Cancer statistics report an increased incidence of OSCC and OPSCC around the world. Though improvements in screening and early diagnosis have dramatically reduced the incidence of this neoplasm in recent years, the 5-year-disease-free survival, is still poor, specially for oropharyngeal cancer, despite the great scientific and financial efforts. Recently, several papers showed that HPV may be involved at least in the pathogenesis of a subgroup of oral and cervical SCC, leading to distinct molecular characteristics compared with HPV-negative ones. Nevertheless, OPSCCs associated with HPV infection seem to show a better prognosis and affect younger patients (< 40 yrs.), especially females. Therefore, there is the need to properly assess oropharyngeal SCC subgroups: 1) not HPV associated/classic oral SCC: less responsive to anticancer drugs: needs novel post-surgical treatment; 2) HPV associated/oral SCC: needs several management options and suitable "target" therapy against the virus, and/or immune-stimulating therapy. Further issues are: 1) the disclosure of putative targets for more efficient molecular therapy, which may work as cervical cancer post-surgical treatment, in anticipation of the effects of "global prevention" performed by WHO anti-HPV vaccination programs; 2) careful identification of precancerous lesions in both sites; dysplasia is currently treated by excisional or ablative procedures, which don't consider the concept of field carcinogenesis. In fact, it is probable that near or far from an excised precancerous lesion new foci of cell transformation may exist, which are not yet macroscopically evident, but, if detected, would put the patient into a high risk subgroup.Comparing findings reported in the recent literature, the data of this state of the art about HPV might add useful informations concerning oropharyngeal carcinogenesis. Moreover, our review would be useful in order to define novel perspectives of treatment choice for Head & Neck cancer patients, by combining well known chemotherapeutical drugs with new molecular "target" therapy. HNC is the eighth most common cause of cancer death worldwide. Its incidence varies widely among different regions. In North America and the EU, HNC accounts for 3% to 4% of all cancer diagnoses. Conversely, in Southeast Asia and Africa, HNC accounts for approximately 8% to 10% of all cancers . Up to nIt is well known that there is a strong association between gene, environment and cancer. Several factors are involved in oral carcinogenesis, such as age, gender, ethnicity, lifestyle, genetic background, status of health and exposure to one or more oncogenic factors . In sevein vitro [Thus, other agents, such as viruses, are being investigated. In particular, with regard to viral involvement, it is still highly controversial whether HPV, widely reported as one of the prominent mechanism behind the development of cervical squamous cell carcinoma, can also be considered an aetiological or a malignant risk factor in oral carcinogenesis . Accordiin vitro ; 4) the in vitro ,19 and, in vitro .In a recent review on epidemiologic and molecular bases, according to Ha and Califano , the Aut- histological grading (well differentiated cancer) and histotype ;- response to chemo-radiotherapy, innovative targeted therapy and/or immunostimulating strategies;- clinical outcome in term of overall survival -24; partBased on recent meta-analysis, besides the classical horizontal transmission during the sexual life, a vertical transmission occurs in approximately 20% of case HPV positive people with HPV-DNA detection in amniotic fluid, foetal membranes, cord blood and placental trophoblastic cells, all suggesting HPV infection in utero, i.e. prenatal transmission .Furthermore, recent studies have proved the existence of a statistically significant synergistic effect between HPV and alcohol. Among heavy alcohol users detected with the virus, the risk of head and neck cancer was statistically significantly increased relative to that of HPV-negative cancer drinkers. Alcohol can biologically modify mucosal tissue, potentially increasing its permeability to viral infection, or it could influence the immune response to HPV . On the However, the protective mechanism by which HPV infection improves the overall survival and prognosis in oral cancer is not yet clearly understood.HPVs are a group of host specific DNA virus with a remarkable epithelial cell specificity. More than 120 different HPV genotypes have been identified and almost 45 subtypes, isolated from the low genital tract, have been grouped into high-(HR) and low-risk (LR) HPV types, considering their risk potential to induce an invasive cervical cancer .In a recent study Munoz et al. classify HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 as high risk viruses, detectable in high grade squamous intraepithelial lesions or in invasive cancer; HPV26, 53 and 66 as potential high risk with a not well known oncogenic potential; while types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 and 89 can be considered as viruses with low oncogenic risk and they can be isolated from low grade epithelial lesions .HPV16, the most common HR HPV type detected in biopsies from women with cervical SCC (55%), was also the most common type detected in biopsies from HNCs (85-95%). In the oropharynx, HPV16 accounted for the overwhelming majority of HPV-positive cases (86.7). Aside from HPV16, other oncogenic HPV types commonly detected in invasive cervical cancer biopsies  were rarely or never detected in HNC biopsies .Conversely, HPV6, which has been designated as ''low-risk'' (LR HPV) or ''non-oncogenic'' to the cervix, was present in a greater number of HNCs than any of the oncogenic types other than HPV16.It is interesting to note that the classically LR HPVs 6 and 11 subtypes have been found in some tonsillar and laryngeal carcinomas. It is well known that in the rare event of benign laryngeal papillomas undergoing malignant transformation, HPV11 has been most commonly implicated. HPV 6 and 11 have also been implicated in malignancies such as Ackerman's tumour . It is clear that in certain rare malignancies, HPV6 and 11 can play a role .Finally, meta-analyses have shown that the HPV subtypes associated with HNC are broadly similar  with those seen in cervical carcinoma. This is likely to reflect a difference in life cycles of the different HPV subtypes in different mucosal locations, with an associated difference in mucosal immune responses .Up to now, data about HPV prevalence in oral infection, risk factors, genetic pathway and molecular pathogenesis, its potential oncogenic role in oral carcinogenesis are very scanty and still dissenting . Since SIt is still unknown how persistent infection gives rice to intraepithelial lesions, and, in turn, to invasive HNC. It is known that oncogenic types of HPV may produce non productive-infection and persist in the cells in low number episomic molecules. It is well known that HPVs exert their oncogenic role after DNA integration, gene expression of E5, E6 and E7 loci and p53/pRb host proteins suppression, leading to increased cell proliferation and contributing to carcinogenesis ,51. ReceAlthough integration of HPV DNA into genomic DNA is a common event in cervical carcinoma and intraepithelial lesions, however, 15-30% of cervical cancer, containing HPV only in the episomal form, shows a plasmidic expression of oncogenic protein E5, E6, E7. The situation in head and neck cancers is not clear, but heterogeneity and the existence of multiple pathways in carcinogenesis is highly likely. Koskinen et al  reportedParadoxically, the low number of HPV DNA copies in integration or episomic status may be underestimated by standard immunopathological studies. When performing the molecular detection of HPV DNA, it is essential that the diagnostic procedures employed are highly sensitive, specific and reliable and it should kept in mind that the efficiency of HPV detection may be affected by several methodological variables. In situ hybridization and in situ oncogenic protein staining techniques have increased sensitivity and specificity of HN diagnostic practices. These techniques have allowed not only detection and identification of low risk/high risk HPV in cytological smears or histopathological immune-sections but, in addition, also the definition of the topographical level of infection, and/or viral integration status . Furthermore, these techniques have provided to calculate the copy number per cell . In addition, by the routinary IHCthe expression of viral HPV proteins E5, E6, E7 as surrogate markers of HPV infection, and the resulting down-regulation of critical tumor suppressor  in histopathologically analyzed HNC can be demonstrated. In any case, we must remember that p16 immunohistochemical positivity is unable to discriminate between HPV integrated vs HPV not integrated OSCCs. IHC and ISH are considered methods with a low sensitivity ,55, becaThe high sensitive and specific SPF10 HPV DNA test, determined by direct sequencing of PCR fragments and genotyping assay, as good screening test, can be performed for HPV detection on paraffin-embedded sections collected during diagnostic procedures as good screening test .PGMY/GP nested PCR system is able to perform consistently at a high level of sensitivity, namely 0.1-1 copy per cell input .However the possibility of overestimation or underestimation of HPV positivity due to technical limitations, the absence of standardization in collection, storing and analyses of tissue samples, the impact in the clinical practice, the cost and the commercial availability are important aspects to consider, in order to establish the exact role of HPV in oral and oropharyngeal lesions and its real tumoral frequency in an anatomical region where HPV has a yet high prevalence.In clinical setting, nowadays we have to perform a sensible, specific and accurate HPV test. In case of false negative results, patients are potentially deprived of important curative tools i.e. radio and chemotherapy. Similarly, false positive patients are potentially deprived of another curative tool: the surgery. Only standardized technical procedures could assist clinicians to provide effective diagnostic test, innovative treatment and more efficient screening systems for OSCC patients. A specific panel of different HPV types, possibly including LR and HR-viruses, should be defined in order to prepare adequate vaccine reducing the risk of HPV-positive oral and oropharyngeal cancers in male and female population and in order to detect an infective pathogenesis in oral and oropharyngeal neoplastic samples.The INK4A locus, harboring the p16INK4A