Background. Next-generation sequencing of cancers has identified important therapeutic targets and biomarkers. The goal of this pilot study was to compare the genetic changes in a human papillomavirus- (HPV-)positive and an HPV-negative head and neck tumor. Methods. DNA was extracted from the blood and primary tumor of a patient with an HPV-positive tonsillar cancer and those of a patient with an HPV-negative oral tongue tumor. Exome enrichment was performed using the Agilent SureSelect All Exon Kit, followed by sequencing on the ABI SOLiD platform. Results. Exome sequencing revealed slightly more mutations in the HPV-negative tumor (73) in contrast to the HPV-positive tumor (58). Multiple mutations were noted in zinc finger genes  and mucin genes . Mutations were noted in MUC12 in both tumors. Conclusions. HPV-positive HNSCC is distinct from HPV-negative disease in terms of evidence of viral infection, p16 status, and frequency of mutations. Next-generation sequencing has the potential to identify novel therapeutic targets and biomarkers in HNSCC. Tobacco use has steadily declined over the last four decades . In paraNext-generation sequencing has yielded important insights into the pathogenesis of other cancers by identifying biomarkers and therapeutic targets. High-throughput sequencing of HNSCC tumors has recently been reported, and NOTCH inactivation was the most significant finding , 7. ThisEthics approval was obtained from Western University Health Sciences Research Ethics Board. Informed consent was obtained from patients undergoing ablative surgery for head and neck cancer to have a portion of their tumor stored, a 10 mL blood sample taken, and their clinical parameters prospectively collected. Two patients were identified for this pilot study: a 49-year-old nonsmoking male with a T2N0 tonsillar cancer treated with transoral robotic surgery and neck dissection and an 81-year-old female with a history of heavy smoking with a T2N0 oral tongue cancer treated with partial glossectomy, neck dissection, and free flap reconstruction. Primary site tumor specimens were taken from the center of the resection specimen. Ten mL of venous blood were drawn intraoperatively into heparinized collection tubes.μm thick). p16 immunohistochemistry was performed as previously described using a mouse monoclonal antibody against p16  at 1 : 500 dilution [For each patient, a portion of the primary tumor was fixed in formalin and embedded in paraffin. The blocks were then sectioned . DNA was extracted from approximately 25 mg of primary tumor using the AllPrep DNA/RNA/Protein kit (Qiagen).μg/mL proteinase K  for 30 minutes, followed by immersion in 0.3% H2O2 in methanol at room temperature for 20 minutes. Slides were then treated for 10 minutes with avidin solution followed by biotin solution. The DakoGenpoint  biotinylated probe that identifies high-risk subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 was added to the slides. The slides were then covered and heated to 92°C for 5 minutes and then incubated at 37°C for 18 hours. DNA-DNA hybrids were detected by successive incubation with 1 : 100 diluted primary horseradish peroxidase-conjugated streptavidin (streptavidin-HRP) for 15 min, with biotinyltyramide for 15 min, and with secondary streptavidin-HRP for 15 min. A cervical cancer was used as a positive control, and a tonsil specimen from a healthy child undergoing tonsillectomy for sleep apnea was used as a negative control. Punctuate hybridization signals localized to the tumor cell nuclei defined an HPV-positive tumor. Scoring was conducted by the two study pathologists (BW and KK).Slides were deparaffinized by immersion in xylene, rehydrated in alcohol, and rinsed in water. Slides were then treated with 20 μL of Arcturus PicoPure extraction buffer, containing proteinase K (Applied Biosystems). The sample was digested at 65°C for 16 hours. The proteinase K was inactivated at 95°C for 10 minutes, and DNA was used directly in PCR. Primers were designed against unique regions of the E6-E7 loci of HPV type 16 and type 18 and synthesized by Sigma Genosys  in 20 μL reactions following the manufacturer's instructions.Patient DNA was extracted from thin section tissue slices. Briefly, a single pathology slide from each patient was deparaffinized, and then the tumor tissue was scraped into a 1.5 mL eppendorf tube containing 50 μg of genomic DNA extracted from matched patient blood and tumor samples was sheared via sonication using the Covaris (S-Series) instrument. The ends of fragmented DNA were repaired and ligated to SOLiD P1 and A1 adapters provided in the Agilent Human All Exon 50 Mb Kit following the manufacturers protocol . The exomes were then captured using the Agilent Human All Exon 50 Mb kit, and the amplified library was purified with AMPure XP beads . Sequencing was performed with the SOLiDToP Paired End Sequencing Kit (Applied Biosystems). The image data collected was analyzed using the ABI corona pipeline to generate DNA reads that were mapped to the reference human genome (UCSC's hg19) using BFAST [Exome libraries were created at The Centre for Advanced Genomics  according to the manufacturer's standard protocol for SOLiD library preparation . Three ng BFAST .Samples were processed as matched sets through the Genome Analysis ToolKit (GATK) v1.3-16 pipeline . Samplesvariant quality equal to or greater than 30,variant confidence/quality by depth (QD) equal to or greater than 2.0,MQ0 < 4 and MQ0/(1.0 ∗ DP)) < 0.1, where MQ0 is the total mapping quality zero reads and DP is the unfiltered read depth.To be considered for further downstream analysis, a tumor variant had to have at least 8x coverage within the target regions 37,038,261 sites (71.86%) for the HPV-positive tumor and 39,150,091 sites (75.96%) for the HPV-negative tumor that met this criterion. In addition to coverage, the following requirements had to be identified by the VariantFiltration walker:http://varianttools.sourceforge.net/Annotation/RefGene), and consequences were identified using ANNOVAR v2012-03-08 [A reference variant required a minimum read depth of 8x within the target region for further consideration  and 38,058,450 sites (73.84%) for HPV-positive and HPV-negative tumors, resp.). This presented 36,020,799 and 37,049,778 comparable sites in the HPV-positive and HPV-negative tumors. Using in-house custom Perl code, somatic variants within the targeted regions were identified. To be classified as a somatic variant the following conditions had to be met: (1) a tumor variant was identified by GATK that met the above filtration requirements and (2) the corresponding position in the normal sample had 8x coverage and did not have a GATK variant call. Somatic variants were annotated with refGene annotations . Only the variants that occurred within exons are listed in Tables No mutations were noted in TP53, CDKN2A (p16), or the NOTCH receptors in either tumor. However, multiple mutations were noted in zinc finger genes  and mucin genes . Mutations were noted in MUC12 in both tumors.in situ hybridization testing, and immunohistochemistry all indicated that patient 1 was HPV type 16 positive (HPV type 18 negative by PCR) and that patient 2 was HPV negative (both type 16 and type 18 negative). When we used our four compiled exome sequences (blood and tumor from both patients) as queries against the HPV type 16 genomic sequence (RefSeq NC_001526.2), the tumor sequence from patient 1 matched numerous regions (39 hits) of the HPV 16 genome . Two large-scale exome sequencing efforts characterizing HNSCC have been reported recently [HPV-positive head and neck squamous cell carcinoma (HNSCC) has been described as molecularly distinct from traditional head and neck cancer . The humrecently .Only a single genetic mutation (Muc12) was shared by both HPV-positive and HPV-negative tumor samples. The cell surface associated Muc12 was the only mucin identified in the HPV-negative tumor. In contrast, the HPV-positive tumor had five mutations in four different mucin genes, including the secreted Muc6, and the transmembrane bound Muc4, Muc12 and Muc16. Stransky et al. reported mutations in all the above mucins except for Muc12 . Mucins We also found multiple mutations in the zinc finger (ZNF) family genes in both tumor types. The ZNF family represents a large group of molecules which are involved in various aspects of transcriptional regulation . There wWe confirmed that the sequence from the human pathogen HPV type 16 was identified within exome sequence of a HNSCC tumor. In order for HPV to be oncogenic, the viral E2 protein, which represses the expression of E6 and E7, must be lost . This onThis study represents a pilot effort to gain experience with this exciting new technology, which was instructive as our group moves forward with large-scale projects. In addition to the small sample size, the quality of data generated limited by the ABI SOLiD platform with an average 30-fold coverage with 50 base pair paired-end reads yielded only 10-fold coverage over approximately two-thirds of the coding sequence. Thus, approximately a third of the exome was not adequately evaluated and important mutations could have been missed. We have recently completed characterizing a panel of head and neck cancer cell lines with 100-fold coverage with 100 base pair paired-end reads, and the results were vastly superior . An averIt should be noted that tremendous insights can be gained by exome sequencing; however, whole genome sequencing offers the advantage to identify other genetic changes that can lead to tumorigenesis including copy number variation and translocations, in addition to point mutations, insertions, and deletions. Alterations in noncoding regions that may be important, such as promoters and miRNAs, would also be identified. The study by Stransky et al. reported whole genome sequencing in two patients and revealed markedly more translocations in the HPV-negative versus the HPV-positive tumors . IdeallyWhole exome sequencing of head and neck cancers can provide important insights into the molecular biology of the disease. HPV-positive and negative head and neck cancers are molecularly distinct, and HPV-negative cancers tend to harbor more mutations. Multiple, integrated HPV 16 sequences were identified in the exome targets from the HPV positive patient. These matches were restricted to the HPV-positive patient's tumor profile providing evidence of the utility of screening exome sequences against pathogen databases.Supplementary Tables S1 and S2. The supplementary tables provide a complete listing of the mutations identified by our bioinformatics pipeline in the HPV-positive (Table S1) and HPV-negative (Table S2) tumor samples but not in the matched blood samples. This listing includes mutations found in both coding and non-coding regions.Click here for additional data file.Click here for additional data file.


