Endoscopic Biopsy Tissues Research Articles

Transformer-based biomarker prediction from colorectal cancer histology: A large-scale multicentric study

Deep learning (DL) can accelerate the prediction of prognostic biomarkers from routine pathology slides in colorectal cancer (CRC). However, current approaches rely on convolutional neural networks (CNNs) and have mostly been validated on small patient cohorts. Here, we develop a new transformer-based pipeline for end-to-end biomarker prediction from pathology slides by combining a pre-trained transformer encoder with a transformer network for patch aggregation. Our transformer-based approach substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. After training and evaluating on a large multicenter cohort of over 13,000 patients from 16 colorectal cancer cohorts, we achieve a sensitivity of 0.99 with a negative predictive value of over 0.99 for prediction of microsatellite instability (MSI) on surgical resection specimens. We demonstrate that resection specimen-only training reaches clinical-grade performance on endoscopic biopsy tissue, solving a long-standing diagnostic problem.

237. DIFFERENTIAL CLONAL EVOLUTION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA IN RESPONSE TO NEO-ADJUVANT CHEMORADIOTHERAPY

Abstract As a major component of esophageal squamous cell carcinoma (ESCC) treatment regimen, neo-adjuvant chemoradiotherapy (NCRT) affects ESCC genome is largely unknown. Deciphering the evolutionary history of ESCC following NCRT would reveal dynamic mutational processes and enable us identify the key molecular features associated with NCRT-resistance. We performed whole exome sequencing (WES) on matched pre-NCRT endoscopic biopsy tissue (n = 29), post-NCRT cystectomy tumor tissue (n = 30). On the basis of a rigorous evaluation of estimated percentage of the estimated percentage of vital residual tumor cells, 29 pre-NCRT cases were classified into two groups: major response (n = 20) and minor response (n = 9). The computational method PyClone was used to define subclones within each tumor. The method Clonality Inference in Tumors Using Phylogeny (CITUP) was subsequently used to infer phylogenetic relationships among subclones. By comparing the subclonal composition of each patient before and after NCRT, we found that resistant subclones were polyclonal and driver mutations improved the fitness of resistant subclones. The number of driver mutations existed in resistant subclones was positively related to the size of subclones. The most frequent driver genes in resistant subclones identified in our research included TP53, NOTCH1 and a novel gene RYR3. Multi resistant subclones result in the resistance of ESCC genome to NCRT. The fitness of resistant subclones is relevant to the number of driver mutations.

1303P Feasibility of endoscopic ultrasound-guided fine needle biopsy tissue sampling for whole exome sequencing in pancreatic cancer

Since most pancreatic cancers are diagnosed at unresectable stage, endoscopic ultrasound guided fine-needle biopsy (EUS-FNB) is the main method to acquire tissue for diagnosis and molecular profiling. We aimed to report the feasibility of whole exome sequencing (WES) with FNB samples and to find factors related with successful WES.

Development of a Machine Learning Model to Distinguish between Ulcerative Colitis and Crohn’s Disease Using RNA Sequencing Data

Crohn’s disease (CD) and ulcerative colitis (UC) can be difficult to differentiate. As differential diagnosis is important in establishing a long-term treatment plan for patients, we aimed to develop a machine learning model for the differential diagnosis of the two diseases using RNA sequencing (RNA-seq) data from endoscopic biopsy tissue from patients with inflammatory bowel disease (n = 127; CD, 94; UC, 33). Biopsy samples were taken from inflammatory lesions or normal tissues. The RNA-seq dataset was processed via mapping to the human reference genome (GRCh38) and quantifying the corresponding gene models that comprised 19,596 protein-coding genes. An unsupervised learning model showed distinct clusters of four classes: CD inflammatory, CD normal, UC inflammatory, and UC normal. A supervised learning model based on partial least squares discriminant analysis was able to distinguish inflammatory CD from inflammatory UC after pruning the strong classifiers of normal CD vs. normal UC. The error rate was minimal and affected only two components: 20 and 50 genes for the first and second components, respectively. The corresponding overall error rate was 0.147. RNA-seq analysis of tissue and the two components revealed in this study may be helpful for distinguishing CD from UC.

