Cory T. Bernadt

Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes

Recent studies have identified large sets of genes in embryonic stem and embryonal carcinoma cells that are associated with the transcription factors Sox2 and Oct-3/4. Other studies have shown that Sox2 and Oct-3/4 work together cooperatively to stimulate the transcription of their own genes as well as a network of genes required for embryogenesis. Moreover, small changes in the levels of Sox2:Oct-3/4 target genes alter the fate of stem cells. Although positive feedforward and feedback loops have been proposed to explain the activation of these genes, little is known about the mechanisms that prevent their overexpression. Here, we demonstrate that elevating Sox2 levels inhibits the endogenous expression of five Sox2:Oct-3/4 target genes. In addition, we show that Sox2 repression is dependent on the binding sites for Sox2 and Oct-3/4. We also demonstrate that inhibition is dependent on the C-terminus of Sox2, which contains its transactivation domain. Finally, our studies argue that overexpression of neither Oct-3/4 nor Nanog broadly inhibits Sox2:Oct-3/4 target genes. Collectively, these studies provide new insights into the diversity of mechanisms that control Sox2:Oct-3/4 target genes and argue that Sox2 functions as a molecular rheostat for the control of a key transcriptional regulatory network.

Improved laboratory resource utilization and patient care with the use of rapid on‐site evaluation for endobronchial ultrasound fine‐needle aspiration biopsy

Endobronchial ultrasound guided (EBUS) fine‐needle aspiration (FNA) biopsy has become widely used to evaluate patients with thoracic abnormalities. Rapid on‐site evaluation (ROSE) can provide the bronchoscopist with immediate evaluation findings during the procedure. This study examines EBUS FNA biopsy procedures with and without ROSE, and investigates the impact of ROSE service on the EBUS procedure and laboratory resource utilization.

Rapid on-site evaluation for endoscopic ultrasound-guided fine-needle biopsy of the pancreas decreases the incidence of repeat biopsy procedures.

Rapid on‐site evaluation (ROSE) for endoscopic ultrasound‐guided fine‐needle aspiration (EUS‐FNA) biopsy of the pancreas provides immediate feedback regarding cellular adequacy to aid in obtaining a definitive diagnosis and has the potential to avoid repeat procedures. The objective of the current study was to measure the impact of ROSE service on the incidence of repeat EUS FNA biopsy procedures.

Differential activity of the FGF-4 enhancer in F9 and P19 embryonal carcinoma cells

Transcription factors Oct‐3/4 and Sox2 behave as global regulators during mammalian embryogenesis. They work together by binding co‐operatively to closely spaced HMG and POU motifs (HMG/POU cassettes). Recently, it was suggested that a critical Sox2:Oct‐3/4 target gene, FGF‐4, is expressed at lower levels in P19 than in F9 embryonal carcinoma (EC) cells, due to lower levels of Sox2 in P19 than in F9 cells. We tested this possibility to better understand how FGF‐4 expression is modulated during development. Although we found that P19 EC cells express ∼10‐fold less FGF‐4 mRNA than F9 EC cells, we determined that Sox2 levels do not differ markedly in F9 and P19 EC cells. We also determined that Sox2 and Oct‐3/4 work together equally well in both EC cell lines. Moreover, in contrast to an earlier prediction based on in vitro binding studies, we demonstrate that the function of the HMG/POU cassettes of the FGF‐4 and UTF1 genes does not differ significantly in these EC cell lines when tested in the context of a natural enhancer. Importantly, we determined that the FGF‐4 promoter is highly responsive to a heterologous enhancer in both EC cell lines; whereas, the FGF‐4 enhancer is 7‐ to 10‐fold less active in P19 than in F9 EC cells. Because F9 and P19 EC cells are likely to represent cells at different stages of mammalian development, we suggest that this difference in FGF‐4 enhancer activity may reflect a mechanism used to decrease, but not abolish, FGF‐4 expression as the early embryo develops. J. Cell. Physiol.

NF-Y Behaves as a Bifunctional Transcription Factor That Can Stimulate or Repress the FGF-4 Promoter in an Enhancer-Dependent Manner

NF-Y is a bifunctional transcription factor capable of activating or repressing transcription. NF-Y specifically recognizes CCAAT box motifs present in many eukaryotic promoters. The mechanisms involved in regulating its activity are poorly understood. Previous studies have shown that the FGF-4 promoter is regulated positively by its CCAAT box and NF-Y in embryonal carcinoma (EC) cells where the distal enhancer of the FGF-4 gene is active. Here, we demonstrate that the CCAAT box functions as a negative cis-regulatory element when cis-regulatory elements of the FGF-4 enhancer are disrupted, or after EC cells differentiate and the FGF-4 enhancer is inactivated. We also demonstrate that NF-Y mediates the repression of the CCAAT box and that NF-Y associates with the endogenous FGF-4 gene in both EC cells and EC-differentiated cells. Importantly, we also determined that the orientation and the position of the CCAAT box are critical for its role in regulating the FGF-4 promoter. Together, these studies demonstrate that the distal enhancer of the FGF-4 gene determines whether the CCAAT box of the FGF-4 promoter functions as a positive or a negative cis-regulatory element. In addition, these studies are consistent with NF-Y playing an architectural role in its regulation of the FGF-4 promoter.

