Method for in vitro diagnosing and prognosing of triple negative breast cancer recurrence
10859577 ยท 2020-12-08
Assignee
- Institut De Cancerologie De L'ouest (Angers, FR)
- Universite D'angers (Angers, FR)
- Institut National De La Sante Et De La Recherche Medicale (Inserm) (Paris, FR)
Inventors
- Catherine Guette (La Possonniere, FR)
- Mario Campone (Nantes, FR)
- Olivier Coqueret (Angers, FR)
- Benjamin Barre (Bain sur Longuenee, FR)
US classification
- 1/1
Cpc classification
G01N2800/52
PHYSICS
International classification
Abstract
The present invention is in the technical field of breast cancer management, and more particularly relates to the diagnosis and/or prognosing of triple-negative breast cancer (TNBC). The invention is more particularly based on the finding that specific biomarkers are abberantly expressed in patients suffering from a triple-negative breast cancer recurrence, and are highly related to the aggressiveness of this disease, and thus to survival of said patient.
Claims
1. An in vitro method for diagnosing and/or prognosing and treating a recurrent triple-negative breast cancer in a subject, comprising the steps of: a) obtaining a biological sample from a subject, b) detecting the expression levels of three biomarkers comprising Desmoplakin, Thrombospondin-1 and Isocitrate dehydrogenase [NADP] in the biological sample, c) diagnosing and/or prognosing a recurrent triple-negative breast cancer when the expression levels of the three biomarkers comprising Desmoplakin, Thrombospondin-1 and Isocitrate dehydrogenase [NADP] are superior to the expression levels of the three biomarkers comprising Desmoplakin, Thrombospondin-1 and Isocitrate dehydrogenase [NADP] in a biological sample of at least one healthy subject, and d) administering an effective amount of a chemotherapeutic agent to the diagnosed subject.
2. The method according to claim 1, further comprising detecting the expression levels of at least two biomarkers selected from the group consisting of Hexokinase-1, 10 kDa heat shock protein, Ig gamma-1 chain C region, SAM domain and HD domain-containing protein 1, and Tryptophanyl-tRNA synthetase in the biological sample in step b).
3. The method according to claim 2, wherein the expression level of Hexokinase-1, 10 kDa heat shock protein, SAM domain and HD domain-containing protein 1, or Tryptophanyl-tRNA synthetase, is superior to a reference expression level obtained from a biological sample of at least one healthy subject.
4. The method according to claim 2, wherein the expression level of Ig gamma-1 chain C region is inferior to a reference expression level obtained from a biological sample of at least one healthy subject.
5. The method according to claim 2, wherein one of said at least two biomarkers is Tryptophanyl-tRNA synthetase.
6. The method according to claim 1, further comprising detecting the expression levels of at least one biomarker selected from the group consisting of Rho GTPase-activating protein 1, Epiplakin, Glucose-6-phosphate 1-dehydrogenase, Keratin type I cytoskeletal 19, Keratin type I cytoskeletal 8, and Dihydropyrimidinase-related protein 3 in the biological sample in step b).
7. The method according to claim 1, further comprising measuring the expression level of the biomarkers Hexokinase-1, 10 kDa heat shock protein, SAM domain and HD domain-containing protein 1, and Tryptophanyl-tRNA synthetase, and Ig gamma-1 chain C region in the biological sample in step b).
8. The method according to claim 1, further comprising detecting the expression level of biomarkers Glucose-6-phosphate 1-dehydrogenase in the biological sample in step b).
9. The method according to claim 1, further comprising detecting the expression level of biomarkers Glucose-6-phosphate 1-dehydrogenase, and Keratin type II cytoskeletal 8 in the biological sample in step b).
10. The method according to claim 1, wherein the expression level is determined by a method selected from the group consisting of Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), enzyme-linked immunospot (ELISPOT), radioimmunoas say (RIA), immunohistochemistry, immunoprecipitation, fluorescence activated cell sorting (FACS), microscopy, flow cytometry, microcytometry, protein binding assay, ligand binding assay, microarray, polyacrylamide gel electrophoresis such as SDS-PAGE, surface plasmon resonance (SPR), Frster resonance energy transfer (FRET), Bioluminescence resonance energy transfer (BRET), chemiluminescence, fluorescent polarization, phosphorescence, mass spectrometry, magnetic resonance imaging (MRI), and any combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
1. Materials and Methods
(10) 1.1. Sample Collection
(11) The inventors selected cases of triple negative breast tumors (TNBC) that were surgically resected with curative intent in the West Cancer Institute tumor bank. All patients provided informed consent for participation and this project was approved by the Institutional Review Board. All specimens were collected immediately after surgery, snap frozen and stored in liquid nitrogen until the time of analysis. We also selected 4 normal macroscopically areas for our control pool. Frozen sections (12 m thick) of either TNBC or normal areas were cut on a cryostat (Bright Instrument Co Ltd, St Margarets Way, UK). Specific sections were stained with toluidine blue for visual reference and each tissue sections from all specimens were evaluated by experienced pathologists for cancer cell proportion determination. Samples containing less than 80% of tumor cells were removed. Clinical features of tissue candidates are summarized in Table 3 below.
(12) TABLE-US-00003 TABLE 3 Clinico-pathological characteristics of patients for tissue proteomic studies Total Non recurrence Recurrence (n = 80) (n = 45) (n = 35) Age Median (range) 56 (28-78) 55 (28-78) 58 (37-76) Grade 1 1 0 1 2 6 6 0 3 73 39 34 pT 08-11 6 5 1 12-15 23 16 7 16-19 11 7 4 20-23 15 10 5 24-27 6 4 2 28-31 5 2 3 32-35 6 4 2 36-70 7 3 4 Adjuvant chemotherapy 3 2 1 Radiotherapy 77 49 28
(13) 1.2. Protein Extraction from Frozen Tissues
(14) Frozen sections (12 m thick) of TNBC or normal breast area were cut on a cryostat (Bright Instrument Co Ltd, St Margarets Way, UK). Specific sections were stained with toluidine blue for visual reference. Ten frozen sections per tumor were lysed in a buffer consisting of 0.1 M Tris-HCl, pH 8.0, 0.1 M DTT, and 4% SDS at 95 C. for 90 min. Detergent was removed from the lysates and the proteins were digested with trypsin using the FASP protocol (Wisniewski et al., 2009) using spin ultrafiltration units of nominal molecular weight cut of 30 000. To YM-30 microcon filter units (Cat No. MRCF0R030, Millipore) containing protein concentrates, 200 L of 8 M urea in 0.1 M Tris/HCl, pH 8.5 (UA), was added and samples were centrifuged at 14 000 g at 20 C for 8 min. This step was performed thrice. Then 6 L of 200 mM MMTS in 8 M urea was added to the filters and the samples were incubated for 20 min. Filters were washed thrice with 200 L of 8 M UA followed by six washes with 100 L 0.5M TEAB. Finally, trypsin (AB sciex) was added in 100 L 0.5M TEAB to each filter. The protein to enzyme ratio was 100:1. Samples were incubated overnight at 37 C. and released peptides were collected by centrifugation. Samples were then dried completely using a Speed-Vac and re-suspended in 100 l of 0.5% trifluoroacetic acid (TFA) in 5% acetonitrile, and were desalted via PepClean C-18 spin columns (Pierce Biotechnology, Rockford, Ill.). Peptide content was determined using Micro BCA Protein Assay Kit (Pierce-Thermo Scientific, Rockford, Ill.).
(15) 1.3. Peptide Labelling with iTRAQ Reagents
(16) Each peptide solution was labelled at room temperature for 2 h with one iTRAQ reagent vial previously reconstituted with 70 l of ethanol for 4plex iTRAQ reagent and reconstituted with 50 l of isopropanol for 8plex iTRAQ reagent. A mixture containing small aliquots from each labeled sample was analyzed by MS/MS to determine a proper mixing ratio to correct for unevenness in peptide yield from Liquid Tissues procedures. Labeled peptides were then mixed in 1:1:1:1 (or 1:1:1:1:1:1:1:1) ratio. Peptide mixture was then dried completely using a Speed-Vac.
(17) 1.4. Peptide OFFGEL Fractionation
(18) For pI-based peptide separation, the inventors used the 3100 OFFGEL Fractionator (Agilent Technologies, Bblingen, Germany) with a 24-well set-up according to the protocol described by Ernoult et al. (2008). Briefly, prior to electrofocusing, samples were desalted onto a Sep-Pak C18 cartridge (Waters). For 24-well set-up, peptide samples were diluted to a final volume of respectively 3.6 mL using OFFGEL peptide sample solution. To start, the IPG gel strip of 24 cm-long (GE Healthcare, Munchen, Germany) with a 3-10 linear pH range was rehydrated with the Peptide IPG Strip Rehydradation Solution according to the protocol of the manufacturer for 15 min. Then, 150 L of sample was loaded in each well. Electrofocusing of the peptides was performed at 20 C. and 50 A until the 50 kVh level was reached. After focusing, the 24 peptide fractions were withdrawn and the wells were washed with 200 L of a solution of water/methanol/formic acid (49/50/1). After 15 min, the washing solutions were pooled with their corresponding peptide fraction. All fractions were evaporated by centrifugation under vacuum and maintained at 20 C. Just prior nano-LC, the fractions were resuspended in 20 L of H.sub.2O with 0.1% (v/v) TFA.
(19) 1.5. Capillary LC Separation
(20) The samples were separated on an Ultimate 3,000 nano-LC system (Dionex, Sunnyvale, USA) using a C18 column (PepMap100, 3 m, 100 A, 75 m id15 cm, Dionex) at 300 nL/min a flow rate. Buffer A was 2% ACN in water with 0.05% TFA and buffer B was 80% ACN in water with 0.04% TFA. Peptides were desalted for 3 min. using only buffer A on the precolumn, followed by a separation for 105 min. using the following gradient: 0 to 20% B in 10 min, 20% to 45% B in 85 min and 45% to 100% B in 10 min. Chromatograms were recorded at the wavelength of 214 nm. Peptide fractions were collected using a Probot microfraction collector (Dionex). We used CHCA (LaserBioLabs, Sophia-Antipolis, France) as MALDI matrix. The matrix (concentration of 2 mg/mL in 70% ACN in water with 0.1% TFA) was continuously added to the column effluent via a micro T mixing piece at 1.2 L/min flow rate. After 12 min run, a start signal was sent to the Probot to initiate fractionation. Fractions were collected for 10 s and spotted on a MALDI sample plate (1,664 spots per plate, Applied Biosystems, Foster City, Calif.).
(21) 1.6. MALDI-MS/MS
(22) MS and MS/MS analyses of off-line spotted peptide samples were performed using the 5800 MALDI-TOF/TOF Analyser (ABsciex) and 4000 Series Explorer software, version 4.0. The instrument was operated in a positive ion mode and externally calibrated using a mass calibration standard kit (ABsciex). The laser power was set between 2800 and 3400 for MS and between 3600 and 4200 for MS/MS acquisition. After screening all LC-MALDI sample positions in MS-positive reflector mode using 1500 laser shots, the fragmentation of automatically-selected precursors was performed at a collision energy of 1 kV using air as collision gas (pressure of 210-6 Torr) with an accumulation of 2000 shots for each spectrum. MS spectra were acquired between m/z 1000 and 4000. For internal calibration, we used the parent ion of Glu1-fibrinopeptide at m/z 1570.677 diluted in the matrix (30 femtomoles per spot). Up to 12 of the most intense ion signals per spot position having a S/N>12 were selected as precursors for MS/MS acquisition. Peptide and protein identification were performed by the ProteinPilot Software V 4.0 (AB Sciex) using the Paragon algorithm as the search engine (Shilov et al., 2007).
(23) Each MS/MS spectrum was searched for Homo sapiens specie against the Uniprot/swissprot database (UniProtKB/Sprot 20110208 release 01, with 525997 sequence entries). The searches were run using with the fixed modification of methylmethanethiosulfate labeled cysteine parameter enabled. Other parameters such as tryptic cleavage specificity, precursor ion mass accuracy and fragment ion mass accuracy are MALDI 5800 built-in functions of ProteinPilot software. The detected protein threshold (unused protscore (confidence)) in the software was set to 1.3 to achieve 95% confidence, and identified proteins were grouped by the ProGroup algorithm (ABsciex) to minimize redundancy. The bias correction option was executed.
(24) A decoy database search strategy was also used to estimate the false discovery rate (FDR), defined as the percentage of decoy proteins identified against the total protein identification. The FDR was calculated by searching the spectral against the Uniprot Homo sapiens decoy database. The estimated low FDR of 0.9% indicated a high reliability in the identified proteins.
(25) 1.7. Quantification of Relative Protein Expression.
(26) The inventors employed a customized software package, iQuantitator (Schwacke et al., 2009; Grant et al., 2009; and Besson et al., 2011) to infer the magnitude of change in protein expression. The software infers treatment-dependent changes in expression using Bayesian statistical methods. Basically, this approach was used to generate means, medians, and 95% credible intervals (upper and lower) for each treatment-dependent change in protein expression by using peptide-level data for each component peptide, and integrating data across the two experiments. For proteins whose iTRAQ ratios were downregulated in tissues, the extent of downregulation was considered further if the higher limit of the credible interval had a value lower than 1. Conversely, for proteins whose iTRAQ ratios were up-regulated in tumors, the extent of upregulation was considered further if the lower limit of the credible interval had a value greater than 1. The width of these credible intervals depends on the data available for a given protein. Since the number of peptides observed and the number of spectra used to quantify the change in expression for a given protein are taken into consideration, it is possible to detect small but significant changes in up- or downregulation when many peptides are available. For each protein, and each peptide associated with a given protein, the mean, median, and 95% credible intervals were computed for each of the protein- and peptide-level treatment effects.
