Serum tumor markers in non-small cell lung cancer. A comparative analysis.

BACKGROUND: The role of serum tumor markers in non-small cell lung cancer (NSCLC) remains undefined. New proposed markers have seldom been rigorously compared with existing standards. The authors prospectively compared the performance of three new monoclonal antibodies (MoAb) (5E8, 5C7, and 1F10) with the established serum markers carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC). METHODS: The cohort consisted of 45 consecutive out-patients with newly diagnosed NSCLC: Control subjects were 38 outpatients with non-neoplastic chronic pulmonary diseases. Blood from each patient and control subject was assayed for all five tumor markers. An enzyme-linked immunosorbent assay (ELISA) was used to determine 5E8, 5C7, and 1F10 reactivity. Commercially available kits were used to measure SCC by radioimmunoassay and CEA by ELISA: Individual and combinations of tumor markers were compared in terms of sensitivity, specificity, and accuracy for NSCLC diagnosis. RESULTS: 5E8 plus 5C7 plus 1F10 significantly surpassed SCC plus CEA in terms of sensitivity (P < 0.05) and proved the most accurate marker combination. Among single markers, 5E8 was most specific, 5C7 most sensitive, and 5C7 and 1F10 each most accurate, but differences from CEA alone were not significant. Subgroup analysis by histologic type and stage demonstrated similar findings, and marker combinations yielded little additional diagnostic benefit. CONCLUSIONS: 5E8, 5C7, and 1F10 performed marginally better than did CEA and SCC in patients with newly diagnosed NSCLC: Many limitations apply in defining a clinical niche for these tumor markers in NSCLC, although 5E8, 5C7, and 1F10 previously have demonstrated a modest prognostic value. An adjunctive role in a few specific clinical contexts remains possible.

Frequency and clinical implications of monoclonal antibody detection of tumor-associated antigens in serum of patients with lung cancer.

We previously showed that a panel of monoclonal antibodies (MAb) (5E8, 5C7, and 1F10) that detect serum tumor-associated antigens (TAA) could distinguish patients with lung cancer from those without to a highly significant degree. However, among patients with lung cancer, the frequency and clinical importance of serum TAA expression were not established. Therefore, we analyzed the serum and initial clinical characteristics of 52 Philadelphia VA patients with newly diagnosed lung cancer seen over a 13-month period. A modified semiquantitative ELISA was employed to determine MAb reactivity. Our cohort was characterized by a mean age of 65 +/- 9 year (SD) and mean Karnovsky score of 74 +/- 10; marked weight loss was present in 28 subjects, and 39 presented with either Stage III or IV disease. The panel detected TAAs in 38 of 52 cases (sensitivity 73%; 95% Cl, 60-83%), including 13 of 22 squamous cell, 9 of 12 adenocarcinoma, 10 of 11 undifferentiated, and 6 of 7 small cell carcinomas. No significant differences were found between the reactive and nonreactive patients in terms of age, stage at presentation, histologic subtype, performance status, or weight loss. However, 1F10 and 5C7 were each associated with a greater risk of early death by Cox proportional hazard analysis (p = 0.017 and 0.006, respectively) even when other prognostic variables are accounted for. We conclude that specific serum TAA can be detected in the majority of lung cancer patients with all major histologic subtypes in a cohort with advanced tumors and poor prognostic indices.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction fragment polymorphism of the cynomolgus monkey major histocompatibility complex.

Among old world monkeys, the major histocompatibility complex (MHC) is defined only in the rhesus (Macaca mulatta), cynomolgus (Macaca fascicularis) and pigtailed (Macaca nemistrina) species. However, little is known about the organization of class I and class II MHC genes or the extent of polymorphism in macaques. In the present study, human and murine class I and class II gene probes were used to analyze the leukocyte antigen (CyLA) system of unrelated and related cynomolgus monkeys. Restriction fragment length polymorphism (RFLP) analysis with a HLA-B7 cDNA probe supports the serologic evidence indicating the existence of a family of class I loci of which several are highly polymorphic. As in the human MHC, the class II beta genes are more polymorphic than class II alpha genes. In a pedigree study, RFLP patterns correlated with CyLA haplotypes as deduced from CyLA-A,B,C and complement factor B(Bf) phenotypes. The RFLP data are consistent with three expressed class I gene loci as well as nonclassical MHC genes potentially related to Qa/T1a in mice. We conclude that the RFLP analysis with cross-hybridizing DNA probes augments the information obtained by serotyping and sets the stage for gene mapping and structural analysis of the CyLA region.

Cell surface glycoprotein associated with human lung tumors that is similar to but distinct from the epidermal growth factor receptor.

