Pan-genomic/Pan-proteomic Approaches to Diseases

2013 Laboratory Investigation  
were Grade 2. The CD45 labeling indices for Grade 1 and 2 tumors were 7.4 ± 19. 7% and 4.7 ± 11.1% (p=0.47). The Ki-67+/CD45+ labeling indices for Grade 1 and 2 tumors were 0.0029 ± 0.10% and 0.60 ± 3.6% (p=0.45). Conclusions: These results suggest that tumor-infiltrating lymphocytes did not affect the Ki-67 labeling index in this series of PanNETs, and will require validation with a larger patient population. 1802 MicroRNAs as Diagnostic Markers Background: Since the discovery of small
more » ... ng RNAs, the analysis of microRNA (miRNA) expression patterns in human cancer have provided new insights into cancer biology. Evidence suggests that deregulated miRNA expression is associated with pancreatic cancer development. In this study, we analyzed the expression of several miRNAs in different types of pancreatic disease to determine if miRNAs expression could aid the diagnosis of pancreatic ductal adenocarcinoma (PDAC) and its precursor -pancreatic intraepithelial neoplasm (PanIN). Design: Resection specimens containing PDAC (n=16), paired pancreatic tissue with negative margin in each case (n=16), chronic pancreatitis (n= 4), normal pancreatic parenchyma (n=5), PanIN (n=5) with different grade of dysplasia (I to III) were selected from our department archive between 2004 and 2011. These formalin-fixed paraffin embedded tissue blocks were evaluated for miR-148a, miR-196 and miR-217 expression by quantitative reverse transcription polymerase chain reaction. Results: Our data show that miR-148a and miR-217 expression levels were significantly down-regulated in PanIN and PDAC compared to the normal pancreatic parenchyma. Comparison of these miRNA levels between the neoplastic lesions and chronic pancreatitis showed that miR-148a (P < 0.05) and miR-217 levels (P < 0.0001) were much lower in PDAC. Dramatic reduction of expression levels in PDAC was further confirmed by comparing their levels in PDAC and paired controls with negative margin which predominantly consists of chronic pancreatitis. In addition, we observed that miR-148a levels were much lower in PanIN than in chronic pancreatitis (P < 0.05). MiR-217 expression level also decreased, although to a lesser extent, in PanIN compared to in chronic pancreatitis (P = 0.09). In contrast, the level of miR-196 expression was significantly overexpressed in PanIN (P<0.0001) and PDAC (P<0.0001) compared to the non-neoplastic pancreatic parenchyma. Interestingly, the degree of deregulation of all three miRNA markers is much higher in PanIN II-III compared to that in PanIN I. Conclusions: Our study demonstrates that miR-148a, miR-217 and miR-196a are significantly deregulated in pancreatic ductal adenocarcinoma, including in the early stage of the pancreatic ductal adenocarcinoma. Thus, these markers can be potentially used as diagnostic markers to distinguish pancreatic ductal adenocarcinoma and its precursor from benign lesions. Background: Intraductal tubulopapillary neoplasm (ITPN) is composed of tubulopapillary masses with high-grade atypia in the pancreatic duct. Intraductal tubular adenoma (ITA) is composed of tubular glands mimicking pyloric glands with low-grade atypia. Some may consider that ITA could be a benign counter part of ITPN, however, whether ITPN and ITA are distinctive or not is not fully assessed. In this study, we compared molecular features between ITPNs and ITAs to know their distinct or similar nature. Design: Formalin-fixed, paraffin-embedded tissues of 14 ITPNs and 15 ITAs were investigated in this study. Foci of tumor and normal tissues were dissected separately from serial sections under microscopic guidance. Genomic DNA was extracted and somatic mutations in exons 10 and 21 of PIK3CA, exons 8 and 9 of GNAS, exons 2 and 3 of KRAS, and exon 15 of BRAF were analyzed. Results: Somatic mutations in PIK3CA were found in 3 of 14 ITPNs (21.4%) but not in any of ITAs (P = 0.0996; Fisher exact test). In contrast, mutations in GNAS were found in none of the ITPNs but were found in 9 of 15 ITAs (60.0%) (P < 0.001; Fisher exact test). Mutations in KRAS were detected in 1 of 14 ITPNs (7.1%) and 12 of 15 ITAs (80.0%) (P<0.001; Fisher exact test). BRAF mutation was found in 1 ITPNs but none of ITAs. Conclusions: These results clearly indicate that ITPNs and ITAs are molecularly distinctive from each other, which suggests that ITPNs do not evolve from ITAs. Furthermore, the molecular features of ITAs were similar to those reported in IPMNs, i.e., prevailed mutations in GNAS and KRAS, which is consistent with the renewed (2010) World Health Organization classification system for intraductal neoplasms of