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Background: Microtubule-associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP-tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker., Methods: Stathmin and MAP-tau expression levels were measured in a breast cancer cohort (n = 651) using the tissue microarray format and quantitative immunofluorescence (AQUA) technology, then correlated with clinical and pathological characteristics and disease-free survival., Results: Univariate Cox proportional hazard models indicated that high stathmin expression predicts worse overall survival (hazard ratio [HR] = 1.48; 95% confidence interval [CI] = 1.119-1.966; P = .0061). Survival analysis showed 10-year survival of 53.1% for patients with high stathmin expression versus 67% for low expressers (log-rank, P < .003). Cox multivariate analysis showed high stathmin expression was independent of age, menopausal status, nodal status, nuclear grade, tumor size, and estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression (HR = 1.19; 95% CI = 1.03-1.37; P = .01). The ratio of MAP-tau to stathmin expression showed a positive correlation to disease-free survival (HR = 0.679; 95% CI = 0.517-0.891; P = .0053) with a 10-year survival of 65.4% for patients who had a high ratio of MAP-tau to stathmin versus 52.5% 10-year survival rate for those with a low ratio (log-rank, P = .0009). Cox multivariate analysis showed the ratio of MAP-tau to stathmin was an independent predictor of overall survival (HR = 0.609; 95% CI = 0.422-0.879; P = .008)., Conclusions: Low stathmin and high MAP-tau are associated with increased microtubule stability and better prognosis in breast cancer., (Copyright © 2012 American Cancer Society.)

Background: The importance of definitive histological subclassification has increased as drug trials have shown benefit associated with histology in nonsmall-cell lung cancer (NSCLC). The acuity of this problem is further exacerbated by the use of minimally invasive cytology samples. Here we describe the development and validation of a 4-protein classifier that differentiates primary lung adenocarcinomas (AC) from squamous cell carcinomas (SCC)., Methods: Quantitative immunofluorescence (AQUA) was employed to measure proteins differentially expressed between AC and SCC followed by logistic regression analysis. An objective 4-protein classifier was generated to define likelihood of AC in a training set of 343 patients followed by validation in 2 independent cohorts (n = 197 and n = 235). The assay was then tested on 11 cytology specimens., Results: Statistical modeling selected thyroid transcription factor 1 (TTF1), CK5, CK13, and epidermal growth factor receptor (EGFR) to generate a weighted classifier and to identify the optimal cutpoint for differentiating AC from SCC. Using the pathologist's final diagnosis as the criterion standard, the molecular test showed a sensitivity of 96% and specificity of 93%. Blinded analysis of the validation sets yielded sensitivity and specificity of 96% and 97%, respectively. Our assay classified the cytology specimens with a specificity of 100% and sensitivity of 87.5%., Conclusions: Molecular classification of NSCLC using an objective quantitative test can be highly accurate and could be translated into a diagnostic platform for broad clinical application., (Copyright © 2011 American Cancer Society.)

Background: Assessment of outcome using a single prognostic or predictive marker is the current basis of targeted therapy, but is inherently limited by its simplicity. Multiplexing has provided better classification, but has only been done quantitatively using RNA or DNA. Automated quantitative analysis is a new technology that allows quantitative in situ assessment of protein expression. The authors hypothesized that multiplexed quantitative measurement of ErbB receptor family proteins may allow better prediction of outcome., Methods: The authors quantitatively assessed the expression of 6 proteins in 4 subcellular compartments in 676 patients using breast carcinoma tissue microarrays. Then using Cox proportional hazards modeling and unsupervised hierarchical clustering, they assessed the prognostic value of the expression singly and multiplexed., Results: Epidermal growth factor receptor (EGFR), HER-2, and HER-3 expression were associated with decreased survival. Multivariate analysis showed high HER-2 and HER-3 expression maintained independence as prognostic markers. Hierarchical clustering of expression data defined a small class enriched for HER-2 expression with 40% 10-year survival, compared with 55% using conventional methods. Clustering also revealed a similarly poor-prognostic subgroup coexpressing EGFR and HER-3 (but low for estrogen receptor, progesterone receptor, and HER-2) with a 42% 10-year survival., Conclusions: This work shows that the combined analysis of protein expression improved prognostic classification, and that multiplexed models were superior to any single-marker-based method for prediction of 10-year survival. These methods illustrate a protein-based, multiplexed approach that could more accurately identify patients for targeted therapies., ((c) 2009 American Cancer Society.)

