Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis.E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology.Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene.Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis. E6 and E7 viral oncogenes [Head and neck cancer is the sixth most common cancer worldwide, with an incidence of around 600,000 cases per year, with rising trends particularly in young people ,2. Despincogenes -6. HPV-pncogenes , and resncogenes . Individncogenes ,11. ThusTP53 as a potential genomic stratifier for HPV status [The causes responsible for the different clinical behavior between HPV+ and HPV- tumors remain poorly understood. Numerous studies comparing gene expression patterns of HPV+ and HPV- cancers have shown different profiles for the two groups -16. It iV status ,18. AnalV status .Changes in DNA methylation play a key role in malignant transformation, leading to the silencing of tumor-suppressor genes and overexpression of oncogenes . In viruBased on these findings, we hypothesized that HPV modulates the epigenome in HNSCC, and set out to test this by comprehensive methylome analysis of HPV+ and HPV- primary tumors and cell lines , and informed consent was obtained where required.We obtained 107 archival formalin-fixed paraffin wax-embedded tissue (FFPE) oropharyngeal cancer samples from the Department of Histopathology (UCLH) and tested for HPV status. Of these, 21 HPV+ and 21 HPV- age-matched samples were selected for methylation analysis PCR on DNA extracted from both FF and FFPE samples. This combination of tests has been shown to have 97% sensitivity and 94% specificity, and to be the best discriminator of favorable outcome  immunostaining of the corresponding FFPE blocks, and confirmed by n = 8) were excluded from further analysis . Sections 3 μm thick were cut from a total of 82 FFPE blocks set from HNSCC samples . The results on all tested samples were negative, and HPV type 16 was the only HPV type that was detected.Primer and probe sequences have been published previously  was carried out on slides  using an automated processor . Depending on tumor size and pathology annotation, clusters of tumor cells were microdissected from one or more slides of the same FFPE block. For the HPV+ and HPV- samples, only the respective p16-positive and p16-negative tumor areas were dissected. The captured cells were estimated to contain 80% or more tumor cells.DNA was extracted using commercial kits from the FF tumor samples  and the laser-dissected FFPE samples .DNAs were prepared in a total volume of 20 μl (1 μg of FF and cell-line DNAs and 2 μg of FFPE DNA per sample) using a previously optimized protocol , in conjP < 0.01), resulting in a raw data matrix of 439,385 probes and 32 samples (18 HPV+ and 14 HPV-). This raw data matrix was then subjected to a principal component analysis to determine the nature of the largest components of variation. We used random matrix theory (RMT) to estimate the number of significant components of variation [R statistical software (version 2.14.0 ) was useariation ,37.a posteriori testing for a potential skew favoring type 1 probes. Although there were only 1,075 type 1 probes among the top 2757, this amounted to an over-enrichment, with an odds ratio of 1.48 (P < 1 × 1016). However, after correcting for differences in CpG density between type 1 and type 2 probes, the enrichment odds ratio favoring type 1 probes was significantly reduced to 1.13 (P = 0.03 approximately). Thus, there was no substantial skew favoring type 1 probes, and we found that normalizing for the design using the peak-based correction method of Dedeurwader only led to overcorrection and increased technical variability  which have slightly different profiles. Although there have been attempts to normalize for that difference , we foun and 2 whAll normalized and raw 450 k methylation data were submitted to the Gene Expression Omnibus  in accordance with the instructions provided .To quantify the strength of the association and to adjust for multiple testing, we estimated the false-discovery rate (FDR) using the q-value procedure . BecauseCopy number variation (CNV) analysis was performed on the DNA of the three HPV+ and three HPV- FF HNSCC samples using a genotyping array . This analysis was required for the normalization of the methylated DNA immunoprecipitation sequencing (MeDIP-seq) data. CNV data were analyzed using Genome Studio software (Illumina).DNAs from three HPV+ and three HPV- fresh-frozen HNSCC samples were subjected to autoMeDIP-seq as previously described , using aThe data were analyzed using the MeDUSA pipeline . Reads wAll normalized and raw MeDIP-seq data were submitted to GEO (NCBI) in accordance with the instructions provided (GEO accession numbers: GSE38263).R statistical software v2.15.1  was usedE7, HPV-16 E6, or both HPV-16 E6 and E7, respectively, were generated by infection of the SCC003 cell line with retroviruses followed by single-cell cloning as follows. Viruses were produced by transfecting human embryonic kidney (HEK)293T cells with pLXSN , together with pHIT- VSVG and MLV-gag/pol (kind gifts from Dr Juan Martin-Serrano) using polyethylenimine , then 72 hours post-transfection, viruses were harvested by removal of the medium and filtration through 0.45 μm surfactant-free filters . The filtered virus stocks were either frozen at -80C or diluted 1:2 in DMEM with 10% FBS and 8 μg/ml hexadimethrine bromide , cells were removed from selection, and plated at limiting dilution in 96-well plates to generate single-cell clones. HPV-16 E6 and E7 qPCR was conducted as described previously [E6 and E7 expression levels in E6-transduced, E7-transduced, and E6+E7-transduced SCC003 cell-line clones (and empty vector controls), qPCR was performed on cDNA following reverse transcription  of total RNA purified from cells , in accordance with the manufacturers' recommendations.HNSCC cell lines UPCI:SCC090 (HPV+), UPCI:SCC003 (HPV-), UPCI:SCC036 (HPV-) and PCI-30 (HPV-) . 93VU-147T (HPV+)  and UM:SCC047 (HPV+)  were used. All cell lines , as described above, were grown in T75 flasks in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin, then RNA was extracted from  in accordance with the manufacturer's recommended protocols, with DNAse digestion. cDNA was synthesized from 500 ng RNA  in accordance with the manufacturer's instructions.CDH8, PCDH10, DNMT1, DNMT3a and DNMT3b using a primer design tool . Sequences are shown in Table Primers were designed for 4 Mastercycler; Eppendorf AG, Hamburg, Germany) for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Relative expression was defined in terms of fold change of expression between the cluster of three negative cell lines relative to three positive cell lines for CDH8 and PCDH10, and vice versa for DNMT3a, DNMT3b, and DNMT1 using the ΔΔCt method on Ct values obtained from qPCR. P values were calculated using the two-tailed Student's t-test across Ct values for the six cell lines from three independent experiments.Master mixes were made up with SYBR Green , 0.2 μmol/l primers and 1 μl cDNA in 10 μl reactions, and qPCR was carried out on a thermal cycler 1500, TSS200, 5' untranslated region (UTR), first exon, body, and 3' UTR). We found that hyper-MVPs in HPV+ samples were preferentially located upstream or near the TSS or in gene bodies but not in first exons, whereas hypo-MVPs in HPV+ samples were preferentially located in gene bodies Figure . Taken tP = 0.001, Figure The enrichment of hyper-MVPs in HPV+ samples and the observation that many of these mapped to CpG islands suggested a possible association with CpG island methylator phenotype (CIMP) in these samples. To investigate this further, we performed consensus clustering over the top 1,000 MVPs, a procedure similar to the one used previously to discover CIMP phenotypes in breast and brain cancer ,52. The t-statistics obtained from the 32 FFPE samples with those obtained from the six FF samples, there was a very strong agreement  samples (three HPV+ and three HPV-). All six samples passed our quality-control criteria (see Methods). We applied the same Bayesian supervised analysis to rank MVPs according to how well they discriminated the three HPV+ from the three HPV- samples. The overwhelming majority of MVPs that were significantly hypermethylated in HPV+ FFPE samples were also hypermethylated (and many significantly hypermethylated) in the HPV+ FF samples relative to HPV- samples. Comparing the regularized t-statistics between the FFPE and HPV cell-line experiments correlated strongly , and again larger than in FF tumor samples  and theE6 and E7. After confirmation of ectopic expression of these HPV oncogenes in three clones of each cell line  . The distribution of methylation changes in E6, E6&E7, and E7 clones compared with controls is illustrated . The skew towards hypermethylation was significantly larger for E6 than for E6&E7, consistent with the lower expression levels of E6 in E6&E7-co-infected clones compared with E6-infected clones  was confirmed to be highly significant in P < 2.2 × 10-16). This suggests that HPV induces a distinct methylation signature that is independent of tissue-specific DNA methylation.To test further the effect of HPV on DNA methylation, methylation data obtained from the 18 HPV+ and 14 HPV- HNSCC samples were integrated with publicly available methylation data on HPV-induced versus smoking-induced cancer, 48 cervical-cancer samples  and 59 lP = 4 × 10-7) enrichment of hyper-MVPs within the cadherin genes. This was also the case in the top 1000 MVPs (Fisher P = 0.0003). We tested the possible biological effects of these cadherins in three separate ways, by showing that 1) their methylation state was sufficient to accurately cluster samples in accordance with HPV status, 2) they have consistent and significant hypermethylation across their promoter regions ; and 3) this promoter hypermethylation associates with decreased gene expression in existing data (see Expression section). Using the k-medoids clustering algorithm , we found that the 11 cadherin-annotated probes within the top 1,000 MVPs were sufficient to detect HPV status (84% correctly classified (27/32), Fisher exact test, P = 0.0002). These 11 MVPs mapped to CpG islands, shores, or shelves of six cadherin genes . The remaining 3.3% hypo-MVPs were enriched for two gene sets previously shown to display upregulation of gene expression in HPV+ head and neck cancers [To find consistent targets across all the data sets , we assigned all of the consistent hyper-MVPs and hypo-MVPs identified above to genes, and ran a gene-set enrichment analysis. The hyper-MVPs, which make up the majority (96.7%) of MVPs, identified several SUZ12 and PRC2 targets  enriched category of the six tested . A sample-permutation approach yielded an expected 4.4 false positives (see Methods). HPV cell lines showed the largest changes in mean TSS200 hypermethylation, significantly larger than FFPE  which showed significant hypermethylation relative to FF  with hypomethylated promoter DMRs.To assess the MVP associations in a more biologically relevant context, we grouped them into DMRs if at least three (range three to seven) had correlated differential methylation levels within the TSS200 promoter region. TSS200 was chosen because it was most significantly positively . These data are consistent with cadherin genes being targets for HPV-mediated hypermethylation and transcriptional silencing in HNSCC.For additional functional evidence of the effect of DNA-methylation changes on gene expression, we compared our methylation differences between HPV+ and HPV- FFPE tumor samples with publicly available gene-expression data . The topCDH8 and PCDH10. We found that CDH8 and PCDH10 were significantly overexpressed in our panel of three HPV- HNSCC cell lines relative to three HPV+ cell lines, correlating with hypermethylation in the latter . However, there was also significant hypomethylation at gene bodies, which, together with promoter hypermethylation, is a clear hallmark of gene silencing . It is wThe inclusion of multiple sample types  in the validation part revealed