A Comparison of the Proteomic Expression in Pooled Saliva Specimens from Individuals Diagnosed with Ductal Carcinoma of the Breast with and without Lymph Node Involvement

Purpose. The objective was to compare the salivary protein profiles of saliva specimens from individuals diagnosed with invasive ductal carcinoma of the breast (IDC) with and without lymph node involvement. Methods. Three pooled saliva specimens from women were analyzed. One pooled specimen was from healthy women; another was from women diagnosed with Stage IIa IDC and a specimen from women diagnosed with Stage IIb. The pooled samples were trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase capillary chromatography on an LC-MS/MS mass spectrometer. Results. The results yielded approximately 174 differentially expressed proteins in the saliva specimens. There were 55 proteins that were common to both cancer stages in comparison to each other and healthy controls while there were 20 proteins unique to Stage IIa and 28 proteins that were unique to Stage IIb.


Introduction
Clinicopathologic factors such as histologic type, tumor size, tumor grade, HER-2/neu over-expression, hormone receptor status, and lymph node involvement are recognized as having prognostic use in breast cancer management [1][2][3][4]. Collectively assessed, axillary lymph node metastasis is the most important prognostic factor predicting breast cancer patient survival [5][6][7]. Currently, the best predictor of axillary lymph node metastasis is the presence or absence of metastasis in the sentinel lymph node.
Current methodologies for this assessment are limited to axillary lymph node dissection and sentinel lymph node biopsy; however, these procedures are not without risks. For example, level I and level II axillary lymph node dissection can be associated with upper extremity lymph edema, wound complications, or nerve injury in a significant proportion of patients. Although sentinel lymph node biopsy is far less morbid than axillary lymph node dissection, it is not without risks or morbidity [8,9]. Sentinel lymph node biopsy, despite its low but measurable false negative rate, provides no information about the presence of additional nonsentinel lymph node metastasis, which may occur in 40% to 70% of cases [10][11][12]. As a consequence, newer, more accurate, and less invasive means of predicting axillary lymph node metastasis would greatly improve breast cancer patient management and quality of life.
Morphological mimicry among human malignancies is a well-known histopathological phenomenon [11]. This is especially true in the case of the neoplastic ductal tissues of the breast and the salivary glands where immunostaining revealed the presence of Her2/neu (c-erbB-2), progesterone receptor, androgen receptor, and GCDFP-15 among these diseased tissues [11][12][13][14]. These studies suggest that similar molecular pathway dysfunctions may also be common within both tissues types [15].
Consequently, research has been performed concerning the presence of cancer related proteins and protein alterations in the secretory by products of these tissues, that is, saliva and nipple aspirate fluid (NAF) [16,17]. Single analyte ELISA-based analyses yielded the presence of Journal of Oncology soluble Her2/neu protein in both saliva and NAF; Her2/neu concentrations were found to be elevated in both fluids secondary to the presence of carcinoma of the breast [16,17]. Remarkably, Her2/neu protein concentrations were also elevated in the contralateral healthy breast within the same subject. Collectively, this line of research suggests that cancer-related cellular signaling may affect healthy exocrine tissues and result in alterations of their secretory by products. Likewise, EGFR and TNF-α were also found to be present in both fluids and altered in the presence of malignant breast disease [18]. Adding further support to this concept, both saliva and NAF were analyzed using mass spectrometry [19][20][21]. These fluid analyses yielded striking similarities with respect to their protein profile in health and were altered in the presence of neoplastic disease [19,21].
Capitalizing on the potential of this possible relationship, significant salivary protein profile comparisons and alterations were reported in early stage breast cancer [21]. As a consequence, the purpose of this paper is to report saliva alterations secondary to late stage IDC with a focus concerning lymph node and nonlymph node involvement among the IDC cohorts.

Design.
The investigators protein profiled three pooled, stimulated whole saliva specimens. One specimen consisted of pooled saliva from 10 healthy subjects, another specimen was a pooled saliva specimen from 10 Stage IIa (T 2 N 0 M 0 ) invasive ductal carcinoma patients (IDC), and the third pooled specimen was from 10 subjects diagnosed with Stage IIb (T 2 N 1 M 0 ) invasive ductal carcinoma [22]. The cancer cohorts were estrogen, progesterone, and Her2/neu receptor status negative as determined by the pathology report. Histological grade was not available for this study. The subjects were matched for age and race and were nontobacco users.
The participating subjects were given an explanation about their participation rights and signed an IRB consent form. The saliva specimens and related patient data are nonlinked and bar coded in order to protect patient confidentiality. This study was performed under the UTHSC IRB approved protocol number HSC-DB-05-0394. All procedures were in accordance with the ethical standards of the UTHSC IRB and with the Helsinki Declaration of 1975, as revised in 1983.