Biportal endoscopic debridement and percutaneous screw fixation technique for spinal tuberculosis: how I do it.

Biportal endoscopy and percutaneous screw fixation are promising techniques that can be applied to treat various degenerative spinal diseases. However, these techniques for spinal tuberculosis have not been reported. Using the biportal endoscopic technique, bilateral decompression, tissue biopsy, and granulation tissue removal were performed using the screw insertion site. Using the percutaneous fixation screw technique, posterior stabilization and sagittal angle restoration were achieved. Paraplegia and radiating pain improved neurologically. Kyphosis was radiologically restored. Spinal tuberculosis (Potts's disease) was histopathologically diagnosed. Minimally invasive endoscopic and percutaneous screw technique can aid the diagnosis and treatment of spinal tuberculosis.

Guidance for diagnosing autoimmune pancreatitis with biopsy tissues.

The biopsy-based diagnosis of autoimmune pancreatitis (AIP) is difficult but is becoming imperative for pathologists due to the increased amount of endoscopic ultrasound-guided biopsy tissue. To cope with this challenge, we propose guidance for the biopsy diagnosis of type 1 AIP. This guidance is for pathologists and comprises three main parts. The first part includes basic issues on tissue acquisition, staining, and final diagnosis, and is intended for gastroenterologists as well. The second part is a practical guide for diagnosing type 1 AIP based on the AIP clinical diagnostic criteria 2018. Inconsistent histological findings, tips for evaluating IgG4 immunostaining and key histological features including the ductal lesion and others are explained. Storiform fibrosis and obliterative phlebitis are diagnostic hallmarks but are sometimes equivocal. Storiform fibrosis is defined as spindle-shaped cells, inflammatory cells and fine collagen fibers forming a flowing arrangement. Obliterative phlebitis is defined as fibrous venous obliteration with inflammatory cells. Examples of each are provided. The third part describes the differentiation of AIP from pancreatic ductal adenocarcinoma (PDAC), focusing on histological features of acinar-ductal metaplasia in AIP, which is an important mimicker of PDAC. This guidance will help standardize pathology reports of pancreatic biopsies for diagnosing type 1 AIP.

Gastric Mucosa Plasma Cells is Unspecific for Diagnosing Helicobacter pylori Infection

Background: A high density of Helicobacter pylori is believed to trigger an accumulation of plasma cells in the gastric mucosa. This accumulation stimulated the production of antibodies, causing high antibodies titer being circulated in the blood. The aims of this study is to identify the correlation between the number of plasma cells and H. pylori density in gastric mucosa.Method: This observational analytic study was performed with cross-sectional approach. The samples were the paraffin blocks which consisted of endoscopic gastric biopsy tissues of chronic gastritis patient in Anatomic Pathology Laboratory Dr. Soetomo General Hospital Surabaya in 2017 period. A total of 30 samples were purposively collected. Endoscopic gastric biopsy tissues were stained by two stains, Haematoxylin-Eosin and Modified Giemsa. The examination was performed by experienced pathologist. The correlation between total plasma cells and H. pylori density in gastric mucosa and the difference of total plasma cells between gastric mucosae with different density of H. pylori were determined.Results: There was no significant correlation between total plasma cells and H. pylori density in gastric mucosa. And there was no significant difference of the number of plasma cells found with different density of H. pylori.Conclusion: The number of plasma cells in the gastric mucosa is unspecific for diagnosing H. pylori infection. Other causes associated with plasma cells need to be assessed in further studies.

Over-Expression of CHD4 Is an Independent Biomarker of Poor Prognosis in Patients with Rectal Cancers Receiving Concurrent Chemoradiotherapy.

