David G. Peters

Leukemia Following Hodgkin's Disease

To investigate the effect of different treatments for Hodgkins disease on the risk of leukemia, we used an international collaborative group of cancer registries and hospitals to perform a case-control study of 163 cases of leukemia following treatment for Hodgkins disease. For each case patient with leukemia, three matched controls were chosen who had been treated for Hodgkins disease but in whom leukemia did not develop. The use of chemotherapy alone to treat Hodgkins disease was associated with a relative risk of leukemia of 9.0 (95 percent confidence interval, 4.1 to 20) as compared with the use of radiotherapy alone. Patients treated with both had a relative risk of 7.7 (95 percent confidence interval, 3.9 to 15). After treatment with more than six cycles of combinations including procarbazine and mechlorethamine, the risk of leukemia was 14-fold higher than after radiotherapy alone. The use of radiotherapy in combination with chemotherapy did not increase the risk of leukemia above that produced by the use of chemotherapy alone, but there was a dose-related increase in the risk of leukemia in patients who received radiotherapy alone. The peak in the risk of leukemia came about five years after chemotherapy began, and a large excess persisted for at least eight years after it ended. After adjusting for drug regimen, we found that patients who had undergone splenectomy had at least double the risk of leukemia of patients who had not, and an advanced stage of Hodgkins disease carried a somewhat higher risk of leukemia than Stage I disease. We conclude that chemotherapy for Hodgkins disease greatly increases the risk of leukemia and that this increased risk appears to be dose-related and unaffected by concomitant radiotherapy. In addition, the risk is greater for patients with more advanced stages of Hodgkins disease and for those who undergo splenectomy.

Molecular Anatomy of an Intracranial Aneurysm Coordinated Expression of Genes Involved in Wound Healing and Tissue Remodeling

Background and Purpose— Approximately 6% of human beings harbor an unruptured intracranial aneurysm. Each year in the United States, >30 000 people suffer a ruptured intracranial aneurysm, resulting in subarachnoid hemorrhage. Despite the high incidence and catastrophic consequences of a ruptured intracranial aneurysm and the fact that there is considerable evidence that predisposition to intracranial aneurysm has a strong genetic component, very little is understood with regard to the pathology and pathogenesis of this disease. Methods— To begin characterizing the molecular pathology of intracranial aneurysm, we used a global gene expression analysis approach (SAGE-Lite) in combination with a novel data-mining approach to perform a high-resolution transcript analysis of a single intracranial aneurysm, obtained from a 3-year-old girl. Results— SAGE-Lite provides a detailed molecular snapshot of a single intracranial aneurysm. These data suggest that, at least in this specific case, aneurysmal dilation results in a highly dynamic cellular environment in which extensive wound healing and tissue/extracellular matrix remodeling are taking place. Specifically, we observed significant overexpression of genes encoding extracellular matrix components (eg, COL3A1, COL1A1, COL1A2, COL6A1, COL6A2, elastin) and genes involved in extracellular matrix turnover (TIMP-3, OSF-2), cell adhesion and antiadhesion (SPARC, hevin), cytokinesis (PNUTL2), and cell migration (tetraspanin-5). Conclusions— Although these are preliminary data, representing analysis of only one individual, we present a unique first insight into the molecular basis of aneurysmal disease and define numerous candidate markers for future biochemical, physiological, and genetic studies of intracranial aneurysm. Products of these genes will be the focus of future studies in wider sample sets.

Noninvasive prenatal diagnosis of a fetal microdeletion syndrome.

This proof-of-principle study shows that it is possible to detect a genetic microdeletion carried by a fetus through analysis of DNA in circulating maternal blood.

