World journal of gastroenterology : WJG, Jan 7, 2014
To characterize tumor necrosis factor receptor-associated protein 1 (TRAP1) expression in the pro... more To characterize tumor necrosis factor receptor-associated protein 1 (TRAP1) expression in the progression of ulcerative colitis (UC)-associated colorectal cancer. Chronic UC is an inflammatory bowel disease that predisposes to colorectal cancer. Immunohistochemical analysis was used to evaluate TRAP1 expression on tissue microarrays containing colonic tissues from 42 UC progressors (patients with cancer or dysplasia) and 38 non-progressors (dysplasia/cancer free patients). Statistical analyses of the TRAP1 immunohistochemistry staining were performed using GraphPad Prism. Differences in the TRAP1 level between non-progressors and progressors were tested for statistical significance using the Mann-Whitney test. Receiver operating characteristic curve method was used to quantify marker performance in distinguishing diseased cases from controls. TRAP1 was up-regulated in the colon tissues from UC progressors, but not in the colon tissues from UC non-progressors. Moreover, up-regulation...
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. Howev... more Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for >10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival <14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFβ-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that 'High-RPS8 and Low-PRELP' was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.
Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk o... more Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma1 that is thought to develop through genomic instability 2 . We considered that the rapid cell turnover and oxidative injury observed in ulcer- ative colitis might accelerate telomere shortening 3 , thereby increasing the potential of chromosomal ends to fuse4, resulting in
Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk o... more Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma 1 that is thought to develop through genomic instability 2 . We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening 3 , thereby increasing the potential of chromosomal ends to fuse 4 , resulting in cycles of chromatin bridge breakage and fusion 5,6 and chromosomal instability associated with tumor cell progression 7,8 . Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges-an intermediate in the breakage and fusion of chromatin bridges 9 -in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.
Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Be... more Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon, including the non-dysplastic mucosa; we hypothesized that the same field defect will include abnormally expressed proteins. Here we applied proteomics to study the protein expression of UC neoplastic progression. The protein profiles of colonic epithelium were compared from 1) UC patients without dysplasia (non-progressors); 2) none-dysplastic colonic tissue from UC patient with highgrade dysplasia or cancer (progressors); 3) high-grade dysplastic tissue from UC progressors and 4) normal colon. We identified protein differential expression associated with UC neoplastic progression. Proteins relating to mitochondria, oxidative activity, calcium-binding proteins were some of interesting classes of these proteins. Network analysis discovered that Sp1 and c-myc proteins may play roles in UC early and late stages of neoplastic progression, respectively. Two overexpressed proteins in the non-dysplastic tissue of UC progressors, CPS1 and S100P, were further confirmed by IHC analysis. Our study provides insight into the molecular events associated with UC neoplastic progression, which could be exploited for the development of protein biomarkers in fields of non-dysplastic mucosa that identify a patient's risk for UC dysplasia.
Proceedings of the National Academy of Sciences, 2009
Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulat... more Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.
Biomarkers that detect pancreatic cancer at earlier stages could improve the outcome of this dead... more Biomarkers that detect pancreatic cancer at earlier stages could improve the outcome of this deadly disease. We investigated a dozen biomarker candidates for their potential as pancreatic cancer blood biomarkers using enzyme-linked immunosorbent assays. Among them, the macrophage migration inhibitory factor and osteopontin blood tests were nearly perfect in distinguishing pancreatic cancer cases from healthy controls (100% and 95% sensitivity, respectively, at 100% specificity). Five biomarker candidates were then tested on an expanded set of diseased controls, which included sera from patients with pancreatitis. The sensitivity dropped significantly for all 5 candidate markers. Our results suggest that biomarker candidates could fail in various steps of biomarker development. Earlier knowledge of candidate biomarker flaws could lead to strategies to overcome the flaw or alternatively lead to earlier termination of biomarkers that are prone to failure in the later phases of validation testing.
The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early s... more The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.
We report a large pedigree in which pancreatic cancer is inherited in an autosomal dominant fashi... more We report a large pedigree in which pancreatic cancer is inherited in an autosomal dominant fashion. Diabetes and exocrine insufficiency was observed in all family members who eventually developed pancreatic cancer. The presence of diabetes, often years before the diagnosis of cancer, allowed identification of those people who had inherited the predisposing allele and who were thus at high risk for the development of malignancy. This family shows that genetic factors can have a striking effect on the development ofpancreatic malignancy and diabetes mellitus. Moreover, preclinical diagnosis of pancreatic cancer in family members provided a unique opportunity to study early molecular changes that accompany the development of human pancreatic cancer.
Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-... more Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fisher's exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer.
Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatm... more Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. Methods: To systematically study protein expression in pancreatic cancer, we used isotopecoded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. Results: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. Conclusions: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.
Patients with pancreatic cancer are usually diagnosed at late stages, when the disease is incurab... more Patients with pancreatic cancer are usually diagnosed at late stages, when the disease is incurable. Pancreatic intraepithelial neoplasia (PanIN) 3 is believed to be the immediate precursor lesion of pancreatic adenocarcinoma, and would be an ideal stage to diagnose patients, when intervention and cure are possible and patients are curable. In this study, we used quantitative proteomics to identify dysregulated proteins in PanIN 3 lesions. Altogether, over 200 dysregulated proteins were identified in the PanIN 3 tissues, with a minimum of a 1.75-fold change compared with the proteins in normal pancreas. These dysregulated PanIN 3 proteins play roles in cell motility, the inflammatory response, the blood clotting cascade, the cell cycle and its regulation, and protein degradation. Further network analysis of the proteins identified c-MYC as an important regulatory protein in PanIN 3 lesions. Finally, three of the overexpressed proteins, laminin beta-1, galectin-1, and actinin-4 were validated by immunohistochemistry analysis. All three of these proteins were overexpressed in the stroma or ductal epithelial cells of advanced PanIN lesions as well as in pancreatic cancer tissue. Our findings suggest that these three proteins may be useful as biomarkers for advanced PanIN and pancreatic cancer if further validated. The dysregulated proteins identified in this study may assist in the selection of candidates for future development of biomarkers for detecting early and curable pancreatic neoplasia.
Microsatellite instability (MIN) has been detected in many cancer types; however, recently we als... more Microsatellite instability (MIN) has been detected in many cancer types; however, recently we also observed it in the nonneoplastic but inflammatory setting of pancreatitis. Consequently, we sought to examine whether MIN was present in another inflammatory condition, ulcerative colitis (UC). MIN was found in 50% of UC patients whose colonie mucosa was negative for dysplasia, 46% of those with high-grade dysplasia, and 40% of those with cancer but in none of the nine ischemie or infectious colitis controls (P < 0.03). Thus, UC patients may have MIN within mucosa that has no histológica! evidence of neoplastic change. MIN in this setting may reflect the inability of DNA repair mechanisms to compensate for the stress of chronic inflammation, and may be one mechanism for the heightened neoplastic risk in UC.
Patients with extensive ulcerative colitis (UC) of longer than 8 years duration are at high risk ... more Patients with extensive ulcerative colitis (UC) of longer than 8 years duration are at high risk for the development of colorectal cancer. The cancers in these patients appear to develop in a stepwise manner with progressive histological changes from negative for dysplasia 3 indefinite for dysplasia 3 dysplasia 3 cancer. The aim of this study was to determine the timing and extent of genomic instability in the progression of UC dysplasia and cancer. Using two polymerase chain reaction (PCR)-based DNA fingerprinting methods, arbitrarily primed PCR and intersimple sequence repeat PCR, we assessed DNA sequence variation in biopsies across the spectrum of cancerous, dysplastic, and nondysplastic mucosa. UC patients with dysplasia/cancer had substantial genomic instability in both their dysplastic and nondysplastic colonic mucosa, whereas instability was not present in the majority of UC patients without dysplasia/cancer. The degree of instability in nondysplastic tissue was similar to that of dysplastic/cancerous mucosa from the same patient, suggesting that this instability was widespread and reached the maximum level early in neoplastic progression. These results suggest that UC patients who develop dysplasia or cancer have an underlying process of genomic instability in their colonic mucosa whereas UC patients who are dysplasia-free do not. (Am J Pathol 2003, 162:665-672)
Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its... more Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: &lt; 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.
No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to ... more No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to undergo neoplastic progression. Recognition of this subset would advance UC cancer surveillance by focusing the available management options onto the highest risk patients. Three different assays of genomic alterations in nondysplastic UC biopsies show promise for distinguishing patients with neoplasia (UC progressors) from those without (UC nonprogressors), including assays of telomere length, anaphase bridges, and chromosomal fluorescence in situ hybridization. Expanding the number of patients and testing of assays simultaneously in the same biopsy further validated their utility. A panel approach also improved testing outcome. A total of 14 UC progressors was readily separable from 15 UC nonprogressors and 6 normal controls. Chromosomal entropy (ie, the extent of alteration diversity) proved to be the most useful test. By receiver-operating characteristic analysis, mean chromosomal entropy in 28 patients over all four chromosomes yielded 100% sensitivity and 92% specificity for distinguishing progressors from nonprogressors with optimum choice of threshold. Moreover, separation was achieved using only nondysplastic and predominantly rectal (82.8%) biopsies that were remote from neoplasia, suggesting that full colonoscopy with extensive biopsies might be avoided for the majority of UC patients, the nonprogressors. These data further strengthen the concept that genomic biomarkers can distinguish UC progressors from nonprogressors and improve cancer sur-veillance in UC.
