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Table S1. 15 Image Analysis Features Utilized by the Risk Classifier Table S2. Performance of Risk Classes Predicted by Test vs. Pathologic Diagnosis in Stratifying BE Patients with Prevalent HGD/EAC from Non-Progressor BE Patients. Figure S1. Representative Whole Slide Scan and Image Analysis Masks.

Table S1. Summary of Progression Endpoints in Incident Progressor Patients. Table S2. Correlation Among Quantitative Image Analysis Features Derived from the Same Candidate Biomarkers. Table S3. Comparison of Predictive Performance of Risk Classes Predicted by Test vs. Clinical Variables in Training Set of BE Patients. Table S4. Comparison of Predictive Performance of Risk Score as a Continuous Variable vs. Clinical Variables in Training Set. Table S5. Comparison of Predictive Performance of Risk Score as a Continuous Variable vs. Clinical Variables in Validation Set. Figure S1. Flowchart of Steps to Train and Validate 3-Tier 15-Feature/Measure Classifier for Risk Prediction in Barrett's Esophagus Biopsies. Figure S2. Multiplexed Biomarker Labeling and Imaging in Incident Progressor BE Cases. Supplementary Methods.

Background: There is a need for improved tools to detect high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus. In previous work, we demonstrated that a 3-tier classifier predicted risk of incident progression in Barrett's esophagus. Our aim was to determine whether this risk classifier could detect a field effect in nondysplastic (ND), indefinite for dysplasia (IND), or low-grade dysplasia (LGD) biopsies from Barrett's esophagus patients with prevalent HGD/EAC.Methods: We performed a multi-institutional case–control study to evaluate a previously developed risk classifier that is based upon quantitative image features derived from 9 biomarkers and morphology, and predicts risk for HGD/EAC in Barrett's esophagus patients. The risk classifier was evaluated in ND, IND, and LGD biopsies from Barrett's esophagus patients diagnosed with HGD/EAC on repeat endoscopy (prevalent cases, n = 30, median time to HGD/EAC diagnosis 140.5 days) and nonprogressors (controls, n = 145, median HGD/EAC-free surveillance time 2,015 days).Results: The risk classifier stratified prevalent cases and non-progressor patients into low-, intermediate-, and high-risk classes [OR, 46.0; 95% confidence interval, 14.86-169 (high-risk vs. low-risk); P < 0.0001]. The classifier also provided independent prognostic information that outperformed the subspecialist and generalist diagnosis.Conclusions: A tissue systems pathology test better predicts prevalent HGD/EAC in Barrett's esophagus patients than pathologic variables. The results indicate that molecular and cellular changes associated with malignant transformation in Barrett's esophagus may be detectable as a field effect using the test.Impact: A tissue systems pathology test may provide an objective method to facilitate earlier identification of Barrett's esophagus patients requiring therapeutic intervention. Cancer Epidemiol Biomarkers Prev; 26(2); 240–8. ©2016 AACR.

Background: Better methods are needed to predict risk of progression for Barrett's esophagus. We aimed to determine whether a tissue systems pathology approach could predict progression in patients with nondysplastic Barrett's esophagus, indefinite for dysplasia, or low-grade dysplasia.Methods: We performed a nested case–control study to develop and validate a test that predicts progression of Barrett's esophagus to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), based upon quantification of epithelial and stromal variables in baseline biopsies. Data were collected from Barrett's esophagus patients at four institutions. Patients who progressed to HGD or EAC in ≥1 year (n = 79) were matched with patients who did not progress (n = 287). Biopsies were assigned randomly to training or validation sets. Immunofluorescence analyses were performed for 14 biomarkers and quantitative biomarker and morphometric features were analyzed. Prognostic features were selected in the training set and combined into classifiers. The top-performing classifier was assessed in the validation set.Results: A 3-tier, 15-feature classifier was selected in the training set and tested in the validation set. The classifier stratified patients into low-, intermediate-, and high-risk classes [HR, 9.42; 95% confidence interval, 4.6–19.24 (high-risk vs. low-risk); P < 0.0001]. It also provided independent prognostic information that outperformed predictions based on pathology analysis, segment length, age, sex, or p53 overexpression.Conclusion: We developed a tissue systems pathology test that better predicts risk of progression in Barrett's esophagus than clinicopathologic variables.Impact: The test has the potential to improve upon histologic analysis as an objective method to risk stratify Barrett's esophagus patients. Cancer Epidemiol Biomarkers Prev; 25(6); 958–68. ©2016 AACR.