(27) The peptide selection criteria for relative quantification were performed as follows. Only peptides unique for a given protein were considered for relative quantification, excluding those common to other isoforms or proteins of the same family. Proteins were identified on the basis of having at least one peptide with an ion score above 95% confidence. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence.
(28) 1.8. Functional Annotation and Network Analysis
(29) Gene ontology (GO) terms for identified proteins were extracted, and overrepresented functional categories for differentially abundant proteins were determined by the high throughput GOMiner tool (National Cancer Institute) (Zeeberg et al., 2005). All proteins that were subjected to iQuantitator analysis served as the background list, and GO terms with at least five proteins were used for statistical calculations. A p value for each term was calculated via the one-sided Fisher's exact test, and FDR was estimated by permutation analysis using 1000 randomly selected sets of proteins sampled from the background list. Statistically significant (FDR <25%) GO terms were clustered based on the correlation of associated proteins to minimize potential redundancy in significant GO terms.
(30) 1.9. Western Blot Analysis of Proteins in TNBC
(31) Freezed tumors were mounted in OCT and cut with a cryostat (Starlet 2212). Forty 12 m sections were then lysed in FASP buffer (4% SDS, 0.1 M Tris) for 90 min at 95 C., sonicated 3 times and centrifugated 10 min RT at 13200 rpm. Protein concentration was evaluated using Pierce BCA protein assay kit (Thermo scientific, #23225). Western blot on tumor lysates were performed as described previously (Besson et al., 2011). Briefly, 50 g of tumor lysates were loaded on 12% polyacrylamide gel and then transferred onto PVDF membrane. After blocking with 5% BSA in TBS (0.1 M, pH 7.4), blots were incubated with the respective primary antibodies at 4 C. overnight (Thrombospondin-1: Abcam Ab1823 1/500, 10 kDa heat shock protein: Abcam Ab108600 1/10000, Tryptophanyl-tRNA synthetase: Abcam Ab 92733 1/10000). The protein abundance of Hsc70 was used as a control for protein loading and was determined with mouse anti Hsc from Santa Cruz sc-7278 1/500 2 h at room temperature. The membranes were incubated with the respective secondary antibody, horseradish peroxidase-conjugated rabbit anti-IgG (goat anti-rabbit IgG-HRP sc-2004, 1:4000; Santa Cruz Biotechnology Inc.) or horseradish peroxidase-conjugated mouse anti-IgG (goat anti-mouse IgG-HRP, sc-2005, 1:4000; Santa Cruz Biotechnology Inc.), for 45 min at room temperature. After each step, blots were washed three times with 0.05% Tween, TBS. The membrane was probed with the indicated antibodies and developed with the ECL on a Chemidoc System (Bio-Rad).
(32) 1.10. ELISA (Enzyme-Linked Immunosorbent Assay)
(33) Commercially available ELISA kits from USCN Life Science Inc. or R&D were used to assay concentrations of Decorin, Asporin and Thrombospondin-1. The kits consisted of 96-well microtiter plates coated with antibody specific to each type of molecule, detection antibodies for identifying the antibody-protein in the plate by streptavidin-biotin labeling and TMB substrate which generated colored product. The sample was added and assay was conducted according to the manufacturer's instruction. The absorbance of the colored product developed at the end of the assay was quantified at wavelength 450 nm on ELISA reader (Tecan Magellan Sunrise). Dilutions of the serums are indicated in Table 4 below.
(34) TABLE-US-00004 TABLE 4 Proteins Supplier Reference serum dilution DECORIN USCN Life E92127Hu in PBS Science Inc. ASPORIN USCN Life E92321Hu in PBS Science Inc. THROMBOSPONDIN-1 R&D Quantikine 1/100 in Systems DTSP10 RD5-33 1X (supplied by Manufacturer)
2. Results
(35) 2.1. Identification of Expressed Proteins: Proteomic Coverage of 80 Triple-Negative Breast Tumors
(36) Using Protein Pilot and iQuantitator softwares, the inventors identified and quantified a total of 2805 non-redundant proteins with at least 2 peptides, according the schematic workflow of experimental design in
(37) TABLE-US-00005 TABLE 5A Enrichment by protein function Percentage Protein In In function Protein data protein in class Actual n R N Expected Ratio p-value z-score set function database Enzymes 690 3099 2724 23844 354 1.949 8.622E78 20.34 22.27% 25.33% 11.42% Phosphatases 58 3099 230 23844 29.89 1.94 3.724E07 5.538 1.87% 25.22% 0.96% Proteases 122 3099 559 23844 72.65 1.679 3.748E09 6.281 3.94% 21.82% 2.34% Kinases 105 3099 654 23844 85 1.235 1.234E02 2.358 3.39% 16.06% 2.74% Ligands 73 3099 514 23844 66.8 1.093 2.230E01 0.8215 2.36% 14.20% 2.16% Transcription 82 3099 959 23844 124.6 0.6579 7.007E06 4.18 2.65% 8.55% 4.02% factors Receptors 83 3099 1565 23844 203.4 0.4081 2.446E25 9.363 2.68% 5.30% 6.56% Other 1891 3099 16691 23844 2169 0.8717 1.285E30 11.7 61.02% 11.33% 70.00%
Columns have the following meaning:
Protein class: a broadly defined protein function;
Actual: number of network objects from the activated dataset(s) for a given protein class;
n: number of network objects in the activated dataset(s);
R: number of network objects of a given protein class in the complete database or background list;
N: total number of network objects in the complete database or background list;
Expected: mean value for hypergeometric distribution (n*R/N);
Ratio: connectivity ratio (Actual/Expected);
z-score: z-score ((Actual-Expected)/sqrt(variance));
p-value: probability to have the given value of Actual or higher (or lower for negative z-score);
In data set: fraction of network objects with a selected function in the activated dataset;
In protein function: fraction of network with a selected function in the activated dataset among; network objects with this function in the complete database or background list;
Protein function in database: fraction of network objects with a selected function in the complete database or background list.
(38) TABLE-US-00006 TABLE 5B Pathway maps Pathway maps p-Value ratio 1 Cytoskeleton remodeling_Cytoskeleton remodeling 1.008e20 55/102 2 Cytoskeleton remodeling_Keratin filaments 5.196e16 27/36 3 LRRK2 in neurons in Parkinson's disease 4.337e15 25/33 4 Cytoskeleton remodeling_TGF, WNT and cytoskeletal remodeling 8.787e15 50/111 5 Cytoskeleton remodeling_Regulation of actin cytoskeleton by Rho GTPases 1.902e14 20/23 6 Cell adhesion_Integrin-mediated cell adhesion and migration 2.581e12 28/48 7 Cell adhesion_Chemokines and adhesion 9.170e11 41/100 8 Apoptosis and survival_Granzyme A signaling 1.152e10 20/30 9 Transport_Clathrin-coated vesicle cycle 1.174e10 33/71 10 Immune response_Alternative complement pathway 1.760e10 23/39
(39) TABLE-US-00007 TABLE 5C Gene Ontology (GO) process Processes p-Value ratio 1 metabolic process 7.084e193 2305/10691 2 cellular metabolic process 4.692e171 2079/9418 3 primary metabolic process 1.426e158 2081/9619 4 cellular process 6.761e152 2875/15962 5 catabolic process 5.562e151 790/2270 6 small molecule metabolic process 1.146e145 910/2880 7 cellular catabolic process 2.285e142 695/1910 8 cellular component organization or 1.902e139 1255/4755 biogenesis 9 cellular component organization or 1.762e131 1090/3947 biogenesis at cellular level 10 cellular component organization 2.660e129 1204/4596
(40) Among these 2805 proteins, 219 proteins met the inventors' definition for differential expression in a comparison between tumor and normal tissues: 126 were overexpressed and 93 were underexpressed (Table 6A). The inventors subjected the 219 identified proteins that were differentially expressed in triple-negative breast tumors to Metacore analysis and categorized them, in a first time, according to molecular function and biological process. When the inventors analyzed the dysregulated proteins for molecular function, they found that the best enrichment score and p-Value were assigned to the GO terms Protein binding, MHC class I receptor activity and GTPase activity (p=2.0 10.sup.13), which included 7 members RAS oncogen family (Table 6B). When the inventors grouped dysregulated proteins based on biological processes, they found that the best GO term score was obtained with cytoskeleton organization (p=9.2 10.sup.16) (Table 6C). Finally, when the inventors analyzed the enrichment of protein function of these dysregulated proteins, they found that the first class with the best z-score was ligands with 14 proteins, indicating potentially biomarker candidates (Table 6D).
(41) TABLE-US-00008 TABLE 6A Proteins underexpressed and overexpressed in triple-negative breast cancer confidence intervals Peptide Gene Protein Mean min max number EEF1A1 Elongation factor 1-alpha 1 3.325 2.191 5.243 12 HLA-A HLA class I histocompatibility antigen. A-33 alpha 2.594 1.935 3.498 4 chain KRT8 Keratin type II cytoskeletal 8 2.698 1.747 4.278 14 MAPK13 Mitogen-activated protein kinase 13 2.9 1.601 5.366 2 TUBA1C Tubulin alpha-1C chain 3.593 1.537 8.237 5 NME1 Nucleoside diphosphate kinase A 2.045 1.462 2.906 6 YBX1 Nuclease-sensitive element-binding protein 1 1.955 1.395 2.714 6 HSPA8 Heat shock cognate 71 kDa protein 5.889 1.372 31.395 27 ITGAM Integrin alpha-M 2.086 1.357 3.218 5 TPM3 Tropomyosin alpha-3 chain 1.886 1.345 2.686 8 NAMPT Nicotinamide phosphoribosyltransferase 1.992 1.341 3.104 12 NME2 Nucleoside diphosphate kinase B 1.791 1.326 2.442 11 RAB5A Ras-related protein Rab-5A 2.095 1.312 3.464 4 USP15 Ubiquitin carboxyl-terminal hydrolase 15 1.887 1.31 2.744 5 KHDRBS1 KH domain-containing. RNA-binding. signal 2.265 1.288 4.013 3 transduction-associated protein 1 STK3 Serine/threonine-protein kinase 3 1.725 1.264 2.336 3 DPYSL3 Dihydropyrimidinase-related protein 3 2.215 1.26 4.311 12 HSP90AB1 Heat shock protein HSP 90-beta 1.878 1.259 2.76 26 SDR16C5 Epidermal retinol dehydrogenase 2 1.943 1.248 3.029 4 DSP Desmoplakin 2.119 1.247 4.138 27 THBS1 Thrombospondin-1 1.682 1.243 2.342 25 SNRPB Small nuclear ribonucleoprotein-associated proteins 1.748 1.236 2.519 10 B and B S100A8 Protein S100-A8 1.652 1.222 2.268 10 TUBA1B Tubulin alpha-1B chain 2.971 1.221 7.506 4 AP1M1 AP-1 complex subunit mu-1 1.869 1.215 2.927 