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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.

Immunohistochemistry testing is highly complex with multiple steps. Assuring the optimum performance of your immunohistochemistry laboratory requires attention to numerous quality monitors. For testing performed on patient specimens, there are also additional regulatory requirements. This chapter answers questions about best practices in quality management in preanalytic, analytic, and postanalytic phases of the total immunohistochemistry test providing examples of possible quality improvement opportunities. It also provides information related to CLIA and FDA regulatory oversight medical devices, in vitro diagnostics (IVD), and analyte-specific reagents (ASR). With regard to immunohistochemistry laboratory accreditation, the final portion of this chapter draws attention to current best practice guidelines of the College of American Pathologists (CAP) relating to immunohistochemistry to prepare for inspection.

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.

Our recent study demonstrated the up-regulation of maspin, IMP3, and S100P and down-regulation of von Hippel-Lindau gene product (pVHL) in ductal adenocarcinoma of the pancreas. Distinction of adenocarcinoma of the gallbladder from benign/reactive glandular epithelium can be challenging if based on hematoxylin and eosin-stained sections alone. Immunohistochemical stains for pVHL, maspin, IMP3, and S100P were performed on 82 gallbladder specimens, including adenocarcinoma (n = 33) and normal/reactive gallbladder (n = 49). The results demonstrated (1) only 6.0% of adenocarcinoma cases were focally positive for pVHL, and all normal and most reactive cases (85%) were diffusely positive for pVHL; (2) maspin, IMP3, and S100P were positive in 100%, 81.8%, and 75.8% of adenocarcinoma cases, respectively; in contrast, 53.1%, 12.2%, and 30.6% of normal/reactive cases were only focally and weakly positive for maspin, IMP3, and S100P, respectively; and (3) 90.3% of adenocarcinoma cases were pVHL-negative and positive for 2 or more positive markers, whereas none of the benign/reactive cases showed this staining profile. This study demonstrates that the immunostaining profile of pVHL-/IMP3+/maspin+/S100P+ is useful in the distinction of adenocarcinoma of the gallbladder from normal/reactive conditions.

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.

The current work assessed the independent contributions of age, comorbidities, tumor features, and treatment approach to the outcome of elderly patients with breast cancer.Records of consecutive women agedor = 70 years with a histologic diagnosis of first invasive breast cancer between 1971 and 2001 and available medical information were reviewed. Restaging to the 2002 TNM system and comorbidity data abstraction were performed. Primary outcomes were overall survival (OS) and breast cancer-specific survival (BCSS).There were 992 patients with a median diagnosis age of 76 years, of whom 840 were approached with a curative intent. Significant comorbidities were recorded as none andor = 3 in 13% and 44% of patients, respectively. The 5- and 10-year OS rates were 59% and 34%; corresponding BCSS rates were 74% and 62%, respectively. Of 693 patients who died during the study period, only 298 (43%) died from their tumors. Stage emerged as the strongest predictor determining OS and BCSS (P = 0.001). In curatively approached patients, age was the next dominant factor affecting survival length (P = 0.001). The comorbidities with significant effect on OS differed by stage and included heart failure, diabetes, and other common comorbidities in early tumors but only extremely debilitating ones in advanced-stage disease (P0.05). Significant favorable effect of systemic therapy emerged only in the univariate model.This study confirms tumor stage as the strongest predictor of survival in elderly patients with breast cancer. It also indicates a significant role for age and comorbidities that varies by stage. Treatment effect should be interpreted with caution because of the retrospective study nature.

The lack of detailed information about antibodies and staining protocols in the literature is quite frustrating, especially when trying to reproduce published results; therefore we have included detailed antibody and staining protocol information, including many recently described antibodies such as GATA3, PAX8, napsin A, p40, ERG, NKX3.1, NKX2.2, SATB2, cadherin 17, arginase-1, pVHL, maspin, UP II, SALL4, SF-1, TROP2, adipophilin, SOX10, Merkel cell polyomavirus, NY-ESO-1, BRAF, and c-MYC, in this second edition. Most antibodies mentioned in this book have been routinely used at Geisinger Medical Laboratories, or have at least been optimized in one or both (Dako and Ventana) staining platforms. Table 4.1 includes antibodies tested with the Dako stainer and Table 4.2 includes antibodies tested with the Ventana staining platform. These two tables provide detailed information for each antibody, including vendor, catalog number, clone, antigen retrieval method, antibody dilution, in vitro diagnostic use (IVD) vs. analyte specific reagents (ASR) vs. research use only (RUO) class, staining pattern, positive control tissue, and the contact information for each vendor. No one in our group has a financial interest in any of these companies.

