Viral infections and the burden of high-grade intraepithelial neoplasias (HIN) and invasive carcinomas (IC) associated to infections by human papillomavirus (HPV) types may be prevented by type-specific anti-HPV vaccines. This study determined the prevalence of HPV types in non-cervical HIN and IC diagnosed from 1999 to 2011 at a general hospital in San Luis Potosí, Mexico.n = 28) or IC (n = 39) confirmed the presence of tumor tissue in 63 of them and changed the diagnosis of 24 from HIN to low-grade intraepithelial neoplasias, that were excluded from the study. HPV DNA was detected with the SPF10-DNA enzyme immunoassay in the 39 cases included, and viral types in the HPV-positive tumors were identified with the INNO-LiPA linear probe array.Review of the 67 formaldehyde-fixed paraffin-embedded non-cervical specimens initially diagnosed as HIN (n = 3) and vulva (n = 1), and 35 IC in the oral cavity (n = 19), penis (n = 8), vagina (n = 7) and vulva (n = 1). There were 13 HPV-positive cases from the vagina (n = 7), vulva (n = 1), penis (n = 1) and oral cavity (n = 1). The viral types identified were the high-risk types HPV16 in the vagina (n = 3) and vulva (n = 3), HPV45 in the vagina (n = 2), HPV59 in the vagina (n = 1) and penis (n = 1), HPV33 in the vagina (n = 1),and HPV35 in the tongue(n = 1); and the low-risk types HPV54 in the vagina (n = 1), and HPV11 in the vulva (n = 1).Among the cases included, four HIN were located in the vagina  and two low-risk types (HPV11 and 54) infect one third of the non-cervical HIN and IC included. Most infections are by a single HPV high-risk type, the most prevalent one being HPV16. Vagina is the most frequent location of the HPV-positive tumors. Vaccination against HPV16 and HPV18 could have prevented around half of the HPV-positive tumors. The order of prevalence of high-risk HPV types associated to cervical carcinomas around the world is HPV16, 18, 31, 33, 35, 45, 52 and 58 [Persistent infection with high-risk HPV types is necessary for neoplastic transformation of the normal cervical epithelium to high-grade intraepithelial neoplasia (HIN) and its progression to invasive carcinoma (IC) –8. Viralet al. [Association of HPV types to non-cervical carcinomas has been demonstrated in recent years. HPV16, 33 and 18 are the most prevalent types in vulvar carcinomas , 12, HPVet al.  found HPIn this study we identified the HPV types present in HIN and IC from the oral cavity, penis, vagina and vulva diagnosed in a 12-year period at the largest general hospital of San Luis Potosí, to estimate the effect that anti-HPV vaccination could have had to prevent them.The protocol of this work was based on that of a multicentric study designed by the ICO and approved by the Delft Diagnostic Laboratory (DDL) . The stuThe study considered the 67 non-cervical tumors whose formalin-fixed paraffin-embedded (FFPE) blocks were recovered. The initial IC and HIN diagnoses were performed between 1999 and 2011 at the Pathology Department of Hospital Central Ignacio Morones Prieto.i.e., containing tumor tissue); sections 2 and 5, 3 μm thick, were placed on individual microscope slides; sections 3 and 4, 5 μm thick, were placed in each of two tubes. In order to avoid cross contamination the microtome blade was replaced after obtaining the sections from each tumor block. Sections on the slides were stained with hematoxylin-eosin and subjected to histopathological review; DNA was extracted from sections 1 and 3 or 5 for HPV detection and typing.The cases included were those in which the ICO Pathology Reference Laboratory confirmed the presence of tumor tissue and made the final (IC or HIN) histopathological diagnoses. FFPE blocks from the 67 cases were analyzed by the sandwich method based on five sections . SectionThe cases were excluded if slides 2 and 4 did not contain tumor tissue. When only the first slide contained tumor tissue, DNA extracted from tube 1 was amplified using the SPF10 (short PCR fragment generated with 10 oligonucleotides) method followed by DNA enzyme immunoassay (DEIA)  to detecet al. [Each 5 μm section from a paraffin block with tumor tissue was mixed with 250 μL of proteinase K (1 mg/mL) dissolved in 10 mM Tris–HCl, 1 mM EDTA (pH 8.0) and 0.5 % Tween-20, and incubated overnight at 60 °C. Proteinase K was then inactivated by heating at 95 °C for 10 min. To determine the presence of HPV DNA, 1 μL of the supernatant from each section treated with proteinase K was added to the SPF10-DEIA PCR mix under the conditions described by Kleter et al. .Samples of the HPV-positive DNA extracts were analyzed with the INNO-LiPA (linear probe array) on-strip reverse hybridization method  which identifies 25 of the 40 anogenital HPV types .The study was carried out in five steps Fig. : 1) 67 fInitial diagnoses of the 63 acceptable cases were 28 HIN and 35 IC Table . There wReview of the slides showed that four FFPE blocks lacked tumor tissue and led to the final diagnoses which determined the inclusion or exclusion of the tumor-containing specimens. Twenty-four cases with initial HIN diagnoses changed to low-grade intraepithelial neoplasia (LIN) and therefore excluded  Table .Agreement between the initial and final diagnoses for IC was 100 % (35/35), but only 14.3 % (4/28) for HIN (Table Fourteen of the cases included were HPV-positive (35.9 %), comprising 10 IC , and four HIN (three from the vagina and one from the vulva).Seven viral types were identified with the INNO-LiPA assay applied to the HPV-positive cases Table : five ofTwelve of the 13 HPV-positive cases (92.3 %) had DNA from a single high-risk type. The only vaginal IC had a double infection by a high-risk and a low-risk type (HPV16 and HPV59). The only vulvar HIN had the low-risk HPV11 type.The SPF10-DEIA-INNOLiPA assay used in this work is highly sensitive and specific for HPV DNA detection and typing. It has been used in several multicenter studies  because The presence of tumor tissue was confirmed in 63 out of the 67 original paraffin blocks and the study included only 39 cases with final HIN or IC diagnoses because the initial diagnoses changed from HIN to LIN in 24 cases. The initial and final diagnoses coincided in all the 35 IC cases included, whereas 24 of the 28 initial HIN cases changed to LIN and were excluded. Since premalignant vaginal squamous epithelial lesions are classified as grade 1 (LIN), grade 2 and grade 3 (the last two grades synonymous with HIN) , the diaThe more stringent diagnostic criteria of the ICO Pathology Reference Laboratory for preinvasive neoplastic lesions resulted in the inclusion of all the initially diagnosed IC but only 14.3 % of the initially diagnosed HIN.LIN samples were excluded from the study because HPV infections are spontaneously cleared in most of them, while they persist in HIN and IC . Most ofet al. [et al. [et al. [Only one third of the cases included were HPV-positive. Nine were IC: six from the vagina, one from the vulva, one from the penis and one from the oral cavity; four were HIN: three from the vagina and one from the vulva. These results are consistent with those of several multicenter studies; Kreimer et al.  showed a [et al.  found pr [et al.  of 16 toet al. [et al. [Seven viral types were identified in the HPV-positive cases: five of high risk , and two of low-risk . Infections with only one low-risk HPV type have been shown in vulvar IC  by Sutton  [et al. .The most prevalent viral type is HPV16 (46.1 %), followed by HPV45 and 59 (15.4 % each). These data are consistent with HPV16 being the most prevalent type in vulvar, vaginal, anal and oral carcinomas , 22.HPV35 was identified in the only oral IC despite the fact that HPV18 is often associated with non-cervical carcinomas  and HPV1A single viral type was detected in 12 of the 13 HPV-positive tumors (92.3 %), and two types in the remaining one. This finding suggest that most infections by a single HPV type are linked to tumor development and agree with a multicenter study which demonstrated single infections in 93 % in cervical and non-cervical HPV-positive carcinomas [The small sample size resulting from the low prevalence of the non cervical carcinomas is an obvious limitation of the study, which includes the corresponding tumors diagnosed for over a decade at a large Mexican hospital, identifies the HPV-positive tumors and the viral types to which they can be attributed, and calculates the ones could that have been prevented by anti-HPV vaccination.The 13 HPV-positive tumors were, in descending order of frequency, nine vaginal, two vulvar, one penile, and one lingual. Assuming that the divalent, quadrivalent and nonavalent HPV vaccines induce complete protection but only against the viral types to which they are directed, the divalent and quadrivalent vaccines could have prevented seven tumors (~54 %) and the nonavalent vaccine 11 tumors (~85 %), mostly vaginal and vulvar.Final histopathological diagnoses leading to the inclusion or exclusion of non-cervical tumors agreed with all the initial IC diagnoses but only one-seventh of the initial HIN diagnoses. Five high-risk viral types  and two low-risk types (HPV11 and 54) infect one third of the tumors included. Twelve of the 13 HPV-positive tumors (92.3 %) are infected by a single viral type. The most prevalent type is HPV16 (46.1 %), followed by HPV45 and 59 (15.4 % each). Vagina is the predominant location of the HPV-positive tumors. Vaccination with the divalent, quadrivalent or nonavalent HPV vaccines in this region could have prevented from half to over two thirds of the HPV-positive non-cervical tumors, mostly vaginal.


