinoma, squamous cell carcinoma, and squamous cell carcinoma in situ, only displayed HPV 16 infection. The infection rate of HPV 18 in small cell carcinoma was significantly higher than in other types of epithelial tumors (P = 0.008). However, similar infection rates of HPV 16 were measured in both groups .Thirty-seven specimens of cervical small cell carcinoma and other types of epithelial tumors were tested for HPV DNA by PCR-RDDH, and all were positive Fig. . The pre P = 0.38, Table 2TP53 missense mutation. Scattered tumor cells illustrated weak or moderate positivity for p53 with rates ranging from 1 to 60% , and there was no difference between TP53 mutation in small cell carcinoma and those in other epithelial neoplasms of uterine cervix (P = 0.682).Strong and diffuse nuclear and cytoplasmic staining for p16 protein was noted in all cases of cervical small cell carcinoma Fig. , squamou60% Fig. , which mSmall cell carcinoma of the uterine cervix is a rare and highly malignant tumor. The etiopathogenetic association between cervical small cell carcinoma and high-risk HPV infections has been well documented in some studies , 2, 7. OThe mean age at diagnosis for women with cervical small cell carcinoma was between 45 and 50 years , which in = 6), adenocarcinoma (n = 5), and adenocarcinoma in situ (n = 1) [n = 7), adenocarcinoma (n = 3), squamous cell carcinoma in situ (n = 4), and adenocarcinoma in situ (n = 1). Our results demonstrate a high frequency of cervical small cell carcinomas associated with squamous or glandular epithelial tumors. They had similar age composition and clinical manifestations as the patients of single cervical tumor. Chan et al. found that pure, rather than mixed histological pattern was a poor prognostic factor for survival [Primary small cell carcinomas of uterine cervix often coexist with squamous cell carcinomas or adenocarcinomas. Wang et al. reported that in 22 cases of primary cervical small cell carcinomas, two exhibited concordant high-grade squamous intraepithelial neoplasia and adenocarcinoma in situ . In the  (n = 1) . Ishida  (n = 1) . Emersonnoma n = , and adesurvival . Howeversurvival . TherefoHigh-risk HPV has been implicated in the carcinogenesis of cervical small cell carcinomas , 7. A meMultiple infections were observed in more than half of the cervical small cell carcinomas in our study. Only one case of multiple infections was found in 7 cases of squamous and glandular epithelial neoplasm. Multiple infections of HPV 18 and 16, rather than pure HPV 18 infection, might play an important role in the pathogenesis of cervical small cell carcinoma. In previous studies, very few cases of multiple infections were reported . A similOverexpression of p16 has been well documented in high-risk HPV-related cervical squamous cell carcinomas and adenocarcinomas, as well as their precursor lesions , 19. ManSince only a single-center retrospective case series was studied, and limited cases were employed, this study should be repeated on a larger scale to confirm our findings. Furthermore, some patients did not complete their follow-up, thus the comprehensive and effective data were not obtained.In summary, our study demonstrated that cervical small cell carcinomas closely correlate with HPV18 and HPV 16 infections. The patients of cervical small cell carcinomas with multiple infection of high-risk HPV may also promote the development of squamous or glandular epithelial neoplasms. In patients with composite cervical neoplasms, multiple infections of HPV 18 and 16 were involved in the development of cervical small cell carcinoma, while the occurrence of cervical squamous cell carcinoma or adenocarcinoma was closely related to HPV 16 infection. Multiple high-risk HPV infection was not related to tumor stage, size, lymphovascular invasion, lymph node metastasis, or prognosis in cervical small cell carcinoma. Similar HPV DNA genotypes, clinicopathological features, and prognosis were observed both in patients with pure small cell carcinomas and those with composite cervical tumors. The small cell carcinoma determined the prognosis of patients with cervical composite tumors. Because of the frequent presence of co-existing tumors, it is important to carefully examine the cervical biopsy specimens in surgical pathological examination.


