Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels.n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets , the expression of immune checkpoints , the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed.In this multi-institutional study, we first examined the histological features displayed by each collected tumour (KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa.Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits. With the exception of rare HPV-negative anal SCC (~5%) that are unresponsive to these standard treatments,6 a complete remission is achieved in the majority (~75%) of patients.7 Beside squamous neoplasms, a few thousands of primary anal canal adenocarcinoma are also diagnosed each year worldwide. More aggressive than SCC and most frequently detected in older patients (sixth decade of life),11 these rare tumours still represent a diagnostic and therapeutic challenge due to the lack of in-depth characterisation. According to a survey conducted by Abel et al.,12 most clinicians only occasionally encounter patients with anal adenocarcinoma. Therefore, the majority of recent descriptive studies are clinical case reports,15 and there is a lack of sufficient information to generate guidelines for uniform treatment recommendations.17 Moreover, molecular perturbations that might serve as targets for therapy are completely unknown.Extending from the anal margin  to the anorectal ring/flexure, the anal canal is the terminal part of the gastrointestinal tract. Despite its small size (~4 cm in length), several types of neoplams may be observed within this anatomical structure, which reflects its embryologic/histologic complexity.n = 74) in order to improve the current controversial diagnosis/management of patients. The histologic, immunologic and mutational profiles of these rare malignant glandular lesions were, for the first time, determined and collected results point to new opportunities for personalised therapeutic intervention.In the present study, we extensively examined a multicentre cohort of anal canal adenocarcinoma , Citadelle Regional Hospital , Jules Bordet Institute , University Hospital Center of Besançon (France), University Hospital of Lausanne (Switzerland) and University of Arkansas for Medical Sciences ) between January 1999 and October 2016 were selected. Tissue specimens (biopsies or tumour resections) from each patient were retrieved from pathology archives with the approval of the ethics committees of the respective institutions. Forty normal tissue samples (haemorrhoidectomies) displaying the three different portions of the anal canal  were also obtained. All cases (haematoxylin and eosin (H&E) staining) were re-examined by senior histopathologists to confirm the diagnosis. Clinicopathological data were collected for all selected patients diagnosed with invasive adenocarcinoma of the anal canal. Patient gender, age at diagnosis, disease stage , HIV status, inflammatory bowel disease, treatment details  and follow-up data were obtained from patient’s medical records. Tumour differentiation was determined using established criteria  7 , anti-Krt16 , anti-Krt20 , anti-CDX2 , anti-p16ink4 , anti-Ki67 , anti-CD4 , anti-CD8 , anti-PD-1 , anti-PD-L1 , anti-Foxp3  and anti-EGFR . The mouse/rabbit Envision (Dako) or Novolink polymer  detection systems were used for the secondary reaction. Mouse and rabbit control IgGs (Santa Cruz Biotechnology) were utilised as negative controls.Immunohistochemical analyses were performed using a standard protocol extensively described previously. > 75%. A similar scoring stratification was used to assess nuclear/cytoplasmic p16ink4 immunoreactivity. T cell subsets  infiltrating the epithelial component of the tumour or the stroma surrounding cancer cells were quantified by computerised counts  and verified by manual counting. The number of positive cells was reported to tumour area yielding a count expressed as number of cells/mm2. As previously described,21 both PD-L1 and EGFR immunolabelled tissues were evaluated by using a semi-quantitative score of the intensity  and extent  of the staining, according to an arbitrary scale. The results obtained with these two scales were multiplied in order to obtain a global score ranged between 0 and 9 for each specimen.All immunolabelled tissues were evaluated independently by experienced histopathologists. Krt7, Krt16, Krt20 and CDX2 immunostainings were scored as positive when a strong/uniform immunoreactivity was detected in more than 90% of epithelial cells. The growth fraction (proliferative index) of a given tumour was determined with the percentage of Ki67-positive cells. Collected results were stratified as follows: 0–5%, 6–25%, 26–50%, 51–75%, and22 this test is highly sensitive for detecting HPV infection in paraffin-embedded tissues.The simultaneous detection of 14 carcinogenic (high-risk) HPV genotypes  was performed using the Abbott RealTime High-Risk HPV assay . As previously demonstrated,23The HPV16 and 18 physical status  was determined by analysing the disruption of the HPV E2 gene expression by quantitative real-time PCR . Both thermal cycling conditions and primer sequences are available in DNA in situ hybridisation was performed according to the manufacturer’s instructions. The most common carcinogenic HPV genotypes  were detected using a probe cocktail . Red Counterstain II  was used in order to facilitate microscopic observation.25 Each collected tissue sample was carefully reviewed by a histopathologist in order to ascertain that the percentage of neoplastic cell was higher than 40%. When cancer cell enrichment was necessary, tumour areas were macrodissected with a scalpel before tissue digestion. Genomic DNA extraction, PCR conditions and primer sequences are detailed in 26 tumours displaying differences in the length of two or more microsatellite sequences (markers) were interpreted as being MSI-High. Lesions showing no instability or one instable repeat were defined as microsatellite stable (MSS) and MSI-Low, respectively.The MSI status of tumour specimens was determined using the pentaplex PCR assay described by Buhard et al. and comprising 5 quasimonomorphic mononucleotide repeats .KRAS , NRAS , BRAF [exons 11 and 15 (full)], PIK3CA [hotspots in exon 9 and 20 ], EGFR , HER2 [exon 22 (full)], KIT , PTEN [exons 5 and 7 (full)], PDGFRA , DDR2 [exon 18 (full)]] were amplified and sequenced using NGS technology. The detailed protocol is available in As described above, all analysed samples contained at least 40% tumour cells. Both tissue lysis and DNA extraction were performed using QIAamp DNA FFPE Tissue Kit  according to supplier’s recommendations. Frequently mutated regions of 10 clinically relevant genes in the gastrointestinal cancer setting   anal squamous (pre)neoplastic lesions. To examine the possible etiological link between HPV infection and glandular carcinoma development within the anal canal, we first analysed the expression of p16ne] Fig. . The prene] Fig.  and PCR ne] Fig.  in 11 , gender (p = 1), tumour differentiation (p = 0.313), TNM classification , tumour stage (p = 0.847) and treatment (p = 0.062). No patient was HIV positive. As mentioned earlier, high-risk HPV infection (HPV16 or HPV18) was only detected in neoplastic lesions arising from the anal glands/transitional zone (p < 0.001). Significantly, these latter tumours displayed a lower proliferative index compared to their counterparts originating from the colorectal mucosa (p = 0.003). Crohn’s disease and ulcerative colitis were also statistically correlated with tumour origin (p = 0.049). With the exception of one case , all these inflammatory conditions were observed in patients with anal gland/transitional zone-type tumours . Three out of 4 (75%) were associated with chronic perianal fistulas. Finally, patients’ characteristics for Krt7-positive anal adenocarcinoma according to HPV status are shown in Supplementary Table Clinicopathological data from 74 patients with histologically confirmed anal adenocarcinoma and treated between January 1999 and October 2016 were collected from 6 University Hospital Centers. As mentioned above, based on medical imaging results, all these glandular tumours were strictly located within the anal canal. Overall, patients were relatively aged  and males slightly outnumbered females . Approximately half  of patients had a lymphatic node involvement and 9 (12.2%) patients had distant metastasis ) at presentation. Most patients  underwent surgery  with neoadjuvant or postoperative chemoradiotherapy. The prescribed radiotherapy dose was usually 45 Gy (in 25 fractions) and 5-Fluorouracil was the most commonly used chemotherapeutic drug. A few patients also received FOLFOX   (mainly as adjuvant treatment) or Capecitabine (Xeloda) . As listed in Table Out of 74 patients diagnosed with adenocarcinoma strictly located in the anal canal, 65 (87.8%) were incorporated in both OS and DFS analysis. Nine (12.2%) patients were excluded because of distant metastasis at the time of diagnosis (UICC stage IV). Furthermore, 5 (55.6%) of these latter metastatic patients did not receive any treatment. Median follow-up was 33.1 months (range: 2–141 months). After treatment completion, local or distant recurrence occurred in 10  patients with invasive neoplasms originating from the anal glands/transitional zone and in 13  patients with colorectal-type anal adenocarcinoma. The two tumour subtypes displayed similar patterns of local/metastatic recurrences and, whatever the tumour origin, all distant recurrences were found either in the liver or the lung. Among the patients who died during the follow-up period, 11  and 13  exhibited a neoplastic lesion who developed from the anal glands/transitional zone or from the colorectal mucosa anatomically located in the anal canal, respectively. Cancer was the cause of the death in three quarter of cases . As illustrated by the Kaplan-Meier survival curves Fig. , patientp = 0.017) ) whereas tumour size (p = 0.031) and age at diagnosis (p = 0.021) were independently associated with reduced DFS and OS, respectively. The prognostic value of patient age (p = 0.017) and N-stage (p = 0.044) for OS was further confirmed by multivariate analysis  associated with DFS and OS were then determined. Forest plots for both univariate and multivariate analysis are shown in Fig. sis Fig. . When al29 Therefore, the prevalence rates of KRAS, NRAS, BRAF, PIK3CA, EGFR, HER2 and PTEN mutations were determined in all collected anal adenocarcinoma