ROC Analysis of p16 Expression in Cell Blocks of Metastatic Head and Neck Squamous Cell Carcinoma

Journal of the American Society of Cytopathology

2021 Apr 01

Wilson, B;Israel, A;Ettel, M;Lott Limbach, A;
| DOI: 10.1016/j.jasc.2021.03.004

Background Oropharyngeal squamous cell carcinoma is associated with human papillomavirus (HPV) and often presents with early metastasis to cervical neck lymph nodes which are amenable to fine needle aspiration (FNA). The most common method of HPV status determination is p16 immunohistochemistry (IHC). The literature suggests that a lower threshold is needed for p16 positivity on cell block. We examined and quantified p16 IHC staining on cell block and used ROC curve analysis to determine an optimal cut off value with high sensitivity and specificity. Methods Thirty-six FNAs of metastatic squamous cell carcinoma from cervical lymph nodes with p16 IHC were evaluated. The p16 stain was quantified in 5% increments and HR-HPV mRNA ISH was performed as a gold standard test. Statistical analysis was performed. Results Interobserver variability was evaluated and was shown to be low with an intraclass correlation coefficient of 0.857. Receiver operating characteristics (ROC) analysis was performed and showed that a cell block p16 IHC cut-off of 15% yielded the highest sensitivity (80%) and specificity (81.8%). Conclusion Our data shows that a threshold of 15% p16 staining in cell block maximizes sensitivity and specificity.

A Case of HPV-Associated Oropharyngeal Squamous Cell Carcinoma with Block-Like, Partial Loss of p16 Expression

Head and neck pathology

2022 Jun 30

Rasmussen, SA;Lewis, JS;Mirabello, L;Bass, S;Yeager, M;Corsten, MJ;Bullock, MJ;
PMID: 35771403 | DOI: 10.1007/s12105-022-01463-4

Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16.A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing.The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes.This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.

Aptima HR-HPV Testing of Cytology Specimens Is an Effective Supplement for p16 Staining to Improve Diagnostic Accuracy for HPV-Related Oropharyngeal Squamous Cell Carcinoma

Acta cytologica

2022 Dec 13

Yang, X;Hu, C;Zhao, H;Zhang, Z;Zhao, L;Yu, J;Ni, X;Guo, H;
PMID: 36513056 | DOI: 10.1159/000527951

Regarding a small proportion of oropharyngeal squamous cell carcinoma (OPSCC) patients who tested p16-positive but human papillomavirus (HPV)-negative, we attempted to perform HPV testing to improve the accuracy of HPV detection in OPSCC patients.We simultaneously performed Aptima HPV testing of cytological specimens and p16 immunohistochemistry (IHC) staining of histologic biopsies from the same cohort of patients with head and neck SCC (HNSCC). The cytological specimens included fine-needle aspiration specimens from patients with enlarged nodes and endoscopic brushing specimens from the primary lesions of patients without enlarged nodes. Cases with discordant results for p16 IHC staining and Aptima HPV testing were reexamined by a third method, RNAscope testing.Sixty patients with HNSCC (39 OPSCC and 21 non-OPSCC) were recruited for examination of HPV status. Among these patients, 28 were p16+/HPV+, 29 were p16-/HPV-, 2 were p16+/HPV-, and 1 was p16-/HPV+. The overall concordance rate between Aptima HPV testing and p16 IHC was 95.0%. Three cases with discordant results for these two methods were reexamined by RNAscope testing, and all were confirmed to be HPV negative. The prevalence of HPV in OPSCC and non-OPSCC patients was 61.5% (24/39) and 19.0% (4/21), respectively. The sensitivity and negative predictive values of Aptima HPV testing and p16 IHC were consistent at 100%, while the specificity and positive predictive values were 96.9% and 96.6% versus 93.8% and 93.3%, respectively. Additionally, 30 OPSCCs were simultaneously examined and diagnosed by both brush cytology and biopsy pathology; six of these SCCs were underdiagnosed by histopathology but accurately diagnosed by supplemental brush cytology.Aptima HPV testing of cytology specimens can be used as an adjuvant examination to identify false-positive OPSCC patients after p16 IHC of biopsies, while brush cytology may be a supplemental method for the histologic diagnosis of malignant oropharyngeal tumors.