The Human Papillomavirus (HPV) genome is divided into early and late coding sequences, including 8 open reading frames (ORFs) and a regulatory region (LCR). Viral gene expression may be regulated through epigenetic mechanisms, including cytosine methylation at CpG dinucleotides. We have analyzed the distribution of CpG sites and CpG islands/clusters (CGI) among 92 different HPV genomes grouped in function of their preferential tropism: cutaneous or mucosal. We calculated the proportion of CpG sites (PCS) for each ORF and calculated the expected CpG values for each viral type.E4, where the majority of CpGs are found within islands/clusters. At least one CGI belongs to each E2/E4 region. We found positive correlations between PCS for each viral ORF when compared with the others, except for the LCR against four ORFs and E6 against three other ORFs. The distribution of CpG islands/clusters among HPV groups is heterogeneous and mucosal HR-HPV types exhibit both lower number and shorter island sizes compared to cutaneous and mucosal Low-risk (LR) HPVs .CpGs are underrepresented in viral genomes. We found a positive correlation between CpG observed and expected values, with mucosal high-risk (HR) virus types showing the smallest O/E ratios. The ranges of the PCS were similar for most genomic regions except E6. L2 and L1 ORF behavior is opposite to that of oncogenes E6 and E7. The first pair possesses relatively low numbers of CpG sites clustered in CGIs while the oncogenes possess a relatively high number of CpG sites not associated to CGIs. In all HPVs, E2/E4 is the only region with at least one CGI and shows a higher content of CpG sites in every HPV type with an identified E4. The mucosal HR-HPVs show either the shortest CGI size, followed by the mucosal LR-HPVs and lastly by the cutaneous viral subgroup, and a trend to the lowest CGI number, followed by the cutaneous viral subgroup and lastly by the mucosal LR-HPVs.There is a difference between viral and cellular CpG underrepresentation. There are significant correlations between complete genome PCS and a lack of correlations between several genomic region pairs, especially those involving LCR and  E for early or L for late) and one regulatory region or LCR. The E region includes six major open-reading frames (ORFs) encoding functional proteins  and oncoproteins , and the L region encodes the two capsid proteins, L1 and L2. E4 is expressed in both early and late stages of the viral life cycle [Human papillomavirus (HPV) constitute a group of over 100 different types. HPV infect stratified epithelia, both mucosal and cutaneous and associate with benign and malignant proliferative disorders. Viral types that preferentially infect mucosal epithelia are grouped into either a low-risk group (LR-HPV) not associated with cancer, or into a high-risk group (HR-HPV) whose members are found in almost all cases of cervical cancer [ins E1, E and E4 aHPV gene expression is mainly regulated at the transcriptional and post-transcriptional levels and several studies have suggested that viral DNA methylation may be associated with viral gene expression and cancer progression -6.Most of the work on HPV methylation has been carried out on the two main viral types involved in cervical cancer (types 16 and 18). In both genomes there is a progressive increase in methylation from asymptomatic carriers, through benign lesions and pre-malignant disease, to cancer tumors. Nevertheless, there is heterogeneity of CpG methylation in viral genomes derived from clinical specimens -7, and tL2 and L1 genes, they have been proposed to be preferentially recognized by the cellular methylation machinery [Furthermore, based on Epstein-Barr virus studies it has been proposed that viral genome methylation may enable a proportion of infected cells to survive cytotoxic T-cell immune surveillance ,9, and sachinery ,12.Methylation is the only known covalent modification of DNA in eukaryotes and plays an important regulatory role in vertebrates by silencing specific genes during development and cell differentiation. Cytosine methylation occurs at the 5'-position of the pyrimidine ring, mainly within a CpG context (m5CpG), although methylation of cytosines in different contexts has recently been described ,14 and 5Usually, the presence of m5CpG in genomic DNA is associated with chromatin condensation and inactivation of gene expression. Nevertheless, it is not clear whether the primary evolutionary role of DNA methylation is transcriptional silencing or a host defense system against endogenous or exogenous parasitic sequence elements .In the genomes of higher eukaryotes, CpG dinucleotides are usually underrepresented, from one third down to 5% of their expected frequency -20. The Similar to their hosts, CpG dinucleotides are underrepresented in the majority of small DNA viruses, although to a lesser extent . It has The general view is that CpG sites in vertebrates, grouped within clusters or islands (CGI), are mostly unmethylated in promoter regions of transcriptionally active genes, and methylated in promoter regions of transcriptionally inactive genes. In contrast, methylation in body genes seems to be evolutionarily conserved and plays an important role in tissue- or cell-specific alternative promoter regulation. Furthermore, tumor cells usually show hypomethylation of the majority of the genome and methylation of CGIs in promoter regions of tumor suppressor genes ,25.According to Gardiner-Garden and Frommer  a CGI isThe majority of the proposals for CGI identification and criteria to analyze methylation predisposition at CpG islands cannot be directly applied to HPV genomes because of their small size. In addition, lack of multiple promoter regions and the scarce data on methylation of different viral types make this scenario difficult.Because in our view, understanding DNA methylation may benefit from our knowledge of CpG distribution along different viral genomes, we have analyzed genomic and regional proportions of CpG sites (PCS) and their arrangement into CGIs, in most human papillomavirus types sequenced to date. To our knowledge, there has been no comprehensive analysis of CGIs in virus genomes. We propose hypotheses about their functional roles, considering HPVs as both a single group or as subgroups in function of their preferential tropism  and their associated risk to induce cancer .After identifying all CpG sites among 92 available HPV DNA sequences, we calculated the expected CpG value for each viral genome. The expected number of CpG sites was calculated as the number of 'C's multiplied by the number of 'G's in the viral genome, divided by the genome size.When we compared the CpG observed vs. the expected values for each viral type, a significant difference was clear . In addition, a positive correlation was found ; only five HR-HPV types  show differences slightly above the average ratio , and that the E2 region (where E4 is co-located) does not show similar PCS values (average E2 PCS without HPV types lacking E4 was 0.028). In three out of 68 viral types, E7 exhibits the highest PCS values while the LCR region shows the highest PCS values in four out of 68 types.From Figure E6, 0.024 (0.002 to 0.045); E7, 0.030 (0.010 to 0.054); E1, 0.018 (0.009 to 0.035); E2, 0.029 (0.014 to 0.050); E4, 0.051 (0.025 to 0.088); L2, 0.023 (0.011 to 0.043); L1, 0.018 (0.009 to 0.036); and LCR, 0.027 (0.009 to 0.054).Regional average PCS (and range) for individual ORFs and the LCR are as follows: E4 ORF PCS averages are the highest when compared to any other region. In fact, E4 ORF PCS averages are quite close, irrespective of type: for cutaneous, 0.054 and for mucosal, 0.049, similar to when mucosal HPVs were grouped in function of risk: low risk, 0.050 and high risk, 0.048. Furthermore, E4 average PCS values are around twice the highest average PCS from any other region in each viral group/subgroup (data not shown).When HPVs are grouped in function of their preferential tropism, the E4 ORF, follow the same trend found in the entire viral genome, however, T (0.68) seems underrepresented and C (1.20) overrepresented, compared to whole genome values. When Arc-sin transformed O/E ratios for single nucleotides in E4 and in the whole genome were compared, we found that the two sets of data are significantly different , even though they are significantly correlated . PCS were Arc-sin transformed in order to properly use statistical procedures, as mentioned in the Methods section.Interestingly, O/E ratios for A (1.16) and G (0.96) individual nucleotides in the E1, E4, L2 and L1 regions, as well as the average of the ORFs; between E6 PCS and those from the E7, E2 and E4 regions; and between E7 PCS and that from the L2 region.HPV types were ordered in function of regional Arc-sin transformed PCS (AsPCS) and compared to each other using a Pearson linear correlation coefficient and two sided t tests. Results are shown in Table E2/E4 region.CGI location is summarized in Table E2/E4; each viral type possessing at least one CGI in this region. It is important to make clear that in the case of E4, as in any other region, we have only taken into account those HPV types where this ORF has been identified. Other regions which most commonly contain CGIs include the E1, E7 and L2 ORFs which have CGIs in at least half of HPV types; the L1 ORF and LCR regions with one CGI in 25 to 50% of viral types, and finally, the region where less than 25% of the viral types possess one CGI is E6.We found that the viral region with the highest number of CGIs (CGIn) is E2 and E4 regions because the E4 ORF is located within the E2 ORF, although in a different reading frame ; all mucosal HPV types, 345 bp (from 205 to 1379 bp); mucosal LR-HPV types, 363 bp (from 205 to 1379 bp), and mucosal HR-HPV types, 275 bp (from 210 to 410 bp). Notably, when CGI frequencies per size were compared, we found significant differences between cutaneous and mucosal HPVs (p < 0.0005) as well as between mucosal LR- and HR-HPVs . Additionally, both cutaneous and mucosal viral types share an average of 3.0 CGIs per viral genome (ranging from 1 to 8), and among mucosal types, LR-HPVs show an average of 3.93 CGIs per viral genome in contrast to HR-HPVs that show only 1.60 CGIs. When compared frequencies per number of CGI, we also found significant differences between cutaneous and mucosal HPVs (p < 0.005) as well as between mucosal LR- and HR-HPVs .Thirty out of 45 cutaneous and 32 out of 47 mucosal types possess 