gene, is a major aberration hotspot in oral and oropharyngeal carcinoma. The p16INK4A gene functions as negative regulator of the cell cycle progression through its inhibition of cdk4/6 and subsequent blockage of the cyclin-dependent phosphorylation of the Rb . GeneticThe current scientific research is aimed at deciphering the molecular alterations involved in p16INK4A down-regulation and to assess the prognostic implications of p16INK4A gene alterations in HNC in order to establish its impact on staging and prognosis compared with the conventional clinical staging parameters .The instability of the CDKN2a/INK4 locus located on 9p21 is reported to be high in carcinoma and the major inactivation of the p16INK4A/p14ARF genes results from promoter methylation, homozygous deletion, loss of heterozygosity and intragenic mutation .These genetic and epigenetic alterations have been detected frequently in a variety of human cancers, including head and neck cancer.According to recent studies, HPV-positive HNCs have intact p16 gene and wild type p53 compared to HPV negative ones, harbouring frequent p16 and p53 gene alterations . This isFor many years, the main prognostic factors of SCC have been the conventional grading, staging and site of tumor. According to recent studies, the viral load of HPV in HNCs as well as p16 expression could be defined the most relevant prognostic markers in HPV positive OSCCs, surpassing the significance of the classical histopathological parameters, and it should be considered for inclusion into official staging system of HNSCC. The viral load of HPV in HNCs appears to vary considerably as reported in a recent update.gold standard test to classify head and neck cancers into 3 distinct profiles: Class I, HPVnegative, p16 low, 0.0 copies; Class II, HPV+, p16 low, 3.6 copies; and Class III, HPV+, p16 high, 46 copies; Class IV, HPV-negative; p16 + Of note, was that Class III tumours had a significantly increased 5 year survival, increased disease-free survival rate and decreased local recurrence rate, compared to tumours in the other classes .Nowadays, the goal of the scientific research is to find new biological markers able to identify set(s) of genes involved in oro-pharyngeal carcinogenesis. This is necessary to evaluate the "tumor fingerprint" that defines the biological behavior of the each single neoplasm. In the last decade the scientific research has shown a growing interest on several biological markers, expressed by cancerous cells in order to use them as indexes of OSCC progression and aggressiveness, also in relation to HPV-DNA detection. The investigation of oncogenic gene expression in HPV-related OSCC and the study of its potential value as predictor of neoplastic progression and clinical outcome could allow to characterize a possible evolutive morphologycal profile of oral cancer and its precancerous lesions. As a rule, the expression of HPV markers and surrogate markers of HPV infection can be easily evaluated by IHC. Different antigens should be used including virus related , and virus induced and/or altered host proteins , caveolin (CAV2), IFNα-inducible protein 6-16 (G1P3), matrix metallopeptidase 12 (MMP12), collagens , and phospholipid scramblase 1 (PLSCR1), and down-regulated for another set II, including other keratins , programmed cell death 4 (PDCD4), protein tyrosine kinase 6 (PTK6), epithelial membrane protein 1 (EMP1), extracellular matrix protein 1 (ECM1), interleukin 1 receptor (IL1R2), and transglutaminase 3 (TGM3). Relative to HPV-HNC HPV+HNC and cervical cancer showed significantly increased expression of gene set III, including PC4/SFRS1-interacting protein 1 (PSIP1), V-myb (MYB), synaptogyrin 3 (SYNGR3), SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin (SMARCA2), synaptonemal complex protein 2 (SYCP2), p16 (CDKN2A), lymphoid-specific heli-case (HELLS), and testicular cell adhesion molecule 1 (TCAM1), whereas expression was decreased for gene set IV, including parathyroid hormone-like hormone (PTHLH), cortactin (CTTN), kallikreins , cyclin D1 (CCND1), caveolin 1 (CAV1), and defensin β4 (DEFB4). E7 oncoproteins of high-risk HPVs induce DNA replication and mitosis by multiple mechanisms, including interacting with pRb, HDACs, and other factors to activate cell cycle-regulated transcription factors such as E2F [CCND1 and CCND2; G1 associated) and cyclin A1 (CCNA1). By contrast, HPV+ cancers up-regulated, relative to HPV-HNC, a much larger set of cell cycle-specific genes such as cyclin E2 , cyclin B1 , and multiple MCMs. Among these, many genes that enhance DNA replication and cell mitosis, including proliferating cell nuclear antigen (PCNA), E2Fs, cdc2, cdc7, and MCMs were significantly up-regulated in HPV+ HNC and cervical cancer relative to HPV- HNC, implying that the HPV+ cancers were more active in cell division. The most striking difference in cell cycle regulatory gene expression was seen with cyclin D1/CCND1 and p16/Ink4a/CDKN2A. In HPV+ cancers, p16 was expressed at high levels and cyclin D1 at low levels, with the converse in HPV- cancers. A recent immunohistochemical study examining just six cell cycle proteins in HNCs confirmed that these changes in p16 and cyclin D1 expression correlate with HPV status and extend to the protein level [SYCP2 and TCAM1. Cell line studies showed that SYCP2 and TCAM1 expression are synergistically up-regulated by E6 and E7. The Peyon's study [SYCP2, STAG3, and TCAM. STAG3 and SYCP2, an SYCP1 homologue, are components of the meiotic synaptonemal complex that promotes recombination, and SYCP1 expression induces formation of a synaptonemal complex-like structure. Aberrant expression of these meiosis-specific proteins in HPV+ cancers may contribute to the genomic instability induced by high-risk HPVs and to further genetic changes during HPV-associated cancer development. According to Slebos' model [On the basis of these tendencies, some Authors ,71 have h as E2F . A signiin level . In HPV+in level . Recentlin level , up-regu's study  found ths' model . Other ss' model . Immunois' model .INK4a; in contrast, HPV+ designated tumors  as compared to HPV-negative HNCs, showing a more restricted panel of cell cycle proteins, Cyclin D1/D2, Cyclin A1. According to the studies from Heidelberg University group, most HPV++ designated OPSCCs  showed reduced pRb, low cyclin-D1 and p-53 and up-regulated p16e tumors . All theThe association of head and neck cancers with clinically significant morbidity and disfiguration makes early detection of the diseases and biomarkers to identify individuals at high risk of great importance. However, most of these markers have been identified either in cancer cell lines or in biopsy specimens from late invasive and metastatic cancers. Moreover, the invasive nature of a biopsy makes it unsuitable for cancer screening in high-risk populations. Exfoliative cytology may be a less invasive method for oral cancer detection. In some recent scientific research has been assessed that HR HPV types detected in oral exfoliated cells could be used as a predictive biomarker of oropharyngeal cancer risk associated with HR HPV infection ,79, becaMoreover we have to remember that saliva can contain different types of HPV reservoirs, and HPV DNA may originate from the following sites:- HPV-positive tumor cells- HR HPV infection that led to the development of oropharyngeal cancer- from an independent HR HPV infection;- phlogistic cells, as giant cell or lymphocytes;- oral epithelium surrounding tumor: normal or dysplastic mucosa;- tonsillar crypts.Generally, the sensitivity of an HPV test in saliva has been reported as low, whereas the specificity is very high. Recent studies report that HPV16 was detected in 50% of the saliva rinse samples from HNSCC patients with detectable HPV16 level in their tissues, in 18% of saliva rinse samples from patients with HPV16 negative primary HNC and in 2.8% of the normal controls. Using a cutoff of HPV 16 >0.001 copies/cell in saliva rinse, the test yielded a sensitivity of 30.4% and a specificity of 98.3%. HPV16 DNA in saliva rinses can reflect HPV16 status of primary HNSCC. As a screening method quantitative analysis of HPV16 DNA in salivary rinses allows for detection of HPV-related HNC; however, specific limitations exist that prevent the application of this as a screening technique for a broad population .Quantitative measurement of salivary HPV16 DNA can be a promise for surveillance and early detection of recurrence. In fact, a very interesting paper proved that the presence of HPV DNA in convalescent salivary rinses is an adverse prognostic marker in HN squamous cell carcinoma, because HPV16 presence in follow-up salivary rinses preceded clinical detection of disease recurrence by an average of 3.5 months . Recent Synthetically, saliva allows to:- detect HR HPV in oral cells- identify its plasmidic vs integrated state- define gene expression patterns by:Genomic MicroarraysEpiGenomic MicroarraysProteomic analysis- be easily used in general population study- be easily used also in selected high rish populations (immune-depression/immune-suppression)- help in decision making as regard the choice of therapy- monitor presence of residual tumor after therapy (cancer progression)Recent reviews were performed in order to establish the evidence for HPV-related malignant disease in regions other than the cervix. One of this studies found sufficient evidence to support a global immunization programme against HPV, irrespective of gender and geography, to help to achieve a reduction in HPV-related malignant diseases in the future .OPSCC: Oro-Pharyngeal Squamous Cell Cancers; OSCC: Oral Squamous Cell Cancer; HPV: Human Papillomavirus; SCC: Squamous Cell Carcinoma; WHO: World Health Organization; HNC: Head and Neck Cancer; EU: European Union; AJCC: American Joint Comittee on Cancer; DSS: Disease Specific Survival; DFS: Disease Free Survival; HR HPV: High Risk HPV; LR HPV: Low Risk HPV; IARC: International Agency on Treatment of Cancer; LSCC: Larynx Squamous Cell Cancer; HN: Head and Neck; IHC: Immunohistochemistry; ISH: in situ hybridization; PCR: Polymerase Chain Reaction.The authors declare that they have no competing interests.GP conceived of the study; PB, AS and SP have made substantial contribution to its design; AS and GP have participated in the acquisition of literature data; GP together with AS and PB helped in the coordination of this work; AS has been involved in drafting the manuscript and in its sequences'alignment, too; finally, PB, GDR and LLM have revised it critically for important intellectual content and they have given final approval of the version to be published. All Authors read and approved the final manuscript.


CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.Cervical lesion grading is critical for effective patient management. A three-tier classification  based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification,  CAP-ASCCP Lower Anogenital Squamous Terminology Standardization (LAST) consensus guidelines have therefore recommended a two-tier approach in which lesions are divided as low- or high-grade squamous intraepithelial lesions (LSIL or HSIL) INK4a immunohistochemistry [IHC]) do not support a standardized three-tier system.The management of patients with pre-invasive cervical lesions is dependent on the assigned histological classification. There is considerable debate over the terminology that best facilitates patient care and consensus usage between pathologists E6/E7 mRNA chromogenic in situ hybridization (CISH) signal patterns as markers of CIN/SIL grade using a novel detection assay  E6/E7 RNA for eighteen different HR HPV genotypes . The test utilizes 10 pairs of oligonucleotide probes per HPV type. Each oligo-probe has a 5′ ∼25 base region that binds specifically with an E6 or E7 sequence and the probe pairs are designed to hybridize contiguously. At the 3′-end of each probe in the pair is a non-HPV hybridizing 14 base sequence: the resulting 28 base sequence hybridizes with the 5′-prime end of ‘pre-amplifier’ oligonucleotides subsequent to the initial HPV hybridization step. Signal amplification is achieved by the sequential hybridization of amplifier sequences that bind to the pre-amplifiers and label-probes conjugated with horseradish peroxidase (HRP) that bind the amplifiers. The theoretical amplification for a given HPV type is 4,000-fold. The primary ‘cooperative’ hybridization step that requires contiguous dual probe binding in order for successful pre-amplifier hybridization helps ensure assay specificity; the use of ∼25 base HPV specific probes supports hybridization to mRNA in formalin-fixed, paraffin-embedded (FFPE) specimens, in which mRNA is typically relatively degraded. Additionally, our investigation of HPV E6/E7 RNA expression has been complemented with the examination of p16 mRNA CISH (RNAscope) expression patterns, HPV DNA CISH signal patterns, p16INK4a IHC staining profiles and HPV DNA genotyping of lesions. Using these combined tests, the aim was to investigate whether distinct HPV-related biological profiles underlie CIN categories and whether RNAscope assays may contribute to CIN/SIL grading.In this study we have investigated high-risk (HR) HPV The causative role of HR HPV genotypes in the development of CIN and cervical carcinoma is well established E6 and E7 oncogenes have transformative properties and impact a wide variety of cellular targets; the best described are degradation of the p53 tumor suppressor protein by E6 protein and abrogation of pRB tumor suppressor protein functioning by E7 protein E6/E7 expression and low levels of HPV DNA replication; productive phase lesions display low-level E6/E7 transcription in the lower third to half of the epithelium and high levels of HPV DNA replication; and, transformative infections demonstrate high levels of E6/E7 expression throughout the full thickness of the epithelium and low levels of HPV DNA replication. E6/E7 over-expression may result by abrogation of the E2 gene, which is involved in E6/E7 regulation, by integration of HPV DNA into the host cell genome that typically involves physical disruption of the E2 gene, or, from E2 silencing by methylation or other epigenetic events The HR-HPV INK4a immunohistochemistry (IHC) is commonly used for distinguishing reactive from neoplastic atypias and for assisting in ≥CIN 2 designation. The p16INK4a protein acts as a regulator of cell cycle control: high levels of p16INK4a  normally prevent S-phase progression by inhibition of cyclin-dependent kinases (CDKs); this results in hypophosphorylated pRB that binds E2F transcription factors. HR HPV E7 protein disrupts pRB binding of E2F: unbound E2F stimulates S-phase progression; p16INK4a over-expression is the observed consequence as E2F also stimulates p16INK4a expression, which no longer acts in a feedback loop to inhibit its own expression INK4a staining and CIN grade and/or latent/productive/transformative phase of infection has been widely reported. Whilst HR-HPV status has clinical utility as a ≥CIN 2 risk biomarker for cervical cytology screening p16This study was conducted with the approval of the University of Vermont (UVM) Institutional Review Board (IRB) Committee on Human Research in the Medical Sciences (CHRMS). The requirement for written informed patient consent was waived by the committee for reasons including the use of archival specimens collected >2 years prior to study initiation (in accordance with College of American Pathologists [CAP] guidelines) and from patients who had received the standard of care required for the treatment of CIN.Eighty-six CIN lesions collected from eighty-three patients were identified by electronic record search : CIN 1 (n = 32), CIN 2 (n = 22), and CIN 3 (n = 32 [2 specimens had concurrent microinvasion]). Lesions were graded on the basis of the original clinical sign-out diagnosis, together with H&E review (KC and KMC) of sections cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Cases were then coded and all subsequent assays were performed masked to the CIN grade assignment. Morphologically normal epithelia adjacent to CIN lesions and/or in separate tissues co-located in an FFPE block were used for internal negative control test purposes. Patient ages ranged from 17 to 57 years .2, 200 µM ACGU dNTP mix (Fermentas), 0.2 units HotStaTaq and 1X HotStaTaq buffer , 1 unit uracil N-glycosylase (UNG) . The reaction mixture was incubated at 37°C for 30 minutes then heated to 95°C for 15 minutes followed by cycles 94°C for one minute, annealing for 1.5 minutes and extension at 72°C for 2 minutes; a touchdown annealing approach was adopted beginning with a temperature of 55°C and decreasing by 0.5°C per PCR cycle until a temperature of 40°C was reached. The detection of a ∼150 base pair amplicon was taken as evidence of HPV positive status. Specific HPV type identity was determined by cycle sequencing of the purified PCR amplicon and NCBI BLAST search. Positive control PCR was performed using PC03/05 primers for the detection of a 209 base pair β-globin sequence A ribbon (∼5–10 sections) of FFPE cervical tissues was cut into a 1.5 mL microfuge tube; the microtome blade was wiped with xylene-substitute and DNA Away  to prevent sample cross-contamination. Tissues were treated with several washes with xylene to remove paraffin-wax and then rehydrated by vortex mixing with 100% and 70% ethanol. Air dried tissues were digested overnight with proteinase K  and purified DNA was recovered using a column clean-up method . DNA was quantified using a NanoDrop 2000 spectrophotometer . PCR for HPV L1 sequences was performed on 100 ng DNA using GP5+/6+ primers as previously described E6/E7 CISH: The RNAscope 2.0 assay was performed according to supplier instructions  using a proprietary probe combination capable of detecting eighteen different HR HPV types. Briefly, 5 µm slide-mounted sections were de-paraffinized through 100% xylene and ethanol washes. Tissues were then treated serially with: Pre-Treatment 1 solution (endogenous hydrogen peroxidase block with Pretreat 1 solution for 10 minutes at room temperature); Pre-Treatment 2 ; and, Pre-Treatment 3 ; rinses with water were performed after each Pre-Treatment step. Tissues were then hybridized in HR 18 HPV hybridization solution, without a cover slip, at 40°C for 2 hours in a HybEZ Oven . After wash buffer steps, signal amplification from the hybridized probe was performed by the serial application of Amp 1 (PreAmplifier step), Amp 2 , Amp 3 (amplifier step), Amp 4 (Label Probe step), Amp 5 and Amp 6 ; wash buffer steps were performed after each Amp step. HRP activity was then demonstrated by the application of 3,3′-diaminobenzidine (DAB) for 10 minutes at ambient temperature. Sections were then counterstained with hematoxylin, dehydrated through graded ethanol  and xylene and then mounted with Cytoseal XYL . Staining data was recorded according to thickness of epithelial staining, presence and amount of diffuse, and/or punctate nuclear staining and cytoplasmic staining as well as signal intensity.HR 18 HPV p16 CISH: CISH for p16 mRNA expression was performed as above using a proprietary oligoprobe combination for p16 mRNA sequences. Staining was recorded in terms of thickness of epithelial staining and intensity of nuclear/cytoplasmic signals.ubiquitin C (to demonstrate detectable mRNA in the FFPE samples) and Bacillus subtilis dapB mRNA targets respectively. Ubiquitin C staining was scored to confirm the presence of cytoplasmic signal and intensity was also noted. Bacillus subtilis dapB staining was reviewed to confirm absence of staining.RNAscope CISH positive and negative tests was performed for every specimen using proprietary probes for human sequence 2, 0.5 mM CaCl2, pH7.5, 30 minutes 40°C). Additionally, samples were hybridized with HPV sense sequence probes that should be expected not to yield HPV mRNA signals.RNA target hybridization assays: Validation of hybridization with mRNA sequences was investigated on a limited set of specimens by tissue pretreatments with RNase A  10 mg/mL in PBS, 30 minutes 40°C) and with DNase I  80 µg/mL in 10 mM TrisCl, 2.5 mM MgClSpecimen control assays: Positive control samples included cervical carcinomas and CIN lesions previously established as HPV positive by GP5+/6+ PCR in conjunction with HPV DNA CISH. Negative control samples include normal cervical epithelium demonstrated as HPV negative by PCR and CISH.HPV DNA CISH was performed as previously described INK4a was performed using clone E6H4  according to kit instructions: following de-paraffinization and rehydration, slides were immersed in epitope retrieval solution at 95–99°C for 10 minutes followed by a 20 minutes cool down period. Peroxidase-blocking reagent was then applied for 5 minutes after which mouse anti-human p16INK4a was applied for 30 minutes followed by visualization reagent for 30 minutes and finally 3,3′-diaminobenzidine (DAB) tetrachloride substrate. Negative control assays were performed using reagents as provided in the CINtec Histology kit. Staining was recorded with reference to thickness of epithelial staining, intensity  and nuclear or cytoplasmic signal.IHC for p16Statistical analyses were performed using GraphPad InStat software .All CIN samples tested positive for β-globin demonstrating amplifiable quality DNA was extracted from the specimens. HPV was detected in 30/32 (93.8%) CIN 1 lesions: high-risk (HR) types were found in 27/32 (84.4%) and low-risk (LR) types were identified in 3/32 (9.4%) samples . Twenty INK4a IHC staining presented as diffuse nuclear and cytoplasmic staining as indicated (CISH and IHC data from the study are summarized in ndicated .Eighty of 86 (93.0%) CINs tested positive for HPV by RNA CISH. HPV was detected in 26/32 (81.3%) CIN 1 s. The six negatives consisted of three LR HPV samples (one HPV-6 and two HPV-11) and three HR infections (two HPV-16 and one HPV-31). The 26 positives were all from specimens infected with HR HPV types. The epithelium from one specimen was thin