Wang H, Wang MX, Su N, Wang LC, Wu X, Bui 1, Nielsen A, Vo HT, Nguyen N, Luo Y, Ma XJ (2014).
PMID: 24637627doi
The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
Otolaryngol Head Neck Surg. 2015 Feb 27.
Stoddard DG Jr, Keeney MG, Gao G, Smith DI, García JJ, O'Brien EK.
PMID: 25724573 | DOI: 0194599815571285.
OBJECTIVE: Assess human papilloma virus (HPV) transcriptional activity in inverted Schneiderian papillomas (IPs). STUDY DESIGN: Case series with chart review. SETTING: Academic tertiary care center. SUBJECTS AND METHODS: Retrospective clinicopathologic review of 19 cases of IP in patients undergoing surgical excision from 1995 to 2013 at Mayo Clinic in Rochester, Minnesota. Surgical pathology archival material was histopathologically reviewed using hematoxylin and eosin-stained slides. Formalin-fixed, paraffin-embedded material from each case was evaluated for p16 expression using immunohistochemistry as well as HPV DNA and E6/E7 messenger RNA (mRNA) transcription using polymerase chain reaction (PCR) and in situ hybridization (via RNAscope technology), respectively. RESULTS: Eight patients were female (42%), with an average age of 53 years (range, 23-82 years). Three demonstrated malignancy, and 5 subsequently recurred. Average follow-up was 49 months (range, 0-200 months), and 1 patient died from squamous cell carcinoma arising from the IP. RNAscope detected HPV mRNA transcripts exclusively within IP in 100% of cases; however, in 11 patients (58%), less than 1% of cells exhibited transcriptional activity. Only 2 of 19 cases (11%) demonstrated mRNA activity in 50% or more cells. HPV DNA was detected in only 2 specimens by PCR. CONCLUSIONS: This study reveals wide prevalence but limited transcriptional activity of HPV in IP. No correlation between HPV transcriptional activity and progression, recurrence, or malignant transformation was identified. These data suggest that transcription of HPV may contribute to the pathogenesis of IP, but prospective data are needed to definitively demonstrate this connection. These results also suggest that RNAscope may be more sensitive than PCR in detecting HPV activity in IP.
PLoS One. 2014 Mar 13;9(3):e91142
Evans MF, Peng Z, Clark KM, Adamson CSC, Ma XJ, Wu X, Wang H, Luo Y, Cooper K
PMID: 24625757 | DOI: 10.1371/journal.pone.0091142.eCollection2014.
Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
Br J Cancer. 2015 Feb 17.
Young RJ, Urban D, Angel C, Corry J, Lyons B, Vallance N, Kleid S, Iseli TA, Solomon B, Rischin D.
PMID: 25688737 | DOI: 10.1038/bjc.2015.59.
Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36-1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26-1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23-2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19-2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.
Head and neck pathology, 7(2):113–21.
Ukpo OC, Thorstad WL, Lewis JS Jr (2012).
PMID: 23179191 | DOI: 10.1007/s12105-012-0406-z.
Human papillomavirus (HPV) is associated with oropharyngeal squamous cell carcinomas. Persistent viral infection is postulated to lead to carcinogenesis, although infection of benign adjacent epithelium is not typically observed. It is known that immune evasive tumor cells can provide an ideal niche for a virus. The B7-H1/PD-1 cosignaling pathway plays an important role in viral immune evasion by rendering CD8+ cytotoxic T cells anergic. We hypothesized that HPV-related oropharyngeal squamous cell carcinomas express B7-H1 as a mechanism for immune evasion. A tissue microarray was utilized, for which HPV E6/E7 mRNA by in situ hybridization was previously performed. Immunohistochemistry was performed to detect B7-H1 and staining was characterized by pattern, distribution, and intensity. B7-H1 was expressed by 84 of the 181 (46.4%) cases. Both tumor cell membranous and cytoplasmic expression were present and cytoplasmic expression was identified in some peritumoral lymphocytes. Expression was analyzed in several different ways and then considered binarily as positive versus negative. Tumors expressing B7-H1 were more likely to be HPV positive (49.2 vs. 34.1 %, p = 0.08). B7-H1 expression showed no correlation with disease recurrence in the entire cohort (OR = 1.09, p = 0.66), HPV positive cohort (OR = 0.80, p = 0.69) or HPV negative cohort (OR = 2.02, p = 0.22). However, B7-H1 expression intensity did correlate with the development of distant metastasis (p = 0.03), and B7-H1 intensity of 3+ (versus all other staining) showed a strong trend towards distant metastasis in the HPV positive (OR = 6.67, p = 0.13) and HPV negative (OR = 9.0, p = 0.13) cohorts. There was no correlation between B7-H1 expression and patient survival for any of the different ways in which staining was characterized, whether binarily, by distribution, intensity, or combined scores. B7-H1 is expressed in the majority of oropharyngeal squamous cell carcinomas with transcriptionally-active HPV. This suggests that B7-H1 expression by tumor cells may play a role in harboring persistent HPV infection.