Supplementary Figure 6 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16INK4A, p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating KrasG12D mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate KrasG12D and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;KrasG12D or Pdx1-Cre;Smad4lox/lox mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16Ink4A expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated KrasG12D allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4lox/lox animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple KrasG12D-dependent disease-associated phenotypes. [Cancer Res 2007;67(17):8121–30]

Supplementary Figure 1 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

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Supplementary Figure 5 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

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Vaccination has been underway in several countries for sexually inactive young girls or women against HPV 16 and 18 to prevent them from infection of these HPV types and concurrent cancer development. However, uterine cervical cancers may remain uncontrolled among some Asian countries, where other types of HPV infection are more frequent. A sensitive cancer screening system would remain important for detection of the earlier stage cervical cancers in Asian countries. In this study, 130 cytotechnologists (CTs) in Asian countries (Taiwan 80, Japan 18, Korea 15, Thailand 11, Singapore 3, Bhutan 2, and Mongolia 1) participated in the vote. Selected 10 cervical Pap smears that would be adequate to identify the diagnostic clues especially for atypical squamous cells (ASC) with two or three representative pictures for each case were displayed on the website. The percentages of consistent diagnosis voted by certified CTs with ≥5 years of experience were compared among 10 cervical cases or among Asian countries enrolled. As results, low consistency for ASC cases and high consistency for squamous intraepithelial lesion (SIL) were observed. Examining specimens for the diagnostic clues of ASC in TBS is crucial to maintain the high sensitivity and positive predictive value of SIL in Asian countries. Diagn. Cytopathol. 2010. © 2010 Wiley-Liss, Inc.

Context.—Establishing adequate interobserver agreement is crucial not only for standardization of patient care but also to ensure validity of findings in multi-institutional trials. Objective.—To evaluate interobserver agreement in assessing chronic hepatitis C (HCV) and acute cellular rejection (ACR) among 17 hepatopathologists involved in the “Hepatitis C 3” trial. Design.—The trial is a randomized multicenter (17 institutions) study involving 312 patients undergoing transplantation for HCV. Patients are randomized to 3 treatment arms. For final data analysis, all biopsy specimens are reviewed by a central pathologist (G.J.N.). Recurrence of HCV is evaluated according to the Batts and Ludwig schema. The 1997 Banff schema is used to evaluate ACR. To assess interobserver agreement, hematoxylin-eosin–stained sections from 11 liver biopsy specimens (6 HCV and 5 ACR) were sent by the central pathologist to 16 local pathologists from 13 institutions. Statistical analysis was performed on raw ACR/HCV data as well as data grouped according to clinically significant primary endpoint cutoffs. Results.—Statistically significant agreement was found among all participating pathologists (P < .001). On κ analysis, the degree of agreement was rated “moderate” for HCV grade and stage and ACR global grading (κ = 0.30, 0.33, and 0.37, respectively). Interobserver agreement was weaker for rejection activity index scoring of ACR (κ = 0.15). A stronger degree of agreement was found when scores were grouped based on endpoint cutoffs (κ = 0.76 “almost perfect” for HCV and 0.62 “substantial” for ACR). Conclusions.—An overall statistically significant interobserver agreement was found among 17 pathologists using the 1997 Banff schema and the Batts and Ludwig schema.

