Cathepsin D and prognosis in breast cancer: one piece of a larger puzzle?
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Biomarkers, Tumor↗
Biomedical subjects
M R Emmert-Buck
Publications and source records attributed to M R Emmert-Buck.
von Hippel-Lindau disease gene deletion detected in microdissected sporadic human colon carcinoma specimens.
The progression of human malignancies is thought to involve the inactivation or loss of tumor suppressor genes. Previous studies have suggested that inactivation of tumor suppressor genes on chromosomes 5q, 17p, 18q, and 8p play a role in the development of colorectal carcinoma. However, chromosome 3p at the von Hippel-Lindau disease (VHL) gene locus (3p25-26) has not been previously implicated in the development or progression of sporadic colorectal carcinoma. The authors have analyzed VHL gene alterations on chromosome 3p in sporadic human colon carcinomas and adenomas using modified microdissection techniques. These techniques allow for procurement and analysis of selected subpopulations of cells from both paraffin-embedded and frozen human tumor specimens. VHL disease gene deletion was detected by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis in microdissected colon carcinoma specimens. Allelic loss of VHL gene was detected in 7 of 11 (64%) informative patients who underwent colectomy for primary sporadic colon carcinoma. However, no allelic loss of VHL gene was shown in colonic adenomas of eight informative patients. These results indicate that VHL disease gene deletion frequently occurs in sporadic colon carcinoma. Because this deletion was not present in adenomas, VHL gene may play a role in colonic carcinogenesis and represent a relatively late event in colonic neoplasia progression. Additionally, microdissection of tissue sections may be especially useful in detecting allelic loss in PCR-based studies of infiltrating tumors, particularly when the tumor cells represent a relatively small percentage of the total cell population.
Adenocarcinoma↗
Genetic analysis of synchronous mucinous tumors of the ovary and appendix.
The coexistence of mucinous ovarian and appendiceal tumors in association with pseudomyxoma peritonei (PP) is well established. However, it has not been determined whether they represent independent or metastatic neoplasms. The authors analyzed microsatellites on chromosome 17q 21.3-22 (nm23), 3p 25-26 (von Hippel Lindau disease [VHL] gene), and 5q 21-22 (D5S346 locus) in 12 synchronous ovarian and appendiceal mucinous lesions. Loss of heterozygosity (LOH) at the nm23 locus has been shown previously in ovarian carcinomas, and genetic alterations at both the 3p and 5q loci have been reported in colorectal carcinomas. The ovarian lesions consisted of nine mucinous tumors of low malignant potential and three invasive adenocarcinomas, and the appendiceal lesions consisted of eight carcinomas without invasion, two invasive carcinomas, and two mucosal hyperplasias. DNA was extracted from microdissected cells obtained from formalin-fixed, paraffin-embedded tissue sections and amplified by polymerase chain reaction. In three specimens, genetic alterations occurred at 17q 21.3-22 in only the ovarian tumors. One of these cases showed LOH on chromosome 5q 21-22 in only the appendiceal tumor. In three other specimens, LOH at the same locus was found in both tumors. Six specimens did not show LOH at any locus. These results suggest that a subset of synchronous mucinos ovarian and appendiceal lesions showing different LOH patterns in both sites most likely represent patients with two separate primary lesions. Another group of specimens with the same allelic loss in both tumors most likely represent patients with a single primary and metastatic spread. Thus, genetic analysis of these lesions may be useful in investigating the origin of histologically similar synchronous tumors.
Adenocarcinoma, Mucinous↗
Loss of heterozygosity on the short arm of chromosome 8 in male breast carcinomas.
