An Ex Vivo Platform for the Prediction of Clinical Response in Multiple Myeloma.

Multiple myeloma remains treatable but incurable. Despite a growing armamentarium of effective agents, choice of therapy, especially in relapse, still relies almost exclusively on clinical acumen. We have developed a system, Ex vivo Mathematical Myeloma Advisor (EMMA), consisting of patient-specific mathematical models parameterized by an ex vivo assay that reverse engineers the intensity and heterogeneity of chemosensitivity of primary cells from multiple myeloma patients, allowing us to predict clinical response to up to 31 drugs within 5 days after bone marrow biopsy. From a cohort of 52 multiple myeloma patients, EMMA correctly classified 96% as responders/nonresponders and correctly classified 79% according to International Myeloma Working Group stratification of level of response. We also observed a significant correlation between predicted and actual tumor burden measurements (Pearson r = 0.5658, P < 0.0001). Preliminary estimates indicate that, among the patients enrolled in this study, 60% were treated with at least one ineffective agent from their therapy combination regimen, whereas 30% would have responded better if treated with another available drug or combination. Two in silico clinical trials with experimental agents ricolinostat and venetoclax, in a cohort of 19 multiple myeloma patient samples, yielded consistent results with recent phase I/II trials, suggesting that EMMA is a feasible platform for estimating clinical efficacy of drugs and inclusion criteria screening. This unique platform, specifically designed to predict therapeutic response in multiple myeloma patients within a clinically actionable time frame, has shown high predictive accuracy in patients treated with combinations of different classes of drugs. The accuracy, reproducibility, short turnaround time, and high-throughput potential of this platform demonstrate EMMA's promise as a decision support system for therapeutic management of multiple myeloma. Cancer Res; 77(12); 3336-51. ©2017 AACR.

Unification of de novo and acquired ibrutinib resistance in mantle cell lymphoma.

The novel Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated high response rates in B-cell lymphomas; however, a growing number of ibrutinib-treated patients relapse with resistance and fulminant progression. Using chemical proteomics and an organotypic cell-based drug screening assay, we determine the functional role of the tumour microenvironment (TME) in ibrutinib activity and acquired ibrutinib resistance. We demonstrate that MCL cells develop ibrutinib resistance through evolutionary processes driven by dynamic feedback between MCL cells and TME, leading to kinome adaptive reprogramming, bypassing the effect of ibrutinib and reciprocal activation of PI3K-AKT-mTOR and integrin-ß1 signalling. Combinatorial disruption of B-cell receptor signalling and PI3K-AKT-mTOR axis leads to release of MCL cells from TME, reversal of drug resistance and enhanced anti-MCL activity in MCL patient samples and patient-derived xenograft models. This study unifies TME-mediated de novo and acquired drug resistance mechanisms and provides a novel combination therapeutic strategy against MCL and other B-cell malignancies.

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry.

Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome c peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H2O2, and their steady-state catalytic properties [ Pearl , N. M. et al. (2008) Biochemistry 47 , 2766 - 2775 ]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome c(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [ Pelletier , H. and Kraut , J. (1992) Science 258 , 1748 - 1755 ], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the "back-side" of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc-CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex.

Handheld ultrasound versus physical examination in patients referred for transthoracic echocardiography for a suspected cardiac condition.