Prevalence of human papillomavirus in head and neck cancers at tertiary care centers in the United States over time

Cancer

2022 Feb 08

Scott-Wittenborn, N;D'Souza, G;Tewari, S;Rooper, L;Troy, T;Drake, V;Bigelow, EO;Windon, MJ;Ryan, WR;Ha, PK;Kiess, AP;Miles, B;Westra, WH;Mydlarz, WK;Eisele, DW;Fakhry, C;
PMID: 35132635 | DOI: 10.1002/cncr.34124

Human papillomavirus (HPV) is responsible for a growing proportion of oropharyngeal squamous cell carcinomas (OPSCCs) among men and White individuals. Whether similar trends apply to women, non-Whites, and non-oropharyngeal squamous cell carcinomas (non-OPSCCs) is unknown.This is a cross-sectional analysis combining 2 multi-institutional case series of incident head and neck squamous cell carcinoma (HNSCC) cases. Incident HNSCCs from 1995 to 2012 were enrolled retrospectively using banked tumor samples and medical record abstraction. Incident HNSCCs from 2013 to 2019 were enrolled prospectively. The prevalence of tumor HPV biomarkers was tested over 3 time periods (1995-2003, 2004-2012, and 2013-2019). Centralized testing was done for p16 immunohistochemistry (p16) and oncogenic HPV in situ hybridization (ISH).A total of 1209 incident cases of HNSCC were included. Prevalence of p16- and ISH-positive tumors increased significantly for oropharynx cancers over time. The majority were positive after 2013 for White patients (p16, 92%; P < .001; ISH 94%; P < .001), Black patients (p16, 72%; P = .021; ISH 67%; P = .011), and Hispanic patients (p16, 100%; P = .04; ISH 100%; P = .013). For women with OPSCC, the prevalence of p16- and ISH-positive tumors increased significantly to 82% (P < .001) and 78% (P = .004), respectively. For non-OPSCCs, there was increased p16 and ISH positivity overall with 24% p16 and 16% ISH positivity in the most recent time period (P < .001 for both).The majority of OPSCCs in US tertiary care centers are now p16 and ISH positive for all sex and race groups. In some populations in the United States, 91% of OPSCCs are now caused by HPV. Few non-OPSCCs are p16 and ISH positive.

HDAC1/2 control proliferation and survival in adult epidermis and pre-basal cell carcinoma via p16 and p53

The Journal of investigative dermatology

2021 Jul 17

Zhu, X;Leboeuf, M;Liu, F;Grachtchouk, M;Seykora, JT;Morrisey, EE;Dlugosz, AA;Millar, SE;
PMID: 34284046 | DOI: 10.1016/j.jid.2021.05.026

HDAC inhibitors show therapeutic promise for skin malignancies; however, the roles of specific HDACs in adult epidermal homeostasis and disease are poorly understood. We find that homozygous epidermal co-deletion of Hdac1 and Hdac2 in adult mouse epidermis causes reduced basal cell proliferation, apoptosis, inappropriate differentiation, and eventual loss of Hdac1/2-null keratinocytes. Hdac1/2 deficient epidermis displays elevated acetylated p53 and increased expression of the senescence gene p16. Loss of p53 partially restores basal proliferation, whereas p16 deletion promotes long-term survival of Hdac1/2-null keratinocytes. In activated GLI2-driven pre-basal cell carcinoma, Hdac1/2 deletion dramatically reduces proliferation and increases apoptosis, and knockout of either p53 or p16 partially rescues both proliferation and basal cell viability. Topical application of the HDAC inhibitor Romidepsin to normal epidermis or GLI2ΔN-driven lesions produces similar defects to genetic Hdac1/2 deletion, and these are partially rescued by loss of p16. These data reveal essential roles for HDAC1/2 in maintaining proliferation and survival of adult epidermal and basal cell carcinoma progenitors and suggest efficacy of therapeutic HDAC1/2 inhibition will depend in part on the mutational status of p53 and p16.

