Engraftment and growth of U266^luciferase cells as monitored by BLI, serum paraprotein and MRI. A.

<p>(i) Dorsal and (ii) ventral BLI acquired from IVIS over weeks 3–7 post-inoculation. <b>B.</b> Quantitative measurement of radiance from BLI. Radiance reflects the intensity of luciferase luminescence and therefore number of luciferase-tagged cells present. Results show that radiance increases in a time-dependent manner over the course of the experiment and that a significant increase in radiance occurs over weeks 5–7 (p<0.05, 1-way ANOVA with Bonferroni post-test). <b>C.</b> Paraprotein levels in the serum increases in a time dependent manner and correlates with the increase seen in BLI. MRI-derived tumour volumes determined at approximately weeks 5 and 10 confirmed tumour progression seen with BLI.</p

CD138 expression changes in response to therapy.

<p><b>A.</b> Percentage of CD138⁺ human myeloma cells measured by flow cytometry in bone aspirates of mice (n = 3), showing a significantly lower percentage of positive cells in both tibias and spine of mice in the two treatment groups than in untreated mice (p<0.05, 2-way ANOVA with Bonferroni post-test). No CD138⁺ cells were observed in the organs of any of the mice. <b>B.</b> Histological analysis of sections from the tibias of mice from each group showed distinct differences. (i) Sections from healthy mice displayed classical architecture, with no CD138⁺ cells. (ii) In comparison, sections from untreated myeloma mice showed a high infiltration of CD138⁺ cells with loss of normal architecture. (iii) Treatment of mice with BZB resulted in the return of normal architecture and loss of CD138⁺ cells. (iv) A similar result was observed in mice treated with tosedostat, but with occasional scattered CD138⁺ cells.</p

Summary of current mouse models for studying the development and treatment of myeloma.

<p>Summary of current mouse models for studying the development and treatment of myeloma.</p

MRI changes in response to therapy.

<p>MRI-derived tumour volumes. <b>A.</b> Tumour was identified as a hyperintense signal enclosed within the cortical bone on T₂-weighted images. MRI images showed a reduction in signal intensity in both treatment groups compared to positive control in both the tibia (T) and femur (F). <b>B.</b> Tumour volume was quantified from regions of interest drawn on the periphery of the hyperintense signal. Data are mean ± SEM, n≥6. Both BZB and tosedostat (TDT) treatment resulted in a significantly lower tumour volume compared to control (p<0.05, 1-way ANOVA with Bonferroni post-test). In addition, there was no significant difference in tumour volume between the BZB treated and negative control group.</p

BLI and serum paraprotein changes in response to therapy.

<p><b>A.</b> (i) Pre-treatment and (ii) post-treatment BLI of mice at weeks 5 and 9. <b>B.</b> Quantitative measurement of radiance from BLI. (i) No significant difference in radiance between treatment groups was seen at the start of the treatment schedule. (ii) Post-treatment radiance levels revealed a significant attenuation of tumour spread by both BZB and tosedostat (p<0.05, 1-way ANOVA with Bonferroni post-test). <b>C.</b> Paraprotein levels during treatment schedule. Positive control mice showed an exponential increase in serum levels of Igλ over 9 weeks. In comparison, both treatment groups did not exhibit the same increase, with significantly lower levels by the end of treatment (p<0.05).</p

Partial Least Squares Discriminant Analysis of NMR spectra acquired on blood serum samples.

<p>Scores (<b>A</b>) and weights (on LV1, <b>B</b>, and LV2, <b>C</b>) plots obtained from OSC-PLS-DA performed on the NMR spectra of 44 blood serum samples. Group A (solid red, 19 samples): patients at diagnosis; group C1 (solid blue, 10 samples): sustained remission and group C2 (empty blue, 15 samples): in relapse after chemotherapy. Cho: choline; Cre: creatinine; Pyr: pyruvate; Ala: alanine; 2HiB: 2-hydroxyisobutyrate; Lac: lactate; 2HB: 2-hydroxybutyrate. H-xan: hypoxantine; Glu: glutamate; Gln: glutamine; Ace: acetate.</p

Patients characteristics.