A cell surface glycoprotein (gp160) present on the surface of non-small cell human lung tumors is characterized and compared with the epidermal growth factor receptor (EGFR). The epitope on gp160 recognized by monoclonal antibody 5E8 is shown to be part of the protein moiety of the molecule and is found to be relatively stable. The epitope is stable over a wide pH range and after treatment with urea as well as most ionic and non-ionic detergents. We have observed that gp160 is similar in several respects to the EGFR. However, despite the similarities, several independent lines of experimental evidence presented here suggest that gp160 and the EGFR are distinct molecules. The first evidence suggesting that these two molecules are different is that the EGFR, but not gp160, is constitutively detectable in the A431 cell tissue culture supernatant, and that a pulse of these cells with epidermal growth factor (under conditions which permit the internalization of the receptor-ligand complexes) significantly reduces the expression of the EGFR without noticeably affecting the level of gp160 on the cell surface. Two very different immunofluorescent patterns marking the position of gp160 and EGFR are observed using monoclonal antibodies specific for each molecule. Using an enzyme-linked immunosorbent assay, it was determined that these same monoclonal antibodies do not cross-inhibit one another, and it was established that gp160, but not EGFR, was retained on an affinity column containing anti-gp160 antibodies immobilized to the solid matrix. An additional finding that supports the notion that gp160 and the EGFR are distinct molecules is that one human lung tumor cell line (Calu-3) has been identified which expresses gp160 but not the EGFR on its surface. These results indicate that there are characteristics which distinguish gp160 from the EFGR, and we establish here that these distinguishing features reflect differences at the protein moiety and not simply differential glycosylation. We conclude from these studies that we have identified and characterized a cell surface molecule that resembles in several respects the epidermal growth factor receptor. This cell surface molecule represents a potentially useful target for the immunotherapy and diagnosis of human non-small cell lung cancer.

Karyotypic evolution of a human undifferentiated large cell carcinoma of the lung in tissue culture.

Serial cytogenetic studies were performed on a cell line derived from a pleural effusion from a patient with undifferentiated large cell carcinoma of the lung. The initial sample had a broad range of chromosome numbers per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid sideline. A sequence consisting of a doubling of chromosome number per cell followed by chromosome loss was observed repeatedly during 40 culture passages. The presence of metaphase spreads showing evidence of endoreduplication suggested this as a likely mechanism for the doubling of chromosome number per cell. Eleven marker chromosomes were observed in the cells of the primary sample; these markers persisted through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16 were consistently overrepresented; each of these chromosomes was involved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22 were consistently underrepresented. Every chromosome, either in its normal form and/or as part of a marker, was represented on the average by at least one copy per diploid cell. Eighteen new marker chromosomes were observed during the course of cell cultivation; one of these evolved into a clonal marker over the course of six cell passages. Of the new marker chromosomes that were formed during the observation period, the majority were found in hypotetraploid cells.

Monoclonal antibody-mediated detection of lung cancer antigens in serum.

We assessed the ability of three monoclonal antibodies (MAbs) (5E8, 5C7, and 1F10) to detect tumor-associated antigens (TAAs) in the sera of patients seen in consultation by the Pulmonary Disease Section at the Philadelphia Veterans Administration Medical Center from September through November 1987. Eighteen of the 61 sera were obtained from patients with histologically established lung cancer. Using a semiquantitative enzyme-linked immunoassay (ELISA), TAAs were detected by the MAb panel in the sera of 12 lung cancer patients, yielding a sensitivity of 67% with a 95% confidence interval of 44 to 84%. The frequency of TAA detection varied among cell types and stages of disease. There were eight false positives and 35 true negatives, giving a specificity of 81% with a 95% confidence interval of 67 to 90%. Two of the false positives came from patients with nonpulmonary tumors known to cross-react with the MAbs (laryngeal and gastric carcinoma). The panel was able to distinguish patients with lung cancer from those without to a highly significant degree (chi 2 = 11.2 with 1 df, p less than 0.001). This study suggests that MAb-mediated detection of serum TAAs may be useful in diagnosing and characterizing lung cancers.

Recombination among protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the protein II family.

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.

Southern blot analysis of major histocompatibility genes of lpr mice.

