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.

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.

HPV-16 infection predicts treatment outcome in oropharyngeal squamous cell carcinoma.

OBJECTIVE: To determine if patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) treated with chemoradiation have improved outcomes. STUDY DESIGN: A retrospective search was used to identify patients with OPSCC treated with concurrent chemoradiation. Pretreatment biopsy specimens were tested for HPV-16 infection and p16 expression. METHODS: Forty-four patients with OPSCC treated with concurrent chemotherapy and intensity-modulated radiation therapy were identified. Eligibility criteria included a minimum two years of follow-up, or biopsy-proven recurrence. In situ hybridization was applied to archival tumor specimens, with HPV-16-positive status defined as positive staining of tumor cell nuclei. p16 expression was assessed by immunohistochemistry. RESULTS: Twenty-seven tumors (61%) were positive for HPV-16 and 29 tumors (66%) expressed p16. HPV-16 infection was highly correlated with p16 expression (P < 10(-7)). Three-year disease-free and overall survival for all patients was 66 percent and 79 percent respectively. Patients with tumors infected with HPV-16 had improved overall (OS) and disease-free survival (DFS) after chemoradiation (OS: hazard ratio [HR] = 0.21, P = 0.01; DFS: HR = 0.30, P = 0.02). CONCLUSION: Patients with OPSCC tumors that are infected with HPV-16 have improved survival after treatment with concurrent chemoradiation.

Benign nodal nevi frequently harbor the activating V600E BRAF mutation.

Mutational activation of the BRAF oncogene is the most common genetic alteration in cutaneous melanoma. Potentially, BRAF mutation analysis of sentinel lymph node (SLN) biopsies could enhance the detection of micrometastases and improve the accuracy of nodal staging for patients with melanoma. Nodal nevi are small aggregates of benign nevus cells that are commonly encountered in the SLNs of patients with melanoma. The status of the BRAF gene in nodal nevi is not known, but this unresolved issue is of critical importance to any future detection strategies that use genetic alterations as biomarkers of metastatic spread. Twenty-six nodal nevi from 26 patients were evaluated for the thymine (T)-->adenine (A) missense mutation at nucleotide 1796 of the BRAF gene using the LigAmp assay, which can detect 1 mutant allele among 10,000 wild-type alleles. For each case, a matching volume of adjacent lymphoid tissue was used as a negative control. BRAF mutations were detected in 13 of the 26 nodal nevi, but in just 1 of the 26 adjacent controls (50% vs. 4%, P<0.0005, Fisher exact). Novel strategies that rely on detection of putative melanoma-specific markers for the diagnosis of micrometastatic melanoma in SLNs need to take into account the molecular genetic profile of the benign nodal nevus. Indeed, these nodal nevi, like melanoma, frequently harbor activating mutations of the BRAF oncogene underscoring the potentially confounding impact of these inclusions on melanoma detection.

Basaloid squamous cell carcinoma of the head and neck is a mixed variant that can be further resolved by HPV status.

Basaloid squamous cell carcinoma (BSCC) of the head and neck is set apart as a distinct subtype of squamous cell carcinoma on the basis of its basaloid appearance and aggressive behavior. The purpose of this study was to determine whether BSCC could be further subdivided on the basis of human papillomavirus 16 (HPV16) status. HPV16 in situ hybridization was performed on 53 BSCCs of the head and neck. Of the 53 BSCCs, 21 (40%) arose in the oropharynx and 32 (60%) arose in nonoropharyngeal sites. HPV16 was detected in 34% of BSCCs overall, but the frequency varied by site. HPV16 was detected in 16 of 21 (76%) BSCCs of the oropharynx, but in only 2 of 32 (6%) BSCCs from nonoropharyngeal sites (P<0.0001, Fisher exact). The absence of HPV16 was significantly associated with decreased overall survival (Hazard ratio=17.1; 95% confidence interval=7.2-40.3, log-rank P=0.0001), even though patients with HPV16-positive carcinomas were more likely to present with lymph nodes metastases (P=0.01, Fisher exact). Morphologic similarities aside, BSCCs are composed of a mixed group of tumors that can be separated on the basis of HPV16 status. The distinction is important. HPV16-positivity in squamous cell carcinomas of the head and neck is now recognized as a powerful indicator of improved patient survival. HPV16 detection thus permits resolution of a less aggressive component within a high-grade subtype of head and neck carcinoma.

Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck.

PURPOSE: Squamous cell carcinomas of the head and neck (HNSCC) often harbor p53 mutations, but p53 protein degradation by the viral oncoprotein E6 may supercede p53 mutations in human papillomavirus 16 (HPV16)-positive tumors. The prevalence of p53 mutations in HPV-positive HNSCCs is indeed lower, but in some tumors these alterations coexist. The purpose of this study was to discern whether HNSCCs differ in the type of p53 mutations as a function of HPV16 status. EXPERIMENTAL DESIGN: The study was nested within a prospective multicenter study (ECOGE 4393/RTOG R9614) of patients with HNSCC treated surgically with curative intent. Tumors from one study center were used to construct a tissue microarray. The tumors were well characterized with respect to p53 mutational status. The tissue microarray was evaluated by HPV16 in situ hybridization. HPV16 analysis was also done on a select group of tonsillar carcinomas known to harbor disruptive p53 mutations defined as stop mutations or nonconservative mutations within the DNA binding domain. RESULTS: HPV16 was detected in 12 of 89 (13%) HNSCCs. By tumor site, HPV16 was detected in 12 of 21 (57%) tumors from the palatine/lingual tonsils, but in none of 68 tumors from nontonsillar sites (P < 0.00001). Both HPV16-positive and HPV16-negative HNSCCs harbored p53 mutations (25% versus 52%), but disruptive mutations were only encountered in HPV16-negative carcinomas. Of seven tonsillar carcinomas with disruptive p53 mutations, none were HPV16 positive, in contrast to HPV16-positive tonsillar carcinomas without disruptive p53 mutations (0% versus 57%; P = 0.008). CONCLUSIONS: Although HPV16 and mutated p53 may coexist in a subset of HNSCCs, HPV16 and disruptive p53 mutations seem to be nonoverlapping events. A less calamitous genetic profile, including the absence of disruptive p53 mutations, may underlie the emerging clinical profile of HPV16-positive HNSCC such as improved patient outcome.

Distribution of BRAF T1799A(V600E) mutations across various types of benign nevi: implications for melanocytic tumorigenesis.

PURPOSE: The BRAF mutation is common in melanomas, but variation in rates across melanoma subtypes points to a complex interplay between BRAF activation and other factors (eg, sun exposure). Nevi also harbor the BRAF mutation. A description of mutation distribution in nevi could provide insight into the significance of this event in melanocytic tumorigenesis. EXPERIMENTAL DESIGN: One hundred thirty-five nevi from 116 patients were evaluated for the T-->A mutation at nucleotide 1799. The nevi were inclusive of congenital (n = 34) and acquired (n = 101) nevi, dysplastic (n = 11) and nondysplastic (n = 124) nevi, and anogenital (n = 24) and common cutaneous (n = 111) nevi. RESULTS: The overall mutation rate was 81%. The rate varied only slightly by anatomic site: BRAF mutations were detected in 21 of 21 (100%) nevi of the head and neck, 62 of 76 (82%) nevi of the trunk, 8 of 14 (62%) nevi of the extremities, and 18 of 24 (75%) anogenital nevi. For acquired nevi, there was no association between BRAF mutations and sun exposure as inferred from anatomic site. There were no significant differences in the mutation rates between congenital and acquired nevi (76% versus 81%; P = 0.5). CONCLUSIONS: The BRAF mutation is uniformly distributed in various types of nevi. Its presence in congenital and anogenital nevi points to mechanisms of induction other than sun exposure. Its ubiquitous presence suggests that it poses no significant threat of malignant transformation, raising doubts about its relevance in melanoma development and its suitability as a target of directed therapy in patients with melanoma.