TTN) mutations, especially in A-band region, represent the most common cause of dilated cardiomyopathy (DCM). Clinical interpretation of these variants can be challenging, as these variants are also present in reference populations. We carried out systematic analyses of TTN truncating variants (TTNtv) in publicly available reference populations, including, for the first time, data from Exome Aggregation Consortium (ExAC). The goal was to establish more accurate estimate of prevalence of different TTNtv to allow better clinical interpretation of these findings.Truncating titin  and 0.19%  for those affecting the A-band. In the A-band region, the prevalences of frameshift, nonsense and essential splice site variants were 0.057%, 0.090%, and 0.047% respectively. Cga/Tga (arginine/nonsense–R/*) transitional change at CpG mutation hotspots was the most frequent type of TTN nonsense mutation accounting for 91.3% (21/23) of arginine residue nonsense mutation (R/*) at TTN A-band region. Non-essential splice-site variants had significantly lower proportion of private variants and higher proportion of low-frequency variants compared to essential splice-site variants .Using data from 1000 Genomes Project, Exome Sequencing Project (ESP) and ExAC, we estimated the prevalence of TTNtv in the population. In the three population datasets, 52–54% of TTNtv were not affecting all TTN A-band suggesting the penetrance of these potentially harmful variants is still poorly understood, and some of these variants do not manifest as autosomal dominant DCM. This calls for caution when interpreting TTNtv in individuals and families with no history of DCM. Considering the size of TTN, expertise in DNA library preparation, high coverage NGS strategies, validated bioinformatics approach, accurate variant assessment strategy, and confirmatory sequencing are prerequisites for reliable evaluation of TTN in clinical settings, and ideally with the inclusion of mRNA and/or protein level assessment for a definite diagnosis.A-band TTNtv are more rare in the general population than previously reported. Based on this analysis, one in 500 carries a truncation in  TTN) is a giant muscle protein expressed in the cardiac and skeletal muscles. It spans half of the sarcomere from Z-line to M-line. Titin is known to play a key role in muscle assembly, force transmission at the Z-line and maintenance of resting tension in the I-band region. The clinically relevant A-band of TTN binds to the thick filament, where it may regulate filament length and assembly, and is thought to be critical for biomechanical sensing and signaling. The C-terminal M-band contains a strain-sensitive kinase, which may have a role in cardiac signal transduction [TTN gene mutations have been found to associate with cardiomyopathies [TTN gene with its ~363 exons. Consequently, TTN mutation frequency and therefore clinical impact were unknown.Titin (sduction . Before opathies –8. This TTN truncating variants (TTNtv)—nonsense, frameshift and essential splice site, to be responsible for approximately 25% of familial cases of idiopathic dilated cardiomyopathy (DCM) and 18% of sporadic cases in a large cohort of subjects. Similarly to Herman et al. [TTN gene as the bulk of the mutations were located predominantly in the A-band region and affecting all transcripts of TTN [TTN truncations are highly enriched in DCM patients when compared to healthy controls [Recently, using a combination of NGS and dideoxy sequencing, Herman et al.  estimaten et al. , in our s of TTN –14. Anotcontrols . Furthercontrols  also concontrols .TTN was estimated to be as high as 3.2% (35/1092) among reference individuals. In the study by Roberts et al. [Utilization of reference population databases has significantly improved our ability to interpret genetic findings. Before the publishing of Exome Aggregation Consortium (ExAC) database in 2014, we have been dependent on the different versions of the 1000 Genomes Project and the National Heart Lung Institutes Exon Sequencing Projects (ESP) , 17. SinTTN domains to better understand the distribution and prevalence of different genetic findings. Our study reveals that identifying a frameshift, nonsense or an essential splice-site variant in the critical A-band of TTN is a rare event. In our study we conclude that in reference population only 1 in 1750 individuals carry a frameshift, 1 in 1100 carry a nonsense, and 1 in 2100 carry an essential splice variant in TTN A-band region that is estimated to affect all isoforms of the gene. We also determine the characteristics of nonsense variants identified and variability in the splice-site regions of TTN gene. We believe our results have a wide application in clinical interpretation of genetic test results and further emphasize that TTNtv in general population exceed significantly the prevalence of DCM associated with TTN truncations.Since the prevalence of TTNtv in reference populations is a critical determinant when interpreting genetic test results, we pursued to analyze this, for the first time, in the over 60 000 ExAC cohort together with the final version of 1000 Genomes project and ESP datasets. Our goal was also to define the specific prevalence of different TTNtv in various TTN gene variant data from the various phases and versions of 1000 Genomes Project were retrieved, and truncating variants comprising of splice, nonsense and frameshift variants were used for analysis.ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521/README.phase1_integrated_release_version3_20120430) and the release of the phase 3 call set, we were keen to re-estimate the frequency of TTN truncating mutations in the population. We downloaded the exonic boundaries of TTN from the Ensembl Genome Browser (www.ensembl.org), and extracted exonic variants from the three versions of the phase 1 variants call sets , and the version 5 (V5) of the phase 3 variants call set available in the 1000 Genomes Project database. Only calls that passed all quality filters were used for downstream analysis.Considering the INDELs filtering on 1000 Genomes Project phase 1 variant call sets  [TTN truncating variants unique to either 1000 Genomes Project or ESP call sets. Variants located in novex-specific exons and exons specific to few transcripts of TTN were filtered before filtering for sequencing coverage/depth. See below for more details on the workflow and results.In a bid to capture a complete map of population ct (ESP)  and ExACct (ESP) , with ovWe extracted TTNtv that passed all quality filters as reported by the 1000 Genomes Project from various phases and versions of the project. Based on the total number of TTNtv in each version of the phase 1, and the version 5 of the phase 3 releases, we estimated the prevalence of TTNtv in the population. Interestingly, we observed a downward trend in both the number of TTNtv and TTNtv prevalence from the oldest to the latest release of the project . Versiondiscussion)  . The inicussion) . After tcussion)  Tables. cussion) . The varTTN truncation call sets suggesting that ESP is enriched with population specific variants, as it is made up of primarily Americans either of European (EA) or African (AA) origin. Furthermore, at least 52% of TTNtv  found in the populations were located in exons that are not present in all transcripts and estimated to have low probability of pathogenicity.In our goal to accurately estimate the prevalence of TTNtv in the general population, we queried TTNtv in 1000 Genomes, ESP and ExAC databases . The worTTN band, 11.0% (19/173) are located in the Z-band; 22.6% (39/173) in the I-band; 52.0% (90/173) in the A-band; and 14.5% (25/173) in the M-band  in ExAC vs 1000 Genomes, we identified lower frequency of nonsense mutations in ExAC cohort compared to 1000 Genomes cohort  . This wae M-band . Based oe M-band . In the TTN A-band region that is estimated to affect all transcripts.The prevalence of TTNtv in clinically relevant A-band was 0.19%, suggesting that 1 in 500 individual carries one of the subtypes of TTNtv. When evaluating by mutation subtype, we conclude that in reference population, only 1 in 1100 individuals carries a nonsense mutation; 1 in 1750 carries a frameshift, and 1 in 2100 carries an essential splice site variant in P = 2.7 × 10−5). AS has the highest frequency of TTNtv while the European population has the least. The frequency of TTNtv in East Asian population (EAS), though lower, is not significantly different from that of the South Asian Population (SAS) . Similarly, the frequency of TTNtv in Finnish European population (FIN) is lower but statistically indistinct when compared with the non-Finnish Europeans (NFE) .Frequencies of TTNtv vary significantly between different ethnic groups  than all other ethnic groups . In this study, we have found a higher frequency of TTNtv in the Asians. Put together, this data confirms the enrichment of not only TTN PAV but also TTNtv in the Asian population (SAS in particular) compared to other ethnic groups.Golbus et al.  recentlyTTN nonsense SNPs in the population. Out of the twenty-three possible ways to change codons into stop codons , twenty-one were found in reference population, with tCg/tAg and tgT/tgA being absent. Nonsense SNPs were more frequent at the first codon position than at the second and third positions . The most frequent type of nonsense mutation in the TTN gene