3 or less CGIs, and 15 out of 45 cutaneous and 15 out of 47 mucosal types possess more than 3 CGIs. In the mucosal group, 14 out of 29 LR- and all 18 HR-HPVs possess 3 or less CGIs, and 15 out of 29 LR-HPVs posses more than 3 CGIs , there is a notable difference between the proportion of CGIs among both mucosal HR- and LR-types , E1 , L2 , L1 , and LCR . In fact, among mucosal HR-HPV types, only HPV 33 contains a CGI in the E6 region; only HPV 45 contains a couple of CGIs in the L2 region, and none of the mucosal HR-HPVs contain CGIs in either the L1 or LCR regions.Although CGIs seem regionally distributed in a similar manner between cutaneous and mucosal types (except for L1 (average 1.71 CGIs on 21 HPV types), followed by L2 (average 1.58 CGIs on 36 HPV types), E1 (average 1.26 CGIs on 61 HPV types) and LCR (average 1.20 CGIs on 25 HPV types). Oncogenes E6 and E7 as well as E2 and E4 ORFs share 1 CGI on 17, 45, 92 and 68 HPV types, respectively.Interestingly, when focusing only on regions where islands were identified , the region possessing the highest CGI number is To our knowledge, this is the first systematic analysis of CGIs, as well as of CpG site distribution performed in 92 HPV types. This analysis provides the main structure underlying the methylation phenomenon, guiding the identification of interesting genomic regions for future epigenetic experimental studies.As anticipated , we founIn this context, it seems necessary to mention that HPVs are small sized viral DNA genomes that do not evolve rapidly, in contrast to other viruses. It has been calculated that approximately 200,000 years are needed for 17 bp to change in these genomes .E6 and LCR regions  and methyl-CpG binding domain protein 4 (MBD4) which are able to remove either uracil or thymine from G·U and G·T miss-pairings where follow-on base excision repair enzymes restore a G·C pair [Nevertheless, the G·C pair ,44.When grouped by tropism, the cutaneous HPV group, shows the greatest CGI average size (446 bp), followed by the LR-HPV group with an intermediate CGI average size 363 bp) and the HR-HPV group shows the smallest average CGI size (272 bp). Additionally, even though CGIn from both cutaneous and mucosal HPV types share similar values , it seems there is a trend toward smaller CGIn in HR- compared to LR- HPVs . These data raise questions about the possible role of CGIs on the interaction between virus and cells. At the same time, this suggests a possible differential susceptibility to methylation in function of either HPV tropism or associated risk. Nevertheless, the only experimental studies on methylomes have been carried out on HPV types 16 and 18, where methylation changes and high heterogeneity were observed along the viral infection phases [63 bp andFinally, it should be interesting to examine virus targeted genes in the human genome and see what their CGI and CpG features are. We hope that our analysis of CpG and CGI distribution among HPV genomes can provide elements for rational experimental approaches related to biological and evolutionary processes of these viruses and their hosts.First, we want to point out that this work is based solely on sequence feature analysis, so the results reported here may need experimental corroboration. In this study, we found that there are both significant differences and correlations between CpG observed and expected values among all sequenced HPV genomes, and that viral genomes have an underrepresentation of only around 50% compared to eukaryotic genomes where underrepresentation is from 30 down to 5%.E6. Furthermore, we found that L2 and L1 ORFs exhibit an opposite behavior to oncogenes E6 and E7; the first pair possessing relatively low numbers of CpG sites which are associated to CGI while the oncogenes possess a relatively high number of CpG sites which are not associated to CGI. We would point out that E4 is the region possessing the highest content of CpGs in every HPV type where an E4 is identified.The regional analysis of CpG site distribution shows that correlations between complete genome PCS are significant, even though there is a lack of correlation between several genomic region pairs, especially those involving LCR and E2/E4 is the only region with at least one CGI in every HPV, and the main difference between this region and the complete genome is that in the E4 region the frequency of Cs seems increased at the expense of Ts. The mucosal HR-HPVs possess the shortest CGI sizes, followed by the mucosal LR-HPVs and finally by the cutaneous viral subgroup. At the same time, the mucosal HR-HPVs show a trend toward lower CGI numbers, followed by the cutaneous viral subgroup and finally by the mucosal LR-HPVs.From the CGI analysis, the Finally, we hope that our analysis of CpG and CGI distribution among HPV genomes can provide support for rational experimental approaches related to the biological and evolutionary processes of these viruses and their hosts.Our CpG analysis comprises two phases: the first one is the comparison of CpG site distribution among HPV types as a single group, and in the second phase we identify possible similarities or differences in CpG site distribution associated to the preferential tropism and/or the risk of cancer caused by the mucosal viral subgroups. The CpG distribution refers to CpG proportions (PCS) and either CGI sizes or numbers.HPV DNA sequences were obtained online from GenBank .Sequence from HPV type 16 includes the changes proposed by the Theoretical Biology and Biophysics Group in Los Alamos National Laboratory. All 92 HPV genomes included in the analysis were reedited so that all sequences begin at the first nucleotide from the E1 and E7 ORFs for HPV types 53 and 3, respectively. In the case of HPV 14D, we found single E6 and E7 ORFs, however these were found to overlap by more than 100 bp. For HPV types without a reported E4 ORF, we identified a single putative ORF in only three viral types: HPV 25, 26 and 32. The other 21 HPV types had more than one identified ORF: we found 11 HPV types with 2, 8 HPV types with 3, 1 HPV type with 4 and 1 HPV type with 5 putative E4 ORFs.Because the absence of data may contribute to bias in the analysis, putative ORFs for each HPV type lacking reported ORFs were identified using the online tool "ORF finder" . The maiThe PCS for the putative ORFs did not notably change the regional PCS averages nor the analysis of the results . Because of the scarce amount of data, we did not include three reported ORFs into the analysis: the E8 ORF has been found only in HPV 1; the E5 ORF is present in only 23 out of 92 HPV types , and the E5B ORF is only reported for two HPV types (11 and 31).We analyzed the entire genomes of 92 viral sequences, as well as dividing them into 8 regions . In summary, the distribution of proportions is often skewed, and the arcsine transformation often makes that distribution more normal.Finally, we calculated the CpG expected values for each viral genome in order to compare them with the CpG observed values.ad hoc Excel macros and corroboration of calculations, using free online statistics software [Possible relationships between data results were identified using rho Spearman Rank Correlation and Chi square tests, as well as Pearson Product Moment Correlation and two sided t tests. Calculations were carried out using software ,49.In order to check for possible mistakes in the calculations, we reviewed the whole procedure by randomly selecting three groups of thirty HPV types each. One group was used to repeat the input data procedures; a second group was used to repeat the output data procedures, and the third group was used to repeat statistical and p value calculations.E(number): viral early ORF; L(number): viral late ORF; LCR: long control region; SRPK1: serine-arginine-rich protein kinase 1.HPV: human papillomavirus; LR-HPV: low-risk HPV types; HR-HPV: high-risk HPV types; ORF: open reading frame; PCS: proportion of CpG sites; AsPCS: Arc-sin transformed proportion of CpG sites; m5CpG: methylated CpG site; CGI: CpG island; CGIn: CpG island number; E(number): viral early protein; L(number): viral late protein; SCG initiated the study, performed the analysis and interpretation, and wrote the manuscript. MMS, VMG and RM obtained the data and revised the manuscript. RAS, GTDC and MAH developed the computational tool and participated in obtaining the data, and AGC provided initial impetus to perform the analysis and participated in writing and reviewing the manuscript. All authors read and approved the final manuscript.SCG is an associate researcher and RAS is a professor at the National University of Mexico; AGC is a researcher at the National University of Mexico and at the National Cancer Institute; VMG is a professor at the Metropolitan University; RM is an associate researcher at the National Cancer Institute; MMS is post doctoral fellow at the AGC lab; GTDC and MAH are students guided by RAS.Table S1. CpG observed and expected values for each HPV type. The table contains the GenBank accession number, the CpG observed and expected numbers, and the CpG O/E ratio, for all 92 HPV types included in the analysis.Click here for fileTable S2. Proportion of CpG sites among different genomic regions. The table contains the proportion of CpGs in the E6, E7, E1, E2, E4, L2 and L1 ORFs, and the LCR, the entire genome and all ORFs together, for every viral type.Click here for fileTable S3. CpG islands per region from each HPV type. The table contains the CpG island number in the E6, E7, E1, E2, E4, L2 and L1 ORFs, and the LCR and of entire genome, the CGI average size and the tropism for every viral type.Click here for fileTable S4. Putative ORFs from HPV types lacking reported ORFs. The table contains every possible coding fragment 200 bp or larger for each HPV type lacking reported ORFs; the starting and ending sites, and the corresponding PCS values.Click here for fileTable S5. p values for correlations between regional AsPCS when putative ORFs are included. The table contains p values for correlations between regional AsPCS values when AsPCS from putative ORFs are included.Click here for file