Background: Multispectral image analysis is an emerging tool that utilizes both spatial and spectral image information to classify images that can be used for the differentiation between benign versus malignant cells. The aim of the current study was to analyze the ability of this tool in differentiating subtle cytologic differences that cannot be appreciated by the human eye. Herein, the authors used fine-needle aspirations (FNAs) of follicular adenoma (FA) and parathyroid adenoma (PA) as a test case., Methods: The Nuance platform was used to collect image stacks that were subsequently analyzed with CRI-MLS software, a neural network-based artificial intelligence system that can classify images using automatically "learned" spatial-spectral features. CRI-MLS was trained on random, well-preserved FA cells and PA cells from the training set (n = 45 cells each). An algorithmic solution was developed and then validated on an independent series comprised of 1904 FA cells from 5 FA cases and 690 PA cells from 5 PA cases., Results: The solution from the CRI-MLS classifier showed 1876 FA cells (98.5%) as true FA and 28 FA cells (1.5%) as false PA, whereas 663 PA cells (96%) were true PA and 27 PA cells (4%) were false FA. The summary result of this solution was a sensitivity of 98.5%, a specificity of 96.1%, and a positive predictive value of 98.6%., Conclusions: The best spatial-spectral imaging solution was able to correctly classify 2534 of 2594 cells (98%) and misclassified only 55 of 2594 cells (2%). These data suggest that this technology may be valuable in a clinical setting to help differentiate and classify morphologically similar lesions., ((c) 2007 American Cancer Society)

Background: Cytotechnologists and pathologists often perform onsite evaluations of thyroid fine-needle aspirations (FNAs) to provide immediate feedback regarding whether adequate material has been obtained for cytologic diagnosis. The current study was designed to determine whether onsite adequacy assessment results in a significant decrease in nondiagnostic specimens between ultrasound (US)-guided FNAs of the thyroid and those performed by palpation alone., Methods: A search was performed to identify in-house thyroid FNAs performed between January 1, 2000 and December 31, 2003 that were obtained under US guidance or by palpation only. It was then recorded whether an onsite adequacy assessment was performed. The submitting physician and final diagnosis also were recorded for each case. Contingency tables were constructed and evaluated using chi-square analysis., Results: Of 1502 in-house thyroid FNAs included in the current study, 981 (65.3%) were performed under US guidance and 521 (34.7%) were performed with palpation alone. Onsite adequacy assessment of the aspirated material was performed in 323 cases (21.5%), whereas 1179 cases (78.5%) were performed without onsite evaluation. Of the 418 palpation-guided FNAs that were performed without adequacy assessment, 70 (16.7%) were reported to be nondiagnostic, whereas of the 103 palpation-guided FNAs with immediate evaluation, only 7 (6.8%) were determined to be inadequate for diagnosis. This difference was statistically significant (P < 0.025). Of 761 US-guided FNAs without immediate adequacy assessment, 54 (7.1%) were nondiagnostic, which was not statistically different from the nondiagnostic rate of 4.5% (10 of 220 cases) for US-guided FNAs with onsite evaluation. However, when these US-guided FNAs were divided further into 2 groups based on the experience of the radiologist performing the FNA, the nondiagnostic rate in the group of experienced radiologists was only 5.4% (or 32 of 592 US-guided FNAs), even though onsite evaluation was not performed. Among radiologists with less experience, adequacy assessment significantly reduced the nondiagnostic rate from 13.0% (22 of 169 FNAs without adequacy assessment) to 4.5% (10 of 220 FNAs with adequacy assessment) (P < 0.01)., Conclusions: The results of the current study demonstrate that onsite adequacy assessment of thyroid FNAs significantly reduces the number of nondiagnostic aspirates. However, the benefit of onsite evaluation, at least for US-guided FNAs, depends on the experience of the radiologist. In the current study, experienced radiologists with a relatively low nondiagnostic rate did not benefit from onsite adequacy assessment. This finding confirms the importance of experience in the performance of FNA, but suggests that onsite adequacy assessment may assist the less experienced operator., (Copyright 2006 American Cancer Society.)