BACKGROUND The objectives of this study were to determine threshold probabilities needed to perform endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and those needed to treat patients suspected of having malignancy and then to compare these thresholds to the pre- and posttest probabilities of malignancy associated with benign, atypical, suspicious, and malignant diagnoses. The goal was to aid endoscopists in making appropriate clinical decisions based on both quantitative and qualitative approaches. METHODS The study included 633 consecutive patients. A decision tree was constructed to estimate the “treatment” threshold. Using treatment threshold and likelihood ratios, the authors determined the “no-test-test” and “test-treatment” thresholds. Pretest probability was compared with no-test-test and test-treatment thresholds, and the post-EUS-FNA probability of malignancy for each diagnostic category with the treatment threshold. Results were stratified by lesion site, lesion size, and cytopathologist. RESULTS EUS-FNA has a wide range of pretest probabilities within which it could be performed (0.06–0.98). The posttest probabilities for malignancy, 0.99 (95% confidence interval [CI], 0.967–0.996) and 0.09 (95% CI, 0.057–0.126), after a positive or a negative result, respectively, were significantly different from the treatment threshold but not those of suspicious, 0.92 (95% CI, 0.767–0.994) diagnosis. The posttest probability of atypical diagnosis, 0.60 (95% CI, 0.407–0.772), was not significantly different from that of pretest probability. Results did not vary by lesion size, organ site, or cytopathologist. CONCLUSION The authors demonstrated the uncertainty associated with EUS-FNA diagnostic categories and used the threshold approach to qualify quantitatively the decision to perform EUS-FNA and the decision to treat patients suspected of having malignancy. Cancer (Cancer Cytopathol) 2006. © 2006 American Cancer Society.

The objective of this study is to assess the utility of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in the diagnosis and staging of colorectal cancer. The study includes patients who underwent EUS-FNA at our institution for staging of colorectal carcinoma or for evaluation peri-rectal masses or distal metastases from August 2000 to November 2010. We assessed the frequency with which EUS-FNA procedure confirms the diagnosis of malignancy and the percent of cases in which it modifies staging of colorectal carcinoma. Using histology as a reference standard, we also assessed the diagnostic performance. We identified 79 cases of EUS-FNA from 77 patients, mean (SD) age of 60 (12.5), 44 males. Twenty-seven (34%) aspirates were from patients with primary rectal/peri-rectal masses, 15 (19%) were from patients with suspected regional lymph node metastasis, and 37 (47%) were cases of suspected of distal metastasis. All lesions were clinically suspicious for primary or metastatic colorectal carcinoma. On cytologic examinations, 43 (54%) cases were confirmed as malignant, 6 (8%) were benign neoplasms, 4 (5%) were suspicious for malignant neoplasm, 2 (3%) showed atypical cells, and the rest 24 (30%) were negative for neoplasms. Fourteen of 27 (52%) of the local rectal masses were confirmed as colorectal carcinoma. Eleven of 15 (73%) regional lymph nodes were positive for metastasis-all, but two of these metastases, were of colorectal origin. Twenty of 37(54%) distal lesions were metastatic neoplasms and 15 of those were colorectal in origin. Diagnosis of primary colorectal carcinoma was confirmed in 52% of the clinically suspicious primary lesions and in 42% regional or distal metastatic lesions. Using histology as a reference standard in 27 of 79 (29%) cases, we calculated an overall sensitivity, specificity, and positive and negative predictive values (C.I) of EUS-FNA of 89% (74-100%), 79% (50-100%) 89% (74-100%), and 79% (51-100%). EUS-FNA is useful for assessing primary and metastatic colorectal lesion. This technique improves staging of suspected nodal or distant metastases.

Since the anti-tumor activity of 5-fluorouracil (5-FU) is due to induction of apoptosis, we assessed the value of expression of key apoptotic molecules (Bax, Bcl-2 and p53) in predicting the efficacy of 5-FU therapy for colorectal adenocarcinomas (CRCs).Archival tissues of CRCs from 56 patients who received a complete regimen of 5-FU-based chemotherapy after surgery, and 56 patients matched for age, gender, ethnicity, tumor stage, tumor location, and tumor differentiation who had undergone only surgery (without any pre- or post-surgery therapy), were evaluated for immunophenotypic expression of Bax, Bcl-2, and p53. Also, these CRCs were evaluated for Bax mutations. The predictive capacity or prognostic value of these markers was assessed by estimating overall survival.The majority of low Bax expressing CRCs have exhibited mutations at the G (8) tract. There was no significant difference in overall survival rates between the categories of surgery alone and 5-FU-treated patients. However, a better survival was observed for patients who received chemotherapy when their CRCs had low Bax/Bcl2 ratio (HR, 1.55; 95% CI: 1.46-31.00). Patients who received surgery alone and whose CRCs lacked Bax expression had 5.33 times higher mortality than those with high Bax expression (95% CI: 1.78-15.94), when controlled for tumor stage and other confounders. Bcl-2 and nuclear p53 accumulation had no predictive value in either patient group.These findings are the first to demonstrate that high Bax expression is a good prognosticator for patients who underwent surgery alone, and that patient with low Bax/Bcl-2 expression ratio benefit from 5-FU-based adjuvant therapies.

