Project description:A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. In this study, we have conducted a prospective sample collection and retrospective blinded validation (PRoBE design) to evaluate the performance and translational utilities of salivary transcriptomic and proteomic biomarkers for the non-invasive detection of breast cancer. The Affymetrix HG U133 Plus 2.0 Array and 2D-DIGE were used to profile transcriptomes and proteomes in saliva supernatants respectively. Significant variations of salivary transcriptomic and proteomic profiles were observed between breast cancer patients and healthy controls. Eleven transcriptomic biomarker candidates and two proteomic biomarker candidates were selected for a preclinical validation using an independent sample set. Transcriptomic biomarkers were validated by RT-qPCR and proteomic biomarkers were validated by quantitative protein immunoblot. Eight mRNA biomarkers and one protein biomarker have been validated for breast cancer detection, yielding ROC-plot AUC values between 0.665 and 0.959. This report provides proof of concept of salivary biomarkers for the non-invasive detection of breast cancer. The salivary biomarkersâ discriminatory power paves the way for a PRoBE-design definitive validation study. Keywords: Salivary biomarker, Breast cancer, Early detection, Salivary transcriptome, Salivary proteome This study, which was approved by the UCLA and Cedars-Sinai Medical Center Institutional Review Boards (#06-07-043 and #3870, respectively), started sample collection in February 2007. The sample collection followed the PRoBE principle (prospective specimen collection). The saliva bank for breast cancer project at the UCLA Dental Research Institute in collaboration with Cedars Sinai Medical Center has collected 200 saliva samples since 2007 with all subjects recruited from the Saul and Joyce Brandman Breast Cancer Center. Of these, 113 samples, including 41 breast cancer patients and 72 healthy control individuals, were selected for the discovery and validation phase of this study. Inclusion criteria of cancer patients consisted of a confirmed diagnosis of breast cancer. Exclusion criteria of cancer patients included therapy/surgery and/or a diagnosis of other malignancies within 5 years prior to saliva collection. Exclusion criteria of control patients included a diagnosis of any malignancies within 5 years prior to saliva collection. Written informed consents and questionnaire data sheets were obtained from all patients who agreed to serve as saliva donors. Unstimulated saliva samples were consistently collected, stabilized, and preserved as previously described. The sample supernatants were reserved at -80 C prior to assay. This study consisted of a discovery phase, followed by an independent preclinical validation phase. Of the 113 samples, 10 breast cancer samples and 10 healthy control samples were chosen for the discovery phase. All breast cancer cases are invasive ductal carcinoma (IDC), the most common type of breast cancer. Biomarkers identified from the discovery studies were first verified using the discovery sample set. An independent sample set, including 31 breast cancer patients and 62 healthy control subjects, was used for the biomarker validation phase.

