Journal of Medical Virology
Phusingha P, Ekalaksananan T, Vatanasapt P, Loyha K, Promthet S, Kongyingyoes B, Patarapadungkit N, Chuerduangphui J, Pientong C.
PMID: 27935063 | DOI: 10.1002/jmv.24744
Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a five-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPVinfection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand.
Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is however not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high risk (HR) HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48,7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either HR HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors.
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.
Lewis JS Jr, Shelton J, Kuhs KL, K Smith D.
PMID: 29190003 | DOI: 10.1007/s12105-017-0871-5
Routine testing for p16 immunohistochemistry (with selective HPV-specific test use) has been recommended for clinical practice in oropharyngeal squamous cell carcinoma (OPSCC). Data suggests that the E6H4 clone performs best for this purpose, yet no studies have evaluated the optimal antibody concentration for OPSCC testing. We evaluated three concentrations (undiluted, 1:5, and 1:10) of the primary antibody solution for E6H4 using tissue microarrays from a cohort of 199 OPSCC patients with a > 70% staining cutoff for positivity. Concordance was evaluated using percent agreement and Cohen's kappa. The concentrations were evaluated for sensitivity and specificity using high risk HPV RNA in situ hybridization (RNA-ISH) and also correlated with Kaplan-Meier overall survival analysis. Inter-rater agreement was very high between p16 results at each concentration and also with RNA in situ hybridization (p < 0.0001 for all). Agreement between p16 undiluted and 1:5 dilution (agreement 98.2%; Kappa 0.943; p < 0.0001) was very high and between p16 undiluted and 1:10 dilution (agreement 79.2%; Kappa 0.512; p < 0.0001) much lower. Intensity of the staining did decrease with the 1:5 and 1:10 dilutions compared to undiluted, but not in a manner that obviously would change test interpretation or performance. Results suggest that the E6H4 antibody performs well at dilutions of up to 1:5 fold with a minor decrease in staining intensity, minimum loss of sensitivity, and no loss of specificity in OPSCC patients. This could result in reagent and cost savings.
ER-positive endocervical adenocarcinoma mimicking endometrioid adenocarcinoma in morphology and immunohistochemical profile: A case report of application of HPV RNAscope detection
Chen, R;Qin, P;Luo, Q;Yang, W;Tan, X;Cai, T;Jiang, Q;Chen, H;
PMID: 33787580 | DOI: 10.1097/MD.0000000000024927
Usual-type endocervical adenocarcinoma (ECA), high-risk HPV associated, is the most common type of glandular carcinoma in the endocervix. Mucin-depleted usual-type ECA is 1 end of morphological lineage of usual-type ECA and morphologically may show endometrioid features, which could cause diagnostic challenge with uterine endometrioid adenocarcinoma (EEC) and primary endometrioid ECA, especially in the setting of small biopsy and endocervical curettage (ECC). A 37-year-old women presented with dyspareunia for 1 year, showing atypical glandular cell on a liquid-based Pap TCT examination and positive for HPV16 detection. ECC showed EEC in another hospital based on its "endometrioid" morphology and immunohistochemical profiles (ER/PR/PAX8 strongly positive, though p16 also strongly positive). The specimen of hysterectomy in our hospital displayed a lesion confined to the uterine cervix showing the same morphology and immunohistochemical profiles as ECC. Finally, we successfully performed HPV RNAscope and detected high-risk human papilloma virus (HPV) E6/E7 mRNA particles in tumor cells in situ, which warranted usual-type ECA with mucin-depleted feature, a rare deviation of usual-type of ECA. The patient underwent total hysterectomy with lymph node dissection. To date, 14 months after surgery, the patient is well without recurrence or distant metastasis, and undergoes regular reexamination. We report a rare case of mucin-depleted usual-type ECA showing overlapping morphological and immunohistochemical profiles with EEC. The pathological diagnosis was confirmed by high-risk HPV RNAscope detection which is superior than immunohistochemistry to identify usual-type ECA, warranting an important role in assisting the diagnosis of morphological vague cases.
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.
Am J Pathol. 2015 Jan 5. pii: S0002-9440(14)00688-9.
Miller DL, Davis JW, Taylor KH, Johnson J, Shi Z, Williams R, Atasoy U, Lewis JS Jr, Stack MS.
PMID: 25572154 | DOI: 10.1016/j.ajpath.2014.11.018.
High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV+ OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV+ OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV+/- OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.
Rajendra, S;Sharma, P;
PMID: 36765833 | DOI: 10.3390/cancers15030873
Esophageal cancer is a relatively common malignancy worldwide with a high mortality (5-year survival of <15%). Despite screening, surveillance, improved imaging and treatment, the exponential rise in OAC continues. The strongest risk factors for OAC are chronic heartburn and metaplastic transformation of the lower third of the esophagus (Barrett's esophagus). The risk profile includes Caucasian race, male gender older age, obesity and smoking. Although the tumor risk in BO has been progressively revised downwards, the exponential rise in OAC remains unchecked. This paradox points to an unidentified missing link. Relatively recently, we provided the world's initial data for a strong association of biologically relevant hr-HPV with BD and OAC. Since then, systematic reviews and meta-analysis have documented HPV DNA prevalence rates in OAC of between 13 to 35%. In this review, we provide some evidence for a probable causal relationship between hr-HPV and OAC. This is challenging given the multifactorial etiology and long latency. Increasingly, high-risk HPV (hr-HPV) is regarded as a risk factor for OAC. This discovery will aid identification of a sub-group of high-risk progressors to esophageal cancer by surveillance and the development of effective preventive strategies including vaccination.
