Virchows Arch. 2015 Jul 31.
Laco J, Sieglová K, Vošmiková H, Dundr P, Němejcová K, Michálek J, Čelakovský P, Chrobok V, Mottl R, Mottlová A, Tuček L, Slezák R, Chmelařová M, Sirák I, Vošmik M, Ryška A.
PMID: 26229021
The aim of the study was to investigate prevalence of high-risk human papillomavirus (HR-HPV) infection in sinonasal carcinomas by immunohistochemistry, in situ hybridization, and polymerase chain reaction, detecting p16INK4a protein (p16) expression and presence of both HPV DNA and HPV E6/E7 messenger RNA (mRNA). The study comprised 47 males and 26 females, aged 23-83 years (median 62 years), mostly (67 %) with a squamous cell carcinoma (SCC). Of the tumors, 53 % arose in the nasal cavity, 42 % in the maxillary sinus, and 5 % in the ethmoid complex. The follow-up period ranged 1-241 months (median 19 months). HPV16, HPV18, or HPV35 were detected in 18/73 (25 %) tumors, 17 SCCs, and 1 small cell neuroendocrine carcinoma. There was a strong correlation between results of HPV detection methods and p16 expression (p < 0.005). HPV-positive SCCs occurred more frequently in smokers (p = 0.04) and were more frequently p16-positive (p < 0.0001) and nonkeratinizing (p = 0.02), the latter occurring more commonly in nasal cavity (p = 0.025). Median survival for HPV-positive SCC patients was 30 months, while for HPV-negative SCC patients was 14 months (p = 0.23). In summary, we confirm that HR-HPV is actively involved in the etiopathogenesis of a significant subset of sinonasal SCCs. p16 may be used as a reliable surrogate marker for determination of HPV status also in sinonasal SCCs. Although we observed a trend toward better overall survival in HPV-positive SCCs, the prognostic impact of HPV status in sinonasal carcinomas needs to be elucidated by further studies.
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.
Mizutani M, Wu JC, Nusse R.
PMID: - | DOI: 10.1161/JAHA.115.002457
Background The adult mammalian heart responds to cardiac injury by formation of persistent fibrotic scar that eventually leads to heart failure. In contrast, the neonatal mammalian heart reacts to injury by the development of transient fibrotic tissue that is eventually replaced by regenerated cardiomyocytes. How fibrosis occurs in the neonatal mammalian heart remains unknown. To start elucidating the molecular underpinnings of neonatal cardiac fibrosis, we investigated Wnt signaling in the neonatal heart after cryoinjury.
Methods and Results Using expression of the Wnt target gene Axin2 as an indicator of Wnt/β‐catenin signaling activation, we discovered that epicardial cells in the ventricles are responsive to Wnt in the uninjured neonatal heart. Lineage‐tracing studies of these Wnt‐responsive epicardial cells showed that they undergo epithelial‐to‐mesenchymal transition and infiltrate into the subepicardial space and exhibit fibroblast phenotypes after injury. In addition, we showed that—similar to adult ischemic injury—neonatal cryoinjury results in activation of Wnt signaling in cardiac fibroblasts near injured areas. Furthermore, through in situ hybridization of all 19 Wnt ligands in injured neonatal hearts, we observed upregulation of Wnt ligands (Wnt2b, Wnt5a, and Wnt9a) that had not been implicated in the adult cardiac injury response.
Conclusions These results demonstrate that cryoinjury in neonatal heart leads to the formation of fibrotic tissue that involves Wnt‐responsive epicardial cells undergoing epithelial‐to‐mesenchymal transition to give rise to fibroblasts and activation of Wnt signaling in resident cardiac fibroblasts.
Pathology - Research and Practice
Wanga D, Fu L, Shah W, Zhang J, Yan Y, Ge X, He J, Wang Y, Xu Li.
PMID: - | DOI: dx.doi.org/10.1016/j.prp.2016.09.009
Background and aims
The causative role of high risk human papillomavirus (HR-HPV) in breast cancer development is controversial, though a number of reports have identified HR-HPV DNA in breast cancer specimens. Nevertheless, most studies to date have focused primarily on viral DNA rather than the viral transcription. The aim of this study was to investigate the presence of HR-HPV in breast cancer tissues at HPV DNA level and HPV oncogenes mRNA level by in situ hybridization (ISH).
Methods
One hundred and forty six (146) cases of breast invasive ductal carcinoma(IDC) and 83 cases of benign breast lesions were included in the study. Type specific oligonucleotide probes were used for the DNA detection of HPV 16,18 and 58 by ISH. HR-HPV oncogenes mRNA was assayed by novel RNAscope HR-HPV HR7 assay ISH. p16 protein expression was evaluated by immunohistochemistry (IHC).
