Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases.

BACKGROUND: Toll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity of checkpoint inhibitors (CPI) in treating liver tumors is limited in part by the distinctive biology of intrahepatic myeloid-derived suppressor cells (MDSC) and challenges with tumor-specific therapeutic delivery. We have shown that the regional delivery of type C TLR9 agonist via pressure-enabled drug delivery (PEDD) system improves delivery to the tumor, enhances depletion of MDSCs and overall, stimulates the immune system in combination with or without CPI. Currently, CPIs are delivered intravenously, although there is a growing interest in its subcutaneous (SQ) administration. We compared nelitolimod formerly known as SD-101 administered using PEDD in combination with systemic (Sys) or SQ CPI in murine liver metastases (LM). METHODS: The LM model was developed by injecting MC38-Luc cells via the spleen of 8-12 week old male C57/BL6 mice followed by splenectomy. After a week, fluorescently labeled nelitolimod (10 µg/mouse) was delivered via PEDD and co-administered anti-programmed cell death-1 (α-PD-1) either via Sys or SQ. Tumor burden was monitored by in vivo imaging system. Serum cytokine levels were analyzed by Luminex. Tissues were harvested on Day 3 (D3) or Day 10 (D10) post-PEDD to enrich CD45+ cells and were analyzed via NanoString targeted transcriptomics (D3) or flow cytometry (FC, D10) to interrogate immune cell populations (D10). For NanoString analysis, the innate immune panels were selected, and for FC, MDSCs (CD11b+Gr1+), B cells (B220+), dendritic cells (DC, CD11c+), T (CD3+) cells, and M1-like macrophages (F4/80+CD38+Egr2-) were quantified. RESULTS: Nelitolimod delivered via PEDD resulted in changes in innate and adaptive immune cells within LM, including depletion of liver MDSC and increased M1-like macrophages in the liver, which are supportive of antitumor immunity. While CPI monotherapy failed to control tumor progression, nelitolimod and CPI combination improved LM control, survival and antitumor immunity beyond the nelitolimod monotherapy effect, irrespective of CPI delivery route. CONCLUSION: The SQ route of CPI delivery was equivalent to Sys in combination with nelitolimod, suggesting SQ-CPI may be a rational choice in combination with PEDD of nelitolimod for liver tumor treatment.

Hemorrhage Sites and Mitigation Strategies After Pancreaticoduodenectomy.

