Localization and characterization of human papillomavirus-16 in oral squamous cell carcinoma

Oral diseases

2021 May 22

Saleh, W;Cha, S;Banasser, A;Fitzpatrick, SG;Bhattacharyya, I;Youssef, JM;Anees, MM;Elzahaby, IA;Katz, J;
PMID: 34022097 | DOI: 10.1111/odi.13920

The role of Human papillomavirus (HPV) in the oral squamous cell carcinoma (OSCC) has not been completely elucidated. The purpose of the present study was to investigate the prevalence and localization of HPV-16 virus in OSCC and to correlate HPV-16 positivity and p16INK4A expression with the clinical and pathological features of OSCC. The archives of Oral Pathology at University of Florida, College of Dentistry were accessed for demographic, clinical, histopathological data and slides of 114 OSCC patients. HPV-16 positivity of OSCC was evaluated by p16INK4A immunohistochemistry (IHC) and HPV-16 E6/E7mRNA by in situ hybridization (ISH). Out of 114 consecutive pathological slides of OSCC, 16 samples (14%) showed positivity for p16INK4A by IHC and 14 samples (12%) were positive for HPV-16 E6/E7mRNA ISH and the Positivity showed a significant correlation with the patients' age, alcohol consumption, and the degree of OSSC differentiation. The hard palate showed the highest positivity of p16INK4A IHC and HPV-16 mRNA ISH (38%,36% respectively). HPV-16 is a significant factor in oral carcinogenesis. We recommend using p16INK4A as a surrogate marker for HPV detection in OSCC, which can be complemented by RNA ISH for the identification of HPV subtype. This article is protected by

Randomised trial of radiotherapy with weekly cisplatin or cetuximab in low risk HPV associated oropharyngeal cancer (TROG 12.01)- a Trans-Tasman Radiation Oncology Group study

International journal of radiation oncology, biology, physics

2021 Apr 24

Rischin, D;King, M;Kenny, L;Porceddu, S;Wratten, C;Macann, A;Jackson, JE;Bressel, M;Herschtal, A;Fisher, R;Fua, T;Lin, C;Liu, C;Hughes, BGM;McGrath, M;McDowell, L;Corry, J;
PMID: 34098030 | DOI: 10.1016/j.ijrobp.2021.04.015

The excellent prognosis of patients with low risk HPV associated oropharyngeal squamous cell carcinoma has led to concerns about overtreatment and excessive toxicity with radiotherapy and cisplatin, leading to interest in de-intensification trials. We investigated whether cetuximab, an EGFR targeting antibody, when combined with radiotherapy would result in a decrease in symptom burden and toxicity with similar efficacy when compared to weekly cisplatin.XXXX, a randomised, multicentre trial involving 15 sites in XXXX enrolled patients with HPV associated oropharyngeal squamous cell carcinoma, AJCC 7th edition Stage III (excluding T1-2N1) or stage IV (excluding T4 and/or N3 and/or N2b-c if smoking history >10 pack years and/or distant metastases). Patients were randomised (1:1) to receive radiotherapy (70Gy in 35 fractions) with either weekly cisplatin, 7 doses of 40mg/m2 or cetuximab, loading dose of 400mg/m2 followed by 7 weekly doses of 250 mg/m2. The primary outcome was symptom severity assessed by the MD Anderson Symptom Inventory Head and Neck Symptom Severity Scale from baseline to 13 weeks post completion of radiotherapy using the area under the curve (AUC). Trial was registered on ClinicalTrials.gov: XXXX RESULTS: Between 17th June 2013 and 7th June 2018, 189 patients were enrolled, with 92 on cisplatin arm and 90 on cetuximab included in the main analysis. There was no difference in the primary endpoint of symptom severity; difference in AUC cetuximab - cisplatin was 0.05 (95%CI: -0.19, 0.30), p= 0.66. The T-score (mean number of ≥ grade 3 acute adverse events) was 4.35 (SD 2.48) in the cisplatin arm and 3.82 (SD 1.8) in the cetuximab arm, p= 0.108. The 3 -year failure-free survival rates were 93% (95% CI: 86-97%) in the cisplatin arm and 80% (95% CI: 70-87%) in the cetuximab arm (hazard ratio = 3.0 (95% CI: 1.2-7.7); p=0.015.For patients with low risk HPV associated oropharyngeal cancer, radiotherapy and cetuximab had inferior failure-free survival without improvement in symptom burden or toxicity compared to radiotherapy and weekly cisplatin. Radiotherapy and cisplatin remains the standard of care.