Burassakarn, A;Phusingha, P;Yugawa, T;Noguchi, K;Ekalaksananan, T;Vatanasapt, P;Kiyono, T;Pientong, C;
PMID: 35454851 | DOI: 10.3390/cancers14081944
Infection by high-risk human papillomaviruses (hrHPVs), including HPV type 16 (HPV16), is a major risk factor for oral squamous cell carcinomas (OSCCs). However, the pathogenic mechanism by which hrHPVs promote oral carcinogenesis remains to be elucidated. Here, we demonstrated that the suppression of a transporter associated with the antigen-processing complex (TAPs; TAP1 and TAP2), which is a key molecule in the transportation of viral antigenic peptides into MHC class-I cells, is affected by the E6 protein of HPV16. Mechanistically, HPV-mediated immune evasion is principally mediated via the signal-transduction network of a lymphotoxin (LT) pathway, in particular LTα1β2 and LTβR. Our analysis of transcriptomic data from an HNSCC cohort from the Cancer Genome Atlas (TCGA) indicated that expression of TAP genes, particularly TAP2, was downregulated in HPV-infected cases. We further demonstrated that LTα1β2 and LTβR were upregulated, which was negatively correlated with TAP1 and TAP2 expression in HPV-positive clinical OSCC samples. Taken together, our findings imply that HPV16 E6 regulates the machinery of the antigenic peptide-loading system and helps to clarify the role of oncogenic viruses in the context of oral carcinoma.
Seminars in Diagnostic Pathology
During the last few decades a phenotypically distinct type of head and neck squamous cell carcinoma (SCC), that is etiologically related to human papillomavirus(HPV), has emerged and its prevalence continues to increase. The tumors are site-specific with special predilection for the oropharynx. They are morphologically and molecularly distinct and are responsive to different types of treatment modalities, with excellent clinical outcome, in spite of early lymph node metastasis. Microscopically, the carcinomas are nonkeratinizing SCCs. More recently, other variants that are believed to be etiologically related to HPV are reported. As a result, several clinical and pathologic questions have emerged. Importantly, whether the virus is biologically active in these tumors and involved in their pathogenesis, and second, what are the clinical implications with regard to patient management and outcome in these HPV-related variants. This review is an attempt to answer some of these questions based on information derived from available yet limited number of publications. The variants to be discussed include; nonkeratinizing SCC (NKSCC), NKSCC with maturation (hybrid type), keratinizing SCC (KSSC), basaloid squamous carcinoma (BSCC), undifferentiated carcinoma (UC), papillary SCC (PSCC), small cell carcinoma, adenosquamous carcinoma (AdSCC) and spindle cell (sarcomatoid) carcinoma.
Am J Otolaryngol. 2014 Jan-Feb;35(1):25-32.
Melkane AE, Mirghani H, Aupérin A, Saulnier P, Lacroix L, Vielh P, Casiraghi O, Griscelli F, Temam S.
PMID: 24112760 | DOI: 10.1016/j.amjoto.2013.08.007.
PURPOSE:
HPV-related oropharyngeal squamous cell carcinomas clearly represent a growing entity in the head and neck with distinct carcinogenesis, clinico-pathological presentation and survival profile. We aimed to compare the HPV prevalence rates and clinico-pathological correlations obtained with three distinct commonly used HPV detection methods.
MATERIALS AND METHODS:
p16-immunohistochemistry (IHC), HPV DNA viral load by real-time PCR (qPCR), and HPV genotyping by a reverse hybridization-based line probe assay (INNO-LiPA) were performed on pretreatment formalin-fixed paraffin-embedded tumor samples from 46 patients treated for single primary oropharyngeal carcinomas.
RESULTS:
Twenty-eight patients (61%) had a p16 overexpression in IHC. Twenty-nine patients (63%) harbored HPV DNA on qPCR. Thirty-four patients (74%) harbored HPV DNA on INNO-LiPA. The concordance analysis revealed a good agreement between both HPV DNA detection methods (κ=0.65); when both tests were positive, the depicted HPV subtypes were always concordant (HPV16 in 27 cases, HPV18 in 1 case). Agreement was moderate between IHC and qPCR (κ=0.59) and fair between IHC and INNO-LiPA (κ=0.22).
CONCLUSIONS:
Certain highly sensitive methods are able to detect the mere presence of HPV without any carcinogenetic involvement while other more specific tests provide proof of viral transcriptional activity and thus evidence of clinically relevant infections. The use of a stepwise approach allows reducing false positives; p16-immunostaining seems to be an excellent screening test and in situ hybridization may overcome some of the PCR limitations.
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology
Ma Y, Patil N, Gagner JP, Miles BA.
PMID: - | DOI: 10.1016/j.ajoms.2016.09.010
Increased testing for human papillomavirus (HPV) in oropharyngeal carcinomas has broadened the range of HPV-associated malignancies identified at this site. While HPV-related oropharyngeal non-keratinizing squamous cell carcinomas (SCC) are known to have a better prognosis than their non-HPV counterparts, HPV positivity may not alter the aggressive nature of HPV-associated small cell neuroendocrine carcinomas (SCNEC). We report a unique case of a mixed non-keratinizing type HPV-associated tonsillar SCC with SCNEC differentiation, and provide a comparison with the rare reported cases of such mixed carcinomas in the literature. Our patient is only the second such case positive for HPV genotype 18 and the only case in which this HPV-related mixed tonsillar tumor occurred in a patient with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The case discussion supports the concept that HPV positivity does not confer a better prognosis in such mixed non-keratinizing type SCC with SCNEC. Our report also alerts pathologists to the need to evaluate for the possibility of a coexisting neuroendocrine component when oropharyngeal squamous cell carcinoma (OPSCC) is diagnosed, as its presence will affect the patients’ clinical management and prognosis