Background Although a majority of studies in cancer biomarker discovery claim to use proportional hazards regression (PHREG) to the study the ability of a biomarker to predict survival, few studies use the predicted probabilities obtained from the model to test the quality of the model. In this paper, we compared the quality of predictions by a PHREG model to that of a linear discriminant analysis (LDA) in both training and test set settings. Methods The PHREG and LDA models were built on a 491 colorectal cancer (CRC) patient dataset comprised of demographic and clinicopathologic variables, and phenotypic expression of p53 and Bcl-2. Two variable selection methods, stepwise discriminant analysis and the backward selection, were used to identify the final models. The endpoint of prediction in these models was five-year post-surgery survival. We also used linear regression model to examine the effect of bin size in the training set on the accuracy of prediction in the test set. Results The two variable selection techniques resulted in different models when stage was included in the list of variables available for selection. However, the proportion of survivors and non-survivors correctly identified was identical in both of these models. When stage was excluded from the variable list, the error rate for the LDA model was 42% as compared to an error rate of 34% for the PHREG model. Conclusions This study suggests that a PHREG model can perform as well or better than a traditional classifier such as LDA to classify patients into prognostic classes. Also, this study suggests that in the absence of the tumor stage as a variable, Bcl-2 expression is a strong prognostic molecular marker of CRC.

We retrospectively studied the use of endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) as a tool for the diagnosis of deep-seated lymphoma. An on-site assessment at the time of EUS-FNAB was performed by a cytopathologist using Diff-Quik (American Scientific Products, McGraw Park, IL) stain. In addition, Papanicolaou stains were performed on EUS-FNAB smears, immunohistochemical stains were performed on cell blocks, and additional samples were sent for flow cytometric analysis. Final cytologic diagnosis was correlated with surgical pathology and/or clinical follow-up. We evaluated EUS-FNAB specimens of deep-seated lymph nodes, spleen, stomach, and pancreas, and 1 EUS-guided needle core biopsy specimen of a lymph node. Thirteen cases of deep-seated lymphoma were diagnosed, including non-Hodgkin lymphomas and Hodgkin lymphoma. One case of hairy cell leukemia was diagnosed. EUS-FNAB is a minimally invasive, cost-effective, and useful tool for the primary diagnosis or staging of deep-seated lymphomas.

BACKGROUND Computerized tomographic (CT)-guided fine-needle aspiration (FNA) cytology is a well-established tool in the diagnosis of hepatic lesions. Endoscopic ultrasound-guided FNA (EUS-FNA), developed recently and used predominantly in evaluating mediastinal and pancreatic lesions, provides access to a significant portion of the liver and to perihepatic structures not readily accessible by a percutaneous approach. METHODS A recent experience (1997-2002) with CT-guided FNA of liver lesions at the University of Alabama Birmingham (UAB) was compared with the first 2.5 years of EUS-FNA experience (2000-2002). Cases were identified using a SNOMED search and all reports and cytologic slides were retrieved for review. RESULTS In 6 years, 34 percutaneous CT-FNA liver biopsies were performed at UAB; in approximately 2.5 years, 16 EUS-FNA liver biopsies were done. In both groups the primary clinical indication was suspected metastatic carcinoma (CT, 41% of cases vs. EUS, 56%). The 2 techniques yielded a similar range of benign, atypical, and malignant diagnoses (CT: 26%, 18%, and 56% vs. EUS: 19%, 25%, and 56%). Because of the clinical setting in which EUS-FNA is usually performed, a much narrower range of neoplasms was sampled by EUS-FNA. Benign gastrointestinal epithelial cells were identified in 60% of the EUS-FNA specimens. CONCLUSIONS Early experience suggests EUS-FNA is comparable to CT-FNA in terms of diagnostic utility for hepatic lesions. Anatomy limits EUS-FNA to only a fraction of the hepatic parenchyma, but that fraction includes the hilum and left lobe of the liver and the proximal biliary tract. The gallbladder, extrahepatic biliary system, and perihilar lymph nodes are readily accessible. Proximate high-resolution ultrasound imaging and cytopathologist involvement in the EUS-FNA process are further advantages. Awareness of artifacts inherent in EUS-FNA sampling (i.e., gut epithelial cells) can minimize a potential diagnostic pitfall. Cancer (Cancer Cytopathol) 2006. © 2006 American Cancer Society.