Saliva Collection and Sample Preparation.
Stimulated whole salivary gland secretion is based on the reflex response occurring during the mastication of a bolus of food. Usually, a standardized bolus (1 gram) of paraffin or a gum base (generously provided by the Wrigley Co., Peoria, IL) is given to the subject to chew at a regular rate. The individual, upon sufficient accumulation of saliva in the oral cavity, expectorates periodically into a preweighed disposable plastic cup. This procedure is continued for a period of five minutes. The volume and flow rate is then recorded along with a brief description of the specimen's physical appearance [23]. The cup with the saliva specimen is reweighed and the flow rate determined gravimetrically. The authors recommend this salivary collection method with the following modifications for consistent protein analyses [24]. A protease inhibitor from Sigma Co (St. Louis, MI, USA) is added along with enough orthovanadate from a 100 mM stock solution to bring its concentration to 1 mM. The treated samples were centrifuged for 10 minutes at top speed in a table top centrifuge. The supernatant was divided into 1 mL aliquots and frozen at −80 • C.

LC-MS/MS Mass Spectroscopy with Isotopic Labeling.
Recent advances in mass spectrometry, liquid chromatography, analytical software, and bioinformatics have enabled the researchers to analyze complex peptide mixtures with the ability to detect proteins differing in abundance by over 8 orders of magnitude [25]. One current method is isotopic labeling coupled with liquid chromatography tandem mass spectrometry (IL-LC-MS/MS) to characterize the salivary proteome [26]. The main approach for discovery is a mass spectroscopy-based method that uses isotope coding of complex protein mixtures such as tissue extracts, blood, urine, or saliva to identify differentially expressed proteins [27]. The approach readily identifies changes in the level of expression, thus permitting the analysis of putative regulatory pathways providing information regarding the pathological disturbances in addition to potential biomarkers of disease. The analysis was performed on a tandem QqTOF QStar XL mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with an LC Packings (Sunny vale, CA, USA) HPLC for capillary chromatography. The HPLC is coupled to the mass spectrometer by a nanospray ESI head (Protana, Odense, Denmark) for maximal sensitivity [16]. The advantage of tandem mass spectrometry combined with LC is enhanced sensitivity and the peptide separations afforded by chromatography. Thus even in complex protein mixtures MS/MS data can be used to sequence and identify peptides by sequence analysis with a high degree of confidence [21,25,26,28].
Isotopic labeling of protein mixtures has proven to be a useful technique for the analysis of relative expression levels of proteins in complex protein mixtures such as plasma, saliva urine, or cell extracts. There are numerous methods that are based on isotopically labeled protein modifying reagents to label or tag proteins to determine relative or absolute concentrations in complex mixtures. The higher resolution offered by the tandem Qq-TOF mass spectrometer is ideally suited to isotopically labeled applications [21,26,29,30].
Applied Biosystems recently introduced iTRAQ reagents [26,29,30], which are amino reactive compounds that are used to label peptides in a total protein digest of a fluid such as saliva. The real advantage is that the tag remains intact through TOF-MS analysis; however, it is revealed during collision-induced dissociation by MSMS analysis. Thus in the MSMS spectrum for each peptide there is a fingerprint indicating the amount of that peptide from each of the different protein pools. Since virtually all of the peptides in a mixture are labeled by the reaction, numerous proteins in complex mixtures are identified and can be compared for their relative concentrations in each mixture. Thus even in complex mixtures there is a high degree of confidence in the identification.