was the change from Cga to Tga (R/*). Of note, the Cga/Tga transitional change at CpG mutation hotspots accounts for 91.3% (21/23) and 62% (21/34) of arginine residue nonsense mutations (R/*) at TTN A-band region and all TTN CpG hotspot nonsense mutations in the population data respectively. Interestingly, 36.1% (13/36) of R/* found in reference population have been reported in Catalogue of Somatic Mutations in Cancer (COSMIC) database [We examined the patterns and the positions of the database .TTN A-band exons. Of note, majority, 88.8% (175/197), of the splice variants affecting A-band exons were non-essential splice-site variants. When analyzing all essential splice site variants, we identified that 83.7% (41/49) were private. In contrast, non-essential splice-site variants had significantly lower proportion of private variants and higher proportion of low-frequency variants when compared to essential splice-site variants . The significance increases with increasing distance away from essential splice site  were non-essential splice variants located >2bp into the intronic region, with 52.8% (197/373) of the variants affecting ice site .TTN, and showed that non-essential splice-site variants in TTN have a tendency of being frequent in general population. In addition, we describe how increased quality of data analysis and larger number of reference individuals in 1000 Genomes Project has markedly reduced the prevalence of TTNtv in this database.By evaluating the ExAC reference population of over 60 000 individuals, we showed that TTNtv affecting all transcripts occur in 0.36%  of the general population. These types of truncations in clinically relevant A-band can be detected in 0.19% of reference population. When evaluating the mutation types separately, the prevalence of each mutation type in A-band was lower than 0.1%, below a commonly used filtering criterion when searching for disease-causing mutation of rare dominantly inherited diseases. Furthermore, we provide insights on the position, type and frequency of nucleotide change(s) leading to nonsense mutations in TTN variants in reference population.The 1000 Genomes Project published the pilot phase of the project in 2010. This release represents data from low-coverage (~2-fold– 6-fold) whole-genome sequencing of 179 individuals, high-coverage (~42-fold) whole-genome sequencing of 6 individuals in 2 trios, and exon-targeted sequencing (> 50-fold coverage) of 8140 exons in 697 individuals . This waTTN. The cytosine-guanine (CpG) dinucleotide, though under-represented in vertebrate genomes, has been reported to be a hotspot for pathological mutations in the human genome [TTN A-band region. Interestingly, 62% of Arg/* CpG hotspot mutations are located at TTN A-band. Furthermore, a significant proportion of TTN Arg/* CpG hotspot mutations have been reported as germ line mutations identified in various cancer cells. This highlights the fact that the CpG is a critical hotspot for both germline and somatic mutations. Considering the high prevalence of Arg/* CpG hotspots in A-band, majority of exons coding for A-band present in all TTN transcripts, and that nonsense mutations in this region do not lead to mRNA decay but seem to work through dominant negative mechanism, it is not surprising that the A-band region is vulnerable to truncating mutations that are highly enriched among DCM patients.Nonsense mutations account for an appreciable proportion of single-basepair substitutions affecting gene-coding region reported in Human Gene Mutation Database (HGMD). Our analysis shows that as in other genes, Cga/Tga  nucleotide substitution at CpG hotspot is the most frequent nucleotide substitution leading to nonsense mutation in n genome , 24. Thin genome . In thisTTN A-band region occurring also in the germ line and manifesting hereditary cardiomyopathy. Epigenetic mechanisms are increasingly being recognized as causes and modulators of human disease. To date, there are few studies on the contribution of DNA methylation to disease onset and progression in cardiomyopathy [MYBPC2) and cardiac myosin binding protein C gene (MYBPC3), a common causal gene for hypertrophic cardiomyopathy. Interestingly, the mean methylation level of CpGs was significantly higher in MYBPC3 than MYBPC2 (P < 0.0001). These probably suggest that there are unique aspects of this cardiac gene that may result in increased genetic mutability. As mechanisms promoting mutability play a role in disease prevalence, evaluation of the methylation levels of TTN, and other DCM-associated genes are warranted.Epigenetic mechanisms could underlie the elevated mutation frequency in the myopathy –28. In 2myopathy  reportedTTN I-band in different tissues. In our goal to characterize essential splice and other splice region variants that might contribute to disease development, we examined and compared the position  and frequency of all TTN essential splice-site and splice-region variants in the population. While essential splice-site variants were predominantly private, we observed an increase in allele copies of non-essential splice-site variants as the distance increases from the essential splice-site. Robert et al. [TTN gene is difficult and these variants should not be claimed deleterious without RNA level evidence of splicing defect.Differential splicing in regions with variable sarcomere ultrastructures accounts for the variable structures of t et al.  recentlyTTN mutations in various skeletal muscle diseases including centronuclear myopathy  [TTN mutations cause early-onset, recessive muscle and cardiac disorder. M-line TTN mutations truncate only part of the M-band portion of TTN [TTN A-band but absent from Z-disk and M-band regions of TTN. The position of TTNtv causing DCM suggests a dominant negative effect. In addition, Herman et al. [TTN transcript levels in patients with or without TTNtv. They also found and reported a comparable allelic expression of TTNtv and SNPs, which does not support substantial nonsense-mediated decay. Using a combination of genetic data, TTN RNA and protein expression in LV tissues, Roberts et al. [Several studies have reported M-line #160150) , tibial #160150) , 31, ear#160150) , with orn et al.  argued tn et al.  reporteds et al.  also conTTN mutations. In these studies, TTNtv co-occurred with a second hit but actually only eight out of 18 patients had two TTNtv, and in two of these cases, bi-allelic status was not confirmed by parental testing [TTN. These observations together with our prevalence estimates highlight that recessive cardiac disease caused by titin dysfunction is possible in rare occasions. Initial evidence exists for recessive inheritance of TTN mutations in neurological disease although clearly more comprehensive segregation studies are warranted to ease interpretation of TTNtv.As majority of TTNtv have been identified at the heterozygous state in patients with sporadic or dominant conditions, such mutations are typically considered autosomal dominant. In majority of the cases, dominant inheritance has been clearly established by co-segregation. To the best of our knowledge, only a few studies have suggested recessive inheritance with  testing , 33, 34. testing  did not TTN truncating mutations being the most common cause for hereditary DCM [TTN gene reliably in clinical setting requires expertise in DNA library preparation, high coverage NGS strategies, validated bioinformatics tools, confirmatory sequencing, skilled interpretation team, and ideally the inclusion of mRNA and/or protein level assessment for definite diagnosis. Current low coverage exome and whole genome sequencing approaches may be sub-optimal for diagnostic work-up in cardiomyopathies as their efficacy for identifying INDELs is weak compared to high coverage targeted sequencing strategies [TTN gene makes it more susceptible than an average sized gene to false positive truncating variant especially when using ‘clinical’ exome sequencing. It can be easily demonstrated by the fact the there is four homozygous TTN variants  in ExAC database and they are found as heterozygote in 1, 5, 10 and 10 individuals respectively, although we would expect to find 245 heterozygotes for one homozygote in this cohort. This indicates a high probability of detected homozygotes being false positives. Further studies are needed to evaluate the reliability of our reference data, and especially to find out how many of the TTNtv in reference populations are true positives. It should be kept in mind that one out of seven low quality score variants (QS<500) in exome sequencing are likely false positives (non-detectable by Sanger sequencing) [Considering the accumulated evidence of tary DCM –11 togettary DCM . Furtherrategies , 36. Moruencing) .S1 TableMarked (†) are the false positives that were filtered out in the subsequent version. Abbreviations: P1V1 –phase 1 version 1; GMAF–Global minor allele frequency.(DOCX)Click here for additional data file.S2 TableMarked (†) are the false positives that were filtered out in the subsequent version. Abbreviations: P1V2 –phase 1 version 2; GMAF–Global minor allele frequency.(DOCX)Click here for additional data file.S3 TableMarked (†) are the variants that were not detected in the phase 3 integrated variant data. Abbreviations: P1V3 –phase 1 version 3; GMAF–Global minor allele frequency.(DOCX)Click here for additional data file.


