The emergence of multiresistant bacterial strains as agents of healthcare-related infection in hospitals has prompted a review of the control techniques, with an added emphasis on preventive measures, namely good clinical practices, antimicrobial stewardship, and appropriate environmental cleaning. The latter item is about the choice of an appropriate disinfectant as a critical role due to the difficulties often encountered in obtaining a complete eradication of environmental contaminations and reservoirs of pathogens. The present review is focused on the effectiveness of hydrogen peroxide vapor, among the new environmental disinfectants that have been adopted. The method is based on a critical review of the available literature on this topic The disinfection of hospital surfaces is a complex operation aimed at reducing the pathogenic microorganism load. An ideal disinfectant must be safe for human health. It may have a good stability in the environment and may be free of toxic activity ,2,3,4. Hydrogen peroxide is a versatile disinfectant, since it can be used in several environmental matrices: air, water, wastewater, surfaces, soil, etc. It may be used in combination with other agents increasing the disinfection times. Hydrogen peroxide is more oxidizing than chlorine and chlorine dioxide. The disinfection mechanism is based on the release of oxygen free radicals, which cause genomic damage in bacterial cells ,6.Hydrogen peroxide in the vaporized form (HPV) is used for surface disinfection. It may be combined with higher or lower concentrations of silver ions ,8,9.the type of microbial strains sensitive or resistant to the compound;the adequate chemical concentrations; andthe contact time.HPV bactericidal activity may be experimentally evaluated in vitro  or in hoThe management of the spread of multidrug-resistant bacteria (MDR) in the hospital setting ,13 is a The incidence of infectious outbreaks from antibiotic resistant microorganisms is becoming one of the main problems in hospital contexts . ConsideAcinetobacter baumannii, and spore-forming bacteria (Clostridium difficile), which are frequently responsible for nosocomial infections [All hospital settings need an accurate evaluation of new disinfection efficacy against bacterial species such as carbapenem-resistant enterobacteria, multidrug-resistant fections .The aim of this review is to evaluate the main studies performed on HPV activity for MDR prevention in vitro and in hospital settings .Escherichia coli strains, mostly in oxyR and Son of Sevenless (SOS) regions, involved in the shelter of genomic damage caused by oxidizing agents [The bactericidal activity of hydrogen peroxide was studied in several in vitro tests. In 1987, a Californian working group published data related to the ability of hydrogen peroxide (30–100 ppm) to cause DNA damage in different g agents ,23.E. coli, Proteus mirabilis, and Klebsiella pneumoniae. The antimicrobial activity of the disinfectant was proved in bacterial suspension and on contaminated steel surfaces (20 cm2), applying 0.3% HPV with 30 ppb silver ions. The microbial growth was monitored at different concentration ranges and for a total time of 24 h. A significant reduction in microbial loads was detected on surfaces .Further in vitro studies were performed by the Asian group of Absalan , who demStaphylococcus aureus American Type Culture Collection (ATCC) 6538 strain, Pseudomonas aeruginosa ATCC 15442 strain, and several clinical isolates of multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa. A good disinfectant activity was obtained in suspension and on surfaces. The efficacy of the biocidal compound varied according to the different action times.An Italian research group  tested iFurther studies ,27 evaluClostridium difficile spores and three antibiotic-resistant strains, MDR Acinetobacter baumannii, vancomycin-resistant (VRE) Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus (MRSA) were selected for the study. The disinfection procedure was divided into three HPV nebulization cycles, for a total of 50–52 min. The HPV concentration peak measured in the air was 500–600 ppm, followed by room aeration, for a total of 2–3 h. Results showed a significant reduction of all microbial species between 4.0 log (VRE) to 5.1 log (MDR A. baumannii). Differences between the different positions and supports (steel and cotton) were not observed. This study demonstrated the effectiveness of HPV on porous surfaces also, a result that was not always guaranteed by disinfectants [Lemmen  assessedfectants .Pseudomonas spp., Enterococcus spp., Staphylococcus spp.). Lower bactericidal activities were observed for the Enterobacteriaceae family  and for Acinetobacter baumannii (about 1 log10).A similar work was carried out by Herruzo , who tesGeobacillus stearothermophilus spores was achieved by using 5% and 10% HP for 70 min [A comparative study evaluating the efficacy of 5%, 10%, and 35% HP, showed a 6 log reduction of MRSA after 30 min of 35% HP treatment. The full kill of r 70 min .Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus) was obtained using a drip-flow reactor. The biofilm was treated with 35% HPV, which allowed a log10 reduction of all the microbial species present in the biofilm in 100 min [A pilot study investigated the role of HPV in biofilm eradication. An artificial biofilm composed by MDR bacteria  values from 0.5 to 20 mM, after disinfectant exposures from 1 min to 24 h. Similar results were showed on the same microorganisms’ associated biofilms.In 2014, some authors  tested tClostridium spp. [Bacillus spp. Starting from data obtained from a previous study (Boyce), which highlighted the ability of HPV in spores’ inactivation of a wide range of Clostridium difficile strains present on metal disks, the Hospital Saint-Antoine group [Clostridium difficile genotype 027/NAP1/BI using 2 cm2 polyvinylchloride (PVC) carriers contaminated with 1 × 106 spores/carriers. Results showed a complete spore eradication after 15 min of 30% HPV treatment.Further research groups tested the HPV activity on spore-forming bacteria ,33,34, sium spp. ,36 and Bne group  tested tBacillus spp. spores. Variable results were obtained for the different study conditions, such as the different tested surfaces (porous and nonporous).A similar work was carried out in 2005 by an American group , who tesMycobacterium tuberculosis was achieved in an in vitro test after HPV treatment [Several studies showed a good HPV activity against standard strains of tuberculous mycobacteria ,40,41. Treatment .Mycobacterium tuberculosis H37Rv (ATCC 27294) strain inside biological safety cabinets (BSCs). Results showed a 3 log reduction of Mycobacterium tuberculosis in less than 30 min of HPV treatment.Hall et al.  tested tPrincipal in vitro tests are summarized in Acinetobacter baumannii strains [Staphylococcus aureus strains [Several studies evaluated the role of HPV in environments contaminated by multidrug-resistant  strains ,45,46,47 strains ,49,50.Acinetobacter baumannii. The same strain was isolated in 7% of the sampled surfaces [Acinetobacter baumannii in some of the tested surfaces after repopulation of the room.Experimental tests  were dirsurfaces . After cAcinetobacter baumannii complex (ACB) [Staphylococcus aureus (MRSA) [In 2011, further studies highlighted the efficacy of HPV in reducing ex (ACB)  and meths (MRSA)  from colIn further studies ,56, HPV Acinetobacter spp. and Enterobacter cloacae [Acinetobacter spp. or Enterobacter cloacae infections occurred in the four months following the procedure, demonstrating the efficacy of the new disinfection method in an endemic situation. It should be noted that the microorganisms reappeared with pulsed-field gel electrophoresis (PFGE) profiles similar to those present before the new disinfection. This occurrence may originate from the mobile medical equipment transported from outside the wards.HPV efficacy on bacteria was also studied by Otter , in a st cloacae  infectio cloacae ,60 and tp = 0.004). After the standard cleaning, 6% of the rooms were still contaminated, while after the HPV treatment this percentage decreased to 0.5%.A working group  publisheClostridium difficile [p < 0.05). The same test was performed using 0.5% sodium hypochlorite, which reduced only 50% of the contamination.A research group  assessedifficile . Surfacep < 0.001) in the Clostridium difficile infection level at the time when 35% HPV disinfection was implemented. Results showed the reduction in the infection rate from 1.0 to 0.4 cases per 1000 patient-days in the 24 months before HPV usage compared with the first 24 months of HPV usage [Another study indicated a significant reduction , proving to be a useful method for the decontamination of critical surfaces such as the curtains.Rutala et al.  tested HExperimental tests performed in hospital settings with HPV are summarized in Literature data published by different research groups highlight a good antimicrobial and sporicidal hydrogen peroxide activity. In vitro experiments show that values from 0.3% to 35% of HP may reduce the microbial loads of various opportunistic pathogens (in suspension and on different types of contaminated surfaces). These results encouraged several tests directly performed in the hospital settings. HPV has been used with encouraging results as an additional method associated with the standard disinfection systems. This approach was mostly implemented in wards with severe environmental contamination levels and endemic cases of MDR infections. The application of devices able to atomize hydrogen peroxide in aerosols, at concentrations of not less than 240 ppm and for different contact times, was effective in reducing the contaminations of all the tested microbial species.Acinetobacter baumannii and Clostridium difficile. In fact, HPV disinfection allowed a 91% reduction of these resistant types of bacterial strains.Moreover, several encouraging results have been obtained on MDR E. coli, Proteus mirabilis, and Klebsiella pneumoniae. A similar efficacy was also observed in hospital wards and intensive care units with surfaces previously colonized by Enterobacter cloacae.Considering the Enterobacteriaceae family, many studies confirmed the in vitro efficacy of HPV against Considering the evidence that normal cleaning/disinfection cycles do not guarantee the same efficacy as HPV treatment, it is possible to consider this system as a recommended method for highly contaminated environments. Moreover, this strategy may be useful in situations where routine sanitization and infection-control measures do not stop the infectious transmission cycle.Klebsiella pneumoniae) suggest the possible application of this system in the hospital environment as a new frontier in the prevention and control of healthcare-related infections, mostly in endemic and prolonged infective conditions.Encouraging results obtained on Enterobacteriaceae (in vitro also on HPV, used in the past as one of the first disinfectants and then forsaken with the introduction of chlorine-based compounds, is therefore proposed today as a highly innovative method for systematic application in the most updated hospital disinfection protocols.


