Biomedical subjects

David L Rimm

Publications and source records attributed to David L Rimm.

64 records · Page 4Linked to original sources

PubMed2002

Quantitative examination of mechanophysical tumor cell enrichment in fine-needle aspiration specimens.

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.

Biopsy, Needle↗

PubMed2002

Automated subcellular localization and quantification of protein expression in tissue microarrays.

The recent development of tissue microarrays-composed of hundreds of tissue sections from different tumors arrayed on a single glass slide-facilitates rapid evaluation of large-scale outcome studies. Realization of this potential depends on the ability to rapidly and precisely quantify the protein expression within each tissue spot. We have developed a set of algorithms that allow the rapid, automated, continuous and quantitative analysis of tissue microarrays, including the separation of tumor from stromal elements and the sub-cellular localization of signals. Validation studies using estrogen receptor in breast carcinoma show that automated analysis matches or exceeds the results of conventional pathologist-based scoring. Automated analysis and sub-cellular localization of beta-catenin in colon cancer identifies two novel, prognostically significant tumor subsets, not detected by traditional pathologist-based scoring. Development of automated analysis technology empowers tissue microarrays for use in discovery-type experiments (more typical of cDNA microarrays), with the added advantage of inclusion of long-term demographic and patient outcome information.

Algorithms↗

PubMed2002

Immunocytochemical analysis of breast cells obtained by ductal lavage.

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.

Antigens, CD↗

PubMed2002

Alterations of Smad signaling in human breast carcinoma are associated with poor outcome: a tissue microarray study.

Based largely on studies of cell lines in vitro and of transgenic mouse models, disruptions of transforming growth factor (TGF) beta signaling are thought to contribute to the development and progression of human breast cancer. However, whether and how TGF-beta signaling becomes disrupted during human breast cancer development in vivo remains largely unknown. To address this question, we have compared the patterns of expression and activation of the postreceptor components of the TGF-beta signaling pathway, the so-called Smads, in human breast cancer cell lines with those in breast carcinoma specimens. None of the breast carcinoma cell lines were growth arrested by TGF-beta in vitro. Each of the tumor cell lines expressed normal levels of Smad2 and -3. Moreover, TGF-beta treatment induced phosphorylation of Smad2 (Smad2P) in each of these lines, except those that lacked TGF-beta type II receptors. Moreover, only one of the cell lines failed to express Smad4. Among 456 cases of human breast carcinoma assembled in tissue microarrays, the majority (92%) expressed Smad2, Smad2P, as well as Smad4, indicating their ability to proliferate within a microenvironment that contains bioactive TGF-beta. Thirty cases (6.6%) failed to express Smad2P, suggesting the loss of TGF-beta receptor signaling. Nine cases (2%) failed to express Smad4, and 3 of these also failed to express Smad2P. Thus, the phenotypes of breast tumors in vivo paralleled that of human breast cancer cell lines in terms of Smad2P and Smad4 expression. Loss of Smad signaling was not associated with any particular histological subtype, histological or nuclear grade, estrogen- or progesterone receptor expression, or HER2/neu expression. Loss of Smad4 was inversely correlated with the presence of axillary lymph node metastases. Most importantly, among patients with stage II breast cancer, lack of Smad2P expression in the tumor was strongly associated with shorter overall survival. Finally, analysis of a small cohort of hereditary breast cancers failed to reveal any association between BRCA1 or BRCA2 genotype and alterations in Smad signaling.

Animals↗

PubMed2002

Use of magnetic enrichment for detection of carcinoma cells in fluid specimens.

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.

Ascites↗

PubMed2002

Subjective differences in outcome are seen as a function of the immunohistochemical method used on a colorectal cancer tissue microarray.

Immunohistochemistry is a useful technique to localize antigens in cell preparations and tissue sections and can be helpful in identifying molecular markers that may be predictive of patient outcomes. Subjective assessment of expression and semiquantitative grading systems are the current standards in pathology literature for the analysis of tissue sections. However, expression levels assessed in this manner may be dramatically affected by the method of visualization. Tissue microarray (TMA) is a recently developed technique for the simultaneous high-throughput evaluation of protein expression on tissue samples from large cohorts of patients. The scoring of TMAs has, in general, mirrored the systems utilized for tissue sections. Here, 4 detection systems (avidin-biotin complex, indirect immunofluorescence, peroxidase-labeled polymer conjugate, and the latter with Cyanine-3-Tyramide amplification) were compared using a beta-catenin antibody on a TMA containing a cohort of colorectal cancer specimens. Peroxidase-labeled polymer with or without tyramide enhancement was found to be the most sensitive method, revealing a greater staining intensity and percentage of nuclear staining, without an apparent increase in background. Subjective assessment of expression is highly dependent on the method of visualization and may illustrate why discrepant data is often seen in literature based on immunohistochemistry.

Avidin↗

PubMedDate not supplied

Direct interaction of the C-terminal domain of alpha-catenin and F-actin is necessary for stabilized cell-cell adhesion.