specimens. The mutational status of three other clinically relevant genes in the gastrointestinal cancer setting  was also assessed. Because of poor DNA quality (high degradation), 6 samples out of 74 (8.1%) were excluded. As revealed by NGS analysis , BRAF (1.5%), PIK3CA (7.6%), PTEN (2.9%)) or completely absent . No case had concurrent KRAS and NRAS mutations  ) compared to their colorectal-type counterparts   Fig. . Finally+, CD8+, Foxp3+, and PD-1+ T cell densities were analysed by immunohistochemistry  relies on a high preexisting T-cell infiltration within tumour microenvironment. In order to determine whether anal glandular neoplasms meet this criteria, PD-L1 expression in epithelial (tumour) cells as well as intratumoural CD4try Fig. . These ltry Fig. . Whatevetry Fig. . Beside try Fig. .Fig. 5To30 Moreover, the restricted expression of Krt7 in tumours arising from the anal glands/transitional zone is consistent with prior case reports33 and parallels the staining pattern of this biomarker in anal SCC located at the squamocolumnar junction.3Anal adenocarcinoma remains a challenge for gastroenterologists, abdominal surgeons and oncologists because of its low incidence, unclear pathogenesis and high recurrence rate. So far, no epidemiologic, genetic and immunologic data are available and only a few studies (mainly case reports) focused on this rare and aggressive disease. Altogether, our findings indicate that the cellular origin  of anal glandular neoplasms influences their natural history, mutational profile and immune microenvironment. Although the tumours were morphologically similar, keratin/CDX2 expression was first shown to be highly specific and sensitive for distinguishing the two adenocarcinoma subtypes described in the present article. The shared expression pattern detected in both normal tissues and invasive cancers further confirms the increasing use of keratin filaments as reliable biomarkers for correlating tumour origin with patient management.34 Every year, about 630,000 SCC attributable to HPV infection are diagnosed worldwide.35 These latter are mainly diagnosed in the uterine cervix but increasing incidences are currently observed in both anal canal and oropharynx. Frequently under-estimated by both virologists and epidemiologists, carcinogenic HPV genotypes are also involved in the development of the majority (~75–80%) of cervical adenocarcinoma.36 Interestingly, transcriptionally active HPV infections were recently found in Barrett’s adenocarcinoma,37 another glandular neoplasm developing within a squamocolumnar junction. To the best of our knowledge, this report provides the first evidence of a causal link between HPV infection and anal adenocarcinoma. Similarly to other HPV-positive adenocarcinoma diagnosed in the uterine cervix or oesophagus, only anal glandular neoplasms  arising from the transitional zone were infected by HPV16 or 18. Although several possible mechanisms were recently highlighted,38 the high susceptibility of junctional/transitional tissues to HPV infection is still the subject of active investigations. Despite a few false-positive (p16ink4 positive/HPV negative) results  that were also shown in the context of both anal and oropharyngeal SCC,39 the reported correlation between high-risk HPV infection and diffuse p16ink4 immunoreactivity was expected. The surprising result was that five  HPV-infected tumours displayed some patches (representing up to one third of the whole tumour area) of p16ink4 negativity. Given that aberrant methylation of p16ink4 promoter is frequently observed in adenocarcinoma arising from the gastrointestinal tract,41 MSP experiments were performed and CDKN2A (p16ink4) promoter hypermethylation was detected in 4 out of 5 tumour samples  was first reported in early 80s.16 Unfortunately, conflicting results exist due to the low number of followed patients in most studies.16 To date, only one study conducted by the Rare Cancer Network analysed more than 50 patients and observed better survival rates after combined chemoradiotherapy compared to radiotherapy/surgery. However, patients treated with this latter combination were significantly older, making difficult the comparison between the two groups.9 Accordingly, our findings demonstrated that patient age was an independent predictor for decreased OS. Therefore, with the exception of local surgery for stage I tumour, as reported in the present study and suggested by the discrepancies in the literature, no standard treatment was shown to clearly improve patient outcome.During the last 20 years, local excision, combined chemoradiotherapy and radical surgery with or without (neo)adjuvant chemotherapy have been successively proposed as the gold standard treatment for these aggressive anal glandular tumours.42 Treatment algorithms of numerous cancers have changed dramatically over the last few years due to the advent of novel targeted drugs. In the context of cancers diagnosed within the gastrointestinal tract, two anti-EGFR monoclonal antibodies (cetuximab and panitumumab) in combination with FOLFIRI were recently approved for the first-line treatment of metastatic colorectal cancers.44 However, their efficacy strongly depends on both EGFR expression and mutational status of its downstream effectors .29 While EGFR expression was detected in the large majority  of both subtypes of anal adenocarcinoma irrespective of the HPV status, interestingly, the frequencies of KRAS/NRAS mutations were significantly higher in HPV-negative tumours compared to their infected counterparts. The low rates of both KRAS and NRAS mutations reported in neoplastic lesions etiologically linked to HPV concurred with the recent results documented for cervical adenocarcinoma.45 Regarding the low prevalence of both BRAF mutations and MSI-high specimens observed in the present study, these latter results are in agreement with those of Yamauchi et al.46 who showed a gradual decrease of these two parameters along the gastrointestinal tract from the ascending colon to the rectum.First suggested by Paul Ehrlich one century ago, the concept of targeted therapy has gained momentum since early 2000s with the clinical success of inhibitors such as trastuzumab (for both breast and gastric cancers) or imatinib (for chronic myeloid leukaemia).47 cancer immunotherapy using immune checkpoint inhibitors  has raised much interest for cancer treatment over the last few years. However, their efficacy for reactivating lymphocyte-mediated cytotoxicity relies on a high pre-existing T cell infiltration of the tumour microenvironment. Importantly, we reported here that T cell densities  detected in the tumour stroma surrounding cancer cells  originating from the anal glands/transitional zone were significantly higher than those found in the vast majority of colorectal-type tumours. Moreover, a similar significant increase was also observed for the expression of PD-L1 by tumour cells. Not related to the MSI status of tumours, these data are likely to be explained by the chronic inflammation frequently observed in squamocolumnar junctions48 and/or by the detection of transcriptionally active HPV infection in approximately half (42.3%) of anal gland/transitional-type adenocarcinoma. Indeed, several recent studies described a significant increased density of both effector (CD4/CD8) and regulatory (Foxp3) tumour infiltrating T lymphocytes in HPV-positive compared to HPV-negative SCC and highlighted the predictive value of CD4/CD8 infiltrates.49 These latter results and those reported in the present article are supportive of the dozen ongoing clinical trials evaluating immune-checkpoint inhibitors in the setting of HPV-positive cancers.Chosen by Science’s editors as the breakthrough of the year for 2013,Taken together, our findings highlight the existence of two subtypes of anal adenocarcinoma with distinct cells of origin. Whatever the tumour subtypes, metastatic patients with wild-type downstream effectors of the EGFR signalling pathway could reasonably be expected to benefit from anti-EGFR monoclonal antibodies. Displaying a strong Krt7 immunoreactivity, tumours arising from the anal glands/transitional zone were shown to be associated with prominent T cell infiltrates and high expressions of PD-1/PD-L1 suggesting that immune checkpoint inhibitors could also represent promising alternatives to current sub-optimal treatment algorithms. Although further studies are still needed, determining the cellular origin of these rare tumours with the specific/sensitive biomarkers validated in the present study could therefore be helpful for clinicians currently struggling with this aggressive neoplastic condition.Supplementary material and methodsSupplementary table 1Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5


The Pap smear has remained the foundation for cervical cancer screening for over 70 years. With advancements in molecular diagnostics, primary high-risk human papillomavirus (hrHPV) screening has recently become an accepted stand-alone or co-test with conventional cytology. However, both diagnostic tests have distinct limitations. The aim of this study was to determine the association between HPV genotypes and cellular epigenetic modifications in three grades of cervical cytology for screening biomarker discovery.ADCY8), cadherin 8, type 2 (CDH8), MGMT, and zinc finger protein 582 (ZNF582)) out of 48 genes screened was quantified by bisulfite-pyrosequencing of genomic DNA. Independent validation of methylation profiles was performed by analyzing data from cervical cancer cell lines and clinical samples from The Cancer Genome Atlas (TCGA).This prospective, cross-sectional study used residual liquid-based cytology samples for HPV genotyping and epigenetic analysis. Extracted DNA was subjected to parallel polymerase chain reactions using three primer sets  for HPV DNA amplification. HPV+ samples were genotyped by DNA sequencing. Promoter methylation of four candidate tumor suppressor genes , LSIL (N = 70), and HSIL (N = 67) also correlated with worsening grade. Similar hypermethylation patterns were found in cancer cell lines and TCGA samples. The combination of four biomarkers, i.e., HPV genotype and three-gene promoter methylation, predicted HSIL (AUC 0.89) better than HPV alone (AUC 0.74) by logistic regression and probabilistic modeling.Two hundred seventy-seven quality cytology samples were analyzed. HPV was detected in 31/100 (31 %) negative for intraepithelial lesion or malignancy (NILM), 95/100 (95 %) low-grade squamous