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Albumin degradation in the renal tubules is impaired in diabetic nephropathy such that levels of the resulting albumin fragments increase with the degree of renal injury. However, the mechanism of albumin degradation is unknown. In particular, fragmentation of the endogenous native albumin has not been demonstrated in the kidney and the enzymes that may contribute to fragmentation have not been identified. To explore this we utilized matrix-assisted laser desorption/ionization imaging mass spectrometry for molecular profiling of specific renal regions without disturbing distinct tissue morphology. Changes in protein expression were measured in kidney sections of eNOSdb/db mice, a model of diabetic nephropathy, by high spatial resolution imaging allowing molecular localizations at the level of single glomeruli and tubules. Significant increases were found in the relative abundances of several albumin fragments in the kidney of the mice with diabetic nephropathy compared with control nondiabetic mice. The relative abundance of fragments detected correlated positively with the degree of nephropathy. Furthermore, specific albumin fragments accumulating in the lumen of diabetic renal tubules were identified and predicted the enzymatic action of cathepsin D based on cleavage specificity and in vitro digestions. Importantly, this was demonstrated directly in the renal tissue with the endogenous nonlabeled murine albumin. Thus, our results provide molecular insights into the mechanism of albumin degradation in diabetic nephropathy.
Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
BACKGROUND - The impact of re-resection of a positive intraoperative bile duct margin on clinical outcomes for resectable hilar cholangiocarcinoma (HCCA) remains controversial. We sought to define the impact of re-resection of an initially positive frozen-section bile duct margin on outcomes of patients undergoing surgery for HCCA.
METHODS - Patients who underwent curative-intent resection for HCCA between 2000 and 2014 were identified at 10 hepatobiliary centers. Short- and long-term outcomes were analyzed among patients stratified by margin status.
RESULTS - Among 215 (83.7%) patients who underwent frozen-section evaluation of the bile duct, 80 (37.2%) patients had a positive (R1) ductal margin, 58 (72.5%) underwent re-resection, and 29 ultimately had a secondary negative margin (secondary R0). There was no difference in morbidity, 30-day mortality, and length of stay among patients who had primary R0, secondary R0, and R1 resection (all p > 0.10). Median and 5-year survival were 22.3 months and 23.3%, respectively, among patients who had a primary R0 resection compared with 18.5 months and 7.9%, respectively, for patients with an R1 resection (p = 0.08). In contrast, among patients who had a secondary R0 margin with re-resection of the bile duct margin, median and 5-year survival were 30.6 months and 44.3%, respectively, which was comparable to patients with a primary R0 margin (p = 0.804). On multivariable analysis, R1 margin resection was associated with decreased survival (R1: hazard ratio [HR] 1.3, 95% confidence interval [CI] 1.0-1.7; p = 0.027), but secondary R0 resection was associated with comparable long-term outcomes as primary R0 resection (HR 0.9, 95% CI 0.4-2.3; p = 0.829).
CONCLUSIONS - Additional resection of a positive frozen-section ductal margin to achieve R0 resection was associated with improved long-term outcomes following curative-intent resection of HCCA.
The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections.
INTRODUCTION - During pancreaticoduodenectomy (PD) for ductal adenocarcinoma, a frozen section (FS) neck margin is typically assessed, and if positive, additional pancreas is removed to achieve an R0 margin. We analyzed the association of this practice with improved overall survival (OS).
METHODS - Patients who underwent PD for pancreatic ductal adenocarcinoma from January 2000 to August 2012 at 8 academic centers were classified by neck margin status as negative (R0) or microscopically positive (R1) on the basis of FS and permanent section (PS). Impact on OS of converting an FS-R1-neck margin to a PS-R0-neck margin by additional resection was assessed.
