- Email: [email protected]
p16 Immunohistochemistry Is a Useful Diagnostic Adjunct in Cases of Metastatic Cervical Carcinoma of Unknown Origin
Accepted Manuscript p16 Immunohistochemistry Is A Useful Diagnostic Adjunct In Cases of Metastatic Cervical Carcinoma of Unknown Origin Marc R. Rohrbach, MD, Christopher J. Britt, MD, Michael Schwalbe, MD, Aaron M. Wieland, MD, Gregory K. Hartig, MD PII:
S0278-2391(16)30763-7
DOI:
10.1016/j.joms.2016.08.029
Reference:
YJOMS 57418
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 29 July 2016 Revised Date:
19 August 2016
Accepted Date: 20 August 2016
Please cite this article as: Rohrbach MR, Britt CJ, Schwalbe M, Wieland AM, Hartig GK, p16 Immunohistochemistry Is A Useful Diagnostic Adjunct In Cases of Metastatic Cervical Carcinoma of Unknown Origin, Journal of Oral and Maxillofacial Surgery (2016), doi: 10.1016/j.joms.2016.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
p16 Immunohistochemistry Is A Useful Diagnostic Adjunct In Cases of Metastatic Cervical Carcinoma of Unknown Origin.
RI PT
Marc R Rohrbach, MD; Corresponding Author Resident, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. K4/719 CSC, 600 Highland Avenue, Madison, WI,
SC
53792-7375, United States. [email protected]; 608-262-6376
M AN U
Christopher J Britt, MD
Resident, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States
TE D
Michael Schwalbe, MD
Resident, Department of Pathology and Laboratory Medicine. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States
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Aaron M Wieland, MD
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Assistant Professor, Section of Head and Neck Surgical Oncology, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States Gregory K Hartig, MD
ACCEPTED MANUSCRIPT
Chief, Section of Head and Neck Surgical Oncology, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland
Keywords:
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Human papillomavirus; HPV; p16 ; Unknown primary; Pathology
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Avenue, Madison, WI, 53792-7375, United States
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Abstract Background: Metastatic cervical carcinoma of unknown primary (MCCUP) is increasing in frequency, in
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part due to rising human papillomavirus (HPV) driven oropharyngeal carcinoma. Identifying the primary site is valuable, as it is associated with increased survival and decreased morbidity. HPV-positive cervical nodal disease focuses attention on the oropharynx for directed biopsies, including tonsillectomy. When the primary is small, carcinoma may not be apparent on traditional hematoxylin and eosin (H&E)
SC
staining alone.
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Methods: We present two cases of p16-positive MCCUP where a small primary carcinoma was not readily identified in surgical specimens using H&E staining.
Results: Additional evaluation of the specimens with p16 immunohistochemistry (IHC) revealed carcinoma in both cases.
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Conclusions: When H&E staining does not reveal carcinoma in cases of MCCUP, p16 IHC should be considered given the high prevalence of HPV-positive MCCUP and the potential for identification of a
AC C
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small primary which might otherwise be missed with H&E staining.
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Introduction
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Metastatic cervical carcinoma of unknown primary (MCCUP) occurs when the primary tumor site cannot be detected despite a thorough history, physical examination, flexible endoscopy, and crosssectional imaging.1,2 Overall, MCCUP accounts for 2 to 9% of head and neck cancers, with a median age
SC
of diagnosis at 60 years, predominantly affecting males.1-3 Squamous cell carcinoma (SCC) accounts for around 90% of MCCUP cases, traditionally in heavy alcohol and tobacco users.1
M AN U
The human papillomavirus (HPV), specifically genotypes 16 and 18, has become a prominent risk factor for oropharyngeal SCC.1 The frequency of HPV-positive oropharyngeal cancer is increasing, and with it, is the frequency of MCCUP diagnoses, possibly because the primary site is often asymptomatic and small.4 Detection of HPV in cervical lymph nodes has value in directing the search for an oropharyngeal primary.