Identification of loss of heterozygosity (LOH) at specific genetic loci in neoplastic cells suggests the presence of a tumor suppressor gene within the deleted region. LOH on chromosome 8p has been identified in colorectal, bladder, hepatocellular, and prostatic carcinomas. Little is currently known about the molecular events occurring during the development of male breast cancer. We studied LOH on chromosome 8p in 23 male breast carcinomas. Five polymorphic DNA markers were used: D8S136 and D8S137 on 8p12-21.3; and D8S254, D8S258, and D8S349 on 8p22. DNA was extracted from microdissected normal and tumor cells obtained from formalin-fixed, paraffin-embedded tissue sections and amplified by the PCR. LOH was identified in 19 of 23 cases (83%) with at least one marker. Seven cases showed LOH only at 8p22, six cases showed LOH only at 8p12-21.3, and six cases showed LOH at both 8p22 and 8p12-21.3. In five of these last six cases, at least one locus was retained between the two deleted regions; thus, the whole short arm of chromosome 8 was not lost in these tumors. Our results show that there are two discrete areas of deletion on chromosome 8p in male breast cancer, suggesting the presence of one or more tumor suppressor genes that may play a role in the development or progression of the disease.
Breast Neoplasms, Male↗
Necropsy as a research method in the age of molecular pathology.
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Autopsy↗
Loss of heterozygosity on chromosome 11q13 in microdissected human male breast carcinomas.
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Breast Neoplasms, Male↗
Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia.
The development and progression of human prostate cancer is associated with genetic abnormalities in tumor cells. Inactivation of tumor suppressor genes due to allelic loss is thought to be an important mechanism of gene alteration in prostatic neoplasms. In this study we examined allelic loss on chromosome 8p12-21 in microdissected samples of normal prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and invasive prostate carcinoma from the same patients. Tissue microdissection under direct microscopic visualization procures pure populations of cells of interest, including small lesions such as PIN. Among 30 patients with concomitant cancer and PIN, we found loss of heterozygosity on chromosome 8p12-21 in 63% (34 of 54) of foci of PIN examined and 90.6% (29 of 32) of tumors, suggesting that abnormalities on chromosome 8p12-21 may be important in the early stages of prostatic carcinoma development. Several cases in which multiple foci of PIN from the same patient were sampled showed different patterns of allelic loss. Fifty-five % (16 of 29) of the prostate carcinomas contained a potential precursor PIN focus based on allelic loss pattern. Our results are consistent with the hypothesis that PIN arises multifocally within the prostate gland, and that a subset of these lesions progress to become carcinoma.
Alleles↗
Cell surface binding of TIMP-2 and pro-MMP-2/TIMP-2 complex.
Tissue inhibitor of metalloproteinases (TIMP-2) is a low molecular weight proteinase inhibitor capable of inhibiting activated matrix metalloproteinases (MMPs). TIMP-2 is found both free and in a 1:1 stoichiometric complex with the pro-enzyme form of MMP-2 (pro-MMP-2/TIMP-2 complex). We have measured the binding of recombinant TIMP-2 to intact HT-1080 and MCF-7 cells. HT-1080 cells in suspension bound 125I-labeled rTIMP-2 with a Kd of 2.5 nM and 30,000 sites/cell. Monolayers of MCF-7 cells were similarly found to bind [125I]rTIMP-2 with a Kd of 1.6 nM and 25,000 sites/cell. Specific binding of MMP-2 alone to HT-1080 cells was not observed; however, pro-MMP-2/TIMP-2 complex was capable of binding to the surface of HT-1080 cells in a TIMP-2-dependent manner. Binding of rTIMP-2 was not competed by the presence of TIMP-1. These results suggest that rTIMP-2 alone binds directly to the cell surface of HT-1080 and MCF-7 cell lines, and TIMP-2 is capable of localizing MMP-2 to the surface of HT-1080 cells via interaction with a specific binding site.
Cell Line↗
Identical allelic loss on chromosome 11q13 in microdissected in situ and invasive human breast cancer.
Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasias to in situ carcinoma and finally to invasive and metastatic cancer. In situ breast carcinoma consists of small, isolated neoplastic foci which cannot be selectively studied by conventional methods. In this study, we used tissue microdissection to examine the loss of heterozygosity (LOH) of chromosome 11q13 in both in situ and invasive lesions of the breast, as compared to normal breast epithelium from the same patients. Forty-one cases of sporadic breast cancer were analyzed. Tissue microdissection allows for procurement and PCR-based analysis of small lesions using either frozen or formalin-fixed, paraffin-embedded tissue sections. LOH on chromosome 11q13 was found in 24 of 36 (67%) of the informative invasive breast cancer cases using two polymorphic DNA markers specific for this region (INT2 and PYGM). Twenty-one of the cases which demonstrated LOH in the invasive tumor also contained in situ carcinoma in the same tissue section. Seventy-one % (15 of 21) of the microdissected in situ lesions showed LOH of chromosome 11q13. Every case (15 of 15) of in situ tumor with LOH showed loss of the same allele in the corresponding invasive tumor cells. The results of this study suggest that a tumor suppressor gene located on chromosome 11q13 may play an important role in the early stages of development of sporadic human breast cancer. This finding provides molecular genetic support for the hypothesis that invasive breast cancer arises from in situ lesions.