TTN) is known as the largest sarcomeric protein that resides within the heart muscle. Due to alternative splicing of TTN, the heart expresses two major isoforms (N2B and N2BA) that incorporate four distinct regions termed the Z-line, I-band, A-band, and M-line. Next-generation sequencing allows a large number of genes to be sequenced simultaneously and provides the opportunity to easily analyze giant genes such as TTN. Mutations in the TTN gene can cause cardiomyopathies, in particular dilated cardiomyopathy (DCM). DCM is the most common form of cardiomyopathy, and it is characterized by systolic dysfunction and dilation of the left ventricle. TTN truncating variants have been described as the most common cause of DCM, while the real impact of TTN missense variants in the pathogenesis of DCM is still unclear. In a recent population screening study, rare missense variants potentially pathogenic based on bioinformatic filtering represented only 12.6% of the several hundred rare TTN missense variants found, suggesting that missense variants are very common in TTN and are frequently benign. The aim of this review is to understand the clinical role of TTN mutations in DCM and in other cardiomyopathies. Whereas TTN truncations are common in DCM, there is evidence that TTN truncations are rare in the hypertrophic cardiomyopathy (HCM) phenotype. Furthermore, TTN mutations can also cause arrhythmogenic right ventricular cardiomyopathy (ARVC) with distinct clinical features and outcomes. Finally, the identification of a rare TTN missense variant cosegregating with the restrictive cardiomyopathy (RCM) phenotype suggests that TTN is a novel disease-causing gene in this disease. Clinical diagnostic testing is currently able to analyze over 100 cardiomyopathy genes, including TTN; however, the size and presence of extensive genetic variation in TTN presents clinical challenges in determining significant disease-causing mutations. This review discusses the current knowledge of TTN genetic variations in cardiomyopathies and the impact of the diagnosis of TTN pathogenic mutations in the clinical setting.Titin ( Dilated cardiomyopathy (DCM) is defined by the presence of left ventricular (LV) or biventricular dilatation and systolic dysfunction in the absence of hypertension, valvular disease, or coronary artery disease sufficient to cause global systolic impairment . The preIn this setting, genetics can justify a significant proportion of DCM cases up to 25%), so the disease can be classified into genetic and non-genetic forms . In DCM,5%, so thTTN) encodes the largest human protein, whose name stems from the word Titans, giants of Greek mythology. Among the genes involved in cardiomyopathies, TTN plays a central role because of its frequency and the key structural, mechanical, and regulatory role within the sarcomere in the striated muscle , and RCM.Truncation mutations of s of DCM . Howeverthogenic , 14. IntCM cases . FurtherTTN gene mutations, previously difficult to analyze due to its size and complexity, has now allowed the identification of more than 60,000 TTN missense variants (reported in the 1000 Genomes Project) , the study of Project) , 17. TheTitin is the largest human protein. Two TTN filaments with opposite polarity span each sarcomere, namely, the contractile unit in striated muscle cells. TTN is responsible for sarcomere passive stiffness generation . TTN is TTN undergo extensive alternative splicing to encode different isoforms. In cardiomyocytes, three different isoforms of titin are expressed: adult N2BA, adult N2B, and the fetal cardiac titin (FCT) isoforms. The I-band sequence defines the different proprieties of each isoform, whereas the Z-disk, A-band, and M-line regions are extremely conserved   . Furthernscripts .TTN gene structure is organized to accommodate extensive splicing events. Roberts et al. defined a percentage spliced in (PSI) score based on RNA sequencing data from end-stage DCM and donor heart in order to find the mean usage of each TTN exon , a RNA splicing factor, plays an important role in this process and a reduced expression of RBM20 can alter TTN splicing and isoform expression in human  plays a role in the pathophysiology of heart disease .TTN in large cohorts of patients with DCM and in the past few years have been prolific in the description of new DCM-related TTN mutations. A comprehensive cohort study by Herman et al. , but with a rate in the healthy volunteers ranging between 1 and 2.9% . In order to address this challenge, a recent multicenter study sequenced the TTN gene in a cohort of 147 DCM patients . Furthermore, the outcome of TTN missense variants carriers did not differ significantly from the other DCM patients  cosegregating with RCM in six affected individuals of a family. The most common genes were excluded due to lack of complete cosegregation. Interestingly, some healthy individuals also harbored the TTN missense variant resulting in an incomplete penetrance  found TTN mutations to be associated with the ARVC phenotype (TTN rare variants (two TTN variants present in one proband) were identified  (TTN rare variants were accounted for in their population of subjects. Among the 67 ARVC affected patients (39 ARVC families), 11 harbored rare TTN variants and 8 desmosomal genes variants. The TTN carriers had increased supraventricular arrhythmias, and conduction disease compared with non-carriers  mainly with autosomal dominant pattern of inheritance . ARVC ishenotype . Among sentified . In addiarriers) . In thiscarriers , while dTTN variants are very frequent; of them, pathogenic mutations are relatively rare and most variants are probably benign. However, a portion of these variants could have a modifier gene effect. For instance, TTN has been proposed as a modifier gene in combination with the Lamin A/C (LMNA) gene . Fourteen individuals harbored LMNA: c.656A>C mutation, and of those five also carried a novel TTN missense mutation (TTN: c.14563C>T) as well  compared with those only harboring the LMNA mutation alone. Furthermore, histological studies showed more evidence that double heterozygote individuals had worse outcomes on a cellular level (TTN variants that contribute to the complexity of the DCM phenotype.NA) gene , 49. A mpression . In a st as well . LMNA gen of DCM . Accordimutation . Upholdie lamins . In a star level . In concTTN truncating variants were also found in ~2% of healthy controls  implantation, such as in other cardiomyopathies at high risk of life-threatening arrhythmias . In. InTTN mhythmias .TTN and other proteins affecting TTN splicing are associated with the development of DCM, but these mechanisms are still not completely understood (TTN splicing have become of great importance in DCM, and are associated with decreasing passive stiffness and increasing chamber compliance (TTN mutations. By genetic approaches or by splicing or posttranslational modifications TTN appears to be a target for future therapeutic interventions (Mutations in derstood . Variablmpliance . Both meventions .TTN missense variants, the situation is even more challenging because TTN missense variants are very common and their real meaning is still unknown. A recent study demonstrated that missense variants did not correlate with the clinical measures of disease severity or progression and indicated that the DCM phenotype caused by TTN missense variants are not distinguishable from other types of DCM (Figure TTN rare missense mutations should not be currently interpreted as disease-causing in most situations (TTN missense mutations may have a modifier role leading to a greater severity of cardiomyopathy (TTN missense variants in DCM will be elucidated with large-scale TTN sequencing and functional investigations on TTN variant domains.Regarding the universe of M Figure . Accordituations . Neverthmyopathy , 48. In Finally, despite the recent advances in genetic studies and in the understanding of the different effects of specific gene mutations in the pathogenesis of DCM, the clinical approach to diagnosing cardiomyopathy affected families remains largely based on the general recommendations for heart failure management, familiar screening programs, and systematic follow-up. The continuous improvement in technologies, such as the increasing evidence concerning the clinical expression of different gene variants might lead in the future to an individualized clinical approach to identifying carriers of different mutations.TTN variants have been shown to cause the following cardiac diseases: DCM, RCM, HCM, and ARVC. The advancement of NGS has allowed researchers to analyze the whole TTN gene, which has revealed the leading role of this gene in DCM. Challenges are the high genetic variability of the gene, the large number of missense and truncation variants found in control populations, and the criteria for clinical diagnosis of many variants demand individualized clinical diagnosis platforms for TTN carriers. Future studies will clarify whether the early identification of TTN-related cardiomyopathies might positively influence the natural history of disease by the early initiation of therapeutic management.Titin is the largest protein in striated muscle. All authors have contributed significantly, read, and approved the manuscript. In particular, MG, RB, and GM: drafting of the manuscript; GS, MT, HG, and LM: revising critically the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papilloma virus (HPV) infection is a major risk factor for a distinct subset of head and neck squamous cell carcinoma (HNSCC). The current review summarizes the epidemiology of HNSCC and the disease burden, the infectious cycle of HPV, the roles of viral oncoproteins, E6 and E7, and the downstream cellular events that lead to malignant transformation. Current techniques for the clinical diagnosis of HPV-associated HNSCC will also be discussed, that is, the detection of HPV DNA, RNA, and the HPV surrogate marker, p16 in tumor tissues, as well as HPV-specific antibodies in serum. Such methods do not allow for the early detection of HPV-associated HNSCC and most cases are at an advanced stage upon diagnosis. Novel noninvasive approaches using oral fluid, a clinically relevant biological fluid, allow for the detection of HPV and cellular alterations in infected cells, which may aid in the early detection and HPV-typing of HNSCC tumors. Noninvasive diagnostic methods will enable early detection and intervention, leading to a significant reduction in mortality and morbidity associated with HNSCC. Head and neck squamous cell carcinoma (HNSCC) includes malignancies in five major anatomic sites, namely, oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx. HNSCC is the sixth most common malignancy with an estimated annual incidence of ∽633,000 and 355,000 deaths worldwide Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most common types of HNSCC, accounting for 263,900 new cases and 128,000 deaths