3 GSTM1 Glutathione S-transferase Mu 1 2.161 1.214 3.872 2 ACTG1 Actin. cytoplasmic 2 1.666 1.206 2.342 8 THBS2 Thrombospondin-2 1.802 1.188 2.753 10 XRCC6 X-ray repair cross-complementing protein 6 1.605 1.186 2.166 22 GGH Gamma-glutamyl hydrolase 2.392 1.186 5.251 3 SORD Sorbitol dehydrogenase 1.763 1.182 2.657 7 NA Small nuclear ribonucleoprotein G-like protein 1.759 1.181 2.659 2 IGLC2 Ig lambda-2 chain C regions 1.601 1.177 2.151 5 HLA-B HLA class I histocompatibility antigen. B-14 alpha 1.55 1.174 2.05 3 chain MSN Moesin 1.514 1.172 1.974 36 MYO6 Myosin-VI 1.62 1.17 2.26 9 TNC Tenascin 1.616 1.164 2.242 44 AP1B1 AP-1 complex subunit beta-1 1.676 1.155 2.446 10 XYLT2 Xylosyltransferase 2 2.337 1.153 4.893 2 H2AFV Histone H2A.V 1.987 1.15 3.472 2 COTL1 Coactosin-like protein 1.703 1.147 2.54 6 ENAH Protein enabled homolog 1.693 1.146 2.496 4 AKR7A3 Aflatoxin B1 aldehyde reductase member 3 1.832 1.146 2.934 2 CAMK2D Calcium/calmodulin-dependent protein kinase type II 1.793 1.144 2.84 4 subunit delta DBI Acyl-CoA-binding protein 1.606 1.143 2.29 7 LRRC59 Leucine-rich repeat-containing protein 59 1.717 1.141 2.559 7 SMARCA5 SWI/SNF-related matrix-associated actin-dependent 1.544 1.14 2.087 13 regulator of chromatin subfamily A member 5 CFL1 Cofilin-1 1.569 1.136 2.194 18 GUSB Beta-glucuronidase 1.59 1.135 2.215 21 XRCC5 X-ray repair cross-complementing protein 5 1.533 1.133 2.06 24 PSME1 Proteasome activator complex subunit 1 1.536 1.124 2.079 11 CFL2 Cofilin-2 1.713 1.124 2.63 2 TUBB Tubulin beta chain 1.481 1.118 1.973 15 NDRG1 Protein NDRG1 1.554 1.117 2.159 6 HYOU1 Hypoxia up-regulated protein 1 2.287 1.117 5.424 21 ARF4 ADP-ribosylation factor 4 1.606 1.114 2.333 6 TOP2B DNA topoisomerase 2-beta 1.566 1.11 2.232 6 CRABP2 Cellular retinoic acid-binding protein 2 1.617 1.11 2.408 8 PPP4R1 Serine/threonine-protein phosphatase 4 regulatory 1.677 1.108 2.532 3 subunit 1 FABP7 Fatty acid-binding protein. brain 2.98 1.106 10.019 6 SET Protein SET 1.596 1.105 2.318 7 TM9SF3 Transmembrane 9 superfamily member 3 1.66 1.105 2.49 6 SARS Seryl-tRNA synthetase. cytoplasmic 1.519 1.103 2.092 12 DSTN Destrin 1.978 1.101 3.997 6 COPA Coatomer subunit alpha 1.496 1.095 2.068 31 PRKCSH Glucosidase 2 subunit beta 1.603 1.095 2.324 15 UAP1L1 UDP-N-acetylhexosamine pyrophosphorylase-like 1.461 1.094 1.946 4 protein 1 ARF1 ADP-ribosylation factor 1 1.624 1.092 2.396 5 EHD1 EH domain-containing protein 1 1.883 1.091 3.34 6 GBP1 Interferon-induced guanylate-binding protein 1 1.753 1.089 2.823 8 TPM1 Tropomyosin alpha-1 chain 1.516 1.086 2.14 14 NA Ig kappa chain V-III region HAH 1.666 1.084 2.577 4 PPP2R4 Serine/threonine-protein phosphatase 2A activator 1.64 1.08 2.468 3 RAB1A Ras-related protein Rab-1A 1.418 1.078 1.872 6 GSTO1 Glutathione S-transferase omega-1 1.586 1.076 2.316 7 EML4 Echinoderm microtubule-associated protein-like 4 1.8 1.075 3.045 8 PLIN2 Perilipin-2 1.774 1.074 2.931 4 DDT D-dopachrome decarboxylase 1.504 1.072 2.129 5 RAB11A Ras-related protein Rab-11A 1.542 1.072 2.259 2 ERH Enhancer of rudimentary homolog 1.828 1.072 3.166 2 CLIC4 Chloride intracellular channel protein 4 1.803 1.07 3.033 3 MCM6 DNA replication licensing factor MCM6 1.498 1.065 2.126 7 MAPRE1 Microtubule-associated protein RP/EB family 1.575 1.06 2.362 5 member 1 DLD Dihydrolipoyl dehydrogenase. mitochondrial 1.887 1.059 3.951 8 NONO Non-P 1.5 1.057 2.099 24 S100A11 Protein S100-A11 1.613 1.057 2.465 4 SAR1A GTP-binding protein SAR1a 1.529 1.054 2.235 6 EPPK1 Epiplakin 1.423 1.052 1.921 14 MYL12B Myosin regulatory light chain 12B 1.579 1.052 2.347 7 FTL Ferritin light chain 1.743 1.051 3.054 9 SEC14L2 SEC14-like protein 2 1.603 1.048 2.499 5 FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4 1.46 1.045 2.025 18 ACSL3 Long-chain-fatty-acid--CoA ligase 3 1.38 1.044 1.838 12 MACF1 Microtubule-actin cross-linking factor 1. isoforms 1.539 1.044 2.276 5 1/2/3/5 CALR Calreticulin 1.539 1.04 2.334 21 C22 UPF0027 protein C22orf28 1.336 1.036 1.72 10 RAP1B Ras-related protein Rap-1b 1.496 1.035 2.158 7 ALDOA Fructose-bisphosphate aldolase A 1.37 1.033 1.816 19 PRCC Proline-rich protein PRCC 1.747 1.032 2.985 2 NAPA Alpha-soluble NSF attachment protein 1.585 1.029 2.451 5 PRKDC DNA-dependent protein kinase catalytic subunit 1.276 1.027 1.587 55 ERO1L ER 1.571 1.027 2.441 6 BAZ1B Tyrosine-protein kinase BAZ1B 1.558 1.026 2.371 5 RAD23B UV excision repair protein RAD23 homolog B 1.389 1.025 1.88 7 ISYNA1 Inositol-3-phosphate synthase 1 1.631 1.025 2.6 3 ACTN1 Alpha-actinin-1 2.196 1.025 4.273 25 FBL rRNA 2- 1.365 1.023 1.846 12 KRT18 Keratin. type I cytoskeletal 18 2.377 1.023 5.583 5 NCF4 Neutrophil cytosol factor 4 1.687 1.021 2.871 4 YWHAZ 14-3-3 protein zeta/delta 1.388 1.02 1.892 13 ECM29 Proteasome-associated protein ECM29 homolog 1.477 1.02 2.169 5 ACTR2 Actin-related protein 2 1.439 1.019 2.037 13 RPLP0 60S acidic ribosomal protein P0 1.527 1.018 2.353 7 RANBP1 Ran-specific GTPase-activating protein 1.554 1.017 2.403 6 GFPT1 Glucosamine--fructose-6-phosphate 1.526 1.016 2.308 9 aminotransferase [isomerizing] 1 PAICS Multifunctional protein ADE2 1.43 1.015 1.996 12 RAN GTP-binding nuclear protein Ran 1.345 1.01 1.77 14 CALD1 Caldesmon 1.434 1.009 2.039 13 CTSB Cathepsin B 1.481 1.009 2.181 7 IMPDH2 Inosine-5-monophosphate dehydrogenase 2 1.444 1.008 2.09 8 KPNA2 Importin subunit alpha-2 2.048 1.007 3.91 5 RUVBL2 RuvB-like 2 1.345 1.006 1.801 15 CMPK1 UMP-CMP kinase 1.332 1.005 1.758 8 KRT6A Keratin. type II cytoskeletal 6A 1.644 1.005 2.705 3 HTATIP2 Oxidoreductase HTATIP2 1.927 1.005 3.78 2 KRT19 Keratin type I cytoskeletal 19 2.235 1.005 4.978 8 ALDH1A1 Retinal dehydrogenase 1 0.717 0.53 0.971 11 MYO1C Myosin-Ic 0.67 0.527 0.849 18 UAP1 UDP-N-acetylhexosamine pyrophosphorylase 0.701 0.526 0.934 5 CFH Complement factor H 0.693 0.518 0.935 23 KIAA1967 Protein KIAA1967 0.677 0.498 0.909 8 KRT5 Keratin. type II cytoskeletal 5 0.66 0.495 0.875 22 CTNNB1 Catenin beta-1 0.66 0.483 0.902 10 ALDH6A1 Methylmalonate-semialdehyde dehydrogenase 0.686 0.48 0.977 7 [acylating]. mitochondrial EIF2C1 Protein argonaute-1 0.686 0.478 0.983 2 PPP3CB Serine/threonine-protein phosphatase 2B catalytic 0.687 0.469 0.999 2 subunit beta isoform KRT10 Keratin. type I cytoskeletal 10 0.656 0.466 0.927 14 S100A10 Protein S100-A10 0.645 0.461 0.912 8 AHNAK Neuroblast differentiation-associated protein AHNAK 0.606 0.453 0.817 96 TINAGL1 Tubulointerstitial nephritis antigen-like 0.673 0.452 0.982 6 ITGB4 Integrin beta-4 0.594 0.45 0.781 17 F2 Prothrombin 0.602 0.448 0.817 18 APOA1 Apolipoprotein A-I 0.602 0.439 0.831 23 HLA-C HLA class I histocompatibility antigen. Cw-15 alpha 0.571 0.424 0.77 5 chain SERPINF1 Pigment epithelium-derived factor 0.563 0.41 0.772 8 PCOLCE Procollagen C-endopeptidase enhancer 1 0.597 0.408 0.86 7 USP11 Ubiquitin carboxyl-terminal hydrolase 11 0.593 0.406 0.857 5 RAB5B Ras-related protein Rab-5B 0.618 0.398 0.949 4 HDAC1 Histone deacetylase 1 0.591 0.394 0.883 5 CAV1 Caveolin-1 0.623 0.392 0.991 3 PYCRL Pyrroline-5-carboxylate reductase 3 0.613 0.384 0.976 4 EHD2 EH domain-containing protein 2 0.613 0.384 0.985 9 APOH Beta-2-glycoprotein 1 0.602 0.382 0.951 9 HIST1H2BK Histone H2B type 1-K 0.546 0.374 0.812 7 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) 0.603 0.373 0.952 2 subunit beta-1 KRT16 Keratin. type I cytoskeletal 16 0.566 0.372 0.855 3 UROS Uroporphyrinogen-III synthase 0.568 0.37 0.865 4 AMBP Protein AMBP 0.528 0.368 0.75 6 LRG1 Leucine-rich alpha-2-glycoprotein 0.587 0.362 0.939 3 CP Ceruloplasmin 0.493 0.36 0.69 13 SERPINA4 Kallistatin 0.579 0.359 0.922 4 LIPE Hormone-sensitive lipase 0.59 0.358 0.977 3 METTL7B Methyltransferase-like protein 7B 0.587 0.357 0.959 3 NES Nestin 0.508 0.354 0.73 14 RAB5C Ras-related protein Rab-5C 0.527 0.354 0.781 5 HLA-A HLA class I histocompatibility antigen. A-2 alpha 0.572 0.354 0.921 2 chain LMO7 LIM domain only protein 7 0.558 0.35 0.883 6 CSNK1A1 Casein kinase I isoform alpha 0.596 0.35 0.989 2 WDR36 WD repeat-containing protein 36 0.581 0.344 0.963 3 SFRP1 Secreted frizzled-related protein 1 0.523 0.333 0.82 4 CBX5 Chromobox protein homolog 5 0.552 0.332 0.919 2 AFM Afamin 0.532 0.33 0.863 5 KNG1 Kininogen-1 0.514 0.327 0.822 10 APOA2 Apolipoprotein A-II 0.556 0.313 0.974 3 GSTM3 Glutathione S-transferase Mu 3 0.523 0.312 0.867 3 PCCB Propionyl-CoA carboxylase beta chain. 0.528 0.309 0.904 2 mitochondrial ACOT2 Acyl-coenzyme A thioesterase 2. mitochondrial 0.494 0.308 0.773 3 LBP Lipopolysaccharide-binding protein 0.52 0.303 0.89 3 APOA4 Apolipoprotein A-IV 0.458 0.302 0.687 9 ABI3BP Target of Nesh-SH3 0.508 0.295 0.869 4 PLIN1 Perilipin-1 0.437 0.293 0.659 8 CAST Calpastatin 0.496 0.29 0.823 5 TNXB Tenascin-X 0.448 0.286 0.696 8 ORM1 Alpha-1-acid glycoprotein 1 0.446 0.281 0.715 7 HIST2H2AC Histone H2A type 2-C 0.391 0.279 0.546 4 OLFML3 Olfactomedin-like 3 0.437 0.278 0.68 4 CLEC3B Tetranectin 0.462 0.27 0.799 4 COL4A1 Collagen alpha-1(IV) chain 0.481 0.269 0.88 3 IGLC1 Ig lambda-1 chain C regions 0.42 0.267 0.656 2 GC Vitamin D-binding protein 0.462 0.265 0.804 12 PIGR Polymeric immunoglobulin receptor 0.488 0.265 0.836 6 KRT1 Keratin. type II cytoskeletal 1 0.453 0.26 0.788 5 PTRF Polymerase I and transcript release factor 0.441 0.253 0.749 6 OLFML1 Olfactomedin-like 1 0.45 0.249 0.82 2 AGR2 Anterior gradient protein 2 homolog 0.413 0.241 0.699 6 MYH11 Myosin-11 0.354 0.234 0.526 13 MFAP4 Microfibril-associated glycoprotein 4 0.402 0.232 0.688 2 CMA1 Chymase 0.363 0.229 0.591 8 TTR Transthyretin 0.327 0.224 0.482 7 CFD Complement factor D 0.38 0.214 0.663 2 SOD3 Extracellular superoxide dismutase [CuZn] 0.368 0.197 0.687 3 HIST3H2A Histone H2A type 3 0.289 0.193 0.433 2 COL6A6 Collagen alpha-6(VI) chain 0.38 0.183 0.823 9 AOC3 Membrane primary amine oxidase 0.335 0.179 0.616 11 ATL2 Atlastin-2 0.419 0.176 0.942 2 SERPIND1 Heparin cofactor 2 0.363 0.167 0.79 2 DPT Dermatopontin 0.282 0.144 0.534 7 TPSAB1 Tryptase alpha-1 0.356 0.139 0.831 2 ADH1C Alcohol dehydrogenase 1C 0.27 0.131 0.533 2 AZGP1 Zinc-alpha-2-glycoprotein 0.172 0.123 0.239 16 OGN Mimecan 0.144 0.104 0.196 15 DCN Decorin 0.503 0.1 0.75 19 ELN Elastin 0.141 0.074 0.271 4 IGHA2 Ig alpha-2 chain C region 0.121 0.054 0.313 8 APOD Apolipoprotein D 0.065 0.043 0.099 10 LGALS4 Galectin-4 0.077 0.035 0.174 3 PIP Prolactin-inducible protein 0.096 0.031 0.289 5 IGJ Immunoglobulin J chain 0.077 0.027 0.214 2 DES Desmin 0.038 0.019 0.079 3