intraepithelial lesion (LSIL), and 71/77 (92 %) high-grade squamous intraepithelial lesion (HSIL) samples. The proportion of IARC-defined carcinogenic HPV types in sequenced samples correlated with worsening grade: NILM 7/29 (24 %), LSIL 53/92 (58 %), and HSIL 65/70 (93 %). Promoter methylation of ADCY8, CDH8, and ZNF582 are correlated with cytological grade. Collectively, these biomarkers may serve as a molecular classifier of Pap smears.HPV genotype and DNA methylation of The online version of this article (doi:10.1186/s13148-016-0263-9) contains supplementary material, which is available to authorized users. In 1941, George Papanicolaou published his landmark paper on the use of vaginal smears for the diagnosis of cervical cancer . Since tSince the isolation and cloning of human papillomavirus (HPV)-16 from cervical carcinoma by zur Hausen et al. in 1983, the HPV is now recognized as a necessary cause of invasive cervical cancer with a prevalence of 99 % in global samples , 5. WithOver the last two decades, our understanding of cancer epigenetics has deepened immensely . The bodTo better understand the trilateral relationship between HPV, genomic DNA methylation, and cervical cytopathology, our goal was to use state-of-the-art molecular techniques to screen and profile HPV genotypes and DNA methylation in normal and precancerous Pap smears. The correlation between the predictors (HPV genotype and extent of cellular DNA methylation) and three cytological grades would then be quantitated and explored for its utility as a molecular classifier of cervical cytology.This study was conducted after gaining approval by the Institutional Review Board of the Brooke Army Medical Center (BAMC), Texas. Inclusion criteria were cervical specimens derived from adult women ≥18 years of age undergoing cervical cytology screening. Exclusion criteria were cervical specimens from patients with conditions that may alter genomic methylation, e.g., pregnancy and non-HPV sexually transmitted infections.N = 100), LSIL (N = 100), and HSIL (N = 77).Liquid-based cytology collected for clinical testing at the Department of Pathology of BAMC was consecutively procured after completion of analysis for cytological diagnosis. Samples were refrigerated at 4 °C until weekly batch DNA extraction. Demographic data were abstracted from the electronic health record (AHLTA) of the Department of Defense (DoD) and code-linked to each specimen. Three categories of the samples, i.e., negative for intraepithelial lesion or malignancy (NILM), low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL), were collected until target accrual numbers were met: NILM  followed by washing with fresh media for 15–30 min at 37 °C. Cells were fixed and permeabilized with the FIX & PERM® kit (Life Technologies). Actin and nuclei were stained with respective reagents, ActinGreen™ 488 and NucBlue® (Life Technologies), washed with PBS, and mounted in ProLong® Gold antifade reagent (Life Technologies). Images were acquired by a Leica TCS SP5 II confocal microscope (Leica Microsystems).Five cervical cancer cell lines  were acquired from the American Type Culture Collection (ATCC) to serve as (+) controls and comparators of methylation. The cell type, tumor site derivation, and HPV status were as follows: SiHa ; HeLa ; Ca Ski ; C33-A ; and DoTc2 . Cells were cultured in flasks for DNA extraction and μ-Slides (Ibidi) for microscopy with appropriate media supplemented with 10 % FBS. EMEM medium (ATCC) was used to grow HeLa, C-33A, and SiHa cells. DMEM and RPMI-1640 media (ATCC) were used to culture DoTc2 and Ca Ski cells, respectively. Cells were grown at 37 °C in a CON = 231) and adenocarcinomas (N = 26). The methylation data  generated with the Illumina HumanMethylation450 platform (HM450) in the level 3 format were used to determine promoter methylation levels of adenylate cyclase 8 (ADCY8), cadherin 8, type 2 (CDH8), O-6-methylguanine-DNA methyltransferase (MGMT), and zinc finger protein 582 (ZNF582). The matched RNA-SeqVersion 2 expression data [N = 3) with matched  DNA methylation (accessed on 15 January 2015) were used to compare within and between subject differences.The cervical cancer cohort of The Cancer Genome Atlas (TCGA) was accessed on 3 October 2014 to acquire DNA methylation data of squamous cell carcinomas  was centrifuged (4000 rpm × 2 min), and the supernatant was removed. The cell pellet 200–250 μL) was transferred into sample tubes (2 mL) and placed in a QIAcube robotic workstation (Qiagen) for DNA extraction using the QIAamp DNA Mini kit (Qiagen). The purified DNA in 150 μL of the eluent was quantified by spectrophotometry and stored at −20 °C prior to amplification. For HPV DNA amplification, three consensus primer sets: (1) MY09/11, (2) FAP59/64, and (3) GP-E6-3F/GP-E7-5B/GP-E7-6B, were used to amplify two regions of HPV L1 and E6/E7 for genotype identification 0–250 μL . AmpliTaPCR products were purified using the GeneRead Size Selection Kit (Qiagen) on the QIAcube robot. Sanger sequencing of the amplicons (~200 ng DNA/sample) was performed by using sequencing primers MY11, FAP59, and GP-E6-3F (Eurofins Operon). Sequence quality was assessed using the Sequence Scanner 2.0 (appliedbiosystems.com), where a “high-quality” trace score (TS)  was defined as ≥20 and a QV20+ value  as ≥100. Quality sequences were filter selected for entry into the Basic Local Alignment Search Tool (BLAST®) and queried against HPV sequences in GenBank® under virus taxonomy ID# 151340 . The HPVTo confirm and discover new hypermethylated genes in cervical carcinoma, 48 genes were selected for testing  was <0.2. Six genes met our screening criteria: ADCY8, CDH8, ZNF582, MGMT, ALK, and NEFL. The best candidates (first four genes) were selected for further testing of individual samples based on documented association with cervical, oral, and/or endometrial carcinoma [N = 33), LSIL (N = 70), and HSIL (N = 67), were subjected to individual locus-specific methylation quantification. Furthermore, the HPV status and methylation levels of these samples were used to construct the multivariable logistic model described below.The screening criterion used to define hypermethylation at each CpG site was ≥2.0× the methylation level (%) of normal cytology samples. This method is comparable to the selection criteria used by Farkas et al.  for β vaarcinoma , 26, 27.For this study, the classification of HPV carcinogenicity was based on the WHO IARC Working Group Reports , 8. SpecADCY8, CDH8, and ZNF582) and a binarized cytological outcome of interest. Outcome model 1 aimed to distinguish normal from abnormal cytology (NILM vs. LSIL/HSIL), whereas model 2 distinguished between non-high and high-grade cytology (NILM/LSIL vs. HSIL). The model equation is as follows:Multivariable logistic regression  was perfe∧(−(b0 + b1X1 + ⋯ + biXi)))Logistic model: Probability of outcome = P(Y = 1) = 1/(1 + Multiple explanatory variables: Model 1 outcome (Y) coding: NILM (0), LSIL/HSIL (1)Model 2 outcome (Y) coding: NILM/LSIL (0), HSIL (1)P value) were selected for cut-point (binarization) determination. The cut-points were chosen at the point of maximum accuracy (∑ sensitivity + specificity). The new binarized methylation variables of these CpG sites, along with HPV carcinogenic status, were entered in a second multivariable logistic regression analysis to select the explanatory variables most predictive of the cytological outcome. The second model equation is as follows:The covariates (CpG position selected from each gene) that had the highest association with the response variable (lowest ∧(−(b0 + b1X1 + ⋯ + b4X4)))Logistic model: Probability of outcome = P(Y = 1) = 1/i methylation X2 = ADCY8 CpG-position i methylation X3 = CDH8 CpG-position i methylation X4 = ZNF582 CpG-position Model 1 outcome (Y) coding: NILM (0), LSIL/HSIL (1)Model 2 outcome (Y) coding: NILM/LSIL (0), HSIL (1)For the final regression models, post-estimation receiver operating characteristic (ROC) curves were constructed and predictions at specified values were computed. After estimating the classification threshold or “cut-point” for each model by using the maximum sum of sensitivity and specificity, diagnostic performance characteristics were determined. The discriminatory performance between multivariable and univariable (HPV carcinogenicity only) models was compared using respective areas under the ROC curve. Pairwise comparisons of predicted probabilities between models were performed with the chi-square test.α = 0.05 and β = 0.20 with an allocation ratio of 2 (N2/N1), a minimum accrual target of N2 = 62 and N1 = 31 per group was required. The quota sampling strategy assured adequate representation from each cytological grade. Furthermore, additional samples were collected to account for potential sample inadequacy and laboratory errors.This study was designed to have an 80 % power to detect a 20 % difference in DNA methylation (%) between normal and abnormal cytology. From the literature, locus-specific promoter methylation levels (%) for NILM, LSIL/HSIL, and cervical cancer have ranged from 0 to 5 %, 15 to 30 %, and 30 to 60 %, respectively , 15, 29.P values <0.05 were considered statistically significant.Data were summarized using means (95 % CI), medians (IQR), and proportions. For hypothesis testing, Wilcoxon rank sum and Kruskal-Wallis tests were used for non-parametric, numerical, or ordinal data. Categorical data were compared using the chi-square test. Correlation between ordinal variables was determined by Spearman’s rho. β value, defined as the ratio of methylated signal over the total signal (methylated + unmethylated)) [ADCY8, CDH8, MGMT, and ZNF582. The median methylation values per locus were stratified by the observation group, i.e., tumor stages and histologic category , and tested for differences by non-parametric methods. All subsequent analyses compared median methylation values across all CpGs per gene as the single sample summary measure. The correlation between methylation  and RNA-SeqV2 expression data  [For TCGA methylation analysis, the pyrosequencing CpG assay for each gene was translated into the Illumina assay by selecting the nearest CpG loci on the HM450K array. Methylation data (ylated))  were usetimates)  was deteN = 400) were collected between January 2013 and 2014. Of all samples, 31 % (N = 123) were excluded because of low quantity, low quality, or sample excess, as described in Table N = 277), the corresponding subjects were composed predominantly of Caucasians (45 %) with a median age