Unknown primary tumors.

An unknown primary tumor (UPT) is defined by the presence of a metastatic cancer without a known primary site of origin despite a standardized diagnostic workup. Clinically, UPTs show rapid progression and early dissemination, with signs and symptoms related to the metastatic site. The molecular bases of their biology remain largely unknown, with no evidence as to whether they represent a distinct biological entity. Immunohistochemistry remain the best diagnostic tool in term of cost-effectiveness, but the time-consuming "algorithmic process" it relies on has led to the application of new molecular techniques for the identification of the primary site of UPTs. For example, several microarray or miRNA classifications of UPTs have been used, with an accuracy in the prediction of the primary site as high as 90%. It should be noted that validating a prediction of tissue origin is challenging in these patients, since most of them will never have a primary site identified. Moreover, prospective studies to determine whether selection of treatment options based on such profiling methods actually improves patient outcome are still missing. In the last few years functional imaging (i.e. FDG-PET/CT) has gained a main role in the detection of the site of origin of UPTs and is currently recommended by the European Association of Nuclear Medicine. However, despite recent refinements in the diagnostic workup, the site of origin of UPT often remains elusive. As a consequence, treatment of patients with UPT is still empirical and inadequate.

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.

Experimental renal disease induced by DNA-anti-DNA immune complexes.

Rabbits immunized with ultraviolet-irradiated DNA (UV-DNA) produced high titers of serum antibody. This experimental model was studied to determine if injection of antigen (UV-DNA) intravenously into immunized animals would induce glomerulonephritis and proteinuria. Proteinuria was observed several days after the start of daily intravenous injections into immunized animals and was sustained as long as injections were continued, but fell to normal values after stopping antigen administration. The kidneys showed glomerulitis sometimes associated with focal proliferative lesions, and immunofluorescence showed rabbit Ig and C3 in glomeruli. By electron microscopy, electron-dense subendothelial deposits were seen. Sucrose density gradient analyses of sera immediately after antigen injections suggested the presence of immune complexes of DNA and antibody since both heavy sedimenting and 7S Ig were detected. After digestion with deoxyribonuclease rabbit Ig could be found only in the 7S sedimenting fractions. Intravenous injection of UV-DNA into normal, nonimmune animals did not produce heavy sedimenting Ig or abnormal sedimentation patterns. These studies with an experimental model might provide insight into pathogenetic mechanisms operating in systemic lupus erythematosus where the importance of DNA-anti-DNA immune complexes have been documented. The studies suggested that gradual accumulation of DNA immune complexes in glomeruli might be one mechanism causing renal functional abnormalities.

Generation and characterization of two human alpha/beta T cell clones. Recognizing autologous breast tumor cells through an HLA- and TCR/CD3-independent pathway.

Cell-mediated immune response to breast tumor has only been marginally investigated. To gain insight into this issue we have developed two clones of distinct phenotype, CD3+ alpha/beta, CD4+, CD8-, CD16-, and CD3+ alpha/beta, CD4-, CD8+, CD16-, respectively, from peripheral blood lymphocytes (PBL) of a breast cancer patient. These effectors, selected on the basis of their cytolytic activity against autologous tumor cells and lack of lysis on NK-sensitive cell lines, preferentially recognize autologous tumor cells. The two clones' cytotoxic activity, while inhibited by anti-LFA-1 mAb, could not be abolished by mAbs to CD3, to class I and class II MHC molecules, and by mAbs to molecules involved in T cell function (i.e., CD4, CD8, CD2). The molecular structure of the alpha and beta T cell receptor chains of the two effector cells, confirmed their clonality and showed that, despite an overlapping killing pattern, they possess distinct TCR alpha and beta chains. These findings demonstrate that breast tumor-specific CTL clones can be generated through current technology and that a alpha/beta effector cell population operating through a HLA-unrestricted and TCR/CD3-independent pathway may be involved in the identification and killing of this tumor.

Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

Intratumor heterogeneity for susceptibility to cytotoxic T lymphocytes (CTL)-mediated lysis represents a major obstacle to cancer adoptive immunotherapy. To overcome the heterogeneity observed in terms of susceptibility of target cells to cell-mediated lysis, in this study we used two purified bispecific monoclonal antibodies (bsmAbs) that recognize molecules expressed by cytotoxic effector cells (CD3 and IgG Fc receptorial molecules), as well as one high molecular weight melanoma-associated antigen (HMW-MAA). The ability of these reagents to enhance or induce a relevant in vitro cytotoxic activity by a CTL clone (CTL 49) isolated from PBL of a melanoma patient was tested on a large panel of autologous and allogeneic melanoma cell lines and clones. Functional studies revealed that the CTL 49 clone lysed all the HMW-MAA+ tumor lines in the presence of bsmAbs and that these reagents affected the target lysis in a cooperative fashion. The effectiveness of bsmAbs in overcoming the heterogeneous susceptibility of human melanoma cells to cell-mediated lysis may find practical implications in cancer adoptive immunotherapy.

Partial characterization of a fetal lung antigen associated with human bronchogenic carcinoma.

