Adam C. Searleman

Anesthesia awareness and the bispectral index.

BACKGROUND Awareness during anesthesia is a serious complication with potential long-term psychological consequences. Use of the bispectral index (BIS), developed from a processed electroencephalogram, has been reported to decrease the incidence of anesthesia awareness when the BIS value is maintained below 60. In this trial, we sought to determine whether a BIS-based protocol is better than a protocol based on a measurement of end-tidal anesthetic gas (ETAG) for decreasing anesthesia awareness in patients at high risk for this complication. METHODS We randomly assigned 2000 patients to BIS-guided anesthesia (target BIS range, 40 to 60) or ETAG-guided anesthesia (target ETAG range, 0.7 to 1.3 minimum alveolar concentration [MAC]). Postoperatively, patients were assessed for anesthesia awareness at three intervals (0 to 24 hours, 24 to 72 hours, and 30 days after extubation). RESULTS We assessed 967 and 974 patients from the BIS and ETAG groups, respectively. Two cases of definite anesthesia awareness occurred in each group (absolute difference, 0%; 95% confidence interval [CI], -0.56 to 0.57%). The BIS value was greater than 60 in one case of definite anesthesia awareness, and the ETAG concentrations were less than 0.7 MAC in three cases. For all patients, the mean (+/-SD) time-averaged ETAG concentration was 0.81+/-0.25 MAC in the BIS group and 0.82+/-0.23 MAC in the ETAG group (P=0.10; 95% CI for the difference between the BIS and ETAG groups, -0.04 to 0.01 MAC). CONCLUSIONS We did not reproduce the results of previous studies that reported a lower incidence of anesthesia awareness with BIS monitoring, and the use of the BIS protocol was not associated with reduced administration of volatile anesthetic gases. Anesthesia awareness occurred even when BIS values and ETAG concentrations were within the target ranges. Our findings do not support routine BIS monitoring as part of standard practice. (ClinicalTrials.gov number, NCT00281489 [ClinicalTrials.gov].).

Activating HER2 Mutations in HER2 Gene Amplification Negative Breast Cancer

UNLABELLED Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. SIGNIFICANCE We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

Prevalence of undiagnosed obstructive sleep apnea among adult surgical patients in an academic medical center.

BACKGROUND Obstructive sleep apnea (OSA) affects approximately 20% of US adults, of whom about 90% are undiagnosed. While OSA may increase risk of perioperative complications, its prevalence among surgical patients is unknown. We tested the feasibility of screening surgical patients for OSA and determined the prevalence of undiagnosed OSA. METHODS In a prospective, observational study adult surgical patients were screened for OSA in an academic hospital. Patients without an OSA diagnosis who screened high-risk were offered a home sleep study to determine if they had OSA. The results were compared with polysomnography (PSG) when available. Charts of high-risk patients were examined for postoperative complications. High-risk patients received targeted interventions as part of a hospital safety initiative. RESULTS There were 2877 patients screened; 661 (23.7%) screened high-risk for OSA, of whom 534 (81%) did not have diagnosed OSA. The portable sleep study detected OSA in 170/207 (82%) high-risk patients without diagnosed OSA. Twenty-six PSGs confirmed OSA in 19 of these patients. Postoperatively there were no respiratory arrests, two unanticipated ICU admissions, and five documented respiratory complications. CONCLUSION Undiagnosed OSA is prevalent in adult surgical patients. Implementing universal screening is feasible and can identify undiagnosed OSA in many surgical patients. Further investigation is needed into perioperative complications and their prevention for patients with undiagnosed OSA.

Long-term cognitive decline in older subjects was not attributable to noncardiac surgery or major illness.

Background:Persistent postoperative cognitive decline is thought to be a public health problem, but its severity may have been overestimated because of limitations in statistical methodology. This study assessed whether long-term cognitive decline occurred after surgery or illness by using an innovative approach and including participants with early Alzheimer disease to overcome some limitations. Methods:In this retrospective cohort study, three groups were identified from participants tested annually at the Washington University Alzheimers Disease Research Center in St. Louis, Missouri: those with noncardiac surgery, illness, or neither. This enabled long-term tracking of cognitive function before and after surgery and illness. The effect of surgery and illness on longitudinal cognitive course was analyzed using a general linear mixed effects model. For participants without initial dementia, time to dementia onset was analyzed using sequential Cox proportional hazards regression. Results:Of the 575 participants, 214 were nondemented and 361 had very mild or mild dementia at enrollment. Cognitive trajectories did not differ among the three groups (surgery, illness, control), although demented participants declined more markedly than nondemented participants. Of the initially nondemented participants, 23% progressed to a clinical dementia rating greater than zero, but this was not more common after surgery or illness. Conclusions:The study did not detect long-term cognitive decline independently attributable to surgery or illness, nor were these events associated with accelerated progression to dementia. The decision to proceed with surgery in elderly people, including those with early Alzheimer disease, may be made without factoring in the specter of persistent cognitive deterioration.

HER2 Activating Mutations Are Targets for Colorectal Cancer Treatment

UNLABELLED The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. SIGNIFICANCE HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.

