Puram, SV;Mints, M;Pal, A;Qi, Z;Reeb, A;Gelev, K;Barrett, TF;Gerndt, S;Liu, P;Parikh, AS;Ramadan, S;Law, T;Mroz, EA;Rocco, JW;Adkins, D;Thorstad, WL;Gay, HA;Ding, L;Paniello, RC;Pipkorn, P;Jackson, RS;Wang, X;Mazul, A;Chernock, R;Zevallos, JP;Silva-Fisher, J;Tirosh, I;
PMID: 37012457 | DOI: 10.1038/s41588-023-01357-3
Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.
Abstract: Background: The recognition of tumor infection by human papilloma virus (HPV) in oropharyngeal squamous-cell carcinoma (OSCC) is emerging as a valid biomarker to more accurate selection of patients for specific treatment, surveillance and tumor staging. To this aim, the HPV detection strategy in OSCC must dissect between HPV that is acting as a driver of malignant transformation, and transcriptionally silent virus involved in productive infection. The aim of this study is to define the better method for the accurate identification of HPV status among OSCC. Patients and Methods: Thirty-six patients were selected for HPV status assessment combining different methods, such as immunohistochemistry (IHC) for p16, in-situ hybridization (ISH) for high risk (HR)-HPV DNA and HR-HPV E6/E7 mRNA along with real-time PCR of HPV16 E6/E7 mRNA. All these cases were originally classified as HPV negative by DNA-based ISH but p16 positive by the IHC. Results: Twenty-six cases showed concordance between methods; whereas, nine cases resulted negative for HPV E6/E7 mRNA RT-PCR but positive for HPV E6/E7 mRNA ISH. Conclusion: By considering that the bright field HPV E6/E7 mRNA ISH could be more sensitive than mRNA-based real-time RT-PCR, and that it provides the precise identification of transcriptionally active HPV infected cells, a randomized analysis to validate the robustness of this preliminary assay will be undertaken.
Doorbar, J;
PMID: 37354969 | DOI: 10.1016/j.tvr.2023.200268
The incorporation of HPV DNA testing into cervical screening programs has shown that many HPV-positive women are cytologically normal, with HPV-positivity fluctuating throughout life. Such results suggest that papillomaviruses may persist in a latent state after disease clearance, with sporadic recurrence. It appears that virus latency represents a narrow slot in a wider spectrum of subclinical and possibly productive infections. Clinical studies, and animal model infection studies, suggested a key role for host immune surveillance in maintaining such asymptomatic infections, and although infections may also be cleared, most studies have used the term 'clearance' to describe a situation where the presence of HPV DNA falls below the clinical detection level. Given our knowledge of papillomavirus immune evasion strategies and the restricted pattern of viral gene expression required for 'basal cell' persistence, the term 'apparent clearance' and 'subclinical persistence' of infection may better summarise our understanding. Subclinical infection also encompasses the lag phase, which occurs between infection and lesion development. This is dependent on infection titre, with multifocal infections developing more rapidly to disease. These concepts can usefully influence patient management where HPV-positivity occurs sometime after the onset of sexual activity, and where vertical transmission is suspected despite a lag period.
Journal of cutaneous pathology
Bartley, B;Cho, WC;Rady, PL;Dai, J;Curry, JL;Milbourne, A;Tyring, SK;Torres-Cabala, CA;
PMID: 36039682 | DOI: 10.1111/cup.14319
Epidermodysplasia verruciformis (EDV) is a rare genodermatosis that predisposes individuals to persistent infection with β-human papillomavirus (HPV) genotypes. The term EDV acanthoma may be applied to lesions with incidental findings of EDV-defining histopathological features without clinical signs of EDV. We report a case of HPV-14- and -21-positive EDV acanthoma arising in association with condyloma in a female patient with a history of low-grade squamous intraepithelial lesion of the cervix positive for high-risk HPV (non-16/18), chronic kidney disease, and systemic lupus erythematosus. The patient had no family or personal history of EDV, but the patient was on immunosuppressive therapy with mycophenolate mofetil and prednisone. A biopsy specimen from one of the perianal lesions revealed histopathologic changes consistent with EDV in the setting of condyloma. Molecular testing showed HPV-14 and -21, which supported the coexistence of condyloma with EDV acanthoma.