Alleles↗
A microdissection technique for archival DNA analysis of specific cell populations in lesions < 1 mm in size.
We have developed a microdissection technique that allows for procurement and analysis of specific, minute cell populations from routine, 5-mu, formalin-fixed, paraffin-embedded histological tissue sections. Lesions < 1 mm in size can be specifically examined. Cells of interest are procured under direct microscopic visualization followed by a single-step DNA extraction and subsequent polymerase chain reaction. Amplification of DNA from selected cell populations was demonstrated by detecting a loss of heterozygosity (LOH) at the von Hippel-Lindau disease (VHL) gene in an atypical renal lesion and a renal cell carcinoma in a kidney of a VHL patient. Moreover, previously unrecognized LOH on the short arm of chromosome 3 (3p25-26) was detected in microdissected colorectal carcinoma cells in a non-VHL patient with sporadic colon carcinoma. This technique should prove useful in DNA studies of small lesions and cell populations. Furthermore, microscopic premalignant, in situ, and invasive lesions can be selectively examined.
Base Sequence↗
Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer.
Cathepsins D, B, and L are acidic lysosomal proteinases involved in intracellular protein turnover. Increased levels of these enzymes have been reported to be indicators of aggressive tumor behavior in human and rodent tumors. In breast cancer increased levels of cathepsin D have been reported to be an independent prognostic factor in women with stage I disease. We used standard immunohistochemical techniques on formalin-fixed, paraffin-embedded tissue to examine the levels of cathepsins D, B, and L in 80 carcinomas of the breast and compared that with other indicators of aggressive tumor behavior, including stage of disease, tumor size, nuclear grade, estrogen receptor status, disease recurrence, and 5-year survival rates. Positive granular cytoplasmic staining was detected for cathepsin D in 90% of the tumors, for cathepsin B in two thirds of the tumors, and for cathepsin L in approximately one half of the tumors. Positive staining also was seen in normal breast epithelium, areas of apocrine metaplasia, stromal fibroblasts, and macrophages. Our results did not show a correlation between the expression of cathepsins D, B, and L and other indicators of aggressive tumor behavior. We conclude that the results obtained using polyclonal anticathepsin antibodies do not support the prognostic usefulness of immunohistochemical analysis of these three proteinases in tumor cells in human breast cancer.
Adult↗
Cathepsin B expression in colorectal carcinomas correlates with tumor progression and shortened patient survival.
Cathepsin B is a lysosomal cysteine proteinase that has the ability to degrade several extracellular matrix components at both neutral and acidic pH and has been implicated in the progression of several human and rodent tumors. We have studied the expression of cathepsin B in human colorectal tissues using a monospecific polyclonal rabbit antibody raised against human liver cathepsin B. In immunoblots of normal and neoplastic colorectal tissues this antibody specifically recognized only cathepsin B. We studied 101 cases of formalin-fixed, paraffin-embedded tissue (15 normal mucosa, 17 adenomas, and 69 carcinomas). Epithelial cells of normal mucosa and adenomas were either negative or showed a weak granular reactivity located in the paranuclear and apical cytoplasm of superficial cells. Small clusters of histiocytes were also positive in the region of the superficial area of the lamina propria. In carcinomas, increased expression of cathepsin B correlated with advanced stage of the disease. Increased immunoreactivity of cathepsin B in malignant cells was associated with either a diffuse cytoplasmic staining or was polarized to the basal pole of the cells. This is in contrast to the punctate paranuclear staining pattern observed in normal colonic mucosal cells. In tumor stromal cells, increased expression of the enzyme correlated with neoplastic progression. Expression of high levels of cathepsin B in the tumor epithelial cells was associated with a significantly shorter survival of the patients. In conclusion, our results indicate that cathepsin B expression is up-regulated in human colorectal carcinomas compared with normal mucosa and adenomas and correlates with tumor progression.
