The utility of high-risk human papillomavirus E6/E7 mRNA in situ hybridization in assessing HPV status on cell block.

Assessment of human papillomavirus (HPV) status is critical to the treatment and prognosis of patients with oropharyngeal squamous cell carcinoma. Patients often present with enlarged cervical lymph nodes which are amenable to fine needle aspiration (FNA) and cell block creation. The most widely used method for assessing HPV status is the surrogate marker p16. Other HPV specific methods such as high-risk HPV E6/E7 mRNA in situ hybridization (ISH) have been shown to perform as well as p16 and are easier to interpret. Our study evaluates the utility of high-risk HPV mRNA ISH in cell block specimens. Thirty-six cases of metastatic squamous cell carcinoma in cervical neck lymph node FNAs were identified over a 3-year period. All cases had p16 immunohistochemistry (IHC) performed on cell block. HR HPV mRNA ISH was performed on the cell block and compared to the p16 results. Additionally, p16 and HR HPV mRNA ISH status was assessed in those cases with corresponding surgical resections. HR HPV mRNA ISH confirmed the p16 IHC (either positive or negative) in 24 of the 36 cases (66.7%). Six false negative cases were p16 negative/HR HPV mRNA ISH positive. HR HPV mRNA ISH was positive in 75% of the four p16 equivocal cases. Two cases were p16 positive/HR HPV mRNA ISH negative. HR HPV mRNA ISH is no more difficult to perform in the IHC lab and is easier to interpret than p16 IHC. HR HPV mRNA ISH is a useful alternative to p16 in cell block specimens.

Wilson BL ，Israel AK ，Lott Limbach AA 《-》

Heterogeneity of p16 immunohistochemistry and increased sensitivity of RNA in situ hybridization in cytology specimens of HPV-related head and neck squamous cell carcinoma.

Many patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HPV-HNSCC) initially present with cervical lymph node metastases. Although p16 immunohistochemistry (IHC) is the most commonly used surrogate marker for HPV, however criteria in cytologic material are not well established. The objective of this study was to better characterize p16 IHC in cell blocks of metastatic HPV-HNSCC, and to evaluate the performance of HPV RNA in situ hybridization (RNA ISH). p16 IHC was performed on cell blocks from 97 metastatic HPV-HNSCC fine-needle aspiration specimens with HPV status confirmed by DNA or RNA ISH or polymerase chain reaction (PCR). Tumor cellularity (<100 cells, 100-500 cells, and >500 cells) and quality (presence of cell clusters, necrosis) were recorded. p16 staining intensity and extent (1%-9%, 10%-69%, and ≥70%) were scored. In addition, RNA ISH was performed on 38 PCR-positive cases. p16 IHC was positive in 90 of 97 cases (93%), demonstrating variable patterns. p16 staining was found to be moderate to strong in 69 cases, with 37 cases (38%) demonstrating positivity in ≥70% of tumor cells. Weak staining occurred in 21 cases (22%) and 7 cases (7%) were negative. Of the 60 cases with weak and/or absent expression or staining in <70% of cells, 30 cases (50%) had <100 tumor cells, 12 (20%) lacked cell clusters, and 19 cases (32%) had extensive necrosis. RNA ISH was positive in 37 of 38 cases (97%) that were HPV positive by PCR. p16 is heterogeneous in cell blocks of metastatic HPV-HNSCC, suggesting that any p16 positivity should prompt confirmatory HPV studies. RNA ISH appears to demonstrate high sensitivity, and laboratories even may consider using RNA ISH as a first-line HPV test in cytologic specimens.

Wong KS ，Krane JF ，Jo VY 《-》

Utility of high-risk HPV RNA chromogenic in situ hybridization in cytology smears and liquid-based preparations from metastatic head and neck squamous cell carcinoma.

High-risk human papillomavirus (HR-HPV) status is critical for the diagnosis, prognosis, and treatment of patients with oropharyngeal squamous cell carcinoma (OPSCC). Patients often present with enlarged cervical nodes, and fine-needle aspiration cytology (FNAC) is frequently the initial diagnostic procedure. Although p16 is the most widely used surrogate marker, problems with interpretation can limit its utility in FNAC. HR-HPV RNA in situ hybridization (ISH) has emerged as a specific way to assess HPV status on cell block preparations of cervical nodes. The authors evaluated the utility of HR-HPV ISH in conventional smears and liquid-based cytology (LBC) preparations of metastatic head and neck squamous cell carcinoma (SCC). Thirty-one aspirates of proven, HPV-related SCC (confirmed by p16 and/or HR-HPV ISH in corresponding surgical specimens) were selected. Ten aspirates of HPV-negative SCC were also retrieved. HR-HPV ISH was performed on 27 smears and 14 LBC preparations. All results were scored as positive, equivocal, or negative. Eighty-four percent of metastatic, HPV-related SCCs were positive for HR-HPV RNA ISH, with high number of signals (n = 19) and low number of signals (n = 7), whereas five HPV-related SCCs were equivocal. All metastatic, HPV-negative SCCs were negative for HR-HPV ISH. HR-HPV ISH can be reliably performed on smears or LBC preparations, particularly when cell blocks are unavailable or paucicellular. Results were easy to interpret when high numbers of signals were present but were challenging in aspirates with low or rare number of signals. The current study suggests that HR-HPV ISH could be used as the initial testing modality for determining HPV status in FNAC specimens of metastatic SCC.

Velez Torres JM ，Alkathery T ，Tjendra Y ，Zuo Y ，Kerr DA ，Gomez-Fernandez C ... -  《-》

Strong SOX10 expression in human papillomavirus-related multiphenotypic sinonasal carcinoma: report of 6 new cases validated by high-risk human papillomavirus mRNA in situ hybridization test.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk HPV (HR-HPV) infection. Using HR-HPV messenger RNA (mRNA) in situ hybridization (ISH), we reported 6 new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma. Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified 6 HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium, and 1 had invasive squamous cell carcinoma (SCC). All HMSC cases were SOX10 positive, whereas the overlying atypical squamous epithelium and the SCC were SOX10 negative. One atypical HMSC-like case was also identified, which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, and SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals compared with HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium and the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

Hsieh MS ，Lee YH ，Jin YT ，Huang WC ... -  《-》

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.

Bishop JA ，Ma XJ ，Wang H ，Luo Y ，Illei PB ，Begum S ，Taube JM ，Koch WM ，Westra WH ... -  《-》