Human Papillomavirus RNA Research Articles

Human papillomavirus and human telomerase RNA component gene in cervical cancer progression

This study aimed to examine hTERC gene in different grades of cervical intraepithelial neoplasia (CIN) and cervical cancer, and the association between hTERC and high risk-human papillomavirus (HR-HPV) infection. Patients who underwent cervical cancer screening at the Second Affiliated Hospital of Kunming Medical University between October 2010 and December 2011 were enrolled. All patients underwent liquid-based cytology test and hybrid capture 2 (HC2) for HPV detection. hTERC was examined using fluorescence in situ hybridization (FISH). Cervical colposcopy biopsy was performed if any of the three results was positive. HC2, FISH, and pathology were compared. A total of 1200 women underwent screening, 150 patients underwent cervical biopsy: 32 in the normal group, 38 in the CIN1 group, 66 in the CIN2/3 group, and 14 in the invasive cervical cancer group. More patients had HR-HPV infection in the CIN2/3 group and ICC group compared with the CIN1 group. hTERC increased with increasing histological dysplasia. There was significant difference in hTERC positive rate between each of the three groups. More patients with hTERC gene amplification were observed in the positive HR-HPV group than in the HR-HPV negative group. In conclusion, hTERC is a potential marker for precancerous cervical cancer lesions. hTERC might be correlated with HR-HPV infection in cervical diseases.

A clinicopathological and molecular analysis of cervical carcinomas with basaloid features.

To investigate the relationship between adenoid basal carcinoma (ABC), adenoid cystic carcinoma (ACC) and squamous cell carcinoma (SCC) in the uterine cervix. We analysed the clinicopathological and molecular features in two pure ABCs, 15 SCCs with ABC-/ACC-like features and seven basaloid SCCs (BSCCs) by chart review, immunohistochemistry, human papillomavirus (HPV) RNA in-situ hybridisation and fluorescence in-situ hybridisation. All patients were alive with no evidence of disease, except for one patient with ACC-like features who died of disease at 18months post diagnosis. The mixed carcinomas comprised variable SCCs and ABC-/ACC-like components displaying vague transitional zones. All components consistently showed diffuse p16, p63 and SOX2, variable cytokeratin (CK)7 and CK17 and rare Ber-EP4 and MYB expression; there was a substantially lower Ki67 index in pure ABCs and the ABC-like components. The ACC-like components showed no myoepithelial differentiation (SMA, calponin and S100) and MYB gene fusions. CK7, CK17 and Ber-EP4 were characteristically stronger in BSCCs than in the mixed carcinomas (P<0.01). High-risk HPV (HR-HPV) E6/E7 mRNA was detected in 12 mixed carcinomas and seven BSCCs, but not in pure ABCs. The HR-HPV mRNA expression was higher in the SCC components and BSCCs than in the ABC-like components of mixed carcinomas (P<0.05). The ACC-like components in mixed carcinomas probably represent the morphological mimics of salivary ACCs. ABC-like components may be the potential precursor of the ACC-like and SCC components. HR-HPV oncogenes may play a role in the pathogenesis of SCCs with ABC-/ACC-like features.

Tissue Fixation Conditions for p16 Immunohistochemistry and Human Papillomavirus RNA In Situ Hybridization in Oropharyngeal Squamous Cell Carcinoma.

Human papillomavirus (HPV) has become a critical prognostic biomarker in oropharyngeal squamous cell carcinoma (OPSCC). While retrospective studies suggest that p16 immunohistochemistry and even HPV RNA in situ hybridization work well on tissues and tumors from a variety of labs and various fixation conditions, no formal study of fixation conditions has been performed to date. We took surgically resected specimens from three p16 and HPV RNA in situ hybridization positive OPSCC patients, divided their fresh tumors into small pieces, and varied the time to formalin fixation as 1, 3, 6, 24, and 48h. Tumors were either held moistened at room temperature or were refrigerated. After fixation and processing, routine hematoxylin and eosin slides were generated and p16 immunohistochemistry and RNA in situ hybridization performed. All three tumors were nonkeratinizing and had strong and diffuse p16 expression at immediate fixation, which, surprisingly, remained positive for all fixation times and conditions and despite significant degeneration at the later points for two of the patients while for one, the nuclear signal dropped out of most cells at early and mid time points, particularly at room temperature, causing false negatives. HPV RNA in situ hybridization stayed positive in all specimens up to 48h of cold ischemic time refrigerated and even at room temperature, except for overtly autolyzed tumor regions. These findings help to establish that, at least for standard nonkeratinizing, p16 and HPV RNA strongly positive OPSCC patients, and using the most common tests in clinical practice, relatively lenient time to fixation may be acceptable if it cannot be avoided. However, for some patients, p16 immunohistochemistry may be sensitive to signal loss with autolysis. HPV RNA in situ hybridization, in particular, seems remarkably resistant to pretest cold ischemic times.