of 28 years . The cytological specimens were stratified proportionately among the three grades: NILM 100/277 (36 %), LSIL 100/277 (36 %), and HSIL 77/277 (28 %). The median concentration of the extracted DNA among the three cytological categories  was statistically equivalent  , whereas the distribution did not vary among NILM. Finally, a high frequency of HPV-58 was noted in HSIL samples.The prevalence of HPV genotypes found in three grades of cytology is shown in Fig. ADCY8, CDH8, and ZNF582  but not MGMT. Pairwise comparison of methylation at each CpG locus between Pap grades revealed higher levels in HSIL than LSIL and NILM with a few exceptions   Fig. . InteresADCY8, CDH8, and ZNF582 was noted in all cell lines except C33A and DoTc2 (which failed the ADCY8 assay). For comparison between cell lines, the methylation levels of all four genes in SiHa (ranging from ~38 % in MGMT to 93 % in ADCY8) were used as the referent. Although some significant differences in DNA methylation levels were detected, e.g., decreased methylation of ADCY8 in HeLa/C33A cells and CDH8 in C33A cells  revealed distinct hypermethylation patterns among ADCY8, ZNF582, and CDH8 . For MGMT, the methylation was consistently low with a median β value of 0.012 across all stages. Moreover, methylation levels were not distinguishable between stages for the four genes . Association analysis between methylation and matched RNA-Seq expression data revealed modest anti-correlation for ZNF582  and MGMT  but not for ADCY8 and CDH8  compared with the three normal samples  were comparable to those of squamous carcinomas; hence, these samples were included in the tumor cohort.TCGA data for the three available tumor/normal matched pairs of cervical tissues were examined for within and between subject promoter methylation differences. Because of the small sample size, formal statistical analysis was not performed. However, increased median methylation (~10×) of les Fig. . NotablyZNF582_CpG-position 3, with an area under the ROC of 0.93. For model 2, the best predictors were HPV carcinogenicity and ADCY8_CpG-position 7, CDH8_CpG-position 3, and ZNF582_ CpG-position 3, with an area under the ROC of 0.89. The discriminatory performance of both multivariable models inclusive of methylation markers was better than that of the univariate predictor (HPV carcinogenicity) model by comparing areas under the ROC .The logistic regression analysis and ROC curves for the univariable and multivariable logit models for cytological outcomes are presented in Additional file ZNF582 over HPV carcinogenicity alone as a predictor of abnormal Pap smear (LSIL/HSIL). More importantly, HPV negativity in conjunction with low ZNF582 methylation was highly indicative of a normal Pap with a negative predictive value (NPV) of 100 %  of predictor variables in model 1 are provided in Additional file ADCY8, CDH8, and ZNF582 promoter methylation over HPV carcinogenicity alone as a predictor of HSIL were significant  of predictor variables in model 2 are tabulated in Additional file The predicted probabilities at representative values over the range of predictor variables are presented as margins plots Fig. . Figure 0 % Fig. . The preant Fig. . The proZNF582 was higher (100 %) than that of HPV (90 %) in detecting abnormal (LSIL/HSIL) cytology. The positive predictive values (PPVs) were comparable at 93–95 %, suggesting that for patients with a positive assay, almost all have abnormal cytology. In contrast, for patients with a negative assay, the chance of finding no disease (NPV) was 100 % for HPV + ZNF582 vs. 66 % for HPV, suggesting that HPV + ZNF582 is a better screening test. For model 2, the PPV was greater for the HPV + three-methylation marker (81 %) vs. HPV (58 %), suggesting that in patients with a positive multi-marker test, almost 80 % will have HSIL. Furthermore, the false-positive rate is lower for the HPV + three-methylation marker (19 %) than for HPV (42 %). Essentially, the results of the two models indicate that (1) HPV + ZNF582 is a better predictor of NILM and (2) HPV + three-methylation marker is a better predictor of HSIL than HPV alone.The diagnostic performance characteristics of models 1 and 2 are presented in Additional file ADCY8, CDH8, and ZNF582; and cytological grade. Our previous experience had shown that parallel PCR testing with multiple primer sets optimizes the sensitivity and breadth of HPV detection; thus, this methodology was used herein [This study aimed to determine the association between HPV genotypes and cellular epigenetic modifications in three grades of cervical cytology. Indeed, our study found positive correlations between HPV carcinogenicity; aberrant DNA methylation in the promoters of d herein . The HPVd herein . Our extd herein . PCR/seqd herein , 34. Furd herein . The racADCY8, CDH8, and ZNF582 concomitant with worsening cytological grade. Conjointly, HPV carcinogenicity and the binarized methylation levels of the three genes were significant predictors of cytological outcome in a multivariable model. Specifically, HPV and ZNF582 demonstrated high discriminatory performance as a screening test to differentiate normal (NILM) from abnormal cytology (LSIL/HSIL) with a NPV of 100 %. In contrast, the lower NPV (66 %) for HPV alone in detecting abnormal cytology may be explained by the elevated false-negative rates of HPV DNA detection in LSIL/HSIL. PCR non-detection may be attributed to several variables, e.g., insufficient DNA template quality or quantity, primer-target mismatch, and loss of HPV viral sequences except for E6 and E7 upon integration into the cellular genome, notably in HSIL and invasive disease [ADCY8, CDH8, and ZNF582 differentiated the <HSIL from HSIL samples with a PPV of 81 %. In terms of clinical utility, the addition of quantitative methylation markers to the probabilistic model significantly improved the diagnostic accuracy of HPV carcinogenicity as a single predictor of cytological outcome.The proportion of carcinogenic HPV genotypes found in the samples after genotyping was highest among the HSIL group. Cellular genomic analyses revealed a significant increase in the promoter methylation of  disease , 30. In  disease . For abnADCY8, CDH8, and ZNF582 was corroborated in five cervical cancer cell lines with two exceptions. C33A cells exhibited low CDH8 methylation levels and DoTc2 failed the ADCY8 assay, presumably because of low levels as well. Both C33A and DoTc2 cells are HPV-negative, which may explain the hypomethylation as previously demonstrated in HPV+/HPV− head and neck squamous cell carcinoma (HNSCC) cell lines and tumors [ADCY8, CDH8, and ZNF582 was markedly elevated across all four stages of cervical carcinoma. The lack of variability between stages suggested that these epimutations occurred early in the neoplastic process. Whether these alterations are tumor “drivers” or “passengers” is unknown. Nonetheless, they serve as informative host biomarkers for epithelial dysplasia/neoplasia. Moreover, within-subject analysis of matched tumor and normal tissues verified differential promoter methylation for ADCY8, CDH8, and ZNF582. It is worth mentioning that when the current study was initiated, there were only 119 cases (year 2011 (N = 66), year 2012 (N = 53)) with 1 matched  sample in TCGA. Furthermore, the targeted CpG loci between pyrosequencing and HM450 methylation assays may not be identical and may thus render different results. Different CpG positions, even in close proximity, within the same CpG-island may exhibit dissimilar methylation levels [Promoter hypermethylation of d tumors . TCGA dan levels .ZNF582, CDH8, and ADCY8 have unique cellular functions that may be repressed via epigenetic modifications and participate in the neoplastic process. First, ZNF582 located on chromosome 19 encodes a nuclear protein belonging to the Cys2His2-(C2H2) zinc finger protein family with a conserved Kruppel-associated box (KRAB) domain [ZNF582 protein is unknown; however, KRAB-ZFPs, in general, are transcriptional repressors that bind to the gene promoter regions via their sequence-specific DNA binding motifs. Some KRAB-ZFPs are known to regulate apoptosis and act as tumor suppressors; thus, inactivation may be involved in tumorigenesis [ZNF582 is frequently methylated in invasive squamous and adenocarcinoma of the cervix, as well as preinvasive disease [CDH8 gene on chromosome 16 encodes cadherin, which is a cell membrane-spanning protein that mediates cell-cell adhesion and recognition [CDH8 were hypermethylated in HPV+ HNSCC [ADCY8 on chromosome 8, encodes a membrane-bound enzyme that catalyzes the formation of cyclic AMP from ATP [ADCY8 hypermethylation and altered expression have also been observed in endometrial cancer [The gene products of ) domain . The exaigenesis . Previou disease , 16, 40.ognition . A recenV+ HNSCC . AdditioV+ HNSCC , 42. Thefrom ATP . This gefrom ATP  elegantll cancer , 45. In carcinogenic and not possibly or not classifiable HPV genotypes. Such dichotomized classification, i.e., high-risk positive or negative HPV, has a significant false-negative rate because of the non-detection of “low-risk” HPV, which may pose a clinical risk. Regarding quantitative DNA methylation, CpG analysis by pyrosequencing was chosen for its accuracy and high quantitative resolution. This method may also be easily translated into a clinically applicable test, i.e., real-time PCR with high-resolution melt analysis [The strength of this study lies in the methodologies used for HPV detection and methylation quantification. HPV detection by parallel PCR/sequencing offers the greatest sensitivity and breadth of HPV detection. This method unleashes the constrained spectrum of HPV genotypes detected by commercial tests to obviate measurement bias. Furthermore, allocating the HPV genotypes by IARC-defined carcinogenicity enumerates oncogenic potential to allow for predictive modeling. In contradistinction, commercially available HPV tests only detect analysis . Essentianalysis . To fillIn conclusion, the results of this study showed that different grades of cervical cytology possess different molecular signatures, which may be translated into a multi-targeted “molecular Pap” for clinical use. With the rapid evolution of molecular technologies, it is foreseeable that cervical cancer screening may become a fully automated, computerized, molecular diagnostic test that may circumvent economic hardships and the non-existence of infrastructures for cytology-based screening programs in developing countries.


Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenicHPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope designfor the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out topredict the epitopes against HPV types 16 and 18 through “reverse vaccinology” approach. We attempted to identify the evolutionarilyconserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within theseregions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that theintertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. Wehad selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 ofHPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA ofL1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK,KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools forvaccine design against HPV. Human papillomaviruses (HPVs) are small in shape, nonenveloped,double stranded DNA viruses whose eight proteinsare encoded by approximately 8 kb circular genome ,2. TheHAmong 15 oncogenic HPV types , HPV 16 and HPV 18 are mostdominant, accounting for about 50% and 20% of cervical cancerrespectively . CumulatThe availability of genomic data, understanding of immuneresponses and immunogenetic variations, new developments incomputer applications and systems biology approach areshowing new directions in vaccine development, away from thetraditional techniques of live-attenuated or inactivated virus orrecombinant VLP vaccines. The primary goal of this study is toinvestigate different conserved epitopes of L1 and L2 capsidproteins through ‘’Reverse Vaccinology’’ approach  andjustThe peptide-based vaccine also has another advantage ofincreased safety, the opportunity to rationally engineeredepitopes for increased potency and the ability to focus immuneresponses on conserved epitopes ,29. ThusAmino acid sequences of the capsid proteins (L1 and L2) of HPVtypes were retrieved from the NCBI database available at(http://www.ncbi.nlm.nih.gov/) updated till December 24, 2016and the accession numbers of those sequences are listed in B cell epitopes of L1 and L2 protein sequences of HPV werepredicted by using Immune Epitope Database (IEDB) AnalysisResource, available at (http://tools.immuneepitope.org/main/html/bcell_tools.html) . KolaskaT cell epitopes of L1 and L2 protein sequences of HPV werecollected by T Cell Epitopes- MHC Binding Prediction Tools byusing Immune Epitope Database (IEDB) web tool available at(http://tools.immuneepitope.org/). Those epitopes, who havepotentiality to bind with MHC class I molecule were determinedby peptide binding to MHC Class I molecules Tool -38. InaThe 3D structures of L1 protein of HPV (types 16 and 18) wereretrieved from the Protein Data Bank (PDB) with folling IDs2R5H and 2R5I, respectively available at (http:www.pdb.org/). The PDB ID (2R5H) and (2R5I) were used atMMDB (Molecular Modeling Database) at(http://www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb.shtml) to deterHPV capsid proteins L1 and L2 usually synthesize late in theinfection cycle and encapsidate the closed circular doublestrandedDNA mini-chromosome. Since L1 and L2 capsidproteins are largely responsible for essential functions includingvirion assembly, virion stability, and virion uncoating, aconsistent pattern of conserved and variable regions is perhapsnot surprising. We had aligned 27 sequences of L1 and 49sequences of L2 proteins of HPV obtained from the NationalCenter for Biotechnology Information database(http://ncbi.nlm.nih.gov) by using CLUSTALW program to findconserved regions with in the sequences. This is because thehighly conserved epitopic regions are less prone to antigenescape and viral mutation . From thTo predict continuous linear B cell epitopes we used Kolaskar &Tongaonkar Antigenicity Method . This meHere T cell MHC-I epitopes were predicted by using Peptidebinding to MHC class I molecules tool [Immune EpitopeDatabase (IEDB)]. This tool considers in an amino acid sequenceor set of sequences and determines each subsequence's ability tobind to a specific MHC class I molecule. Consistent dysregulationof HLA expression has been reported in cervical neoplasia, which can influence the prospects for cell-mediated vaccine therapies.HLA-A*0201 allele’s expression has been found to be increased inHPV-associated cervical cancer . TherefoT cell MHC-II epitopes were predicted by using Peptide bindingto MHC class II molecules tool [Immune Epitope Database(IEDB)]. This tool provides a consensus approach to predict MHCClass II epitopes based upon Sturniolo, ARB, and SMM align.Several HLA class II alleles have been found to increase the riskof cervical cancer when associate with it ,44. DRB1Vaccines composed of longer peptide sequences generate bothCD4 and CD8 specific T cell responses against HPV oncogenes. Itresults in more vigorous CD8 CTL responses than vaccinationwith exact minimal CTL (Cytotoxic T lymphocytes) epitopelength . We had Furthermore, three dimensional structure of L1 protein of HPVtype 16 and type 18 were retrieved by using PDB (ProteinDatabank) ID 2r5h and 2r5i respectively .Finally we had choosen those B and T cell linear epitopes whohave aromatic amino acids , as aromatic amino acids have been found to increase immunogenecity. Epitope sequences are shown inpurple color in the Epitopes 48EEYDLQFIFQLCKITLTA65, and 45RHGEEYDLQFIFQLCKITLTA65 of L1 protein of HPV type 16, Figure 3and epitopes 63LPDPNKF69, 79PETQRLVWAC88, 36PVPGQYDA43, 77YNPETQRLVWAC88, 188DTGYGAMD195, 36PVPGQYDATK45, 45KQDIPKVSAYQYRVFRV61, 130RDNVSVDYKQTQLCI144 and 49YSRHVEEYDLQFIF62 of L1 protein ofHPV type 18, Worldwide HPV-induced dysplasia and cancer cause significantmorbidity . AlthougFrom the multiple sequence alignment results, we found that theintertype variability of the L2 proteins is higher than L1. Thus, L2is less consistent in the positions of variable regions than the L1.Conserved regions of L1 and L2 revealed that more epitopespresent in conserved regions within the sequences of L1 than L2protein and these regions would be major targets for neutralizingantibodies. Thus, L1 protein could be a better target for vaccinedesign than L2. We preferentially selected B cell and T cell linearepitopes with longer length because vaccination with longerpeptides generates high levels of neutralizing antibodies thanvaccination with exact minimal epitope length . SeveralFinally, we hypothesized that epitopes 48EEYDLQFIFQLCKITLTA65, and 45RHGEEYDLQFIFQLCKIT LTA65 Figure 3 of L1protein of HPV type 16, and epitopes 63LPDPNKF69,79PETQRLVWAC88, 36PVPGQYDA43, 77YNPETQRLVWAC88,188DTGYGAMD195, 36PVPGQYDAT K45, 45KQDIPKVSAYQYRVFRV61, 130RDNVSVDYKQTQ LCI144 and49YSRHVEEYDLQFIF62 Immunogenicity than carbohydrates, lipids and nucleic acids andhere we had designed epitopes of a protein. Primary structure ofproteins found to have lesser immunogenicity than secondarystructure of same protein. Since epitopes of HPV L1 protein wereshown in 3D structures they could be potential immunogen.Heteropolymers (composed of different amino acids) are moreimmunogenic than homopolymers. Since epitopes of HPV L1protein are heteropolymer, they could be effective immunogen.Several studies had been reported some epitopes of HPV L1protein such as epitope sequences MVLILCCTLAILFCVA, LAILFCVADVNVFHIF, DVNVFHIFLQMSVWRP andQMSVWRPSEATVYLPP of HPV type 58 ; epitopeTherapeutic vaccines play a vital role in preventing the metastaticspread of cervical cancer with immediate impact rather thanprophylactic vaccines, which normally takes many years toreduce deaths from this disease. We have proposed an alternativestrategy of peptide vaccines for conditions where VLP (Virus likeParticle) vaccines like Gardasil and Cervarix may not be ideal.Our exercise yielded a set of epitopes of L1 protein for HPV type16 and 18. Because of the nature of the viral coat proteins, theepitopes proposed in this paper may not provide the high level ofefficiency that the VLP vaccines can provide, but with suitableadjuvants may enhance the neutralizing antibody production.Hence, we conclude that, epitopes 48EEYDLQFIFQLCKITLTA65,45RHGEEYDLQFIFQLCKITLTA65, 63LPDPNKF69, 79PETQRLVWAC88, 36PVPGQYDA43, 77YNPETQRLVWAC88, 188DTGYGAMD195, 36PVPGQYDATK45, 45KQDIPKVSAYQYRVFRV61, 130RDNVSVDYKQTQLCI144 and 49YSRHVEEY DLQFIF62 of L1 protein might be used as potential tools fordesignation of a new vaccine against HPV type 16 and 18.However, it is necessary to perform wet laboratory experimentsto test the efficacy of the results of studies like this. At last suchpreparations should be strongly recommended due to the widespread of papillomavirus-induced cervical, genital, and othercancers and the sufferings these cause.


