A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Phenotyping of lesions of melanocyte origin with monoclonal antibodies to melanoma-associated antigens and to HLA antigens.

The antigenic profiles of a large number of surgically removed human benign and malignant lesions of melanocyte origin have been analyzed with the use of monoclonal antibodies (MoAb) against la antigens, against the HLA-A,B,C-beta 2-microglobulin molecular complex, against a cytoplasmic melanoma-associated antigen (MAA), and against membrane-bound MAA. Membrane-bound MAA include a high-molecular-weight MAA (HMW-MAA), a 115K MAA, and a 100K MAA. Appearance of the HMW-MAA and of the cytoplasmic MAA, as well as cytoplasmic distribution or loss of HLA-A,B,C antigens, occurs in benign lesions. Additional appearance of Ia antigens is associated with malignant transformation of melanocytes. The antigenic profile defined by the battery of MoAb used displays differences among benign lesions of different histogenesis, between benign and malignant lesions, and among malignant lesions with different histopathologic properties. These results suggest that phenotyping of surgically removed lesions with anti-MAA and anti-HLA MoAb may contribute to the understanding of the steps involved in tumor progression of melanocytes and may aid in the diagnosis of lesions with unusual histopathologic features.

Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens.

Surgically removed benign and malignant human skin lesions of nonmelanocyte origin have been tested with monoclonal antibodies to la antigens, to the HLA-A,B antigenic molecular complex, and to melanoma-associated antigen(s) (MAA). MAA include a high-molecular-weight (HMW) MAA, a 115,000-molecular-weight MAA, a 94,000-molecular-weight MAA, and a cytoplasmic MAA. Indirect immunofluorescence was used as the assay system because of the limited amount of tissue available. When the amount of tissue available was sufficient, double determinant immunoassays (DDIA) were used to quantitate the level of the HMW MAA and of the cytoplasmic MAA. The results of the DDIA were in agreement with those of indirect immunofluorescence in more than 75% of the cases. Malignant skin tumors of various histiotypes displayed three types of changes: 1) appearance of la antigens and cytoplasmic MAA, 2) increase in the level of the HMW MAA, of a 115,000- and a 100,000-molecular-weight MAA, and 3) reduction in the level of HLA-A,B antigens and beta 2-microglobulin. A significant heterogeneity was found in the antigenic profile among various lesions of a given histiotype as well as among tumor cells within a given lesion.

Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen.

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.

Distribution of human Class I (HLA-A,B,C) histocompatibility antigens in normal and malignant tissues of nonlymphoid origin.

Indirect immunofluorescence and immunoperoxidase staining of surgically removed tissues of nonlymphoid origin with monoclonal antibodies to the heavy and light chain of HLA-A,B,C antigens have shown that they have a more restricted tissue distribution than previously assumed. HLA-A,B,C antigens were not detected in brain cortex, cerebellum, sympathetic ganglia, hypophysis, parathyroid gland, thyroid, exocrine pancreas, hepatocytes, sperm, seminiferous tubules, or skeletal or smooth muscle. Malignant transformation of cells may be associated with appearance, changes in cellular distribution of HLA-A,B,C antigens, and/or dissociation in the expression of the two subunits. Analysis of primary tumors and of autologous metastases showed heterogeneity in the expression of HLA-A,B,C antigens among lesions removed from different sites. The degree of heterogeneity did not correlate with the site of origin of metastases.

Differential expression of intercellular adhesion molecule 1 in primary and metastatic melanoma lesions.