an important observation, with direct implications for projects with an epigenetic biomarker component, such as ICGC , IHEC 6, OncoTraE-Cadherin (CDH1), T-Cadherin (CDH13) and Proto-Cadherin 10 (PCDH10) which are recognized tumor-suppressor genes, and have been found to be hypermethylated in a number of human cancers [CDH8 and CDH13  was also found, and both are likely to contribute to the different clinical behavior of HPV+ and HPV- HNSCC with regard to survival and response to therapy. Linking these two lines of evidence suggests a possible mechanism whereby HPV could drive tumor progression by promoting EMT [We obtained two lines of evidence with respect to functional support for the identified hypermethylation signature. First, we were able to partially phenocopy the signature by ectopic expression of the two HPV oncogenes ion data . Among tting EMT  through This work significantly advances our understanding of the epigenetic dynamics at genomic loci targeted by oncogenic viruses as shown here for loci associated with the infection of HPV in HNSCC. Based on the previously established finding that patients with HPV+ HNSCC have a better prognosis than do patients with HPV- HNSCC, it is tempting to speculate that this advantage may be partly epigenetically mediated. Our results certainly implicate DNA methylation in this process. If confirmed, targeted reprogramming of the identified HPV-mediated hypermethylation signature (or parts of it) in patients who suffer from HPV-cancer offers a potential translational application for our findings. Although still at an early experimental stage, targeted reprogramming has recently been reported, including in cancer cells ,66. In tCIMP: CpG island methylator phenotype; DMEM: Dulbecco's modified Eagle's medium; DMR: Differentially methylated region; DNMT: DNA methyltransferase; EMT: Epithelial to mesenchymal transition; FBS: Fetal bovine serum; FDR: False-discovery rate; FF: fresh-frozen; FFPE: Formalin-fixed, paraffin wax-embedded; GADPH: Glyceraldehyde-3-phosphate dehydrogenase; HEK: Hhuman embryonic kidney; HNSCC: Head and neck squamous cell cancer; HPV: Human papillomavirus; HPV+: HPV-positive; HPV-: HPV-negative; MDS: Multi-dimensonal scaling; MVP: Methylation variable position; PcG: Polycomb group proteins; PRC2: Polycomb repressive complex 2; qPCR: Quantitative polymerase chain reaction; RMT: Random matrix theory; TSS: transcription start site; UTR: untranslated region.The authors declare that they have no competing interests.All authors contributed to the interpretation of data and to the writing of the manuscriptML, AET, CB, and SB were responsible for tudy design and conceptualization of the study; ML, AF, TF, CT, HKT, LB, NP, FG, FV, PO'F, and NK for sample preparation, tumor collection, and technical work; AJ for histology: AET, GW, ML, and JW for computational biology; and ML, AET, GW, and JW for figures and tables. All authors read and approved the final version.Supplemental figures.Click here for fileSupplemental tables.Click here for file


Despite the availability of the PAP test, cervical cancer continues to cause considerable morbidity and mortality. Many women default cervical cytology for a variety of reasons. This demands the development of alternative screening strategies, such as HPV testing on self-procured cervical-vaginal specimens in order to capture this group of women. We investigated the self-procured air-dried vaginal discharge for HPV testing. We recruited 82 patients with HPV-associated cervical lesions and 36 patients with normal cervical pathology. Participants were briefed and informed consents obtained. Each was then given a kit containing written instructions, a slim napkin, an empty zip-lock plastic bag for soiled napkin specimen, and a return envelope. After wearing the napkin for the day, the patient removes it, dries it, and returns the specimen by mail. Specimens were batched and a 0.5 cm area of each stained napkin was tested for HPV by PCR. Specimens from all 26 patients with high-grade (CIN 2 or above) HPV-induced cervical lesions and 4 of 36 normal subjects tested positive for HPV, giving a sensitivity and specificity of 100% and 88.9%, respectively. We propose offering to women who refuse cervical cytology the alternative screening strategy of testing of self-procured air-dried vaginal discharge for HPV. This method of cervical cancer screening is also suitable for people living in remote regions of the world. For half a century, the PAP smear was the only practicable way to screen for cervical pre-neoplasias and neoplasias. However, the inadequacies of the PAP smear is well known to practitioners of cytology , 2. It iHigh-risk HPV is found in almost all cervical cancers. With the discovery of the necessary role of human papillomavirus (HPV) in cervical carcinogenesis , 5, HPV There are considerable interests in exploring alternative specimens and methods for their collection for the diagnosis of HPV-induced cervical lesions (HCL). Thus, urine, vaginal tampons, and other means of self-procurement of cervical cells have been tested, with various successes and limitations -18.In 1869 Friedrich Miescher discovered DNA from pus that had collected on soiled surgical bandages . RecentlPatients referred to our hospital with abnormal PAP smears  or above) were screened by clinical history, repeat PAP smear, and colposcopy, with or without biopsy. During follow-up visits, patients with confirmed HCL Table  were invTo determine the value of HPV testing in patients who underwent ablative surgery for HCL, we recruited nine patients at 6 week after surgery for cervical intraepithelial neoplasia (CIN) by loop electrosurgical excision procedure (LEEP).The study was approved by the Clinical Research Ethics Committee of Princess Margaret Hospital, Hong Kong. Participants were briefed and consent obtained. Patients were then given a kit containing the following items: demographics and registration form, written instructions, commercially available slim sanitary napkin, zip-lock plastic bag, and return envelope. Patients were instructed to use the napkin on any day between periods. The napkin was introduced in the morning. At the end of the day, the sanitary napkin was removed, placed inside the zip-lock bag and gently blown dry. The specimen was then mailed to our laboratory for testing. We collected all the specimens and stored them in the laboratory at room temperature until testing. When all specimens have been collected, we accessioned the specimens and commenced batch testing.Researchers are blinded to the pathology results. Care was taken when handling specimens to prevent cross-contamination. All specimens were opened in the biological safety cabinet and visually inspected for stains. A 0.5 cm piece of napkin containing stains was cut out from each specimen and inserted into an Eppendorf tube for DNA extraction. Gloves were changed between cases. As a check against contamination, we intercalated an unused napkin for every ten specimens.DNA extraction was carried out by the addition of 600 ul cell lysis solution  and 3 ul proteinase K (20 mg/ml). The specimen was vortexed and incubated overnight at 56°C. After cooling to room temperature, the napkin fragments were removed. 200 ul protein precipitation solution was added to the cell lysate and the tube vortexed vigorously at high speed for 20 seconds. Centrifugation was carried out at 14,000 rpm for 5 minutes to pellet the precipitated proteins. This step was repeated if the pellet was not tight. The supernatant containing extracted DNA was pipetted into a clean 1.5 ml microfuge tube containing 600 ul 100% isopropanol. One ul of 20 mg/ml glycogen solution (DNA carrier) was added. The tube was inverted 50 times to mix the sample thoroughly, followed by centrifugation at 14,000 rpm for 2 minutes to pellet the DNA. After pipetting off the supernatant as far as possible, 300 ul 70% ethanol was added and the tube inverted several times to wash the DNA pellet. This was followed by centrifugation at 14,000 rpm for 5 minutes and the pipetting and discarding of the supernatant. The pellet was then air-dried for 15 minutes with the cap open. This was followed by the addition of 30 ul of DNA hydration solution.The specimen was then aliquoted into two separate reaction tubes for PCR, using GP5+/GP6+ primers for one tube and MY09/MY11 for the other tube as previously described , 22. NegSelected cases were typed by DNA sequencing and/or flow-through hybridization .Eighty-two patients with HCL were recruited. They represented a cross section of lesions ranging from condyloma to invasive squamous carcinoma, adenocarcinoma and clear cell carcinoma of cervix Table . VisibleAll specimens with CIN 2 or above, with the exception of a small cell carcinoma tested positive in at least one PCR reaction , 83 and CP6108  . HPV typNine patients had HPV testing six weeks after LEEP Table . PatientMost other methods of cervical specimen procurement for HPV testing have assumed the need for preserved (in preservation solution) specimens. Others have emphasized the advantage of reflex cytology on liquid-preserved specimens. We favor air-drying of the specimens to enhance the ease of handling and compliance, having previously detected HPV in air-dried vaginal discharge . Here weAlthough we did not systematically conduct opinion polls, patients have not expressed dissatisfaction with this method of specimen collection. In fact, the compliance was excellent. All recruited patients submitted the requested specimens. We have also demonstrated that it is feasible to receive specimens in the mail and that specimen processing can be done rapidly and without cross-contamination. In addition, we found that specimen storage in room temperature without preservatives or refrigeration is compatible with HPV detection even after several months (data not shown).Our results showed that all 26 cases of CIN 2 or above, including 9 cases of various types of invasive carcinoma were positive for HPV DNA on PCR-testing of self-procured air-dried vaginal discharge. The importance of high-risk HPV in the pathogenesis of such lesions cannot be overemphasized , 26. TheSome institutions have explored the role of HPV testing following surgical ablation of HCL , 28. We The advantages of air-dried specimens are manifold. Patients can collect such specimens by themselves without the need for any special tools or the purchase of special equipments, which add to the cost, complexity, and stigmatization associated with it. Because the specimens can be mailed to the testing facility, the pre-analytic cost is further reduced. It also permits anonymity, thus facilitating the inclusion of as many women as possible – reporting can be performed on the internet if the patient self-assigns unique user identification. Finally, even women living in under-serviced localities may be extended the benefits of cervical cancer screening. This is particularly relevant to people living in rural areas in many continents, especially Asia, Africa, Australasia, and the Americas.Our study is restricted by the referral nature of our clinic population. The true value of HPV testing in cervical cancer screening using our approach can only be realized in a population screening setting involving much larger number of subjects. In addition, other methods of HPV testing may be explored to enhance automation and further reduce the cost.In conclusion, we demonstrated that self-procured air-dried vaginal discharge is a preferred specimen type for the identification of high-grade HPV-induced cervical lesions. Researchers can now focus on two issues, determining whether this approach to cervical cancer screening is acceptable to women and validating this approach as being applicable to the general population. Women who underwent LEEP for CIN and who did not recur were also negative for HPV, suggesting that eradication of CIN by LEEP also eradicates HPV. Like cutaneous wart, cervical HPV infection also seems to be a surgical disease. Resection or extirpation of HPV-associated cervical lesions might be employed more liberally to eradicate HCL as wells as HPV.


