Probes for HPV

Rising prevalence rates of high-risk human papillomaviruses (hrHPV) infection in oropharyngeal carcinoma (up to 80 %) have been reported in North America and Scandinavia. We have analysed 424 German and 163 Brazilian head and neck squamous cell carcinomas (HNSCC) from the oral cavity (OSCC), oropharynx (OPSCC) and hypopharynx (HPSCC) using p16 immunohistochemistry, HPV DNA PCR and sequencing, hrHPV DNA in situ hybridisation (ISH) and hrHPV E6/E7 RNA ISH. In the German series, 52/424 cases (12.3 %) were p16-positive/hrHPV-positive (OSCC 3.8 % [10/265], OPSCC 34.4 % [42/122], HPSCC 0 % [0/37]). In addition, there were 9 cases that were p16-positive/hrHPV-negative (5 OPSCC and 4 OSCC). In the Brazilian series, the overall hrHPV DNA prevalence by PCR was 11.0 % ([18/163]; OSCC 6 % [5/83], OPSCC 15.5 % [11/71], HPSCC 22.2 % [2/9]). Ten of these cases were hrHPV-positive/p16-positive. The remaining 8 hrHPV-positive/p16-negative cases were also negative in both ISH assays. Furthermore, 5 p16-positive/hrHPV-negative cases (2 OPSCC and 3 OSCC) were identified. In both series, HPV16 was by far the most common HPV type detected. We confirm that regardless of geographical origin, the highest hrHPV prevalence in HNSCC is observed in oropharyngeal carcinomas. The proportion of HPV-associated OPSCC was substantially higher in the German cohort than in the Brazilian series (34.4 vs. 15.5 %), and in both groups, the prevalence of hrHPV in OPSCC was much lower than in recent reports from North America and Scandinavia. We suggest, therefore, that it may be possible to define areas with high (e.g. USA, Canada, Scandinavia), intermediate (e.g. Germany) and low (e.g. Brazil) prevalences of HPV infection in OPSCC.

There is emerging evidence that vulvar squamous cell carcinoma (VSCC) can be prognostically subclassified into 3 groups based on human papillomavirus (HPV) and p53 status: HPV-associated (HPV+), HPV-independent/p53 wild-type (HPV-/p53wt), or HPV-independent/p53 abnormal (HPV-/p53abn). Our goal was to assess the feasibility of separating VSCC and its precursors into these 3 groups using p16 and p53 immunohistochemistry (IHC). A tissue microarray (TMA) containing 225 VSCC, 43 usual vulvar intraepithelial neoplasia (uVIN/HSIL), 10 verruciform acanthotic vulvar intraepithelial neoplasia (vaVIN), and 34 differentiated VIN (dVIN), was stained for p16 and p53. Non-complementary p16 and p53 patterns were resolved by repeating p53 IHC and HPV RNA in-situ hybridization (ISH) on whole sections, and sequencing for TP53. Of 82 p16-positive VSCC, 73 (89%) had complementary p16 and p53 patterns and were classified into the HPV+ group, 4 (4.9%) had wild-type p53 staining, positive HPV ISH, and were classified into the HPV+ group, while 5 (6.1%) had p53 abnormal IHC patterns (1 null, 4 overexpression), negativity for HPV ISH and harboured TP53 mutations (1 splice-site, 4 missense); they were classified as HPV-/p53abn. Of 143 p16-negative VSCC, 142 (99.3%) had complementary p53 and p16 patterns; 115 (80.4%) HPV-/p53abn and 27 (18.9%) HPV-/p53wt. One had a basal-sparing p53 pattern, positivity for HPV ISH, and was negative for TP53 mutations; it was classified into the HPV+ category. The use of IHC also led to the following revised diagnoses: HSIL to dVIN (3/43), dVIN to vaVIN (8/34), and dVIN to HSIL (3/34). Overall, 215/225 VSCC (95.6%) could be easily classifiable into 3 groups with p16 and p53 IHC. We identified several caveats, with the major caveat being that 'double positive' p16/p53 should be classified as HPV-/p53abn, and propose an algorithm which will facilitate the application of p16 and p53 IHC to classify VSCC in pathology practice.

As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.