CK19, the lowest molecular weight keratin, is also expressed in basal layer of squamous epithelia of mucosal surfaces. Previous studies have shown that High Risk-Human Papilloma Virus (HR-HPV) epithelial infection induces cell immortalization via E6 and E7 viral proteins and this, in turn, impairs cytokeratin expression in cancerous cells lines derived from uterine cervix. Here, we demonstrate the possible relationship between HR-HPV−) and UPCI-SCC-154 (HPV+) cell lines by immunocytochemistry (ICC) and flow cytometry.We analyzed 38 cases of Oral Squamous Cell Carcinomas/ Oro-Pharyngeal Squamous Cell Carcinomas (OSCCs/OPSCCs) by Immunohistochemistry (IHC) using specific antibody (Ab) detecting CK19, by In Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR) based methods in order to define the HPV infectious status. We also evaluated the variation of CK19 expression in UPCI-SCC-131 (HPV+ (mean: 288.0 ± 24.3) and HPV− cancers (mean: 66.2 ± 96.9). This difference was statistically significant (p < 0.001) with a strong evidence of correlation . ROC curve analysis was performed on CK19 expression index related to HPV positivity. Heterogeneous areas of immunoreactivity varying in percentage value, intensity and/or localization were observed in normal epithelium, both perilesional and distant from the tumor with important differences between HR-HPV+ and HR-HPV− carcinomas. By ICC and flow cytometry, the two analyzed cell lines were both CK19 positive but showed a different level of expression, in particular it should be noted that the UPCI-SCC-154 (HPV+) cell line had a higher expression than UPCI-SCC-131 (HPV−).CK19 OSCC/OPSCC score has been identified multiplying percentage of cancer expressing cells to staining intensity. CK19 expression score in OSCCs/OPSCCs was very different between HPV+ OSCCs/OPSCCs. This test has a good accuracy. We identified ROC curve with a cut-off > 195 for HR-HPV positive results . Furthermore, in OSCC/OPSCC, the CK19 test may be useful in identifying HR-HPV infection, the latter being related to HPV E7 potential to disrupt normal cytokeratin expression pattern.In this study we demonstrated, for the first time, strong association between CK19 up-regulation and HR-HPV According to epidemiological studies, a real shift in OSCCs/OPSCCs aetiology may be one of the main reasons of the recent reported improvements in survival of a specific subgroup of patients, treated with radiotherapy . From thAccording to Bernard et al. the heterogeneous Papillomaviridae family now contains 29 genera formed by 189 papillomavirus types, isolated from humans, non-human mammals, birds and reptiles. In particular 120 fully sequenced genotypes have been isolated from humans . More reHPVs exert their oncogenic potential by expressing E6 and E7 viral proteins in infected host cells.These proteins affect cell cycle in terms of proliferation induction and deregulation by targeting, respectively, p53 and pRB tumor suppressors, up to promote carcinogenesis. In particular, it appears plausible that E7 oncogene is able to redirect terminally differentiating epithelial cells to support viral DNA amplification, and in this way the generation and the maintenance of progeny virus .Integration of HPV DNA into the host DNA, as key-mechanism to induce high grade lesions and invasive cancer , 19, is The exact molecular mechanisms used by HPVs to control the replication of their own genome and to promote and preserve their transcriptional activity to the detriment of host infected cell are extremely tangled. Although the viral mRNA translation process is intricately tied to the differentiation program of the host epithelial tissue, it is broadly acknowledged that the expressed viral oncoproteins are highly able to induce keratinocytes immortalization and to enhance the disruption of the normal cytokeratin (CK) expression pattern in stratified squamous epithelium , in thisCytokeratins are proteins containing 10 nm intermediate filaments found in the intracytoplasmic cytoskeleton of epithelial tissue, with a molecular weight of 40-68 kDa . They seAccording to the reported literature, normal oral mucosa expresses cytokeratins 4, 5, 13, and 14 . This paAn early study by Kellokoski J, et al. evaluated by IHC the distribution of cytokeratins 19, 14, 16 and 17 and 8 and 18 in 96 oral mucosal biopsies taken from women with genital HPV infections. They observed a more efficient and intense CK19 expression in the basal cell layer of HPV DNA-positive samples, suggesting for the first time that viral infection disturbs the keratinocyte differentiation in the basal cells of oral epithelium accelerating the production of LMW cytoskeletal protein .Up to now analyses of the specific epithelial keratin profile in HPV infected oral mucosa and in HPV related oral and oropharyngeal cancers are scanty and questionable. Although it is well known that CK19 and/or its fragment CYFRA 21–1 correlate with tumor progression both in the uterine cervix and in the upper aerodigestive tract –33 and a+ oral/oropharyngeal cancer and the high levels of CK19 expression.Aims of the present work have been firstly to detect HR-HPV DNA in OSCC/OPSCC, secondly to value the pattern of expression of CK19, and finally to demonstrate the CK19 overexpression in neoplastic cells  in order to prove the possible relationship between HR-HPVOur study-cohort was composed of 38 patients , with a mean age of 64.83 years (range:46–89). Characteristics of the patients group are shown in Table The study also included 10 negative control cases of non-neoplastic oral (n.5) and oropharyngeal mucosa (n.5): these specimens were negative for HPV-DNA by ISH assay using Inform HPV family-III (Ventana - Roche) and Inform HPV family-II (Ventana - Roche) and consensus primer PCR.We have also included some control cases of uterine cervix HR-HPV positive lesions ), all of them previously characterized for HPV by PCR followed by direct sequencing.Regarding immunohistochemical analysis, overall, the average expression of CK19 in OSCC/OPSCC was 40.7 % which resulted statistically significant when compared to the corresponding normal, distant and/or peritumoural epithelium (p < 0.05). Furthermore, considering the estimated statistical cut-off value of 67 % of stained cells, we have identified two cancer groups: CK19highOSCCs/OPSCCs (13 cases) and CK19lowOSCCs/OPSCCs (25 cases). Considering the staining intensity of CK19 expressing cells, we observed 9 cases scored as 0, 3 cases scored as 1+, 3 cases scored as 2+ and 23 cases with score 3+. On this basis we subdivided OSCCs/OPSCCs in two groups: CK19faintOSCCs/OPSCCs (12 cases with score 0–1) and CK19strongOSCCs/OPSCCs (26 cases with score 2–3). Moreover, according to the final score (0–300), OSCCs/OPSCCs have been divided in two classes: CK19high scoreOSCCs/OPSCCCs (15 cases with score > 150) and CK19low scoreOSCCs/OPSCCs (23 cases with score < 150). Finally, a wide variability in the intensity and a prevalent distribution in the basal layers have been observed in the controls of normal oral and oropharyngeal mucosa.Immunohistochemical results were statistically correlated with the clinico-pathological findings  and evaluated by statistical univariate analysis (ANOVA). No statistically significant correlations have been observed.Regarding HPV detection in OSCC/OPSCC, among 38 cases, 10 were HR-HPV+ cancers  , 1 case was HPV31+ and 1 case HPV56+; the remaining positive case, at the ISH evaluation, showed an integrated status for HR-HPV, but it has not been possible to assess the viral genotype. Among the HR-HPV+ cases as resulted by consensus PCR and/or ISH, 6 cases were investigated by ISH also to determine the integrated and/or episomic status of the virus: in 5 cases the HR-HPV was associated to an integrated status; only 1 case resulted negative with cytoplasmic signals, but the HR-HPV positivity was established by consensus PCR. Finally, among the 28 HR-HPV− cancers, 2 were HPV6+ , 1 was HPV53+  and the remaining cases (n.25) were HPV−.R) Table : 6 casesRegarding the correlation between CK19 expression and HR-HPV infection in OSCC/OPSCC, we have noted that CK19 expression scores were very different in the two groups of analyzed cancers (HR-HPV+/HR-HPV−). We obtained higher values in HR-HPV positive group (mean: 288.0 ± 24.3) than in negative one (mean: 66.2 ± 96.9)  with a strong evidence of correlation . We described ROC curve with a cut-off > 195 for HR-HPV positive result   Fig. . High scHeterogeneous areas of immunoreactivity varying in percentage value, intensity and/or localization were observed in normal epithelium, both perilesional and distant from the tumor.In perilesional areas, in HR-HPV−cancers, we observed CK19 positivity in 13 out 19 valuable cases (68.4 %) and a generally moderate-strong staining, localized in all epithelial layers .In HR-HPV+cancers we observed a constant expression of CK19  and a generally moderate-strong staining, mostly localized to the upper layers.p = 0.017)  Fig. , Table 4In mucosal areas distant from OSCCs/OPSCCs, in HR-HPV−cancers we observed a prevalent absence of CK19 expression (58.9 %) and when present , we noted a wide variability in the intensity of CK19 staining, distributed mostly in the basal layers.In HR-HPV+OSCCs CK19 staining was always present , generally moderate-strong in intensity, localized equally at basal and superficial epithelial layers. The differences regarding CK19 staining intensity .ty Table  and locaty Table  were staFinally, we measured the variation of CK19 expression in UPCI-SCC-131 and UPCI-SCC-154 cell lines by ICC and flow cytometry. The two cell lines were both positive to CK19 antibody but showed a different level of expression, in particular it should be noted that the UPCI-SCC-154 cell line had a higher level of expression, characterized by strong intensity and a diffuse pattern; on the other hand, UPCI-SCC-131 cell line showed a lower level of expression, with mostly moderate, focally strong intensity of the staining  had a greater fluorescence value. Although the two studied cell lines were both positive for CK19 expression, different rates of positivity have been reported. The relative extent of CK19 expression, as revealed by the mean fluorescence intensity (MFI), varied among the analyzed cell lines, being lower for UPCI-SCC-131  in comparison to the UPCI-SCC-154   Fig. . The abs92) Fig. : both ceSince the HR-HPV subtypes are always more frequently linked to aetiology of many human cancers including oral and oropharyngeal cancer, an innovative approach to the study of OSCC/OPSCC should be based on a better understanding of the molecular viral background and the relative interaction between virus and host cell. It has well established that some HR-HPVs (in particular HPV16) induce immortalization of keratinocytes and at the same time the deregulation of the normal CK expression pattern in stratified squamous epithelium . The invCytokeratin 19 (CK19), a 40 kDa acidic cytokeratin (Type I), is normally expressed in stratified squamous epithelium and it has been considered a marker of pre-malignancy and susceptibility to cancers like the OSCC .Although we have not observed any type of correlation between immunohistochemical CK19 expression and the analyzed clinico-pathological findings, a recent study has confirmed CK19 as a valid prognostic marker in human OSCC and its over-expression as an important molecular event in pathogenesis of oral carcinoma .Beforehand and similarly, other Authors have evaluated the role of CK19 in OSCC, reporting different percentages of expression –42.highOSCCs/OPSCCs and CK19lowOSCCs/OPSCCs. According to staining intensity, generally, it was mainly moderate or strong (3 cases scored as 2+ and 23 cases with score 3+).In our study, considering an average CK19 expression in cancer of 40.7 % and an estimated statistical cut-off value of 67 % of stained cells, we have identified two cancer groups: CK19Heterogeneous areas of immunoreactivity varying in percentage value, intensity and/or localization were also observed in normal epithelium, both perilesional and distant from the tumor.