Probes for INS

Abstract

OBJECTIVE:

The Silva invasion pattern-based classification system stratifies endocervical adenocarcinomas (ECAs) into 3 categories corresponding to risk of metastasis and recurrence, but has only been evaluated for HPV-associated ECAs of usual type. We examined whether the Silva system is applicable to all endocervical adenocarcinomas, especially those not associated with HPV.

METHODS:

Complete slide sets from 341 surgical specimens of ECA were collected from 7 institutions worldwide. All specimens were associated with clinical records covering at least 5 years of follow-up. Tumors were classified as HPV-associated (HPVA) or not (NHPVA) by both morphology and detection of HPV using in situ hybridization. Recurrence and survival were analyzed by multivariate Mantel-Haenszel methods.

RESULTS:

Most specimens (292; 85.6%) were HPVA, while 49 (14.3%) were NHPVA. All NHPVAs were Silva pattern C, while 76.0% of HPVAs were pattern C, 14.7% pattern A, and 9.3% pattern B. Including both HPVAs and NHPVAs, lymphovascular invasion (LVI) was detected in 0% of pattern A, 18.5% of pattern B and 62.6% of pattern C cases (p < 0.001). None of the pattern A or B cases were associated with lymph node metastases (LNM), in contrast to pattern C cases (21.8%). Among patients with Silva pattern C ECA, those with HPVA tumors had a lower recurrence rate and better survival than those with NHPVA; however, when adjusted for stage at diagnosis, the difference in recurrence and mortality was small and not statistically significant.

CONCLUSIONS:

Application of the Silva system is only relevant in HPVA cervical adenocarcinoma.

Abstract

Importance  
High-risk human papillomavirus (HPV) has been associated with Barrett dysplasia and esophageal adenocarcinoma. Nevertheless, the prognostic significance of esophageal tumor HPV status is unknown.

Objective  
To determine the association between HPV infection and related biomarkers in high-grade dysplasia or esophageal adenocarcinoma and survival.

Design, Setting, and Participants  
Retrospective case-control study. The hypothesis was that HPV-associated esophageal tumors would show a favorable prognosis (as in viral-positive head and neck cancers). Pretreatment biopsies were used for HPV DNA determination via polymerase chain reaction, in situ hybridization for E6 and E7 messenger RNA (mRNA), and immunohistochemistry for the proteins p16INK4A and p53. Sequencing of TP53 was also undertaken. The study took place at secondary and tertiary referral centers, with 151 patients assessed for eligibility and 9 excluded. The study period was from December 1, 2002, to November 28, 2017.

Main Outcomes and Measures  
Disease-free survival (DFS) and overall survival (OS).

Results  
Among 142 patients with high-grade dysplasia or esophageal adenocarcinoma (126 [88.7%] male; mean [SD] age, 66.0 [12.1] years; 142 [100%] white), 37 were HPV positive and 105 were HPV negative. Patients who were HPV positive mostly had high p16INK4A expression, low p53 expression, and wild-type TP53. There were more Tis, T1, and T2 tumors in HPV-positive patients compared with HPV-negative patients (75.7% vs 54.3%; difference, 21.4%; 95% CI, 4.6%-38.2%; P = .02). Mean DFS was superior in the HPV-positive group (40.3 vs 24.1 months; difference, 16.2 months; 95% CI, 5.7-26.8; P = .003) as was OS (43.7 vs 29.8 months; difference, 13.9 months; 95% CI, 3.6-24.3; P = .009). Recurrence or progression was reduced in the HPV-positive cohort (24.3% vs 58.1%; difference, −33.8%; 95% CI, −50.5% to −17.0%; P < .001) as was distant metastasis (8.1% vs 27.6%; difference, −19.5%; 95% CI, −31.8% to −7.2%; P = .02) and death from esophageal adenocarcinoma (13.5% vs 36.2%; difference, −22.7%; 95% CI, −37.0% to −8.3%; P = .01). Positive results for HPV and transcriptionally active virus were both associated with a superior DFS (hazard ratio [HR], 0.33; 95% CI, 0.16-0.67; P = .002 and HR, 0.44; 95% CI, 0.22-0.88; P = .02, respectively [log-rank test]). Positivity for E6 and E7 mRNA, high p16INK4Aexpression, and low p53 expression were not associated with improved DFS. On multivariate analysis, superior DFS was demonstrated for HPV (HR, 0.39; 95% CI, 0.18-0.85; P = .02), biologically active virus (HR, 0.36; 95% CI, 0.15-0.86; P = .02), E6 and E7 mRNA (HR, 0.36; 95% CI, 0.14-0.96; P = .04), and high p16 expression (HR, 0.49; 95% CI, 0.27-0.89; P = .02).

Conclusions and Relevance  
Barrett high-grade dysplasia and esophageal adenocarcinoma in patients who are positive for HPV are distinct biological entities with a favorable prognosis compared with viral-negative esophageal tumors. Confirmation of these findings in larger cohorts with more advanced disease could present an opportunity for treatment de-escalation in the hope of reducing toxic effects without deleteriously affecting survival.

