A novel panel of protein biomarkers for predicting response to thalidomide-based therapy in newly diagnosed multiple myeloma patients

Multiple myeloma (MM) is a heterogeneous group of disorders both genotypically and phenotypically. Response to thalidomide-based induction therapy in newly diagnosed patients varies significantly in published clinical trials. Proteomic analysis was performed on 39 newly diagnosed MM patients treated with a thalidomide-based regimen (22 responders; 17 non-responders) using immunodepletion, 2-D DIGE analysis and mass spectrometry. Zinc-α-2-glycoprotein (ZAG), vitamin D-binding protein (VDB), serum amyloid-A protein (SAA) and β-2-microglobulin (B2M) had statistically significant higher concentrations in non-responders compared to responders, while haptoglobin (Hp) had a lower concentration. ELISAs were used to validate the candidate protein biomarkers using unfractionated serum from 51 newly diagnosed MM patients (29 responders; 22 non-responders). Using logistic regression, the best possible area under the curve (AUC) was 0.96 using ZAG, VDB and SAA in combination. Leave-one-out-cross-validation (LOOCV) indicated an overall predictive accuracy of 84% with associated sensitivity and specificity values of 81.8 and 86.2%, respectively. Subsequently, 16 of 22 thalidomide-refractory patients successfully achieved complete response or very good partial response using second-line treatment suggesting that the biomarker profile is specific to thalidomide response rather than identifying patients with MM refractory to all therapies. Using a novel panel of predictive biomarkers, the feasibility of predicting response to thalidomide-based therapy in previously untreated MM has been demonstrated.     

Identification of a panel of novel serum osteoarthritis biomarkers

Osteoarthritis (OA) is the most common rheumatic pathology. Because currently available diagnostic methods are limited and lack sensitivity, the identification of new specific biological markers for OA has become a focus. The purpose of this study was to identify novel protein biomarkers for moderate and severe OA in serum. Sera were obtained from 50 moderate OA patients, 50 severe OA patients, and 50 nonsymptomatic controls. Serum protein levels were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF mass spectrometry. We identified 349 different proteins in the sera, 262 of which could be quantified by calculation of their iTRAQ ratios. Three sets of proteins were significantly (p < 0.05) changed in OA samples compared to controls. Of these, 6 were modulated only in moderate OA, 13 only in severe OA and 7 in both degrees. Although some of these proteins, such as cartilage oligomeric matrix protein, have a previously reported putative biomarker value for OA, most are novel biomarker candidates for the disease. These include some complement components, lipoproteins, von Willebrand factor, tetranectin, and lumican. The specificity and selectivity of these candidates need to be validated before new molecular diagnostic or prognostic tests for OA can be developed.     

Identification of serum biomarkers for aging and anabolic response

Objective

With the progressive aging of the human population, there is an inexorable decline in muscle mass, strength and function. Anabolic supplementation with testosterone has been shown to effectively restore muscle mass in both young and elderly men. In this study, we were interested in identifying serum factors that change with age in two distinct age groups of healthy men, and whether these factors were affected by testosterone supplementation.

Methods

We measured the protein levels of a number of serum biomarkers using a combination of banked serum samples from older men (60 to 75 years) and younger men (ages 18 to 35), as well as new serum specimens obtained through collaboration. We compared baseline levels of all biomarkers between young and older men. In addition, we evaluated potential changes in these biomarker levels in association with testosterone dose (low dose defined as 125 mg per week or below compared to high dose defined as 300 mg per week or above) in our banked specimens.

Results

We identified nine serum biomarkers that differed between the young and older subjects. These age-associated biomarkers included: insulin-like growth factor (IGF1), N-terminal propeptide of type III collagen (PIIINP), monokine induced by gamma interferon (MIG), epithelial-derived neutrophil-activating peptide 78 (ENA78), interleukin 7 (IL-7), p40 subunit of interleukin 12 (IL-12p40), macrophage inflammatory protein 1β (MIP-1β), platelet derived growth factor β (PDGFβ) and interferon-inducible protein 10 (IP-10). We further observed testosterone dose-associated changes in some but not all age related markers: IGF1, PIIINP, leptin, MIG and ENA78. Gains in lean mass were confirmed by dual energy X-ray absorptiometry (DEXA).

Conclusions

Results from this study suggest that there are potential phenotypic biomarkers in serum that can be associated with healthy aging and that some but not all of these biomarkers reflect gains in muscle mass upon testosterone administration.     