Currently, the role of human papillomavirus (HPV)-58 in southwestern China has been unexplored. Although there is some controversy, it is proposed that the viral load of HPV correlates with the severity of intraepithelial lesions.We identified 7747 patients from south Sichuan and adjacent regions who were diagnosed with HPV between 2013 and 2017. The HR-HPV subtype distribution was analyzed and the patient’s viral loads were quantified using real-time RT-PCR.P < 0.001, P = 0.016, P = 0.026, respectively). Using receiver operating characteristic (ROC) curve analysis, the optimum thresholds for predicting severe intraepithelial lesions of cases  with HPV-16, HPV-58, and HPV-33, respectively, were obtained, which were 1, 0.93, and 0.25, respectively.Among all 7747 patients screened for HPV genotypes, 1728 patients (22.31%) were identified as having HR-HPV subtypes. In patients without intraepithelial lesions (12.41%), HPV-52, HPV-16, and HPV-58 were the three most prevalent HR-HPV subtypes. Moreover, HPV-16, HPV-58, and HPV-33 were the most prevalent subtypes in patients with cervical intraepithelial neoplasia grade II (CINII) (42.86%) and grade III (CINIII) (59.81%), and accounted for the majority of invasive cervical cancer (ICC) (69.34%). Thus, viral loads of HPV-58, HPV-16, and HPV-33 positively correlated with the severity of cervical lesions  contains supplementary material, which is available to authorized users. Human Papillomaviruses (HPVs) belong to the highly heterogeneous family of DNA viruses, and can cause intraepithelial neoplasias in skin and mucosal cells. Roughly 40 high-risk (HR)-HPV types are found in the female genital tract, which have the potential to drive the evolution of high-grade premalignant lesions into cervical carcinomas . Thus, fThe distribution of HPV types varies geographically. In a previous report, covering millions of cases from five continents, it was indicated that HPV-16 was the most prevalent and carcinogenic genotype worldwide, followed by HPV-18 in many regions . HoweverIntegration of HR-HPV into the host genome is vital for malignant transformation , 9. TherA total of 7747 specimens from patients in southern Sichuan and the adjacent areas were collected between January 2013 and November 2017 in the First Affiliated Hospital of Southwest Medical University, China. The majority of the patients showed lesions whereas the remainder visited the hospital for routine cervical exams. The age of the patients ranged from 16 to 86 years with an average of 40.33 ± 9.36 years. All patients first underwent routine cytological screening . Patients with intraepithelial lesions, malignancy or who showed other morphologic abnormalities by TCT, or who had a HPV infection underwent histological (biopsy) and histopathological procedures, then a final diagnosis was made. The results of each procedure were interpreted by two experts, and if they did not agree on a diagnosis, a third expert was consulted to finalize the diagnosis. Patients with malignancies outside the reproductive system were excluded from this study.For the HPV test, cervical samples were obtained by gynecologists via a cytobrush, and resuspended in 20 mL of liquid-based cytology medium. To extract DNA from these samples, 50 μL of the liquid-based cytology sample was pelleted, and 200 μL of the denaturing reagent  was added to the pellet. Then, samples were incubated at 100 °C for 10 min, and centrifuged for 5 min at 10,000 RPM. Supernatant was collected and the DNA concentration was measured using a NanoDrop 2000 spectrophotometer.Cervical specimens were examined for HPV DNA using a Slan-96P Real time PCR Systemassay . The following 13HR-HPV genotypes were evaluated: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The PCR program was performed as described previously . The duaClassifications of lesions in TCT were performed in conformity with the Bethesda 2001 criteria, including negative for intraepithelial lesion or malignancy (NILM), which includes normal and inflammatory tissues; atypical squamous cells of undetermined significance/cannot exclude high grade lesion (ASC-US/H); low grade squamous intraepithelial lesion (LSIL); high grade squamous intraepithelial lesion (HSIL); cervical squamous cell carcinoma (SCC); atypical glandular cells (AGC); endocervical adenocarcinoma (ECA).Patients with intraepithelial lesions or who showed other morphologic abnormalities by TCT, or had a HPV infection underwent a pathological procedure for final diagnosis. The biopsy specimens obtained were fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin. Pathological identifications were performed in conformity with the 2014 World Health Organization (WHO) (Fourth Edition) classification criteria and described as follows: a normal cervix describes those who are negative for intraepithelial lesions or malignancy and do not have inflammation or other benign lesions. Inflammation describes those with inflammatory or other benign lesions, including chronic cervicitis, cervical hypertrophy, nabothian cysts, erosion, bleeding, and hyperplasia. CINI describes cervical intraepithelial neoplasia grade I (low dysplasia), corresponding to LSIL. CINII and CINIII describe cervical intraepithelial neoplasia grade II (moderate dysplasia) and III (severe dysplasia), respectively, corresponding to HSIL. ICC describes invasive cervical cancer, whereas others describe cases after surgery or treatment of hysteromyoma, and so on.2 test was performed to evaluate the significance of differences between designated groups. All analyses were two-sided. Pearson’s correlation analysis was employed to evaluate the relationship between viral load and the severity of intraepithelial lesions. In the analysis, CINI, CINII, and CINIII cases were given a score of 1, 2, and 3, respectively, cancer cases were given a score of 4, whereas others without definite intraepithelial lesions were given a score of 0. ROC curves were calculated to indicate the optimum thresholds for predicting intraepithelial lesions and severe intraepithelial lesions in cases with HPV-16, HPV-58, and HPV-33 subtypes.Analyses were carried out using SPSS software. A Pearson’s χOf the samples obtained, 6735 (86.94%) cases were negative for intraepithelial lesions , 618 (7.98%) cases had intraepithelial lesions or cancer, and 394 (5.09%) cases showed other characteristics , α-6 (HPV-56), α-7 , and α-9 . The HPV infection rates of α-5, α-6, and α-7 in patients were much lower compared to that of α-9. Cases with α-9 infection accounted for 13.78, 13.05, 36.74, 76.47, 68.42, and 72% in patients with no symptoms, inflammation, CINI, CINII, CINIII, and ICC, respectively.P < 0.01), and accounted for 25.12, 42.86, 59.81, and 69.34% in CINI, CINII, CINIII, and ICC patients, respectively  (Table P = 0.027) and negatively correlated to the viral load of HPV-59 (P = 0.022). The severity of the cervical lesions positively correlated to the viral load (P < 0.001). For specific genotypes, viral loads of HPV-16, HPV-58, and HPV-33 significantly correlated with the severity of intraepithelial lesions, with correlation coefficients of 0.121 (P = 0.016), 0.189 (P < 0.001), and 0.209 (P = 0.026), respectively. The viral loads of other subtypes did not significantly correlate with the severity of lesions.In general, no significant correlation was observed between the total viral load and the age of patients (4) Table . For speTo quantitatively evaluate intraepithelial lesions , the ROC curves for viral loads and intraepithelial lesions in cases with HPV-16, HPV-58, and HPV-33 were calculated Fig. . OptimumThe correlation between HPV subtype and cervical pathology status presented in this study may supply guidance for HPV vaccination programs and preventative strategies. The overall HR-HPV prevalence among the 7747 patients was 1728 (22.31%), which was similar to the findings of a previous meta-analysis of the Chinese population . The thrHR-HPV prevalence rates increased with the severity of the intraepithelial lesions –19. HoweHPV-52 has been reported as the most prevalent genotype in many regions, including eastern China , 6, 17, P < 0.001), which was in line with the results described by Dalstein et al. [Viral load determined by quantitative methods has been performed to predict the development of high-grade cervical lesions , 25, 26.n et al. . A majorn et al. . The clen et al. . This man et al. . Our resHildesheim et al.  found a In this study, a broad summary is presented of the distribution of the HPV type in the southern Sichuan province in women with cervical lesions. The most prevalent HPV types in cases with severe cervical lesions were HPV-16, HPV-58, and HPV-33. HPV-16 was the most common carcinogenic HPV type in southwestern China, followed by HPV-58. The viral load of HPV-58 subtype correlated with the severity of cervical lesions, as was the case for HPV-16, and HPV-33. Additional intensive studies are needed to fully evaluate the carcinogenicity of HPV-58 for HPV prevention in southern Sichuan and adjacent areas in China.Additional file 1:Table S1. Original data of final diagnoses and quantitative PCR identification. (XLS 902 kb)