The past decade has produced major innovations and a great variety of features available in automated staining instruments. This chapter is a “buyer’s guide” for automated IHC instruments. For those new to the topic of automated staining, it begins with discussions of the advantages and disadvantages of automated versus manual staining techniques to help you decide if automation is the right choice for your laboratory. The basics of the general types of mechanics that differentiate the platforms are illustrated. Industry jargon about “open” and “closed” systems are better defined. To help with creating a thoughtful business plan to justify the budget expense of automation, the considerations that include the cost and potential savings of operating the equipment over and above the purchase price are presented. The different strategies for slide capacity and continuous processing that affect overall test throughput are described. A comprehensive feature comparison table is included to reveal how the current clinical instruments stack up side by side. With the information in this chapter you will know how to evaluate whether an instrument is right for you and understand the value of technological advancements as they arrive in the future.

With the new role of immunohistochemistry as a test to guide the use of specific therapies, new demands for quantitative accuracy and reproducibility have been placed on laboratories. In response to this need for increased quality of IHC testing, the College of American Pathologists (CAP) has released updated guidelines for the validation, performance, and ongoing quality management of receptor testing in breast cancer cases. This chapter reviews the current requirements of these testing guidelines to help laboratories adopt these best practices. Information is offered on the FDA approval of current antibodies and probes for this testing. Diagrams illustrate the recommended testing algorithm combining results of immunohistochemical and in situ hybridization testing for HER2. Photomicrographs of positive, negative, and equivocal staining reactions are provided to aid in the interpretation of immunohistochemical and in situ hybridization assays. Figures demonstrating the use of appropriate and inappropriate internal control reactions serve to help decide, if results are valid and can be reported or should be rejected.

Objectives Estrogen receptor (ER), gross cystic disease fluid protein 15 (GCDFP-15), and mammaglobin (MGB) are commonly used breast-specific immunomarkers; however, about half of metastatic breast carcinomas are negative for all three. GATA-binding protein 3 (GATA-3) has emerged recently as a sensitive and relatively specific immunomarker for breast and urothelial carcinomas, but the data documenting its expression in ER-negative breast carcinomas are limited; this often poses a dilemma in the setting of metastases. The purpose of this study is to investigate expression of GATA-3 in ER-negative breast carcinomas. Methods Immunohistochemical evaluation of GATA-3, GCDFP-15, and MGB on 96 ER-negative breast carcinomas was performed. Results Overall, 69% (66/96), 15% (14/96), and 35% (34/96) of ER-negative breast carcinomas expressed GATA-3, GCDFP-15, and MGB, respectively. Conclusions Our data suggest that GATA-3 is, so far, the best breast-specific immunomarker, especially when encountering ER-negative metastatic breast carcinomas. GATA-3 should be included in the panel of immunomarkers in the workup of tumors of unknown primary.

Background Solitary cysts occur in approximately 10% of patients with autosomal dominant polycystic kidney disease (ADPKD), and fine needle aspiration cytology (FNAC) can be beneficial in evaluating complications related to the cysts and in excluding malignancies and other cystic lesions that can occur in these patients. Case FNAC was performed on a benign epithelial cyst in a symptomatic, 25-year-old, white female with ADPKD. The aspirate consisted of scattered small, flat groups of uniform epithelial cells arranged in a honey-comb fashion. Conclusion To the best of our knowledge, this is the first FNAC report of a pancreatic cyst in ADPKD.