Project description:Pancreatic cancer is the fourth leading cause of cancer death. Lack of early detection technology for pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. Oral fluid (saliva) meets the demand for non-invasive, accessible, and highly efficient diagnostic medium. The level of salivary analytes, such as mRNA and microflora, vary upon disease onset; thus possess valuable signatures for early detection and screening. In this study, we evaluated the performance and translational utilities of the salivary transcriptomic and microbial biomarkers for non-invasive detection of early pancreatic cancer. Two biomarker discovery technologies were used to profile transcriptome in saliva supernatant and microflora in saliva pellet. The Affymetrix Human Genome U133 Plus 2.0 Array was used to discover altered gene expression in saliva supernatant. The Human Oral Microbe Identification Microarray (HOMIM) was used to investigate microflora shift in saliva pellet. Biomarkers selected from both studies were subjected to an independent clinical validation using a cohort of 30 early pancreatic cancer, 30 chronic pancreatitis and 30 healthy matched-control saliva samples. Two panels of salivary biomarkers, including eleven mRNA biomarkers and two microbial biomarkers were discovered and validated for pancreatic cancer detection. The logistic regression model with the combination of three mRNA biomarkers (ACRV1, DMXL2 and DPM1) yielded a ROC-plot AUC value of 0.974 (95% CI, 0.896 to 0.997; P < 0.0001) with 93.3% sensitivity and 90% specificity in distinguishing pancreatic cancer patients from healthy subjects. The logistic regression model with the combination of two bacterial biomarkers (Neisseria elongata and Streptococcus mitis) yielded a ROC-plot AUC value of 0.895 (95% CI, 0.784 to 0.961; P < 0.0001) with 96.4% sensitivity and 82.1% specificity in distinguishing pancreatic cancer patients from healthy subjects. Importantly, the logistic regression model with the combination of four biomarkers (mRNA biomarkers, ACRV1, DMXL2 and DPM1; bacterial biomarker, S. mitis) could differentiate pancreatic cancer patients from all non-cancer subjects (chronic pancreatitis and healthy control), yielding a ROC-plot AUC value of 0.949 (95% CI, 0.877 to 0.985; P < 0.0001) with 92.9% sensitivity and 85.5% specificity. This study comprehensively compared the salivary transcriptome and microflora between pancreatic cancer and control subjects. We have discovered and validated eleven mRNA biomarkers and two microbial biomarkers for early detection of pancreatic cancer in saliva. The logistic regression model with four salivary biomarkers can detect pancreatic cancer specifically without the complication of chronic pancreatitis. This is the first report demonstrating the value of multiplex salivary biomarkers for the non-invasive detection of a high impact systemic cancer. Keywords: Salivary biomarker, pancreatic cancer, early detection, salivary transcriptome, salivary microflora This study, which was approved by the UCLA Institutional Review Board, started sample collection in February 2006 and ended in April 2008. It had a discovery and verification phase, followed by an independent validation phase. All patients with clinically diagnosed early or locally advanced pancreatic cancer, chronic pancreatitis and matched healthy control were recruited from the UCLA Medical Center. The controls were matched for gender, age, ethnicity and smoking history. We chose early and locally advanced pancreatic cancers because they were considered early stage diseases. All patients were recently diagnosed with primary disease, and had not received any prior treatment in the form of chemotherapy, radiotherapy, surgery, or alternative remedies. No subjects had a history of prior malignancy, immunodeficiency, autoimmune disorders, hepatitis, or HIV infection. Written informed consent was obtained from all patients who agreed to serve as saliva donors. Unstimulated saliva samples were collected and processed as previously described. The saliva bank of pancreatic disease at the UCLA Dental Research Institute has collected 283 saliva samples since 2006. Of these, 114 samples, from 42 pancreatic cancer patients, 30 chronic pancreatitis patients and 42 healthy control subjects, met the following eligibility criteria: the disease subjectsâ?? samples were obtained immediately after primary diagnosis from patients without evidence of metastasis; the saliva sample was free of blood (Table 1 in accompanying manuscript). Of the 114 samples, 12 pancreatic cancer samples and 12 healthy control samples were chosen for the discovery and verification phase. The transcriptomic approach profiled the saliva supernatant samples from 12 pancreatic cancer patients and 12 healthy control subjects using the Affymetrix Human Genome U133 Plus 2.0 Array platform.