Background: Measures of vascular endothelial growth factor (VEGF) expression in pancreatic cancer typically have been qualitative or semiquantitative. The objective of this study was to use a series of algorithms called AQUA that quantitatively assesses protein expression on tissue microarrays (TMAs) to compare in situ expression of VEGF and its primary receptors, VEGF receptor 1 (FLT-1) and VEGF receptor 1 (FLK-1), on a pancreatic cancer TMA., Methods: TMAs were constructed by arraying 1.5-mm cores from 76 samples of pancreatic adenocarcinoma (1996-2002) that were obtained from the archives of the Yale Department of Pathology. The staining for AQUA was similar to standard immunohistochemistry and involved antigen retrieval and the application of primary antibodies, but with epifluorescence detection. Slides were counterstained with 4',6-diamidino-2-phenylindole for nuclear visualization and cytokeratin for membrane visualization. The primary antibodies used were VEGF, FLT-1, FLK-1, and cytokeratin., Results: Disease stage was highly prognostic for outcome, as expected. Total amounts of VEGF and its receptors were assessed within the tumor mask and were divided into quartiles. Kaplan-Meier survival curves showed that VEGF and FLK-1 were not associated clearly with outcome. However, the expression of FLT-1 was correlated significantly, and the patients who had tumors with the lowest expression FLT-1 levels had the worst survival (P = .0038). In multivariate analysis, FLT-1 expression was an independent prognostic factor for overall survival (P = .0044)., Conclusions: VEGF and its 2 principal receptors were expressed to varying degrees in tumors of the pancreas. A significant association was found between low expression of FLT-1 and both poor prognosis and advanced stage, suggesting that tumor expression of this VEGF receptor is a marker of less aggressive disease., (2006 American Cancer Society)

Background: Since its introduction more than 45 years ago, the pelvic wash has gained widespread acceptance and is used routinely use at most centers. However, widely varying figures have been reported regarding its sensitivity for peritoneal involvement in ovarian tumors. In the current study, the authors evaluated a consecutive group of pelvic (peritoneal or abdominopelvic) washings performed in the evaluation of adnexal masses to determine whether histologic subtype significantly affects the tumor detection rate using this procedure., Methods: Reports from all washes performed over a 5-year period in the evaluation of adnexal masses were evaluated and correlated with those of the synchronously obtained histologic specimens. The sensitivity for each histologic subtype was calculated, with ovarian surface involvement and/or tumoral involvement of any peritoneal surface defined as the criterion standard. Cases with cytologic and histologic concordance were defined as true-positive or true-negative. Statistical significance was determined using the Fisher exact test., Results: In the current study, 185 of 846 (21.9%) total washes were associated with malignant (n = 161) or borderline (n = 24) tumors involving the ovary. For the malignancies, the overall cytology detection rate was 25%. A comparison of the cytology detection rates for the individual histologic subtypes with the overall rate demonstrated that the serous carcinomas were more likely (P = 0.0144) and the clear cell carcinomas were less likely (P = 0.0452) to be detected in pelvic washings. Cytology detection rates for mucinous, endometrioid, and undifferentiated carcinomas did not appear to differ significantly (P > 0.05) from the average detection rate. The cytohistologic correlation rate (efficiency), sensitivity, and specificity for the 5 most common histologic subtypes (n = 130) were 79.23%, 50.77%, and 93.33%, respectively. Differences also were observed in the calculated sensitivity for each subtype: serous (n = 57), 71.4%; endometrioid (n = 30), 58.33%; clear cell (n = 19), 20%; mucinous (n = 13), 50%; and undifferentiated (n = 11), 50%. Borderline tumors demonstrated a sensitivity and specificity of 80% and 100%, respectively., Conclusions: In the current study, the pelvic wash was found to be a specific, but only moderately sensitive, technique for detecting peritoneal involvement in ovarian tumors. The histologic subtype of the underlying ovarian tumor was found to have an effect on the likelihood of detection of peritoneal involvement using this diagnostic assay., (Copyright 2004 American Cancer Society.)

