1/10/2016

HPV-Related Oropharyngeal - “Catch Me If You Can!”

James S. Lewis Jr. MD Associate Professor Vanderbilt University Medical Center March 16, 2016

Disclosures I have no financial or other conflicts of interest to report.

(just that I think way too much about this topic!)

1 1/10/2016

THE “STORY”

HPV “Epidemic” in Oropharyngeal SCC - OPSCC incidence up in most countries, particularly in economically more developed ones:

- US, UK, Japan, NorthernHPV + Europe, Brazil, but not Asia or South/Central AmericaOropharynx HPV ‐ only

- In US, 225% increase in HPV+ oropharyngeal SCC (1984-2004) - 50% decrease in HPV- over same time period

*Chaturvedi et al. J Clin Oncol 2012; 29: 4294. *Chaturvedi et al. J Clin Oncol 2013; 31: 4550.

2 1/10/2016

HPV “Epidemic” in Oropharyngeal SCC

By 2020, overall oropharyngeal SCC burden in US predictedHPV + to surpass cervical SCC Already Happened!!! ~13,000 patients with OPSCC in 2012*

*Chaturvedi et al. J Clin Oncol 2012; 29: 4294. *Chaturvedi et al. J Clin Oncol 2013; 31: 4550.

Transmission of HPV

• HPV is sexually transmitted

• Increased sex partners and practicing oral sex is associated with:

– Prevalent and incident oral high risk HPV detection

– Increased rates of HPV-related OPSCC

• Oral high risk HPV is 1-10% and usually cleared, but can persist

– Increased rates and persistence in men and smokers

• Risk of HPV from open mouthed kissing suggested

Fakry/D’Souza –Oral Oncol 2013; 49(9): 863‐71

3 1/10/2016

Epidemic!!!

Well, at least a modest one, anyway…

Typical Patient with HPV-Related Oropharyngeal SCC

– Caucasian Insert Head – Male (~95%) Shot of Aging Hollywood – 50-60’s Actor Here… – History of smoking (mostly light or former)

– Higher than average sex history, particularly oral sex, many partners

4 1/10/2016

Survival Data by /HPV Status

The Presence of Transcriptionally- Active HPV in Oropharyngeal SCC is Highly and Independently Predictive of Better Patient Survival

(patients have ~2-5 times lower risk for overall and disease specific mortality)

5 1/10/2016

Transcriptionally-active HPV-related OPSCC is a clinically, biologically, morphologically, and molecularly distinct form of head and SCC SCC Variant

Given the prognostic (and emerging treatment) differences, it is critical for us to diagnose them correctly and communicate the findings clearly in our reports All patients with newly diagnosed oropharyngeal squamous cell carcinoma should get some form of p16 +/- HPV testing

6 1/10/2016

Testing should also be performed on:

1) Tissue (and/or FNA cell blocks) from neck metastases of known oropharyngeal primary

2) Tissue (and/or FNA cell blocks) from neck metastases of unknown primary

HPV DNA PCR DNA ISH p16 IHC

Nonkeratinizing Morphology HPV E6/E7 mRNA ISH

7 1/10/2016

In the oropharynx, all HPV specific and surrogate marker tests shown to be roughly equivalent prognostic markers…

So which one(s) to use and what “approach”?

Strategies for p16/HPV Testing in Oropharyngeal SCC: Three “Schools” of Thought:

1) p16 immunohistochemistry alone

2) Morphology-informed strategy: nonkeratinizing SCC with

+ p16 IHC sufficient; all others also get HPV-specific testing

3) Across the board p16 IHC and HPV-specific testing

8 1/10/2016

Expert Opinions/Recommendations

College of American Pathologists Committee Pharynx:

Checklist item for p16 IHC (with suggestions for when to add HPV specific testing)

p16 p16

HPV Why p16?

Sign here ______

p16 overexpression indicates that the HPV is causing “favorable” signaling in the tumor cells

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p16

“The Practical Thing” Reproducible

p16 Immunohistochemistry Alone Many caveats!

(you should know what these are

for your practice)

10 1/10/2016

Caveats for p16 IHC Alone

• 1) Only in oropharyngeal

• 2) Extensive nuclear+cytoplasmic staining cutoff (>70%) for calling positivity

• 3) Strict rules on use in neck metastases and CUP – (Level II-III and nonkeratinizing morphology)

Which p16 Antibody? Which HPV-Specific Test?