TE D
This can be performed with HPV genome specific polymerase chain reaction (PCR) or in-situ hybridization (ISH) techniques, or more commonly, using p16 immunohistochemistry (IHC) as a screening tool for the presence of HPV-driven disease.3-5 This is because HPV protein E7 leads to
EP
degradation of the retinoblastoma protein, ultimately leading to over-expression of the cyclindependent kinase inhibitor, p16.6 Therefore, p16 over-expression correlates with the presence of
AC C
transcriptionally-active HPV and is accepted as an effective surrogate immunomarker for HPV-driven carcinoma .6
Reported prevalence of HPV-positive MCCUP varies, primarily due to inconsistent definitions for
MCCUP and the extent of workup performed.5 Motz et al found that for patients initially diagnosed with MCCUP, meaning no clinical evidence of a primary lesion on physical exam, radiographic, or endoscopic evaluation, as many as 90% were HPV-positive. The overall primary site detection rate in their cohort for patients with MCCUP was 59.5%, although only 54.2% of those underwent palatine or lingual
ACCEPTED MANUSCRIPT
tonsillectomy.4 Trans-oral robotic surgery (TORS) and trans-oral laser microsurgery (TLM), which allow magnified views of the oropharynx and facilitate lingual tonsillectomy, are thought to increase detection rates of primary tumors. Graboyes et al employed an approach of initial magnified inspection, with
RI PT
directed biopsies and subsequent ipsilateral palatine and lingual tonsillectomy if frozen section
evaluation of the directed biopsies was unrevealing. With this approach 89% of MCCUP primaries were identified.7 However, Motz et al found that the detection rate before and after these techniques was
SC
similar in HPV-positive tumors.4 Even with the most exhaustive efforts employing TORS or TLM to identify the primary site, advanced techniques which may not be used in many practice settings,
M AN U
approximately 10% go undetected, of which, 28-52% are HPV-positive.3,5,7
In this case report, we introduce two patients who presented with MCCUP. Both underwent surgical sampling of their oropharynx to identify the primary. Initial pathology of their surgical oropharyngeal specimens using standard hematoxylin and eosin (H&E) staining did not reveal
primary tumor.
EP
Case One
TE D
carcinoma, yet repeat evaluation of the same specimens with p16 IHC resulted in identification of the
A 48-year-old male presented to the University of Wisconsin Otolaryngology Clinic with fullness
AC C
in his right neck that was noted following an upper respiratory infection six months prior. He was a nonsmoker with only occasional ethanol use. Physical examination and flexible endoscopy did not reveal any concerning lesions. A positron emission tomography (PET) scan and computed-tomography (CT) scan obtained prior to his visit showed a hypermetabolic 6.3 cm neck mass but no primary lesion. An excisional biopsy of the neck mass was performed at an outside institution, which confirmed metastatic non-keratinizing squamous cell carcinoma. No p16 IHC was performed. Due to his younger age and non-
ACCEPTED MANUSCRIPT
smoking status, a request was made by our institution to have the outside institution perform p16 IHC on the outside excisional biopsy, and this showed diffuse nuclear and cytoplasmic positivity for p16.
RI PT
He then underwent a right completion selective neck dissection as well as ipsilateral robotic palatine and lingual tonsillectomy. Initial pathology review with H&E was negative for carcinoma in the palatine and lingual tonsil specimens. However, given the high probability for an ipsilateral
oropharyngeal primary site based on the p16 status of the cervical nodes, p16 IHC was performed on
SC
the palatine and lingual tonsil specimens. This revealed a six by seven millimeter focus of carcinoma within his right inferior palatine tonsil. He went on to complete adjuvant chemoradiotherapy, and
M AN U
identification of the primary allowed for a reduced radiation dose and sparing of the contralateral neck. Case Two
A 53-year-old male presented to the University of Wisconsin Otolaryngology clinic with a one
TE D
month history of right neck pain, right otalgia and an enlarging right neck mass. He did have a 40 pack year smoking history. A fine needle aspirate of the neck mass was performed and confirmed metastatic non-keratinizing squamous cell carcinoma. No p16 staining was performed, likely because of the
EP
significant smoking history. Physical examination and flexible endoscopy did not reveal any concerning lesions. A PET and CT scan showed significant hypermetabolic right-sided cervical lymphadenopathy, but
AC C
did not reveal a primary lesion.