Background: Although cytologic evaluation of urine specimens is a standard procedure in the diagnosis and follow-up of bladder carcinoma, its sensitivity and specificity are low. Cytopathologic diagnoses are driven primarily by spatial relations or morphology. Although color enhances the pathologist's perception of the specimen, spectral information plays a minimal role in diagnostic processes. Recently, methods have been developed to capture and analyze spectral information from clinical specimens. In the current study, the authors determined the classification value of spectral information by testing its ability to discriminate between malignant and benign urothelial cells in cytology specimens., Methods: Multiple images of benign urothelial cells (n = 39) and urothelial carcinoma cells (n = 35) were collected at serial wavelengths using a liquid crystal tunable optical filter and composited into a mosaic using ENVI (Environment for Visualizing Images) software. Through minimum noise fractionation and principal component analysis, the spectral information in the mosaic was compressed into a 29-dimensional scatter plot. The data generated were analyzed using visual and spectral end member extraction on both the original data set and a second independent data set (test set)., Results: One area of spectral clustering in the scatter plot segmented with carcinoma cells exclusively (100% specific), but was not present in every cell (approximately 50%), which may indicate that these spectral profiles are present in a subpopulation of malignant cells or at specific points of their cell cycle. Using ENVI algorithms, the authors found that a particular classification spectrum (end member 9) and its closest relatives identified malignant cell clusters, with a sensitivity and specificity that reached 82% and 81%, respectively. To validate this mechanism in a test set, a second mosaic comprised of 15 benign and 15 malignant clusters was analyzed using end member 9, resulting in a combined sensitivity and specificity of 73%., Conclusions: The results of the current study demonstrate that spectral information, in the complete absence of morphologic or spatial information, allows discrimination of benign and malignant urothelial cells in routine urine cytology specimens. The authors believe that this novel technology, combined with spatial analysis, has the potential to serve as an ancillary test for improved detection of bladder carcinoma., (Copyright 2004 American Cancer Society.)

Background: It was found previously that alphaB-crystallin, a small heat-shock protein, was overexpressed in a metastatic variant of the GI101A human breast carcinoma cell line. The objective of the current study was to determine whether the expression of alphaB-crystallin in primary breast carcinomas was associated with lymph node metastasis and survival., Methods: Expression of alphaB-crystallin was measured in human breast carcinoma cell lines by immunoblotting. Expression in human breast carcinomas was evaluated by immunohistochemical staining of tissue microarrays that contained samples from 317 patients with lymph node-negative breast carcinoma and 291 patients with lymph node-positive breast carcinoma., Results: It was found that alphaB-crystallin was expressed constitutively in certain breast carcinoma cell lines, including those that were capable of metastasizing in immunodeficient mice. Expression of alphaB-crystallin in human tissue samples was associated strongly with lymph node involvement (P < 0.0001; chi-square test) and, to a lesser degree, with high nuclear grade (P = 0.05). Increased intensity of expression was correlated with shorter survival (P = 0.0091; log-rank test). Multivariate analysis indicated that alphaB-crystallin expression was not independent of lymph node status as a predictor of survival., Conclusions: The data obtained in the current study revealed a strong association between high expression levels of alphaB-crystallin in primary breast carcinoma specimens and lymph node involvement. Further studies will be needed to prospectively elucidate the role of this novel tumor marker as a clinical prognostic marker in local and locally advanced breast carcinoma as well as its potential status as a new target for therapy in patients with breast carcinoma., (Copyright 2004 American Cancer Society.)

