Single-Institution Learning Curve for Management of Mega-Fistulae Revision.

BACKGROUND: Mega-fistulae are generalized aneurysmal dilations of a high flow (1500-4000 mL/min) autogenous arteriovenous (AV) access which may result in hemorrhage and/or high-output cardiac failure. Current treatments include ligation, ligation with prosthetic jump graft, and imbrication; however, these may not be suitable for advanced disease, or may result in loss of functioning access, poor cosmesis, or recurrence. We describe our early experience with a technique of complete mega-fistula resection and replacement with an early use prosthetic graft that both maintains existing AV access and eliminates the need for long-term catheter (LTC) placement; including lessons learned. METHODS: A single-center, retrospective review of medical records was conducted from March 2018-February 2021. Outcomes were technical success, LTC use, time to cannulation, and complications. Mega-fistulae were completely resected from the proximal to distal aneurysmal segment, including all pseudoaneurysms, followed by tunneling a prosthetic graft (Propaten later converted to Acuseal; W.L. Gore Assoc.) with an end-to-end anastomosis to the remaining arterial and venous ends of the previous AV access. RESULTS: We had 100% immediate technical success (n=12). Pre-operative long-term catheters were placed in all eight Propaten patients; one was already placed in an Acuseal patient.  Average time to cannulation was six weeks with Propaten and 4.5 days with Acuseal. At 30 days, three Propaten patients developed complications including one instance of skin necrosis, one seroma, and one hematoma. Two Acuseal patients developed complications including one central venous occlusion (CVO) and one graft infection. Of the six patients with long-term follow-up, five continue to use their access, however, two required thrombectomies and central venous angioplasties. One patient required a new contralateral access due to CVO. CONCLUSIONS: Complete mega-fistula resection and replacement with Acuseal graft maintains existing AV access and may eliminate the need for long-term catheter placement. Our early experience with this technique is encouraging, but further follow-up is required to determine the durability of this approach.

A Novel Angiography Scoring System Predicts Improvement After Isolated Common Femoral Endarterectomy With Profundaplasty.

BACKGROUND: Numerous angiography-based peripheral arterial disease classification schemes have been developed to stratify severity of preoperative patient disease, but few studies have correlated angiography-based anatomic classification schemes to postoperative outcomes. This study examined whether a proposed pre-operative angiography scoring system was predictive of outcomes after isolated common femoral endarterectomy with profundaplasty (CFEP). METHODS: A retrospective review was conducted of patients treated with isolated CFEP for claudication and/or rest pain at a single institution from 2016-19. Pre-operative angiograms were assessed quantitatively by 4 blinded surgeons across 3 domains: profunda stenosis, profunda disease length, and outflow disease severity. Table I describes the proposed angiography scoring system. Internal consistency reliability of rater scores was calculated using Cronbach alpha. Outcomes included clinical improvement, further interventions, major amputations, mortality, and mean increase in ankle-brachial index (ABI) at 30 days, and 6 months. McNemar tests, between-group t-tests, Pearson correlations, and linear regression were used. RESULTS: Clinical Outcomes 88% of patients (n = 22) had clinical improvement at 30 days; the remaining 12% of patients (n = 3) required further interventions. One patient (4%) required major amputation between 30 days and 6 months for recurrence of rest pain that had initially resolved after isolated CFEP. There was 0% mortality during the study period. Mean ABI increased by 0.15 ± 0.21 at 30 days, and by 0.06 ± 0.21 at 6 months. Angiography Scoring System Profunda stenosis score was associated with clinical improvement at 6 months (P = 0.04). A profunda stenosis score of ≥2.6 was strongly associated with 6-month clinical improvement (64% of those ≥ 2.6 improved, versus 15% of those <2.6, P = 0.15). Profunda stenosis score was associated with ABI improvement at 30 days (r = 0.73, P = 0.01) and 6 months (r = 0.82, P = 0.007). Profunda disease length score was associated with clinical improvement at 30 days (P = 0.002). 100% of patients with a profunda disease length score of ≥1.5 clinically improved at 30 days, versus 67% of those with <1.5 (P = 0.04). Angiography scores were not found to be associated with further intervention, major amputation, or mortality. Cronbach alpha for profunda stenosis, profunda disease length, and outflow severity scores were 0.90, 0.90, and 0.79, respectively, indicating strong internal consistency. CONCLUSIONS: This institutional angiography scoring system successfully predicts clinical improvement following CFEP.  Higher profunda stenosis and profunda disease length scores were most predictive of operative success within 6 months. Future validation studies will investigate these outcomes in a larger population, and over a longer period.