worldwide in 2008 The significant increase in HPV-associated OPSCC over the last decade among nonsmoking, young individuals Most HPV infections do not progress to cancer, however, delayed clearance of infection has been shown to be a risk factor for the development of anogenital cancer The incidence of HPV-positive OPSCC is the highest among middle-aged, nonsmoking white males (40–59 years old) with higher socioeconomic status and multiple sexual partners HPV positivity in OPSCC is associated with a reduction in the risk of death compared to patients with HPV-negative tumors. A study has shown that patients with HPV-positive OPSCC had a higher 5-year disease-free survival (75% vs. 14%) and overall survival (79% vs. 19%) compared to patients with HPV-negative tumors HPV L1, the major capsid protein that can self-assemble into virus-like particles (VLP) is the basis for HPV vaccines that generate neutralizing antibodies against the VLP proteins HPV is a member of the Papillomaviridae family, small DNA viruses that are commonly detected in various species including birds and mammals The HPV genome consists of a small double-stranded DNA of ∽8000 base pairs, divided into three major regions. The early genes (E1–E7) are expressed early in the viral infectious cycle for the regulation of transcription, plasmid replication, and transformation. The late genes code for the major (L1) and minor (L2) capsid proteins, involved in the packaging of viral genome and virus release. The long control region (LCR) contains the regulatory elements for transcription and replication. E6 and E7 encode the main HPV oncoproteins that promote cell cycle progression and viral DNA replication. As most people with HPV infection do not develop cancers, expression of E6 and E7 is necessary but not sufficient for malignant transformation. However, increased proliferative capacity and evasion of apoptosis induced by E6 and E7 can lead to the accumulation of DNA damage and mutations that can ultimately result in malignant transformation and carcinogenesis.INK4a, which is routinely used as a biomarker for HPV-associated lesions and cancers. p16INK4a is a tumor suppressor in normal cells, but it has been shown to be essential for the survival of HPV-positive cervical cancer cell lines The major role of high-risk E7 protein is to reprogram terminally differentiated epithelial cells at the surface epithelium in order for the host cells to re-enter the cell cycle, which is required for viral DNA replication. E7 binds to the retinoblastoma  and other members of the Rb family, such as p107 and p130. Proteins from the Rb family regulate the G1-S phase transition through interaction with the E2F family of transcription factors, which in turn control many genes that are involved in regulating cell cycle progression, differentiation, mitosis, and apoptosis α (TNFα). TNFα activates the extrinsic apoptotic pathway through TNF receptor 1 (TNFR1), Fas cell surface death receptor (FAS) and the TNF-related apoptosis-inducing ligand (TRAIL) receptors. E6 abrogates the apoptotic effect of TNFα by binding to TNFR1, which inhibits the subsequent transduction of apoptotic signals Aberrant cell proliferation and DNA synthesis in the absence of sufficient growth signals as observed in differentiating HPV-infected cells can trigger p53-dependent apoptotic programs. The inactivation of Rb by E7 protein sensitizes cells to p53-dependent apoptosis, but E6 protein targets p53 for degradation, thus inhibiting the proapoptotic functions of p53 The expression of E6 and E7 can result in the immortalization of host cells but it is insufficient to directly transform cells. HR E6 and E7 independently induce genomic instability in normal cells Currently, there is no consensus on the optimal way to identify HPV-positive HNSCC. Different methods include the detection of p16 protein expression using immunohistochemistry (IHC) as well as HPV-related genetic material using polymerase chain reaction (PCR) and in situ hybridization (ISH) in tumor biopsy samples. In addition, the presence of HPV-specific antibodies in serum has also been associated with increased risk of developing OPSCC INK4A in HNSCC tumor biopsies has been shown to serve as a surrogate marker to identify HPV infection in histologic preparations from HNSCCs INK4A-positive/HPV-negative and 8% were p16INK4A-negative/HPV-positive INK4A is also overexpressed in a subset of HNSCC lacking HPV DNA, with close to 14% of tumors that were p16-positive were negative by HPV-specific ISH and PCR INK4A was observed in 3/14 (21.4%) patients who were positive for HPV DNA and mRNA During immortalization of host cells, the E7 protein of HR-HPV binds to Rb, resulting in the compensatory overexpression of the tumor suppressor gene p16 in HPV-infected tumor cells PCR is a highly sensitive and cost-effective method for the detection of HPV. Due to the high number of HPV strains, primers targeting the conserved L1 open reading frame are commonly used to detect a broad spectrum of HPV, such as the MY09/MY11 and GP5/GP6 primer pairs E6 and E7 regions have been shown to harbor many sequence variations between HPV types Another method of HPV DNA detection in tumor samples is ISH. The advantage of ISH over a PCR method is the high specificity due to the reliable detection and identification of HPV in topographical relationship with their pathological lesions The expression of mRNA from integrated and episomal HPV DNA indicates that viral oncogenic transcripts are crucial in tumor initiation and progression The specificity of HPV-specific DNA and RNA ISH for HPV infection in tumor samples in combination with the high sensitivity of p16 IHC or HPV DNA PCR can be an effective diagnostic strategy for HPV-associated HNSCC. It has been suggested that screening tumors with p16 IHC to be performed first, and upon positive result, to be followed by HPV-specific test, such as ISH or PCR The detection of HPV-specific IgG in serum is a useful biomarker to determine previous and current HPV infection status Most of the current diagnostic assays are designed for excised tumor tissues obtained at the time of surgical biopsy or resection. More recently, the use of biological markers in oral fluids for the detection of HPV-associated HNSCC has been gathering a lot of attention due to the noninvasive and cost-effective nature, as well as the proximity to oral tumors, which allows for early cancer detection and monitoring of disease progression n = 201) revealed that there was a significant correlation between HR-HPV detected in oral rinse and the HR-HPV types present in tumor tissues An earlier study of oral exfoliated cells and tumor tissues from HNSCC patients (P < 0.001). However, the authors noted that some tumors that were HPV-positive did not yield HPV positivity in the oral rinses, and that the oral rinse level of HPV16 was significantly lower than that of the tumor, presumably due to the diluting effect of normal exfoliated cells unrelated to the tumor cells HPV DNA detection in tumor tissue and oral rinse from patients with tumors demonstrated a significant correlation  without such lesions. Another study also included the HR group of HIV-positive patients and found that HPV DNA was detected in saliva of 7/68 (10.3%) of oral cancer patients and in 12/34 (35.3%) HIV-positive individuals A study by Fakhry et al. aimed to develop a cytology test for the detection of HPV-associated HNSCC from populations with elevated risk of developing OPSCC, namely patients with abnormal oropharyngeal lesions and HIV-infected patients A recent study analyzed and correlated the presence of HPV DNA in oral samples  in patients with the incidence of HPV-positive tonsillar and base of tongue cancer Studies to date have collectively demonstrated promising data for the utilization of oral fluid as a valid specimen for the detection of HR HPV as well as other prognostic markers. Currently, there is only one laboratory-based salivary diagnostic test available to detect HPV  that determines the risk of developing HPV infection by detecting different strains of HPV such as HPV8, 11, 16, and 18 via PCR using oral rinse samples. However, the clinical performance interims of the sensitivity and specificity of this test and other oral fluid-based detection methods lack experimental evidence. It is also important to experimentally validate if HPV infection is current/active or in the past. Oral fluid based tests to determine HPV infection require further improvements due to the different origins of the cells being tested in oral fluid, namely HPV-positive tumor cells, any associated HR-HPV infection that can lead to the development of oral cancers or an independent HR HPV infection, as well as the percentage of host healthy exfoliated cells and immune cells HPV-associated HNSCC represents a distinct entity from tobacco and alcohol-related HNSCC. The expression of HR-HPV oncoproteins E6 and E7 upon viral infection and integration results in the immortalization of host cells and subsequent malignant transformation through increased proliferative capacity, upregulation of antiapoptotic pathways and increased genomic instability. Currently, the treatment plans for nonoropharyngeal HNSCC patients do not change with respect to HPV status. However, the improved outcome in patients with HPV-positive OPSCC has been shown in many studies and the de-escalation of treatment for this subset of HNSCC has been proposed