World journal of gastroenterology : WJG, Jan 7, 2014
To characterize tumor necrosis factor receptor-associated protein 1 (TRAP1) expression in the pro... more To characterize tumor necrosis factor receptor-associated protein 1 (TRAP1) expression in the progression of ulcerative colitis (UC)-associated colorectal cancer. Chronic UC is an inflammatory bowel disease that predisposes to colorectal cancer. Immunohistochemical analysis was used to evaluate TRAP1 expression on tissue microarrays containing colonic tissues from 42 UC progressors (patients with cancer or dysplasia) and 38 non-progressors (dysplasia/cancer free patients). Statistical analyses of the TRAP1 immunohistochemistry staining were performed using GraphPad Prism. Differences in the TRAP1 level between non-progressors and progressors were tested for statistical significance using the Mann-Whitney test. Receiver operating characteristic curve method was used to quantify marker performance in distinguishing diseased cases from controls. TRAP1 was up-regulated in the colon tissues from UC progressors, but not in the colon tissues from UC non-progressors. Moreover, up-regulation...
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. Howev... more Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with a dismal prognosis. However, while most patients die within the first year of diagnosis, very rarely, a few patients can survive for &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;10 years. Better understanding the molecular characteristics of the pancreatic adenocarcinomas from these very-long-term survivors (VLTS) may provide clues for personalized medicine and improve current pancreatic cancer treatment. To extend our previous investigation, we examined the proteomes of individual pancreas tumor tissues from a group of VLTS patients (survival ≥10 years) and short-term survival patients (STS, survival &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;14 months). With a given analytical sensitivity, the protein profile of each pancreatic tumor tissue was compared to reveal the proteome alterations that may be associated with pancreatic cancer survival. Pathway analysis of the differential proteins identified suggested that MYC, IGF1R and p53 were the top three upstream regulators for the STS-associated proteins, and VEGFA, APOE and TGFβ-1 were the top three upstream regulators for the VLTS-associated proteins. Immunohistochemistry analysis using an independent cohort of 145 PDAC confirmed that the higher abundance of ribosomal protein S8 (RPS8) and prolargin (PRELP) were correlated with STS and VLTS, respectively. Multivariate Cox analysis indicated that &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;High-RPS8 and Low-PRELP&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; was significantly associated with shorter survival time (HR=2.69, 95% CI 1.46-4.92, P=0.001). In addition, galectin-1, a previously identified protein with its abundance aversely associated with pancreatic cancer survival, was further evaluated for its significance in cancer-associated fibroblasts. Knockdown of galectin-1 in pancreatic cancer-associated fibroblasts dramatically reduced cell migration and invasion. The results from our study suggested that PRELP, LGALS1 and RPS8 might be significant prognostic factors, and RPS8 and LGALS1 could be potential therapeutic targets to improve pancreatic cancer survival if further validated.
Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk o... more Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma1 that is thought to develop through genomic instability 2 . We considered that the rapid cell turnover and oxidative injury observed in ulcer- ative colitis might accelerate telomere shortening 3 , thereby increasing the potential of chromosomal ends to fuse4, resulting in
Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk o... more Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma 1 that is thought to develop through genomic instability 2 . We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening 3 , thereby increasing the potential of chromosomal ends to fuse 4 , resulting in cycles of chromatin bridge breakage and fusion 5,6 and chromosomal instability associated with tumor cell progression 7,8 . Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges-an intermediate in the breakage and fusion of chromatin bridges 9 -in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.
Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Be... more Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon, including the non-dysplastic mucosa; we hypothesized that the same field defect will include abnormally expressed proteins. Here we applied proteomics to study the protein expression of UC neoplastic progression. The protein profiles of colonic epithelium were compared from 1) UC patients without dysplasia (non-progressors); 2) none-dysplastic colonic tissue from UC patient with highgrade dysplasia or cancer (progressors); 3) high-grade dysplastic tissue from UC progressors and 4) normal colon. We identified protein differential expression associated with UC neoplastic progression. Proteins relating to mitochondria, oxidative activity, calcium-binding proteins were some of interesting classes of these proteins. Network analysis discovered that Sp1 and c-myc proteins may play roles in UC early and late stages of neoplastic progression, respectively. Two overexpressed proteins in the non-dysplastic tissue of UC progressors, CPS1 and S100P, were further confirmed by IHC analysis. Our study provides insight into the molecular events associated with UC neoplastic progression, which could be exploited for the development of protein biomarkers in fields of non-dysplastic mucosa that identify a patient's risk for UC dysplasia.
