[PPT] - HPV Testing in Head and Neck Think before you order p16 Cancer PowerPoint Presentation

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HPV Testing in Head and Neck Cancer

Annemieke van Zante MD/PhD Head & Neck Pathology and Cytopathology

Think before you order p16 Disclosures

I have nothing to disclose

The WHO Head and Neck 2017

Oropharygeal squamous cell carcinoma:

HPV-positive HPV-negative

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Why?

Despite reductions in smoking… the incidence of cancer of the

ropharynx has increased

SEER DATA

Cervical Cancer Oral Cavity and Pharynx

Incidence of HPV-attributed Oropharyngeal cancer now exceeds Cervical cancer

Cancer Average annual no. Attributable to any HPV type No. Cervical 11,771 10,700 (90.6%) All oropharyngeal cancers 15,738 11,000 (70.1%)

Viens LJ, Henley SJ, Watson M, et al. Human Papillomavirus–Associated Cancers — United States, 2008–

2012. MMWR Morb Mortal Wkly Rep 2016;65:661–666.

Histomorphology

Morphological spectrum of HPV+

rorpharyngeal SCC:
Basaloid
Papillary
Lymphoepithelioma-like
Sarcomatoid

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Histomorphology Histomorphology Why test?

As a group, patients with HPV+

ropharynx SCC have improved clinical
utcomes compared to patients with

conventional, HPV-negative SCC.

Given significant implications on patient

prognosis, HPV status will be integrated into the American Joint Committee on Cancer (AJCC) staging in new 8th Ed.

Why test?

Many HPV+ patients may be cured by single

modality therapy (surgery or radiation alone).

NCCN Treatment Guidelines are currently the

same for both HPV+ and HPV- tumors, but many clinicians will take a less aggressive approach in HPV+ cases.

While patients with HPV+ disease have a good

prognosis, the side effects from multi-modality therapy are significant.

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Clinical utility

If metastatic SCC of unknown primary involves and groin node and is HPV+ Look in the anogenital tract If metastatic SCC of unknown primary involves an upper cervical lymph node and is HPV+ Look in the oropharynx

Where is the oropharynx? How should we test?

Need a technically practical, reproducible,

and easily interpreted marker for high risk HPV within tumor cells

Widely available, inexpensive,

standardized

Utilization and interpretation should

follow best practice guidelines and consensus statements for consistent reporting

What HPV test is the ”best”?

Assay Method Sensitivity Specificity PPV NPV P16 IHC 97% 82% 80% 97%

* Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive

ropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang,

S Bui, Y Luo, P Sloan, R J Shaw and M Robinson.

P16: High sensitivity, lower specificity

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What HPV test is the ”best”?

Assay Method Sensitivity Specificity PPV NPV P16 IHC 97% 82% 80% 97% hrHPV DNA ISH 94% 91% 89% 95%

* Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive

ropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang,

S Bui, Y Luo, P Sloan, R J Shaw and M Robinson.

DNA ISH: Lower sensitivity, better specificity, technically challenging

What HPV test is the ”best”?

Assay Method Sensitivity Specificity PPV NPV P16 IHC 97% 82% 80% 97% hrHPV DNA ISH 94% 91% 89% 95% DNA qPCR 91% 87% 83% 93%

* Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive

ropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang,

S Bui, Y Luo, P Sloan, R J Shaw and M Robinson.

DNA PCR: Lower sensitivity/specificity

Cannot distinguish passenger virus from driver

What HPV test is the ”best”?

Assay Method Sensitivity Specificity PPV NPV P16 IHC 97% 82% 80% 97% hrHPV DNA ISH 94% 91% 89% 95% DNA qPCR 91% 87% 83% 93% hrHPV RNA ISH* 97% 93% 91% 98%

* Br J Cancer. 2013 Apr 2; 108(6): 1332–1339. Validation of a novel diagnostic standard in HPV-positive

ropharyngeal squamous cell carcinoma. A G Schache, T Liloglou, J M Risk, T M Jones, X-J Ma, H Wang,

S Bui, Y Luo, P Sloan, R J Shaw and M Robinson.

mRNA ISH detects transcriptionally active HPV oncogenes E6/E7 with high sensitivity and specificity…. But technically challenging

High risk HPV RNA in situ

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hrHPV RNA in situ

High sensitivity, high specificity New protocols and reagents for

automated platforms

Standardization will be challenging

Reconsidering P16

The correlation between hrHPV RNA ISH and

p16 IHC is very high.*

An endogenous cell cycle protein

verexpressed in tumor cells with

transcriptionally-active high risk HPV.

E6H4, MTM Lab, ER 1 20’ (BOND) Predilute Widely available, technically practical,

reproducible, and easily interpreted

* Mirghani H, Casiraghi O, Amen F, et al. Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice. Mod Pathol. 2015;28(12):1518-1527.

