In situ detection of basic fibroblast growth factor by highly specific antibodies.

Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

Image analysis confirmation of DNA aneuploidy in flow cytometric DNA distributions having a wide coefficient of variation of the G0/G1 peak.

With nuclei extracted from paraffin-embedded tissues, resolution of normal diploid DNA and abnormal near-diploid/aneuploid populations by flow cytometry (FCM) is especially difficult. These samples, compared with fresh tissue, tend to show a broader DNA distribution, appearing as a wide (high) coefficient of variation (CV) of the G0/G1 peak. To address the question of whether there may be aneuploid populations hidden in wide CV diploid G0/G1 peaks, the authors measured DNA content in nuclei extracted from paraffin-embedded tumor tissue by morphometric image analysis (IA) in addition to FCM. Of 29 samples showing little evidence of DNA aneuploidy by FCM, in 20 of 20 with G0/G1 CVs greater than or equal to 5.50% there was an aneuploid population when analysis was performed by IA. Of the remaining nine samples with CVs less than or equal to 4.41%, all were diploid in the G0/G1 region by both FCM and IA. The presence of aneuploid populations in FCM distributions with wide CV G0/G1 peaks can be confirmed by IA.

Prognostic factors and clinical trials.

The identification of prognostic factors is an important area of cancer research. This article contains an introduction to the uses of prognostic factors, statistical methods for their identification, and a summary of the literature concerning the evolution of serum beta 2 microglobulin as a prognostic factor in multiple myeloma.

Immunohistochemistry in surgical pathology. The case of the undifferentiated malignant neoplasm.

An ideal immunohistochemical screening panel would be one in which each antibody is 100% sensitive and specific for the target cell type (e.g., markers for epithelial neoplasms, lymphomas, sarcomas, etc.). Anyone who has practiced immunochemistry is well aware that this situation does not exist. High sensitivity is hindered by the loss of key antigens through formalin fixation and routine tissue processing. Although sensitivity can be improved by dealing with fresh-frozen, lyophilized, or plastic-embedded specimens, these procedures are often perceived as inconvenient by pathologists. Specificity is a more insidious problem. With the advent of monoclonal antibody technology, many individuals equated the monospecificity (e.g., marking one antigen a determinant) with tissue specificity. This, of course, is not the case as determinants recognized by one monoclonal antibody may be expressed on cells of different lineage. High sensitivity and high specificity are important for different reasons. By definition, an undifferentiated neoplasm lacks morphologic features to unequivocally substantiate sarcoma, lymphoma, carcinoma, or melanoma. Thus, antibodies with low sensitivity that fail to mark a significant percent of cases will provide inconclusive or erroneous information. The failure of an antibody to stain a particular tissue could be a true-negative (valuable information) or a false-negative (misleading information) result. Obviously, when antibodies have a sufficiently low sensitivity, their use is a liability rather than an advantage. Specificity is obviously important. When an undifferentiated neoplasm is found to be "positive" for a particular marker, there is a tendency to immediately categorize the neoplasm. In this setting, when histologic features of cellular differentiation are totally lacking, an extreme degree of trust is being placed on the immunohistochemical technique. From our earlier discussion it is apparent that perfect sensitivity and specificity do not exist among most immunohistochemical reagents. Accordingly, the safest approach is to use a panel of antibodies that will disclose anomalous immunohistochemical reactions (e.g., a neoplasm positive for both keratin and LCA). Specimens from such cases should be carefully evaluated with additional monoclonal antibodies and scrutinized by light microscopy. Furthermore, while immunohistochemistry provides for rapid and cost-effective diagnosis, electron microscopy may still contribute valuable information. Despite our best intentions and desires, it is also clear that a small percentage of "undifferentiated" neoplasms will remain undifferentiated. Quality control and quality assurance are two final, but important, issues to address. An extraordinary large number of variables in tissue selection, fixation, and processing can skew results.(ABSTRACT TRUNCATED AT 400 WORDS)

The application of monoclonal antibodies in the cytologic evaluation of tumors.

The focus of this review has been the application of MAbs as adjuncts in the interpretation of cytology specimens. It is evident that most if not all of the MAbs studied thus far are neither completely tumor-specific nor -sensitive; however, when used to address a directed clinical question, they may be "operationally specific." More important, there continues to be no current substitute for the understanding and practice of sound diagnostic cytopathologic principles. Ultimately, the application of MAbs resides in the importance of tumor-associated antigen expression and phenotyping of tumors with therapeutic and prognostic implications.

Immunohistochemistry of solid tumors. Brief review of selected problems.

The immunohistochemical approach to tumor typing has dramatically improved our possibilities in the objective diagnosis of neoplasms. Use of optimal material and careful techniques will help to maintain good sensitivity, specificity, and reproducibility of immunohistochemistry. However, the complexity of antigen patterns in tumors, and lack of comprehensive knowledge about them requires caution in the interpretation of results, and may prohibit the simple use of diagnostic algorithms. Especially it is not certain whether the results obtained from typical representatives of various tumor entities will pertain to borderline cases and to undifferentiated variants of the same entities. Use of panels of antibodies rather than the use of single "diagnostic" tests will help to avoid these diagnostic pitfalls. However, all tumor types do not have immunohistochemically distinctive features. This emphasizes the need to use other techniques in such cases, and also suggests that some entities, such as malignant fibrous histiocytoma, are from the point of view of immunohistochemistry diagnoses only made by exclusion rather than being specifically diagnosable entities. All diagnostic immunohistochemistry has to be interpreted in the context of standard histological examination.

Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues.

The human homolog of the rat neu oncogene, HER2 (also termed c-erbB2) has been demonstrated in amplified form in human breast tumors with poor prognosis. Although amplification of the gene correlates with expression of a 185-kDa transmembrane glycoprotein, no extensive information is available regarding the extent of tissue and tumor specificity of this gene product. We have addressed this issue by immunohistochemically evaluating the expression of p185 HER2 in normal tissue and various tumors using monoclonal antibodies (MAbs) to distinct epitopes of its extracellular domain. No detectable levels of p185 HER2 were found in fetal tissues analyzed, with the exception of renal tubules in 2 out of 3 specimens tested and in intestinal epithelium. In adult tissues, detectable levels of this glycoprotein were found in a restricted number of cell types, the expression being heterogeneous among individuals and cell histotypes. Among the neoplasms assayed p185 HER2 was expressed in 46% of primary breast cancers, in 28% of ovarian tumors and in 30% of colon rectum malignancies. No male breast adenocarcinomas were p185-positive. A large number of other tumors tested revealed only a low incidence of expression of the p185. In metastatic breast tumors p185 HER2 was demonstrated homogeneously among multiple autologous lesions and almost invariably (80%) the expression of p185 in the primary lesion correlated with that of the deriving metastases. Our findings indicate that the expression of the p185 HER2 represents a tumor marker of clinical relevance in breast cancer. Whether this holds true for other malignancies remains to be explored.

Evaluation of receptors for somatostatin in various tumors using different analogs.

The binding characteristics of several somatostatin (SS-14) analogs developed in our laboratory were examined in various human and animal tumors and normal tissues. In rat cerebral cortex and human breast cancer membranes the interaction of SS-14 with its binding sites was rapid, specific, saturable, linear with protein concentrations, and dependent on time and temperature. Analysis of kinetic and equilibrium experimental data showed that the interaction of [125I-Tyr11]SS-14 with the binding sites in all normal and tumoral tissue specimens was consistent with the presence of a single class of noncooperative binding sites. Superactive octapeptide analogs of somatostatin-containing hexapeptide sequences Cys-Phe-D-Trp-Lys-Thr-Cys or Cys-Tyr-D-Trp-Lys-Val-Cys showed significant binding affinities to SS-14 receptors. Among these analogs, D-Trp-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2 (RC-98-I) showed the highest binding affinity to normal human pancreatic tissue and human pancreatic adenocarcinoma. In contrast, Sandostatin (SMS 201-995) bound only to normal pancreas, not to human pancreatic cancers. Analog RC-98-I also showed a high binding to human and rat prostate cancers. In human epithelial ovarian cancers and an arrhenoblastoma, analogs D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Trp-NH2 (RC-95-I), D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121) and D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) appeared to be the most potent in displacing labeled SS-14. Analogs Ac-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-NH2 (RC-101-I) as well as RC-121, RC-160, and RC-95-I, but not SMS-201-995, showed high binding affinity in human breast cancers. In specimens of human meningioma the highest binding was found with analogs RC-121, RC-95-I, and RC-101-I. Since marked variations in binding affinities were noted for several analogs in the tissues of origin and the tumors, this suggest that differences may exist between somatostatin receptors not only in normal vs. cancerous tissues, but also among various tumors. Our findings also imply that some analogs could be therapeutically superior to others in the treatment of certain tumors.

Screening test for hormone sensitivity by the autoradiographic method using cell mats.

We developed an autoradiographic screening test for hormone sensitivity of single cell suspensions of tumor tissues on cell mats, which inhibited the growth of normal cells alone. We applied this method to our newly established KSE-1 line derived from esophageal carcinoma and compared this method with well-established cytoplasmic and nuclear assays. Our assay, though taking longer to implement and providing only qualitative information, requires significantly smaller specimens than the current biochemical assay and will predict the hormone sensitivity of only viable neoplastic cells.

Quality control test for immunoreactivity of radiolabeled antibody.

A rapid method for the measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment has been developed. This may be used as a quality control test prior to patient administration of the radiolabeled antibody preparation. The test employs solid phase antigens and the assay is conducted under conditions of antigen excess. Assay parameters have been evaluated and a standardized procedure has been developed. The assay has been compared to a standard extrapolation method and found to give approximately the same result. The test has been used on four different radiolabeled antibodies currently in clinical trials in patients with colorectal cancer. Mean immunoreactive fractions for these radiolabeled antibodies ranged from 35 to 65% and the variability of the immunoreactive fraction ranged from 140 to 240% for different antibodies. We conclude that the quality, defined as the immunoreactive fraction, of radiolabeled antibodies is both low and highly variable, indicating the need for a quality control test of these radiopharmaceuticals in the clinic prior to patient administration.