The patient was taken to the operating room for direct laryngoscopy with right tongue base
biopsy and palatine tonsillectomy. Initial review of the tongue base biopsies using H&E showed some concerning cells, but was non-diagnostic. Knowing the high prevalence of HPV related disease in cases of MCCUP, and the non-keratinizing morphology of the nodal disease which is classic for HPV-related carcinoma, pathology then re-evaluated the tongue base biopsies using p16 IHC. This revealed a small
ACCEPTED MANUSCRIPT
focus of carcinoma amongst a dense lymphocytic background. He is currently undergoing chemoradiotherapy.
RI PT
Discussion: In cases of MCCUP, the initial goal is to determine the location of the occult primary site.
Identification of the primary site is associated with improvement in survival.8,9 Furthermore, lack of
SC
identification of the primary site necessitates wider-field radiation with consequent increased
morbidity.10 HPV-positive nodal disease suggests an oropharyngeal primary, an association that has led
M AN U
to improved discovery of the primary lesion by guiding surgeons to perform directed biopsies and tonsillectomy.8,10,11 For those initially diagnosed as HPV-positive MCCUP, as many as 89% of primary tumors may be identified in the oropharynx utilizing exhaustive sampling of the ipsilateral oropharynx.7 Even with comprehensive sampling of the oropharynx, 10 percent or more of patients will ultimately go
TE D
without identification of the primary site, and these patients are classified as true, or definitive, MCCUP. Estimates for the rate of true MCCUP vary, ranging from less than 10% to over 40% in some series, with more than 50% of these true, or definitive, MCCUP cases still being HPV-positive.4,5,7 It is
EP
unclear why such high numbers of undetected primaries remain for HPV-positive disease. The techniques using TORS and TLM are resource intense and may not be generalizable to hospitals that
AC C
aren’t high volume academic centers, which may explain some of the variation in reported rates of true MCCUP. Additionally, a small number of these cases may be outside of the oropharynx, such as the nasopharynx or hypopharynx, and thus not discovered through oropharyngeal biopsies. However, it is also known that HPV-related malignancies arise from crypt epithelium of the palatine and lingual tonsils and may be only millimeters in size, such that they are not only missed clinically, but also missed by the pathologist.3-5 There are several histopathologic characteristics which may contribute to the inconspicuousness of a small tumor. The first may be the subtleness of a non-keratinizing carcinoma,
ACCEPTED MANUSCRIPT
which lacks the distinctive keratinization and conspicuous eosinophilic tinctorial quality of keratinizing squamous cell carcinoma. In addition, tonsillar tissue in general is unique due to the abundance of lymphoid tissue, which complicates pathologic review. As in both of these cases, a reactive dense
RI PT
lymphocytic background may hide a small focus of carcinoma, as the small basaloid cells of non-
keratinizing carcinoma may look similar to lymphocytes, endothelial cells of high-endothelial venules, or sinus histiocytes (Figure 1A). In addition, lymphoid germinal centers and nests of non-keratinizing
SC
squamous cell carcinoma may look similar, so that small nests of carcinoma go unnoticed amongst numerous germinal centers. In both of these cases, p16 IHC highlighted nests of carcinoma, making
M AN U
them much more conspicuous amongst the dense lymphocytic background (Figure 1B). In certain instances, when there is concern for carcinoma which cannot easily be diagnosed on H&E alone, pathologists use a pan-cytokeratin marker such as AE1/AE3 to better highlight carcinoma cells within the stroma. While this may highlight carcinoma, it highlights all epithelial derived cells,
TE D
including that of the native epithelium. Because tonsillar tissue is heavily convoluted, tangential planes of section often include normal crypt epithelium which may appear as a nest of epithelial cells within the stroma. These foci of non-neoplastic crypt epithelium may look very similar to foci of carcinoma, and
EP
both are often present on the same cut (Figure 2A). While pan-cytokeratin markers such as AE1/AE3 would highlight all epithelial cells non-selectively, p16 would highlight only the p16 positive carcinoma
AC C
and thus allow the pathologist to more easily differentiate p16 positive carcinoma from normal crypt epithelium, which would not be highlighted by p16 IHC (Figures 2B and C). In the case of these two patients, despite tonsillectomy and directed oropharyngeal biopsies,
initial histopathologic review did not reveal a primary focus of carcinoma when using only H&E staining. Given the high prevalence of HPV positive disease in MCCUP, p16 staining was performed, resulting in identification of the primary site and ultimately the ability to de-intensify therapy. In cases of MCCUP,
ACCEPTED MANUSCRIPT
when traditional H&E staining of oropharyngeal specimens does not reveal a focus of carcinoma, p16 IHC should be considered given the high prevalence of HPV-positive disease and the potential for
RI PT
identification of a small primary which might otherwise be missed with standard H&E staining. References:
1. Arrangoiz R, Galloway TJ, Papavasiliou P, Ridge JA, Lango MN. Metastatic cervical carcinoma
SC
from an unknown primary: literature review. Ear Nose Throat J 2014; 93(4-5):E1-E10.
