An effective approach for BRAF V600E mutation analysis of routine thyroid fine needle aspirates.

INTRODUCTION: Molecular testing for genetic alterations in thyroid neoplasms, including BRAF V600E (BRAF) mutation, are often applied to thyroid aspirates falling into the Bethesda System for Reporting Thyroid Cytopathology indeterminate categories. Current methods typically use dedicated aspirated material, without morphological determination of containing the cells of interest and may be of elevated cost. We describe our experience with BRAF mutation analysis on material obtained from Papanicolaou (PAP)-stained ThinPrep® (TP) slides. METHODS: Eighty-three cases collected between 2012 and 2019 with more than 100 cells were selected. An electronic record of a whole slide scan was made for each case before testing. The coverslips were removed, and DNA was extracted from material scraped from each slide using the Qiagen QIAamp DNA FFPE Tissue Kit. BRAF testing was performed using a highly sensitive mutation detection assay, either COLD-PCR, castPCR, or droplet digital PCR. RESULTS: Fourteen out of 83 cases had a BRAF mutation. Of these, 8 were classified as atypia of undetermined significance or suspicious for malignancy in which follow-up showed conventional papillary thyroid carcinoma in 5 out of 6 cases. The specificity and positive predictive value were 97% and 91%, respectively. CONCLUSIONS: BRAF mutation analysis can be performed on material obtained from routine clinical PAP-stained TP slides. As a first step, this unconventional effective approach may reduce costs related to the molecular evaluation of thyroid nodule aspirates and provides the opportunity for cytomorphological confirmation that the cells of interest are present in material submitted for BRAF mutation analysis.

Adenoid cystic carcinoma of the salivary gland metastasizing to the pericardium and diaphragm: Report of a rare case.

BACKGROUND: Adenoid cystic carcinoma (AdCC) is an uncommon malignancy of the salivary gland characterized by slow growth, increased risk of recurrence and poor prognosis. The annual incidence in the United States is approximately 1200 cases per year and rarely involves the body cavities. CASE PRESENTATION: We present a case of a 48-year-old male diagnosed with AdCC of the left submandibular gland. He received his last chemotherapy in 2006 and presented with pleural metastasis. After undergoing pleurectomy and decortication procedure, pericardial fluid and biopsies from the chest wall, sixth rib, diaphragm, pleural cavity and pericardium were sent for pathologic evaluation. A diagnosis of metastatic adenoid cystic carcinoma was confirmed, including in the pericardium, pericardial fluid and diaphragm. CONCLUSION: AdCC of the submandibular gland is a malignant tumor with a high mortality rate. It is very rare for AdCC to metastasize to the pericardium and diaphragm. Metastasis to uncommon sites such as seen in our case with metastases to the pericardium and diaphragm shows the aggressive and unpredictable nature of this tumor, requiring close follow up, and indicating the need for molecular profile analysis and biomarker-stratified clinical trials.

Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles.

Type II alveolar epithelial cells (AEC2s) play an essential role in the function and maintenance of the pulmonary epithelium. Several transgenic mice have been developed to study the function of these cells in vivo by using the human SFTPC promoter to drive expression of Cre recombinase. The precise activity of each of these transgenic alleles has not been studied, and previous reports suggest that their activity can depend on breeding strategies. We bred mice with a conditional allele of the essential telomere capping protein TRF2 with two different SFTPC-Cre-transgenic strains and observed opposite phenotypes (100% lethality vs. 100% viability). We characterized the Cre recombinase activity in these two transgenic lines and found that the contrasting phenotypes were driven by difference in embryonic expression of the two transgenes, likely due to position effects or differences in the transgenic constructs. We also tested if SFTPC-Cre activity was dependent on maternal or paternal inheritance. When paternally inherited, both SFTPC-Cre alleles produced offspring with constitutive reporter activity independent of the inheritance of the Cre allele, suggesting that Cre recombinase was expressed in the male germline before meiosis. Immunohistochemical analysis of the testis showed reporter activity during spermatogenesis. Analysis of single-cell RNA sequencing data from murine and human testis demonstrated SFTPC expression uniquely during human spermatogenesis, suggesting that use of the human promoter in these constructs is responsible for male germline activity. Our data highlight the importance of careful analysis of transgenic allele activity and identify an SFTPC-Cre allele that is useful for panepithelial targeting in the mouse.