Neoadjuvant concurrent chemoradiotherapy (CCRT), followed by radical proctectomy, is the standard treatment for locally advanced rectal cancer. However, a poor response and therapeutic resistance continue to occur despite this treatment. In this study, we analyzed the microarray datasets (GSE68204) of rectal cancer from the Gene Expression Omnibus database, and identified CHD4 as one of the most significantly up-regulated genes among all subunits of the nucleosome remodeling and histone deacetylation (NuRD) complex, in non-responders to CCRT, among locally advanced rectal cancer (LARC) patients. We confirmed the predictive and prognostic significance of CHD4 expression in CCRT treatment, and its correlation with other clinicopathological features, such as tumor regression grade (TRG), therapeutic response, and patient survival. This was carried out by immunohistochemical studies on endoscopic biopsy tissues from 172 rectal cancer patients, receiving neoadjuvant concurrent chemoradiotherapy (CCRT). A high expression of CHD4 was significantly associated with pre-treatment tumor status (p < 0.001) and lymph node metastasis (p < 0.001), post-treatment tumor status (p < 0.001), and lymph node metastasis (p < 0.001), vascular invasion (p = 0.042), and tumor regression grade (p = 0.001). A high expression of CHD4 could also predict poor disease-specific survival and metastasis-free survival (log-rank test, p = 0.0373 and p < 0.0001, respectively). In multivariate Cox proportional-hazards regression analysis, CHD4 overexpression was an independent factor of poor prognosis for metastasis-free survival (HR, 4.575; 95% CI, 1.717–12.192; p = 0.002). By in vitro studies, based on cell line models, we also demonstrated that, the overexpression of CHD4 induced radio-resistance in microsatellite instability-high (MSI-H) colorectal cells (CRCs). On the contrary, the knockdown of CHD4 enhanced radiosensitivity in microsatellite stable (MSS) CRCs. Altogether, we have identified CHD4 as an important regulator of radio-resistance in both MSI-H and MSS CRC cell lines.

A Comparison of Homogenization vs. Enzymatic Lysis for Microbiome Profiling in Clinical Endoscopic Biopsy Tissue Samples

Identification of the human microbiome has proven to be of utmost importance with the emerging role of bacteria in various physiological and pathological processes. High throughput sequencing strategies have evolved to assess the composition of the microbiome. To identify possible bias that may exist in the processing of tissue for whole genome sequencing (WGS), it is important to evaluate the extraction method on the overall microbial content and composition. Here we compare two different methods of extraction, homogenization vs. enzymatic lysis, on gastric, esophageal and colorectal biopsies and survey the microbial content and composition using WGS and quantitative PCR (qPCR). We examined total bacterial content using universal 16S rDNA qPCR as well as the abundance of three phyla (Actinobacter, Firmicutes, Bacteroidetes) and one genus (Fusobacterium). We found minimal differences between the two extraction methods in the overall community structure. Furthermore, based on our qPCR analysis, neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different, primarily due to variations in detection of low abundance genus. We also demonstrate that the homogenization extraction method provides higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract.

Three-gene immunohistochemical panel predicts progression and unfavorable prognosis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly invasive disease with a poor long-term survival rate. Although there has been progress in understanding the pathogenesis of ESCC, there are currently no molecular biomarkers that are used in routine clinical practices to determine prognosis. Therefore, the aim of this study was to determine a small immunohistochemical panel that could predict the prognosis of patients with ESCC. Phospholipase C epsilon-1 (PLCE1), IKKα, IKBα, p65, and p53 were highly expressed in ESCC tissues. The high expression level of PLCE1, IKBα, and p53 showed a significant positive correlation with a short overall survival (P = .022, .009, and .024, respectively). This 3-biomarker panel (ie, PLCE1, IKBα, and p53) was found to be a predictor of ESCC, with a worse overall survival as each positive marker was added (hazard ratio, 1.553; 95% confidence interval, 1.166-2.067; P = .003). In another cohort (including 1922 esophageal endoscopic biopsy tissues), the lesions of 28 patients were aggravated. Three proteins (PLCE1, 12/28 [42.86%]; IKBα, 16/28 [57.14%]; p53, 16/28 [57.14%]) were immunoreactive in patients with progressive disease. Our study identified and validated that this immunohistochemical biomarker panel of 3 proteins, consisting of PLCE1, IKBα, and p53, is not only independently associated with an unfavorable outcome for ESCC patients but also able to predict disease progression to precancerous lesions.