Flow cytometric measurement of glutathione content of human cancer biopsies.

Rice et al. (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase. We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2-2.0 fmol cell-1. Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCl and examined by flow cytometry. There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with Coulter volume. By comparing tumour cell fluorescence to that of calibration beads, and assuming that the relationship with GSH content for EMT6 holds for other cells, a mean GSH content of 0.95 fmol cell-1 was derived for nine carcinomas, and 0.21 fmol cell-1 for five non-Hodgkin's lymphomas. Although this semi-quantitation needs further validation, the method used here is rapid, gives an indication of heterogeneity of tumour cell GSH content, and can be applied to fine needle biopsy samples. It therefore shows promise as a means for studying prospectively the relationship of GSH content to clinical drug and radiation sensitivity, and for monitoring the effects of agents such as buthionine sulphoximine which are intended to improve treatment results through tumour cell GSH depletion.

Presence of growth hormone-releasing hormone-like immunoreactivity in human tumors: characterization of immunological and biological properties.

The distribution and physical and biological properties of GH-releasing hormone-like immunoreactivity (GHRH-IR) in human tissues and tumors was investigated using a specific GHRH RIA, gel chromatography, immunoaffinity chromatography, and bioassay with cultured rat anterior pituitary cells. Variable concentrations of GHRH-IR, ranging from 1.4-39.0 ng/g wet wt, were found in normal liver, lung, placenta, and pancreas. In the latter tissue, however, a different chromatographic profile and a marked decrease in GHRH-IR after immunoaffinity occurred, suggesting that GHRH-IR in pancreatic extracts is not native GHRH. In all tumors examined (n = 35) GHRH-IR could be detected, and four tumors (three carcinoids and one jejunal carcinoma) contained a very high amount of GHRH-IR (greater than 1000 ng/g wet wt). Affinity chromatography of tumor extracts led to a significant loss (greater than 50%) of GHRH-IR in nine tumors. The four tumors containing large amounts of GHRH-IR were obtained from two patients with active acromegaly and two patients who had no clinical evidence of acromegaly. Using antibodies with different specificities for GHRH-(1-44) and GHRH shortened at the C-terminus, varying concentrations of GHRH-(1-44) in these tumors were found, ranging from 10-87% of the total GHRH-IR. The biological activity of GHRH in the four tumor extracts was similar to that of synthetic GHRH alone or GHRH added to control tissue subjected to extraction. These results demonstrate the presence of GHRH-IR in the majority of normal tissues and tumors, which, though they may produce large amounts of biologically active GHRH, do not always lead to acromegaly.

A panel of monoclonal antibodies to keratin no. 7: characterization and value in tumor diagnosis.

Reactivity patterns of seven mouse monoclonal antibodies to human keratin 7 were compared using immunoblotting and immunohistochemistry on cultured cells and normal human and animal tissues. Differences in keratin specificities as determined by two-dimensional immunoblots and interspecies cross-reactivity data on 8 mammalian species suggest that at least six nonidentical epitopes of the keratin 7 molecule are recognized by this panel of reagents. Immunohistochemical examination of a panel of various human neoplasms with monoclonal antibodies monospecific for keratins 7, 18 and 19 revealed potential value of keratin subtyping in differential diagnosis of tumors in general and in subclassification of carcinomas in particular.

P-glycoproteins in pathology: the multidrug resistance gene family in humans.

Many cancers do not respond to chemotherapy on primary exposure to drugs, thus manifesting intrinsic drug resistance. Other cancers that do initially respond subsequently become resistant to the same drugs and simultaneously to other drugs to which the patient has had no previous exposure. This is a form of acquired drug resistance. There is a pressing need to better understand the mechanisms of drug resistance and to use this information to develop strategies for the chemosensitization of drug-resistant tumors. A goal of the pathology laboratory is to offer chemosensitivity tests that identify intrinsic or acquired resistance of tumors to specific drugs or classes of drugs to enable the clinician to tailor therapy to the biology of cancers in individual patients. Multidrug resistance is one type of drug resistance. It can be present in either an intrinsic or acquired form. The human gene that confers human multidrug resistance, the MDR1 gene, has been cloned and classified as a member of the MDR gene family. Its encoded protein, called Mdr1, is an energy-driven membrane efflux transporter that maintains intracellular concentrations of certain chemotherapeutic drugs at nontoxic levels. Useful model systems for studying multidrug resistance have been developed in several research laboratories. Applying selection pressure by exposing cultured cancer cells to escalating doses of natural product anti-cancer drugs allows cross-resistant cell lines to be produced which share patterns of drug resistance with human cancers. A common feature of these drug-resistant lines is the expression of Mdr1. Using techniques of genetic engineering, molecular probes have been developed that can be used to measure MDR1 mRNA and MDR1 gene amplification. Mdr can be measured by immunochemistry methods. Currently, such measurements are being used to stratify patients in clinical trials designed to determine if chemosensitization by inhibition of the pump function of Mdr is a clinically useful therapeutic strategy. If successful, Mdr/MDR1 mRNA laboratory testing might significantly increase the clinical laboratory's role in cancer patient management.