Human papilloma virus testing in oropharyngeal squamous cell carcinoma: What the clinician should know.

Oral oncology, 50(1):1–9.

Mirghani H1, Amen F2, Moreau F3, Guigay J4, Ferchiou M5, Melkane AE6, Hartl DM7, Lacau St Guily J (2014).
PMID: 24169585 | DOI: 10.1016/j.oraloncology.2013.10.008.

High risk Human Papilloma virus (HR-HPV) associated oropharyngeal cancers are on the increase. Although, the scientific community is aware of the importance of Human Papilloma Virus (HPV) testing, there is no consensus on the assays that are required to reliably identify HR-HPV related tumors. A wide range of methods have been developed. The most widely used techniques include viral DNA detection, with polymerase chain reaction (PCR) or In Situ Hybridization, and p16 detected by immunohistochemistry. However, these tests provide different information and have their own specific limitations. In this review, we summarize these different techniques, in light of the recent literature. p16 Overexpression, which is an indirect marker of HPV infection, is considered by many head and neck oncologists to be the most important marker for patient stratification. We describe the frequent lack of concordance of this marker with other assays and the possible reasons for this. The latest developments in HPV testing are also reported, such as the RNAscope™ HPV test, and how they fit into the existing framework of techniques. HPV testing must not be considered in isolation, as there are important interactions with other parameters, such as tobacco exposure. This is an important and rapidly evolving field and is likely to become pivotal to staging and choice of treatment of oropharyngeal carcinoma in the future.

Human papillomavirus-mediated carcinogenesis and tumor progression

Genome Instability & Disease

2021 Apr 01

Abboodi, F;Delva, N;Emmel, J;Renrick, A;Buckhaults, P;Banister, C;Creek, K;Pirisi, L;
| DOI: 10.1007/s42764-021-00038-x

Full size image [/article/10.1007/s42764-021-00038-x/figures/1] The findings described above support the statement that HPV infection is common, but, in comparison, cervical cancer is quite rare, leading to the conclusion that HPV infection alone is not sufficient to produce cancer, as tumor development and progression require the contribution of multiple factors. Among the risk factors for cancer development and progression in women infected with HR HPV are the determinants of persistent infection, as it is well established that only women in whom HR HPV infection persists are at risk for cervical lesions that may progress to cancer (Banister et al. 2015 and references therein). HPV persistence has also been linked to HPV-mediated disease in men (Bettampadi et al. 2020 [/article/10.1007/s42764-021-00038-x#ref-CR16]). This is an important area of study, because in principle, if we were able to determine at a single visit whether or not an incident HR HPV infection will persist, we could target HPV-mediated cancer surveillance resources to the people who present with persistent infection. Our own (unpublished) findings support the concept that women with persistent HPV infection fail to mount a strong immune response to HPV. In turn, immune responses to HPV are likely to be influenced by HLA and SNP profiles, both of which have been linked with cervical cancer susceptibility (Chen et al. 2014; Das Gosh et al. 2017). Among the many SNPs that have relevance for cervical cancer development, the Arg/Pro TP53 polymorphism at codon 72 has received considerable attention, as the homozygote Arg/Arg phenotype is associated with a higher risk of developing cervical cancer, at least in certain populations (Ojeda et al. 2003; Chuery et al. 2017). TP53 codon 72 polymorphism has been connected with higher HPV E6/E7 expression, which appears to correlate with the Arg/Arg genotype (Chuery et al. 2017). Despite the continuing controversies in this area, there is evidence that this particular polymorphism plays a role in cervical cancer development, albeit with additional intervening factors that may modulate its impact in different populations.

The presence of high-risk human papillomavirus (HPV) E6/E7 mRNA transcripts in a subset of sinonasal carcinomas is evidence of involvement of HPV in its etiopathogenesis.

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.

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

Cancer Cytopathol.

2018 Jul 05

Coppock JD, Willis BC, Stoler MH, Mills AM.
PMID: 29975461 | DOI: 10.1002/cncy.22027

Abstract

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.

Adenosquamous Carcinoma of the Head and Neck: Relationship to Human Papillomavirus and Review of the Literature.

Head & Neck Pathology, 5(2):108–116.

Masand RP, El-Mofty SK, Ma XJ, Luo Y, Flanagan JJ, Lewis JS Jr (2011).
PMID: 21305368 | DOI: 10.1007/s12105-011-0245-3.