Heterogeneity of p16 immunohistochemistry and increased sensitivity of RNA in situ hybridization in cytology specimens of HPV-related head and neck squamous cell carcinoma.

Many patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HPV-HNSCC) initially present with cervical lymph node metastases. Although p16 immunohistochemistry (IHC) is the most commonly used surrogate marker for HPV, however criteria in cytologic material are not well established. The objective of this study was to better characterize p16 IHC in cell blocks of metastatic HPV-HNSCC, and to evaluate the performance of HPV RNA in situ hybridization (RNA ISH). p16 IHC was performed on cell blocks from 97 metastatic HPV-HNSCC fine-needle aspiration specimens with HPV status confirmed by DNA or RNAISH or polymerase chain reaction (PCR). Tumor cellularity (<100 cells, 100-500 cells, and >500 cells) and quality (presence of cell clusters, necrosis) were recorded. p16 staining intensity and extent (1%-9%, 10%-69%, and ≥70%) were scored. In addition, RNA ISH was performed on 38 PCR-positive cases. p16 IHC was positive in 90 of 97 cases (93%), demonstrating variable patterns. p16 staining was found to be moderate to strong in 69 cases, with 37 cases (38%) demonstrating positivity in ≥70% of tumor cells. Weak staining occurred in 21 cases (22%) and 7 cases (7%) were negative. Of the 60 cases with weak and/or absent expression or staining in <70% of cells, 30 cases (50%) had <100 tumor cells, 12 (20%) lacked cell clusters, and 19 cases (32%) had extensive necrosis. RNA ISH was positive in 37 of 38 cases (97%) that were HPV positive by PCR. p16 is heterogeneous in cell blocks of metastatic HPV-HNSCC, suggesting that any p16 positivity should prompt confirmatory HPV studies. RNA ISH appears to demonstrate high sensitivity, and laboratories even may consider using RNA ISH as a first-line HPV test in cytologic specimens.

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since "low" or "high" HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Detection of HPV RNA molecules in stratified mucin-producing intraepithelial lesion (SMILE) with concurrent cervical intraepithelial lesion: a case report

BackgroundStratified mucin-producing intraepithelial lesion (SMILE) is a rare precursor lesion in the uterine cervix that is considered a variant of adenocarcinoma in situ (AIS). Although human papillomavirus (HPV) is thought to be related to the development of SMILE, there is little information available on the detection of HPV integrated into the lesion.Case presentationA 30-year-old female underwent a routine uterine cervical cancer screening, and her Pap smear indicated the possible existence of atypical glandular cells. A cervical biopsy with endocervical curettage was performed. The histopathological analysis showed that she had SMILE and high-grade squamous intraepithelial lesion (HSIL) on her cervix. The lesion was found to be positive for HPV genotypes 52 and 68 by multiplex PCR. In situ hybridization with HPV RNA probes revealed that these HPV types were involved in the onset of HSIL and SMILE, respectively.ConclusionsRare, high-risk HPV genotypes may contribute to the development of SMILE, and their detection can be useful for preventing the progression to carcinoma and ensuring adequate patient management.

Genome-wide miRNA profiling reinforces the importance of miR-9 in human papillomavirus associated oral and oropharyngeal head and neck cancer

Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV− OPSCC, 62 to HPV− OSCC, and 31 shared with both. The results for 9 miRNAs (miR-9, -21, -29a, -100, -106b, -143 and -145) were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV− HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA.

