Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method

Abstract

Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification-a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of an unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94% vs. 9%, P<0.0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16/HPV DNA), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.

Figure 1

In this non-keratinizing squamous cell carcinoma of the oropharynx (A, hemotoxylin and eosin stain), HPV DNA in situ hybridization establishes the presence of virus (B), HPV mRNA in situ hybridization confirms transcriptional activation of mRNA E6/E7 (C), and overexpression of the cyclin D kinase inhibitor p16 disruption indicates disruption of the retinoblastoma tumor suppressor pathway (D, inset at higher magnification highlights the granular nature of the hybridization signals).

Figure 2

In this HPV-related oropharyngeal squamous cell carcinoma (A, hemotoxylin and eosin stain), HPV was not detected using the Ventana Inform HPV III Family16 probe for HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 66 (B), but the presence of HPV at low viral copy numbers was evident using the type 16 specific probe (DAKO GenPoint, Carpinteria, CA) (C, arrows point to small hybridization signals within nuclei of tumor cells). The mRNA transcripts are seen as numerous granular signals (D).

Figure 3

The frequency of P16 positive and E6/E7 mRNA negative squamous cell carcinomas by anatomic subsites of the head and neck. The relative uniform frequency of these discrepant cases across all anatomic subsites including those that do not appear to be targeted by HPV suggests that non-viral mechanisms may play a role in p16 overexpression in some head and neck squamous cell carcinomas.

Figure 4

The HPV status of the oropharyngeal carcinoma (case 13 of Table 2) was equivocal due to the presence of strong p16 staining (A) but the absence of hybridization signals by HPV DNA in situ hybridization (not shown). Despite the inability to detect HPV DNA, abundant E6/E7 mRNA transcripts are detected using the E6/E7 mRNA method (B).

Figure 5

The biology of HPV infection and malignant transformation provides several sequential points for HPV recognition. Concurrent evidence for HPV DNA, transcriptional activation, and disruption of key growth regulatory pathways (e.g. retinoblastoma pathway) confirms the presence of clinically and biologically relevant HPV.