Adenosquamous carcinoma (ADSC) of the head and neck is an aggressive variant of squamous cell carcinoma (SCC). Certain variants of head and neck SCC are human papillomavirus (HPV)-related and have better prognosis. The relationship of HPV to head and neck ADSC has not been investigated. We searched our files for the term "adenosquamous" and head and neck subsites and found cases from 1998 to 2009. The requisite histologic criteria were the presence of SCC combined with distinct gland formation and/or intracellular mucin. DNA in situ hybridization for high-risk HPV, RNA in situ hybridization for high risk HPV E6 and E7 transcripts, and immunohistochemistry for p16 and p53 were performed. The existing literature on ADSC was also reviewed. Of the 18 cases, eight were from the larynx and hypopharynx, four from the oral cavity, three from the oropharynx, and three from the nasal cavity. Three cases (16%) showed both high risk HPV E6 and E7 and p16 expression, one from the nasal cavity and two from the oropharynx. Both oropharyngeal carcinoma patients were alive and disease free at 34 and 103 months, respectively. ADSCs of the head and neck are a heterogeneous group of tumors. A small minority of cases harbor HPV and most of these, particularly those occurring at sites with known high prevalence of HPV, show active viral transcription with detectable E6 and E7 and overexpression of p16. The HPV-related oropharyngeal cases, though rare, appear to do very well clinically, while the remaining cohort of ADSC patients do quite poorly. Head and neck ADSC appears to be a mixed variant that can be further classified according to its HPV status.

p16-positive lymph node metastases from cutaneous head and neck squamous cell carcinoma: No association with high-risk human papillomavirus or prognosis and implications for the workup of the unknown primary.

Cancer

2016 Feb 16

McDowell LJ, Young RJ, Johnston ML, Tan TJ, Kleid S, Liu CS, Bressel M, Estall V, Rischin D, Solomon B, Corry J.
PMID: 26881928 | DOI: 10.1002/cncr.29901.Abstract BACKGROUND: The incidence of p16 overexpression and the role of human papillomavirus (HPV) in cutaneous head and neck squamous cell carcinoma (cHNSCC) are unclear. METHODS: One hundred forty-three patients with cHNSCC lymph nod

Abstract

BACKGROUND:

The incidence of p16 overexpression and the role of human papillomavirus (HPV) in cutaneous head and neck squamous cell carcinoma (cHNSCC) are unclear.

METHODS:

One hundred forty-three patients with cHNSCC lymph node metastases involving the parotid gland were evaluated for p16 expression by immunohistochemistry. The detection of 18 high-risk HPV subtypes was performed with HPV RNA in situ hybridization for a subset of 59 patients. The results were correlated with clinicopathological features and outcomes.

RESULTS:

The median follow-up time was 5.3 years. No differences were observed in clinicopathological factors with respect to the p16 status. p16 was positive, weak, and negative in 45 (31%), 21 (15%), and 77 cases (54%), respectively. No high-risk HPV subtypes were identified, regardless of the p16 status. The p16 status was not prognostic for overall (hazard ratio, 1.08; 95% confidence interval [CI], 0.85-1.36; P = .528), cancer-specific (hazard ratio, 1.12; 95% CI, 0.77-1.64; P = .542), or progression-free survival (hazard ratio, 1.03; 95% CI, 0.83-1.29; P = .783). Distant metastasis-free survival, freedom from locoregional failure, and freedom from local failure were also not significantly associated with the p16 status.

CONCLUSIONS:

p16 positivity is common but not prognostic in cHNSCC lymph node metastases. High-risk HPV subtypes are not associated with p16 positivity and do not appear to play a role in this disease. HPV testing, in addition to the p16 status in the unknown primary setting, may provide additional information for determining a putative primary site.

Evaluation of the efficacy of the four tests (p16 immunochemistry, PCR, DNA and RNA In situ Hybridization) to evaluate a Human Papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.

Hum Pathol.

2018 Apr 20

Augustin J, Outh-Gauer S, Mandavit M, Gasne C, Grard O, Denize T, Nervo M, Mirghani H, Laccourreye O, Bonfils P, Bruneval P, Veyer D, Péré H, Tartour E, Badoual C.
PMID: 29684499 | DOI: 10.1016/j.humpath.2018.04.006

It is now established that HPV plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), notably oropharyngeal squamous cell carcinomas (SCCs). However, it is not clear which test one should use to detect HPV in oropharyngeal (OP) and non-OP SCCs. In this study, using 348 HNSCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: PCR, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (PCR) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive standalone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%) but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities below 50%, and RNA-CISH, DNA-CISH and p16 + DNA-CISH had respectively 100%, 97% and 99% specificities. As a standalone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances.

Human papillomavirus status and prognosis of oropharyngeal high-grade neuroendocrine carcinoma

Oral oncology

2023 Jan 24

de Sousa, LG;Lazar Neto, F;Dal Lago, EA;Sikora, A;Hanna, E;Moreno, A;Phan, J;Glisson, BS;Bell, D;Ferrarotto, R;
PMID: 36702015 | DOI: 10.1016/j.oraloncology.2023.106311

