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.
Sathasivam HP, Santambrogio A, Andoniadou CL, Robinson M, Thavaraj S.
PMID: 30101315 | DOI: 10.1093/annonc/mdy313
Oral Oncology, 2014 - Elsevier
Westra WH
PMID: 24932529 | DOI: 10.1016/j.oraloncology.2014.05.004
Much recent attention has highlighted a subset of head and neck squamous cell carcinomas (HNSCCs) related to human papillomavirus (HPV) that has an epidemiologic, demographic, molecular and clinical profile which is distinct from non-HPV-related HNSCC. The clinical significance of detecting HPV in a HNSCC has resulted in a growing expectation for HPV testing of HNSCCs. Although the growing demand for routine testing is understandable and appropriate, it has impelled an undisciplined approach that has been largely unsystematic. The current state of the art has now arrived at a point where a better understanding of HPV-related tumorigenesis and a growing experience with HPV testing can now move wide scale, indiscriminant and non-standardized testing towards a more directed, clinically relevant and standardized approach. This review will address the current state of HPV detection; and will focus on why HPV testing is important, when HPV testing is appropriate, and how to test for the presence of HPV in various clinical samples. As no single test has been universally accepted as a best method, this review will consider the strengths and weaknesses of some of the more commonly used assays, and will emphasize some emerging techniques that may improve the efficiency of HPV testing of clinical samples including cytologic specimens.
International Journal of Cancer, 132(4), 882–890.
Gao G, Chernock RD, Gay HA, Thorstad WL, Zhang TR, Wang H, Ma XJ, Luo Y, Lewis JS Jr, Wang X (2013).
PMID: 22821242 | DOI: 10.1002/ijc.27739.
Oropharyngeal squamous cell carcinoma (SCC) is strongly associated with human papillomavirus (HPV) infection, which is distinctively different from most other head and neck cancers. However, a robust quantitative reverse transcription PCR (RT-qPCR) method for comprehensive expression profiling of HPV genes in routinely fixed tissues has not been reported. To address this issue, we have established a new real-time RT-PCR method for the expression profiling of the E6 and E7 oncogenes from 13 high-risk HPV types. This method was validated in cervical cancer and by comparison with another HPV RNA detection method (in situ hybridization) in oropharyngeal tumors. In addition, the expression profiles of selected HPV-related human genes were also analyzed. HPV E6 and E7 expression profiles were then analyzed in 150 archived oropharyngeal SCC samples and compared with other variables and with patient outcomes. Our study showed that RT-qPCR and RNA in situ hybridization were 100% concordant in determining HPV status. HPV transcriptional activity was found in most oropharyngeal SCC (81.3%), a prevalence that is higher than in previous studies. Besides HPV16, three other HPV types were also detected, including 33, 35 and 18. Furthermore, HPV and p16 had essentially identical expression signatures, and both HPV and p16 were prognostic biomarkers for the prediction of disease outcome. Thus, p16 mRNA or protein expression signature is a sensitive and specific surrogate marker for HPV transcriptional activity (all genotypes combined).
Rettig EM, Gooi Z, Bardin R, Bogale M, Rooper L, Acha E, Koch WM.
| DOI: 10.1177/2473974X18818415
Abstract Objective. Oral human papillomavirus (HPV) infection is the precursor for a growing subset of oropharyngeal squamous cell carcinomas (OPSCCs) in the developed world. This study was designed to characterize oral HPV infection and OPSCC in a region with high rates of HPV-driven cervical cancer. Study Design. Cross-sectional cohort study, retrospective case series. Setting. Northwest Cameroon referral hospital. Subjects and Methods. Individuals infected with human immunodeficiency virus attending an outpatient clinic were evaluated for oral HPV infection with oral swabs or rinses that were tested for 51 HPV types. HNSCCs diagnosed and/or treated at the same hospital from 2011 to 2017 were retrospectively reviewed to ascertain demographic and tumor characteristics, and available OPSCCs were tested for HPV. Results. The oral HPV infection study population comprised 101 participants. Most (69%) were female and neversmokers (84%). Participants had median 4 lifetime sexual partners (interquartile range, 3-7; range, 1-100). Five participants (5%) had oral HPV infection; one had 2 HPV types. HPV types detected were HPV68 (n = 2), HPV82 (n = 2), HPV32 (n = 1), and unknown (n = 1). No significant demographic or behavioral differences were detected among individuals with vs without oral HPV infection. OPSCCs comprised just 8% (n = 11) of 131 HNSCCs in the retrospective study population. Two of 7 OPSCCs were HPV positive. Conclusion. The low prevalence of OPSCC observed in northwest Cameroon together with the rarity of oral HPV infection suggests low rates of HPV-driven oropharyngeal carcinogenesis in the region. Future research should examine how geographic differences in oral HPV infection are influenced by cultural norms and affect HPV-OPSCC epidemiology
Dreyer JH, Hauck F, Oliveira-Silva M, Barros MH, Niedobitek G. (2013).
PMID: 23503925 | DOI: 10.1007/s00428-013-1393-5.