A human lung tumor-associated fetal antigen (LTFA) has been partially isolated and characterized. The antigen that differs in several immunochemical parameters from previously described lung cancer antigens was shared by fetal lung and liver tissue. The neoantigen migrated in immunoelectrophoresis as an alpha2-beta globulin, had an average molecular size of 7S, and was soluble in 50% saturated ammonium sulfate. Whereas LTFA was insensitive to both DNase and RNase treatment, its antigenicity was completely abolished by pronase. The biologic significance of this antigen and its possible clinical use were discussed.

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.

A 94,000-dalton glycoprotein expressed by human melanoma and carcinoma cells.

The IgG2a monoclonal antibody (MoAb) 376.96S, secreted by a hybridoma derived from a mouse immunized with cultured human melanoma cells COLO 38, reacts with a single 94,000-dalton glycoprotein that is peripherally associated with the plasma cell membrane of cultured melanoma cells. Indirect immunofluorescence analysis with cryostat thin sections of human tissues showed that this antigen is absent from a large variety of normal tissues but is readily detectable on melanomas, nevi, and several different carcinomas. The MoAb 376.96S binds with cultured melanoma and carcinoma cell lines to a similar extent and can mediate both complement-dependent and cell-dependent lysis of these cells. The 94,000-dalton glycoprotein detected by MoAb 376.96S is distinct in its tissue distribution, antigenicity, and molecular profile from several structures previously identified with monoclonal antibodies that have similar molecular weights.

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.

Structural properties and tissue distribution of the antigen recognized by the monoclonal antibody 653.40S to human melanoma cells.

The hybridoma 653.40S, constructed with splenocytes from an inbred BALB/c mouse immunized with cultured human melanoma cells, secreted an antibody that had been shown to recognize an antigenic determinant restricted to human melanoma cell lines. The monoclonal antibody (MoAb) 653.40S showed immunoprecipitation of two glycopolypeptides synthesized by melanoma cells, one with the apparent molecular weight of 280,000 and the other one with a molecular weight larger than 500.000. These two glycopolypeptides were not bridged by disulfide bonds and were peripheral rather than integral to the plasma cell membrane. Comparison of the reactivity of cells of the melanocyte lineage with the MoAb 653.40S and with the MoAb Q5/13 to human Ia-like antigens showed that the former reacted with proliferating melanocytes and melanoma cells, whereas the latter reacted only with melanoma cells. The MoAb 653.40S did not react with a large variety of surgically removed normal and tumor tissues except for some instances of basal cell and squamous cell carcinomas. These results suggested that double staining of pigmented skin lesions with the MoAb 653.40S and with an MoAb to Ia-like antigens may help to solve controversial diagnosis of melanoma. Furthermore, the MoAb 653.40S may be useful for radioimaging and immunotherapy of melanoma.

Tissue distribution, molecular profile, and shedding of a cytoplasmic antigen identified by the monoclonal antibody 465.12S to human melanoma cells.

The mouse immunoglobulin G2 monoclonal antibody (MoAb) 465.12S reacts with a cytoplasmic antigen present in human melanoma cells but not detectable in melanocytes. Indirect immunofluorescent staining of a large number of surgically removed normal adult and fetal tissues with the MoAb 465.12S detected the cytoplasmic antigen in epithelial cells from several organs. The intensity of staining was greater in adult tissues than in the corresponding fetal tissues. Furthermore, the MoAb 465.12S stained nearly all of the surgically removed tumors tested but did not stain many of the normal tissues from which they originated. In almost all cases, the intensity and frequency of staining wa greater for tumor cells than for corresponding normal tissues. From cultured carcinoma and melanoma cells, the MoAb 465.12S immunoprecipitated four glycopolypeptides with molecular weights of 94,000, 75,000, 70,000, and 25,000. Incorporation of 3H-labeled sugars into the various components of the cytoplasmic antigen revealed that the M.W. 75,000 component was unusual in that it contained only glucosamine and mannose. The antigenic determinant defined by the MoAb 465.12S appears to be protein rather than carbohydrate in nature since it is heat sensitive and is expressed on the antigens synthesized by cells in presence of tunicamycin. Analysis of the spent culture medium of carcinoma and melanoma cell lines revealed that the cytoplasmic antigen is readily shed by these cells and consists of a major M.W. 94,000 and a minor M.W. 72,000 component. Treatment of cultured melanoma cells with the antibiotic tunicamycin showed that glycosylation of the cytoplasmic antigen is required for its shedding and/or stability in the spent culture medium.

Expression of CAR-3 and TAG-72 macromolecules in normal and transformed endometrium: potential diagnostic application in postmenopausal patients.