A significant subset of carcinomas developed in the head and neck (H&NCs) are associated with specific human papillomaviruses (HPV) genotypes. In particular, 40–60% of oropharyngeal carcinoma cases are linked to HPV. Epidemiological studies have demonstrated that HPV oral infections are predominantly sexually transmitted and are more frequent among men (10–18%) than women (3.6–8.8%). Although there is a large diversity of HPV genotypes associated with H&NCs, HPV16 lineage represents 83% of the reported cases. The prognostic value of HPV as a biological parameter is well recognized. However, the use of HPV DNA as a diagnostic and/or predictive marker is not fully developed. Recent data reporting the physical state of the HPV genome in tumors have shown that HPV DNA integration into the tumor cell genome could lead to the alteration of cellular genes implicated in oncogenesis. Most importantly, HPV DNA corresponds to a tumor marker that can be detected in the blood of patients. Profile of the HPV DNA molecular patterns in tumor cells using New Genome Sequencing-based technologies, allows the identification of highly specific tumor markers valuable for the development of innovative diagnostic and therapeutic approaches. This review will summarize recent epidemiological data concerning HPV-associated H&NCs, the genomic characterization of these tumors, including the presence of HPV DNA in tumor cells, and will propose perspectives for developing improved care of patients with HPV-associated H&NCs, based on the use of viral sequences as personalized tumor markers and, over the longer term, as a therapeutic target. The first aim of this review is to summarize recent data concerning the pathology of head and neck carcinomas (H&NCs) associated with the human papillomaviruses (HPV), including prevalence and viral epidemiology which characterize these tumors as well as the specificities of HPV DNA as a tumor marker. In the second aim, we will explore specific perspectives focused on the use of HPV DNA as a prognostic tumor marker in the blood of patients with H&NCs and on the developments of new applications in clinical oncology related to the introduction of New Genome Sequencing (NGS) approaches as tools for the optimized characterization of viral DNA in the tumor cells of HPV-positive H&NCs.The reported worldwide prevalence of HPV-associated H&NCs varies between 25.9  and 30% In the general population, the prevalence of asymptomatic HPV infection was assessed by viral analyses of oral rinse specimens. The reported infection statistics ranged from 6.9% in the United States  to 13.1%vs no sexual relations (7.5 vs 0.9%). The prevalence increased with the number of partners but no evidence for increased risk related to particular sexual behavior was observed. However, one explanation for the observed higher prevalence among men could be attributed to the higher probability of HPV transmission through oral sex with women vs men. Indeed, oral HPV prevalence increased more sharply with the number of sexual partners for men than women   is signi identified thousands of unique HPV16 viral isolates , and PIK3CA and focal amplification of E2F1 whereas HPV-negative cases are characterized by a high rate of TP53 mutations and abrogation of the G1/S checkpoint via CDKN2A/B deletion and/or CCND1 amplification. In line with an enhancement of cell proliferation induced by the E7 HPV protein   to 71.4%and TTC6 , 40.Many strategies for HPV characterization in H&NCs have been used , 43 inclThe outcome of HPV-associated H&NCs is better than that of HPV-negative cases . In a meversus 88% for viral-negative cases  the development of the detection of circulating tumor DNA (ctDNA) using HPV sequences as a tumor marker, (b) the introduction of NGS methods for the optimal molecular characterization of viral DNA in clinical oncology, and (c) the analysis of the tumor immune microenvironment in the frame of immunotherapeutic protocols.via the presence of somatic mutations, but the low rate of target molecules, especially in early stage tumors, implies that their detection can be affected by stochastic sampling leading to a lack of target molecules in some specimens  High rates of ctHPV DNA positivity are observed at diagnosis in patients with HPV-associated H&NCs; (II) The ctHPV DNA level is poorly related to tumor volume; (III) ctHPV DNA is already detectable in patients with subclinical disease. In clinical oncology, the first potential application is the possibility of using ctHPV DNA detection as an alternative approach to histology for the diagnosis of HPV-associated invasive carcinoma. This new approach could notably be used for the diagnosis of relapse in patients previously treated for an HPV-associated tumor and presenting abnormal imaging. In this situation, the detection of ctHPV DNA should be a sufficient criterion to confirm the diagnosis of relapse, avoiding unnecessary biopsy procedure that may cause morbidity. However, the question of the specificity of this approach has to be considered since some cases may be difficult to interpret. We previously provided a proof of concept showing that the highly specific viral insertional signature could be detected in the blood of patients  and thatThe comparison between virological and clinical data showed that ctHPV DNA load at diagnosis is poorly indicative of disease outcome , 77. ThiThe high level of sensitivity and specificity of ctHPV DNA as a tumor marker in patients with HPV-associated carcinomas advocates for use of this marker to improve the biological follow-up of patients. The clinical relevance of this approach has been documented in colon carcinomas . HoweverThe detection of HPV DNA in biological specimens is commonly performed using q-PCR technique. However, this technology is not sufficient to allow the identification of the specific molecular markers useful for the diagnostic or follow-up purposes described above. In contrast, NGS is a powerful tool able to provide, in a single experiment, the extensive molecular characterization of HPV DNA in tumors: the complete nucleotide sequence of the viral genome (genotyping), its host chromosomal modifications , physical status, integration signature, chromosomal integration site(s), and identification of the gene(s) located in the vicinity , 81–84 Figure 1Importantly, the NGS-based method developed for the molecular characterization of HPV DNA in cervical tumors can be successfully applied to the detection and characterization of ctHPV DNA . The NGSThe important role of the immune microenvironment as a major feature for tumor response to therapies of subsequent disease outcome has been underlined. Among the various parameters that can be analyzed, includes the density of the effector T cells (CD8+) and of PD-L1 in both immune cells and tumor cells that represent key prognostic and predictive parameters recurrently found in several studies –60. The versus negative)  . HoweverHPV-associated tumors represent a privileged model for the analysis of ctDNA and viral sequences constitute a very convenient biological role model to assess the course of the disease. ctHPV DNA is a sensitive tumor marker and this should facilitate the clinical validation of the “liquid biopsy approach” for the diagnosis of invasive carcinomas, for instance in case of suspicion of relapse. This validation will require the analysis of various tumors differing in size, localization, and viral genotype before the ctHPV DNA approach could be implemented as a recognized diagnostic tool in clinical oncology, avoiding more invasive procedures such as biopsies or fine needle aspirations. The ctDNA load at diagnosis is a poor prognostic marker, but its dynamic during the treatment is a promising predictive surrogate of tumor response. During the follow-up procedures, the clinical validation of ctHPV DNA as a predictive marker of relapse will solicit prospective studies demonstrating that all of the biological relapses precede clinical relapses. Difficulties need to be overcome. For instance, current studies report that advanced cases of HPV-positive tumors remain ctHPV DNA negative and the identification of the parameters accounting for this discrepancy is a prerequisite for a large use of ctDNA as a surrogate marker of the course of H&NCS.Once these clinical validations are obtained, a major challenge remains to document the clinical utility of the diagnosis of sub-clinical disease, which depends mainly on the possibility of treatment. New tools will be necessary for the care of subclinical relapses and viral-associated tumors represent an attractive model for the development of immunotherapy. As an example, the treatment of high grade cervical intra-epithelial neoplasia using therapeutic HPV16/18 vaccine is currently in evaluation and provides encouraging results . Such anThe analysis of a large series of H&NCs using NGS approaches should enhance our knowledge about the biology of these tumors and favor further developments in diagnosis, follow-up, and treatments. The implementation of large data bases collecting the biological and clinical data obtained will be a powerful common tool favoring these advances.XS-G wrote the manuscript and AH reviewed the manuscript. All authors contributed to the article and approved the submitted version.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.


Vulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections.INK4a.VSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type-specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed for HPV RNA and for upregulation of the cellular protein p16INK4a-positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110.The rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16A small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely. Vulvar squamous cell carcinoma (VSCC) is a rare tumor of the female genital tract, accounting for about 5% of all gynecological malignancies , 2. In NBased on a pooled prevalence rate of HPV DNA, 34% of vulvar cancers have been reported to be attributable to HPV .INK4a (p16) or/and detection of viral RNA, may enable more precise estimation of the proportion of VSCC that may be attributable to HPV, as previously shown in head and neck cancer  age, 73 [11.8] years) collected during 2013–2016 were retrieved from the archive of the Department of Obstetrics and Gynecology, University of Turin, Sant’Anna Hospital, Turin, Italy. According to the International Federation of Gynecology and Obstetrics (FIGO) classification of vulvar cancer , the VSCTen sections of 10 μm from each FFPE tissue block were processed at the Department of Pathology, University of Turin, Italy. Sections S1 and S10 were used for hematoxylin and eosin histology, and S2 and S9 were used for p16 IHC. In addition, S3–S5 and S6–S8 were collected for DNA and RNA extraction, respectively. To avoid cross-contamination between different samples, each time a new case was sectioned, the microtome was extensively washed with DNA Away  and a new microtome blade was used. Empty paraffin blocks were cut after every 10 cancer specimens and blindly analyzed. Tumor size/status was documented by routine pathologic evaluation, in accordance with the World Health Organization (WHO) classification .Total DNA was prepared by incubating overnight at 37 °C three internal FFPE sections (S3–S5) from each VSCC/MTS tissue in 250 μl of digestion buffer . Then, to inactivate the proteinase K and to separate paraffin from the aqueous phase, samples were incubated at 95 °C for 10 min, centrifuged, and chilled on ice . The aquTotal RNA was prepared from tissue sections S6–S8 using the PureLink FFPE Total RNA Isolation Kit . DNase I  treatment was carried out on the RNA purifying columns during sample processing as previously described . ExtractHPV DNA was detected by E7 type-specific multiplex genotyping (E7-MPG), which combines multiplex polymerase chain reaction (PCR) and hybridization to type-specific oligonucleotide probes on fluorescent beads  , 34. E7-The sensitivity has been evaluated using serial dilutions of DNA from HPV types. This multiplex PCR protocol is highly sensitive, with the ability to detect only 10 copies of the viral genome , 40.All HPV DNA-positive VSCC/MTS tissues, including HPV16 and non-HPV16 DNA-positive samples, and a group of 10 randomly selected HPV DNA-negative tissues were analyzed for HPV16 E6*I mRNA and, as a control for tissue and RNA quality, for human UbC mRNA. Tissues that were DNA-positive for HR- and pHR-HPV types other than HPV16 were also analyzed for E6*I mRNA of the specific HR- and pHR-HPV types(s) detected. In addition, tissues that were DNA-positive for LR-HPV6 or 11 were analyzed for the presence of unspliced E6 mRNA of HPV6 and 11, respectively. RNA detection from FFPE sections was performed as previously described . BrieflySamples that were negative in duplicate assays for HPV E6*I or E6 and for UbC mRNA were considered RNA-invalid.Expression of p16 was evaluated manually by IHC in FFPE sections using the CINtec p16 Histology Kit  according to the manufacturer’s instructions and as previously described . Expressn = 3) and p16 (n = 2) data, or to the absence of cancer tissue in the block (n = 1). Thus, a total of 102 VSCC and 7 MTS tissues were included in the analysis. The 102 VSCC cases had a mean age of 70 years ; FIGO stages were IB (52.9%), II (1%), IIIA (20.6%), IIIB (10.8%), IIIC (4.9%), and IVA (9.8%). Mean tumor diameter was 30 mm  and mean depth of stromal invasion 8 mm , with 40.2, 47.1 and 12.7% of tumors showing depth of invasion of < 5 mm, 5–12 mm, and > 12 mm, respectively. Furthermore, MTS selected from 7 cases were analyzed .Of 107 VSCC and 8 MTS cases initially selected, five VSCC cases and one MTS case were excluded due to invalid RNA (n = 1), HPV6 (n = 3), and HPV56 (n = 1). Multiple HPV infections were detected in two MTS cases; both were positive for HPV6 and HPV16 (Table n = 2) and HPV16 (n = 1). There was no concordance for the HPV types in the remaining MTS cases (n = 4) and the corresponding VSCCs (Supplementary Table STable n = 6), HPV53 (n = 1), HPV56 (n = 1), and HPV58 (n = 1). One VSCC case simultaneously tested positive for HPV DNA and RNA from HPV16 and HPV56. Of the 7 mucosal HPV DNA-positive MTS cases, only one (14.3%) had E6*I mRNA of the same type (HPV16). None of the HPV6 DNA-positive cases tested positive for full-length HPV6 E6 mRNA.To evaluate the etiological role of the mucosal HPV detected, all 17 HPV DNA-positive VSCC and all 7 MTS cases were analyzed for the presence of HR- and pHR-HPV E6*I and unspliced E6 for LR-HPV (Table n = 6) were HPV DNA-positive and HPV RNA-negative (n = 2) or HPV DNA-negative and HPV RNA-negative (n = 2), or were not tested for HPV DNA and RNA (n = 2). Only one MTS case showed p16 overexpression , and was also positive for HPV16 DNA and RNA. The corresponding VSCC from the same case was also positive for all three markers.Overexpression of the cell-cycle inhibitor p16 is a well-validated surrogate marker of HPV transformation in the cervix . We deteThe presence of DNA of cutaneous beta and gamma HPV in cytological samples of the genital tract has been reported , 37, 44.Mucosal HR-HPV types have been clearly associated with the development of a small subset of VSCC. However, the contribution of the viral infectious agents appears to vary in different geographical regions . In partIn the first step, we investigated the presence of HPV DNA in VSCCs by using a sensitive HPV genotyping approach covering all oncogenic and probably/possibly oncogenic HPV types. The prevalence of HPV DNA-positive cases was observed to be nearly 17%. Previous studies on VSCC reported HPV DNA frequencies ranging from 29 to 45% , 45, 46.CDKN2A, the gene coding for p16 [Because HPV transcriptional activity may reflect its oncogenic activity, we applied a HPV-type-specific E6*I mRNA detection method, established for 20 HR- or pHR-HPV types and validated for use in FFPE tissues, to evaluate the presence of HPV RNA in HPV DNA-positive VSCCs. We observed viral RNA in 47.1% 8/17) of HPV DNA-positive VSCC cases, with HPV16 RNA showing the highest prevalence. Eight of 102 VSCC cases (7.8%) were likely to be HPV-driven, i.e. positive for both HPV DNA and RNA. None of the HPV6 DNA-positive samples tested positive for HPV RNA. The upregulation of p16 has been suggested as a surrogate marker for oncogenic HR-HPV infections, and p16 IHC has frequently been used not only to determine and/or confirm positivity for HPV in various tumors but also to define HPV-related tumors . Therefo of HPV D for p16 , or the  for p16 .Previous reports showed higher concordance between the HR-HPV transcript expression and p16 overexpression, ranging from 83 to 90% , 21, 49.In this study, we were also able to retrieve MTSs for a small number of patients with VSCC. Only one case showed concordance in HPV DNA/RNA positivity and p16 expression between VSCC and MTS.The investigation of the presence of HR-HPV E6 mRNA instead of analyzing E6 oncoprotein, which may be more informative on the oncogenic implications of HR-HPVs in VSCC development, may be considered a limitation of this study. However, we focused our attention on evaluating the expression of the p16 protein, which is considered a surrogate marker for all transforming HR-HPV infections and represents a single target molecule, different from the E6 proteins with type-specific polypeptide sequence variability.Finally, although independent studies have reported that cutaneous beta and gamma HPV types can be detected in the genital tract, our study does not provide evidence for their involvement in the development of VSCC.We characterized the HPV-independent and HPV-related VSCCs in a cohort of more than 100 VSCC cases from Italy. We identified as putative HPV-related tumors a small proportion of VSCC cases that tested positive for both HPV DNA and RNA. Our results support the causative role of HPV in vulvar carcinogenesis in a distinct subset of cases and highlight the prevalence of HPV16, thus reinforcing the importance of HPV vaccination programs.Additional file 1: Table S1. HPV DNA, HPV RNA and p16 status in 102 VSCC and 7 MTS individual cases.


A “concerning post” is a display of a user’s emotional crisis on a social media platform. A better understanding of concerning posts is relevant to suicide prevention, but little is known about social media users’ attitudes and responses to concerning posts. Military veterans in the United States are disproportionately affected by suicide, often use social media, and may have exposure to individuals with elevated suicide risk via concerning posts.The objective of the study was (1) to obtain insight into whether and how US military veterans respond to members of their social network on social media  who are experiencing substantial emotional distress, and (2) to identify potential interventions that could assist in users’ response to concerning posts.We recruited veterans through Facebook and conducted semistructured interviews with 30 participants between June and December 2017. We used a summary template for rapid analysis of each interview, followed by double-coding using a codebook based on topic domains from the interview guide. Members of the research team met regularly to discuss emerging patterns in the data, generate themes, and select representative quotes for inclusion in the manuscript.Veterans were reluctant to disclose emotional and health issues on Facebook, but they were open to reaching out to others’ concerning posts. There was a complex calculus underlying whether and how veterans responded to a concerning post, which involved considering (1) physical proximity to the person posting, (2) relationship closeness, (3) existing responses to the post, and (4) ability to maintain contact with the person. Veterans desired additional training, backed by community-based veteran organizations, in how to respond to concerning posts from peers.There is a need to incorporate features that will help veterans effectively respond to concerning posts from peers into suicide prevention training and to expand access for veterans to such training. As social media has become embedded into our daily lives, researchers have taken on the task of unpacking its myriad implications for our health. Studies of social media have often focused on examining psychosocial harms from exposure to social media, such as loneliness , negativAn especially delicate but important health concern in the social media landscape is suicide prevention. Suicidal individuals are more likely than nonsuicidal individuals to spend time online . ExpressAmidst this contemporary reality, the notion of a “concerning post” has emerged, which we define as a display of a user’s emotional crisis or other indication of an acute mental health concern on a social media platform. Little is known about individuals’ attitudes and responses to concerning posts. Among the available studies, results have conflicted at times. For instance, one study found that youth lacked confidence in how to respond to a concerning post , while aWhile much of the research in this area has focused on youth, military veterans are another key target population for suicide interventions. Veterans in the United States are disproportionately affected by suicide. Their rate of suicide is 50% (1.5 times) higher than that of nonveterans, according to the most recent available data . SuicideUsing data from interviews with veterans who use social media, our objectives in this study were (1) to obtain insight into whether and how veterans respond to “friends” and family members on social media who are experiencing substantial emotional distress, and (2) to identify potential interventions that could assist veterans in responding to concerning posts.This research was conducted as part of a mixed-methods study focused on understanding opportunities to use social media platforms, particularly Facebook, to reach and provide support to veterans at risk of psychiatric problems. We interviewed Operation Enduring Freedom, Operation Iraqi Freedom, and Operation New Dawn veterans with a service separation date after October 1, 2001, who were in the metropolitan area of Portland, Oregon, United States. Thirty participants were interviewed—a number chosen based on the literature, feasibility, and our research team’s experience reaching thematic saturation with such a sample size ,19.Participants who had responded to Facebook advertisements and completed an online survey were eligible for participation in the current study. The survey that was administered has been previously described ,21; in bFifty individuals were contacted to participate, and 30 of them completed an approximately 40- to 60-minute interview between June and December 2017. Twenty-three interviews were completed in person; the remaining seven interviews were conducted over the phone, which allowed for participation by individuals who could not present in person to participate in the study. Two research team members (HM and AT) conducted the interviews using a semistructured interview guide . QuestioSurvey responses were used for participants’ descriptive characteristics. For qualitative analysis, we first created a summary template for rapid analysis of each interview ,23. We tA summary of participants’ characteristics is presented in Veterans were reluctant to disclose emotional and health issues on social media but were open to reaching out in response to others’ concerning posts. Interviews revealed multiple reasons for veterans’ reluctance to share negative emotional states or information about their health. First, Facebook was viewed as a venue for positive self-presentation; thus, they were disinclined to use it as a place to “air dirty laundry.” Second, when veterans did need support, they usually preferred to obtain it from other avenues in the “real world” that involve “person-to-person interaction,” or they preferred to be self-reliant. Also, veterans believed that sharing personal health information was “risky” to their privacy. Lastly, participants noted that Facebook friends were not necessarily “real friends.”While most veterans were cautious about disclosing their own health issues, a number of veterans were understanding of others who chose to post about