Use of monoclonal antibodies in immunohistochemistry.
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Animals↗
Biomedical subjects
A K Tandon
Publications and source records attributed to A K Tandon.
Hepatitis A virus-associated cholecystitis.
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Acute Disease↗
Clinicoepidemiological study of drug resistance in Indian kala-azar.
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Antimony Sodium Gluconate↗
Cathepsin D by western blotting and immunohistochemistry: failure to confirm correlations with prognosis in node-negative breast cancer.
PURPOSE: We attempted to replicate and improve on our previous study (N Engl J Med 322:297-302, 1990) that showed that 34-kd cathepsin D levels as determined by Western blotting strongly correlated with disease-free survival (DFS) and overall survival (OS) of axillary node-negative (N-) breast cancer patients. We also examined the prognostic significance of cathepsin D measured by immunohistochemistry (IHC) in these patients. PATIENTS AND METHODS: Western blotting was performed on cytosols from frozen tumor specimens of 927 N- breast cancer patients in the San Antonio Breast Tumor Bank. The monoclonal antibody M1G8 was used to detect cathepsin D (in previous study, a polyclonal antiserum had been used). The same monoclonal antibody was also used for frozen-section IHC staining of tumor specimens from 562 N- patients from the same tumor bank. Levels of cathepsin D expression were then correlated with DFS and OS. RESULTS: Although the levels of cathepsin D expression as measured by Western blotting and IHC correlated with each other and with levels of cathepsin D measured in previous work using Western blotting with the polyclonal antiserum, in this present study, using the monoclonal antibody M1G8, we were unable to demonstrate that cathepsin D expression (measured by either Western blotting or by IHC) correlates with DFS or OS. CONCLUSION: In this study, cathepsin D expression as determined by either Western blotting or IHC using the monoclonal antibody M1G8 failed to improve the prognostic evaluation of N- breast cancer patients. The role of cathepsin D expression as a prognostic factor is still not precisely defined, raising questions about its use in the routine evaluation of N- breast cancer patients.
Analysis of Variance↗
Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications.
BACKGROUND: Cell synthesis of heat shock (stress-response) proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kd heat shock protein (hsp70) is thought to be involved in protein-protein interactions including those of the protein products of the human c-myc oncogene and the p53 (also known as TP53) tumor suppressor gene. PURPOSE: The purpose of this study was to investigate whether elevated hsp70 expression may be an indicator of biological stress experienced by a breast cancer and may, therefore, predict disease outcome. METHODS: Levels of hsp70 were determined by Western blot analysis in primary breast tumors from patients with negative axillary lymph nodes. We performed exploratory data analyses on a set of 162 primary breast cancers and constructed prognostic indexes of hsp70 expression levels. The optimal cutpoint for hsp70 expression was considered to be the value yielding the greatest separation for disease-free survival for the resulting two groups of patients. That cutpoint was then validated in a set of 345 tumors by univariate and multivariate analyses. Data were analyzed for overall survival, disease-free survival, tumor size, and patient age, as well as estrogen receptor and progesterone receptor status, ploidy (DNA content), and percentage of cells in S phase as determined by flow cytometry. RESULTS: Expression of hsp70 emerged as a useful prognostic factor, both in univariate and in multivariate analyses. Patients whose tumors had high expression of hsp70 had significantly shorter disease-free survival (P = .006). The other statistically significant factors were S-phase fraction (P = .008) and tumor size (P = .01). For patients who received adjuvant therapy, hsp70 was the only independent predictor of disease recurrence (P = .05). For those with tumors 1-3 cm in diameter, hsp70 (P = .008) and S-phase fraction (P = .02) were statistically significant predictors of recurrence. CONCLUSIONS: Measurement of hsp70 expression in primary tumors from patients with node-negative breast cancer may be useful in identifying patients at high risk for disease recurrence and thus may affect decisions regarding treatment after surgery. IMPLICATIONS: Future studies should be performed to determine if detection of hsp70 by immunohistochemistry can be used to predict clinical outcome and to better understand the relationships between hsp70 and the effects of various treatment modalities.
Analysis of Variance↗
In situ hybridization and immunohistochemical analysis of cytomegalovirus-associated ileal perforation.
