Colon Cancer Samples Research Articles

Variable Levels of Long Noncoding RNA Expression in DNA Mismatch Repair-Proficient Early-Stage Colon Cancer.

Long noncoding RNAs (lncRNAs) have been suggested to be biomarkers for diagnosis and prognosis of sporadic colorectal cancer. This study aimed to characterize the expression profile of lncRNAs in DNA mismatch repair-proficient (pMMR) early-stage colon cancer (CC). The microsatellite instability (MSI) status was examined by a multiplex PCR. The expression of lncRNA and mRNA was analyzed by microarrays. The differentially expressed lncRNAs and mRNAs were determined by bioinformatic analyses and validated in 44 CC samples and 32 non-tumor colonic specimens by quantitative real-time polymerase chain reaction (qRT-PCR). We found that 16 out of 67 CC had MSI-L CC and 7 with MSI-H. In comparison with that in five non-tumor colonic samples, microarray indicated that 1492 lncRNAs and 1639 mRNAs were upregulated while 1804 lncRNAs and 1073 mRNAs downregulated in four pMMR early-stage CC. Bioinformatic analyses revealed that the differentially expressed mRNAs were involved in the process of cell division, angiogenesis, apoptotic, differentiation, the PI3K-Akt/p53/TNF pathways and others. The co-expression lncRNA and mRNA networks indicated five hot spots with significantly high co-expression degrees. Further quantitative RT-PCR revealed that 4 out of 6 lncRNAs were significantly upregulated while the other 2 lncRNAs were downregulated in the CC. Stratification analysis demonstrated that 5 out of 6 lncRNAs were significantly associated with TNM stage and/or distant metastasis in this population. Differentially expressed lncRNAs were significantly associated with clinical features of patients with pMMR CC and may participate in the tumorigenesis of pMMR CC.

Characterization of the number and site of APC mutations in sporadic colorectal cancer.

630 Background: Truncating mutations in the adenomatous polyposis coli ( APC) gene are well-described events in the carcinogenesis of colorectal carcinomas (CRC) and may impact one or both APC alleles. These aberrations often fall within the mutation cluster region (MCR) of the APC gene to preserve a “just right” number of beta-catenin binding sites in the resulting mutant APC protein. Further clinical and genotypic characterization of CRCs based on number and site of mutations in APC determined using modern next generation sequencing methods is needed. Methods: Next generation sequencing of 70 CRC tumors was performed at a single institution via UNCseq to determine mutations in a panel of 247 oncogenes and tumor suppressors, including APC. RNASeq, DNA sequencing, and clinical characteristics from 224 colon and rectal cancer samples in The Cancer Genome Atlas (TCGA) project were also obtained. Results: In the UNCseq cohort, 58 patients (83%) had at least one inactivating APC mutation, and 33 (47%) had two mutations. Of those with at least one mutation, 81% had a mutation in the MCR (residues 1281-1556), but only 5/33 (15%) with two mutations had both in the MCR. In the TCGA cohort, 162 (72%) had at least one inactivating APC mutation, and 52 (23%) had two mutations. Of those with at least one mutation, 59% had a mutation in the MCR, but only 3/52 (6%) with two mutations had both in the MCR. Gene expression of APC was higher in those with no APC mutations vs. 1-2 mutations (p = 0.015), but was not significantly different between those with one vs. two mutations (p = 0.29). The absence of APC mutations was associated with microsatellite instability (p < 0.001) and with right-sided primary tumors (p = 0.001 by chi-square). Conclusions: CRCs have high frequency of biallelic APC mutations, and the majority of tumors with APC mutations had a single mutation within the MCR region of the APC gene. These genotypic factors may impact tumor biology and clinical features.