health issues. One participant summarized this distinction:I think for some people they find that they can post about their bad day, and they get a bunch of people “like” it, a bunch of people put the sad face on, a bunch of people say, “You’re amazing. You’re brilliant. You go girl.” That kind of thing. I’ve definitely seen a ton of people do that, but at the same time I would never do that. And it’s not like I don’t want support, but I feel like I would be negatively judged for that.OscarFurther, many veterans were open to responding when others disclosed personal information in a post, particularly emotional struggles or suicidality.I’ve seen other friends do it, and I’ve come to the aid of other friends because they have shared it on Facebook out in the open just to their timeline or whatever. I’ve also helped friends who have reached out to me personally and [sent] Facebook messages and said, “I’m having this crisis. I can use some words of encouragement or some words of advice or some help just in general.”BryanParticipants identified a post as concerning in several ways. Most obviously, this was direct language expressing emotional distress, particularly when the distressed person had a known mental health condition , when the person was acting out of character , or if the person had one or more recent psychosocial stressor, such as divorce/breakup, death of a loved one, or loss of a job. Several veterans also expressed a feeling of concern without an identifiable cause, noting, for instance, “[The post] just seems off” [Seth]. Veterans occasionally did not interpret a post as concerning if they felt the post was “venting” [Natasha] or that the person was seeking attention.There was a complex calculus behind whether and how individuals responded to a concerning post. Once veterans had determined a post to be concerning, they wanted to be there for a peer in distress, but they did not always know if and how they should respond. Before they reached out, veterans went through a series of considerations. Elements of this complex calculus included (1) physical proximity, (2) relationship closeness, (3) existing responses to the post, and (4) ability to follow up with the person in crisis. While the weight given to each consideration varied across situations and circumstances, each factor influenced decisions related to whether veterans responded, how they chose to reach out, and how urgently and directly they responded.When a friend posted something on Facebook that appeared to indicate suicidal ideation, participants reported that the preferred response was to attempt to make direct, one-on-one contact to talk with the friend. This direct contact could be through Facebook itself (using a private message), email, a phone call or text, or meeting up in person. Multiple methods of contact could be combined, typically with the goal of having the online contacts lead to an in-person contact.When veterans saw a concerning post on Facebook, one of the first considerations for how they chose to respond was their physical proximity to the individual in distress. If the friend was within a reasonable travel distance, veterans often indicated it was optimal to visit them or call immediately to arrange an in-person meetup as soon as possible. If a friend expressed loneliness or concerning thoughts, participants felt responding through Facebook alone was insufficient. By meeting up in person, veterans felt that they could “get them out and let them know that there are still people for them” [Jamie] and “read more in a person by watching their body language” [Robert]. If the friend was not in close proximity and the level of concern was high, some veterans noted that they might try to get a next-of-kin or other person who was physically closer to check in on the friend.Depends on physical proximity, if it’s somebody within driving distance, within a few hours I’ll be at their house banging on the fucking door. If not, there’s going to be phone calls and emails and text messages and that’s the way I would react.AlyssaVeterans varied in their responsiveness to concerning posts depending on the closeness of their relationship with the person in crisis. For instance, if the concerning post was from an acquaintance or someone that was on the periphery of their life, they were more likely to respond in a less direct or less immediate fashion.I'll send them the phone number for the veteran's suicide hotline, that’s really about as far as I'm willing to go unless I know the person.AndreaIf I don’t know them personally, I try to give an encouraging thought.NatashaFor a close relationship, efforts to respond were more direct and more intensive. One veteran said that in the case of a close friend or family member, he would respond as follows:I probably wouldn’t even be on social media in that case. I would’ve just gone to the person and, you know, physically gone to see them.ConradFor relationships that fall somewhere between acquaintance and close friend or family, participants tended to communicate their availability but ultimately left the onus of further connection on the person in crisis. These efforts were often made under the assumption that there was someone closer who could help the person in crisis but also acknowledged that they were unsure of this and were thus willing to provide support if needed.…you can reach out to them and just give them a little love and then you instant message him and say, “Here’s my phone number and call me if you need to.”JerryA third consideration in the response calculus related to the interactions and activity in response to a concerning post, including “likes,” comments, and shares that were visible to others. Often, the timing of the post relative to when it was being seen was a factor. Veterans stressed the importance of timing for two reasons: first, the circumstances surrounding the crisis may have changed, and second, others may have already intervened.I’ve seen it a couple times or I’ve been, for lack of better term, late to the conversation and you’ve seen other people comment before that and someone said, “Hey, I’ve talked to ‘em.”NealRecognizing that timing of a post and actions by others might have resulted in resolution by the time they saw a concerning post, participants also described assessing if there had already been direct and supportive responses to the friend when deciding if they should respond.I’m usually not the first person to come along by it, so usually I’ll check the comment section and see what’s being done. Typically, someone will comment and then they’ll say: I’ve got ahold of them, or got them talking.RichardAnd after somebody posts, there's some likes and dislikes and maybe a “hey, what's up, call me.” I imagine in those situations there's four or five personal conversations going on with that person. And then they decide who is understanding the situation, talk with them.YolandaSome veterans emphasized both the need to initially reach out and the importance of following up with a friend in crisis. Veterans who discussed the importance of follow-up considered whether they had the capacity to continue to maintain contact and actively provide support.One veteran responded that she had a friend who posted on Facebook “reaching out about how lonely they were.” She went on to say the following:…they’re obviously looking for that public acknowledgment, so, I did that. And kept it… you know, more… general, I guess, and supportive, and then, messaged them more. You know, specifically relating to it and then scheduling a follow-up visit with them.JamieShe let the friend know they weren’t alone and scheduled a time to meet up with them.it was the next day or something. Just for a quick coffee date, you know, just to get them out and let them know that there are still people for them. [Jamie]…you don’t just listen and walk away. You still have to follow-up with them and keep talking with them. It might not even be someone that you actually really know, but if you come across someone in that state of mind, it’s usually kind of obvious that something’s not quite right so don’t ignore them. [Bryan]…Veterans desired suicide prevention training that would prepare them to assist others in emotional crisis. The suggestion for training on how to respond to concerning posts, specifically if suicidal ideation was suspected or indicated, was raised by participants as well as asked about by the interviewer. Veterans indicated a desire for training in suicide prevention skills to help a peer in crisis. A common reason they cited for wanting training was a sense of being unprepared to assist others in crisis or even worry about exacerbating the situation.I have no idea what to say but just trying to make myself available, trying to say as little damaging stuff as I can… It’s mostly that I have no idea what should I do to help? …I got pretty much nothing else.AlyssaI don’t know how to sometimes get the point across to them without sounding judgmental.LynnEven veterans who indicated having prior suicide prevention training, often during military service, felt that the training was inadequate or insufficient. This seemed especially vital for veterans who were interested in helping others but worried about how to initiate conversations with more socially distant individuals.Would I want more? Yes. Just to let me know I’ve got the basics down. I’m not a licensed mental health professional so I can’t go about helping them solve their problems, but I can get [someone in crisis] in the right direction.EdwardI’d like to recognize more warning signs, and I would like to have some conversation items that I could take as far as how to talk about it, or how to bring it up.LewisVeterans identified two features of suicide prevention training that would make it especially appealing. First, training sponsored or delivered by community-based veteran organizations was believed to “carry more weight” [Neal] and be “a good source of credibility” [Edward]. Many participants who followed at least one veteran-centric Facebook page or group  felt these groups could act as trusted sponsors of suicide prevention training. One veteran described the kind of support he’s encountered in one such private Facebook group:…every 22nd of every month, we do a check in. We’ll post a thing, check in with our veteran friends because it comes out that there’s 22 vets that commit suicide every day. And we started a program called “22 to None,” so we support each other. I’ve made a couple friends with some other vets, and other Navy vets. And, one guy… he told me, gave me his number, he says you ever start feeling really bad, give me a call. He was a trained counselor as well.KeithSecond, veterans were most willing to receive training targeted at helping their veteran peers. Their shared experiences, military background, and camaraderie as a result of service made a difference in their level of comfort and trust when talking about the topic of suicide with other veterans.It’s very easy for me to have a relationship or an exchange of information between another person who’s in the service. It’s easier than it is for me to sometimes talk with my wife, or members of my family because they don’t have that background like we have—those who’ve served.MeghanHave you ever had two different people tell you the same piece of information? One is Bob who you don’t really know particularly well, but the other one is somebody who is close to you—[you] know and respect—they repeat that same information, you place a lot more weight on it. With somebody who you’ve had a lot of those shared experiences with, I may not know them particularly well, but they’ve been in the same places I have, they’ve faced the same problems I’ve had, and if they say that this is helpful, I place a lot more weight on it.AlyssaIn the daily course of their lives, veterans are likely to encounter others in emotional crisis, and this can manifest through concerning posts on social media platforms such as Facebook. When presented with a concerning post, veterans go through a complex calculus—involving an assessment of physical proximity, relationship closeness, activity on the post, and ability to follow-up—which informs the extent and manner in which they reach out to provide support. The order of the complex calculus and the weight of each element varies in each situation. Veterans are often inclined to help their peers in some way, but they often feel unprepared as to how to respond to a concerning post. Given these circumstances, veterans desire suicide prevention training that extends beyond what they received during military service. They seek additional training that is germane to responding to concerning posts on social media, backed by community-based veteran organizations, and tailored to providing assistance to their veteran peers.Our finding that veterans are thoughtful and potentially motivated to respond positively to concerning posts suggests a key exception to prior research on displays of negative emotions on social media. Experimental and observational studies have shown that emotions are transmitted through social media posts ,26. IndiOur findings complement the use of two education and training intervention models in the literature: gatekeeper training and bystander education. Both gatekeeper training and bystander education train individuals to recognize the signs of crisis and develop skills to triage such a situation and assist with accessing subsequent professional assistance ,32.Gatekeeper training has been specifically designed to support “upstream” suicide prevention. Reviews have found evidence of gatekeeper training’s ability to increase knowledge and foster self-efficacy and other beliefs adaptive to suicide prevention . In a reOur findings are constrained by several study limitations. Participants were recruited from a sample of locally recruited veterans from Facebook, and thus our findings may not be entirely generalizable to veterans nationally, individuals less inclined to participate in research or to use Facebook, or individuals who have experiences on other social media platforms. The last constraint is particularly relevant given rapid shifts in the social media landscape. There are other facets of Facebook posts, such as posts within private Facebook groups or automatic identification of concerning posts using machine learning models , that weSocial media is an important venue through which many connect with their social network. One consequence of this, particularly among veterans who are disproportionately affected by and exposed to suicide, is encountering concerning posts. Our findings call for expanding access to ongoing suicide prevention training opportunities—in partnership with community-based veteran organizations—and incorporating features into existing suicide prevention training programs that will help veterans in their response to concerning posts from their veteran peers.