Abstract 1518: Gene expression of trefoil proteins, gastrokines and mucins in Indian gastric cancer patients

Abstract Background: Gastric cancer is the (GC) third most cause of cancer related mortality worldwide. Histopathologically, GC is sub classified into two major histopathological subtypes- diffuse and intestinal. The trefoil factor family - TFF1, TFF2 and TFF3 is known to play a pivotal role in gastrointestinal mucosal defence and repair. Gastrokines (GKN1, GKN2) are stomach specific secretary proteins which play fundamental roles in regulating gastric epithelial homeostasis. Similarly, secreted mucins are large O-glycosylated proteins that participate in the protection/defence of underlying mucosa in normal adults. The loss of expression of trefoil proteins, gastrokines and several mucins in retinoblastoma and colorectal cancer are reported in recent studies. Aims: The molecular underpinnings and gene expression of Indian GC patients are largely unknown. In this study, we evaluated the gene expression of TFF1, TFF2, TFF3, GKN1, GKN2, MUC3A, MUC5AC, MUC13 in gastric cancer patients from India. Additionally, we wanted to see whether these gene expressions are different in two histopathological groups. Materials and Methods: Endoscopic biopsy tissue from gastric tumour and adjacent normal regions were collected with ethical approval and informed consent from 42 patients . After histopathological confirmation by two pathologists, total RNA were isolated from tumour and corresponding normal tissue and gene expression was measured by qRT-PCR. Statistical analysis was performed by R package. Result and Discussion: We identified that TFF1, TFF2, TFF3, GKN1, GKN2 are significantly differentially expressed in gastric tumour tissue with respect to adjacent normal tissue whereas mucins had no significant deregulation. TFF1, TFF2, GKN1 and GKN2 are found to be down regulated in most tumour tissues compared to adjacent normal. Only GKN2 expression is significantly different among diffuse and intestinal subtypes . Our statistical analysis also revealed that gene expression of TFF1, TFF2, GKN1 and GKN2 are highly correlated with the gene expression of MUC5AC. Further study with larger patient sample is required to fully understand the complex interplay of trefoil, gastrokines and mucins in GC. Citation Format: Dipjit Basak, Pronoy Kanti Mondal, Brishti Mondal, Ranajoy Ghosh, Aniket Halder, Gopal Krishna Dhali, Abhijit Chowdhury, Indranil Mukhopadhyay, Shalini Datta. Gene expression of trefoil proteins, gastrokines and mucins in Indian gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1518.

RPN2 is effective biomarker to predict the outcome of combined chemotherapy docetaxel and cisplatin for advanced gastric cancer.

