Genetic and transcriptomic analyses in a rare case of HPV-related oropharyngeal squamous cell carcinoma combined with small cell carcinoma
Cold Spring Harbor molecular case studies
Sato, K;Nishiyama, K;Taguchi, K;Jiromaru, R;Yamamoto, H;Matsunaga, A;Nagata, R;Rikimaru, F;Toh, S;Higaki, Y;Oda, S;Nakagawa, T;Masuda, M;
PMID: 34462366 | DOI: 10.1101/mcs.a006102
Human papillomavirus (HPV)-related oropharyngeal small cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas employing a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes upregulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 upregulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA dataset. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.Cold Spring Harbor Laboratory Press.
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.
Ye R, Quinlan MA, Iwamoto H, Wu HH, Green NH, Jetter CS, McMahon DG, Veestra-VanderWeele J, Levitt P, Blakely RD.
PMID: 26793096 | DOI: 10.3389/fnsyn.2015.00020
The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] modulates many key brain functions including those subserving sensation, emotion, reward, and cognition. Efficient clearance of 5-HT after release is achieved by the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). To identify novel SERT regulators, we pursued a proteomic analysis of mouse midbrain SERT complexes, evaluating findings in the context of prior studies that established a SERT-linked transcriptome. Remarkably, both efforts converged on a relationship of SERT with the synaptic adhesion protein neuroligin 2 (NLGN2), a post-synaptic partner for presynaptic neurexins, and a protein well-known to organize inhibitory GABAergic synapses. Western blots of midbrain reciprocal immunoprecipitations confirmed SERT/NLGN2 associations, and also extended to other NLGN2 associated proteins [e.g., α-neurexin (NRXN), gephyrin]. Midbrain SERT/NLGN2 interactions were found to be Ca(2+)-independent, supporting cis vs. trans-synaptic interactions, and were absent in hippocampal preparations, consistent with interactions arising in somatodendritic compartments. Dual color in situ hybridization confirmed co-expression of Tph2 and Nlgn2 mRNA in the dorsal raphe, with immunocytochemical studies confirming SERT:NLGN2 co-localization in raphe cell bodies but not axons. Consistent with correlative mRNA expression studies, loss of NLGN2 expression in Nlgn2 null mice produced significant reductions in midbrain and hippocampal SERT expression and function. Additionally, dorsal raphe 5-HT neurons from Nlgn2 null mice exhibit reduced excitability, a loss of GABAA receptor-mediated IPSCs, and increased 5-HT1A autoreceptor sensitivity. Finally, Nlgn2 null mice display significant changes in behaviors known to be responsive to SERT and/or 5-HT receptor manipulations. We discuss our findings in relation to the possible coordination of intrinsic and extrinsic regulation afforded by somatodendritic SERT:NLGN2 complexes.
Eur Arch Otorhinolaryngol. 2014 Dec 27
Strojan P, Zadnik V, Šifrer R, Lanišnik B, Didanović V, Jereb S, Poljak M, Kocjan BJ, Gale N.
PMID: 25542247
Incidence trends in head and neck squamous cell carcinoma in Slovenia, 1983-2009: role of human papillomavirus infection.
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.
Histopathology. May; 60(6):982-91.
Lewis JS Jr1, Ukpo OC, Ma XJ, Flanagan JJ, Luo Y, Thorstad WL, Chernock RD (2012)
PMID: 22360821 | DOI: 10.1111/j.1365-2559.2011.04169.x.
AIMS:
Human papillomavirus is well established in oropharyngeal squamous cell carcinoma as both causative and prognostic, but its significance in non-oropharyngeal tumours is unclear. In particular, the significance of finding viral DNA is not known. We sought to evaluate nonoropharyngeal squamous cell carcinomas for transcriptionally-active human papillomavirus and to compare this with the presence of viral DNA.
METHODS:
We evaluated an 87 patient tissue microarray cohort of oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas for high risk human papillomavirus DNA and E6 and E7 mRNA transcripts by in situ hybridization, and for p16 expression by immunohistochemistry.