Primary squamous cell carcinoma (SCC) of the breast is an extremely rare tumor. It is diagnosed when the malignant cells are entirely of squamous type, the tumor is independent from overlying skin, and other primary SCC sites are excluded. This report presents clinical, radiologic, and histologic correlative findings of a patient diagnosed with pure SCC of the breast. Literature review suggests that primary SCC tumors of the breast are large, often cystic lesions, with a low rate of nodal involvement, no expression of estrogen and progesterone receptors, and resistance to the chemotherapy regimens commonly used in breast cancer.

Etanercept, a systemic inhibitor of α-TNF, is used for treatment of various autoimmune disorders. We report a case of spontaneous resolution of etanercept-induced lupus nephritis.A 57-year-old female patient taking etanercept for psoriasis presented with laboratory-and histology-confirmed lupus nephritis. After stopping etanercept, there was normalization of proteinuria, hematuria, serum complements, anti-dsDNA antibody, and resolution of the acute glomerular inflammatory process on repeat kidney biopsy.This case demonstrates serology- and biopsy-confirmed resolution of active lupus nephritis upon withdrawal of etanercept.

With the new role of immunohistochemistry as a test to guide the use of specific therapies, new demands for quantitative accuracy and reproducibility have been placed on laboratories. In response to this need increased quality of IHC testing, the College of American Pathologists (CAP) has released guidelines for the validation, performance, and ongoing quality management of receptor testing in breast cancer cases. This chapter reviews the new requirements of these testing guidelines to help laboratories adopt these best practices. Information is offered on the FDA approval of ­current antibodies for this testing. Diagrams illustrate the recommended testing algorithm combining results of immunohistochemical and in situ hybridization testing for HER2. Photomicrographs of ­positive, negative, and equivocal staining reactions are provided to aid in the interpretation of immunohistochemical and in situ hybridization assays. Figures demonstrating the use of appropriate and inappropriate internal control reactions serve to help decide, if results are valid and can be reported or should be rejected.

Immunohistochemistry testing is highly complex with multiple steps. Assuring the optimum performance of your immunohistochemistry laboratory requires attention to numerous quality monitors. For testing performed on patient specimens, there are also additional regulatory requirements. This chapter answers questions about best practices in quality management in preanalytic, analytic, and postanalytic phases of the total immunohistochemistry test providing examples of possible quality improvement opportunities. It also provides information related to CLIA and FDA regulatory oversight medical devices, in vitro diagnostics (IVD), and analyte-specific reagents (ASR). With regard to immunohistochemistry laboratory accreditation, the final portion of this chapter draws attention to current best practice guidelines of the College of American Pathologists (CAP) relating to immunohistochemistry to prepare for inspection.

Immunohistochemistry (IHC) is an indispensable tool for pathological diagnosis of both neoplastic and non-neoplastic diseases of the liver and biliary tract. It has a wide spectrum of application ranging from pathogen detection to differentiation of primary from metastatic tumors. The focus of this chapter is to address applications regarding diagnosis and differential diagnosis of liver and biliary tract tumors. Commonly used markers or antibodies are listed in various tables detailing their staining patterns in benign, reactive and neoplastic conditions. Suggested panels of markers are provided to address specific diagnostic challenges such as distinguishing benign vs. malignant biliary proliferation and hepatocellular lesions, differentiating intrahepatic cholangiocarcinoma from extrhepatic cholangiocarinoma and metastatic adenocarcinomas from other organs. A process is defined utilizing an initial screening panel and subsequent confirmatory markers for unknown primary tumors in liver. Photomicrographs are provided to illustrate characteristic staining patterns.

71-year-old white man with no prior history of malignancy and a 2-week history of obstipation, increasing abdominal girth, and abdominal cramping sought medical attention after he began passing bright red blood per rectum. He underwent colonoscopy, which revealed a fungating and hemorrhagic rectosigmoid mass lesion. Shortly after colonoscopy, the patient experienced a rapid decrease in oxygen saturation and required intubation with mechanical respiration. During the next several days, he developed acute renal failure leading to multiple organ system failure and eventually death. Before he died, the patient was sent for computed tomographic scans of the chest, abdomen, and pelvis, which revealed extensive ‘‘metastatic disease’’ involving the lungs, liver, and multiple bony sites. While in radiology, a computed tomographic scan‐guided needle biopsy of a tumor nodule in the bony pelvis was conducted. The needle aspiration slides were relatively hypocellular and contained a mixture of discohesive cells. Some of these cells displayed overt cytologic features of malignancy, including large irregular nuclei and prominent eosinophilic nucleoli (Figure 1). Osteoclast-type giant cells were admixed with the malignant cells. The core biopsy demonstrated a sheetlike expanse of the same malignant cells, some of which possessed long, tapering cytoplasm. A smattering