and partially denuded and was discounted from further descriptive review. Most (21/31 [67.7%]) CIN 1 lesions were characterized by the presence of intense diffusely stained nuclei mostly in the top 1/3-1/2 of the epithelium with weak to strong intensity dot-like nuclear and cytoplasmic signals mainly in the lower layers . Four 1; in the All 22 CIN 2 lesions stained positive for HPV: one (4.5%) showed only nuclear and cytoplasmic dot-like signals confined to the lower 1/2 of the epithelium; 21/22 (95.5%) were characterized by medium to strong nuclear and cytoplasmic dot-like signals throughout the epithelial thickness ; 14  presented with full-thickness epithelial layer staining with medium to strong nuclear and cytoplasmic dot-like signals ; in fourSignificant trends  were notEight of the initial 86 specimens selected for study were discounted from DNA CISH review; four specimens were un-interpretable due to high-background staining and in the other four samples the lesion was no longer present. HPV was detected by biotinyl-tyramide-based CISH in 38/78 (48.7%) CIN lesions (vs. 80/86 [93.0%] RNAscope HR HPV [P<0.0001]). Additionally, in contrast to RNAscope, HPV DNA CISH signals were frequently focal and sporadic, whereas the RNAscope signals were profuse and detected in a high percentage of cells within a lesion.Ten (34.5%) of 29 CIN 1 specimens were DNA CISH positive: 5 (20.7%) showed superficial layer diffuse nuclear staining only, one (3.4%) showed superficial diffuse nuclear signals and sporadic punctate signals at all levels of the epithelium. Four (13.8%) specimens showed only punctate signals: 1 (3.4%) showed relatively abundant strong signals in the lower two thirds of the epithelium, 1 sporadic weak signals in the lower two thirds, 1 sporadic weak signals in the lower half of the epithelium, and one sample showed frequent strong punctate signals in the lower portion of the epithelium but the superficial layers missing. Of the CIN 2 s, 13/20 (65.0%) were positive: superficial diffuse nuclear staining was detected in 8 (40.0%) samples, of which 3 (15.0%) also showed sporadic punctate signals at all levels of the epithelium and 1 showed superficial punctates  CIN lesions; signals were predominantly cytoplasmic in all positive samples. Among CIN 1 s, p16 expression was detected in 24/30 (80.0%) specimens; (1/32 specimen excluded for high background staining and one for loss of lesion). Staining was confined to the lower 1/3–1/2 of the epithelium in 11/30 (36.7%) samples; l0 were weak and 1 medium in staining intensity. Thirteen (43.3%) samples showed signals in the lower 2/3 to full epithelial thickness; 10 weak and 3 medium in intensity. Twenty-one (95.5%) of 22 CIN 2 s demonstrated p16 staining. In four (18.2%) specimens signals  were confined to the lower 1/3 to 1/2 of the epithelium and in 17 (77.3%) samples staining was present in the lower 2/3 to full epithelial thickness . All 32 CIN 3 specimens showed lower 2/3 (9 [28.1%]) to full thickness (23 [71.9%]) epithelial staining. Signals were weak in 10, medium in 10 and strong in 12 specimens. p16 expression detectable in basal up to and including superficial layers increased with CIN grade  was detected in 80/85 (94.1%) of specimens. Of 32 CIN 1 specimens, 29 (90.6%) showed staining; a thin epithelial layer was noted in one samples and discounted from further descriptive analysis. Twenty-four (77.4%) of 31 samples demonstrated lower 1/3 −1/2 staining; weak intensity in 7, moderate in 8, and strong in 9 specimens. Four (12.9%) samples showed lower 2/3 to 3/3 epithelial thickness staining that was moderate to strong in intensity. p16INK4a staining was found in 21/22 (95.5%) CIN 2 specimens: in 3 (13.6%) samples confined to the lower 1/3 −1/2 ; in 18 (81.8%) cases, 2/3–3/3 moderate to strong epithelial staining was noted. One CIN 3 sample was discounted from the study through loss of the lesion. Of 31 CIN 3 s, 30 (96.8%) showed 2/3-full thickness epithelial staining that was moderate to strong in intensity; one samples was negative. p16INK4a staining patterns were in general distinguished CIN 2+ for CIN 1 but did not support the differentiation of CIN2 from CIN3 (p16rom CIN3 –S3.Ubiquitin C (UBC) mRNA expression was detectable in all specimens as abundant moderate to intense cytoplasmic signals demonstrating the preservation of RNAscope detectable mRNA in the FFPE specimens. Staining with the Bacillus subtilis dapB mRNA probe was noted in several samples (mainly CIN 1 s) as fine/weak dot-like signals in all tissue compartments: this was interpreted as non-specific assay-related staining . [Technical note: further to this study a newer version (v2.0) of the RNAscope assay has been marketed showing a cleaner background with the dapB probe.].staining  and S6; in situ hybridization. To confirm this application in the detection of HPV infections, a few selected tissues were pretreated with RNase A or DNase I prior to hybridization with antisense and sense-strand HPV probes. DNase I exposure confirmed that HPV mRNA signals are detectable in epithelial cell cytoplasm and nuclei  of 29 HR HPV positive CIN 1 lesions and 100% of 22 CIN 2 and 32 CIN 3 s stained positive by HPV RNA CISH. CISH staining in lesions was readily identifiable: within the epithelial stratum showing CISH signals ≥50% cells stained; moderate to strong signal intensity was noted in nearly all cases; staining was judged as weak in 4 (13.8%) CIN1 s, 1 (4.6%) CIN2 s and 1 (3.1%) CIN3 s. Staining was absent from morphologically normal tissues. This high level of HPV detection and the staining patterns detected (intense nuclear and cytoplasmic dots as well as diffusely staining nuclei) directly parallels data obtained from HPV RNA Tyramide-based CISH for HPV DNA was significantly less effective at staining CIN lesions than RNAscope . In a prE6/E7 transcripts. HPV RNA CISH may also be helpful for distinguishing normal/CIN mimics  from true CIN. Clearly, overtreatment may result if non-CIN is mistaken as CIN and under-treatment when CIN goes unrecognized These data support the use of HPV RNA CISH as a gold standard assay for determining whether an anogenital specimen (or even a head and neck tumor) is high-risk HPV positive and actively expressing oncogenic The HPV RNA CISH staining patterns can be interpreted as markers of HPV phase of infection : latent,p16 mRNA was detected by CISH in 87.1% of the lesions and extended past basal-mid (BME+) layers in 38.7% cases whereas p16 protein expression was detected in 90.6% of cases and was detected in BME+ layers in 12.9% of specimens. These findings are consistent with CIN 1 morphology harboring several HPV related biologies. Firstly, latent/early or possibly regressing lesions showing a low viral load and an absence of productive phase HPV replication  with a relatively heterogeneous group (n = 13) of mainly HR HPV types. Just over a third of these infections were detectable by HPV DNA CISH and sixty percent of these included the detection of abundant diffuse staining cells in the superficial layers. HR HPV mRNA CISH showed signals in 81.3% of the specimens with superficial signals in two-thirds of cases. p16 mRNA and protein expressions were detectable through the greater part of the epithelial thickness in most cases. Of the remaining CIN 2 s, 27.3% showed only abundant intense full-epithelial thickness dot-like signals consistent with a transformative phase of HPV infection . Top row: H&E; second row: HPV E6/E7 RNA CISH; third row: p16 RNA CISH; fourth row: p16INK4a IHC; fifth row: UBC RNA CISH; sixth row: E.Coli dap B RNA CISH. Column groups a-d . All images were originally taken using a 20X objective lens. Scale bar: 50 µm. a-d see  legend. (PDF)Click here for additional data file.Figure S2CIN 2 staining (expanded data). Top row: H&E; second row: HPV E6/E7 RNA CISH; third row: p16 RNA CISH; fourth row: p16INK4a IHC; fifth row: UBC RNA CISH; sixth row: E.Coli dap B RNA CISH. Columns a-d Click here for additional data file.Figure S3CIN 3 staining (expanded data). Top row: H&E; second row: HPV E6/E7 RNA CISH; third row: p16 RNA CISH; fourth row: p16INK4a IHC; fifth row: UBC RNA CISH; sixth row: E.Coli dap B RNA CISH. Columns a-d (see INK4a IHC staining was negative in one instance (S7). All images were originally taken using 20X objective lens. Scale bar: 50 µm. a-d see  legend. (PDF)Click here for additional data file.Figure S4HPV DNA CISH. a. CIN 1 lesion superficial diffuse staining nuclei. b. CIN 2 lesion superficial diffuse staining nuclei. c. CIN 3 lesion showing diffuse and punctate signals through the thickness of the lesion. All images were originally taken using 20X objective lens. Scale bar: 50 µm.(PDF)Click here for additional data file.Figure S5HPV E6/E7RNA CISH control tests: productive phase HPV 16 infection. a. HPV diffuse nuclear staining signal patterns and fine nuclear and cytoplasmic dot-like signals after hybridization with antisense probe. b. HPV diffuse nuclear staining signal patterns after hybridization with sense-strand E6/E7 probe. These indicate that some diffuse nuclear staining may be HPV DNA. c. Reduced HPV diffuse nuclear staining signal patterns and absence of fine nuclear and cytoplasmic dot-like signals after hybridization with antisense probe on tissues pretreated with RNase A. These data suggest diffuse nuclear staining is  due to hybridization with HPV DNA and that the fine nuclear and cytoplasmic signals are HPV RNA. d. HPV diffuse nuclear staining signal patterns after hybridization with sense-strand HPV probe on tissues pretreated with RNase A suggestive of hybridization with HPV DNA. e. Absence of HPV staining signal patterns after hybridization with anti-sense-strand HPV probe on tissues pretreated with DNase I confirming diffuse signal patterns involve probe hybridization with DNA. f. Reduced intensity diffuse HPV staining signal patterns after hybridization with sense-strand HPV probe on tissues pretreated with DNase I confirming diffuse signal patterns involve probe hybridization with DNA. Detected signals may be due to incomplete DNase digestion of abundant productive phase HPV DNA. g. & h. Abundant UBC RNA staining (g) eliminated (h) after tissue pretreatment with RNase A. All images were originally taken using 40X objective lens. Scale bar: 20 µm.(PDF)Click here for additional data file.Figure S6HPV E6/E7RNA CISH control tests: transformative phase HPV 16 infection. a. HPV nuclear and cytoplasmic dot-like signals after hybridization with antisense probe. b. Absence of staining after hybridization with sense-strand HPV probe. This finding shows that the signals detected in (a) represent HPV RNA targets and not DNA. c. & d. Absence of any staining with anti-sense or sense-stand HPV probes on tissues pretreated with RNase A indicating the non-detection of HPV DNA targets. e. HPV positive staining after hybridization with antisense HPV probe on tissues pretreated with DNase I confirming signal patterns result from probe hybridization with RNA. f. Absence of staining in DNase I treated tissue hybridized with sense-strand HPV probe confirming that the anti-sense probes detect HPV E6/E7 RNA. g. & h. Abundant UBC RNA staining (g) eliminated (h) after tissue pretreatment with RNase A. All images were originally taken using 40X objective lens. Scale bar: 20 µm.(PDF)Click here for additional data file.