Faster and less invasive tools to identify patients with ileal colonization by adherent-invasive E. coli in Crohn's disease.

BACKGROUND AND AIMS: The identification of Crohn's disease (CD)-associated adherent and invasive Escherichia coli (AIEC) is time-consuming and requires ileal biopsies. We aimed to identify a faster and less invasive methods to detect ileal colonization by AIEC in CD patients. METHODS: CD patients requiring ileo-colonoscopy were consecutively enrolled in this prospective multicenter study. Samples from saliva, serum, stools, and ileal biopsies of CD patients were collected. RESULTS: Among 102 CD patients, the prevalence of AIEC on ileal biopsies was 24.5%. The abundance and global invasive ability of ileal-associated total E. coli were respectively ten-fold (p = 0.0065) and two-fold (p = 0.0007) higher in AIEC-positive (vs. AIEC-negative), while abundance of total E. coli in the feces was not correlated with AIEC status in the ileum. The best threshold of ileal total E. coli was 60 cfu/biopsy to detect AIEC-positive patients, with high negative predictive value (NPV) (94.1%[80.3-99.3]), while the global invasive ability (>9000 internalized bacteria) was able to detect the presence of AIEC with high positive predictive value (80.0% [55.2-100.0]). Overall, 78.1% of the AIEC + patients were colonized by two or less different AIEC strains. The level of serum anti-total E. coli antibodies (AEcAb) was higher in AIEC-positive patients (p = 0.038) with a very high negative predictive value (96.6% [89.9-100.0]) (p = 0.038) for a cut-off value > 1.9 × 10-3 . CONCLUSIONS: More than two thirds of AIEC-positive CD patients were colonized by two or less AIEC strains. While stools samples are not accurate to screen AIEC status, the AEcAb level appears to be an attractive, rapid and easier biomarker to identify patients with Crohn's disease harboring AIEC.

Isolation, Gastroprotective Effects and Untargeted Metabolomics Analysis of Lycium Minutifolium J. Remy (Solanaceae).

Lycium minutifolium J. Remy (Solanaceae) is commonly used as an infusion in traditional medicine to treat stomach pain, meteorism, intestinal disorders, stomach ailments, and other severe problems including prostate cancer and stomach cancer. From the EtOAc extract of L. minutifolium bark five known metabolites were isolated using chromatographic techniques. The gastroprotective effects of the EtOAc fraction and edible infusion extract of the bark were assayed on the hydrochloric acid (HCl)/EtOH induced gastric ulcer model in mice to support the traditional use of the plant. The EtOAc extract and the edible infusion showed gastroprotective effect at dose of 100 mg/kg reducing lesions by 31% and 64%, respectively. The gastroprotective action mechanisms of the edible infusion at a single oral dose of 100 mg/kg were evaluated suggesting that prostaglandins, sulfhydryl groups, and nitric oxide are involved in the mode of gastroprotective action. The UHPLC analysis coupled to high-resolution mass spectrometry of the edible infusion showed the presence of twenty-three compounds. Our results can support the gastroprotective properties of the edible infusion extract, and at least can validate in part, the ethnopharmacological uses of the plant.

Racial disparities in late-stage prostate cancer: a SEER analysis 2005-2015.

INTRODUCTION: To evaluate the impact of prostate cancer screening guidelines on different racial and ethnic populations. MATERIALS AND METHODS: Data was collected from the 2005-2015 Surveillance, Epidemiology, and End Results (SEER) program. Incidence of prostate cancer diagnosis was categorized and analyzed by stage, race/ethnicity, and age group. Appropriate univariate and multivariable statistical analysis was performed. RESULTS: The odds of being diagnosed with regional-stage prostate cancer in 2013-2015 were 1.3 times higher for black men, 1.3 times higher for Asian American/Pacific Islander (AAPI) men, and 1.2 times higher for white men when compared to 2005-2008. The odds of being diagnosed with distant-stage prostate cancer in 2013-2015 were 1.6 times higher for black men, 1.8 times higher for AAPI men, and 2.1 times higher for white men when compared to 2005-2008. In 2005-2008, 2009-2012, and 2013-2015 respectively, the odds of being diagnosed with distant-stage prostate cancer were 1.8 times higher, 1.7 times higher, and 1.4 times higher for black men compared to white men, and 1.5 times higher, 1.5 times higher, and 1.4 times higher for AAPI men compared to white men (all respective p < .001). CONCLUSIONS: The proportion of late-stage prostate cancer has increased significantly in all US males regardless of race and/or ethnicity. From 2013-2015, all men had a higher chance of being diagnosed with regional or distant stage disease compared to years prior. Newly-diagnosed regional-stage disease increased the most over time in AAPI and black men, while distant prostate cancer increased the most over time in white men.