<p>Footnotes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056422#pone-0056422-t001" target="_blank">table 1</a>. Serum reference ranges:- beta2 microglobulin 0.5–4.0 mg/l; serum creatinine 45–110 µmol/l (0.5 to 1.2 mg/dl). Paraprotein types detected by immunofixation of serum and urine: GLO IgG lambda, no flc in urine; GKU IgG kappa, flc in urine; NS non secretory, no Ig detected in blood or urine; KUO kappa flc only detected in urine but not serum; AKU IgA kappa, kappa flc in urine; ALO IgA lambda, no flc in urine; KUS kappa flc only detected in serum and urine; ALU IgA lambda, flc in urine; DLU IgD lambda, flc in urine.</p

Proton NMR spectra and Partial Least Squares Discriminant Analysis of blood serum samples.

<p>Representative sections of proton NMR spectra at the diagnosis (red), remission (green) and prolonged remission (blue) of one multiple myeloma patient. (<b>A</b>) up-field region (0.75–4.25 ppm), (<b>B</b>) down-field region (5.1–8.9 ppm), 20 times increased intensity compared to (A). (<b>C</b>) Scores plot obtained from OSC-PLS-DA performed on the NMR spectra of 71 blood serum samples. Group A (solid red, 19 samples): patients at diagnosis; group B (solid green, 27 samples): patients after chemotherapy; group C1 (solid blue, 10 samples): sustained remission and group C2 (empty blue, 15 samples): in relapse after chemotherapy.</p

Proton NMR-Based Metabolite Analyses of Archived Serial Paired Serum and Urine Samples from Myeloma Patients at Different Stages of Disease Activity Identifies Acetylcarnitine as a Novel Marker of Active Disease

<div><p>Background</p><p>Biomarker identification is becoming increasingly important for the development of personalized or stratified therapies. Metabolomics yields biomarkers indicative of phenotype that can be used to characterize transitions between health and disease, disease progression and therapeutic responses. The desire to reproducibly detect ever greater numbers of metabolites at ever diminishing levels has naturally nurtured advances in best practice for sample procurement, storage and analysis. Reciprocally, since many of the available extensive clinical archives were established prior to the metabolomics era and were not processed in such an ‘ideal’ fashion, considerable scepticism has arisen as to their value for metabolomic analysis. Here we have challenged that paradigm.</p> <p>Methods</p><p>We performed proton nuclear magnetic resonance spectroscopy-based metabolomics on blood serum and urine samples from 32 patients representative of a total cohort of 1970 multiple myeloma patients entered into the United Kingdom Medical Research Council Myeloma IX trial.</p> <p>Findings</p><p>Using serial paired blood and urine samples we detected metabolite profiles that associated with diagnosis, post-treatment remission and disease progression. These studies identified carnitine and acetylcarnitine as novel potential biomarkers of active disease both at diagnosis and relapse and as a mediator of disease associated pathologies.</p> <p>Conclusions</p><p>These findings show that samples conventionally processed and archived can provide useful metabolomic information that has important implications for understanding the biology of myeloma, discovering new therapies and identifying biomarkers potentially useful in deciding the choice and application of therapy.</p> </div

Partial Least Squares Discriminant Analysis of NMR spectra acquired on blood serum samples.

<p>Scores (<b>A</b>) and weights (on LV1; <b>B</b>) plots obtained from OSC-PLS-DA performed on the NMR spectra of 52 blood serum samples. Group B (solid green, 27 samples): patients after chemotherapy; group C1 (solid blue, 10 samples): sustained remission; group C2 (empty blue, 15 samples): in relapse after chemotherapy. Lac: lactate; Gly: glycine; Tau: taurine; Cho: choline; Suc: succinate; Pyr: pyruvate; Ace: acetate; 2HiB: 2-hydroxyisobutyrate.</p