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

INTRODUCTION: BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. METHODS: A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. RESULTS: BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 µM to greater than 50 µM and 0.039 µM to greater than 5 µM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. CONCLUSIONS: BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

BACKGROUND: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. PROCEDURE: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. RESULTS: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. CONCLUSIONS: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

Serologic Profile of Antiparietal Cell Antibodies, Pepsinogens, and H. pylori and Risk of Upper Gastrointestinal Cancer: A Nested Case-Control Study in China.

BACKGROUND: Autoimmune gastritis is understudied and possibly associated with gastric noncardia adenocarcinoma (GNCA) and esophageal squamous cell carcinoma (ESCC) in Western populations when it presents as pernicious anemia. METHODS: A nested case-control study within a Chinese cohort included 100 ESCC, 200 gastric cardia adenocarcinoma (GCA), and 200 GNCA cases diagnosed between 1986 and 2001 and 400 controls. Serostatus of antiparietal cell antibodies (APCA), Helicobacter pylori antibodies, and pepsinogens were measured using commercial kits and serum collected at baseline. We used logistic regression to calculate odds ratios (OR) and 95% confidence interval (CI) for associations between serologic biomarkers and cancer risk adjusted for numerous potential confounders. RESULTS: There was an average interval of 8 years between baseline blood draw and cancer diagnosis. The baseline prevalence of APCA seropositivity was 10.0% and 14.5% in subjects who developed GCA and GNCA, respectively. APCA seropositivity was inversely associated with later development of GCA (OR = 0.42; 95% CI, 0.24-0.75), but not significantly associated with later development of GNCA (OR = 0.82; 95% CI, 0.50-1.36) or ESCC (OR = 1.05; 95% CI, 0.58-1.88). APCA seropositivity was significantly associated with low pepsinogen I/II ratios (OR = 3.69; 95% CI, 1.66-8.21), and individuals with low pepsinogen I/II ratios who were seronegative for APCA had the highest risk of both GCA and GNCA. CONCLUSIONS: APCA seropositivity measured years prior to diagnosis was associated with prevalent atrophic gastritis but inversely associated with incident GCA in this Chinese population. IMPACT: APCA may contribute to a growing list of serologic markers that can improve risk stratification for gastric cancer.

GDF15 is an epithelial-derived biomarker of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

Composite Graft Pretreatment With Hydrogen Sulfide Delays the Onset of Acute Rejection.

INTRODUCTION: Vascularized composite allotransplantation can reconstruct devastating tissue loss by replacing like-with-like tissues, most commonly in the form of hand or face transplantation. Unresolved technical and ethical challenges have meant that such transplants remain experimental treatments. The most significant barrier to expansion of this field is the requirement for systemic immunosuppression, its toxicity and effect on longevity.Hydrogen sulfide (H2S) has been shown experimentally to ameliorate the ischemia reperfusion injury associated with composite tissue autotransplantation, which has been linked to acute rejection in solid organ transplantation. In this protocol, a large-animal model was used to evaluate the effect of H2S on acute rejection after composite tissue allotransplantation. MATERIALS AND METHODS: A musculocutaneous flap model in SLA-mismatched swine was used to evaluate acute rejection of allotransplants in 2 groups: control animals (n = 8) and a treatment group in which the allografts were pretreated with hydrogen sulfide (n = 8). Neither group was treated with systemic immunosuppression. Acute rejection was graded clinically and histopathologically by an independent, blinded pathologist. Data were analyzed by t tests with correction for multiple comparisons by the Holm-Sídák method. RESULTS: Clinically, H2S-treated tissue composites showed a delay in the onset of rejection that was statistically significant from postoperative day 6. Histopathologically, this difference between groups was also apparent, although evidence of a difference in groups disappeared beyond day 10. CONCLUSIONS: Targeted hydrogen sulfide treatment of vascularized composite allografts immediately before transplantation can delay acute rejection. This may, in turn, reduce or obviate the requirement for systemic immunosuppression.