Preoperative chemotherapy, often using docetaxel and cisplatin, is a famous treatment option for advanced gastric cancer in Japan. But, there are no effective biomarkers that predict the therapeutic outcome on gastric cancer. Ribophorin II (RPN2) silencing, which decreases glycosylation of P-glycoprotein (P-gp) and membrane localization, restores the sensitivity to docetaxel and cisplatin. We inquired whether RPN2 expression in advanced gastric cancer biopsy tissues may be a predictive biomarker for docetaxel and cisplatin combination preoperative chemotherapy.We judged RPN2 expression immunohistochemically in upper endoscopic biopsy tissues from 40 advanced gastric cancer patients, who received the combination preoperative chemotherapy of docetaxel and cisplatin and gastrectomy with D2 resection during 2008-2014, and compared clinicopathological effects between RPN2-positive and RPN2-negative groups. We also examined sensitivity of RPN2-knockout gastric cancer cells by genome editing to docetaxel and cisplatin.RPN2 expression was observed in 19 of 40 gastric cancer cases. The RPN2-negative group had better clinicopathological responses to docetaxel and cisplatin combination chemotherapy than the RPN2-positive group, especially, in assessment of the histopathological criteria to preoperative chemotherapy. And RPN2-negative group had a significantly higher overall survival and progression-free survival compared to the RPN2-positive group. We also found RPN2-knockout to change docetaxel and cisplatin sensitivity in vitro.RPN2 expression in upper endoscopic biopsy tissues can be an effective predictive biomarker for the treatment outcome to docetaxel and cisplatin combination preoperative chemotherapy in advanced gastric cancer.

Retention Esophagitis as a Significant Clinical Predictor of Progression to Esophageal Cancer in Achalasia.

Background/AimsChronic liquid and/or food stasis caused by retention esophagitis (RE) in achalasia is a notable endoscopic finding because of the presence of a thickened or whitish esophageal mucosa and histologically altered squamous hyperplasia. We aimed to identify the clinical features of RE associated with achalasia and to clarify the clinical definition of RE in achalasia as a precancerous lesion identified by analyzing biomarker expressions.MethodsFrom 2006 to 2015, we retrospectively reviewed 37 patients with achalasia without previous treatment. Among them, 21 patients had diagnostic findings of RE (RE+) and 16 patients had no diagnostic findings of RE (RE–). Immunohistochemical staining of p53, p16, and Ki-67 was performed on the endoscopic biopsy tissues from the patients with achalasia and 10 control patients with non-obstructive dysphagia.ResultsThe symptom duration and transit delay were significantly longer in the RE+ group than in the RE– group. We found particularly high p53 positivity rates in the RE+ group (p<0.001). The rate of p16 expression was also significantly higher in the RE+ group than in the other two groups (p=0.003).ConclusionsA high p53 expression rate was more frequently found in the RE+ group than in the other two groups. RE could be a meaningful clinical feature of achalasia for predicting esophageal carcinogenesis.

Plasma ctDNA RAS/RAF mutations analysis for monitoring overall survival (OS) and heterogeneity in metastatic colorectal cancer patients (mCRC).

701 Background: The study was designed to estimate the clinical usefulness of genetic analysis for circulating tumor DNA (ctDNA) by digital polymerase chain reaction in patients with mCRC. Methods: We compared B-RAF and K/N/H-RAS mutations detected in surgical or endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctDNA from 63 patients with mCRC. p53 mutational status and PD-L1 expression in the serum and in tissues respectively (positivity H score &gt; 55) were also analyzed with a comparison with survival rates. Results: The frequencies of K-RAS mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 18 of 63 samples (28.57%), 5 of 63 samples (7.93%), and 6 of 63 samples (9.52%), respectively. Conversely, the rates of the mutations in ctDNA were 22 of 63 samples (34.92%), 6 of 63 samples (9.52%), and 9 of 63 samples (14.28%), respectively. No H-RAS or N-RAS mutation were detected. 2 tissue samples harbored a BRAF mutation (3.17%) in a complete discordance with same mutation detected in 6 different serum ctDNA (9.52%). Overall, the K-RAS mutation rates in tissue and ctDNA were 45.72% and 58.72%, respectively, and the concordance rate between them was 29 of 37 samples (78,37%). OS did not appear to differ by the presence of K-RAS mutations in tissue or in the serum DNA, but the OS of patients with B-RAF mutations in ctDNA was significantly shorter than that of patients with tissue K-RAS/B-RAF mutations (11 vs 19 months, p &lt; 0.0001). PD-L1 positivity was significantly associated with p53 mutational status (concordance: 82%) and with a worse outcome for OS. Conclusions: Analysis of ctDNA is a new useful procedure for detecting mutations in mCRC patients. This non invasive method may have great potential to understand tumor heterogeneity and appear a new strategy for the planning therapies as well as for predicting survival.