Mass cytoscreening for the early diagnosis of endometrial carcinoma has thus far been hampered by the low diagnostic accuracy of current cytopathology. In this study we have analyzed the reactivity of the two monoclonal antibodies, AR-3 and B72.3, recognizing two distinct glycosylated high molecular weight carcinoma associated antigens on histological specimens from normal, hyperplastic, and transformed endometrium with the aim of establishing their diagnostic potential. Because women with a high risk of endometrial cancer are frequently postmenopausal, where normal endometrium is characterized by atrophy and cystic glandular hyperplasia, the following findings were of interest. Both antibodies reacted with variable and apical staining patterns with a minority of specimens of normal cycling endometrium from premenopausal women. However, they were constantly negative when tested on normal atrophic postmenopausal endometrium, and only monoclonal antibody AR-3 occasionally stained glandular cystic hyperplasia. By contrast, lesions with atypical hyperplasia, which represent a preneoplastic condition, were stained by both antibodies in 89 or 67% of the cases depending on the monoclonal antibody used (100% if used in combination). Furthermore, 98% of the endometrial carcinomas tested were found to react with the combination of two monoclonal antibodies. If these findings are confirmed in a multicentric study, the use of the two reagents could be a valuable adjunct in the cytodiagnosis of endometrial cancer, especially in providing a guideline to selecting patients for endometrial curettage and additional diagnostic procedures.

Class I major histocompatibility complex enhancement by recombinant leukocyte interferon in the peripheral blood mononuclear cells and plasma of melanoma patients.

In vivo administration of escalation doses of recombinant alpha-interferon (IFN-alpha) during a phase I trial in malignant melanoma patients caused dose-dependent increases in the mRNA accumulation, synthesis, steady state cellular content, and plasma membrane expression of class I major histocompatibility complex molecules in peripheral blood mononuclear cells. In addition, circulating levels of class I molecules were also enhanced. These findings show that (a) antigenic enhancement by biomodifiers may occur in vivo, in humans and (b) the mechanism of class I major histocompatibility complex enhancement by IFN-alpha is similar in vitro and in vivo. Furthermore, because peripheral blood mononuclear cells of different melanoma patients display different susceptibility to IFN-alpha, the entity of their antigenic modulation may represent a useful parameter to evaluate the efficacy of different therapeutic regimens and/or assess the individual susceptibility to the molecular changes induced by IFN-alpha.

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.

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.

A cytoplasmic human melanoma associated antigen as a marker of activation in lymphoid cells.

A cytoplasmic glycoprotein, originally identified by the monoclonal antibody 465.12S in melanoma tumors, is significantly increased in epithelial cells of different histotype following transformation. In the present study we show that the cytoplasmic melanoma associated antigen (cyt-MAA) is drastically enhanced in lymphoid cells by polyclonal and allogeneic stimulation, as well as by transformation. Normal T-cells with helper and suppressor phenotype are far more susceptible than B-cells to this enhancement. However, among transformed lymphoid cells, the expression of the cyt-MAA does not correlate with lineage, but rather with stage of differentiation. Acute lymphoblastic leukemias represent the only exception, since in these lymphoid malignancies cyt-MAA levels are highly heterogeneous even within groups of phenotypically similar lesions. Thus, the expression of the cyt-MAA is shared by cells of distant embryological origin in early stages of their differentiation and/or during proliferation. Quantitation of the cyt-MAA may provide useful information for the classification of some lymphoid malignancies.

Double determinant immunoassay to measure a human high-molecular-weight melanoma-associated antigen.

Using monoclonal antibodies to distinct determinants of a human high-molecular weight melanoma-associated antigen (HMW-MAA), a double determinant immunoassay has been developed. The assay is specific and reproducible. Its sensitivity is influenced by the incubation time of antibodies with antigen sources and the combination of antibodies, as well as by the pH of the buffer and the incubation time used to coat plates with antibodies. Testing with the double determinant immunoassay of Nonidet P-40 extracts of human cell lines and of surgically removed normal and malignant tissues has confirmed the restricted tissue distribution of the HMW-MAA. In addition, significant differences have been found in the level of HMW-MAA in melanoma cell lines, as well as in melanoma lesions removed from different patients and from different sites of a given patient. The amount of HMW-MAA shed by various melanoma cell lines does not correlate with their cell surface expression and with their level in Nonidet P-40 extracts. Interferon and hyperthermia increase the shedding of the HMW-MAA by melanoma cells.

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.

Level of a membrane-bound high-molecular-weight melanoma-associated antigen and a cytoplasmic melanoma-associated antigen in surgically removed tissues and in sera from patients with melanoma.

Utilizing double-determinant immunoassays (DDIAs), the high-molecular-weight melanoma-associated antigen (HMW-MAA) was detected only in fetal skin and in one nipple of 54 normal tissues from adults tested, while the cytoplasmic MAA recognized by the monoclonal antibody 465. 12S was found in most of the normal tissues tested. Among malignant lesions, the HMW-MAA was found in melanomas, astrocytomas, and skin carcinomas; the cytoplasmic MAA was found in all of the malignant lesions tested, even those which originated from normal tissues without detectable cytoplasmic MAA. The levels of the HMW-MAA and of the cytoplasmic MAA showed marked variations in malignant lesions removed from various patients, as well as in autologous metastatic lesions removed from four patients with melanoma. No relationship was found between the degree of expression of the two MAA analyzed and the clinical stage of the disease. Both types of MAA were found in sera from patients with melanoma or other types of cancers, as well as in sera from healthy donors. The level of the HMW-MAA tended to be higher in patients with Stage IV melanoma.