A patient with the acquired immunodeficiency syndrome (AIDS) with a cytomegalovirus (CMV) infection localized in the area of an ileal Kaposi sarcoma resulting in perforation is described. Because only one case of such an association of Kaposi sarcoma with CMV leading to perforation has been reported, the presence and distribution of CMV-related nucleic acids and proteins in the affected segment of intestine were evaluated. By using in situ hybridization and immunocytochemical analyses the presence of CMV was shown within epithelial, endothelial, smooth muscle, and inflammatory cells at the site of perforation. This study not only confirmed that CMV can be detected in virtually all components of the intestinal wall despite the absence of distinctive cytomegalic changes, but also generated critical information that illustrates the usefulness of immunohistochemistry and in situ hybridization in elucidating the pathogenesis of CMV-associated lesions. These findings lend further support to the concept that CMV plays a pivotal role in the pathogenesis of intestinal perforation and emphasize the critical importance of in situ hybridization in gaining insight into the mechanisms of CMV-induced injury.
Antigens, Viral↗
Multiple-marker immunohistochemical phenotypes distinguishing malignant pleural mesothelioma from pulmonary adenocarcinoma.
The diagnostic distinction between malignant pleural epithelial mesothelioma and pulmonary adenocarcinoma is often difficult and partially reliant on the use of immunohistochemistry (IHC). While there are several available IHC markers preferentially expressed in adenocarcinomas compared with mesotheliomas, there are no readily available or well-validated markers preferentially expressed in mesotheliomas and no markers are absolutely specific for either tumor. Thus, there always will be an element of doubt regarding the identity of lesions diagnosed by IHC using these markers and an undesirable reliance on negative results if the lesion is truly a mesothelioma. To help optimize the ability of currently available but imperfect markers to resolve adenocarcinoma and mesothelioma and to minimize the impact of false-negative results on their diagnosis, a systematic study was undertaken to identify multiple-marker immunostaining phenotypes that are the most specific and sensitive for each type of tumor. The study involved staining a series of malignant epithelial mesotheliomas (n = 34) and pulmonary adenocarcinomas (n = 103) with eight markers that were selected on the basis of previous reports of their relatively restricted specificities for one or the other of these lesions (and included carcinoembryonic antigen [CEA], tumor-associated glycoprotein 72 [B72.3], Leu-M1, vimentin, thrombomodulin, secretory component, carcinoma antigen-125, and periodic-acid-Schiff with diastase [for mucin]. Considering the markers one, two, and three at a time, all possible combinations of results were analyzed by computer to identify the phenotype most sensitive and specific for adenocarcinoma or mesothelioma. The best single marker was CEA (positive: 97% specific and sensitive for adenocarcinoma; negative: 97% specific and sensitive for mesothelioma). However, because examples of both tumor types expressed CEA, none of the study cases were unequivocally resolved and the risk of false-negative results was relatively high. The best two markers were CEA and B72.3 (both positive: 100% specific and 88% sensitive for adenocarcinoma; both negative: 99% specific and 97% sensitive for mesothelioma). The four possible combinations of results for these two markers resolved 68% of cases (93 of 137 cases), with less risk of false-negative results than a single marker. In general, several three-marker panels resulted in sensitivities and specificities similar to the two-marker panel of CEA and B72.3. In particular, the three-marker panel of CEA, B72.3, and Leu-M1 resulted in eight possible phenotypes that resolved 74% of cases (101 of 137 cases), with even further reduced risk of false-negative results.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenocarcinoma↗
Glutathione transferase GST pi in breast tumors evaluated by three techniques.
The glutathione transferases are involved in intracellular detoxification reactions. One of these, GST pi, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GST pi expression in 60 human breast tumors by three techniques, immunohistochemistry. Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GST pi expression and steroid receptor status with all of the techniques utilized. In addition, there was a trend toward higher GST pi expression in poorly differentiated tumors, but no correlation was found between tumor GST pi content and DNA ploidy or %S-phase. GST pi expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GST pi measurements using either Northern hybridization or Western blot analysis. These results suggest that immunohistochemistry is the method of choice for measuring GST pi in breast tumors.
Blotting, Northern↗
Immunohistochemical study of intermediate filament proteins on routinely processed, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval.