− cancers than in HR-HPV+ ones (68.4 % vs 100 %) where the marker preferentially was located to the upper layers. In more distant areas, in HR-HPV− OSCCs/OPSCCs CK19 staining was observed in 41.1 % of cases with a wide variability in the intensity and distributed mostly in the basal layers, as observed in normal oral/oropharyngeal mucosa; differently, in HR-HPV+ OSCCs/OPSCCs CK19 was present in 100 % of cases, generally moderate-strong in intensity and localized in all epithelial layers.In perilesional areas, we mostly observed a moderate-strong positivity for CK19, distributed in all epithelial layers  and with lower percentages in HR-HPVConsidering its low molecular weight, CK19 in normal conditions should be mostly located in the basal/parabasal layers . In our Already in the past, it was suggested that when CK19 appears in the suprabasal cell layers in oral epithelium, it might be a consequence of a delay or a disturbance in the terminal differentiation, probably indicating retention of hyper-proliferative potential and acquisition of cellular atypia associated to premalignancy . On the Anyway, it still remains to understand the exact role of CK19 in the genesis of HPV related oral and oropharyngeal cancer and how keratinocyte differentiation could be involved in pathogenesis of HPV infection . In 1991Considering that HPV unrelated OSCCs/OPSCCs are generally quite resistant to chemotherapeutic agents while HPV related cancers are characterized by radio-chemo sensitivity, HPV detection and in particular HR-HPV identification are of basic importance in HNSCC patients clinical management. Up to now, in spite of the significant number and the types of molecular available methods or their combinations, there is not a general recognized agreement about the ‘golden standard’ identification test.The currently performed techniques of viral detection in clinical practice such as ISH, routinely IHC for viral oncoprotein (E5-6-7) and p16-IHC test  are cons+ versus HPV− OSCCs/OPSCCs innovative techniques, as well as SPF10 HPV DNA test, PGMY/GP nested PCR system, quantitative E6 RNA PCR [Recently, in order to distinguish HPV RNA PCR , 56 and  RNA PCR , 58, areIn an our previous study we have clearly demonstrated that a combined triple method, consisting in p16-IHC/ISH/Consensus PCR test, preserves the morphological context of HPV-DNA signals in formalin-fixed, paraffin-embedded (FFPE) samples and increases the overall specificity and sensitivity in HPV detection .+ and HPV− OSCCs/OPSCCs. We have reported higher values of CK19 expression in HR-HPV+ cancers  than in negative ones . This statistically significant difference (p < 0.001) suggests a possible association between HR-HPV and CK19 expression.The presented study adds CK19 as a new interesting marker able to determine a differential diagnosis among HPVSimilar results have been obtained also by immunocytochemical analyses performed on two SCC-cell lines (differing for the HPV infectious status) and by flow cytometry. Finally, these observations can be regarded as a possible evidence of an interplay existing between HPV oncogenic activity and keratinocyte differentiation.We conclude that the routinely evaluation of an immunohistochemical panel including p16, and CK19 should be assessed by further investigations since these markers, exclusively and in combination, could add useful informations concerning oral and oropharyngeal carcinogenesis and improve the current diagnostic tools, allowing to characterize a possible clinic-morphologycal profile of oral/oropharyngeal cancer and its precancerous lesions.Upon approval by the Ethical Committee of the University of Foggia, 38 subjects affected from OSCC/OPSCC were randomly selected from 4 different Hospitals of the Middle and South of Italy and enrolled in this study . All patients or their relatives gave their informed written consent.The cases have been randomly chosen from cohort known for the HPV prevalence as previously published –61 and wFinally, we have also selected an adequate group of non-neoplastic oral (n.5) and oropharyngeal mucosa (n.5) samples and some control cases of uterine cervix HR-HPV HSILs (n.3) for the comparative statistical evaluations. Characteristics of the group of the patients are shown in Table Finally, at the Institute of Pathological Anatomy, University of Foggia, a total of 38 archival FFPE OSCCs/OPSCCs were investigated by standard IHC for CK19, by consensus HPV-DNA PCR methods to detect HPV infection and by ISH to study the viral integration status into the host DNA.highOSCCs/OPSCCs and CK19lowOSCCs/OPSCCs. Then, we have also evaluated the staining intensity, that has been scored as follows: negative (0), faint (1), moderate (2), strong (3). Finally, intensity staining has been multiplied to IHC percentage in order to obtain the final molecular expression score, ranging from zero to 300 units. Appropriate positive and negative controls were run for the tested antibody. On our OSCC/OPSCC samples, serial sections, in addition to the neoplastic area, also included perilesional areas and mucosal areas > 5 cm distant from the tumor. Moreover, negative controls were performed on other sections that comprised normal areas of removed oral and oropharyngeal mucosa for surgical non-neoplastic diseases; negative control slides without primary antibody were also included. Positive control was executed on sections obtained from a case of infiltrating colon cancer. Inter-rate reliability between the two investigators blindly and independently examining the immunostained sections was assessed by the Cohen’s K test, yielding K values higher than 0.7.0 in almost all instances.IHC was performed on 4 μm paraffin sections mounted on poly-L-lysine-coated glass slides, by standard linked streptavidin-biotin horseradish peroxidase (LSAB-HRP) technique, using a specific monoclonal antibody against CK19 (mAb clone A53-B/A2.26), by the Benchmark XT autostainer . Cases were evaluated on the basis of percentage of positive cells and, considering a statistical cut-off of 67 %, they were distinguished into two categories: CK19HPV DNA was researched by nested PCR (MY/GP primers), and HPV genotype was determined by direct sequencing of PCR fragments. Three types of control were included in each reaction series: blank control, HPV-neg.ve Wi cells as neg.ve control and HPV18 DNA-pos.ve HeLa cells, in dilutions from 20,000–50,000 down to 2–5 HPV DNA copies, as pos.ve control. HPV DNA was amplified by PCR assay using primers useful for samples with a low copy number of HPV (MY09–MY11 primer pair in combination with GP5–GP6 primer pair) as previously demonstrated in current literature  and amplhttp://www.ijbcb.org/HPV/).HPV genotyping was based on direct sequencing of MY or MY/GP PCR fragments. Amplification products were purified by Microcon YM-100 ; the sequence of both DNA strands was determined by the BigDye Ready Reaction Kit in the automatic sequencer ABI Prism 310 Analyzer . Alignments were obtained from the GenBank on-line BLAST server and HPV sequences downloaded from the HPV database ; and the following probes for LR-HPV: 6, 11 .ISH signals have been evaluated on at least ten High power fields at Olympus BX-41 optical microscope . OSCC/OPSCC cases showing prominent nuclear punctuated (discreet dot-like) signals have been considered as integrative (I). Cases with exclusive nuclear cluster signals as been evaluated as episomal (E). Cases showing a prevalent nuclear cluster signals along with also focal punctuated signals of integration have been evaluated as mixed episomic-integrative (E-I). According to manufacturer instructions, artifacts or non-specific findings as been considered the followings: non-cellular stromal precipitates; cytoplasms of polymorphonuclear leukocytes (PMNs), eosinophils, lymphocytes and endothelial cells; non-specific staining of nucleoli.+) . The cell lines were purchased from the DSMZ, Braunschweig Germany.Cell lines derived from OSCCs and normal keratinocytes have been used for ICC and cytofluorimetric analyses. The cell lines used were (1) UPCI-SCC-131  (2) UPCI-SCC-154  supplemented with non-essential amino acids, 10 % fetal bovine serum (FBS), 2 mM L-glutamine,1 % penicillin and 1 % streptomycin in a humidified incubator containing 5 % CO2 at 37 °C.Cells were grown on four-chamber tissue culture-treated glass slides  pre-coated with poly-L-lysine to enhance cell attachment at a density of 5000 cells per well. Cultured cells were rinsed with PBS, and finally fixed in alcohol. Immunostaining was performed using LSAB-HRP technique and specific mouse monoclonal Ab anti CK19 (mAb clone A53-B/A2.26). Evaluations of the immunocytochemical staining were performed separately by two observers with at least 10 HPFs using optical microscopy.To detect CK19 nonconfluent cultures were trypsinized into single cell suspension, counted, washed with phosphate-buffered saline (PBS), fixation and permeabilization by intracell kit . Then the cells were incubated with FITC conjugated CK19 antibody ab87014 or isotope antibody ab81197 (both from Abcam) as negative control for 1 h. After washed twice with PBS, samples were analyzed by FACSCalibur (Becton Dickinson).All data have been analyzed by MedCalc 12.2.1.0 (for Windows), SOFA Statistics 1.4.3 and R 2.11.1 (for Linux) statistical softwares using Debian 7 and Windows Operating Systems.t-test of average. Spearman’s method and Point-biserial correlation coefficient were used to study linear correlation and to determine the relationship between CK19 expression and HR-HPV positivity . In order to select a relevant CK19 cut-off score to identify HPV positive samples, Receiver Operating Characteristic (ROC) curve analysis was carried out. The point on the curve, maximizing sensitivity and specificity of CK19 expression score, was selected as the cut-off score above which it was considered positive marker of HPV positivity. The expected average value of the ROC area is 0.5 if there is no discrimination between the groups. In order to distinguish a real discrimination between the groups from the case of no discrimination, a p-value was calculated. A small p-value makes it unlikely that the ROC area can be reconciled with the case of no discrimination. Only values of p < 0.01 were considered significant.Differences between groups were determined using the Independent Samples