Utility of high-risk HPV RNA chromogenic in situ hybridization in cytology smears and liquid-based preparations from metastatic head and neck squamous cell carcinoma

Cancer cytopathology

2022 Nov 09

Velez Torres, JM;Alkathery, T;Tjendra, Y;Zuo, Y;Kerr, DA;Gomez-Fernandez, C;
PMID: 36350307 | DOI: 10.1002/cncy.22659

High-risk human papillomavirus (HR-HPV) status is critical for the diagnosis, prognosis, and treatment of patients with oropharyngeal squamous cell carcinoma (OPSCC). Patients often present with enlarged cervical nodes, and fine-needle aspiration cytology (FNAC) is frequently the initial diagnostic procedure. Although p16 is the most widely used surrogate marker, problems with interpretation can limit its utility in FNAC. HR-HPV RNA in situ hybridization (ISH) has emerged as a specific way to assess HPV status on cell block preparations of cervical nodes. The authors evaluated the utility of HR-HPV ISH in conventional smears and liquid-based cytology (LBC) preparations of metastatic head and neck squamous cell carcinoma (SCC).Thirty-one aspirates of proven, HPV-related SCC (confirmed by p16 and/or HR-HPV ISH in corresponding surgical specimens) were selected. Ten aspirates of HPV-negative SCC were also retrieved. HR-HPV ISH was performed on 27 smears and 14 LBC preparations. All results were scored as positive, equivocal, or negative.Eighty-four percent of metastatic, HPV-related SCCs were positive for HR-HPV RNA ISH, with high number of signals (n = 19) and low number of signals (n = 7), whereas five HPV-related SCCs were equivocal. All metastatic, HPV-negative SCCs were negative for HR-HPV ISH.HR-HPV ISH can be reliably performed on smears or LBC preparations, particularly when cell blocks are unavailable or paucicellular. Results were easy to interpret when high numbers of signals were present but were challenging in aspirates with low or rare number of signals. The current study suggests that HR-HPV ISH could be used as the initial testing modality for determining HPV status in FNAC specimens of metastatic SCC.

High-Risk Human Papillomavirus Testing in Cytology Aspiration Samples from the Head and Neck Part 1: A Review of the Literature on Available Testing Options

Journal of the American Society of Cytopathology

2022 Jun 01

Manucha, V;Adeniran, A;Asiry, S;Hoda, R;Johnson, D;van Zante, A;VandenBussche, C;Griffith, C;
| DOI: 10.1016/j.jasc.2022.05.003

Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma is increasing in incidence and is often first diagnosed on a cytology fine needle aspiration (FNA) specimen of metastatic nodal disease of the neck. In the setting of oropharyngeal squamous cell carcinoma, HPV status defines the disease with HPV-associated tumors having better overall prognosis than those that are HPV negative. Furthermore, metastatic squamous cell carcinoma of the neck of unknown origin requires testing for HPV as a positive result suggests an oropharyngeal primary. As a result, HPV testing in aspirate samples is increasingly important for the proper diagnosis and treatment of patients with head and neck squamous cell carcinoma. Although HPV testing in cervicovaginal cytology specimens is common and well-established, testing in head and neck FNA samples remains challenging. p16 immunohistochemistry is an excellent surrogate marker for HPV in tumors of known or suspected oropharyngeal origin, but the criteria used in histologic specimens may not be appropriate in cytology samples. FNA samples are more frequently hypocellular, and cytology cell blocks have variable fixation and processing steps, limiting the utility of p16 immunohistochemistry. Other potential testing options have been reported in the literature including staining of aspirate smears and molecular testing of liquid-based samples. The American Society of Cytopathology Clinical Practice Committee recently surveyed the American Society of Cytopathology membership to determine the current state of HPV testing in aspirate samples, and this review article is designed to provide a summary of the current literature on various testing options in FNA samples.