Salivary Protein Analyses with iTRAQ.
Briefly, the saliva samples were thawed and immediately centrifuged to remove insoluble materials. The supernatant was assayed for protein using the Bio-Rad protein assay (Hercules, CA, USA) and an aliquot containing 100 μg of each specimen was precipitated with 6 volumes of −20 • C acetone. The precipitate was resuspended and treated according to the manufacturers instructions. Protein digestion and reaction with iTRAQ labels was carried out as previously described and according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Briefly, the acetone precipitable protein was centrifuged in a table-top centrifuge at 15,000 × g for 20 minutes. The acetone supernatant was removed and the pellet resuspended in 20 uL dissolution buffer. The soluble fraction was denatured and disulfides reduced by incubation in the presence of 0.1% SDS and 5 mM TCEP (tris-(2carboxyethyl) phosphine)) at 60 • C for one hour. Cysteine residues were blocked by incubation at room temperature for 10 minutes with MMTS (methyl methane-thiosulfonate). Trypsin was added to the mixture to a protein : trypsin ratio of 10 : 1. The mixture was incubated overnight at 37 • C. The protein digests were labeled by mixing with the appropriate iTRAQ reagent and incubating at room temperature for one hour. On completion of the labeling reaction, the four separate iTRAQ reaction mixtures were combined. Since there are a number of components that can interfere with the LCMSMS analysis, the labeled peptides are partially purified by a combination of strong cation exchange followed by reverse phase chromatography on preparative columns. The combined peptide mixture is diluted 10-fold with loading buffer (10 mM KH 2 PO 4 in 25% acetonitrile at pH 3.0) and applied by syringe to an ICAT Cartridge-Cation Exchange column (Applied Biosystems, Foster City, CA) column that has been equilibrated with the same buffer. The column is washed with 1 mL loading buffer to remove contaminants. To improve the resolution of peptides during LCMSMS analysis, the peptide mixture is partially purified by elution from the cation exchange column in 3 fractions. Stepwise elution from the column is achieved with sequential 0.5 mL aliquots of 10 mM KH2PO4 at pH 3.0 in 25% acetonitrile containing 116 mM, 233 mM, and 350 mM KCl, respectively. The fractions are evaporated by Speed Vac to about 30% of their volume to remove the acetonitrile and then slowly applied to an Opti-Lynx Trap C18 100 uL reverse phase column (Alltech, Deerfield, IL) with a syringe. The column was washed with 1 mL of 2% acetonitrile in 0.1% formic acid and eluted in one fraction with 0.3 mL of 30% acetonitrile in 0.1% formic acid. The fractions were dried by lyophilization and resuspended in 10 uL 0.1% formic acid in 20% acetonitrile. Each of the three fractions was analyzed by reverse phase LCMSMS.

Reverse Phase LCMSMS.
The desalted and concentrated peptide mixtures were quantified and identified by nano-LCMS/MS on an API QSTAR XL mass spectrometer (ABS Sciex Instruments) operating in positive ion mode. The chromatographic system consists of an UltiMate nano-HPLC and FAMOS autosampler (Dionex LC Packings). Peptides were loaded on a 75 cm x 10 cm, 3 mm fused silica C18 capillary column, followed by mobile phase elution: buffer (A) 0.1% formic acid in 2% acetonitrile/98% Milli-Q water and buffer (B): 0.1% formic acid in 98% acetonitrile/2% Milli-Q water. The peptides were eluted from 2% buffer B to 30% buffer B over 180 minutes at a flow rate 220 nL/min. The LC eluent was directed to a NanoES source for ESI/MS/MS analysis. Using information-dependent acquisition, peptides were selected for collision induced dissociation (CID) by alternating between an MS (1 second) survey scan and MS/MS (3 seconds) scans. The mass spectrometer automatically chooses the top two ions for fragmentation with a 60second dynamic exclusion time. The IDA collision energy parameters were optimized based upon the charge state and mass value of the precursor ions. In each saliva sample set there are three separate LCMSMS analyses.
2.6. Bioinformatics. The accumulated MSMS spectra are analyzed by ProQuant and ProGroup software packages (Applied Biosystems) using the SwissProt database for protein identification. The ProQuant analysis was carried out with a 75% confidence cutoff with a mass deviation of 0.15 Da for the precursor and 0.1 Da for the fragment ions. The ProGroup reports were generated with a 95% confidence level for protein identification. Protein Pilot software package was used to assess the data produced from the mass spectrometry analyses. The Venn diagrams were constructed using the NIH software program (http://ncrr.pnl.gov). Graphic comparisons with log conversions and error bars for protein expression were produced using the ProQuant software. Descriptive statistics were performed using SPSS statistical software.