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

Objective: To observe the clinicopathologic features of oropharyngeal squamous cell carcinoma associated with human papilloma virus (OPSCC-HPV) and discuss the role and value of different in situ hybridization (ISH) detection methods for HPV in pathologic diagnosis. Methods: Fifteen cases of OPSCC-HPV were collected from Department of Pathology, Beijing Tongren Hospital, Capital Medical University from January 2016 to August 2018. These cases were diagnosed in accordance with the WHO classification of head and neck tumors. The histopathologic features and the clinicopathologic data were retrospectively analyzed. Immunohistochemistry (two-step EnVision method) was done to evaluate the expression of p16, Ki-67 and p53. ISH was used to detect HPV DNA (6/11 and 16/18). RNAscope technology was used to evaluate the presence of HPV mRNAs (16 and 18). Results: The mean age for the 15 patients (8 males, 7 females) was 47 years (range from 30 to 69 years). OPSCC-HPV typically presentedat an advanced clinical stage, six patients had cervical lymphadenopathy (large and cystic), seven had tonsillar swelling, one had tumor at base of tongue, and one had odynophagia. Microscopically the tumors exhibited distinctive non-keratinizing squamous cell carcinoma morphology. Cervical nodal metastases were large and cystic, with thickening of lymph node capsules. OPSCC-HPV raised from crypt epithelium and extended beneath the tonsillar surface epithelial lining as nests and lobules, often with central necrosis. Tumor cells displayed a high N: C ratio, and high mitotic and apoptotic rates. Tumor nests are often embedded within lymphoid stroma, and may be infiltrated by lymphoid cells.Fifteen cases (15/15) were strongly positive for p16; Ki-67 index were 60%-90%; they were focally positive or negative for p53. Ten cases (10/10) were negative for HPV 6/11 DNA, and one case(1/10) was focally positive for HPV16/18 DNA. Eleven cases (11/11) were strongly positive for HPV16 mRNA, one case was focally positive for HPV18 mRNA. Conclusions: OPSCC-HPV is a pathologically and clinically distinct form of head and neck squamous cell carcinoma. OPSCC-HPV is associated with high-risk HPV (type 16) in all cases. Detection of high-risk HPV16 mRNA by RNAscope is of great significance in the final diagnosis and pathogen identification.

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk human papillomavirus (HR-HPV) infection. Using HR-HPV mRNA in situ hybridization (ISH), we reported six new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma (ACC). Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified six HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium and one also had invasive squamous cell carcinoma (SCC). All HMSC 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, 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 than HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium as well as 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.

We sought to classify endocervical adenocarcinomas (ECAs) based on morphologic features linked to etiology (ie, human papillomavirus [HPV] infection), unlike the World Health Organization 2014 classification. The International Endocervical Adenocarcinoma Criteria and Classification (IECC criteria), described herein, distinguishes between human papillomavirus-associated adenocarcinoma (HPVA), recognized by the presence of luminal mitoses and apoptosis seen at scanning magnification, and no or limited HPVA features (nonhuman papillomavirus-associated adenocarcinoma [NHPVA]). HPVAs were then subcategorized based on cytoplasmic features (mostly to provide continuity with preexisting classification schemes), whereas NHPVAs were subclassified based on established criteria (ie, gastric-type, clear cell, etc.). Complete slide sets from 409 cases were collected from 7 institutions worldwide. Tissue microarrays representing 297 cases were constructed; immunohistochemistry (p16, p53, vimentin, progesterone receptor) and chromogenic in situ hybridization using an RNA-based probe set that recognizes 18 varieties of high-risk HPV were performed to validate IECC diagnoses. The 5 most common IECC diagnoses were usual-type (HPVA) (73% of cohort), gastric-type (NHPVA) (10%), mucinous adenocarcinoma of HPVA type, including intestinal, mucinous not otherwise specified, signet-ring, and invasive stratified mucin-producing carcinoma categories (9%), clear cell carcinoma (NHPVA) (3%) and adenocarcinoma, not otherwise specified (2%). Only 3 endometrioid carcinomas were recognized and all were NHPVA. When excluding cases thought to have suboptimal tissue processing, 90% and 95% of usual-type IECC cases overexpressed p16 and were HPV, whereas 37% and 3% of NHPVAs were p16 and HPV, respectively. The 1 HPV gastric-type carcinoma was found to have hybrid HPVA/NHPVA features on secondary review. NHPVA tumors were larger and occurred in significantly older patients, compared with HPVA tumors (P<0.001). The high-risk HPV chromogenic in situ hybridization probe set had superior sensitivity, specificity, and positive and negative predictive values (0.955, 0.968, 0.992, 0.833, respectively) compared with p16 immunohistochemistry (0.872, 0.632, 0.907, 0.545, respectively) to identify HPV-related usual carcinoma and mucinous carcinoma. IECC reliably segregates ECAs into HPVA and NHPVA types using morphology alone. This study confirms that usual-type ECAs are the most common type worldwide and that mucinous carcinomas comprise a mixture of HPVA and NHPVA, with gastric-type carcinoma being the major NHPVA type. Endometrioid and serous carcinomas of the endocervix are extraordinarily rare. Should clinical outcomes and genomic studies continue to support these findings, we recommend replacement of the World Health Organization 2014 criteria with the IECC 2017.