Malignant Transformation of Fanconi Anemia Complementation Group D2-deficient (Fancd2 -/-) Hematopoietic Progenitor Cells by a Single HPV16 Oncogene.

To demonstrate that Fanconi anemia complementation group D2-deficient (Fancd2-/-) hematopoietic progenitor cell lines can be transformed by transfection with a plasmid containing either the E6 or E7 oncogene of human papillomavirus (HPV) to generate malignant plasmacytoma-inducing cell lines. In order to determine whether a single HPV type 16 (HPV16) oncogene induced malignant transformation, Fancd2-/- and Fancd2+/+ interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines were transfected with plasmids containing E6 or E7 oncogene, or control empty plasmid. Fancd2-/- but not Fancd2+/+ cells were transformed into malignant IL3-independent cells by both E6, and E7 oncogenes, but not by empty plasmid. Hematopoietic cell lines and tumors induced by Fancd2-/- E6 and Fancd2-/- E7 cell lines were positive for each respective HPV RNA and protein. A single HPV16 oncogene is adequate to produce malignant transformation of Fancd2-/- hematopoietic cells.

Epstein-Barr virus and human papillomaviruses as favorable prognostic factors in nasopharyngeal carcinoma: A nationwide study in Finland.

BackgroundNasopharyngeal carcinoma (NPC) is related to Epstein‐Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors.MethodsWe analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records.ResultsIn our white patient cohort, 93 of 150 (62%) patients were EBV‐positive and 21/150 (14%) patients were HPV‐positive with no coinfections. Thirty‐six (24%) tumors were negative for both viruses. The 5‐year disease‐specific survival for patients with EBV‐positive, HPV‐positive, and EBV/HPV‐negative tumors was 69%, 63%, and 39%, respectively. In multivariable‐adjusted analysis, overall survival was better among patients with EBV‐positive (P = .005) and HPV‐positive (P = .03) tumors compared to patients with EBV/HPV‐negative tumors.ConclusionsIn our low‐incidence population, EBV and HPV are important prognostic factors for NPC.

Reducing the Radiation Therapy Dose Prescription for Elective Treatment Areas in Human Papillomavirus–Associated Oropharyngeal Carcinoma Being Treated With Primary Chemoradiotherapy at Memorial Sloan Kettering Cancer Center

AbstractData on the biology and radiosensitivity of human papillomavirus–related cancers have led many expert radiation oncologists to decrease the radiation dose to target areas in selected situations. At Memorial Sloan Kettering Cancer Center, we have altered our radiation guidelines to allow substantial dose reduction to elective treatment regions and to omit radiation completely in other regions that have historically been included in the elective treatment volume. Here we summarize our current dose prescriptions and briefly explain the rationale behind these choices, which we anticipate will change when additional data mature. These recommendations are based on the American Joint Committee on Cancer seventh edition TNM Staging System and applicable only to patients who receive concurrent platinum-based chemotherapy and in whom both p16 immunohistochemistry and human papillomavirus RNA in situ hybridization tests are positive.

Detection of High-Risk Human Papillomavirus in Oral Cavity Squamous Cell Carcinoma Using Multiple Analytes and Their Role in Patient Survival.

