Timothy J. Geddes

Changes in renal markers and acute kidney injury after marathon running

Background:  The impact of marathon running on kidney function has not been previously described.

Leukemia Mediated Endothelial Cell Activation Modulates Leukemia Cell Susceptibility to Chemotherapy through a Positive Feedback Loop Mechanism.

In acute myeloid leukemia (AML), the chances of achieving disease-free survival are low. Studies have demonstrated a supportive role of endothelial cells (ECs) in normal hematopoiesis. Here we show that similar intercellular relationships exist in leukemia. We demonstrate that leukemia cells themselves initiate these interactions by directly modulating the behavior of resting ECs through the induction of EC activation. In this inflammatory state, activated ECs induce the adhesion of a sub-set of leukemia cells through the cell adhesion molecule E-selectin. These adherent leukemia cells are sequestered in a quiescent state and are unaffected by chemotherapy. The ability of adherent cells to later detach and again become proliferative following exposure to chemotherapy suggests a role of this process in relapse. Interestingly, differing leukemia subtypes modulate this process to varying degrees, which may explain the varied response of AML patients to chemotherapy and relapse rates. Finally, because leukemia cells themselves induce EC activation, we postulate a positive-feedback loop in leukemia that exists to support the growth and relapse of the disease. Together, the data defines a new mechanism describing how ECs and leukemia cells interact during leukemogenesis, which could be used to develop novel treatments for those with AML.

Isolation and genomic characterization of stem cells in head and neck cancer.

This study investigated the use of 3 different established cell‐sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines.

SPIN: Development of Sample-specific Protein Integrity Numbers as an Index of Biospecimen Quality

It is widely accepted that variable biorepository specimen handling conditions can significantly alter outcomes of clinical research studies, suggesting the need for a metric for sample analyte protein integrity. In line with the National Cancer Institute (NCI) Best Practices, it is vital that the integrity of specimens used for biomarker studies are of the highest standard to ensure validity of the data they generate and confidence in the application of new findings to clinical management. We describe the creation of a program to discover proteins in biorepository samples that can be utilized to assess the integrity of stored specimens for protein-based biomarker studies, similar to the universally accepted quality metric for RNA, the RNA Integrity Number, or RIN. The study mimics potential variation in pre-analytical conditions which may result in proteolysis and other proteome-associated changes and employs surface-enhanced laser desorption time-of-flight mass spectrometry (SELDI-TOF MS) to assess changes in multiple proteins and peptides in a high-throughput manner. Candidate peaks from SELDI spectra of representative sample types (e.g., serum, urine, tissue extracts) which demonstrate differing but reproducible sensitivity to suboptimal processing and storage were selected and quantified in a series of specimens stored in the BioBank within the Beaumont Health System. We then assigned a relative index known here as Sample-specific Protein Integrity Number, or SPIN, which is derived from a ratio of nonstable vs. stable proteins for each sample type in the investigation. This methodology can be applied to every sample type and, once refined and established, the SPIN could be used by any biobank or laboratory using biobanked samples without specialized equipment and irrespective of the sample pre-analytical collection conditions.

Gene expression changes associated with the progression of intraductal papillary mucinous neoplasms.

Objectives The diagnosis of high-grade intraductal papillary mucinous neoplasm (IPMN) is difficult to distinguish from low-grade IPMN. The aim of this study was to identify potential markers for the discrimination of high-grade and invasive (HgInv) IPMN from low- and moderate-grade dysplasia IPMN. Methods Laser capture microdissection was used to isolate distinct foci of low-grade, moderate-grade, high-grade, and invasive IPMN from paraffin-embedded archival tissue from 14 patients who underwent resection for IPMN. Most samples included multiple grades in the same specimen. Affymetrix Human Exon microarrays were used to compare low- and moderate-grade dysplasia IPMN with HgInv IPMN. Results Sixty-two genes were identified as showing significant changes in expression (P ⩽ 0.05 and a 2-fold cutoff), including up-regulation of 41 in HgInv IPMN. Changes in gene expression are associated with biological processes related to malignant behavior including cell motion, cell proliferation, response to hypoxia, and epithelial-to-mesenchymal transition. In addition, altered signaling in several transforming growth factor &bgr;–related pathways was exhibited in the progression of IPMN to malignancy. Conclusions This study identifies a set of genes associated with the progression of IPMN to malignancy. These genes are potential markers that could be used to identify IPMN requiring surgical resection.

Gene Expression Characterization of HPV Positive Head and Neck Cancer to Predict Response to Chemoradiation

Human papillomavirus (HPV) has been shown to have a causal role in the development of head and neck squamous cell carcinoma. While HPV-positive head and neck cancer is associated with a better response to treatment in the majority of patients, there is a subset who does not respond favorably to current therapy. Identification of these patients could prevent unnecessary morbidity and indicate the need for alternative therapeutic options. Tissue samples were obtained from 19 patients with HPV-positive head and neck squamous carcinoma treated with chemoradiation therapy. HPV status was confirmed by polymerase chain reaction analysis through detection of HPV16 E7 in both DNA and RNA. RNA was isolated from tissue samples and subjected to microarray gene expression analysis. In addition to identification of potential genetic biomarkers (including LCE3D, KRTDAP, HMOX1, KRT19, MDK, TSPAN1), differentially expressed genes associated with genomic stability, cell cycle, and DNA damage were detected between responders and non-responders. These results were further validated with publicly available gene expression studies. This pilot study suggests prospective biomarkers that predict response to therapy. The importance of genes involved with genomic stability is highlighted in both development and progression of head and neck squamous cell carcinoma but also recurrence. Potential development of an assay may prove beneficial to clinicians, assisting them to provide alternative care sooner thus lowering morbidity.