OBJECTIVES: The purpose of this study was to test the hypothesis that handheld ultrasound (HHU) provides a more accurate diagnosis than physical examination in patients with suspected cardiovascular abnormalities and that its use thus reduces additional testing and overall costs. BACKGROUND: Despite the limitations of physical examination and the demonstrated superiority of HHU for detecting cardiac abnormalities, it is not routinely used for the bedside diagnosis of cardiac conditions. METHODS: Patients referred for a standard echocardiogram for common indications (cardiac function, murmur, stroke, arrhythmias, and miscellaneous) underwent physical examination and HHU by different cardiologists, who filled out a form that also included suggestions for additional testing, if necessary, based on their findings. RESULTS: Of 250 patients, 142 had an abnormal finding on standard echocardiogram. Of these, HHU correctly identified 117 patients (82%), and physical examination correctly identified 67 (47%, p < 0.0001). HHU was superior to physical examination (p < 0.0001) for both normal and abnormal cardiac function. It was also superior to physical examination in correctly identifying the presence of substantial valve disease (71% vs. 31%, p = 0.0003) and in identifying miscellaneous findings (47% vs. 3%, p < 0.0001). Of 108 patients without any abnormalities on standard echocardiography, further testing was suggested for 89 (82%) undergoing physical examination versus only 60 (56%) undergoing HHU (p < 0.0001). Cost modeling showed that HHU had an average cost of $644.43 versus an average cost of $707.44 for physical examination. This yielded a savings of $63.01 per patient when HHU was used versus physical examination. CONCLUSIONS: When used by cardiologists, HHU provides a more accurate diagnosis than physical examination for the majority of common cardiovascular abnormalities. The finding of no significant abnormality on HHU is also likely to result in less downstream testing and thus potentially reduce the overall cost for patients being evaluated for a cardiovascular diagnosis.

A preclinical assay for chemosensitivity in multiple myeloma.

Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma, an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating three-dimensional extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used to convert experimental data into dose-exposure-response "surfaces," which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical multiple myeloma treatments, in three multiple myeloma cell lines and seven patient-derived primary multiple myeloma cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology.

Comparison of left and right atrial volume by echocardiography versus cardiac magnetic resonance imaging using the area-length method.

Increased atrial volumes predict adverse cardiovascular events. Accordingly, accurate measurement of atrial size has become increasingly important in clinical practice. The area-length method is commonly used to estimate the volume. Disagreements between atrial volumes using echocardiography and other imaging modalities have been found. It is unclear whether this has resulted from differences in the measurement method or discrepancies among imaging modalities. We compared the right atrial (RA) and left atrial (LA) volume estimates using the area-length method for transthoracic echocardiography and cardiovascular magnetic resonance (CMR) imaging. Patients undergoing echocardiography and CMR imaging within 1 month were identified retrospectively. For both modalities, the RA and LA long-axis dimension and area were measured using standard 2- and 4-chamber views, and the volume was calculated using the area-length method for both atria. The echocardiographic and CMR values were compared using the Bland-Altman method. A total of 85 patients and 18 controls were included in the present study. The atrial volumes estimated using the area-length method were significantly smaller when measured using echocardiography than when measured using CMR imaging (LA volume 35 ± 20 vs 49 ± 30 ml/m², p <0.001, and RA volume 32 ± 23 vs 43 ± 29 ml/m², p = 0.012). The mean difference (CMR imaging minus echocardiography) was 14 ± 14 ml/m² for the LA and 10 ± 16 ml/m² for the RA volume. Similar results were found in the healthy controls. No significant intra- or interobserver variability was found within each modality. In conclusion, echocardiography consistently underestimated the atrial volumes compared to CMR imaging using the area-length method.

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain.

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site.

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.

Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry.

Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.

Muscle activation patterns in subjects with and without low back pain.

OBJECTIVE: To analyze muscle activation patterns during various footplate perturbations, used as proprioceptive challenges in patients with low back pain (LBP) and in controls. DESIGN: A prospective and controlled comparative study. SETTING: Outpatient clinic. PARTICIPANTS: Twenty subjects with chronic LBP and 20 age- and sex-matched controls. INTERVENTIONS: The subjects underwent 5 sets of footplate perturbations in 3 directions with 16 perturbations for each set. MAIN OUTCOME MEASURES: Latency, frequency, and asymmetry of muscle activation of the erector spinae, rectus abdominus, anterior tibialis, and gastrocnemius muscles were measured bilaterally with surface electromyography. RESULTS: In the toes-up movements, subjects with LBP were significantly less likely to activate their rectus abdominus muscles (P=.02), and they were more likely to exhibit asymmetric muscle activation in the smaller forward movements (odds ratio=4.1, P=.03). The latter result appears to be driven by asymmetric contraction of the erector spinae and rectus abdominus. CONCLUSIONS: Significantly more subjects with LBP than control subjects exhibited absent firing of trunk muscles during 2 of the 5 footplate perturbations. These results suggest an abnormality of the neuromuscular loop and may represent altered proprioception.