(42) TABLE-US-00009 TABLE 6B Gene ontology (GO) molecular function - triple-negative breast cancer Molecular functions pValue Ratio 1 protein binding 8.478e25 193/8829 2 MHC class I receptor activity 3.771e19 14/31 3 binding 9.638e17 232/13778 4 GTPase activity 2.003e13 23/257 5 polysaccharide binding 2.689e11 20/239 6 pattern binding 2.689e11 20/239 7 glycosaminoglycan binding 3.064e11 19/214 8 nucleoside-triphosphatase activity 3.074e11 37/840 9 heparin binding 3.601e11 17/166 10 small molecule binding 4.286e11 75/2773 11 receptor binding 6.439e11 51/1506 12 GTP binding 7.220e11 25/407 13 carbohydrate binding 8.160e11 28/514 14 pyrophosphatase activity 1.081e10 37/878 15 hydrolase activity, acting on acid 1.190e10 37/881 anhydrides, in phosphorus-containing anhydrides 16 hydrolase activity, acting on acid 1.268e10 37/883 anhydrides 17 guanyl nucleotide binding 1.631e10 25/423 18 guanyl ribonucleotide binding 1.631e10 25/423 19 structural molecule activity 2.884e10 32/699 20 purine ribonucleoside triphosphate binding 1.339e9 57/1960 21 purine ribonucleotide binding 2.570e9 57/1995 22 ribonucleotide binding 2.617e9 57/1996 23 structural constituent of cytoskeleton 2.943e9 12/96 24 nucleotide binding 3.295e9 67/2573 25 nucleoside phosphate binding 3.348e9 67/2574 26 purine nucleotide binding 3.440e9 57/2011 27 actin binding 1.710e8 21/381 28 cytoskeletal protein binding 2.807e7 27/703 29 catalytic activity 4.501e7 114/6069 30 hydrolase activity 1.013e6 62/2676 31 protein complex binding 5.122e6 20/496 32 phosphatidylcholine-sterol 1.922e5 03/05 O-acyltransferase activator activity 33 cholesterol binding 2.334e5 05/28 34 glutathione transferase activity 5.354e5 05/33 35 identical protein binding 5.658e5 28/992 36 ATP binding 9.852e5 38/1592 37 opsonin binding 1.047e4 03/08 38 sterol binding 1.076e4 05/38 39 collagen binding 1.154e4 06/61 40 peptidase regulator activity 1.202e4 13/301 41 adenyl ribonucleotide binding 1.428e4 38/1621 42 transferase activity, transferring alkyl or 1.510e4 06/64 aryl (other than methyl) groups 43 intermediate filament binding 1.556e4 03/09 44 rRNA primary transcript binding 1.571e4 02/02 45 D-dopachrome decarboxylase activity 1.571e4 02/02 46 adenyl nucleotide binding 1.679e4 38/1634 47 microfilament motor activity 2.120e4 04/24 48 steroid binding 2.381e4 07/98 49 extracellular matrix binding 2.438e4 05/45 50 cysteine-type endopeptidase inhibitor 2.481e4 06/70 activity
(43) TABLE-US-00010 TABLE 6C Gene ontology (GO) process - triple-negative breast cancer Process networks pValue Ratio 1 Cytoskeleton_Intermediate filaments 9.295e16 21/81 2 Cell adhesion_Integrin-mediated cell-matrix 8.568e9 23/214 adhesion 3 Cell adhesion_Cell junctions 4.510e8 19/162 4 Cytoskeleton_Regulation of cytoskeleton 6.496e8 20/183 rearrangement 5 Immune response_Phagosome in antigen 4.017e7 22/243 presentation 6 Protein folding_Protein folding nucleus 2.395e6 10/58 7 Cytoskeleton_Actin filaments 3.764e6 17/176 8 Protein folding_ER and cytoplasm 1.997e5 08/45 9 Immune response_Antigen presentation 6.401e5 16/197 10 Cell cycle_Mitosis 7.642e5 15/179
(44) TABLE-US-00011 TABLE 6D Enrichment by protein function - triple-negative breast cancer Percentage Protein In In function Protein data protein in class Actual n R N Expected Ratio p-value z-score set function database Ligands 14 271 514 23844 5.842 2.396 2.400E03 3.432 5.17% 2.72% 2.16% Proteases 10 271 559 23844 6.353 1.574 1.066E01 1.472 3.69% 1.79% 2.34% Enzymes 48 271 2724 23844 30.96 1.55 1.353E03 3.273 17.71% 1.76% 11.42% Kinases 10 271 654 23844 7.433 1.345 2.125E01 0.9602 3.69% 1.53% 2.74% Phosphatases 3 271 230 23844 2.614 1.148 4.867E01 0.2412 1.11% 1.30% 0.96% Receptors 18 271 1565 23844 17.79 1.012 5.143E01 0.05253 6.64% 1.15% 6.56% Transcription 4 271 959 23844 10.9 0.367 1.428E02 2.145 1.48% 0.42% 4.02% factors Other 164 271 16691 23844 189.7 0.8645 5.086E04 3.427 60.52% 0.98% 70.00%
(45) 2.2. A Proteomic Coverage of the Different Status
(46) The inventors used the iQuantitator software to quantify protein expression between the different status relapse and no relapse. For the relapse group, 295 proteins were significantly differentially expressed: 165 were overexpressed and 130 were underexpressed (Table 7A). The Metacore analysis of this list of proteins indicated a cytoskeleton remodeling with a pValue=9.2 10-12 for the Process Network Regulation of Cytoskeleton Rearrangement and a best enrichment score and p-value for Binding (p=9.4 10-26) in the GO Molecular Functions term. It should be noted that 26 ligands were found in this list characterizing the Relapse group (Table 7D).
(47) TABLE-US-00012 TABLE 7A Proteins underexpressed and overexpressed in recurrence of triple-negative breast cancer Confidence intervals Peptide Gene Protein Mean min max number CYP2A6 Cytochrome P450 2A6 5.532 3.695 8.322 7 ALOX15B Arachidonate 15-lipoxygenase B 5.031 3.61 7.064 10 SDR16C5 Epidermal retinol dehydrogenase 2 5.394 3.079 9.449 4 HLA-A HLA class I histocompatibility antigen. A-33 alpha 3.504 2.388 5.128 4 chain GUSB Beta-glucuronidase 2.962 2.209 3.971 18 S100A7 Protein S100-A7 2.952 1.93 4.509 2 HPGD 15-hydroxyprostaglandin dehydrogenase [NAD+] 2.917 1.896 4.542 5 DHRS2 Dehydrogenase/reductase SDR family member 2 3.134 1.885 5.283 7 FKBP5 Peptidyl-prolyl cis-trans isomerase FKBP5 3.329 1.822 6.087 6 ACSL3 Long-chain-fatty-acid--CoA ligase 3 2.536 1.803 3.581 12 NAMPT Nicotinamide phosphoribosyltransferase 2.566 1.788 3.695 11 KRT8 Keratin type II cytoskeletal 8 2.782 1.678 4.622 14 EEF1A1 Elongation factor 1-alpha 1 2.348 1.674 3.317 12 NA Small nuclear ribonucleoprotein G-like protein 2.505 1.669 3.818 2 DBI Acyl-CoA-binding protein 2.266 1.595 3.249 6 SCP2 Non-specific lipid-transfer protein 2.129 1.571 2.881 10 MDH2 Malate dehydrogenase. mitochondrial 2.226 1.567 3.166 11 CALR Calreticulin 1.998 1.564 2.531 21 TM9SF3 Transmembrane 9 superfamily member 3 2.576 1.538 4.319 5 UAP1L1 UDP-N-acetylhexosamine pyrophosphorylase-like 2.268 1.529 3.401 3 protein 1 SNRPB Small nuclear ribonucleoprotein-associated proteins 2.079 1.517 2.834 10 B and B SEC14L2 SEC14-like protein 2 2.471 1.476 4.225 4 YBX1 Nuclease-sensitive element-binding protein 1 2.086 1.464 3.001 6 IPO9 Importin-9 2.527 1.42 4.424 3 KPNB1 Importin subunit beta-1 2.1 1.409 3.081 10 HLA-B HLA class I histocompatibility antigen. B-14 alpha 2.154 1.386 3.375 3 chain MCCC2 Methylcrotonoyl-CoA carboxylase beta chain. 1.905 1.377 2.66 12 mitochondrial RAB5A Ras-related protein Rab-5A 2.185 1.366 3.557 3 TPM3 Tropomyosin alpha-3 chain 2.182 1.344 3.648 7 AKR1C2 Aldo-keto reductase family 1 member C2 2.442 1.337 4.435 5 IDI1 Isopentenyl-diphosphate Delta-isomerase 1 2.071 1.335 3.198 6 CNN2 Calponin-2 2.131 1.335 3.388 4 GOT1 Aspartate aminotransferase. cytoplasmic 1.983 1.33 2.99 7 THBS1 Thrombospondin-1 1.728 1.316 2.285 23 DPYSL3 Dihydropyrimidinase-related protein 3 1.892 1.313 2.712 12 DSP Desmoplakin 1.818 1.311 2.531 25 CRABP2 Cellular retinoic acid-binding protein 2 1.923 1.31 2.816 8 HSP90AB1 Heat shock protein HSP 90-beta 1.927 1.309 2.921 25 KPNA2 Importin subunit alpha-2 2.158 1.308 3.501 5 DDT D-dopachrome decarboxylase 2.116 1.301 3.485 5 IDH2 Isocitrate dehydrogenase [NADP]. mitochondrial 1.733 1.299 2.304 20 G6PD Glucose-6-phosphate 1-dehydrogenase 1.751 1.298 2.376 18 S100A11 Protein S100-A11 2.031 1.289 3.176 3 PLIN2 Perilipin-2 2.216 1.288 3.846 4 GPI Glucose-6-phosphate isomerase 1.739 1.277 2.349 16 NME2 Nucleoside diphosphate kinase B 1.678 1.268 2.215 11 NDRG1 Protein NDRG1 1.838 1.268 2.663 5 ALCAM CD166 antigen 1.865 1.255 2.789 7 ITGAM Integrin alpha-M 2.019 1.246 3.283 5 FTL Ferritin light chain 1.892 1.24 3.011 9 IPO5 Importin-5 1.9 1.238 2.936 8 TUBA1C Tubulin alpha-1C chain 1.752 1.236 2.503 4 SORD Sorbitol dehydrogenase 2.005 1.227 3.329 4 USP15 Ubiquitin carboxyl-terminal hydrolase 15 1.931 1.209 3.115 3 TOP2B DNA topoisomerase 2-beta 1.671 1.206 2.333 6 FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4 1.715 1.193 2.489 18 AKR1B15 Putative aldo-keto reductase family 1 member B15 1.969 1.193 3.263 3 SAR1A GTP-binding protein SAR1a 1.725 1.184 2.498 5 STK3 Serine/threonine-protein kinase 3 1.746 1.184 2.563 2 CFL2 Cofilin-2 1.673 1.181 2.4 2 PPA2 Inorganic pyrophosphatase 2. mitochondrial 2.403 1.179 4.955 2 AP1M2 AP-1 complex subunit mu-2 1.778 1.178 2.732 4 KRT7 Keratin. type II cytoskeletal 7 2.271 1.178 4.914 26 SET Protein SET 1.678 1.175 2.438 6 PPP2R4 Serine/threonine-protein phosphatase 2A activator 1.969 1.171 3.321 2 PRKCSH Glucosidase 2 subunit beta 1.592 1.167 2.18 15 XRCC5 X-ray repair cross-complementing protein 5 1.502 1.164 1.936 23 CALD1 Caldesmon 1.676 1.158 2.426 12 ENAH Protein enabled homolog 1.911 1.158 3.183 3 ACOX1 Peroxisomal acyl-coenzyme A oxidase 1 1.774 1.156 2.804 7 CRAT Carnitine 1.862 1.156 3.019 4 THBS2 Thrombospondin-2 1.628 1.148 2.323 10 EEF1D Elongation factor 1-delta 1.631 1.147 2.356 10 DHCR24 24-dehydrocholesterol reductase 1.936 1.146 3.379 3 MSN Moesin 1.412 1.145 1.724 35 H2AFV Histone H2A.V 1.995 1.142 3.485 2 PPP4R1 Serine/threonine-protein phosphatase 4 regulatory 1.888 1.14 3.209 2 subunit 1 CRABP1 Cellular retinoic acid-binding protein 1 1.677 1.137 2.467 7 S100A8 Protein S100-A8 1.522 1.134 2.046 9 ATP6V1A V-type proton ATPase catalytic subunit A 1.551 1.133 2.128 10 TMED7 Transmembrane emp24 domain-containing protein 7 2.07 1.126 3.85 3 ARF4 ADP-ribosylation factor 4 1.692 1.123 2.576 6 TKT Transketolase 1.584 1.122 2.208 21 PSME1 Proteasome activator complex subunit 1 1.519 1.118 2.057 11 TNC Tenascin 1.574 1.116 2.269 44 NQO1 NAD(P)H dehydrogenase [quinone] 1 1.542 1.108 2.152 6 SOD1 Superoxide dismutase [CuZn] 1.615 1.108 2.347 6 CAMK2D Calcium/calmodulin-dependent protein kinase type II 1.739 1.108 2.769 4 subunit delta MAPK13 Mitogen-activated protein kinase 13 1.942 1.107 3.547 2 FASN Fatty acid synthase 1.563 1.106 2.223 56 HSD17B11 Estradiol 17-beta-dehydrogenase 11 2.016 1.106 3.671 4 RAB1A Ras-related protein Rab-1A 1.551 1.099 2.203 5 NAPA