Results: A total of 309 women were included in the analysis. One hundred fifty-two women received the vaccine. HPV vaccination reduced in a statistically significant manner (p < 0.05) HPV DNA positivity rates for genotypes 16, 18, and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18, and 31 genotypes, respectively, prior to vaccination. A less pronounced, statistically insignificant reduction was shown for women who tested positive for both HPV DNA and mRNA E6 and E7 expression for HPV16, 18, and 33 subtypes. Statistically significant reduction in HPV mRNA positivity was solely documented for genotype 31 (p = 0.0411). Conclusions: HPV vaccination appears to significantly affect the rates of HPV16, 18, and 31 DNA-positive infections in the population testing HPV DNA-positive for the aforementioned genotypes. The above findings deserve verification in larger cohorts.The objective of this study was to investigate the hypothesis that HPV vaccination administered in patients with low-grade (LG) cytology shortly after an initial colposcopic assessment could prospectively alter HPV-related biomarkers. This was a prospective pilot observational study involving women attending a colposcopy clinic for evaluation of abnormal LG cytology that were advised to undergo HPV vaccination and proceeded accordingly. These women were compared with a matched unvaccinated group. Women requiring cervical biopsies or CIN treatment were excluded. Intervention: A full three-dose HPV vaccination was undertaken with either the 2-valent or the 4-valent anti-HPV VLP vaccine. LBC samples were obtained prior and after the completion of the vaccination regimen and tested for HPV DNA genotyping (CLART-2 HPV test) and E6 and E7 mRNA (NASBA technique). Results: Alterations of HPV-related biomarkers at a colposcopy reassessment appointment 12 months later. Analysis: The  The consistent implementation of cervical cancer screening programs in the mid-1980s led to significant reductions in this neoplasm’s morbidity and mortality rates in several developed countries. More recently, the global launch of prophylactic HPV vaccination programs materialized primary cervical cancer prevention and empowered the aspiration of a possible disappearance of cervical cancer in the near future . HoweverIn Greece, despite several initiatives, no national cervical cancer screening program is currently in place, and most women are being opportunistically screened, predominantly by cytology and lately by HPV DNA and mRNA co-testing. Individuals with cytological abnormalities are referred for further assessment mainly to specialized colposcopy centers. Commencing in 2007, Greece was among the first countries to incorporate the anti-HPV vaccine  vaccines) free of charge in the national immunization program. Throughout the study period, a vaccination strategy was ongoing with a primary cohort being adolescent girls from 12 to 15 years of age, while a catch-up cohort of older females up to the age of 26 was discontinued in December 2016 with the introduction of the 9-valent HPV vaccine. However, of note is that no vaccination registry or any other similar system is in place in Greece to monitor vaccination uptake; all relevant information is based on unpublished data from the Ministry of Health. Therefore, it seems that only approximately 30–35% of the eligible target group individuals have received the full regimen until today. There are several reasons for this disappointing coverage; predominantly, an active anti-vaccine lobby and ineffective counseling by physicians )). The preliminary results of a study of population subgroups with different inclusion criteria were presented at the annual meeting of the British Society for Colposcopy and Cervical Pathology, BSCCP, in 2013 .In all the women, just prior to the colposcopic evaluation, a liquid-based cytology (LBC) sample was obtained and sent for cytological and biomolecular analysis for established HPV-related biomarkers, in particular:The HPV DNA genotyping using CLART® HUMAN PAPILLOMAVIRUS 2 allows simultaneous detection of 35 different HPV genotypes  by PCR amplification of a fragment within the highly conserved L1 region of the virus , while tThe cytological examination was expressed according to the Bethesda classification (TBS2001 system) ,42. As sAt the follow-up visit, 12 months post-recruitment, all women’s history forms were updated regarding details focusing on aspects of sexual history . Women were asked if they had completed the full three-dose HPV vaccination regimen or not, commencing no later than 1-month post-recruitment. In this case, they were asked to provide specific information regarding the vaccine brand and a detailed administration calendar; all these data were recorded. Then, a new LBC vial was obtained and sent for cytological and biomolecular evaluation for the same HPV-related biomarkers as during the recruitment and subsequently underwent a repeat colposcopic examination. Women with disease progression  were scheduled for biopsies or treatment during a subsequent visit, outside the timeframe of the study.The flowchart of the study population is depicted in In order to explore the alterations of HPV-related biomarker positivity rates after HPV vaccination, we compared the vaccinated population with a matched control cohort, performing two groups of analyses.The 1st analysis focused on those individuals who had HPV DNA-positive and mRNA-negative infections; these groups were vaccinated group 1 (V1) and non-vaccinated group 1 (N-V1).The 2nd analysis group was based on the population that was found to have both HPV DNA test and the mRNA test positive at the first visit. The groups analyzed in the 2nd analysis were named: vaccinated group 2 (V2) and non-vaccinated group 2 (N-V2).p-value) was set to 0.05, thus, the statistically significant difference between the compared groups was with p < 0.05. We assessed HPV DNA genotyping and mRNA E6 and E7 positivity rates after administration or not of the vaccine in theV1 and N-V1 groups as well as in the V2 and N-V2 groups. Accuracy parameters like the p-value, odds ratios (OD), relative risk (RR), absolute relative risk (ARR), and number needed to treat (NNT) with confidence intervals (CI 95%) were calculated for each group.Statistical analysis was performed with the SAS for Windows 9.4 software platform . Descriptive values were expressed as the means ± standard deviation (SD) or percentages. Comparisons between groups in terms of the qualitative parameters were made using the chi-squared test. For the continuous parameters , it was not possible to ensure normality, therefore, non-parametric tests were applied, specifically, the Kruskal–Wallis test. Furthermore, we calculated the power of the study for the cumulative results of HPV DNA and mRNA positivity rates in the vaccinated and non-vaccinated groups. The significance level  with no statistical significance was documented, with the exception of HPV31 mRNA E6/E7 which reached statistical significance. Despite the pilot character of our study in an emerging field, we strongly believe that larger patient cohorts and more robust data are necessary in order to elucidate which oncogene’s expression represents the most suitable biomarker to assess vaccines’ efficacy.