The Transcriptional Activity of LAMP3 Gene Involved in Autophagocytosis in Colorectal Cancer LAMP3 Expression in Colorectal Cancer

Background: Autophagocytosis is a biological process involving the controlled distribution of cell fragments and organelles in order to obtain an additional source of energy. LAMP3 (lysosome-associated membrane protein 3) is a heavily glycosylated integral membrane protein located mainly in the lysosomal membrane. Recent studies have shown that it participates in tumor metastasis and drug resistance. Its main role is contribution to tumor cells proliferation, migration and invasion. The aim of this study was to determine changes in transcriptional activity of LAMP3 at various stages of colorectal cancer. In addition, an attempt was made to select miRNAs potentially regulating LAMP3 expression using bioinformatic databases. Methods: The study was conducted on healthy colon samples and colon cancer samples in four stages. Molecular analysis included the extraction of total RNA, purification of the obtained extracts, expression profile analysis using oligonucleotide microarray technique and in silico determination of miRNAs potentially regulating the LAMP3 expression. Results: The level of LAMP3 expression is higher in colorectal adenocarcinoma cells than in non-cancerous cells and depends on the stage of the disease. Conclusions: LAMP3 may promote cancer progression, metastasis and cause the resistance to treatment.

Decreased expression of MS4A12 inhibits differentiation and predicts early stage survival in colon cancer.

Lack of normal differentiation was considered as a common defect in cancer cells. MS4A12, a colon-specific gene, belongs to MS4A family that plays an important role in differentiation, proliferation and cell cycle regulation. The aim of the study was to investigate MS4A12 role in colon cancer cell differentiation and its prognostic value in colon cancer. We used sodium butyrate (BS) to set up differentiated model of colon cancer cell line LoVo. Cell differentiation was evaluated with ALP activity and E-cadherin. We used BS (4 mmol/L) inducing differentiation of LoVo cell and found after BS treated over 48h MS4A12 variant-1 (one of MS4A12 gene transcripts) as well as ALP and E-cadherin of LoVo cells were all increased significantly. When silence MS4A12 variant-1, the elevation of ALP and E-cadherin in BS-treated cells were all inhibited. Besides, after silence MS4A12 variant-1, the cells showed significant resistances to BS function of induction cell cycle arrest and apoptosis. Survival analysis used GEO datasets GSE39582 and GSE38832 that include 681 distinct colon cancer samples. Log-rank test and Cox's proportional hazards regression were applied to analyzing single and multiple prognostic variables, respectively. In early stage colon cancer, the patients with low MS4A12 expression had a poor survival (HR=1.72; p=0.036), while in advanced stage colon cancer MS412 had little prognostic value (HR=0.89; p=0.601). These results indicated MS4A12 might relate to colon cancer cell differentiation and supposed to be a risk classification marker for early stage colon cancer.

MicroRNA-98 Suppress Warburg Effect by Targeting HK2 in Colon Cancer Cells.

Warburg effect is a hallmark of cancer cells. Accumulating evidence suggests that microRNAs (miRs) could regulate such metabolic reprograming. Aberrant expression of miR-98 has been observed in many types of cancers. However, its functions and significance in colon cancer remain largely elusive. To investigate miR-98 expression and the biological functions in colon cancer progression. miR-98 expression levels were determined by quantitative RT-PCR in 215 cases of colon cancer samples. miR-98 mimic or inhibitor was used to test the biological functions in SW480 and HCT116 cells, followed by cell proliferation assay, lactate production, glucose uptake, and cellular ATP levels assay and extracellular acidification rates measurement. Western blot and luciferase assay were used to identify the target of miR-98. miR-98 was significantly down-regulated in colon cancer tissues compared to adjacent colon tissues and acted as a suppressor for Warburg effect in cancer cells. miR-98 inhibited glycolysis by directly targeting hexokinase 2, or HK2, illustrating a novel pathway to mediate Warburg effect of cancer cells. In vitro experiments further indicated that HK2 was involved in miR-98-mediated suppression of glucose uptake, lactate production, and cell proliferation. In addition, we detected HK2 expression in colon cancer tissues and found that the expressions of miR-98 and HK2 were negatively correlated. miR-98 acts as tumor suppressor gene and inhibits Warburg effect in colon cancer cells, which provided potential targets for clinical treatments.