Response to thalidomide in multiple myeloma: impact of angiogenic factors

Thalidomide has antiangiogenic and immunomodulatory effects, mediated by several cytokines such as vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF-2), hepatocyte growth factor (HGF), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Although extramedullary plasmacytomas (EMP) have a high vascularization, the response of these patients to thalidomide is controversial. Thirty-eight patients with refractory/relapsed MM were treated with thalidomide. Eleven patients had EMP when therapy was initiated. Serum specimens were obtained in patients before treatment was started and at the time of maximum response in responding patients or at thalidomide discontinuation in non-responders. Serum levels of VEGF, HGF and FGF-2 were determined in 18 patients whereas IL-6 and TNF-alpha were measured in 19 patients. Sixteen of the 38 patients (42%) responded to thalidomide. The response rate was significantly higher in patients without EMP (59% vs 0%, p = 0.0006 ). VEGF serum levels were significantly higher in responding patients. In contrast, baseline serum levels of HGF were significantly lower in responders. Neither VEGF nor HGF serum levels showed correlation with the presence of EMP. Baseline TNF-alpha serum levels were significantly lower in responding patients and in those without EMP. The serum levels of FGF-2 and IL-6 did not correlate with response to treatment or presence of EMP.     

Exploration of serum biomarkers for predicting the response to Inchinkoto (ICKT), a Japanese traditional herbal medicine

Introduction

In patients with obstructive jaundice, biliary drainage sometimes fails to result in improvement. A pharmaceutical-grade choleretic herbal medicine, Inchinkoto (ICKT), has been proposed to exert auxiliary effects on biliary drainage; however, its effects are variable among patients.

Objectives

The aim of this study is to explore serum biomarkers that are associated with pharmaceutical efficacy of ICKT.

Methods

Obstructive jaundice patients who underwent external biliary decompression were enrolled (n?=?37). ICKT was given orally 3 times a day at daily dose of 7.5 g. Serum and bile samples were collected before, 3 h after, and 24 h after ICKT administration. The concentrations of total bilirubin, direct bilirubin, and total bile acid in bile specimens were measured. Metabolites in serum samples were comprehensively profiled using LC–MS/MS and GC–MS/MS. Pharmacokinetic analysis of major ICKT components was also performed.

Results

ICKT administration significantly decreased serum ALT and increased bile volume after 24 h. The serum concentrations of ICKT components were not well correlated with the efficacy of ICKT. However, the ratio of 2-hydroxyisobutyric acid to arachidonic acid and the ratio of glutaric acid to niacinamide, exhibited good performance as biomarkers for the efficacy of ICKT on bile flow and ALT, respectively. Additionally, comprehensive correlation analysis revealed that serum glucuronic acid was highly correlated with serum total bilirubin, suggesting that this metabolite may be deeply involved in the pathogenesis of jaundice.

Conclusions

The present study indicates that ICKT is efficacious and provides candidates for predicting ICKT efficacy. Further validation studies are warranted.

     

Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma

Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 μM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.     

Identification of a select metabolite panel for measuring metabolic perturbation in response to heavy exertion

Introduction and objective

Databases from three global metabolomics-based studies (N?=?59) (PMID: 25409020, 26561314, 29566095) were evaluated for metabolite shifts following heavy exertion (75-km cycling) to generate a representative, select panel of metabolites identified by variable importance in projection (VIP) scores.

Methods and results

OPLS-DA was used to separate samples at pre- and post-exercise during the water-only trial in one of the studies (PMID: 26561314), and of 590 metabolites, 26 (all but one from the lipid pathway) had a VIP?>?2 and were selected for the panel. A second OPLS-DA based on the 26 metabolites was performed to separate pre- and post-exercise samples, and this model performed as well as the one with 590 metabolites (Q2Y?=?0.923, 0.925 respectively); this model also showed a complete separation using OPLS-DA plots between pre- and post-exercise samples for the other two studies. A latent variable t1 (a linear combination of the 26 metabolites), was generated and the metabolite data at each time point were projected to t1 with the relative distance on t1 and area under the curve (AUC) determined from the three databases. Acute carbohydrate compared to water-only ingestion was linked to a 28–47% reduction in AUCs following exercise depending on the carbohydrate source and recovery time period.

Conclusions

These data support that a panel of 26 metabolites can be used to represent global metabolite increases induced by prolonged, intensive exercise. This select panel includes metabolites primarily from the lipid super pathway, and exercise-induced increases are sensitive to the moderating effect of acute carbohydrate ingestion.