The incidence of endocervical adenocarcinoma, the second most common cervical cancer in the world, has been on the rise. While most cervical cancers are squamous cell carcinomas and associated with high-risk oncogenic human papillomavirus (HPV), approximately 15% of endocervical adenocarcinomas, which now represent about one quarter of all cervical cancers, are HPV-independent. In this review, we will focus on the shortcomings of historical histologic classification systems of female genital tract tumors as they pertain to endocervical adenocarcinomas, and we will highlight the advantages of the new International Endocervical Adenocarcinoma Criteria and Classification system, which forms the basis for the WHO 2020 classification. We will cover the various histologic types, subtypes, and variants of endocervical adenocarcinoma with regard to morphology, immunophenotype, molecular genetics, HPV status and differential diagnosis, and we will provide International Society of Gynecological Pathologists recommendations for diagnosing these tumors. Endocervical adenocarcinomas are a heterogeneous group of neoplasms, contrary to what was previously thought. Screening strategies designed for and effective in detecting squamous cell carcinoma precursors are less effective in detecting endocervical glandular precursor lesions. Because of this and other reasons, the real and relative incidence of endocervical adenocarcinoma has increased in recent years from 5% to up to 20-25%, particularly in patients 30 yr of age or older, according to studies from the United States and Europe Endocervical adenocarcinomas are morphologically heterogeneous and have traditionally been diagnosed based on morphology, primarily on tumor architecture and the presence of intracytoplasmic mucin (WHO 2014 classification system). The diagnosis of many tumor types included in the WHO 2014 classification were poorly reproducible Usual-type endocervical adenocarcinoma was defined as “the most common type of adenocarcinoma with relative mucin depletion” but without any specific defining or quantitative criteria.Endometrioid endocervical adenocarcinoma was defined as an adenocarcinoma that morphologically resembled endometrial endometrioid adenocarcinomas, just like mucin-depleted usual types, but there are no criteria for differentiating usual-type from endometrioid-type endocervical adenocarcinomas based on morphology. Subsequent studies, discussed later in the text, have shown that this is an extraordinarily rare type of primary endocervical adenocarcinoma Mucinous endocervical adenocarcinomas included gastric-type, intestinal-type, and signet ring cell-type tumors, which is problematic as tumors of various etiologies (HPV-associated or HPV-independent) can be present within these groups. For example, adenocarcinomas with goblet cells  can be either HPV-associated or HPV-independent. In addition, it is now well established that gastric-type mucinous adenocarcinomas is a highly aggressive tumor etiologically unrelated to HPV infection, yet this is not taken into account in the classification of these tumors TP53 mutant in older patients, effectively combining 2 seemingly different tumors into a single category. Finally, serous carcinoma is currently not categorized as a type of primary endocervical adenocarcinoma.Serous endocervical adenocarcinomas were defined as morphologically identical to endometrial or adnexal serous carcinomas, yet the WHO 2014 classification system states that they can be HPV-positive “serous-like” in younger patients and The previous WHO 2014 classification categorized endocervical adenocarcinomas into the following types: usual, mucinous, villoglandular, clear cell, serous, endometrioid, and mesonephric The IECC, which was first described in 2018, forms the basis for the WHO 2020 classification The IECC system is based on identifying mitotic figures and apoptotic bodies at 40 to 100× magnification on hematoxylin & eosin (H&E)-stained slides and, therefore, is easy to apply in daily practice. When easily identifiable mitoses and apoptotic bodies are seen at this power, the tumor is likely HPV-associated; when mitoses and apoptotic bodies are absent or found with difficulty at high magnification, the tumor is likely HPV-independent Endocervical adenocarcinomas should be classified according to the forthcoming WHO 2020 classification system, which incorporates the IECC system.Both classification systems categorize endocervical adenocarcinomas into HPV-associated and HPV-independent types using morphology alone.The WHO 2020 and IECC systems includes newly described microscopic variants of HPV-associated endocervical adenocarcinomas.Ancillary testing for diagnosis (such as p16) does not need to be reflexively performed, as the morphology is tightly linked to HPV status. p16 and high-risk HPV testing should be reserved for difficult or ambiguous cases, such as the occasional HPV-associated adenocarcinoma lacking easily identifiable mitoses and apoptotic bodies.If interpretation is difficult, a diagnostic algorithm based on the amount of intracytoplasmic mucin and ancillary testing  may be useful in the differential diagnosis of the various histologic types and histologic mimics RNA-based in situ hybridization for high-risk HPV exhibits higher sensitivity and specificity compared with HPV DNA polymerase chain reaction (PCR) RNA-based in situ hybridization for high-risk HPV, although not available in most institutions, may have superior sensitivity, specificity, and positive and negative predictive values compared with p16 in identifying HPV-associated endocervical adenocarcinomas A recent workshop dedicated to endocervical adenocarcinoma was organized by the ISGyP in Los Angeles before the 2020 United States and Canadian Academy of Pathology (USCAP) annual meeting. Key points and recommendations from the workshop included the following:HPV-associated endocervical adenocarcinomas include the usual type, mucinous type, and HPV-associated adenocarcinoma NOS. These tumor types may arise from an in situ component, such as usual-type endocervical AIS and stratified mucin-producing intraepithelial lesion, but this is not seen in every case.The usual type is the most common HPV-associated endocervical adenocarcinoma, accounting for 75% to 80% of all endocervical adenocarcinomas When lacking intracytoplasmic mucin, a “pseudoendometrioid” morphology can pose problems in the differential diagnosis with endometrioid adenocarcinoma of the cervix or corpus.Villoglandular and micropapillary architectural patterns are variants of usual-type tumors and may be associated with more typical areas.They may have a “serous-like” morphology, and therefore, metastatic serous carcinomas from other organs may be considered.They rarely contain benign-appearing squamous elements, making it difficult to distinguish them from adenosquamous carcinomas or endometrioid adenocarcinomas with squamous differentiation of the corpus.Characteristically, the tumor cells of usual-type endocervical adenocarcinomas are columnar, with pseudostratified elongated and hyperchromatic nuclei. The cytoplasm is usually mucin-depleted, and the presence of apical mitotic figures and basal apoptotic bodies is virtually pathognomonic . Intracytoplasmic mucin can still be seen, sometimes extensively within parts of a tumor, although they do not reach the 50% threshold for mucinous-type tumors. In rare cases, the presence of benign squamous metaplasia, particularly when extensive, can mimic adenosquamous carcinomas or endometrioid adenocarcinomas with squamous differentiation. The stroma is often, but not always desmoplastic, and sometimes there is an accompanying inflammatory infiltrate, necrosis, or pools of mucin Fig. .Architecturally, usual-type endocervical adenocarcinomas are predominantly composed of glands with irregular shapes and sizes and smooth luminal borders. Papillary, cribriform, and solid patterns can also occur. Uncommonly, villoglandular, micropapillary, macrocystic, microcystic, trabecular, and single-cell patterns (mimicking breast lobular carcinoma) can occur Fig.  30.Villoglandular architecture deserves special mention, as it had been previously considered a distinct type of endocervical adenocarcinoma with an excellent prognosis A micropapillary pattern, occurring alone or admixed with other HPV-associated patterns, can also be seen in HPV-associated endocervical adenocarcinomas . This recently recognized architectural variant is microscopically represented by small, tightly cohesive papillary groups of neoplastic cells with eosinophilic cytoplasm and atypical nuclei, typically surrounded by clear spaces resembling vascular channels Usual-type tumors almost always exhibit diffuse p16 positivity  and are positive for HPV by RNA in situ hybridization, the most sensitive method for detecting high-risk HPV-associated types An important differential diagnostic consideration for usual-type HPV-associated endocervical adenocarcinoma is endometrioid-type adenocarcinoma of the cervix or corpus. Cervical endometrioid adenocarcinoma, discussed later, is very rare, thought to arise in the setting of endometriosis, and is HPV-negative. As a rule, mucin-depleted invasive adenocarcinomas of the cervix should not be diagnosed as endometrioid type in the presence of prominent apical mitotic figures and apoptotic bodies, as these lesions are almost invariably mucin-depleted usual-type HPV-associated adenocarcinomas. Distinguishing HPV-associated adenocarcinomas from endometrial endometrioid adenocarcinomas is particularly important, as clinical management differs. This differential diagnosis can be complicated in biopsy material and curettings, which often contain limited material. ER, PR, and vimentin are generally positive in endometrioid adenocarcinomas of the endometrium, whereas p16 is patchy/negative and HPV is negative. These markers should be interpreted with caution, as high-grade endometrioid adenocarcinomas of the corpus can be negative for ER and PR and rarely p16 block-positive. Furthermore, usual-type HPV-associated tumors of the cervix can uncommonly be ER, PR, and vimentin-positive, and very rarely p16-negative. Loss of one or more mismatch repair (MMR) proteins  in tumor cells is strongly suggestive of an endometrioid adenocarcinoma of the corpus with microsatellite instability. CEA has limited diagnostic usefulness, as focal staining can be observed in both tumors. In difficult cases, mRNA high-risk HPV in situ hybridization, together with clinical features, can be diagnostically helpful.Usual-type HPV-associated endocervical adenocarcinomas with papillary architecture and high-grade nuclear features (including the micropapillary variant) can also mimic serous carcinoma of the cervix, and the differential diagnosis may include direct involvement or a drop metastasis of tubo-ovarian or endometrial serous carcinoma. Serous carcinoma of the cervix is no longer accepted as a primary tumor type, as all such tumors are p16-negative and HPV-positive, and almost always p53 wild-type, in contrast to true serous carcinomas such tumors are