EZH2 cooperates with E2F1 to stimulate expression of genes involved in adrenocortical carcinoma aggressiveness.

BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.

A ZNRF3-dependent Wnt/ß-catenin signaling gradient is required for adrenal homeostasis.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/ß-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike ß-catenin gain-of-function models, which induce high Wnt/ß-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/ß-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing ß-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/ß-catenin activation, which is regulated by ZNRF3.

Reducing Electrolyte Testing in Hospitalized Children by Using Quality Improvement Methods.

BACKGROUND AND OBJECTIVES: Despite studies indicating a high rate of overuse, electrolyte testing remains common in pediatric inpatient care. Frequently repeated electrolyte tests often return normal results and can lead to patient harm and increased cost. We aimed to reduce electrolyte testing within a hospital medicine service by >25% within 6 months. METHODS: We conducted an improvement project in which we targeted 6 hospital medicine teams at a large academic children's hospital system by using the Model for Improvement. Interventions included standardizing communication about the electrolyte testing plan and education about the costs and risks associated with overuse of electrolyte testing. Our primary outcome measure was the number of electrolyte tests per patient day. Secondary measures included testing charges and usage rates of specific high-charge panels. We tracked medical emergency team calls and readmission rates as balancing measures. RESULTS: The mean baseline rate of electrolyte testing was 2.0 laboratory draws per 10 patient days, and this rate decreased by 35% after 1 month of initial educational interventions to 1.3 electrolyte laboratory draws per 10 patient days. This change has been sustained for 9 months and could save an estimated $292 000 in patient-level charges over the course of a year. Use of our highest-charge electrolyte panel decreased from 67% to 22% of testing. No change in rates of medical emergency team calls or readmission were found. CONCLUSIONS: Our improvement intervention was associated with significant and rapid reduction in electrolyte testing and has not been associated with unintended adverse events.

DNA Methylation Is an Independent Prognostic Marker of Survival in Adrenocortical Cancer.

CONTEXT: Adrenocortical cancer (ACC) is an aggressive tumor with a heterogeneous outcome. Prognostic stratification is difficult even based on tumor stage and Ki67. Recently integrated genomics studies have demonstrated that CpG islands hypermethylation is correlated with poor survival. OBJECTIVE: The goal of this study was to confirm the prognostic value of CpG islands methylation on an independent cohort. DESIGN: Methylation was measured by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). SETTING: MS-MLPA was performed in a training cohort of 50 patients with ACC to identify the best set of probes correlating with disease-free survival (DFS) and overall survival (OS). These outcomes were validated in an independent cohort from 21 ENSAT centers. PATIENTS: The validation cohort included 203 patients (64% women, median age 50 years, 80% localized tumors). MAIN OUTCOME MEASURES: DFS and OS. RESULTS: In the training cohort, mean methylation of 4 genes (PAX5, GSTP1, PYCARD, PAX6) was the strongest methylation marker. In the validation cohort, methylation was a significant prognostic factor of DFS (P < 0.0001) and OS (P < 0.0001). Methylation, Ki67, and ENSAT stage were combined in multivariate models. For DFS, methylation (P = 0.0005) and stage (P < 0.0001) but not Ki67 (P = 0.19) remained highly significant. For OS, methylation (P = 0.0006), stage (P < 0.0001), and Ki67 (P = 0.024) were independent prognostic factors. CONCLUSIONS: Tumor DNA methylation emerges as an independent prognostic factor in ACC. MS-MLPA is readily compatible with clinical routine and should enhance our ability for prognostication and precision medicine.

Boron Trifluoride Gas Adsorption in Metal-Organic Frameworks.

Coordinatively unsaturated metal-organic frameworks (MOFs) were studied for boron trifluoride (BF3) sorption. MOF-74-Mg, MOF-74-Mn, and MOF-74-Co show high initial uptake (below 6.7 × 10-3 bar) with negligible deliverable capacity. The BF3 isotherm of MOF-74-Cu exhibits gradual uptake up to 0.9 bar and has a deliverable gravimetric capacity that is more than 100% higher than activated carbon. Two other Cu2+ MOFs, MOF-505 and HKUST-1, have slightly lower deliverable capacities compared to MOF-74-Cu.

PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development.

Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates ß-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling.

EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progression.

Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.

BAP1 Immunohistochemistry and p16 FISH in the Diagnosis of Sarcomatous and Desmoplastic Mesotheliomas.