Serial analysis of the vascular endothelial transcriptome under static and shear stress conditions

We have utilized serial analysis of gene expression (SAGE) to analyze the response of human coronary artery endothelial cells (HCAECs) to laminar shear stress (LSS). Primary cultures of HCAECs were exposed to 15 dyn/cm(2) LSS for 24 h in a parallel plate flow chamber and compared with identical same passage cells cultured under static conditions. The expression levels of a number of functional categories of genes were reduced by shear stress including those encoding proteins involved in cell proliferation (CDC10, CDC20, CDC23, CCND1, CCNB1), angiogenesis (ANGPTL4, CTGF, CYR61, ENG, EPAS1, EGFR, LGALS3, PGK1, and SPARC), extracellular matrix and cell-matrix adhesion (EFEMP1, LOXL2, P4HB, FBN1, FN1, ITGA5, ITGAE, ITGAV, ILK, LAMR1) and ATP synthesis (ATP5G3, ATP5J2, ATP5L, ATP5D). We also observed an increase in the LSS-responsive expression of genes encoding stress response proteins, including HMOX1, which is significant since HMOX1 may have anti-inflammatory and vasodilatory vascular effects. The autosomal dominant polycystic kidney disease (ADPKD) genes PKD1 and PKD2 were also elevated by LSS. ADPKD is associated with vascular malfunction, including the impairment of vasoreactive processes. To our knowledge, this is the first SAGE-based analysis of the shear stress-responsive endothelial cell transcriptome. These immortal data provide a resource for further analyses of the molecular mechanisms underlying the biological response to LSS and contribute to the expanding collection of publicly available SAGE data.

A microarray‐based approach for the identification of epigenetic biomarkers for the noninvasive diagnosis of fetal disease

We describe a novel microarray‐based approach for the high‐throughput discovery of epigenetic biomarkers for use in the noninvasive detection of fetal genetic disease.

Comparative Gene Expression Analysis of Ovarian Carcinoma and Normal Ovarian Epithelium by Serial Analysis of Gene Expression

Despite the poor prognosis of ovarian cancer and the importance of early diagnosis, there are no reliable noninvasive biomarkers for detection in the early stages of disease. Therefore, to identify novel ovarian cancer markers with potential utility in early-stage screening protocols, we have undertaken an unbiased and comprehensive analysis of gene expression in primary ovarian tumors and normal human ovarian surface epithelium (HOSE) using Serial Analysis of Gene Expression (SAGE). Specifically, we have generated SAGE libraries from three serous adenocarcinomas of the ovary and, using novel statistical tools, have compared these to SAGE data derived from two pools of normal HOSE. Significantly, in contrast to previous SAGE-based studies, our normal SAGE libraries are not derived from cultured cell lines. We have also compared our data with publicly available SAGE data obtained from primary tumors and “normal” HOSE-derived cell lines. We have thus identified several known and novel genes whose expressions are elevated in ovarian cancer. These include but are not limited to CLDN3, WFDC2, FOLR1, COL18A1, CCND1, and FLJ12988. Furthermore, we found marked differences in gene expression patterns in primary HOSE tissue compared with cultured HOSE. The use of HOSE tissue as a control for these experiments, along with hierarchical clustering analysis, identified several potentially novel biomarkers of ovarian cancer, including TACC3, CD9, GNAI2, AHCY, CCT3, and HMGA1. In summary, these data identify several genes whose elevated expressions have not been observed previously in ovarian cancer, confirm the validity of several existing markers, and provide a foundation for future studies in the understanding and management of this disease.

Gene expression is altered in perfused arterial segments exposed to cyclic flexure ex vivo

AbstractCertain regions of coronary and other arteries undergo cyclic flexure due to attachment to the heart or crossing of joints. Such motion gives rise to fluctuations in transmural stress and luminal shear stress. It is well known that cyclic variation of these biomechanical forces influences many aspects of vascular cell biology including gene expression. The purpose of this work was to investigate the hypothesis that cyclic flexure of arterial segments influences their gene expression. Bilateral porcine femoral arteries were obtained fresh from the abattoir. One vessel was mounted in an ex vivo perfusion system and subjected to an intraluminal pressure of 60 mm Hg and flow of 50 ml/min to serve as a control. The other vessel was mounted in a second perfusion system with similar hemodynamic conditions, but also subjected to controlled cyclic bending consistent with that found in coronary arteries in vivo. Reverse transcriptase-polymerase chain reaction analysis demonstrated that E-selectin and matrix metalloproteinase-1 (MMP-1) were consistently and significantly downregulated in the specimens subjected to 4 h of cyclic bending as compared to the control (n=8, p < 0.05). Our results show that cyclic flexure of arterial segments in vitro may influence their gene expression. Further investigation should follow this novel observation and focus on other known mediators to more carefully elucidate the consequence of cyclic flexure on arterial pathobiology.