• No current data supporting one p16 antibody/clone/platform over another*

(*but see USCAP abstract ___)

• No current data dictating one HPV-specific test over another, either*.

*But HPV RNA ISH probably emerging…

11 1/10/2016

Systematic Literature Review

CAP Pathology and Laboratory Quality Center: Human Papillomavirus Testing in Head and Neck Squamous Cell Carcinomas Expert Panel

HPV-Related Oropharyngeal Carcinoma = The Bad Guy

HPV-Related Oropharyngeal Carcinomas Have Many Morphologies and Presentations = Masquerading as Other Lesions

12 1/10/2016

Pathologists = “Tissue Detectives”

A Pathologist’s Job?

To Recognize “The Bad Guy” in All His Many Forms and to Call Them Out

13 1/10/2016

Today’s Aim:

TeachYou Many of the “” of HPV-Related Oropharyngeal Carcinoma

Case #1:

57 year old man with and a granular and ill-defined mass in the

right base of tongue.

14 1/10/2016

Reverse Maturation

15 1/10/2016

Retraction Artifact

Brisk Mitotic Activity

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p16 Positive

Diagnosis:

Oropharynx, base of tongue, biopsy:

- p16 Positive (or HPV-Related) Squamous Cell Carcinoma, NonkeratinizingType with Maturation

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Is this tumor:

Well-Differentiated

Moderately-Differentiated

Poorly-Differentiated

Differentiation Not Applicable

Answer:

Differentiation Not Applicable

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Morphology in Oropharyngeal Squamous Cell Carcinoma

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20 1/10/2016

“Reverse Keratinizing Areas Maturation”

Nonkeratinizing Areas

Clefting/Retraction

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20‐25% 20‐25% 50‐60%

Keratinizing Nonkeratinizing Nonkeratinizing w/Maturation (100% maturing (>10 but <100% (<10% maturing squamous maturing squamous squamous differentiation) differentiation) differentiation)

22 1/10/2016

Nonkeratinizing Morphology and HPV

• Four Years of Clinical Practice –Washington University

– 435 OPSCC Cases

• Nonkeratinizing (NK) SCC = 53% of total cases

• 99.1% were p16 positive (226/228)

• Combine surrogate markers:

• All 48 NKSCC that were “audited” for HPV mRNA (by RTPCR and RNA ISH) were positive (100%)

Dibe‐Gondim et al, USCAP Meeting Abstract ‐ 2015

Strictly defined nonkeratinizing SCC (minimal maturing squamous differentiation) is almost implicitly related to transcriptionally-active high risk HPV

Chernock. Head Neck Pathol 2009; 3: 186. Lewis Jr. Histopathol 2012; 60: 427.

23 1/10/2016

College of American Pathologists Cancer Committee Pharynx:

Protocol for the Examination of Specimens From Patients With Carcinomas of the Pharynx

2013 Modification: p16 IHC sufficient alone for “nonkeratinizing or predominantly nonkeratinizing oropharyngeal SCC”

24 1/10/2016

“Grading” of Oropharyngeal Squamous Cell Carcinoma

Literature Descriptions

“Patients with HPV positive tumors had a higher prevalence of high grade lesions”

“The HPV positive cases were more likely to be poorly differentiated than those that were negative”

25 1/10/2016

Morphology in Oroph SCC • Turns out that “poorly differentiated/high grade” tumors actually have much better prognosis – Nonkeratinizing, partially keratinizing, lymphoepithelial, and basaloid tumors consistently related to transcriptionally-active high risk HPV – Genetically less complex and better treatment response – Paradoxical – and not at all what is clinically conveyed by the terms

Let’s come back to the crypts…

Nonkeratinizing SCC

26 1/10/2016

*Avoid terms about grading and/or differentiation for p16/HPV positive tumors

Fight that urge!!!

Now Something Even More Confusing!!!

27 1/10/2016

Nonkeratinizing SCC with Multinucleation

Nonkeratinizing SCC with maturation and nuclear anaplasia

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Nonkeratinizing SCC with multinucleation and anaplasia

Nonkeratinizing SCC with Nuclear Anaplasia

p16

29 1/10/2016

p16 Positive Patients vs p16 Negative

p16 positive & A/MN negative

p16 positive & A/MN positive

p16 negative

Overall Survival (years) Lewis Jr. et al. Am J Surg Pathol 2012; 36: 1036

Anaplasia and Multinucleation:

Only one study to date

Only surgically-resected patients

Pathologist reproducibility in classification?