BACKGROUND Endoscopic ultrasound (EUS) is a powerful imaging modality to identify and determine the extent of a lesion. In addition, EUS is superior to a computed tomography scan in detecting lesions < 3 cm. The objective of the current study was to determine whether small lesions (≤ 25 mm) affected the specimen adequacy and the diagnostic accuracy for lesions aspirated under EUS guidance. METHODS In the current study, 209 consecutive EUS-guided fine-needle aspiration biopsy (EUS-FNAB) samples ≤ 25 mm (100 samples) or > 25 mm (109 samples) as determined by EUS were obtained from 151 patients with a mean age of 62 years (range, 39–94 years). A cytopathologist present in the endoscopy suite determined specimen adequacy. Yield of adequate samples for diagnosis, number of passes, and operating characteristics of EUS-FNAB for small (≤ 25 mm) and large lesions (>25 mm) were compared. RESULTS The overall yield of obtaining adequate samples for diagnosis was 96% (201 of 209). There was no difference noted with regard to the yield of obtaining samples (96% vs. 96%) from small or large lesions. A mean of 2.5 passes (range, 1–9 passes) was needed to obtain adequate samples from lesions ≤ 25 mm, whereas a mean of 4.5 passes (range, 1–11 passes) was needed to obtain adequate samples from lesions > 25 mm. The sensitivity (96% vs. 96%), specificity (100% vs. 100%), and diagnostic accuracy (98% vs. 97%) for EUS-FNAB were comparable whether the lesion was ≤ 25 mm or > 25 mm. CONCLUSIONS EUS-FNAB was a highly effective technique to obtain samples from small (≤ 25 mm) and large (> 25 mm) lesions without affecting the sensitivity, specificity, or diagnostic accuracy. Cancer (Cancer Cytopathol) 2004. © 2004 American Cancer Society. Cancer (Cancer Cytopathol) 2004. © 2004 American Cancer Society.

Endoscopic ultrasound (EUS) is used to detect and delineate the extent of lesions in the gastrointestinal tract, periluminal lymph nodes, pancreas and hepatobiliary tree, left kidney, spleen, and adrenal glands. EUS-guided fine-needle aspiration (FNA) has added a new dimension to the capabilities of EUS because it permits characterization of the lesion, thereby enabling triage of patients for more efficient and effective management. This review focuses on the advantages and limitations of EUS-FNA, including a discussion of potential pitfalls in the diagnosis of commonly aspirated deep-seated lesions, such as those of the pancreas and lymph nodes. It also addresses the practical considerations associated with establishing an effective service and the importance of an integrated approach in which the cytopathologist undertakes a key role, interacting extensively with the endoscopist and the patient management team. EUS-FNA is a sensitive modality that enables specific and accurate diagnosis of deep-seated lesions. Samples can be obtained effectively from small lesions (25 mm), irrespective of the organ site. On-site assessment permits a highly accurate preliminary diagnosis of malignancy for samples obtained by EUS-FNA and provides an opportunity to increase the diagnostic yield of samples.

It is our hypothesis that if Helicobacter pylori could be demonstrated conclusively to have transgressed the mucosal surface into the lamina propria, this would help explain how H pylori recruits inflammatory cells. We report our immunohistochemical and electron microscopic findings that demonstrate that H pylori can be detected in the lamina propria of the stomach, offering evidence of its invasive potential. We stained 67 endoscopic gastric biopsy specimens with Warthin-Starry silver and immunoperoxidase stains for H pylori. In addition, transmission electron microscopy was performed on 1 case. The presence of surface H pylori was associated significantly with active (P.0001) and chronic (P.0001) inflammation. H pylori could not be identified in the lamina propria using the Warthin-Starry silver stain alone. Immunoreactivity for H pylori in the lamina propria was detected in 20 (30%) of 67 gastric biopsy specimens. Transmission electron microscopy confirmed the immunohistochemical findings. H pylori can infiltrate the lamina propria of the gastric mucosa, thereby proving morphologic evidence of its invasive capability.