Impact of a novel phosphoinositol-3 kinase inhibitor in preventing mitochondrial DNA damage and damage-associated molecular pattern accumulation: Results from the Biochronicity Project.

BACKGROUND: Despite improvements in the management of severely injured patients, development of multiple organ dysfunction syndrome (MODS) remains a morbid complication of traumatic shock. One of the key attributes of MODS is a profound bioenergetics crisis, for which the mediators and mechanisms are poorly understood. We hypothesized that metabolic uncoupling using an experimental phosphoinositol-3 kinase (PI3-K) inhibitor, LY294002 (LY), may prevent mitochondrial abnormalities that lead to the generation of mitochondrial DNA (mtDNA) damage and the release of mtDNA damage-associated molecular patterns (DAMPs). METHODS: Sixteen swine were studied using LY, a nonselective PI3-K inhibitor. Animals were assigned to trauma only (TO, n = 3), LY drug only (LYO, n = 3), and experimental (n = 10), trauma + drug (LY + T) groups. Both trauma groups underwent laparotomy, 35% hemorrhage, severe ischemia-reperfusion injury, and protocolized resuscitation. A battery of hemodynamic, laboratory, histological, and bioenergetics parameters were monitored. Mitochondrial DNA damage was determined in lung, liver, and kidney using Southern blot analyses, whereas plasma mtDNA DAMP analysis used polymerase chain reaction amplification of a 200-bp sequence of the mtDNA D-loop region. RESULTS: Relative to control animals, H + I/R (hemorrhage and ischemia/reperfusion) produced severe, time-dependent decrements in hepatic, renal, cardiovascular, and pulmonary function accompanied by severe acidosis and lactate accumulation indicative of bioenergetics insufficiency. The H-I/R animals displayed prominent oxidative mtDNA damage in all organs studied, with the most prominent damage in the liver. Mitochondrial DNA damage was accompanied by accumulation of mtDNA DAMPs in plasma. Pretreatment of H + I/R animals with LY resulted in profound metabolic suppression, with approximately 50% decreases in O2 consumption and CO2 production. In addition, it prevented organ and bioenergetics dysfunction and was associated with a significant decrease in plasma mtDNA DAMPs to the levels of control animals. CONCLUSIONS: These findings show that H + I/R injury in anesthetized swine is accompanied by MODS and by significant mitochondrial bioenergetics dysfunction, including oxidative mtDNA damage and accumulation in plasma of mtDNA DAMPs. Suppression of these changes with the PI3-K inhibitor LY indicates that pharmacologically induced metabolic uncoupling may comprise a new pharmacologic strategy to prevent mtDNA damage and DAMP release and prevent or treat trauma-related MODS. LEVEL OF EVIDENCE: Therapeutic study, level III.

Structure-Cytotoxicity Relationships of Analogues of N14-Desacetoxytubulysin H.

Herein we report structure-cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.

Inducing metabolic suppression in severe hemorrhagic shock: Pilot study results from the Biochronicity Project.