Background: Aberrant activation of the beta-catenin signaling pathway has been implicated in several malignancies, including breast carcinoma. Recently, it was shown that p53 down-regulated beta-catenin in a complex fashion. The authors examined the expression of beta-catenin, key members of its signaling pathway, and p53 in a large cohort of breast tumors., Methods: The authors conducted an immunohistochemical analysis of the expression of beta-catenin, upstream modulators (HER-2/neu, Met, and epidermal growth factor receptor [EGFR]), downstream target genes (cyclin D1 and matrix metalloproteinase-7 [MMP7]), and p53 on a tissue microarray of 346 lymph node-negative breast carcinomas. The results were correlated with one another and with standard clinicopathologic parameters., Results: beta-Catenin expression was observed in the membrane and/or cytoplasm without any significant nuclear expression. HER-2/neu and EGFR were observed on the membrane in 21% and 6% of tumors, respectively, and Met stained in a membrane/cytoplasm distribution in 28% of cases. Cyclin D1 was expressed in the nucleus and MMP7 was expressed in the cytoplasm in 26% and 75% of tumors, respectively. Nuclear expression of p53 was noted in 31% of tumors. When each marker was analyzed separately, only p53 and Met demonstrated a significant correlation with survival. However, patients who had tumors that coexpressed high levels of beta-catenin and p53 had markedly worse overall survival (P = 0.0026). In multivariate analysis, only tumor size, Met, and the coexpression of beta-catenin and p53 retained statistical significance., Conclusions: The current findings support a potential synergistic effect of abnormal beta-catenin regulation and p53 status in the pathogenesis and natural history of lymph node-negative breast carcinoma. Furthermore, the results show that a combined analysis of multiple markers, notably beta-catenin and p53, may enhance the prognostic capabilities compared with individual markers., (Copyright 2004 American Cancer Society.)

Background: It has been shown that receptor tyrosine kinases (RTKs) predict outcome in patients with breast carcinoma. Although RTKs are a large family, HER-2, epidermal growth factor receptor (EGFR), Met (hepatocyte growth factor receptor), and others all have shown the ability to predict outcome. However, it remains unclear whether these markers are defining the same subpopulation of patients with breast carcinoma. In this study, the authors attempted to determine the correlation between RTKs on the basis of their ability to stratify a population according to outcome., Methods: The authors used tissue microarray technology to study 324 patients with lymph node negative breast carcinoma who had 20-40 years of follow-up. Expression was assessed using immunohistochemical stains for Met, EFGR, fibroblast growth factor receptor (FGFR), and HER-2. Expression levels were assessed by two observers, and correlations were analyzed. Standard pathology information, including tumor size, nuclear grade, Ki-67 receptor status, and estrogen and progesterone receptor expression levels, also was collected., Results: RTK expression in the study cohort revealed two strong correlations. Specifically, HER-2 and EGFR showed similar expression patterns (P < 0.0001), and Met cytoplasmic domain and FGFR cytoplasmic staining showed similar expression patterns (P < 0.0001), but no correlation was found between the two groups. Of these RTKs, only high levels of Met cytoplasmic domain showed significance as a prognostic marker defining a shortened survival compared with the rest of the population (P = 0.0035; relative risk, 2.04). In the same group of patients, HER-2, hormone receptor status, and other RTK family receptors were not correlated with outcome. In multivariate analysis, only Met cytoplasmic domain and tumor size showed independent predictive value., Conclusions: The current results indicate that the cytoplasmic domain of Met shows a unique staining pattern and defines a set of patients unique from the set of patients defined by overexpression of HER-2, EGFR, or hormone receptors. Furthermore, this group of patients is associated tightly and independently with worse outcome., (Copyright 2003 American Cancer Society.)

Background: The advent of cDNA microarrays and other molecular technologies necessitates the acquisition of tumor cell-enriched material because nonmalignant cells often decrease the sensitivity of the assays. Fine-needle aspiration (FNA) specimens from carcinoma have long been noted to be enriched in malignant cells. The current study quantitated the relative representation of tumor versus nontumor cells in FNA specimens compared with tissue sections using breast carcinoma as a model., Methods: Five patients who had undergone both a diagnostic FNA and a surgical excision for breast carcinoma between January and July of 1996 were selected. Five random cellular fields from representative slides of the FNA (using the ThinPrep preparation of the wash) and surgical excision specimens were photographed digitally at x20 power. The cells were judged as tumor or nontumor cells and then were counted manually in each field., Results: The calculated percentage of malignant cells in the FNA specimen (as represented on the ThinPrep slide) ranged from 66-93% compared with the calculated percentage of 37-78% noted in histologic sections. The average of all 5 fields from all 5 cases showed that 83.1% of the total cells were malignant in the ThinPrep preparation compared with 62.3% in the histologic sections. This difference was highly statistically significant when analyzed using the chi-square test (P = 0.0009)., Conclusions: The percentage of malignant cells on FNA specimens from breast carcinoma, as assessed by ThinPrep, was found to be significantly higher than that obtained by surgical excision. The results of the current study quantitatively confirm the impression of practicing cytopathologists, but also suggest that FNA will provide a good substrate for cDNA microarray and other molecular analyses., (Copyright 2002 American Cancer Society.)