P<0.001; OR 1.49 (1.08–2.07), P 0.017, respectively] and a decreased risk with the 942CT genotype . Most 844CT genotypes were linkage CT(844)-CC(942), which increased the risk of cervical carcinoma and CIN3 . Most 942CT were linkage CC(844)-CT(942), which decreased the risk of cervical carcinoma . In some grouping, the 844CT and 942CT were further enriched; especially HR-HPV-positive subjects both in the CIN3 and the cervical carcinoma, the 844CT had greater enrichment. These results included that CT(844)-CC(942) was associated with a high risk of cervical carcinoma and CIN3, and the CC(844)-CT(942) decreased the risk. The 844CT had a higher level of enrichment in HR-HPV positive individuals, which is probably related to HR-HPV susceptibility. There was no significant difference of the MLH3 mRNA expression and these two amino acid substitutions did not impact on the protein function.To investigate the association between MLH3 Pro844Leu, Thr942Ile polymorphisms and potential linkage with the risk of cervical carcinoma and potential effect on protein function, we carried out a case-control study with 400 cervical squamous cell carcinoma, 400 CIN3 and 1200 normal controls in a Chinese population. The results showed that there was an increased risk of cervical carcinoma and CIN3 associated with the genotype 844CT [OR 2.17 (1.61–2.94);  Cervical carcinoma is the second most common type of cancer among women worldwide The DNA mismatch repair (MMR) system has a crucial role in genomic stability by correcting errors acquired during DNA replication and recombination MLH3 might have a role in colorectal cancer tumorigenesis In this study, we investigated the distribution of the common Pro844Leu and Thr942Ile genotypes in Chinese women, and assessed the association of these polymorphisms or linkage disequilibrium with the risk of cervical squamous cell carcinoma (CSCC), CIN III and their interaction with HR-HPV infection, sexual and reproductive risk factors, and analyzed the MLH3 gene mRNA expression level and the effect on protein function.400 CSCC patients, 400 CIN III patients and 1200 normal controls were recruited from Zhejiang Province, China. The diagnosis was confirmed by two pathologists. Normal controls were collected from healthy women volunteers during gynecologic examinations between June 2004 and December 2008. The selection criteria for healthy controls included no positive cytological finding, no gynecological tumor, no endometriosis, no other cancer history, no immune disease. Of these, 201 patients with CSCC, 357 patients with CIN III and 637 controls agreed to provide cervical brush-off samples for HR-HPV test.This study was approved by the Medical Ethical Committee of Women’s Hospital, School of Medicine, Zhejiang University (No.2004002). All patients signed informed consent to allow molecular research on specimen obtained.Genomic DNA was extracted from peripheral blood using a DNA extraction kit . All DNA samples were dissolved in water and stored at –20°C.5′-CAGAAGTAGAGGAAAGTAATGG-3′ and 5′-CTGTGGCATCTTCTACCG-3′, respectively, which produce a 732 bp fragment. PCR was done in a 25 µl reaction mixture. The reaction conditions were: 94°C 5 min, 35cycles of 94°C 30 s, 55°C 30 s, 72°C for 45 s and 72°C for 5 min.MLH3 Pro844Leu (CCT2645CTT) or Thr942Ile (ACA2939ATA) genotypes were determined by bidirectional sequencing. The forward and reverse primers for MLH3 were The PCR product was purified and amplified with a BigDye terminator 3.1 kit . Amplified dye-terminated products were sequenced using forward and reverse primers with the ABI PRISM 3100 Genetic Analyzer.HR-HPV infection was assayed using the HC II kit . Cervical sampling for HPV DNA was done with the Digene Cervical Sampler by measuring the relative light unit (RLU)/positive control ratio:RLU of specimen/mean RLU of two positive controls.Histological examination of 282 freshly frozen tumor tissue samples confirmed cervical squamous cell carcinoma. Total RNA was extracted by using TRIzol reagent  and subjected to reverse transcription after digestion with RNase-free DNase I. The following primers were used for qRT-PCR; 95°C 30 s, 40 cycles at 95°C 5 s followed with 60°C 35 s.MLH3 primers:5′-TGCCCGTTATCCAGAGGTTG-3′.forward: 5′-TATACGCTCATGGGCAGCG-3′.reverse: GAPDH primers:5′-GAGAAGGCTGGGGCTCATTT-3′.forward: 5′-AGTGATGGCATGGACTGTGG-3′.reverse: Ct method, in which ΔCt is the difference in value between each Ct value of the target gene minus that of GAPDH.The MLH3 and GAPDH PCR products were 244 bp and 231 bp, respectively. qRT-PCR was done with the SYBR Premix Ex Taq kit with an Applied Biosystems 7900 HT system. All samples were run in triplicate. The relative mRNA expression levels were calculated according to the 2–Δhttp://sift.bii.a-star.edu.sg). Amino acid substitutions are reported as TOLERATED or DAMAGING. SIFT scores were designated as tolerant (0.201–1.00), borderline (0.101–0.20), potentially intolerant (0.051–0.10) or intolerant (0.00–0.05).Predicting the effects of nonsynonymous SNPs on protein function was done with the SIFT algorithm , 95% confidence intervals(CIs) and P-values were obtained by binary logistic regression analysis. The control was set as the reference group for analysis. For mRNA expression analysis, we used the 1-sample KS nonparametric test to detect the normal distribution of 2Ct–Δ values and Levene’s test for equality of variances to verify the homogeneity of variance and then we chose the independent sample t-test to define the differences.Differences in lifestyle habits and genotype frequencies were evaluated with Pearson’s χP≤0.05. All statistical analysis was done with SPSS for Windows software 10.0 version.All reported values are two-tailed. The level of statistically significant difference was set at The genomic DNA showed a single band corresponding to a length of 30∼50 kb, which indicated it was not degraded. There was sufficient PCR product for sequencing and each showed a single band. The sequencing results gave sharp peaks with no significant overlap, which showed the nucleotide sequence was very reliable.P<0.001) and the CIN III group had significantly more individuals <40 years old (P<0.001) compared to the control. There was no significant difference beside the increase of the proportion of individuals with number of parities >3 in both the carcinoma and CIN III groups. The HR-HPV infection rate was 88.6% in the carcinoma and 86.8% in the CIN III but only 31.4% in the control, which indicated the infection rate in both patient groups was significantly higher compared to the control (P<0.001). These indicated that smoking and HR-HPV infection in CIN III and cervical carcinoma patients were more than controls.In the control, CIN III and carcinoma groups, 602/598, 258/142 and 160/240 individuals were >40 years old/<40 years old, respectively. The carcinoma group had significantly more individuals >40 years old (P 0.017] and CSCC . The frequency of the Thr942Ile (942CT) genotype was significantly different between patients and controls. Women heterozygous for the Thr942Ile (942CT) genotype had a decreased risk of CIN III  and CSCC . In the HR-HPV positive group, the heterozygous Pro844Leu (844CT) genotype increased the risk of CSCC significantly . The heterozygous Thr942Ile (942CT) genotype might decrease the risk of CIN III.The frequency of Pro844Pro(844CC) and Pro844Leu(844CT) was 89.3% and 10.8% in the control, 84.8% and 15.3% in the CIN III and 79.3% and 20.8% in CSCC, respectively. These results revealed that women with the heterozygous Pro844Leu (844CT) genotype had a significantly increased risk of CIN III , whereas the CT(Pro844Leu)-CC(Thr942Thr) genotype increased the risk of CIN III or CSCC. (Given that the frequencies of both Pro844Leu (844CT) and Thr942Ile (942CT) genotypes change the risk of CIN III or carcinoma significantly, we next examined the linkage disequilibrium between Pro844Leu (844CT) and Thr942Ile (942CT). The frequencies of the four most common genotypes were significantly different between patients and controls. Compared to the most common genotype CC(Pro844Pro)-CC(Thr942Thr), women with the CC(Pro844Pro)-CT(Thr942Ile) genotype had a significantly decreased risk for CIN III or CSCC. Table 4., Additionally, most Pro844Leu (844CT) genotypes were linkage genotype CT-CC (55/61 and 77/83) and most Thr942Ile (942CT) genotypes were linkage genotype CC-CT (18/24 and 29/35) in the CIN III and CSCC, respectively. These indicated that the majority of CT genotype distributions of MLH3 polymorphisms are caused by the linkage disequilibrium with the corresponding alleles , 4. TherP 0.031; OR 4.00(2.01–7.98), P 0.001, respectively]. However, the CC-CT or CT-CT haplotype was not associated with the risk of CIN III or CSCC in women with HR-HPV infection. , fection. Table 4.P was >0.05 when grouped according to HR-HPV infection . . Table 5.P-value of more than one group was >0.05, whereas those of the other groups were <0.05 . Meanwhile, we analyzed the HR-HPV infection grouping and we found the CT-CC haplotype, whether in CIN III or CSCC, had a more significant enrichment  in the HR-HPV-negative group.  , Thr942Ile (942CT) or haplotype were found further enrichment in some groupings, and not in other groupings according to our data. These indicated that some behavior may be affected by some genetic factors. We found obvious enrichment of Pro844Leu (844CT) and CT-CC in the HR-HPV-positive group but no further enrichment for the Thr942Ile (942CT) locus. These results indicated the Pro844Leu (844CT) locus play an important role in pathogenicity cervical cancer or precancerous lesions, and an essential role in the HR-HPV infection. The Thr942Ile(942CT) locus in the HR-HPV-positive group showed no enrichment, indicating it had an effect on the pathogenicity of cervical cancer and precancerous lesions but no effect in the HR-HPV infection process. A number of studies showed that HR-HPV positive participants had a significantly increased risk for the development of cervical carcinoma According to our results, it is plausible that the Pro844Leu genotype decreased the MLH3 function, leading to genomic instability and consequent increased susceptibility to cancer, whereas the Thr942Ile genotype might further improve MLH3 function, which implied Pro844Leu and Thr942Ile have opposite effects on MLH3 function. Although both Pro844Leu and Thr942Ile variants are related to the risk of cervical carcinoma, the CT-CC and CC-CT, but not the CT-CT, linkage haplotypes change the risk of cervical carcinoma. Thus, it is more accurate to examine the association between the linkage disequilibrium of two MLH3 SNPs and the risk of cervical carcinoma, because the function of a certain protein is determined by two or more amino acid sites Taylor et al. reported MLH3 Pro844Leu and Thr942Ile polymorphism will not impact MMR protein expression but predicted these two SNPs will change the structure and function of MLH3 in endometrial cancer. They concluded that MLH3 participated in the endometrial carcinoma pathogenicity by changing the function of protein MLH3 instead of altering its expression


