Breast cancer related proteins are present in saliva and are modulated secondary to ductal carcinoma in situ of the breast.

OBJECTIVE: The objective of this study was to determine if protein-by-products secondary to cancer related oncogenes appear in the saliva of breast cancer patients. METHODS: Three pooled (n = 10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women diagnosed with a benign breast tumor and the other one pooled specimen was from women diagnosed with ductal carcinoma in situ (DCIS). Differential expression of proteins was measured by isotopically tagging proteins in the tumor groups and comparing them to the healthy control group. Experimentally, saliva from each of the pooled samples was 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 (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. RESULTS: The results of the salivary analyses in this population of patients yielded approximately 130 proteins in the saliva specimens. Forty-nine proteins were differentially expressed between the healthy control pool and the benign and cancer patient groups. CONCLUSIONS: The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to DCIS. Additionally, there may be salivary protein profiles that are unique to both DCIS and fibroadenoma tumors.

A Catalogue of Altered Salivary Proteins Secondary to Invasive Ductal Carcinoma: A Novel In Vivo Paradigm to Assess Breast Cancer Progression.

The objective of this manuscript is to introduce a catalogue of salivary proteins that are altered secondary to carcinoma of the breast. The catalogue of salivary proteins is a compilation of twenty years of research by the authors and consists of 233 high and low abundant proteins which have been identified by LC-MS/MS mass spectrometry, 2D-gel analysis and by enzyme-linked immunosorbent assay. The body of research suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be useful in the study of breast cancer progress, treatment efficacy and the tailoring of individualized patient care.

Salivary Protein Profiles among HER2/neu-Receptor-Positive and -Negative Breast Cancer Patients: Support for Using Salivary Protein Profiles for Modeling Breast Cancer Progression.

Purpose. The objective of this study was to compare the salivary protein profiles from individuals diagnosed with breast cancer that were either HER2/neu receptor positive or negative. Methods. Two pooled saliva specimens underwent proteomic analysis. One pooled specimen was from women diagnosed with stage IIa HER2/neu-receptor-positive breast cancer patients (n = 10) and the other was from women diagnosed with stage IIa HER2/neu-receptor-negative cancer patients (n = 10). The pooled samples were trypsinized and the peptides labeled with iTRAQ reagent. Specimens were analyzed using an LC-MS/MS mass spectrometer. Results. The results yielded approximately 71 differentially expressed proteins in the saliva specimens. There were 34 upregulated proteins and 37 downregulated proteins.

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.

Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity.

Proteomic analyses by mass spectrometry are propelling the field of medical diagnostics forward at unprecedented rates because of its ability reliably to identify proteins that are at the femtomole level in concentration. These advancements have also benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of malignant tumors that are remote from the oral cavity. Saliva is easy to collect and may provide diagnostic information about a variety of cancers. In particular, proof-of-principle has been demonstrated for salivary biomarker research. This article reviews the literature, discusses the theories associated with saliva-based tumor diagnostics, and presents the current research focused on the use of saliva as a diagnostic medium for the detection of cancer.

The use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to detect putative breast cancer markers in saliva: a feasibility study.

BACKGROUND: Technologies are now available enabling saliva to be used to diagnose disease, predict disease progression, and monitor therapeutic efficacy. This pilot study describes the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) to detect putative breast cancer markers in saliva. METHODS: Salivary specimens were analyzed as either pooled cancer saliva specimens, or individual specimens from healthy women and women diagnosed with carcinoma of the breast. The specimens were applied to a variety of protein chip arrays, washed extensively to remove unbound analytes and analyzed on a SELDI mass spectrometer. RESULTS: The results of this initial study suggest that the WCX protein chip array prepared and washed at pH 3.5 yielded the most promising results. Additionally, the analyses revealed a number of proteins that were higher in intensity among the cancer subjects when compared with controls. These salivary proteins were present at the 18, 113, 170, 228 and 287 km/z ranges using SELDI analyses. CONCLUSIONS: The study suggests that saliva may be useful for high-throughput biomarker discovery.

Aging and salivary cytokine concentrations as predictors of whole saliva flow rates among women: a preliminary study.