BACKGROUND: Suspended animation-like states have been achieved in small animal models, but not in larger species. Inducing metabolic suppression and temporary oxygen independence could enhance survivability of massive injury. Based on prior analyses of key pathways, we hypothesized that phosphoinositol-3-kinase inhibition would produce metabolic suppression without worsening organ injury or systemic physiology. METHODS: Twenty swine were studied using LY294002 (LY), a nonselective phosphoinositol-3-kinase inhibitor. Animals were assigned to trauma only (TO, n = 3); dimethyl sulfoxide only (DMSO, n = 4), LY drug only (LYO, n = 3), and drug + trauma (LY + T, n = 10) groups. Both trauma groups underwent laparotomy, 35% hemorrhage, severe ischemia/reperfusion injury, and protocolized resuscitation. Laboratory, physiologic, cytokine, and metabolic cart data were obtained. Histology of key end organs was also compared. RESULTS: Baseline values were similar among the groups. Compared with the TO group, the LYO group had reversible decreases in heart rate, mean arterial pressure, cardiac output, oxygen consumption, and carbon dioxide production. Compared with TO, LY + T showed sustained decreases in heart rate (113 vs. 76, p = 0.03), mean arterial pressure (40 vs. 31 mm Hg, p = 0.02), and cardiac output (3.8 vs. 1.9 L/min, p = 0.05) at 6 hours. Metabolic parameters showed profound suppression in the LY + T group. Oxygen consumption in LY + T was lower than both TO (119 vs. 229 mL/min, p = 0.012) and LYO (119 vs. 225 mL/min, p = 0.014) at 6 hours. Similarly, carbon dioxide production was decreased at 6 hours in LY + T when compared with TO (114 vs. 191 mL/min, p = 0.043) and LYO (114 vs. 195 mL/min, p = 0.034) groups. There was no worsening of acidosis (lactate 6.4 vs. 8.3 mmol/L, p = 0.4) or other endpoints. Interleukin 6 (IL-6) showed a significant increase in LY + T when compared with TO at 6 hours (60.5 vs. 2.47, p = 0.043). Tumor necrosis factor α and IL-1ß were decreased, and IL-10 increased in TO and LY + T at 6 hours. Markers of liver and kidney injury were no different between TO and LY + T groups at 6 hours. CONCLUSIONS: Phosphoinositol-3-kinase inhibition produced metabolic suppression in healthy and injured swine without increasing end-organ injury or systemic physiologic markers and demonstrated prolonged efficacy in injured animals. Further study may lead to targeted therapies to prolong tolerance to hemorrhage and extend the "golden hour" for injured patients.

High-Throughput Microdissection for Next-Generation Sequencing.

Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.

WU polyomavirus in respiratory epithelial cells from lung transplant patient with Job syndrome.

We detected WU polyomavirus (WUPyV) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with Job syndrome by using immunoassays specific for the WUPyV viral protein 1. Co-staining for an epithelial cell marker identified most WUPyV viral protein 1-positive cells as respiratory epithelial cells.

Hydrogen sulfide mitigates reperfusion injury in a porcine model of vascularized composite autotransplantation.

BACKGROUND: Devastating extremity injuries are prevalent but often survivable on the modern battlefield. These complex injuries require advanced methods of reconstruction, involving prolonged ischemic periods and reperfusion injury. Using our group's validated porcine model of gracilis myocutaneous flap transplantation, this study demonstrates that an interim perfusion of hydrogen sulfide (H2S) mitigates the effects of reperfusion injury in the setting of delayed restoration of blood flow. METHODS: A gracilis myocutaneous flap (200-400 g; surface area, 250 cm²) was procured from the hind limb of a Yorkshire swine (70-90 kg, n=16). The right external carotid artery and the internal jugular vein are the recipient axis. Group 1 (control, n = 6) underwent delayed anastomosis with a 3-hour ischemic period. Group 2 (n=10) underwent a similar delayed anastomosis with an interim perfusion of H2S during the ischemic period. The animals survived for 14 days. Systemic biomarker assays for skeletal muscle tissue injury (creatine kinase, lactate dehydrogenase, and aspartate transaminase) and proinflammatory markers (tumor necrosis factor α and interleukin 6) provide assessment of reperfusion injury at the cellular level. RESULTS: The control animals (3 hours of ischemia with an interim perfusion of heparinized saline) demonstrated increased levels of injury biomarkers and proinflammatory cytokines compared with the animals receiving H2S infusion and identical ischemic interval. The control flaps had a mean creatine kinase level of 280³×10 U/L (±80×10³), compared with the H2S group, which had a mean of 99×10³ U/L (±14×10³; P=0.0007 at postoperative day 2). lactate dehydrogenase levels (mean) were 26×10³ U/L (±8×10³) versus 9×10³ U/L (±3×10³; P=0.0004) and aspartate transaminase levels (mean) were 1651 U/L (±324) versus (873 U/L [±279]; P=0.0013) for the control and treatment groups, respectively. Similarly, an intergroup difference in IL-6 was found, although not statistically significant. Tumor necrosis factor α levels (mean) were 93 pg/mL (±14) versus 39 pg/mL (±4; P=0.0013) for the control and treatment groups, respectively. CONCLUSIONS: This study demonstrated the mitigating properties of H2S on reperfusion injury. Interim perfusion with H2S resulted in diminution of ischemia-dependent biomarkers after 3 hours of ischemia. Follow-up studies will translate these findings as an evolving method for reconstructing previously unreconstructable injuries.