Immunochemical studies have shown that the monoclonal antibody (MoAb) CL203.4, elicited with immune interferon treated cultured human melanoma cells Colo 38, recognizes intercellular adhesion molecule 1 (ICAM-1). The determinant defined by MoAb CL203.4 is distinct and spatially distant from that defined by anti-ICAM-1 MoAb RR1/1, which had been elicited with Epstein-Barr virus-transformed B-lymphocytes from a lymphocyte function associated antigen 1 deficient patient. Immunohistochemical testing with MoAb CL203.4 of surgically removed lesions of melanocyte origin has shown a markedly lower reactivity with benign than with malignant lesions. Among the latter, a higher percentage of metastatic than of primary lesions was stained by MoAb CL203.4. The higher expression of ICAM-1 in metastases than in primary lesions is unique to melanoma, since no difference was found in its distribution in primary and metastatic lesions of a variety of malignancies of different embryological origin. Reactivity with MoAb CL203.4 of primary lesions removed from patients with stage I melanoma showed a highly significant correlation with the lesion thickness and with the clinical course of the disease. The disease free interval in patients without detectable reactivity of their primary lesion with MoAb CL203.4 was significantly (P = 0.004) longer than that of patients whose primary lesion was stained with MoAb CL203.4. These results suggest that ICAM-1 may be a useful marker in the analysis of the molecular mechanism underlying the association between lesion thickness and clinical course of the disease.

Heterogeneity in the expression of HLA and tumor-associated antigens by surgically removed and cultured breast carcinoma cells.

Surgically removed normal and malignant mammary tissues and human breast carcinoma cell lines were tested in binding assays with monoclonal antibodies to HLA-A,B,C antigens, beta 2-microglobulin, HLA-DR antigens, and tumor-associated antigens; the latter included a Mr 280,000, a Mr 94,000, and a Mr 85,000 membrane-bound glycoprotein and a cytoplasmic antigen. HLA-A,B antigens, beta 2-microglobulin, HLA-DR antigens, and the cytoplasmic antigen are expressed by normal mammary cells. Their malignant transformation may be associated with quantitative changes in the expression of these antigens and with the appearance of Mr 94,000 and Mr 85,000 glycoproteins. The Mr 280,000 glycoprotein was detected on only one of the breast carcinoma cell lines tested. Analysis of primary tumors and autologous axillary lymph node metastasis from 13 patients has shown differences in the expression of all the antigens tested between primary and metastatic lesions.

Transformation of thyroid epithelium is associated with loss of c-kit receptor.

The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.

Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.

The protooncogene c-kit encodes a tyrosine kinase with a molecular weight of 145,000, highly related to the platelet derived growth factor/colony stimulating factor receptors. Mutations of the murine gene result in impairment of hematopoiesis, gametogenesis, and of the melanocyte cell lineage. In order to elucidate c-kit functions in development and oncogenesis we have analyzed immunohistochemically its expression in human normal and transformed nonlymphoid tissues. The receptor has been detected in spermatogonia, melanocytes, and unexpectedly, in astrocytes, renal tubules, parotid cells, thyrocytes, and breast epithelium. While the gene product is expressed in seminoma, lung tumors, and melanoma of low invasiveness, no detectable levels have been detected in thyroid and breast carcinomas, astrocytomas, and invasive melanomas. In breast tumors these findings were confirmed by paired, Northern blot analysis of RNA preparations from normal and transformed tissue. The present results demonstrate that the c-kit receptor plays a role in the development of a larger spectrum of cell lineages. Furthermore, on the basis of the transformation associated changes, we speculate that, while in some cell types, c-kit expression positively regulates mitogenesis and is selected for in neoplastic transformation, in other tissues the c-kit pathway is involved in morphogenesis and differentiation and is, therefore, negatively selected in the course of tumor progression.

Analysis of the antigenic profile of uveal melanoma lesions with anti-cutaneous melanoma-associated antigen and anti-HLA monoclonal antibodies.