The control arm of PATRICIA  was used to investigate the risk of progression from cervical HPV infection to cervical intraepithelial neoplasia (CIN) or clearance of infection, and associated determinants.Women aged 15-25 years were enrolled. A 6-month persistent HPV infection (6MPI) was defined as detection of the same HPV type at two consecutive evaluations over 6 months and clearance as ≥2 type-specific HPV negative samples taken at two consecutive intervals of approximately 6 months following a positive sample. The primary endpoint was CIN grade 2 or greater (CIN2+) associated with the same HPV type as a 6MPI. Secondary endpoints were CIN1+/CIN3+ associated with the same HPV type as a 6MPI; CIN1+/CIN2+/CIN3+ associated with an infection of any duration; and clearance of infection. The analyses included 4825 women with 16,785 infections (3363 womenwith 6902 6MPIs). Risk of developing a CIN1+/CIN2+/CIN3+ associated with same HPV type as a 6MPI varied with HPV type and was significantly higher for oncogenic versus non-oncogenic types. Hazard ratios for development of CIN2+ were 10.44 (95% CI: 6.96-15.65), 9.65 (5.97-15.60), 5.68 (3.50-9.21), 5.38 (2.87-10.06) and 3.87 (2.38-6.30) for HPV-16, HPV-33, HPV-31, HPV-45 and HPV-18, respectively. HPV-16 or HPV-33 6MPIs had ~25-fold higher risk for progression to CIN3+. Previous or concomitant HPV infection or CIN1+ associated with a different HPV type increased risk. Of the different oncogenic HPV types, HPV-16 and HPV-31 infections were least likely to clear.Cervical infections with oncogenic HPV types increased the risk of CIN2+ and CIN3+. Previous or concomitant infection or CIN1+ also increased the risk. HPV-16 and HPV-33 have by far the highest risk of progression to CIN3+, and HPV-16 and HPV-31 have the lowest chance of clearance. Persistent infection with oncogenic human papillomavirus (HPV) is necessary for the development of cervical cancer . ApproxiChlamydia trachomatis and herpes simplex virus [Many sexually active individuals become infected with HPV after beginning their first sexual relationship . HPV infex virus –11. CIN ex virus . ®Cervarix) in over 18,000 young women.A better understanding is needed of the natural history of progression of a cervical HPV infection to a CIN or to clearance of the infection. Such analyses can be performed using the control arm of large trials of prophylactic HPV vaccines where data on HPV types, histological lesions and potential determinants of disease progression are extensively collected. The present paper reports the analysis of HPV infection and its clearance or progression to CIN in the PApilloma Trial against Cancer In young Adults (PATRICIA), a phase III trial of the HPV-16/18 AS04-adjuvanted vaccine , controlled, 4-year PATRICIA trial (NCT00122681/580299/008). The objectives were to investigate the time between detection of an HPV infection (through HPV genotyping) and development of a CIN lesion associated with the same HPV type, and to evaluate determinants associated with disease progression or natural clearance of infection.The clinical trial methodology, including full inclusion and exclusion criteria, trial locations and dates, has been described previously ,14. BrieSubjects completed a behavioural questionnaire  includinWritten informed consent or assent was obtained from all participants or their parents, and the protocol and other materials were approved by independent ethics committees or institutional review boards , a 12-month persistent infection (12MPI), a less than 6MPI, and an infection detected only at the last visit of the study. A transient infection was defined as detection of a specific HPV type in a cervical sample at any single point during the follow-up period, followed by a negative sample for the same HPV type at the next evaluation. A 6MPI was defined as detection of the same HPV type at two consecutive evaluations over a 6-month period or greater i.e. a sequence of positive samples with the same HPV type not interrupted by negative samples over a total range of >5 months (>150 days). The start of the 6MPI was defined as the date of the first positive sample in the sequence. A 12MPI was defined in the same way, over a 12-month period. A less than 6MPI was defined as an infection with duration ≤150 days, again with the same HPV type, not interrupted by negative samples .Clearance was defined as the occurrence of at least two consecutive type-specific negative HPV DNA samples taken at approximately a 6-month interval following a positive sample. Although we recognise that apparent clearance could in reality be an inability to detect the infection, we use the term clearance for convenience. Histologically confirmed lesions were detected in colposcopically-directed biopsy samples or post-excision samples following treatment and were categorised as CIN grade 1 or greater (CIN1+), CIN grade 2 or greater (CIN2+) and CIN grade 3 or greater (CIN3+). CIN1+ included CIN1, CIN2, CIN3 and adenocarcinoma in situ (AIS) identified by standard methods. The primary endpoint was CIN2+ associated with the same HPV type as a 6MPI. Secondary endpoints were CIN1+ and CIN3+ associated with the same HPV type as a 6MPI; CIN1+, CIN2+ and CIN3+ associated with the same HPV type as an infection of any duration; and clearance of HPV infections.The analysis was performed in the control group of the total vaccinated cohort for efficacy (TVC-E) . The TVCChlamydia trachomatis during the past 12 months, marital/partner status, previous pregnancy, use of hormones for contraception or other indication, and use of an intrauterine device. The main determinants of interest were HPV type and duration of HPV infection. Covariates known to influence the risk of acquiring HPV infection were also accounted for in the analyses. Eight potential behavioural characteristics were included in the models: tobacco exposure measured as number of pack-years , age at first sexual intercourse, number of sexual partners during the past 12 months, history of In addition, the models accounted for previous cervical HPV infection with an oncogenic HPV type or with only non-oncogenic HPV types (i.e. preceding the onset of the reference infection), cervical HPV co-infection with an oncogenic HPV type or with only non-oncogenic HPV types (i.e. following the onset of the reference infection and preceding detection of the lesion), previous CIN1+ lesion associated with any oncogenic or non-oncogenic HPV type different to the reference infection (i.e. CIN1+ preceding the onset of the reference infection), and concomitant CIN1+ lesion associated with any oncogenic or non-oncogenic HPV type different to the reference infection (i.e. CIN1+ following the onset of the reference infection and preceding its end). Cervical HPV co-infections and CIN1+ lesions associated with an HPV type different to the reference HPV infection were included in the models as time-varying covariates.The time between a cervical HPV infection and development of a CIN lesion was analysed using the Kaplan-Meier method as well as univariate and multivariable Cox regression models. The statistical unit was the infection, and variance estimates adjusted for the correlation within subjects were obtained using the robust estimation method ,18. The A total of 9256 women in the TVC-E were aged 15–25 years and had negative or low-grade cytology . Of thesChlamydia trachomatis (90.3%) and had used hormonal contraception (65.4%)  age was 19.7 (3.1) years, most were single (74.4%), with no tobacco exposure (63.4%), had started sexual intercourse between the ages of 15 and 17 years (51.8%), had one or no sexual partner during the past 12 months (71.3%), had not been pregnant (73.1%), had no history of  (65.4%) . CharactThe number of women with a 6MPI who developed a CIN1+, CIN2+ or CIN3+ lesion during the study is shown in A previous infection with an oncogenic HPV type different from the 6MPI significantly increased the risk of CIN1+  . PresencResults of the univariate and multivariable analyses of the risk of developing a CIN lesion that account for behavioural determinants are shown in Tables S2–S4 in HPV-16, HPV-51 and HPV-52 were the most frequent HPV types observed , with HPV-16 being the most frequently detected in CIN1+ and CIN2+. As observed for 6MPIs, in the multivariable analysis, the association between an infection of any duration with an oncogenic HPV type and development of a lesion became stronger as lesion severity increased . Again, The duration of the HPV infection had a marked impact on the risk of developing a lesion, with a 6MPI showing the highest risk for CIN lesion development, followed by a persistent infection of less than 6 months duration, while a transient HPV infection showed the lowest risk . CompareCo-infection with an oncogenic HPV type different from the reference infection significantly increased the risk of developing CIN1+, CIN2+ or CIN3+ . A previResults of the univariate and multivariable analyses of the risk of developing a CIN lesion that account for behavioural determinants are shown in Tables S5–S7 in Overall, 53%, 79%, 87% and 89% of all HPV infections were cleared at 12, 24, 36 and 48 months, respectively . The medResults of the univariate and multivariable analyses of clearance of HPV infection that account for behavioural determinants are shown in Table S8 in The results for prevalence, risk of progression to CIN and chance of clearance are summarised according to HPV type in The analysis evaluated the risk of progression to CIN lesion and the chance of clearance according to HPV type. The role of HPV types and persistent infection as the main risk factors for CIN lesion were confirmed and the results also showed the roles of previous infection and co-infection with an oncogenic HPV type, as well as previous or concomitant CIN1+, as risk factors for progression to CIN in young women. Our large sample size allowed for the first time calculation of the risk of progression of HPV infection to CIN3+. Previous studies have been limited in their sample size and number of infections evaluated, and largely focused on development of invasive cervical cancer in adult women.The five most prevalent HPV types were HPV-16, HPV-31, HPV-18, HPV-33 and HPV-45. The risk of developing a CIN lesion was higher following infection with oncogenic HPV types than with non-oncogenic types, the highest risk being with HPV-16 and HPV-33 (up to 10-fold higher for CIN2+ and up to 25-fold higher for CIN3+ compared with non-oncogenic types), followed by HPV-31, HPV-45 and HPV-18. The association between an infection with an oncogenic HPV type and the risk of CIN became stronger as lesion severity increased, reflecting the increasing prevalence of HPV types belonging to the A7 and A9 HPV species as lesion severity increases .Our findings reflect those of other studies evaluating prevalence of different HPV types in cervical cancer and the risk of progression from infection to pre-cancerous lesions or cancer. A US-based case-control study showed that the most prevalent HPV types in ICC and carcinoma in situ are HPV-16/18/45 and HPV-16/31/33, respectively . An inteHPV-18 is the second most prevalent HPV type in ICC after HPV-16 ,19. The The risk of developing a CIN was increased by co-infection. Similarly, risk was increased by the presence of a separate CIN1+; this was true regardless of whether the CIN1+ lesion preceded the onset of the reference infection or whether it was concomitant with the reference infection. Some studies have shown that there is a higher chance of acquiring a new HPV type if already infected –27, and Consistent with the risk of progression to CIN, HPV-16 and HPV-31 had the least chance of being cleared, followed by HPV-33, HPV-18, HPV-45 and any other oncogenic type. HPV-16 and HPV-31 have been previously reported to have the lowest rates of clearance . PreviouSeveral behavioural determinants were associated with higher risk of progression from infection to lesion  and lower