A 15-gene signature for prediction of colon cancer recurrence and prognosis based on SVM

ObjectiveTo screen the gene signature for distinguishing patients with high risks from those with low-risks for colon cancer recurrence and predicting their prognosis. MethodsFive microarray datasets of colon cancer samples were collected from Gene Expression Omnibus database and one was obtained from The Cancer Genome Atlas (TCGA). After preprocessing, data in GSE17537 were analyzed using the Linear Models for Microarray data (LIMMA) method to identify the differentially expressed genes (DEGs). The DEGs further underwent PPI network-based neighborhood scoring and support vector machine (SVM) analyses to screen the feature genes associated with recurrence and prognosis, which were then validated by four datasets GSE38832, GSE17538, GSE28814 and TCGA using SVM and Cox regression analyses. ResultsA total of 1207 genes were identified as DEGs between recurrence and no-recurrence samples, including 726 downregulated and 481 upregulated genes. Using SVM analysis and five gene expression profile data confirmation, a 15-gene signature (HES5, ZNF417, GLRA2, OR8D2, HOXA7, FABP6, MUSK, HTR6, GRIP2, KLRK1, VEGFA, AKAP12, RHEB, NCRNA00152 and PMEPA1) were identified as a predictor of recurrence risk and prognosis for colon cancer patients. ConclusionOur identified 15-gene signature may be useful to classify colon cancer patients with different prognosis and some genes in this signature may represent new therapeutic targets.

Adequately defining tumor cell proportion in tissue samples for molecular testing improves interobserver reproducibility of its assessment.

Gene mutation status assessment of tumors has become standard practice in diagnostic pathology. This is done using samples comprising tumor cells but also non-tumor cells, which may dramatically dilute the proportion of tumor DNA and induce false negative results. Increasing sensitivity of molecular tests presently allows detection of a targeted mutation in a sample with a small percentage of tumor cells, but assessment of tumor cellularity remains essential to adequately interpret the results of molecular tests. Comprehensive tumor cell counting would provide the most reliable approach but is time consuming, and therefore rough global estimations are used, the reliability of which has been questioned in view of their potential clinical impact. The French association for quality assurance in pathology (AFAQAP) conducted two external quality assurance schemes, partly in partnership with the French group of oncology cytogenomics (GFCO). The purpose of the schemes was to (1) evaluate how tumor cellularity is assessed on tissue samples, (2) identify reasons for discrepancies, and (3) provide recommendations for standardization and improvement. Tumor cell percentages in tissue samples of lung and colon cancer were estimated by 40-50 participants, on 10 H&E virtual slides and 20 H&E conventional slides. The average difference between lowest and highest estimated percentage was 66. This was largely due to inadequate definition of cellularity, reflecting confusion between the percentage of tumor cells and the percentage of the area occupied by tumor in the assessed region. The widest range of interobserver variation was observed for samples with dense or scattered lymphocytic infiltrates or with mucinous stroma. Estimations were more accurate in cases with a low percentage of tumor cells. Macrodissection of the most homogeneous area in the tissue reduced inter-laboratory variation. We developed a rating system indicating potential clinical impact of a discrepancy. Fewer discrepancies were clinically relevant since the study was conducted. Although semi-quantitative estimations remain somewhat subjective, their reliability improves when tumor cellularity is adequately defined and heterogeneous tissue samples are macrodissected for molecular analysis.

Protein and glycomic plasma markers for early detection of adenoma and colon cancer

ObjectiveTo discover and confirm blood-based colon cancer early-detection markers.DesignWe created a high-density antibody microarray to detect differences in protein levels in plasma from individuals diagnosed with colon cancer <3 years...