Incidences of oral tongue, base of the tongue, and tonsil cancers have been increasing steadily in many parts of the world in spite of declining rates of tobacco use over the last four decades. A better understanding of the etiology, interactions between risk factors, and new approaches to prevention and treatment are necessary to change this course. This paper will present evidence supporting a potential role of chronic inflammation in the etiologies of oral human papillomavirus infection and head and neck squamous cell carcinoma, and it will discuss the implications for prevention and treatment. Over 500 000 new cases of head and neck squamous cell carcinoma (HNSCC) occur annually in the world . The Natα (IFN-α), cyclo-oxygenase-2 (COX-2), and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, usually have obstacles including toxicity and reversal of their effects after cessation of treatment [HNSCC is largely preventable, yet the majority of current research has focused on treatment and diagnosis. Most of the existing prevention strategies for HNSCC have been secondary and tertiary with limited success , 7. Chemreatment , 9. Primreatment , 2. Oralreatment –12. A hireatment . Howeverreatment . A largeThis short review will (1) present evidence supporting a potential role of chronic inflammation on the etiologies of oral HPV infection and HNSCC and (2) discuss the implications for prevention and treatment.Clinical, epidemiologic, and animal studies support an association between chronic inflammation and cancer in several organs –18. ChroIn the oral cavity, periodontitis is a chronic inflammatory disease of the structures around teeth associated with dental biofilm . The ensA link between poor oral hygiene and HNSCC has been suggested for over two decades –46 but tP < 0.001). After adjustment for age, gender, race/ethnicity, marital status, smoking status, alcohol use, and missing teeth, each millimeter of ABL was associated with >4-fold increased odds of HNSCC . The strength of the association was greatest in the oral cavity , followed by oropharynx  and larynx  (P = 0.038) [In a recent hospital-based case-control study with histologically confirmed 266 primary incident HNSCC cases and 207 controls in Upstate New York, ABL was significantly higher in cases compared to controls  . Further= 0.038) .The potential association between chronic inflammation and HNSCC is further supported by two recent case control studies suggesting a beneficial effect of nonsteroidal anti-inflammatory drugs (NSAID) against HNSCC , 51. In in vivo and in vitro studies have confirmed the associations of several genes and proteins involved in different stages of inflammation with carcinogenesis [The biological mechanism of the association between chronic inflammation and cancer has been described extensively but is also evolving continuously since both inflammation and cancer are complex processes under the control of many driving forces –18. Bactogenesis –71. In aogenesis , 73.The steady increase in the incidence of oropharyngeal cancers over the last four decades has been mainly attributed to oral HPV infection which has been accepted as an etiological factor for a subset of HNSCC –14. HPV HPV is a small DNA virus with a specific tropism for squamous epithelia. More than 120 different HPV types have been isolated to date. Low-risk HPVs, including HPV-6 and HPV-11, induce benign hyperproliferations of the epithelium such as papillomas or warts which rarely progress to cancer . High-riPopulation-based studies estimate that the prevalence of HPV DNA in normal oral mucosa is 5–10% , 76. In HPV-positive HNSCCs have a unique risk factor profile. These tumors are more common in younger patients, have a male predominance, are often staged higher, yet have a survival advantage. These patients are less likely to have used tobacco and alcohol excessively . PatholoAlthough the natural history of cervical HPV infection is well established, the mechanism of transmission for oral HPV infection, which has a distinct immunologic environment, is largely unknown. There is increasing evidence that oral HPV transmission may be related to sexual history, and particularly oral sexual behavior. Case-control studies have shown associations of oral HPV infection with the number of oral and genital sexual partners, age at first of oral-genital contact, frequency of sexual contacts, and history of genital warts –85. Vertα, modulate proliferation of HPV and expression of its oncogenes E6 and E7 in cervical epithelial cells [in vitro study has shown that a nonsteroidal anti-inflammatory drug strongly induced degradation of the HPV oncoproteins causing growth arrest and apoptosis in cervical carcinoma cells in a concentration-dependent manner [Few epidemiological studies suggest that chronic inflammation of the cervix increases the risks of HPV infection and cervical cancer –90. In aal cells –96. Suppt manner . Continut manner . The orat manner .Chlamydia trachomatis increased the persistence of cervical HPV infection and the risk of cervical cancer [An association between chronic inflammation and HPV infection is biologically plausible. HPV infects basal cells of the epithelium exclusively, and it usually gains access through microabrasions. In addition, replication of the virus is closely associated with basal cell proliferation , 75. Mucl cancer , 102. Pel cancer , 104. Hol cancer –109.The potential role of chronic inflammation in the etiology of HNSCC is summarized in Understanding the interactions among risk factors may be the most important step towards effective prevention and treatment of HNSCC. It is well established that carcinogenesis is a multifactorial process and the presence of a single risk factor is usually not sufficient to cause cancer. Yet, most studies have focused on independent effects of specific risk factors , 7. ReceSaliva provides a means of interaction between different carcinogens. It also provides a means of transport from one surface to another promoting disease at distant sites. Salivary enzymes also metabolize tobacco, alcohol, and dietary components into carcinogens . This maIt is critical to understand the characteristics of the dental biofilm to effectively treat periodontitis. Biofilms are sessile communities of bacteria embedded in a polymeric matrix irreversibly attached to a surface. The bacteria in the biofilm differ profoundly from their free-floating counterparts and are resistant to antimicrobials as well to host defense . Thus, eIf prospective studies confirm that chronic local inflammation is in fact a significant player in the natural histories of oral HPV infection and HNSCC, this may have important implications, not only for prevention, but also for treatment. Chronic local inflammation, such as periodontitis, is easy to detect and may represent a clinical high-risk profile for both oral HPV infection and HNSCC. Subjects with sources of chronic inflammation in the oral cavity may be screened for oral HPV infection and HNSCC for early diagnosis.Chronic local inflammation, both independently and by facilitating oral HPV infection, may be an important player in the etiology of HNSCC.