the pancreas that describes that intraductal neoplasms are classified into IPMNs and ITPNs, and ITA is a variant of IPMNs. Background: Pancreatic cystic lesions constitute a broad spectrum of entities ranging from non-mucinous to mucinous and benign to malignant cysts. Endoscopic ultrasound -guided fine needle aspiration (EUS-FNA) cytology is routinely utilized for pre-operative diagnosis. Cyst fluid obtained from EUS-FNA is commonly submitted for chemical analysis of tumor markers to aid in the diagnosis. However, the value of cytologic interpretation is frequently limited by low cellularity of aspirated fluid and the reliability of cyst fluid tumor marker is variable. Recently, molecular tests of cyst fluid have been applied and show high specificity in the differentiation of the pancreatic cystic lesions. In this study, we investigated the usefulness of molecular analysis of pancreatic cyst fluid in the setting of inconclusive cytology and non-contributory tumor markers test. Design: We retrospectively reviewed pancreatic cystic lesions with EUS-FNA cytology, cyst fluid tumor markers (CEA and amylase) and molecular tests (K-ras mutation and loss of heterozygocity) from 2009-2011. The inconclusive cytology diagnosis was defined as "non-diagnostic" or "atypical cytology". Non-contributory cyst fluid chemical analysis includes 2 situations: 1) amylase<250U/L and CEA <192 ng/ml or 2) amylase >250 U/L and CEA >192ng/ml. The molecular tests and the histopathologic results of the resection specimens were analyzed. Results: The cohort consists of 32 pancreatic cyst EUS-FNA cytology cases, which shows 20 cases (62.5%) with non-diagnostic (34.4%) or atypical (28.1%) cytology. Among the 20 cases, non-contributory tumor marker analysis was found in 11 cases (55%). The molecular analysis shows K-ras mutation or loss of heterozygocity in 5 out of 11 cases (45%), which also indicates a mucinous cystic neoplasm with potential for neoplastic progression. Interestingly, 3 out of 4 pancreatic cyst resection specimens showed pancreatic intraepithelial neoplasias (PanIN)-1A or 1B with benign molecular features; while elevated cystic fluid CEA and amylase were identified in all the cases with PanIN-1A and 1B. Conclusions: Inconclusive pancreatic cyst cytology and non-contributory cystic fluid tumor markers were frequently encountered, making accurate diagnosis challenging. Our study indicates that cyst fluid molecular tests for K-ras mutation and allelic imbalance provide extra information and may help with the diagnosis and clinical management. Our novel finding is that PanIN-1A and 1B might be related to both elevated cyst fluid CEA and amylase without K-ras mutation. Background: MicroRNAs (miRNAs) are highly conserved RNAs that serve as master regulators of gene expression. They are exciting biomarkers and therapeutic targets. Studies have found altered expression of miRNAs in tissue disease states; however, the interpretation of these studies may be fundamentally flawed because the changing cellular composition of tissues in disease states is not adequately accounted for. Techniques to control for this source of variability should increase the relevance of changes in miRNA species identified in profiling studies of diseased tissue. Design: Our approach characterizes both factors that can affect miRNA expression levels: altered cellular composition of tissue and disease specific expression changes. We have utilized miRNA expression profiles from a panel of cell types to deconvolute observed miRNA profiles generated from various tissues. We have then explored the impact of this two factor analysis via a modeling study of ulcerative colitis. Results: We utilized publicly available data from 16 cell types including inflammatory, endothelial, stromal, and epithelial cells to predict the cellular composition and model the observed expression of the miRNA expression profile from 4 tissues (Fig. 1A) . The predicted cellular compositions are consistent with the expected composition of these tissues and the modeled miRNA levels are highly correlated to the observed signals. We next performed a modeling experiment of ulcerative colitis using image analysis to predict cellular ratio changes (Fig. 1B) . We generated hypothetical miRNA profiling data of normal colon and ulcerative colitis in which we manually altered only 3 miRNAs at the cell level -potential disease specific changes. We then analyzed the data via routine univariate hypothesis testing with and without correction for changes in tissue composition. Without tissue composition correction, most miRNAs identified are false positives (Fig. 1C) .
doi:10.1038/labinvest.2013.32 fatcat:rfx7n7yfync3rbiqdd4mb5dvze