2. Grau C, Johansen LV, Jakobsen J, Geertsen P, Andersen E, Jensen BB. Cervical lymph node
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metastases from unknown primary tumours. Results from a national survey by the Danish Society for Head and Neck Oncology. Radiother Oncol 2000; 55 (2): 121-9. 3. Boscolo-Rizzo P, Schroeder L, Romeo S, Pawlita M. The prevalence of human papillomavirus in squamous cell carcinoma of unknown primary site metastatic to neck lymph nodes: a systematic review. Clin Exp Metastasis. 2015 Dec;32(8):835-45. doi: 10.1007/s10585-015-9744-z. Epub
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2015 Sep 10. PubMed PMID: 26358913.
4. Motz K, Qualliotine JR, Rettig E, Richmon JD, Eisele DW, Fakhry C. Changes in Unknown Primary Squamous Cell Carcinoma of the Head and Neck at Initial Presentation in the Era of Human
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Papillomavirus. JAMA Otolaryngol Head Neck Surg. 2016 Jan 14:1-7. doi:
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10.1001/jamaoto.2015.3228. [Epub ahead of print] PubMed PMID: 26769661. 5. Sivars L, Tani E, Näsman A, Ramqvist T, Munck-Wikland E, Dalianis T. Human Papillomavirus as a Diagnostic and Prognostic Tool in Cancer of Unknown Primary in the Head and Neck Region. Anticancer Res. 2016 Feb;36(2):487-93. Review. PubMed PMID: 26851001.
6. Lewis JS Jr. p16 Immunohistochemistry as a standalone test for risk stratification in oropharyngeal squamous cell carcinoma. Head Neck Pathol. 2012 Jul;6 Suppl 1:S75-82.
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7. Graboyes EM, Sinha P, Thorstad WL, Rich JT, Haughey BH. Management of human papillomavirus-related unknown primaries of the head and neck with a transoral surgical approach. Head Neck. 2015; 37 (11): 1603-1611.
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8. Davis KS, Byrd JK, Mehta V, et al. Occult primary head and neck squamous cell carcinoma: Utility of Discovering Primary Lesions. Otolaryngol Head Neck Surg. 2014;2:151:272-278.
9. Haas I, Hoffmann TK, Engers R, Ganzer U. Diagnostic strategies in cervical carcinoma of an
SC
unknown primary (CUP). Eur Arch Otorhinolaryngol. 2002;6:259:325-333.
10. Zhang MQ, El-Mofty SK, and Davilla RM. Detection of Human Papillomavirus-related Squamous
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Cell Carcinoma Cytologically and by In Situ Hybridization in Fine-needle Aspiration Biopsies of Cervical Metastasis. Cancer Cytopathology; American Cancer Society. 2008: 118-123 11. Park JM, Jung CK, Choi YJ, et al. The use of an immunohistochemical diagnostic panel to determine the primary site of cervical lymph node metastases of occult squamous cell
Figure Legend
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carcinoma. Hum Pathol 2010; 41 (3): 431-7.
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Figure 1: A: Lymphoid hyperplasia and small vessels with reactive endothelium conceal inconspicuous nests of carcinoma on H&E (100X magnification). B: Immunohistochemistry for p16 highlights nests of squamous carcinoma (100X magnification). Figure 2: A: A tangential section demonstrates well-circumscribed foci of carcinoma (arrow) juxtaposed with non-neoplastic crypt epithelium (arrowheads) at 200X magnification with H&E. B: Pancytokeratin highlights both the neoplastic and non-neoplastic epithelium at 200X magnification. C: p16
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immunohistochemistry reveals block-like positivity in the tumor nests, while the crypt epithelium
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EP
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SC
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remains negative at 200X magnification.
