MGP Panel is a comprehensive targeted genomics panel for molecular profiling of multiple myeloma patients

PURPOSE: We designed a comprehensive multiple myeloma (MM) targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. EXPERIMENTAL DESIGN: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNAs). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical fluorescence in situ hybridization (FISH) (translocations), multiplex ligation probe analysis (MLPA) (CNAs), whole genome sequencing (WGS) (CNAs, mutations, translocations) or droplet digital PCR (ddPCR) of known standards (mutations). RESULTS: Canonical IgH translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for one patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2=0.969. VAFs for 74 mutations were compared between sequencing and ddPCR with concordance of R2=0.9849. CONCLUSIONS: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost effective, comprehensive, clinically actionable and can be routinely deployed to assist risk stratification at diagnosis or post-treatment to guide sequencing of therapies

Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression

Multiple, complex molecular events characterize cancer development and progression(1,2). Deciphering the molecular networks that distinguish organ- confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high- throughput liquid- and- gas- chromatography- based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer ( 42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N- methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non- invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine- N- methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.Early Detection Research Network ; National Institutes of Health ; MTTC ; Clinical Translational Science Award ; Fund for Discovery of the University of Michigan Comprehensive Cancer Center ; University of Michigan Cancer Biostatistics Training Grant ; Doris Duke Charitable FoundationWe thank J. Granger for help in manuscript preparation, J. Siddiqui and R. Varambally for help with the clinical database, and A. Vellaichamy and S. Pullela for technical assistance. We thank K. Pienta for access to metastatic prostate cancer samples from the University of Michigan Prostate SPORE rapid autopsy programme. This work is supported in part by the Early Detection Research Network (A.M.C., J.T.W.), National Institutes of Health (A.S., S.P., J.B., T.M.R., D.G., G.S.O. and A.M.C.) and an MTTC grant (G.S.O. and A.S.). A.M.C. is supported by a Clinical Translational Science Award from the Burroughs Welcome Foundation. A. S. is supported by a grant from the Fund for Discovery of the University of Michigan Comprehensive Cancer Center. L. M. P. is supported by the University of Michigan Cancer Biostatistics Training Grant. A. M. C and S. P. are supported by the Doris Duke Charitable Foundation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62661/1/nature07762.pd

Membrane Transition Temperature Determines Cisplatin Response.

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients

Synthesis, conformational and pharmacological studies of glycosylated chimeric peptides of Met-enkephalin and FMRFa

Our previous study showed that a chimeric peptide of Met-enkephalin and FMRFamide, YFa (YGGFMKKKFMRFa) not only caused antinociception and potentiated morphine analgesia but also blocked the development of tolerance and physical dependence. In the continuation of that study three chimeric analogues of YFa, [Ser5]YFa, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa, were synthesized. To increase the bioavailability and penetration of blood brain barrier (BBB), glycosylated analogues, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa, have been synthesized by solid phase peptide synthesis by building block method using anomeric acetate activation method. Circular dichroism studies showed that all the three chimeric peptides are stable and have a propensity for adopting helical conformation in the presence of membrane mimicking solvent. In comparison of parent chimeric peptide YFa, helicity of [Ser5]YFa, [O-Glu-Ser5]YFa and [O-Gal-Ser5]YFa has decreased. Pharmacological studies using tail-flick latency in mice showed that [O-Glu-Ser5]YFa have increased analgesia and bioavailability in comparison of [O-Gal-Ser5]YFa and non-glycosylated analogue [Ser5]YFa. Exhibition of enhanced analgesia by [O-Glu-Ser5]YFa as compared to [O-Gal-Ser5]YFa seems to be due to preference of GLUT-1 transporter system for glucose

Co-incubation of cisplatin with isopropanol leads to enhanced apoptosis without an increase in intercellular cisplatin concentration.

<p>(A) Expression levels of cleaved PARP, an apoptotic marker, as measured by western blot for cells incubated with 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone along with cisplatin free controls (B) Levels of intracellular cisplatin were measured using optical emission spectrometry for the three treatments, 50mM isopropanol plus 10μM cisplatin, with 100μM menthol plus 10μM cisplatin or with 10μM cisplatin alone. The differences between the three treatments are not statistically significant (n = 8 trials). Also shown are predicted levels of cisplatin obtained by assuming that intracellular cisplatin that is directly proportional to the external concentration determines the extent of cell death (as described in Methods). The solid line denotes the predicted mean theoretical value and corresponding dashed lines denote error bounds. (C) Levels of intracellular cisplatin for UMSCC1 and the more resistant cell line Me-180pt treated with 10μM cisplatin. The solid line as described previously denotes predicted levels given the assumptions stated in 4B and dashed lines indicate error bounds.</p

Changes in transition temperature in GPMVs correlate with the cell lines resistance to cisplatin.

<p>(A) GPMVs were isolated from four cell-lines as described in the Methods section. The transition temperature shifts are reported by comparing the transition temperatures of GPMVs probed in the presence of 10 μM cisplatin to untreated GPMVs. (B) Data points in panel A were plotted against a previously reported measure of surviving fraction to cisplatin obtained using clonogenic assays for the same four cell lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.ref008" target="_blank">8</a>]. Surviving fraction was measured using a clonogenic survival assay. Surviving fractions were measured 72 hours after treatment with 10uM cisplatin. The straight line is drawn to visually distinguish sensitive and resistant celllines. Transition temperature shifts upon incubation with 10 μM cisplatin or exposure to 10 Gy irradiation for GPMVs isolated from ME-180 pt cells (C) and RBL cells (D). In all cases, points represent the average of at least 3 independent measurements and error bounds represent the standard error of the mean. Significance between transition temperature shift measurements were evaluated using t-tests.</p

Modulating transition temperature affects cisplatin mediated cellular response in UMSCC1 cells.

<p>(A) Transition temperature shifts measured for GPMVs isolated from UMSCC1 in the presence of 50mM isopropanol or 100 μM menthol or each of these treatments in combination with 10μM cisplatin. (B) Efficacy of 50mM isopropanol and 100μM menthol action on UMSCC1 cells calculated as the number of cells present after 24h of treatment divided by the number of cells present in an untreated control. (C) Efficacy of cisplatin action as a function of the transition temperature shift effected by the treatment in isolated GPMVs shown in A. Efficacy of cisplatin action on UMSCC1 cells as above was computed as above in (B) by dividing the number of cells present after 24h of treatment compared to the number of cells present in an untreated control. (D) Plots show relative cell counts as a function of cisplatin concentration either in the presence or absence of 50mM isopropanol or 100μM menthol. Each point represents the average and SEM of at least 4 independent measurements, and lines are fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a>. (E) Average IC₅₀ values as determined by fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140925#pone.0140925.e001" target="_blank">Eq 1</a> to individual dose response curves. Values represent an average and SEM over at least 4 independent measurements.</p