PurposeAccurate detection of human papillomavirus (HPV) in oral cavity squamous cell carcinoma (OSCC) is essential to understanding the role of HPV in disease prognosis and management of patients. We used different analytes and methods to understand the true prevalence of HPV in a cohort of patients with OSCC with different molecular backgrounds, and we correlated HPV data with patient survival.MethodsWe integrated data from multiple analytes (HPV DNA, HPV RNA, and p16), assays (immunohistochemistry, polymerase chain reaction [PCR], quantitative PCR [qPCR], and digital PCR), and molecular changes (somatic mutations and DNA methylation) from 153 patients with OSCC to correlate p16 expression, HPV DNA, and HPV RNA with HPV incidence and patient survival.ResultsHigh prevalence (33% to 58%) of HPV16/18 DNA did not correlate with the presence of transcriptionally active viral genomes (15%) in tumors. Eighteen percent of the tumors were p16 positive and only 6% were both HPV DNA and HPV RNA positive. Most tumors with relatively high copy number HPV DNA and/or HPV RNA, but not with HPV DNA alone (irrespective of copy number), were wild-type for TP53 and CASP8 genes. In our study, p16 protein, HPV DNA, and HPV RNA, either alone or in combination, did not correlate with patient survival. Nine HPV-associated genes stratified the virus-positive from the virus-negative tumor group with high confidence (P < .008) when HPV DNA copy number and/or HPV RNA were considered to define HPV positivity, and not HPV DNA alone, irrespective of copy number (P < .2).ConclusionIn OSCC, the presence of both HPV RNA and p16 is rare. HPV DNA alone is not an accurate measure of HPV positivity and therefore may not be informative. HPV DNA, HPV RNA, and p16 do not correlate with patients’ outcome.

Epstein-Barr virus, but not human cytomegalovirus, is associated with a high-grade human papillomavirus-associated cervical lesions among women in North Carolina.

Human papillomavirus (HPV) infection is known to contribute to cervical carcinogenesis, yet other cofactors that may contribute to oncogenesis are poorly understood. Herein, we examine whether the Epstein-Barr virus (EBV) and human cytomegalovirus (CMV), two oncomodulatory viruses, are associated with HPV-mediated cervical neoplastic progression. Sixty patient cervical brush samples from a study of North Carolina women were obtained. HPV RNA positivity was determined by Aptima testing (Hologic Corporation, Marlborough, MA). The level of viral transcripts for EBV and CMV was quantified (reverse transcription polymerase chain reaction analysis), and the coinfection status with HPV was then compared with the patient's cervical cytology grade. Over one-third (38.3%) of the study population was CMV positive, whereas 43.3% was EBV positive. When sample data were stratified by the cytology grade, 36.5% (19/52) of normal patients, 75% (3/4) of low-grade squamous intraepithelial lesions (LSIL), and 100% (4/4) of patients with high-grade SIL (HSIL) were EBV positive. Conversely, 35.2% (18/52) of normal patients, 25% (1/4) of patients with LSIL, and 50% (2/4) of patients with HSIL were CMV positive. When examining only HPV positive-associated HSIL, 100% (4/4) were positive for both HPV and EBV detection. This suggests that co-viral detection with HPV and EBV is associated with more advanced HSIL cervical lesions, while CMV displayed no clear association with a higher grade of cervical cytology. Co-viral detection with EBV may increase the oncogenicity and/or serve as a viral marker of progression to HPV-associated high-grade cervical dysplasia.

HPV RNA in situ hybridization can inform cervical cytology-histology correlation.

In situ hybridization for human papillomavirus (HPV) messenger RNA (HPV RNA ISH) recently was introduced as an ancillary tool in the diagnosis of cervical squamous intraepithelial lesions, and can aid in the distinction between low-grade squamous intraepithelial lesions (LSILs) versus reactive/negative biopsies. Prior work has shown that up to one-half of cases originally diagnosed as LSIL are reclassified as negative/reactive by expert consensus review of morphology, and negative HPV RNA ISH results most often correlate with an expert diagnosis of negative/reactive. Given that LSIL overdiagnoses on biopsy may result in the erroneous clinical impression that a cervical lesion has been sampled appropriately, the authors proposed that HPV RNA ISH can inform cytology-histology correlation for challenging LSIL biopsies. A total of 92 cervical biopsies originally diagnosed as LSIL were reviewed by 3 gynecologic pathologists and reclassified based on consensus opinion of morphology. ISH was performed for high-risk and low-risk HPV E6/E7 mRNA. Prior/concurrent cytology results were collected. Based on expert consensus morphologic review, 49% of biopsies (45 of 92 biopsies) originally diagnosed as LSIL were reclassified as negative, 6.5% (6 of 92 biopsies) were reclassified as high-grade squamous intraepithelial lesion, and 44.5% (41 of 92 biopsies) were maintained as LSIL. The majority of LSIL biopsies reclassified as negative (80%; 36 of 45 biopsies) were HPV RNA negative, whereas 93% of LSIL biopsies (39 of 41 biopsies) and 100% of high-grade squamous intraepithelial lesion biopsies were HPV RNA positive. LSIL often is overdiagnosed by morphology on biopsy, potentially leading to the false impression that a lesion identified on cytology has been sampled. Performing RNA ISH on biopsies decreases histologic LSIL overdiagnosis, and potentially can prompt further sampling when there is cytology-histology discordance. Cancer (Cancer Cytopathol) 2018. © 2018 American Cancer Society.