Over the last 10 years, it has become clear that patients with head and neck cancer can be stratified into two distinct subgroups on the basis of the etiology of their disease. Patients with human papillomavirus-related cancers have significantly better survival rates and may necessitate different therapeutic approaches than those with tobacco and/or alcohol related cancers. This review discusses the various biomarkers currently in use for identification of patients with HPV-positive cancers with a focus on the advantages and limitations of molecular and nano-scale markers. Optimal treatment for these patients usually involves evaluation in a multidisciplinary setting with the coordination of surgery, chemotherapy, and radiation therapy. In many cases combinations of therapy are used .Over the last 10 years, a growing proportion of patients with head and neck cancer have been found to have tumors attributable to the human papillomavirus (HPV). This virus has long been known to cause cancers of the uterine cervix in women and of the anal canal in both men and women. Initially described in head and neck cancer by Syrjanen and colleagues due to histologic similarities between oropharyngeal cancers and cervical cancers , HPV is 2.There are currently more than 100 HPV subtypes that can be classified into low-risk and high-risk on the basis of their association with invasive malignancies. Just over a dozen HPV types have been classified as high risk. Of these, head and neck cancer is almost exclusively caused by HPV-16 which accounts for >90% of cases . This isi.e., tonsil, base of tongue, and soft palate) where the rate of HPV positivity has been as high as 80% in some studies [vs. retromolar trigone) and the difficulty in assigning a site of origin in some large primary tumors, it is also possible that HPV-positive cancers arising in other head and neck subsites are actually misclassified and truly arise from either the base of tongue or the tonsil.In addition, HPV does not appear to affect all mucosal head and neck sites equally . The vas studies ,10,11. Gvs. 28%, respectively) [There appears to be significant geographic and temporal variation in the rates of HPV-positive head and neck cancers with higher rates in the US compared to Europe 47% ctively)  and sevectively) ,14. Wher% ctivelyctively) .HPVs are DNA viruses that are encoded by approximately 8000 base pair genomes. The double stranded circular DNA encodes eight proteins: E1, E2, E4, E5, E6, E7, L1, and L2. Carcinogenesis is thought to be driven by expression of E5, E6 and E7 with the other early genes playing important roles in viral gene transcription and viral DNA replication. L1 and L2 encode the capsid proteins which form the “coat” of the virus and which are targeted by HPV-vaccines (a topic beyond the scope of this review).Interestingly, while patients with HPV-associated head and neck cancers commonly present with more advanced disease, they have significantly improved outcomes compared with stage and comorbidity matched HPV-negative patients. Differences in five year overall survival between HPV-positive and HPV-negative patients exceed 30% in a number of retrospective analyses ,16–19. TThese large differences in outcome have arisen in an era during which patients with HPV-positive cancers were treated no differently from those with HPV-negative cancers. However, in the past several years the oncology community has begun to think about HPV-positive head and neck cancer as a different disease than traditional tobacco/alcohol related head and neck cancer –22. It i3.3.1.Originally described by Edwin Southern in 1975, the “Southern Blot” is the gold standard test to measure the number of copies of a given gene or to analyze stretches of DNA that are too repetitive for PCR amplification or classical sequencing methods ,24. WhilAt this time, these steps are not easily amenable to automation. In addition, and of concern to a clinical lab, they typically involve multiple wash steps that produce large volumes of dilute radioactive waste. While Southern blots are labor intensive, they have an important role in studying tumor viruses such as HPV as they allow one to detect integration of the viral genome into the host genome. Additionally, assays can be developed with wash parameters and probes that are well suited to screening for multiple HPV subtypes. Southern blots can also be used to confirm the presence of specific HPV subtypes . When co3.2.In situ hybridization (ISH) is a commonly used in the diagnostic lab setting to test for HPV [i.e., fluorescent ISH or FISH) that can be visualized using a standard pathology microscope  allows for amplification of DNA isolated from tumor cells. Briefly, a DNA polymerase recognizes an oligonucleotide primer bound to a specific DNA sequence . By usinThe resulting PCR products from one or more HPV subtype can be detected by oligonucleotide array. Briefly, type-specific probes are plated onto an array. The PCR product is hybridized to the chip and resulting signals are visualized with a DNA chip scanner. This type of assay can have a sensitivity approached 95% and has the added benefit of being able to detect multiple HPV types within a single specimen ,29. ThisUnlike standard PCR that amplifies genomic DNA, reverse transcriptase PCR (RT-PCR) utilizes RNA that is first reverse transcribed into cDNA. Following the generation of cDNA, PCR is performed as described above.i.e., does the viral DNA present result in production of mRNA and viral proteins). Due to the high dynamic range of qRT-PCR (>7 logs of input), one can detect RNA sequences present at very low concentrations that may not be identified by conventional PCR [Quantitation of products for both standard PCR and RT-PCR can be performed by agarose gel electrophoresis. Alternatively, products can be detected in real-time with the use of DNA sequence-specific probes or fluorescent dyes . qRT-PCRonal PCR .Both PCR and RT-PCR are highly sensitive tests owing to the exponential amplification of target sequences that lie between two priming sequences. In theory these techniques can detect a single copy of a target sequence within a given sample. In reality, this high sensitivity can lead to false positive results either through the inclusion of random HPV genomes (particularly troublesome in labs which commonly study HPV) or by detection of HPV genomes within the investigated tissues, but that are not causative for the malignancy. Probe based qRT-PCR, which utilizes a third probe that lies within the amplified region can significantly decrease the risk of false positives.These tests can also be used to estimate the integration status of the HPV genome. Upon integration of the viral genome, both the L1 and E2 genes are typically disrupted and lost. Samples that retain E6 and E7 expression, but do not express L1 or E2 are considered to harbor integrated HPV ,33 while3.3.2.Next-generation sequencing (NGS) is a promising method for assessment of HPV-status on a nano scale. NGS utilizes high-throughput sequencing technology to simultaneously sequence many thousands of DNA or expressed RNA fragments . A numbeSeveral groups have recently published proof-of-concept studies using two of the available next-generation technologies to assess for HPV status in cytology samples  and form3.4.3.4.1.While not testing whether a given patient's tumor is related to HPV infection, exposure to HPV can be determined through serology. Using serum antibody detection systems, monoclonal antibodies raised against HPV type-specific epitopes are used to compete with a patient's antibodies produced in response to HPV infection or HPV immunization. Due to high background rates in the general population, HPV serology is not currently useful as a screening tool  and at t3.4.2.Ink4A or p16) is a protein involved in cell cycle regulation. Expression of p16 has been shown to correlate with significantly improved outcomes in patients with head and neck squamous cell carcinomas [Cyclin-dependent kinase inhibitor 2A (a.k.a., p16rcinomas ,40. Thisrcinomas . Overexprcinomas . Howeverrcinomas .Currently, whether patients with p16 positive, but HPV-negative cancers derive the same benefit from a given treatment as those with p16 positive, HPV-positive cancers remains unknown. In many centers, p16 testing is currently used as a surrogate for HPV status without actually detecting HPV DNA. To overcome this limitation, it has been proposed to couple p16 IHC with a secondary assay to directly detect HPV DNA or RNA .in situ hybridization, PCR, or RT-PCR, the detection of p16 by IHC requires no specialized equipment or tissue handling. Nearly all pathology laboratories are well equipped for IHC and proprietary kits are commercially available for p16 staining in formalin-fixed, paraffin-embedded tissue [In comparison to southern blot hybridization, d tissue . Shi andd tissue . In theid tissue .4.Our ability to detect HPV infection in head and neck cancer samples has greatly outpaced our ability to use this information to alter therapy. However, ongoing clinical trials and improved understanding of the molecular mechanisms underlying HPV-associated cancers promise to enable personalization of therapy on the basis of HPV-infection. At the current time limitations of alternative methods for detection of HPV result in the use of IHC for p16 as the de facto test for HPV-positivity . However


Databases were used to identify 32 female patients with AIS treated for various conditions between 2009 and 2012 at the Department of Gynecology, Uppsala University Hospital  and previous cytological, HPV and histological results. Of the individuals in the study, 64.3% had a previously recorded cytological result showing squamous cell abnormalities; five had glandular cell abnormalities (18%) and two had AIS (7.1%). Among the patients with available HPV results, 95% were HR-HPV-positive; HPV18/45 predominated (77%), followed by HPV16 (27%). The patients with multiple HPV infections were aged ≤32 years, while patients aged ≥38 years were only infected with HPV18/45. All but three patients had cervical intraepithelial neoplasia (CIN) in the AIS-adjacent squamous mucosa, 79% of which was CIN2 or worse. The present retrospective database study suggests that AIS is detected at screening mainly due to simultaneous squamous precursor lesions and that HPV18/45 infection is an increasing cofactor for AIS in older patients. HPV analyses of glandular precursor lesions aid in the identification of female individuals at risk of progression to invasive disease, and thus have a favorable effect on adenocarcinoma prevention, together with vaccination.The incidence rates of cervical adenocarcinoma have been increasing over the last two decades, contrary to those of squamous cell carcinoma. This trend is particularly evident among females aged <40 years and has occurred despite extensive cytology-based screening programs. The aim of the present retrospective database study was to investigate adenocarcinoma  Cervical cancer is a disease that mainly affects females in the fourth or fifth decade of life. According to the International Agency for Research on Cancer, cervical cancer is the second most common cancer in females worldwide and the most common in low- and middle-income countries . High-riin situ (AIS)  . AIS is tu (AIS) . Howevertu (AIS) . HPV testu (AIS) –16 and ttu (AIS) ,18.The aim of the present study was to investigate AIS with respect to cytological results, HPV infections and histological results from the AIS-adjacent squamous mucosa using data from the SymPathy database of the Department of Pathology, Uppsala University Hospital  and the database of the organized cervical cancer screening program of the County of Uppsala.The SymPathy database  contains information on results obtained at the Department of Clinical Pathology and Cytology, Uppsala University