Probes for CDKN2A

Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.

Tertiary lymphoid structure (TLS) reflects an intense immune response against cancer, which correlates with favorable patient survival. However, the association of TLS with tumor-infiltrating lymphocytes (TILs) and clinical outcomes has not been investigated comprehensively in pancreatic ductal adenocarcinoma (PDAC).We utilized an integrative molecular pathological epidemiology database on 162 cases with resected PDAC, and examined TLS in relation to levels of TILs, patient survival, and treatment response. In whole-section slides, we assessed the formation of TLS and conducted immunohistochemistry for tumor-infiltrating T cells (CD4, CD8, CD45RO, and FOXP3). As confounding factors, we assessed alterations of four main driver genes (KRAS, TP53, CDKN2A [p16], and SMAD4) using next-generation sequencing and immunohistochemistry, and tumor CD274 (PD-L1) expression assessed by immunohistochemistry.TLSs were found in 112 patients with PDAC (69.1%). TLS was associated with high levels of CD4+ TILs (multivariable odds ratio [OR], 3.50; 95% confidence interval [CI] 1.65-7.80; P = 0.0002), CD8+ TILs (multivariable OR, 11.0; 95% CI 4.57-29.7, P < 0.0001) and CD45RO+ TILs (multivariable OR, 2.65; 95% CI 1.25-5.80, P = 0.01), but not with levels of FOXP3+ TILs. TLS was associated with longer pancreatic cancer-specific survival (multivariable hazard ratio, 0.37; 95% CI 0.25-0.56, P < 0.0001) and favorable outcomes of adjuvant S-1-treatment. TLS was not associated with driver gene alterations but tumor CD274 negative expression.Our comprehensive data supports the surrogacy of TLS for vigorous anti-tumor immune response characterized by high levels of helper and cytotoxic T cells and their prognostic role.

Gastric-type cervical adenocarcinoma (GCA) is a rare and aggressive type of endocervical adenocarcinoma (ECA) with distinct histopathologic features and unfavorable treatment outcomes, but no genomic prognostic factor has been revealed. We aimed to systematically investigate the somatic alterations of GCA at genome-wide level and evaluate their prognostic value.We performed whole-exome sequencing (WES) on 25 pairs of tumor and matched normal samples to characterize the genomic features of Chinese patients with GCA and investigated their relations to histopathological characterizations and prognosis. The prognostic value of the genomic alterations was evaluated in a total of 58 GCA patients.Mutations were commonly observed in reported GCA-related driver genes, including TP53 (32%), CDKN2A (20%), SKT11 (20%), BRCA2 (12%), SMAD4 (12%), and ERBB2 (12%). Recurrent novel trunk mutations were also observed in PBRM1 (12%), FRMPD4 (12%), and NOP2 (8%) with high variant allele frequency. Moreover, enrichment of the APOBEC signature was attributed to frequent gain of somatic copy number alteration (SCNA) of APOBEC3B (20%), which perfectly matched the nuclear-positive staining of APOBEC3B through immunohistochemistry. In contrast, APOBEC3B alteration was absent in patients with conventional type of ECA (N = 52). Notably, positive APOBEC3B was consistently enriched in patients with favorable prognosis in both the discovery cohort and an additional 33 GCA patients, thus indicating a significant association with lower relapse risk of GCA independent of cancer stage (P = 0.02).Our results can aid understanding of the molecular basis of GCA in the Chinese population by providing genomic profiles and highlighting the potential prognostic value of APOBEC3B for GCA through routine clinical IHC.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is an age-related progressive and fatal lung disease with limited therapeutic options. IPF exhibits several pathological features of epithelial reprogramming, including cellular senescence. Moreover, distal airway remodeling and bronchiolization can result in honeycomb cysts. The molecular and cellular mechanisms that lead to this prominent phenotype, however, still remain poorly characterized. Here, we aimed to decipher the IPF distal bronchiole and alveolar cell subtypes and their potential contribution to IPF development and progression. METHODS: EpCAM+ cells were isolated and enriched from human lung tissue (IPF/age-matched donors, each n=3) followed by single-cell RNA sequencing on 10x Genomics platform. Single cell RNA-sequencing (scRNA-Seq) data was pre-processed on Scanpy pipeline, including filtered, ambient gene correction, batch correction, clustering and annotation. RNA scope and fluorescent immunolabeling were used to confirm the gene expression and further localization of proteins to specific cell types. Air-liquid interface cultures (ALI) were used to generate a functional in vitro 3D airway model and to conduct functional studies. RESULTS: We generated a dataset of 46199 cells and found distinct cell clusters, including rare cell types, such as suprabasal cells, recently reported in the healthy lung. We identified features of distal alveolar epithelial cells enriched in healthy donor while airway epithelial cells were enriched in IPF patients. We further analyzed our data set for marker that are enriched in aberrant basal cells in IPF and identified Gprotein coupled receptor (GPR) 87 as a novel surface marker of distal Keratin (KRT)5 + basal cells. Correlation analysis showed GPR87 expression was highly correlated with the expression of CDKN2A (ρ= 0.84), a senescence marker. KEGG pathway analysis further revealed genes positive correlated with GPR87 expression were enriched in P53 signaling pathway further corroborating a link with cellular senescence. GPR87 expression was localized to distal bronchioles and honeycomb cysts in IPF in situ by RNA Scope and immunolabeling. ALI cultures stimulated with TGF- β, a fibrotic inducer, led to increased GPR87 expression. Furthermore, modulation of GPR87 in primary human bronchial epithelial cell cultures resulted in impaired airway cell maturation and ciliogenesis. CONCLUSION: GPR87 is a novel surface marker of KRT5 + basal progenitor cells likely contributing to senescence, bronchiole remodeling and honeycomb cyst development in IPF. Further studies are ongoing to elucidate whether GPR87 is a potential druggable target of fibrosis and senescence.