Alpha-soluble NSF attachment protein 1.752 1.099 2.864 5 CDK1 Cell division protein kinase 1 1.92 1.099 3.38 3 C1S Complement C1s subcomponent 2.216 1.099 4.614 4 TUBB Tubulin beta chain 1.463 1.098 1.942 15 FAH Fumarylacetoacetase 1.953 1.097 3.452 3 SEC23B Protein transport protein Sec23B 1.497 1.092 2.062 10 IMPDH2 Inosine-5-monophosphate dehydrogenase 2 1.596 1.088 2.341 8 RAP1B Ras-related protein Rap-1b 1.46 1.083 1.972 7 ECM29 Proteasome-associated protein ECM29 homolog 1.611 1.081 2.401 5 TUBA1B Tubulin alpha-1B chain 1.604 1.08 2.415 3 SMARCA5 SWI/SNF-related matrix-associated actin-dependent 1.458 1.077 1.981 12 regulator of chromatin subfamily A member 5 SQRDL Sulfide:quinone oxidoreductase. mitochondrial 1.415 1.076 1.861 14 PMVK Phosphomevalonate kinase 1.549 1.075 2.239 8 MYO6 Myosin-VI 1.634 1.074 2.472 9 IARS Isoleucyl-tRNA synthetase. cytoplasmic 1.458 1.073 2.004 14 NDUFS8 NADH dehydrogenase [ubiquinone] iron-sulfur 1.671 1.072 2.652 2 protein 8. mitochondrial RBM25 RNA-binding protein 25 1.627 1.07 2.479 3 MCM6 DNA replication licensing factor MCM6 1.488 1.069 2.085 7 PTGES3 Prostaglandin E synthase 3 1.56 1.067 2.304 5 GGH Gamma-glutamyl hydrolase 1.775 1.067 2.933 3 COPE Coatomer subunit epsilon 1.589 1.066 2.37 5 PRKDC DNA-dependent protein kinase catalytic subunit 1.279 1.063 1.537 55 GLO1 Lactoylglutathione lyase 1.477 1.062 2.062 6 IDH1 Isocitrate dehydrogenase [NADP] cytoplasmic 1.483 1.06 2.085 11 GSTO1 Glutathione S-transferase omega-1 1.54 1.059 2.244 7 CSE1L Exportin-2 1.588 1.059 2.381 7 PPA1 Inorganic pyrophosphatase 1.518 1.056 2.194 8 SSBP1 Single-stranded DNA-binding protein. mitochondrial 1.467 1.055 2.041 8 PTBP1 Polypyrimidine tract-binding protein 1 1.472 1.052 2.1 14 CCT7 T-complex protein 1 subunit eta 1.528 1.051 2.267 17 RPL8 60S ribosomal protein L8 1.475 1.05 2.087 13 GOLGB1 Golgin subfamily B member 1 1.674 1.05 2.662 3 PPP3CA Serine/threonine-protein phosphatase 2B catalytic 1.672 1.05 2.685 2 subunit alpha isoform GFPT1 Glucosamine--fructose-6-phosphate 1.629 1.049 2.499 8 aminotransferase [isomerizing] 1 SFRS1 Splicing factor. arginine/serine-rich 1 1.569 1.047 2.373 8 SOD2 Superoxide dismutase [Mn]. mitochondrial 1.545 1.046 2.277 5 PGRMC1 Membrane-associated progesterone receptor 1.662 1.041 2.592 3 component 1 DERL1 Derlin-1 1.902 1.041 3.49 2 SERBP1 Plasminogen activator inhibitor 1 RNA-binding 1.512 1.04 2.211 9 protein CANX Calnexin 1.416 1.039 1.942 18 PPIB Peptidyl-prolyl cis-trans isomerase B 1.499 1.038 2.186 17 PEPD Xaa-Pro dipeptidase 1.842 1.038 3.357 2 SARS Seryl-tRNA synthetase. cytoplasmic 1.405 1.037 1.914 11 CALU Calumenin 1.45 1.035 2.015 8 STAT3 Signal transducer and activator of transcription 3 1.482 1.034 2.126 8 UGDH UDP-glucose 6-dehydrogenase 1.358 1.033 1.779 17 POR NADPH--cytochrome P450 reductase 1.392 1.033 1.859 9 ISYNA1 Inositol-3-phosphate synthase 1 1.739 1.032 2.962 3 DNASE2 Deoxyribonuclease-2-alpha 2.018 1.03 3.973 2 CLIC4 Chloride intracellular channel protein 4 1.781 1.029 3.075 3 PGAM1 Phosphoglycerate mutase 1 1.374 1.026 1.866 15 EIF2C2 Protein argonaute-2 1.474 1.024 2.108 3 KRT18 Keratin. type I cytoskeletal 18 2.195 1.024 4.949 5 DPP3 Dipeptidyl peptidase 3 1.514 1.023 2.236 11 RAP2B Ras-related protein Rap-2b 1.719 1.023 2.872 3 PAICS Multifunctional protein ADE2 1.366 1.021 1.825 12 RAN GTP-binding nuclear protein Ran 1.304 1.02 1.678 14 EPPK1 Epiplakin 1.44 1.015 2.047 13 DDB1 DNA damage-binding protein 1 1.487 1.015 2.203 10 KRT19 Keratin type I cytoskeletal 19 2.044 1.013 4.534 8 NONO Non-P 1.259 1.012 1.584 23 SERPINH1 Serpin H1 1.272 1.011 1.59 20 RPL35 60S ribosomal protein L35 1.521 1.01 2.291 6 CALML5 Calmodulin-like protein 5 1.554 1.009 2.377 7 HIST1H1C Histone H1.2 1.468 1.008 2.12 9 NUDT3 Diphosphoinositol polyphosphate phosphohydrolase 1 1.639 1.008 2.662 3 TES Testin 1.669 1.008 2.833 5 EIF4A1 Eukaryotic initiation factor 4A-I 1.366 1.007 1.83 14 SLC25A6 ADP/ATP translocase 3 1.65 1.006 2.667 4 LRRC59 Leucine-rich repeat-containing protein 59 1.489 1.003 2.217 7 IL4I1 L-amino-acid oxidase 1.507 1 2.243 3 ERH Enhancer of rudimentary homolog 1.863 1 3.455 2 LMNA Prelamin-A/C 0.8 0.662 0.966 26 AHNAK Neuroblast differentiation-associated protein AHNAK 0.671 0.578 0.779 96 SPTAN1 Spectrin alpha chain. brain 0.711 0.577 0.874 58 ACADVL Very long-chain specific acyl-CoA dehydrogenase. 0.736 0.546 0.992 12 mitochondrial HSPG2 Basement membrane-specific heparan sulfate 0.67 0.535 0.842 53 proteoglycan core protein UBC Polyubiquitin-C 0.696 0.524 0.923 5 RBM39 RNA-binding protein 39 0.7 0.516 0.949 7 CFH Complement factor H 0.642 0.512 0.808 23 SPTBN1 Spectrin beta chain. brain 1 0.635 0.505 0.797 34 HIST1H2BL Histone H2B type 1-L 0.669 0.501 0.911 8 ITGB4 Integrin beta-4 0.661 0.498 0.875 17 KRT10 Keratin. type I cytoskeletal 10 0.676 0.493 0.934 14 RAB1B Ras-related protein Rab-1B 0.674 0.483 0.941 6 SELENBP1 Selenium-binding protein 1 0.639 0.479 0.853 12 NA Ras-related protein Rap-1b-like protein 0.67 0.471 0.959 2 KIAA1967 Protein KIAA1967 0.664 0.47 0.941 7 CAD CAD protein 0.666 0.467 0.966 12 MAGOHB Protein mago nashi homolog 2 0.669 0.463 0.973 4 APCS Serum amyloid P-component 0.659 0.461 0.941 6 UTRN Utrophin 0.677 0.46 0.987 6 NES Nestin 0.589 0.457 0.753 14 APOB Apolipoprotein B-100 0.603 0.45 0.808 19 ASPN Asporin 0.657 0.435 0.976 16 CTNNB1 Catenin beta-1 0.602 0.427 0.852 10 LAMA5 Laminin subunit alpha-5 0.605 0.424 0.87 7 HRG Histidine-rich glycoprotein 0.637 0.424 0.977 11 S100A10 Protein S100-A10 0.592 0.423 0.818 7 TST Thiosulfate sulfurtransferase 0.622 0.422 0.926 8 CNN1 Calponin-1 0.636 0.413 0.983 3 CES1 Liver carboxylesterase 1 0.625 0.409 0.943 6 IGLC6 Ig lambda-6 chain C region 0.61 0.404 0.899 2 LAMB2 Laminin subunit beta-2 0.562 0.403 0.79 6 LRG1 Leucine-rich alpha-2-glycoprotein 0.619 0.402 0.935 3 EIF2C1 Protein argonaute-1 0.609 0.401 0.93 2 MYO1C Myosin-Ic 0.553 0.4 0.767 18 F13A1 Coagulation factor XIII A chain 0.542 0.398 0.737 11 APOH Beta-2-glycoprotein 1 0.59 0.398 0.871 9 CAST Calpastatin 0.607 0.396 0.935 5 SLC4A1 Band 3 anion transport protein 0.618 0.395 0.964 5 SUCLG2 Succinyl-CoA ligase [GDP-forming] subunit beta. 0.62 0.395 0.965 4 mitochondrial F2 Prothrombin 0.512 0.391 0.667 17 USP11 Ubiquitin carboxyl-terminal hydrolase 11 0.597 0.391 0.906 3 AMBP Protein AMBP 0.555 0.388 0.801 6 NID1 Nidogen-1 0.559 0.387 0.797 9 CCDC22 Coiled-coil domain-containing protein 22 0.618 0.387 0.975 4 DDX60 Probable ATP-dependent RNA helicase DDX60 0.617 0.382 0.976 2 HDAC1 Histone deacetylase 1 0.584 0.378 0.905 5 FABP4 Fatty acid-binding protein. adipocyte 0.602 0.374 0.972 7 ALG5 Dolichyl-phosphate beta-glucosyltransferase 0.604 0.369 0.976 3 LMO7 LIM domain only protein 7 0.568 0.368 0.877 6 HPDL 4-hydroxyphenylpyruvate dioxygenase-like protein 0.595 0.364 0.966 5 CAMK2B Calcium/calmodulin-dependent protein kinase type II 0.596 0.364 0.981 2 subunit beta KRT15 Keratin. type I cytoskeletal 15 0.558 0.363 0.855 8 GSTM3 Glutathione S-transferase Mu 3 0.593 0.362 0.963 3 SERPING1 Plasma protease C1 inhibitor 0.525 0.359 0.766 7 PCOLCE Procollagen C-endopeptidase enhancer 1 0.551 0.355 0.867 7 SERPINA4 Kallistatin 0.562 0.355 0.885 4 SERPINF1 Pigment epithelium-derived factor 0.513 0.353 0.735 8 OLFML3 Olfactomedin-like 3 0.537 0.35 0.819 4 CP Ceruloplasmin 0.473 0.346 0.643 13 ACTB Actin. cytoplasmic 1 0.481 0.343 0.675 8 WDR36 WD repeat-containing protein 36 0.565 0.336 0.947 2 METTL7B Methyltransferase-like protein 7B 0.567 0.335 0.952 3 LBP Lipopolysaccharide-binding protein 0.57 0.333 0.978 3 MRPL13 39S ribosomal protein L13. mitochondrial 0.559 0.332 0.928 2 S100B Protein S100-B 0.562 0.331 0.95 2 APOA1 Apolipoprotein A-I 0.422 0.327 0.549 23 TINAGL1 Tubulointerstitial nephritis antigen-like 0.492 0.324 0.756 5 ACOT2 Acyl-coenzyme A thioesterase 2. mitochondrial 0.518 0.324 0.818 3 GNB1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) 0.553 0.324 0.927 2 subunit beta-1 PCCB Propionyl-CoA carboxylase beta chain. mitochondrial 0.558 0.324 0.95 2 CTSG Cathepsin G 0.522 0.323 0.86 3 CSNK1A1 Casein kinase I isoform alpha 0.561 0.322 0.963 2 CFD Complement factor D 0.554 0.317 0.951 2 SAP30BP SAP30-binding protein 0.567 0.317 0.996 2 KNG1 Kininogen-1 0.508 0.307 0.857 10 APOA2 Apolipoprotein A-II 0.5 0.294 0.853 3 UAP1 UDP-N-acetylhexosamine pyrophosphorylase 0.435 0.288 0.654 4 PLS1 Plastin-1 0.517 0.285 0.911 2 ARF3 ADP-ribosylation factor 3 0.483 0.284 0.831 3 MYO1G Myosin-Ig 0.519 0.281 0.937 3 AFM Afamin 0.482 0.276 0.838 5 RAB5C Ras-related protein Rab-5C 0.418 0.275 0.632 4 APOA4 Apolipoprotein A-IV 0.387 0.271 0.55 9 PTRF Polymerase I and transcript release factor 0.415 0.269 0.645 6 CPB1 Carboxypeptidase B 0.391 0.268 0.571 5 CD36 Platelet glycoprotein 4 0.476 0.267 0.841 2 ORM1 Alpha-1-acid glycoprotein 1 0.405 0.265 0.625 7 SFRP1 Secreted frizzled-related protein 1 0.418 0.254 0.676 3 CLEC3B Tetranectin 0.434 0.251 0.75 3 TUBGCP2 Gamma-tubulin complex component 2 0.497 0.25 0.976 2 EHD2 EH domain-containing protein 2 0.352 0.247 0.506 9 HLA-C HLA class I histocompatibility antigen. Cw-15 alpha 0.387 0.247 0.601 5 chain PLIN4 Perilipin-4 0.45 0.244 0.84 3 GC Vitamin D-binding protein 0.436 0.234 0.838 11 AGR2 Anterior gradient protein 2 homolog 0.385 0.231 0.641 6 CMA1 Chymase 0.312 0.221 0.446 8 PIGR Polymeric immunoglobulin receptor 0.339 0.218 0.531 6 PLIN1 Perilipin-1 0.343 0.218 0.543 8 OLFML1 Olfactomedin-like 1 0.399 0.217 0.739 2 ATL2 Atlastin-2 0.455 0.215 0.89 2 ABI3BP Target of Nesh-SH3 0.404 0.212 0.75 3 PRELP Prolargin 0.32 0.192 0.57 16 HP Haptoglobin 0.275 0.191 0.396 16 CPA3 Mast cell carboxypeptidase A 0.309 0.189 0.507 4 MYH11 Myosin-11 0.262 0.179 0.382 13 TNXB Tenascin-X 0.301 0.179 0.497 6 HIST2H2AC Histone H2A type 2-C 0.259 0.177 0.376 4 LTF Lactotransferrin 0.243 0.164 0.359 41 AOC3 Membrane primary amine oxidase 0.232 0.161 0.328 11 DPT Dermatopontin 0.253 0.161 0.397 7 TTR Transthyretin 0.271 0.159 0.466 6 ORM2 Alpha-1-acid glycoprotein 2 0.276 0.154 0.505 3 TPSAB1 Tryptase alpha-1 0.284 0.153 0.52 2 HIST3H2A Histone H2A type 3 0.241 0.149 0.386 2 AZGP1 Zinc-alpha-2-glycoprotein 0.192 0.133 0.276 15 SOD3 Extracellular superoxide dismutase [CuZn] 0.227 0.132 0.391 3 COL6A6 Collagen alpha-6(VI) chain 0.257 0.132 0.483 9 IGHA2 Ig alpha-2 chain C region 0.23 0.128 0.415 8 MFAP4 Microfibril-associated glycoprotein 4 0.231 0.121 0.433 2 OGN Mimecan 0.135 0.102 0.178 15 IGHA1 Ig alpha-1 chain C region 0.112 0.081 0.152 14 ELN Elastin 0.141 0.08 0.248 4 APOD Apolipoprotein D 0.107 0.075 0.152 10 DCN Decorin 0.103 0.07 0.153 19 LGALS4 Galectin-4 0.106 0.059 0.191 3 IGJ Immunoglobulin J chain 0.122 0.049 0.291 2 PIP Prolactin-inducible protein 0.077 0.039 0.152 5 DES Desmin 0.03 0.017 0.051 3 ARHGAP1 Rho GTPase-activating protein 1 1.289 1.002 1.716 8
