AI-guided histopathology predicts brain metastasis in lung cancer patients.

Brain metastases can occur in nearly half of patients with early and locally advanced (stage I-III) non-small cell lung cancer (NSCLC). There are no reliable histopathologic or molecular means to identify those who are likely to develop brain metastases. We sought to determine if deep learning (DL) could be applied to routine H&E-stained primary tumor tissue sections from stage I-III NSCLC patients to predict the development of brain metastasis. Diagnostic slides from 158 patients with stage I-III NSCLC followed for at least 5 years for the development of brain metastases (Met+, 65 patients) versus no progression (Met-, 93 patients) were subjected to whole-slide imaging. Three separate iterations were performed by first selecting 118 cases (45 Met+, 73 Met-) to train and validate the DL algorithm, while 40 separate cases (20 Met+, 20 Met-) were used as the test set. The DL algorithm results were compared to a blinded review by four expert pathologists. The DL-based algorithm was able to distinguish the eventual development of brain metastases with an accuracy of 87% (p < 0.0001) compared with an average of 57.3% by the four pathologists and appears to be particularly useful in predicting brain metastases in stage I patients. The DL algorithm appears to focus on a complex set of histologic features. DL-based algorithms using routine H&E-stained slides may identify patients who are likely to develop brain metastases from those who will remain disease free over extended (>5 year) follow-up and may thus be spared systemic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Development and validation of a decision tree for distinguishing pulmonary adenocarcinomas with mucinous features and metastatic colorectal adenocarcinoma.

BACKGROUND: Diagnosis of mucinous carcinomas in the lung on transbronchial biopsy or fine-needle aspiration (FNA) samples can be difficult for the pathologist, because primary and metastatic tumors can have similar morphological, immunohistochemical, and molecular characteristics. Correct diagnosis is key to determine appropriate therapy and to distinguish primary from metastatic disease. This distinction often falls to the pathologist in patients with a history of mucinous adenocarcinoma of the colon. Despite its drawbacks, immunohistochemistry is often employed to help assign a primary site for mucinous adenocarcinomas in the lung. However, the published data in this regard is limited to studies that use only a handful of markers. METHODS: The authors examined the staining characteristics and heterogeneity of CK7, TTF-1, NapsinA, CK20, CDX2, and SATB2 in resection specimens of pulmonary adenocarcinomas with mucinous features and metastatic colorectal adenocarcinoma. RESULTS: Based on the heterogeneity, sensitivity, and specificity in this cohort, the authors developed a decision tree based on TTF-1, SATB2, CDX2, and CK7 to categorize tumors as primary or metastatic lesions. Validation of the decision tree in FNA specimens from the lungs and lung-draining lymph nodes showed 84% concurrence in cases from the lung and 100% concurrence in cases from the lymph node. In cases where the algorithm assigned a primary site, it was 95% accurate compared to the multidisciplinary diagnosis. CONCLUSIONS: This method holds promise in distinguishing primary versus metastatic lesions in resection, biopsy, and FNA samples from the lungs.

Fluorescence in situ hybridization of thyroid fine-needle aspiration biopsy distinguishes between neoplastic and non-neoplastic Hürthle cell lesions.

