Open Access

Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients

  • Authors:
    • Ayodele A. Alaiya
    • Mahmoud Aljurf
    • Zakia Shinwari
    • Fahad Almohareb
    • Hafiz Malhan
    • Hazzaa Alzahrani
    • Tarek Owaidah
    • Jonathan Fox
    • Fahad Alsharif
    • Said Y. Mohamed
    • Walid Rasheed
    • Ghuzayel Aldawsari
    • Amr Hanbali
    • Syed Osman Ahmed
    • Naeem Chaudhri
  • View Affiliations

  • Published online on: July 7, 2016     https://doi.org/10.3892/ijo.2016.3618
  • Pages: 913-933
  • Copyright: © Alaiya et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual's best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as ‘personalized-medicine-model’.

Introduction

Chronic myeloid leukemia (CML) is unequivocally distinguishable from other myeloproliferative disorders by the presence of a reciprocal translocation of chromosomes 9 and 22 (13). Although the Philadelphia chromosome is detected in 90–95% of CML patients, evidence of the BCR-ABL rearrangement is also usually detected in the subgroup of Philadelphia chromosome-negative CML patients (46).

The presence of BCR-ABL in CML patients and the requirement of kinase activity for BCR-ABL function make this an attractive target for selective kinase inhibitors.

The old traditional therapy of newly diagnosed chronic phase-CML patients includes busulfan and hydroxyurea and most of the patients will stay in a chronic phase for approximately 3–5 years (7,8). Treatment of CML later evolved to where the goal was prolongation of the chronic phase through induction of karyotypic remission and possibly molecular remission using Alfa-interferon therapy with or without cytosine arabinoside. Thereafter, imatinib mesylate (IM) a tyrosine kinase inhibitor (TKI) was introduced as potential molecular therapy for CML (7,9). IM is capable of inhibiting BCR-ABL kinase activity by blocking ABL tyrosine kinase action through the binding and subsequent inactivation of the ATP-binding sites of ABL tyrosine kinase in leukemic cells (9,10). Since its introduction, several clinical trials have demonstrated the efficacy of IM and new generation TKIs in the treatment of CML, including patients with interferon-refractory CP-CML, as well as patients with CML in blast crisis (11).

Approximately more than 50% of CML patients treated with imatinib achieve a complete cytogenetic response (11,12). CML progression while on imatinib is usually due to the emergence of imatinib-resistant BCR-ABL mutant cells.

The relatively unpredictable biological behavior is a major challenge in its management as the chronic phase of CML is less aggressive and has very favorable prognosis with an excellent 5-year survival rate. By contrast, the biologically aggressive blast phase of CML is often rapidly fatal (2). Currently, there is no recognized prognostic value for the baseline BCR-ABL level, furthermore, there are variations in sensitivity or dependability of RQ-PCR assays across different laboratories (13). There is therefore a need to develop molecular markers for selection of choice of therapy at the time of diagnosis and to identify patients that are more likely to achieve a sustained remission, and patients who are more likely to develop resistance to imatinib therapy.

New analytical tools in proteomics are emerging that give new insights into biological processes that may speed up the discovery of potential biomarkers. Quantitative molecular variations may be used for the development of methods for tumor classification based on large amounts of gene expression data generated by 2-DE analysis of proteins (14,15).

The main aim of the present study is towards discovery of objective markers that predict patients’ response status and selection of appropriate choice of therapy at the onset of disease diagnosis. It focuses on the analysis of global peripheral blood plasma and bone marrow plasma protein expression profiles among CP-CML patients who achieved LT-MMR on imatinib compared with patients without MMR as well as whether or not they remain on TKI or switch to second generation TKI or requiring alternative therapy.

The endpoint is to identify disease-specific/disease-associated protein biomarkers seen in bone marrow tissue as well as in peripheral blood plasma. This would subsequently allow monitoring of such biomarker proteins in peripheral blood, rather than bone marrow, demanding less invasive procedures for objective prediction of individual’s best treatment options and prognostic monitoring of CML patients.

Materials and methods

All bone marrow samples were obtained by aspiration procedure via posterior iliac crest under local anesthesia. Because of limited amount of materials for analysis, the cells were not flow cytometry sorted, rather unsorted bone marrows as well as unsorted peripheral blood plasma were collected and prepared for analysis.

Bone marrow and plasma, samples obtained at diagnosis and prior to initiation of treatment from 37 patients with newly diagnosed CP-CML were subjected to expression proteome analysis using combined gel-based 2-DE and label-free in-solution quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Patients selections into those that achieved or did not achieve MMR was based on patients with serial positive or negative responses to treatment at different time-points (3, 6, 12 and 24 months, respectively). Patients that responded at a time-point but failed to respond at the next time-point were not included in the analysis. However, patients that did not achieve MMR at 3 months, but subsequently achieved MMR at 6, 12 and 24 months were included. Because there was fewer number of patients with MMR at 3 months, the focus of our analyzed time-points were at 6, 12 and 24 months. Twenty-five patients consisting 13 with major molecular response and 12 without major molecular response were analyzed. In addition, patients that failed tyrosine kinase inhibitor (TKI) were analyzed. Four additional patients samples not included in the proteomics analysis were used in the western blot analysis. The overview of experimental design is shown in Fig. 1 and the clinical characteristics of all patients were as indicated in Table I.

Table I

Clinical characteristics of analyzed samples.

Table I

Clinical characteristics of analyzed samples.

TKI-failMMR at 6 monthsMMR at 12 monthsMMR at 18 monthsMMR at 24 months





SamplesGenderAge (years)NoYesNoYesNoYesNoYesNoYes
CML1Female14
CML2Female14
CML3Female26
CML4Male18
CML5Male50
CML6Female50
CML7Male41
CML8Female64
CML10Male27
CML13Male44
CML15Male21
CML16Male44
CML17Female18
CML18Female65
CML19Male26
CML21Male39
CML22Female67
CML23Male47
CML24Male18
CML25Male40
CML26Female30
CML27Female36
CML28Female37
CML29Female33
CML30Female44
CML31Female48
CML32Female38
CML33Female32
CML34Male52
CML38Male37
CML40Male61
CML41Male47
CML43Female51
CML44Female14
CML45Female45
CML46Female45
CML47Male32
Total1916171816171120822
Sample preparation protocols for proteomic analysis

All the patients with primary diagnosis of CML were recruited in Oncology Center at KFSH&RC. From each of the patients, 10 ml of peripheral EDTA-anti-coagulated blood (plasma) was taken. Where possible, bone marrow aspirations were obtained from the same patients in addition to peripheral blood samples.

All samples were subjected to extensive pre-analysis cleanup using human albumin removal protocols (Agilent Technologies). Written and signed informed consents were obtained from all patients and the Institution’s Research Advisory Council, under the Office of Research Affairs, approved the study (RAC# 2050-040).

Protein separation by high resolution two dimensional gel electrophoresis, (2-DE) scanning and image analysis

Equivalent amount of 50 mg total proteins for each analyzed sample was dissolved in 350 ml volume of rehydration buffer [2% (v/v) IPG-buffer 4–7 linear] and loaded onto an 11-cm IPG-strip 4–7 linear (Bio-Rad Laboratories). This gave better overview of gel separated protein spots across the entire chosen pH window and gel images were visualized by SYPRO Ruby fluorescent staining. Stained gels were scanned using a Typhoon Trio Imager (GE) and data were analyzed using the Progenesis SameSpots software (version 7.1.0; Nonlinear Dynamics, Ltd., Newcastle, UK). Gel images were compared for qualitative and quantitative differences. In addition, the protein expression profiles were used to assess the level of individual variability and only samples with similar phenotypic changes were used for sample pools for LC/MS/MS (due to low through-put analysis) as detailed below. Polypeptide quantities were calculated based on the normalized total integrated density volume.