Establishment and genomic characterizations of patient-derived esophageal squamous cell carcinoma xenograft models using biopsies for treatment optimization

BackgroundSquamous cell carcinoma is the dominant type of esophageal cancer in China with many patients initially diagnosed at advanced stage. Patient-derived xenografts (PDX) models have been developed to be an important platform for preclinical research. This study aims to establish and characterize PDX models using biopsy tissue from advanced esophageal cancer patients to lay the foundation of preclinical application.MethodsFresh endoscopic biopsy tissues were harvested from patients with advanced esophageal cancer and implanted subcutaneously into NOD/SCID mice. Then, the PDXs were serially passaged for up to four generations. Transplantation was analyzed and genomic characteristics of xenografts were profiled using next-generation sequencing.ResultsTwenty-five PDX models were established (13.3%, 25/188). The latency period was 75.12 ± 19.87 days (50–120 days) for the first passage and it decreased with increasing passaging. Other than tumor stages, no differences were found between transplantations of xenografts and patient characteristics, irrespective of chemotherapy. Histopathological features and chemosensitivity of PDXs were in great accordance with primary patient tumors. Each PDX was assessed for molecular characteristics including copy number variations, somatic mutations, and signaling pathway abnormalities and these were similar to patient results.ConclusionsOur PDX models were established from real time biopsies and molecularly profiled. They might be promising for drug development and individualized therapy.

Clinical features of negative pathologic results after gastric endoscopic submucosal dissection.

Endoscopic submucosal dissection (ESD) is widely used for the treatment of gastric superficial neoplasms. Negative pathologic results after ESD can confuse the endoscopists and patients. The aim of this study was to evaluate the reasons for and the factors associated with negative pathologic results after ESD. From December 2008 to July 2015, a total of 1379 lesions diagnosed as definite dysplasia or adenocarcinoma by endoscopic forceps biopsy (EFB) were removed with an ESD procedure. The initial endoscopic and pathologic findings were analyzed. The incidence of negative pathology after ESD was 2.0% (28/1379). Compared with positive pathologic lesions, negative pathologic lesions were smaller and had less surface area (P<0.001). The reasons for negative pathologic lesions after ESD were complete removal by EFB (n=20), over-estimations of the EFB specimen (n=5), and different ESD site (n=3). Small tumor size and surface area are associated with negative pathologic results after ESD. When negative pathologic results are reported after ESD, we should review the previous endoscopic biopsy tissue specimen and compare the previous EFB site to the ESD site. Thereafter, regular endoscopic examination of the lesion is needed.

Expressions and its clinical significance of microRNA-21 and microRNA-146a in colorectal neoplasms

Objective To investigate the differences of miRNA-21 and miRNA-146a expression between colorectal neoplasms tissues and serum of the patients with colorectal neoplasm, and the clinical significance was analyzed. Methods The endoscopic biopsy tissues and serum samples of 100 colorectal cancer (CRC), 80 colorectal adenoma (CRA) patients and 65 healthy controls were collected. The expressions of miRNA-21 and miRNA-146a were detected by quantitative real time polymerase chain reaction. The relationship between the expression and clinicopathological features were analyzed. And then, the diagnostic value of expression difference of serum miRNA in colorectal neoplasm were evaluated. The Mann-Whitney U test and Kruskal-Wallis test were performed for comparisons between groups, and the correlation of miRNA expression between tissues and serum was analyzed by Spearman test. Results The expressions of miRNA-21 in the tissues of CRC and CRA were 8.573±0.898 and 7.746±1.183, respectively, which were significantly higher than that of healthy controls 6.160±0.835 (U=120.129 and 33.230, both P 0.05). The expression of miRNA-146a in the tissues and serum of CRC patients were both lower than those of CRA patients (U=73.809 and 21.123, both P<0.01). The degree of decreased expression of miRNA-146a in CRC patients was correlated with TNM staging and tumor differentiation degree, however there was no correlation between the expression in CRA and clinical features. According to receiver operating characteristic (ROC) curve analysis, the AUC of miRNA-21, miRNA-146a and a combination of them in CRC and health individuals was 0.889, 0.791 and 0.863, respectively; in CRA and health individuals was 0.784, 0.692 and 0.761, respectively; in CRC and CRA was 0.705, 0.820 and 0.713, respectively. Conclusion The different expressions of miRNA-21 and miRNA-146a had potential values in early detection of colorectal cancer. Key words: microRNA; microRNA-21; microRNA-146a; Colorectal neoplasms; Tissues; Serum; Real-time polymerase chain reaction