INTRODUCTION: Fine-needle aspiration (FNA) biopsy of Hürthle cell proliferations can be difficult to characterize based purely on morphologic features. Studies have shown Hürthle cell neoplasms often demonstrate gains in chromosomes 5, 7, and 12. This study examined fluorescence in situ hybridization (FISH) performance characteristics in non-neoplastic and neoplastic Hürthle cell proliferations sampled by FNA biopsy in order to assess chromosome patterns. MATERIALS AND METHODS: FNA biopsies of Hürthle cell proliferations, including nodular hyperplasia (NH), Hürthle cell adenoma (HCA), and Hürthle cell carcinoma (HCC), that had subsequent surgical excision were selected. FISH was performed on an air-dried, modified Wright-Giemsa-stained, aspirate smear slide from each case using a 3-color panel consisting of 1 subtelomeric and 2 centromeric probes for chromosomes 5, 7, and 12. Chromosomal probe patterns were recorded in up to 50 cells. A positive result was considered when >15% of cells showed a polysomy in 2 or more chromosomes. RESULTS: A total of 25 cases were included in the study. All cases of NH were negative, and 7 of 9 (78%) HCAs and 8 of 12 (67%) HCCs were positive. Of the positive cases, 2 of the 7 (29%) HCAs showed >50% of cells with polysomy, and 5 of the 8 (63%) HCCs showed >50% of the cells with polysomy. CONCLUSION: Thyroid FNA biopsy can identify Hürthle cell proliferations; risk stratification based on morphology is difficult, however. FISH chromosomal evaluation of thyroid FNA biopsies is useful to distinguish neoplastic from non-neoplastic Hürthle cell proliferation.

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. © 2016 Wiley Periodicals, Inc.

Aptima HR-HPV testing from Diff-Quick-stained fine-needle aspiration smears of oropharyngeal squamous cell carcinoma.

INTRODUCTION: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a biologically unique form of carcinoma that is important to identify for prognosis and treatment. The objective of this study was to evaluate the performance of the Aptima HPV assay using Diff-Quick (DQ) stained smears from fine-needle aspiration (FNA) of HPV-related oropharyngeal SCC. MATERIALS AND METHODS: Patients with a diagnosis of head and neck SCC who also had FNA sample demonstrating metastatic disease were identified. Using a mounting media-based cell transfer technique, approximately 200 tumor cells were selected and harvested from DQ-stained aspirate smeared slides. The selected cells were tested for high risk HPV using the Aptima HPV assay, an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA from high-risk types of HPV. These results were compared with the p16 immunohistochemical staining of the corresponding surgical pathology specimens. RESULTS: Twenty-eight of 32 (87.5%) FNAs of p16-positive oropharyngeal SCC were positive for high-risk HPV by the Aptima assay and 18 of 18 (100%) FNAs of p16-negative SCC were negative for high-risk HPV by the Aptima assay. CONCLUSIONS: DQ-stained FNA smears can be used by the Aptima HPV assay to accurately detect high-risk HPVs in oropharyngeal SCCs with a sensitivity of 87.5% and a specificity of 100%. This provides an alternative to p16 immunohistochemical staining of FNA cell block material, which may not be available on all specimens.

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 liver and pancreas endoscopic ultrasonography-guided fine-needle aspiration biopsy in patients with a primary pancreatic lesion.

BACKGROUND: Endoscopic ultrasonography (EUS) is commonly used in the evaluation of pancreas masses, and when a liver lesion is visualized, it can undergo a fine-needle aspiration (FNA). This can provide diagnostic and staging information. The purpose of the study was to correlate the findings of patients who underwent EUS FNA biopsy of a pancreas lesion and a liver lesion during the same procedure. MATERIALS AND METHODS: The pathology database at Washington University Medical Center was searched for EUS FNA biopsy cases where biopsy of both the pancreas and liver were performed over a consecutive 10-year period (2003-2013). All pathology reports were reviewed, and clinical information and diagnostic results were recorded. RESULTS: A total of 102 cases were identified. For pancreas cases, 79.4% were malignant and for liver cases, 58.8% were malignant. In pancreas lesions categorized as suspicious for malignancy (9%), the liver biopsy provided a diagnosis of malignancy in 67% of cases. A malignant pancreatic cohort demonstrated a 62.9% liver malignancy. A malignant liver cohort corresponded to a malignant pancreas diagnosis in 86.6% of cases and a suspicious-malignant group of 98.3%. CONCLUSIONS: The 102 cases with concomitant EUS FNA biopsy of the pancreas and liver demonstrated the ability to provide a diagnosis of pancreas malignancy and correlate regional metastatic malignancy in the liver. In patients with a pancreas mass and in the appropriate clinical setting, a liver EUS FNA biopsy has the ability to provide a diagnosis of malignancy and demonstrate a high positive predictive value of malignancy in the pancreas (98.3%).