Proceedings of the National Academy of Sciences, 2009
Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulat... more Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.
Biomarkers that detect pancreatic cancer at earlier stages could improve the outcome of this dead... more Biomarkers that detect pancreatic cancer at earlier stages could improve the outcome of this deadly disease. We investigated a dozen biomarker candidates for their potential as pancreatic cancer blood biomarkers using enzyme-linked immunosorbent assays. Among them, the macrophage migration inhibitory factor and osteopontin blood tests were nearly perfect in distinguishing pancreatic cancer cases from healthy controls (100% and 95% sensitivity, respectively, at 100% specificity). Five biomarker candidates were then tested on an expanded set of diseased controls, which included sera from patients with pancreatitis. The sensitivity dropped significantly for all 5 candidate markers. Our results suggest that biomarker candidates could fail in various steps of biomarker development. Earlier knowledge of candidate biomarker flaws could lead to strategies to overcome the flaw or alternatively lead to earlier termination of biomarkers that are prone to failure in the later phases of validation testing.
The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early s... more The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.
We report a large pedigree in which pancreatic cancer is inherited in an autosomal dominant fashi... more We report a large pedigree in which pancreatic cancer is inherited in an autosomal dominant fashion. Diabetes and exocrine insufficiency was observed in all family members who eventually developed pancreatic cancer. The presence of diabetes, often years before the diagnosis of cancer, allowed identification of those people who had inherited the predisposing allele and who were thus at high risk for the development of malignancy. This family shows that genetic factors can have a striking effect on the development ofpancreatic malignancy and diabetes mellitus. Moreover, preclinical diagnosis of pancreatic cancer in family members provided a unique opportunity to study early molecular changes that accompany the development of human pancreatic cancer.
Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-... more Although gastric cancer is one of the highest mortality cancers in the world, no methods for non-invasive diagnosis have been described. Aberrant hypermethylation, an epigenetic mechanism of inactivation of tumor suppressor genes, is an emerging approach to noninvasive diagnosis of gastric cancer. We have previously shown high prevalence of aberrant hypermethylation of Reprimo (RPRM) a novel tumor suppressor gene, in plasma from gastric cancer but not in plasma from healthy donors (Clin Cancer Res. 2008;14:6264-9). This finding suggested that RPRM promoter methylation could be a sensitive and specific biomarker for the non-invasive detection of gastric cancer. Here we explore in a prospective and blind fashion specificity and sensitivity of RPRM for non-invasive diagnoses of gastric cancer. Methods. We prospectively collect 83 gastric cancer patients from two hospitals and 223 healthy donors from corresponding blood banks. Detection of RPRM was perform by methylation specific PCR (MSP). We perform endoscopy in healthy donors that were RPRM(+) in plasma. Results. Aberrant hypermethylation of RPRM was identified in 67/74 (90.5%) and 75/79 (94.9%) of tumor and plasma from gastric cancer patients, respectively. Among healthy donors, methylation of RPRM was identified in 29.1% (65 of 223) of tested cases. These differences were statistically significant (p< 0.001 by Fisher's exact test). The sensibility and specificity of RPRM detection in plasma were 89% (95% CI: 81%-98%) and 67% (95% CI: 58%-75%) respectively. We perform endoscopy in 5 RPRM positive healthy donors, and no cancer was found. Only one healthy donor was diagnosed with chronic gastritis. Discussion. Our data indicate that detection of RPRM promoter methylation in cell free plasma DNA is a promissory method for non-invasive detection of gastric cancer. RPRM could be included in the serologic biopsy along with pepsinogen and gastrin 17 not only for the identification of gastric atrophy, the precursor lesion of gastric cancer but also for direct identification of gastric cancer.
Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatm... more Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. Methods: To systematically study protein expression in pancreatic cancer, we used isotopecoded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. Results: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. Conclusions: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.