Protein in solution-digestion

The plasma samples were diluted and protein concentrations of all samples were normalized as previously described (16). Briefly, for analytical runs, equal amount of protein was taken from each sample to generate a pool of patient as one group. The samples within same sample cohort were pooled due to low through-put of LC/MS/MS analysis platform. However, samples were initially screened using 2-DE for homogeneity within the same analysis group. For each analysis sample group, 200 μg complex protein mixture was taken and exchanged twice with 500 μl of 0.1% RapiGest (Waters Corp., Manchester, UK). Protein concentrations of between 0.50 and 1 μg/μl was achieved at the end of digestion. Details of digestion protocols are as previously described (16,17). Briefly, proteins were denatured in 0.1% RapiGest SF at 80°C for 15 min, reduced in 10 mM DTT at 60°C for 30 min, and alkylated in 10 mM Iodoacetamide (IAA) for 40 min at room temperature in the dark. Samples were trypsin digested at 37°C overnight. Samples were diluted with aqueous 0.1% formic acid prior to LC/MS analysis in order to achieve a load of ~2 μg on analytical column. All samples were spiked with yeast alcohol dehydrogenase (ADH; P00330) as internal standard to the digests in order for absolute quantitation.

Protein identification by mass spectrometry: LC-MSE analysis

The digested peptides were subjected to 1-Dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on Synapt G2 (Waters Corp.). Expression proteomics data were generated between sample groups using both qualitative and quantitative protein changes. The ESI-MS analysis and instrument settings were optimized on the tune page as previously reported (16).

A total of 2 μl sample injection representing ~1 μg protein digests was loaded on-column and samples were infused using the Acquity sample manager with mobile phase consisting of A1 99% water +1% acetonitrile + 0.1% formic acid and B1 acetonitrile + 0.1% formic acid with sample flow rate of 0.450 μl/min. Data acquisition using iron mobility separation experiments (HDMSE) were performed and data were acquired over a range of m/z 50–2000 Da with a total acquisition time of 115 min. All samples were analyzed in triplicate runs (triplicate runs were repeated on two different occasions as a measure of reproducibility) and data were acquired using the MassLynx programs (version. 4.1, SCN833; Waters) operated in resolution and positive polarity modes. ProteinLynx Global Server (PLGS) 2.2 and Progenesis QI for proteomics (Progenesis QIfp version 2.0.5387) (Nonlinear Dynamics/Waters) were used for all automated data processing and database searching. The generated peptide masses were searched against two-unified non-redundant databases (Uniprot/Swiss-Prot Human protein sequence database) using the PLGS 2.5 and Progenesis QIfp for protein identification (Waters).

Data analysis and informatics

Progenesis QI v.2.0.5387 for proteomics was used to process and search the data to accurately quantify and identify proteins that are significantly changing between sample groups. The human database containing thousands of reviewed non-redundant entries were downloaded from UniProt/Swiss-Prot and search algorithm was applied as previoudly described (18). The criteria used for the database search were as previously described (16). Normalized label-free quantification was achieved using Progenesis QI software. The generated differentially expressed data was filtered to show only statistically (ANOVA), significantly regulated proteins (P≤0.05) and a fold change >1.5. In addition, ‘Hi3’ absolute quantification was performed using ADH as an internal standard to give an absolute amount of each identified protein. These options are available as incorporated into the Progenesis QIfp (Nonlinear Dynamics/Waters).

Results

Changes in protein expression between patients with/without major molecular response at 6 months

A total of 73 protein spots on 2-DE gels differed significantly between patients that achieved MMR from those who did not achieve MMR (P<0.05 and at least 1.5-fold difference). The locations of these protein spots are shown as marked on a representative 2-DE map derived from a sample with MMR in Fig. 2A. Even though the identifications of these protein spots were not done, their quantitative expression fingerprints from 2-DE analysis pattern accurately predicts 13 individuals that achieved MMR at 6 months from 12 subjects without MMR (No-MMR) using principal component analysis (PCA) (Fig. 2B).

These findings are similar to what was observed with PCA plot generated from non-gel LC/MS/MS analysis platform, as some of the results were independently validated using the label free quantitative liquid chromatography tandem mass spectrometry as detailed below.

LC/MS/MS analysis of peripheral blood for prognostic monitoring of early CML treatment response

Peripheral blood samples were evaluated for early treatment response at 6 month and prediction of treatment options towards personalized medicine. Approximately 115 protein species were identified, of which only 64 were significantly differentially expressed between MMR and No-MMR sample groups. (> 1.5- to ∞-fold change, p<0.05). These proteins predict accurately patients with MMR vs. No-MMR patients using unsupervised Hierarchical Cluster Analysis (Fig. 3).

Evaluation of bone marrow and peripheral blood protein profiles for prognostic monitoring of prolonged and sustained treatment response vs. persistent no-major molecular response

Some of the patients have been followed for more than 24 months. Patients who have been consistent over a long-term in achieving and maintaining MMR from 6 months until 24 months were labeled as LT-MMR, while patients that have been persistent with No-MMR from 6 months until 24 months were called P-No-MMR. We believe that the ability to select early responders from 6 months all through 24 months would be very helpful to identify markers that would accurately predict patients with risk of delayed or suboptimal response further than 6 months. These cohorts of patients were considered as important in an effort to provide the possibility to identify surrogate biomarkers to evaluate long-term treatment response and discovery of disease-specific/disease-associated proteins for objective prognostic monitoring of CML patients.

Equal amounts of total peripheral blood plasma proteins from 10 LT-MMR patients were pooled and compared for their protein expressions among 10 other samples from P-No-MMR patients using quantitative label-free LC/MS/MS expression proteome analysis.

Approximately 700 proteins representing 280 unique protein species were identified (due to different protein isoforms). Only 164 of the 280 proteins were significantly differentially expressed between LT-MMR and P-No-MMR sample groups (>1.5- to ∞-fold change; P<0.05) and accurately predict patients with major molecular response (LT-MMR) vs. No-major molecular response (P-No-MMR) using unsupervised principal component analysis (Fig. 4A). The list of identified differentially expressed proteins in PBP is described in Table IIA.

Table II

The identified differentially expressed proteins in peripheral blood plasma (PBP) and bone marrow plasma (BMP) from CML patients with major molecular response (MMR), No-MMR, On-tyrosine kinase inhibitor (On-TKI) and NOT-on-TKI.

Table II

The identified differentially expressed proteins in peripheral blood plasma (PBP) and bone marrow plasma (BMP) from CML patients with major molecular response (MMR), No-MMR, On-tyrosine kinase inhibitor (On-TKI) and NOT-on-TKI.