Journal of Investigative Dermatology
Kolitz, E;Lucas, E;Hosler, G;Kim, J;Hammer, S;Lewis, C;Xu, L;Day, A;Mauskar, M;Lea, J;Wang, R;
| DOI: 10.1016/j.jid.2021.10.009
Vulvar squamous cell carcinoma (VSCC) pathogenesis is traditionally defined by the presence or absence of human papillomavirus (HPV), but the definition of these groups and their molecular characteristics remains ambiguous across studies. Here, we present a retrospective cohort analysis of 36 patients with invasive VSCC where HPV status was determined using RNA in situ hybridization (ISH) and polymerase chain reaction (PCR). Clinical annotation, p16 immunohistochemistry (IHC), programmed death ligand-1 (PD-L1) IHC, HPV16 circular E7 RNA (circE7) detection, and RNA-sequencing (RNA-seq) of the cases was performed. A combination of ISH and PCR identified 20 cases (55.6%) as HPV-positive. HPV-status did not impact overall survival (HR: 1.36, 95% CI: 0.307 to 6.037, p=0.6857) or progression-free survival (HR: 1.12, 95% CI: 0.388 to 3.22, p=0.8367), and no significant clinical differences were found between the groups. PD-L1 expression did not correlate with HPV status, but increased expression of PD-L1 correlated with worse overall survival. Transcriptomic analyses (n=23) revealed distinct groups, defined by HPV status, with multiple differentially expressed genes previously implicated in HPV-induced cancers. HPV-positive tumors showed higher global expression of endogenous circular RNAs (circRNAs), including several circRNAs that have previously been implicated in the pathogenesis of other cancers.
Chinese journal of pathology
Zhao YH, Bai YP, Mao ML, Zhang H, Zhao XL, Yang DM, Wan HF, Liu HG.
PMID: 30695865 | DOI: 10.3760/cma.j.issn.0529-5807.2019.02.010
Objective: To observe the clinicopathologic features of oropharyngeal squamous cell carcinoma associated with human papilloma virus (OPSCC-HPV) and discuss the role and value of different in situ hybridization (ISH) detection methods for HPV in pathologic diagnosis. Methods: Fifteen cases of OPSCC-HPV were collected from Department of Pathology, Beijing Tongren Hospital, Capital Medical University from January 2016 to August 2018. These cases were diagnosed in accordance with the WHO classification of head and neck tumors. The histopathologic features and the clinicopathologic data were retrospectively analyzed. Immunohistochemistry (two-step EnVision method) was done to evaluate the expression of p16, Ki-67 and p53. ISH was used to detect HPV DNA (6/11 and 16/18). RNAscope technology was used to evaluate the presence of HPV mRNAs (16 and 18). Results: The mean age for the 15 patients (8 males, 7 females) was 47 years (range from 30 to 69 years). OPSCC-HPV typically presentedat an advanced clinical stage, six patients had cervical lymphadenopathy (large and cystic), seven had tonsillar swelling, one had tumor at base of tongue, and one had odynophagia. Microscopically the tumors exhibited distinctive non-keratinizing squamous cell carcinoma morphology. Cervical nodal metastases were large and cystic, with thickening of lymph node capsules. OPSCC-HPV raised from crypt epithelium and extended beneath the tonsillar surface epithelial lining as nests and lobules, often with central necrosis. Tumor cells displayed a high N: C ratio, and high mitotic and apoptotic rates. Tumor nests are often embedded within lymphoid stroma, and may be infiltrated by lymphoid cells.Fifteen cases (15/15) were strongly positive for p16; Ki-67 index were 60%-90%; they were focally positive or negative for p53. Ten cases (10/10) were negative for HPV 6/11 DNA, and one case(1/10) was focally positive for HPV16/18 DNA. Eleven cases (11/11) were strongly positive for HPV16 mRNA, one case was focally positive for HPV18 mRNA. Conclusions: OPSCC-HPV is a pathologically and clinically distinct form of head and neck squamous cell carcinoma. OPSCC-HPV is associated with high-risk HPV (type 16) in all cases. Detection of high-risk HPV16 mRNA by RNAscope is of great significance in the final diagnosis and pathogen identification.