Objective. The purpose of this study was to assess the knowledge level about HPV and screening of cervical cancer in women from the metropolitan region of Natal, Brazil. Materials and Methods. A descriptive cross-sectional study involving sexually active women was conducted. The participants were submitted to a face-to-face interview, using a structured questionnaire that permitted the quantification of data and opinions of the respondents. Results. Most participants (70.9%) had poor knowledge about HPV and also the Pap test (53.0%). The high level of knowledge about HPV was associated with age, education, marital status, household income, and pregnancy, while the high level of knowledge about the Pap test proved to be associated only with education and household income. Conclusion. The results highlight the need for performing educational campaigns emphasizing the role of HPV in the etiology of cervical lesions of different degrees, including cervical cancer, as well as the importance of having a Pap test regularly to prevent these diseases. Cervical cancer is the third most common cancer affecting women, after nonmelanoma skin cancer and breast cancer, and is the third leading cause of death by cancer among women worldwide, with higher incidence in developing countries , 2. It iThe natural history of cervical cancer reveals that, despite its high incidence, this malignant neoplasm stands out among those with the greatest potential for prevention and cure in view of their infectious nature . CervicaThere is no doubt that when women face participation decisions, it is ideal for them to have a good understanding and perception of the risk of acquiring cervical cancer and the benefits of preventing it . In BrazHuman papillomavirus (HPV) is one of the most common causes of sexually transmitted disease in both men and women around the world, especially in developing countries. The prevalence of asymptomatic infection varies from 2 to 44%, depending on the population and studied region . Most HPHPV infection is more prevalent in young adults, at the beginning of their sexual activity, with a subsequent decline in the prevalence rate with increasing age, likely as a result of the development of an immune response against the virus and reduction of sexual activity , 11. CurToday, more than 150 different HPV types have been cataloged and about 40 can infect the epithelial lining of the anogenital tract and other mucosal areas of the human body. Based on their association with cervical cancer and precursor lesions, HPVs are classified as high-risk (HR-HPV) and low-risk (LR-HPV) oncogenic types. LR-HPV types, such as HPV 6 and 11, can cause common genital warts or benign hyperproliferative lesions with very limited tendency to malignant progression, while infection with HR-HPV types, highlighting HPV 16 and 18, is associated with the occurrence of premalignant and malignant cervical lesions . HR-HPV The recognition of the central role of HPV in the etiology of cervical cancer has dramatically changed the vision of how to prevent this cancer. Among the strategies, introduction of HPV testing for primary screening or as an adjunct test and the introduction of HPV vaccines to prevent HPV infection are being evaluated in different settings around the world . Indeed, these advances are available in many countries that have approved the introduction of the HPV vaccine. However, gaps in knowledge about the causal role of HPV in cervical cancer and the benefits of preventing HPV infection may hamper the successful introduction of the technologies. The greater the women's knowledge of HPV and its role in the development of cervical cancer, the greater will be the adherence to preventive measures. The objective of this study was to evaluate the knowledge level about HPV and the Pap smear screening among high school students and women treated in the health units of the metropolitan region of Natal, Brazil.This was a descriptive cross-sectional study carried out during the period of January 2009 to December 2011 involving sexually active women of the metropolitan region of Natal, capital of Rio Grande do Norte State, Northeastern Brazil. We chose basic health units and public schools, distributed throughout all the 10 municipalities that make up the metropolitan region of Natal, to participate in the research. The interviewers approached potential female participants among the high school students of the selected schools and among women attending basic health units during the study period. In the schools, some classes were randomly chosen and the students were invited by interviewers to participate in the study. In the health units, women who sought by spontaneous demand the screening test for cervical cancer were invited to participate in the study. After agreeing to participate in the study, the women aged 18 years old or older signed an informed consent before survey administration. Those under 18 years had their participation authorized by parents or guardians. A total of  706 participants, aged 14 to 59 years, were included in the study. The only inclusion criteria were to be of female gender, to have already initiated sexual activity, and to agree to participate in the research. This study was approved by the Research Ethics Committee of Federal University of Rio Grande do Norte.All interviews were conducted face-to-face by trained enumerators using a paper structured questionnaire, containing 25 questions including 15 questions about age, ethnicity, marital status, education, household income, sexual activity, and reproductive health history. There were six questions regarding Pap smear knowledge, including such questions as (1) Have you previously heard about this exam? (2) What is the purpose of this exam? and (3) What is the frequency with which the exam must be done? Other questions concerned the level of awareness of the respondents on the advantages and benefits of the examination for the health of women. The remaining four questions were related to HPV knowledge, including questions about the potential consequences of HPV and the awareness of the signs and symptoms, and mode of transmission. All the Pap test and HPV knowledge questions were accompanied by multiple-choice answers, one of which was considered the correct answer to the question. The interviewers were instructed to recite all multiple-choice answers to the participants and then ask for the answer to each question.We defined the level of knowledge according to the number of correct responses. Knowledge about Pap screening was categorized as high, medium, or low based on the six Pap-related questions in the survey. A participant with a high level of knowledge answered at least five of the six Pap knowledge questions correctly; participants with a medium level of knowledge answered three or four of the six questions correctly, and participants with a low level of knowledge answered none or at most two of the questions correctly. Women who responded correctly to all four HPV-related questions were categorized as having a high level of knowledge on the virus, those who answered two or three questions correctly were categorized as having a medium level of knowledge, and the women who responded correctly to only one or none of the HPV-related questions were categorized as having a low knowledge level. The participant ethnicity was defined based on self-reports according to the criterion of the Instituto Brasileiro de Geografia e Estatística (IBGE), which classifies ethnicity into five categories: white, black, mulatto, Asian, and “native,” which were combined into a nonwhite category. We considered as having low income those participants who reported a household income of up to the minimum wage (678.00 reais/month), equivalent to US$ 320.00; as middle income, those with a household income between one and four times the minimum wage, and as high-income, women who reported a household income higher than four times the minimum wage. For the purposes of analysis, women with high Pap screening knowledge were compared against those with low and medium knowledge. Similarly, women with a high level of knowledge about HPV were compared with those who demonstrated a low level of knowledge on the virus.χ2 test.  P  value ≤ 0.05 was considered statistically significant. To verify the association between the variables studied and the knowledge level for HPV and Pap test, we used the odds ratio (OR) and its 95% confidence intervals (CIs), according to the univariate regression model, using the software SPSS, version 17.0, and PEPI. Statistical significance within each group was evaluated by Pearson's The profile of the segment of the population studied is presented in Most (51.8%) of the studied population consisted of young women aged 14 to 28 years, 52.3% were of nonwhite ethnicity, and 64.2% were either married or living in a stable relationship with her partner. Regarding the level of education, 37.1% reported that they had only elementary school level or less, 52.4% were attending high school or had completed high school, and only 10.5% reported being enrolled in or having completed some type of education above high school. The vast majority (78.9%) of the women interviewed reported a low income (household income of up to R$ 678.00 reais/month), equivalent to US$ 320.00. Most of them (52.8%) had never had a pregnancy, and among those who had been pregnant, the majority (81.0%) had between one and three children.Participants of this study showed a low level of knowledge about Pap test screening. When asked if they knew what a Pap test was, 53.0% of the respondents indicated that they knew about the exam and were able to say what it means, and 48.0% of them were able to provide the correct frequency with which the test should be done and to demonstrate awareness of the advantages and benefits of performing the procedure regularly. According to the level of knowledge presented, participants were categorized as having low, medium, or high level of knowledge about the Pap test screening, based on the correct number of responses. Of the women surveyed, 22.4% exhibited a high level of knowledge about the Pap test, 24.6% had a medium