30 1/10/2016

Case #1 p16 Positive/HPV-Related OPSCC Mimicking a Poorly Differentiated Tumor

Case #2:

42 year old man with a large neck mass. On clinical examination, he has a subtle irregularity of the left tonsil.

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p16

Copyright © 2013 by ASCP 63

Diagnosis?

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Concept of Tonsillar ‘Crypt Dysplasia’ • Theoretical as precursor lesion

• Electron microscopy studies of normal tonsil – “reticulated” crypt

Normal Tonsillar Crypts

Copyright © 2013 by ASCP 66

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C Normal Tonsillar Crypts L

E Capillaries within reticulated crypt epithelium

Discontinuous Basement C Membrane E

Perry ME. J Anat 1994; 185: 111

What percentage of patients with HPV-related oropharyngeal SCC have nodal metastases at presentation? 20%

35%

50%

80%

95%

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Answer:

80%

Nodal metastases are present at presentation in ~80 to 85%+ of all HPV-related oropharyngeal squamous cell carcinomas

Ang et al. NEJM 2010; 363: 24. Jordan et al. Am J Surg Pathol 2012; 36: 945. Lewis Jr. et al. Am J Surg Pathol 2010: 1044:38.

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Tonsillar Crypt Epithelium a Poor Barrier to the Spread of Carcinoma

NKSCC Along Tonsillar Crypts Mimicking In Situ Only

36 1/10/2016

Overtly invasive foci also lacking stromal reaction

“The tissue doesn’t lie…but it may try to deceive you!”

Louis “Pepper” Dehner, MD Washington University in St. Louis

http://news.wustl.edu/news/Pages/13804.aspx

37 1/10/2016

Recommended Diagnosis

p16 positive (or HPV-related) squamous cell carcinoma, nonkeratinizing type*

*No morphologic difference from clearly metastasis-capable tumor

*Caveats

• Tonsillar crypt epithelium (overlying/adjoining lymphoid tissue) – Not isolated to tonsillar surface epithelium

• Clinical “mass” lesion – Not flat, erythroplakic, or extensive

38 1/10/2016

Not the Normal Tonsil surface

Not the surface Here

Here

Here

And See USCAP Abstract # 19553:

“Nonkeratinizing Carcinoma In Situ of the Upper AerodigestiveTract: An HPV- Related Entity”

39 1/10/2016

Courtesy of: James Netterville M.D.

NK SCC in situ of the oropharyngeal surface mucosa

40 1/10/2016

NK SCC in situ of the oropharyngeal surface mucosa

p16 Positive

41 1/10/2016

High Risk HPV RNA ISH Positive

Clinical Follow Up

Patient had 3 years of unresectable disease without invasion, and subsequently two separate T1 invasive in the field of SCCis

(oroph and hypoph)

Currently alive with no evidence of

invasive disease

42 1/10/2016

Case #2 Tonsillar Crypt HPV-Related OPSCC Mimicking SCC In Situ

Case #3:

51 year old man with a large, cystic, level II- III neck mass.

On clinical examination and imaging, no primary tumor is apparent.

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Level II-III Neck Mass

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Glands with Mucin

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Apical Cilia

p16 Positive

46 1/10/2016

Diagnosis Case #3

Metastatic p16 Positive Ciliated Adenosquamous Carcinoma*

* An oropharyngeal primary tumor is suspected.

Clinical Follow Up:

An ipsilateral tonsillectomy and completion neck dissection was performed.

Histologically, a 0.4 cm mass was identified:

47 1/10/2016

48 1/10/2016

Apical Cilia

p16 Positive

49 1/10/2016

Subsequent Research High Risk HPV DNA ISH Testing Was:

Positive

7 Cases

3 Cases

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Ciliated Adenosquamous Carcinoma

• 10 cases reported

–All with neck metastases

–5 established oropharyngeal primaries

–5 remain unknown primary

–All p16 diffusely positive and hrHPV DNA positive

Ciliated Cells in Cancer??? Don’t believe it? Well, ok then…

But you have to be con-cilia-tory about it!