Hospital. Data therein are identified by specific topographical and morphological codes . The database of the organized population-based cervical cancer screening program of the County of Uppsala includes cytological, HPV and histological results from all laboratories in the county. Cytological and histological samples from the screening program are stored at the Department of Clinical Pathology and Cytology, where all cytological, HPV and histological testing for the screening program is performed. The study was approved by the Regional Ethical Review Board in Uppsala, Sweden.A total of 32 female patients with histologically confirmed AIS, who received treatment for various conditions at the Department of Gynecology at Uppsala University Hospital between 2009 and 2012, were identified in the SymPathy database, and the previous cytological, HPV and histological results for these patients were retrieved from the database of the cervical cancer screening program of the County of Uppsala. Cytological results were available for 28 patients, two of whom had provided cytological samples due to clinical symptoms . HPV results were available for 22 patients, of whom three had participated in primary screening with HPV tests  and one All the cytological results in the database were classified according to the classification of cervical intraepithelial neoplasia (CIN) by the Swedish Society for Clinical Cytology . For theμl distilled H2O. The water was then carefully removed with a pipette. The DNA elution was performed in 50 μl distilled H2O at 95°C for 30 min in a heating block (with heated lid). The DNA extract (3 μl) was used as the template in each real-time PCR  ,24 for HHomo sapiens hydroxymethylbilane synthase (HMBS); GenBank accession no. M95623.1] and the following HPV types, partly in groups: 16, 18/45, 31, 33/52/58, 35, 39, 51, 56 and 59. In order to be able to determine if a sample contained sufficient amounts of material for an HPV test to be considered informative, a threshold of 10 copies of the nuclear single copy gene was used per PCR, based on the analysis of HMBS. In addition, for the HPV test itself, the sample had to contain a minimum of 10 HPV copies to be typed positive  patients with AIS and an available cytological result showed signs of squamous cell changes, including 10 patients with atypical squamous cells of undetermined significance (ASCUS), three with LSIL and five with high-grade squamous intraepithelial lesions (HSIL). Only five patients had a cytological result of atypical glandular cells of undetermined significance  and two exhibited AIS 7.1%; .The HPV tests recorded in the database were performed on average 4.5 months  prior to the histological AIS diagnosis. A total of 22 patients were tested for HPV, 95% of whom were HR-HPV-positive. HPV18/45 was markedly predominant and occurred in 77% (n=17) of the HPV-positive patients, followed by HPV16 . Multiple HPV infections were recorded in five patients; three had double infections  and two exhibited triple infections (HPV16+18/45+33/52/58 and HPV16+33/52/58+56). The patients with multiple infections were all ≤32 years of age, while the patients aged ≥38 years were only infected with HPV18/45.The AIS-adjacent squamous mucosa showed various grades of CIN in all but three of the 32 patients with AIS, in whom the squamous epithelium did not show signs of atypical cells. Notably, CIN2 or worse was identified in the AIS-adjacent squamous mucosa of 25 patients with AIS 78.1%; .Cervical carcinogenesis is a stepwise process. Between 15 and 20 years are usually required for the increasingly abnormal (dysplastic) cervical epithelial lesions to progress to invasive cancer. Persistent infection has a critical role in the oncogenic HPV types –30. Sincet al  the percentage of adenocarcinomas of the uterine cervix varied between 2 and 7% across a prescreening cohort. During the screening period, adenocarcinoma cases increased significantly and accounted for 25.7% of cases on average (P<0.001), with a marked difference observed between younger and older individuals. In the screening cohort, the percentages of adenocarcinoma among younger patients and patients ≥70 years were approximately six- and three-fold higher, respectively, compared with the prescreening cohort .Although the present study was based on a relatively small number of cases, it is notable that the relative proportion of HPV18/45 infections in AIS increased with age. All patients ≥38 years of age were only infected with HPV18/45. In addition, the majority of patients ≥44 years of age were admitted for colposcopy due to a positive HPV test or a cytological result of abnormal glandular cells, not atypical squamous cells. The reason for this age-related difference remains unclear.et al showed that infections with HPV16 and 18 detected 15 years prior to diagnosis when no cytological changes were evident, were associated with an increased risk of developing in situ and invasive cervical adenocarcinoma (It is noteworthy that almost all patients with AIS had a recorded HPV infection with an oncogenic type. Cytology screening has a low sensitivity for identifying pre-malignant squamous alterations compared with primary HPV testing. It has been reported that HPV testing may be as much as twice as sensitive as cytology and that in older patients the difference may be even more pronounced ,38. When


Primary human papilloma virus (HPV) screening is more effective than cytology in reducing the risk of cervical cancer, but screening intervals should be extended in HPV-negative women. However, some Markov models predicted that long intervals are associated with an excess risk of cervical cancer. The aim of this analysis was to estimate the real-life risks and benefits of annual Papanicolaou (Pap) screening in HPV-negative women with normal cytology.Women with negative Hybrid Capture 2 (HC2) results and normal cytology at the time of inclusion in the Hannover HPV screening trial underwent annual Pap smears for 5 years. A subgroup was randomly selected for retesting with cytology, HC2, and colposcopy 60–68 months after recruitment.Of 4236 women included, 3406 had at least one Pap smear, but only 1185 attended all five annual screening visits. The proportion of women with at least one abnormal smear was 14.4% in 60 months. The probability of abnormal smears increased continuously over time. No case of ≥ CIN2+ was observed during 5 years. Of 605 women selected for subgroup analysis, 292 agreed to be retested (48.3%). The rate of high-risk HPV at 60–68 months was 3.0% (9/296).The long-term risk of high-grade neoplasia after an initial negative HC2 test and normal cytology result was low, while the rate of false-positive abnormal Pap smears was significant and increased constantly over time. Pap smear screening of HPV-negative women more frequently than every 5 years could be potentially harmful and seems to be of little clinical value. There is a high level of evidence that human papilloma virus (HPV) testing is more effective than cytology in reducing the risk of CIN3 and cervical cancer and allows for the extension of screening intervals up to 7 years ,2. OrganWhile there is general agreement among experts that the benefits of short-interval Pap smear testing may be outweighed by the risks of overdiagnosis, overtreatment and other kinds of harm, there is limited direct evidence to support this approach. In a prospective cohort study including 8466 women in Hannover and Tuebingen, Germany, we demonstrated that a negative HPV test result, even in combination with a positive Pap result, virtually excluded any risk of underlying high-grade disease . HoweverBetween December 1998 and September 2001, women (age 30 years or older) attending routine cervical cancer screening were recruited from 15 urban, suburban or rural, office-based gynecology practices in Hannover and the surrounding areas. Women were eligible for inclusion in the original trial  if they At the first gynecological examination, the cervix was visualized and a sample was taken for routine cervical cytology following the procedures normally used in each gynecological practice. A second sample was then obtained with a Digene Cervical Sampler , and suspended in 1 mL of specimen transport medium  for HPV DNA testing. Samples for HPV testing were stored at 4°C for a maximum of 4 weeks prior to testing at the University of Hannover. HPV testing was performed for high-risk types only, using the HC2 test (HR-HC2/Qiagen Inc.) following the manufacturer’s instructions. Samples were considered positive if they attained or exceeded the FDA approved threshold of 1.0 pg HPV DNA/mL.All cervical smears were analyzed at one of eight cytology laboratories routinely used by each participating gynecology office. In this study, smears were considered: a) ‘positive’ if any degree of cytological abnormality was observed ; b) ‘low-grade squamous intraepithelial lesion (LSIL) or more’ if classified as Pap3d (CIN1 or 2) or more. Pap3d (CIN 1) is equivalent to LSIL, whereas Pap3d (CIN2) is equivalent to high-grade squamous intraepithelial lesion (HSIL) in the Bethesda classification.The ‘Pap2w’ (W = wiederholen or repeat) is a widely used category although it is not part of official German (Munich II) cytology classification . Pap2w iWomen with negative HPV tests and normal Pap smear results (double negative) at recruitment were followed with annual Pap smear screening in private gynecology offices according to German guidelines. Pap smear results and all interventions related to cervical disease were reported for inclusion in the central study database.After approximately 5 years’ follow up (50–68 months) a representative subgroup of women with initially double-negative test results (every seventh participant) was invited for colposcopy, Pap smears and HC2 retesting. Punch biopsies or endocervical curettage were performed if appropriate.The aim of the follow-up subgroup analysis was to determine the proportion of women developing abnormal cytology and the proportion with HR-HPV infection at the end of the study.The rationale for the selection of every seventh double-negative participant was based on our previous experience showing that in the first year of a study only 50% of such subjects will accept the invitation for repeat screening. In Germany in women aged 30+ years the average rate of CIN3+ is 0.3–0.9% and of CIN2+ 1.0–1.6% [For data analysis we used SPSS Statistics version 20 . The Kaplan-Meier method was used for survival analysis to determine the proportions of events of ≥ Pap2w during the follow-up period . To inveAt enrolment, 4236 of 4737 women had negative HC2 results and normal Pap smear findings, of whom 3406 (80.4%) had at least one further routine Pap smear in the following 1–6 years. Mean age was 40.8 years (SD 8.0). In total, 281 women developed an abnormal cytology, of whom 202  were diagnosed with ASC-US and 79  with LSIL or more during follow-up. Kaplan-Meier cumulative 1-minus survival curves  had hysterectomies without colposcopy outside the study protocol because of persisting abnormal smears. No case of CIN2+ was reported among these 33 women with histological assessment.From 605 women randomly selected from the population that tested HC2 negative and showed normal Pap smears at study entry, 296 (48.9%) agreed to undergo colposcopy 60–68 months after recruitment  during Pap smear follow-up, including 33 women with histological assessment. We conclude that HPV screening allows early detection of clinically relevant lesions, and that the incidence of true high-grade lesions is low among women with a double-negative cotest (cytology and HPV), although likely to be higher in the routine setting compared with study conditions.Our data identify disadvantages of the current annual Pap smear screening concept in Germany. In participants who attended annually for all five screening