Alpha-catenin functions to anchor adherens junctions to the filamentous actin (F-actin) cytoskeleton, through direct and indirect binding mechanisms. When truncated at amino acid 865, alpha-catenin exhibited a markedly reduced F-actin binding affinity compared to wild-type. Expression of the truncated mutant in the alpha-catenin deficient colon carcinoma cell line, Clone A, could not restore an adhesive phenotype when compared. Furthermore, the truncated alpha-catenin fusion protein failed to concentrate at sites of cell-cell contact, to promote morphological changes associated with epithelial monolayers, and to stimulate resistance to shearing forces in a hanging drop aggregation assay. Subsequent attempts to isolate single residues governing the direct F-actin interaction, using neutralizing charge or reverse charge mutations of basic residues within a homology modeled alpha-catenin C-terminal 5-helix bundle, had no effect on F-actin cosedimentation. We conclude that direct attachment of alpha-catenin to F-actin is required to promote cadherin-mediated contact formation and strong cell-cell adhesive states.

Actins↗

PubMedDate not supplied

Loss of Smad signaling in human colorectal cancer is associated with advanced disease and poor prognosis.

PURPOSE: Based largely on in vitro investigations and animal studies, investigators believe that disruptions of transforming growth factor-beta (TGF-beta) signaling contribute to the development and progression of human colorectal cancer. The purpose of this study was to directly assess the status of the TGF-beta signaling pathway in colorectal cancer and determine the effects of its disruption on clinical behavior and outcome. MATERIALS AND METHODS: Smad proteins are the principal intracellular components of the TGF-beta signaling pathway. We conducted a high-throughput analysis of the expression patterns of Smad2, phosphorylated (activated) Smad2 (pSmad2), and Smad4 in more than 600 human colorectal cancer specimens assembled in tissue microarrays. RESULTS: The vast majority (93.8%; 95% CI: 92%-96%) of colorectal cancers expressed phosphorylated Smad2, indicating the ability of the tumors to survive and proliferate within a microenvironment that contains bioactive TGF-beta. Twelve of 633 (1.9%; 95% CI: 1%-3%) cases failed to express Smad2, and 15 of 641 (2.3%; 95% CI: 1%-4%) cases failed to express Smad4. Moreover, 29 of 615 (4.7%; 95% CI: 3%-7%) of cases expressed Smad2 but not its activated form (pSmad2), suggesting the presence of a TGF-beta receptor defect. Based on an analysis of 577 cases for which clinical outcome information was available, failure to express Smad2, pSmad2, or Smad4 was associated with advanced-stage disease, the presence of lymph node metastases, and a significantly shorter overall survival (median survival: 35 vs 58 months). DISCUSSION: Loss of Smad activation and/or expression occurs in approximately 10% of colorectal cancers. This subset has a poor prognosis because of its association with advanced disease and the presence of lymph node metastases at diagnosis.

Cell Division↗

PubMedDate not supplied

Immunohistochemical biomarkers in patients with early-onset breast carcinoma by tissue microarray.

UNLABELLED: Young women with breast cancer have a poor prognosis, and the role of biologic markers in young women is not well defined. We investigated the association of estrogen receptor, progesterone receptor, Bcl-2, HER-2/neu, p53, and Ki-67 with clinicopathologic features and outcome in young women with breast cancer. METHODS: A cohort of 103 patients with early-onset breast cancer treated with conservative surgery and radiotherapy were entered in this study. Age range was 25-45 years, and median follow-up was 8.7 years. Each of the paraffin-embedded specimens was immunologically stained for six biomarkers expression by a recently developed tissue microarray method. RESULTS: The 10-year overall breast relapse-free and distant relapse-free survival rates were 82.7%, 84.6.4%, and 66.7%, respectively, with 14 local relapses and 26 distant metastases among the 103 patients evaluated. Positive expression of estrogen receptor, progesterone receptor, bcl-2, HER-2/neu, p53, and Ki-67 were 42.7%, 48.5%, 35.6%, 28.0%, 36.9%, and 39.7%, respectively. Tumor stage and nodal status were significantly associated with overall survival and distant metastasis-free rate in univariate and multivariate analysis. Progesterone receptor negativity and Ki-67 positivity were associated with distant metastasis. There was no statistically significant correlation between the six biomarkers and local relapse. CONCLUSIONS: Progesterone receptor, Ki-67, tumor stage, and nodal status were prognostic factors for distant failure in early-stage breast cancer in young patients. Further studies are needed to find other biologic markers associated with local failure in this group of patients.

Adenocarcinoma, Mucinous↗

PubMedDate not supplied

p53 mutations as tumor markers in fine needle aspirates of palpable breast masses.

OBJECTIVE: Mutations in p53 exons 5-8 are found in 40-50% of breast carcinomas. We performed a retrospective analysis of p53 mutations in fluid-based, archival fine needle aspirates (FNAs) of breast masses to determine their potential diagnostic utility as breast tumor cell markers. STUDY DESIGN: Residual, fluid-based, archival FNAs of 27 breast masses were retrospectively evaluated by polymerase chain reaction (PCR), single-strand conformational polymorphism analysis (SSCP) and sequencing for p53 exons 5-8. Results were compared with the morphologic diagnoses and genotyping of available excisional biopsy tissue. RESULTS: Six of the twenty-seven cases were found to have a clonal mutation in p53; all six mutated cases showed carcinoma on subsequent excisional biopsy. Definitive cytologic diagnosis of cancer had been possible in only four of the six cases. Identical mutations were found in the excised carcinomas in the five cases with available tissue. None of the 14 aspirates with benign cytology had detectable mutations in p53. CONCLUSION: p53 Mutational analysis by PCR/SSCP/sequencing deserves to be critically studied as a diagnostic criterion in patients with indeterminate or suspicious cytology. Validation studies should be performed to test p53 mutations as molecular diagnostic markers in breast cytology specimens.

Abscess↗