Results
Tables 1-5 summarize the results of the mass spectrometry analysis of the pooled salivary specimens and illustrate protein comparisons between Stage IIa versus healthy and Stage IIb versus healthy. The results identified and compared approximately 174 differentially expressed proteins in the saliva specimens. Of the 174 proteins, 158 (91%) were significant at an alpha level of P < .05 with a 95% confidence level. The mean percent peptide coverage for the complete panel proteins was 63.5% (±19.6) with a range of 35% to 96.5% coverage. The median value was 67.7% coverage.
The pie chart in Figure 1 illustrates the percentage of proteins according to protein function. There were 55 proteins that were common to both cancer stages in comparison to each other while there were 20 proteins unique to Stage IIa and 28 proteins that were unique to Stage IIb (Table 1). Figure 2 represents a Venn diagram of the overlapping proteins between the three groups of women. Figure 3 illustrates the comparison of the log ratio of the relative intensity (cancer/control) of the proteins which were common to both Stage IIa and Stage IIb while Figure 4 shows the proteins that were different between the two groups. It is worth noting that in Figure 3 Tables 2 and 3, 40 of the 75 proteins (53%) were significant at the P < .001 to P < .0001 levels.
Tables 4 and 5 are a list of the up-(n = 38) and down-(n = 45) regulated proteins observed in the Stage IIb cancer as compared to healthy controls. Of these 83 differentially expressed proteins, 54 (65%) were significant at the P < .001 to P < .0001 levels. There were 6 proteins that exhibited a 2.0 or greater fold increase in protein level in the Stage IIb cancer cohort as compared to the control subjects.

Discussion
To the best of our knowledge this is the first attempt to determine salivary protein profile alterations related to lymphovascular invasion. As a consequence we have only a few references by which to compare our data.
The proteins listed in Tables 2-4 are common to saliva and are listed in references concerning salivary proteomics of whole saliva and those constituents contributed by individual gland secretions [21,[31][32][33][34]. Likewise, many of the proteins are common to those identified in proteomic studies of cancer cell lines and serum or plasma from individuals diagnosed with IDC [21,27,[35][36][37][38][39]. Additionally, there is a proteomic study by Pei et al. [40] that compared proteomic tissue protein profiles from paired normal tissue to malignant tissues with and without lymphovascular invasion  A second by Li et al. [41] used metastatic (lymph nodes) breast cancer cell lines that they developed in order to produce protein profile comparisons [41]. Comparative S10AB S10A9 TGM3  BPIL1  TPM3  CRIS3  CAH6  DMBT1  TKT  S10A6  FIBA  B2MG  S10A4  K1C9  KV205  FETUA  FABPE  fAM25  CYTA  1433Z  AMYC  ANXA1  ANXA3  APOA1  CAH1  CAH6  CALM  CLUS  DSC2  FETUA  G6PI  GDIS  HBA  HBB  HEMO  HV301  IGHA2  IGHG2  ILEU  K2C6A  PDIA1  PPIA  TALDO  THIO  TPM3  TRFE  proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein gel spots were altered more than three-fold between the variant and its parental counterpart. Using SEQUEST with uninterpreted tandem mass raw data, they found eleven differentially expressed protein spots that were identified with high confidence. The proteins were identified as Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (sent in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parTPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54. The proteins were preental counterpart [41]. As shown in Tables 2-5, we identified a number of the same proteins. For example, the 14-3-3, tropomyosin and the peroxiredoxin family of proteins were found to be altered in the saliva of our late stage cancer profiles as well. Of particular interest is the fact that these proteins were not altered in the profiles of early stage cancer, that is, Stage 0 and Stage I performed by the authors of this manuscript [21,42].

Conclusions
The authors have examined the salivary proteome that is altered in the presence of carcinoma of the breast with and without lymph node metastasis. We do not want to over emphasize the findings at this point, but we are encouraged to find that these protein profiles are found to be altered in the supernants from cancer tissues which provide additional support to our findings. The authors urge the exploration of saliva proteomics for in vivo systems modeling of carcinoma of tissues of ectodermal origin. Saliva can also be described as a media which provides "real-time" results [43]. The fluid is continually produced and excreted in an open-ended circuit, unlike blood which exists in a "closed-loop." Blood, a circulating media, may contain proteins that are a day, a week, or a month old as well as proteins which have passed numerous times through many organ systems or have been excreted [43]. Saliva, with its continuous flow, is not subject to the aforementioned effects. Consequently, saliva and nipple aspirates may be a more useful than blood as the protein profiles of these fluids easier to assay than blood and are both altered in the presence of malignant diseases [16,43].
Further study is required to determine their diagnostic utility. The authors plan to validate the protein biomarkers by western blot using commercially available antibodies. ELISA will also be used to assay a larger sample size and determine the sensitivity and specificity of the biomarkers. It is the hope of the investigators that this preliminary research will establish the foundation for a "point-of-care" test for clinical decision making in the treatment of carcinoma of the breast.