The reactivity of 12 surgically removed uveal melanoma lesions with monoclonal antibodies (MoAb) to 14 membrane-bound and 2 cytoplasmic cutaneous melanoma-associated antigens (MAA), to the 2 subunits of HLA Class I antigens and to the gene products of the HLA-D region was compared with that of cutaneous melanoma lesions and correlated with their histiotype. The membrane-bound determinants defined by the anti-Mr 92,000 and 45,000 MAA MoAb TP39.1, anti-Mr 110,000 MAA MoAb M111, anti-Mr 118,000 MAA MoAb TP36.1, anti-Mr 115,000 MAA MoAb 345.134, anti-ICAM-1 MoAb CL203.4 and anti-Mr 31,000 MAA MoAb M2590, and the cytoplasmic determinants defined by the anti-MAA MoAb 465.12 and 2G-10 display a distribution in uveal melanoma lesions similar to that in cutaneous melanoma lesions. On the other hand, membrane-bound determinants defined by the anti-Mr 100,000 MAA MoAb 376.96, anti-9-O-acetyl-GD3 ganglioside MoAb ME311 and anti-GD2-GD3 ganglioside MoAb ME361 were not detected in the uveal melanoma lesions tested. Furthermore, the membrane-bound determinants defined by the anti-GD3 MoAb R24, anti-nerve growth factor receptor MoAb ME20.4, anti-Mr 97,000 MAA MoAb 140.240, anti-carcinoembryonic antigen MoAb B1.1 and anti-HMW-MAA 149.53, 225.28, and 763.74 have a markedly lower expression in uveal than in cutaneous melanoma lesions. Incubation of uveal melanoma lesions with the pool of the MoAb 149.53, 225.28, and 763.74 recognizing distinct and spatially distant determinants of the HMW-MAA increased the intensity of staining of six lesions and stained four lesions which were not stained by the individual monoclonal antibodies. The distribution of HLA Class I antigens in uveal melanoma lesions resembles that in cutaneous melanoma lesions, since they are expressed in all the lesions of the mixed and epithelioid type but were not detected in those of the spindle type, i.e., the counterparts of nevocellular nevi. HLA Class II antigens are expressed with a lower frequency in uveal than in cutaneous melanoma lesions, since they were detected only in 2 of the 12 lesions. One of them is of the mixed type and the other one of the epithelioid type. Besides HLA antigens the determinants defined by the anti-carcinoembryonic MoAb B1.1, anti-ICAM-1 MoAb CL203.4, and anti-GD3 MoAb R24 displayed a differential distribution in the different histiotypes of uveal melanoma, since they are preferentially expressed in lesions of the mixed and epithelioid type.

Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions.

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.01). ET(A)R mRNA was also detected in 84% of the carcinomas examined, whereas ET(B)R mRNA was expressed in 50% of the tumors. The in vivo presence of mature ET-1 and ET(A)R was confirmed by immunohistochemistry, demonstrating a higher expression in primary and metastatic cells. Ten primary cultures of ovarian tumors secreted ET-1 and were positive for ET-1 and ET(A)R mRNA, whereas only 40% expressed ET(B)R mRNA. Radioligand binding studies showed that ET-1-producing cells also expressed functional ET(A)R, whereas no specific ET(B)R could be demonstrated. ET-1 stimulated dose-dependent [3H]thymidine incorporation and enhanced the mitogenic effect of epidermal growth factor. The ET(A)R-selective antagonist BQ 123 strongly inhibited ET-1-stimulated growth and substantially reduced the basal growth rate of unstimulated cells, whereas the ET(B)R-selective antagonist BQ 788 had no effect. In conclusion, the present data demonstrate a novel mechanism in the growth control of ovarian carcinoma in vivo mediated by the ET-1 autocrine loop that selectively occurs via the ET(A)R.

Low prevalence of selective human leukocyte antigen (HLA)-A and HLA-B epitope losses in early-passage tumor cell lines.

The down-regulation of human leukocyte antigen (HLA) class I molecules, especially the selective down-regulation of certain allelic products, is believed to represent a major mechanism of tumor escape from immune surveillance. In the present report, an original approach is described to precisely evaluate and classify HLA class I epitope losses in 30 cancer patients with malignant melanoma and lung, breast, endometrium, ovary, and colon carcinoma tumors. Early-passage tumor cell lines were established in culture from the corresponding metastatic tumor lesions obtained in each patient. Both the cell lines and the tumor lesions were compared, in their HLA-A and -B expression, to the peripheral blood mononuclear cells (PBMCs) obtained from the same patient (autologous PBMCs). On the basis of HLA-genotyping data, the appropriate monoclonal antibodies identifying mono- and poly-morphic HLA-A and HLA-B epitopes were selected from a panel of 34 antibodies for a total of 24 testable alleles. The selected antibodies were used not only in immunohistochemical assays on cryostatic tumor sections and cytospins of PBMCs but also in quantitative, sensitive flow cytometry assays on early-passage tumor cells and PBMC suspensions. With this latter method, a low overall HLA expression was detected in 26 tumor cell explants and a complete, generalized HLA-A, HLA-B, HLA-C loss in the remaining 4 cases. However, no complete, selective loss of any of the 45 tested HLA-A and HLA-B allomorphs was observed. Sequences from all of the HLA class I alleles could be detected at the genomic DNA level in tumor cells and tissues. At variance from the literature and the results of immunohistochemical experiments performed in parallel on the corresponding tumor lesions, the relative proportions of the various HLA epitopes were relatively preserved in each early-passage cell line/PBMC pair, and selective increases, rather than decreases, in the expression of polymorphic HLA epitopes had the highest prevalence and greatest magnitude. Our data suggest an alternative tumor stealth strategy in which up- and down-regulation are equally important. This alternative model of tumor-host interaction better fits the available models of tumor cell recognition by CTLs and natural killer cells bearing activatory and inhibitory receptors for HLA-A, HLA-B, HLA-C molecules.