We assessed whether infection with chlamydia increases the incidence of carcinogenic human papillomavirus (HPV) infections and if HPV persistence is affected by chlamydia co-infection. For 1982 women (16–29 years-old) participating in two consecutive rounds of a chlamydia screening implementation trial, swabs were polymerase chain reaction tested to detect chlamydia and 14 carcinogenic HPV genotypes. HPV type-specific incidence and persistence rates were stratified for chlamydia positivity at follow-up. Associations were assessed by multilevel logistic regression analyses with correction for sexual risk factors. HPV type-specific incidence ranged from 1.4% to 8.9% and persistence from 22.7% to 59.4% after a median follow-up of 11 months (interquartile range: 11–12). Differences in 1-year HPV persistence rates between chlamydia -infected and noninfected women were less distinct than differences in HPV incidence rates . The effect of chlamydia co-infection on HPV-infection risk did not significantly differ by HPV genotype. In conclusion, infection with chlamydia increases the risk of infection by carcinogenic HPV types and may enhance persistence of some HPV types. Although these findings could reflect residual confounding through unobserved risk factors, our results do give reason to explore more fully the association between chlamydia and HPV type-specific acquisition and persistence. Chlamydia trachomatis  infection, an STI that often remains asymptomatic. The implication of chlamydia in the pathogenesis of cervical carcinoma is controversial, as its interference has not been demonstrated at a particular stage of the carcinogenic process. Alternatively, chlamydia could increase susceptibility to HPV co-infection Cervical carcinoma is the third most common cancer among women worldwide, and most cases occur in developing countries The presence of chlamydia antibodies has been associated with an increased risk for invasive squamous cell carcinoma of the cervix, although no association was seen with adeno- or adenosquamous cell carcinoma Epidemiological studies on the impact of chlamydia on HPV acquisition and/or persistence have yielded equivocal results due to the difficulty in separating biological from behavioral effects. If a biological association exists, it might differ among HPV types, which differ in clearance time www.chlamydiatest.nl) with a personal code to complete a voluntary questionnaire and request a home-sampling test package. Questions addressed ethnicity, educational level, the number of sexually active years (continuous variable), number of sexual partners in the 6 months prior to follow-up measurement (continuous variable), and condom use with a steady and casual partner in the 6 months prior to follow-up measurement .The study design is detailed elsewhere In total, 16,679 women participated in two or more rounds. Of participants in rounds 1 and 2, 5% tested chlamydia-positive at round 1, of whom 7% were also positive at round 2. These percentages were similar for participants in rounds 2 and 3 , the Netherlands.Self-collected vaginal swabs were sent to three regional laboratories in an envelope suited for mailing biological materials. Participants were instructed to post the sample immediately after self-collection. The samples were tested for chlamydia within 7 days after reception of the material in the laboratory with three commercially available nucleic acid amplification tests according to manufacturers' guidelines . One laboratory pooled five samples for testing and, if a pool tested positive, all five were retested. The two laboratories that did not pool confirmed each chlamydia-positive sample by retesting the specimen. Infections found at baseline were treated following CSI protocol; thus chlamydia infections found at follow-up were defined as newly acquired. Remaining material from the swab was prepared for future STI testing and stored in the Biobank at −80°C.The HPV testing protocol is described elsewhere To assess the association of chlamydia with 1-year HPV persistence, we selected all women of whom a chlamydia and HPV test result for two consecutive study rounds was available and the time between both rounds was between 8 and 16 months.P < 0.05. When numbers were smaller than five, Fisher's exact test was used instead of the chi-square test.For the descriptive analysis, we calculated the percentage of incident and persistent HPV infections among women with and without a chlamydia infection. The reference group for HPV incidence was negative for a certain type at both rounds; the reference group for HPV persistence was being positive for a certain type at baseline but negative for that type at follow-up. Note that each person was included in either analysis, but not both, for a particular HPV type. Due to the type-specific reference groups, numbers differed among the HPV types with corresponding power fluctuations. The Pearson chi-square test was used to compare percentages and to test if observed differences were significant, Type-specific and pooled associations between HPV incidence or persistence and chlamydia were analyzed using multilevel logistic regression models using generalized estimating equations. To model associations between HPV type-specific responses on the level of the participants, we made use of the alternating logistic regression algorithm of the SAS procedure GENMOD, assuming an exchangeable correlation structure between pairs of responses As not all participants completed a questionnaire, sexual risk variables were missing for about 25% of women at follow-up Table. Taking Results of chlamydia testing and HPV genotyping were available for 2051 women participating in two consecutive rounds. Of these, 1982 (97%) women were retested within the required 8- to 16-month interval with a median interval of 11 months (interquartile range [IQR]: 11–12). At follow-up the median age was 25 years (IQR: 22–27), most women were of Dutch origin and highly educated. In more than half, first sexual intercourse took place at age 17, resulting in a median number of eight sexually active years (IQR: 5–10). Sixty per cent reported having one sexual partner in the last 6 months prior to follow-up, 18% had two partners, and 16% had three or more. Characteristics are given in TableOf the 1982 women, 108 (5.5%) tested positive for chlamydia at baseline, 66 (3.3%) at follow-up. Five women were chlamydia positive at both rounds. For women with and without baseline infection, the median follow-up time was similar: 11 months (IQR: 11–12).At baseline, 1324 (66.8%) of the 1982 women were infected with HPV. Of these, 513 women had a single HPV infection, 640 women were infected with multiple types and of 171 women the specific type could not be identified. At follow-up 1419 (71.6%) women were HPV infected of which 538 with a single HPV type, 708 with multiple HPV types and 173 with an undetectable type.-positive women than among -negative women . Results from the analysis on imputed data did not differ from complete case analysis, as shown in The five most incident HPV types over the follow-up period were HPV16 (6.4%), HPV31 (6.1%), HPV51 (8.9%), HPV52 (6.8%), and HPV66 (6.7%), whereas the least incident was HPV35 (1.4%) .P = 0.80). Results from analyses of complete cases and imputed data are shown in The 1-year persistence rate ranged from 22.7% for HPV59 to 59.4% for HPV31 over the follow-up period  was found. This suggests that women with a chlamydia infection at baseline have less chance of persistent HPV infection than women without a chlamydia infection at baseline.In a large follow-up study of young and sexually active women, we assessed the possible association between chlamydia infection and the subsequent acquisition and persistence of infection with carcinogenic HPV types. We found that HPV incidence rates were significantly higher among women with chlamydia co-infection as compared to those without. However, chlamydia co-infection had no significant effect on clearance of pre-existing HPV infections in our analyses.Our study is unique in that we analyzed the associations between chlamydia and HPV incidence and persistence in a type-specific manner. However, we could not confirm whether associations between chlamydia and HPV differ among types. This negative finding could reflect insufficient statistical power (as the study was not designed to detect type-specific differences) or indicate a truly uniform effect of chlamydia co-infection on the acquisition and persistence of carcinogenic HPV types. The effect of chlamydia on HPV incidence was studied in substantially more women than its effect on HPV persistence. Still, the relative risks of HPV infection among chlamydia-positive versus -negative women showed little variation across HPV types, suggesting that type-specific deviations from the overall association between chlamydia and HPV acquisition are small. A side note to be made is that it could be possible that some of the infections defined as incident infections are in reality latent infections which reactivated again.A possible biological explanation for the increased risk of HPV infection among women with chlamydia co-infection is that chlamydia causes local inflammation leading to damage of the epithelial tissue which could make a woman more susceptible for HPV infection, irrespective of HPV type Lack of power is manifest in our analysis of type-specific 1-year HPV persistence, as we observed sizeable differences among relatively small numbers regarding the effect of chlamydia co-infection on clearance of pre-existing HPV infection across types. Moreover, the follow-up that we used may have been too short to detect an increased persistence of HPV infections among chlamydia-positive as compared to chlamydia-negative women as our sampling interval was of the same order as the natural duration of HPV infection Our interpretation is consistent with Shew et al., who looked at the effect of chlamydia on the duration of HPV persistence C. trachomatis. If a long-lasting chlamydia infection is needed to hamper clearance of an HPV infection, our intervention study may have been too short to observe an effect of chlamydia co-infection on HPV clearance. The optimal next step would be the retrospective testing of half-yearly samples from a longitudinal study, with sufficient length of follow-up. Using samples already collected for other purposes would solve the ethical problem of including long-lasting chlamydia infections.Further research is needed into the relation between chlamydia and type-specific HPV persistence. In prospective studies like ours, a detected chlamydia infection is always treated, limiting its duration. In a recent publication, Seraceni et al. used molecular techniques to distinguish between recently acquired and chronic chlamydia infections in a cross-sectional study design In conclusion, chlamydia is positively associated with an incident infection with a carcinogenic HPV type. This could reflect enhancement in HPV acquisition by chlamydia or residual confounding through unobserved risk factors. Although no significant association was found for chlamydia and HPV persistence, our results point toward an increased persistence of carcinogenic HPV types in the presence of chlamydia co-infection. Our study was not designed to assess possible interactions between chlamydia and HPV, which may have hampered the strength of some of the findings. However, the results do give more than sufficient reason to explore more fully the association between chlamydia and HPV type-specific persistence.