Measuring telomere length for the early detection of precursor lesions of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is the sixth leading cause of cancer death worldwide; current early detection screening tests are inadequate. Esophageal balloon cytology successfully retrieves exfoliated and scraped superficial esophageal epithelial cells, but cytologic reading of these cells has poor sensitivity and specificity for detecting esophageal squamous dysplasia (ESD), the precursor lesion of esophageal squamous cell carcinoma (ESCC). Measuring telomere length, a marker for chromosomal instability, may improve the utility of balloon cytology for detecting ESD and early ESCC. METHODS: We examined balloon cytology specimens from 89 asymptomatic cases of ESD (37 low-grade and 52 high-grade) and 92 age- and sex-matched normal controls from an esophageal cancer early detection screening study. All subjects also underwent endoscopy and biopsy, and ESD was diagnosed histopathologically. DNA was extracted from the balloon cytology cells, and telomere length was measured by quantitative PCR. A receiver operating characteristic (ROC) curve was plotted for telomere length as a diagnostic marker for high-grade dysplasia. RESULTS: Telomere lengths were comparable among the low- and high-grade dysplasia cases and controls, with means of 0.96, 0.96, and 0.92, respectively. The area under the ROC curve was 0.55 for telomere length as a diagnostic marker for high-grade dysplasia. Further adjustment for subject characteristics, including sex, age, smoking, drinking, hypertension, and body mass index did not improve the use of telomere length as a marker for ESD. CONCLUSIONS: Telomere length of esophageal balloon cytology cells was not associated with ESCC precursor lesions. Therefore, telomere length shows little promise as an early detection marker for ESCC in esophageal balloon samples.

Mechanism and in vitro pharmacology of TAK1 inhibition by (5Z)-7-Oxozeaenol.

Transforming growth factor-ß activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.

A multi-day environmental study of polycyclic aromatic hydrocarbon exposure in a high-risk region for esophageal cancer in China.

Linzhou, China has one of the highest rates of esophageal squamous cell carcinoma in the world. Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), may have a role in this increased risk. To better understand PAH sources, we measured PAHs in the air and food of 20 non-smokers over multiple days and compared the concentrations with a urinary PAH biomarker, 1-hydroxypyrene glucuronide (1-OHPG). Sampling occurred over 4 consecutive days. Kitchen air samples (days 2-3) and duplicate diet samples (days 1-4) were analyzed for 14 or more unique PAHs, including BaP. Daily urine samples (days 1-3) were analyzed for 1-OHPG. Mixed-effects models were used to evaluate the associations between air or food PAH concentrations and urine 1-OHPG concentrations. The median kitchen air BaP concentration was 10.2 ng/m(3) (interquartile range (IQR): 5.1-20.2 ng/m(3)). The median daily food BaP concentration and intake were 0.08 ng/g (IQR=0.04-0.16 ng/g) and 86 ng/day (IQR=41-142 ng/day), respectively. The median 1-OHPG concentration was 3.36 pmol/ml (IQR=2.09-6.98 pmol/ml). In mixed-effects models, 1-OHPG concentration increased with same-day concentration of food BaP (P=0.07). Although PAH concentrations in air were not associated with 1-OHPG concentrations, the high concentrations of PAHs in both air and food suggest that they are both important routes of exposure to PAHs in this population. Further evaluation of the role of PAH exposure from air and food in the elevated rates of esophageal cancer in this region is warranted.