Patients with pancreatic cancer are usually diagnosed at late stages, when the disease is incurab... more Patients with pancreatic cancer are usually diagnosed at late stages, when the disease is incurable. Pancreatic intraepithelial neoplasia (PanIN) 3 is believed to be the immediate precursor lesion of pancreatic adenocarcinoma, and would be an ideal stage to diagnose patients, when intervention and cure are possible and patients are curable. In this study, we used quantitative proteomics to identify dysregulated proteins in PanIN 3 lesions. Altogether, over 200 dysregulated proteins were identified in the PanIN 3 tissues, with a minimum of a 1.75-fold change compared with the proteins in normal pancreas. These dysregulated PanIN 3 proteins play roles in cell motility, the inflammatory response, the blood clotting cascade, the cell cycle and its regulation, and protein degradation. Further network analysis of the proteins identified c-MYC as an important regulatory protein in PanIN 3 lesions. Finally, three of the overexpressed proteins, laminin beta-1, galectin-1, and actinin-4 were validated by immunohistochemistry analysis. All three of these proteins were overexpressed in the stroma or ductal epithelial cells of advanced PanIN lesions as well as in pancreatic cancer tissue. Our findings suggest that these three proteins may be useful as biomarkers for advanced PanIN and pancreatic cancer if further validated. The dysregulated proteins identified in this study may assist in the selection of candidates for future development of biomarkers for detecting early and curable pancreatic neoplasia.
Microsatellite instability (MIN) has been detected in many cancer types; however, recently we als... more Microsatellite instability (MIN) has been detected in many cancer types; however, recently we also observed it in the nonneoplastic but inflammatory setting of pancreatitis. Consequently, we sought to examine whether MIN was present in another inflammatory condition, ulcerative colitis (UC). MIN was found in 50% of UC patients whose colonie mucosa was negative for dysplasia, 46% of those with high-grade dysplasia, and 40% of those with cancer but in none of the nine ischemie or infectious colitis controls (P < 0.03). Thus, UC patients may have MIN within mucosa that has no histológica! evidence of neoplastic change. MIN in this setting may reflect the inability of DNA repair mechanisms to compensate for the stress of chronic inflammation, and may be one mechanism for the heightened neoplastic risk in UC.
Patients with extensive ulcerative colitis (UC) of longer than 8 years duration are at high risk ... more Patients with extensive ulcerative colitis (UC) of longer than 8 years duration are at high risk for the development of colorectal cancer. The cancers in these patients appear to develop in a stepwise manner with progressive histological changes from negative for dysplasia 3 indefinite for dysplasia 3 dysplasia 3 cancer. The aim of this study was to determine the timing and extent of genomic instability in the progression of UC dysplasia and cancer. Using two polymerase chain reaction (PCR)-based DNA fingerprinting methods, arbitrarily primed PCR and intersimple sequence repeat PCR, we assessed DNA sequence variation in biopsies across the spectrum of cancerous, dysplastic, and nondysplastic mucosa. UC patients with dysplasia/cancer had substantial genomic instability in both their dysplastic and nondysplastic colonic mucosa, whereas instability was not present in the majority of UC patients without dysplasia/cancer. The degree of instability in nondysplastic tissue was similar to that of dysplastic/cancerous mucosa from the same patient, suggesting that this instability was widespread and reached the maximum level early in neoplastic progression. These results suggest that UC patients who develop dysplasia or cancer have an underlying process of genomic instability in their colonic mucosa whereas UC patients who are dysplasia-free do not. (Am J Pathol 2003, 162:665-672)
Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its... more Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: &lt; 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.
No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to ... more No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to undergo neoplastic progression. Recognition of this subset would advance UC cancer surveillance by focusing the available management options onto the highest risk patients. Three different assays of genomic alterations in nondysplastic UC biopsies show promise for distinguishing patients with neoplasia (UC progressors) from those without (UC nonprogressors), including assays of telomere length, anaphase bridges, and chromosomal fluorescence in situ hybridization. Expanding the number of patients and testing of assays simultaneously in the same biopsy further validated their utility. A panel approach also improved testing outcome. A total of 14 UC progressors was readily separable from 15 UC nonprogressors and 6 normal controls. Chromosomal entropy (ie, the extent of alteration diversity) proved to be the most useful test. By receiver-operating characteristic analysis, mean chromosomal entropy in 28 patients over all four chromosomes yielded 100% sensitivity and 92% specificity for distinguishing progressors from nonprogressors with optimum choice of threshold. Moreover, separation was achieved using only nondysplastic and predominantly rectal (82.8%) biopsies that were remote from neoplasia, suggesting that full colonoscopy with extensive biopsies might be avoided for the majority of UC patients, the nonprogressors. These data further strengthen the concept that genomic biomarkers can distinguish UC progressors from nonprogressors and improve cancer sur-veillance in UC.
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Papers by David Crispin