Detecting human papillomavirus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) is clinically relevant, but there is no agreement about the most appropriate methodology. We have studied 64 oropharyngeal carcinomas using p16 immunohistochemistry, HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR) followed by pyrosequencing. We have also evaluated a new assay, RNAscope, designed to detect HPV E6/E7 RNA transcripts. Using a threshold of 70 % labelled tumour cells, 21 cases (32.8 %) were p16 positive. Of these, 19 cases scored positive with at least one HPV detection assay. Sixteen cases were positive by HPV DNA-ISH, and 18 cases were positive using the E6/E7 RNAscope assay. By PCR and pyrosequencing, HPV16 was detected in 15 cases, while one case each harboured HPV33, 35 and 56. All p16-negative cases were negative using these assays. We conclude that p16 expression is a useful surrogate marker for HPV infection in HNSCC with a high negative predictive value and that p16-positive cases should be further evaluated for HPV infection, preferably by PCR followed by type determination. Using RNase digestion experiments, we show that the RNAscope assay is not suitable for the reliable discrimination between E6/E7 RNA transcripts and viral DNA.
Pathology - Research and Practice
Cao M, Shah W, Qi J, Zhou Y, Wang Y, Chen H.
PMID: - | DOI: 10.1016/j.prp.2016.06.011
Abstract
Purpose
High-risk human papillomavirus (HR-HPV) infections was the causal factor in the development of cervical cancer, but the significance of HPV viral load in the prediction of the response to current therapeutic approaches had not reached consensus. The present study was performed to assess the high risk HPV viral load of cervical cancer patients who underwent radiotherapy alone or in combination with chemotherapy or hyperthermotherapy or both in correlation to long-term survival.
Methods
116 cervical cancer patients were recruited and assigned into four groups of different therapeutic modalities. The prevalent high risk types of HPV 16, 18, 58 were detected by type specific in situ hybridization (ISH), and HPV mRNA was detected by RNA scope assay using RNA scope 2.0 FFPE Reagent Kit. Semi-quantification of the HR-HPV viral load was measured based on the intensity of ISH signal captured from the tumor nests in the grey scale.
Results
The HR-HPV viral load had a significant negative correlation with survival (rs = −0.368,P = 0.001). The 15-year survival rate of low viral load group was 68.18%, moderate viral load group was 52.17%, and high viral load group was 34.69% (P = 0.001). HPV mRNA expression was strongly consistent with HPV viral load. The 15-year survival rates of different therapeutic groups were 39.29%, 58.62%, 50.00%, 55.17%, respectively (P = 0.545). Combinatorial treatment modalities improved the actual survival, which demonstrated no significant difference among 5,10 and 15 years comparison. Cox regression analysis showed that the relative risk of death was obviously higher in the HPV 18 single positive group and high HPV viral load group.
Conclusions
The semi-quantitive viral load assessment in situ is a feasible approach in clinical practice. The more the HPV viral load was, the worse the survival of patients would be. The combinational treatments were in favor of the disease-stabilization.
Discrepancy of p16 immunohistochemical expression and HPV RNA in penile cancer. A multiplex in situ hybridization/immunohistochemistry approach study
Infectious agents and cancer
Zito Marino, F;Sabetta, R;Pagliuca, F;Brunelli, M;Aquino, G;Perdonà, S;Botti, G;Facchini, G;Fiorentino, F;Di Lauro, G;De Sio, M;De Vita, F;Toni, G;Borges Dos Reis, R;Neder, L;Franco, R;
PMID: 33789689 | DOI: 10.1186/s13027-021-00361-8
The high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30-50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue. We collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC. The multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 - 15 %- versus 6 out of 60 - 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 - 10 %- versus 4 out of 60 - 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity. Our findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %. Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.
Detection of HPV infection in urothelial carcinoma using RNAscope: Clinicopathological characterization
Musangile, FY;Matsuzaki, I;Okodo, M;Shirasaki, A;Mikasa, Y;Iwamoto, R;Takahashi, Y;Kojima, F;Murata, SI;
PMID: 34164940 | DOI: 10.1002/cam4.4091
Human papillomavirus (HPV) is a well-established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV-related urothelial carcinoma (UC).Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high-risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA-PCR.The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV-positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six-fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, p = 0.002). In addition, HPV infection showed a significant association with tumor grade (OR =9.8, p = 0.03) and stage (OR =4.7, p = 0.03) of UC. Moreover, the metastatic rate was higher in HPV-positive than in negative UC (OR =3.4).These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays.
Gelwan E, Malm IJ, Khararjian A, Fakhry C, Bishop JA, Westra WH.