Expression of VEGF-D, SMAD4, and SMAD7 and Their Relationship with Lymphangiogenesis and Prognosis in Colon Cancer

AimThe vascular endothelial growth factor (VEGF) and TGF-β1 pathways play important roles in cancer. However, few studies have evaluated the expression and roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, and the conclusions remain controversial. To clarify the roles of VEGF-D, SMAD4, and SMAD7 in colon cancer, we examined their expression and evaluated correlations with lymphangiogenesis, prognosis, and chemotherapeutic outcome. MethodsThe expression of VEGF-D, SMAD4, and SMAD7 was immunohistochemically examined in 251 primary colon cancer samples obtained from the Harbin Medical University. ResultsThe expression of VEGF-D, SMAD4, and SMAD7 was identified in 71.7, 41.0, and 69.7 % of samples, respectively. Positive expression of VEGF-D and SMAD7 and lost expression of SMAD4 were significantly correlated with lymph node metastasis and high lymphatic vessel density. VEGF-D and SMAD7 were found to be independent indicators of prognosis and chemotherapy outcome, and positive expression of either VEGF-D or SMAD7 was associated with significantly shorter overall survival and disease-free survival (OS and DFS) than negative expression in all 251 patients (P < 0.001 for OS and DFS) and patients following chemotherapy (P < 0.001 for OS and DFS). ConclusionVEGF-D, SMAD4, and SMAD7 were involved in lymphangiogenesis and lymph node metastasis. VEGF-D and SMAD7 can serve as predictors of prognosis and chemotherapeutic outcome in colon cancer.

Genomic profiling of stage II and III colon cancers reveals APC mutations to be associated with survival in stage III colon cancer patients.

Tumor profiling of DNA alterations, i.e. gene point mutations, somatic copy number aberrations (CNAs) and structural variants (SVs), improves insight into the molecular pathology of cancer and clinical outcome. Here, associations between genomic aberrations and disease recurrence in stage II and III colon cancers were investigated. A series of 114 stage II and III microsatellite stable colon cancer samples were analyzed by high-resolution array-comparative genomic hybridization (array-CGH) to detect CNAs and CNA-associated chromosomal breakpoints (SVs). For 60 of these samples mutation status of APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS was determined using targeted massive parallel sequencing. Loss of chromosome 18q12.1-18q12.2 occurred more frequently in tumors that relapsed than in relapse-free tumors (p < 0.001; FDR = 0.13). In total, 267 genes were recurrently affected by SVs (FDR < 0.1). CNAs and SVs were not associated with disease-free survival (DFS). Mutations in APC and TP53 were associated with increased CNAs. APC mutations were associated with poor prognosis in (5-fluorouracil treated) stage III colon cancers (p = 0.005; HR = 4.1), an effect that was further enhanced by mutations in MAPK pathway (KRAS, NRAS, BRAF) genes. We conclude that among multiple genomic alterations in CRC, strongest associations with clinical outcome were observed for common mutations in APC.

Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

Single-cell SNP analyses and interpretations based on RNA-Seq data for colon cancer research.

Single-cell sequencing is useful for illustrating the cellular heterogeneities inherent in many intricate biological systems, particularly in human cancer. However, owing to the difficulties in acquiring, amplifying and analyzing single-cell genetic material, obstacles remain for single-cell diversity assessments such as single nucleotide polymorphism (SNP) analyses, rendering biological interpretations of single-cell omics data elusive. We used RNA-Seq data from single-cell and bulk colon cancer samples to analyze the SNP profiles for both structural and functional comparisons. Colon cancer-related pathways with single-cell level SNP enrichment, including the TGF-β and p53 signaling pathways, were also investigated based on both their SNP enrichment patterns and gene expression. We also detected a certain number of fusion transcripts, which may promote tumorigenesis, at the single-cell level. Based on these results, single-cell analyses not only recapitulated the SNP analysis results from the bulk samples but also detected cell-to-cell and cell-to-bulk variations, thereby aiding in early diagnosis and in identifying the precise mechanisms underlying cancers at the single-cell level.

Prognostic role of tissue transglutaminase 2 in colon carcinoma.