Project description:Pancreatic cancer is the fourth leading cause of cancer death. Lack of early detection technology for pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. Oral fluid (saliva) meets the demand for non-invasive, accessible, and highly efficient diagnostic medium. The level of salivary analytes, such as mRNA and microflora, vary upon disease onset; thus possess valuable signatures for early detection and screening. In this study, we evaluated the performance and translational utilities of the salivary transcriptomic and microbial biomarkers for non-invasive detection of early pancreatic cancer. Two biomarker discovery technologies were used to profile transcriptome in saliva supernatant and microflora in saliva pellet. The Affymetrix Human Genome U133 Plus 2.0 Array was used to discover altered gene expression in saliva supernatant. The Human Oral Microbe Identification Microarray (HOMIM) was used to investigate microflora shift in saliva pellet. Biomarkers selected from both studies were subjected to an independent clinical validation using a cohort of 30 early pancreatic cancer, 30 chronic pancreatitis and 30 healthy matched-control saliva samples. Two panels of salivary biomarkers, including eleven mRNA biomarkers and two microbial biomarkers were discovered and validated for pancreatic cancer detection. The logistic regression model with the combination of three mRNA biomarkers (ACRV1, DMXL2 and DPM1) yielded a ROC-plot AUC value of 0.974 (95% CI, 0.896 to 0.997; P < 0.0001) with 93.3% sensitivity and 90% specificity in distinguishing pancreatic cancer patients from healthy subjects. The logistic regression model with the combination of two bacterial biomarkers (Neisseria elongata and Streptococcus mitis) yielded a ROC-plot AUC value of 0.895 (95% CI, 0.784 to 0.961; P < 0.0001) with 96.4% sensitivity and 82.1% specificity in distinguishing pancreatic cancer patients from healthy subjects. Importantly, the logistic regression model with the combination of four biomarkers (mRNA biomarkers, ACRV1, DMXL2 and DPM1; bacterial biomarker, S. mitis) could differentiate pancreatic cancer patients from all non-cancer subjects (chronic pancreatitis and healthy control), yielding a ROC-plot AUC value of 0.949 (95% CI, 0.877 to 0.985; P < 0.0001) with 92.9% sensitivity and 85.5% specificity. This study comprehensively compared the salivary transcriptome and microflora between pancreatic cancer and control subjects. We have discovered and validated eleven mRNA biomarkers and two microbial biomarkers for early detection of pancreatic cancer in saliva. The logistic regression model with four salivary biomarkers can detect pancreatic cancer specifically without the complication of chronic pancreatitis. This is the first report demonstrating the value of multiplex salivary biomarkers for the non-invasive detection of a high impact systemic cancer. Keywords: Salivary biomarker, pancreatic cancer, early detection, salivary transcriptome, salivary microflora

Project description:Background: The biological role of microRNAs (miRNAs) in field cancerization is unknown. To investigate the involvement of miRNAs in gastric field cancerization, we evaluated the expression profile of ten miRNAs and their diagnostic value. Methods: We used three groups of FFPE gastric samples: non-cancer (NC), cancer adjacent (ADJ) and gastric cancer (GC). The expression profiles of hsa-miR-10a, -miR-21, -miR-29c, -miR-135b, -miR-148a, -miR-150, -miR-204, -miR-215, -miR-483 and -miR-664a were investigated using qRT-PCR. The results obtained by qRT-PCR were validated in Small RNA-Seq data from the TCGA database. The search for target genes of the studied miRNAs was performed in the miRTarBase public database and miRTargetLink tool, using experimentally validated interactions. In addition, we also performed the functional analysis of these genes using enrichment in KEGG pathways. The potential as biomarker was evaluated using a receiver operating characteristic (ROC) curve and the derived area under the curve (AUC>0.85) analysis. Results: The miRNAs hsa-miR-10a, -miR-21, -miR-135b, hsa-miR-148a, -miR-150, -miR-215, -miR-204, -miR-483 and -miR-664a were up-regulated in ADJ and GC compared to NC (P<0.03); and hsa-miR-21 and -miR-135b were up-regulated in GC compared to ADJ (P<0.01). Hsa-miR-148a, -miR-150, -miR-215, -miR-483 and -miR-664a were not differentially expressed between GC and ADJ, suggesting that both share similar changes (P>0.1). The TS-miR hsa-miR-29c was up-regulated in ADJ compared to NC and GC (P<0.01); we did not observe a significant difference in the expression of this miRNA between NC and GC. This feature may be an antitumor mechanism used by cancer-adjacent tissue because this miRNA regulates the BCL-2, CDC42 and DMNT3A oncogenes. The expression level of hsa-miR-204 was associated with Helicobacter pylori infection status (P<0.05). Functional analysis using the genes regulated by the studied miRNAs showed that they are involved in biological pathways and cellular processes that are critical for the establishment of H. pylori infection and for the onset, development and progression of GC. hsa-miR-10a, -miR-21, -miR-135b, -miR-148a, -miR-150, -miR-215, -miR-483 and -miR-664a were able to discriminate NC from other tissues with great accuracy (AUC>0.85). Conclusion: The studied miRNAs are closely related to field cancerization, regulate genes important for gastric carcinogenesis and can be potentially useful as biomarkers in GC.

