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Dive into the research topics where Gregory M. Hayes is active.

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Featured researches published by Gregory M. Hayes.


Nature Protocols | 2007

Measurement of cell proliferation by heavy water labeling

Robert Busch; Richard A. Neese; Mohamad Awada; Gregory M. Hayes; Marc K. Hellerstein

DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.


Blood | 2009

In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia

Carlo Calissano; Rajendra N. Damle; Gregory M. Hayes; Elizabeth Murphy; Marc K. Hellerstein; Carol Moreno; Cristina Sison; Matthew Kaufman; Jonathan E. Kolitz; Steven L. Allen; Kanti R. Rai; Nicholas Chiorazzi

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium ((2)H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+ cells compared with CD38- counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+ fraction than in CD38- cells; when analyzed on an individual patient basis, CD38+ (2)H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.


Molecular Medicine | 2011

Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells.

Carlo Calissano; Rajendra N. Damle; Sonia Marsilio; Xiao Jie Yan; Sophia Yancopoulos; Gregory M. Hayes; Claire Emson; Elizabeth Murphy; Marc K. Hellerstein; Cristina Sison; Matthew Kaufman; Jonathan E. Kolitz; Steven L. Allen; Kanti R. Rai; Ivana Ivanovic; Igor Dozmorov; Sergio Roa; Matthew D. Scharff; Wentian Li; Nicholas Chiorazzi

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.


Nature Protocols | 2007

Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio–mass spectrometric analysis

Jason Voogt; Mohamad Awada; Elizabeth Murphy; Gregory M. Hayes; Robert Busch; Marc K. Hellerstein

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio–mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (≥0.002% atom percent excess 2H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of 2H2O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (>∼107). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.


Leukemia Research | 2010

Isolation of malignant B cells from patients with chronic lymphocytic leukemia (CLL) for analysis of cell proliferation: Validation of a simplified method suitable for multi-center clinical studies

Gregory M. Hayes; Robert Busch; Jason Voogt; Iche M. Siah; Tracy A. Gee; Marc K. Hellerstein; Nicholas Chiorazzi; Kanti R. Rai; Elizabeth Murphy

BACKGROUND Heavy water ((2)H(2)O) labelling of DNA enables the measurement of low-level cell proliferation in vivo, using gas chromatography/pyrolysis isotope ratio mass spectrometry (GC/P/IRMS), but the methodology has been too complex for widespread use. Here, we report a simplified method for measuring proliferation of malignant B cells in patients with chronic lymphocytic leukemia (CLL). DESIGN AND METHODS Patients were labelled with (2)H(2)O for 6 weeks; blood samples were obtained at 0, 3, and 6 weeks during (2)H(2)O labelling and 9, 12, and 16 weeks thereafter. Bone marrow was sampled at week 6. Phlebotomy was performed at multiple, non-research clinical sites. CLL cells were isolated in a central laboratory, using a novel RosetteSep-based method; DNA labelling was analyzed by GC/P/IRMS. RESULTS In 26 of 29 patients, CLL cell isolation resulted in > or =95% purity for malignant CD5+ B cells; in one patient, malignant cells expressed marginal levels of CD5, and in two others, further sorting of CD5hi malignant cells was required. Cell yields correlated with white blood cell counts and exceeded GC/P/IRMS requirements ( approximately 10(7) cells) >98% of the time; high-quality DNA labelling data were obtained. RosetteSep isolation achieved adequate CLL cell purity from bone marrow in only 64% of samples, but greatly reduced subsequent sort time for impure samples. CONCLUSION This method enables clinical studies of CLL cell proliferation outside of research settings, using a shorter (2)H(2)O intake protocol, a minimal sampling protocol, and centralised sample processing. The CLL cell isolation protocol may also prove useful in other applications. (clinicaltrials.gov identifier: NCT00481858).


Clinical Cancer Research | 2012

Regional Cell Proliferation in Microdissected Human Prostate Specimens after Heavy Water Labeling In Vivo: Correlation with Prostate Epithelial Cells Isolated from Seminal Fluid

Gregory M. Hayes; Jeff Simko; Daniel Holochwost; Kyle Kuchinsky; Robert Busch; Lisa M. Misell; Elizabeth Murphy; Peter R. Carroll; June M. Chan; Katsuto Shinohara; Marc K. Hellerstein