(48) TABLE-US-00013 TABLE 7B Gene Ontology (GO) molecular function - recurrence of triple-negative breast cancer Molecular functions pValue Ratio 1 binding 9.410e26 324/13778 2 protein binding 5.514e25 249/8829 3 MHC class I receptor activity 3.277e17 14/31 4 glycosaminoglycan binding 4.145e13 24/214 5 polysaccharide binding 6.496e13 25/239 6 pattern binding 6.496e13 25/239 7 carbohydrate binding 1.109e12 36/514 8 heparin binding 1.190e12 21/166 9 structural molecule activity 8.422e12 41/699 10 small molecule binding 1.884e11 95/2773 11 catalytic activity 2.085e11 164/6069 12 oxidoreductase activity, acting on the 3.350e11 18/139 CHOH group of donors, NAD or NADP as acceptor 13 oxidoreductase activity, acting on 1.883e10 18/154 CHOH group of donors 14 receptor binding 3.434e10 61/1506 15 nucleotide binding 4.193e9 84/2573 16 nucleoside phosphate binding 4.270e9 84/2574 17 collagen binding 6.695e9 11/61 18 isomerase activity 1.890e8 16/160 19 hydrolase activity 1.140e7 82/2676 20 GTPase activity 1.211e7 19/257 21 actin binding 2.305e7 23/381 22 superoxide dismutase activity 4.297e7 04/05 23 oxidoreductase activity, acting on 4.297e7 04/05 superoxide radicals as acceptor 24 oxidoreductase activity 5.270e7 36/839 25 GTP binding 7.314e7 23/407 26 structural constituent of cytoskeleton 8.242e7 11/96 27 guanyl nucleotide binding 1.416e6 23/423 28 guanyl ribonucleotide binding 1.416e6 23/423 29 pyrophosphatase activity 1.521e6 36/878 30 hydrolase activity, acting on acid 1.645e6 36/881 anhydrides, in phosphorus-containing anhydrides 31 hydrolase activity, acting on acid 1.733e6 36/883 anhydrides 32 purine ribonucleoside triphosphate binding 2.038e6 62/1960 33 calmodulin binding 2.914e6 14/179 34 purine ribonucleotide binding 3.624e6 62/1995 35 ribonucleotide binding 3.683e6 62/1996 36 purine nucleotide binding 4.683e6 62/2011 37 cytoskeletal protein binding 5.434e6 30/703 38 monocarboxylic acid binding 5.647e6 09/75 39 coenzyme binding 6.287e6 15/218 40 high-density lipoprotein particle binding 1.025e5 04/09 41 nucleoside-triphosphatase activity 1.032e5 33/840 42 extracellular matrix binding 1.108e5 07/45 43 cofactor binding 2.797e5 17/308 44 intramolecular oxidoreductase activity 2.944e5 07/52 45 phosphatidylcholine-sterol 4.962e5 03/05 O-acyltransferase activator activity 46 steroid binding 4.998e5 09/98 47 carboxylic acid binding 7.754e5 13/210 48 lipoprotein particle binding 8.777e5 05/27 49 protein-lipid complex binding 8.777e5 05/27 50 cholesterol transporter activity 1.023e4 04/15
(49) TABLE-US-00014 TABLE 7C Gene Ontology (GO) process - recurrence of triple-negative breast cancer Networks pValue Ratio 1 Cytoskeleton_Intermediate filaments 9.234e12 19/81 2 Cytoskeleton_Regulation of cytoskeleton 1.330e7 22/183 rearrangement 3 Cell adhesion_Integrin-mediated cell-matrix 7.312e6 21/214 adhesion 4 Immune response_Phagosome in antigen 1.609e5 22/243 presentation 5 Cytoskeleton_Actin filaments 1.919e5 18/176 6 Cell cycle_Mitosis 2.418e5 18/179 7 Cell adhesion_Cell-matrix interactions 2.075e4 18/211 8 Immune response_Antigen presentation 2.719e4 17/197 9 Cytoskeleton_Spindle microtubules 9.227e4 11/109 10 Cell adhesion_Cell junctions 9.275e4 14/162
(50) TABLE-US-00015 TABLE 7D Enrichment by protein function - recurrence of triple-negative breast cancer Protein In function Protein In data protein in class Actual n R N Expected Ratio p-value z-score set function database Ligands 26 382 514 23844 8.235 3.157 2.918E07 6.309 6.81% 5.06% 2.16% Phosphatases 8 382 230 23844 3.685 2.171 3.259E02 2.277 2.09% 3.48% 0.96% Proteases 17 382 559 23844 8.956 1.898 9.214E03 2.742 4.45% 3.04% 2.34% Enzymes 80 382 2724 23844 43.64 1.833 5.155E08 5.895 20.94% 2.94% 11.42% Kinases 12 382 654 23844 10.48 1.145 3.572E01 0.4808 3.14% 1.83% 2.74% Receptors 22 382 1565 23844 25.07 0.8775 3.034E01 0.6399 5.76% 1.41% 6.56% Transcription 3 382 959 23844 15.36 0.1953 1.170E04 3.246 0.79% 0.31% 4.02% factors Other 215 382 16691 23844 267.4 0.804 7.684E09 5.898 56.28% 1.29% 70.00%
(51) For the no relapse group, 189 proteins were significantly differentially expressed: 98 were overexpressed and 91 were underexpressed (Table 8A). For this group, the best score for the Process Network was obtained for Cell adhesion_Integrin-mediated cell-matrix adhesion (p=7.5 10.sup.11) (Table 8C). The protein class ligands was found to have to best z-score in the module Enrichment for Protein Function with 15 proteins (Table 8D).
(52) TABLE-US-00016 TABLE 8A Proteins underexpressed and overexpressed in non-recurrence of triple- negative breast cancer Confidence intervals Peptide Gene Protein Mean min max number APOD Apolipoprotein D 0.049 0.03 0.08 10 PIP Prolactin-inducible protein 0.069 0.016 0.224 5 IGHA2 Ig alpha-2 chain C region 0.07 0.044 0.112 8 COL1A1 Collagen alpha-1(I) chain 0.087 0.054 0.128 21 IGJ Immunoglobulin J chain 0.087 0.037 0.201 2 AZGP1 Zinc-alpha-2-glycoprotein 0.1 0.068 0.147 15 DCN Decorin 0.155 0.096 0.269 19 OGN Mimecan 0.16 0.118 0.214 11 TF Serotransferrin 0.165 0.132 0.206 41 LTF Lactotransferrin 0.18 0.088 0.373 36 COL6A6 Collagen alpha-6(VI) chain 0.187 0.078 0.42 3 HMGCS2 Hydroxymethylglutaryl-CoA synthase. mitochondrial 0.215 0.119 0.391 4 DES Desmin 0.22 0.111 0.428 2 ADH1C Alcohol dehydrogenase 1C 0.299 0.12 0.681 2 AKR1C1 Aldo-keto reductase family 1 member C1 0.306 0.164 0.565 2 TTR Transthyretin 0.327 0.222 0.484 6 DPT Dermatopontin 0.333 0.2 0.562 4 OLFML3 Olfactomedin-like protein 3 0.35 0.197 0.617 4 DHRS2 Dehydrogenase/reductase SDR family member 2 0.353 0.186 0.668 7 HIST3H2A Histone H2A type 3 0.359 0.242 0.529 2 PLIN1 Perilipin-1 0.361 0.217 0.585 7 CLEC3B Tetranectin 0.38 0.173 0.804 2 BCCIP BRCA2 and CDKN1A-interacting protein 0.381 0.209 0.678 2 IGHG1 Ig gamma-1 chain C region 0.398 0.275 0.571 12 LBP Lipopolysaccharide-binding protein 0.407 0.24 0.686 3 HPGD 15-hydroxyprostaglandin dehydrogenase [NAD+] 0.41 0.251 0.654 5 OAT Ornithine aminotransferase. mitochondrial 0.416 0.301 0.565 20 CFD Complement factor D 0.417 0.218 0.781 2 HP Haptoglobin 0.421 0.302 0.58 13 HPX Hemopexin 0.421 0.214 0.912 16 GC Vitamin D-binding protein 0.423 0.291 0.605 9 LRG1 Leucine-rich alpha-2-glycoprotein 0.426 0.247 0.723 3 SERPINA4 Kallistatin 0.435 0.234 0.792 4 AOC3 Membrane primary amine oxidase 0.436 0.3 0.633 10 APOA4 Apolipoprotein A-IV 0.438 0.256 0.754 6 PTRF Polymerase I and transcript release factor 0.443 0.26 0.738 5 ALOX15B Arachidonate 15-lipoxygenase B 0.448 0.285 0.702 10 MYH11 Myosin-11 0.457 0.343 0.61 13 MFAP4 Microfibril-associated glycoprotein 4 0.459 0.259 0.807 2 ORM1 Alpha-1-acid glycoprotein 1 0.46 0.26 0.8 4 IDI1 Isopentenyl-diphosphate Delta-isomerase 1 0.463 0.263 0.798 5 SFRP1 Secreted frizzled-related protein 1 0.464 0.311 0.699 3 NA Ig kappa chain V-II region GM607 (Fragment) 0.465 0.267 0.803 2 MCCC1 Methylcrotonoyl-CoA carboxylase subunit alpha. 0.465 0.228 0.917 2 mitochondrial CP Ceruloplasmin 0.467 0.349 0.624 11 CYP2A6 Cytochrome P450 2A6 0.469 0.3 0.726 7 AHSG Alpha-2-HS-glycoprotein 0.471 0.191 0.997 4 AACS Acetoacetyl-CoA synthetase 0.474 0.235 0.916 2 AMBP Protein AMBP 0.475 0.334 0.674 5 CMA1 Chymase 0.48 0.344 0.679 6 IGLC6 Ig lambda-6 chain C region 0.482 0.333 0.697 2 MCCC2 Methylcrotonoyl-CoA carboxylase beta chain. 0.482 0.322 0.712 11 mitochondrial BGN Biglycan 0.483 0.365 0.631 19 UROS Uroporphyrinogen-III synthase 0.483 0.281 0.808 3 AKR1B15 Putative aldo-keto reductase family 1 member B15 0.487 0.308 0.773 3 HAAO 3-hydroxyanthranilate 3.4-dioxygenase 0.496 0.287 0.846 2 ASPN Asporin 0.497 0.364 0.672 15 IGHM Ig mu chain C region 0.498 0.369 0.682 15 PIGR Polymeric immunoglobulin receptor 0.505 0.307 0.81 5 HBA1 Hemoglobin subunit alpha 0.51 0.29 0.881 10 F2 Prothrombin 0.516 0.397 0.673 14 TNXB Tenascin-X 0.519 0.309 0.897 6 SDCBP Syntenin-1 0.522 0.337 0.812 5 ACSS3 Acyl-CoA synthetase short-chain family member 3. 0.526 0.285 0.961 2 mitochondrial LPP Lipoma-preferred partner 0.529 0.355 0.787 10 SOD3 Extracellular superoxide dismutase [CuZn] 0.54 0.314 0.886 3 OPLAH 5-oxoprolinase 0.546 0.332 0.923 13 DNAJA3 DnaJ homolog subfamily A member 3. 0.548 0.325 0.908 4 mitochondrial APOH Beta-2-glycoprotein 1 0.555 0.323 0.948 5 PCOLCE Procollagen C-endopeptidase enhancer 1 0.556 0.36 0.848 6 ALDH6A1 Methylmalonate-semialdehyde dehydrogenase 0.559 0.356 0.862 7 [acylating]. mitochondrial SLC9A3R1 Na(+)/H(+) exchange regulatory cofactor NHE-RF1 0.567 0.368 0.865 4 TWF1 Twinfilin-1 0.579 0.375 0.886 4 CTBP2 C-terminal-binding protein 2 0.589 0.362 0.954 3 CYB5A Cytochrome b5 0.594 0.397 0.879 4 AHNAK Neuroblast differentiation-associated protein 0.597 0.48 0.739 61 AHNAK PLG Plasminogen 0.6 0.409 0.875 12 CLU Clusterin 0.605 0.384 0.973 8 ERLIN2 Erlin-2 0.607 0.385 0.952 4 EFEMP1 EGF-containing fibulin-like extracellular matrix 0.608 0.391 0.937 3 protein 1 ERLIN2 Erlin-2 0.609 0.383 0.959 4 KRT1 Keratin. type II cytoskeletal 1 0.612 0.408 0.955 13 SERPING1 Plasma protease C1 inhibitor 0.616 0.397 0.961 6 HIST1H2AC Histone H2A type 1-C 0.619 0.415 0.926 2 ASS1 Argininosuccinate synthase 0.619 0.388 0.981 4 HIST2H2AC Histone H2A type 2-C 0.62 0.429 0.898 4 SCP2 Non-specific lipid-transfer protein 0.643 0.467 0.888 10 HIST1H2BK Histone H2B type 1-K 0.646 0.466 0.874 6 MGST1 Microsomal glutathione S-transferase 1 0.648 0.421 0.999 4 C4BPA C4b-binding protein alpha chain 0.669 0.448 0.997 4 SELENBP1 Selenium-binding protein 1 0.765 0.6 0.976 11 ADAR Double-stranded RNA-specific adenosine 1.324 1.002 1.738 11 deaminase CHD4 Chromodomain-helicase-DNA-binding protein 4 1.344 1.029 1.757 11 SAMHD1 SAM domain and HD domain-containing protein 1 1.347 1.055 1.713 13 PSME1 Proteasome activator complex subunit 1 1.347 1.022 1.786 9 NONO Non-POU domain-containing octamer-binding 1.366 1.073 1.731 19 protein DDX5 Probable ATP-dependent RNA helicase DDX5 1.372 1 1.864 7 NCL Nucleolin 1.376 1.019 1.891 19 GRB2 Growth factor receptor-bound protein 2 1.409 1.036 1.941 8 CCT2 T-complex protein 1 subunit beta 1.416 1.064 1.877 13 HNRNPA2B1 Heterogeneous nuclear ribonucleoproteins A2/B1 1.441 1.024 2.14 21 FBL rRNA 2-O-methyltransferase fibrillarin 1.444 1.047 1.99 9 PRKAR2A cAMP-dependent protein kinase type II-alpha 1.445 1.043 2.019 6 regulatory subunit TRIM28 Transcription intermediary factor 1-beta 1.449 1.13 1.857 17 DYNLL1 Dynein light chain 1. cytoplasmic 1.46 1.042 2.066 4 HK1 Hexokinase-1 1.463 1.002 2.228 12 DNM2 Dynamin-2 1.481 1.071 2.035 9 STIP1 Stress-induced-phosphoprotein 1 1.49 1.076 2.102 11 S100A8 Protein S100-A8 1.5 1.106 2.024 9 SNRPB Small nuclear ribonucleoprotein-associated proteins 1.505 1.071 2.146 10 B and B EZR Ezrin 1.522 1.05 2.208 9 P4HB Protein disulfide-isomerase 1.522 1.033 2.274 28 ATP6V1A V-type proton ATPase catalytic subunit A 1.529 1.073 2.203 9 HSP90B1 Endoplasmin 1.53 1.212 1.954 32 CALR Calreticulin 1.533 1.168 2.03 18 HSP90B1 Endoplasmin 1.537 1.213 1.979 32 PAICS Multifunctional protein ADE2 1.538 1.108 2.14 10 MAP2K2 Dual specificity mitogen-activated protein kinase 1.539 1.031 2.33 2 kinase 2 H2AFY Core histone macro-H2A.1 1.542 1.078 2.199 10 DEK Protein DEK 1.545 1.13 2.127 8 SET Protein SET 1.55 1.099 2.189 6 ACTR3 Actin-related protein 3 1.552 1.129 2.13 12 ACTG1 Actin. cytoplasmic 2 1.559 1.103 2.197 6 GAA Lysosomal alpha-glucosidase 1.568 1.078 2.297 10 LCP1 Plastin-2 1.569 1.126 2.193 20 XRCC5 X-ray repair cross-complementing protein 5 1.583 1.103 2.286 16 SSRP1 FACT complex subunit SSRP1 1.588 1.067 2.356 8 KRT14 Keratin. type I cytoskeletal 14 1.588 1.011 2.549 5 HSPE1 10 kDa heat shock protein. mitochondrial 1.591 1.083 2.363 6 TNC Tenascin 1.609 1.114 2.496 38 EIF2S3 Eukaryotic translation initiation factor 2 subunit 3 1.61 1.127 2.307 3 WARS Tryptophanyl-tRNA synthetase. cytoplasmic 1.615 1.001 2.598 9 HSPA5 78 kDa glucose-regulated protein 1.623 1.177 2.298 30 SSR1 Translocon-associated protein subunit alpha 1.626 1.016 2.583 3 CAMK2D Calcium/calmodulin-dependent protein kinase type 1.631 1.07 2.574 3 II subunit delta PPIA Peptidyl-prolyl cis-trans isomerase A 1.633 1.012 2.56 14 MRPL19 39S ribosomal protein L19. mitochondrial 1.635 1.087 2.459 4 ERH Enhancer of rudimentary homolog 1.642 1.01 2.699 2 CALU Calumenin 1.649 1.133 2.418 7 LRRC59 Leucine-rich repeat-containing protein 59 1.656 1.14 2.419 7 ERH Enhancer of rudimentary homolog 1.657 1.017 2.724 2 PAPSS1 Bifunctional 3-phosphoadenosine 5- 1.677 1.113 2.551 13 phosphosulfate synthase 1 CFL1 Cofilin-1 1.681 1.054 2.543 17 MSN Moesin 1.695 1.349 2.133 32 ISYNA1 Inositol-3-phosphate synthase 1 1.698 1.016 2.853 3 RBMX Heterogeneous nuclear ribonucleoprotein G 1.706 1.08 2.706 6 MAT2B Methionine adenosyltransferase 2 subunit beta 1.711 1.002 2.923 3 AKR7A3 Aflatoxin B1 aldehyde reductase member 3 1.711 1 2.994 2 RAN GTP-binding nuclear protein Ran 1.722 1.3 2.29 9 ARF1 ADP-ribosylation factor 1 1.722 1.176 2.553 5 NME2 Nucleoside diphosphate kinase B 1.725 1.191 2.515 9 RPS27A Ubiquitin-40S ribosomal protein S27a 1.733 1.192 2.533 4 TUBB Tubulin beta chain 1.758 1.118 2.706 15 SLC2A1 Solute carrier family 2. facilitated glucose 1.782 1.081 3.003 3 transporter member 1 SMARCA5 SWI/SNF-related matrix-associated actin- 1.796 1.164 2.832 4 dependent regulator of chromatin subfamily A member 5 YBX1 Nuclease-sensitive element-binding protein 1 1.797 1.176 2.765 6 PAK2 Serine/threonine-protein kinase PAK 2 1.804 1.079 3.009 2 CTNNBL1 Beta-catenin-like protein 1 1.834 1.054 3.211 4 MACF1 Microtubule-actin cross-linking factor 1. isoforms 1.838 1.109 3.039 3 1/2/3/5 USP15 Ubiquitin carboxyl-terminal hydrolase 15 1.853 1.263 2.737 4 RPS9 40S ribosomal protein S9 1.862 1.195 3.145 14 AP1B1 AP-1 complex subunit beta-1 1.877 1.2 2.947 10 CPNE3 Copine-3 1.914 1.221 3.052 4 EIF4A1 Eukaryotic initiation factor 4A-I 1.921 1.446 2.559 13 EIF4A1 Eukaryotic initiation factor 4A-I 1.927 1.457 2.614 13 TUBA1B Tubulin alpha-1B chain 1.929 1.063 3.529 2 CPNE1 Copine-1 1.932 1.214 3.123 4 MYL12B Myosin regulatory light chain 12B 1.943 1.43 2.651 7 MAN2B1 Lysosomal alpha-mannosidase 1.952 1.22 3.123 5 MAPRE1 Microtubule-associated protein RP/EB family 1.973 1.217 3.188 3 member 1 CSDE1 Cold shock domain-containing protein E1 1.975 1.057 3.613 4 SOD2 Superoxide dismutase [Mn]. mitochondrial 1.991 1.332 2.986 5 GSTM1 Glutathione S-transferase Mu 1 1.993 1.1 3.682 2 MRPL9 39S ribosomal protein L9. mitochondrial 1.994 1.163 3.469 3 AARS Alanyl-tRNA synthetase. cytoplasmic 2.001 1.33 3.056 9 KRT6A Keratin. type II cytoskeletal 6A 2.004 1.09 3.811 3 KRT15 Keratin. type I cytoskeletal 15 2.049 1.271 3.401 8 KDM1A Lysine-specific histone demethylase 1A 2.069 1.065 3.93 2 LSP1 Lymphocyte-specific protein 1 2.077 1.047 4.086 2 TPM1 Tropomyosin alpha-1 chain 2.089 1.296 3.316 12 NME1 Nucleoside diphosphate kinase A 2.162 1.395 3.349 4 ACTN4 Alpha-actinin-4 2.47 1.088 4.765 22 AP1M1 AP-1 complex subunit mu-1 2.492 1.437 4.336 3 EEF1A1 Elongation factor 1-alpha 1 2.508 1.538 4.473 11 ACP2 Lysosomal acid phosphatase 2.543 1.451 4.436 3 ITGAM Integrin alpha-M 2.555 1.486 4.406 3 MAPK13 Mitogen-activated protein kinase 13 2.761 1.514 5.106 2 SSB Lupus La protein 2.884 1.705 4.803 5 AGR2 Anterior gradient protein 2 homolog 6.456 3.28 12.577 3
(53) TABLE-US-00017 TABLE 8B Gene Ontology (GO) molecular function - non-recurrence of triple-negative breast cancer Molecular functions pValue Ratio 1 protein binding 1.134e22 168/8829 2 binding 1.241e21 210/13778 3 small molecule binding 5.986e10 65/2773 4 nucleotide binding 2.893e8 58/2573 5 nucleoside phosphate binding 2.933e8 58/2574 6 oxidoreductase activity, acting on 3.779e8 12/139 the CHOH group of donors, NAD or NADP as acceptor 7 structural constituent of cytoskeleton 8.925e8 10/96 8 oxidoreductase activity, acting on 1.177e7 12/154 CHOH group of donors 9 structural molecule activity 6.414e7 24/699 10 catalytic activity 1.507e6 99/6069 11 purine ribonucleotide binding 3.651e6 44/1995 12 ribonucleotide binding 3.699e6 44/1996 13 aldo-keto reductase (NADP) activity 4.047e6 05/23 14 alditol:NADP+ 1-oxidoreductase activity 4.128e6 04/11 15 purine nucleotide binding 4.489e6 44/2011 16 purine ribonucleoside triphosphate binding 5.512e6 43/1960 17 cell surface binding 5.773e6 07/64 18 identical protein binding 1.006e5 27/992 19 enzyme binding 1.157e5 32/1302 20 oxidoreductase activity 1.449e5 24/839 21 actin binding 1.843e5 15/381 22 alcohol dehydrogenase (NADP+) activity 2.182e5 04/16 23 cytoskeletal protein binding 2.724e5 21/703 24 receptor binding 3.475e5 34/1506 25 heterocyclic compound binding 3.705e5 92/5912 26 organic cyclic compound binding 3.731e5 92/5913 27 adenyl ribonucleotide binding 6.522e5 35/1621 28 serine-type endopeptidase activity 7.581e5 10/203 29 adenyl nucleotide binding 7.650e5 35/1634 30 ATP binding 1.038e4 34/1592 31 methylcrotonoyl-CoA carboxylase activity 1.162e4 02/02 32 androsterone dehydrogenase (B-specific) 1.162e4 02/02 activity 33 glycosaminoglycan binding 1.173e4 10/214 34 unfolded protein binding 1.445e4 8/140 35 carbohydrate binding 1.586e4 16/514 36 serine-type peptidase activity 2.261e4 10/232 37 serine hydrolase activity 2.507e4 10/235 38 hydrolase activity 2.554e4 48/2676 39 polysaccharide binding 2.868e4 10/239 40 pattern binding 2.868e4 10/239 41 chaperone binding 3.159e4 05/55 42 nucleoside-triphosphatase activity 3.259e4 21/840 43 actin filament binding 4.356e4 06/90 44 heparin binding 4.590e4 8/166 45 pyrophosphatase activity 5.807e4 21/878 46 hydrolase activity, acting on acid 6.067e4 21/881 anhydrides, in phosphorus-containing anhydrides 47 hydrolase activity, acting on acid 6.246e4 21/883 anhydrides 48 complement binding 6.284e4 03/16 49 ketosteroid monooxygenase activity 6.876e4 02/04 50 17-alpha,20-alpha-dihydroxypregn-4-en- 6.876e4 02/04 3-one dehydrogenase activity
(54) TABLE-US-00018 TABLE 8C Gene Ontology (GO) process - non-recurrence of triple-negative breast cancer Process networks pValue Ratio 1 Cell adhesion_Integrin-mediated 7.479e11 24/214 cell-matrix adhesion 2 Cytoskeleton_Intermediate filaments 3.098e9 14/81 3 Cytoskeleton_Regulation of cytoskeleton 5.148e9 20/183 rearrangement 4 Cytoskeleton_Actin filaments 4.775e7 17/176 5 Immune response_Phagosome in antigen 2.548e6 19/243 presentation 6 Immune response_Phagocytosis 1.175e5 17/222 7 Inflammation_Amphoterin signaling 7.625e5 11/118 8 Cell cycle_Mitosis 2.233e4 13/179 9 Inflammation_Kallikrein-kinin system 3.088e4 13/185 10 Inflammation_IL-6 signaling 3.807e4 10/119
(55) TABLE-US-00019 TABLE 8D Enrichment by protein function - non-recurrence of triple-negative breast cancer Percentage Protein In In function Protein data protein in class Actual n R N Expected Ratio p-value z-score set function database Ligands 15 235 514 23844 5.066 2.961 1.925E04 4.484 6.38% 2.92% 2.16% Enzymes 49 235 2724 23844 26.85 1.825 2.103E05 4.565 20.85% 1.80% 11.42% Kinases 10 235 654 23844 6.446 1.551 1.139E01 1.427 4.26% 1.53% 2.74% Phosphatases 3 235 230 23844 2.267 1.323 3.958E01 0.4918 1.28% 1.30% 0.96% Proteases 7 235 559 23844 5.509 1.271 3.141E01 0.6458 2.98% 1.25% 2.34% Receptors 5 235 1565 23844 15.42 0.3242 1.553E03 2.759 2.13% 0.32% 6.56% Transcription 3 235 959 23844 9.452 0.3174 1.365E02 2.153 1.28% 0.31% 4.02% factors Other 143 235 16691 23844 164.5 0.8693 1.631E03 3.076 60.85% 0.86% 70.00%
(56) 2.3. Classification Based on Relapse Status
(57) The inventors investigated if they could detect differences between relapse and no relapse groups in terms of protein levels in the triple-negative tumors by OPLS analysis. This analysis was performed on 549 proteins for which quantitative informative was available in all the tumors. The OPLS model, initially based on all 549 proteins, was optimized by stepwise removal of proteins with small VIP (Variable Importance in the Projection) value. This was performed until the model did not improve anymore as judged by the CV-ANOVA p-value, indicative of the probability that the model is the result of chance alone. The optimized OPLS model included 58 proteins (p=2.1 10.sup.15) (