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EP
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M AN U
SC
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EP
TE D
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SC
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S0278-2391(16)30763-7
DOI:
10.1016/j.joms.2016.08.029
Reference:
YJOMS 57418
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 29 July 2016 Revised Date:
19 August 2016
Accepted Date: 20 August 2016
Please cite this article as: Rohrbach MR, Britt CJ, Schwalbe M, Wieland AM, Hartig GK, p16 Immunohistochemistry Is A Useful Diagnostic Adjunct In Cases of Metastatic Cervical Carcinoma of Unknown Origin, Journal of Oral and Maxillofacial Surgery (2016), doi: 10.1016/j.joms.2016.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
p16 Immunohistochemistry Is A Useful Diagnostic Adjunct In Cases of Metastatic Cervical Carcinoma of Unknown Origin.
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Marc R Rohrbach, MD; Corresponding Author Resident, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. K4/719 CSC, 600 Highland Avenue, Madison, WI,
SC
53792-7375, United States. [email protected]; 608-262-6376
M AN U
Christopher J Britt, MD
Resident, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States
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Michael Schwalbe, MD
Resident, Department of Pathology and Laboratory Medicine. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States
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Aaron M Wieland, MD
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Assistant Professor, Section of Head and Neck Surgical Oncology, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland Avenue, Madison, WI, 53792-7375, United States Gregory K Hartig, MD
ACCEPTED MANUSCRIPT
Chief, Section of Head and Neck Surgical Oncology, Division of Otolaryngology-Head & Neck Surgery, Department of Surgery. University of Wisconsin School of Medicine and Public Health. 600 Highland
Keywords:
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M AN U
SC
Human papillomavirus; HPV; p16 ; Unknown primary; Pathology
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Avenue, Madison, WI, 53792-7375, United States
ACCEPTED MANUSCRIPT
Abstract Background: Metastatic cervical carcinoma of unknown primary (MCCUP) is increasing in frequency, in
RI PT
part due to rising human papillomavirus (HPV) driven oropharyngeal carcinoma. Identifying the primary site is valuable, as it is associated with increased survival and decreased morbidity. HPV-positive cervical nodal disease focuses attention on the oropharynx for directed biopsies, including tonsillectomy. When the primary is small, carcinoma may not be apparent on traditional hematoxylin and eosin (H&E)
SC
staining alone.
M AN U
Methods: We present two cases of p16-positive MCCUP where a small primary carcinoma was not readily identified in surgical specimens using H&E staining.
Results: Additional evaluation of the specimens with p16 immunohistochemistry (IHC) revealed carcinoma in both cases.
TE D
Conclusions: When H&E staining does not reveal carcinoma in cases of MCCUP, p16 IHC should be considered given the high prevalence of HPV-positive MCCUP and the potential for identification of a
AC C
EP
small primary which might otherwise be missed with H&E staining.
ACCEPTED MANUSCRIPT
Introduction
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Metastatic cervical carcinoma of unknown primary (MCCUP) occurs when the primary tumor site cannot be detected despite a thorough history, physical examination, flexible endoscopy, and crosssectional imaging.1,2 Overall, MCCUP accounts for 2 to 9% of head and neck cancers, with a median age
SC
of diagnosis at 60 years, predominantly affecting males.1-3 Squamous cell carcinoma (SCC) accounts for around 90% of MCCUP cases, traditionally in heavy alcohol and tobacco users.1
M AN U
The human papillomavirus (HPV), specifically genotypes 16 and 18, has become a prominent risk factor for oropharyngeal SCC.1 The frequency of HPV-positive oropharyngeal cancer is increasing, and with it, is the frequency of MCCUP diagnoses, possibly because the primary site is often asymptomatic and small.4 Detection of HPV in cervical lymph nodes has value in directing the search for an oropharyngeal primary.