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

BACKGROUND: 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. METHODS: In total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high-risk HPV messenger RNA was performed on 31 of the FNA cell blocks. RESULTS: Although partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high-risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%). CONCLUSIONS: There was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false-negative results if applied to FNA cell blocks.

Transcriptional regulation of the murine Elf3 gene in embryonal carcinoma cells and their differentiated counterparts: requirement for a novel upstream regulatory region.

The transcription factor Elf3, which is one of over 25 Ets family members, is expressed in a wide variety of carcinomas and has been shown to promote the transcription of many genes implicated in cancer. To understand how the Elf3 gene is regulated at the transcriptional level, we probed its 5'-flanking region, and we report here the identification of both proximal and distal regions that regulate murine Elf3 promoter activity. In addition to mapping the transcription start site of the Elf3 gene, the work described in this study identifies four cis-regulatory elements in the proximal promoter region of the gene. These include a cis-regulatory element previously designated ESE, a kappaB site, a POU motif, and a CCAAT box. In addition, we demonstrate that a novel 94 bp region 2 kb upstream of the transcription start site significantly elevates Elf3 promoter activity in F9-differentiated cells, but not in the parental F9 embryonal carcinoma (EC) cells. This region appears to be largely responsible for the increase in Elf3 promoter activity that accompanies the differentiation of embryonal carcinoma cells.

Targeted next-generation sequencing using fine-needle aspirates from adenocarcinomas of the lung.

BACKGROUND: Molecular testing of cancer is increasingly critical to medicine. Next-generation sequencing (NGS) provides comprehensive, unbiased, and inexpensive mutation analysis of multiple genes with a single test. However, to the authors' knowledge, the usefulness of NGS in fine-needle aspiration (FNA) specimens, which may be the only specimens available, is unknown. Non-small cell lung cancer (NSCLC) is an ideal model in which to evaluate cytopathologic applications of NGS because FNA is used for diagnosis and staging and specific molecular therapeutic targets in NSCLC are known. Herein, the performance and quality of targeted NGS in FNA specimens from a small series of lung adenocarcinomas is evaluated. METHODS: Sequence data were generated from FNA specimens and paired formalin-fixed paraffin-embedded (FFPE) tissues from 5 patients with lung adenocarcinoma. DNA was isolated from FNA aspirate smears and cores of FFPE tissue. Multiplex sequencing of 27 cancer-related genes was performed after hybrid capture enrichment. Read-quality metrics and single-nucleotide variant calls were compared. RESULTS: The overall concordance of total reads across specimens was > 99% and the average concordance of single-nucleotide variants was 99.5%. The total reads generated, as well as the percentages of mapped, on-target, and unique reads were statistically indistinguishable (P > 0.05) between FFPE and FNA preparations. There also was no difference in the depth of sequencing coverage, including exon-level coverage in known lung cancer mutation hotspots. CONCLUSIONS: DNA isolated from FNA slides yields comprehensive, accurate, and statistically indistinguishable sequence information compared with that obtained from FFPE tissue. These results support the integration of NGS technologies into the standard cytopathology workflow. Cancer (Cancer Cytopathol) 2014;122:104-13. © 2013 American Cancer Society.

Predictive value of intra-abdominal lymph nodes in pancreatic endoscopic ultrasonography-guided fine-needle aspiration biopsy.