visits, the risk of having at least one atypical Pap smear result consistently increased during the second to the fifth screening rounds. If every participant attended all five annual screening rounds (100% attendance), the overall specificity of the Pap smear screening program would be less than 86% for CIN2+. However, only approximately one-third of women attended at least four follow-up visits. The high rate of participants lost during follow-up is only partly explained by migration and women failing to attend routine visits. Another contributing factor is that women frequently changed their gynecologists and were lost to follow-up if the new practice was not participating in the study. As private practices act as independent units, the new practice was usually unaware of previous screening results. Therefore, we cannot exclude the possibility that some cases of high-grade neoplasia may have been missed, but based on the overall follow-up data, the rate of missed diagnosis seems to be very low.In fact, our study protocol may even underestimate adverse events associated with annual Pap smear tests. We actively discouraged excisional therapy because we assumed a very low risk of CIN2+ in the initially HPV-negative study population and, because we relied completely on voluntary submission of information about any kind of invasive procedures, we cannot exclude a substantial under-reporting of such measures. The practice of histological assessment of women with atypical Pap smears with the direct use of cold knife conization without prior colposcopy is still widespread and adds to the relatively high cost of cervical cancer screening as demonstrated in a recent study in Germany . In addiGuidelines recommend screening intervals can be safely extended to 3 years in women aged over 30 years when HPV and Pap tests are combined . For womWe found a low rate of new HPV infections among women who were initially tested negative for HR-HPV. The rate of new infections was just 3.0% based on HC2 results at 60–68 months of follow-up. This makes repeated HPV-screening with intervals of every 5 years an even more attractive option to prevent cervical cancer. It seems very likely that the HPV-prevalence will be much lower in subsequent screening rounds compared with the prevalence found at first recruitment.In conclusion, in women aged over 30 years with normal cytology and a negative HPV test result, Pap smear screening of HPV-negative women more frequently than every 5 years could be potentially harmful and seems to be of little clinical value.KUP received speaker’s honorarium from Roche Diagnostics and Becton Dickinson. GB received speaker’s honorarium from Hologic and Roche Diagnostics. TI received institutional grants from Gen-Probe and Hologic. The other authors declare that they have no competing interest.TI and KUP were responsible for the overall study design and trial coordination. KUP was responsible for the data analysis, interpretation, writing of the manuscript and data collection. FR, BH, GB were responsible for field work, data collection and interpretation. NB and MB were responsible for statistical analyses and interpretation. AL was responsible for quality control and data collection. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/379/prepub


Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas (BCs) around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV-16 viral load/physical status and E6/E7 expressions were determined.The amplification of a housekeeping gene showed that 46/55 samples (84%) had amplifiable DNA. HPV-16 was detected in 4/46 BCs (8.7%) and EBV was detected in 3/46 (6.5%) BCs. The analysis of HPV-16 physical status showed that this virus was integrated in all of the tumors with a relatively low viral load (range: 0.14 to 33.8 copies/cell). E6 and E7 transcripts, however, were not detected in any HPV-16 positive specimens. Using a Cox-regression model, we found a statistically significant association between EBV presence and poor survival (p = 0.013).The findings in this study suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of BC. Breast cancer (BC) is a leading cause of death in women around the world. In Chile, BC is the second cause of death by cancer with a mortality rate of 11.0/100.000 habitants . Thus, eIn this study, HPV and EBV sequences were searched in BCs from patients living in Santiago of Chile. Fifty-five formalin-fixed paraffin-embedded BCs from different public and private hospitals were collected and the extracted DNAs analyzed for integrity by amplifying a 110 bp fragment of the betaglobin gene. Forty-six samples out of 55 were betaglobin positive (84%). Using conventional PCR for a 65 bp fragment of L1 region, HPV was detected in 4/46 (8.7%) BCs. Using qRT-PCR for EBV EBNA-1, EBV was present in 3/46 (6.5%) BCs. Co-infection with both viruses was only detected in 1/46 2.1%) cases. In addition, ISH for EBER-1 showed that all of the BCs were EBV negative. A non-statistically significant association between HPV presence and age, tumor size, histology and differentiation grade was found  BCs [Other authors have analyzed HPV in BC specimens, suggesting a causal association. Hennig et al. proposed that HPV can be transported by circulation (bloodstream/lymphatic system) from cervix to the breast . In addi.6%) BCs . On the .6%) BCs .INK4a by a negative feedback mechanism [The loss of E2 gene function by HPV integration is known to be one of the major genetic events facilitating transformation and transition to malignancy . The E2 echanism . High-riechanism . The anaechanism . So, wheEven though, HPV-16 was present and integrated in BCs, we were unable to detect E6 and E7 transcript expressions in the specimens. The successful amplification of a fragment of β2-microglobulin was used as control of cDNA preparation and amplification from paraffin-embedded tissues. It has been reported that in general HPV load in extragenital tumors is heterogeneous and lower that in cervical cancer ,24,25 soOn the other hand, there is a relative consensus that for detection of EBV latent infection, the "gold standard" method is ISH for EBER-1 . InteresIn conclusion, we reported here that HPV and EBV prevalence in breast cancer from Chile is relatively low, thus a possible direct etiological role of these virus is unlikely. However, additional studies are warranted to elucidate the function of HPV/EBV in a subset of breast carcinomas from Chile.Fifty-five BCs from patients of different Hospitals in Santiago of Chile were considered for this study. The Hospitals were Las Condes Clinic; San Juan de Dios Hospital; Instituto Nacional del Cáncer; San Borja Arriaran Hospital; José Joaquín Aguirre Hospital; Arturo Lopez Perez Foundation and Santa María Clinic. The average age of the patients was 58.7 years (range: 48.1-69.3). The histological types of BC were: 48 ductal, 6 lobulillar and 1 mucinous. In the present study, histological classification was made using the guidelines of the Japan Cancer Society , which f-1 of proteinase K  and incubated overnight at 56°C. The solution was heated at 100°C for 10 min and subsequently the DNA was purified by phenol-chloroform extraction and precipitated with cold ethanol. DNA quality was tested by PCR for betaglobin using PCO3 5'-ACACAACTGTGTTCACTAGC-3' and PCO4 5'-CAACTTCATCCACGTTCACC-3' primers under the following PCR conditions: denaturation at 95°C for 15 min, 40 cycles with the cycling profile of 95°C for 1 min, 52°C for 1 min, 72°C for 1 min and final extension at 72°C for 5 min. To detect HPV genomes, broad-spectrum SPF10-biotinylated primers were used. The PCR products, 65 bp of the L1 gene, were characterized on a 4% agarose gel and visualized under UV radiation with ethidium bromide staining. HPV genotyping was performed using the Inno-Lipa HPV Genotyping V2 test  [Sections of 10 μm thickness were cut and collected in sterile tubes. For DNA extraction, each sample was treated with 1 mL of xylene and subsequently washed with 1 mL of ethanol. The sediment was resuspended in 50 mM Tris-Cl pH 8.0, 1 mM EDTA, pH 8.0, and 0.5% Tween 20 containing 200 μgmLBelgium) . As negaTo determine the presence, physical status and viral load of HPV-16, real-time PCR was performed with the ABI Prism 7700 Sequence Detection System  and 2 × QuantiTect SYBR Green PCR kit . The HPV-16 primers for E6 ORF amplification were as follows: E6F: 5'-GAGAAACTGCAATGTTTCAGGACC-3' and E6R: 5'-TGTATAGTTGTTTGCAGCTCTGTGC-3'. The HPV-16 primers for E2 ORF amplification were as follows: E2F: 5'-AACGAAGTATCCTCTCCTGAAATTATTAG-3' and E2R: 5'-CCAAGGCGACGGCTTTG-3'. The PCR conditions were 2 min at 50°C, 15 min at 95°C, and a two-step cycle of 95°C for 15 s, and 60°C for 60 s for a total of 40 cycles . The siz2. The conditions of amplification were: denaturation at 95°C for 10 min, 45 cycles of 95°C for 15 s, 55°C for 20 s, 72°C for 20 s and a final extension at 72°C for 20 s. The sequence of primers was: E6F: 5'-CAACAAACCGTTGTGTGAT-3'; E6R: 5'-CGTGTTCTTGATGATCTGC-3'. A melting curve analysis between 62°C to 95°C with a variation of 0.5°C was made. The melting temperature (Tm) of the amplification product from a positive control was 79.85°C. For E7, the reaction mixture was composed by 1X SensiMix, 0.8 μM primers and 3 mM MgCl2. The conditions of amplification were initial denaturation at 95°C for 10 min followed by 45 cycles consisting of denaturation at 95°C for 15 s, annealing at 57°C for 20 s and extension at 72°C for 20 s. The sequence of primers was E7F: ATGCATGGAGATACACCTAC; E7R: 5'-CATTAACAGGTCTTCCAAAG-3'. The amplification of β2-microbulin gene was carrying out as follow: 0.8 μM Primer F; 0.8 μM primer R, 5 mM MgCl2, 1X Sensimix and 50 ng cDNA. The amplification program was initial denaturation at 95°C for 10 min, followed by 45 cycles consisting of denaturation at 95°C for 15 s, annealing at 55°C for 20 s and extension at 72°C for 20 s. The sequence of primers was: B2MF: 5'-TGTAAGCAGCATCATGGA-3' and B2MR: 5'-AGTGTAAGTGTATAAGCATATCAA-3'. The melting point of the amplified product was 81.15°C. All the amplifications were made using Rotor Gene 6000 (Corbett Research) real-time PCR equipment.The RNA purification was carrying out using the High Pure RNA paraffin kit (Roche), according to instructions of the manufacturer. The obtained RNA was resuspended in 50 μL of TE  and stored at -80°C until use. The cDNA preparation was made as follows: 100 ng of purified RNA were transformed to cDNA using RNAsin 1 U/μL ; 1× buffer TR ; 10 μg/μL random primers ; 20 U/μL MMLV  and 2 mM dNTPs in a final volume of 20 μL. The reaction mixture was incubated at 37°C for 1 h, 70°C for 15 min and stored at -20°C. Ten microliters of cDNA were used for qRT-PCR. The amplification reaction was carrying out separately for HPV-16 E6 and E7 transcripts using the SensimixSYBR (Bioline) kit in a final volume of 25 μL. For E6, the reaction mixture was composed by 1X SensiMix, 0.8 μM primers and 5 mM MgClFifty-seven specimens of BC were used to construct a tissue array as previously described . The tumIn situ hybridization (ISH) with a complementary digoxigenin-labeled 30-base probe was used to detect EBER-1 expression according to the procedure previously published . BrieflyFisher's exact test was used for determine differences between groups. P-value < 0.05 was considered statistically significant. For survival analysis, a Cox-regression model was used.The authors declare that they have no competing interests.FA, SAk, AC and CK conceived of the study, analyzed the data and participated in the redaction of the manuscript. CG and NK made the analysis of the clinical specimens and analyzed the data. AC and LS got specimens for analysis, gave clinical information, and gave clinicopathologic support. OP gave statistical support and analysis. YE, CK and SAm gave analytical support for analysis. All authors read and approved the final manuscript.