51 1/10/2016

Nodal metastases are present at presentation in ~80 to 85%+ of all HPV-related oropharyngeal squamous cell carcinomas

Ang et al. NEJM 2010; 363: 24. Jordan et al. Am J Surg Pathol 2012; 36: 945. Lewis Jr. et al. Am J Surg Pathol 2010: 1044:38.

Tumors Arising in Crypts + Crypt Architecture Permissive to Spread + Large Neck Nodal Mets ______= “Clinically Occult Primary Tumors”

52 1/10/2016

Cancer of Unknown Primary (CUP)*

*Unknown at clinical presentation UNKNOWN!*

Cancer of Unknown Primary (CUP) • ~75% Squamous Cell Carcinoma

• Locations when primary found

– Oropharynx (palatine tonsils and base of tongue)

• Represents ~90% of all tumors

– Most HPV related

• Uncommon: Hypopharynx (pyriform sinus)

• Rare: Nasopharynx and supraglottic larynx

Zengel et al. Virchows Archiv 2012; 119: 283. Tribius et al. Oral Oncol 2012; 48: 1178. Cianchetti et al. Laryngoscope 2009; 119: 2348.

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Branchial Cleft Carcinoma

Squamous Cell Carcinoma Can Arise in a Branchial Cleft : True

False

54 1/10/2016

Answer:

True*

(*at least theoretically…

but vanishingly rare)

Bradley PT, Bradley PJ. Curr Opin Ontolaryngol Head Neck Surg 2013; 21: 118‐23

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Branchial Cleft Carcinoma • Continues to be reported • Criteria proposed by Martin et al. (1950): – 1) Occurrence along anterior SCM – 2) Histopathology c/w origin from a tissue know to be present with branchial cleft – 3) Demonstration of carcinoma arising in association with a benign, epithelial- lined cyst (i.e. transition) – 4) At least 5 years of clinical follow up with periodic examinations w/o identification of a primary tumor. • Fewer than 40 published cases meet these

Martin et al. Ann Surg 1950; 132: 867.

Case #3 Metastatic HPV-Related SCC Mimicking a Benign Cyst (with Cilia) in the Neck

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Case #4:

63 year old man with a painless right neck mass and, on clinical examination, a 1-2cm submucosal mass in the ipsilateral tonsil.

57 1/10/2016

Is Cancer Present in This Image?

Yes

No

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Atypical Cells With Prominent Nucleoli

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p40 Positive

p16 Positive

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EBER Negative

Diagnosis:

Oropharynx, right tonsil, biopsy:

- p16 positive (or HPV-related) carcinoma, lymphoepithelial type

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Another example: Lymphoepithelial carcinoma

Oropharyngeal Lymphoepithelial Carcinoma • Several case series:

– Variant of SCC (immunohistochemistry)

– Favorable prognosis (radiosensitive)

– 2 series with p16 & HPV DNA ISH (38 cases)

• 36 (95%) p16 pos and 36 (95%) HPV DNA pos

• All negative for EBV (EBER -)

Singhi et al. Am J Surg Pathol. 2010;34(6):800. Carpenter et al. Mod Pathol. 2011;24(10):1306.

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Oropharyngeal Lymphoepithelial Carcinoma • Histologically can be VERY subtle!!!

• DDx includes

– Reactive crypts (no apoptosis/mitosis)

(different IHC)

• Use p16 diagnostically (and prognostically)

For p16 Positive/HPV-Related Oropharyngeal Tumors…

“Lymphoepithelial” Terminology

Better Than Calling Them“Undifferentiated”

63 1/10/2016

Other SCC Variants in the Oropharynx

In Oropharynx:

Nonkeratinizing SCC = HPV-Related But…

HPV-Related SCC Not Necessarily = Nonkeratinizing

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Histologic Types Among p16 Positive OPSCC – 332 Patients 13, 4% 11, 3% 10, 3% Nonkeratinizing

Nonkeratinizing with 26, 8% Maturation Keratinizing

45, 14% 226, 68% Lymphoepithelial Basaloid

Papillary

Basaloid SCC Papillary SCC

~3% 80% HPV+ ~3% 80% HPV+

Adenosquamous Carcinoma Spindle Cell Carcinoma = Rare (rare cases p16 and HPV positive)