The prognostic impact of human papillomavirus (HPV) infection or smoking on oropharyngeal high-grade neuroendocrine carcinoma (HG-NEC) is not established.Retrospective study with patients with oropharyngeal HG-NEC seen at MD Anderson Cancer Center from 1997 to 2020, and previously reported patients with oropharyngeal HG-NEC and known p16 and HPV status from the literature review. Survival was estimated with the Kaplan-Meier method, and survival differences assessed with the log-rank test and Cox proportional hazards models.Thirty patients were included; most had a heavy (≥10 pack-years) smoking history (52%), locoregional disease (stage III-IVB; 77%), and p16-positive tumor (92%). HPV was positive in 65% of tested samples (15/23). Of 24 patients treated with curative intent, the objective response rates was 90% (9/10) and 81% (17/21), respectively, for induction chemotherapy and definitive radiotherapy. During follow-up, 54% (13/24) recurred, mostly (11/13) at distant sites. Median overall survival (OS) was 46 months (95% CI, 14.3 - NA). OS was not associated with HPV status (HR 0.73, P = 0.6) or smoking (HR 1.16, P = 0.8). Among 63 patients with known HPV status after the literature review (19 HPV- and 44 HPV + ), HPV status remained unassociated with OS (P = 0.92).This is the largest retrospective cohort of oropharyngeal HG-NEC. Distant recurrence rate after curative treatment was high, suggesting that multimodality treatment including systemic therapy may benefit patients with locally advanced disease. HPV infection did not affect survival outcomes, therefore should not lead to therapy de-intensification for this histology.

ITEM

RNAscope™ Probe- HPV 6-C2

Cat No. 407501-C2

Manual Assay RNAscope

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ 2.5 HD Duplex Assay, RNAscope™ Multiplex Fluorescent Assay
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ Probe - HPV 6-C2 - Human papillomavirus 6, E6-E7 mRNA
Channel :	2
RNAscope™ Assay Platform :	Manual Assay RNAscope

Assay Compatibility

RNAscope™ 2.5 HD Duplex Assay, RNAscope™ Multiplex Fluorescent Assay
Recommended Controls
Product Insert/Data Sheet

No Related Documents...

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ 2.5 HD Duplex Assay, RNAscope™ Multiplex Fluorescent Assay
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ Probe - HPV 6-C2 - Human papillomavirus 6, E6-E7 mRNA
Channel :	2
RNAscope™ Assay Platform :	Manual Assay RNAscope

ITEM

RNAscope™ Probe- HPV 6-C3

Cat No. 407501-C3

Manual Assay RNAscope

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ Multiplex Fluorescent Assay
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ Probe - HPV 6-C3 - Human papillomavirus 6, E6-E7 mRNA
Channel :	3
RNAscope™ Assay Platform :	Manual Assay RNAscope

Assay Compatibility

RNAscope™ Multiplex Fluorescent Assay
Recommended Controls
Product Insert/Data Sheet

No Related Documents...

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ Multiplex Fluorescent Assay
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ Probe - HPV 6-C3 - Human papillomavirus 6, E6-E7 mRNA
Channel :	3
RNAscope™ Assay Platform :	Manual Assay RNAscope

ITEM

RNAscope™ 2.5 LS Probe- HPV 6-C3

Cat No. 407508-C3

Automated Assay for Leica Systems - RNAscope

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ LS Multiplex Fluorescent Reagent Kit
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ 2.5 LS Probe - HPV 6-C3 - Human papillomavirus 6, E6-E7 mRNA
Channel :	3
RNAscope™ Assay Platform :	Automated Assay for Leica Systems - RNAscope

Assay Compatibility

RNAscope™ LS Multiplex Fluorescent Reagent Kit
Recommended Controls
Product Insert/Data Sheet

No Related Documents...

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	RNAscope™ LS Multiplex Fluorescent Reagent Kit
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ 2.5 LS Probe - HPV 6-C3 - Human papillomavirus 6, E6-E7 mRNA
Channel :	3
RNAscope™ Assay Platform :	Automated Assay for Leica Systems - RNAscope

ITEM

RNAscope™ 2.5 LS Probe- HPV 6-C4

Cat No. 407508-C4

Automated Assay for Leica Systems - RNAscope

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	N/A
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ 2.5 LS Probe - HPV 6-C4 - Human papillomavirus 6, E6-E7 mRNA
Channel :	4
RNAscope™ Assay Platform :	Automated Assay for Leica Systems - RNAscope

Assay Compatibility

RNAscope™ LS Multiplex Fluorescent Reagent Kit
Recommended Controls
Product Insert/Data Sheet

No Related Documents...

Specifications

Gene :	N/A
Species^* :	N/A
Species (common) :	N/A
Entrez Gene ID :	N/A
Gene Alias :	N/A
Accession No:	JN252323.1
Target Region [Base Pairs (bp)] :	135 - 685
No. of Pairs :	9
Assay Compatibility :	N/A
Shipping Temp :	2-8 C
Storage Temp :	2-8 C
Shelf Life :	24 months from the date of manufacturing * Please check expiration dates on the reagent package
Probe description :	RNAscope™ 2.5 LS Probe - HPV 6-C4 - Human papillomavirus 6, E6-E7 mRNA
Channel :	4
RNAscope™ Assay Platform :	Automated Assay for Leica Systems - RNAscope

Pages

	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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