[Primary ovarian squamous cell carcinoma: clinicopathological features and prognostic analysis of fifteen cases]

Zhonghua bing li xue za zhi = Chinese journal of pathology

2022 Apr 08

Xi, Y;Zhang, ML;He, C;Cheng, GP;Jin, JY;Fang, XH;Zhu, T;Su, D;
PMID: 35359045 | DOI: 10.3760/cma.j.cn112151-20210719-00516

Objective: To assess the clinical features and treatment outcomes in patients with primary ovarian squamous cell carcinoma (POSCC). Methods: Fifteen patients with primary ovarian squamous cell carcinoma diagnosed from January 2009 to December 2018 in Cancer Hospital of the University of Chinese Academy of Sciences were collected. The expression of p16, hMLH1, hMSH2, hMSH6 and PMS2 in POSCC was detected by immunohistochemistry, and the status of high-risk human papillomavirus (HPV) by RNAscope test. Results: Squamous cell carcinoma with different degrees of differentiation was found in 15 cases, including three cases with high differentiation and 12 cases with medium to low differentiation. There were four cases with in situ squamous cell carcinoma, four cases with teratoma, one case with endometrial carcinoma/atypical hyperplasia, and one case with endometriosis. p16 was expressed in five cases (5/15), indicating coexisting high-risk HPV infection. There was no high-risk HPV infection in the remaining 10 cases, and p16 staining was negative. There was no deficient mismatch repair protein in all cases. The overall survival time (P=0.038) and progression free survival (P=0.045) of patients with high-risk HPV infection were longer than those without HPV infection. Conclusions: POSCC is more commonly noted in postmenopausal women and often occurs unilaterally. Elevated serological indexes CA125 and SCC are the most common finding. Morphologically, the tumors show variable degrees of differentiation, but the current data suggest that the degree of differentiation cannot be used as an independent prognostic index. High-risk HPV infection may be associated with the occurrence of POSCC, and that the prognosis of POSCC patients with HPV infection is better than that of patients without infection.

TP53 positivity combined with high fibrinogen expression defines a subtype of oral squamous cell carcinoma with an unfavorable prognosis

Human Pathology

2022 Oct 01

Inoue, A;Matsumoto, T;Ito, Y;Saegusa, M;Takahashi, H;
| DOI: 10.1016/j.humpath.2022.10.008

The number of deaths due to oral squamous carcinoma (OSCC), a malignant tumor of the oral cavity, is on the increase. We examined fibrinogen (FIB) expression in patients with OSCC and developed novel immunoprofile classification methods that include FIB. The plasma FIB level in patients with OSCC was elevated compared with that in patients with non-tumor oral disease (non-T); using a cut-off point of 342 mg/dL, we found the area under the curve-receiver operating characteristic level for OSCC was 0.745. Similarly, FIB expression in OSCC tissues was significantly higher compared with that in non-T tissues. Hierarchical clustering based on the immunoprofile of several markers including FIB, p53, and p16 revealed four groups that could be used to categorize OSCC cases (referred to as immunoprofile subtypes, [IPS], I-IV). Tumors in IPS-II, which were FIB+/p53+, were associated with a significantly worse overall survival (OS) when compared with the other subtypes. We conclude that our IPS classification system can facilitate prognostic evaluation in OSCC, and that quantification of FIB is an important component of the classification strategy for this disease.

Novel In Situ Hybridization Assay for Chromogenic Single-Molecule Detection of Human Papillomavirus E6/E7 mRNA

Microbiology spectrum

2023 Feb 21

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.

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.

Cervical Adenosquamous Carcinoma: Detailed Analysis of Morphology, Immunohistochemical Profile, and Outcome in 59 Cases

International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists

2022 Aug 31

Stolnicu, S;Hoang, L;Zhou, Q;Iasonos, A;Terinte, C;Pesci, A;Aviel-Ronen, S;Kiyokawa, T;Alvarado-Cabrero, I;Oliva, E;Park, KJ;Soslow, RA;
PMID: 36044310 | DOI: 10.1097/PGP.0000000000000921

Although both the 2014 and 2020 World Health Organization (WHO) criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma (ASC), in practice, ASC diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphologic, and clinical features and outcomes associated with ASCs, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed ASCs (including glassy cell carcinoma and related lesions) to confirm an ASC diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as ASCs, 34 retained their ASC diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or ASCs), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy adenocarcinomas were reclassified as poorly differentiated HPV-associated carcinomas based on morphology and immunophenotype. There were no significant immunophenotypic differences between ASCs and pure invasive stratified mucin-producing carcinomas with regard to HPV and other markers including p16 expression. Although limited by a small sample size, survival outcomes seemed to be similar between all groups. ASCs should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The 2 putative glassy cell carcinomas studied did not meet our criteria for ASC and categorizing them as such should be reconsidered.

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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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