Verrucous Carcinoma = Rarer! (all cases p16 and HPV negative) <1% ???% HPV+

65 1/10/2016

Best data available (limited numbers) suggests that oropharyngeal SCC variants, when related to transcriptionally-active HPV, still retain favorable prognosis

Case #4 Oropharyngeal HPV-related lymphoepithelial carcinoma mimicking lymphoma and also an EBV- related tumor

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OK…, here is yet one more

A B

Two Tumors in the Oropharynx

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A B

p16

A B

Cytokeratin 5/6

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A B

Courtesy of Justin Bishop M.D. p40

A B

Synaptophysin

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Diagnosis: Oropharyngeal Small Cell Carcinoma

Positive HR HPV RNA ISH

Oropharyngeal Small Cell Carcinoma

Bishop et al. Am J Surg Pathol 2011; 35: 1679 Kraft et al. Am J Surg Pathol 2012; 36: 321 Bates et al. Head Neck Pathol 2014; 8: 127

70 1/10/2016

Oropharyngeal Small Cell Carcinoma

• Quite rare: Only 14 HPV+ cases reported

• Very similar histologically to nonkeratinizing SCC

– And 3 of 14 cases mixed NKSCC and small cell

• Always p16 positive regardless of HPV status

• Occasionally p63 positive (p40 preferable)

Oropharyngeal Small Cell Carcinoma

–Prognosis – Very poor (especially relative to HPV+ squamous cell carcinoma) – ~66% dead of disease, most with distant metastases

– But is it better than for HPV negative small cell carcinoma??? – 33% long term survivors…

Bishop et al. Am J Surg Pathol 2011; 35: 1679 Kraft et al. Am J Surg Pathol 2012; 36: 321 Bates et al. Head Neck Pathol 2014; 8: 127

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Recommendation: Have a low threshold in oropharyngeal SCC for confirming with IHC!!!

- p40 - synaptophysin - chromogranin - cytokeratin 5/6

Summary • We are amidst an epidemic of HPV-related OPSCC in the developed world – Risk of death 2 to 5 times lower than HPV unrelated • All new oropharyngeal primary tumors (or their neck metastases) should be tested for p16 IHC (+/- HPV) • Most tumors nonkeratinizing in morphology – “Differentiation” status not applicable (paradoxical)

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Summary • Range of morphology in HPV-related tumors very broad – Nonkeratinizing to keratinizing – Metastasis-capable tumors mimicking carcinoma in situ – Anaplasia/multinucleation – Ciliated – Almost all other SCC variants including lymphoepithelial – Small cell carcinoma • Cystic neck metastases common (and a pitfall)