knowledge level, and the majority (53.0%) were categorized as having a low level of knowledge about the exam . P  values association between the considered variables and Pap knowledge. We found that the high knowledge level of the Pap test was associated with level of education (P < 0.001) and household income (P < 0.001), while the middle-level knowledge of this exam was associated with marital status (P < 0.001) and pregnancy (P < 0.001). Association between the Pap test knowledge level according to the considered variables and odds ratio (OR) can be seen in P < 0.001) and having a middle or high household income versus low household income (P < 0.001) were related to an increase of the Pap test knowledge score. The odds ratio of having a high Pap knowledge score was 10.2 times greater among women with high school or above, compared with those with elementary school or less (95% CI 5.534–8.825), and 2.68 times greater for the women with a middle or high household income, compared with those with a low household income (95% CI 1.810–3.987).Having or not having the high-level knowledge of the Pap test was considered the dependent variable, and independent variables were those identified as significant in With regard to the level of knowledge of the Pap test, among the 706 women surveyed, 96.2% had heard about it, but only 47.0% had medium or high knowledge related to the procedure. The main source of information about the Pap test was health care staff, mentioned by 45.2% of the respondents, followed by friends and family with 22.5%. We found that 87.4% of the interviewed women reported that they had had at least one Pap test during their lifetime; 69.4% had had the procedure done with the frequency of at least once every three years, as recommended by the Ministry of Health; 18% said they had not had the test in the last 3 years, and 12.6% reported that they had never had the test throughout their life. When asked about the reasons for not having the procedure, the responses most commonly mentioned by these women were that they felt that nothing was wrong (28.6%), neglect (23.4%), had fear of pain (12.2%), and felt ashamed of exposing their intimacy (6.8%).Participants of this study presented a very low level of knowledge related to HPV. Only a minority of the women interviewed were able to quote correctly the transmission mode of the HPV and identify the signs and symptoms of the infection caused by this virus, as well as consciously recognize the relationship between the genital HPV infection and the occurrence of cervical lesions including cancer. A significant portion of respondents (64.2%) said they had heard about HPV, but a much smaller proportion (29.1%) demonstrated having some knowledge about the virus. Only 20.0% of the women respondents knew that HPV is a sexually transmitted agent, and a very small percentage (9.1%) of participants were able to establish the correlation between HPV and the occurrence of anogenital warts and of premalignant cervical lesions and cervical cancer. P  values confirmed the existence of association between knowledge level of the virus with age, (P < 0.001), education (P < 0.001), household income (P < 0.001), pregnancy (P < 0.001), and marital status (P = 0.006).Thus, when subjects were analyzed according to the level of knowledge presented about HPV, it was found that most (70.9%) of the participants were categorized as having low knowledge about the virus; 20.0% exhibited a medium knowledge level, and only 9.1% were categorized as having a high level of knowledge about HPV .  Table P < 0.001) and 49 to 59 years (P = 0.001) versus those aged between 14 and 28 years; in those having high school education or above versus elementary school education or less (P < 0.001); in the married or accompanied versus single (P = 0.003); and in those with middle or high household income versus low household income (P < 0.001). The odds ratio of having a high HPV knowledge score was 4.50 times greater in women aged 39–48 years (95% CI 2.289–8.859) and 3.71 times greater in those aged between 49 and 59 years (95% CI 1.718–8.001), compared with those aged between 14 and 28 years. Furthermore, we observed values of the odds ratio of 43.16 times greater in women with high school or above, compared with those with elementary school or less (95% CI 5.534–8.825); of 1.92 times greater in women married or accompanied, compared with single women (95% CI 1.056–3.507); 3.11 times greater in those with a middle or high household income compared to those with a low household income (95% CI 1.824–5.305); and 3.81 times greater in women who had at least one pregnancy, compared with those who had never been pregnant (95% CI 2.116–6.844).Association between HPV knowledge level according to the considered variables and odds ratio (OR) can be seen in Cervical cancer screening is a diagnosis resource of fundamental importance to women's health in that it allows early detection of the precursor lesions and the therapeutic intervention, before they progress to the malignant form. Because of its infectious nature, cervical cancer has a high potential for prevention, which can be put into practice by avoiding risky behaviors for acquisition of the HPV infection, as well as through the use of available vaccines. Thus, knowledge about the role of HPV in the development of the disease becomes of paramount importance for improving the degree of awareness of women to adopt measures to reduce the risks of infection by this virus, as well as to have early diagnosis of the existing infections. In this study we evaluated the degree of knowledge about HPV and the Pap test in a segment of the female population of the metropolitan region in Natal, Brazil.According to the official data, the metropolitan region of Natal consists of 10 municipalities including the capital, with a female population of approximately 367,768 women aged 14 to 59 years. This study focused only on the women in this age group, enrolled in public high schools, and the women being treated in basic health units in the municipalities involved, in the period from January 2009 to December 2011.Our study showed that 87% of the surveyed women reported having had a preventive Pap test at least once in their lifetime and 69.4% said they had had the procedure according to the recommended frequency suggested by the Ministry of Health of Brazil. This coverage rate of the Pap test is similar to that found by Fernandes et al.  in a stuThe results show that the surveyed women had a very low knowledge level about the screening test for cervical cancer, since only 47.0% had a medium or high level of knowledge about the exam, compared with the women from Medellin [Regarding the knowledge of the women from this study about HPV, although it was very low with only 29.1% of those interviewed showing a medium or high degree of knowledge on the virus, this proportion is above that described for the women of Medellin , but farAlthough almost all participants said that they had heard about the Pap test, only a minority could talk about its purpose but, nonetheless, were unaware of the advantages and benefits of having the procedure regularly to prevent cervical cancer. However, a significant proportion (69.4%) of the respondents said they had undergone the examination with the frequency recommended by the Health Ministry. This suggests that most of the women had the examination because they were recommended to do so, but they did not know the purpose it served, nor did they possess any degree of awareness about the advantages and benefits of the procedure for their health. On the other hand, a significant portion of respondents had heard about the HPV, and a smaller proportion knew that it is transmitted through sexual contact; however, the majority of them were unable to establish any correlation between genital infection with this virus and the occurrence of cervical lesions, including cancer.The Brazilian public health system consists of the SUS , which aims to provide full health coverage to every citizen of Brazil. Actions directed to family planning are inserted within the SUS through the Family Health Program (PSF). However, due to its wide scope, this system has limitations, which sometimes may make access to the health service difficult. Moreover, there is a low degree of knowledge and awareness of the women in relation to HPV and the importance of a periodical Pap test, aimed at the early detection and treatment of cervical lesions associated with HPV and cervical cancer prevention. This is reflected in the low levels of coverage obtained by the cervical cancer screening program. In this context, the findings of this study may serve to arouse the attention of professionals working at PSF about the need to intensify educational activities, aiming to improve the level of knowledge and awareness about the role of HPV in the etiology of uterine cervical lesions, including cervical cancer, and the importance of performing periodic cytologic exams to prevent these diseases.The present study has some limitations. First, the target population included only women studying in public schools or attending public health units, a population who generally present a lower socioeconomic status and who may not be so representative of the women population of Natal city. Secondly, questions about the HPV vaccine were not included in this research. Although the vaccine is the most effective way to prevent HPV infection, it is not accessible to the majority of the population, since it is not yet effectively included in the Brazilian official program of vaccination.Our results show that the majority of women of the segment of the studied population have low levels of knowledge and of conscientization of the role of HPV as a causative agent of precancerous and cancerous lesions of the anogenital tract. Furthermore, the majority of women respondents do not have adequate knowledge about the screening test for cervical cancer or about the advantages and benefits of doing so regularly to prevent the disease. Thus, it is clear there is a critical need to improve the knowledge of the local female population on the role of HPV in the etiology of cervical cancer, as well as about the screening method of this disease. Therefore, we suggest educational campaigns emphasizing the role of HPV in the etiology of the cervical lesions of different degrees, including cancer, as well as the importance of regular Pap tests to prevent these diseases.