Importance: Postpancreatectomy hemorrhage is an uncommon but highly morbid complication of pancreaticoduodenectomy. Clinical evidence often draws suspicion to the gastroduodenal artery stump, even without a clear source. Objective: To determine the frequency of gastroduodenal artery bleeding compared to other sites and the results of mitigation strategies. Design, Setting, and Participants: This cohort study involved a retrospective analysis of data for consecutive patients who had pancreaticoduodenectomy from 2011 to 2021 at Memorial Sloan Kettering Cancer Center (MSK) and Thomas Jefferson University Hospital (TJUH). Exposures: Demographic, perioperative, and disease-related variables. Main Outcomes and Measures: The incidence, location, treatment, and outcomes of primary (initial) and secondary (recurrent) hemorrhage requiring invasive intervention were analyzed. Imaging studies were re-reviewed by interventional radiologists to confirm sites. Results: Inclusion criteria were met by 3040 patients (n = 1761 MSK, n = 1279 TJUH). Patients from both institutions were similar in age (median [IQR] age at MSK, 67 [59-74] years, and at TJUH, 68 [60-75] years) and sex (at MSK, 814 female [46.5%] and 947 male [53.8%], and at TJUH, 623 [48.7%] and 623 male [51.3%]). Primary hemorrhage occurred in 90 patients (3.0%), of which the gastroduodenal artery was the source in 15 (16.7%), unidentified sites in 24 (26.7%), and non-gastroduodenal artery sites in 51 (56.7%). Secondary hemorrhage occurred in 23 patients; in 4 (17.4%), the gastroduodenal artery was the source. Of all hemorrhage events (n = 117), the gastroduodenal artery was the source in 19 (16.2%, 0.63% incidence in all pancreaticoduodenectomies). Gastroduodenal artery hemorrhage was more often associated with soft gland texture (14 [93.3%] vs 41 [62.1%]; P = .02) and later presentation (median [IQR], 21 [15-26] vs 10 days [5-18]; P = .002). Twenty-three patients underwent empirical gastroduodenal artery embolization or stent placement, 7 (30.4%) of whom subsequently experienced secondary hemorrhage. Twenty percent of all gastroduodenal artery embolizations/stents (8/40 patients), including 13% (3/13 patients) of empirical treatments, were associated with significant morbidity (7 hepatic infarction, 4 biliary stricture), with a 90-day mortality rate of 38.5% (n = 5) for patients with these complications vs 7.8% without (n = 6; P = .008). Ninety-day mortality was 12.2% (n = 11) for patients with hemorrhage (3 patients [20%] with primary gastroduodenal vs 8 [10.7%] for all others; P = .38) compared with 2% (n = 59) for patients without hemorrhage. Conclusions and Relevance: In this study, postpancreatectomy hemorrhage was uncommon and the spectrum was broad, with the gastroduodenal artery responsible for a minority of bleeding events. Empirical gastroduodenal artery embolization/stent without obvious sequelae of recent hemorrhage was associated with significant morbidity and rebleeding and should not be routine practice. Successful treatment of postpancreatectomy hemorrhage requires careful assessment of all potential sources, even after gastroduodenal artery mitigation.

GCM2 Variants in Familial and Multiglandular Primary Hyperparathyroidism.

CONTEXT: Multiglandular and familial parathyroid disease constitute important fractions of primary hyperparathyroidism (PHPT). Germline missense variants of GCM2, a regulator of parathyroid development, were observed in familial isolated hyperparathyroidism and sporadic PHPT. However, as these previously reported GCM2 variants occur at relatively high frequencies in the population, understanding their potential clinical utility will require both additional penetrance data and functional evidence relevant to tumorigenicity. OBJECTIVE: Determine the frequency of GCM2 variants of interest among patients with sporadic multigland or familial parathyroid disease and assess their penetrance. DESIGN AND PATIENTS: DNA-encoding PHPT-associated GCM2 germline variants were polymerase chain reaction-amplified and sequenced from 107 patients with either sporadic multigland or suspected/confirmed familial parathyroid tumors. RESULTS: GCM2 variants were observed in 9 of 107 cases (8.4%): Y282D in 4 patients (6.3%) with sporadic multigland disease; Y394S in 2 patients (11.1%) with familial PHPT and 3 (4.8%) with sporadic multigland disease. Compared with the general population, Y282D was enriched 5.9-fold in multigland disease, but its penetrance was very low (0.02%). Y394S was enriched 79-fold in sporadic multigland disease and 93-fold in familial PHPT, but its penetrance was low (1.33% and 1.04%, respectively). CONCLUSIONS: Observed in vitro-activating GCM2 variant alleles are significantly overrepresented in PHPT patients with multiglandular or familial disease compared to the general population, yet penetrance values are very low; that is, most individuals with these variants in the population have a very low risk of developing PHPT. The potential clinical utility of detecting these GCM2 variants requires further investigation, including assessing their possible role as pathogenic/low-penetrance alleles.

A Novel Treatment for Metastatic Serous Cystadenocarcinoma Using a Microwave Ablation: Case Report and Review of the Literature.