TE D
This can be performed with HPV genome specific polymerase chain reaction (PCR) or in-situ hybridization (ISH) techniques, or more commonly, using p16 immunohistochemistry (IHC) as a screening tool for the presence of HPV-driven disease.3-5 This is because HPV protein E7 leads to
EP
degradation of the retinoblastoma protein, ultimately leading to over-expression of the cyclindependent kinase inhibitor, p16.6 Therefore, p16 over-expression correlates with the presence of
AC C
transcriptionally-active HPV and is accepted as an effective surrogate immunomarker for HPV-driven carcinoma .6
Reported prevalence of HPV-positive MCCUP varies, primarily due to inconsistent definitions for
MCCUP and the extent of workup performed.5 Motz et al found that for patients initially diagnosed with MCCUP, meaning no clinical evidence of a primary lesion on physical exam, radiographic, or endoscopic evaluation, as many as 90% were HPV-positive. The overall primary site detection rate in their cohort for patients with MCCUP was 59.5%, although only 54.2% of those underwent palatine or lingual
ACCEPTED MANUSCRIPT
tonsillectomy.4 Trans-oral robotic surgery (TORS) and trans-oral laser microsurgery (TLM), which allow magnified views of the oropharynx and facilitate lingual tonsillectomy, are thought to increase detection rates of primary tumors. Graboyes et al employed an approach of initial magnified inspection, with
RI PT
directed biopsies and subsequent ipsilateral palatine and lingual tonsillectomy if frozen section
evaluation of the directed biopsies was unrevealing. With this approach 89% of MCCUP primaries were identified.7 However, Motz et al found that the detection rate before and after these techniques was
SC
similar in HPV-positive tumors.4 Even with the most exhaustive efforts employing TORS or TLM to identify the primary site, advanced techniques which may not be used in many practice settings,
M AN U
approximately 10% go undetected, of which, 28-52% are HPV-positive.3,5,7
In this case report, we introduce two patients who presented with MCCUP. Both underwent surgical sampling of their oropharynx to identify the primary. Initial pathology of their surgical oropharyngeal specimens using standard hematoxylin and eosin (H&E) staining did not reveal
primary tumor.
EP
Case One
TE D
carcinoma, yet repeat evaluation of the same specimens with p16 IHC resulted in identification of the
A 48-year-old male presented to the University of Wisconsin Otolaryngology Clinic with fullness
AC C
in his right neck that was noted following an upper respiratory infection six months prior. He was a nonsmoker with only occasional ethanol use. Physical examination and flexible endoscopy did not reveal any concerning lesions. A positron emission tomography (PET) scan and computed-tomography (CT) scan obtained prior to his visit showed a hypermetabolic 6.3 cm neck mass but no primary lesion. An excisional biopsy of the neck mass was performed at an outside institution, which confirmed metastatic non-keratinizing squamous cell carcinoma. No p16 IHC was performed. Due to his younger age and non-
ACCEPTED MANUSCRIPT
smoking status, a request was made by our institution to have the outside institution perform p16 IHC on the outside excisional biopsy, and this showed diffuse nuclear and cytoplasmic positivity for p16.
RI PT
He then underwent a right completion selective neck dissection as well as ipsilateral robotic palatine and lingual tonsillectomy. Initial pathology review with H&E was negative for carcinoma in the palatine and lingual tonsil specimens. However, given the high probability for an ipsilateral
oropharyngeal primary site based on the p16 status of the cervical nodes, p16 IHC was performed on
SC
the palatine and lingual tonsil specimens. This revealed a six by seven millimeter focus of carcinoma within his right inferior palatine tonsil. He went on to complete adjuvant chemoradiotherapy, and
M AN U
identification of the primary allowed for a reduced radiation dose and sparing of the contralateral neck. Case Two
A 53-year-old male presented to the University of Wisconsin Otolaryngology clinic with a one
TE D
month history of right neck pain, right otalgia and an enlarging right neck mass. He did have a 40 pack year smoking history. A fine needle aspirate of the neck mass was performed and confirmed metastatic non-keratinizing squamous cell carcinoma. No p16 staining was performed, likely because of the
EP
significant smoking history. Physical examination and flexible endoscopy did not reveal any concerning lesions. A PET and CT scan showed significant hypermetabolic right-sided cervical lymphadenopathy, but
AC C
did not reveal a primary lesion.