HPV-16 modifies the overall survival (OS) of patients with oropharyngeal cancer (OPSCC). HPV-16 has been established as risk factor for OPSCC, but HPV-16 infection may also reside in the larynx and oral cavity. We evaluated HPV-16 status on OS of Head and Neck Squamous Cell Carcinoma (HNSCC) patients.2. Survival analysis was completed using Kaplan-Meier estimator and multivariate Cox regression analysis.HPV-16 infection was confirmed by amplification of E6 and E7 viral oncogenes through PCR assay and E6 IHC in 185 HNSCC samples. Associations between HPV-16 status and clinicopathological parameters were performed using Fisher’s exact test and xP = 0.002). HPV-16 positive tumors of the larynx (LSCC) and pharynx (PSCC) showed improved OS compared to HPV-16 negative tumors. Also, HPV-16 positive patients exposed to radiotherapy presented a better survival.OS of HPV-16 positive patients was longer compared to HPV-16 negative patients (HPV-16 status has a positive prognostic value in HNSCC. Addition of HPV-16 status to the TNM staging can provide better assessment in prognosis and guide treatment for HNSCC patients. HNSCC is the seventh most common type of cancer diagnosed, and it is ranked as the eighth cause of cancer death worldwide , 2. ThisIn the United States, Puerto Rican Hispanics, African-Americans, and economically disadvantaged Whites are at greater risk of developing HNSCC. The incidence of HNSCC in Puerto Ricans is 2.5 higher than Hispanics in the US . The incThe etiology of HNSCC involves a variety of toxic, environmental, and viral agents . StudiesThe complex anatomical structure of the head and neck area makes it very challenging for clinicians to determine the primary site of HNSCC . DetectiThis is a retrospective study where patients meeting the following criteria were eligible for inclusion: histologically proven squamous cell carcinoma arising from the pharynx , oral cavity, and larynx treated surgically between 1993 and 2005. Fresh-frozen tumor tissue was collected from all HNSCC accrued patients. Additionally, genomic DNA of HNSCC patients had been previously tested for HPV-16 status by Gp5+/6+ primer region within the L1 gene consensus PCR , HPV-16 Genomic DNA from all tumor samples was isolated using the DNA Isolation kit for Cells and Tissues  according to the manufacturer instructions. DNA concentration was measured with NanoDrop 8000 UV–vis Spectrophotometer .β-actin gene primers with a forward primer 5′-gcccatctacgaggggta-3′, a reverse primer 5′-ccttaatgtcacgcacga-3′, and a probe oligo 5′-accaccacggccgagcgg-3′. Reaction mixtures with SiHa DNA (1–2 copies of HPV-16) and K562 DNA (HPV-16 negative) were used as positive and negative control, respectively. qPCR reactions were carried out in a 96-well optical tray with a final volume of 25 μL. Each reaction consisted of 600 nM of each primer, 200 nM of each probe , 1X of TaqMan Universal PCR Master Mix (Applied Biosystems), which contains the Taq Polymerase, dNTPs, and ROX reference dye, and 75 ng of genomic DNA. DNA was amplified in a 7500 Real Time PCR System . Thermal cycling conditions were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and an annealing temperature of 60 °C for 1 min. All HNSCC samples classified as HPV-16 positive had amplification of E6 and E7 viral oncogenes through qPCR assay.HPV-16 status had been pre-screened by Gp5+/6+ consensus PCR followed by HPV-16 E6/E7 type-specific PCR, and results were confirmed for this study with a TaqMan-based qPCR targeted at HPV-16 E6 and E7 viral oncogenes. The HPV-16 E6 specific primer set included a forward primer 5′-gcacagagctgcaaacaactataca-3′, a reverse primer 5′-tcccgaaaagcaaagtcatatacc-3′, and a probe oligo 5′-tgtactgcaagcaacagttactgcgacgt-3′. The HPV-16 E7 specific primer set included a forward primer 5′-gatgaaatagatggtccagc-3′, a reverse primer 5′-gctttgtacgcaaccgaagc-3′, and a probe oligo 5′-cggacagagcccattacaatattgtaacc-3′. Quality and amount of input DNA samples were tested in each qPCR assay with 2, as appropriate. Odds ratio (OR) calculations for clinicopathological parameters were performed using binary logistic. Overall survival (OS) was measured in months from the date of diagnosis until death, if occurred. Survival analyses were performed using Kaplan-Meier curves. Log-rank Mantel-Cox and Gehan-Breslow Wilcoxon tests were used to determine the significance between two survival curves. Established prognostic factors having an impact on HNSCC survival were analyzed in a multivariate Cox regression analysis. Statistical significance was established to be p ≤ 0.05. Statistical analyses were performed using IBM SPSS Version 22 .Data from independent groups was compared using Fisher exact test or xPatient characteristics are shown in Table When we distribute the study cohort by HPV-16 status, 52.4 % were HPV-16 positive and 47.6 % were HPV-16 negative. The HNSCC sub-site distribution of the HPV-16 positive patients was 42.3 % LSCC, 37.1 % were OSCC and 20.6 % were PSCC. No statistically significant association was found between HPV-16 status and gender, age, risk factors and staging (Table P = 0.002). Cox regression analysis was performed and showed that HPV-16 positive HNSCC patients have an improved survival compared to HPV-16 negative patients after adjustment for established prognostic factors such as nodal status, tumor stage, cell differentiation, tumor site, heavy smoking and heavy drinking usage, age, and sex . As well, the OS of PSCC patients improves in HPV-16 positive cases (129.2 months) compared to HPV-16 negative cases (20.4 months) (P = <0.001). In contrast, the OS of OSCC patients was not significantly different between HPV-16 positive and HPV-16 negative cases    Fig. . Likewis11) Fig. .Fig. 3OvRecent studies have observed that HPV-16 positivity is a strong prognostic marker for OS in patients treated with primary radio-chemotherapy . We compP = <0.001). Likewise, the OS of HPV-16 positive patients with a history of heavy alcohol consumption was better (99.9 months) than HPV-16 negative patients (65.1 months) (P = 0.012) , we evaluated if HPV-16 status affects the OS of a HNSCC smoker population. As shown on Fig. Our study shows that HPV-16 positivity modifies the OS of HNSCC patients in two anatomical sub-sites, PSCC and LSCC, but not in OSCC Fig. . PreviouTP53 mutations, and lower expression of EGFR [The key mechanism for which HPV-16 infection gives a better prognosis to HNSCC patients is still unknown. However, it has been proposed that HPV-16 positive cells have an increased sensitivity to cancer therapies, a slower cellular growth rate, an enhanced immune response towards the virus, or a combination of these factors . Additio of EGFR . Because of EGFR –37. In cp53 mutations or an increase in EGFR expression [It has been proposed that HPV-16 positivity in HNSCC produces distinct tumor sub-site differences when exposure risks are combined, suggesting that different molecular pathways are involved . When wepression . If suchpression . Also, ipression .HPV-16 status is an important factor for establishing the prognosis and treatment of HNSCC. Our results showed that HPV-16 positive patients had a better response to radiotherapy when compared to HPV-16 negative patients. This increase in radiosensitivity could be mediated through wild–type p53-mediated apoptosis in HPV-16 positive cells and a lower chromosomal loss .The primary limitations of this study are the small number of early stages (I and II) HNSCC samples, and a small group of PSCC’s in our study cohort. These limitations may explain why we did not observe a statistical difference in early stage tumors by HPV-16 status. Our HNSCC patient population is composed mostly of late stage tumors , predominantly from LSCC and OSCC. This issue occurs because our head and neck cancer service is attached to a supraterciary level hospital care center which is responsible for the management of complex cancer cases for the whole island of Puerto Rico.In this study we showed that, HPV-16 presence, in HNSCC tumors, causes an increase in OS and increases radiosensitivity of tumor cells. Interestingly, we have shown that HPV-16 is present, not only in OPSCC as previously described, but it was also detected in LSCC and OSCC. Although the TNM classification has been effective for prognostication of HNSCC, HPV-16 detection may serve as a potential biomarker, in combination with the TNM, to better establish the prognosis of LSCC and PSCC and guide treatment. Future work should be directed to understand how HPV-16 affects HNSCC carcinogenesis, and how its infection modifies the disease progression increasing the OS in these patients.HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; IHC, immunohistochemistry; LSCC, larynx squamous cell carcinoma; OPSCC, oropharynx squamous cell carcinoma; OS, overall survival; OSCC, oral cavity squamous cell carcinoma; PSCC, pharynx squamous cell carcinoma; qPCR, quantitative real-time polymerase chain reaction; RTX, radiotherapy; TNM, TNM classification of malignant tumors


Of the 92 Invader positive samples, 58 (63%) were positive with the Cobas assay, while 34 (37%) were negative. Of the 134 Invader negative samples, 6 (4%) were positive with the Cobas while 128 (96%) were negative. The observed discordance may be attributed to the previously described false positive rate of the Invader ASR assay. The Cobas 4800 high-risk HPV assay is a viable new tool for use in the clinical setting to identify high-risk HPV.As new platforms for high-risk strains of human papillomavirus (HR HPV) testing are introduced into the clinical laboratory, it is important to verify their performance and agreement. In this validation study, post-aliquot cervical cytopathology specimens (n = 226) were used to analyze agreement between the Invader HPV ASR assay (Hologic) and the recently FDA-approved Cobas 4800 high-risk HPV assay (Roche). Residual sample from 92 Invader positive and 134 Invader negative samples were analyzed with the Cobas 4800 test. Discordant results were further analyzed by Linear Array HPV genotype testing (Roche). To assess intra- and inter-run precision, 31 Invader positive samples were run in duplicate on the Cobas 4800 by different operators over multiple days and purchased HR HPV DNA control was run in ten replicates. Cross-contamination during cytology processing was evaluated by spiking 6 Invader negative samples with different volumes of Acrometrix HPV High Risk Positive Control and analyzed on the Cobas with 4 negative samples in between. There was significant discordance between the assays (p < 0.001; exact McNemar X Cervical cancer is the second most common cause of cancer in women worldwide . Over thCurrent US Food and Drug Administration (FDA)-approved molecular methods of HR HPV screening in these specific populations include the Hybrid Capture 2 HPV DNA test , the Cervista HPV HR test , the Cervista HPV 16/18 test , the Cobas 4800 HPV test  and the Aptima HPV assay . Cervical cancer screening guidelines were written based on the performance of the Hybrid Capture 2 assay in the late 1990s and early 2000s. As new testing methods for HR HPV are approved and introduced into the clinical laboratory, it is important to verify their performance and agreement between tests and technologies. The Cobas 4800 high-risk HPV test is approved to detect HPV16, HPV18 and a pool of 12 other high-risk strains in pre-aliquot PreservCyt media  or Cobas PCR Cell Collection Media . The ATHENA HPV study and several smaller studies have compared the Cobas test with the Hybrid Capture 2 assay ,8,9. How2 test), with overall agreement of only 82%. Of the 20 randomly-selected Invader positive discordant samples analyzed by Linear Array HPV genotype testing, 4 were low-risk HPV and 12 were negative for HPV DNA. One sample lacked sufficient cellularity to obtain a result. The remaining 3 samples genotyped as HPV types of uncertain oncogenic risk . Of the 226 specimens tested by the Invader and Cobas for HR HPV, 92 were previously positive and 134 negative by the Invader assay. No specimens were excluded for inadequate cellularity/DNA content. Of the 92 Invader positive samples, 58 (63%) were positive on the Cobas 4800, while 34 (37%) were negative. Of the 134 Invader negative samples, 6 (4%) were positive by the Cobas 4800 while 128 (96%) were negative see . In totaThere was 100% concordance between the 31 positive samples run in duplicate on the Cobas 4800. The mean difference between the cycle threshold (Ct) of the two runs was 0.4 with a standard deviation of 1.4. The run-to-run coefficient of variation was 3.5%. There was not a significant difference between the runs (p = 0.9). All 10 HR HPV control samples tested positive for HR HPV with a mean Ct of 33.33 and standard deviation of 0.50 and a coefficient of variation of 1.5%. All interspersed negative samples tested negative. Of 10 the samples re-processed on the Cytyc T-3000, the 6 spiked positive HR control samples all tested positive for HR HPV on the Cobas 4800 while the 4 interspersed negative samples all tested negative.et al., show a lower potential false positive rate with the Cervista assay  . The assAt the time of this study, our laboratory performed reflex testing for HR HPV using the Invader HPV ASR assay on specimens with a cytologic diagnosis of ASC-US as well as co-testing for women over the age of 30 years upon clinician request. To perform this HR HPV testing, 4 mL of residual  cytology specimen in PreservCyt medium were aliquotted from the original collection vial and 1 mL of this sample was then used in the assay. Before HR HPV testing, these samples had been previously processed with the Cytyc T-3000 , an automated slide preparation technology for liquid-based cytology. In this study, we identified consecutive cervical cytopathology specimens (n = 226) between October 2011 and December 2011 that had already undergone Invader HPV testing and that had adequate residual volume in the PreservCyt medium for additional testing. Each of the residual PreservCyt cytology specimens was analyzed with the Cobas 4800 assay within three months of initial collection. PreservCyt specimens were stored at 4 °C. Twenty Invader-positive discordant samples were randomly selected and analyzed using the Roche HPV Linear Array to detect the presence or absence of HPV DNA and to genotype positive samples. To assess precision, 31 Invader positive samples were run in duplicate on the Cobas 4800 by different operators over multiple days. Additionally, HPV High Risk control material was purchased from Acrometrix, Inc and was analyzed on the Cobas 4800 in 10 replicates within the same run with previously tested negative samples between each positive control.To control for possible sample-to-sample cross-contamination during cytology processing by the Cytyc T-3000, 6 Invader negative samples were spiked with different volumes of Acrometrix HPV High Risk Positive Control: 2 samples with 4 mL, 2 with 2 mL and 2 with 1 mL. These 6 spiked samples were re-processed on the Cytyc T-3000 with 4 previously negative samples interspersed between the controls in the following order: 4 mL, blank, 2 mL, 1 mL, blank, 4 mL, blank, 2 mL, 1 mL, blank. Each sample was analyzed for HR HPV on the Cobas 4800.In summary, poor agreement between the Cobas 4800 and Invader HPV ASR assays can be attributed to the false positive rate of the Invader assay as determined by HPV genotyping. We conclude that the Cobas 4800 assay offers an important advantage in HR HPV sensitivity on a platform with excellent precision. The Cobas 4800 also offers other advantages such as simultaneous genotyping of HR HPV types 16 and 18, lower false positive rate than the Invader assay, smaller batch size (between 24 and 96 samples) and more rapid turn-around time (5 hours), thus providing a viable new tool for use in the clinical setting to identify HR HPV.


