J Int J Clin Exp Pathol (2018)
Cui L, Qu C, Liu H.
| DOI: ISSN:1936-2625/IJCEP0085220
Abstract: Aims: To investigate the frequency and transcriptional activity of HPV and its correlation to p16 and p21 expression in basaloid squamous cell carcinoma (BSCC) of the larynx. Methods: We evaluated tissues from 29 patients with BSCC of the larynx for the expressions of p16 and p21 proteins by immunohistochemistry (IHC) and for HPV E6 and E7 mRNA by RNA in situ hybridization (ISH). The presence of genotype-specific HPV DNA was evaluated using PCR-RDB in formalin-fixed paraffin-embedded tissues. P16 and p21 expression and HPV DNA status were correlated with clinicopathological features. Results: HPV DNA was detected in 8 of 29 (27.59%) patients, with HPV-16 being the predominant genotype. P16 and p21-positivity were observed in 7/29 (24.14%) and 8/29 (27.59%) patients, respectively. HPV was not correlated with p16 expression (P > 0.05). However, p21 expression was significantly higher in HPV-positive tumors than in HPV-negative tumors (P < 0.05). No cases exhibited transcriptionally active HPV in our series. Conclusion: Our findings suggest that a small fraction of BSCC of the larynx is HPV DNA-positive in this Chinese population, p21 expression was significantly higher in HPV-positive tumors, and no cases were HPV transcriptionally active in this small cohort. Further research of HPV and its role in BSCC of the larynx are warranted.
Annals of oncology : official journal of the European Society for Medical Oncology
Rischin, D;Mehanna, H;Young, RJ;Bressel, M;Dunn, J;Corry, J;Soni, P;Fulton-Lieuw, T;Iqbal, G;Kenny, L;Porceddu, S;Wratten, C;Robinson, M;Solomon, BJ;Trans-Tasman Radiation Oncology Group and the De-ESCALaTE HPV Trial Group, ;
PMID: 35525376 | DOI: 10.1016/j.annonc.2022.04.074
High CD103+ intratumoral immune cell (ITIC) abundance is associated with better prognosis in unselected patients with human papilloma virus associated oropharyngeal squamous cell carcinoma(HPV-associated OPSCC) treated with cisplatin and radiotherapy(CIS/RT). Substituting cetuximab(CETUX) for CIS with RT in HPV-associated OPSCC resulted in inferior efficacy. Our aim was to determine if quantification of ITIC CD103 could be used to identify a population of HPV-associated OPSCC with superior prognosis.We pooled data from the TROG 12.01 and De-ESCALaTE randomised trials that compared CETUX/70GyRT with CIS/70GyRT in low risk HPV-associated OPSCC: AJCC 7th Stage III (excluding T1-2N1) or stage IV (excluding N2b-c if smoking history >10 pack years and/or distant metastases), including all patients with available tumor samples. The primary endpoint was failure-free survival (FFS) in patients receiving CETUX/ RT comparing CD103+ ITIC high (>30%) versus low (<30%). High/low CD103 were compared using Cox regression adjusting for age, stage and trial.Tumor samples were available in 159/182 patients on TROG 12.01 and 145/334 on De-ESCALaTE. CD103+ ITIC abundance was high in 27% of patients. The median follow-up was 3.2 years. The 3-year FFS in patients treated with CETUX/RT were 93% (95% CI: 79-98%) in high CD103 and 74% (95% CI: 63-81%) in low CD103, adjusted HR 0.22 (95% CI: 0.12-0.41); p<0.001. The 3-year overall survival in patients treated with CETUX/RT was 100% in high CD103 and 86% (95% CI: 76-92%) in low CD103, p<0.001. In patients treated with CIS/RT there was no significant difference in FFS.CD103+ ITIC expression separates CETUX/RT treated low risk HPV-associated OPSCC into excellent and poor prognosis subgroups. The high CD103 population is a rational target for de-intensification trials.
Rooper LM, Bishop JA, Westra WH.
PMID: 28181187 | DOI: 10.1007/s12105-017-0779-0
The role of human papillomavirus (HPV) as an etiologic and transformational agent in inverted Schneiderian papilloma (ISP) is unclear. Indeed, reported detection rates of HPV in ISPs range from 0 to 100%. The true incidence has been confounded by a tendency to conflate high- and low-risk HPV types and by the inability to discern biologically relevant from irrelevant HPV infections. The recent development of RNA in situ hybridization for high-risk HPV E6/E7 mRNA now allows the direct visualization of transcriptionally active high-risk HPV in ISP, providing an opportunity to more definitively assess its role in the development and progression of ISPs. We performed p16 immunohistochemistry and high-risk HPV RNA in situ hybridization on 30 benign ISPs, 7 ISPs with dysplasia, 16 ISPs with carcinomatous transformation, and 7 non-keratinizing squamous cell carcinomas (SCCs) with inverted growth that were unassociated with ISP. Transcriptionally active HPV was not detected in any of the 52 ISPs including those that had undergone carcinomatous transformation, but it was detected in two of seven (29%) non-keratinizing SCCs that showed inverted growth. There was a strong correlation between high-risk HPV RNA in situ hybridization and p16 immunohistochemistry (97%; p < 0.01). These results indicate that transcriptionally active high-risk HPV does not play a common role in either the development of ISP or in its transformation into carcinoma.