Project description:Pancreatic cancer is the fourth leading cause of cancer death. Lack of early detection technology for pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. Oral fluid (saliva) meets the demand for non-invasive, accessible, and highly efficient diagnostic medium. The level of salivary analytes, such as mRNA and microflora, vary upon disease onset; thus possess valuable signatures for early detection and screening. In this study, we evaluated the performance and translational utilities of the salivary transcriptomic and microbial biomarkers for non-invasive detection of early pancreatic cancer. Two biomarker discovery technologies were used to profile transcriptome in saliva supernatant and microflora in saliva pellet. The Affymetrix Human Genome U133 Plus 2.0 Array was used to discover altered gene expression in saliva supernatant. The Human Oral Microbe Identification Microarray (HOMIM) was used to investigate microflora shift in saliva pellet. Biomarkers selected from both studies were subjected to an independent clinical validation using a cohort of 30 early pancreatic cancer, 30 chronic pancreatitis and 30 healthy matched-control saliva samples. Two panels of salivary biomarkers, including eleven mRNA biomarkers and two microbial biomarkers were discovered and validated for pancreatic cancer detection. The logistic regression model with the combination of three mRNA biomarkers (ACRV1, DMXL2 and DPM1) yielded a ROC-plot AUC value of 0.974 (95% CI, 0.896 to 0.997; P < 0.0001) with 93.3% sensitivity and 90% specificity in distinguishing pancreatic cancer patients from healthy subjects. The logistic regression model with the combination of two bacterial biomarkers (Neisseria elongata and Streptococcus mitis) yielded a ROC-plot AUC value of 0.895 (95% CI, 0.784 to 0.961; P < 0.0001) with 96.4% sensitivity and 82.1% specificity in distinguishing pancreatic cancer patients from healthy subjects. Importantly, the logistic regression model with the combination of four biomarkers (mRNA biomarkers, ACRV1, DMXL2 and DPM1; bacterial biomarker, S. mitis) could differentiate pancreatic cancer patients from all non-cancer subjects (chronic pancreatitis and healthy control), yielding a ROC-plot AUC value of 0.949 (95% CI, 0.877 to 0.985; P < 0.0001) with 92.9% sensitivity and 85.5% specificity. This study comprehensively compared the salivary transcriptome and microflora between pancreatic cancer and control subjects. We have discovered and validated eleven mRNA biomarkers and two microbial biomarkers for early detection of pancreatic cancer in saliva. The logistic regression model with four salivary biomarkers can detect pancreatic cancer specifically without the complication of chronic pancreatitis. This is the first report demonstrating the value of multiplex salivary biomarkers for the non-invasive detection of a high impact systemic cancer. Keywords: Salivary biomarker, pancreatic cancer, early detection, salivary transcriptome, salivary microflora This study, which was approved by the UCLA Institutional Review Board, started sample collection in February 2006 and ended in April 2008. It had a discovery and verification phase, followed by an independent validation phase. All patients with clinically diagnosed early or locally advanced pancreatic cancer, chronic pancreatitis and matched healthy control were recruited from the UCLA Medical Center. The controls were matched for gender, age, ethnicity and smoking history. We chose early and locally advanced pancreatic cancers because they were considered early stage diseases. All patients were recently diagnosed with primary disease, and had not received any prior treatment in the form of chemotherapy, radiotherapy, surgery, or alternative remedies. No subjects had a history of prior malignancy, immunodeficiency, autoimmune disorders, hepatitis, or HIV infection. Written informed consent was obtained from all patients who agreed to serve as saliva donors. Unstimulated saliva samples were collected and processed as previously described. The saliva bank of pancreatic disease at the UCLA Dental Research Institute has collected 283 saliva samples since 2006. Of these, 114 samples, from 42 pancreatic cancer patients, 30 chronic pancreatitis patients and 42 healthy control subjects, met the following eligibility criteria: the disease subjects’ samples were obtained immediately after primary diagnosis from patients without evidence of metastasis; the saliva sample was free of blood (Table 1 in accompanying manuscript). Of the 114 samples, 12 pancreatic cancer samples and 12 healthy control samples were chosen for the discovery and verification phase. The transcriptomic approach profiled the saliva supernatant samples from 12 pancreatic cancer patients and 12 healthy control subjects using the Affymetrix Human Genome U133 Plus 2.0 Array platform.

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Salivary microRNAs as promising biomarkers for detection of esophageal cancer.

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Salivary microRNAs as promising biomarkers for detection of esophageal cancer.

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