Background: Intraductal breast fluids containing exfoliated mammary epithelial cells can be harvested from the breast by ductal lavage to screen for disease-associated cytologic abnormalities. In addition to epithelial cells, breast fluids contain large numbers of mammary foam cells, and the tissue of origin of these foam cells has been the subject of controversy for many years. Immunocytochemical, morphologic, and molecular studies variously have supported a mammary epithelial origin versus a histiocytic origin for this cell type. In the current study, the authors performed immunocytochemical analysis with epithelial specific and macrophage specific antibodies to characterize and quantify breast cells obtained by ductal lavage., Methods: Breast fluids were harvested from 19 individual breast ducts in 15 female patients by ductal lavage. Cells from each specimen were processed for immunocytochemical staining using the AE1/AE3 multicytokeratin and CD68 (clone KP1) monoclonal antibodies. Cells were classified as mammary epithelial cells or mammary foam cells on the basis of morphologic criteria, and the cells were counted and evaluated for immunoreactivity with epithelial specific and macrophage specific antibodies., Results: The CD68 macrophage specific antibody stained all ductal lavage cells that exhibited foam cell morphology. The AE1/AE3 multicytokeratin antibody demonstrated strong, positive staining of cells that exhibited epithelial morphology but failed to demonstrate significant staining of mammary foam cells. The lavage specimens contained a range of 3040-278,850 epithelial cells and 2230-90,480 foam cells. The median numbers of epithelial cells and foam cells per lavage sample were 15,680 and 29,200, respectively. The ratio of epithelial cells to foam cells varied among specimens ranging from 3.4 to 0.09 (median, 0.8). Seven of 19 lavage specimens contained more epithelial cells than foam cells, whereas 12 samples contained a greater proportion of foam cells., Conclusions: Immunocytochemical analysis using the AE1/AE3 multicytokeratin and CD68 antibodies supports a histiocytic origin for the majority of mammary foam cells harvested from the ductal system of the human breast by ductal lavage. Although mammary foam cells constitute a significant proportion of the cellular population obtained by ductal lavage, thousands of epithelial cells also are harvested.

Background: Ascites fluid or a pleural effusion are common events in metastatic carcinoma, but they also can be associated with several other medical conditions. The standard for determination of malignancy in these situations is cytologic evaluation of these fluids. Although this method is frequently successful, there are times when it fails, even when the patient has a malignancy, either because of insufficient cells in the fluid or for other reasons. This study addresses this problem taking advantage of the recent advances in technology for detection of rare epithelial cells in liquid specimens., Methods: The authors examined fluid specimens from 59 patients to determine the frequency of recovery of epithelial cells compared with that achieved by conventional cytopathology. The Dynal CELLection Epithelial Enrich (Dynal AS, Oslo, Norway) method was used. This method is based on immunomagnetic selection of cells binding to EpCAM antibodies. Carcinoma cells were confirmed by morphology and, when there was sufficient material, by E-cadherin staining., Results: Grouping the cases by cytologic diagnosis, the authors found malignant cells using the cell enrichment assay in 11 of 12 malignant cases, 2 of 5 atypical cases, and 3 of 42 negative cases. Further investigations were conducted on the five cases that were cytologically negative or atypical but yielded epithelial cells after immunomagnetic enrichment. Four cases ultimately were proven malignant by other methods and one had incomplete follow-up., Conclusions: The new methods available for epithelial cell enrichment in liquids may be used successfully on cytologic fluid specimens and may lead to increased sensitivity for detection of malignancy, and consequently more accurate staging., (Copyright 2002 American Cancer Society.)

The use of molecular techniques in cytopathology has become an accepted and billable practice for certain applications. Other applications still are in the developmental or experimental stages but may soon be clinically useful. The current study provides a review of these techniques including molecular detection of clonality, in situ hybridization, loss of heterozygosity, and single base mutation detection. A number of new molecular techniques recently have been described that may have a dramatic impact on diagnostic pathology. These techniques, in situ detection of single base mutations and microarray technology, are introduced and discussed in the context of potential applications to cytopathology in the future.