ABSTRACT: The incidence of pancreatic cystic neoplasms has grown because of increased detection. Among these lesions, serous cystadenoma was traditionally thought to be universally benign and indolent. However, there is an exceedingly rare malignant variant of serous cystadenoma known as serous cystadenocarcinoma (SCAC) that can exhibit local invasion into adjacent structures, hepatic implants, and metastatic spread to the abdominal viscera. Diagnosis of SCAC can be challenging as it is histologically identical to serous cystadenoma. To better understand this entity, a review of all published accounts of SCAC was performed in which tumor and patient factors were characterized. In addition, we present the case of a 49-year-old woman who was found to have a solitary hepatic metastasis due to SCAC, 11 years after a distal pancreatectomy for serous cystadenoma. She was successfully treated with percutaneous microwave ablation and has no evidence of recurrence 3 years later. This report details the first published account of percutaneous ablation in such a setting. Compared with hepatectomy, hepatic ablation may offer a less invasive but equally effective treatment option in well-selected patients.

A Critical Appraisal of the July Effect: Evaluating Complications Following Pancreaticoduodenectomy.

BACKGROUND: Reports of higher rates of medical errors in the month of July have generated concern regarding major surgery at academic institutions early in the yearly promotion cycle. This study was designed to evaluate perioperative outcomes in patients undergoing pancreaticoduodenectomy (PD) at different times of the year. MATERIALS AND METHODS: Outcomes were retrospectively evaluated for patients treated in July versus the rest of the year and in the first quarter (July-September) versus the remaining quarters. The primary outcome was operative morbidity as measured by Clavien-Dindo grade, a classification system of surgical complications. Secondary outcomes included mortality, operative blood loss, pancreatic fistula formation, delayed gastric emptying, intraabdominal abscess, anastomotic leak, reoperation, and other variables of interest. RESULTS: From January 2003 to September 2015, 472 patients underwent PD by a single academic surgeon. Overall, 77.1% of PDs were performed for malignancy. The number of patients did not significantly vary by month or by quarter. The incidence of major morbidity (Clavien-Dindo grade ≥ III) in patients who had a PD was 12.2% in July and 17.5% in all other months (P = 0.79). The rate of pancreatic fistula, intraabdominal abscess, reoperation, readmission, and mortality did not differ significantly by month or by quarter (P > 0.05 for all). CONCLUSIONS: The current study does not find any correlation between time of year and operative morbidity or mortality, suggesting that PD can be safely performed irrespective of timing.

Analysis of Activating GCM2 Sequence Variants in Sporadic Parathyroid Adenomas.

CONTEXT: Sporadic, solitary parathyroid adenoma is the most common cause of primary hyperparathyroidism (PHPT). Apart from germline variants in certain cyclin-dependent kinase inhibitor genes and occasionally in MEN1, CASR, or CDC73, little is known about possible genetic variants in the population that may confer increased risk for development of typical sporadic adenoma. Transcriptionally activating germline variants, especially within in the C-terminal conserved inhibitory domain (CCID) of glial cells missing 2 (GCM2), encoding a transcription factor required for parathyroid gland development, have recently been reported in association with familial and sporadic PHPT. OBJECTIVE: To evaluate the potential role of specific GCM2 activating variants in sporadic parathyroid adenoma. DESIGN AND PATIENTS: Regions encoding hyperparathyroidism-associated, activating GCM2 variants were PCR amplified and sequenced in genomic DNA from 396, otherwise unselected, cases of sporadic parathyroid adenoma. RESULTS: Activating GCM2 CCID variants (p.V382M and p.Y394S) were identified in six of 396 adenomas (1.52%), and a hyperparathyroidism-associated GCM2 non-CCID activating variant (p.Y282D) was found in 20 adenomas (5.05%). The overall frequency of tested activating GCM2 variants in this study was 6.57%, approximately threefold greater than their frequency in the general population. CONCLUSIONS: The examined, rare CCID variants in GCM2 were enriched in our cohort of patients and appear to confer a moderately increased risk of developing sporadic solitary parathyroid adenoma compared with the general population. However, penetrance of these variants is low, suggesting that the large majority of individuals with such variants will not develop a sporadic parathyroid adenoma.

Interdisciplinary Approach to Spatiotemporal Population Dynamics: The North Orkney Population History Project.