A, The identified differentially expressed proteins in PBP of CML patients

AccessionPeptide countAnova (p)Max fold changeHighest mean conditionLowest mean conditionDescription
P5019720.0005342.41067CML-PBP-TKI-YCML-PBP-MMR 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase
P1628149.90E-082.92498CML-PBP-TKI-NCML-PBP-TKI-Y23 kDa protein
P4931341.97E-079.09421CML-PBP-TKI-YCML-PBP-No-MMR30 kDa ribonucleoprotein, chloroplast precursor
O8653534.48E-1222.9885CML-PBP-TKI-NCML-PBP-TKI-Y3-isopropylmalate dehydratase small subunit
P4235212.83E-1212.8902CML-PBP-TKI-NCML-PBP-MMR50S ribosomal protein L9.
O6619030.00192115.3266CML-PBP-No-MMRCML-PBP-TKI-N60 kDa chaperonin (Protein Cpn60) (groEL protein)
P5017410.0001482.33176CML-PBP-TKI-YCML-PBP-MMRAcetyl-CoA acetyltransferase
P4134151.37E-093.82215CML-PBP-TKI-NCML-PBP-No-MMRActin 11
P5345842.59E-1025.5243CML-PBP-TKI-YCML-PBP-No-MMRActin 5 (Fragment)
P5350641.85E-056.06449CML-PBP-TKI-YCML-PBP-No-MMRActin, cytoplasmic type 8
P53466a40.0001784.16358CML-PBP-TKI-NCML-PBP-TKI-YActin, cytoskeletal 2 (LPC2)
P0732611.50E-1433782.8CML-PBP-TKI-YCML-PBP-MMRAllophycocyanin beta chain
P7250511.97E-1150.0172CML-PBP-TKI-YCML-PBP-TKI-NAllophycocyanin beta chain
P0276398.94E-052.16961CML-PBP-TKI-YCML-PBP-MMRAlpha-1-acid glycoprotein 1 precursor (AGP 1)
P1965278.07E-103.8292CML-PBP-TKI-YCML-PBP-MMRAlpha-1-acid glycoprotein 2 precursor (AGP 2)
P01009357.33E-062.57662CML-PBP-TKI-YCML-PBP-TKI-N Alpha-1-antitrypsin precursor
P04217a174.44E-112.21378CML-PBP-TKI-YCML-PBP-MMR Alpha-1B-glycoprotein
P01023714.34E-093.03669CML-PBP-TKI-YCML-PBP-No-MMR Alpha-2-macroglobulin precursor (Alpha-2-M)
P3970120.00185717.1724CML-PBP-MMRCML-PBP-TKI-Y Alpha-ribazole-5′-phosphate phosphatase
P41361a,b62.78E-072.68159CML-PBP-TKI-NCML-PBP-TKI-YAntithrombin-III (ATIII)
P01008151.56E-125.19919CML-PBP-TKI-YCML-PBP-TKI-NAntithrombin-III precursor (ATIII) (PRO0309)
P32262a62.65E-06InfinityCML-PBP-MMRCML-PBP-TKI-YAntithrombin-III precursor (ATIII)
P3226187.40E-06InfinityCML-PBP-No-MMRCML-PBP-TKI-YAntithrombin-III precursor (ATIII)
P1549748.88E-1632.5405CML-PBP-MMRCML-PBP-TKI-YApolipoprotein A-I precursor (Apo-AI)
P1864836.73E-082.76435CML-PBP-MMRCML-PBP-TKI-YApolipoprotein A-I precursor (Apo-AI)
P02648127.81E-062.49354CML-PBP-TKI-NCML-PBP-TKI-YApolipoprotein A-I precursor (Apo-AI)
P0265264.96E-103.48432CML-PBP-TKI-YCML-PBP-No-MMRApolipoprotein A-II precursor (Apo-AII) (ApoA-II)
P06727a120.000632.06242CML-PBP-TKI-YCML-PBP-TKI-NApolipoprotein A-IV precursor (Apo-AIV)
P0265523.46E-117.42195CML-PBP-TKI-YCML-PBP-No-MMRApolipoprotein C-II precursor (Apo-CII)
P02649108.01E-083.13115CML-PBP-TKI-YCML-PBP-No-MMRApolipoprotein E precursor (Apo-E)
P4377311.03E-083.21196CML-PBP-TKI-NCML-PBP-MMRATP-dependent hsl protease ATP-binding subunit
P0188412.13E-09InfinityCML-PBP-TKI-YCML-PBP-MMR Beta-2-microglobulin precursor
P3162514.44E-1629.2811CML-PBP-TKI-YCML-PBP-MMRBifunctional protease/dUTPase [Includes: Aspartic]
Q0859525.42E-072.36202CML-PBP-TKI-NCML-PBP-No-MMRBR1 protein
P06702a32.35E-125.10685CML-PBP-No-MMRCML-PBP-MMRCalgranulin B (Migration inhibitory factor-related
P0709029.28E-094.35593CML-PBP-TKI-YCML-PBP-MMRCalretinin (CR)
P00450336.96E-102.07132CML-PBP-TKI-YCML-PBP-TKI-NCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
P13635193.89E-072.06575CML-PBP-TKI-YCML-PBP-No-MMRCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
Q61147196.29E-055.77271CML-PBP-No-MMRCML-PBP-TKI-NCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
O3400210.00013768.1783CML-PBP-MMRCML-PBP-TKI-YCitrate synthase (EC 4.1.3.7)
P2352816.64E-0917.4873CML-PBP-No-MMRCML-PBP-MMRCofilin, non-muscle isoform (18 kDa phosphoprotein)
Q0370822.25E-07InfinityCML-PBP-MMRCML-PBP-TKI-NColicin E7 immunity protein (ImmE7)
P0073641.06E-113.2943CML-PBP-TKI-YCML-PBP-No-MMRComplement C1r component precursor
P0987142.38E-072.92284CML-PBP-TKI-YCML-PBP-TKI-NComplement C1s component precursor
P01027a221.58E-118.12844CML-PBP-TKI-NCML-PBP-TKI-YComplement C3 precursor (HSE-MSF)
P01024835.80E-102.95796CML-PBP-TKI-YCML-PBP-TKI-NComplement C3 precursor [Contains: C3a anaphylatox]
P01030a200.0014792.42252CML-PBP-No-MMRCML-PBP-TKI-NComplement C4 precursor [Contains: C4A anaphylatox]
P0418670.0001662.2386CML-PBP-TKI-NCML-PBP-MMRComplement factor B precursor (C3/C)
P0515634.54E-073.80805CML-PBP-TKI-YCML-PBP-No-MMRComplement factor I precursor (EC 3.4.21) (C3B/)
Q3343916.13E-1176.1488CML-PBP-TKI-YCML-PBP-TKI-NCytochrome c oxidase polypeptide I
P1453218.42E-0811.1984CML-PBP-TKI-NCML-PBP-TKI-YCytochrome C551 peroxidase precursor
Q3873215.73E-0816.099CML-PBP-TKI-NCML-PBP-TKI-YDAG protein, chloroplast precursor
P5775935.60E-135.45666CML-PBP-TKI-NCML-PBP-TKI-YEndoplasmic reticulum protein ERp29 precursor
P2071011.19E-0824.9012CML-PBP-TKI-NCML-PBP-TKI-Y Excisionase
Q4576510.00058213.2686CML-PBP-TKI-NCML-PBP-No-MMRFerric uptake regulation protein
P026712305.53907CML-PBP-TKI-YCML-PBP-TKI-NFibrinogen alpha/alpha-E chain precursor
P02675361.52E-092.8323CML-PBP-TKI-YCML-PBP-TKI-NFibrinogen beta chain precursor
P02679266.02E-063.07718CML-PBP-TKI-YCML-PBP-TKI-NFibrinogen gamma chain precursor
P11276110.0002012.27101CML-PBP-No-MMRCML-PBP-TKI-NFibronectin precursor (FN) (Fragments)
P0804114.74E-054.36822CML-PBP-MMRCML-PBP-TKI-YGas vesicle protein C
P4780520.0051066.89488CML-PBP-TKI-YCML-PBP-MMRGastrulation specific protein G12
P1302032.96E-062.44545CML-PBP-TKI-YCML-PBP-MMRGelsolin (Actin-depolymerizing factor)
P0639630.0001024.00281CML-PBP-TKI-YCML-PBP-TKI-NGelsolin precursor, plasma (Actin-depolymerizing)
P0622825.86E-072.30924CML-PBP-TKI-YCML-PBP-TKI-NGene 27 protein
P1575111.74E-072.52369CML-PBP-TKI-YCML-PBP-MMRGeneral secretion pathway protein L
P2372240.0048173.55572CML-PBP-MMRCML-PBP-TKI-NGlyceraldehyde 3-phosphate dehydrogenase
P5504221.22E-084.00025CML-PBP-TKI-YCML-PBP-TKI-NGTP-binding protein RAD (RAS associated)
P00739133.99E-115.55201CML-PBP-TKI-YCML-PBP-TKI-NHaptoglobin-related protein precursor
P9195311.37E-074.42879CML-PBP-TKI-YCML-PBP-No-MMRHatching enzyme precursor (HE) (HEZ)
P0192266.01E-1410.9884CML-PBP-TKI-YCML-PBP-MMRHemoglobin α chain
P0741420.00154822.3314CML-PBP-No-MMRCML-PBP-TKI-NHemoglobin α chain
P19002a22.15E-052.87378CML-PBP-No-MMRCML-PBP-MMRHemoglobin α-1, α-2, and α-3 chains
P0205448.10E-1554.1252CML-PBP-TKI-YCML-PBP-MMRHemoglobin β chain
P1439154.48E-115.10044CML-PBP-TKI-NCML-PBP-No-MMRHemoglobin β chain
P1898581.04E-092.8812CML-PBP-TKI-YCML-PBP-No-MMRHemoglobin β chain
P0213422.66E-0919.544CML-PBP-MMRCML-PBP-TKI-YHemoglobin β chain
P1898454.21E-093.66515CML-PBP-TKI-YCML-PBP-MMRHemoglobin β chain
P0204953.19E-05976.807CML-PBP-TKI-YCML-PBP-No-MMRHemoglobin β chain