Endoglin is a suitable target for efficient imaging of solid tumors: in vivo evidence in a canine mammary carcinoma model.

Increasing evidence suggests that endoglin (CD105) is a new powerful marker of neovascularization in solid malignancies; thus, using breast cancer as a model, we investigated whether targeting of CD105 by monoclonal antibody (mAb) MAEND3 can be used for in vivo imaging of solid tumors. Immunohistochemistry and flow cytometry identified differential expression of CD105 on breast cancer and endothelial cells; in fact, neoplastic cells were weakly and rarely stained by mAb MAEND3, which in contrast, strongly and invariably stained blood vessel endothelia within the breast adenocarcinomas investigated and cultured endothelial cells. Moreover, in contrast to CD31, which currently represents the reference marker to assess angiogenetic activity, CD105 expression was highest in semiconfluent and actively proliferating endothelial cells, and it progressively decreased as cells reached tight confluency and low [3H]thymidine uptake. i.v. administration of 18 MBq of 125I-labeled mAb MAEND3 efficiently imaged spontaneous mammary adenocarcinomas in two dogs; the uptake of radiolabeled mAb was rapid and intense because tumor: background ratios of 8.2:1 and 9.3:1 were reached 8 h after mAb administration, in the absence of immediate and/or long-term clinical side effects. Altogether, our present data suggest that targeting of CD105 on tumor-associated blood vessels may represent a new strategy for in vivo imaging of solid malignancies, regardless of their histological origin.

Production and characterization of the murine monoclonal antibody 2G10 to a human T4-tyrosinase epitope.

Employing as immunogen a short-term passaged, highly pigmented human melanoma cell line, we have produced the murine MoAb 2G10 of the IgG1 isotype. The antibody immunoprecipitated from 35S-methionine and 3H-glucosamine metabolically labeled human melanoma cells with a single-chain glycoprotein of 75 kD molecular weight. No such molecule could be precipitated from murine melanomas. To further investigate the fine specificity of the MoAb, immunochemical and immunohistochemical studies were performed. These studies demonstrated that MoAb 2G10 binds a significant fraction of tyrosinase activity from cell lysates, completely immunodepletes soluble cell extract of T4-tyrosinase molecules, and produces immunostaining patterns superimposable on those obtained with anti-T4-tyrosinase antibodies. Thus, MoAb 2G10 appears to recognize a human-specific determinant carried by either T4-tyrosinase or a closely related molecule. The functional relevance of this epitope remains to be evaluated.

Recombinant anti-human melanoma antibodies are versatile molecules.

The low cost, high versatility, and reliable production of bacterially produced recombinant antibody fragments speeds up the development of tumor-targeting agents. High-quality recombinant anti-melanoma antibodies are much sought after in the scientific community. We cloned the murine antibody 225.28S, currently used in radioimmunoimaging of human melanoma lesions, in single-chain Fv configuration (scFv) for soluble expression in bacteria. The recombinant antibody fragment conserved the binding specificity of the parental antibody. In order to arm the scFv(225.28S) with biologically useful effector functions, we developed vectors for soluble expression of scFv(225.28S) in bacteria that allow both covalent and noncovalent chemical antibody modification at positions that do not interfere with antigen binding. An expression vector was developed that appends a cysteine residue at the C-terminal extremity of the recombinant antibody, thus allowing reaction with thiol-specific reagents, including 99mTc labeling, at a position that does not interfere with antigen binding. The scFv(225.28S) was also successfully expressed with a casein kinase II substrate tag that enables efficient and stable 32P labeling. For noncovalent antibody modification, we developed an expression vector that appends the human calmodulin gene at the C-terminal extremity of scFv(225.28S). The calmodulin domain is poorly immunogenic and can be targeted with chemically modified high-affinity calmodulin ligands. The recombinant anti-human melanoma antibodies described in this article should prove useful "building blocks" for the development of anti-melanoma diagnostic and therapeutic strategies.