chance of clearance of infection . An increased risk of cervical abnormalities and/or cancer associated with these determinants has been shown in other studies –8.The analyses had some strengths and limitations. We included only women with a confirmed HPV infection including 6MPI. The analysis therefore focused on factors that may influence the risk of progression from infection to lesion, and was not confounded by the risk of HPV acquisition. Another major strength was that the PATRICIA study included a large population with well-characterised virological and histological samples and high follow-up rates over 48 months. Although the study was multinational, involving women from many ethnic groups, all participants followed the same protocol. The high number of participants and length of follow-up allowed analysis of CIN3+ lesions, which are a better predictor of ICC than CIN2+ lesions . PreviouA limitation of the analysis is that misclassification of HPV infection below the threshold for detection by PCR  might have overestimated rates of transient HPV infection and underestimated 6MPI. Therefore, analyses of clearance were done on subjects with two consecutive negative PCR results (“confirmed clearance”). However, a sensitivity analysis using a single negative PCR result gave very similar results (data not shown), demonstrating that the risk of misclassification is low. As mentioned earlier, apparent clearance could actually represent inability to detect the infection. In addition, because pre-cancerous lesions may develop over an extended period, the duration of which is influenced by the HPV type the lesion is associated with, a follow-up of 48 months will not allow detection of all lesions. This may have led to underestimation of the progression rate of some HPV types. A further limitation is that many women started sexual activity before study enrolment and thus some previous HPV infections would not have been recorded. Finally, the study included only women aged 15–25 years, with six or fewer lifetime sexual partners and no history of immunosuppressive disease, limiting its generalizability; in particular, age may have an important influence on the rate of progression or clearance of HPV infection.In conclusion, we have used data from the control arm of the large, well-controlled PATRICIA study to evaluate the natural history of progression of HPV infection to CIN lesion or to clearance according to individual HPV type. Importantly, we included CIN3, the immediate precursor lesion of cervical cancer. A better understanding of the natural mechanisms of progression to CIN lesions could contribute towards effective implementation of HPV testing as part of screening for early detection and prevention of cervical cancer, as recommended by recent US guidelines . InfectiFile S1Supporting FilesTable S1. List of Independent Ethics Committees/ Institutional Review Boards.Table S2. Risk of progression of a 6-month persistent cervical HPV infection to a CIN1+ lesion associated with the same HPV type.Table S3. Risk of progression of a 6-month persistent cervical HPV infection to a CIN2+ lesion associated with the same HPV type.Table S4. Risk of progression of a 6-month persistent cervical HPV infection to a CIN3+ lesion associated with the same HPV type.Table S5. Risk of progression of an HPV infection of any duration to a CIN1+ lesion associated with the same HPV type.Table S6. Risk of progression of an HPV infection of any duration to a CIN2+ lesion associated with the same HPV type.Table S7. Risk of progression of an HPV infection of any duration to a CIN3+ lesion associated with the same HPV type.Table S8. Clearance and median duration of a cervical HPV infection.(DOCX)Click here for additional data file.File S2Protocol and STROBE documents.(ZIP)Click here for additional data file.


Two clinically relevant high-risk HPV (HR-HPV) types 16 and 18 are etiologically associated with the development of cervical carcinoma and are also reported to be present in many other carcinomas in extra-genital organ sites. Presence of HPV has been reported in breast carcinoma which is the second most common cancer in India and is showing a fast rising trend in urban population. The two early genes E6 and E7 of HPV type 16 have been shown to immortalize breast epithelial cells in vitro, but the role of HPV infection in breast carcinogenesis is highly controversial. Present study has therefore been undertaken to analyze the prevalence of HPV infection in both breast cancer tissues and blood samples from a large number of Indian women with breast cancer from different geographic regions.The presence of all mucosal HPVs and the most common high-risk HPV types 16 and 18 DNA was detected by two different PCR methods - (i) conventional PCR assays using consensus primers  or HPV16 E6/E7 primers and (ii) highly sensitive Real-Time PCR. A total of 228 biopsies and corresponding 142 blood samples collected prospectively from 252 patients from four different regions of India with significant socio-cultural, ethnic and demographic variations were tested.All biopsies and blood samples of breast cancer patients tested by PCR methods did not show positivity for HPV DNA sequences in conventional PCRs either by MY09/11 or by GP5+/GP6+/HPV16 E6/E7 primers. Further testing of these samples by real time PCR also failed to detect HPV DNA sequences.Lack of detection of HPV DNA either in the tumor or in the blood DNA of breast cancer patients by both conventional and real time PCR does not support a role of genital HPV in the pathogenesis of breast cancer in Indian women. BRCA1 and BRCA2 [Breast cancer is the second most common cancer in the world, after the cancer of the lung, affecting one in eight women during their lifetime, but it is the leading cancer among women worldwide . In Indind BRCA2 ,4, sex-sHigh-risk human papillomaviruses (HR-HPVs) are carcinogenic viruses which are primarily associated with cervical cancer but are also linked with other anogenital cancers and cancers of other organ sites  such as Reports on the distribution of HPV infection in breast cancer are not only limited but also highly controversial. Several authors -31 incluA total of two hundred fifty two breast cancer patients (n = 252) were recruited for analysis of HPV infection from different geographic regions of India. The region wise distribution of samples is presented in Table Cellular DNA from freshly collected breast tumor biopsies and peripheral venus blood were isolated using standard Proteinase K digestion, phenol-chloroform extraction and ethanol precipitation method routinely being used in our laboratory ,49,50. TApproximately, 100-200 ηg cellular DNA was utilized for conventional PCR using the protocol routinely followed in our laboratory ,49,51. F2, 12.5 μM of each dNTP , 5 pmoles of each oligonucleotide primer and 0.5 U Taq DNA polymerase . The temperature profile used for amplification constituted an initial denaturation at 95°C for 5 min followed by 30 cycles with denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec which was extended for 7 min in the final cycle. The HPLC-purified oligonucleotide primers were custom synthesized commercially by M/s Microsynth GmbH .Briefly, the method involved a 25 μl reaction mix containing 100-200ηg DNA, 10 mM Tris-Cl (pH 8.4), 50 mM KCl, 1.5 mM MgCl4-101) supplied under WHO Global HPV LabNet program. Background genomic DNA from HPV-negative cell line C33a was used as negative control and for dilution of standards as prescribed by WHO. Briefly, the amplification was performed in 25 μl reaction mix containing 5 pmol each of GP5+ and GP6+ primers and 30-50ηg of genomic DNA in 5 ul of test volume. The amplification ramp included first step for 3 min at 95°C for initial denaturation, followed by denaturation cycle of 20 sec at 95°C, an annealing cycle of 15 sec at 40°C and an elongation and readout cycle of 30 sec at 72°C for 45 cycles. The specificity was verified by a dissociation curve analysis. Linear plots of the log copy number vs the number of the threshold cycles was consistently obtained for HPV16 L1 amplification and the correlation coefficient was between 0.995 to 1.000 in each run.Highly sensitive qRT-PCR was performed for detection of low copy number HPV infection. qRT-PCR employed in the study had the capability of detecting as low as 5 copies of HPV genome per reaction which is equivalent to 0.005 HPV copies per cell equivalent using GP5+/GP6+ consensus primers with Biorad SYBR Green Supermix kit and iCycler PCR . p53 exon 5 primer sequences Table  were useOf 252 patients studied, 62% were in pre-menopausal stage while the remaining 38% formed the post-menopausal group. Clinical staging showed 7.14% (18/252), 14.3% (36/252), 23.01% (58/252), 23.8% (60/252), 26.6% (67/252) and 5.15% (13/252) of the patients belonging to stage I, IIa, IIb, IIIa, IIIb and IV respectively, whereas histopathologic grading revealed 22.60% (57/252), 50.8% (128/252) and 26.6% (67/252) of the tumors in grade I, II and III respectively (Table No HPV DNA was detected using consensus primers located within the conserved L1 region of HPV genome by performing 3 to 4 times repeated conventional PCR in any of the 228 breast tumor and 142 blood DNA specimens, while β-globin showed amplification of 268 bp product in all the cases Figure . The repDespite breast cancer being one of the most frequently diagnosed cancers in women, the etiology and molecular pathobiology of breast carcinogenesis is still not clearly understood. In addition to various exogenous, endogenous, hormonal and genetic risk factors responsible for breast carcinogenesis, its viral etiology particularly implicating HPV remains highly controversial. Although HPV infection, particularly the HR-HPV types 16 and 18, have been strongly implicated in the development of cervical  and otheOur previous study on a limited number of patients  as well Of 22 previous studies on HPV in breast cancer till date, 15 reported presence of HPV infection in breast cancer and the authors have tried to put forward their possible explanation for detection of HPV DNA in breast cancer. Although the spreading of genital HPV infection from one site to the other is not established, a few studies have reported detection of genital HPVs in peripheral blood lymphocytes of patients with urino-genital cancers -67 and hIt is also interesting to note that several authors found both positive and negative results from the same region ,29,43,44Similarly, from Austria, Widschwendter et al  analyzedInterestingly, in Brazilian women, Damin et al  could noSeveral other authors from different parts of the world reported presence of HPV infection in breast cancer cases. It is, however, most important to note that not many studies have indicated or analyzed if the women with breast cancer recruited for HPV detection also had anogenital or oropharynheal lesions with HPV infection. Although the route of HPV transmission from one organ to the other is not clear, Hening et al  demonstrIn spite of exceedingly higher prevalence of genital HPV infection in India as compared to that in Europe and in the USA including presence of other conducive risk factors such as early age marriage, multiple pregnancy, malnutrition, poor hygiene and healthcare, the complete absence of HPV infection in Indian women with breast cancer in the present study does not suggest the role of HPV in breast carcinogenesis in Indian women. It is suggested that although HPV DNA is undetectable, serologic assay may be useful in revealing exposure by detecting presence of antibodies against HPV. But natural HPV infection being poorly immunogenic, the antibody titer will be too low to be detected with the existing serological tests. HPV serology is presently not available in our laboratory and is being developed under the WHO LabNet programme. We however, do not preclude the possibility of yet unidentified HPV playing important role in breast carcinogenesis in India. Furthermore, due to the changing social and psyco-sexual behavior particularly in urban Indian population, it may not be unlikely to find, presence of HPV in breast tissue in future due to an exterior transfer of HPV form genital organs to the breast.Complete absence of HPV DNA either in the tumor tissue or in the blood of the same breast cancer patients recruited from four different regions of the country by employing the highly sensitive qRT-PCR does not support the etiological role of oncogenic HPV in the pathogenesis of breast cancer in Indian women.HPV: human papilloma virus; PCR: polymerase chain reaction and Breast carcinomaThe authors declare that they have no competing interests.SH - carried out experiments, analyzed the data, and drafted the manuscript. S Hussain, DB, MMM - provided breast cancer biopsy samples, clinical diagnosis and information from J & K. UK, SP, AT, RK - to perform some of the experiments and critical reading of the manuscript. NJ, SS - has done all work related to real time PCR. TD - provided blood samples of breast cancer patients, diagnosis and clinical information from Goa. SC - provided breast cancer biopsy and blood samples, diagnosis and clinical information from Silchar. YMS - provided clinical breast cancer biopsy and blood samples, diagnosis and clinical information from Imphal. ACB - co-designed the study and revised the manuscript. KS - collection of blood and biopsy samples from Bangalore and critical reading of the manuscript. BCD - conceived and designed the study, interpreted the data and critically corrected and communicated the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/27/prepub