Journal of Neuroendocrinology
Watanabe, Y;Fisher, L;Campbell, R;Jasoni, C;
| DOI: 10.1111/jne.13302
Polycystic ovary syndrome (PCOS) is a female endocrine disorder that is associated with prenatal exposure to excess androgens. In prenatally androgenized (PNA) mice that model PCOS, GABAergic neural transmission to and innervation of GnRH neurons is increased. Evidence suggests that elevated GABAergic innervation originates in the arcuate nucleus (ARC). We hypothesised that GABA-GnRH circuit abnormalities are a direct consequence of PNA, resulting from DHT binding to androgen receptor (AR) in the prenatal brain. However, whether prenatal ARC neurons express AR at the time of PNA treatment is presently unknown. We used RNAScope _in situ_ hybridization to localize AR mRNA (_Ar_)-expressing cells in healthy gestational day (GD) 17.5 female mouse brains and to assess co-expression levels in specific neuronal phenotypes. Our study revealed that less than 10% of ARC GABA cells expressed _Ar_. In contrast, we found that ARC kisspeptin neurons, critical regulators of GnRH neurons, were highly co-localised with _Ar_. Approximately 75% of ARC _Kiss1_-expressing cells also expressed _Ar_ at GD17.5, suggesting that ARC kisspeptin neurons are potential targets of PNA. Investigating other neuronal populations in the ARC we found that approximately 50% of pro-opiomelanocortin (_Pomc_) cells, 22% of tyrosine hydroxylase (_Th_) cells, 8% of agouti-related protein (_Agrp_) cells and 8% of somatostatin (_Sst_) cells express _Ar_. Lastly, RNAscope in coronal sections showed _Ar_ expression in the medial preoptic area (mPOA), and the ventral part of the lateral septum (vLS). These _Ar_-expressing regions were highly GABAergic, and 22% of GABA cells in the mPOA and 25% of GABA cells in the vLS also expressed _Ar_. Our findings identify specific neuronal phenotypes in the ARC, mPOA and vLS that are androgen sensitive in late gestation. PNA-induced functional changes in these neurons may be related to the development of impaired central mechanisms associated with PCOS-like features.
Rao, X;Zheng, L;Wei, K;Li, M;Jiang, M;Qiu, J;Zhou, Y;Ke, R;Lin, C;
PMID: 36809088 | DOI: 10.1128/spectrum.03896-22
RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA in situ hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel in situ hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock probing and rolling circle amplification, combined with chromogenic readout. We designed padlock probes for 14 types of high-risk HPV and demonstrated that E6/E7 mRNA could be visualized in situ as discrete dot-like signals using bright-field microscopy. Overall, the results are consistent with the clinical diagnostics lab's hematoxylin and eosin (H&E) staining and p16 immunohistochemistry test results. Our work thus shows the potential applications of RNA in situ hybridization for clinical diagnostics using chromogenic single-molecule detection, offering an alternative technical option to the current commercially available kit based on branched DNA technology. IMPORTANCE In situ detection of viral mRNA expression in tissue samples is of great value for pathological diagnosis to access viral infection status. Unfortunately, conventional RNA in situ hybridization assays lack sensitivity and specificity for clinical diagnostic purposes. Currently, the commercially available branched DNA technology-based single-molecule RNA in situ detection method offers satisfactory results. Here, we present our padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression in formalin-fixed paraffin-embedded tissue sections, providing an alternative yet robust method for viral RNA in situ visualization that is also applicable to different types of diseases.
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.
Detection of Human Papillomavirus in Non-Small Cell Carcinoma of the Lung
Chang SY, Keeney M, Law M, Donovan J, Aubry MC, Garcia J.
High-risk human papillomavirus (hrHPV) is an etiologic agent in squamous cell carcinoma (SqCC) arising in the oropharynx and cervix, and a proven prognostic factor in oropharyngeal SqCC. Many studies have found HPV in non-small cell lung carcinoma (NSCLC). Recent studies advocate the detection of mRNA transcripts of E6/E7 as more reliable evidence of transcriptively active HPV in tumor cells. The clinical significance of finding HPV remains unclear in NSCLC. This study sought to determine the prevalence of biologically active HPV infection in NSCLC comparing different methodologies. Surgical pathology material from resected primary lung adenocarcinoma (ADC; n = 100) and SqCC (n = 96) were retrieved to construct tissue microarrays. In-situ hybridization (ISH) for hrHPV DNA (DNA-ISH), hrHPV E6/E7 RNA (RNA-ISH), and p16 immunohistochemistry (IHC) were performed. Cases of oropharyngeal SqCC with known HPV infection were used as positive controls. Expression of p16 was scored as positive if at least 70% of tumor cells showed diffuse and strong nuclear and cytoplasmic staining. Punctate nuclear hybridization signals by DNA-ISH in the malignant cells defined an HPV-positive carcinoma. Of the 196 patients (range 33-87 years; 108 men), p16 was positive in 19 ADC and 9 SqCC, but HPV DNA-ISH and RNA-ISH were negative in all cases. Our study did not detect HPV infection by DNA-ISH or RNA-ISH in any cases of primary NSCLC despite positive p16 expression in a portion of ADC and SqCC. p16 should therefore not be used as a surrogate marker for HPV infection in NSCLC.