Human papillomavirus testing in metastatic squamous cell carcinoma of the neck with unknown primary using PCR on fine-needle aspiration smears: a prospective clinical study

European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery

2021 Oct 24

Channir, HI;Lomholt, AF;Gerds, TA;Charabi, BW;Kiss, K;von Buchwald, C;
PMID: 34689237 | DOI: 10.1007/s00405-021-07133-5

Squamous cell carcinoma metastasis of the head and neck with unknown primary tumor (CUP) comprises a diagnostic challenge. Human papillomavirus (HPV) testing on cytologic specimens is gaining increasing focus as this may facilitate an early diagnosis of HPV-induced oropharyngeal carcinoma. This study aimed to prospectively assess PCR-based HPV-DNA testing on FNA smears in a clinical setting.Patients referred to a tertiary Head and Neck Cancer Center with suspected CUP were included from November 2016 to November 2018. Scraped cell material from FNA smears was analyzed for HPV-DNA with PCR using general primers (GP5 + /GP6 +) and correlated with the origin and histology of the primary tumor (oropharynx vs. outside oropharynx or benign tumor). The turn-around time reflecting the workflow for HPV-DNA testing by PCR was also calculated.A total of 93 patients were enrolled in the study. The sensitivity and specificity were 86.7% [95% CI 75.4-94.1%] and 92.0% [95% CI 74.0-99.0%], and the positive and negative predictive values were 96.3% [95% CI 87.3-99.0%] and 74.2% [95% CI 59.9-84.7%], respectively. The turn-around time for HPV testing was a mean four calendar days.HPV-DNA testing on FNA smears can be performed within a reasonable timeframe and can guide for the detection of an HPV-positive oropharyngeal primary tumor in the clinical setting for patients presenting with CUP of the head and neck.

RTOG-0129 risk groups are reproducible in a prospective multicenter heterogeneously treated cohort

Cancer

2021 Jun 18

Fakhry, C;Tewari, SR;Zhang, L;Windon, MJ;Bigelow, EO;Drake, VE;Rooper, LM;Troy, T;Ha, P;Miles, BA;Mydlarz, WK;Eisele, DW;D'Souza, G;
PMID: 34143891 | DOI: 10.1002/cncr.33682

Recursive partitioning analysis (RPA) from the Radiation Therapy Oncology Group (RTOG)-0129 has identified a low-risk group of patients with oropharynx cancer (OPC) who might benefit from therapeutic de-intensification. These risk groups have not yet been reproduced in an independent cohort treated heterogeneously. Therefore, the objective of this analysis was to validate the RPA risk groups and examine the prognostic impact of novel factors.Patients with OPC were enrolled in a prospective study at 3 academic medical centers from 2013 to 2018. Medical record abstraction was used to ascertain clinical variables including staging and survival according to the 7th edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual. Human papillomavirus-positive tumor status was determined by p16 immunohistochemistry and/or HPV RNA in situ hybridization. Kaplan-Meier and log-rank methods were used to compare survival. Cox proportional hazards were used to generate univariate and multivariable hazard ratios (HRs).Median follow-up time was 3.2 years. The low-, intermediate-, and high-risk groups had significant differences in 2-year overall survival (OS, 99.1%; 95% CI, 94.4%-99.9% vs OS, 93.0%; 95% CI, 74.7%-98.2% vs OS, 80.0%; 95% CI, 40.9%-94.6%; Poverall = .0001) and 2-year progression-free survival (PFS, 97.5%; 95% CI, 92.4%-99.2% vs PFS, 89.3%; 95% CI, 70.3%-96.4% vs PFS, 80.0%; 95% CI, 40.9%-94.6%; Poverall < .002). After adjustment for age, sex, and level of educational attainment, OS and PFS were significantly lower for the intermediate- (OS adjusted hazard ratio [aHR], 5.0; 95% CI, 1.0-23.0; PFS aHR, 3.4; 95% CI, 1.0-11.5), and high- (OS aHR, 7.3; 95% CI, 1.4-39; PFS aHR, 5.0; 95% CI, 1.2-21.6) risk groups compared with the low-risk group. Lower education was also independently significantly associated with worse OS (aHR, 8.9; 95% CI, 1.8-44.3) and PFS (aHR, 3.1; 95% CI, 1.0-9.6).In patients with OPC, the RTOG-0129 RPA model is associated with OS and PFS in a heterogeneously treated cohort.