Characterization of clear cell renal cell carcinoma by gene expression profiling.

OBJECTIVES Use global gene expression to characterize differences between high-grade and low-grade clear cell renal cell carcinoma (ccRCC) compared with normal and benign renal tissue. METHODS Tissue samples were collected from patients undergoing surgical resection for ccRCC. Affymetrix gene expression arrays were used to examine global gene expression patterns in high- (n = 16) and low-grade ccRCC (n = 13) as well as in samples from normal kidney (n =14) and benign kidney disease (n = 6). Differential gene expression was determined by analysis of variance with a false discovery rate of 1% and a 2-fold cutoff. RESULTS Comparing high-grade ccRCC with each of normal and benign kidney resulted in 1,833 and 2,208 differentially expressed genes, respectively. Of these, 930 were differentially expressed in both comparisons. In order to identify genes most related to progression of ccRCC, these differentially expressed genes were filtered to identify genes that showed a pattern of expression with a magnitude of change greater in high-grade ccRCC in the comparison to low-grade ccRCC. This resulted in the identification of genes such as TMEM45A, ceruloplasmin, and E-cadherin that were involved in cell processes of cell differentiation and response to hypoxia. Additionally changes in HIF1α and TNF signaling are highly represented by changes between high- and low-grade ccRCC. CONCLUSIONS Gene expression differences between high-grade and low-grade ccRCC may prove to be valuable biomarkers for advanced ccRCC. In addition, altered signaling between grades of ccRCC may provide important insight into the biology driving the progression of ccRCC and potential targets for therapy.

Utilizing In-House Resources to Correct Sample Mixups in a Medium Throughput Biorepository: A Case Study.

Given the high demands on a multidisciplinary approach to sample collection, and despite the rigorous quality assurance and quality control measures designed to minimize sample misidentification in our state of the art biorepository, the potential for error is still a concern. Measures to deal with potential uncertainties are a necessary part of every successful biobanking operation. The Beaumont BioBank and associated Core Molecular Laboratory have developed procedures to address these rare incidents. Here we present a case study of occurrences in the Beaumont BioBank in which the identity of samples was uncertain and a resolution workflow was implemented to quickly remove the ambiguity. Using Core Molecular Laboratory in-house resources, including Mass Array technology and the valuable insight and experience from a multidisciplinary team, a comprehensive troubleshooting schema has been developed and applied toward resolving sample identification uncertainties. As per standard operating procedures, each step of these incidents was recorded and a final report prepared.

Gene Expression Differences Predict Treatment Outcome of Merkel Cell Carcinoma Patients

Due to the rarity of Merkel cell carcinoma (MCC), prospective clinical trials have not been practical. This study aimed to identify biomarkers with prognostic significance. While sixty-two patients were identified who were treated for MCC at our institution, only seventeen patients had adequate formalin-fixed paraffin-embedded archival tissue and followup to be included in the study. Patients were stratified into good, moderate, or poor prognosis. Laser capture microdissection was used to isolate tumor cells for subsequent RNA isolation and gene expression analysis with Affymetrix GeneChip Human Exon 1.0 ST arrays. Among the 191 genes demonstrating significant differential expression between prognostic groups, keratin 20 and neurofilament protein have previously been identified in studies of MCC and were significantly upregulated in tumors from patients with a poor prognosis. Immunohistochemistry further established that keratin 20 was overexpressed in the poor prognosis tumors. In addition, novel genes of interest such as phospholipase A2 group X, kinesin family member 3A, tumor protein D52, mucin 1, and KIT were upregulated in specimens from patients with poor prognosis. Our pilot study identified several gene expression differences which could be used in the future as prognostic biomarkers in MCC patients.

SELDI-TOF-MS Serum Profiling Reveals Predictors of Cardiac MRI Changes in Marathon Runners.

Purpose. To utilize proteomics to discover proteins associated with significant cardiac magnetic resonance imaging (MRI) changes in marathon runners. Methods. Serum from 25 runners was analyzed by surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Proteomic profiles were compared in serum samples obtained prior to the race, at the finish line and within 7 hours after race to identify dynamic proteins correlated with cardiac MRI changes. Results. 693 protein/peptide clusters were identified using two ProteinChip surface chemistries and, of these, 116 were significantly different between the three time points. We identified 7 different patterns of protein expression change within the runners and 5 prerace protein peaks, 16 finish-line protein levels, and 15 postrace proteins which were correlated with significant postrace cardiac MRI changes. Conclusions. This study has identified baseline levels of proteins which may be predictive of risk of significant cardiac damage following a marathon race. Preliminary identification of the significant proteins suggested the involvement of cytokines and other proteins involved in stress and inflammatory response.