Detection of K‐ras gene mutation by liquid biopsy in patients with pancreatic cancer

Cell-free circulating tumor DNA (ctDNA) in serum has been considered to be a useful candidate for noninvasive cancer diagnosis. The current study was designed to estimate the clinical usefulness of genetic analysis for ctDNA by digital polymerase chain reaction in patients with pancreatic cancer. The authors compared K-ras mutations detected in endoscopic ultrasound-guided fine-needle aspiration biopsy tissue DNA and in ctDNA from 75 patients with pancreatic cancer. K-ras mutations in the serum of 66 independent, consecutive patients with pancreatic cancer were also analyzed and the authors compared the results with survival rates. The frequencies of the mutations in tissue samples at G12V, G12D, and G12R in codon 12 were 28 of 75 samples (37.3%), 22 of 75 samples (29.3%), and 6 of 75 samples (8.0%), respectively. Conversely, the rates of the mutations in ctDNA were 26 of 75 samples (34.6%), 29 of 75 samples (38.6%), and 4 of 75 samples (5.3%), respectively. Overall, the K-ras mutation rates in tissue and ctDNA were 74.7% and 62.6%, respectively, and the concordance rate between them was 58 of 75 samples (77.3%). Survival did not appear to differ by the presence of K-ras mutations in tissue DNA, but the survival of patients with K-ras mutations in ctDNA was significantly shorter than that of patients without mutations in both a development set (P = .006) and an independent validation set (P = .002). The difference was especially evident in cases with a G12V mutation. Analysis of ctDNA is a new useful procedure for detecting mutations in patients with pancreatic cancer. This noninvasive method may have great potential as a new strategy for the diagnosis of pancreatic cancer as well as for predicting survival.

Abstract B211: Comparison of detection of FGFR2 amplification by quantitative real-time-PCR (qPCR) and fluorescent in situ hybridization (FISH) in gastric cancer.