Verrucous Carcinoma of the Esophagus Is A Genetically Distinct Subtype of Esophageal Squamous Cell Carcinoma

Histopathology

2021 May 07

Isidro, RA;Dong, F;Hornick, JL;Wee, JO;Agoston, A;Patil, DT;Deshpande, V;Zhao, L;
PMID: 33960520 | DOI: 10.1111/his.14395

Esophageal verrucous carcinoma (VSCC) is a rare and morphologically distinct type of esophageal squamous cell carcinoma (SCC). Diagnosing VSCC on biopsy material is challenging given the lack of significant atypia and the presence of keratinizing epithelium and exophytic growth. The molecular pathogenesis of VSCC remains unclear. The aim of this study was to characterize the genomic landscape of VSCC in comparison to conventional esophageal SCC. Three cases of VSCC from the Brigham and Women's Hospital pathology archive were identified. Formalin-fixed, paraffin-embedded (FFPE) tumor tissue was used for p16 immunohistochemistry (IHC), high-risk HPV in situ mRNA hybridization (ISH), and DNA isolation. Tumor DNA was sequenced using a targeted massively parallel sequencing assay enriched for cancer-associated genes. Three additional cases of VSCC were identified by image review of The Cancer Genome Atlas (TCGA) esophageal SCC cohort. VSCC cases were negative for p16 IHC and high-risk HPV ISH. TP53 mutations (p<0.001) and copy number variants (CNVs) for CDKN2A (p<0.001), CDKN2B (p<0.01) and CCND1 (p<0.01) were absent in VSCC and significantly less frequent in comparison to conventional SCC. Five VSCC cases featured SMARCA4 missense mutations or inframe deletions compared to only 4/88 conventional SCC cases (p<0.001). VSCC featured driver mutations in PIK3CA, HRAS, and GNAS. Recurrent CNVs were rare in VSCC. VSCC is not only morphologically but also genetically distinct from conventional esophageal SCC, featuring frequent SMARCA4 mutations and infrequent TP53 mutations or CDKN2A/B CNVs. Molecular findings may aid in establishing the challenging diagnosis of VSCC. This article is protected by

Oncolytic adenoviral H101 synergizes with radiation in cervical cancer cells

Current cancer drug targets

2021 Mar 07

Duan, Y;Bai, H;Li, X;Wang, D;Wang, Y;Cao, M;Zhang, N;Chen, H;Wang, Y;
PMID: 33687882 | DOI: 10.2174/1568009621666210308103541

A major challenge in cervical cancer radiotherapy is to tailor the radiation doses efficiently to both eliminate malignant cells and to reduce the side effects to normal tissue. Oncolytic adenoviral drug H101 is recently tested and approved for topical adjuvant treatment of several malignancies. This study is to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 combined with radiation in different cervical cancer cells. Human cervical cancer cells C33a, SiHa, CaSki, and Hela were treated with varying concentrations of H101 alone or combined with radiation (2Gy or 4Gy). Cell viability and apoptosis were measured at indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration were measured by qRT-PCR. RNA scope in-situ detect HPV E6 status. P53 protein alteration are detected by Western blot. Cell viability and apoptosis show the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anti-cancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV negative C33a cells (P<0.05). Low HPV16 viral load SiHa cell was more sensitive to combination therapy than high HPV16 viral load CaSki cell (P<0.05). The combined treatment could reduce HPV16 E6 expression and increase cellular P53 level compared to radiation alone in SiHa and CaSki (P<0.05). Oncolytic adenoviral H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.

RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.

The Journal of Molecular Diagnostics, 14(1), 22–29.

Wang, F, Flanagan, J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y. (2012).
PMID: 22166544 | DOI: 10.1016/j.jmoldx.2011.08.002.

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.

Durable response in a patient with recurrent respiratory papillomatosis treated with immune checkpoint blockade

Head & neck

2022 Jul 11

Bai, K;Norberg, SM;Sievers, C;Meyer, T;Friedman, J;Hinrichs, C;Allen, CT;
PMID: 35815785 | DOI: 10.1002/hed.27144

Immune checkpoint blockade can provide clinical benefit for patients with advanced cancer. Here, we report durable disease control over many years following PD-L1 blockade through induction of a viral antigen-specific T cell response in an adult patient with recurrent respiratory papillomatosis.Antigen-specific T cell response assays, single cell RNA-sequencing, and RNA-scope was used to study clinical tissues.An HPV6 E2-specific T cell clone restricted to HLA-B*55, present at low frequency in the pre-treatment papilloma, significantly expanded after six doses of PD-L1 blockade and remained present and functional at the site of initial response in the larynx as a tissue resident memory T cell for 4 years. An associated reduction in E2 target gene was observed following treatment.Although demonstrated in a single exceptional responder, these results highlight that immune checkpoint blockade may induce durable, viral antigen-specific immunity of sufficient magnitude to control disease in patients with nonmalignant disorders.

