Head and neck squamous cell carcinomas of unknown primary: Can ancillary studies help identify more primary tumor sites?

Abstract

A subset of head and neck squamous cell carcinomas present solely as metastatic disease in the neck and are of unknown primary origin (SCCUP). Most primary tumors will ultimately be identified, usually in the oropharynx. In a minority of cases, the primary site remains elusive. Here, we examine the role of ancillary testing, including mutational signature analysis (MSA), to help identify likely primary sites in such cases. Twenty-two cases of SCCUP in the neck, collected over a 10-year period, were classified by morphology and viral status; including human papillomavirus (HPV) testing by p16 immunohistochemistry (IHC) and RT-qPCR, as well as Epstein-Barr virus (EBV) testing by EBER-ISH. CD5 and c-KIT (CD117) IHC was done to evaluate for possible thymic origin in all virus-negative cases. Whole exome sequencing, followed by MSA, was used to identify UV signature mutations indicative of cutaneous origin. HPV was identified in 12 of 22 tumors (54.5%), favoring an oropharyngeal origin, and closely associated with nonkeratinizing tumor morphology (Fisher's exact test; p = 0.0002). One tumor with indeterminant morphology had discordant HPV and p16 status (p16+/HPV-). All tumors were EBV-negative. Diffuse expression of CD5 and c-KIT was identified in 1 of 10 virus-negative SCCUPs (10%), suggesting a possible ectopic thymic origin rather than a metastasis. A UV mutational signature, indicating cutaneous origin, was identified in 1 of 10 (10%) virus-negative SCCUPs. A cutaneous auricular primary emerged 3 months after treatment in this patient. Primary tumors became clinically apparent in 2 others (1 hypopharynx, 1 hypopharynx/larynx). Thus, after follow-up, 6 tumors remained unclassifiable as to the possible site of origin (27%). Most SCCUPs of the neck in our series were HPV-associated and thus likely of oropharyngeal origin. UV signature mutation analysis and additional IHC for CD5 and c-KIT for possible thymic origin may aid in further classifying virus-negative unknown primaries. Close clinical inspection of hypopharyngeal mucosa may also be helpful, as a subset of primary tumors later emerged at this site.

Keywords: Epstein–Barr virus; Human papilloma virus; Mutational signature analysis; Squamous cell carcinoma; UV mutational signature; cancer of unknown primary.

Fig. 1. –

Representative tumors showing keratinizing (A), nonkeratinizing (B), and indeterminate (C) morphologies (images at 400× magnification). Positive p16 immunostaining in tumor with indeterminate morphology (HPV-negative) (200× magnification) (D).

Fig. 2. –

Categorization and breakdown of all SCCUP cases by viral testing (HPV and EBV), IHC (c-KIT and CD5), MSA, and eventual emergence of a primary site.

Fig. 3. –

Representative images of case 13 showing keratinizing-type squamous cell carcinoma at 100× magnification (A) and 200× magnification (B) with relatively monotonous cytological features and a dense lymphocytic infiltrate (panel B represents a magnified portion of panel A). This case showed diffuse positive staining for both CD5 (C) and c-KIT (D), raising the possibility of thymic origin (images at 200× magnification).

Fig. 4. –

Proposed algorithm for the potential use of viral testing and MSA in the evaluation of SCCUP. MSA would likely provide the most potential benefit for viral negative level II/III lymph nodes. MSA would additionally potentially benefit cases in which there is clinical suspicion of a cutaneous primary.