Background: Lymph node metastasis is the oldest and most reliable prognostic indicator in breast carcinoma. In the absence of tumor metastasis, draining lymph nodes can undergo hyperplasia, resulting in increases in the number and size of detectable lymph nodes. The prognostic value of this process has never been established. Lymph node negative breast carcinoma provides a unique opportunity to study the downstream effects of increased lymphatic drainage and lymph node hyperplasia., Methods: The authors studied 290 cases of lymph node negative breast carcinoma and provided patients with a median of 103 months of follow-up. The number of tumor free lymph nodes in ipsilateral axillary resections, as well as 10 traditional histopathologic markers, were analyzed for their prognostic value., Results: The cohort was divided into quartiles according to the number of tumor negative lymph nodes. The 5-year survival for patients with 20 or more tumor free lymph nodes (top quartile) was 84.7%, compared with 96.3% for patients with fewer than 20 tumor free lymph nodes. The 5-year relative risk of dying of metastatic disease in the top quartile was 3.61 (95% confidence interval, 1.37-9.52, P = 0.01), independent of necrosis, tumor size, patient age, nuclear and histologic grade, lymphocytic infiltrate, and lymphovascular invasion. The absolute lymph node number was highly associated with the presence of necrosis in invasive tumor., Conclusions: The number of tumor free lymph nodes is a novel, independent predictor of aggressive disease in cases of lymph node negative breast carcinoma. This finding may be a biologic function of host-derived, and possibly tumor-derived, lymphangiogenic cytokines., (Copyright 2000 American Cancer Society.)

Background: Activation of Met, the receptor for scatter factor/hepatocyte growth factor, is associated with mitogenesis, motogenesis, and decreased cell adhesion. Elevated expression of Met has been shown in advanced cases of carcinoma of the prostate, stomach, pancreas, and thyroid. The authors previously demonstrated that Met expression is an independent prognostic marker associated with decreased survival in patients with breast carcinoma., Methods: Expression of Met in 113 archival breast carcinoma specimens from patients with axillary lymph node negative invasive ductal carcinoma was evaluated using a standard immunoperoxidase technique. Cases were scored by two pathologists using an H-score algorithm and then analyzed for correlation with known prognostic factors and survival., Results: Expression of Met showed a bimodal distribution with 25% of cases showing high levels of expression. In contrast to previous studies, the results of the current study showed a significant association between Met expression and nuclear and histologic grade. The 5-year survival rate for Met negative patients with tumors with low Met expression was 95% in this cohort, compared with 80% for patients with tumors showing high Met expression. Patients whose tumors had a high level of Met expression were found to have a 5-year relative risk (RR) of dying of metastatic disease of 5.05 (P = 0.03) independent of patient age and tumor size. Combined analysis of Met and nuclear grade resulted in a marked increase in independent predictive value (RR = 33.4; P < 0.01)., Conclusions: The results of the current study found that high levels of Met expression were associated with death due to metastatic disease in patients with axillary lymph node negative breast carcinoma. Expression of Met may be a useful prognostic indicator of more aggressive disease in patients whose prognosis is not easily stratified by current histopathologic markers., (Copyright 1999 American Cancer Society.)

Background: Fine-needle aspiration (FNA) of breast specimens can be difficult and between 10-25% of the lesions ultimately are classified as "atypical," even by the most experienced cytopathologist. The goal of this study was to identify a molecular mechanism that reliably distinguishes benign and malignant (or pre-malignant) lesions and that could be used as an adjunct in these morphologically ambiguous cases., Methods: Because all malignancies represent clonal proliferations, assessment of clonality represents a potential molecular mechanism for making this distinction. Excess material preserved from breast FNAs was examined using the human androgen receptor locus clonality assay. This assay allows determination of clonality on the basis of X chromosome inactivation as detected by polymerase chain reaction analysis of genomic DNA after methylase-sensitive restriction digestion., Results: In this pilot study, 25 cases showed reproducible results. All malignant cases (9 of 9) were monoclonal, whereas 10 of 12 benign cases were polyclonal. Of four atypical cases, two were monoclonal and both were found to be malignant after surgical resection. Monoclonality was observed in two benign cases that were hyperplastic lesions., Conclusions: These preliminary results suggest that this test may provide a non-morphologic molecular mechanism for the objective categorization of breast FNAs.