Altered arterial homeostasis and cerebral aneurysms: a molecular epidemiology study.

OBJECTIVE:We hypothesized that patients with intracranial cerebral aneurysms (IAs) harbor a molecular defect in the process responsible for maintaining arterial integrity (arterial homeostasis). In this study, we undertook a preliminary assessment of differential expression of key molecules involved with each phase of homeostasis: arterial flow modulation, arterial tear and repair, and the ensuing extracellular matrix. METHODS:Key molecules from each phase of the arterial homeostatic process were selected: prostacyclin-stimulating factor, implicated with arterial flow modulation; PNUT and RAI, involved with tissue repair and arterial remodeling; and Type III collagen and fibronectin, which are key constituents of the extracellular matrix. A small sample of the IA dome was harvested at the time of surgical repair from both ruptured and unruptured domes. Pericranial vascular tissue was harvested from a sample of the superficial temporal artery (STA) or occipital artery from aneurysmal and nonaneurysmal patients undergoing craniotomy for unrelated conditions. Statistical analysis examining expression of each marker was performed initially using dichotomous analysis (presence or absence of expression), followed by an assessment of quantitative differences in expression. Initial analysis was restricted to the pair consisting of dome and STA harvested from each individual patient. This was followed by a pooled analysis in which all domes and STAs were respectively pooled. RESULTS:A total of 86 tissue samples were studied, including 24 IA domes, STA samples from 43 aneurysmal patients, and STA samples from 19 nonaneurysmal patients. We found that the degree of prostacyclin-stimulating factor and RAI expression was reduced in ruptured aneurysm domes when compared with STAs from IA patients (odds ratio, 0.26; 95% confidence interval [CI], 0.08–0.89; and odds ratio, 0.18; 95% CI, 0.03–0.94, respectively). Type III collagen expression also was reduced among ruptured domes when compared with STA (P = 0.042). These differences were found to be independent of the effects of smoking with adjusted odds ratios of 0.25 (95% CI, 0.08–0.77) and 0.18 (95% CI, 0.04–0.79), respectively, for prostacyclin-stimulating factor and RAI. No statistically significant differences were noted among the unruptured domes. CONCLUSION:These preliminary data suggest an impaired ability to express proteins responsible for flow modulation and arterial repair within the ruptured domes when compared with control pericranial tissue. The study generates a hypothesis of impaired arterial homeostasis with a reduced ability to modulate hemodynamic flow with perhaps increased microinjury. This is exacerbated further by an impaired molecular ability to repair the vessel wall, culminating in aneurysm rupture. The study has limitations based on the use of pericranial tissue as the control and the relatively small sample size. Nevertheless, this study suggests that altered arterial homeostasis warrants further investigation in hopes of better understanding IA pathogenesis.

Maternal cell-free DNA-based screening for fetal microdeletion and the importance of careful diagnostic follow-up

Background:Noninvasive prenatal screening (NIPS) by next-generation sequencing of cell-free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21 in high risk pregnancies. NIPS can identify fetal genomic microdeletions; however, sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus who tested positive for atypical 22q deletion via maternal plasma cfDNA testing.Methods:We performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. Fluorescence in situ hybridization and chromosomal microarray diagnostic genetic tests were done postnatally.Conclusion:NIPS detected a 22q microdeletion that, upon diagnostic workup, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.Genet Med 17 10, 836–838.

High resolution non-invasive detection of a fetal microdeletion using the GCREM algorithm.

The non‐invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non‐invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM).