Proteomics-Based Identification of HSP60 as a Tumor-Associated Antigen in Early Stage Breast Cancer and Ductal Carcinoma in situ C. Desmetz,†,‡,§ F. Bibeau,| F. Boissie`re,| V. Bellet,§ P. Rouanet,⊥ T. Maudelonde,†,‡,§ A. Mange´,†,‡,§,# and J. Solassol*,†,‡,§,# Department of Cellular Biology, CHU Montpellier, Hoˆpital Arnaud de Villeneuve, Montpellier, France, University of Montpellier I, Montpellier, France, Department of Clinical Oncoproteomics, CRLC Val d’Aurelle, Montpellier, France, Department of Pathology, CRLC Val d’Aurelle, Montpellier, France, and Department of Surgery, CRLC Val d’Aurelle, Montpellier, France Received February 18, 2008
The detection of autoantibodies in cancer patients has been shown to constitute an excellent tool for early diagnosis. Because breast cancer still lacks early diagnostic markers, we investigated novel tumorassociated antigens and related autoantibodies in sera from patients with early stage breast cancer compared to autoimmune disease, other cancers, and healthy volunteers, using a proteomics-based approach. Among the 26 protein antigens specifically recognized by early stage breast cancer sera, we focused on Heat Shock Protein 60 (HSP60). Using ELISA, we investigated the frequency of autoantibodies directed against this protein in the sera of 240 individuals, comprising patients with either ductal carcinoma in situ (DCIS) (n ) 49) or early stage breast cancer (n ) 58), other cancers (n ) 20), autoimmune disease (n ) 20), and healthy subjects (n ) 93). Autoantibodies directed against HSP60 were present in 16/49 (31%) early stage breast cancer and 18/58 (32.6%) DCIS patients, compared to 4/93 (4.3%) healthy subjects. In particular, autoantibodies were present in 11/23 patients (47.8%) with high-grade DCIS, compared to 5/26 (19.2%) with low-grade DCIS. HSP60 mRNA levels were significantly higher in primary breast cancer compared to healthy breast tissues. Using immunohistochemistry, we found that HSP60 expression gradually increases from normal through DCIS to invasive tissues. Our results indicate that HSP60 autoantibodies may be of interest in terms of clinical utility for the early diagnosis of breast cancer and more particularly in DCIS. Moreover, HSP60 overexpression during the first steps of breast carcinogenesis may be functionally correlated to tumor growth and/or progression. Keywords: tumor-associated antigen • autoantibodies • serum marker • early breast cancer • HSP60
Introduction Early detection and diagnosis is of great importance in breast cancer management. The measurement of serum biomarkers has been the most widely used approach. Two such markers for breast cancer are CEA (carcinoembryonic antigen) and CA15-3.1 However, the lack of specificity and sensitivity of both these markers preclude their general use in breast cancer early diagnosis. It is therefore essential to identify clinically reliable biomarkers and to develop an effective approach for early diagnosis. The identification of tumor specific markers capable of eliciting an immune response early in tumor development could provide an effective strategy in this regard. At early stages of cancer, the amount of tumor antigens in tumor cells or in * To whom correspondence should be addressed. Dr. Je´roˆme Solassol, Baˆtiment de Recherche CRLC Val d’Aurelle, Parc Eurome´decine, 208 rue des Apothicaires, 34298 Montpellier Cedex 5, France. Tel: (33) 4 67 61 24 12. Fax: (33) 4 67 33 95 90. E-mail:
[email protected]. † Hoˆpital Arnaud de Villeneuve. ‡ University of Montpellier I. § Department of Clinical Oncoproteomic, CRLC Val d’Aurelle. | Department of Pathology, CRLC Val d’Aurelle. ⊥ Department of Surgery, CRLC Val d’Aurelle. # Both authors should be considered as last coauthors.
3830 Journal of Proteome Research 2008, 7, 3830–3837 Published on Web 08/07/2008
the circulation is usually too low for detection using classical methods.2 However, the humoral response to such antigens through the synthesis of autoantibodies generates a remarkable biological amplification that allows the detection of cancer many months to years prior to the clinical diagnosis of a tumor2 and subtle alterations in antigen proteins that might otherwise go undetected.3 Several approaches are currently available for the identification of tumor-associated antigens (TAAs). In contrast to SEREX, which allows the identification of TAAs based on the analysis of recombinant proteins, serological proteomics analysis (SERPA) permits the identification of autoantibodies to proteins as they occur in their natural state.4 This approach enables the detection of antigens associated with aberrant post-translational modifications of tumor cell proteins. Although the range of reported possible serological tumor markers linked to a humoral response is broad,4–18 only a few have been incorporated into routine oncological practice such as analyses of p53 autoantibodies,7 and none have proven valuable for the diagnosis of early stage breast cancer or breast ductal carcinoma in situ (DCIS). In this study, we screened the reactivity of early stage breast cancer sera against proteins from primary 10.1021/pr800130d CCC: $40.75
2008 American Chemical Society
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HSP60 Autoantibodies in Early Breast Cancer breast cancer tissues resolved by two-dimensional gel electrophoresis (2-DE) and analyzed by Western blotting. The reactivity of autoantibodies from early stage breast cancer sera was compared to that of sera from patients with autoimmune diseases or other cancers and healthy volunteers. Proteins specifically recognized by sera from early stage breast cancer were then identified by mass spectrometry. Among the identified antigens, the molecular chaperone HSP60, potentially implicated in carcinogenesis and tumor progression,19,20 elicited a specific immunoreactivity. We further report the presence of autoantibodies against HSP60 in 31 and 32.6% of early stage breast cancer and DCIS sera, respectively, compared to 4.3% in healthy controls. HSP60 was found upregulated at the mRNA, and protein levels in early stage breast cancer tissue compared to healthy breast tissue. These results demonstrate the potential usefulness of detecting HSP60 autoantibodies in the screening and diagnosis of early invasive and more importantly in situ breast cancers. Moreover, our data suggest that this protein may play a role in early breast carcinogenesis and could be validated as an immunotherapeutic target.
Materials and Methods Sera, Tissues, and Cell Lines. All human samples were collected prospectively between 2005 and 2007 in the cancer institute CRLC Val d’Aurelle, Montpellier, at the time of diagnosis after obtaining written informed consent. For 2-DE Western blot experiments, sera were obtained from patients with breast cancer (clinical stage I, lymph node negative, and grade I, n ) 20), other cancers (ovarian cancer, n ) 10; prostate cancer, n ) 10), autoimmune disease (rheumatoid arthritis, n ) 10; systemic lupus erythematosus, n ) 10), and healthy volunteers (n ) 20). Mean age of subjects who donated sera for this study was 55.4 years (range, 35-71 years). For ELISA experiments, sera were obtained from 49 patients with DCIS, 58 patients with early stage breast cancer of different histotypes, and 93 matched healthy controls exempted of any breast disease. Detailed clinicopathologic information of the DCIS and invasive breast cancer patients are listed in Table 1. DCIS and early stage breast cancer were classified according to the WHO classification Tumors and were graded according to Bloom and Richardson. For 2-DE experiments, two primary invasive breast tumor tissues (T3N0) were obtained during surgical resection and samples were immediately frozen at -80 °C and stored until use. For RT-PCR experiments, early invasive (T1N0, n ) 7, T1N1, n ) 7) and healthy (n ) 14) breast tissues were obtained during surgical resection. Samples were immediately frozen at -80 °C and stored until use. For immunohistochemistry (IHC) experiments, breast tumor tissues (n ) 10) were formol-fixed and paraffin-embedded. 2-DE Western Blot Analysis. All reagents and materials for 2-DE experiments were purchased from GE Healthcare (Uppsala, Sweden), unless otherwise indicated. Twenty micrometer thick sections from two breast tumors were lyzed (7 M urea, 2 M thiourea, 4% CHAPS, cocktail protease inhibitors-Roche), centrifuged at 15 000g for 15 min at 4 °C, and supernatants were pooled. Total proteins were then precipitated with acetone and centrifuged at 13 000g for 30 min. Proteins (150 µg) were solubilized in 700 µL isoelectrofocusing medium (urea, 7 M; thiourea, 2 M; CHAPS, 4%; ampholines, preblended, pI 3.5-9.5, 8 mg/ml; DTT, 100 mM; tergitol NP7, 0.2%; and traces of bromophenol blue). Sample loading onto 18 cm, pH 3-10 nonlinear, Immobiline DryStrips was performed by passive gel
Table 1. Clinicopathological Characteristics of Early-Invasive Breast Carcinomas and DCIS characteristics
Early stage breast cancer Median age, y (range), 64 (43-84) Histotype Invasive ductal carcinoma Invasive lobular carcinoma Histologic grade Grade 1 Grade 2 Grade 3 Tumor size T1 Lymph node status Negative ERR Negative Positive PgR Negative Positive Her-2 overexpression Negative Positive DCIS Median age, y (range), 60 (45-80) Histotype Low grade High grade
no. cases (%)
58
51 (87.9) 7 (12.1) 17 (29.4) 27 (46.5) 14 (24.1) 58 (100) 58 (100) 40 (69) 18 (31) 27 (46.5) 31 (53.5) 46 (79.3) 12 (20.7) 49
26 (53.1) 23 (47.9)
rehydratation. Isoelectrofocusing was performed for 50 000 V/h using the IPGPhor IEF System. After the first dimension, the IPG strips were equilibrated for 10 min in a buffer containing urea (6 M), Tris-HCl (50 mM, pH 6.8), glycerol (30%), SDS (2%), DTT (10 mg/ml), and bromophenol blue and then for 15 min in the same buffer containing 15 mg/ml iodoacetamide instead of DTT. For the second dimension, the strips were loaded onto vertical 10-17% SDS polyacrylamide gradient gels. The gels were silver stained according to the procedure of Shevshenko et al.21 Separated proteins were transferred onto a Hybond P polyvinylidene fluoride (PVDF) membrane (Millipore) for 1 h at 300 mA, using a Trans-Blot system (Bio-Rad), or visualized by silver staining. After transfer, PVDF membranes were incubated with blocking buffer (PBS 5% skimmed milk), and then with 4 pools of 5 sera from patients with early stage breast cancer or with 4 pools of 5 sera from patients of each control groups at a 1:200 dilution O/N at 4 °C. After 3 washings, membranes were reacted with horseradish peroxidase-conjugated goat antihuman IgG antibody (109-035-003, Jackson ImmunoResearch Laboratories) at a 1:3000 dilution for 1 h at RT. Membranes were washed 3 times and immunodetection was accomplished using Super Signal West Pico Chemiluminescent Substrate kit (Pierce), followed by autoradiography on Hyperfilm ECL (Amersham). All pool of sera was assayed in triplicate. A mouse anti-HSP60 monoclonal antibody (ab5479; Abcam) was used at 1:5000 dilution for Western blotting and was processed as for incubations with patient sera, with antirabbit immunoglobulin horseradish peroxidase-conjugated goat antimouse IgG (115-035-146, Jackson ImmunoResearch Laboratories) at a 1:3000 dilution. In-Gel Enzyme Digestion and Mass Spectrometry. Spots from 2-DE silver stained gels were excised and in-gel digested Journal of Proteome Research • Vol. 7, No. 9, 2008 3831
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Table 2. List of 38 Selected Reactive Protein Spots Subjected to MALDI-TOF MS Analysis spot no.
1 2 3 4 5,6 7 8 9 10 11 12 13
accession no.
P08107 P10809 P08670 Q02790 P30101 P05787 P61158 Q96GX7 P05783 P11177
14,15 16 17 18 19,20 21 22 23 24 25 26 27 28 29,30 31 32 33,34 35,36 37,38
P06733 Q06323 P07339 P52565 P32119 P15531 P29373 P04792 Q8WZ68 P30041 P60174 P18669 P00915 P30043 P05092 P61978
protein name
Undefined Heat shock 70 kDa protein 1 60 kDa Heat shock protein, mitochondrial precursor Vimentin FK506-binding protein 4 (peptidyl-prolyl cis-trans isomerase) Protein disulfide isomerase A3 precursor Keratin, type II cytoskeletal 8 (cytokeratin 8) Actin-like protein 3 (Actin-related protein 3) Similar to selenium binding protein 1 Keratin, type I cytoskeletal 18 (cytokeratin 18) Undefined Pyruvate dehydrogenase E1 component beta subunit, mitochondrial precursor Undefined Alpha enolase (Non neural enolase) Proteasome activator complex subunit 1 Cathepsin D precursor Undefined Rho GDP-dissociation inhibitor 1 (Rho GDI alpha) Peroxiredoxin 2 (thioredoxin peroxidase 1) Nucleoside diphosphatase kinase A (NDKA) Cellular retinoic acid-binding protein II CRABPII) Undefined Heat-shock protein beta-1 27 kDa Interferon alpha 1b Peroxiredoxin 6 (antioxidant protein 2) Triosephosphate isomerase (TIM) Phosphoglycerate mutase 1 Carbonic anhydrase I (carbonate dehydratase I) Flavin reductase (NAPDH-dependent diaphorase) Peptidyl-propyl cis-trans isomerase A (cyclophilin A) Heterogeneous nuclear ribonucleoprotein K (hnRNP K)
using trypsin (Gold, Promega), as previously described.22 Digest products were completely dehydrated in a vacuum centrifuge and resuspended in 10 mL formic acid (2%), desalted using ZipTips C18 (Millipore), eluted with 10 mL ACN-TFA (50-0.1%) and concentrated to 2 mL. Aliquots (0.5 mL) were mixed with
MW (kDa)
pI
score
70294 61187 53579 51926 57146 53510 47797 52928 47897
5.48 5.70 5.07 5.35 5.98 5.52 5.61 5.93 5.34
168 157 169 179/88 132 255 134 262 205
39536
6.2
79
47350 28876 45037
6.99 5.78 6.10
152 189 77
23250 22049 17309 15723
5.02 5.66 5.83 5.43
67 112 95 76
22836 19611 25002 26807 28769 28778 22088 18098 51230
5.98 5.17 6.02 6.51 6.75 6.63 7.31 7.82 5.39
87 64 144 113/130 63 63 94/94 175/133 91/90
the same volume of R-cyano-4-hydroxy-trans-cinnamic acid (Sigma, 10 mg/ml in ACN-TFA, 50-0.1%) and loaded on the target of an Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonik). Analyses were performed in reflectron mode with an accelerating voltage of 25 kV and a delayed extraction of 50
Figure 1. Screening of autoantibodies directed against TAAs in the sera of early stage breast cancer patients. The proteins from primary breast cancer were separated by 2-DE and silver stained (A) or transferred to a PVDF membrane and probed with the sera from early stage breast cancer patients (B), healthy controls (C), autoimmune diseases (D), and other cancers (E). The numbered circles indicate the spots specifically recognized by early stage breast cancer sera. Their identities are listed in Table 2. 3832
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HSP60 Autoantibodies in Early Breast Cancer
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Figure 2. Specific detection of HSP60. (A) Specific detection of TAAs using early stage breast cancer sera. Enlargements of the regions containing the HSP60 protein revealed with breast cancer, other cancers, autoimmune disease, and healthy volunteers sera are shown. An antihuman IgG was used as a secondary antibody. (B) Specific detection of HSP60 using anti-HSP60 antibody. Spot corresponding to HSP60 is indicated by black arrows.
ns. Spectra were analyzed using the Flex-Analysis software (version 2.4, Bruker Daltonik). Identification of proteins was performed using the MASCOT software (version 2.1, Matrixscience) against the Swiss-Prot or TrEMBL databases. The following parameters were used for database interrogation: mass tolerance of 50 ppm (even if the mass accuracy of our analyses was usually better than 20 ppm); fixed chemical modification, carbamidomethylation of cysteins; variable chemical modification, oxidation of methionines; matching peptides with one missed cleavage accepted only when they included two consecutive basic residues or when arginine or lysine residues were followed by one or several acidic residues inside the peptide amino acid sequence. MASCOT scores greater than 63 and 68 were considered significant (p < 0.01) for Swiss-Prot and TrEMBL database interrogation, respectively. Mass spectrometry analyses were performed by the proteomic platform of the Institute of Functional Genomics in Montpellier. Detection of HSP60 Autoantibodies by ELISA. HSP60 autoantibodies were detected in the sera of patients using the antihuman HSP60 (total) ELISA kit from Stressgen (Assays Designs, Inc., Ann Arbor, MI), following manufacturer’s recommendations. Sera were diluted 1/500. All samples were assayed in duplicate and the results were the mean value of the reading. The cutoff value of reactivity was defined as mean OD (absorbance at 450 m) of sample plus 2 folds of standard deviation (SD) of normal sera. Detection of HSP60 by ELISA. HSP60 was quantitated in the sera of patients using the HSP60 ELISA kit from Stressgen (Assays Designs, Inc., Ann Arbor, MI), following manufacturer’s recommendations. Sera were diluted 1/10. All samples were assayed in duplicate, and the results were the mean value of the reading. RNA Isolation, Reverse Transcription, and Real Time Quantitative PCR Analysis. Total RNA from 14 breast tumors and 14 healthy breast frozen tissues were extracted from 15
20-µm thick sections using RNeasy Mini kit (Qiagen). RNA integrity was verified using a 2100 bioanalyser (Agilent technologies). For first strand cDNA synthesis, 5 µg of total RNA, 200 ng of random hexamers, and 1 µL of dNTP mix (10 mM) were incubated 5 min at 65 °C in a total volume of 13 µL in RNase free water and then 4 µL of 5× first strand buffer, 1 µL of 1 M DTT, 40 UI of RNaseOUT. Recombinant Ribonuclease Inhibitor (Invitrogen) and 200 U of Superscript III (Invitrogen) were added to make a total volume of 20 µL. The mix was then incubated 5 min at 25 °C and 60 min at 60 °C, and the reaction was stopped at 70 °C during 15 min. Complementary DNAs were frozen at -20 °C until use. Real time quantitative PCR was performed using the LightCycler 2.0 system (Roche). PCR conditions were an initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing for 10 s (see below), and extension at 72 °C for 10 s. Data concerning HSP60 were normalized according to data from TBP89 and RS9 housekeeping genes. Sequences of forward and reverse primers for HSP60 (Tm 60 °C) were 5′-TGGTGACAATAGAAAGAACCAGCTT-3′ and 5′-GTCAATCCCTCTTCTCCAAACACT-3′, respectively. Sequences of forward and reverse primers for TBP89 (Tm 65 °C) were 5′-TTTTCTTGCTGCCAGTCTGGAC3′ and 5′-CACGAACCACGGCACTGATT-3′, respectively. Sequences of forward and reverse primers for RS9 (Tm 62 °C) were 5′-AAGGCCGCCCGGGAACTGCTGAC-3′ and 5′-ACCACCTGCTTGCGGACCCTGATA-3′, respectively. Immunohistochemistry Staining of HSP60. Four micrometer thick sections of breast tissue were deparaffinized with xylene and rehydrated with several graded ethanols before Hematoxylin-Eosin-Saffron staining or immunohistostaining. Immunohistochemical analyses were performed by using the Dako autostainer (Dako). Tissue sections were treated for 45 min at 95 °C with citrate (pH ) 6) for antigen retrieval. Slides were then incubated with a rabbit anti-HSP60 polyJournal of Proteome Research • Vol. 7, No. 9, 2008 3833
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clonal antibody (ab31115; Abcam) during 30 min at RT at a 1:2000 dilution. Slides were treated with a peroxidase inhibitor (Dako) for 10 min to quench the endogenous peroxidase activity. The detection of the antibody binding was visualized with a peroxidase-conjugated polymer backbone (Envision+ dual link, Dako) using diaminobenzidine as a chromogen. The sections were then counterstained with hematoxylin. Statistical Analysis. Statistical analysis was done using StatsDirect (Version 2.6.2) and GraphPad InStat (version 3.06) software. Quantitative PCR data were analyzed with the unpaired Mann-Whitney test. The level of statistical significance was set at the value of P < 0.05. Data in the text are presented as the mean ( SEM. ELISA results were analyzed using one sided Fischer’s exact test.
Results Breast Tumor Proteins Recognized Specifically by Early Stage Breast Cancer Sera. Sera obtained from 20 newly diagnosed patients with early stage breast cancer, 20 patients with autoimmune disease (rheumatoid arthritis and systemic lupus erythematosus), 20 patients with other cancers (prostate and ovary), and 20 matched healthy subjects were investigated for the presence of autoantibodies against breast tumor tissue proteins separated by 2-DE. Sera from patients with breast cancer reacted with numerous spots, some of which were observed in the control groups and thus considered representative of nonspecific reactivity (Figure 1). Thirty-eight spots were specifically recognized by sera from breast cancer patients but not by sera from the control groups. These spots were excised from the gel, trypsindigested, and subsequently analyzed by MALDI-TOF MS. We successfully identified 26 of these protein spots (Table 2) as those implicated in cellular metabolism (NDKA, triosephosphate isomerase, cathepsin D precursor, flavin reductase, proteasome activator complex subunit 1, pyruvate dehydrogenase E1, phosphoglycerate mutase 1, carbonic anhydrase I), cytoskeletal regulation (vimentin, cytokeratin 8, cytokeratin 18, HSP27), intracellular trafficking (cytokeratin 18, similar to selenium binding protein 1, FKBP4, HSP60, protein disulfide isomerase A3 precursor), signal transduction (hnRNPK, CRABPII, protein disulfide isomerase A3 precursor, Rho GDI alpha), apopotosis (HSP60, peroxiredoxin 2, HSP27, HSP70.1, RhoGDI alpha), and the folding of proteins (HSP60, HSP70.1, PPIA, FKBP4). Figure 1A shows the location of the 26 protein spots on the 2-DE silver-stained gel. In particular, we focused on the protein spot 3, which exhibited a strong reactivity with all breast cancer sera pools, though not with controls (Figure 2A). We identified this spot as the 60 kDa heat shock protein (HSP60), with a MASCOT score of 157, a 34% protein coverage, a molecular weight of 61.187 kDa, and a pI value of 5.70 (Figure 3 and Table 2). Identification of HSP60 was confirmed with 2-DE Western blotting using an anti-HSP60 mouse monoclonal antibody (Figure 2B). Frequency of HSP60 Autoantibodies in the Sera of DCIS, Early Stage Breast Cancer Patients, and Control Groups. We next performed ELISA experiments to determine the frequency of HSP60 autoantibodies in the sera of breast cancer patients (Table 3). We screened 240 prospectively obtained sera from DCIS patients (n ) 49), early stage breast cancer patients (n ) 58), healthy controls (n ) 93), other cancers (n ) 20), and autoimmune disease (n ) 20). The frequencies of HSP60 autoantibodies were 32.6% (16/49) and 31% (18/58) in DCIS and early stage breast cancer, respectively, compared to 4.3% 3834
Journal of Proteome Research • Vol. 7, No. 9, 2008
Figure 3. Mass spectrometry identification of HSP60. (A) Matrixassisted laser desorption ionization mass spectrum obtained from HSP60 after trypsin digestion. Marked numbers indicate the m/z for tryptic digests of HSP60. (B) Matched peaks were searched against the nonredundant Swiss-Prot and TrEMBL database interrogation. (C) The sequence coverage is shown in bold.
(4/93) in healthy controls and 0% in other control groups. This corresponded to a significant difference between DCIS (p < 0.0001) or early stage breast cancer patients (p < 0.0001) and healthy controls. Interestingly, high-grade DCIS showed a more elevated frequency of HSP60 autoantibodies (47.8%) (p < 0.0001) than low-grade DCIS (19.2%) (p ) 0.0233) when compared to healthy controls. The relation between the frequency of HSP60 autoantibodies in tumor tissues and histomorphological and biological variables were statistically analyzed. The frequency of HSP60 antibodies was significantly lower in low-grade DCIS compared to high-grade DCIS (p ) 0.0188). We observed no significant relation between the frequency of HSP60 autoantibodies and all other parameters such as estrogen receptor, progesterone receptor, and Her-2 status, variables with known association to therapy response and survival of patients with neoplasia.23 Finally, performance of the diagnostic value of HSP60 autoantibodies were estimated (Table 4). Results indicated an overall classification accuracy of 61.5%, specificity of 95.7%, sensitivity of 31.8%, positive predictive value of 89.5%, negative predictive value of 54.9%, and AUC of 63.7% for discriminating healthy and cancer patients. mRNA Expression of HSP60 in Breast Cancer. To examine the possibility that the reactivity of breast cancer sera is due to an elevated expression of HSP60 mRNA, we performed real time quantitative PCR on 14 early breast tumors and 14 healthy breast tissue samples. We used 2 different genes, RS9 and TBP89, as internal controls and calculated the ratio
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HSP60 Autoantibodies in Early Breast Cancer
Table 3. Frequency of HSP60 Autoantibodies Detected in the Sera of Early Breast Cancer Patients by ELISA number of cases
Early stage breast cancer Histotype Invasive ductal carcinoma Invasive lobular carcinoma Histologic grade Grade 1 Grade 2 Grade 3 ERR Negative Positive PgR Negative Positive Her-2 overexpression Negative Positive
number of positive HSP60 autoantibody sera (%)
p value healthy vs cancer