RESULTS - A total of 1399 patients had FS neck margins analyzed. Median OS was 19.7 months. On FS, 152 patients (10.9%) were R1, and an additional 51 patients (3.6%) had false-negative FS-R0 margins. PS-R0-neck was achieved in 1196 patients (85.5%), 131 patients (9.3%) remained PS-R1, and 72 patients (5.1%) were converted from FS-R1-to-PS-R0 by additional resection. Median OS for PS-R0-neck patients was 21.1 months versus 13.7 months for PS-R1-neck patients (P < 0.001) and 11.9 months for FS-R1-to-PS-R0 patients (P < 0.001). Both FS-R1-to-PS-R0 and PS-R1-neck patients had larger tumors (P = 0.001), more perineural invasion (P = 0.02), and more node positivity (P = 0.08) than PS-R0-neck patients. On multivariate analysis controlling for adverse pathologic factors, FS-R1-to-PS-R0 conversion remained associated with significantly worse OS compared with PS-R0-neck patients (hazard ratio: 1.55; P = 0.009).
CONCLUSIONS - For patients who undergo pancreaticoduodenectomy for pancreatic ductal adenocarcinoma, additional resection to achieve a negative neck margin after positive frozen section is not associated with improved OS.
BACKGROUND - The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has been anticipated to improve communication between pathologists and clinicians and thereby patient outcomes. In the current study, the impact of TBSRTC on various quality and outcome measures was assessed.
METHODS - The current study included all patients who underwent fine-needle aspiration (FNA) of the thyroid between April 2006 and April 2009. Before implementation, the authors used generic diagnostic categories; after implementation, TBSRTC was used. Quality of reporting, diagnostic categories, rate of surgery, rates of frozen section, the "risk" of malignancy after a cytologic diagnosis, and errors before and after implementation of TBSRTC were compared using the chi-square and Fisher exact tests. Multilevel likelihood ratios and the receiver operating characteristic were used to compare the accuracy of FNA before and after implementation.
RESULTS - A total of 1671 FNAs (957 obtained before and 714 obtained after implementation of TBSRTC) were obtained from 1339 patients. Of these, 301 patients (191 before and 110 after implementation) underwent subsequent surgical resection. Before implementation, the reports were more ambiguous (3.7% vs 0.5%; P < .05) and implicit (5.1% vs 2.7%; P < .05) than after implementation. The overall rate of surgery decreased after implementation of TBSRTC (24.5% vs 19.6%; P < .05). The overall risk of malignancy did not appear to be affected by implementation of TBSRTC, but it decreased significantly after a benign FNA diagnosis compared with a diagnosis of an atypical lesion or follicular neoplasm. The rate of frozen section remained unchanged. The diagnostic accuracy was not found to be significantly different before compared with after implementation of TBSRTC.
CONCLUSIONS - Implementation of TBSRTC appears to improve the quality of reporting by lowering the number of ambiguous and implicit diagnoses and decreases the overall surgery rates, particularly for benign lesions, but it does not appear to have any effect on the accuracy of FNA of the thyroid, false-positive rates, or the frequency of intraoperative consultations.
Copyright © 2011 American Cancer Society.
The pathologic classification of nonsmall cell lung cancer (NSCLC) is evolving. Lung adenocarcinoma is morphologically heterogeneous, with mixtures of acinar, papillary, bronchioloalveolar, and solid patterns in more than 80% of cases. In case of synchronous or metachronous multiple NSCLC, the distinction of intrapulmonary metastases from independent primary tumors is of great clinical importance as it influences staging and potentially the therapeutic strategy. Here we took advantage of a cohort of 20 patients with 42 multiple NSCLC tumors (24 potential pair comparisons) that were annotated molecularly using genomic and mutational profiling to evaluate the value of comprehensive histologic assessment in this setting. Using the Martini-Melamed criteria, paired tumors were characterized as multiple primary NSCLCs in 21 cases and as intrapulmonary metastases in 3 cases. Genomic and mutational data led to a diagnosis of multiple primaries in 14 cases and of metastases in 8 cases; 2 cases could not be assessed. This molecular characterization contradicted the Martini-Melamed diagnosis in 7 (32%) of the 22 assessable comparisons. Adenocarcinoma was found in 32 (76%) of the 42 tumors. After review in a blinded fashion, semiquantitative comprehensive histologic assessment of paired tumors was different in 16 and similar in 8 paired tumors. We found that comparing adenocarcinomas is a complex issue that requires assessment not only of percentages of the histologic subtypes, but also the recording of additional histologic details such as cytologic features, patterns of stroma, necrosis, discrete nodularity versus miliary growth and variants such as clear cell, signet ring, mucinous, and fetal patterns. We also found that paired squamous cell carcinomas could be compared based on histologic subtyping in addition to cytologic and stromal characteristics. Considering histologically different tumors as multiple primaries, and similar tumors as metastases, comprehensive histologic subtyping was consistent with the molecular characterization in 20 (91%) of the 22 pairs comparisons. In summary, based on a well characterized cohort with detailed clinical, pathologic and molecular data, we found comprehensive histologic assessment is a powerful tool that seems to be a promising way to determine whether multiple lung adenocarcinomas or squamous cell carcinomas are metastatic or multiple primaries. This has great clinical implications for staging and therapeutic management of lung cancer patients with multiple tumors. Given its high correlation with molecular characterization of such tumors, it may provide a much cheaper and faster method to address this problem.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 microg of protein yielded approximately 400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from approximately 17% after one year of storage to approximately 25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.