Warm autoimmune hemolytic anemia (WAIHA), a rare disease (0.2-1 per 100,000 population), ranges from an indolent form with mild hemolysis to a life-threatening condition that necessitates transfusion of incompatible red cells. WAIHA can be either idiopathic or secondary to medications or to a lymphoproliferative disorder. We report a case of profound hemolytic anemia in a liver-transplant eligible patient who was diagnosed with cirrhosis secondary to non-alcoholic steatohepatitis (NASH). The patient initially was treated with red cell transfusion, iv immunoglobulin, and steroids. He developed acute renal failure that required dialysis. Subsequent management included plasmapheresis and rituximab therapy. The patient developed hepatorenal syndrome and died from progressive hepatic failure. To our knowledge, this is the first report of an association between NASH and WAIHA.

Morphoproteomic analysis reveals the constitutive activation of the mTOR, ERK, and NF-kappaB pathways in high risk neuroblastoma (HRN) cases as evidenced by (a) collective commonalities of: phosphorylated (p)-mTOR, p70S6K, ERK 1/2, and NF-kappaBp65 protein analytes using phosphospecific probes directed against sites of activation; (b) nuclear translocation of p-p70S6K, p-ERK 1/2, and p-NF-kappaBp65; and (c) correlative expression of the S phase-associated kinase Skp-2 (at a relatively high percentage in tumoral nuclei) and of the anti-apoptotic protein bcl-2. Based on a review of the literature, these preliminary observations appear to be the first morphoproteomic study on primary neuroblastomas.

CCI-779 (temsirolimus), an ester of rapamycin and an inhibitor of the mammalian target of rapamycin (mTOR), is currently in phase II trials for treatment of patients with solid cancers. The mTOR functions as a checkpoint for cell proliferation, with upstream Akt and downstream p70S6K serving as its most important mediators. The aim of this study was to evaluate the expression and activation of the Akt-mTOR-p70S6K pathway in renal cell carcinoma (RCC), seeking to strengthen the rationale for targeted therapy of RCC using rapamycin or a rapamycin analogue. Tissue microarray sections containing 128 primary RCCs, 22 metastatic RCCs, and 24 non-neoplastic (normal) kidneys (NK) were immunostained with monoclonal antibodies to phosphorylated (p)-Akt (Ser473), p-mTOR (Ser2448), and p-p70S6K (Thr389). Western blotting was performed on 3 cases of clear cell RCC (CRCC) and the corresponding non-neoplastic (normal) renal tissues using the same antibodies. The immunostain scoring system included: (a) location; (b) distribution; and (c) intensity. The normal kidneys provided baseline scores for comparison. Expression of p-Akt, p-mTOR, and p-p70S6K was seen in 100% (n = 24) of NKs and nearly 100% (n = 150) of both primary and metastatic RCCs. The p-p70S6K was located in the nucleus in both NKs and RCCs. The p-Akt was observed in the nucleus and cytoplasm of NKs and in the nucleus and cytoplasm/ membrane (plasmalemma) of RCCs. The p-mTOR was identified in the membrane of NKs and the membrane/nucleus of RCCs. The levels of expression of p-p70S6K, p-mTOR, and p-Akt were significantly higher in RCC than in NK in the overall pattern (intensity and distribution, p0.05). Western blotting also showed higher expression of p-p70S6K, p-mTOR, and p-Akt in RCCs compared to the corresponding normal kidney tissues (p0.05). These findings indicate that correlative over-expression and activation of p-Akt, p-mTOR, and p-p70S6K are commonly observed in RCCs. After considering these findings in the context of other established protein circuitries and pathways in RCC, we propose therapeutic approaches that incorporate rapamycin-like agents and other small molecule inhibitors in a combinatorial fashion in future clinical trials for RCC.