Tissue transglutaminase 2 (TG2) is involved in many biological processes, from wound healing to neurodegeneration. Recently, there has been an increasing interest in this enzyme as a potential prognostic marker or therapy target in human neoplasms. The aim of this study was to analyze expression of TG2 messenger RNA (mRNA) and protein in colon cancer samples and to evaluate the potential value of TG2 as prognostic marker. We investigated not only expression level but also location of the protein in a series of human tumors. In silico analysis using the GSE39582 dataset showed that TG2 mRNA expression is associated with earlier relapse. The results of qPCR in our cohort showed TG2 mRNA to be up-regulated in 25 out of 70 samples (34%). Kaplan-Meier plots and log-rank test showed that patients with high TG2 mRNA expression have significantly worse prognosis in terms of overall survival (OS) and a trend to earlier recurrence. Immunohistochemical staining of tumor sections for TG2 revealed stromal staining in 152 cases (88%) and epithelial cell staining in 105 cases (62%). In stage II patients, stromal expression showed a significant association with disease-free survival (DFS). In patients with metastatic disease, TG2 expression was also associated with poor prognosis. Cox multivariate analysis showed that TG2 expression in epithelial cells is significantly and independently associated with OS, together with node involvement and presence of metastasis. Stromal TG2 expression was associated with DFS. In summary, in non-metastatic colorectal cancer patients, stromal TG2 expression is significantly associated with DFS and epithelial TG2 expression with OS, independently of node involvement and metastasis.

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer.

The role of adjuvant chemotherapy in stage T2-T3N0 colon cancer (CC) is controversial and there are currently no reliable factors allowing for individual selection of patients with high risk of relapse for such therapy. We searched for microRNA-based signature with prognostic significance in this group. We assessed by qRT-PCR expression of 754 microRNAs (miRNAs) in tumour samples from 85 stage pT2-3N0 CC patients treated with surgery alone. MiRNA expression was compared between two groups of patients: 40 and 45 patients who did and did not develop distant metastases after resection, respectively. Additionally, miRNA expression was compared between CC and normal colon mucosa samples and between the mismatch repair (MMR) competent and deficient tumours. Low expression of miR-1300 and miR-939 was significantly correlated with shorter distant metastasis-free survival (DMFS) in Cox univariate analysis (p.adjusted = 0.049). The expression signature of five miRNAs (miR-1296, miR-135b, miR-539, miR-572 and miR-185) was found to be prognostic [p = 1.28E−07, HR 8.4 (95 % CI: 3.81–18.52)] for DMFS and cross-validated in a leave-one-out analysis, with the sensitivity and specificity of 74 and 78 %, respectively. The expression of miR-592 was significantly associated with the MMR status (p.adjusted <0.01). The expression of several novel miRNAs were found to be tumour specific, e.g. miR-888, miR-523, miR-18b, miR-302a, miR-423-5p, miR-582-3p (p < 0.05). We developed a miRNA expression signature that may be predictive for the risk of distant relapse in early stage CC and confirmed previously reported association between miR-592 expression and MMR status.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-016-9810-1) contains supplementary material, which is available to authorized users.

Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment.

Abstract 5166: Gene expression microarray analysis identifies association of genes in the integrin/fak signaling pathway with claudin-7 in human colon cancer tissue

Abstract The role and mechanism of the integrin/focal adhesion kinase (FAK)/ERK signaling pathway in the progression of many malignant tumor types, including colon cancer, have been widely reported. The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in the regulation of the integrin/FAK/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, in colon cancer and adjacent normal tissues. Quantitative real-time RT-PCR verified the results for mRNA expression, and immunohistochemistry verified the results for protein expression. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway members. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples. The observed changes in the expression of all proteins except Cldn-7 were in agreement with the changes in the corresponding mRNA levels. Cldn-7 was predicted not to directly regulate any genes in the integrin/FAK/ERK signaling pathway, but these factors may participate in a common network that regulates cancer progression, in which the MAPK pathway is the central node. Citation Format: Lu Kong, Lei Ding. Gene expression microarray analysis identifies association of genes in the integrin/fak signaling pathway with claudin-7 in human colon cancer tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5166.