We aimed to clarify the possible role of human papillomavirus (HPV) infection in the malignant transformation of sinonasal inverted papilloma (IP).INK4a gene products were investigated by immunohistochemistry.Subjects comprised 32 patients with chronic rhinosinusitis (CRS), 17 with IP, 5 with IP and squamous cell carcinoma (IP + SCC), and 16 with primary sinonasal SCC. HPV presence, viral loads, and physical status were investigated using polymerase chain reaction. Retinoblastoma (pRb), p53, and p16p = 0.04). High-risk HPV-16 was the most frequently encountered subtype . HPV-16 viral loads varied from 2.5 to 7953 E6 copies/50 ng genomic DNA. Patients in the SCC and IP + SCC groups had significantly higher viral loads than those in the IP and CRS groups (p < 0.01). All SCC and IP + SCC patients with HPV-16 demonstrated mixed-type integration, whereas 4 of 5 HPV-16 patients in the IP and CRS groups showed episomal type infection (p = 0.04). Positivity to pRb was found in 78.1 % of CRS, 35.3 % of IP, and 68.8 % of SCC cases. The presence of HPV DNA negatively correlated with pRb expression in SCC (p = 0.029) and IP (P = 0.049) groups. Although 62.5 % of SCC cases exhibited p53 positivity, only 5.9 % of IP, and no CRS cases were positive. Regardless of HPV status, p16INK4a positivity was frequently detected in IP cases (82.4 %), less in SCC (12.5 %) cases, and was not detected in the CRS group. Neither the IP nor SCC cohorts showed any correlation between HPV presence and the expression of either p53 or p16INK4a.HPV DNA was detected in 6.3 % of cases with CRS, 29.4 % with IP, 40 % with IP + SCC, and 25 % with SCC. IP cases had significantly higher HPV presence than CRS cases (INK4a is not a reliable surrogate marker for HPV infection in IP.HPV infection was more frequent in the IP, IP + SCC, and SCC groups than the CRS group. Higher viral loads and integration observed in the IP + SCC and SCC groups, and an inverse correlation between HPV presence and positive pRb indicated that persistent infection and integration play a part in tumorigenesis and malignant transformation in certain IP cases. However, p16 A variety of benign and malignant tumors may arise in the sinonasal cavity. Of these, inverted papilloma (IP), a benign neoplasm, has unique clinical characteristics; of note are its high rates of recurrence and malignant transformation. According to several systematic reviews and meta-analyses, the malignant transformation rate of IP is estimated to be approximately 10 % , 17, 20.Human papillomavirus (HPV) infection in the sinonasal tract is estimated to be present in 37.8 % of IPs  and 27 %In our previous study, viral loads and physical status of HPV were examined using fresh frozen samples of IP, SCC, and inflammatory mucosa from the sinonasal tract to clarify the clinical importance of HPV in IP. HPV genomes were detected in 46.1 % of IPs, 27.3 % of SCCs, and 7.6 % of the inflammatory group, respectively . BecauseINK4a and cyclin D1 genes are components of the pRb cell cycle control pathway. The active hypophosphorylated form of pRb binds and blocks the action of the transcription factor, inhibiting the transition from G1 to S phase. Cyclin D1 stimulates the phosphorylation of pRb by associating itself with cyclin-dependent kinases (CDKs). Binding of p16INK4a to CDK 4 and 6 blocks their association with the D-type cyclins. Another well-known cellular tumor suppressor, p53, is involved in processes such as cell cycle progression, DNA repair, chromatin remodeling, differentiation, apoptosis and senescence. HPV-mediated tumorigenesis is mainly due to the activities of two viral oncoproteins, E6 and E7. HPV E6 can induce the degradation of p53 by direct binding to the ubiquitin ligase E6AP, inhibit p53-dependent signaling upon stress stimuli, and contribute to tumorigenesis [The retinoblastoma (pRb), p16igenesis , 27, 36.INK4a gene products—were investigated to reinforce our assumptions from the previous study.Several reports concerning cell cycle protein expression in IP, IP + SCC, and SCC have been published , 36, 37.The study protocol was approved in advance by the Institutional Review Board of the University of the Ryukyus. This study was conducted to conform to the principles of the Declaration of Helsinki.We have obtained consent to publish histologic pictures from the participants.Patient characteristics Table Table 1PaAccording to Krouse classification  of IP, T2)HPV detection Table Table 2HPMetachronous SCC occurred in 3 cases and synchronous SCC in 2 cases of the IP + SCC group. The periods between the initial treatments for IP to malignant transformation in the 3 metachronous SCC cases were 5 months, 6 years, and 9 years. Patients in the IP + SCC group received surgery and subsequent radiotherapy as adjuvant treatments for cancerous lesions. Although 1 case recurred after surgery combined with radiotherapy in the IP + SCC group, salvage endoscopic resection for the recurrent lesion was performed successfully and the lesion has not recurred for 9 years. The patient had HPV-16 infection with mixed integration.p = 0.04, Fisher’s exact test).HPV genomes were detected in 5 of the 17 patients in the IP group . In addition, all patients with HPV-16 infection in the SCC and IP + SCC groups demonstrated mixed type integration, whereas 4 of the 5 patients with HPV-16 infection in the IP and CRS groups showed episomal type infection .3)INK4a by immunohistochemistry  and multiple HPV infections were not detected. Of the 13 HPV patients, HPV-16 was present in 10, therefore viral loads and physical status of HPV-16 were investigated in the positive samples. Viral loads varied from 2.5 to 7953 INK4a. The expression of pRb was observed in 78.1 % of the CRS group . A similar relationship between the presence of HPV DNA and pRb expression was observed in the IP group .Figure oup Fig. , 35.3 % The expression of p53 was observed in 10 of the 16 patients (62.5 %) in the SCC group and 1 of the 5 patients (20 %) in the IP + SCC group, whereas it was expressed rarely in the IP (5.9 %) and CRS . HPV infection in the IP group was not related to p16INK4a expression  samples. However, the accumulation of p53 to levels detectable by immunohistochemistry is associated with p53 mutations . In the  with IP . AlthougINK4a has been proposed as a surrogate marker for HPV infection [INK4a as a biomarker. Smith et al. found no concordance between p16INK4a expression and HPV detection in 20 % of head and neck cancers [INK4a activation [INK4a overexpression was 53.2 % for the detection of HPV DNA in HNSCC, and was considerably better in oropharyngeal SCC at 80 % [INK4a overexpression was identified in only 1 of the 4 SCC cases with HPV, but in more than 80 % of IP cases. These findings are consistent with previous reports [INK4a in IP are unknown, these results revealed that, in contrast to OPSCC, p16INK4a immunoreactivity is not a surrogate marker for HPV infection in IP.Expression of the tumor suppressor p16nfection . Overexp cancers , possibltivation . In our  reports . AlthougIn summary, the abovementioned immunohistochemical characteristics in specimens from patients with HPV infection in the IP, IP with SCC, and SCC groups were similar to other HPV-related tumors. These immunohistochemical findings and current PCR-based results suggest that HPV infection is involved in inducing malignant transformation of IP through alteration of cell cycle protein expression.INK4a is not a reliable surrogate marker for HPV infection in IP.HPV infection was more frequently observed in IP, IP + SCC, and SCC groups than in the CRS group. The higher viral loads and integration observed in the IP + SCC and SCC groups and the inverse correlation between HPV presence and positive pRb in immunohistochemistry indicated that persistent HPV infection and integration are involved in tumorigenesis and malignant transformation in certain IP cases. However, p16Patient characteristics.The clinical features in each group, such as age, sex, and tumor stage, were reviewed from medical records.Detection of HPV genome and identification of HPV types.The subjects in the present study consisted of 17 patients with IP (IP group), 5 with IP associated with SCC (IP + SCC group), 16 with primary sinonasal SCC (SCC group), and 32 with chronic rhinosinusitis (CRS group). Surgical treatments were performed on all patients in the IP and IP + SCC groups. Specimens from 17 patients in the IP group, 1 in the IP + SCC group, 15 in the SCC group, and 32 in the CRS group were collected and stored in liquid nitrogen until analysis. FFPE samples were used for the analysis of only 4 of the 5 cases from the IP + SCC group, and fresh frozen samples were used for the analysis of all other cases in the CRS, IP, and SCC groups, and 1 case in the IP + SCC group.Fresh frozen samples were analyzed for HPV and HPV types as previously reported . A GentrThe presence of HPV DNA was analyzed by PCR using the general consensus primer sets GP5+/GP6+ and MY09/11 [E6 and E2 genes of HPV-16 with the ABI Prism 7300 Sequence Detection System (Applied Biosystems) and TaqMan PCR Master Mix II . The procedures, including experimental conditions, primers and TaqMan probes, were identical to a previous report [E6 and E2 genes were generated by amplification of serial 10-fold dilutions  of a plasmid pB-actin carrying the complete HPV-16 early region . For cellular DNA quantification, an external standard curve was generated using known serial dilutions  of human genomic placental DNA , and β-globin was amplified as described by van Duin et al. [t (threshold cycle) values against the logarithm of the standard curve. The relative viral load was identified by calculating the copy numbers of specimen E6 in 50 ng cellular DNA. The physical status of HPV-16 was determined according to a previously described method [E6 was calculated by subtracting the copy numbers of E2  from the total copy numbers of E6 . Ratios of E2 copy number/total E6 of <1 indicate the presence of both integrated and episomal forms (mixed-type integration). An E2/E6 ratio nearly equal to 1 indicates predominance of the episomal form, whereas a ratio of 0 indicates the presence of only the integrated form.3)INK4a.Immunohistochemistry for pRb, p53, and p16To investigate HPV-16 viral loads and physical status, quantitative real-time PCR using HPV-16 DNA-positive samples was performed to measure the quantities of the s report . Two stan et al. . The amod method . The amoINK4a antibody . After extensive washing in phosphate-buffered saline, the slides were incubated for 30 min at room temperature with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (MTM Laboratories). Immunolabeling was visualized by incubation in 3-3′-diaminobenzidine and stained slides were counterstained with hematoxylin.Serial 4 μm-thick sections from FFPE samples were deparaffinized in xylene and hydrated in a graded series of alcohol. Epitope retrieval was performed by heating at 95–99 °C for 10 min in Tris/EDTA buffer (pH 9.0). Endogenous peroxidase activity was quenched by incubating the sections in 3 % hydrogen peroxide and 15 mM sodium azide for 5 min. The sections were subsequently incubated overnight at 4 °C with primary monoclonal mouse anti-pRb antibody  for pRb, primary monoclonal mouse anti-p53 antibody  for p53 staining, and primary monoclonal mouse anti-p16INK4a-positive when intense nuclear and/or cytoplasmic reactivity was present. Positive expression was defined as pRb and p53 staining in more than 25 % [INK4a staining in 40 %, of 2000 tumor cells [Cases were considered pRb-, p53-, or p16han 25 % , or p16Ior cells .