Adenoma↗
Pleomorphic rhabdomyosarcoma arising in association with the right pulmonary artery.
Pulmonary artery rhabdomyosarcomas are rare lesions, with a small number of cases reported in the literature. We report a case of pulmonary artery rhabdomyosarcoma that arose in the right pulmonary artery. The patient presented with cough, chest pain, and hemoptysis and was found to have a sarcoma arising in the right pulmonary artery and extending distally within the pulmonary vasculature into the right lung. The gross pathologic features, microscopic appearance, immunohistochemical profile, and computed tomographic and electron microscopic findings are presented. The site of origin and differential diagnosis of these lesions are discussed.
Adult↗
Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples.
Gelatinase A (MMP-2) and cathepsin B are proteinases which have been proposed to participate in human tumor invasion and metastasis. Precise quantitation of the activity of these enzymes in invading tumors has not been previously described. We utilized a novel tissue microdissection technique to determine levels of enzyme activity in specific microscopic areas of invasive human colon cancer. Tissue specimens smaller than one high power field can be extracted from the samples and analyzed. Increased levels of pro-enzyme and active enzyme forms of gelatinase A (MMP-2) and increased cathepsin B activity were localized in regions of tumor invasion as compared with a matched number of normal epithelial cells from the same patient. Levels of progelatinase B (MMP-9) were also increased in the tumors; however, we did not observe activation of this enzyme. To investigate the mechanism of gelatinase A activation, we amplified DNA of specific microdissected tumor cell populations using polymerase chain reaction. We did not detect a mutation in the activation locus of the enzyme in any of the tumors studied, which suggests that activation may be due to up-regulation of a tumor-associated gelatinase A activating species. Microdissection of frozen tissue sections may prove valuable in the study of proteinases in human tumor invasion as well as in the detection of genetic alterations in human cancers.
Base Sequence↗
Analysis of mRNA quality in freshly prepared and archival Papanicolaou samples.
OBJECTIVE: To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN: To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month-old samples. Smears of approximately 500 base pairs were observed in the 1-year-old and 10-year-old samples. Successful mRNA amplification was possible from microdissected cell populations. CONCLUSION: Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.
Cervix Uteri↗
The role of tissue microdissection in cancer research.
Tissue microdissection is a laboratory method that is used to procure specific cells or cell populations from a histology slide under direct microscopic visualization. The recovered cells can be studied with a variety of DNA, messenger RNA, and protein analysis methods, including new high-throughput gene expression and proteomics technologies. This approach is permitting investigators to comprehensivelyexamine the molecular anatomy of cells in tissue sections forthe first time. This article reviews the development and evolution of tissue microdissection techniques, summarizes examples of research studies, and discusses related challenges that the research community must address. Additional information and complete laboratory protocols are available on a website at http://cgap-mf.nih.gov/.
DNA, Neoplasm↗
Detection of the von Hippel-Lindau gene deletion in cytologic specimens using microdissection and the polymerase chain reaction.
Previous studies have demonstrated von Hippel-Lindau (VHL) gene abnormalities in renal cell carcinoma. Archival cytologic samples of renal cell carcinomas (RCCs) from two patients were evaluated for allelic loss of the VHL gene. Small clusters of renal carcinoma cells were dissected from cytocentrifuge preparations under direct microscopic visualization followed by single-step DNA extraction and subsequent polymerase chain reaction (PCR). Amplification of DNA from the harvested tumor cells demonstrated a loss of heterozygosity at the VHL gene in five RCC specimens obtained from two patients. The combination of microdissection and PCR enabled DNA studies to be performed on a pure population of tumor cells isolated from heterogeneous samples containing admixed normal cellular elements. Selected genetic studies performed on microscopically targeted cells will help to further the biologic and cytomorphologic characterization of the targeted cell population.
Alleles↗