A biobank is a depository for biomaterials from a representative portion of a human population and acts as a vault with intricate detailed information pertaining to the individuals from whom biological materials have been collected. Biobanks can be classified into population biobanks, biobanks for molecular epidemiology and biobanks for disease biology.RGCB has been involved in biobanking with respect to three large studies in India namely i)Molecular Epidemiology of HPV in India, ii) A Randomized 2 versus 3 dose HPV Vaccination Clinical trial and iii) HPV AHEAD- : Role of Human Papilloma Virus Infection and other co-factors in the aetiology of Head & Neck Cancer.A cervical cytology biobank has been established at the centre which is an almost inexhaustible resource for fundamental and applied biological research. It helps to understand the natural history of HPV infection and HPV induced lesions and cancers, screening effectiveness, exploration of new biomarkers, surveillance of the short- and long-term effects of the introduction of HPV vaccination. However legal and ethical principles concerning personal integrity and data safety must be respected strictly and biobank based studies require approval of ethical review boards.The HPV vaccination study involves 10 different sites & different collaborating institutes across the country and all samples are shipped to RGCB, Trivandrum, which acts as a Central storage facility/ biobank. Sample shipment was an important procedure involving communication between base laboratory and biobank, import of registration database, strict temperature control, tracking of shipment and ready storage freezers on arrival. The sample verification process involves a temperature logger, freeze control tubes and bar code verification. The amount of clinical data linked to the samples determinate the availability and biological value of the sample. Thus acquiring the background information of each sample and cataloguing the available information systematically and meticulously is a must.The major problems faced in the management of cervical cytology biobanks have been reclassification of smears before entry into database, failure of temperature logger, bar codes slipping off and maintaining uninterrupted power supply.Publicly funded biobanks aim to promote the development of new knowledge by giving the research community access to data and samples. The most efficient way to acquire these benefits is to first maximize the use of biobanks in research and, second, to maximize the dissemination of knowledge developed by the research projects that used the biobanks. However there are issues with regard to the use of such stored materials especially when the demand occurs from the private sector. The decision is finally made by a scientific management committee consisting of members of World Health Organization (WHO) and the international agency for research on cancer (IARC).


Objective: It has been documented that human papilloma virus (HPV) DNA replication requires proliferating cell nuclear antigen (PCNA). However the association between them in tumors is still controversial. Up to now, the role of HPV in laryngeal squamous cell carcinoma (LSCC) has not been clearly established, and the correlation between HPV and PCNA in LSCC remains poorly explored.Methods: We retrospectively reviewed the clinicopathological features and follow-up data of 71 patients with LSCC. The lesions were examined for PCNA using immunohistochemistry, and for HPV using in situ hybridization. Results: 31 (43.7%) cases showed infection of HPV and 38 (53.5%) showed overexpression of PCNA. No significant difference of HPV status in clinicopathological features was found. While there was a significant difference of PCNA expression in histology grade but no significant difference of PCNA expression in other clinicopathological features could be detected, and the expression of PCNA is not a significant predictor of survival in LSCC patients. However, HPV infection is a favorable prognostic factor in LSCC patients. Moreover, HPV infection is associated with PCNA overexpression.Conclusion: Human papilloma virus (HPV) infection is an indicator of better prognosis in LSCC and associated with the expression of PCNA.  But to date the correlation between HPV and LSCC is still disputed. Many studies have reported that HPV infection is associated with a better prognosis in patients with head and neck squamous cell carcinoma (HNSCC).2,3 However, there are frequent controversial and contradictory results.4Laryngeal squamous cell carcinoma (LSCC) is one of the most common forms of head and neck cancer. The incidence is increasing continuously. Since three decades ago, human papillomavirus (HPV) has been considered as a causative agent and linked to LSCC.5 The association has been shown between PCNA expression and cell proliferation in head and neck tumors.6The proliferating cell nuclear antigen (PCNA) is a nuclear polypeptide essential for DNA synthesis and replication of mammalian cells. Its expression reaches a maximum point in the S-phase of the cell cycle. The level of PCNA is one of the methods to define the tumor proliferation. PCNA has been evaluated as an unfavorable prognostic factor and might be associated with radiosensitivity.7 Some studies have investigated the correlation between the PCNA expression and HPV infection. However, the results remain highly controversial.8,9 To date, the relationship between the expression of PCNA and the presence of HPV in patients with LSCC remains poorly explored. In the present study, we evaluated the PCNA, HPV and clinicopathological features in the patients suffering from LSCC, and investigated the relationships between them.It has been documented that HPV DNA replication requires the presence of PCNA.This study consisted of 71 patients with LSCC who were diagnosed and treated with surgical management in our department between 1997 and 2008. Adjuvant radiotherapy was given in addition to surgical treatment to 39 patients. To assess the advancement of the disease, the TNM classification according to 2002 UICC was used. A minimum of two years follow-up was required, unless the patient died from the disease.Immunohistochemistry: All resection specimens were fixed in 10% formalin and rountinely processed for paraffin embedding. Five microns thick sections from the tissue blocks were placed on coated glass slides, and for immunohistochemical examination the streptavidin peroxidase method was used. The sections were deparaffinized and rehydrated, and then microwaved for an antigen retrieval procedure. Tissue sections were immunostained applying the antibody PCNA  in a dilution 1:100. Counterstaining was performed with hematoxylin. In negative controls the primary antibody was omitted, and a section previously shown to be positive was used as a positive control. The expression of PCNA was assessed in the cell nuclei and evaluated using a five-point grading system. Less than 10% tumor cells positive was scored 0, 10% to 25% scored as 1+, 25% to 50% scored as 2+, 50% to 75% scored as 3+, and more than 75% scored as 4+. At least 500 cells per high-power (objective lens×40) field and three fields were observed, and grades of 3+ and 4+ were regarded as overexpression.In Situ Hybridization (ISH): In situ hybridization was performed according to the protocol of HPV In Situ Hybridization/Detection Kit, (Types 16/18) . In brief, the tissue sections (5 μm) were baked at 73℃ overnight. After deparaffinizing, the sections were digested with proteinase K, DNase and RNase solution. Then hybridization probe was added and heated at 95℃ for 10 minutes to denature the DNA. To allow hybridization of the probe with the target nucleic acid, the sections were incubated at 37℃ overnight. After hybridization finished, the sections were blocked with protein block solution, and combined with the Biotin-Antigen and streptavidine-AP conjugate. In negative controls the hybridization solution (negative control) provided by the Kit was used. HPV staining was documented as either positive or negative.The differences between the values of different groups were evaluated by the chi-square test. Disease-free survival and overall survival curves were calculated using the Kaplan-Meier method. And the significance of the differences between the curves was estimated by the log-rank test. Multivariate analysis was performed using the Cox logistic regression model by Wald’s backward method. P≤0.05 was considered significant. SPSS 12.0 statistical software was used to conduct all statistical analysis.The present study was approved by the Ethics Committee of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University. The clinicopathological features of the patients are summarized in Disease-free survival: During the follow-up, 20 patients relapsed. Among these patients, 9 recurred at the primary site, 9 recurred in the neck and two patients had distant metastasis. In univariate analyses, stage (p=0.017) and HPV infection (p=0.029) were the statistically significant predictors of DFS , N status (p=0.015) and stage (p=0.002) (p=0.002) . While Hp=0.002) . In mult10 also detected HPV infection in LSCC. Similar to our results, they indicated that no significant correlation was found between the incidence of HPV and the epidemiological and histological grade, and clinical stage of tumors. In their results, HPV is also detected more frequently in T2-T4 stage and in patients with nodal relapse, but more HPV-positive patients are younger.Recently, many studies have focused on the relationship between HPV and LSCC. In the present study, we found that HPV infection is more frequent in elderly patients than younger patients, and more frequent in T2-T4 stage than T1 stage, and detected more frequently in patients with nodal metastasis. However, none of these differences is statistically significant. Morshed11 explored the presence of HPV in LSCC and found that there was no significant correlation between the presence of HPV in laryngeal carcinomas and clinicopathological variables such as age, site, grade, lymph node status, and stage of the disease. While Hoshikawa et al12 showed that laryngeal carcinomas of glottic origin had higher HPV positive rates  than those of other sites. Nichols et al13 detected HPV-16 infection in oropharyngeal squamous