P1175860.00227713.0218CML-PBP-MMRCML-PBP-TKI-NHemoglobin β chain
P02094a20.0043667.02752CML-PBP-MMRCML-PBP-TKI-NHemoglobin β-major chain
Q2822040.00023530.7953CML-PBP-TKI-NCML-PBP-TKI-YHemoglobin ɛ chain
P05546130.0057742.11422CML-PBP-TKI-YCML-PBP-TKI-NHeparin cofactor II precursor (HC-II)
P3343350.0005773.03464CML-PBP-MMRCML-PBP-TKI-NHistidine-rich glycoprotein (Histidine-proline rich)
Q2864050.0010286.73632CML-PBP-MMRCML-PBP-TKI-NHistidine-rich glycoprotein precursor
P1145712.09E-1043.477CML-PBP-TKI-NCML-PBP-TKI-YHistone-like protein HLP-1 precursor (DNA-binding)
P09631a18.27E-146.74686CML-PBP-MMRCML-PBP-TKI-YHomeobox protein Hox-A9 (Hox-1.7)
Q10521a12.13E-053.30175CML-PBP-TKI-YCML-PBP-TKI-NHypothetical 16.9 kDa protein Rv2239c
P37506a18.12E-103.91542CML-PBP-TKI-YCML-PBP-MMRHypothetical 20.4 kDa protein in COTF-TETB
Q106161 1.93E-062.87092 CML-PBP-TKI-N CML-PBP-TKI-YHypothetical 56.0 kDa protein Rv1290c
P0708310.00041511.8324CML-PBP-No-MMRCML-PBP-TKI-NHypothetical 9.8 kDa protein in Gp55-nrdG intergenic region
Q9KD4521.21E-103.97407CML-PBP-MMRCML-PBP-TKI-YHypothetical protein BH1374
P4767920.0005074.0852CML-PBP-TKI-YCML-PBP-TKI-NHypothetical protein MG441
P42962a20.0005549.91114CML-PBP-TKI-YCML-PBP-TKI-NHypothetical protein ycsE
P5446222.28E-1360.8113CML-PBP-MMRCML-PBP-TKI-YHypothetical protein yqeV
P01876b141.04E-124.48826CML-PBP-TKI-NCML-PBP-MMRIg alpha-1 chain C region
P01862a20.001527InfinityCML-PBP-No-MMRCML-PBP-TKI-NIg gamma-2 chain C region
P01860110.0005424.16369CML-PBP-TKI-YCML-PBP-No-MMRIg gamma-3 chain C region (Heavy chain)
P01861143.90E-092.35422CML-PBP-TKI-YCML-PBP-No-MMRIg gamma-4 chain C region
P19181a40.0055722.28883CML-PBP-MMRCML-PBP-TKI-NIg heavy chain V region 5A precursor
P01765a24.91E-095.63765CML-PBP-TKI-NCML-PBP-TKI-YIg heavy chain V-III region TIL
P01620a50.00058911.6515CML-PBP-No-MMRCML-PBP-TKI-NIg kappa chain V-III region SIE
P0184260.0003942.20304CML-PBP-TKI-YCML-PBP-TKI-NIg lambda chain C regions
P0171425.10E-123.83063CML-PBP-No-MMRCML-PBP-TKI-YIg lambda chain V-III region SH
P04220127.49E-063.79369CML-PBP-TKI-NCML-PBP-TKI-YIg MU heavy chain disease protein (BOT)
P0159150.0005495.43077CML-PBP-No-MMRCML-PBP-TKI-NImmunoglobulin J chain
P1581429.08E-065.19282CML-PBP-MMRCML-PBP-TKI-YImmunoglobulin lambda-like polypeptide 1
P3622810.0001793.92057CML-PBP-MMRCML-PBP-TKI-YInfection structure-specific protein 56
P5628933.29E-072.32089CML-PBP-TKI-NCML-PBP-No-MMRInitiation factor EIF-5A-1
P0131412.90E-095.68794CML-PBP-TKI-NCML-PBP-TKI-YInsulin
O0283362.32E-09183.422CML-PBP-MMRCML-PBP-TKI-YInsulin-like growth factor binding protein complex
P19827a132.04E-072.19294CML-PBP-TKI-NCML-PBP-TKI-YInter-alpha-trypsin inhibitor heavy chain H1 precursor
P5665115.41E-1118.9887CML-PBP-MMRCML-PBP-TKI-Y Inter-alpha-trypsin inhibitor heavy chain H2
P19823170.0013772.02663CML-PBP-TKI-YCML-PBP-TKI-N Inter-alpha-trypsin inhibitor heavy chain H2
P0275071.91E-122.51124CML-PBP-TKI-NCML-PBP-MMRLeucine-rich alpha-2-glycoprotein (LRG)
P0626721.32E-124.06168CML-PBP-TKI-NCML-PBP-No-MMRLight-independent protochlorophyllide reductase
P1842827.86E-082.56066CML-PBP-TKI-YCML-PBP-MMR Lipopolysaccharide-binding protein precursor (LBP)
P13796a49.06E-137.72276CML-PBP-No-MMRCML-PBP-TKI-YL-plastin (Lymphocyte cytosolic protein 1) (LCP-1)
P2871712.95E-074.88405CML-PBP-TKI-YCML-PBP-TKI-NMating pheromone 3 precursor
Q9RV6218.32E-072.27719CML-PBP-TKI-NCML-PBP-MMRNADH pyrophosphatase (EC 3.6.1.-)
P4121112.57E-062.48053CML-PBP-MMRCML-PBP-TKI-YNeuron specific calcium-binding protein
P7056310.00053713.799CML-PBP-No-MMRCML-PBP-TKI-NNucleoside diphosphate-linked moiety X motif 6
P1428715.51E-05142.537CML-PBP-MMRsCML-PBP-TKI-NOsteopontin precursor (Bone sialoprotein 1)
P9708522.31E-062.01262CML-PBP-TKI-YCML-PBP-MMROuter membrane protein U precursor (Porin ompU)
P3154420.00065149.286CML-PBP-MMRCML-PBP-TKI-YPhoH protein (Phosphate starvation-inducible protein
P5709314.74E-105.0011CML-PBP-No-MMRCML-PBP-TKI-YPhytanoyl-CoA dioxygenase, peroxisomal
P0395225.36E-103.76097CML-PBP-TKI-YCML-PBP-No-MMRPlasma kallikrein precursor
P02753a45.90E-133.91711CML-PBP-TKI-YCML-PBP-No-MMRPlasma retinol-binding protein precursor (PRBP)
P2192210.00023536.2475CML-PBP-TKI-YCML-PBP-No-MMRPrecorrin-4 C11-methyltransferase
Q0625321.39E-094.17508CML-PBP-MMRCML-PBP-TKI-YPrevent host death protein
P07737a33.18E-1414.753CML-PBP-TKI-YCML-PBP-MMRProfilin I
P2660410.001614InfinityCML-PBP-No-MMRCML-PBP-TKI-YProtein hdeA precursor (10K-S protein)
Q9SM4115.77E-086.67068CML-PBP-TKI-NCML-PBP-TKI-YProtein translation factor SUI1 homolog.
P00734150.0004793.44209CML-PBP-TKI-YCML-PBP-TKI-NProthrombin precursor (EC 3.4.21.5)
Q5579422.35E-138.13328CML-PBP-TKI-NCML-PBP-MMRPutative arsenical pump-driving ATPase
Q1541840.0048056.05567CML-PBP-TKI-NCML-PBP-TKI-YRibosomal protein S6 kinase alpha 1
P0058032.27E-094.02263CML-PBP-TKI-NCML-PBP-TKI-YRNA polymerase sigma-32 factor (Heat shock regulator)
P1407210.000233168.597CML-PBP-No-MMRCML-PBP-TKI-NRubredoxin (Rd)
P5840229.27E-069.67406CML-PBP-TKI-NCML-PBP-TKI-YSensor protein evgS precursor
Q9ZK1426.65E-1218.9567CML-PBP-TKI-NCML-PBP-TKI-YSerine acetyltransferase (SAT)
P02787a532.49E-052.63861CML-PBP-TKI-YCML-PBP-TKI-NSerotransferrin precursor (Siderophilin)
P49064a45.43E-05InfinityCML-PBP-TKI-YCML-PBP-MMRSerum albumin precursor (Allergen Fel d 2)
Q28522435.22E-115.61756CML-PBP-TKI-YCML-PBP-No-MMRSerum albumin precursor (Fragment)
P027681201.15E-092.87802CML-PBP-TKI-YCML-PBP-No-MMRSerum albumin precursor
P0274311.17E-126.80911CML-PBP-TKI-YCML-PBP-TKI-NSerum amyloid P-component precursor (SAP)
P2716952.21E-052.43474CML-PBP-TKI-YCML-PBP-MMRSerum paraoxonase/arylesterase 1
P0427828.55E-094.0875CML-PBP-TKI-YCML-PBP-No-MMRSex hormone-binding globulin precursor (SHBG)
P95340a13.77E-1516.6343CML-PBP-TKI-YCML-PBP-No-MMRShikimate 5-dehydrogenase
P5767511.56E-0724.6905CML-PBP-TKI-YCML-PBP-MMRStanniocalcin 2 (STC-2) (Fragments)
Q9R0K822.68E-106.96573CML-PBP-TKI-YCML-PBP-MMRStanniocalcin 2 precursor (STC-2)
P4169134.82E-1119.1566CML-PBP-TKI-YCML-PBP-TKI-NSuperfast myosin regulatory light chain 2 (MYLC2)
P0372912.18E-1211.1468CML-PBP-TKI-NCML-PBP-TKI-YTail assembly protein K
P4369139.61E-113.55237CML-PBP-No-MMRCML-PBP-TKI-YTranscription factor GATA-4(GATA binding factor-4)
O2234710.00213212.1326CML-PBP-MMRCML-PBP-TKI-NTubulin alpha-1 chain (Alpha-1 tubulin)
P1245918.40E-149.68647CML-PBP-TKI-NCML-PBP-No-MMRTubulin beta-1 chai
P02774a172.45E-072.6983CML-PBP-TKI-YCML-PBP-No-MMRVitamin D-binding protein precursor (DBP) (Group-s)
P0400496.06E-092.12057CML-PBP-TKI-YCML-PBP-MMRVitronectin precursor (Serum spreading factor)