The scientific progresses achieved, during the twenty-first century, in molecular biology, in cytogenetics, in the use of stem cells in the treatment of various pathologies by reprograming them , in medi2 laser. The advent of new energies, i.e., RF, YAG laser, Argon laser, plasma energy, excimer, etc., opened new perspectives for surgical management of diseases.Born in the 70s, the laparoscopic surgery, now offers a better topographic approach of lesions, an almost perfect macroscopic depiction of colors. Hence, surgeons can spare more healthy tissues such as vessels and nerves, and thus adapt a more conservative procedure while restoring normal anatomy that is essential for preserving fertility. Endometriosis is one of the most obvious examples , 3. ThesThe miniaturization of instruments in hysteroscopy coupled with the development of hydrosonography allowed a better exploration of the uterine cavity, endometrial biopsies, and resection of intra-uterine lesions. Thus preventing invasive and radical treatments and reducing post-operative morbidity.However, the greatest advancements have been realized in cellular and molecular biology, in cytogenetics, and in proteomics. Indeed, the recent knowledge in the field of stem cells and their specific reprograming expand horizons to gene and cell therapy. A simple cell could be considered as a treatment by re-colonizing unhealthy tissues, thus preventing transplantation , 4, 5. Tin vitro culture of pathological cells, disease modeling, and collection of withdrawn tissues pre-operatively allows researchers to understand etiophysiopathogeny, and natural evolution of the disease as in endometriotic tissues , the progress in the knowledge of genetic mapping offers much hope to patients and couples suffering from family and hereditary diseases.Owing to progress in the field of infertility and assisted reproductive techniques (ART), i.e., intra cytoplasmic spermatozoid injection (ICSI) and vitrification of oocytes and embryos, increasing numbers of patients could be treated. 2 conization) prevents their transformation toward an invasive cervical cancer. The use of CO2 laser coupled with colposcopy allowed “see-and-treat” management of cervical, vaginal, vulvar, and perineal lesions related to HPV in an ambulatory way with a minimal morbidity. Eradication of cervical cancer during this century is possible and conceivable, only if national programs of information of gynecologists, family practitioners, as well as a program of HPV vaccination are settled.A better knowledge of human papilloma viruses (HPV) and the conception of vaccination against those HPV contribute in the reduction of the prevalence of cervical cancer, since HPV is involved in the genesis of cervical dysplasia and thus in that of cervical cancer. A radical treatment of cervical dysplasia , thus allowing an optimal management with less morbidity and less psychological disturbance in the respect of national legal frames. These great achievements could not be possible without progress in genetics and biology. Many markers are currently studied to screen earlier patients predisposed to develop preeclampsia during pregnancy or to predict premature rupture of membranes and labor, thus preventing wet lung syndrome. Progresses in neonatal resuscitation decreased perinatal morbidity and mortality. The detection of fetal DNA in the maternal blood with the hope of the complete genome fetal sequencing represents another breakthrough.in utero fetal surgery as a vesico-amniotic shunting (a twin–twin transfusion syndrome with Laser ablation of vessels), the treatment of fetal bladder obstructions, an aortic or pulmonary valvuloplasty   , an atriEpidural anesthesia, the new standardized criteria of fetal heart rate analysis, the advent of the STAN, the analysis of scalp pH contributed to the improvement of perinatal prognosis. Techniques of embolization contributed to a less aggressive, but effective, treatment of post-partum hemorrhage, thus saving lives and sparing uteri.With such a therapeutic arsenal, there should be no more fatality. However questions arise, among them “should we go back to a more physiological or natural practice of obstetrics?” or “why the rate of cesarean sections increased, and all this technology is available?” Even though we cannot compromise progresses, the question is to be debated.In both gynecology and obstetrics, optimal training of the nurses, midwifes, and residents is essential. In these fields, specific simulators are ideal tools to start, maintain, and develop skills. Finally, internet and other media are obvious tools to enhance knowledge for patients, doctors, midwives, and nurses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Human papillomaviruses (HPVs) have been proved as one of the etiological factors of oropharyngeal squamous cell carcinoma (OPSCC). Patients with tumors of viral etiology have a lower recurrence rate and better prognosis. OPSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. The aim of this study was to determine whether HPV infection and the immunological status influence patient prognosis individually or in concurrence. Material and Methods. Sixty patients with oral and oropharyngeal carcinomas were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA. Basic lymphocyte subpopulations were determined in the peripheral blood by means of flow cytometry. Results. Significantly better disease-specific survival (DSS) was observed in patients with HPV-positive tumors. Nodal status, tumor grade, recurrence, and CD8+/Tregs ratio were identified as factors influencing DSS. A higher level of Tregs and a lower ratio of CD8/Tregs influenced overall survival (OS) independently of HPV status and age. Patients with HPV-positive tumors and high levels of Tregs survived significantly better than patients from the other groups. Conclusion. Better survival is associated with HPV positivity and elevated Tregs levels. Our data suggest that HPV infection and Tregs do not influence patient prognosis in concurrence. Head and neck squamous cell carcinoma (HNSCC) arising in the oral cavity, oropharynx, hypopharynx, and larynx is the sixth leading cancer by incidence worldwide, with more than 550,000 cases annually , 2. The HPV infection is controlled by the host immune system and most of the infections are cleared within two years . HNSCC iNatural killer (NK) cells are part of the innate immune system and have been shown to participate mainly in the early control against virus infected cells and tumors. The activation of NK cells occurs via the integration of signaling molecules with activating or inhibitory receptors on the NK cell surface or can be mediated by proinflammatory cytokines, which are released in response to viral infection. A direct interaction between HPV infection and the innate immune system has been observed in patients with cervical cancer. The authors suggest that NK cells might be activated by HPV viral particles. It has been shown that NK cells are abundantly present in the tissue of preneoplastic cervical lesions but much less cells have been detected in cervical squamous cell carcinomas , 16. In Regulatory T cells (Tregs) are able to suppress antitumor immunity and in many patients with solid tumors; their increased levels in the tumor tissue as well as in the peripheral blood have been documented –24. SimiWe have previously shown that in HNSCC patients, HPV positivity is a strong prognostic factor for both better survival and less frequent recurrent disease , 36. AnoPatients with primary squamous cell cancer of the oral cavity or oropharynx  treated within a four-year interval at the Department of Otolaryngology and Head and Neck Surgery, 1st Medical Faculty Charles University and Motol University Hospital, Prague, who signed the informed consent form were enrolled into the study. The study received official institutional and ethical approval from the participating institutions. Data on demographics, risk factors for oral cavity and oropharyngeal cancer, and risks related to HPV exposure were collected by a questionnaire. The medical and pathology reports were completed for each patient. Altogether 60 patients were included in the study. From all patients, the tumor tissue and blood samples were obtained.All but three patients underwent surgical treatment and the tumor tissue was sent on dry ice to the Pathology Department. In the three patients who had not been treated surgically, tumor biopsies were performed. The pathologist obtained two side-by-side sections of the tumor from the primary site. One of the paired sections from each anatomical location was then labeled, snap frozen in liquid nitrogen, and stored for future analysis. The other paired section from each anatomical site was fixed in 10% neutral formalin and paraffin embedded. From each paraffin block, the first and last sections were histologically analyzed to confirm that the sections in between—assigned for the detection of viral nucleic acids and immune histochemical (IHC) analysis—contained at least 10% of tumor cells in the entire volume of the sample. Nucleic acids, both DNA and RNA, were extracted from the tumor tissue by means of the Ambion Recover All TM Total Nucleic Acid Isolation Kit for FFPE Tissues  as specified before . Care wa GAPDH internal control gene [All procedures have been described in detail previously , 36. HPVrol gene . As a corol gene .IHC examination was performed as specified before . BrieflyIn all studied patients, the following immunological parameters were determined: CD3+, CD4+, CD8+, Tregs, CD8+/Tregs ratio, CD4+/CD8+ ratio, CD4+ CD8+ sum, CD19, CD4+CD45RA+, and CD3−CD56+CD16+ cells. Samples of peripheral blood were analyzed by flow cytometry  after lysis of erythrocytes by FACS Lysing Solution  and staining with antibody-fluorochrome conjugates. We strictly adhered to the instructions in the manufacturer's protocol for the respective reagents. Antibodies anti-CD45 FITC/CD14 PE (to correctly set the lymphocyte gate), anti-CD3 FITC/CD19 PE, anti-CD3 FITC/CD16CD56 PE, anti-CD4 FITC/CD8 PE, anti-CD45RA FITC/anti-CD4 PE, and anti-CD3 FITC/CD4 PE/CD25 APC  were used. Ten thousand cells in the lymphocyte gate were acquired for analysis and the data were analyzed using the CellQuest software. Results are expressed as the percentages of the respective cell subpopulations of all lymphocytes and Tregs are expressed as the percentages of CD25+ cells of the CD3+CD4+ cells.9/L and ≤8 mg/L.The mean values of these parameters were compared between the groups of HPV-positive and HPV-negative HNSCC cases and between patients with good and bad prognosis (see below). Elevated Tregs levels were those with more than 10% of Tregs in the peripheral blood. To exclude the influence of acute inflammation on the immunological parameters, the standard “inflammatory parameters” were established. White blood count (WBC) and C-reactive protein (CRP) were detected using a routine laboratory procedure with the respective cut-off values of ≤10 t-test with 95% confidence intervals (CI) and two-tailed P values. All tests were two-sided and the significance level was α = 0.05.The mean levels of the immunological parameters as measured in the peripheral blood were compared between the groups of patients with HPV-positive and HPV-negative tumors and between the groups of patients with good and bad prognosis. Patients with good prognosis were those who did not have any recurrence and were alive at the end of the followup, while patients with bad prognosis were those who either relapsed or died before the end of the followup. The peripheral blood lymphocytes were treated as continuous variables in all analyses. Additionally, for the Tregs cells, two groups were distinguished: Tregs-high patients of patients with more than 10% of Tregs in the peripheral blood and Tregs-low patients with less than 10% of Tregs in the peripheral blood. The comparison was performed byP < 0.1). To compare the qualitative characteristics we used the Pearson χ2 test for 2 × 2 contingency tables and to compare the quantitative characteristics, we applied the Mann-Whitney test. The analyses were performed using the statistical program SPSS . The Kappa statistics was calculated to measure the agreement for simultaneous positivity for HPV DNA and expression of HPV E6 mRNA.Survival was measured in days from the date of diagnosis to the date of death or to the date the patient was last known to be alive. Patients