Abstract 3035: Development and characterization of CCK2R-targeted SMDCs and identification of GIST as a potential therapeutic indication

Abstract Cholecystokinin B receptor (CCKBR/CCK2R) is a GPCR of the cholecystokinin receptor family that is primarily expressed in the CNS and the gastrointestinal tract where it binds the ligands cholecystokinin and gastrin, respectively, and mediates their downstream actions. Since it has been reported that CCK2R is overexpressed in certain cancer types, we have explored the potential of utilizing CCK2R as a receptor system to deliver potent therapeutic warheads. To this end, we designed a series of CCK2R-targeted small-molecule drug conjugates (SMDCs) containing either a non-peptidic antagonist (Z-360) or a peptidic agonist (CCK8) as their ligand moiety and explored their activity in in vitro and in vivo models. While EC1812 (Z-360-tubulysin B) failed to elicit either competable cytotoxicity in vitro or robust therapeutic response in vivo (n = 5; 1 PR) when tested against a HEK293-CCK2R overexpressing cell line, the addition of a PEG36 linker to generate EC1977 greatly improved therapeutic efficacy in vivo (n = 5; 1 CR, 4 cures) despite having no activity in vitro. In contrast, while EC1868 (CCK8-tubulysin B) demonstrated potent, competable cytotoxicity in vitro it had only modest activity in vivo (n = 5; 5 CRs). To explore the clinical relevance of a CCK2R-targeted SMDC, we performed mRNA profiling of a human cancer array to identify indications that have high CCK2R mRNA expression. Of all the indications present on the array, those representing gastrointestinal stromal tumor (GIST) were identified as expressing the highest level of CCK2R mRNA. To further elucidate the clinical relevance of this finding, we procured human clinical samples from GIST patients and determined CCK2R-specific radioligand binding and mRNA levels. These data revealed that 50% of GIST samples express high levels of CCK2R mRNA and exhibit functional ligand binding. Interestingly, although 50% of normal stomach samples had similar mRNA levels as the high-expressing GIST samples, they failed to exhibit detectable radioligand binding. In contrast, HCC, colon cancer, and pancreatic cancer samples were all negative for both mRNA expression and ligand binding. Subsequent screening of a series of GIST patient-derived xenograft (PDX) models for functional binding and mRNA levels failed to identify an appropriate model for further in vivo testing. In conclusion, we have developed and characterized a series of CCK2R-targeted SMDCs and tested their activity in vitro and in vivo. Given the expression and functional binding of CCK2R in GIST we propose that GIST may represent an indication for CCK2R-targeted therapies. Citation Format: Jonathan M. Shillingford, Joseph A. Reddy, Melissa Nelson, Marilynn Vetzel, Garth L. Parham, Ryan Dorton, John Guan, Nikki Parker, Haiyan Chu, Iontcho A. Vlahov, Christopher P. Leamon. Development and characterization of CCK2R-targeted SMDCs and identification of GIST as a potential therapeutic indication. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3035.