B, The identified differentially expressed proteins in BMP of CML patients with MMR, No-MMR, On-TKI and NOT-on-TKI

AccessionPeptide count used for quantificationAnova (p)Max fold changeHighest mean conditionLowest mean conditionDescription

Q9ZEY820.008661.5676CMR-NTKI-N2-isopropylmalate synthase (EC 4.1.3.12)
P49313a,b10.000862.8992TKI-NCMR-Y30 kDa ribonucleoprotein, chloroplast precursor
P02578b10.000232.4784TKI-NCMR-YActin 1
Q03341b10.0003319.7447CMR-NTKI-YActin 2
P02580b20.0000116.5471CMR-YCMR-NActin 3
P0782910.018323.2349CMR-YTKI-NActin 3-SUB1
P9358410.013761.5206CMR-NCMR-YActin 82 (Fragment)
P5346010.009288.5512TKI-NCMR-NActin, muscle 1A
P50138b10.0043188.6922CMR-YTKI-YActin
Q9P4D110.010993.7590CMR-YTKI-YActin
P43652b130.000032.0878CMR-YTKI-NAfamin precursor (Alpha-albumin) (Alpha-Alb)
P19652b60.001631.5175CMR-YTKI-NAlpha-1-acid glycoprotein 2 precursor (AGP 2)
P01010b10.004212.2484CMR-YCMR-NAlpha-1-antitrypsin precursor (Alpha-1 protease inhibitor)
P01009270.020491.7589CMR-YTKI-N Alpha-1-antitrypsin precursor (Alpha-1 protease inhibitor)
P08697b70.002312.7616CMR-YTKI-N Alpha-2-antiplasmin precursor (Alpha-2-plasmin inhibitor)
Q9N2D010.031474.9779CMR-YTKI-N Alpha-2-HS-glycoprotein precursor (Fetuin-A)
P01023a670.001301.5666CMR-YTKI-N Alpha-2-macroglobulin precursor (Alpha-2-M)
P01019110.022951.4615CMR-YCMR-NAngiotensinogen precursor [Contains: Angiotensin I
P00896b10.000015.0581CMR-NTKI-NAnthranilate synthase component I (EC 4.1.3.27)
P01008a90.003201.4376CMR-YTKI-YAntithrombin-III precursor (ATIII) (PRO0309)
P32261b20.000845.0712TKI-NCMR-NAntithrombin-III precursor (ATIII)
P0980920.024211.5680TKI-NCMR-YApolipoprotein A-I precursor (Apo-AI)
P15497a20.038984.7003CMR-YCMR-NApolipoprotein A-I precursor (Apo-AI)
P06727140.013992.0475CMR-YTKI-YApolipoprotein A-IV precursor (Apo-AIV)
P02655a,b20.000012.0801CMR-YTKI-NApolipoprotein C-II precursor (Apo-CII)
P4169710.004231.9243TKI-YCMR-NBud site selection protein BUD6 (Actin interacting protein)
P0510920.046179.0518CMR-YTKI-NCalgranulin A (Migration inhibitory factor-related protein)
P2585420.013681.5390TKI-YCMR-NCalmodulin-1 (Fragment)
Q9NZT110.000881.9462CMR-NTKI-YCalmodulin-like skin protein
Q00371b10.0000223.1103TKI-NCMR-NCAP22 protein
P00915b60.000725.4236CMR-NTKI-NCarbonic anhydrase I (EC 4.2.1.1) (Carbonate dehydrase)
P25773b10.000006.6740CMR-YCMR-NCathepsin L (EC 3.4.22.15) (Progesterone-dependent)
P00450200.007271.5284CMR-YCMR-NCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
P1363560.022861.5201CMR-YTKI-NCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
Q6114750.030542.4399TKI-NTKI-YCeruloplasmin precursor (EC 1.16.3.1) (Ferroxidase)
P1090960.000121.5866CMR-YCMR-NClusterin precursor (Complement-associated protein)
P2595830.007471.9061TKI-YTKI-NComG operon protein 6
P0274720.0405228.8755CMR-YTKI-YComplement C1q subcomponent, C chain precursor
P01026100.000011.8285TKI-NCMR-YComplement C3 precursor [Contains: C3A anaphylatox]
P1238770.000101.8101CMR-NCMR-YComplement C3 precursor [Contains: C3A anaphylatox]
P01024a680.000881.6430CMR-YTKI-NComplement C3 precursor [Contains: C3a anaphylatox]
P01028b420.000202.0579CMR-YTKI-YComplement C4 precursor [Contains: C4A anaphylatox]
P1064330.047121.4974CMR-YCMR-NComplement component C7 precursor
P02748b70.001312.5543CMR-YTKI-NComplement component C9 precursor
P08603300.003651.4060CMR-YTKI-NComplement factor H precursor (H factor 1)
P48416b30.000003.5184TKI-NCMR-YCytochrome P450 10 (EC 1.14.-.-) (CYPX)
Q92I25b10.000072.6454TKI-YCMR-NDihydrodipicolinate synthase (EC 4.2.1.52) (DHDPS)
P31073b10.000102.2735TKI-NCMR-NDihydrofolate reductase (EC 1.5.1.3)
P20861b10.0000016.7020TKI-NCMR-YFan G protein precursor
P02671a,b210.000032.2257CMR-YTKI-YFibrinogen alpha/alpha-E chain precursor
P02675a,b240.000102.4767CMR-YCMR-NFibrinogen beta chain precursor [Contains: Fibrinogen]
Q02020b20.004612.5361CMR-YCMR-NFibrinogen beta chain precursor [Contains: Fibrinogen]
P1448070.005422.0499CMR-NCMR-YFibrinogen beta chain precursor [Contains: Fibrinogen]
P02679a,b130.001102.1792CMR-YCMR-NFibrinogen gamma chain precursor
Q92T2720.000301.5959TKI-NCMR-NGlucokinase (EC 2.7.1.2) (Glucose kinase)
Q92J7410.007122.6314CMR-YCMR-N Glutamyl-tRNA(Gln) amidotransferase subunit C
Q6075940.003011.8431TKI-NCMR-YGlutaryl-CoA dehydrogenase, mitochondrial precursor
P23722a30.003801.5602TKI-NCMR-YGlyceraldehyde 3-phosphate dehydrogenase
Q9ZKP0a,b20.002922.4902CMR-YTKI-N Glycerol-3-phosphate dehydrogenase [NAD(P)+]
P5015010.033275.9505TKI-NCMR-YGuanine nucleotide-binding protein G(I)/G(S)/G(O)
P07736b10.001892.7741TKI-NCMR-YGuanyl-specific ribonuclease U1 (EC 3.1.27.3) (Rna)
P5041710.007645.7455CMR-YTKI-NHaptoglobin precursor
P0073840.048342.6291CMR-YTKI-YHaptoglobin-2 precursor