INTRODUCTION: Endoscopic ultrasonography (EUS)-guided fine-needle aspiration (FNA) biopsy is a commonly used method for the evaluation of pancreatic lesions. EUS-guided FNA of the intra-abdominal lymph nodes (LNs) can provide critical diagnostic information that is important for clinical management and tumor staging. This study examines the predictive value of intra-abdominal LN EUS-guided FNA biopsy associated with pancreatic lesions. MATERIALS AND METHODS: Over a 10-year period, the pathology database was searched for patients with concurrent pancreas and intra-abdominal LN EUS-guided FNA biopsy. The corresponding reports were reviewed, and clinical information and diagnostic results were recorded. RESULTS: There were 252 cases where both a pancreas lesion and intra-abdominal LN were biopsied. Of this group, 182 LNs were classified as negative (72%), 47 as positive (19%), and 23 as atypical (9%). Within the negative LN cohort, the pancreas FNAs fell into the following diagnostic categories: benign (47%), malignant (30%), and atypical/suspicious (23%). Within the positive LN cohort, the pancreas lesion correlated with the following diagnostic categories: malignant (89%), atypical (4%), and suspicious (6%). A positive LN EUS-guided FNA biopsy had a 98% positive predictive value for malignancy. Within the atypical LN cohort, the pancreas correlated with the following diagnostic categories: malignant (57%), atypical/suspicious (26%), and benign (17%). CONCLUSIONS: An atypical LN diagnostic category is strongly associated with a malignant pancreas lesion. A positive LN EUS-guided FNA biopsy has a 98% positive predictive value for pancreatic malignancy. A positive diagnostic category for an intra-abdominal LN can provide strong predictive evidence of a corresponding malignancy of the pancreas.

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

BACKGROUND: 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. METHODS: The cytopathology database at Washington University Medical Center, St. Louis, Missouri, was searched for EBUS FNA biopsy cases before and after introduction of ROSE service, and a matched cohort was collected. Reports were reviewed and pertinent data was collected, such as sites biopsied, ROSE performance, slide smears, cell blocks, and diagnostic categories. Statistical analysis of the results was performed. RESULTS: A matched case-controlled EBUS FNA cohort of 340 patients (680 total) for each category of non-ROSE and ROSE service were identified. There was a 33% reduction in the number of sites biopsied with ROSE. A total of 68% of patients with ROSE had just one biopsy site compared to only 36% of non-ROSE patients. There was a 30% decrease in total slides (mean, 5.27 slides) after the introduction of ROSE. All of these improvements were statistically significant. CONCLUSIONS: EBUS FNA biopsy ROSE service benefits patients by contributing to significantly fewer biopsies and improved utilization of health care resources. ROSE service results in substantially fewer total slides, which has a significant impact on the cytopathology laboratory work effort. The use of ROSE for EBUS FNA biopsy provides significant improvements in patient care 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.

BACKGROUND: 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. METHODS: Over a consecutive 3-year period, the pathology database at Washington University Medical Center was searched for patients with both an initial and subsequent EUS FNA biopsy demonstrating a solid lesion of the pancreas. These were divided temporally between the time before and after the introduction of ROSE service. Reports were reviewed and results were recorded. RESULTS: A total of 379 patients underwent ROSE service and 377 patients did not. The percentage of repeat non-ROSE EUS FNA cases was 5.8% and the percentage of repeat ROSE EUS FNA cases was 2.9%. The use of the ROSE service was found to decrease the number of repeat procedures by approximately 50% (P = .024). For those patients who underwent a repeat EUS-FNA procedure, the ROSE service provided a higher rate of definitive diagnosis among patients undergoing repeat procedures (67%) versus the non-ROSE cohort (27%). CONCLUSIONS: The use of ROSE for EUS-FNA biopsy of the pancreas was found to result in fewer patients undergoing repeat procedures. Patients who required a repeat procedure with the use of ROSE had a higher percentage of definitive diagnostic categorization on the repeat biopsy. Initial use of ROSE for EUS-FNA of solid pancreatic lesions was found to decrease the number of patients who required a repeat procedure.

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 approximately 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.

Roles of the conserved CCAAT and GC boxes of the human and mouse type II transforming growth factor-beta receptor genes.