     

Identification of serum biomarkers for premature ovarian failure

Premature ovarian failure (POF) is defined when a female achieves menopause before the age of 40. Although many conditions are known to be causative for POF, the most common one is idiopathic. This study was undertaken to investigate the pathogenesis of POF using proteomic tools. Two-dimensional electrophoresis (2-DE) analysis was performed to screen for proteins differentially expressed in patients with POF. Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified 11 significant proteins differentially expressed in the serum of POF patients: 5 proteins with expression increased more than two folds, 5 proteins with expression decreased more than two folds, and 1 protein expressed specifically in the serum of patients with POF. The results of the 2-DE analysis were further validated by Western blotting and ELISA analyses, which 5 reproductive system-related proteins (Ceruloplasmin, Complement C3, Fibrinogen α, Fibrinogen β, and SHBG) were selected. The different expression levels for these proteins were confirmed and demonstrated the possibility of using them as biomarkers to screen POF. These pre-clinical data provide plausible translational implications for targeting the pathogenesis of POF for each protein.     

Identification of novel serum peptides biomarkers for female breast cancer patients in Western China

This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre‐ and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic‐bead‐based separation followed by MALDI‐TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1–6 upregulated and Peaks 7–10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605‐629, ITIH4 347–356, and APOA2 43–52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

Assessment of serum thiol/disulfide homeostasis in multiple myeloma patients by a new method

Objectives: The etiology of multiple myeloma (MM) is not exactly known. This study investigated the role of thiol/disulfide homeostasis in the etiopathogenesis of MM.

Methods: Some 50 patients with MM (aged 39–84 years) and 50 sex-matched healthy volunteer controls (aged 50–91 years) participated in this study. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfide were measured.

Results: Native and total thiol levels in the control group were determined to be higher than in the study and patient groups (P<0.001). Disulfide levels were found to be higher in the control group than in the study group and higher in newly diagnosed patients than in outpatients who were undergoing treatment (P=0.002). The ratios of thiol levels were found to be similar in both the study and control groups (P>0.05).

Discussion: The results of the study show that although there was a decrease in the levels of disulfide, native thiol, and total thiol, the balance of thiol/disulfide was maintained. This is the first study to research the homeostasis of dynamic thiol/disulfide from the perspective of the new method that was used. We hope that this study will encourage and facilitate further studies in this area.     

Exonic variants in multiple myeloma patients associated with relapsed/ refractory and response to bortezomib regimens

Novel treatment in multiple myeloma represented by proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies have produced a deep response. However, relapses are possible, and all classes of drugs are refractory to patients. Next-generation sequencing has improved our understanding of the multiple myeloma genome related to drug resistance and has discovered many genomic variants. Therefore, this study was conducted to investigate new variants associated with drug resistance in MM patients who relapsed and refractory to bortezomib regimen and daratumumab treatment using next-generation sequencing for whole-exome sequencing. Peripheral blood samples were collected in EDTA tubes from six patients; four were in relapsed and refractory to bortezomib regimens and daratumumab; two patients responded to bortezomib regimens. Whole-exome sequencing was performed by the MGI-DNBSEQ-G400 instrument. We identified 21 variants in multiple myeloma patients. Seventeen variants were found in relapsed and refractory multiple myeloma in 11 genes (GNAQ, PMS1, CREB1, NSUNS2, PIK3CG, ROS1, PMS2, FIT4, KDM5A, STK11 and ZFHX3). And four variants were identified in two patients with response to bortezomib regimens in 4 genes (RAF1, CREB1, ZFHX3 and INSR). We have observed several genetic variants in many genes that may have been associated with the poor prognosis and poor response to treatment in these patients. These values should be further confirmed in large sample studies using the RNA-seq technique to identify genome expression.     

Identification of a panel of mitotic spindle-related genes as a signature predicting survival in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is one of the most malignant tumor types worldwide. Our objective was to identify a genetic signature that could predict the prognosis of patients with LUAD. We extracted gene data sets from The Cancer Genome Atlas and obtained differentially expressed genes that were highly expressed at every stage. These genes were analyzed using gene set enrichment analysis to obtain four biological processes associated with LUAD. Subsequently, Cox univariate and multivariate analyses were performed to generate four optimized models (G2M checkpoint, E2F targets, mitotic spindle, and glycolysis). We identified a mitotic spindle-related signature (KIF15, BUB1, CCNB2, CDK1, KIF4A, DLGAP5, ECT2, and ANLN), which could be an independent prognostic indicator, to predict the prognosis of patients with LUAD. This new discovery should offer opportunities to explore the pathogenesis of LUAD and prove clinically useful in predicting LUAD patient prognosis.     

Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients

In the present study, we screened proteomic and cytokine biomarkers between patients with adenomatous polyps and colorectal cancer (CRC) in order to improve our understanding of the molecular mechanisms behind turmorigenesis and tumor progression in CRC. To this end, we performed comparative proteomic analysis of plasma proteins using a combination of 2DE and MS as well as profiled differentially regulated cytokines and chemokines by multiplex bead analysis. Proteomic analysis identified 11 upregulated and 13 downregulated plasma proteins showing significantly different regulation patterns with diagnostic potential for predicting progression from adenoma to carcinoma. Some of these proteins have not previously been implicated in CRC, including upregulated leucine‐rich α‐2‐glycoprotein, hemoglobin subunit β, Ig α‐2 chain C region, and complement factor B as well as downregulated afamin, zinc‐α‐2‐glycoprotein, vitronectin, and α‐1‐antichymotrypsin. In addition, plasma levels of three cytokines/chemokines, including interleukin‐8, interferon gamma‐induced protein 10, and tumor necrosis factor α, were remarkably elevated in patients with CRC compared to those with adenomatous polyps. Although further clinical validation is required, these proteins and cytokines can be established as novel biomarkers for CRC and/or its progression from colon adenoma.     

Proteomic biomarkers predicting lymph node involvement in serum of cervical cancer patients. Limitations of SELDI-TOF MS

ABSTRACT: BACKGROUND: Lymph node status is not part of the staging system for cervical cancer, but provides important information for prognosis and treatment. We investigated whether lymph node status can be predicted with proteomic profiling. Material & methods Serum samples of 60 cervical cancer patients (FIGO I/II) were obtained before primary treatment. Samples were run through a HPLC depletion column, eliminating the 14 most abundant proteins ubiquitously present in serum. Unbound fractions were concentrated with spin filters. Fractions were spotted onto CM10 and IMAC30 surfaces and analyzed with surface-enhanced laser desorption time of flight (SELDI-TOF) mass spectrometry (MS). Unsupervised peak detection and peak clustering was performed using MASDA software. Leave-one-out (LOO) validation for weighted Least Squares Support Vector Machines (LSSVM) was used for prediction of lymph node involvement. Other outcomes were histological type, lymphvascular space involvement (LVSI) and recurrent disease. RESULTS: LSSVM models were able to determine LN status with a LOO area under the receiver operating characteristics curve (AUC) of 0.95, based on peaks with m/z values 2,698.9, 3,953.2, and 15,254.8. Furthermore, we were able to predict LVSI (AUC 0.81), to predict recurrence (AUC 0.92), and to differentiate between squamous carcinomas and adenocarcinomas (AUC 0.88), between squamous and adenosquamous carcinomas (AUC 0.85), and between adenocarcinomas and adenosquamous carcinomas (AUC 0.94). CONCLUSIONS: Potential markers related with lymph node involvement were detected, and protein/peptide profiling support differentiation between various subtypes of cervical cancer. However, identification of the potential biomarkers was hampered by the technical limitations of SELDI-TOF MS.     

Renal safety of zoledronic acid with thalidomide in patients with myeloma: a pharmacokinetic and safety sub-study

Background  

Cases of impaired renal function have been reported in patients who had been treated with both zoledronic acid and thalidomide for myeloma. Hence, we conducted a safety study of zoledronic acid and thalidomide in myeloma patients participating in a trial of maintenance therapy.     

Diurnal variations of serum beta 2-microglobulin in multiple myeloma

The chronobiological circadian behaviour in serum levels of beta 2-microglobulin has been investigated in three groups of subjects: (A) 6 healthy controls; (B) 6 patients with untreated multiple myeloma; (C) 6 patients with multiple myeloma in complete remission after polychemotherapy. From all subjects, under the same standard life conditions, venous blood samples were drawn at 4-hour intervals starting from midnight during the span of a whole day. Circulating serum beta 2-microglobulin levels were determined by RIA method. The time-related data were analyzed by chronograms and the "mean-group cosinor" method. A significant circadian rhythm for serum beta 2-microglobulin was detected in the control group, with a peak in the morning hours, and in untreated patients, with a peak in the afternoon hours. No significant rhythm was found in treated patients with multiple myeloma. A significant mesor reduction was noted in patients with complete remission, correlated with the absence of circadian rhythm, in respect to untreated patients. These data suggest that serum levels of beta 2-microglobulin could be related to the neoplastic plasma cell proliferation and to the effect of therapy, and that the circadian evaluation could be used as a guide in monitoring myeloma patients.