Women living with HIV (WHIV) are disproportionately impacted by cervical dysplasia and cancer. The burden is greatest in low-income countries where limited or no access to screening exists. The goal of this study was to describe knowledge and intentions of WHIV towards HPV self-collection for cervical cancer screening, and to report on factors related to HPV positivity among women who participated in testing.A validated survey was administered to 87 HIV positive women attending the Kisenyi Health Unit aged 30–69 years old, and data was abstracted from chart review. At a later date, self-collection based HPV testing was offered to all women. Specimens were tested for high risk HPV genotypes, and women were contacted with results and referred for care. Descriptive statistics, Chi Square and Fischer-exact statistical tests were performed.The vast majority of WHIV (98.9%) women did not think it necessary to be screened for cervical cancer and the majority of women had never heard of HPV (96.4%). However, almost all WHIV found self-collection for cervical cancer screening to be acceptable. Of the 87 WHIV offered self-collection, 40 women agreed to provide a sample at the HIV clinic. Among women tested, 45% were oncogenic HPV positive, where HPV 16 or 18 positivity was 15% overall.In this group of WHIV engaged in HIV care, there was a high prevalence of oncogenic HPV, a large proportion of which were HPV genotypes 16 or 18, in addition to low knowledge of HPV and cervical cancer screening. Improved education and cervical cancer screening for WHIV are sorely needed; self-collection based screening has the potential to be integrated with routine HIV care in this setting. Cervical cancer is a largely preventable and curable disease that affects over half a million women each year, with the greatest impact in low and middle income countries (LMIC) where access to screening, diagnosis, and treatment are extremely limited . As a reIn most LMIC, visual inspection with acetic acid (VIA) is the standard of care for cervical cancer screening, which requires trained health personnel to perform pelvic exams, and willingness by women to attend a clinic and undergo an invasive examination. This is a barrier to effective cervical cancer screening for many women in LMIC settings . VIA briThe study design was a cross-sectional survey of knowledge, attitudes and behaviours towards self-collected sampling for cervical cancer screening in WHIV, who were then invited to participate in self-collection based screening. Interviews with WHIV and screening were conducted at a satellite HIV clinic of the Infectious Disease Institute (IDI) located at the Kisenyi Health Unit in an impoverished area of Kampala, Uganda. WHIV aged 30–69 years old attending a routine appointment for their HIV care were invited to participate by a member of the Friends Council, an HIV positive peer group between July and August 2013. An additional inclusion criterion was access to a mobile telephone. Women were excluded if they had previously had a hysterectomy, cervical cancer or they were unable to provide consent. Ethics approval for the study was obtained from the Research Ethics Board of the University of British Columbia (H09-02935-A013) and Makerere University (H13-02117).A structured questionnaire was created based on a previously validated survey using the Theory of Planned Behaviour exploring women’s intentions to provide a self-collected specimen for cervical cancer screening . Items wIn November 2013, local research assistants fluent both in English and Luganda were identified by the Ugandan ASPIRE research coordinator and trained in collaboration with the Canadian ASPIRE research team. In a private location that ensured patient confidentiality and allowed one-on-one interviews by the local research assistant, the survey participant’s primary language was used to explain the study, obtain informed consent, and complete the survey. After participants completed initial knowledge survey questions, the procedure of self-collecting a sample for HPV testing was explained by the local research assistant using a diagram of the swab, the genital tract, and cartoons of how to obtain self-collected sample. Surveys were completed by consenting study participants over a three-week period. Following completion of all survey and data recording, a workshop tailored from survey responses was held for study participants as an educational intervention about HIV and cervical cancer, HPV, and cervical cancer screening. This workshop also functioned to inform participants of relevant findings.Neisseria gonorrhoeae and Chlamydia trachomatis with real-time PCR. Women who tested HPV positive were contacted by phone with results and scheduled a colposcopy appointment for assessment at Mulago Hospital, a tertiary care center. Women who tested positive for N. gonorrhoeae and C. trachomatis were offered antibiotic treatment and counseling.In February–June 2014, all participants were contacted by a nurse from the HIV clinic by phone and invited to make an appointment to participate in self-collection based HPV testing at the HIV clinic. Women who could not be reached by phone were approached at their next HIV follow up visit. At the time of their clinic visit, women were instructed on how to use the self-collected vaginal swab and asked to provide a sample in a private room at the clinic. Specimens were tested for high risk HPV , p > 