The North Orkney Population History Project is a multidisciplinary data collection, digitization, and analysis effort that aims to reconstruct longitudinal demographic, environmental, and economic change. We describe the motivation, methodological approach, data sources, and some initial findings of the project. Detailed contextual information about a single community allows for the joint analysis of the changing population and changing landscape. The combination of diverse data sources and disciplinary approaches has resulted in findings that would not have been possible if each source had been considered in isolation. The approach adopted by the project offers a way to examine the interaction of a population with its landscape over a period of change.

TERT promoter hypermethylation is associated with poor prognosis in adrenocortical carcinoma.

Telomere maintenance, most commonly achieved by telomerase activation through induction of the telomerase reverse transcriptase (TERT) gene, is required for cell immortalization, a hallmark of cancer. Adrenocortical carcinoma (ACC) is an endocrine tumor for which TERT promoter mutations and telomerase activation have been reported. The present study assessed alterations of the TERT gene locus and telomere length in relation to clinical characteristics in ACC. In total, 38 cases of ACC with known TERT promoter mutational status were included. TERT promoter methylation densities were assessed by pyrosequencing, and TERT copy numbers and telomere length were determined by quantitative polymerase chain reaction analysis, followed by comparison of the mRNA expression of TERT and clinical parameters. The ACC tissue samples showed increased TERT copy numbers, compared with normal adrenal tissue (NAT) samples (P=0.001). Mutually exclusive TERT copy number gains or promoter mutation were present in 70% of the ACC samples. The ACC tissues exhibited higher levels of CpG promoter methylation of all eight CpG sites investigated within the ­578 to ­541 bp (Region A), compared with the NATs (P=0.001). High methylation density at this region was associated with metastatic disease and/or relapse, poor survival rates and higher European Network for the Study of Adrenal Tumor stage (P<0.05). The mRNA expression of TERT was inversely correlated with methylation density at ­162 to ­100 bp (Region B). Correlation was observed between relative telomere length and the gene expression of TERT. It was concluded that epigenetic alterations of the TERT promoter are frequent and associated with advanced disease and poorer clinical outcome in ACC.

SLC12A7 alters adrenocortical carcinoma cell adhesion properties to promote an aggressive invasive behavior.

BACKGROUND: Altered expression of Solute Carrier Family 12 Member 7 (SLC12A7) is implicated to promote malignant behavior in multiple cancer types through an incompletely understood mechanism. Recent studies have shown recurrent gene amplifications and overexpression of SLC12A7 in adrenocortical carcinoma (ACC). The potential mechanistic effect(s) of SLC12A7 amplifications in portending an aggressive behavior in ACC has not been previously studied and is investigated here using two established ACC cell lines, SW-13 and NCI-H295R. METHODS: SW-13 cells, which express negligible amounts of SLC12A7, were enforced to express SLC12A7 constitutively, while RNAi gene silencing was performed in NCI-H295R cells, which have robust endogenous expression of SLC12A7. In vitro studies tested the outcomes of experimental alterations in SLC12A7 expression on malignant characteristics, including cell viability, growth, colony formation potential, motility, invasive capacity, adhesion and detachment kinetics, and cell membrane organization. Further, potential alterations in transcription regulation downstream to induced SLC12A7 overexpression was explored using targeted transcription factor expression arrays. RESULTS: Enforced SLC12A7 overexpression in SW-13 cells robustly promoted motility and invasive characteristics (p < 0.05) without significantly altering cell viability, growth, or colony formation potential. SLC12A7 overexpression also significantly increased rates of cellular attachment and detachment turnover (p < 0.05), potentially propelled by increased filopodia formation and/or Ezrin interaction. In contrast, RNAi gene silencing of SLC12A7 stymied cell attachment strength as well as migration and invasion capacity in NCI-H295R cells. Transcription factor expression analysis identified multiple signally pathways potentially affected by SLC12A7 overexpression, including osmotic stress, bone morphogenetic protein, and Hippo signaling pathways. CONCLUSIONS: Amplification of SLC12A7 observed in ACCs is shown here, in vitro, to exacerbate the malignant behavior of ACC cells by promoting invasive capacities, possibly mediated by alterations in multiple signaling pathways, including the osmotic stress pathway.