Blastic NK cell lymphoma is a rare hematolymphoid neoplasm. This report illustrates an unusual presentation of this entity, namely as a primary leukemia, but without skin lesions.

Follicular lymphoma is classified into grades (G)1, 2, and 3 based on the number of centroblasts in neoplastic follicles. However, the accuracy of manually counting these centroblasts is limited by certain cells (large centrocytes, follicular dendritic cells, and histiocytes) that could mimic centroblast morphology. The reproducibility of follicular lymphoma grading is dependent upon observer experience; therefore, significant variations occur. This study is to explore a more objective, reliable way of grading follicular lymphoma using a quantitative imaging system in conjunction with immunostains with antibodies to proliferation markers MIB-1 and S-phase kinase-associated protein 2 (SKP2). Fifty-eight follicular lymphomas (G1, n = 23; G2, n = 18; and G3, n = 17) were studied on formalin-fixed, paraffin-embedded sections. Positive nuclear staining of both Ki-67 and SKP2 was recorded using the quantitative Clarient ACIS II system (Aliso Viejo, CA, USA). Ten high-power fields (x400) from randomly selected neoplastic follicles were counted by a pathologist blinded to the previously assigned morphologic grade. The results show that the percentages of Ki-67+ and SKP2+ cells significantly differ among the different grades of follicular lymphoma. A higher grade of follicular lymphoma is associated with a higher percentage of Ki-67+ and SKP2+ cells. The overall SKP2+% cells are substantially lower than Ki-67+% cells in the same grade of follicular lymphoma. Statistical significance is observed in Ki-67+ cells between follicular lymphoma G1 and follicular lymphoma G3 and between G1 and G2. In contrast, statistical significance is noted in SKP2+% cells between follicular lymphoma G1 and follicular lymphoma G3 and between follicular lymphoma G2 and follicular lymphoma G3. The findings suggest that the SKP2 expression has better discrimination with grades of follicular lymphoma than Ki-67 expression. Compared with traditional methods, quantitation of SKP2 expression using a quantitative image analysis system might be a useful and objective approach in grading follicular lymphoma.

Determination of Her-2/neu oncogene amplification is important in the current treatment of breast carcinoma. In addition to fluorescence in situ hybridization (FISH) and immunohistochemical stain (HercepTest), chromogenic in situ hybridization (CISH) has been shown to be a sensitive and specific method to determine the Her-2/neu status of surgical specimens. The effectiveness of CISH in detecting the Her-2/neu oncogene in cytologic specimens has not been well documented. Twenty-five cases of fine needle aspirate smears and touch imprints from infiltrating ductal carcinomas were examined. Both CISH and FISH were performed on each case using a digoxigenin-labeled Her-2 DNA probe for CISH (Zymed) and both Her-2 and chromosome 17 probes for FISH (Vysis). Sixty tumor cells were evaluated in each case. The scoring system and interpretation of CISH were as follows: (1) no amplification (5 brown dots/nucleus), (2) amplification (10 brown dots/nucleus), and (3) low-level amplification (5-9 brown dots/nucleus). Of the 25 cases analyzed, 23 (3 amplified and 20 nonamplified) showed similar results for both methods. Two cases were discordant. In these cases, low-level amplification was suggested by CISH but nonamplification by FISH. One of the cases can be explained by polysomy for chromosome 17 by FISH. In conclusion, our preliminary data suggest that CISH is a useful technique to determine Her-2/neu oncogene status in cytologic specimens. In a case of low-level amplification, a CISH chromosome 17 probe should be used, or FISH is recommended for confirmation.