European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery
Channir, HI;Lomholt, AF;Gerds, TA;Charabi, BW;Kiss, K;von Buchwald, C;
PMID: 34689237 | DOI: 10.1007/s00405-021-07133-5
Squamous cell carcinoma metastasis of the head and neck with unknown primary tumor (CUP) comprises a diagnostic challenge. Human papillomavirus (HPV) testing on cytologic specimens is gaining increasing focus as this may facilitate an early diagnosis of HPV-induced oropharyngeal carcinoma. This study aimed to prospectively assess PCR-based HPV-DNA testing on FNA smears in a clinical setting.Patients referred to a tertiary Head and Neck Cancer Center with suspected CUP were included from November 2016 to November 2018. Scraped cell material from FNA smears was analyzed for HPV-DNA with PCR using general primers (GP5 + /GP6 +) and correlated with the origin and histology of the primary tumor (oropharynx vs. outside oropharynx or benign tumor). The turn-around time reflecting the workflow for HPV-DNA testing by PCR was also calculated.A total of 93 patients were enrolled in the study. The sensitivity and specificity were 86.7% [95% CI 75.4-94.1%] and 92.0% [95% CI 74.0-99.0%], and the positive and negative predictive values were 96.3% [95% CI 87.3-99.0%] and 74.2% [95% CI 59.9-84.7%], respectively. The turn-around time for HPV testing was a mean four calendar days.HPV-DNA testing on FNA smears can be performed within a reasonable timeframe and can guide for the detection of an HPV-positive oropharyngeal primary tumor in the clinical setting for patients presenting with CUP of the head and neck.
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Novack, R;Zhang, L;Hoang, LN;Kadhim, M;Ng, TL;Poh, CF;Kevin Ko, YC;
PMID: 36906072 | DOI: 10.1016/j.modpat.2023.100153
The diagnosis of oral epithelial dysplasia is based on the degree of architectural and cytologic atypia in the squamous epithelium. The conventional grading system of mild, moderate, and severe dysplasia is considered by many the gold standard in predicting the risk of malignant transformation. Unfortunately, some low-grade lesions, with or without dysplasia, progress to squamous cell carcinoma (SCC) in short periods. As a result, we are proposing a new approach to characterize oral dysplastic lesions that will help identify lesions at high risk for malignant transformation. We included a total of 203 cases of oral epithelial dysplasia, proliferative verrucous leukoplakia, lichenoid, and commonly observed mucosal reactive lesions to evaluate their p53 immunohistochemical (IHC) staining patterns. We identified 4 wild-type patterns, including scattered basal, patchy basal/parabasal, null-like/basal sparing, mid-epithelial/basal sparing, and 3 abnormal p53 patterns, including overexpression basal/parabasal only, overexpression basal/parabasal to diffuse, and null. All cases of lichenoid and reactive lesions exhibited scattered basal or patchy basal/parabasal patterns, whereas human papillomavirus-associated oral epithelial dysplasia demonstrated null-like/basal sparing or mid-epithelial/basal sparing patterns. Of the oral epithelial dysplasia cases, 42.5% (51/120) demonstrated an abnormal p53 IHC pattern. p53 abnormal oral epithelial dysplasia was significantly more likely to progress to invasive SCC when compared with p53 wild-type oral epithelial dysplasia (21.6% vs 0%, P < .0001). Furthermore, p53 abnormal oral epithelial dysplasia was more likely to have dyskeratosis and/or acantholysis (98.0% vs 43.5%, P < .0001). We propose the term p53 abnormal oral epithelial dysplasia to highlight the importance of utilizing p53 IHC stain to recognize lesions that are at high risk of progression to invasive disease, irrespective of the histologic grade, and propose that these lesions should not be graded using the conventional grading system to avoid delayed management.