(58) TABLE-US-00020 TABLE 9 Maps pValue Ratio No 1 Blood coagulation_Blood 4.471E06 4 39 relapse coagulation 2 Chemotaxis_Lipoxin inhibitory 3.141E04 3 46 action on fMLP-induced neutrophil chemotaxis relapse 1 Cytoskeleton remodeling_Keratin 7.957E07 4 36 filaments
(59) 2.4. Proteomic Signature of Relapse Group Triple-Negative Breast Tumors
(60) By combining protein lists obtained from the univariate (with iQuantitator) and the multivariate analyses (OPLS), two lists of proteins were generated that characterized the Relapse (9 proteins) and No Relapse (5 proteins) groups of triple negative breast tumors (Tables 10A and B). It is not possible to assign a significant pathway for the No-Relapse group with a FDR<0.05; on the other side, the pathway Cytoskeleton remodeling_Keratin filaments (p=1.9 10.sup.8) was found for the Relapse group, according our previous analyses for this group. Among these proteins, Thrombospondin-1 is known to be a secreted ligand. While the Desmoplakin is a plasma membrane protein, it has already been described as a protein detectable in serum (Lpez-Farr A. J. et al, 2012).
(61) TABLE-US-00021 TABLE 10A Proteins characterizing the no relapse for triple-negative breast cancer Biomarkers for iTRAQ TNBC no Relapse GO uniprot expression ratio WARS Tryptophanyl- mitochondria over-expressed 1.6 tRNA synthetase SAMHD1 SAM domain and nucleus over-expressed 1.4 HD domain- containing protein 1 HSPE1 10 kDa heat mitochondria over-expressed 1.6 shock protein IGHG1 Ig gamma-1 chain secreted under-expressed 0.4 C region HK1 Hexokinase-1 cytosol over-expressed 1.5
(62) TABLE-US-00022 TABLE 10B Proteins characterizing the relapse for triple-negative breast cancer iTRAQ Biomarkers for TNBC Relapse GO uniprot expression ratio DSP Desmoplakin plasma over-expressed 1.8 membrane ARHGAP1 Rho GTPase- cytosol over-expressed 1.3 activating protein 1 EPPK1 Epiplakin cytoplasm over-expressed 1.4 KRT19 Keratin type I cell over-expressed 2.1 cytoskeletal 19 periphery THBS1 Thrombos- secreted over-expressed 1.7 pondin-1 KRT8 Keratin type II keratin over-expressed 2.7 cytoskeletal 8 filament IDH2 Isocitrate mitochondria over-expressed 1.7 dehydrogenase [NADP] G6PD Glucose-6- cytoplasm over-expressed 1.7 phosphate 1-dehydro-genase DPYSL3 Dihydro- cytoplasm over-expressed 1.9 pyrimidinase- related protein 3
(63) 2.5. Pathways Analysis of Individual Tumor
(64) Pathway analysis was also performed on each of the 80 tumor samples separately. By this, the inventors obtained a fingerprint of affected pathways for each tumor. All proteins with a level significantly differing from the mean protein level among all Relapse group tumors or No-relapse group tumors were included in the individual tumor analysis; in total 1078 proteins. The relative intensities of these tumor specific proteins were mapped to the pathway maps of the Metacore database. The analysis identified the pathways that were the most significant in each of the 80 individual tumors data sets, measured by Fisher's exact test. The inventors used the association ranks as input variables to the multivariate analysis for sample comparison, thus based on pathway enrichment. To detect pathway alterations connected to relapse risk, the inventors performed OLPLS analysis on the pathway association data. The OPLS model was optimized as described in the group analysis. The inventors performed stepwise removal of variables (i.e. pathways) with less influence on the separation performance of the model until the model did not improve anymore. The most significant pathway for No-relapse group is Glycolysis. The top-ranked pathways in relapse group were cytoskeleton Remodeling_Keratins-filaments and Gap junctions.
(65) 2.6. Validation of Dysregulated Protein Expression
(66) To proceed with the first steps in validating our MS analysis, the inventors confirmed the differential expression of two dysregulated proteins by Western-Blot analysis using samples from the same triple-negative breast tumors cohort. The proteins Thrombospondin-1 and Tryptophanyl-tRNA synthetase were selected for validation based on their potential significance in breast cancer tumorigenesis. The expression of Thrombospondin-1 was found to be elevated in primary breast tumors of the recurrent group when compared to the non-recurrent group tumors. Inversely, Tryptophanyl-tRNA synthetase was found to be elevated in the No Relapse group when compared to the Relapse group (
(67) 2.7. Pronostic Value of the Markers
(68) The prognostic value of the markers was evaluated through estimation of overall survival (OS) using Kaplan-Meier method. The patients were divided into two categories based on the median iTraq expression data for each marker: high versus low expression.
(69) For the No relapse group, patients' tumor with high expression levels of Tryptophanyl-tRNA synthetase (WARS), 10 kDa heat shock protein (HSPE1), SAM domain and HD domain-containing protein 1 (SAMHD1) and Hexokinase-1 (HK1) experienced a significantly better DFS (Disease Free Survival) compared with those with low expression (p=0.0017, p=0.0042, p=0.0073, p=0.0124 respectively), and inversely, patients' tumor with low expression levels of Ig gamma-1 chain C region (IGHG1) showed a significant better DFS compared with those with high expression (p=0.0339), in agreement with iTraq results (
(70) For the Relapse group, patients' tumors with high expression level of any of the 9 proteins experienced a significantly worse DFS compared with those with low expression (p<0.0001, p=0.0001, p=0.0005, p<0.0001, p=0.021, p=0.0051, p=0.020, p=0.0457 and p=0.031) for pourquoi Desmoplakin (DSP), Thrombospondin-1 (THBS1), Glucose-6-phosphate 1-dehydrogenase (G6PD), Isocitrate dehydrogenase [NADP] (IDH2), Keratin type I cytoskeletal 19 (KRT19), Keratin type II cytoskeletal 8 (KRT8), Epiplakin (EPPK1), Rho GTPase-activating protein 1 (ARHGAP1), and Dihydropyrimidinase-related protein 3 (DPYSL3), respectively (
(71) The
(72) 2.8. Preliminary Validation in Patient-Derived Serum Samples
(73) The inventors then questioned whether any of secreted protein could be measured in the serum of TNBC patients, notably in TNBC relapsing patients.
(74) The determination of the amount of Desmoplakin in the serum of the TNBC cohort corresponding to the triple-negative tumors studied in this paper, showed that Desmoplakin is more concentrated in the Relapse group (p=0.01) than in the No-Relapse group, according to the proteomic results disclosed herein (
(75) As very little serum samples were available in this cohort, the inventors extended the determination of Desmoplakin expression in a newer TNBC serum cohort. Based on this new cohort, the inventors demonstrated a significant overexpressed amount of Desmoplakin in TNBC serums compared with healthy controls (p=0.0008).
3. Discussion
(76) Despite the many recent advances in breast tumors treatments through targeted therapies, no specific treatment exists for the triple-negative breast tumors and there are no prognostic molecular markers that would predict whether a tumor will behave aggressively or remain indolent. It is abundantly clear that tumor biology plays a significant role in resultant tumor behaviour. Unfortunately, triple-negative breast primary tumors that are placed in the same prognostic category based on currently used parameters, may behave differently. It is the inventors' hypothesis that the underlying biology of these tumors and differences in its detail will determine a particular tumor's potential for aggressiveness. In addition, these biological differences can be used to identify novel molecular markers that may be useful for diagnostic, prognostic, or predictive purposes, the success of which would pave the road to a new era of personalized medicine in breast cancer.
(77) In this study, the inventors performed quantitative proteomic profiling of 80 triple-negative breast tumors to identify first differential protein expression between triple-negative breast tumors and normal tissues and second, to identify potential prognostic markers of recurrence. To the inventors' knowledge, this study represents the largest proteomic analysis of triple-negative breast tumors ever realized.
(78) From all the triple negative breast tumors, 219 proteins with significant differential expression in tumors compared to normal tissues were identified. Among these proteins, 58 proteins had previously been reported to be involved in breast tumors.
(79) Another aim of this study was to identify differential protein expression between no relapse patients' group and relapse patients' group. The inventors characterized 5 proteins associated with the no-relapse group (i.e. Hexokinase-1, 10 kDa heat shock protein, Ig gamma-1 chain C region, SAM domain and HD domain-containing protein 1, and Tryptophanyl-tRNA synthetase) and 9 proteins associated with the relapse group (i.e. Desmoplakin, Rho GTPase-activating protein 1, Epiplakin, Glucose-6-phosphate 1-dehydrogenase, Isocitrate dehydrogenase [NADP], Keratin type I cytoskeletal 19, Keratin type I cytoskeletal 8, Dihydropyrimidinase-related protein 3, and Thrombospondin-1). It is thus proposed herein that the above group of proteins is a protein signature of the no-relapse group and relapse group of triple negative breast tumors, respectively.
(80) Among these proteins, it was shown that Desmoplakin could be easily detected in serum of patients, and was also differentially expressed in Relapse group compared to No-Relapse group. As previously mentioned, this protein is a key component of desmosomes, and belong to the pathway cytoskeleton Remodeling_Keratins-filaments and Gap junctions found in Metacore analysis, that is in agreement with the fact that this pathway is the top-ranked pathway characterizing the Relapse group in our proteomic approach. Expanding the assays on a larger cohort sera of patients with breast cancer, the inventors demonstrated that this protein is differentially over-expressed in TNBC sera compared to healthy controls.
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