Abstract 1383: NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues

Abstract Background: HTG Molecular (HTG) developed a targeted Next Generation Sequencing (NGS)-based gene expression assay that measures mRNA levels of 2,560 genes (2,532 oncology-related biomarker genes). The HTG EdgeSeq Oncology Biomarker Panel Assay (OBP) is based on a novel derivative of our quantitative nuclease protection chemistry (qNPA) that enables extraction-free quantitation (detection) of mRNA from variety of sample types including formalin-fixed, paraffin embedded (FFPE) tissue. Purpose: To determine (1) linearity across a range of sample inputs, (2) recommended sample input amounts for FFPE, cells, and extracted RNA, and (3) reproducibility of the HTG EdgeSeq Oncology Biomarker Panel Assay in measuring the mRNA expression of 2,560 genes. Methods: Lysates of 5 micron sections of FFPE tissues (lung, breast, prostate, and colon carcinoma; melanoma; 25 mm2 to 0.78 mm2 per reaction), THP-1 and HCC78 cell lines (7500 cells to 234 cells per reaction), and Universal RNA (URNA; 25 ng to 1.56 ng per reaction) were used for the linearity and sample input studies. URNA (25 ng per reaction) was used to demonstrate reproducibility of the assay across multiple days and processors (one processor across three days and three processors on one day). Sequencing libraries were generated from the qNPA reactions and run on an Illumina MiSeq sequencing platform. The HTG EdgeSeq Parser was used for post-sequencing data processing. Linear regression (R2) and Pearson correlation coefficients (r) were used to assess linearity and reproducibility of the assay. Results: The R2 for linearity across four concentration points for lung FFPE tissue (6.25-0.78 mm2), cell lines (1875-234 cells), and URNA (12.5-1.56 ng) were &amp;gt;0.97, 0.99, and 0.99, respectively. The (r) between low (1.56 mm2) and high (12.5 mm2) sample inputs for each FFPE tissue type was &amp;gt; 0.98. The (r) for intra-run, inter-day, and inter-run reproducibility were &amp;gt; 0.95, &amp;gt; 0.98, and &amp;gt; 0.98 respectively. In addition, differential expression of tissue-specific genes was identified in the respective FFPE tissues, including NKX2 and MUC1 in lung, ERBB2 in breast, NKX3, KLK2, and KLK3 in prostate, and SPP1 and PRAME in melanoma. Conclusions: The HTG EdgeSeq Oncology Biomarker Panel Assay for a 2,560-gene panel of oncology-related biomarkers is linear over a wide range of sample inputs, can comprehensively analyze very small, clinically relevant tissues, and is highly reproducible. The demonstrated performance of the assay in breast, lung, colon, and prostate cancer and melanoma FFPE samples enables multiplex oncology biomarker profiling of these and other malignant neoplasms. Citation Format: Monica M. Reinholz, Debrah M. Thompson, Ihab Botros, Matt Rounseville, Patrick C. Roche. NGS-based measurement of gene expression of 2560 oncology-related biomarkers in formalin-fixed, paraffin-embedded (FFPE) tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1383.

Abstract 3967: Amplicon-based NGS detects targeted variants in paired tissue and ctDNA samples

Abstract Circulating tumor DNA (ctDNA) in blood stream has been recognized as an essential sample source for non-invasive biopsy of cancer patients. With next generation sequencing (NGS) technology it is possible to detect multiple cancer relevant mutation spots simultaneously. In order to understand the correlation of mutation spectrum between DNA from original solid tumor and DNA from plasma, we analyzed paired DNA samples with amplicon-based NGS. Paired frozen tissue and plasma samples were collected from 17 colon cancer patients under the IRB protocol. Tissue DNA was extracted with NucleoSpin Tissue kit of Clontech. The ctDNA was extracted from 0.5ml of plasma with Quick-cfDNA™ Serum &amp; Plasma Kit from Zymo Research. The library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences, Inc. This panel generates 263 amplicons covering 56 clinically relevant oncology-related genes. Input ctDNA from plasma was 0.5 to 10 ng depending on the sample yield. The sequencing was conducted on Illumina's Miseq with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter. Non-synonymous variant calls by the tissue DNA sequencing were defined by coverage depth (30), frequency (&amp;gt;5%), and forward/reverse balance (≠0). The defined non-synonymous variant calls were searched from variant calls by the paired ctDNA sequencing with the cutoff frequency at 0.1%. In 17 paired colon cancer samples, 86 non-synonymous variant calls were identified by tissue DNA sequencing. The variant calls of tissue DNA samples showed 56 of 86 variants matched variant calls in the paired ctDNA samples. Among 56 variant calls in ctDNA, 44 variant calls were assumed as germline variants according to the 1000 genome reference and similar frequency of the variant call between paired tissue DNA and ctDNA. There were 12 variant calls deemed somatic variant calls by the literature references and the differential frequency of the variant call between paired tissue DNA and ctDNA. The 12 detected somatic variant calls were clustered in seven cases. Twelve somatic variant calls with frequency (%) in tissue and plasma DNA samples are shown as APC S1456 frame shift (41.8 vs 6.17), KRAS G12D(37.0 vs 5.0),TP53 G105R(56.0 vs 11.3), HNF1A P291 frame shift(36.8 vs 2.41), HNF1A P291 frame shift(27.1 vs 1.2) KRAS G12D(23.1 vs 0.3), ERBB4 I377T(8.3 vs 0.5),PTEN L267 frame shift (12.4 vs 0.4), MAKP2K1 L57N(23.9 vs 0.7),TP53 R273C(25.8 vs 0.3), TP53 H178D(12.1 vs 1.1),TP53 T377 frame shift(11.2 vs 0.1). Remaining 30 somatic variant calls were detected only in tissue DNA samples. Our results indicated that the simultaneous detection of somatic variants of multiple genes in ctDNA samples with the amplicon-based DNA sequencing is applicable with variant frequency over 0.1%. The NGS analysis of paired tissue DNA and ctDNA is a reliable evaluation for cancer related mutations in ctDNA. Citation Format: Adam H. Greer, Glenn Mills, Hong Yin. Amplicon-based NGS detects targeted variants in paired tissue and ctDNA samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3967.