P0741420.007538.8724CMR-NTKI-NHemoglobin alpha chain
P0193210.04336InfinityCMR-YTKI-YHemoglobin alpha chain
P01948b10.004322.0401TKI-YCMR-YHemoglobin alpha-1 and alpha-2 chains
Q9XSN310.008341.3880CMR-YTKI-NHemoglobin alpha-1 chain
P19002b20.000003.9434CMR-NCMR-YHemoglobin alpha-1, alpha-2, and alpha-3 chains
P02037b10.001665.3495CMR-NTKI-YHemoglobin beta chain
P1175820.037623.2576CMR-YTKI-YHemoglobin beta chain
P0202710.0445616.1529CMR-NCMR-YHemoglobin beta chain
P0206410.022022.3093TKI-NCMR-NHemoglobin beta-1 chain (Major)
P02074b10.000004.1199CMR-NCMR-YHemoglobin beta-III chain
P19886b20.000082.0278CMR-NCMR-YHemoglobin delta chain
P2005820.036191.8809TKI-NCMR-NHemopexin precursor
P4596510.0402913.7398CMR-YCMR-NHypothetical 19.4 kDa protein T09A5.5 in chromosome
Q0510710.025052.0311CMR-YCMR-NHypothetical 23.6 kDa protein
O3471720.013551.4268TKI-YCMR-YHypothetical oxidoreductase ykuF (EC 1)
P44030b10.000004.4405TKI-YCMR-YHypothetical protein HI0659
P42968b10.000034.3060TKI-NCMR-NHypothetical transcriptional regulator ycsO
P01876a,b10.000133.1121CMR-YCMR-NIg alpha-1 chain C region
P0185980.000151.8808TKI-YTKI-NIg gamma-2 chain C region
P01860a30.000181.4555TKI-YTKI-NIg gamma-3 chain C region (Heavy chain disease protein)
P01861a50.024951.4049CMR-YTKI-NIg gamma-4 chain C region
P0177920.020522.4688CMR-YCMR-NIg heavy chain V-III region TUR
P0161710.000161.9790CMR-YTKI-NIg kappa chain V-II region TEW
P0162530.014641.8173CMR-YCMR-NIg kappa chain V-IV region Len
P01842a50.007631.4632CMR-YCMR-NIg lambda chain C regions
P01591a50.034302.1773CMR-YTKI-YImmunoglobulin J chain
P0133510.005142.4827TKI-NCMR-YInsulin precursor
O0266810.0104113.1392CMR-YTKI-YInter-alpha-trypsin inhibitor heavy chain H2 precursor
P9727920.034232.0472TKI-YTKI-NInter-alpha-trypsin inhibitor heavy chain H2 precursor
Q42891b10.000022.2505TKI-NCMR-NLactoylglutathione lyase (EC 4.4.1.5) (Methylglyoxal)
P02750a90.017981.3841TKI-YCMR-NLeucine-rich alpha-2-glycoprotein (LRG)
P06267a,b10.000053.9296CMR-NTKI-N Light-independent protochlorophyllide reductase iron-sulfur ATP-binding protein
Q6123320.015943.5492CMR-YTKI-YL-plastin (Lymphocyte cytosolic protein 1) (LCP-1)
P5216210.0102725.2703CMR-YTKI-NMAX protein
P48310b10.000242.4866CMR-YTKI-NMinor capsid protein VI precursor
O03698b10.000412.9113CMR-NCMR-YNADH-ubiquinone oxidoreductase chain 4 (EC 1.6.5.3)
Q4387510.013424.0047CMR-YCMR-NNonspecific lipid-transfer protein 4.2 precursor
P2305110.000023.3474TKI-YTKI-NNucleocapsid protein
P39115b10.000003.4012CMR-NCMR-YNucleotide binding protein ExpZ
P32119b30.000004.3238CMR-NCMR-YPeroxiredoxin 2 (Thioredoxin peroxidase 1)
Q42858b10.000074.2693CMR-NTKI-NPhenylalanine ammonia-lyase (EC 4.3.1.5)
O07125b10.000992.7853CMR-NTKI-NPhosphocarrier protein HPr (ptsH)
P0941110.018861.5949TKI-YTKI-NPhosphoglycerate kinase 1 (EC 2.7.2.3)
Q9KDM420.005131.6582TKI-NCMR-NPhosphoserine aminotransferase (serC) (PSAT)
P0275330.011951.5216CMR-NTKI-NPlasma retinol-binding protein precursor (PRBP)
P7615910.005381.7156TKI-NCMR-YProbable lysozyme from lambdoid prophage Qin
O6702410.03110 InfinityCMR-YTKI-NProbable peroxiredoxin
P0773720.008701.8459CMR-YCMR-NProfilin I
P0053620.006971.5076TKI-NCMR-NProto-oncogene serine/threonine-protein kinase mos
P4560410.000211.9033CMR-NCMR-YPTS system, N-acetylglucosamine-specific EIIABC component
Q5948210.005194.2028CMR-YTKI-NPurine nucleoside phosphorylase (deoD)
P55429b10.000042.5979CMR-NCMR-YPutative integrase/recombinase Y4EF
Q9AB8030.000011.5354TKI-YCMR-YPutative outer membrane protein CC0351 precursor
Q9X48020.001131.8668CMR-NCMR-YPutative signal peptide peptidase sppA
P3444330.029052.3131CMR-YTKI-YRas-like protein F54C8.5
P3429520.024741.4695TKI-YCMR-NRegulator of G protein signaling rgs-1
Q9CG17a10.000031.7092CMR-YTKI-NRibonuclease HII (EC 3.1.26.4) (RNase HII)
P56566b20.004783.4601TKI-NCMR-NS100 calcium-binding protein A3 (S-100E protein)
P12346b20.000002.4638TKI-YTKI-NSerotransferrin precursor (Siderophilin) (Beta-1-metal-binding globulin)
P19134b110.003472.2136TKI-NCMR-NSerotransferrin precursor (Siderophilin) (Beta-1-metal-binding globulin)
P02787440.005741.4954CMR-YCMR-NSerotransferrin precursor (Siderophilin) (Beta-1-m-b-g)
P02769b50.000032.4650TKI-YCMR-NSerum albumin precursor (Allergen Bos d 6)
Q2852270.041082.6927CMR-YTKI-NSerum albumin precursor (Fragment)
P49065a,b20.000166.1150CMR-NTKI-YSerum albumin precursor
P27169a,b30.004162.1032TKI-YTKI-NSerum paraoxonase/arylesterase 1 (EC 3.1.1.2)
Q9CES7b10.000062.0972TKI-YTKI-NShikimate 5-dehydrogenase (EC 1.1.1.25)
P29950b20.002972.6116CMR-YTKI-YUracil-DNA glycosylase (EC 3.2.2.-) (UDG) (Fragment)
P02774240.000131.9884CMR-YTKI-NVitamin D-binding protein precursor (DBP) (VDB)
P7306910.007651.8377CMR-NCMR-YYcf48-like protein