Comprehensive Genetic Analysis of Follicular Thyroid Carcinoma Predicts Prognosis Independent of Histology.

Context: Follicular thyroid carcinoma (FTC) is classified into minimally invasive (miFTC), encapsulated angioinvasive (eaFTC), and widely invasive (wiFTC) subtypes, according to the 2017 World Health Organization guidelines. The genetic signatures of these subtypes may be crucial for diagnosis, prognosis, and treatment but have not been described. Objective: Identify and describe the genetic underpinnings of subtypes of FTC. Methods: Thirty-nine tumors, comprising 12 miFTCs, 17 eaFTCs, and 10 wiFTCs, were whole-exome sequenced and analyzed. Somatic mutations, constitutional sequence variants, somatic copy number alterations, and mutational signatures were described. Clinicopathologic parameters and mutational profiles were assessed for associations with patient outcomes. Results: Total mutation burden was consistent across FTC subtypes, with a median of 10 (range 1 to 44) nonsynonymous somatic mutations per tumor. Overall, 20.5% of specimens had a mutation in the RAS subfamily (HRAS, KRAS, or NRAS), with no notable difference between subtypes. Mutations in TSHR, DICER1, EIF1AX, KDM5C, NF1, PTEN, and TP53 were also noted to be recurrent across the cohort. Clonality analysis demonstrated more subclones in wiFTC. Survival analysis demonstrated worse disease-specific survival in the eaFTC and wiFTC cohorts, with no recurrences or deaths for patients with miFTC. Mutation burden was associated with worse prognosis, independent of histopathological classification. Conclusions: Though the number and variety of somatic variants are similar in the different histopathological subtypes of FTC in our study, mutational burden was an independent predictor of mortality and recurrence.

Predictors for Use of Sentinel Node Biopsy and the Association with Improved Survival in Melanoma Patients Who Have Nodal Staging.

BACKGROUND: It is unknown how many patients with localized melanoma undergo sentinel lymph node biopsy (SLNB) or if there is a therapeutic effect from performing nodal staging. We evaluated predictors for SLNB use and assessed if there was an association with improved survival in melanoma patients who had SLNB. METHODS: The Surveillance, Epidemiology, and End Results database was queried for clinically node-negative melanoma cases ≥ 0.75 mm in thickness treated from 2010 to 2012. Clinicopathologic factors were correlated with SLNB use, overall survival (OS), and melanoma-specific survival (MSS). RESULTS: Overall, 13,703 cases were included. SLNB was performed in 1479 of 3439 thin cases (43.0%), 5810 of 8522 intermediate-thickness cases (68.2%), and 916 of 1742 thick cases (52.6%). On multivariable analysis, age ≥ 70 years, thickness < 1 or > 4 mm, head/neck or trunk tumor location, being unmarried, African American race, and residing in a county with a lower level of education were significantly associated with a lower likelihood of performing SLNB (p < 0.05). Patients with intermediate-thickness or thick melanoma who had a SLNB had significantly improved OS and MSS compared with patients who did not have a SLNB (p < 0.05). On multivariable analysis, SLNB use significantly predicted for improved OS and MSS (p < 0.01). CONCLUSIONS: Only 68.2% of intermediate-thickness and 52.6% of thick melanomas are treated with SLNB. Age, thickness, tumor location, race, marital status, and socioeconomic factors appear to influence the performance of SLNB. This data becomes more relevant with the finding that SLNB use is potentially associated with improved survival.

Cataract Surgery in Patients with Diabetes: Management Strategies.