Results
HR-HPV 16,18 and 58 DNA were detected in 52 out of 146 (35.6%) IDC and in 3 out of 83 (3.6%) benign breast lesions by ISH. The HR-HPV mRNAs was detected only in a few specimens with strong HPV DNA positivity(4/25) in a few scattered cancer cells with very weak punctate nuclear and/or cytoplasmic staining. p16 over-expression did not correlate with the HPV DNA positive breast cancer samples(17/52 HPVDNA+ vs 28/94 HPV DNA-, p = 0.731).
Conclusions
HR-HPVs certainly exist in breast cancer tissue with less active transcription, which implies that the causal role of HPV in breast cancer development need further study.
Swangphon P, Pientong C, Sunthamala N, Bumrungthai S, Azuma M, Kleebkaow P, Tangsiriwatthana T, Sangkomkamhang U, Kongyingyoes B, Ekalaksananan T.
PMID: 29064053 | DOI: 10.1007/s12307-017-0200-2
HPV infected cervical cells secrete mediators that are gradually changed and have influence on infiltrating M2 phenotypic monocytes in cervical lesions. However, profiles of circulating immune cells in women with cervical lesions and M2 phenotypic monocyte activity in HPV infected cervical lesions are limited. This study aimed to investigate circulating monocyte populations correlated with M2 phenotype density and its activity in HPV infected cervical lesions. HPV DNA was investigated in cervical tissues using PCR. High risk HPV E6/E7 mRNA was detected using in situ hybridization. CD163 immunohistochemical staining was performed for M2 macrophage. CD163 and Arg1 mRNA expression were detected using real-time PCR. Circulating monocyte subpopulations were analyzed using flow cytometry. CD163 and Arg1 mRNA expression were increased according to cervical lesion severity and corresponding with density of M2 macrophage in HSIL and SCC in stroma and peri-tumoral areas. Additionally, the relationship between M2 macrophage infiltration and high risk HPV E6/E7 mRNA expression was found and corresponded with cervical lesion severity. Circulating CD14+CD16+ and CD14+CD163+ monocytes were elevated in No-SIL and cervical lesions. Interestingly, CD14+CD64+ monocyte was greatly elevated in HSIL and SCC, whereas intracellular IL-10+monocytes were not significantly different between cervical lesions. The correlation between increasing ratio of circulating CD64+/CD163+monocyte and density of infiltrating CD163+ monocytes was associated with severity of HPV infected cervical lesions. The elevated circulating CD64+/CD163+ monocyte ratio correlates to severity of HPV infected cervical lesions and might be a prognostic marker in cervical cancer progression.
Head Neck Pathol. 2014 Apr 5
Bishop JA, Yonescu R, Batista D, Yemelyanova A, Ha PK, Westra WH
PMID: 24706055 | DOI: 10.1007/s12105-014-0541-9
High risk human papillomavirus (HPV) is firmly established as an important cause of oropharyngeal carcinoma. Recent studies have also implicated HPV as a cause of mucoepidermoid carcinoma (MEC)—a tumor of salivary gland origin that frequently harbors MAML2 translocations. The purpose of this study was to determine the prevalence of transcriptionally active HPV in a large group of MECs and to determine whether HPV infection and the MAML2 translocation are mutually exclusive events. Break-apart fluorescence in situ hybridization for MAML2 was performed on a tissue microarray containing 92 MECs. HPV testing was performed using RNA in situ hybridization targeting high risk HPV mRNA E6/E7 transcripts. Of the 71 MECs that could be evaluated by FISH, 57 (80 %) harbored the MAML2 rearrangement. HPV was not detected in any of the 57 MECs that contained a MAML2 rearrangement, in any of the 14 MECs that did not contain the rearrangement, or in any of the 21 MECs where MAML2 status was unknown. High risk HPV does not appear to play any significant role in the development of MEC. It neither complements nor replaces MAML2 translocation in the tumorigenesis of MEC
American J of Surgical Pathology, 35(9):1343–1350.
Ukpo OC, Flanagan JJ, Ma XJ, Ma XJ, Luo Y, Thorstad WL, Lewis JS Jr (2011).
PMID: 21836494 | DOI: 10.1097/PAS.0b013e318220e59d.