Embryonal carcinoma (EC) cells are used widely to study the molecular mechanisms that regulate the transcription of genes during mammalian embryogenesis. The type II transforming growth factor-beta receptor (TbetaR-II) gene is expressed at very low levels by mouse EC cells prior to differentiation. Differentiation of EC cells results in increases of both the steady-state levels of TbetaR-II mRNA and the activity of the TbetaR-II promoter. Several cis-regulatory elements have been shown previously to regulate the TbetaR-II gene. This study focuses on the role of a CCAAT box and three GC boxes in the regulation of the human and mouse TbetaR-II promoters in EC-differentiated cells. We demonstrate that the CCAAT box and two flanking GC boxes, Sp A and Sp B, function as positive regulatory elements in the human TbetaR-II promoter, and that the transcription factor complex NF-Y positively regulates the human TbetaR-II promoter through the CCAAT box motif. We also show that the CCAAT box and the downstream GC box Sp B, which are conserved between the human and mouse promoters, behave as positive regulatory elements in the mouse TbetaR-II promoter. In addition, we demonstrate that the transcription factor Sp1 can bind to the Sp B GC box in vitro. Finally, we show that a GC box located 25 bp upstream of the major transcription start site of the TbetaR-II gene plays a minimal role in the function of the TbetaR-II promoter in EC-differentiated cells. Together, our studies highlight important differences and similarities in the cis-regulatory elements that regulate the human and mouse TbetaR-II promoters.

Transcription factor Sox-2 inhibits co-activator stimulated transcription.

Previous studies have shown that transcription of the fibroblast growth factor-4 (FGF-4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the transcription start site within the untranslated region of the last exon. The transcription factors Sox-2 and Oct-3 cooperatively bind to critical cis-regulatory elements within the enhancer to synergistically activate transcription. Moreover, the co-activator p300 can mediate the synergistic activity of Sox-2 and Oct-3, and p300 associates with the FGF-4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF-4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox-2 expression in F9 EC cells limit the transcription of the FGF-4 gene. The studies presented in this report argue that Sox-2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox-2 in F9 EC cells decreases FGF-4 promoter activity. In addition, overexpression of Sox-2 in these cells inhibits activation by the co-activators p300, CBP, and OCA-B in a manner that requires the transactivation domain of Sox-2. These findings suggest that Sox-2 levels in F9 EC cells are regulated carefully to avoid interference with the transcription of critical genes.

Stimulation of the murine type II transforming growth factor-beta receptor promoter by the transcription factor Egr-1.

Previous studies have demonstrated that differentiation of murine embryonal carcinoma (EC) cells leads to the appearance of high affinity receptors for transforming growth factor-beta (TGF-beta). Subsequently, it was demonstrated that differentiation of F9 EC cells leads to increases in the transcription of the type II TGF-beta-receptor gene (TbetaR-II) and leads to significant increases in the steady-state levels of TbetaR-II mRNA. Analysis of the human TbetaR-II promoter in F9-differentiated cells identified several cis-regulatory elements that influence the activity of the promoter, including a CRE/ATF site and a CCAAT box motif. In the work described in this report, we focused on the effect of the transcription factor Egr-1 on the murine TbetaR-II promoter. We have identified an Egr-1 response-element approximately 150 bp upstream of the major transcription start site of the murine TbetaR-II gene. We demonstrate by electrophoretic mobility shift analysis (EMSA) that this cis-regulatory element binds Egr-1, and we demonstrate that disruption of this site eliminates the response to Egr-1. As part of this analysis, we also examined the effect of Egr-1 on human TbetaR-II promoter. In contrast to a previous report, which reported that Egr-1 inhibits expression of human TbetaR-II promoter/reporter gene constructs, we did not observe an inhibitory effect of Egr-1 that was specific for the human TbetaR-II promoter. Taken together, the findings described in this report identify important differences between the human and the murine TbetaR-II promoter, and our findings identify an Egr-1 cis-regulatory element that is capable of stimulating the activity of the murine TbetaR-II promoter.