Fakhry C, Westra WH, Wang SJ, van Zante A, Zhang Y, Rettig E, Yin LX, Ryan WR, Ha PK, Wentz A, Koch W, Richmon JD, Eisele DW, D'Souza G.
PMID: 28241096 | DOI: 10.1002/cncr.30353
Abstract
BACKGROUND:
Human papillomavirus (HPV) is a well-established prognostic marker for oropharyngeal squamous cell cancer (OPSCC). Because of the limited numbers of women and nonwhites in studies to date, sex and racial/ethnic differences in prognosis have not been well explored. In this study, survival differences were explored by the tumor HPV status among 1) patients with OPSCCs by sex and race and 2) patients with nonoropharyngeal (non-OP) head and neck squamous cell cancers (HNSCCs).
METHODS:
This retrospective, multi-institution study included OPSCCs and non-OP HNSCCs of the oral cavity, larynx, and nasopharynx diagnosed from 1995 to 2012. Race/ethnicity was categorized as white non-Hispanic, black non-Hispanic, Asian non-Hispanic, and Hispanic of any race. Tumors were centrally tested for p16 overexpression and the presence of HPV by HPV16 DNA and high-risk HPV E6/E7 messenger RNA in situ hybridization. Kaplan-Meier and Cox proportional hazards models were used to evaluate overall survival (OS).
RESULTS:
The study population included 239 patients with OPSCC and 621 patients with non-OP HNSCC with a median follow-up time of 3.5 years. After adjustments for the tumor HPV status, age, current tobacco use, and stage, the risk of death was lower for women versus men with OPSCC (adjusted hazard ratio, 0.55; P = .04). The results were similar with p16. In contrast, for non-OP HNSCCs, HPV positivity, p16 positivity, and sex were not associated with OS.
CONCLUSIONS:
For OPSCC, there are differences in survival by sex, even after the tumor HPV status has been taken into account. For non-OP HNSCC, the HPV status and the p16 status are not of prognostic significance.
Belobrov S, Cornall AM, Young RJ, Koo K, Angel C, Wiesenfeld D, Rischin D, Garland SM, McCullough M.
PMID: 29024035 | DOI: 0.1111/jop.12649
Abstract
BACKGROUND:
The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC.
METHODS:
Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus.
RESULTS:
HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH.
CONCLUSION:
We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.
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.
Am J Surg Pathol. Dec;36(12):1874–1882.
Bishop JA, Ma XJ, Wang H, Luo Y, Illei PB, Begum S, Taube JM, Koch WM, Westra WH (2012).
PMID: 23060353 | DOI: 10.1097/PAS.0b013e318265fb2b.
Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification-a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of an unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94% vs. 9%, P<0.0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16/HPV DNA), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.
Mod Pathol. 2013 Feb;26(2):223-31.
Chernock RD, Wang X, Gao G, Lewis JS Jr, Zhang Q, Thorstad WL, El-Mofty SK.
PMID: 22996374 | DOI: 10.1038/modpathol.2012.159.