a Fifty-four differentially expressed proteins that were common between the two body fluid compartments (i.e. the 164 and 138 datasets from PBP and BMP respectively) as described in Fig. 4. This set of 54 proteins was then used in the unsupervised hierarchical clustering analysis as shown in Fig. 7. The proteins that are in bold in part A are also identified in BMP samples.

b Sixty-four significantly differentially expressed proteins (>1.5- to ∞-fold change, P<0.05) between MMR and No-MMR sample groups used for the generation of dendrogram in Fig. 3. These proteins predict accurately patients with MMR vs. No-MMR patients using unsupervised Hierarchical Cluster Analysis. (Due to resolution problem, the list was cropped from the dendrogram plot). The proteins that are in bold in part B are also identified in PBP samples.

Similar to peripheral blood samples, >700 proteins representing 250 unique protein species were identified when similar analysis was done on bone marrow pooled samples from 8 LT-MMR patients and 8 P-No-MMR patients. One hundred and thirty-eight of the total identified proteins were significantly differentially expressed between LT-MMR and P-No-MMR bone marrow sample groups (>1.5- to ∞-fold change, P<0.05; Table IIB). These proteins predict accurately LT-MMR patients vs. P-No-MMR patients using unsupervised principal component analysis (Fig. 4B). These results were subsequently evaluated for comparisons with the patterns obtained in early treatment response at 6 months. Notably, the pattern and accuracy of clustering of samples is very similar to that observed with the hierarchical cluster analysis plots at 6 months (Fig. 3).

Protein fingerprinting for prediction of treatment options for individualized therapy

Towards achieving the goal of personalized medicine, the above observed differentially expressed proteins between samples derived from LT-MMR patients vs. P-No-MMR patients were evaluated for their potential for objective prediction of treatment options for some of these cohorts of CML patients. Interestingly, the panel of 164 and 138 differentially expressed protein datasets derived from peripheral blood plasma (PBP) and bone marrow (BM) respectively, also discriminates patients that stay on IM after 1 year of treatment from patients that ultimately required alternative treatment options (second generation TKI/others) (Fig. 5). Following >2 years of follow-up of these patients the same dataset of potential protein biomarkers could still accurately separate all analyzed sample groups into their respective molecular response and treatment sub groups, indicating their usefulness for treatment monitoring as well as prediction of best choice of therapy for individual patient. Some of the identified proteins were implicated in hematological diseases as potential biomarkers using ingenuity pathway analysis (IPA) (Fig. 6). Functional annotations/disease affiliations of some of these proteins implicated in CML are further described under discussion below.

Figure 6

(A) Pathway analysis of network signaling of some of the identified proteins as represented in the ingenuity pathway analysis database. The analysis of the identified proteins is composed of 2 hematological disease related networks with over 100 associated molecules that were merged into one as shown above. The connections and the expression profiles of some of the identified proteins are as indicated. Red indicates an upregulated protein, and pink color is indicative of downregulation. A direct connection is by solid line and broken lines indicate an indirect interaction between different molecules. Other molecules outside the identified in this study are in grey color. (B) The functional characteristics and disease relatedness of some of the identified proteins were mapped in Ingenuity database. The majority of these molecules are located mostly in the plasma membrane, cytoplasm and extracellular space, while only a few are located in the nucleus. Some these molecules functions as enzymes, transporters, transcription regulator, or G-protein coupled receptor. Others act as kinases, peptidase or growth factor. Furthermore, some of these molecules as represented in multiple sub-signaling networks mostly regulate among others: Cell-To-Cell Signaling and Interaction, Hematological System Development and Function. Other implicated functional annotations include, aggregation of blood cells, coagulation, quantity of aggregate cells as well as quantity of granulocytes. [The network analysis was done and figure generated in ingenuity pathway analysis program (IPA v8.7)].

Identification of protein changes in BM as a reflection of detectable changes in peripheral blood

One of the main goals of this study was to identify/develop disease-specific/disease-associated protein biomarkers seen in bone marrow tissue as well as in peripheral blood plasma. This would subsequently allow monitoring of such biomarker proteins in peripheral blood, rather than bone marrow, demanding less invasive procedures for objective prediction of individual’s best treatment options and prognostic monitoring of CML patients. We therefore explored the possibility whether the proteins that are significantly differentially expressed in bone marrow do also show similar expression pattern in peripheral blood. With this in mind, we calculated how many of the 164 differentially expressed proteins in peripheral blood and the 138 protein dataset in bone marrow are common to both body compartments. We found that only 54 proteins (~35%) were in common between the two 164 and 138 datasets as described above. This set of 54 proteins was then subjected to unsupervised hierarchical clustering and correspondence analyses. As shown in Fig. 7, all sample groups were distinctively separated into four response subtypes using unsupervised hierarchical cluster analysis. The common proteins between the two body fluid compartments were highlighted in bold in Table II.

Validation by western blot analysis of some of the identified proteins

In an attempt to validate some of the differentially expressed proteins, we have used immunoblotting analysis. Nine individual samples consisting of 4 samples not included in the proteomics analysis and 5 other samples from the proteomics analyzed sample groups were tested for their expression of haptoglobin and hemoglobin using specific antibodies against these proteins. The expression levels of these proteins across all sample groups were consistent with the average protein normalized levels seen with label-free quantitative LC/MS/MS analysis (Fig. 8). Large scale validation of the majority of these proteins was beyond the scope of this study in order to develop limited panel of markers for clinical trial in a later study.

Discussion

Clinical and molecular diagnosis of most hematological malignancies including CML can be accurately made; however, prediction of treatment response elude the currently available tools for patient care.

A subset of significantly differentially expressed proteins from both peripheral blood and bone marrow were selected for their ability to discriminate samples derived from CML patients that responded differently to initial first line treatment with imatinib. Our strategy of proteomics mining of BM and PBP from the same individual patient would provide unique possibility to identify biomarkers from both sources thus, entailing less invasive procedures.