Role of mucosal high-risk human papillomavirus types in head and neck cancers in Romania.

BackgroundLimited information is available about the involvement of human papillomavirus (HPV) in head and neck squamous cell carcinomas (HNSCCs) in Romanian patients.ObjectiveTo evaluate the HPV-attributable fraction in HNSCCs collected in Northeastern Romania.Materials and methodsIn total, 189 formalin-fixed paraffin-embedded tissue samples (99 oral cavity tumors, 28 oropharynx, 48 pharynx, and 14 larynx/hypopharynx) were analyzed for HPV DNA and RNA using Luminex-based assays, and for overexpression of p16INK4a (p16) by immunohistochemistry.ResultsOf the 189 cases, 23 (12.2%) were HPV DNA-positive, comprising half of the oropharyngeal cases (14/28, 50.0%) and 9/161 (5.6%) of the non-oropharyngeal cases. HPV16 was the most prevalent HPV type (20/23, 86.9%), followed by HPV18 (5/23, 21.7%) and HPV39 (1/23, 4.3%). Only two (2/189, 1.1%) HNSCC cases were HPV-driven, i.e. positive for both HPV DNA and RNA.ConclusionA very small subset of HNSCC cases within this cohort from Northeastern Romania appeared to be HPV-driven.

Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

Immediate histopathologic follow-up of cervista and aptima high-risk HPV assays in women with LSIL cytology.

Recent guidelines recommended the optional deferral of colposcopy for postmenopausal women with low-grade squamous intraepithelial lesion (LSIL) Papanicolaou (Pap) test results and negative human papillomavirus (HPV) testing. The objective of the current study was to assess the histopathologic follow-up of Cervista and Aptima high-risk HPV (hrHPV) testing in patients with LSIL cytology. Women with LSIL Pap test results and Cervista or Aptima hrHPV testing results were retrospectively identified from June 2013 through July 2017. Histological follow-up results within 6 months after LSIL Pap tests were analyzed. A total of 1731 and 1906 cases of LSIL Pap tests, respectively, were tested on Cervista and Aptima platforms. Among the 2119 cases with histopathologic follow-up, cervical intraepithelial neoplasia of types 2/3 (CIN2/3) was diagnosed in 184 women (8.9%) and the detection rate was significantly higher in women with positive HPV testing compared with those with a negative result on both assays. Both methods demonstrated comparable performance for detecting CIN2/3 lesions. However, in women aged ≥50 years, the specificity for the detection of CIN2/3 lesions by the Aptima assay was statistically significantly higher than that of the Cervista test (48.7% vs 23.1%; P<.01), although there were no significant differences in the sensitivity, positive predictive value, and negative predictive value between these 2 assays in this age group. The Aptima assay was found to be statistically significantly more specific than the Cervista test for detecting CIN2/3 lesions among women aged ≥50 years. These findings not only further support the recommendations by the American Society for Colposcopy and Cervical Pathology that hrHPV triage is an acceptable option for postmenopausal women with LSIL cytology, but also provide additional evidence that HPV RNA testing may be more useful in clinical risk stratification due to its specificity in the postmenopausal population. Cancer Cytopathol 2018. © 2018 American Cancer Society.

Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.

Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.

