Molecular Diagnostics of Head and Neck Tumors Justin A. Bishop, M.D. Associate Professor of Pathology The Johns Hopkins University Baltimore, Maryland
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1 Molecular Diagnostics of Head and Neck Tumors Justin A. Bishop, M.D. Associate Professor of Pathology The Johns Hopkins University Baltimore, Maryland Two Main Topics Molecular insights in salivary gland neoplasia Human papillomavirus related tumors Salivary Gland Carcinomas 1
2 Salivary Gland Pathology One of the most difficult areas of ENT pathology. Rare tumors few pathologists see high volumes. Tremendous variety. 2016: 42 tumors or tumor like lesions. Even a single tumor type (e.g., pleomorphic adenoma) may show marked morphologic variability. New molecular findings have driven changes in classification New entities Consolidation of others Translocations in Salivary Gland Tumors Tumor Translocation Gene fusion Mammary analogue t(12;15) ETV6 NTRK3 secretory carcinoma Polymorphous adenocarcinoma Hyalinizing clear cell carcinoma Mucoepidermoid carcinoma Adenoid cystic carcinoma t(1;14) t(x;14) t(12;22) t(11;19) t(11;15) t(6;9) t(8;9) ARID1A PRKD1 DDX3X PRKD1 Other PRKD2 and PRKD3 rearrangements EWSR1 ATF1 CRTC1 MAML2 CRTC3 MAML2 MYB NFIB MYBL1 NFIB Modified from McCord C, et al. Diagn. Histopathol (6):
3 (Mammary analogue) secretory carcinoma (MASC) First 16 cases described in 2010 by Skálová, et al. > 200 additional published cases to date. Included as a new entity in 2017 WHO as secretory carcinoma. Mammary analogue secretory carcinoma Parotid gland most common (~2/3), but also oral cavity and submandibular gland. Rarely weird places like skin and thyroid! Usually adults (range 10 86, mean 47). Men = women. 3
4 4
5 Mammary analogue secretory carcinoma Positive for S100, mammaglobin, GATA3, CKs. Variably positive for GCDFP. Negative for ER, PR, Her2, DOG 1. S100 Mammaglobin 5
6 Mammary analogue secretory carcinoma Characteristically harbors t(12;15) (p13;q25) resulting in the formation of the ETV6 NTRK3 fusion gene. Same translocation as secretory breast cancer, congenital mesoblastic nephroma, infantile fibrosarcoma, some leukemias, PTCs, and IMTs. Rare examples of ETV6 X Mammary analogue secretory carcinoma Most cases were previously called acinic cell carcinoma. MASC is S100 and mammaglobin positive, DOG 1 negative. MASC lacks serous acinar cells (i.e., zymogen granules). Other considerations include mucoepidermoid carcinoma, low grade salivary duct carcinoma, adenocarcinoma NOS. 6
7 Mammary analogue secretory carcinoma Prognosis appears to be similar to acinic cell carcinoma, a low grade malignancy that may occasionally behave aggressively. Very rare examples with high grade transformation. Translocation represents an attractive potential therapeutic target. Some leukemias are responsive to tyrosine kinase inhibitors targeting ETV6 fusion proteins Polymorphous Adenocarcinoma Formerly polymorphous low grade adenocarcinoma (PLGA) Oral cavity, especially palate. Mean 59 years, M:F ratio 1:2 Painless mass, +/ ulcer 7
8 8
9 Cribriform Adenocarcinoma of the Tongue and Minor Salivary Glands (CATMSG) First described in 1999 by Michal, et al. Originally limited to tongue (n=8), origin from thyroglossal duct was questioned. Later expanded to other oral cavity sites CATMSG Cribriform Adenocarcinoma of the Tongue and Minor Salivary Glands (CATMSG) Most commonly tongue (usually base), occasionally other oral sites like palate, lips, floor of mouth. Adults, 21 to 85 (mean 57). Men = women. Frequently metastasizes (~70%) to cervical lymph nodes, often as presenting feature. 9
10 10
11 11
12 S100 12
13 p63 p40 CAT and PAC CAT: 75% harbor rearrangements of PRKD1, PRKD2, and PRKD3. PAC: hot spot mutations of PRKD genes. But. Significant % are in a mixed group that can have either alteration. WHO: not enough evidence (yet) to separate from polymorphous adenocarcinoma. (Hyalinizing) clear cell carcinoma Described in 1994 as HCCC. Variably named Clear cell adenocarcinoma (AFIP) Clear cell carcinoma, NOS (WHO 2005) Now clear cell carcinoma in WHO
14 Hyalinizing clear cell carcinoma Originally regarded as a diagnosis of exclusion. Discovery of consistent EWSR1 ATF1 gene fusion has helped define this tumor type more precisely. Same translocation as OCCC, clear cell sarcoma, myoepithelial carcinoma of ST. Hyalinizing clear cell carcinoma Most common in oral cavity (especially base of tongue and palate), but may be seen in other minor or major salivary glands. Slight female predominance. Usually 5 th 8 th decades, rare in children. Submucosal swelling, +/ ulcer and pain. 14
15 15
16 16