Abstract 2159: In vitro characterization of PTAU and FU interactions in colon cancer cells

Abstract Background: Fluorouracil (FU) remains a main anticancer therapy for colorectal cancer (CRC) and other solid tumors. The mechanism of action of FU is associated with inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. High toxicity and wide-spread chemoresistance, however, have limited the utilization of FU-based therapies. Five intracellular enzymes, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), thymidine phosphorylase (TP), and uridine phosphorylase (UP) are key determinants of FU sensitivity or resistance. Combination therapies aimed at improving the bioavailability of FU and reducing host toxicity has been evaluated. Recently, our group developed a new uridine phosphorylase inhibitor, 5-phenylthio-acyclouridine (PTAU) that has shown promise in reducing host toxicity and increasing FU efficacy in vivo. The current studies were to characterize interactions of PTAU and FU in vitro and to assess its effects on key metabolic enzymes in human colon cancer cells. Hypothesis: We hypothesized that inhibition of uridine phosphorylase by PTAU would ameliorate the toxic effects of FU and enhance its efficacy by permitting increased doses of FU. Methods: Three colon cancer cell lines with differing p53 status, HCT116-p53wt (wild-type), HCT116-p53null, and HT-29 (p53-mutant); and a normal colonic epithelial cell line, CRL-1790, were used. Doubling times were determined, and IC50 values for FU were derived from kill curves using MTT assay. Cells were treated for 72 hours with IC50 doses of FU, PTAU (100 μM) or uridine (25 μM), alone or in combination, or with the solvent, DMSO. mRNA levels for DPD, TS, TP, and UP were quantitated by qRT-PCR; for intracellular protein expression, cells were fixed and stained with monoclonal antibodies and analyzed by flow cytometry. Results: FU sensitivity correlated with cell doubling times. Basal expression of FU metabolic enzymes differed between CRC and normal cells. mRNAs for DPD and UP were higher in normal relative to cancer cells, but TS mRNA was higher in cancer cells. FU treatment upregulated mRNA expression of DPD, UP, and TS in cancer cells but not in normal cells. Only in cancer cells, DPD induction by FU was potentiated by PTAU. In contrast, treatment with FU decreased protein levels of DPD, TP, and TS in cancer cells. PTAU induced TS protein expression in cancer cells, an effect that was reduced by co-treatment with FU. Conclusions: FU induces mRNA levels of DPD, UP, and TS in cancer cell lines, but protein levels are reduced; the discrepancy between mRNA and protein levels following FU treatment underscores the need for evaluation of colon cancer samples to assess response to FU therapy. This work was supported by a Supplement of the MSM/TU/UABCCC U54 Partnership grant (3U54-CA118948-09S1) from NIH. Citation Format: Esther A. Suswam, Gaurav Kumar, Hyung-Gyoon Kim, Mohamed Osmar, Mahmoud H. el Kouni, Upender Manne. In vitro characterization of PTAU and FU interactions in colon cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2159.