The separation of sarcomatous and desmoplastic mesotheliomas from benign organizing pleuritis can be morphologically very difficult. Deletion of p16 (CDKN2A) by fluorescence in situ hybridization (FISH) testing appears to be a reliable marker of malignancy in mesothelial proliferations, and more recently it has been reported that, in this setting, loss of BAP1 by immunohistochemistry is only seen in malignant mesotheliomas. To determine how useful these tests are with sarcomatous and desmoplastic mesotheliomas, we examined 20 such tumors. Loss of BAP1 was seen in 3/20 (15%) and deletion of p16 by FISH was seen in 16/20 (80%) cases. Loss of one or the other marker was observed in 17/20 (85%). We also examined 13 sarcomatoid carcinomas, an important differential diagnosis of sarcomatoid mesotheliomas, and found that BAP1 was never lost, but p16 was deleted in 3/11 (27%). We conclude that: (1) BAP1 immunohistochemistry is relatively insensitive in the context of sarcomatous and desmoplastic mesotheliomas, but as a matter of time and cost efficiency may nonetheless be a useful first approach to the problem; (2) deletion of p16 by FISH is considerably more sensitive, but there remain a proportion of cases in which p16 is not deleted; (3) a small improvement in sensitivity can be achieved by using both markers; (4) in the context of a spindle cell malignant tumor in the pleura or peritoneum, which morphologically might be a metastatic sarcomatoid carcinoma or a mesothelioma, the finding of BAP1 loss favors mesothelioma, but p16 FISH cannot be used to separate sarcomatous mesotheliomas from sarcomatoid carcinomas.

Utility of BAP1 Immunohistochemistry and p16 (CDKN2A) FISH in the Diagnosis of Malignant Mesothelioma in Effusion Cytology Specimens.

The diagnosis of malignant mesothelioma in effusion cytology specimens is controversial. BAP1 immunohistochemistry and p16 fluorescence in situ hybridization (FISH) have recently been reported as reliable markers of malignancy in biopsies of mesothelioma. To determine whether these markers, singly or in combination, might also be useful in effusion cytology specimens, we examined 15 biopsies of epithelial mesotheliomas and 3 benign mesothelial reactions and corresponding effusion cytology paraffin-embedded cell blocks. Four cytology specimens were too scanty for p16 FISH analysis but were interpretable for BAP1 immunohistochemistry. Overall, loss of BAP1 and/or deletion of p16 was seen in 11/11 (100%) of matched cytology and tissue biopsy specimens. BAP1 loss alone was seen in 10/15 (67%) biopsies and 10/15 (67%) cytology specimens. Homozygous deletion of p16 by FISH was found in 12/15 (80%) biopsy specimens and 8/11 (73%) evaluable cytology specimens. Seven of 15 (47%) biopsies and 5/11 (42%) cytology specimens showed loss of both markers. All mesothelioma biopsy/cytology pairs showed exactly the same pattern of BAP1 or p16 retention or loss in the biopsy and cytology specimens. The 2 peritoneal mesothelioma cases demonstrated loss of BAP1 but not p16. None of the benign mesothelial reactions or corresponding cytology specimens showed loss of either marker. We conclude that both BAP1 immunohistochemistry and p16 FISH analysis provide reliable markers of mesothelial malignancy in effusion cytology specimens, especially where the atypical mesothelial proliferation is well sampled. BAP1 is easier to interpret with scanty specimens. On the basis of small numbers of cases, use of both markers appears to increase sensitivity.

BAP1 immunohistochemistry and p16 FISH to separate benign from malignant mesothelial proliferations.

A variety of immunohistochemical (IHC) stains have been proposed to mark either benign or malignant mesothelial proliferations. Loss of the p16 tumor suppressor (CDKN2A), through homozygous deletions of 9p21, is a good marker of mesotheliomas but lacks sensitivity. Recent reports indicate that some mesotheliomas are associated with loss of BRCA-associated protein 1 (BAP1) expression. Here we investigate BAP1 and p16 as potential markers of malignancy and compare test characteristics with previously proposed markers using a well-characterized tissue microarray. BAP1 protein expression was interrogated by IHC. The p16 locus was examined by fluorescence in situ hybridization (FISH) directed toward chromosome 9p21. Loss of BAP1 was identified in 7/26 mesotheliomas and 0/49 benign proliferations. Loss of p16 was identified in 14/27 mesotheliomas and 0/40 benign proliferations, yielding 100% specificity and positive predictive value for each marker. Together, BAP1 IHC and p16 FISH were 58% sensitive for detecting malignancy. Various combinations of p53, EMA, IMP3, and GLUT1 showed reasonably high specificity (96% to 98%) but poor to extremely poor sensitivity. Combined BAP1 IHC/p16 FISH testing is a highly specific method of diagnosing malignant mesotheliomas when the question is whether a mesothelial proliferation is benign or malignant and is particularly useful when tissue invasion by mesothelial cells cannot be demonstrated. However, combined BAP1/p16 FISH testing is not highly sensitive, and negative results do not rule out a mesothelioma. The test characteristics of previously proposed markers EMA, p53, GLUT1, IMP3 suggest that, even in combination, these markers are not useful tools in this clinical setting.