Diabetes is a chronic systemic disease that affects nearly one in eight adults worldwide. Ocular complications, such as cataract, can lead to significant visual impairment. Among the worldwide population, cataract is the leading cause of blindness, and patients with diabetes have an increased incidence of cataracts which mature earlier compared to the rest of the population. Cataract surgery is a common and safe procedure, but can be associated with vision-threatening complications in the diabetic population, such as diabetic macular edema, postoperative macular edema, diabetic retinopathy progression, and posterior capsular opacification. This article is a brief review of diabetic cataract and complications associated with cataract extraction in this population of patients.

Epigenetic modifications in poorly differentiated and anaplastic thyroid cancer.

Well-differentiated thyroid cancer accounts for the majority of endocrine malignancies and, in general, has an excellent prognosis. In contrast, the less common poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) are two of the most aggressive human malignancies. Recently, there has been an increased focus on the epigenetic alterations underlying thyroid carcinogenesis, including those that drive PDTC and ATC. Dysregulated epigenetic candidates identified include the Aurora group, KMT2D, PTEN, RASSF1A, multiple non-coding RNAs (ncRNA), and the SWI/SNF chromatin-remodeling complex. A deeper understanding of the signaling pathways affected by epigenetic dysregulation may improve prognostic testing and support the advancement of thyroid-specific epigenetic therapies. This review outlines the current understanding of epigenetic alterations observed in PDTC and ATC and explores the potential for exploiting this understanding in developing novel therapeutic strategies.

Suppression of Forkhead Box Protein O1 (FOXO1) Transcription Factor May Promote Adrenocortical Tumorigenesis.

Despite recent comprehensive genetic analyses, molecular evidence for a pathophysiological continuum linking benign adrenocortical adenoma (ACA) and highly aggressive adrenocortical carcinoma (ACC) is still elusive. Using human tumor samples and the established ACC cell line SW-13, this study investigated potential regulatory roles for FOXO transcription factors, in modulating adrenocortical tumorigenesis. Adrenocortical tumor specimens (20 ACAs, 10 ACCs, and 9 normal adrenal tissue samples) obtained from 30 patients were analyzed for ubiquitously expressed FOXO transcription factors, FOXO1 and FOXO3 using qRT-PCR and immunohistochemistry. The SW-13 ACC cells were used to study the phenotypic effects of FOXO regulation in vitro. While FOXO3 expression remained unchanged in ACCs, FOXO1 expression was found to be significantly downregulated in 19/20 ACAs and 9/10 ACCs (p<0.0001 and p<0.05, respectively), suggesting a global role for FOXO1 suppression in promoting and maintaining adrenocortical dedifferentiation. Silencing of FOXO1 in SW-13 cells resulted in significant loss of viability (p<0.001) mediated by apoptosis as determined by quantitative Annexin V immunofluorescence analysis (p<0.01). FOXO1 silencing also augmented the migratory behavior of SW-13 cells (p<0.0001), suggesting distinct roles for FOXO1 in promoting viability and controlled motility of adrenocortical cells.

A Systematic Review of Proinsulin-Secreting Pancreatic Neuroendocrine Tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) are a heterogeneous group of islet cell-derived neoplasms with a propensity toward hormone production. Among PNETs, proinsulin-secreting tumors (proinsulinomas) are exceedingly rare. The objective of this study is to collect and summarize the existing literature to provide a comprehensive evaluation of this uncommon disease. METHODS: A systematic review was performed to characterize the clinicopathologic features of proinsulinoma. Using the electronic biomedical databases PubMed, Ovid Medline, and Embase, 316 publications were screened for relevance of which 14 were selected. We also present two patients with proinsulinoma treated at Yale New Haven Hospital. RESULTS: Of the 16 patients included in the study, the mean age was 56.8 and there was a 2:1 female predominance. The majority of patients presented with symptomatic hypoglycemia with normal or low insulin levels. Median tumor diameter was 1.2 cm and 80% were located in the body and tail of the pancreas. Following resection, most patients had normalization of hormonal levels without recurrence (75%; 12/16). CONCLUSION: Proinsulinomas are rare pancreatic neuroendocrine tumors that have the potential to cause hypoglycemia. While insulinomas and proinsulin-secreting tumors have many physiologic parallels, these cases illustrate several key distinctions in their diagnosis and management.

A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.

BACKGROUND: Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex. METHODS: We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex. RESULTS: While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring ß-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability. CONCLUSIONS: DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.

Overexpression of cytochrome P450 2A6 in adrenocortical carcinoma.

BACKGROUND: Cytochrome P450-mediated metabolism of chemotherapeutic agents contributes to chemotherapy resistance in multiple malignancies. Adrenocortical carcinoma is known to have a poor response to adjuvant therapies; however, the mechanism remains unknown. Recent comprehensive genetic analyses of adrenocortical carcinomas demonstrated recurrent copy number gains in multiple cytochrome P450 genes prompting investigation into whether cytochrome P450 overexpression potentiates adrenocortical carcinoma chemoresistance. METHODS: We determined the expression patterns of 6 cytochrome P450 genes (CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2S1, and CYP4F2) predicted to be amplified in adrenocortical carcinoma (n = 29) relative to normal adrenal cortex (n = 10). Gene copy numbers were determined with the TaqMan copy number assay. Gene silencing was performed via small interfering RNA (siRNA) in the adrenocortical carcinoma cell line NCI-H295R and treated with mitotane and cisplatin. RESULTS: Of the 6 cytochrome P450 genes tested, CYP2A6 was overexpressed with a 55-fold mean increase compared to normal adrenal samples (P < .05). Immunohistochemical analysis confirmed protein overexpression. Copy gains of CYP2A6 were found in 26% (7/27) of adrenocortical carcinoma specimens. Silencing of CYP2A6 in NCI-H295R cells resulted in decreased cell viability and increased chemosensitivity (P < .05). CONCLUSION: Frequent upregulation in adrenocortical carcinomas and the reversal of chemoresistance in adrenocortical carcinoma cells via enforced silencing suggest a role for CYP2A6 in adrenocortical malignancy.

Suppression of cytochrome P450 4B1: An early event in adrenocortical tumorigenesis.

BACKGROUND: Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Conversely, adrenocortical adenomas are common and benign. Despite their shared histologic origin, little evidence exists to suggest that adrenocortical adenoma arises from adrenocortical carcinoma. Recent genetic analyses of adrenocortical carcinoma have shown recurrent gene copy deletion of CYP4B1, a cytochrome P450 isozyme. This study investigates a potential role for CYP4B1 in modulating adrenocortical tumorigenesis and/or conferring chemoresistance to adrenocortical carcinomas. METHODS: Using TaqMan, real-time quantitative polymerase chain reaction techniques, we investigated CYP4B1 expression in normal adrenal cortex (n = 10), histologically confirmed adrenocortical adenomas (n = 10), and adrenocortical carcinomas (n = 10). Adrenocortical carcinoma cell lines were enforced to express CYP4B1, and effects on cell death and enhanced mitotane and cisplatin sensitivity were tested. RESULTS: Gene expression analyses demonstrated suppression of CYP4B1 in 100% of both the adrenocortical adenomas (10/10) and adrenocortical carcinomas (10/10) tested. Average relative expression of CYP4B1 was decreased at 0.19 (0.01-0.50; P < .01) in adrenocortical adenomas and nearly absent in adrenocortical carcinomas (0.01; 0.00-0.05; P < .01). Protein expression correlated with mRNA expression. Ectopic expression of CYP4B1 promoted cytotoxicity and increased chemosensitivity in adrenocortical carcinoma cell lines. CONCLUSION: CYP4B1 is silenced in both benign and malignant adrenocortical tumors and may contribute to tumorigenesis and chemoresistance. Sensitization of adrenocortical carcinoma cells engineered to overexpress CYP4B1 further supports this notion.