Patients with HPV-positive cancer were young and lacked traditional risk factors.Oropharyngeal Cancer Epidemic and HPV A growing body of research shows that human papillomavirus (HPV) is a common and increasing cause of oropharyngeal squamous cell carcinoma (OSCC). Thus, the International Agency for Research against Cancer has acknowledged HPV as a risk factor for OSCC, in addition to smoking and alcohol consumption. Recently, in Finland, the United Kingdom, the Netherlands, the United States, and Sweden, incidence of OSCC has increased, and an increase in the proportion of HPV-positive tumors was noted. On the basis of these data and reports indicating that patients with HPV-positive cancer have their first sexual experience at a young age and have multiple partners, we postulate that increased incidence of OSCC in the United States and some countries in northern Europe is because of a new, primarily sexually transmitted HPV epidemic. We also suggest that individualized treatment modalities and preventive vaccination should be further explored. HNSCC is the sixth most common type of cancer in the world; almost 600,000 cases are reported annually, and of these, ≈10% (or more for some geographic locations) are OSCC , E6 and E7 deregulate cell cycle control by E6 binding and degradation of p53, and E7 binds and inhibits the function of the retinoblastoma protein (Rb)  performed primarily by using formalin-fixed, paraffin-embedded tissue, in which the DNA can be partially degraded. It is now widely accepted that it is easier to detect longer HPV DNA fragments in fresh or fresh-frozen material, although newer techniques are more sensitive. Many early studies during the 1980s were based on Southern blot techniques or in situ hybridization for detection of HPV.––Since the 1990s, virology laboratories used PCR for detection of HPV DNA  in the incidence of tonsillar cancer in the Stockholm area (,,In continuation of the above study, we followed the incidence of tonsillar and base of tongue cancer in Stockholm in the Swedish Cancer Registry and demonstrated a substantial increase for both tumor types during 1970–2006, as shown in Shortly afterwards, we demonstrated that the prevalence of HPV-positive base of tongue cancer also had increased in the Stockholm area during 1998–2007 (The strength of the above studies is that the Swedish Cancer Registry covers all cancer cases in Sweden and that we have analyzed all available pretreatment diagnostic biopsy specimens for HPV. The limitation of our study is that Sweden is a small country with only 9 million inhabitants.Covering almost the same time period as above, Chaturvedi et al. reported an increase in the proportion of potentially HPV-related OSCC in the United States (The possible causes for this increase have been discussed extensively and have focused on changes in sexual patterns, such as increased oral sex or increasing numbers of sex partners. A significant association has been shown between HPV-positive tonsillar cancer and early initial sex or number of oral or vaginal sex partners (Furthermore, in a recent study by D’Souza et al., it was shown that the risk of developing oral HPV infection increased with increases in lifetime oral or vaginal sex partners (Several reports show an increase in OSCC and the proportion of HPV-positive OSCC and an association of the latter to early sex debut and many partners. Thus, we suggest that we are encountering a slow epidemic of mainly sexually transmitted HPV-induced OSCC.,The possibility that we are dealing with an HPV-induced epidemic of OSCC warrants special attention. For example, in Stockholm, the incidence of HPV-positive tonsillar cancer has increased 7-fold over 30 years (–,,,Several reports have been published and other studies are ongoing to assess which molecular factors, such as p16, p53, and others, besides the presence of HPV in OSCC, can best predict clinical outcome and which treatments are optimal according to the same predictive markers (It has been shown in an experimental setting that HPV-positive tumors were not more curable on the basis of increased epithelial sensitivity to cisplatin or radiation therapy (Summarizing treatment of OSCC patients, it is obvious that additional information will be required before it will be possible to guide treatment decisions for the individual patient on the basis of HPV status. Nevertheless, there is accumulating evidence that HPV status and overexpression of p16, and having never smoked, is of benefit. Future prospective clinical studies, including diagnostics of HPV, molecular and immunologic profiles, history of smoking, cessation of smoking during therapy, and effects of different treatment modalities and their side effects on quality of life, will be of benefit for personalized treatment.–,–Finally, it is also essential to keep in mind that we now have vaccines directed against HPV16, which accounts for ≈80%–90% of all HPV-positive OSCC, at least in Europe and the United States (Few if any of other studies have focused on performing isolated health economic analysis of the effect of HPV on OSCC. However, a recent study pointed out that there is an improvement of the present cost-effectiveness of HPV vaccines when the effects on other HPV-associated tumors cancers are included (


Pap screening, an effective method for cervical cancer prevention, is now supported by molecular human papillomavirus (HPV) testing. Recently commercialised preventive vaccines also provide new tools for the primary prevention of cervical cancer. To determine appropriate prevention strategies, the Health General Direction, Lombardy Region, funded a project that aims to characterize and monitor HPV infections and related cervical diseases in high-risk women.VALHIDATE is a 5-year multicentre open prospective cohort study. It will recruit 7000 consenting women aged 13–65 years to provide information about the local biomolecular epidemiology of HPV infection and cervical diseases in high-risk women recruited from nine clinical centres and one faith-based organisation. The study will estimate the overall and type-specific prevalence of HPV infection and cervical abnormalities. It also aims to compare standard Pap screening with biomolecular screening, and to assist in the design of targeted regional prevention programs directed specifically at high-risk groups. Three groups of high-risk women: 1000 HIV-infected women (aged 26–65 years), 1000 recent migrant women (aged 26–65 years) and 3000 young women (aged 13–26 years) and 1 control group: 2000 women (aged 26–45 years) attending a spontaneous screening program, will be recruited. Sample sizes will be revised after the first year. Adult participants will undergo conventional cervical cytology, HPV DNA screening and genotyping. Paediatric participants will undergo HPV DNA testing and genotyping of urine samples. HPV DNA, cytological abnormalities and HPV types will be analysed according to demographic, epidemiological, behavioural, and clinical data collected in an electronic case report form. Overall and stratified prevalences will be estimated to analyse the associations between HPV infection and selected characteristics. Logistic regression models will be used to estimate crude and adjusted odds ratios. Cox proportional hazard models will be used to estimate hazard ratios over time and between groups.This study will provide substantial insight into HPV infections and related cervical diseases in high-risk groups and will help determine appropriate regional cervical cancer prevention strategies. Cervical cancer screening with cervical cytology is a proven and effective secondary prevention strategy that can achieve a significant reduction in cervical cancer incidence in the industrialized world -3. HowevRecently commercialized cervical cancer vaccines, moreover, provide new tools for the primary prevention of cervical cancer with a very high rate of protection for more than eight years when used in women previously uninfected by the genotypes included in the vaccines -30. The To determine appropriate strategies for cervical cancer prevention and to optimize the allocation of resources, the Health General Direction, Lombardy Region, funded a project that aims to characterize and monitor HPV infections and related cervical diseases in high-risk women across a wide range of ages.VALHIDATE  is a multicentre open cross-sectional and prospective cohort study conducted over a 5-year period (Dec. 2010–Dec. 2015) in the Lombardy Region. It aims to provide information about the local epidemiology of HPV infections and related cervical diseases as well as the distribution of HPV genotypes across ages and in high-risk groups of women.The study was commissioned and funded by the Health General Direction of the Lombardy Region and has been approved by the ethical committees of the hospitals involved in the research. Approval for the storage of residual biological samples for further analysis was also obtained from the hospitals’ ethical committees.The study will recruit 7000 consenting women aged 13–65 years through nine clinical centres in four general hospitals  and one faith-based voluntary organization that offers primary health care for migrants lacking health insurance.One university virology laboratory, four microbiology laboratories and four pathology laboratories located in the hospitals will analyse the samples collected from the enrolled women.The cross-sectional study was designed to estimate the prevalence of HPV infection, cervical abnormalities and HPV type-specific prevalence across a wide range of age and risk groups. The prospective cohort study was designed to compare the standard screening procedure (Pap test) with biomolecular HPV-testing across different age and risk groups and to assist in the design of targeted regional prevention programs directed specifically at high-risk groups.The secondary objectives of the study are to: a) identify the geographical distributions of HPV 16 and HPV 18 variants and their association with clinical aspects; b) identify the prevalence, distribution and incidence of potential HPV 16 and HPV 18 vaccine escape mutants; c) monitor the HPV type distribution over time for an early identification of the type replacement phenomenon following the beginning of the vaccination campaign for 12-year-old girls in the Lombardy Region (September 2008).Written informed consent for participation in the study and for the conservation of the collected samples in a bio-bank will be asked from all eligible women and will be essential for participation in the study.Two thousand women aged 26–45 years will be recruited from those attending a spontaneous Pap screening program (SPSW).One thousand HIV-infected women (HIVW) will be recruited from those followed up for HIV infection in three infectious disease units. One thousand recent migrant women (less than 1 year) (RMW) will be recruited mostly through a faith-based organization, and 3000 young women (aged 13–26 years) will be recruited from those asking for a Gynaecologic (1326G) or Paediatric consultation (1318P). Those vaccinated with anti-HPV vaccines prior to enrolment or during the follow-up period will not be excluded from the study. Instead, the vaccinated participants will be considered a subgroup to be monitored and evaluated over time.Those with a history of histologically proven grade II or higher CIN requiring treatment, who were pregnant at the time of enrolment or who were unable to provide informed consent are excluded from the study. For participants aged < 18 years, written informed consent will be required from their parents or legal guardian.Participants will be recruited for 12 months; this will be extended if necessary. They will be followed up for 3 years, and the study is scheduled for completion 3 years after the last enrolment.Adult participants will undergo a medical assessment appropriate to their group , cytological screening following the Italian Guidelines  for CervHPV-DNA testing will be performed by the reference virology laboratory. HPV DNA-positive samples will be genotyped using a commercial kit in the four microbiology laboratories. Oncogenic risk associated with HPV types will be defined according to the 2011 IARC classification . SamplesAn additional analysis for the characterization of HPV 16 and HPV 18 variants that could potentially elude the immune response induced by the vaccination (escape mutants) will be performed.Participants with discordant results (HPV-positive/cytology-negative) will be recalled after 12 months for reassessment with cytology and HPV testing.Paediatric participants will undergo a paediatric evaluation and HPV DNA