Correlation of p16 immunohistochemistry in FNA biopsies with corresponding tissue specimens in HPV‐related squamous cell carcinomas of the oropharynx

Human papillomavirus (HPV)‐related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin‐dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high‐risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine‐needle aspirations (FNAs) with surgical pathology specimens of HPV‐related oropharyngeal SCC.

Utilization of p40 (ΔNp63) with p63 and Cytokeratin 5/6 Immunohistochemistry in Non-Small Cell Lung Carcinoma Fine-Needle Aspiration Biopsy

Objective: Specific subclassification of pulmonary non-small cell carcinoma (NSCCA) is clinically necessary, and the aim of this study is to examine the utilization of p40 (ΔNp63) in fine-needle aspiration (FNA) biopsy for lung NSCCA. Study Design: Database files of the Washington University Medical Center were searched. Patients who underwent endobronchial ultrasound and CT FNA of a primary lung neoplasia were selected and immunohistochemistry (IHC) was performed. A panel of markers was utilized, including p40, p63, cytokeratin (CK) 5/6, thyroid transcription factor, and napsin. Results: One hundred patients were identified and comprised 38 squamous cell carcinomas (SCCA), 46 adenocarcinomas (AdCA), and 16 NSCCA. For SCCA, p40 was positive in 34/38 cases (89%) and negative in 4/38 cases (11%); p63 was positive in 33/38 cases (87%) and negative in 5/38 cases (13%); CK5/6 was positive in 38/38 cases. For AdCA cases, p40 was negative, p63 was positive in 2 cases (5%) and CK5/6 was negative in 43/46 cases (92%). Conclusion: For NSCCA, p40 had 89% sensitivity and 100% specificity compared to p63 with 86% sensitivity and 96% specificity and CK5/6 with 100% sensitivity and 96% specificity. In the evaluation of FNA biopsy for pulmonary NSCCA, p40 is a useful IHC marker for neoplastic subclassification, with better specificity in comparison to p63.

Fine-needle aspiration biopsy of HPV-related squamous cell carcinoma of the head and neck: Current ancillary testing methods for determining HPV status

Human papillomavirus (HPV)‐related head and neck squamous cell carcinoma (HNSCC) is a unique form of carcinoma that largely arises from the tonsillar tissue in the oropharynx. These tumors often present with cervical lymphadenopathy resulting in a fine needle aspiration (FNA) biopsy. Use of the cytology specimen to determine the HPV‐status has significant prognostic and treatment implications as HPV‐related tumors have a more favorable prognosis and response to nonsurgical therapies. While several different ancillary testing methods are available that have proven effective for determining HPV status in FNA specimens from HNSCCs, there is currently no consensus regarding HPV testing in this setting. Diagn. Cytopathol. 2017;45:221–229.

Primary Squamous Cell Carcinoma of the Pancreas: a Case Report and Literature Review

The patient is a 79-year-old Caucasian man with a history of T2N0M0 moderately differentiated adenocarcinoma of the gastroesophageal junction, which was diagnosed in October 2014; he was treated with definitive chemoradiation therapy and had evidenced no disease during 24months of subsequent follow-up. In November 2016, he presented to his primary care physician with an unintentional weight loss of 9 kg and upper abdominal pain. Laboratory evaluation was unrevealing. A computed tomography (CT) of the chest, abdomen, and pelvis demonstrated a large, heterogeneously enhancing, predominately hypoenhancing mass within the pancreatic tail, which was invading both the splenic hilum and the body of the stomach, with involvement of the splenic artery, as well as liver lesions suspicious for metastasis (Fig. 1a, b). In light of the CT findings, the patient underwent upper gastrointestinal endoscopy (EGD) and EUS-FNA for a tissue diagnosis. The EGD with careful attention to GE junction and stomach was inconclusive. The EUS demonstrated an illdefined hypoechoic mass in the tail of the pancreas, which was invading the muscularis propria layer of the gastric wall (Fig. 2). Cytology revealed poorly differentiated SCC (Fig. 3). Given his age, marginal performance status, and metastasis of the disease, the patient underwent palliative chemotherapy; he passed away after 3 months of treatment.

Fine-Needle Aspiration Biopsy of Hürthle Cell Neoplasms of the Thyroid

Abstract Fine-needle aspiration (FNA) biopsy of Hürthle cell containing thyroid nodules can be challenging because of a broad differential that contains both benign and malignant entities. Most nodules diagnosed by FNA as a follicular neoplasm, Hürthle cell type, are benign. This results in unnecessary surgeries and the risk of complications that accompany it. Numerous studies have tried to determine the most useful cytomorphologic features to best make the distinction between benign and malignant Hürthle cell nodules. While there are reproducible cytomorphologic features that can favor one over the other, there does not appear to be a single cytologic feature that can entirely exclude a neoplasm. While morphologically Hürthle cell carcinoma is considered a variant of follicular carcinoma, there is evidence to support it may be a distinct entity. In the future, molecular studies might be able to complement the cytomorphologic findings of FNA to provide a more refined and specific diagnosis for Hürthle cell containing thyroid nodules.