PMID: 28877058 | DOI: 10.1097/PAS.0000000000000929
The oral cavity and oropharynx have historically been viewed as a single anatomic compartment of the head and neck. The practice of combining the oral cavity and oropharynx has recently been revised, largely owing to the observation that human papillomavirus (HPV)-related carcinogenesis has a strong predilection for the oropharynx but not the oral cavity. The purpose of this study was to determine whether HPV is evenly distributed across squamous cell carcinomas of the oropharynx including those sites that do not harbor tonsillar tissues such as the soft palate. A search of the medical records of the Johns Hopkins Hospital identified 32 primary squamous cell carcinomas of the soft palate (n=31) and posterior pharyngeal wall (n=1). All were evaluated with p16 immunohistochemistry and high-risk HPV in situ hybridization (ISH) (29 by RNA ISH and 3 by DNA ISH). For comparison, we also reviewed the medical records to obtain the HPV status of patients who had undergone HPV testing of primary tonsillar carcinomas over the same time interval as part of their clinical care. High-risk HPV as detected by ISH was present in just 1 (3.1%) of the 32 oropharyngeal squamous cell carcinomas, including 1 of 2 p16-positive carcinomas. The difference in HPV detection rates between tonsillar and nontonsillar sites was significant (1/32, 3.1% vs. 917/997, 92%; P<0.0001). HPV is not frequently detected in squamous cell carcinomas arising from nontonsillar regions of the oropharynx. Indeed, squamous cell carcinomas of the soft palate more closely resemble those arising in the oral cavity than those arising in areas of the oropharynx harboring tonsillar tissue. This finding not only further sharpens our understanding of site-specific targeting by HPV, but may have practical implications regarding HPV testing and even the way the oral vault is oncologically compartmentalized to partition HPV-positive from HPV-negative cancers.
Zhonghua bing li xue za zhi = Chinese journal of pathology
Xi, Y;Zhang, ML;He, C;Cheng, GP;Jin, JY;Fang, XH;Zhu, T;Su, D;
PMID: 35359045 | DOI: 10.3760/cma.j.cn112151-20210719-00516
Objective: To assess the clinical features and treatment outcomes in patients with primary ovarian squamous cell carcinoma (POSCC). Methods: Fifteen patients with primary ovarian squamous cell carcinoma diagnosed from January 2009 to December 2018 in Cancer Hospital of the University of Chinese Academy of Sciences were collected. The expression of p16, hMLH1, hMSH2, hMSH6 and PMS2 in POSCC was detected by immunohistochemistry, and the status of high-risk human papillomavirus (HPV) by RNAscope test. Results: Squamous cell carcinoma with different degrees of differentiation was found in 15 cases, including three cases with high differentiation and 12 cases with medium to low differentiation. There were four cases with in situ squamous cell carcinoma, four cases with teratoma, one case with endometrial carcinoma/atypical hyperplasia, and one case with endometriosis. p16 was expressed in five cases (5/15), indicating coexisting high-risk HPV infection. There was no high-risk HPV infection in the remaining 10 cases, and p16 staining was negative. There was no deficient mismatch repair protein in all cases. The overall survival time (P=0.038) and progression free survival (P=0.045) of patients with high-risk HPV infection were longer than those without HPV infection. Conclusions: POSCC is more commonly noted in postmenopausal women and often occurs unilaterally. Elevated serological indexes CA125 and SCC are the most common finding. Morphologically, the tumors show variable degrees of differentiation, but the current data suggest that the degree of differentiation cannot be used as an independent prognostic index. High-risk HPV infection may be associated with the occurrence of POSCC, and that the prognosis of POSCC patients with HPV infection is better than that of patients without infection.
Craig SG, Anderson LA, Schache AG, Moran M, Graham L, Currie K, Rooney K, Robinson M, Upile NS, Brooker R, Mesri M, Bingham V, McQuaid S, Jones T, McCance DJ, Salto-Tellez M, McDade SS and James JA
PMID: 30890775 | DOI: 10.1038/s41416-019-0414-9
BACKGROUND: TNM8 staging for oropharyngeal squamous cell carcinomas (OPSCC) surrogates p16 immunohistochemistry for HPV testing. Patients with p16+ OPSCC may lack HPV aetiology. Here, we evaluate the suitability of TNM8 staging for guiding prognosis in such patients. METHODS: HPV status was ascertained using p16 immunohistochemistry and high-risk HPV RNA and DNA in situ hybridisation. Survival by stage in a cohort of OPSCC patients was evaluated using TNM7/TNM8 staging. Survival of p16+/HPV- patients was compared to p16 status. RESULTS: TNM8 staging was found to improve on TNM7 (log rank p = 0.0190 for TNM8 compared with p = 0.0530 for TNM7) in p16+ patients. Patients who tested p16+ but were HPV- (n = 20) had significantly reduced five-year survival (33%) compared to p16+ patients (77%) but not p16- patients (35%). Cancer stage was reduced in 95% of p16+/HPV- patients despite having a mortality rate twice (HR 2.66 [95% CI: 1.37-5.15]) that of p16+/HPV+ patients under new TNM8 staging criteria. CONCLUSION: Given the significantly poorer survival of p16+/HPV- OPSCCs, these data provide compelling evidence for use of an HPV-specific test for staging classification. This has particular relevance in light of potential treatment de-escalation that could expose these patients to inappropriately reduced treatment intensity as treatment algorithms evolve.