who died of causes other than head and neck tumor were considered censored observations in the disease-specific survival (DSS) analyses. Time-to-event measures were analyzed by the Kaplan-Meier method, Log-rank test, and Cox multivariate regression. Ninety-five percent confidence intervals for odds ratios were based on the normal approximation. The variables considered in the Cox regression models were the presence of HPV, age, gender, smoking, alcohol consumption, tumor size, nodal status , tumor grade, tumor location, peripheral blood Tregs level, CD4+, CD8+, CD4+ CD8+ sum, CD4+/CD8+ ratio, CD8+/Tregs ratio, CD19, and CD3−56+16+ cells. A forward stepwise procedure was performed to find significant covariates (Ninety percent (54/60) of HNSCC cases were males and 10.0% (6/60) were females . The meaOut of 60 patients, 50.0% (30/60) had HPV DNA detected in the tumor tissue (data not shown). Only HR HPV types were found. The most frequently detected type in a single infection was HPV 16, found in 96.6% (29/30) of patients, followed by HPV 58 in 3.3% (1/30) of patients. Multiple infections were revealed in one patient (3.3%) who was positive for HPVs 16 and 18. HPV DNA was detected more often in oropharyngeal tumors than in tumors of the oral cavity (96.6% versus 3.3%). In the oropharyngeal location, tonsillar tumors were the most frequently positive, 68.9% (20/29). All but four HPV-DNA positive tumors expressed HPV 16 E6 mRNA and one sample was not available for RNA extraction. One of the samples negative for viral mRNA expression was positive for HPV type 58 and another one was copositive for HPVs 16 and 18. The correlation of the simultaneous positivity for HPV 16 DNA and expression of HPV 16 E6 mRNA was substantial (Kappa = 0.786). The samples positive for viral mRNA expression were considered HPV positive for future analyses.In 54.2% (32/59) of cases, the p16 oncoprotein was detected. Three samples positive for HPV 16 E6 mRNA were negative for p16 expression. One sample was not available for the analysis. The correlation of the expression of viral mRNA and p16 positivity was substantial (Kappa = 0.672) as well as the correlation of HPV DNA and p16 positivity (Kappa = 0.617). HPV DNA detection in this study had substantially higher sensitivity (93.0% versus 75.0%) and lower specificity (86.0% versus 88.0%) in comparison to the detection of p16 when samples positive for viral mRNA were considered positive (data not shown).P = 0.005) than patients with HPV-negative tumors (mean = 6.4%). No difference in other immunological parameters studied was detected between the two groups of patients. Then, to evaluate the prognostic value of immunological markers, we compared the prevalence of lymphocyte populations at enrollment between patients with good and bad prognosis . No statistically significant differences were observed (P = 0.052)  were compared in the peripheral blood of patients with HPV-positive and HPV-negative tumors. The analysis of these immunological parameters showed that patients with HPV-positive tumors had significantly higher levels of CD3−CD56+CD16+ NK cells  . No patiP = 0.373 and P = 1.000).Elevated Tregs levels were found in 61.6% (37/60) of the study patients. The mean rate of Tregs was 15.3% (data not shown). Higher numbers of Tregs were found more often in patients with tumors of the oral cavity  than in those with oropharyngeal tumors . Among patients with the oropharyngeal tumors, elevated Tregs were most frequently associated with tonsillar tumors . Elevated Tregs levels were more often detected in patients with well-differentiated tumors  and higher tumor stages  but the differences were not statistically significant (P = 0.018) (data not shown). Patients with elevated Tregs levels (P = 0.039) had significantly better overall survival (OS). Furthermore, the survival of patients was assessed depending on HPV16 E6 presence and Tregs level was assessed. Four groups of patients were compared: HPV+/Tregs high, HPV−/Tregs low, HPV−/Tregs high, and HPV+/Tregs low (P = 0.087). Better OS was also detected in patients with smaller tumors (P = 0.019) and in nonsmokers (P = 0.023) (data not shown).The mean follow-up period was 4.3 years. At the end of the followup, 33 patients were free of the disease. Of 27 patients who died, 15 died of primary tumor and seven died of unrelated diseases and for five patients, data were not available for analyses. Significantly better DSS was observed in patients with HPV16 E6-positive tumors in comparison to HPV-negative tumors, using the Kaplan Meier analysis and Log Rank test (regs low . In concP = 0.909) (data not shown).Higher counts of Tregs did not affect the incidence of recurrence (P = 0.929), tumor size (P = 0.780), tumor grade (P = 0.116), and nodal status (P = 0.253), and occurrence of recurrence (P = 0.251) was found (data not shown). In the multivariate Cox regression analysis (P = 0.001), lower tumor grade (adjusted P = 0.046), absence of lymph nodes metastases (adjusted P = 0.041), and lower CD8+/Tregs ratio (adjusted P = 0.012), with an advantage at the borderline of significance in patients with smaller tumors (T1 + T2) (adjusted P = 0.058). Improved overall survival was found in patients with smaller tumors (T1 + T2) (adjusted P = 0.003), absence of lymph node metastases (adjusted P = 0.041), no recurrences (adjusted P = 0.0001), higher Tregs counts (adjusted P = 0.035), and lower CD8+/Tregs ratio (adjusted P = 0.011).No difference was found between the levels of Tregs and HPV status  as a specific marker for human Tregs . The colhigh Tregs and tumor infiltrating CD4+ CD25high Tregs in HNSCC patients. In contrast, Wamsom et al. [Lau et al.  and Greem et al.  who compHuman papillomaviruses are now, together with smoking and alcohol consumption, an established risk factor for head and neck cancer . HPVs arWe observed increased mean levels of NK cells and B cell lymphocytes in patients with HPV-positive tumors; however, only the former difference was marginally significant. No differences in the levels of immunological parameters in the peripheral blood were detected when comparing the patients without recurrence and/or those who died were compared. We have previously reported decreased numbers of NK cells in HNSCC patients in comparison to healthy controls . In the While it has been shown that for the initial steps of HNSCC tumor development one of the known etiological factors, smoking, alcohol, and HR HPV infection, is necessary, the growth and tumor progression is associated with the local failure of the host immune system. NK cells are part of the innate immune defense against pathogens and cancer . RecentlRenoux et al.  have repOur results are consistent with the findings of Schaefer et al. , StraussOnly a limited number of studies have analyzed the relationship between HPV-associated HNSCC, the immune profile, and prognosis of these patients. Wamsom et al.  have detBetter survival of HNSCC patients is associated with HPV etiology of the tumor as well as with the elevated levels of regulatory T cells and lower CD8+/Tregs ratio. These virological and immunological parameters do not influence patient prognosis in concurrence. The combination of HPV positivity and increased levels of Tregs may have a prognostic value in patients with head and neck carcinoma.


WIG-1 (ZMAT3) is located in chromosomal region 3q26, that is frequently amplified in human tumors, including cervical cancer. We have examined the status of WIG-1 and the encoded Wig-1 protein in cervical carcinoma cell lines and tumor tissue samples. Our analysis of eight cervical cancer lines  by spectral karyotype, comparative genomic hybridization and Southern blotting revealed WIG-1 is not the primary target for chromosome 3 gains. However, WIG-1/Wig-1 were readily expressed and WIG-1 mRNA expression was higher in the two HPV-negative cervical cell lines  than in HPV-positive lines. We then assessed Wig-1 expression by immunohistochemistry in 38 cervical tumor samples. We found higher nuclear Wig-1 expression levels in HPV-negative compared to HPV positive cases (p = 0.002) and in adenocarcinomas as compared to squamous cell lesions (p<0.0001). Cases with moderate nuclear Wig-1 staining and positive cytoplasmic Wig-1 staining showed longer survival than patients with strong nuclear and negative cytoplasmic staining (p = 0.042). Nuclear Wig-1 expression levels were positively associated with age at diagnosis (p = 0.023) and histologic grade (p = 0.034). These results are consistent with a growth-promoting and/or anti-cell death function of nuclear Wig-1 and suggest that Wig-1 expression can serve as a prognostic marker in cervical carcinoma.The p53 target gene  Cervical cancer ranks the third among the most common cancers in women worldwide and is the most common type of cancer in Eastern Africa, South-Central Asia and Melanesia HR-HPV encodes two early proteins, E6 and E7, that target two major cellular tumor suppressor pathways. E6 targets the p53 tumor suppressor for degradation, resulting in loss of p53-dependent apoptosis and/or senescence in situ to invasive cancer PIK3CA) at 3q26.32 TERC) at 3q26.2 RBP1-RBP2) at 3q23, the TP63 gene at 3q28 SOX2) at 3q26.33 Genomic alterations such as gene amplifications are important features of cervical carcinogenesis WIG-1  maps to 3q26.32 WIG-1 is a bona fide p53 target gene WIG-1 is highly conserved from amoeba to human TP53 mRNA which is then stabilized. Thus, Wig-1 forms a positive feedback loop with p53 that enhances p53 expression MYCN oncogene mRNA CDKN1A mRNA (encoding p21) through recruitment of the RISC complex WIG-1 is amplified and/or over-expressed in human papillary thyroid carcinoma (www.ebi.ac.uk/gxa/), lung squamous cell carcinoma www.cbioportal.org), suggesting an oncogenic function.Human WIG-1 gene in cervical carcinoma cell lines and Wig-1 expression in both cervical carcinoma cell lines and tumor samples. We investigated the expression of Wig-1 in relation to different clinical parameters such as: histological tumor type, grade and stage, HPV status, age at diagnosis, and survival. Our results indicate that high nuclear Wig-1 expression is a marker for poor prognosis in cervical carcinoma.In this study we have examined the 2.The 8 human cervical cancer cell lines Ca Ski, SiHa, C-4I, C-33A, ME-180, HT-3, MS751 and SW756 were purchased from ATCC . The SaoWIG-1 locus relative to a control probe from 3p23 was determined by Southern blot analysis using standard methods. The methods used are described in detail in the Supplementary material.Cell lines were HPV typed using a broad spectrum PCR assay WIG-1 cDNA and a 500 bp PCR fragment of beta-actin labeled with the Megaprime DNA labeling System . Hybridization was performed in ULTRA hybridization buffer  and the signals were detected and analyzed in a FLA-3000 phosphorimager . Normal human fibroblasts were used as control with 18S RNA as a reference for the analysis.For Northern