Lung cancer is the leading cause of cancer deaths in both men and women in the United States. The LIFE (Light Induced Fluorescence Endoscopy) Project was initiated at the University of North Carolina Medical Center in November, 1999, for the dual purposes of (1) detecting pre-invasive lung cancer in high-risk patients and (2) studying the molecular biology of pre-invasive lesions of the bronchus for possible development of molecular biomarkers. Of the 47 patients enrolled, all were current or former tobacco smokers, except for 1. Fluorescence endoscopy was utilized, in addition to white light bronchoscopy, to increase the detection of intraepithelial lesions. Adjacent biopsies were submitted for permanent and frozen sections, respectively, from four predetermined sites as well as from any abnormal areas. The snap-frozen specimens were cryostat sectioned, and the mucosal epithelial cells laser capture microdissected for DNA analysis. The great majority of specimens yielded sufficiently abundant and intact DNA to accomplish the molecular objectives. Histologic concordance of adjacent permanent and frozen sections was equivalent to the concordance of adjacent permanent sections, suggesting that frozen section diagnosis was adequate for the research purpose of correlating histology with molecular analysis.
OBJECTIVES - To evaluate the utility of routine intraoperative frozen-section histologic analysis during partial nephrectomy to ensure negative surgical margins. Partial nephrectomy has gained acceptance for surgical treatment of small renal cancers. Many surgeons send specimens for intraoperative frozen section histologic analysis to ensure negative margins.
METHODS - We reviewed the records of 78 patients who underwent partial nephrectomy for presumed malignancy. Patient demographics, intraoperative findings, and pathologic and clinical outcomes were analyzed.
RESULTS - Seventy-nine partial nephrectomies were performed in 78 patients. Frozen sections were obtained intraoperatively in 76 cases. In 1 case (1.3%), a single margin was interpreted as positive for carcinoma, prompting deeper resection. The final histopathologic finding was interpreted as angiomyolipoma rather than carcinoma. The final pathologic examination revealed renal cell carcinoma in 52 (66%) of 79 cases. The mean oncologic follow-up was 16.2 months. One local recurrence was noted (1.9%). It arose in the resection bed 19 months after removal of a 4.5-cm tumor (pathologic Stage T3a). Both intraoperative frozen section margins and final pathologic margins were negative in this case. One patient developed pulmonary metastases and represented the only metastatic recurrence, as well as the only cancer-related death in our cohort (1.9%).
CONCLUSIONS - Our data suggest that when partial nephrectomy is performed with attention to excising a perimeter of grossly normal-appearing parenchyma, sending specimens for intraoperative frozen section analyses may provide an unnecessary expense without providing meaningful, reliable information. Additional studies, including larger cohorts and extended follow-up, are needed to support these results.
A method has been developed for obtaining visual pigment absorbance spectra from fixed, frozen sections of the albino rat retina. The retinas are transversely sectioned at various thicknesses on a cryo-microtome and the resulting slices are examined with a photon-counting microspectrophotometer. Problems caused by the limited pigment content of small receptors are reduced and measurements can be made at well-specified locations along the retinal section. This method is both precise and accurate and permits comparisons across sections and across animals.