testing of urine samples. The algorithms for the follow-up of adult and paediatric participants are presented in Figure The Pap smear sample will be obtained by using a brush . Cervical specimens for conventional cytology will be processed by spreading the cells onto a glass slide, fixing cells within a few minutes, and then sending them to the reference pathology laboratory for investigation.The same cervical brush used for cytology will be then immersed and rinsed in a vial filled with 20 ml of PreservCyt® solution , and stored at room temperature (RT) until processing. Ten millilitres of each PreservCyt solution containing cervical cells will be centrifuged at 3800 × g for 15 min at RT. After centrifugation, the pellet will be resuspended in 1 ml of phosphate buffered saline (PBS), transferred into a new 1.5 ml collection test tube and stored at −20 °C until nucleic acid isolation.First-void urine samples will be collected in sterile containers, kept at RT and processed within 6–8 hours of collection. At least 15 ml of each urine sample will be centrifuged at 3800 × g for 20 min at RT. After centrifugation, the opaque phase will be recovered, transferred into a new 1.5 ml collection test tube and then centrifuged at 16,000 × g for 15 min at RT. The pellets obtained will be resuspended in 700 μl of PBS and stored at −20 °C until nucleic acid isolation.Referral for colposcopy will be driven by the Pap results. Cytology showing atypical squamous cells or worse will be referred for colposcopy evaluation and biopsies will be taken from suspicious areas of the cervix, according to the standard procedures in Italy . Biopsy ® EasyMAG™, bioMérieux bv, France) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA will be evaluated through a Thermo Scientific NanoDrop 2000 spectrophotometer . DNA integrity will be assessed by amplification of a 268-base pair (bp) fragment in the ubiquitous β-globin gene using the primer pair PCO4 (5′–CAACTTCATCCACgTTCACC–3′) and GH20 (5′–gAAgAgCCAAggACAggTAC–3c′) [DNA will be extracted from cervical and urine samples using an automated system for total nucleic acid extraction (NucliSENSTAC–3c′) .HPV DNA will be detected by PCR amplification of a 450-bp segment of ORF L1 using the degenerate primer pair ELSI-f (5′–gCNCARggHCATAAYAATgg–3′) and ELSI-r (5′–CgNCCHAADggAAAYTgATC–3′). An equiExtra , a line probe assay based on the principle of reverse hybridization. Part of the L1 region of the HPV genome will be amplified using SPF10 primers. The resulting biotinylated amplicons will be subjected to denaturation and hybridization with sequence-specific DNA probes for the identification of 28 different HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74, and 82. To establish the genotype/s present in the sample, the line patterns will be compared with the interpretation chart supplied with the assay.The 450-bp fragments will be genotyped using INNO-LiPA HPV Genotyping alpha of Papillomaviruses.Samples for which the obtained line pattern cannot be assigned to any genotype pattern or that have no type-specific lines, but at least one positive HPV control line will be scored as HPV-positive, but untypeable (HPV X). These samples will be referred to the reference virology laboratory for genotype analysis by RFLP. The 450-bp fragments obtained by the ELSI-PCR protocol will be tested by the RFLP technique with RsaI, DdeI and HaeIII digestion enzymes . Briefly, 10 μl of the amplified products will be added to three final mixtures of 15 μl containing 1U of each enzyme in a specific buffer. The restriction reaction will be performed at 37 °C for 1 hour and the enzymes will be then inactivated at 80 °C for 20 minutes. Fragments of different lengths will be separated on 3% agarose gel. The pattern of fragments generated by the restriction enzymes will allow for identification of all types in the genus For all HPV16- and HPV18-positive samples, a 730-bp fragment of the LCR region will be amplified  ,38. The For all HPV 16-positive samples, a 911-bp and 879-bp fragment of the L1 gene will be amplified  . The oveFor all HPV 18-positive samples, a 911-bp and 870-bp fragment of the L1 gene will be amplified  . The ovePhylogenetic analysis of the sequences will be conducted by the bioinformatics programs described above. The analysis of codon-specific non-synonymous and synonymous substitutions and the selection pressures will be inferred by MEGA  and the Residual biological samples collected in the PreservCyt solution that are positive for HPV DNA and/or for cytological abnormalities will be stored at −20 °C for further studies. Concordant HPV DNA-negative/cytology-negative samples and residual urine samples will be processed as follows: after centrifugation, the pellet obtained will be stored at −20 °C and will then be available for future molecular investigationsDescriptive statistics (means ± SD or median) and frequency analyses will be used to describe the whole study sample and each cohort with regard to demographic, epidemiological, behavioural, clinical, immunological, and HPV vaccination status data. Data will be collected with a specially developed e-CRF released on a web platform built with PHP 5.3 and JQuery. The database resides on MySql 5.0. The application is hosted at the Mario Negri Institute  with security features according to the current laws. Anonymous data extraction will be performed at the data storage centre.Overall and HPV type-specific prevalence will be estimated with 95% confidence intervals (CI), according to the Poisson approximation, in the four participant groups (three high-risk groups and control group). Logistic regression models will be used to estimates odds ratios (OR) and their corresponding 95% CI to quantify the associations between HPV infection and major demographic, clinical and behavioural characteristics in the four participant groups. All variables will be analysed in both univariate and multivariate models to control for major confounding factors.Sensitivity, specificity, and positive and negative predictive values of the HPV test compared with the Pap test will be estimated overall and by group. Test performance in matched pairs will be estimated with McNemar’s tests, and proportions will be compared using Fisher’s exact tests. Exact methods will be applied when expected cell frequencies are less than five.The sample size was calculated by taking published data as well as economic feasibility into account to allow the estimation of differences in the prevalence of type-specific HPV between the high-risk and control groups. From preliminary results obtained from the first 1300 women enrolled , the prevalence varied from 0.5%–2%  in the control group, and was 2–3 fold higher in the high-risk groups . The planed sample of 1000 women for each high-risk group and 2000 low-risk women will be adequate to detect differences for genotypes with a prevalence of 1%–1.5% in the control group.We will analyze the incidence of new cytological abnormalities, progression or regression of baseline lesions, incidence, clearance and persistence of HPV infection for all women who had at least one follow-up visit. The proportion of women lost to follow-up is expected to be different in each participant group. The RMW group may have more difficulty in returning for a follow-up visit. Socio-demographic differences between women who return and who are lost to follow-up will be analysed. Conversely, women in the HIV cohort, who will be recruited from the centre they attend for HIV treatment, may be more likely to attend the study follow-up visits.The proportions of women who we expect to be lost to follow-up are therefore estimated at around 10%, 20% and 60% in the HIVW, SPSW and RMW groups, respectively.Single and multiple infections will be defined without considering uncharacterized HPV types (HPV X). The Kaplan-Meier method will be used to evaluate differences in the incidence of abnormal cytology at follow-up between women in the high-risk groups versus the control group. When appropriate, a Cox model will be used to account for the effects of major confounding factors. For the paediatric group, we will recruit about 250 girls for each year of age between 13 and 18 years to estimate the age at first HPV infection, time to clearance or persistence of HPV infection, and the change after 3 years of follow-up related to major behavioural characteristics.All the analyses will be performed using SAS software for Windows version 9.2 .In the last 50 years, the introduction of screening methods has substantially reduced the incidence of cervical cancer in the industrialised world. However, inequalities in the distribution of cervical cancer prevalence and mortality are still present at national, regional or local levels. In general, high-risk women are those who are not reached for any reason by prevention and screening programs. This study will provide substantial insight on HPV infections and related cervical diseases, which will enable us to define the risk of cervical cancer and its precursor lesions in high-risk women compared with the general population living in the Lombardy Region. In turn, this information will assist health care providers and policy makers to improve regional strategies and optimize cervical cancer prevention in high-risk groups.VALHIDATE: eVALuation and monitoring of HPV Infections and relATEd cervical diseases in high-risk women; CIN: Cervical Intraepithelial Neoplasia; SPsW: Spontaneous Pap Screening Program Women; HIVW: HIV infected women; RMW: Recent Migrant Women; 1326G: women aged 13–26 referred to gynaecologic consultation; 1326P: women aged 13–26 referred to paediatric consultation; RFLP: Restriction Fragment Length Polymorphism; eCRF: electronic Case Report Form; RT: Room Temperature; PBS: Phosphate Buffered Saline.The authors declare that they have no competing interests.GO, ET, MG, GR contributed to the conception and design of the study. GO, LC, ET conceived, designed and refined the data collection process and database structure and helped in developing the eCRF. GO, GR, MG, ET secured funding for the study. LC, PB will develop and conduct the statistical analysis. AMC, CG, MF will perform cytology. NZ, GL, GC will perform HPV genotyping. ET, SB will perform molecular and phylogenetic studies. MMF, IC, GZ, EB, IA, VB, SE, GT, and AM will recruit participants and carry out the screening and follow-up processes, contributing substantially to the acquisition of clinical and epidemiological data. All authors read, revised and approved the final manuscript.Maria Concetta Antonacci, Pathology Unit, L Sacco University Hospital, Milan, Italy - antonacci.concetta@hsacco.itIrene Arcidiacono, Division of Infectious and Tropical Diseases, Hospital of Lodi, Italy - irene.arcidiacono@ao.lodi.itPaola Bertuccio, Lifestyle Habits and Prevention Unit, Laboratory of Epidemiology, Department of Epidemiology, Pharmacological Research Institute “Mario Negri”, Milan, Italy - paola.bertuccio@marionegri.itSilvia Bianchi, Department of Biomedical Sciences for Health, University of Milan, Italy - silvia.bianchi@unimi.itVeronica Boero, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Mangiagalli, Regina Elena Milan, Italy - vboero@gmail.comGiuseppe Cambiè, SIMT, Hospital of Lodi - giuseppe.cambie@ao.lodi.itIrene Cetin, Gynaecology Unit, L Sacco University Hospital, Milan, Italy - Irene.cetin@unimi.itSusanna Esposito, Department of Maternal and Pediatric Sciences, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy - susanna.esposito@unimi.itMarcella Falchetti, Department of Patology- Spedali Civili di Brescia, - marcella.falchetti@spedalicivili.brescia.itMichela Maria Fasolo, STD Unit, Infectious Diseases II, L Sacco University Hospital, Milan, Italy - fasolo.michela@hsacco.itCarlo Galli, Pathology Unit, Hospital of Lodi - carlo.galli@ao.lodi.itEmanuela Bertazzoli, Hospital of Lodi, Italy - emanuela.bertazzoli@virgilio.itGiovanna Lunghi, Virology Unit, Clinical Chemistry and Microbiology Lab - Ca’ Granda Hospital, Milan - lab.viro@policlinico.mi.itAlberto Matteelli, Institute of Infectious and Tropical Diseases, University of Brescia, Italy - matteelli@med.unibs.itGiancarlo Tisi, Gynaecology Unit, Spedali Civili Brescia, Italy - dott.tisi@gmail.comNadia Zanchetta, Microbiology Unit, L Sacco University Hospital, Milan, Italy - zanchetta.nadia@hsacco.itGianvincenzo Zuccotti, Pediatric Unit, L. Sacco Hospital, Università degli Studi di Milano, Milan, Italy - gianvincenzo.zuccotti@unimi.itrd Feb 2010), Sacco Hosp General Direction Resolution n174/2010 (9 March 2010).Sacco Hospital Ethical Committee resolution n35 (3The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/204/prepub