regarded as HPV-associated endocervical adenocarcinomas rather than serous carcinomas.Recent studies have suggested that it may be important to differentiate between usual- and mucinous-type HPV-associated endocervical adenocarcinomas due to possible differences in clinical behavior and survival outcomes (mucinous-type tumors are associated with worse outcomes); this should be confirmed by future study of additional cohorts PIK3CA, KRAS, and PTEN, and abnormalities in segments of the PI3K/Akt/mTOR signaling cascade, some of which have predictive and prognostic value Prevalent molecular alterations in usual-type HPV-associated endocervical adenocarcinomas include mutations in Mucinous NOS: ≥50% of tumor cells have intracytoplasmic mucin in a usual-type background.Intestinal: goblet cells, representing ≥50% of cells in a usual-type or mucinous background. Paneth-like cells can also be seen occasionally.Signet-ring: round cells with a mucinous vacuole that displaces the nucleus to the periphery, representing ≥50% of cells in a usual-type background.ISMC: invasive nests of stratified columnar cells with variable amounts of intracytoplasmic mucin and peripheral palisading The WHO 2014 classification system grouped HPV-positive and HPV-negative tumors (such as gastric-type adenocarcinomas) with abundant intracytoplasmic mucin in the mucinous category. The IECC and WHO 2020 systems, however, include mucinous (non–gastric-type) adenocarcinoma as an HPV-associated adenocarcinoma, with the following subcategories:These tumors, which are not often encountered in routine practice, can display various architectures, such as glandular, insular, solid, trabecular, and single-cell patterns. As they are all HPV-associated, they pathognomonically display mitotic activity and apoptotic bodies at 40 to 100× magnification. The stroma can display mucin pools and inflammatory infiltrates Fig. .All subtypes can display a wide range of architectural and cytologic diversity.The signet-ring subtype is very rare and, if pure, may indicate metastasis to the cervix.The signet-ring subtype may be admixed with usual-type.Intestinal differentiation is also found in gastric-type tumors, which are HPV-independent (see below).Because of the intracytoplasmic mucin, mucinous-type can be misdiagnosed as gastric-type adenocarcinomas, endometrioid adenocarcinomas with mucinous differentiation, and metastases from other organs.ISMC can occur in pure form or admixed with usual-type or mucinous-type HPV-associated endocervical adenocarcinomas, adenosquamous, or neuroendocrine carcinomas.The recently described ISMC type of HPV-associated mucinous endocervical adenocarcinoma has been reported to have a poorer prognosis than that of other HPV-associated types Essentially all mucinous-type HPV-associated endocervical adenocarcinomas exhibit block-type positivity for p16 and HPV positivity Various HPV-associated mucinous-type endocervical adenocarcinomas are sometimes misdiagnosed as gastric-type endocervical adenocarcinoma, and the distinction may be particularly difficult with a small biopsy specimen. Most HPV-associated mucinous-type tumors have easily detectable mitotic figures and apoptotic bodies at 40 to 100× magnification, whereas mitoses and apoptotic bodies are typically rare in gastric-type tumors. Intestinal differentiation can occur in both types. p16 and HPV testing can assist with diagnosis in problematic cases, but p16 is rarely negative in HPV-associated carcinomas and rarely positive in gastric type carcinomas.In addition, HPV-associated mucinous-type endocervical adenocarcinomas can be misdiagnosed as endometrioid adenocarcinoma of the endometrium with extensive mucinous differentiation, and an algorithm for diagnosis has been proposed for tumors with abundant intracytoplasmic mucin using HPV testing and ER immunohistochemistry This very rare tumor is HPV-associated but cannot be classified morphologically into any of the known categories Fig. B, C. Of Usual-type tumors lacking intracytoplasmatic mucin should not be diagnosed as endometrioid-type tumors.HPV-associated endocervical adenocarcinomas with villoglandular and micropapillary patterns can be designated as usual-type tumors, but these patterns should be noted on the pathology report.A diagnosis of primary cervical serous carcinoma should not be made when dealing with serous-like morphology; most examples will represent an HPV-associated endocervical adenocarcinoma with serous-like morphology or a metastasis from the uterine corpus or fallopian tube/ovary.A micropapillary component of any percentage has a propensity for aggressive behavior and should be reported.Similarly, ISMC of any percentage has a propensity for aggressive behaviour and should be reported.Mucinous-type (including NOS) tumors are likely associated with a worse survival compared with usual-type tumors, so keeping these 2 categories distinct and reporting them separately is recommended until more studies are conducted.Whereas p16 and HPV testing are not always needed, they may be useful in problematic cases, and in the absence of block-type p16 staining or HPV, a diagnosis of HPV-associated endocervical adenocarcinoma should be questioned.HPV-independent endocervical adenocarcinomas account for ~15% of endocervical adenocarcinomas in most Western countries (the incidence may be higher in East Asia due to a relatively high frequency gastric-type tumors in this region). These tumors include gastric, clear cell, mesonephric and endometrioid-type carcinomas and adenocarcinoma NOS. Preinvasive premalignant lesions associated with gastric-type endocervical adenocarcinoma are lobular endocervical glandular hyperplasia (LEGH), specifically atypical LEGH and gastric-type adenocarcinoma in situ. There are no identified precursor lesions for clear cell and endometrioid-type tumors Gastric-type endocervical adenocarcinoma, initially described by Kojima and colleagues, is the second most common adenocarcinoma of the cervix, accounting for >20% of all cervical adenocarcinomas in Japan The minimal deviation pattern  can be difficult to diagnose and differentiate from a benign glandular lesion, especially with a small biopsy specimen.Gastric-type tumors have a morphology and immunohistochemical profile similar to pancreatic ductal adenocarcinoma and cholangiocarcinoma.Gastric-type tumors can have variable architectures and cytologic features mimicking other neoplasms.There are currently no totally reliable “positive” markers to establish a diagnosis of gastric-type endocervical adenocarcinoma.p16 is usually negative or focally positive but can be diffusely positive (block-type) in a minority of cases.In contrast to usual-type tumors, which typically contain elongated and pseudostratified hyperchromatic nuclei, gastric-type tumors are characterized by basally located rounded nuclei when well differentiated. The nuclei have clear or delicate, diffuse chromatin, sometimes with a distinct nucleolus, and typically appear pale in comparison to the hyperchromatic nuclei of usual-type tumors. Atypia ranges from minimal to marked. Architecturally, the tumor forms glands or solid areas, but papillary, trabecular, or single-cell patterns can be present. The glands vary in size and shape from small, simple tubular forms to cystically dilated with irregular contours and sometimes intraluminal papillary infoldings. The glands infiltrate the stroma, eliciting a desmoplastic response, and lymphovascular invasion is common. Minimal deviation adenocarcinoma of mucinous type , which is included in the spectrum of gastric-type endocervical adenocarcinomas, is characterized by low-grade morphology, with very well-differentiated glands with a “claw-like” pattern, lined by cells with abundant intracytoplasmic mucin and minimally atypical nuclei. The glands are distributed haphazardly within the stroma, sometimes with minimal or no desmoplasia Fig. . The terGastric-type endocervical adenocarcinoma produces neutral gastric (pyloric-type) mucin that stains magenta (pink/red) on PAS/Alcian blue histochemical stain, whereas normal endocervical mucins are acidic and stain dark blue. As previously mentioned, gastric-type endocervical adenocarcinomas morphologically resemble adenocarcinomas of the pancreatobiliary tract, with a similar immunohistochemical profile; one difference is that most, but not all, gastric-type endocervical adenocarcinomas express PAX8 (see below) As stated previously, gastric-type endocervical adenocarcinoma is usually negative or exhibits patchy, non–block-type immunoreactivity with p16 and is HPV-negative; however, occasional examples exhibit block-type immunoreactivity with p16 When gastric-type endocervical adenocarcinoma is difficult to distinguish from usual-type endocervical adenocarcinoma, the presence of prominent mitotic and apoptotic activity favors a usual-type diagnosis. MUC6, HIK1083, p16, and HPV testing may be of value. High-grade squamous intraepithelial lesions and HPV-associated AIS will favor a usual-type diagnosis, although small numbers of gastric-type endocervical adenocarcinomas may contain such lesions coincidentally. Atypical LEGH or gAIS will favor a gastric-type diagnosis. Of interest, carcinomas exhibiting overlapping morphology with areas resembling both usual-type (with prominent apoptosis and mitoses) and gastric-type tumors have been described. In these difficult cases, HPV and p16 testing can usually identify whether it is an HPV-associated mucinous carcinoma or gastric-type tumor with morphologic mimicry rather than a true mixed carcinoma, which likely does not occur Gastric-type adenocarcinoma may be particularly difficult to differentiate from clear cell carcinoma, especially with biopsy material. HNF1beta and Napsin A can be positive in both tumors, but HNF1beta is particularly likely to be positive in gastric-type endocervical adenocarcinomas, as discussed previously When distinguishing between morphologically well-differentiated variants of gastric-type endocervical adenocarcinoma  and a variety of benign cervical glandular lesions, such as deep Nabothian cysts or endocervical glandular hyperplasia, ER/PR staining can be useful, as gastric-type tumors are usually negative and most benign glandular lesions, with the exception of LEGH and mesonephric remnants, are positive for these markers. The “claw-like” shape and deep placement of glands along with at least mild nuclear atypia and the presence of at least focal stromal desmoplasia are also more typical of gastric-type endocervical adenocarcinomas. These features are helpful in differentiating gastric-type endocervical adenocarcinoma from LEGH, in which a preserved lobular architecture with minimal cytologic atypia is observed. Immunohistochemistry is of limited value in distinguishing between LEGH and well-differentiated variants of gastric-type endocervical adenocarcinoma, emphasizing the value of H&E morphology to separate these entitites. Some authors have suggested that smooth muscle actin staining may be useful, as this marker is positive in cervical stromal cells adjacent to invasive glands of gastric-type endocervical