Immunohistochemical detection of high-affinity nerve growth factor receptor in neuroblastoma.

High levels of mRNA (as assessed by northern blot) for the high-affinity nerve growth factor receptor (p140TRK) are predictive of favourable outcome in neuroblastoma. The feasibility of determining p140trk on frozen sections using a recently developed monoclonal antibody was evaluated, and immunohistochemical findings were compared to those obtained from northern blot analysis. Primary tumour samples from 28 untreated patients were quick frozen and an indirect immunofluorescence assay was performed on 4-microns acetone-fixed cryostat sections. 9 cases were positive with immunohistochemistry, and these were among the 15 cases also positive by northern blot. None of the cases negative by northern blot were positive with immunohistochemistry. The concordance rate was 79% (P < 0.03), with a sensitivity of 60% and a specificity of 100%. Immunohistochemistry can thus be rather reliable for assessing p140trk expression, even when only very small amounts of tissue are available, such as with needle biopsy.

Expression of Ia-like antigens in normal human nonlymphoid tissues.

Recent results have demonstrated that Ia-like antigen expression in animal tissues is not restricted to cells associated with immune functions. Whether this phenomenon occurs in normal human tissues has been investigated in this study by indirect immunofluorescence on cryostat sections using monoclonal antibodies to framework determinants of Ia-like antigens. The results of this study show that Ia-like antigens are expressed on several normal human tissues of different embryonic origin. These include the epithelium of the gastrointestinal tract, urinary bladder, bronchial glands, thymic reticuloepithelial cells (entodermic origin), epithelium of mammary gland, acinar cells of parotid, astrocytes (ectodermic origin), alveolar macrophages, Kupffer cells, glomerular and peritubular renal endothelium, endometrium, and Langerhans cells (mesodermic origin).

First-trimester human trophoblast is class II major histocompatibility complex mRNA+/antigen.

Lack of expression of the polymorphic class I and class II MHC antigens in the cytotrophoblast is one of the major factors determining the privileged immunologic status of the placenta. In this report, we show that first-trimester human placental cytotrophoblast cells display moderate to strong expression of class II MHC (HLA-DR alpha and -DR beta) and Ii chain transcripts, apparently in absence of detectable class II antigens and Ii chain. In addition, DR alpha, DR beta, and Ii mRNAs, but not antigens, are consistently upregulated by IFN-gamma. Constitutive expression and upregulation of mRNAs are detectable in trophoblast cells kept in short term as well as prolonged (2-3 weeks) culture. These results are reminiscent of an analogous mRNA+/antigen- dissociation occurring, in the case of class I MHC gene products, in a subpopulation of first-trimester cytotrophoblast cells. Thus, analogous mechanisms prevent the expression of potentially hazardous class I and II allodeterminants at early stages of semiallogeneic pregnancy.

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin.

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region. HLA-DR antigens appear more frequently than the other two types of HLA class II antigens and HLA-DP antigens more frequently than HLA-DQ antigens. Differential expression of the gene products of the HLA-D region was also found in autologous metastases removed from different anatomic sites from patients with melanoma. The HLA class II phenotype of surgically removed malignant lesions did not correlate with the degree of differentiation of tumor cells and/or with the expression and/or cellular distribution of HLA class I antigens. Furthermore, in melanoma lesions, no relationship was found between the HLA class II phenotype and the expression of 3 membrane bound and 1 cytoplasmic melanoma associated antigen recognized by monoclonal antibodies. The functional significance and the practical implications of the differential expression of the gene products of the HLA-D region by tumor cells are discussed.