We report the case of a 46-yr-old man with a 16-yr history of type I diabetes mellitus who developed rapid onset of nephrotic syndrome. Renal biopsy revealed diabetic nephropathy, characterized by thickened glomerular basement membranes (GBM), mild nodular glomerulosclerosis, and focal arteriolar hyalinization. Immunofluorescent (IF) studies showed strong granular IgM staining along glomerular loops, with subepithelial and intramembranous immune complex deposits along glomerular capillary loops demonstrated by electron microscopy (EM). These findings are consistent with membranous glomerulopathy with IgM as the predominant immunoglobulin. In addition, there were large aggregates of electron-dense material composed of numerous ring or spherical particles, ranging from 200 to 400 nm, in Bowman's space, which corresponded to eosinophilic aggregates on light microscopy (LM) and strong IgM stained materials by IF studies.

The aim of this study was to elucidate protein circuitry in breast cancer based on the profiling of hormone-receptor-negative breast carcinomas using morphoproteomic and pharmacoproteomic techniques. Three human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were reacted by immunohistochemical (IHC) procedures to detect several categories of protein analytes. Immunoreactivities and cell compartmentalizations were scored from 0 to 3+ positivity using bright-field microscopy. An automated cellular imaging system (ACIS) was used to obtain a final combined score of staining intensity and positive cells in the IHC reactions, to enable comparisons with the visual scores and the rates of inhibition by pharmaceutical agents. FDA-approved inhibitors that target the protein circuitry were added to the cultures for 2-4 days. Proliferation assays were conducted, and in vitro inhibition rates were calculated as (control - treated)/control. Western blot analyses of whole cell lysates assessed the effects of the pharmaceutical agents on selected aspects of protein circuitry. Good to excellent correlation was observed between visual scores and ACIS scores (r values from 0.732 to 0.996 in 10 of 11 trials). Gleevec produced growth inhibition that correlated with the composite expressions of the platelet-derived growth factor (PDGF) family of ligands and receptors; captopril inhibited only MDA-175, consistent with its unique expression of plasmalemmal angiotensin-converting enzyme (ACE); and interferon (IFN)-alpha effected growth inhibition in accordance with the degree of conventional (c) protein kinase C (PKC)-alpha and phosphorylated (p)-PKCalpha/betaII expressions. Western blot analyses revealed correlative changes of several intracellular signals following incubation with these inhibitors. This study shows (a) a close association between the immunohistochemical expression of signal transduction markers and in vitro inhibition by pharmaceutical agents, and (b) correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone-receptor-negative breast cancer cell lines. Such morphoproteomic and pharmacoproteomic profiling of individual tumors may enable the pathologist and oncologist to design antitumor therapy that is customized for an individual patient.

Background: Current histologic methods for diagnosis are limited by intra- and inter-observer variability. Immunohistochemistry (IHC) methods are frequently used to assess biomarkers to aid diagnoses, however, IHC staining is variable and nonlinear and the manual interpretation is subjective. Furthermore, the biomarkers assessed clinically are typically biomarkers of epithelial cell processes. Tumors and premalignant tissues are not composed only of epithelial cells but are interacting systems of multiple cell types, including various stromal cell types that are involved in cancer development. The complex network of the tissue system highlights the need for a systems biology approach to anatomic pathology, in which quantification of system processes is combined with informatics tools to produce actionable scores to aid clinical decision-making. Aims: Here, we describe a quantitative, multiplexed biomarker imaging approach termed TissueCypher™ that applies systems biology to anatomic pathology. Applications of TissueCypher™ in understanding the tissue system of Barrett's esophagus (BE) and the potential use as an adjunctive tool in the diagnosis of BE are described. Patients and Methods: The TissueCypher™ Image Analysis Platform was used to assess 14 epithelial and stromal biomarkers with known diagnostic significance in BE in a set of BE biopsies with nondysplastic BE with reactive atypia (RA, n = 22) and Barrett's with high-grade dysplasia (HGD, n = 17). Biomarker and morphology features were extracted and evaluated in the confirmed BE HGD cases versus the nondysplastic BE cases with RA. Results: Multiple image analysis features derived from epithelial and stromal biomarkers, including immune biomarkers and morphology, showed significant differences between HGD and RA. Conclusions: The assessment of epithelial cell abnormalities combined with an assessment of cellular changes in the lamina propria may serve as an adjunct to conventional pathology in the assessment of BE.