As a result of the Human Papillomavirus (HPV) vaccination program, the prevalence of precancerous dysplasia and invasive cervical cancer has substantially decreased. In this brief report, we present a case of a young patient who was diagnosed with in situ adenocarcinoma of the cervix. This 30-year-old female had completed the HPV vaccination after she became sexually active and has been undergoing annual gynecological assessments, including clinical examination and Pap test, all of which had been negative. This year, her Pap test revealed a low grade squamous intraepithelial lesion (LGSIL) and additionally a colposcopy was performed. Given the extent of the lesion and since the colposcopy was inadequate, the patient underwent a type 3 large loop excision of the transformation zone and a curettage of the endocervix under local anesthesia. The pathological diagnosis from cervical biopsy revealed an in situ adenocarcinoma of the endocervix with negative limits. The HPV subtypes 16 and 83 were detected with PCR. After proper consultation she decided to preserve her fertility and to undergo a regular follow-up, postponing hysterectomy after the completion of her family planning. In conclusion, this case report highlights the need for diagnostic surveillance regarding HPV-related cervical cancer even after vaccination. Human Papillomavirus (HPV) is a DNA virus, member of the papillomavirus family, which causes genital warts, precancerous dysplasia, and cervical, anal, and other anogenital and oropharyngeal cancers. HPV infection is the most common sexually transmitted infection with prevalence of up to 80% among sexually active individuals of both sexes . GenitalThis is the case of a 30-year-old sexually active nulligravida female presenting with an abnormal Pap test. The patient had a free gynecological and family medical history, she has been smoking since the age of 15 , and she had completed the HPV vaccination (three doses of the HPV vaccine) 6 years ago, after she became sexually active. There was no known history of STDs reported from her sexual partners  and she had the same sexual partner for the last 3 years.The patient had been undergoing gynecological assessment by her treating specialist, including clinical examination and Pap test in a private practice, annually, since the age of 20, and all of them had been negative; however, at the age of 29, the Pap test revealed for the first time a low grade squamous intraepithelial lesion (LGSIL). One year later, the patient was referred to the Cervical Pathology Department, Third Department of Obstetrics and Gynecology, General University Hospital “Attikon”, University of Athens; after the clinical and gynecological examination, liquid phase cytological examination and a colposcopy were performed. The cytological assessment revealed a high-grade squamous intraepithelial lesion (HGSIL) and the PCR HPV test  was positive for types 16 and 83. The dual staining immunocytochemical evaluation, using the CINtec® cytology double staining (p16/Ki67) kit, revealed p16 and ki-67 positivity.Because of the extent and the immunocytochemical features of the lesion, and since the colposcopy was inadequate (the transformation zone was type 3 and the squamocolumnar junction was not entirely visible), the patient underwent a type 3 large loop excision of the transformation zone (LLETZ) and a curettage of the endocervix under local anesthesia. The pathological diagnosis from cervical biopsy revealed an in situ adenocarcinoma of the endocervix with negative limits. HPV typing in the biopsy sample confirmed the positivity for HPV 16 and 83 was detected. In addition, there was positive immunostaining of the protein p16 and Ki-67 expression was 30%.After the counseling, the patient decided to preserve her fertility since she wishes to procreate. She was informed about the possible risks and decided to perform a hysterectomy by the completion of her family planning. One year after LLETZ, she remains without evidence of local recurrence or invasive cancer. Written informed consent was obtained by the patient for the publication of this case report.We present a case of cervical adenocarcinoma in situ (AIS) in a 30-year-old female with a positive HPV 16 typing test though fully vaccinated against HPV after the onset of her sexual life. This is a rare incident that is raising many questions about the efficacy and the safety of HPV vaccines. What remains unknown about this case is the time of the infection since the patient's annual Pap smears had been negative. One possible explanation may therefore be the efficacy of the vaccine. Since the two vaccines became available and were included in the National Vaccination Program of many countries including Greece, a variety of studies have been published concerning the impact of the vaccines on the prevalence of HPV 16 and 18 related precancerous lesions, in situ carcinomas, and invasive cancers.Villa et al.  in the 5In 2007 the members of the FUTURE II study group  publisheWong et al.  presenteIn 2012, Szarewski et al.  identifiIn another study by Gertig et al.  there arThe aforementioned studies may indicate that the VE may not always be 100%. Therefore, it might be helpful to monitor the immunological response of the patient after the vaccination by controlling the antibodies against the HPV viruses, although the clinical relevance of this test has not been fully established. In conclusion, vaccinated females should not consider themselves totally secure. It is highly recommended that they continue their annual gynecological examination, since the Pap smear and/or HPV typing have been proved an excellent method of secondary prevention.


Penile cancer (PeCa) is a rare malignancy with potentially devastating effects. Squamous cell carcinoma is the most common variant with distinct precancerous lesions before development into invasive disease. Involvement of the inguinal lymph nodes is the most important prognostic factor in PeCa, and once disease is present outside the groin, prognosis is poor. Metastatic PeCa is challenging to treat and often requires multidisciplinary approaches in management. Due to its rarity, molecular understanding of the disease continues to be limited with most studies based on small, single center series. Thus far, it appears PeCa has diverse mechanisms of carcinogenesis affecting similar molecular pathways. In this review, we evaluate the current landscape of the molecular carcinogenesis of PeCa and explore ongoing research on potential actionable targets of therapy. The emergence of anti-epidermal growth factor receptor (EGFR) and other immunotherapeutic strategies may improve outcomes for PeCa patients. Penile cancer (PeCa) is a rare malignancy in Europe and North America . The oveAlthough rare, PeCa is known for early locoregional and angiolymphatic spread . Thus, tIn the era of precision medicine, there has been increased interest in the use of targeted therapies, given that the response to standard chemotherapy in advanced PeCa is short-lived; however, the rarity of the disease makes it difficult to perform prospective randomized trials. In addition, the paucity of knowledge of PeCa molecular drivers presents an obstacle in developing novel therapeutic agents. In this study, we review the current understanding of the molecular pathogenesis of penile SCC and explore ongoing research and clinical trials on potential actionable targets of therapy that may help delineate future therapeutic paradigms for advanced PeCa.Advanced PeCa is challenging to treat and often requires a multimodal approach involving systemic therapies. Evaluation of the inguinal and pelvic nodes is an essential component during initial evaluation. Even for those presenting with clinically negative groins (cN0), the likelihood of metastatic disease approaches 25% . ChemothCurrent literature suggests immediate and prophylactic inguinal lymph node dissection (ILND) carries improved survival rather than a delayed or therapeutic ILND . For thoInPACT is a multinational, multidisciplinary collaboration which plans on recruiting 400 patients (200 from the UK and 200 from USA and Europe) with locally advanced SCC of the penis over a five year period. The study will help clarify the role of surgery and its integration with multimodal therapies. Patients will be divided into one of the following three treatment arms: 1. ILND with no neodjuvant treatment, 2. neoadjuvant chemotherapy followed by ILND, or 3. neoadjuvant chemoradiotherapy followed by ILND . AdditioPenile cancer even in advanced stages can be responsive to several chemotherapeutic agents. Presently, cisplatin-based regimens  are the most active first-line chemotherapy agents ,16,17. TAlthough unique aberrant pathways have been found in various malignancies, molecular mechanisms underlying PeCa carcinogenesis remain poorly understood. At present, PeCa is thought to arise from progression of precursor lesions, and can be subdivided into human papillomavirus (HPV)-dependent and HPV-independent pathways . An overHPV is a DNA virus with more than 100 different known genotypes and has been implicated in the development of penile carcinoma ,23,24. VE5, E6, and E7 oncogenes. However, only E6 and E7 oncogenes are necessary for malignant transformation and maintenance of malignant phenotype in host cells [E5 oncogene is not necessary for malignant transformation; however, it may contribute to carcinogenesis by manipulating viral uptake of host target cells. The E5 gene product is a transmembrane protein that regulates activation of epidermal growth factor receptor (EGFR). EGFR upregulation leads to a decrease in E-cadherin expression and associated increase in matrix metalloproteinase (MMP)-9 resulting in decreased cell-to-cell adhesion [E6 and E7 oncogenes contribute to carcinogenesis by disrupting centrosome synthesis required for mitosis. Thus, the development of multipolar mitosis is a hallmark feature of both HPV-mediated premalignant and malignant lesions. Additionally, E6 and E7 oncoproteins target the tumor suppressors p53 and retinoblastoma-1 (RB1) genes, respectively [HPV encodes the st cells . The actadhesion . The E6 ectively . These tE7 binds to the Rb tumor-suppressor with much higher affinity than low-risk HPV subtypes, such as HPV-6 and HPV-11. One of the major functions of Rb is to bind and inhibit transcription factors of the E2F-family which leads to downregulation of products involved in DNA and chromosomal replication [p16INK4A to accumulate in the nucleus and inhibiting G1 cyclin-dependent kinase 4 (CDKN4) and CDKN6; thus, leading to phosphorylation (inactivation) of the Rb tumor-suppressor protein [p16INK4A overexpression can serve as a surrogate immunohistochemistry (IHC) marker of disease, and could be a target for antigen-specific immunotherapy for men at significant risk of disease recurrence [There are 20 HPV serotypes which are known to infect the genital tract and are generally classified between low-risk (lr) or high-risk (hr) depending on their correlation with cervical malignancy. HPV-16 and HPV-18 have been found in around 31% of penile tumors with HPV-16 being the predominant subtype . In caselication . This in protein . Hence, currence . AlthougPenile carcinomas that are not a result of HPV infection are thought to be a consequence of precursor lesions in areas of chronic irritation/injury  that progress