A pharmacologic inhibitor of the protease Taspase1 effectively inhibits breast and brain tumor growth.

The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.

Statistically identifying tumor suppressors and oncogenes from pan-cancer genome-sequencing data

MOTIVATION Several tools exist to identify cancer driver genes based on somatic mutation data. However, these tools do not account for subclasses of cancer genes: oncogenes, which undergo gain-of-function events, and tumor suppressor genes (TSGs) which undergo loss-of-function. A method which accounts for these subclasses could improve performance while also suggesting a mechanism of action for new putative cancer genes. RESULTS We develop a panel of five complementary statistical tests and assess their performance against a curated set of 99 HiConf cancer genes using a pan-cancer dataset of 1.7 million mutations. We identify patient bias as a novel signal for cancer gene discovery, and use it to significantly improve detection of oncogenes over existing methods (AUROC = 0.894). Additionally, our test of truncation event rate separates oncogenes and TSGs from one another (AUROC = 0.922). Finally, a random forest integrating the five tests further improves performance and identifies new cancer genes, including CACNG3, HDAC2, HIST1H1E, NXF1, GPS2 and HLA-DRB1. AVAILABILITY AND IMPLEMENTATION All mutation data, instructions, functions for computing the statistics and integrating them, as well as the HiConf gene panel, are available at www.github.com/Bose-Lab/Improved-Detection-of-Cancer-Genes. CONTACT rbose@dom.wustl.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer.

Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer‐based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2‐driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC‐MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC‐MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well‐known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA‐approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens.

Taspase1-dependent TFIIA cleavage coordinates head morphogenesis by limiting Cdkn2a locus transcription

Head morphogenesis requires complex signal relays to enable precisely coordinated proliferation, migration, and patterning. Here, we demonstrate that, during mouse head formation, taspase1-mediated (TASP1-mediated) cleavage of the general transcription factor TFIIA ensures proper coordination of rapid cell proliferation and morphogenesis by maintaining limited transcription of the negative cell cycle regulators p16Ink4a and p19Arf from the Cdkn2a locus. In mice, loss of TASP1 function led to catastrophic craniofacial malformations that were associated with inadequate cell proliferation. Compound deficiency of Cdkn2a, especially p16Ink4a deficiency, markedly reduced the craniofacial anomalies of TASP1-deficent mice. Furthermore, evaluation of mice expressing noncleavable TASP1 targets revealed that TFIIA is the principal TASP1 substrate that orchestrates craniofacial morphogenesis. ChIP analyses determined that noncleaved TFIIA accumulates at the p16Ink4a and p19Arf promoters to drive transcription of these negative regulators. In summary, our study elucidates a regulatory circuit comprising proteolysis, transcription, and proliferation that is pivotal for construction of the mammalian head.

Abstract 132: HER2 activating mutations are potential targets for colorectal cancer treatment

Background: Cancer genome sequencing is identifying new genetic alterations and new driver events in human cancers. The Cancer Genome Atlas (TCGA) colorectal cancer project found that 7% of colorectal cancer (CRC) patients have HER2 somatic mutations or HER2 gene amplification. HER2 gene amplification in CRC is known to produce resistance to the EGFR monoclonal antibodies, cetuximab and panitumumab. However, the impact of HER2 somatic mutations in CRC has not been studied and it is open question as to whether HER2 mutations are clinically important in CRC. Results: Introduction of the HER2 mutations, S310F, L755S, V777L, and V842I, into immortalized colon epithelial cells increased cell signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of HER2 V842I mutation into colorectal cancer cell lines produced resistance to the EGFR antibodies, cetuximab and panitumumab. HER2 mutations are potently inhibited by low nanomolar doses of the second generation, irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple WT (KRAS, NRAS, BRAF, and PIK3CA WT) colorectal cancer patient derived xenografts (PDX’s) identified 4 PDX9s with HER2 mutations (4/48 = 8.3%). Treatment of these PDX9s with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus a tyrosine kinase inhibitor produced durable shrinkage of the tumors in mice. After the final timepoint in each PDX experiment, the mice were sacrificed and the tumors excised. The tumor histology demonstrated that dual HER2 targeted therapy caused reduction in tumor cellularity and acquisition of more differentiated features. Conclusions: These data demonstrate that HER2 activating mutations are a new drug resistance mechanism to EGFR monoclonal antibodies. More importantly, these data suggest that HER2 activating mutations may themselves be a drug target for the treatment of colorectal cancer. These data form a strong pre-clinical rationale for clinical trials targeting HER2 activating mutations in metastatic CRC patients. The NSABP Foundation is in the design stage for such a clinical trial and will use its living patient biorepository (MPR-1 trial) that contains 1350 colorectal cancer patients to identify subjects for this trial. Citation Format: Shyam M. Kavuri, Naveen Jain, Francesco Galimi, Francesca Cottino, Adam C. Searleman, Wei Shen, Livio Trusolino, Samuel A. Jacobs, Andrea Bertotti, Ron Bose. HER2 activating mutations are potential targets for colorectal cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 132. doi:10.1158/1538-7445.AM2015-132