The utility of high-risk human papillomavirus in situ hybridization in cytology cell block material from cystic head and neck lesions

Cancer cytopathology

2021 Nov 10

Han, LM;Saunders, TA;Calkins, SM;
PMID: 34758205 | DOI: 10.1002/cncy.22529

Human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) presents frequently as metastasis in a neck lymph node that may be cystic or necrotic. Fine-needle aspiration (FNA) biopsies are often first-line diagnostic procedures. p16 immunohistochemistry (IHC) is a surrogate marker for high-risk HPV (hrHPV) infection but can be challenging to interpret. This study evaluated the use of hrHPV in situ hybridization (ISH) in cytology cell blocks of cystic neck lesions.Twenty-four FNA cases with cell blocks and surgical correlates were evaluated. p16 IHC and hrHPV ISH were assessed on cell blocks (C-p16 and C-hrHPV ISH), and hrHPV ISH on surgical samples (S-hrHPV ISH). All results were classified as negative, positive, or equivocal.Two cases were excluded because of insufficient tissue on recut. On the basis of C-hrHPV ISH cases, 12 were positive, 5 were negative, and 5 were equivocal. All 12 positive C-hrHPV ISH cases had concordant S-hrHPV ISH with no false positives. Of the 5 negative C-hrHPV ISH cases, 4 had concordant S-hrHPV ISH, and 1 had a discordant S-hrHPV ISH. Of the 5 equivocal C-hrHPV ISH cases, S-hrHPV ISH were both positive and negative. Fourteen cases were equivocal by C-p16; 9 cases were reliably classified by C-hrHPV ISH (5 positive, 4 negative; 64%).C-hrHPV ISH can be reliably used, especially when positive. A negative or equivocal interpretation of C-hrHPV ISH may warrant repeat testing. Compared to C-p16, C-hrHPV ISH is more frequently diagnostic and could be helpful for HPV-OSCC diagnosis and management.

Effectiveness of radiation therapy on brain invasion by human papillomavirus-related multiphenotypic sinonasal carcinoma: A case report

Neuropathology : official journal of the Japanese Society of Neuropathology

2021 Dec 21

Kuroda, N;Kawaji, H;Arai, Y;Otsuki, Y;Miura, K;Minato, H;Kuroda, K;Nakatogawa, H;Yamazoe, T;Tanaka, T;Inenaga, C;
PMID: 34933397 | DOI: 10.1111/neup.12762

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is newly suggested and characterized by HPV-related tumors. HMSC has a relatively good prognosis. No cases of brain invasion have been reported to date. We encountered a case of brain invasion by HMSC, in which we assessed the effectiveness of radiotherapy in comparison with biopsy and autopsy. A 69-year-old man was referred to a hospital three months after intracerebral hemorrhage (ICH). Contrast magnetic resonance imaging revealed a tumor in the ethmoid sinus involving the brain. We performed transnasal biopsy and intensity-modulated radiotherapy for sinonasal and intracranial lesions. Despite radiotherapy, the patient died on day 41 after radiation. Biopsy specimens displayed mixed findings of epithelial and mesenchymal components. The tumor was immunoreactive for p16, and the RNA in situ hybridization for HPV was positive. Finally, we diagnosed the patient as having HMSC. Autopsy of the sinonasal tissue revealed a reduction in the number of tumor cells. There was a marked reduction in the number of tumor cells in the sinonasal tissue compared to that in the invaded brain tissue. The effectiveness of radiotherapy could depend on the histopathological components and location of the lesion, even in the same patient.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1

Proceedings of the National Academy of Sciences of the United States of America

2021 Jan 19

Liu, H;Xu, J;Yang, Y;Wang, X;Wu, E;Majerciak, V;Zhang, T;Steenbergen, RDM;Wang, HK;Banerjee, NS;Li, Y;Lu, W;Meyers, C;Zhu, J;Xie, X;Chow, LT;Zheng, ZM;
PMID: 33436409 | DOI: 10.1073/pnas.2014195118

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Assessment of HER2 in Gastric Type Endocervical Adenocarcinoma and its Prognostic Significance

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

2023 Feb 23

Wang, S;Zhou, X;Niu, S;Chen, L;Zhang, H;Chen, H;Zhou, F;
PMID: 36841435 | DOI: 10.1016/j.modpat.2023.100148

As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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