Although immunohistochemical studies of intermediate filament proteins have been carried out on temporal bone sections by using modified fixation/embedding techniques to preserve antigenicity, there have been no light microscopic studies concerning immunohistochemical staining on routinely formalin-fixed celloidin-embedded human temporal bone sections. A method for immunostaining routinely processed celloidin-embedded tissues would be extremely valuable in that it would permit study of the extensive collections of formalin-celloidin temporal bone specimens that exist in major centers of otopathologic research. Recently, we have developed a new technique which can be used to retrieve the antigenicity masked by formalin fixation and decalcification. This method requires immersing slides for 30 min at room temperature in a solution of saturated sodium hydroxide in methanol before immunostaining. Using this method, 45 celloidin-embedded human temporal bone sections were stained with monoclonal antibodies to keratin, vimentin, neurofilament, glial fibrillary acidic protein and desmin as primary antibodies using a sensitive streptavidin-biotin procedure. The results obtained by using this technique are at least equivalent to those obtained with modified fixatives, cryosections or immuno-electron microscopy. This new method may provide a useful approach for studying routinely processed, celloidin-embedded human temporal bone sections and open a new field in immuno-otopathology.
Adult↗
Prognosis and treatment decisions in patients with breast cancer without axillary node involvement.
BACKGROUND: Every month, treatment decisions must be made for more than 6000 patients with breast cancer without axillary node involvement in the United States. Approximately 70% of these patients will survive more than 10 years after surgery and/or radiation treatment without additional systemic adjuvant therapy. If we had good methods to identify patients who are destined to have a recurrence of their disease, only those patients should receive adjuvant therapy. METHODS: The authors reviewed the literature supporting the use of currently available prognostic factors for patients with node-negative breast cancer, and formulated a framework on which prognostic factor information can be based to help make these treatment decisions. RESULTS: The steps involved in making treatment decisions are: use prognostic factors to determine the recurrence probability; calculate the expected treatment benefit; and weigh the expected benefits against the potential risks. CONCLUSIONS: Prognostic factors can be used to help make treatment decisions for patients with breast cancer without axillary node involvement. However, the final treatment decision must take into account all aspects of the patient and her disease, and the physician must help the patient evaluate her prognostic factors, arrive at an understanding of her particular risk of recurrence, and weigh the potential benefits and risks of adjuvant therapy.
Axilla↗
Correlation of HER-2/neu amplification with expression and with other prognostic factors in 1103 breast cancers.
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Breast Neoplasms↗
Inhibition of breast cancer cell growth in vitro by a tyrosine kinase inhibitor.
Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of breast cancer.
Breast Neoplasms↗
Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer.
Using permanent-section immunohistochemistry, we investigated the role of HER-2/neu in the development and progression of human breast cancer by measuring its overexpression in a series of hyperplastic (n = 30), dysplastic (n = 15), and malignant neoplastic (n = 708) lesions of ductal epithelium and by evaluating the relationships between overexpression and clinicopathologic features known to have prognostic significance in these lesions. The neoplasms included pure ductal carcinoma in situ (DCIS; n = 59) and infiltrating ductal carcinoma (IDC; n = 649). The latter were all node negative and stratified into IDC combined (n = 237) or not combined (n = 412) with a "significant amount" of DCIS (defined as DCIS greater than or equal to 10% of total tumor cellularity). Overexpression of HER-2/neu was not observed in any of the hyperplastic or dysplastic lesions. In contrast, it was present in 56% of pure DCIS and in 77% of the comedo subtype of this group. Only 15% of IDC overexpressed HER-2/neu. However, the rate of overexpression was significantly higher in the subset of IDC combined with DCIS compared with the subset of IDC not combined with DCIS (22% v 11%, respectively; P less than .0001). These results are consistent with the hypothesis that HER-2/neu plays a more important role in initiation than in progression of ductal carcinomas. They also suggest that overexpression decreases within individual tumors as they evolve from in situ to increasingly invasive lesions or, alternatively, that many invasive carcinomas arise de novo (ie, without progressing through a significant in situ stage) by mechanisms not involving HER-2/neu. In addition, overexpression of HER-2/neu was associated with several poor prognostic features (younger patient age, premenopause, negative estrogen receptor status, negative progesterone receptor status, and high nuclear grade) in the subset of IDC combined with DCIS. With one exception (negative estrogen receptor status) these associations were lost in IDC not combined with DCIS, also suggesting that the role of HER-2/neu changes during the progression of human breast cancer.
Biomarkers, Tumor↗
A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.
The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.
Antibodies, Monoclonal↗
HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma.