HNSCC Gene Expression Subtypes, Including a HPV Subtype, Demonstrate Differential Immune Cell and Biomarker Associations

Human papilloma virus (HPV) RNA expression in combination with a gene expression signature of 144 genes was used to classify head and neck squamous cell carcinoma (HNSCC) into 5 distinct subtypes. The subtypes are investigated for differential associations with clinical features, immune infiltration, and immune marker expression. Using previously published Bindea et al. immune cell gene signatures (24 in total) and the TCGA Head & Neck cancer gene expression dataset (HNSCC n=520), we examined immune cell expression in relation to 5 HNSCC gene expression subtypes (atypical, classical, basal, mesenchymal, and HPV subtype). The HPV tumor subtype was determined by alignment of RNAseq with HPV sequences and by evaluation of its gene expression profile. Signatures of multiple immune cells as well as single immune-biomarkers, (CTLA4, PDCD1(PD-1), and CD274(PD-L1) were examined across the 5 subtypes. Differential gene expression was examined using the Kruskal-Wallis test. Immune cell signature associations with tumor subtype and with CD274 expression were evaluated using linear regression. Survival-based associations were evaluated with cox proportional hazard models. Immune cell expression was significantly different across the subtypes (Tcells KW P=1.7e-17) and in general was highest in HPV positive tumors. The classical expression subtype demonstrated lower Tcell immune expression. CD274 (PD-L1) expression was less variable across the subtypes but lowest expression was again seen in the classical subtype (KW P=3.7e-06). Mutation burden varied across the subtypes with the highest mutation burden observed in the atypical subtype and the lowest mutation burden in the HPV tumors (KW P=1.7e-10). Subtype and HPV status were more strongly associated with immune expression than CD274 (PD-L1) expression (median F-test P-value and adjusted R-squared were 2.0e-16 and 0.14 for subtype versus 6.7e-07 and 0.04 for CD274). Cox models with adjustment for stage suggested higher immune cell expression is associated with better survival, and most strongly in the HPV tumor subtype including T gamma delta cells HR 0.2 (95% CI 0.06-0.68) and T cells HR 0.33 (95% CI 0.14-0.79). Biologic subtypes of HNSCC reveal key differences in immune cell expression, which were not always correlated with CD274 (PD-L1) expression. Evaluation of subtypes as potential biomarkers for immunotherapy response in HNSCC should be investigated.

The role of human papillomavirus in p16-positive oral cancers.

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. 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 insitu hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. 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. 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.

HPV RNA In Situ Hybridization can Inform Cervical Cytology-Histology Correlation

Background In situ hybridization for human papillomavirus (HPV) messenger RNA (HPV RNA ISH) recently was introduced as an ancillary tool in the diagnosis of cervical squamous intraepithelial lesions, and can aid in the distinction between low-grade squamous intraepithelial lesions (LSILs) versus reactive/negative biopsies. Prior work has shown that up to one-half of cases originally diagnosed as LSIL are reclassified as negative/reactive by expert consensus review of morphology, and negative HPV RNA ISH results most often correlate with an expert diagnosis of negative/reactive. Given that LSIL overdiagnoses on biopsy may result in the erroneous clinical impression that a cervical lesion has been sampled appropriately, the authors proposed that HPV RNA ISH can inform cytology-histology correlation for challenging LSIL biopsies. Methods A total of 92 cervical biopsies originally diagnosed as LSIL were reviewed by 3 gynecologic pathologists and reclassified based on consensus opinion of morphology. ISH was performed for high-risk and low-risk HPV E6/E7 mRNA. Prior/concurrent cytology results were collected. Results Based on expert consensus morphologic review, 49% of biopsies (45 of 92 biopsies) originally diagnosed as LSIL were reclassified as negative, 6.5% (6 of 92 biopsies) were reclassified as high-grade squamous intraepithelial lesion, and 44.5% (41 of 92 biopsies) were maintained as LSIL. The majority of LSIL biopsies reclassified as negative (80%; 36 of 45 biopsies) were HPV RNA negative, whereas 93% of LSIL biopsies (39 of 41 biopsies) and 100% of high-grade squamous intraepithelial lesion biopsies were HPV RNA positive. Conclusions LSIL often is overdiagnosed by morphology on biopsy, potentially leading to the false impression that a lesion identified on cytology has been sampled. Performing RNA ISH on biopsies decreases histologic LSIL overdiagnosis, and potentially can prompt further sampling when there is cytology-histology discordance. Cancer (Cancer Cytopathol) 2018. © 2018 American Cancer Society.