The patient was taken to the operating room for direct laryngoscopy with right tongue base
biopsy and palatine tonsillectomy. Initial review of the tongue base biopsies using H&E showed some concerning cells, but was non-diagnostic. Knowing the high prevalence of HPV related disease in cases of MCCUP, and the non-keratinizing morphology of the nodal disease which is classic for HPV-related carcinoma, pathology then re-evaluated the tongue base biopsies using p16 IHC. This revealed a small
ACCEPTED MANUSCRIPT
focus of carcinoma amongst a dense lymphocytic background. He is currently undergoing chemoradiotherapy.
RI PT
Discussion: In cases of MCCUP, the initial goal is to determine the location of the occult primary site.
Identification of the primary site is associated with improvement in survival.8,9 Furthermore, lack of
SC
identification of the primary site necessitates wider-field radiation with consequent increased
morbidity.10 HPV-positive nodal disease suggests an oropharyngeal primary, an association that has led
M AN U
to improved discovery of the primary lesion by guiding surgeons to perform directed biopsies and tonsillectomy.8,10,11 For those initially diagnosed as HPV-positive MCCUP, as many as 89% of primary tumors may be identified in the oropharynx utilizing exhaustive sampling of the ipsilateral oropharynx.7 Even with comprehensive sampling of the oropharynx, 10 percent or more of patients will ultimately go
TE D
without identification of the primary site, and these patients are classified as true, or definitive, MCCUP. Estimates for the rate of true MCCUP vary, ranging from less than 10% to over 40% in some series, with more than 50% of these true, or definitive, MCCUP cases still being HPV-positive.4,5,7 It is
EP
unclear why such high numbers of undetected primaries remain for HPV-positive disease. The techniques using TORS and TLM are resource intense and may not be generalizable to hospitals that
AC C
aren’t high volume academic centers, which may explain some of the variation in reported rates of true MCCUP. Additionally, a small number of these cases may be outside of the oropharynx, such as the nasopharynx or hypopharynx, and thus not discovered through oropharyngeal biopsies. However, it is also known that HPV-related malignancies arise from crypt epithelium of the palatine and lingual tonsils and may be only millimeters in size, such that they are not only missed clinically, but also missed by the pathologist.3-5 There are several histopathologic characteristics which may contribute to the inconspicuousness of a small tumor. The first may be the subtleness of a non-keratinizing carcinoma,
ACCEPTED MANUSCRIPT
which lacks the distinctive keratinization and conspicuous eosinophilic tinctorial quality of keratinizing squamous cell carcinoma. In addition, tonsillar tissue in general is unique due to the abundance of lymphoid tissue, which complicates pathologic review. As in both of these cases, a reactive dense
RI PT
lymphocytic background may hide a small focus of carcinoma, as the small basaloid cells of non-
keratinizing carcinoma may look similar to lymphocytes, endothelial cells of high-endothelial venules, or sinus histiocytes (Figure 1A). In addition, lymphoid germinal centers and nests of non-keratinizing
SC
squamous cell carcinoma may look similar, so that small nests of carcinoma go unnoticed amongst numerous germinal centers. In both of these cases, p16 IHC highlighted nests of carcinoma, making
M AN U
them much more conspicuous amongst the dense lymphocytic background (Figure 1B). In certain instances, when there is concern for carcinoma which cannot easily be diagnosed on H&E alone, pathologists use a pan-cytokeratin marker such as AE1/AE3 to better highlight carcinoma cells within the stroma. While this may highlight carcinoma, it highlights all epithelial derived cells,
TE D
including that of the native epithelium. Because tonsillar tissue is heavily convoluted, tangential planes of section often include normal crypt epithelium which may appear as a nest of epithelial cells within the stroma. These foci of non-neoplastic crypt epithelium may look very similar to foci of carcinoma, and
EP
both are often present on the same cut (Figure 2A). While pan-cytokeratin markers such as AE1/AE3 would highlight all epithelial cells non-selectively, p16 would highlight only the p16 positive carcinoma
AC C
and thus allow the pathologist to more easily differentiate p16 positive carcinoma from normal crypt epithelium, which would not be highlighted by p16 IHC (Figures 2B and C). In the case of these two patients, despite tonsillectomy and directed oropharyngeal biopsies,
initial histopathologic review did not reveal a primary focus of carcinoma when using only H&E staining. Given the high prevalence of HPV positive disease in MCCUP, p16 staining was performed, resulting in identification of the primary site and ultimately the ability to de-intensify therapy. In cases of MCCUP,
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when traditional H&E staining of oropharyngeal specimens does not reveal a focus of carcinoma, p16 IHC should be considered given the high prevalence of HPV-positive disease and the potential for
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identification of a small primary which might otherwise be missed with standard H&E staining. References:
1. Arrangoiz R, Galloway TJ, Papavasiliou P, Ridge JA, Lango MN. Metastatic cervical carcinoma
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from an unknown primary: literature review. Ear Nose Throat J 2014; 93(4-5):E1-E10.