cell carcinoma and demonstrated that HPV-16 infected tumors are correlated with smaller primary site size. Although the relationships between HPV infection and clinical characteristics of patients with LSCC have been explored in multiple studies, the results are still controversial.Moreover, same as our results, no obvious difference of HPV infection between supraglottic and glottic tumor was found. Jacob et al14 reported that HPV positive HNSCCs have higher survival rates than HPV negative cases. Fakhry et al3 evaluated the association of HPV status with therapeutic response and survival in patients with head and neck squamous cell carcinomas, and demonstrated that patients with HPV positive tumors had higher response rates after chemoradiation treatment and significantly improved overall survival. Ang KK et al2 analysed the association between tumor HPV status and survival among patients with stage III or IV oropharyngeal squamous cell carcinoma and found HPV status is a strong and independent prognostic factor. In present study, the data indicated that the HPV infection is a favorable predictor of either disease-free survival or overall survival of patients with LSCC. However, there are still controversial and contradictory results. Stephen et al15 detected HPV in 79 patients with primary LSCC and indicated that no differences in survival between HPV positive and HPV negative patients is found. Morshed10 investigated HPV status in patients with LSCC, and found that HPV positive laryngeal cancer patients had no significantly better overall survival and disease specific survival when compared with patients with HPV negative tumors.HPV infection is considered as an indicator of better prognosis in patients with HNSCC in many studies. Pintos et al16,17 E7, which is a viral oncoprotein, can be produced by HPV, and binds to pRb leading to releasing the E2F transcription factor which can activate PCNA gene. Thus, PCNA can be regarded as a surrogate marker of viral early gene activity in papillomavirus infected tissue.18 Branca et al19 observed that in patients with cervical intraepithelial neoplasia (CIN) or cervical carcinomas PCNA expression intensity was significantly associated with the detection of high-risk HPV in the lesions, being significantly more intense in high-risk HPV positive lesions. Similar correlation between HPV and PCNA was also found by Noya et al,20 who showed that HPV E7 protein induces PCNA protein in differentiated keratinocytes by reactivating the transcription of the PCNA.PCNA is an E2F-regulated gene product which is induced in papillomas by the presence of E7.9 evaluated PCNA expression and HPV status in patients with cervical cancer and no significant correlation was found between these two. To our knowledge, there has been little study to report PCNA expression and HPV presence in the patients with LSCC. Jacob et al11 used PCR to detect the infection of HPV and immunohistochemistry to detect the PCNA expression in 41 patients with LSCC. Their results showed that HPV infection was correlated with increased expression of PCNA. However, they did not explore the role of HPV infection in prognosis of patients with LSCC.While other studies failed to establish any direct association between the PCNA expression and HPV infection. Wang et al21 Compared with PCR method, ISH can identify the HPV infection in the tumor cell nuclei, which could provide more reliable results. In this study, we used in situ hybridization to explore HPV infection and more patients were included. The result showed that HPV infection was associated with PCNA overexpression. Moreover, in present study, there was a significant difference of PCNA expression in histology grade but no significant difference of PCNA expression in other clinicopathological features could be detected, and the expression of PCNA is not a significant predictor of disease-free survival or overall survival in LSCC patients. Similar results were also demonstrated by other studies.22Moreover, although PCR is more sensitive than other approaches, as total DNA are extracted from tissue samples, so the detected HPV DNA can be derived from non-cancerous cells, a tumor surface contamination or just only few cancer cells.Either HPV or PCNA has been investigated as a LSCC tumor marker in many studies. However, the relationship between HPV infection and PCNA expression in LSCC tumors has not been well documented. The present study showed that HPV infection is a favorable prognostic factor in patients with LSCC although no significant difference of HPV infection in the clinicopathological features was detected. Furthermore, the infection of HPV is significantly correlated with PCNA overexpression in LSCC patients.


Testing for high-risk human papillomavirus (HPV) in primary screening for cervical cancer is considered more sensitive, but less specific, in comparison with Pap-smear cytology. Women with persistent HPV infections have a higher risk of developing cervical intraepithelial neoplasia 2+ (CIN2+) lesions. This study was performed to evaluate the gain in specificity for detection of histologically confirmed CIN2+ lesions achieved by short-time repeat testing for high-risk HPV in women aged 30–65 years, with the primary sample for HPV analysis taken by self-sampling.A total of 8000 women in Uppsala County, aged 30–65 years, who had not attended organised screening for 6 years or longer, were offered self-sampling of vaginal fluid at home and the samples sent for HPV typing. Of these, 8% (669) were not possible to contact or had performed hysterectomy. Women positive for high-risk HPV in the self-sampling test were invited for a follow-up HPV test and a cervical biopsy on average 3 months after the initial HPV test.In all, 39% (2850/7331) of invited women chose to perform self-sampling of vaginal fluid at home. High-risk HPV infection was found in 6.6% (188) of the women. In all, 89% of the women testing HPV positive performed a follow-up examination, on average 2.7 months, after the first test and 59% of these women were HPV positive in the follow-up test. The prevalence of CIN2+ lesions in women with an initial HPV-positive test was 23% (95% CI 18–30%) and in women with two consecutive HPV-positive tests was 41% (95% CI 31–51%). In women with two positive HPV tests, the prevalence of CIN2+ lesions varied from 49% in women at age 30–39 years to 24% in women at age 50–65 years. Short-time repeat HPV testing increased the specificity for detection of CIN2+ lesions from about 94.2% to 97.8%. The most prevalent HPV types were HPV16 (32%), followed by HPV18/45 (19%) and HPV 33/52/58 (19%).The short-time persistence of high-risk HPV infection in this age group was about 60%. Repeat testing for high-risk HPV using self-sampling of vaginal fluid can be used to increase the specificity in the screening for cervical cancer in women aged 30–65 years. The introduction of organised cytological screening in Sweden in the late 1960s has resulted in a decrease of the prevalence of cervical cancer by ∼50% (Infection with high-risk types of human papillomavirus (HPV) is the dominant risk factor for development of cervical carcinoma , and it The use of testing for high-risk HPV in primary screening is complicated by the low specificity of a single HPV-positive test as an indictor of cervical cancer risk. Many women have transient infections, and HPV testing in primary screening could therefore result in inconvenience if no premalignant cell alterations are diagnosed in the cytological smear and no treatment is available for HPV infection. The value of HPV testing can be increased by triaging with cytology or using p16 staining. However, women with a persistent HPV infection have been shown to be at higher risk of developing cervical cancer  from Uppsala County, between the ages of 30–65 years and who had not attended organised cytological screening for 6 years or longer according to the data registry of the organised screening, were offered self-sampling of vaginal fluid at home using the Qvintip device , 2850 (39%) chose to participate and sent a vaginal fluid sample to Aprovix for high-risk HPV analysis. The samples were analysed using the hybrid capture II (HC2) method, which detects the presence of any of 13 high-risk HPV types . The result of the HPV analyses was communicated to the participating women within 2–3 weeks after the arrival of the vaginal sample to the laboratory. Women testing positive for HPV were offered an attendance to a gynaecological surgery for a follow-up examination within 1–3 months. At the follow-up visit, the physician performed a colposcopy, collected biopsy samples from the portio  and sampled cervical fluid for a repeat HPV analysis. The second HPV test was performed using the hpVIR method, which detects the following high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59  of participating women performed self-sampling of vaginal fluid at home. No difference in attendance rate was seen for different age groups (data not shown). In all, 6.6% (188/2850) of the participants were positive for high-risk HPV in the self-sample. The prevalence of high-risk HPV decreased with age, from 11.5% in women aged 30–39 years to 4.7% in women aged 50–65 years . The CINOf the 188 women that were positive for high-risk HPV in the self-sample, 168 performed a follow-up examination, giving a compliance of 89% (168/188). Eleven of the women chose to visit a midwife reception for sampling of cervical smear for cytological screening and HPV analysis. These women are not further described. In three additional women, cervical biopsy samples were collected but no sample for HPV analysis was obtained, and these were also excluded from further analyses.In the remaining 154 women, cervical biopsies were performed in combination with sampling for a repeat HPV testing at a gynaecological surgery. Five HPV tests were regarded as invalid because of low amount of cells in the sample. In the remaining 149 women with two valid consecutive high-risk HPV tests, of which 88 were positive and 61 negative, the presence of CIN2+ lesions could be evaluated. The mean time between the first and second HPV test was 2.7 months, ranging from 4.0 months for women in the age group of 30–39 years to 1.9 months for women in the age group of 50–65 years.P=0.350). Among the 88 women with a persistent HPV infection, 36  had histologically defined CIN2+ lesions . The specificity of using a single HPV test in screening in comparison with two consecutive HPV tests for detection of histologically defined CIN2+ is shown in Information on the infecting HPV type was obtained for 88 women participating in the second HPV test. HPV16 was the most prevalent type (32%), followed by HPV18/45 (19%) and the group HPV33/52/58 (19%). Among other types, HPV31 occurred in 9%, HPV35 in 3%, HPV 39 in 3%, HPV51 in 3%, HPV6 in 4% and HPV59 in 10% of women positive in the second HPV test. The prevalence of HPV16 decreased with age, from 34% in premenopausal women to 23% in postmenopausal (⩾50 years old) women  relative to the women aged 30–39 years (4 months). Another explanation is that the transformation zone between squamous and glandular mucosa moves upwards in the cervical canal after menopause and it is possible that a number of CIN2+ lesions are not detected in older women despite gynaecological examination, colposcopy and cervical biopsy.We used two different high-risk HPV tests in the present investigation. Although the hpVIR test is favoured by its ability to be type specific, the self-sampling device Qvintip has been validated in combination with the FDA-approved HC2 method . The prevalence of HPV16 decreased with age from 34% in premenopausal women to 23% in postmenopausal women. This observation may have some significance with respect to the long-term effect of human HPV vaccination, in particular for countries with an organised screening, in which most cervical cancer cases occur in women 50 years of age and older.