blot analyses cells were harvested by trypsination and total RNA was isolated using Trizol reagent  according to manufacturer's protocol. RNA was separated in 1% agarose/formamide gels and blotted to Zeta-Probe GT Genomic Tested Blotting membranes . Probes used for hybridization were the coding part of WIG-1 expression was also analysed by real-time PCR using HPRT1 as reference gene and SYBR Green based LightCycler PCR as further described in the Supplementary material.Cells were harvested by trypsination and lysed in a buffer containing 100 mM Tris pH 8.0, 150 mM NaCl, and 1% NP-40 and 1% Protease Inhibitor Cocktail . The protein lysates were separated in 10% SDS-PAGE gels and blotted at 20 V for 30 min to Hybond ECL-membranes  using a transblot SD semi dry transfer cell . Primary antibodies used a human Wig-1 polyclonal antibody raised against a GST-Wig-1 fusion protein 1∶2000,  and β-acin situ (CIS n = 4), and 25 cases had adenocarcinoma (ADCA). The 25 ADCAs were all without any squamous cell component. Furthermore, the HPV-negative tumors were histopathologically re-evaluated which revealed typical morphology in agreement with the initial diagnosis. Ethical approval was obtained from the Regional Ethical Review Board in Stockholm. Written informed consent was given by participants for their clinical records to be used in this study and patient records/information was anonymized and de-identified prior to analysis. All patients were retrospectively followed-up from the time of diagnosis until January 2012. Clinical information was obtained for each case concerning age at diagnosis, tumor classification, histology grade, stage and outcome after follow-up  or cancer ollow-up . Serial Total protein was extracted from a cervical carcinoma sample by homogenization with polytron system PT1200CL  and purified with AllPrep RNA/Protein Kit . Protein concentrations were determined using Bradford  and 30 to 50 µg were loaded on 10% NuPAGE Novex Bis-Tris Gels  in loading buffer and reducing agent (Invitrogen) and run in Mops buffer (Invitrogen). Proteins were blotted to nitrocellulose membranes using the iBlot Dry Blotting System (Invitrogen) and incubated with a rabbit polyclonal Wig-1 antibody  followed by anti-β-actin antibody . Primary antibodies were visualized using HPR-conjugated secondary antibodies  and signals were detected as above.For the purpose of immunohistochemistry the antibody was tested in a dilution serie from 1∶200 to 1∶1,600 which revealed that 1∶400 was the optimal dilution to assess Wig-1 expression. Furthermore different batches of the Wig-1 antibody from Gentex gave similar staining patterns in the cervical carcinoma samples.2O2) in distilled water for 30 minutes. Non-specific staining was blocked by incubating the sections with 4% normal goat serum  in Tris buffer (TBS) for 30 minutes at room temperature. Sections were incubated with Wig-1 antibody  at 4°C overnight. On the following day the sections were washed in TBS and first incubated with goat anti-rabbit IgG  and subsequently with ABComplex/HRP  in TBS with Triton (2∶2∶100) for 30 minutes at room temperature to visualize reaction products. Diaminobenzidine was used as chromogen. After counterstaining with hematoxylin, the slides were dehydrated and mounted with a xylene-soluble medium.Immunohistochemistry was performed on all 38 cervical carcinoma samples. Sections were deparaffinized, rehydrated and microwaved in 10 mM citrate buffer (pH 6.0) for 20 minutes. Endogenous peroxidase activity was inhibited using 0.5% hydrogen peroxide   . This shWIG-1 is not a primary target for frequent gains. This is well illustrated by the fact that WIG-1 is located telomeric of the commonly amplified 3q23–24 interval in Me180 and SiHa cells, and centromeric of the amplified 3q27-ter interval in ME-180 cells. Similarly, these 3q amplifications detected by CGH were not accompanied by WIG-1 locus amplifications as determined by Southern blotting.Taken together, our analyses showed that 3q amplifications occur in cervical cancer cells, but WIG-1 mRNA and Wig-1 protein expression in all cell lines  were identified in all cell lines, with predominance of the 10 kb mRNA  than in the HPV-positive lines . We also investigated whether the expression levels of Wig-1 differed between HPV-positive and HPV-negative adenocarcinomas (ADCA) and found a statistically significant difference in nuclear Wig-1 staining intensity between HPV-positive and HPV-negative ADCA samples . In general, HPV-negative cervical tumors showed higher nuclear Wig-1 expression levels than HPV-positive tumors.In this panel of cervical carcinomas we observed a possible correlation between Wig-1 expression levels and HPV status. HPV DNA is known to almost invariably be present in SCC p = 0.023) and histologic grade (p = 0.034) (p<0.0001).Univariate analysis showed significant positive associations between nuclear Wig-1 staining intensity and clinical parameters, including age at diagnosis (= 0.034) . HoweverInterestingly, we found that moderate nuclear Wig-1 expression was associated with better overall survival . Positivp = 0.042) . We found that this subgroup of patients had a significantly better prognosis than patients whose tumors showed strong nuclear and negative cytoplasmic Wig-1 staining  .WIG-1/Wig-1 is amplified and/or overexpressed in many human tumors, including lung squamous cell carcinoma www.cbioportal.org), which is consistent with an oncogenic function. WIG-1 has been mapped to chromosome 3q26.32, a region that shows frequent copy number gain in various human tumors WIG-1 drives 3q gain in cervical cancer. We therefore examined WIG-1 and its expression in cervical carcinoma cell lines, and assessed Wig-1 expression patient tumor biopsies.WIG-1 is not a main target for the frequent gains. WIG-1 is located telomeric of the commonly amplified 3q23–24 region in Me180 and SiHa cells, and centromeric of the amplified 3q27-ter interval in ME-180 cells. Importantly, the 3q amplifications were not accompanied by WIG-1 gene amplification as determined by Southern blotting or FISH. Thus, we conclude that WIG-1 is not the critical gene that drives 3q gain in cervical cancer. Moreover, our analysis of WIG-1 mRNA and protein levels in the cervical carcinoma cell lines did not reveal any significant correlation between WIG-1 expression and copy number alterations at the DNA level. We noted, however, that WIG-1 mRNA expression was higher in the two HPV-negative cervical carcinoma lines  than in the HPV-positive lines that only showed low or moderate WIG-1 mRNA levels  and adenocarcinomas (ADCA). The prevalence of HPV infection in SCCs is almost 100% MYCN, c-FOS, c-JUN and CYCLIN D1, and downregulation of the pro-apoptotic FASInterestingly, while all cervical tumors showed positive Wig-1 staining we observed several distinct Wig-1 immunostaining patterns, including moderate nuclear and positive cytoplasmic staining (pattern A) and strong nuclear and negative cytoplasmic staining (pattern B) . We founWIG-1 is a p53 target gene, the question arises as to whether Wig-1 expression levels correlate with p53 status of the tumors and/or presence of HPV, which encodes the E6 protein that targets p53 for degradation. The TP53 gene is more frequently mutated in HPV-negative than in HPV-positive cervical cancer cell lines and tumors TP53 mutation , several other potential transcription factors, for instance SOX2, can potentially induce Wig-1 expression Since mutation . The TP5From the clinical standpoint it is very important to identify women at risk of developing cervical cancer by finding early markers that permit rapid and focused preventive action, as well as to elucidate the factors that influence development of cancer in HPV-infected women and HPV-negative women.It is also important to increase the biological understanding of the factors that influence the development of cervical cancer in women with and without HPV infection, and to understand the molecular mechanisms of cervical carcinogenesis, in order to improve early diagnosis, differential diagnosis, and evaluation of tumor aggressiveness. Molecular markers will help to differentiate between benign disease and malignancy, and thereby prove useful in clinical practice. Our demonstration that Wig-1 expression levels are higher in HPV-negative cervical carcinoma suggests a possible role of Wig-1 in HPV-negative cervical carcinogenesis. Moreover, the finding that moderate nuclear Wig-1 expression levels and positive cytoplasmic Wig-1 staining are associated with better survival indicates that Wig-1 expression levels as assessed by immunohistochemistry could represent a novel prognostic maker for cervical cancer. Our results may contribute to further optimization of current tumor diagnostics and pave the way for the development of new treatment of cervical cancer in the future.In our study, HPV infection was not detected in all cases, which could either reflect true HPV negativity or represent ‘false negatives’ owing to technical difficulties in HPV genome detection. However, in the current literature, HPV infection has not been reported in the same proportion in adenocarcinomas as in squamous cervical carcinomas HPV transmission is increasingly common in young women worldwide and several biological factors may help us to develop more effective preventive screening strategies for detection and treatment of women with cervical carcinomas. Should we be able to identify women at risk of developing such cancers based on a single marker, or a combination of different markers, such analyses may become a substitute for, or complement to, cytological screening. Studies indicate that first-generation HPV vaccines might prevent at least two-thirds of cervical cancers. By combining improved detection of precursor lesions of cervical cancer using molecular markers in screening programs with vaccination against HPV, cervical cancer may become the most preventable cancer globally.Table S1Clnical and Wig-1 immunohistochemistry data for cervical carcinoma cases in the study.(XLS)Click here for additional data file.File S1Figure S1, Western blot analysis of Wig-1 protein expression. Western lot analysis showing Wig-1 expression levels in eight cervical carcinoma cell lines and one osteosarcoma cell line (Saos2). Beta-actin was used as loading control. * Saos2 was used as a control cell line. Figure S2, WIG-1 mRNA expression in HPV-positive and HPV-negative cervical carcinoma cell lines. The scatter plot shows relative WIG-1 mRNA expression levelsas assessed by qRT-PCR. HPV-positive lines: Ca Ski, ME-180, MS751, SiHa, SW756 and C-41; HPV-negative lines: C-33A and HT-3. Figure S3, Specificity of the Wig-1 antibody . Western blot analysis showing a single band of expected size at 34 kDa for endogenous Wig-1 in cervical carcinoma tissue. Molecular size markers are shown to the right in kDa. Beta-actin served as a loading control.(DOC)Click here for additional data file.