73 USCAP 2016 Long Course

Handout – James S. Lewis Jr. MD

HPV-Related Oropharyngeal Carcinoma – “Catch Me If You Can!” – 3/16/2015

Key Points: 1) As many as 60-80% of all oropharyngeal SCC in the US are related to transcriptionally active high risk HPV, ~90% of which is HPV type 16. 2) The prognosis for these tumors is much better than for HPV negative tumors with approximately 2 to 5 times lower risk of death from disease or from any cause. 3) All new oropharyngeal SCC (and variants), as well as cervical nodal metastasis with known clinical lesion in the oropharynx and those of clinically unknown primary, should at least be tested for p16 expression by immunohistochemistry with a cutoff of nuclear and cytoplasmic staining in >70% of tumor cells. Intensity of staining is usually strong but does not necessarily have to be. Many recommend HPV specific testing of all p16 positive tumors, regardless of the situation. Most pathologists, though (and including this author) recognize that p16 IHC positivity alone is sufficient as a prognostic marker and surrogate for active HPV in oropharyngeal SCC and its cervical nodal metastases in most situations. HPV specific testing may be indicated, however, for p16 IHC positive tumors in certain specific situations. Experts do not agree upon when, however. Examples of recommendations are the CAP Pharynx Cancer Committee which recommends p16 for all oropharyngeal SCC and its metastases, and that, if the tumor is nonkeratinizing or partially nonkeratinizing, then no additional testing is needed (this because p16 positive nonkeratinizing oropharyngeal SCC harbors transcriptionally active high risk HPV in virtually all cases obviated need for testing for it). They recommend HPV specific testing of all p16 positive keratinizing oropharyngeal SCC and for any p16 negative nonkeratinizing or partially nonkeratinizing tumors. Some recommend HPV specific testing of all p16 positive oropharyngeal SCC, regardless of other features. There is currently no agreement on the best HPV specific testing type. HPV DNA PCR, DNA, and now RNA ISH are functional, with various pluses and minuses. RNA- based ISH testing probably performs better than DNA-based tests, but is not yet widely available. The test or tests performed should be defined by the pathologists and the clinicians in their respective practices until more formal recommendations arrive. Pathologists should be well informed on these issues. A evidence-based CAP committee is currently in place that is processing literature data to make specific recommendations for HPV testing in head and neck cancers, with results expected in mid 2016. 4) HPV-related oropharyngeal SCC can morphologically mimic a great many other processes from benign to non-neoplastic to malignant and lead the pathologist to diagnose lesions incorrectly or to use inappropriate diagnostic terminology, including: a. Cancer of “high grade” or of “poor differentiation” – most oropharyngeal SCC are nonkeratinizing in appearance (50-60%) with little cytoplasm, brisk mitotic activity, and frequent necrosis. However, although these tumors look “aggressive”, they are proven to be the ones most likely to harbor transcriptionally active HPV and to have markedly better treatment responses and prognosis than typical head and neck SCC. As such, HPV- related oropharyngeal SCC should not be “graded”. b. SCC in situ of the tonsillar crypt epithelium - based on EM studies of the tonsillar crypt epithelium showing intraepithelial capillaries and a discontinuous basement membrane, in situ appearing foci of nonkeratinizing SCC cannot be discerned from metastasis-capable tumor, so should not be diagnosed as in situ. Rather, simply diagnose these as “squamous cell carcinoma”. This only applies for foci clearly lying along the invaginated tonsillar crypt epithelium, with a close cuff of organized lymphoid tissue and when the clinicians note a clinical “mass lesion”. This is because rare examples of extensive tonsillar, laryngeal, and oral cavity nonkeratinizing HPV-related severe dysplasia/SCC in situ have been recently described that line surface epithelium and which may or may not harbor foci of invasive cancer.

c. Cystic neck metastasis mimicking benign branchial cleft cysts or branchial cleft cysts with carcinoma developing in them – HPV-related oropharyngeal SCC very frequently develops nodal metastases (~80-85%) despite often very small primary tumors. These are frequently cystic and necrotic. Further, gland formation (adenosquamous carcinoma) and ciliated cells lining the cystic spaces (both in primary tumors and in the cystic neck metastases) have been described (so-called “ciliated adenosquamous carcinoma”). One half of the described cases lacked any clinically identifiable primary mucosal lesion and thus strongly mimicked benign cystic neck lesions. The distinction lies in the presence of obviously malignant squamous epithelium/cells in metastases versus benign cysts. Further, such lesions are consistently HPV-related so have diffuse p16 positivity and also high risk HPV DNA and mRNA positivity. Carcinoma arising in a branchial cleft cyst can occur, albeit extremely rarely. Very strict criteria for it have been proposed, and these criteria will consistently exclude metastatic cystic HPV-related SCC. d. HPV-related oropharyngeal carcinomas with a lymphoepithelial pattern resembling EBV-related – A minority of oropharyngeal SCC (~1%) will be histologically lymphoepithelial (or undifferentiated). These tumors have a syncytial growth pattern with vesicular nuclei and prominent nucleoli along with extensive tumor- infiltrating lymphocytes and plasma cells. In the oropharynx, >90% of such tumors are transcriptionally active HPV-related, and they have a favorable treatment responses and prognosis, despite the somewhat concerning histologic features. e. HPV-related basaloid, papillary, and adenosquamous SCC variants, similar in histology to those arising at non-oropharyngeal sites - These together represent ~5% of oropapharyngeal SCC and most of them (on the order of 80%) are related to transcriptionally active high risk HPV. Although limited by small numbers of cases, the available data suggests that they have the same favorable prognosis as other HPV-related SCCs. Verrucous carcinomas are rare in oropharynx and are consistently HPV negative. Rare spindle cell carcinomas in the oropharynx have been shown to be related to transcriptionally active HPV but the clinical significance is unknown.

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answers. Oral Oncol. 2013; 49(9): 863-71.

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