MYB RNA detection by in situ hybridisation has high sensitivity and specificity for the diagnosis of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common primary salivary gland cancers. ACC has several benign and malignant mimics amongst salivary gland neoplasms. An accurate diagnosis of ACC is essential for optimal management of the patients and their follow-up. Upregulation of MYB has been described in 85-90% of ACC, but not in other salivary gland neoplasms. In ACC, MYB upregulation can occur as a result of a genetic rearrangement t(6;9) (q22-23;p23-24), MYB copy number variation (CNV), or enhancer hijacking of MYB. All mechanisms of MYB upregulation result in increased RNA transcription that can be detected using RNA in situ hybridisation (ISH) methods. In this study, utilising 138 primary salivary gland neoplasms including 78 ACC, we evaluate the diagnostic utility of MYB RNA ISH for distinguishing ACC from other primary salivary gland neoplasms with a prominent cribriform architecture including pleomorphic adenoma, basal cell adenoma, basal cell adenocarcinoma, epithelial myoepithelial carcinoma, and polymorphous adenocarcinoma. Fluorescent in situ hybridisation and next generation sequencing were also performed to evaluate the sensitivity and specificity of RNA ISH for detecting increased MYB RNA when MYB gene alterations were present. Detection of MYB RNA has 92.3% sensitivity and 98.2% specificity for a diagnosis of ACC amongst salivary gland neoplasms. The sensitivity of MYB RNA detection by ISH (92.3%) is significantly higher than that of the FISH MYB break-apart probe (42%) for ACC. Next generation sequencing did not demonstrate MYB alterations in cases that lacked MYB RNA overexpression indicating high sensitivity of MYB RNA ISH for detecting MYB gene alterations. The possibility that the sensitivity may be higher in clinical practice with contemporary samples as compared with older retrospective tissue samples with RNA degradation is not entirely excluded. In addition to the high sensitivity and specificity, MYB RNA testing can be performed using standard IHC platforms and protocols and evaluated using brightfield microscopy making it a time and cost-efficient diagnostic tool in routine clinical practice.

Viral Integration Plays a Minor Role in the Development and Prognostication of Oral Squamous Cell Carcinoma.

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.