2. Grau C, Johansen LV, Jakobsen J, Geertsen P, Andersen E, Jensen BB. Cervical lymph node
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metastases from unknown primary tumours. Results from a national survey by the Danish Society for Head and Neck Oncology. Radiother Oncol 2000; 55 (2): 121-9. 3. Boscolo-Rizzo P, Schroeder L, Romeo S, Pawlita M. The prevalence of human papillomavirus in squamous cell carcinoma of unknown primary site metastatic to neck lymph nodes: a systematic review. Clin Exp Metastasis. 2015 Dec;32(8):835-45. doi: 10.1007/s10585-015-9744-z. Epub
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2015 Sep 10. PubMed PMID: 26358913.
4. Motz K, Qualliotine JR, Rettig E, Richmon JD, Eisele DW, Fakhry C. Changes in Unknown Primary Squamous Cell Carcinoma of the Head and Neck at Initial Presentation in the Era of Human
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Papillomavirus. JAMA Otolaryngol Head Neck Surg. 2016 Jan 14:1-7. doi:
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10.1001/jamaoto.2015.3228. [Epub ahead of print] PubMed PMID: 26769661. 5. Sivars L, Tani E, Näsman A, Ramqvist T, Munck-Wikland E, Dalianis T. Human Papillomavirus as a Diagnostic and Prognostic Tool in Cancer of Unknown Primary in the Head and Neck Region. Anticancer Res. 2016 Feb;36(2):487-93. Review. PubMed PMID: 26851001.
6. Lewis JS Jr. p16 Immunohistochemistry as a standalone test for risk stratification in oropharyngeal squamous cell carcinoma. Head Neck Pathol. 2012 Jul;6 Suppl 1:S75-82.
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7. Graboyes EM, Sinha P, Thorstad WL, Rich JT, Haughey BH. Management of human papillomavirus-related unknown primaries of the head and neck with a transoral surgical approach. Head Neck. 2015; 37 (11): 1603-1611.
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8. Davis KS, Byrd JK, Mehta V, et al. Occult primary head and neck squamous cell carcinoma: Utility of Discovering Primary Lesions. Otolaryngol Head Neck Surg. 2014;2:151:272-278.
9. Haas I, Hoffmann TK, Engers R, Ganzer U. Diagnostic strategies in cervical carcinoma of an
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unknown primary (CUP). Eur Arch Otorhinolaryngol. 2002;6:259:325-333.
10. Zhang MQ, El-Mofty SK, and Davilla RM. Detection of Human Papillomavirus-related Squamous
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Cell Carcinoma Cytologically and by In Situ Hybridization in Fine-needle Aspiration Biopsies of Cervical Metastasis. Cancer Cytopathology; American Cancer Society. 2008: 118-123 11. Park JM, Jung CK, Choi YJ, et al. The use of an immunohistochemical diagnostic panel to determine the primary site of cervical lymph node metastases of occult squamous cell
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carcinoma. Hum Pathol 2010; 41 (3): 431-7.
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Figure 1: A: Lymphoid hyperplasia and small vessels with reactive endothelium conceal inconspicuous nests of carcinoma on H&E (100X magnification). B: Immunohistochemistry for p16 highlights nests of squamous carcinoma (100X magnification). Figure 2: A: A tangential section demonstrates well-circumscribed foci of carcinoma (arrow) juxtaposed with non-neoplastic crypt epithelium (arrowheads) at 200X magnification with H&E. B: Pancytokeratin highlights both the neoplastic and non-neoplastic epithelium at 200X magnification. C: p16
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immunohistochemistry reveals block-like positivity in the tumor nests, while the crypt epithelium
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