adenocarcinoma and negative in the stromal cells surrounding LEGH Theoretically, in all gastric-type endocervical adenocarcinomas, a metastasis, especially from the pancreas or biliary tree, should be ruled out by clinical means, as the morphology and the immunophenotype of gastric-type endocervical adenocarcinomas and pancreatic/biliary adenocarcinomas can be very similar, if not identical. PAX8 positivity favors a primary cervical tumor diagnosis, but not all gastric-type endocervical adenocarcinomas of the cervix are PAX8-positive. The differential diagnosis with clear cell carcinoma is discussed later in the text.STK11. Somatic mutations of STK11 have also been identified in some sporadic cases TP53, CDKN2A, and ERBB2/ERBB3, and less commonly, mutations in GNAS, SMAD4, and PIK3CAThe molecular mechanisms underlying the pathogenesis of gastric-type endocervical adenocarcinoma are still emerging. These tumors sometimes develop in patients with Peutz-Jeghers syndrome, an autosomal dominant disorder caused by germline mutation of This rare tumor of the cervix, which accounts for ~4% of all cervical adenocarcinomas, occurs in young women exposed to diethylstilbestrol in utero  and sporadically in young or older postmenopausal women (when the tumor predominantly involves the endocervix) The precursor lesion of cervical clear cell carcinoma is still unknown.The tumor cells can have clear or eosinophilic cytoplasm, which may mimic gastric-type tumors.Clear cell carcinomas often show patchy staining with p16, although only rare cases have block-type staining.HNF1beta is a nonspecific marker, as it is positive in many other types of endocervical adenocarcinoma.The main malignant differential diagnoses are gastric-type and mesonephric-type endocervical adenocarcinomas.Before diagnosing a primary cervical clear cell carcinoma, an endometrial primary should be excluded.Clear cell carcinoma can also mimic benign glandular lesions, such as microglandular hyperplasia and Arias-Stella reaction.Similar to clear cell carcinomas of the uterine corpus or ovary, clear cell carcinoma of the cervix is characterized by solid, papillary, and/or tubulocystic architecture with polygonal or hobnail cells with abundant clear or eosinophilic cytoplasm. A tubulocystic architecture is especially common in primary cervical clear cell carcinoma, and a hyalinized stroma is often present. Mitotic activity is often, but not always, low Fig. .Clear cell carcinomas are negative for HPV and usually exhibit negative or focal p16 staining but can occasionally demonstrate block-type positivity The differential diagnosis of clear cell endocervical adenocarcinoma includes gastric-type and mesonephric-type endocervical adenocarcinomas, and useful morphologic features and ancillary tests to differentiate clear cell from gastric-type carcinomas have been detailed above. To summarize, although both clear cell carcinoma and gastric-type adenocarcinomas typically have low nuclear to cytoplasmic ratios with prominent cytoplasmic borders, the absence of characteristic clear cell carcinoma growth patterns and presence of obvious intracytoplasmic mucin would be more in keeping with a diagnosis of gastric-type adenocarcinoma. Furthermore MUC6, HIK1083, and carbonic anhydrase IX are significantly more commonly positive in gastric-type adenocarcinoma, whereas Napsin A positivity will favor a clear cell carcinoma diagnosis. None of these markers are totally specific. It is important to remember that these tumors are HPV-independent. Clear cell carcinomas can also mimic benign glandular lesions, such as Arias-Stella reaction and microglandular hyperplasia.KRAS, HRAS, or TP53 were found on molecular analysis in an older study Microsatellite instability has been reported in cervical clear cell carcinomas, whereas no mutations in Mesonephric endocervical adenocarcinoma of the cervix is very rare, accounting for <1% of all cervical adenocarcinomas and originating from mesonephric remnants deep in the lateral cervical wall A well-defined precursor lesion has not been described, although some mesonephric adenocarcinomas likely arise from mesonephric remnants/hyperplasia.Characteristically, these tumors exhibit an admixture of various architectural growth patterns.The tumor cells generally have scant cytoplasm and atypical nuclei that may resemble those of papillary thyroid carcinomas.These tumors can mimic endometrioid or serous carcinoma, clear cell carcinoma, or usual-type HPV-associated tumors, and ancillary tests can assist in their differentiation.A spindle-cell component can sometimes be seen; a diagnosis of carcinosarcoma should be reserved for those rare tumors with heterologous elements.Morphologically, these tumors characteristically exhibit an admixture of growth patterns, including ductal, tubular, glomeruloid, papillary, retiform, solid, sex cord-like, and spindled (sarcomatoid). A small tubular pattern, sometimes containing intraluminal eosinophilic colloid-like material, is especially characteristic. The tumor cells usually have scant cytoplasm, and many nuclei may be oval, optically clear, grooved, and overlapping, similar to papillary thyroid carcinoma. Other nuclei are hyperchromatic. Adjacent benign mesonephric remnants are identified in a minority of cases. The tumor usually develops in the lateral aspect of the cervix, but when discovered, this origin in the lateral walls is often not apparent with circumferential cervical involvement Fig. .These tumors are negative for HPV, and are usually negative (or exhibit non–block-type positivity) for p16. ER and PR tend to be completely negative An important differential diagnostic consideration is the usual-type endocervical adenocarcinoma. HPV testing, p16, and GATA3 immunohistochemistry can be diagnostically useful in these cases, as mesonephric endocervical adenocarcinoma will be HPV-negative and p16-negative (or non–block-type) and typically positive for GATA3.In contrast to mesonephric-type tumors, endometrioid adenocarcinomas of the endometrium are mostly ER-positive and PR-positive, GATA3-negative and TTF1-negative, and show confirmatory endometrioid features Florid mesonephric hyperplasia can mimic mesonephric adenocarcinoma, but it usually does not form a mass lesion. Nuclear atypia, mitotic activity, morphologic patterns other than small tubular, a stromal reaction, lymphovascular invasion, and spread outside the cervix favor a mesonephric-type adenocarcinoma diagnosis.KRAS mutations in most mesonephric adenocarcinomas of the cervix, whereas a smaller number show activating NRAS mutations. Demonstration of a KRAS or NRAS mutation may be useful in diagnosing a mesonephric adenocarcinoma and distinguishing this from florid mesonephric hyperplasia, which does not exhibit mutations ARID1A/B and chromosomal abnormalities with copy number gains in 1q, loss of 1p, and gain of chromosomes 10 and 12 have also been reported Recent molecular studies have demonstrated This is a very rare primary tumor of the cervix that is said to arise in the setting of endometriosis. An endometrioid-type endocervical adenocarcinoma diagnosis should be made with caution; previously, in many institutions, a diagnosis of primary endometrioid adenocarcinoma of the cervix was often made for usual-type adenocarcinomas with minimal intracytoplasmic mucin. When strict diagnostic criteria are used, this is an extremely rare tumor, accounting for <1% of cervical carcinomas Primary cervical endometrioid-type endocervical adenocarcinomas are very rare, and tumors of the cervix with an endometrioid appearance are almost all HPV-positive, with most cases being of usual type.Cervical endometrioid adenocarcinoma is thought to arise in the setting of endometriosis; however, a premalignant lesion is not well defined.Before diagnosing a primary cervical endometrioid adenocarcinoma, spread from the corpus should be excluded.The immunohistochemical profile is expected to be similar to that of endometrioid adenocarcinoma of the uterine corpus.According to IECC/WHO 2020, endometrioid adenocarcinoma of the cervix must display endometrioid morphology with “confirmatory endometrioid features,” such as: (a) at least focally identified low-grade endometrioid glands, (b) lined by columnar cells, (c) pseudostratified nuclei, (d) no more than moderate atypia, (e) with or without squamous differentiation, (f) and/or endometriosis present, and (g) lacking HPV-associated features, such as prominent mitotic figures and apoptotic bodies Fig.  13.p16 is usually negative or non–block-type; HPV is negative The main differential diagnostic consideration is usual-type endocervical adenocarcinoma and, as mentioned above, a reflexive diagnosis of endometrioid-type endocervical adenocarcinoma should not be made in the presence of a mucin-depleted adenocarcinoma. The other main differential diagnostic consideration is an endometrioid adenocarcinoma arising from the corpus or ovary, with spread into the cervix. The differential diagnosis can be difficult on a biopsy with minimal tissue, as the morphology and immunohistochemical profiles of endometrioid carcinomas arising at these sites are identical, and HPV is negative. Correlation with clinical, radiologic, and macroscopic features is essential, as their management depends largely on the site of origin.These HPV-independent tumors cannot be classified into one of the other types Fig. A. MorphoImmunohistochemistry is of limited value in distinguishing between LEGH and well-differentiated variants of gastric-type endocervical adenocarcinoma, and this is a predominantly morphologic diagnosis.Serous carcinoma of the cervix does not exist.True endometrioid adenocarcinoma of the endocervix is extremely rare and should not be reflexively diagnosed in the presence of a mucin-poor endocervical adenocarcinoma.Immunohistochemical markers are useful for differentiating between the various HPV-independent histologic types, and HPV testing for differentiating between HPV-associated and -independent endocervical adenocarcinomas.Endocervical adenocarcinomas represent a very heterogeneous group of tumors. Usual and mucinous types are associated with HPV infection; gastric, clear cell, mesonephric, and endometrioid types are not driven by HPV. The distinction between HPV-associated and HPV-independent endocervical adenocarcinomas has important clinical implications, as HPV-independent tumors tend to develop in older patients, present at a higher clinical stage, have a worse prognosis, have different and unusual patterns of spread, and respond differently to typical oncologic treatment for cervical cancer. Moreover, the fact that some endocervical adenocarcinomas are HPV-independent is important for HPV vaccination strategies and HPV-based screening programs.As HPV-associated and HPV-independent endocervical adenocarcinomas have different molecular drivers, it is imperative to understand their unique features, which is necessary for the development of future targeted therapies, ongoing genomic studies, and clinical trials, which hopefully will add more to our understanding of this uncommon group of tumors.