Purpose: Prostate cancer is detected with increasing frequency but has a highly variable natural history and prognosis and active surveillance of men with low-risk prostate cancer would benefit greatly from minimally invasive methods to identify progression. We describe here two novel in vivo metrics of cell proliferation in men with prostate neoplasia. Experimental Design: Three groups of men drank heavy water, a nonradioactive, stable isotopic tracer for 14 to 28 days: (i) healthy men, (ii) men scheduled for transrectal core needle biopsy, and (iii) men scheduled for radical prostatectomy. Prostate epithelial cells (PEC) were isolated from ejaculated seminal fluid in all subjects. Histologically graded lesions were microdissected from tissue slides obtained from subjects undergoing surgery and proliferation rates were measured from isolated cells via mass spectrometry. Results: Proliferation rates of seminal PEC in healthy men (0.10%–0.27%/d) were stable on repeat sampling. Rates above 0.34%/d were seen only in patients with cancer where rates increased progressively from normal tissue through benign prostate hyperplasia, prostate intraepithelial neoplasia, and tumor grades III and IV in all subjects. Seminal PEC kinetics correlated highly with the most proliferative microdissected region in each subject (r2 = 0.94). Conclusions: Prostate cell proliferation can be measured in vivo from microdissected histopathology sections or noninvasively from seminal fluid where the latter reflects the most proliferative region of the gland. This approach may allow monitoring of progression in men with low-risk prostate cancer. Clin Cancer Res; 18(12); 3250–60. ©2012 AACR.


Leukemia | 2017

Leukemia-cell proliferation and disease progression in patients with early stage chronic lymphocytic leukemia

Elizabeth Murphy; Donna Neuberg; Laura Z. Rassenti; Gregory M. Hayes; Robert Redd; Claire Emson; K. Li; Jennifer R. Brown; William G. Wierda; Scott M. Turner; Clive S. Zent; John C. Byrd; C. McConnel; Jaqueline C. Barrientos; Neil E. Kay; Marc K. Hellerstein; Nicholas Chiorazzi; Thomas J. Kipps; Kanti R. Rai

The clinical course of patients with recently diagnosed early stage chronic lymphocytic leukemia (CLL) is highly variable. We examined the relationship between CLL-cell birth rate and treatment-free survival (TFS) in 97 patients with recently diagnosed, Rai stage 0–II CLL in a blinded, prospective study, using in vivo 2H2O labeling. Birth rates ranged from 0.07 to 1.31% new cells per day. With median follow-up of 4.0 years, 33 subjects (34%) required treatment by NCI criteria. High-birth rate was observed in 44% of subjects and was significantly associated with shorter TFS, unmutated IGHV status and expression of ZAP70 and of CD38. In multivariable modeling considering age, gender, Rai stage, expression of ZAP70 or CD38, IGHV mutation status and FISH cytogenetics, only CLL-cell birth rate and IGHV mutation status met criteria for inclusion. Hazard ratios were 3.51 (P=0.002) for high-birth rate and 4.93 (P<0.001) for unmutated IGHV. The association between elevated birth rate and shorter TFS was observed in subjects with either mutated or unmutated IGHVs, and the use of both markers was a better predictor of TFS than either parameter alone. Thus, an increased CLL birth rate in early stage disease is a strong predictor of disease progression and earlier treatment.


Blood | 2014

Kinetic Measurement of Leukemia-Cell Proliferation Rate By Deuterium Labeling Predicts Time to Initial Treatment of Patients with Chronic Lymphocytic Leukemia

Elizabeth Murphy; Donna Neuberg; Kanti R. Rai; Laura Z. Rassenti; Claire Emson; Kelvin Li; Neil E. Kay; William G. Wierda; Gregory M. Hayes; Jennifer R. Brown; John C. Byrd; Coline McConnel; Jacqueline C. Barrientos; Robert Redd; Scott Turner; Robert Busch; Marc K. Hellerstein; Nicholas Chiorazzi; Thomas J. Kipps


Blood | 2008

In Vivo Labeling of Dividing Chronic Lymphocytic Leukemia B Cells Suggests That CXCR4 and CD5 Define the Clonal Subfraction That Recently Proliferatead and Emigrated from a Solid Tissue

Carlo Calissano; Rajendra N. Damle; Gregory M. Hayes; Elisabeth J. Murphy; Marc Hellerstein; Barbara Sherry; Matthew Kaufman; Steven L. Allen; Kanti R. Rai; Nicholas Chiorazzi


Blood | 2007

Expression of CD38 and High Levels of CD5 Identifies Members of CLL Clones That Have Progressed beyond G1 and Proliferated.

Carlo Calissano; Rajendra N. Damle; Marc K. Hellerstein; Elisabeth J. Murphy; Gregory M. Hayes; Kaufman Matthew; Steven L. Allen; Kanti R. Rai; Nicholas Chiorazzi

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Elizabeth Murphy

National Institutes of Health

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Kanti R. Rai

North Shore-LIJ Health System

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Nicholas Chiorazzi

The Feinstein Institute for Medical Research

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Robert Busch

University of Cambridge

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Carlo Calissano

The Feinstein Institute for Medical Research

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Rajendra N. Damle

The Feinstein Institute for Medical Research

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Claire Emson

University of California

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