Self-pMHCII complexes are variably expressed in the thymus and periphery independent of mRNA expression but dependent on the activation state of the APCs.

Self-peptide MHCII ligands are critical for selection of CD4+ T cells in the thymus, and maintenance in the periphery. To date, no investigation as to the exact thymic and peripheral expression of a naturally occurring positive selecting self-peptide MHCII (self-pMHCII) complex has taken place. We have generated a sensitive T cell hybridoma to functionally detect the endogenous presentation of a confirmed positive selecting self-pMHCII complex for a CD4+ transgenic T cell. Using this tool to survey and quantify the expression selecting of self-pMHCII, we have shown unequivocal proof that a known CD4+ selecting ligand can be presented on both positive and negative selecting thymic APCs. We also show that peripheral presentation of this same selecting ligand is affected by the activation state of the APCs. Furthermore, discrepancies between the gene expression and self-pMHCII complex presentation of this bona fide selecting ligand suggest that functional detection self-ligand complexes will be required to establish a complete view of the naturally presented endogenous self-pMHC landscape.

Integrated genomic characterization of adrenocortical carcinoma.

Adrenocortical carcinomas (ACCs) are aggressive cancers originating in the cortex of the adrenal gland. Despite overall poor prognosis, ACC outcome is heterogeneous. We performed exome sequencing and SNP array analysis of 45 ACCs and identified recurrent alterations in known driver genes (CTNNB1, TP53, CDKN2A, RB1 and MEN1) and in genes not previously reported in ACC (ZNRF3, DAXX, TERT and MED12), which we validated in an independent cohort of 77 ACCs. ZNRF3, encoding a cell surface E3 ubiquitin ligase, was the most frequently altered gene (21%) and is a potential new tumor suppressor gene related to the ß-catenin pathway. Our integrated genomic analyses further identified two distinct molecular subgroups with opposite outcome. The C1A group of ACCs with poor outcome displayed numerous mutations and DNA methylation alterations, whereas the C1B group of ACCs with good prognosis displayed specific deregulation of two microRNA clusters. Thus, aggressive and indolent ACCs correspond to two distinct molecular entities driven by different oncogenic alterations.

p16 FISH deletion in surface epithelial mesothelial proliferations is predictive of underlying invasive mesothelioma.

An atypical mesothelial proliferation along the pleural or peritoneal surface without evidence of invasive tumor poses a diagnostic challenge. Homozygous deletion of p16 (CDKN2A) by fluorescence in situ hybridization (FISH) has been shown to be a good marker of malignancy in mesothelial proliferations, but correlations of p16 status between atypical surface proliferations and underlying mesothelioma have not been described. We used p16 FISH to investigate 11 pleural and 7 peritoneal mesotheliomas that had both an invasive component and a separate surface mesothelial proliferation. In 5/11 pleural samples and 1/7 peritoneal samples, the invasive mesotheliomas showed homozygous deletion of p16 (all cases in excess of 90% of cells deleted); the surface proliferation in all 6 cases with deletion in the invasive tumor was also p16 deleted. Conversely, the 12 tumors that did not show p16 deletion in the invasive compartment also did not have deletion in the surface component. We conclude that (1) surface mesothelial proliferations near invasive mesotheliomas show the same pattern of p16 by FISH as the underlying tumor and may represent in situ disease or growth of the underlying mesothelioma along the serosal surface; (2) p16 deletion in mesothelial surface proliferations is strongly associated with p16 deletion in underlying mesotheliomas, and biopsies consisting of pure surface mesothelial proliferations that are p16 deleted allow a diagnosis of mesothelioma without an additional biopsy if there is clinical (thoracosopic/laparoscopic) or radiologic evidence of diffuse pleural or peritoneal tumor; (3) however, the absence of p16 deletion in surface proliferations does not rule out underlying invasive mesothelioma.

ARMC5 mutations in macronodular adrenal hyperplasia with Cushing's syndrome.

BACKGROUND: Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushing's syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. METHODS: We genotyped blood and tumor DNA obtained from 33 patients with corticotropin-independent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. RESULTS: The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. CONCLUSIONS: Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumor-suppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.).

Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography-tandem mass spectrometry.

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 µL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.