The relation between HPV and head and neck cancer has recently and extensively been investigated. The purpose of this study was to indentify HPV genotypes, as well as E6/E7 mRNA expression of high-risk HPVs  in oral squamous cell carcinomas (OSCCs) from 45 Greek patients. The overall prevalence of HPV DNA positive OSCCs was 11.1% (5/45), while high-risk HPV DNA was found in 6.7% (3/45) of OSCCs. E6/E7 mRNA expression was detected in 8.9% (4/45) of the oral cavity samples. Our data indicated that HPV 16 was the commonest genotype identified in HPV-positive OSCCs by both DNA and RNA tests. This study confirms the prevalence of HPV infections among patients with OSCCs. Future analysis and followup of more OSCCs will enable us to correlate HPV detection and clinical outcome. Cervical infection by one of approximately 15 high-risk HPV types is generally accepted as the necessary causative agent of cervical cancer . The uppHNSCC is usually etiologically linked to tobacco and/or alcohol or other lifestyle habits, but a minority of patients develop HNSCC in the absence of exposure to these factors or any other obvious predisposing genetic defect. Several investigators have reported detection of HPV infection in healthy oral mucosa as well as in squamous cell carcinoma of the oral cavity (OSCC) and oropharynx (OPSCC) , 5, whicIn the cervix, the persistence of HPV infection may increase the likelihood of viral integration and concomitant deregulation of viral protein expression, leading to overexpression of E6/E7 oncoproteins . In oralThe purpose of our study is to define HPV detection in patients with OSCCs and to evaluate the detection of E6/E7 high-risk HPV mRNA as a possible biomarker for posttreatment surveillance.Sixty-four (64) cytological samples were obtained from patients with HNSCC of the oral cavity,  who visited the outpatient clinic of the Radiotherapy Department of the Regional Anticancer Oncology Hospital “Saint Savvas”, Athens, Greece, between November 2008 and November 2011. Patients were eligible if they had not received any previous cancer treatment. Samples were obtained upon diagnosis. Ethical approval was granted by the ethics committee of Regional Anticancer Oncology Hospital of Athens “St. Savvas,” and all participants provided written, informed consent. The samples were collected using a brush and swabbing the mouth, maxilla and mandible, oral tongue, and buccal mucosa and were preserved in Thin Prep .μL of elution buffer. DNA quality test was carried out using Human Globin, Beta, Primer set kit  according to manufacturer's instructions. To assess RNA integrity, 5 μg of RNA per sample was separated on 1% formaldehyde-agarose gel.Cytological samples were transferred in lysis buffer  for 30 minutes, then total nucleic acid was extracted by the offboard protocol with the NucliSENS easyMAG platform (bioMérieux Hellas S.A), according to the manufacturer's instructions. The nucleic acids were eluted in 55 μL PapilloCheck MasterMix, 0.2 μL HotStarTaq plus DNA polymerase , and 5 μL DNA from the tissue sample. Hybridization is followed by mixing 30 μL of the PapilloCheck hybridization buffer in a new reaction tube with 5 μL of the PCR product at room temperature and transferring 25 μL of the hybridization mix into each compartment of the chip. We incubated the chip for 15 minutes at room temperature in a humid atmosphere. The chip was washed in 3 washing solutions, centrifuged for 3 minutes at 5000 rpm, and scanned on the CheckScannerTM.Then, the PapilloCheck HPV-Screening  was used for the type-specific identification of 24 types of HPV . This technology is based on a DNA chip for the type-specific identification of 24 types of HPV. E1-based PCR was performed according to the manufacturer's guidelines. For each sample, we mixed 19.8 2). In each premix, we added U1A/HPV 16, HPV 33/45, or HPV 18/31 primer and molecular beacon mixes, KCl stock solution, and NASBA water. Secondly, 10 μL of this premix was distributed to each well in a reaction plate, and the addition of 5 μL RNA followed. The plates were incubated for 4 minutes at 65°C to destabilize secondary structures of RNA, followed by cooling down to 41°C. The reaction was started by addition of enzymes  and measured in real time using the Lambda FL 600 fluorescence reader  at 41°C for 2 hours and 30 minutes.A commercial real-time NucliSENS EasyQ assay  was performed for the qualitative detection of HPV E6/E7 mRNA of five high-risk HPV types  according to the manufacturer's instructions. Firstly, three premixes were made by adding reagent sphere diluent  into reagent spheres  OF < 70%. Furthermore, patients with severe renal, liver, cardiovascular, or pulmonary diseases and patients with hematological malignancies were excluded.Following diagnosis and molecular analysis, patients with positive histology of HNSCC of the oral cavity, with a Karnofsky Index (KI) ≥ 70%, and with normal renal, hepatic and bone marrow function were included. Baseline laboratories studies requirements included neutrophils greater than 1.5 × 10All participants underwent an extensive presurgical evaluation that included clinical examination, panendoscopy, complete blood count and biochemical profile, clearance creatinine, cardiovascular examination, chest X-ray or CT, and CT and/or MRI of head and neck. 2N1M0) were treated surgically. Patients with locally advanced disease , stage III-IVA, were treated either with induction chemotherapy or radical chemoradiation. Depending on response after induction chemotherapy, patients underwent either surgery followed by postoperative chemoradiation or radical combined radiation therapy with chemotherapy.Patients with early stage resectable disease (stage I-II) and selected patients of stage III  were marked by at least four points, which were 0.5 cm away. Tumor response was evaluated after the end of induction chemotherapy using a clinical examination and CT or MRI of the head and neck with tumor volumetry. Clinical tumor response to chemotherapy was defined by the RECIST criteria. Complete response (CR) was defined as a reduction of the tumor volume ≥ 70% up to the complete disappearance. Partial response (PR) was defined as at least a 30% decrease in the sum of diameters of tumor, taking as reference the baseline sum diameters. Progressive disease (PD) was defined as enlargement of tumor volume ≥ 20% or new tumor manifestations. Stable disease (SD) was defined as insufficient shrinkage to qualify for PR and insufficient increase to qualify for PD.The median Follow-up interval was 18 months (range 4 to 36 months). Patients are monitored every two months in the first two years, every three to four months in the next 3 to 5 years, and once a year thereafter until death. Overall survival was calculated from the date of diagnosis to the date of death. Disease-free survival was estimated from the end of treatment to tumor recurrence, distant metastasis, or death from any cause.P values of <0.05 were considered statistically significant.Data were analysed using SAS v9.0. Absolute and relative frequencies were used to present the HPV positivity. Chi-squared tests were performed to assess statistical significance of any differences in prevalence. 2 × 2 contingency tables Fisher's exact test was performed along with odds ratio and 95% confidence intervals calculation. We collected 64 samples from the oral cavity, 19 of which were unsatisfactory for molecular analysis due to insufficient quantity of DNA, thus 45 samples were finally analysed . The patFurther analysis using the NucliSENS EasyQ assay detected E6/E7 mRNA expression from the five high-risk HPV types  in 4/45 of the total samples (8.9%) . HPV 16 was the commonest type revealed by the RNA test as shown in 1N0M0), 5 Stage II (T2N0M0), 2 Stage III (T2N1M0)) underwent surgical excision . 4 of them had no adverse features, and they did not undergo any further treatment. The rest of the 4 patients with adverse features  received external beam radiation therapy (EBRT) 54–60 Gy.8 of 45 patients . However, one of them developed a second primary tumor of the lung and so far is disease-free from both primaries .2, (d1) Cisplatin 75 mg/m2 (d1), and 5-FU 500 mg/m2 (d1-d5) (referred as treatment protocol A) and 9 patients underwent radical external beam radiation therapy (EBRT: 68–72 Gy) with concurrent chemotherapy (Cisplatin) (d1–d22) and weekly cetuximab due to medical contraindications for induction chemotherapy with TPF (referred as treatment protocol B). From the 37 patients with locally advanced  disease (stage III-IVA), 28 patients underwent induction chemotherapy with 2 cycles of TPF  and weekly cetuximab. For the patients with high-risk features, such as R1 resection, extra capsular nodal spread, and vascular space invasion, perineural invasion total dose of 66 Gy was granted.From 28 patients treated with protocol A, 18 had complete response. 