RESULTS:
We found only two of the 73 (2.7%) evaluable cases to harbour transcriptionally-active human papillomavirus. Both of these tumours were from the larynx, one was positive for human papillomavirus DNA by in situ hybridization, and both were extensively positive for p16. All oral cavity and hypopharyngeal tumours were negative for human papillomavirus.
CONCLUSIONS:
Transcriptionally-active human papillomavirus appears to be rare in laryngeal, hypopharyngeal, and oral cavity squamous cell carcinomas. As such, it appears unlikely to be a 'driver' or to be clinically significant in most established tumours.
Ruuskanen M, Irjala H, Minn H, Vahlberg T, Randen-Brady R, Hagström J, Syrjänen S, Leivo I.
PMID: 30549170 | DOI: 10.1002/hed.25450
Abstract BACKGROUND: Nasopharyngeal 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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: In our low-incidence population, EBV and HPV are important prognostic factors for NPC.
Annals of Diagnostic Pathology (2018)
Nicol AF, de Andrade CV, Gomes SC, Brusadelli MG, Lodin HM, Wells SI, Nuovo GJ.
| DOI: 10.1016/j.anndiagpath.2018.12.001
Importin-β, exportin-5, p16, Ki-67, Mcl1, PDL1, and cFLIP are each over-expressed in the majority of CIN 1 lesions. These biomarkers, plus HPV E6/E7 RNA, were analyzed in carcinoma-in-situ (CIS), microinvasive, and squamous cell carcinoma (SCC) of the uterine cervix and cervical carcinoma cell lines. Only p16 and Ki-67 continued to be over-expressed in CIS, with a concomitant marked increase in E6/E7 RNA. There was a highly significant increase in PDL1 expression and decrease in Ki-67 (each p < 0.001) in microinvasive cancer compared to CIS whereas p16 and E6/E7 remained stable. As the lesion progressed to SCC, p16 and E6/E7 RNA remained strongly overexpressed with a concomitant over expression of importin-β and Ki67. HPV positive Caski cells showed significant elevations of p16, importin-β, exportin-5 and PDL1 compared to the HPV negative cervical cancer cell line C33A, consistent with viral induction of these biomarkers. The data suggest that PDL1 may be a useful biomarker to differentiate CIS from microinvasive cancer and, thus, anti-PDL1 therapy may inhibit the progression of CIS to the invasive stage.
Archives of Medical Science - Civilization Diseases
Nascimento, M;Silva Galbiatti-Dias, A;Oliveira-Cucolo, J;Pavarino, É;Goloni-Bertollo, E;
| DOI: 10.5114/amscd.2022.119197
The presence of human papillomavirus (HPV) in patients with head and neck squamous cell carcinoma (HNSCC) can guide medical management. The aim of this study was to highlight the importance of HPV diagnosis, verifying which diagnostic techniques are most used in identifying HPV and the differences between these techniques, in the research aimed at establishing a consensus on the gold standard method. We verify that HPV infection is associated with the development of HNSCC. The techniques most commonly used for diagnosis of HPV are immunohistochemistry (IHC), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Our study concludes that detection of E6/E7 DNA by PCR is the most accepted method of diagnosis. The standardization of an accurate HPV diagnostic method can reduce morbidity and mortality in HNSCC, especially in emerging countries, where few screenings are performed, in addition to improving the social and economic impact of the disease.
Yu, XD;Zhu, Y;Sun, QX;Deng, F;Wan, J;Zheng, D;Gong, W;Xie, SZ;Shen, CJ;Fu, JY;Huang, H;Lai, HY;Jin, J;Li, Y;Li, XM;
PMID: 36446933 | DOI: 10.1038/s41593-022-01200-8
Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.
MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus
Development (Cambridge, England)
Kaiser, K;Jang, A;Kompanikova, P;Lun, MP;Prochazka, J;Machon, O;Dani, N;Prochazkova, M;Laurent, B;Gyllborg, D;van Amerongen, R;Fame, RM;Gupta, S;Wu, F;Barker, RA;Bukova, I;Sedlacek, R;Kozmik, Z;Arenas, E;Lehtinen, MK;Bryja, V;
PMID: 34032267 | DOI: 10.1242/dev.192054
The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.
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.