into neoplastic lesions. Even though the initial source of these precursor lesions has not been entirely elucidated, inflammation is understood to be fundamental to tumor development in these cases as many penile cancers arise from sites of inflammation . Inflamm. Other important mediators in inflammation-induced penile carcinogenesis are cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). COX-2 has been shown to be greatly expressed in PeCa [COX-2 is overexpressed, there is an overproduction of prostaglandins and thromboxanes, with PGE2 specifically playing a critical role in proliferation, angiogenesis, and activation of EGFR [PGE2 activates β-catenin-T-cell factor, which supports replicative potential and immortalization; and PI3K, which assists in cell migration and invasion [A key tumor suppressor gene of ROS/RNS damage is p16. Loss of heterozygosity of the p16 gene has been frequently observed in penile carcinomas; thus, it is possible this pathway plays a crucial role in penile carcinogenesis, specifically in the context of chronic inflammation . Other i in PeCa . When CO of EGFR . Additioinvasion ,34.p16INK4a/cyclinD/Rb and p14ARF/MDM2/p53 pathways [p16INK4a promotor region (inactivation) has been observed in 15% of hr-HPV negative cases [Evidence suggests that gene alterations  are more frequent in HPV-independent than HPV-mediated tumors ,36. As mpathways ,38. Hypeve cases . When coA few recently discovered molecular targets have been reported in the literature with encouraging findings. However, validation continues to be a struggle due to lack of preclinical PeCa systems to validate results. Nevertheless, these initial findings justify clinical trials investigating these novel targets. E6/E7 pathways appear to be promising actionable targets of therapy. In the cervical HPV experience, one phase II therapeutic vaccination study using a combination of synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 caused a 40% histopathological regression in women with HPV-16-positive or HPV-18-positive lesions [E6 and E7 reactive TILs [Targeting the ive TILs . AlthougPD-1)/PD-1 ligand (PD-L1) axis has been demonstrated to play an important role in tumor immune escape, and immunotherapies targeting this pathway have shown great success in other urologic malignancies [PD-L1 expression in penile cancer [PD-L1 and associated with poor disease specific survival (DSS) (p = 0.011) and lymph node metastasis (p = 0.024). One large study validated this high proportion of PD-L1 expression in which 48% (96/200) of patients stained positive, and mainly HPV-negative tumors [PD-L1 immunohistochemical assessment and scoring, these findings indicate PD-1/PD-L1 as a potential target in PeCa. Trials are being planned using anti-PD-1 (NCT02837042) or PD-L1 antibodies (NCT02721732) that may clarify the role of checkpoint inhibition in PeCa. The molecular link between unique genomic features and response to checkpoint inhibitors require further investigation. The program death-1  family is composed of EGFR, HER2, HER3, and HER4 transmembrane tyrosine kinase receptors [EGFR to be a common feature of penile carcinomas independent of histologic subtype, grade, and HPV status [EGFR is associated with increased risk of recurrence  and shorter overall survival in N0-1 patients  [EGFR monoclonal antibody panitumumab [EGFR-targeted drugs cetuximab, erlotinib or gefitinib, alone or in combination, showed a 23.5% partial response with cetuximab having increased antitumor activity for those treated with cisplatin-based chemotherapy [p = 0.077) when compared to those who received taxane-based regimens alone [EGFR pathway plays an initial role in penile carcinogenesis, those with advanced disease continue to benefit from standard chemotherapy regimens in addition to anti-EGFR therapy. A prospective randomized trial evaluating cetuximab and TIP (NCT02014831) was initially set to begin enrollment but was withdrawn due to lack of industry drug supply.The human epidermal growth factor receptor  in 6 patients who had previously received at least two chemotherapy regimens. One partial response and 4 stable disease responders were observed . AdditioPI3K pathway have been implicated in various malignancies [PTEN and amplification of PIK3CA and Akt. One study found HPV-negative tumors expressed more activated EGFR than HPV-positive ones and this expression correlated with activated Akt, implicating EGFR as an upstream regulator of Akt signaling in penile cancer [HER3 expression was significantly more common in HPV-positive cases and positively correlated with cytoplasmic Akt1 expression. Currently two companion trials targeting the HER pathway in advanced penile SCC are underway. One 1st-line/neoadjuvant trial using Dacomitinib (NCT0172833), a potent, irreversible kinase inhibitor of human EGFR/HER1, HER2 and HER4, has shown anti-tumor activity in N2-3 and M1 patients. Preliminary results have demonstrated a 42.8% (6/14) progression free rate with median PFS of 4.47 months and median overall survival (OS) of 11.9 months. The most common side effects were skin toxicity in 7 patients, diarrhea in 2 and bleeding cutaneous metastasis in one [EGFR amplification in 4 responders and mutations in HRAS, BRAF, PIK3CA, PTEN, and STK11 in 47% of non-responders with potential associated resistance to EGFR inhibitors [HER pathway with preliminary data thus far linking molecular alterations with clinical response.Genetic alterations of genes in the gnancies . Such ale cancer . Converss in one . Updatedhibitors . AnotherMYC amplification was first reported with integration of HPV DNA sequences [EGFR than HPV-positive tumors with corresponding increases in pAkt expression [Other than HPV-driven transformation, little is known about the molecular alterations during the development of PeCa. Significant advances in genetic sequencing have allowed discovery of multiple genomic alterations occurring during PeCa progression. For those HPV-positive cases, equences . Converspression . FurtherCDKN2A locus which codes the tumor suppressors p16INK4A and p14ARF. Co-inactivating mutations in CDKN2A and p53 were observed more frequently in lichen sclerosus-derived tumors than in HPV-derived cases (p = 0.053) in one study . Another study identified p53 expression along with p16INK4A negativity in HPV-negative tumors [CDKN2A was correlated with negative and weak expression of p16 in one study, with all of the HPV-negative cases having weak or no p16 expression [p16INK4A [p16 expression and methylation differences of CDKN2A between HPV-positive and negative tumors support the hypothesis that PeCa develops from distinct molecular pathways with likely inherent differences in therapeutic approaches.Epigenetic modifications are potentially reversible alterations in DNA methylation or chromatin that are not associated with changes in DNA sequence . Publishe tumors . Hypermepression . Lymph np16INK4A . These dp16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or survival. Another study using whole exome sequencing of 27 PeCa samples revealed 810 genes containing somatic mutations with a mean somatic mutation rate of 30 per sample [p < 0.05). Another study of 25 patients treated with first-line cisplatin-based chemotherapy evaluated expression of 738 genes using NanoString technology [MAML2 (p = 0.004), KITLG (p ≤ 0.0001), and JAK1 (p = 0.029) genes were associated with poor OS, and upregulated FANCA was associated with better OS (p = 0.024). Acquired mutational changes in these genes may help explain mechanisms of resistance to first-line chemotherapy, thus warranting further evaluation as therapeutic targets.With the arrival of deep targeted sequencing, more studies on genetic alterations have been possible in the last few years. In one of the first molecular studies, next-generation sequencing identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample . Furtherr sample . Of notechnology . In univ4p15.2, 9p22.3, 19p13.2, 19p12 and 19q13.2. Distinct patterns of copy number gains were noted for both the HPV-positive  and lymph node positive samples (3q26.2 and 11q22.2). These copy number variations included known oncogenes MYC and FGFR3 as well as tumor suppressors CDKN2A, CCND1, RB1and p53. Another study used array comparative genomic hybridization combined with HPV genotyping [3p and 8p were related to worse prognostic features including advanced T stage, recurrence, and death from disease. Losses of 3p21.1–p14.3 and gains of 3q25.31–q29 were associated with reduced DSS. Loss of DLC1 was an independent risk factor for recurrence on multivariate analysis. The study was significant in showing that recurrent copy-number alterations have a prognostic value in PeCa. Copy number variation, in which a considerable number of base pairs are duplicated or deleted, is a relatively new field in genomics. One group analyzed copy-number aberrations in 24 patients using high density genome wide methylation arrays . Signifinotyping . GenomicMMP1 and MMP12 may be regulated by hsa-miR-145-5p, which was down-expressed in PeCa tissues [MMP1 showed increased expression in patients with lymph node metastasis. A recent study reported loss of miR-1, miR-101, and miR-204 associated with lymph node metastasis and unfavorable prognosis [Studies have suggested miRNA, which modulates gene expression at posttranscriptional level, is frequently dysregulated and aberrantly expressed in human cancers ,68. One  tissues . Althougrognosis . In termMetastatic penile cancer has a poor prognosis after treatment with standard chemotherapy agents. The incorporation of molecular panels has the potential to increase available prognostic and therapeutic capabilities. It is clear penile cancer has distinct molecular pathways with diverse genetic and epigenetic changes with potential therapeutic implications. More translational research and collaboration is needed to continue to develop novel diagnostic biomarkers and effective therapeutic strategies for PeCa patients. Thus far, preliminary data on molecular alterations linked to clinical benefits are being reported. A better understanding of the basic biology of penile cancer can help design future prospective trials and offer insights into potential precision medicine approaches for this rare disease.