INTRODUCTION: How aging influences the secretion of saliva is not entirely clear. Postmortem studies have demonstrated that, with age, the parenchyma of the salivary glands is gradually replaced by fat, connective tissue, and oncocytes. However, functional studies among healthy individuals indicate that aging per se does not lead to a diminution in the capacity of these glands to produce saliva. To date, the ambiguity between the morphometric and the clinical evidence cannot be explained. The purpose of this study was to determine if there was any relationship between the cytokine concentrations, salivary flow rates and aging. METHODS: The study group consisted of 50 racially mixed women between the ages of 18 and 90. SWS were collected for 5 min using a 1-cm(3) cube of paraffin as a stimulant. The samples were aliquoted, then frozen until assayed. Cytokine concentrations were determined using ELISA kits. The statistical analysis consisted of descriptive statistics, mean comparisons, and regression analyses. RESULTS: The results suggest a reduction in SWS in association with an increase of salivary TGF-beta1 concentrations. The overall model demonstrated an ability of the independent variables to predict the dependent variable (SWS) at the R(2) = 0.53 level. The addition of the salivary cytokines to the model suggests that TGF-beta1 (p < 0.045) and a near significant IL-2 (p < 0.07) concentration best explain the decrease in salivary function. The statistical relationship is supported by the physiological relationship between TGF-beta1, IL-2, apoptosis, and aging. CONCLUSION: The results suggest a reduction in SWS flow in association with an increase of salivary TGF-beta1 concentrations. This reduction concurs with the histomorphometric investigations, which indicate an age-related reduction of acinar cells in salivary gland tissues.

The expression of the c-erbB-2 receptor protein in glandular salivary secretions.

BACKGROUND: As the maintenance medium of the oral cavity, saliva is secreted from exocrine glands that include the parotid, submandibular, sublingual, and minor salivary glands. Considering that saliva is a fluid suffused with protein, it is possible that the solubilized by-products of oncogenic expression may be present in saliva. Recent studies suggest the presence of solubilized extracellular domain portion of the c-erbB-2 protein in serum, nipple aspirates, and saliva. As a consequence, the purpose of this study was to determine the presence and concentration of c-erbB-2 in major salivary gland secretions. METHODS: Fifteen healthy women had serum, stimulated whole (SWS), parotid (SP), and submandibular/sublingual (SS) salivary secretions collected. The specimens were analyzed for c-erbB-2 using enzyme linked immunosorbent assays (ELISAs). Western blots using c-erbB-2 were also performed on these specimens. RESULTS: The ELISAs revealed the presence of c-erbB-2 in SWS (24.50 Units/ml), SP (19.66 Units/ml), SS (15.59 Units/ml) and serum (1472.15 Units/ml). Western blots confirmed the presence of these 185 kDa proteins. CONCLUSIONS: These results suggest that the protein, c-erbB-2, is present in relatively equal amounts in both SP and SS glandular secretions. Elevated glandular salivary c-erbB-2 concentrations could be useful as a preliminary, non-invasive test in clinical decision making when diagnosing salivary gland carcinomas. Additionally, this marker may have utility in distinguishing between oral lesions that are benign, pre-malignant and malignant in the oral cavity. Further research is required to determine if these findings have clinical utility.

The potential use of saliva to detect recurrence of disease in women with breast carcinoma.

BACKGROUND: Approximately 1 woman in every 10 will develop breast cancer in her lifetime. It has been shown that screening for breast cancer can reduce breast cancer mortality. The use of a saliva-based test could prove to be very useful in post-operative and/or adjunctive therapy management of breast cancer patients. METHODS: The following study was undertaken to establish the possible usefulness of the salivary protein product of the oncogene c-erbB-2 in following patients diagnosed with carcinoma of the breast. Included in this study were 25 patients with a mean age of 54 years with varying histological diagnoses and stages of carcinoma of the breast. ELISA assays for c-erbB-2 and CA 15-3 were performed on serum and stimulated whole saliva samples collected on all patients prior to any adjunct therapy or surgery and sequentially during therapy. RESULTS: The results of the GLM analyses using marker concentration as the dependent variable and treatment regimen and the serial assessments as independent variables yielded a significant overall model for both the serum (P < 0.007) and salivary (P < 0.017) c-erbB-2 markers. The model for serum c-erbB-2, however, exhibited a significant difference for treatment regimen (P < 0.001) with the chemotherapy and radiation treatment regimen being significantly different (P < 0.001) from the other treatment therapies. Time (serial assessments) was not significant. The model for the salivary c-erbB-2 marker was reversed. Treatment regimen was not significant for this model; however, time (serial assessments) was significant (P < 0.002). The serum and salivary CA 15-3 marker models yielded no significant results. Paired t-test analyses indicated that only the salivary c-erbB-2 concentrations exhibited a significant difference between the pre- and post-therapy values (t = 4.245, P < 0.0001). Additionally, salivary c-erbB-2 displayed greater percent reductions across all therapies as compared to the other markers. CONCLUSIONS: This preliminary study appears to indicate that c-erbB-2 protein expression in saliva may be a very useful diagnostic tool for measuring patient response to chemotherapy and/or surgical treatment of their disease.