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.
The Journal of physiology
Peltekian, L;Gasparini, S;Fazan, FS;Karthik, S;Iverson, G;Resch, JM;Geerling, JC;
PMID: 37291801 | DOI: 10.1113/JP283169
In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.
Liu Y, Huang Y, Liu T, Wu H, Cui H, Gautron L.
PMID: 27111742 | DOI: -
While Agouti-related peptide (AgRP) neurons play a key role in the regulation of food intake, their contribution to the anorexia caused by pro-inflammatory insults has yet to be identified. Using a combination of neuroanatomical and pharmacogenetics experiments, this study sought to investigate the importance of AgRP neurons and downstream targets in the anorexia caused by the peripheral administration of a moderate dose of lipopolysaccharide (LPS; 100 μ g/kg, ip). First, in the C57/Bl6 mouse, we demonstrated that LPS induced c-fos in select AgRP-innervated brain sites involved in feeding, but not in any arcuate proopiomelanocortin neurons. Double immunohistochemistry further showed that LPS selectively induced c-Fos in a large subset of melanocortin 4 receptor-expressing neurons in the lateral parabrachial nucleus. Secondly, we used pharmacogenetics to stimulate the activity of AgRP neurons during the course of LPS-induced anorexia. In AgRP-Cre mice expressing the designer receptor hM3Dq-Gq only in AgRP neurons, the administration of the designer drug clozapine-N-oxide (CNO) induced robust food intake. Strikingly, CNO-mediated food intake was rapidly and completely blunted by the coadministration of LPS. Neuroanatomical experiments further indicated that LPS did not interfere with the ability of CNO to stimulate c-Fos in AgRP neurons. In summary, our findings combined together support the view that the stimulation of select AgRP-innervated brain sites and target neurons, rather than the inhibition of AgRP neurons themselves, is likely to contribute to the rapid suppression of food intake observed during acute bacterial endotoxemia.
Journal of Neuroendocrinology
Decourt, C;Connolly, G;Ancel, C;Inglis, M;Anderson, G;
| DOI: 10.1111/jne.13190
Agouti-related peptide (AgRP) neurons are thought to indirectly regulate the activity of hypothalamic gonadotrophin-releasing hormone neurons which control fertility. AgRP neurons also drive caloric intake and are modulated by metabolically-relevant hormones, providing a link to the hypothalamic-pituitary-gonadal axis. In mice expressing Cre-dependant designer receptors (DREADDs) in AgRP neurons, we activated or silenced these neurons in vivo using the synthetic ligand clozapine-N-oxide (CNO) to observe the effect of AgRP neuron activity on timing of puberty. To validate these animals, we chronically treated both stimulatory (hM3Dq) and inhibitory (hM4Di) DREADD × AgRP-Cre mice with CNO, observing a pronounced increase and decrease of food intake, respectively, consistent with the known orexigenic effects of these neurons. RNAscope was performed to visually confirm the activation of AgRP neurons. Puberty onset was assessed in males and females. There was no effect on preputial separation in males or vaginal opening and first oestrus in females after CNO treatment from day 26 to 30 to chronically modulate AgRP neurons. Next, to determine whether the delay in puberty onset occurring in response to neonatal underfeeding could be overcome by inhibiting AgRP neuronal activity, mice were raised in large (neonatally underfed) or normal litter sizes. The delay in puberty from underfeeding was completely reversed in CNO-treated AgRP-hM4Di male mice. These data highlight the inhibitory role of AgRP neurons to delay puberty onset when undernutrition occurs during the neonatal period, at least in male mice.
Journal of cellular physiology
Zhang, CL;Lin, YZ;Wu, Q;Yan, C;Wong, MW;Zeng, F;Zhu, P;Bowes, K;Lee, K;Zhang, X;Song, ZY;Lin, S;Shi, YC;
PMID: 35312067 | DOI: 10.1002/jcp.30719
Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt-induced hypertension. Here, we demonstrate that wild-type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY-GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain-derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV-Cre recombinase into the Arc only of the NPY-targeted mutant mice carrying a loxP-flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt-dependent blood pressure. In summary, our study uncovers an important Arc NPY-originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.