Estimation of pre-immunisation prevalence of HPV and distribution of HPV types is fundamental to understanding the subsequent impact of HPV vaccination. We describe the type specific prevalence of HPV in females aged 20–21 in Scotland who attended or defaulted from cervical screening using three specimen types; from attenders liquid based cytology and from defaulters urine or self-taken swabs.n = 2148), collected from women aged 20–21 attending for their first smear were genotyped for HPV. A sample (n = 709) from women who had defaulted from screening was also made available for HPV testing through the use of postal testing kits (either urine samples (n = 378) or self-taken swabs (n = 331)). Estimates of prevalence weighted by deprivation, and for the postal testing kit, also by reminder status and specimen type were calculated for each HPV type. The distribution of HPV types were compared between specimen types and the occurrence of multiple high-risk infections examined. The influence of demographic factors on high-risk HPV positivity and multiple infections was examined via logistic regression.Residual liquid based cytology samples  or those associated with cross-protection  was common. Individuals were more likely to test positive for high-risk HPV if they resided in an area of high deprivation or in a rural area. The overall distribution of HPV types did not vary between defaulters and attenders. Multiple infections occurred in 48.1% of high-risk HPV positive individuals. Excluding vaccine types the most common pairing was HPV 56 and 66.Understanding of the pre-immunisation prevalence of HPV in young women puts Scotland in a prime position to assess the early effect of vaccination as the first highly vaccinated cohorts of individuals enter the screening programme. Differences in results with different specimen types must be taken into account when monitoring the impact of vaccination programmes. The prevalence and circulating genotypes of human papillomavirus (HPV) infection in different study populations has recently been reported -3. The iA better understanding of the natural history and epidemiology of HPV feeds into models of vaccine impact and cost effectiveness to inform decision-making on HPV immunisation and future changes to cervical screening. Defining a baseline against which to monitor the impact of vaccination provides a means to measure the earliest expected change - which is a decrease in the prevalence of vaccine-specific HPV.The range of study populations, severity and types of associated pathology in target age groups means that few of the studies on HPV prevalence are generalizable. Estimates of HPV prevalence in the general population often come from studies that consider samples from cervical screening programmes due to ease of sampling and availability of a link to cytology/disease status ,3. TheseFor an estimate of Cervarix® vaccine impact it is important to ascertain the effect of the vaccination programme on the whole population, with particular focus on the age group where these changes will be observed first. In Scotland, cervical screening currently begins at age 20, therefore it is one of the few countries in the world able to detect early impacts of the vaccine which was introduced in September 2008. The vaccine was implemented as routine in-school vaccination of those aged 12–13 with a catch-up campaign for all those aged 13–17 years old at the time  and achiThis paper describes the methods used to establish representative population-based estimates of HPV prevalence in unvaccinated women aged 20–21 in Scotland. By using two sampling methods, we examined the prevalence of HPV in those who attended for cervical screening and in those who did not and the distribution of HPV types detected. We aimed to improve the representativeness of the baseline HPV prevalence estimates and compare patterns between those who default from and those who attend for screening. In addition, we examined the effect of different specimen types upon estimates of HPV prevalence. The occurrence of multiple HPV types is examined with particular emphasis on co-incidence of high-risk vaccine types (16 and 18) with other high-risk types. Such estimates are fundamental to understanding the impact of the HPV vaccination in the general population, while validating the public health approach taken in Scotland.Scotland has a population of approximately 5.3 million  and an oIn 2009, anonymised residual liquid based cytology (LBC) samples from young women aged 20–21 years attending their first screening appointment were collected from all (11) NHS cytopathology laboratories in Scotland, which have national coverage. To achieve the desired sample size of 2,000 specimens, each laboratory collected over a two month period, staggered throughout the year, and selected samples from women born after 1st January 1988, with the number of samples collected proportionate to the population which the laboratory served.Self-taken specimens were obtained using a postal testing kit (PTK) for women who defaulted from attending their first screening appointment in 2008. From an anonymised data file of 15367 women, 5500 were randomly selected across all Health Board administration areas ensuring a geographically representative sample. Half of the randomly selected women were sent a urine test kit and half a self-taken vaginal swab kit. Details of the methodology are reported elsewhere . National surveillance has been approved through the NHS National Clinical Governance committees and Caldicott Guardians at individual NHS Boards. Data linkage of information was approved by the NHS National Services Scotland (NSS) CHI advisory group.Characteristics of the cervical screening and self-sampling groups are shown in Table p = 0.001) and similarly for the urine samples , p < 0.0001)  indicating little difference in HPV types between those who defaulted and those who attended screening. Within the PTK sample, urine and self-taken swabs were found to have a different distribution of types (p = 0.032) with higher detection of HPV types 16, 56, 18, 51, and 45 in the self-taken swabs compared to urine.Comparison of the distributional spread of HPV types, excluding the “HPV other” category, showed that the aggregated PTK sample (urines and swabs together) did not have a significantly different distribution compared to the LBC samples . No significant interactions were found . In the multivariable model, the linear effect of quintile of non-white residents became statistically non-significant (p = 0.497) and was removed from the model.Adjusting for the effect of specimen type, urban/rural classification, SIMD and proportion of non-white residents were all found individually to have a statistically significant linear trend effect on positivity  and of urban/rural residence (p = 0.0014) and a difference between the specimen types (p < 0.0001). The adjusted odds ratios indicate that individuals in SIMD4 and SIMD5 (least deprived) had lower odds of high-risk HPV positivity than those in SIMD1 (most deprived) (Table p = 0.477).The reduced model showed a linear effect of SIMD (d) Table . The oddn = 2771). Of those infected with more than one high-risk HPV type, 58.4% were infected with two types, 25.6% with three, 9.9% with four, 4.3% with five and 1.8% with more than five. The maximum number found in an individual was 8 high-risk HPV types.Overall, 1271 individuals tested positive for any HPV and 1021 for a high-risk HPV type. Of these 1021, 491 (48.1%) were infected with more than one high-risk type, 17.7% of the total samples , urban/rural status (p = 0.46) or ethnicity quintile (p = 0.62) and the odds of having a multiple high-risk HPV infection compared to a single high-risk HPV. Differences were found in the odds of detecting multiple high-risk HPV infection between specimen types (p = 0.016) with fewer multiple infections detected in self-taken samples in line with the reduced sensitivity of these collection methods ; Urine OR = 0.60 ).Amongst those positive for any high-risk HPV no significant association was found between SIMD quintile  individuals compared to those who attend for screening  we wouldHPV infection was common in this population of young women and multiple infections were detected in approximately half of all HPV positive samples. Baseline pre-immunisation levels of the vaccine specific types were high, with HPV 16 the more common than HPV 18. The aggregate occurrence of non-vaccine types, in particular those deemed to be high-risk, is more prevalent than the vaccine specific types and our results are comparable to those of other recent UK based studies ,17.Individual risk factors associated with HPV infection status, such as sexual activity and ethnic minority background , were noThere was no overall difference in the distribution of HPV types between the aggregate PTK sample and the LBC samples, implying that HPV patterns are similar in both populations. In terms of prevalence estimates, urine and self-taken swabs are not comparable to LBC samples nor to each other but if used consistently could allow trends to be monitored for surveillance purposes especially in those defaulting from screening .Multiple high-risk HPV infections were common, with around half of those infected having more than one type detected. Quantifying and defining the extent of multiple HPV infection is important as it may limit or enhance the effectiveness of the immunisation programme. With regard to the former, any increase in the prevalence of non-vaccine high-risk genotypes due to type replacement rather than unmasking could erode the programme’s cost-effectiveness. On the other hand, cross protection of the vaccine may somewhat mitigate against type replacement, particularly against 31, 33 & 45. There is some evidence suggesting that Cervarix® may confer relatively high levels of cross-protection .Sexual behaviour has been shown to influence the risk of having multiple HPV infections and overall there are differences between younger and older women in factors that influence high-risk HPV positivity . There iOur study estimated the prevalence of HPV in unvaccinated young women living in Scotland. This study was essential in order to determine the baseline burden of HPV infection before vaccination. Specimens were tested using the same HPV assay at the same testing laboratory ensuring consistency of results. Although the varying sensitivity of the different specimen types in detecting HPV does not allow direct comparison between the prevalence of HPV in those attending screening and those defaulting, the similarity in the type specific HPV prevalence distributions and the consistent differences in the prevalence estimates obtained from the three sample types suggest that the infection pattern between defaulters and attenders is unlikely to be substantially different.Due to the low uptake of defaulters to the PTK  the effeWe are now in a prime position to ascertain the early effects of the HPV vaccine on young women who were part of the initial vaccination campaign and who were called for cervical screening from 2010. Having a systematic surveillance programme which has included the characterisation of a pre-vaccine baseline will allow us to robustly determine these effects.The authors declare that they have no competing interests.KK conducted the statistical analysis and led the drafting of the paper with all authors. CR, KS, KC and HC designed the study. KC undertook HPV testing of the samples. JL managed and linked the data. KS, AP, KP and MD managed the programme and provided epidemiological interpretation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/519/prepub