Report of microarray analysis of peripheral blood and bone marrow of CML samples in blast crisis cells, has been shown with demonstrable biological changes between two bodily fluids (19). Our analysis of peripheral blood samples of 164 differentially expressed proteins show that all samples were correctly classified and similar result was observed with 138 protein changes in bone marrow samples as shown in Fig. 4. Only 54 proteins were shown to be commonly differentially expressed between blood dataset and bone marrow protein dataset in the present study, supporting our notion that it might be possible to identify significant changes in the bone marrow of CML patients that are measurable at peripheral blood compartment for routine diagnostics.

We have attempted to use both the BMP and PBP data-sets that accurately predict patients MMR status for possible prediction of patients that continue to stay on IM after 1 year of treatment vs. those that ultimately required alternative treatment options (second generation TKI/others). Thus, the expression of the 158 protein changes in BM between MMR and No-MMR were further evaluated in 16 unrelated patients that stay on TKI after 1 year of imatinib treatment from patients that ultimately required alternative treatment options (second generation TKI/others). We found four distinct clusters with samples with MMR and No-MMR being very closely separated (not as distinct as in Fig. 4), while patients that stay on TKI (i.e. after 1 year of imatinib) treatment were distantly separated from patients that ultimately required alternative treatment options (second generation TKI/others) as shown in Fig. 5, meaning that it will be challenging to construct a universal model for management of CML patients and that prognostic datasets need to be created for each specific response type.

We have used two independent proteomics analysis platforms in the present study. The expression profiles of 2-DE protein spots successfully discriminated two sample groups of CML patients with MMR and No-MMR. We recognized the inherent limitation of 2-DE based studies (2022) hence, we have in addition used label-free quantitative protein expression using high definition liquid chromatography tandem mass spectrometry (LC/MS/MS) to extensively map the proteome of bone marrow as well as peripheral blood samples.

Previous studies have used multivariate statistical algorithms and artificial learning models to predict cancer prognosis and for grading different solid tumors (15,2328). The majority of these studies reported varying degrees of sensitivity and specificity based on evaluation of different clinical parameters (20,24).

Gene expression studies on hematological disease have been largely carried out by analysis of DNA or RNA microarrays. These genomics studies have indicated the potentials of large scale analysis of gene expression towards better understanding the molecular basis of leukemogenesis and that this information could potentially be useful in the classification of subtypes of hematological malignancies (19,29,30). In a recent study of CLL samples, Alsagaby and colleagues used combined transcriptomics and proteomics analyses to unravel the heterogeneity of gene expression patterns as well attempting to identify proteins that are implicated in prognosis of chronic lymphocytic leukemia (31). Recent studies have attempted to evaluate protein changes between imatinib sensitive and resistance samples (32) as well as to better understand the molecular mechanism in therapy resistance at the level of bone marrow extracellular fluid in CML (33).

Our initial analysis of 64 differentially expressed proteins of peripheral blood for prognostic monitoring of early CML treatment response at 6 months was encouraging and led us into extensive analysis of samples with sustained long-term MMR against patients that persistently could not achieve MMR.

Some of the identified proteins in the bone marrow of the 138 dataset for the prolonged and sustained MMR vs. persistent No-MMR were further evaluated for their functional characteristics and their hematological disease relevance using ingenuity pathway analysis (IPA). In the canonical pathway analysis of network signaling of identified proteins, only 48 of the 138 identified differentially expressed proteins were represented in the IPA database. The analysis of the identified proteins is composed of multiple networks of which, one is implicated in hematological disorders. The cellular localization, interconnections and functional annotation as well as the expression profile of some of these 48 identified molecules are as detailed in Fig. 6A. A review of some of these molecules showed that they mostly regulate among others: cell-to-cell signaling and interaction, hematological system development and function, aggregation of blood cells, coagulation, as well as quantity of granulocytes as indicated in Fig. 6. Among the identified proteins in this study is TYRO3 protein tyrosine kinase, a member of TAM family of receptor tyrosine kinases (RTKs) and known for their role as regulator of cellular proliferation, migration and survival processes, as well as maintenance of blood coagulation equilibrium (34). We observed connection of TYRO 3 in AKT/P13K pathway; similar to that previously described (3436).

The S100A8 is a calcium-binding protein of the S100 family and have been described to be associated with myeloid differentiation (37). We observed a more than 9-fold differential expression of S100A8 and in the network connecting with RAS, TGFb, MAPK and MMP. The S-100 protein has been previously reported as a useful marker in juvenile chronic myeloid leukemia (JCML) as well as myeloid leukemia cutis (LC) (38,39).

Overexpression of MYC has been associated with CML with poor response to imatinib (40,41). We observed a more than 25-fold differential expression of MYC associated factor x in this study.

Altogether our findings indicate that rather than the use of a single marker, analyses of a panel of protein markers have the potential to provide better insight into complex biologic processes towards better prognostication of CML patients.

We recognize the limitation of this study as samples were prospectively collected and patients observed over the years for their treatment responses. One other issue with this study is the low number of patients enrolled in different clinical and molecular response groups; hence we have limited the analysis to evaluation of patients based on MMR and whether or not they are on IM or alternative treatment option (second generation TKI/others).

In conclusion, we have identified protein signatures capable of prediction of molecular response and choice of therapy for CML patients at 6 months and beyond using expression proteomics as objective stratification of CML patients for treatment options.

Although these results are very promising, we recognized that analysis of much larger materials of patients with similar treatments and responses will be necessary to validate if clustering analysis can be used as a routine prognostic tool for CML patients.

These proteins might be valuable once validated, to complement the currently existing parameters for reliable and objective prediction of disease progression, monitoring treatment response and clinical outcome of CML patients as a model of personalized medicine.

Acknowledgements

We thank Dr Abdelilah Aboussekhra for critical review, as well as Mr. Melvin Velasco, Mr. Parvez Siddiqui, Mr. Romeo Caracas and Ms. Tusneem Elhassan for technical assistant. We acknowledge the assistant and support of Mr. Faisal Al Otaibi and the logistics and purchasing department, RC, KFSH&RC. The present study was supported by the Research Center Administration, KFSH&RC, Riyadh, Saudi Arabia (RAC# 2050 040).

Abbreviations:

CML

chronic myeloid leukemia

CMR

complete molecular response

CP

chronic phase

DAS

dasatinib

IM

imatinib mesylate

MCyR

major cytogenetic response

MMR

major molecular response

No-MMR

no-major molecular response

LT-MMR

long-term-MMR

P-No-MMR; persistent-no-MMR; TKI

tyrosine kinase inhibitors

2-DE

two-dimensional gel electrophoresis

LC-MS/MS

liquid chromatography coupled with tandem mass spectrometry

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September 2016
Volume 49 Issue 3

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APA
Alaiya, A.A., Aljurf, M., Shinwari, Z., Almohareb, F., Malhan, H., Alzahrani, H. ... Chaudhri, N. (2016). Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients. International Journal of Oncology, 49, 913-933. https://doi.org/10.3892/ijo.2016.3618
MLA
Alaiya, A. A., Aljurf, M., Shinwari, Z., Almohareb, F., Malhan, H., Alzahrani, H., Owaidah, T., Fox, J., Alsharif, F., Mohamed, S. Y., Rasheed, W., Aldawsari, G., Hanbali, A., Ahmed, S. O., Chaudhri, N."Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients". International Journal of Oncology 49.3 (2016): 913-933.
Chicago
Alaiya, A. A., Aljurf, M., Shinwari, Z., Almohareb, F., Malhan, H., Alzahrani, H., Owaidah, T., Fox, J., Alsharif, F., Mohamed, S. Y., Rasheed, W., Aldawsari, G., Hanbali, A., Ahmed, S. O., Chaudhri, N."Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients". International Journal of Oncology 49, no. 3 (2016): 913-933. https://doi.org/10.3892/ijo.2016.3618