Background: The c-met protooncogene encodes the met protein, the receptor for scatter factor/hepatocyte growth factor, a growth factor that modulates the motility and stable interaction of the epithelial cells. This study assesses the expression of met receptor in breast carcinoma and its prognostic value with respect to survival., Methods: Immunofluorescence was used to evaluate 91 archival breast carcinoma specimens using a polyclonal antibody to the cytoplasmic domain of the receptor. Cases were scored by two pathologists on a percentage basis and then converted to binary scores (positive or negative) on the basis of a bimodal distribution., Results: Strong expression of met was found in 20 invasive ductal breast tumor specimens (22%). The 5-year survival of patients whose tumors showed decreased met expression was 89%, in contrast to a 52% 5-year survival rate in patients whose tumors expressed met (P = 0.008). This trend also was observed in patients without lymph node metastases at presentation, in whom met negative patients had a 95% 5-year survival compared with only 62% for met positive patients (P = 0.006) Multivariate analysis using the Cox proportional hazards model showed met expression to be an independent predictor of survival, with a predictive value nearly equivalent to that associated with lymph node status., Conclusions: The authors conclude that expression of met in patients with invasive ductal carcinoma of the breast is a strong, independent predictor of decreased survival and may be a useful prognostic marker with which to identify a subset of patients with more aggressive disease.

Background: The distinction between benign reactive mesothelial cells and well differentiated carcinoma can be difficult in pleural, peritoneal, and especially pericardial fluids. E-cadherin is an adhesion protein that is specifically expressed in cells of epithelial lineage. In this study, anti-E-cadherin antibodies were used to identify and distinguish carcinoma cells from reactive mesothelial cells., Methods: Pleural, peritoneal, and pericardial fluids were prepared using the Cytyc Thin Prep processor. The specimens were comprised of a mix of 45 cases that were diagnosed as carcinoma, suspicious, or reactive by Papanicolaou staining of routine material seen by the authors' service. Routine immunologic techniques were used with a commercially available E-cadherin antibody., Results: In most cases of carcinoma, tumor cells showed a strong positive membranous reaction product (32 of 37). This included four cases that were not cytomorphologically diagnosed as malignant, but subsequently proved to be malignant. E-cadherin staining was not observed in five tumors, two of which were not expected to express this protein. One benign case showed cells staining for E-cadherin, although the cells were not malignant by morphologic criteria. Because this case was a surgical pelvic washing, these cells more likely were epithelial contaminants than true false-positives., Conclusions: The epithelial specific cell-cell adhesion marker E-cadherin reliably distinguishes reactive mesothelial cells from carcinoma and is a useful adjunctive test to distinguish benign reactive mesothelial cells from well differentiated carcinoma cells in fluid specimens.

Background: A pathologic diagnosis is usually required to determine definitive management for a palpable lesion. In this era of cost control, fine-needle aspiration (FNA) provides a low-cost alternative to open excisional biopsy. Using a broad range of cases collected over 20 years, the authors of this study sought to quantify the savings resulting from the use of FNA on superficial palpable lesions to obtain a pathologic diagnosis., Methods: 12,452 cases collected by the cytopathology service at the Medical College of Virginia during the period 1972-1991 were used to produce a profile of case type, diagnosis, and indication for surgery. Charge-based cost estimations or Relative Value Units were calculated using the 1995 Physicians' Fee Reference or published Medicare participant fees. The charges for the FNA procedure and open surgical biopsy were compared, and all other biopsy-related costs were omitted., Results: FNA provided a sufficient pathologic diagnosis to obviate open surgical biopsy in 63-85% of the cases. Estimation of cost savings on the basis of the distribution of cases and indications for surgery suggested a savings of $250,000 to $750,000 per 1000 FNA performed, or approximately 5500 Relative Value Units., Conclusions: This study quantifies the substantial savings that result from obtaining a pathologic diagnosis by the FNA procedure rather than open surgical biopsy.