Abstract Background: Fibroblast growth factor receptor 2 (FGFR2) amplification is associated with tumorigenesis of gastric cancer and can be a promising molecular target for the treatment of FGFR2-amplified gastric cancer. So far, the most optimal single method to screen patients with FGFR2 amplification has not been determined. To screen patients with gastric cancer harboring FGFR2 amplification, we aim to investigate whether qPCR can replace the FISH method which is the golden standard but less sensitive and much more expensive than qPCR. Methods: FISH (Abnova, #FG0018) and qPCR (Applied Biosystems, HS05182482_cn) method for FGFR2 amplification were performed with formalin-fixed paraffin-embedded (FFPE) tissues of patients with gastric cancer who were diagnosed from 2007 to 2012 in Asan Medical Center, Seoul, Korea, and whose FFPE tissues contained at least 70% of tumor cells. qPCR was conducted initially in 26 patients who had both endoscopic biopsy and surgical tissues in the diagnosis to figure out which samples are better between biopsy and surgical tissues. According to the results, 182 patients with endoscopic biopsy tissues were further included. FISH was defined as positive in case of FGFR2 to CEP10 ratio &amp;gt; 2.0. Results: In 26 patients who had paired endoscopic biopsy and surgical samples, the qPCR-based copy number assay for FGFR2 amplification was more sensitive in biopsy samples; i.e., FGFR2 copy number by qPCR was higher in biopsy samples in 13 (50%) patients, while it was higher in surgical samples only in 3 (11.5%) patients. In a total of 208 endoscopic biopsy FFPE samples including 182 patients with biopsy tissues, copy number of FGFR2 ranged from 0.8 to 399.0 (median 15.9) by qPCR and from 0.7 to 166.9 (median 6.1) by FISH. 16 biopsy samples showing FGFR2 copy number &amp;gt; 10 by qPCR were all FISH-positive, while 192 biopsy samples showing FGFR2 copy number &amp;lt; 10 by qPCR were all FISH-negative. In cases of FGFR2 copy number &amp;gt; 10 in biopsy tissues by qPCR, the copy numbers were very well correlated between qPCR and FISH in all patients, and were also over 10 in surgical tissues regardless of methods in 26 patients. The positive rate of FGFR2 amplification was 7.7% with a cut-off value of 10 by qPCR. Conclusion: This study suggests that it is better to use biopsy samples than surgical tissues to detect FGFR2 amplification by qPCR; and for patient screening in gastric cancer, the optimal cut-off value for definite FGFR2 amplification by qPCR is 10 in comparison with the results of FISH. Clinical relevance of intermediate FGFR2 copy number elevation &amp;lt; 10 by qPCR needs to be addressed in future clinical trials using FGFR2 inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B211. Citation Format: Young-Soon Na, Young Soo Park, Min-Hee Ryu, Chae-Won Lee, Hye Jin Park, Ju-Kyung Lee, Sook Ryun Park, Baek-Yeol Ryoo, Yoon-Koo Kang. Comparison of detection of FGFR2 amplification by quantitative real-time-PCR (qPCR) and fluorescent in situ hybridization (FISH) in gastric cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B211.

Prevalence of gene amplifications of SOX-2, c-MET, and FGFR1 in Asian patients with esophageal squamous cell carcinoma.

e15127 Background: Amplifications of SOX-2, c-MET, and FGFR1 have recently been recognized as important molecular derangements with therapeutic implication in squamous cell carcinoma of lung. We investigated the significance of these molecular derangements in esophageal squamous cell carcinoma (ESCC). Methods: Locally advanced ESCC patients who had received curative- intent loco-regional therapy and had available pre-treatment endoscopic biopsy tissues at National Taiwan University Hospital, Taipei, Taiwan, were enrolled. DNA was extracted from formalin-fixed paraffin-embedded tissues. Amplifications of SOX-2, c-MET, and FGFR1 were evaluated by quantitative PCR (TaqMan Copy Number Assay, Applied Biosystems, CA, USA), using TERT as a reference gene. Amplification of specific gene was defined as a copy number more than 4. Results: Forty locally advanced ESCC patients, M: F= 39: 1 with a median age of 56 years (range: 37 to 80), were included. The primary tumors located at cervical, upper thoracic, middle thoracic, and lower thoracic esophagus in 2, 9, 23, and 6 patients, respectively; the tumor staging according to AJCC 2002 version was T3N0, AnyTN1, and M1a in 1, 37, and 2 patients, respectively. Amplifications of SOX-2 and c-MET were identified in 14 (35%) and 7 (17.5%) patients, respectively. No FGFR1 amplification was detected. The amplifications of SOX-2 and c-MET were not associated with any known clinical features of the patients, including gender, age (&lt;60 vs ≧60), primary tumor location, clinical stage, and performance status (ECOG 0, 1). Conclusions: Amplifications of SOX-2 and c-MET, existing in significant portions of Asian patients with ESCC, are worthy of exploration as therapeutic targets of ESCC.