Zhonghua bing li xue za zhi = Chinese journal of pathology
Li, LL;Cui, YY;Gao, PY;Xia, L;Liu, GZ;Liu, H;
PMID: 35785831 | DOI: 10.3760/cma.j.cn112151-20220301-00137
Objective: To investigate the clinicopathological characteristics, immunophenotype, molecular characteristics, differential diagnosis, clinical treatment and prognosis of mixed carcinoma of cervix with adenoid cystic pattern. Methods: Three cases of mixed cervical carcinoma with adenoid cystic pattern were collected at the Affiliated Hospital of Xuzhou University Medical School from 2018 to 2021.The clinicopathological characteristics were analyzed, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed. The related literature was reviewed. Results: The three patients were postmenopausal women with a median age of 74.7 years. The clinical symptom was vaginal bleeding without obvious causes. One case was an endophytic tumor, and the others were exophytic. The median diameter of the three cases was 3.3 cm. Two patients underwent hysterectomy, the tumors infiltrated the external 1/3 and middle 1/3 of the cervix respectively. All the lymph nodes were negative. One patient had a previous biopsy. Microscopically, all three tumors were characterized by a cribriform structure, which were filled with basophilic myxoid substance and surrounded by tubules lined by two layers of cells. The tumor cells had scanty cytoplasm and showed the characteristics of cervical basal-like cells. All three cases were accompanied by high-grade squamous intraepithelial lesions and squamous cell carcinoma, and one also showed a non-specific spindle cell sarcomatoid component. Within the double-layered epithelial structure, the outer epithelium was positive for p63, CD117, p16INK4a (clone E6H4) and MYB protein and negative for S-100 by IHC. The combined positive score of PD-L1 (clone 22C3) was less than 1 in all three cases. Human papillomavirus (HPV) types 16 and 18 were detected in one patient preoperatively, while high-risk HPV were positive in the other two patients by RNAscope ISH postoperatively. None of the three cases showed MYB gene rearrangement by FISH. The mean follow-up time was 23.3 months (36, 28 and 6 months, respectively). Two patients underwent hysterectomy and radiotherapy survived without disease. One patient survived with tumor just by radiotherapy and drug therapy. Conclusions: Mixed cervical carcinoma with adenoid cystic pattern is extremely rare. It is a high-grade malignancy with poor prognosis. The tumor is associated with high-risk HPV infection, without MYB gene rearrangement, and with low PD-L1 immunoreactivity. Radical surgery combined with radiotherapy and chemotherapy is the mainstay of treatment at present.
Hsieh MS, Lee YH, Jin YT, Huang WC.
PMID: 30071233 | DOI: 10.1016/j.humpath.2018.07.026
HPV-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk human papillomavirus (HR-HPV) infection. Using HR-HPV mRNA in situ hybridization (ISH), we reported six new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma (ACC). Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified six HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium and one also had invasive squamous cell carcinoma (SCC). All HMSC were SOX10-positive whereas the overlying atypical squamous epithelium and the SCC were SOX10-negative. One atypical HMSC-like case was also identified which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals than HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium as well as the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.
Archives of pathology & laboratory medicine
Haqshenas, G;Molano, M;Phillips, S;Balgovind, P;Garland, SM;Hawkes, D;Brotherton, JM;Machalek, DA;Murray, G;
PMID: 37226838 | DOI: 10.5858/arpa.2022-0317-OA
Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored.To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples.We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 and that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples.Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3).Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.
Koster, S;Gurumurthy, RK;Kumar, N;Prakash, PG;Dhanraj, J;Bayer, S;Berger, H;Kurian, SM;Drabkina, M;Mollenkopf, HJ;Goosmann, C;Brinkmann, V;Nagel, Z;Mangler, M;Meyer, TF;Chumduri, C;
PMID: 35210413 | DOI: 10.1038/s41467-022-28569-1
Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression.