10 of them underwent radical surgery and 8 of them underwent radical radiotherapy with concurrent chemotherapy. The inception of postoperative radiotherapy was recommended not later than 8 weeks after surgery. Depending on histological findings, patients underwent EBRT 54–60 Gy with or without concurrent chemotherapy , 11 HPV DNA negative), 2 died from lung metastasis (1 HPV DNA positive (HPV 16), 1 HPV DNA negative), and 2 are still alive with local recurrence (2 HPV DNA negative) .The rest of the 10 patients who were treated with protocol A had partial response . All of From 9 patients with stage III-IVA who were treated with protocol B, 5 of them are free of disease, 1 died from metastatic disease, and 3 are still alive with local recurrence. All of them were HPV DNA negative. Of the approximately 120 HPV types so far known to infect the mucosal surfaces of the genital tract, 14 are considered to be “high risk” or “oncogenic” , 8, 12. The great variation in HPV prevalence found in OSCCs in different studies may be due to differences among the analyzed population, but also due to differences in the samples tested , the methods of DNA extraction, and, most importantly, the HPV detection methods used , 16. SevOur study shows that the use of DNA and RNA detection methods confirms the prevalence of HPV infection among patients with OSCCs. HPV 16 infection has been found in 2 cases. In both cases infected by HPV 16, the E6/E7 transcripts have been detected by the RNA test. Coinfections were not completely absent. In one case with HPV 6 infection, what is interesting is that the RNA test revealed HPV 16 E6/E7 mRNA. This may be explained by the fact that total viral DNA of HPV 16 has been integrated into the host genome, and, therefore, it cannot be detected by the DNA test.In 75% of the HPV-positive SCCs of the oral tongue, NucliSENS EasyQ assay detected E6/E7 mRNA expression of HPV 16 and HPV 18. HPV implication in oral tongue cancer has already been stated by many authors . P = 0.4215). The prevalence of HPV among currently disease-free patients was also not statically significant (P = 1.000).On the other hand, clinical results indicated that the association between patient mortality and HPV detection was not statistically significant (As already stated, E6/E7 mRNA expression from high-risk HPV types is probably a less common event in HNSCC patients , 14. NevAlthough authors support better prognosis for HPV-positive oral cancer cases , 16, 18,Sexual transmission of oncogenic HPV genotypes is universally accepted for the anogenital region, whereas the route of infection for the oropharyngeal region is unclear , 18. AltThe tumor biology of HPV-positive OSCC is still under investigation . HPV detThe ability to control head and neck cancer will therefore depend on three basic cornerstones: prevention, detection, and early diagnosis. The recent development of an HPV vaccine might offer hope for the prevention of cervical and anogenital carcinomas and possibly also represent an additional prevention option for a substantial number of patients with HNSCC.This study described the detection rates and attribution of HPV genotypes as well as the E6/E7 mRNA expression of site-specific OSCCs in 45 Greek patients. HPV 16 accounted for most HPV-positive OSCCs. DNA and RNA assays detected the same genotypes in all high-risk HPV infected samples. This study confirms the prevalence of HPV infection among patients with OSCCs. Future analysis and followup of more OSCCs will enable us to correlate HPV detection and clinical outcome.


The problem to be addressed in this paper is a phase-modulated waveform design for the detection of extended targets contaminated by signal-dependent noise (clutter) and additive noise in practical radar systems. An optimal waveform design method that leads to the energy spectral density (ESD) of signal under the maximum signal-to-clutter-and-noise ratio (SCNR) criterion is introduced first. In order to make full use of the transmission power, a novel phase-iterative algorithm is then proposed for designing the phase-modulated waveform with a constant envelope, whose ESD matches the optimal one. This method is proven to be able to achieve a small SCNR loss by minimizing the mean-square spectral distance between the optimal waveform and the designed waveform. The results of extensive simulations demonstrate that our approach provides less than 1 dB SCNR loss when the signal duration is greater than 1 μs, and outperforms the stationary phase method and other phase-modulated waveform design methods. In actual radar systems, it is necessary to select the operating band, transmit waveform modulation, and receiver processing strategy judiciously in order to maximize the probability of detecting the presence of a target while maintaining a prescribed rate of false alarms . AdditioMuch earlier work has been presented on the techniques of waveform design for detecting targets –4 and onIn this paper, we only consider zero Doppler targets that represent the worst-case scenario, and describe an approach that yields the applicable phase-modulated waveform for extended target detection in the presence of clutter. The scattering behavior of extended target that can be described by the impulse response function ,8,9, whiThe main contribution of this paper is the method of appropriate phase-modulated waveform design for extended target detection based on the optimal waveform derived from the maximum SCNR constraint. Considering the general requirements of waveform on finite duration and energy , an analThe paper is organized as follows. In Section 2, we present the system model and define the problem statement. In Section 3, considering the finite waveform energy and duration time, we introduce an optimal waveform design method for extended target detection according to SCNR criteria. In Section 4, we introduce a phase-iterative algorithm for designing the appropriate phase-modulated waveform. In Section 5, we present the performance results and discuss the proposed waveform design algorithms. Our conclusions are given in Section 6.2.u(t) is a complex-valued finite duration baseband transmit waveform for −T/2 ≤ t ≤ T/2, where T is the signal duration time. The scattering of target and clutter are viewed as linear processes that are modelled by the system impulse response h(t) and w(t) respectively, here h(t) is the impulse response of the extended target and is assumed to be deterministic and integrable and w(t) is a zero-mean complex wide sense stationary (WSS) Gaussian random process with known power spectral density (PSD) wwP(f). The transmit signal u(t) is modulated to the transmission carrier frequency cf and passes through the target channel h(t) and clutter random channel w(t), the return echo is demodulated to baseband signal v(t) and then passes the ideal low-pass filter (LPF) with passband . In addition, n(t) is the zero-mean additive WSS Gaussian noise with known PSD nnP(f) and is supposed to be independent of u(t), h(t) and w(t). Because the impulse response h(t) and w(t) are assumed to be independent in practical applications, the target echo s(t) and the clutter returns c(t) are also independent when the transmitted signal u(t) is deterministic and accurately known.The block diagram in y(t) is:LPF[·] represents the ideal low-pass filter with passband  and sy(t), cy(t) and ny(t) are the target echo component, clutter returns and additive noise, respectively. Additionally, the transmit waveform u(t) must satisfy the finite duration and energy constraints . In nnP(f) and the spectrum of extended target impulse response H(f + cf). Because the ESD difference G(ϕ(t)) can converge to zero when the signal duration time T is infinite T.Initialize the phase vector p = 0 and calculate the ESD difference G(0).Let the initial number be p = p + 1.k from 0 to N – 1 and find the solution Set up)( = cejϕp)( = G(ϕp) and δ = Gp)( – Gp−1); otherwise, up) decreases monotonically with each iterative step, the phase-modulated waveform ESD pmɛ(f) can approximate the optimal transmit ESD optɛ(f) as much as possible in the mean-square sense.Since 5.In this section, numerical results are presented and some discussions are conducted to evaluate the performance of the optimal waveform design for extended target detection and the proposed phase-iterative algorithm for the phase-modulated waveform design.5.1.5.1.1.cf is 10 GHz, the bandwidth B is 10 MHz, the energy of transmit signal E is 106 joules, and the additive noise is white with PSD nnP(f) = 1 for f ∈ . A target with multiple reflection centers according to the multiple reflection model . The optimal transmit ESD for extended target detection in f ∈ . The additive noise is assumed to be white with PSD nnP(f) = N0, where N0 is a constant. To simplify the discussion, the value of A is assumed to be E is large enough so as to make λ ≤ 1. The optimal transmit ESD |U(f)|2 in Under this assumption, the impulse response of a point target can be represented by a delta function; therefore, the amplitude spectrum of the target impulse response will be a specified value For the transmit energy constraint in U(f)|2 = ξ/wwP(f + cf), where ξ is a constant. The PSD of clutter returns c(t) will be ccP(f + cf) = |U(f)|2 wwP(f + cf) = ξ, it means the optimal signal can whiten the clutter. This result is the same as Kay’s given in . T is assumed to be 2 μs. As seen in pmu(t) by the optimal phase, which is the angle of the optimal signal optu(t), can achieve a better ESD approximation with fewer iterations.The initialization of the phase vector 6.The problem of detecting targets in clutter has been the subject of much research and has led to the development of clutter rejecting waveforms. In this paper, we perform the phase-modulated waveform design for approximating to the optimum performance of the extended target detection. A simple theoretical solution of the optimal ESD of complex-valued transmit waveform is obtained. Based on this, we present a novel phase-iterative method for designing the phase-modulated waveform, aiming to minimize the mean-square spectral distance between the optimal waveform and the designed waveform, which can promise the approximation of the output SCNR to the maximum SCNR. The numerical simulations show that the advantage of the proposed phase-iterative waveform design technique is substantial, and that our approach outperforms the stationary phase method and other phase-modulated waveform design methods.