0.05.Descriptive statistics for all survey questions, chart review data, and screening results were generated for all participants. Chi-square or Fisher’s exact test were used to compare factors of interest between HPV+ and HPV- women that participated in screening. Unadjusted odds ratios (OR) were calculated for all variables that reached significance of In total 84 of 87 study participants completed the survey. Details for the demographic characteristics and chart review data of WHIV are captured in Table The survey responses for the knowledge, attitudes and behaviours among WHIV in this study are captured in Table Overall 98.8% of WHIV intended to provide HPV self-collected specimens for cervical cancer screening. Only one woman indicated that she would not be willing to self-collect, and this was because she was pregnant at the time of the study. Despite the high acceptability of self-collection among WHIV, the majority of women did not think it was necessary to be screened for cervical cancer (98.8%). Similarly, perceived risk of HPV was low (8.3%), while 44% of WHIV were unsure if they were at risk. The vast majority of women (95.2%) did not see embarrassment or concerns of how to collect the specimen properly as barriers to screening; nor were they concerned others would perceive them to have cervical cancer if they were tested for HPV (97.6%), and 69.9% found it acceptable for an outreach worker drop off a swab at their home.N. gonorrhoeae or C. trachomatis in the study population. Factors associated with HPV positivity are included in Table p = 0.03) and WHIV who have had blood work within the past 6 months were more likely to be HPV positive   were able to attend the clinic to provide a self-collected sample for high risk HPV testing. A total of 18/40 (45.0%) tested positive for one of the 12 high risk HPV genotypes and of those 6 (15.0%) women were positive for HPV strains 16 or 18. Among HPV positive women, 5/18 attended follow up colposcopy assessment, one of whom was positive for HPV 16/18, and all women were negative for cervical dysplasia. There were no cases of Among WHIV who participated in the study but did not attend screening, 2 of 47 could not be reached by phone, 5 of 47 indicated that they had screened for cervical cancer elsewhere, and 40 of 47 refused to attend the clinic. The main reasons for refusal were that distance to travel was too far, not having time to attend screening, or did not show up for the scheduled appointment.Although our population in Kisenyi was highly engaged in HIV care, less than 20% had ever received any education around cervical cancer, 96% had never heard of HPV, and almost 99% did not feel it was necessary to be screened. These findings reflect a potential lack of cervical cancer training among HIV care providers, and competing health priorities in HIV positive populations. The low percentage of women in our study who had ever had a pelvic exam (14.5%) is further evidence of the potential impact that offering HPV self-collection as part of routine HIV care could have on WHIV to enhance the uptake of cervical cancer screening. Others have emphasized the need to integrate cervical cancer screening into routine HIV care for WHIV and have documented the impact of missed opportunities for education about cervical cancer by HIV care providers .In this group of WHIV engaged in care, there was a high prevalence of oncogenic HPV types (45.0%), a large proportion of which were HPV 16 or 18 (15%). This is much higher than other studies that ASPIRE has conducted in Kisenyi where HPV positivity rates among HIV negative women was only 28.9%, of which 5.3% were HPV 16 or 18 . Our HIVIn our population, women who were taking oral contraceptives were more likely to be HPV positive. This is consistent with other studies, although the literature around the role of oral contraceptive on HPV positivity is unclear and in need of further study , 26. WomThis study was limited by its small sample size, which prevented us from conducting further statistical testing such as logistic regression. Another limitation was that a large proportion of our original survey population was unavailable to attend screening. Due to limitations on the study timeline, we were unable to offer screening during a routine HIV clinical visit to demonstrate an integrated approach. Despite this, we feel our results demonstrate the feasibility of a self-collection based approach to screening, while underscoring the challenges of offering interventions in a siloed manner.Despite alarmingly low levels of knowledge of HPV, global expansion of the HPV vaccine in countries including Uganda offers the opportunity for community health workers to engage and educate girls, women and their families on cervical cancer prevention. Self-collection for HPV screening has been demonstrated as a highly acceptable and effective method of cervical cancer screening, particularly in susceptible populations such as WHIV , 15. ThiTo not integrate prevention of cervical cancer in HIV care would do a great disservice to the accomplishments that have been made in the management of HIV. The overwhelming and growing need for accessible and effective screening in WHIV at risk for cervical cancer should compel us to look at feasible methods on a large scale. Our study of WHIV in this LMIC setting demonstrates the potential for HPV self-collection to be integrated into routine HIV care at a community level.