To determine the distribution of cervical high-risk human papillomavirus genotypes and their association to cellular abnormalities in women from Fez and its neighborhood.Women attending the Hassan II University Hospital for cervical pap smears were recruited after an informed consent. Interviews and two cervical samples were performed for each woman. Cervical samples were used for cytological analysis and HPV DNA detection. HPV was typed using a method based on multiplex PCR with fluorescently labeled specific primers followed by capillary electrophoresis. The study was approved by the ethics committee of the Faculty of Medicine and Pharmacy of Fez.The HPV prevalence in the studied population was 43.1% and the most prevalent types were HPV 53 (23 cases); HPV 16 (20 cases); HPV 35 (18 cases); HPV 51 (10 cases) and HPV 56 (7 cases). From the 619 confirmed pap smears, 20% were abnormal. The cytological abnormalities were significantly associated to HPV infection, women age, number of pregnancies and parity (p < 0.05).More attention should be given to HPV in Morocco because it represents an important public health concern. The distribution of carcinogenic HPV types in the studied population is different from the data in other regions but epidemiological studies in other Moroccan regions are required. Human papillomavirus (HPV) is a group of double-stranded circular DNA virus that affects human epithelia including ano-genital mucosa. Most genital HPV infections remain asymptomatic and undergo spontaneous clearance without leaving any lesion. However, in some cases, persistent infection by at least one high-risk HPV genotype is responsible for precancerous lesions that can evolve to invasive cervical cancer , 2.Cervical cancer prevention programs in western countries allowed a decrease in the incidence of cervical cancer cases and cervical cancer related deaths –7. In coIn the particular context of Morocco, little is known about the burden of HPV and the distribution of high-risk HPV types. However, some studies have been made and the prevalence of HPV infections in Moroccan women has been determined in some regions –13. Two The aims of this study were i) to evaluate the prevalence of HPV infection in a population of women who consulted the University Hospital of Fez; ii) to determine the distribution of HPV genotypes using a newly developed HPV genotyping method that targets a total of sixteen HPV types including all the confirmed high-risk types; iii) to determine the risk factors associated to HPV infections or cellular abnormalities.All the recruited women were informed about the study objectives, methods and outcomes. Interviews and tests were performed only for consenting women. Due to the high rate of illiteracy, verbal consents were accepted and mentioned on the interview forms. All the participating women were assigned a code which is used to identify samples without individual information. All the procedures in this study were approved by the IRB of the Hassan II University Hospital, Fez.Women were recruited from November 2013 through December 2014 in the Laboratory of Anatomopathology of Hassan II University Hospital in Fez, Morocco. These women were referred by gynecologists  for cervical smears. A face-to-face interview was performed to collect socio-demographic and sexual behavior data from consenting women. The questions included age, educational level, profession, marital status, area of residence, number of sex partners, use of contraception, history of gynecological diseases, menopause, number of pregnancy and parity.Two different cervical samples were performed successively. The first was performed with Cytoprep Brush and the cells were stored in CYTOfast preservative solution for liquid-based cytological smears. The smears were observed by three different pathologists and cellular lesions were classified according to the Bethesda 2001 system . The secHPV DNA was detected using the broad spectrum primer set GP5+/GP6+ as described previously . PCR proDescriptive analyses were performed for all variables. Prevalence values of HPV infections and cytological abnormalities were calculated. The relationship between different variables and HPV infection or cellular abnormality was checked using Fisher exact test or Khi square test when applicable. After the univariate analysis, variables with p ≤ 0.20 were used in a stepwise selection procedure to determine the factors associated to HPV infection or cellular abnormalities in a logistic regression model. In all tests, the limit of significance was set to a P value of 0.05. All the statistical analyses were performed using SPSS statistics version 20.A total of 633 participants coming from Fez and its neighborhood were included in this study. All the women were Moroccans, Muslims and aged 20 to 85 years old with a mean age of 46.55 (± 10.88) years. The women were referred from peripheral health care services where they first consulted for gynecological check-up (55.3%), control after the diagnosis of breast or ovary neoplasic lesions (17.2%), leucorrhoea (8.8%), metrorrhagia (7%), dysmenorrheal (4.1%), skin lesions (2.7%), sterility (2.4%), menstrual disorders , prolapse  and ectopic pregnancy . All the women had never been tested for HPV infection and never received HPV vaccine. None of the women was an active smoker but 30.3% were passive smokers. Most of them (68%) were illiterate and 26.1% had already used oral contraception. Most of the women (93.3%) have only one lifetime sexual partner who is their husband and 70.9% had their first sexual intercourse under the age of 20 years old. Among these women, 49.5% bore more than three pregnancies and 25.7% experienced at least one miscarriage .Among 633 samples, 14 did not contain enough cells for cytological interpretation. Cytological examination results were confirmed for 619 samples from which 495 (80%) showed a normal cytology and 122 abnormalities. The abnormal Pap smears include 30 (4.8%) atypical squamous cells of undetermined significance (ASCUS), 81 (13.1%) low-grade squamous intraepithelial lesions (LSIL) and 13 (2.1%) high-grade squamous intraepithelial lesions (HSIL). The prevalence of cellular abnormalities increases with women age: from 5.3% in women under 30 years old, it reaches a value of 31.3% in women above 59 years old . The higThe quality of DNA extracted from all the 633 samples was good enough for HPV DNA detection and typing. The overall HPV prevalence in the studied population was 43.1%. HPV prevalence decreased from 35.9% in women under 30 years old to 33.6% in women between 40 and 49 years old and then increased progressively to reach the highest value (58.8%) in women of 60 years old and above . AccordiStatistical analysis was made to determine factors associated with HPV infection. In the univariate analysis, HPV infection was found to be significantly associated with age, lifetime number of sexual partners and menopausal status (p < 0.05). The educational level, the age at first sexual intercourse, the use of oral contraceptives, passive smoking, history of miscarriage and the use of public bathrooms seemed not determinant in HPV acquisition . The logHPV positive samples underwent HPV typing using the method described above. Among HPV positive samples, 187 (68.5%) harbored low risk types and 86 (31.5%) harbored at least one carcinogenic HPV type. The five most frequent types are HPV 53, 16, 35, 51 and 56 which are identified respectively in 3.6%; 3.2%; 2.8%; 1.6% and 1.1% of cervical samples. Double infections were identified in 5 cases (0.8%). The distribution of HPV genotypes according to cytological results is presented in The relationship between cellular abnormality and other variables was also tested. In the univariate analysis, it was found that cytological abnormalities are significantly related to HPV positivity, women age, menopause, number of pregnancies, high parity and illiteracy (p < 0.05). The use of oral contraceptives, the age at first sexual intercourse, the lifetime number of sexual partners, the use of public bathrooms, passive smoking and history of miscarriage were not statistically related to cytological abnormalities . After lType-specific prevalence in abnormal smears were compared to those in women with normal cytology. The prevalence of HPV 16, 35, 45 and 56 were significantly higher in women with abnormalities . When thThe studied population is not representative of the whole female population in Morocco. However, the results provide a first insight on the distribution of mucosal high-risk HPV types in women from Fez. A relatively high HPV prevalence (43.1%) was recorded confirming the value of 42.5% obtained in the previous study conducted on women with normal cytology in the period between February 2007 and December 2008 in the same region . Most HPHPV prevalence and genotype distribution in the present study are different from the results obtained in women attending the department of children and mothers’ pathology of Ibn Sina hospital in Rabat where HPV prevalence was 15.7% with HPV 16 and 18 as the most prevalent types . It is iThe HPV infection profile in the studied population shows some similarities with HPV epidemiology in many other regions. Among these similarities are the high HPV prevalence and the bimodal shape of the HPV age-specific prevalence. In effect, high HPV prevalence rates were recorded in countries such as Australia (38.7); Cameroon (38.5%); Gabon (46%); Mexico (67.1%) and Saudi Arabia (31.6%) –24. MoreThe most prevalent genotype in this study is HPV 53. This genotype was also reported as the most prevalent type in Gabon . The relThe comparison between normal and abnormal pap smears regarding the distribution of HPV types shows clearly the difference between HPV 53 and the group 1 carcinogenic types. In effect, despite the high prevalence of HPV 53, it was not associated with cellular abnormalities whereas HPV 16, 35, 45 and 56 showed significant relationship with abnormalities. However, HPV 53 should be considered in future cervical cancer prevention programs in Morocco because molecular evidence of HPV 53 carcinogenicity has been provided . FurtherThe association of HPV infections with cytological abnormalities is highly significant. This shows that HPV infections in the studied region constitute a real public health concern. In effect, even if invasive cervical cancer cases are not recorded in this study, intra epithelial lesions of high significance were recorded. Among women with HSIL, the worst abnormality recorded in this study, HPV DNA was detected in 76.9% cases. This value is lower than HPV prevalence in HSIL cases in Africa (85%), Asia (78%), Europe (88%), Latin America (85%) and North America (86%) . HoweverThe logistical regression models show clearly that HPV infections are responsible for cervical abnormalities. Increased number of pregnancies and menopause are both significantly related to HPV infections and cervical abnormalities. Most of women (93.3%) in the present study have only one lifetime sexual partner but women who had two or more lifetime sexual partners have an increased risk of HPV infection. However, the lifetime number of sexual partners was not associated to cellular abnormalities. This result shows that having two or more sexual partners increases the risk of HPV infection but it is not responsible for HPV persistence and cellular transformation. Cellular lesions result from HPV persistence after escaping from the immune system which is related to the hormonal balance. Pregnancy and menopause are responsible for many psychological, physiological and hormonal changes that can weaken the immune system . The redBased on the results of this study, it is clear that HPV is a real public health concern in Morocco. A high rate of women harbor HPV infections and some of them even present precancerous lesions. The distribution of carcinogenic HPV genotypes in the studied region is very specific and shows that more epidemiological investigations are required. The next step could be the evaluation of HPV genotypes distribution in the general population of women from all the regions in Morocco and the distribution of HPV genotypes in cervical cancer cases collected from all the oncologic centers in Morocco.Even if the studied population does not represent the whole Moroccan population, the results show that the future HPV prevention programs should be based on the epidemiological profile of HPV infections in the whole country. Nonetheless, women education, HPV screening in old women and appropriate monitoring of pregnant women could be helpful in preventing cellular lesions development. Finally, women in menopause should be given a particular attention.