Human papillomavirus (HPV) is established as causative in oropharyngeal squamous cell carcinomas (OSCCs), being detected in 50% to 80% of tumors by DNA in situ hybridization (ISH) and/or polymerase chain reaction. However, these tests do not assess viral transcription. Many consider E6/E7 messenger ribonucleic acid (mRNA) the best indicator of HPV status, but it has not been detected in situ in OSCC. We constructed tissue microarrays (TMAs) from a cohort of OSCC for which p16 immunohistochemistry and HPV DNA ISH were previously performed on whole sections. We utilized a novel, chromogenic RNA ISH assay called RNAscope to detect E6/E7 mRNA of HPV-16 and other high-risk types on these TMAs. RNA ISH results were obtained for 196 of 211 TMA cases, of which 153 (78.1%) were positive. p16 immunohistochemistry and HPV DNA ISH were positive in 79.0% and 62.4% of cases, respectively. Concordance between RNA and p16, DNA and p16, and RNA and DNA were 96.4%, 78.7%, and 83.5%, respectively. Only 7 cases (3.6%) were discrepant between RNA ISH and p16. In univariate analysis, all 3 tests correlated with better overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) (all P<0.001). In multivariate analysis, OS correlated significantly with RNA (hazard ratio=0.39, P=0.001), DNA (0.53, P=0.03), and p16 (0.30, P<0.001), but DSS and DFS correlated significantly only with p16 (DSS: 0.36, P=0.006; DFS: 0.42, P=0.016). RNA ISH is more sensitive than DNA ISH in detecting HPV in OSCC, and it correlates strongly with p16. Although both tests were comparable, p16 more strongly stratified patient outcomes.
The American journal of surgical pathology, 37(9):1349–1356.
Mehrad M, Carpenter DH, Chernock RD, Wang H, Ma XJ, Luo Y, Luo J, Lewis JS Jr, El-Mofty SK. (2013).
PMID: 23797720 | DOI: 10.1097/PAS.0b013e318290427d.
A relationship between human papillomavirus (HPV) infection and papillary squamous cell carcinoma (PSCC) has been suggested. However, to date, no studies have thoroughly and directly evaluated for transcriptional activity of the virus or the clinicopathologic significance of HPV-positive PSCC. Forty-eight cases of PSCC were retrieved from our surgical pathology database and were reviewed by 4 study pathologists, with tumors defined as SCC with a significant component of papillary growth in the tumor. Immunohistochemical analysis for p16 and p53 was performed. Overexpression of p16 was used as a surrogate marker of transcriptionally active HPV. Transcriptional activity was also directly evaluated using RNA in situ hybridization to detect high-risk HPV E6/E7 mRNA. Clinical follow-up data were obtained by chart review. Seven cases were located in the oral cavity, 19 in the oropharynx, and 22 in the larynx. Two morphologic types of PSCC were identified: keratinizing type, in which the epithelial cells showed a maturation trend with minimal surface parakeratin, and nonkeratinizing type, in which the papillae were completely covered by immature basaloid cells. Transcriptionally active HPV was present in 23 of 43 (53.4%) tumors. The majority of tumors harboring transcriptionally active HPV arose in the oropharynx, showed nonkeratinizing morphology, were p16 positive, and p53 negative. Transcriptionally active HPV was also present in many laryngeal and oral cavity PSCCs. Overall survival, disease-specific survival, and disease-free survival were favorable and did not significantly differ by anatomic subsite. However, HPV-related tumors showed a trend toward better survival.
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.
Cancer Cytopathol. 2015 Aug 4.
Jalaly JB, Lewis JS Jr, Collins BT, Wu X, Ma XJ, Luo Y, Bernadt CT.
PMID: 26242494 | DOI: 10.1002/cncy.21600.
Abstract BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high-risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine-needle aspirations (FNAs) with surgical pathology specimens of HPV-related oropharyngeal SCC. METHODS: In total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high-risk HPV messenger RNA was performed on 31 of the FNA cell blocks. RESULTS: Although partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high-risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%). CONCLUSIONS: There was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false-negative results if applied to FNA cell blocks.
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.
Jiang RT, Wang JW, Peng S, Huang TC, Wang C, Cannella F, Chang YN, Viscidi RP, Best SRA, Hung CF, Roden RBS.
PMID: 28515303 | DOI: 10.1128/JVI.00699-17
Mus musculus Papillomavirus1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not common laboratory strains. To facilitate study of immune control, we sought an outbred and immune competent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) by scarification on their tail with MmuPV1 resulted in three clinical outcomes: 1) persistent (>2 months) papillomas (∼20%), 2) transient papillomas that spontaneously regress typically within 2 months (∼15%), 3) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated using a candidate preventive/therapeutic naked DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and E7 (mE7) early proteins and residues 11-200 of late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and failure of disease to recur after CD3 T cell depletion. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice, although significantly lower in mice that initially presented with persistent warts as compared with those that spontaneously cleared their infection. An HPV16-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with HPV16 pseudovirus. Thus MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearance of HPV-related disease.IMPORTANCE High risk type human papillomaviruses (hrHPV) cause 5% of all cancer cases worldwide, notably cervical, anogenital and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries, and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas, and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Further an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16 suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.