Although a strong etiologic relationship between human papillomavirus (HPV) and a majority of oropharyngeal squamous cell carcinomas has been established, the role of HPV in non-oropharyngeal head and neck carcinomas is much less clear. Here, we investigated the prevalence and clinicopathologic significance of HPV and its reported biomarkers, CDKN2A(p16) and CDKN1A(p21), in laryngeal squamous cell carcinomas in patients treated either with primary surgery and postoperative radiation or with definitive radiation-based therapy. Nearly all of 76 tumors were keratinizing and none displayed the nonkeratinizing morphology that is typically associated with HPV infection in the oropharynx. However, CDKN2A(p16) immunohistochemistry was positive in 21 cases (28%) and CDKN1A(p21) in 34 (45%). CDKN2A(p16) and CDKN1A(p21) status strongly correlated with each other (P=0.0038). Yet, only four cases were HPV positive by DNA in situ hybridization or by reverse transcriptase PCR E6/E7 mRNA (all four were CDKN2A(p16) and CDKN1A(p21) positive). Unexpectedly, 9 additional tumors out of 20 CDKN2A(p16) positive cases harbored high-risk HPV DNA by PCR. For further investigation of this unexpected result, in situ hybridization for E6/E7 mRNA was performed on these nine cases and all were negative, confirming the absence of transcriptionally active virus. Patients with CDKN1A(p21)-positive tumors did have better overall survival (69% at 3 years) than those with CDKN1A(p21)-negative tumors (51% at 3 years) (P=0.045). There was also a strong trend towards better overall survival in the CDKN2A(p16)-positive group (P=0.058). Thus, it appears that the role of HPV is more complex in the larynx than in the oropharynx, and that CDKN2A(p16) and CDKN1A(p21) expression may not reflect HPV-driven tumors in most cases. Because of this, CDKN2A(p16) should not be used as a definitive surrogate marker of HPV-driven tumors in the larynx.
Chuerduangphui J, Pientong C, Patarapadungkit N, Chotiyano A, Vatanasapt P, Kongyingyoes B, Promthet S, Swangphon P, Bumrungthai S, Pimson C, Ekalaksananan T.
PMID: 28741068 | DOI: 10.1007/s12032-017-1010-6
Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.
Mehrad M, Dupont WD, Plummer WD Jr, Lewis JS Jr.
PMID: 29235037 | DOI: 10.1007/s12105-017-0874-2
The favorable features of high-risk human papillomavirus (HPV) in the head and neck are limited to those harboring transcriptionally-active HPV, which occur predominantly in the oropharynx (OP). Factors rendering the OP susceptible to HPV oncogenesis are largely unexplored. The role of cytokeratin 7 (CK7) in predisposition to HPV and cancer in the cervix has been evaluated. However, its significance in the H&N is unknown. CK7 immunohistochemistry was performed on a tissue microarray cohort of OP and non-oropharyngeal (NOP) squamous cell carcinomas (SCC) with known clinical follow-up and HPV E6/7 mRNA status. Expression was graded based on the distribution (1 ≤ 33%, 2 = 33-66%, 3 ≥ 66%) and intensity (1 = weak, 2 = strong) with combined score of ≥ 2 considered positive. Survival analysis was performed. Seventy-four NOPSCCs and 204 OPSCCs were studied. HPV was positive in 2.7% of NOPSCCs and 70.9% of OPSCCs. CK7 was positive in 23.0% of OPSCCs and 14.8% of NOPSCCs (p = 0.2), and in 24.1% of HPV positive versus 17.2% of negative patients (p = 0.2). There was no correlation with age, race, gender, HPV status, histologic type, tumor subsite, treatment, stage, or co-morbidities, and CK7 expression was not significantly associated with overall or disease specific survival. In our series, CK7 is positive in ~ 25% of H&N SCCs, although usually only focally. While CK7 has been suspected to be overexpressed selectively in HPV-related OPSCCs due to their origination from tonsillar crypt epithelium, we did not find any significant difference by anatomic H&N subsite, nor by HPV status, for its expression and found no association with patient survival.