17 Hyalinizing clear cell carcinoma Squamous immunophenotype: diffusely positive for p40, p63, CK5/6. Negative for S100, calponin, actin, GFAP. 17
18 Hyalinizing clear cell carcinoma Differential diagnosis: Myoepithelial carcinoma. Epithelial myoepithelial carcinoma. Mucoepidermoid carcinoma. Squamous cell carcinoma. Calponin 18
19 CK5/6 p16 19
20 Hyalinizing clear cell carcinoma Low grade by definition. Good prognosis, only occasional recurrences or lymph node metastases. Distant metastases and tumor related deaths are rare. Translocations in Salivary Gland Tumors Tumor Translocation Gene fusion Mammary analogue t(12;15) ETV6 NTRK3 secretory carcinoma Polymorphous adenocarcinoma Hyalinizing clear cell carcinoma Mucoepidermoid carcinoma Adenoid cystic carcinoma t(1;14) t(x;14) t(12;22) t(11;19) t(11;15) t(6;9) t(8;9) ARID1A PRKD1 DDX3X PRKD1 Other PRKD2 and PRKD3 rearrangements EWSR1 ATF1 CRTC1 MAML2 CRTC3 MAML2 MYB NFIB MYBL1 NFIB Modified from McCord C, et al. Diagn. Histopathol (6):
21 Overview of HPV Related Head and Neck Carcinoma Oropharynx 80 90% Pai SI and Westra WH. Ann. Rev. Mech. Dis. 2009;4:
22 PD L1 Lyford Pyke S, et al. Clin Cancer Res. 2013; 73: Oropharynx is a separate chapter in the new edition of the WHO 22
23 2017 Oropharygeal SCCs are sub classified by HPV status in the new WHO edition HPV+ vs. HPV OPSCC HPV HPV+ Incidence Falling Rising Age Older Younger Socio economic status Low High Risk factors Tobacco, alcohol Sexual behavior Survival Worse Better HPV+ vs. HPV OPSCC Ang K et al. N Engl J Med 2010; 363(1):
24 HPV Squamous Cell Carcinoma HPV+ Squamous Cell Carcinoma HPV+ Squamous Cell Carcinoma 24
25 HPV+ Squamous Cell Carcinoma Metastatic HPV+ Squamous Cell Carcinoma HPV+ Squamous Cell Carcinoma 25
26 TCGA Data Beck TN and Golemis EA. Cancers Head Neck. 2016; 1: Issues Unique to HPV+ OPSCC Grading Invasion Tumor Grading Semi quantitative measurement of differentiation, expressed as the degree to which a tumor resembles the normal tissue from which it arises Well differentiated Moderately differentiated Poorly differentiated Undifferentiated May correlate with tumor behavior 26
27 Oropharyngeal HPV+ SCC should not be graded Invasive? All oropharyngeal HPV+ SCC are invasive Variants of HPV+ Oropharyngeal Carcinoma Basaloid Lymphoepithelial like Papillary Adenosquamous Adenocarcinoma, NOS Sarcomatoid Neuroendocrine carcinoma Small cell Large cell neuroendocrine 27
28 Neuroendocrine variants of oropharyngeal HPV+ carcinoma are aggressive HPV+ Small Cell Carcinoma HPV+ Large Cell NE Carcinoma HPV Testing Tumor classification Prognosis Eligibility for clinical trials Diagnostic When to test for HPV All oropharyngeal SCC and variants Cervical lymph node SCC metastases of OP or unknown primary NOT non oropharyngeal ENT sites (routinely) 28
29 How to test for HPV? High risk types only. 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 HPV types 6 and 11 are low risk. Cause papillomas and warts. Can cause morbidity (e.g., laryngeal papillomatosis) but not a significant cause of HPV related OPSCC HPV 6/11 How to test for HPV? Methods: p16 immunohistochemistry PCR for HPV DNA PCR for HPV E6/E7 mrna DNA in situ hybridization RNA in situ hybridization Cytology based techniques Combinations/algorithms Widely available, easy to perform Highly sensitive Diffuse (>70%), strong, nuclear and cytoplasmic ~80% specific in oropharynx p16 Poor surrogate outside of oropharynx Rautava J and Syrjanen S. Head Neck Pathol. 2012;6(1s):
30 Was widely available, easy to perform and interpret Highly specific Tissue context Suboptimal sensitivity HPV 16 DNA HPV 16 E6/E7 RNA Highly sensitive Highly specific Tissue context Detects transcriptionally active virus Not widely available on automated platforms (yet) Bishop JA,et al. Am. J. Surg. Pathol. 2012;36(12): What about cyto material? Often the first material available. All of the tissue based testing methods can be done on cell blocks. BUT p16 is often more patchy in FNA material than it is in tissue. % difficult to determine, threshold not standardized.. p16 RNA ISH 30
31 Also p16 often positive in branchial cleft cysts, lung and skin SCCs. Be careful with p16 in FNAs! More specific testing methods often needed. p16 Liquid phase assays Hybrid Capture II, Cervista TM HPV HR, Roche cobas HPV test, and APTIMA HPV assay. Already in wide use for cervical cytology. Obviates the need for creating a cell block. Provides a quantitative result with clear cut scoring. A few studies with promising results Widespread clinical validation still needed before these assays can be routinely used 31
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