Breast cancer is one of the most important neoplasia among women. To reduce its incidence and mortality impact it would be desirable to early identify risk factors associated with its development. It was recently suggested that biological agents could be the etiological cause, particularly Human Papilloma Virus (HPV). No specific relationship with different breast cancer types has been demonstrated until now. In particular, the triple-negative breast cancer (TNBC), characterized by a receptor negative pattern (ER/PgR/HER2–negative) and poor prognosis, can represent one of the most relevant clinical and public health priority in terms of observational research.Aim of the study was to evaluate the HPV-positivity prevalence in two breast cancer series (TNBC vs. non-TNBC) in Northern Sardinia, Italy. The sample size of each group was represented by 40 formalin-fixed and paraffin-embedded specimens. The mean age was 60.3 years. The majority of the cancers were ductal (84%). The grading distribution was different: G2 was the most prevalent grade in the non-TNBC series, whereas G3 was the most frequent in the TNBC series . Six biological samples were HPV-positive (7.5%): the positivity was assessed only in the TNBC group . The isolated genotypes were: 16, 31, 45, 52, 6, and 66. Only one co-infection was found .The prevalence of HPV-positivity in TNBC specimens was 15%. On the basis of its carcinogenetic ability, an etiological role in the pathogenesis of the cancer could be supposed. This association should be confirmed with longitudinal studies to better assess the role of the HPV infection in TNBC and non-TNBC tumors. Moreover, risk factors may be not recognized in a large proportion of patients [Breast cancer is the most common cancer and the principle cause of death from cancer in women worldwide . Althougpatients . Therefopatients  from genpatients ; howeverpatients -10 but opatients -13. Incopatients -16; besipatients -19.In clinical practice, according to a recent molecular classification, immunohistochemical expression of Estrogen (ER) and Progesterone (PgR) receptors and HER2 allows to categorize breast cancers in different prognostic subgroups , with distinct therapeutic approaches . In partRecently, our group performed a case–control investigation, which described a high overall prevalence of HPV cervical infection in women living in Northern Sardinia, Italy. In particular, HPV types −16 and −51 DNA were detected in 12.5 and 6.3% of atypical squamous cells of undetermined significance; 24.0% and 16.0% of low-grade squamous intraepithelial lesions; 50.0% and 50.0% of high-grade squamous intraepithelial lesions, respectively . MoreoveOn this basis, we carried out a case–control study aimed at identifying the presence of HPV-DNA in embedded tissues series of TNBC diagnosed in women from Northern Sardinia.Ethical approval and informed consent for this study was unnecessary, according to the Italian legislation concerning the guidelines for the performance of observational studies (G.U. n. 76. 31-3-2008). However, a formal approval of the study protocol was requested in 2013 to the Ethical Committee of the Azienda Sanitaria Locale n°1 of Sassari, Italy (PN-1137/L 2013).A case–control study, based on a case–control design, was performed to evaluate the prevalence of HPV infection in a subset of 40 embedded TNBC and 40 embedded non-TNBC, as controls; both groups of breast cancer series belonged to women admitted to a tertiary University Hospital located in Sassari, Italy.An ad-hoc case report form was created to record demographic, histopathological and virological information. Data were anonymized in compliance with the Law Decree No. 196/2003, article 24 .http://www.who.int/biologicals/areas/vaccines/hpv_labnet/en/).Based on a sample size calculation, 40 formalin-fixed and paraffin-embedded (FFPE) tissues from TNBC specimens were consecutively and retrospectively selected from the pool of those collected in the Pathology Unit, Department of Clinical and Experimental Medicine, University of Sassari. The selection of every TNBC specimen was associated with a FFPE non-TNBC specimen, as control, paired by age (±5 years). The enrolled series was diagnosed by a pathologist who was blinded to the final diagnosis. The same methodological blinding procedure was followed for the virological analysis, carried out in triplicate at the WHO quality-assured laboratory of the Hygiene and Preventive Medicine Unit, Department of Biomedical Sciences, University of Sassari  in accordance with the manufacturer’s instructions. To obtain genomic DNA 10ul of RNase A  were applied directly to the silica membrane to digest contaminating RNA. The quantity and the quality of nucleic acids were assessed spectrophotometrically  using Nanodrop ND1000 . Positive and negative controls were represented by HPV-positive samples and water blanks, respectively HPV DNA sequences were detected with the INNO-LiPA HPV Genotyping Extra kit , based on the reverse hybridization principle, according to manufacturer’s instructions. The INNO-LiPA assay covers all currently known high-risk HPV genotypes, such as high-risk , low-risk HPV genotypes , and some additional types , while HPV 13, 34 and 67 genotypes are interpreted as HPV X. The INNO-LiPA assay utilizes a cocktail of biotinylated consensus primers (SPF10) to amplify a 65-bp region within the L1 Open Reading Frame of multiple HPV types. The biotinylated PCR products are then genotyped by hybridization to HPV type-specific oligonucleotide probes bound to nitrocellulose membranes and detected by an alkaline phosphatase-streptavidin conjugate and colorimetric detection. Post-PCR hybridization and colorimetric detection were performed using the Auto-LiPA 48 instrument in accordance with the manufacturer’s recommendations. At the end of color development, the strips were scanned and evaluated by the Line Reader and Analysis software (LiRAS) to determine if defined bands for a particular genotype probe were visible. A biological sample is considered HPV positive if at least one of the type-specific lines or one of the HPV control lines is positive. An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. When positivity for HR genotypes −16, −18, −31, −45, −51, and −52 was found, confirmation was obtained testing biological samples with an “in-house” Real-Time quantitative TaqMan PCR assay [The sample size, calculated considering a first type error of 0.05, based on a case control ratio of 1:1, and an estimated exposure prevalence of 0.134, computed pooling proportions of 7 studies conducted in Europe , with a A total of 80 embedded breast cancer specimens were analyzed: 40 were TNBC and 40 were embedded non-TNBC, namely 38 “luminal A” and 2 “HER2 overexpressing” variants. The mean (standard deviation) age of the patients was 60.3 14.2) years; in particular, it was 60.3 (15.8) and 60.3 (12.6) for TNBC cases and controls, respectively . Patients’ characteristics and pathology features are described in Table 4.2 years1 7.6% (n = 6), G2 49.4% (n = 39), and G3 43.0% (n = 34); in particular, G1 0.0% (n = 0), G2 28.2% (n = 11), and G3 71.8% (n = 28) for the TNBC cases; conversely, the proportion for the non-TNBC controls was G1 15.0% (n = 6), G2 70.0% (n = 28), and G3 15.0% (n = 6) .According to histopathological classification, the majority of the cancers were ductal , followed by lobular , and others . The proportional grading distribution in the entire series was the following: GOut of 80 patients six  were positive for at least one HPV genotype. In particular, HPV positivity was detected only among the 40 TNBC . The most frequent genotypes were HPV-16 (28.6%), −31 (14.3%), −45 (14.3%), −52 (14.3%), −6 (14.3%), −66 (14.3%); the only co-infection detected was HPV-6 and HPV-66.Logistic regression analysis did not identify covariates associated with HPV-positivity (results not showed).HPV genome was detected in approximately 8% of the cases suggesting a viral pathogenesis of the cancer on the basis of its well known role in the carcinogenesis of the epithelial cells , even ifThe difference in terms of cellular differentiation was striking: the proportion of G3 was significantly higher in the TNBC group than in the non-TNBC category. However, no differences for the histological type were found, since the majority of the carcinomas showed ductal differentiation.TNBC were the most undifferentiated carcinomas: they were the only neoplastic type found positive to the molecular analysis aimed at diagnosing HPV DNA. The non-TNBC series were HPV negative, and the difference in terms of positivity was statistically significant.A more prevalent genotype was not identified: those detected were genotypes usually identified in the geographical context where this epidemiological study was carried out ,23.Even if the study carried out in our center detected only HPV-positivity in the TNBC, the probability of virological positive results in other cancers showing different receptor patterns could be hypothesized . NevertAt our best knowledge, our research is one of the first observational studies proving an association between TNBC phenotype and HPV-positivity. Moreover, our findings strengthen the hypothesis of an association between HPV-positivity and poorly differentiated variants. Therefore, our results, which can represent the basis for future studies, should be carefully evaluated because only a few HPV positive cases were identified and other studies found HPV in breast tissues with estrogen-receptor expression . MoreoveFurthermore, the explanation of the role played by the host susceptibility will be necessary, proving if the genetic background can predispose to an imbalance of the native and/or adaptive immunity favoring the infection in that specific anatomical area, as well as the activation of an uncontrolled proliferation cycle.The demonstration of the etiologic role of the HPV infection could be relevant, particularly because of the availability of highly efficacious preventive vaccines; in case of a research concordance on the role of HPV, this primary prevention strategy could increase the armamentarium of the current tools implemented in the breast cancer prevention.i.e. the case/control design, is the one suggested in case of rare events. The sample size is smaller than the statistical population size but, in order to avoid unreliable results related to unpowered studies, we computed the hypothesized sample size representative of the current population on the basis of several epidemiology-based assumptions. Furthermore, we could not assess the pathogenetic role of the HPV genotypes: new molecular hybridization in situ studies are needed to evaluate the cellular location and the pattern of hybridization of the HPV genome, that is to evaluate if the HPV-DNA is integrated in the human DNA or shows episomal replication in the neoplastic nuclei. It is straightforward that the integration in the human genome can alter the control of the cellular duplication cycle. Another study we are performing will try to answer to that relevant pathogenetic issue. The risk of a cross-contamination was avoided adopting sterile procedures during the preparation of the biological samples; the methodological efficacy of the molecular procedures was indirectly demonstrated by the detection of six different genotypes out of six positive TNBC samples.This study shows some limitations. Its retrospective nature cannot assess if HPV infection precedes the cancer development. However, the epidemiological nature of the study, Another unsolved problem is the origin of the HPV: to understand the original site of infection is important for the identification of preventive measures. On this basis, a concomitant virological study on cervical and/or oral-laryngeal samples could have allowed to appreciate if the sexual transmission has a role in the human spread of HPV types . UnfortuIt is clear that the immunization programs, carried out in young adults, could play a role in the near future in the protection against those genotypes involved in the cancer pathogenesis and included in the currently available vaccines. However, the low prevalence of HPV suggests that other epidemiological, demographic , clinical, virological risk factors should be assessed to better understand the potential synergistic or additive pathogenetic role of HPV genotypes.