PURPOSE: Amplification and/or overexpression of the HER-2/neu oncogene have been shown to correlate with poor clinical outcome in patients with axillary node-positive breast cancer. In contrast, the prognostic significance of HER-2/neu in node-negative disease is controversial. This study was undertaken to evaluate further the relationship between HER-2/neu and clinical outcome in node-negative disease. PATIENTS AND METHODS: Overexpression of HER-2/neu was evaluated by permanent-section immunohistochemistry in tumors from 613 patients with long-term clinical follow-up enrolled in the Intergroup Study 0011. Patients were stratified into low-risk (n = 307) and high-risk (n = 306) groups on the basis of tumor size and estrogen-receptor (ER) status. Low-risk patients were defined as having small (less than 3 cm), ER-positive tumors and were observed without additional treatment after initial surgery. High-risk patients had either ER-negative or large (greater than or equal to 3 cm), ER-positive tumors and were randomized to be observed (n = 146) or to receive adjuvant chemotherapy (n = 160) after surgery. RESULTS: The rate of HER-2/neu overexpression was 14.3% in all tumors combined and was higher in invasive carcinomas with (21.5%) than without (11.2%) a significant noninvasive or in situ histologic component (P less than .0001). There was no relationship between overexpression and clinical outcome in the natural history setting of combined low-risk and high-risk patients not receiving adjuvant therapy (n = 453). Based on the reasoning that the influence of HER-2/neu may have been obscured by high-risk features and/or the presence of noninvasive carcinoma, we also analyzed the subset of patients with low-risk lesions not containing a significant in situ component (n = 179). Patients of this group with HER-2/neu-positive tumors showed only 40% disease-free survival (DFS) at 5 years, compared with over 80% in patients with HER-2/neu-negative tumors (P less than .0001). A similar inverse correlation was observed between overexpression and overall survival in the same group of patients (P = .0001). In a separate analysis involving patients receiving adjuvant chemotherapy, those with HER-2/neu-negative tumors showed significantly improved DFS in response to therapy compared with patients with HER-2/neu-positive tumors. CONCLUSION: Overexpression of HER-2/neu is associated with poor clinical outcome in a subset of node-negative patients with small, ER-positive, predominantly invasive tumors and may play a role in resistance to adjuvant chemotherapy.
Breast Neoplasms↗
S-100 protein in human inner ear: use of a novel immunohistochemical technique on routinely processed, celloidin-embedded human temporal bone sections.
The authors recently developed a new antigen retrieval technique which allows immunohistochemical detection of proteins in routinely processed, celloidin-embedded human temporal bone sections. This technique was used in the present study, which reports the occurrence of S-100 protein in the human inner ear. Fifteen celloidin-embedded human temporal bone sections, obtained from the Eastern Temporal Bone Bank at the Massachusetts Eye and Ear Infirmary, were tested with the monoclonal antibody to S-100. This protein was found in the spiral ligament, in Reissner's membrane, in the spiral limbus, and in the basement membrane of the cochlea. S-100-positive, thin fibers could be seen as supporting elements in the acoustic nerve and the facial nerve. This protein was localized along the surface of vestibular wall. The new technique provides an immunohistochemical approach to studying routinely processed human temporal bone sections and may prove useful in the field of immuno-otopathology.
Collodion↗
Treatment decisions in axillary node-negative breast cancer patients.
Treatment decisions must be made on 9000 axillary node-negative breast cancer patients each month in the United States. Which of these patients will benefit from adjuvant therapy is a major question. Valid methods are needed to distinguish those patients who are "cured" from those who will suffer a cancer recurrence. A complex network of prognostic variables enters into the treatment decision, together with a risk-versus-benefit assessment. We are using a neural-network-based form of artificial intelligence that, once "trained" with data representing an event and its outcome, can identify subsets of patients with low recurrence risks. Larger data sets are being evaluated with the hope of introducing the neural-network technique to routine clinical practice.
Breast Neoplasms↗
Variant human breast tumor estrogen receptor with constitutive transcriptional activity.
Since progesterone receptor (PgR) is normally induced by estrogen, breast cancer lacking estrogen receptor (ER) would also be expected to lack PgR. However, a small percentage of breast cancers are ER- yet PgR+. These tumors might possess an ER which is defective in estrogen binding but is still functional in stimulating estrogen-responsive genes such as PgR. We have now detected such a variant, lacking exon 5 of the hormone-binding domain, using complementary DNA amplified by the polymerase chain reaction. This variant was the predominate ER RNA expressed in three ER-/PgR+ tumors. Furthermore, the variant ER constitutively activates transcription of a normally estrogen-dependent gene construct in yeast cells. The variant ER could explain the expression of PgR in certain tumors and have therapeutic implications.
Base Sequence↗