P16 Immunohistochemistry What is positive?

P16 IHC is positive in tissue specimens

(non-cytology) when there is at least 70% nuclear and cytoplasmic expression with at least moderate to strong intensity.

Staining must be both nuclear and

cytoplasmic to be considered positive.

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P16 interpretation What is positive?

With these criteria, sensitivity of p16

approaches 100%.

The specificity of p16 in the oropharynx is

~85-95%.

Excellent inter-rater agreement (κ = .97)

Keratinizing HPV+ SCC CAP guidelines coming soon…

1. Pathologists should use p16 IHC as a

surrogate for hrHPV on all new

ropharynx cancers.
2. Additional HPV-specific testing is at the

discretion of the pathologist.

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CAP guidelines coming soon…

3. Pathologists should not routinely

perform low-risk HPV testing on patients with head and neck carcinomas.

4. HPV status is not a reliable marker for

aggressive behavior in non-SCC. Don’t test other tumor types.

What about other head and neck sites?

The PPV of p16 IHC for HPV in non-

ropharyngeal SCC is low (25-50%).

There is no proven prognostic difference

based on HPV status outside of the

ropharynx.

DO NOT routinely test non-oropharyngeal SCC.

What about other head and neck sites?

But I’ve heard that HPV DNA is present in 25% of laryngeal and sinonasal carcinomas?

Using RNA-based HPV detection methods

HPV may be an etiologic agent in 2% of SCC outside the oropharynx.

The prognostic significance is unknown. P16 status is misleading.

What about recurrences?

Recurrence can occur outside of the boundaries of the oropharynx. P16 status can be helpful to distinguish between a new primary tumor and recurrence.

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What about metastases?

Two scenarios:

1. New diagnosis of cancer. Primary site

has not been established

2. Diagnosis has been established and

patient has a new metastatic lesion.

1. New diagnosis:

HPV testing is indicated in patients with metastatic SCC of unknown primary in a cervical upper or mid jugular chain lymph node (levels 2 and 3).

Note that p16 status is suggestive of

ropharyngeal origin.

20 to 30% of aggressive head and neck cutaneous SCCs overexpress p16 unrelated to high risk HPV.

2. New metastasis

High risk HPV testing is indicated for patients with oropharyngeal SCC and a new metastatic lesion:

When the original tumor was not tested. When there is diagnostic uncertainty

regarding recurrence vs. new primary. P16 is positive in a ≈25% lung SCC. HPV specific testing is indicated if p16 is positive.

Testing FNAs

1. FFPE cell block from FNA

P16 IHC

hrHPV in situ

2. Liquid based specimens

Published sensitivities and specificities >90%

Roche cobas (Roche Molecular Systems) Cervista HR and HPV16/18 (Hologic) Hybrid-Capture 2 (Qiagen)

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P16 on FNA cell block

How should p16 be interpreted on cell block

specimens?

Recent studies suggest that thresholds as

low as 10-15% positive cells may be valid for cell blocks.

Xu B, Ghossein R, Lane J, Lin O, Katabi N. The utility of p16 immunostaining in fine needle aspiration in

p16-positive head and neck squamous cell carcinoma. Hum Pathol. 2016;54193-200.

Holmes BJ, Maleki Z, Westra WH. The Fidelity of p16 Staining as a Surrogate Marker of Human

Papillomavirus Status in Fine-Needle Aspirates and Core Biopsies of Neck Node Metastases: Implications for HPV Testing Protocols. Acta Cytol. 2015;59(1):97-103.

Jalaly JB, Lewis JS, Jr., Collins BT, et al. Correlation of p16 immunohistochemistry in FNA biopsies with

corresponding tissue specimens in HPV-related squamous cell carcinomas of the oropharynx. Cancer

Cytopathol. 2015;123(12):723-731.

Oropharyngeal Carcinoma: A Unique Human Papillomavirus-Associated Tumor of the Head and Neck. Jordan, Richard; Gillison, Maura; van Zante, Annemieke. Pathology Case Reviews: July/August 2011 - Volume 16 - Issue 4 - pp 173-175

P16 on FNA cell block

1. Obtain viable tumor cells in intact

fragments.

2. Do not interpret anucleate squamous

debris or necrotic specimens.

3. Equivocal specimens should be sent for

HPV specific testing (ISH or PCR).

Conclusions

1. Perform hrHPV testing on all new
ropharyngeal SCC by p16 IHC.
Reporting p16 status for non-oropharyngeal

SCC may be misleading and is not recommended.

2. The cutoff for positive p16 on tissue is 70%.

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P16+ Oropharyngeal SCC

Histological grading or subtyping is not

currently advocated.

The diagnosis should include “HPV positive”

r “P16 positive.”