Several recent functional and immunofluorescent studies have suggested that abnormal major histocompatibility genes may be expressed in mice homozygous for the autoimmune disease-accelerating lpr (lymphoproliferation) gene. In an effort to establish the molecular basis of the expression of abnormal MHC molecules in these mice, we used MHC class I- and class II-specific cDNAs to probe endonuclease-digested genomic DNA from strains expressing lpr to look for restriction fragment length polymorphisms (RFLPs). The RFLP pattern of MRL/Mp-lpr/lpr (MRL/lpr) DNA, as determined using five restriction enzymes, was identical to that of MRL/MP-+/+ and typical of the H-2k haplotype exhibited by C3H/HeSnJ and AKR/J, which are the two H-2k haplotype strains from which MRL was derived. DNA from C57BL/6-lpr/lpr (B6/lpr) exhibited a pattern similar to C57BL/6, again indicating that the lpr gene did not alter MHC genes, within the limitations of this approach. Because macrophages derived from lpr mice had been specifically reported to express altered MHC haplotypes, DNA was prepared from MRL/lpr peritoneal macrophages and Southern blotting was performed using probes for I-A beta and I-E beta. The patterns observed were typical of H-2k DNA. The data thus indicate that the abnormalities of MHC genes previously observed in lpr mice may be due to the influence of other genes on MHC gene products, but are probably not due to alterations in MHC genes themselves.

The major histocompatibility complex of primates.

The major histocompatibility complex (MHC) encodes cell surface glycoproteins that function in self-nonself recognition and in allograft rejection. Among primates, the MHC has been well defined only in the human; in the chimpanzee and in two species of macaque monkeys the MHC is less well characterized. Serologic, biochemical and genetic evidence indicates that the basic organization of the MHC linkage group has been phylogenetically conserved. However, the number of genes and their linear relationship on the chromosomes differ between species. Class I MHC loci encode molecules that are the most polymorphic genes known. These molecules are ubiquitous in their tissue distribution and typically are recognized together with nominal antigens by cytotoxic lymphocytes. Class II MHC loci constitute a smaller family of serotypes serving as restricting elements for regulatory T lymphocytes. The distribution of class II antigens is limited mainly to cell types serving immune functions, and their expression is subject to up and down modulation. Class III loci code for components C2, C4 and Factor B (Bf) of the complement system. Interspecies differences in the extent of polymorphism occur, but the significance of this finding in relation to fitness and natural selection is unclear. Detailed information on the structure and regulation of MHC gene expression will be required to understand fully the biologic role of the MHC and the evolutionary relationships between species. Meanwhile, MHC testing has numerous applications to biomedical research, especially in preclinical tissue and organ transplantation studies, the study of disease mechanisms, parentage determination and breeding colony management. In this review, the current status of MHC definition in nonhuman primates will be summarized. Special emphasis is placed on the CyLA system of M. fascicularis which is a major focus in our laboratory. A highly polymorphic cynomolgus MHC has been partially characterized and consists of at least 14 A locus, 11 B locus, 7 C locus class I allelic specificities, 9 Ia-like class II antigens and 6 Bf (class III) variants.

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene.

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.

Failure of PFC inhibition assays to distinguish idiotypically between clonotypes that are readily distinguishable by RIA analysis.

Two different hemolytic plaque assay protocols that are commonly used to quantitate idiotype-positive antibody-secreting cells have been compared to a standard radioimmunoassay (RIA) to test for their ability to discriminate between related, but idiotypically distinct, clonotypes. The idiotype proband used in this analysis is the individual specific idiotype associated with the dextran-binding myeloma protein M104E (M104E IdI). Antibodies specific for this private idiotype (anti-M104E IdI) were purified by a combination of adsorption and affinity chromatography of the immunoglobulin fraction isolated from the sera of rabbits repeatedly immunized with M104E. The same affinity-purified anti-M104E IdI antibodies were used in the hemolytic plaque assays and in the RIA. In one of the plaque assays, the putative idiotype-positive antibody-forming cells were scored by lysis of target erythrocytes to which the anti-idiotype had been covalently coupled. In the other plaque assay, the idiotype-positive cells were determined indirectly by anti-idiotype inhibition of PFC produced on dextran-coupled target erythrocytes. The fidelity of these two assays to quantitate the M104E private idiotype expression in individual BALB/c mice after a single immunization with dextran B-1355S was determined by comparing the plaque assay data to the data generated by a double-antibody, post-precipitation RIA of either the antibodies in the serum or of monoclonal antibodies produced by hybridomas. Our data indicate that both plaque assay protocols reflect an overestimate of the actual expression of M104E private idiotype. By using a library of dextran-specific hybridomas (that have been characterized in an RIA with respect to their M104E IdI cross-reactivity), we have shown that the PFC overestimate of the M104E expression observed in dextran-immune mice is due to the inability of both plaque assay protocols to distinguish between dextran-specific clonotypes that express idiotypes cross-reactive with, but not identical to, the 104E IdI. We conclude that the plaque assay should be used only in conjunction with an RIA to estimate the idiotype expression. This is especially true in situations where closely related cross-reactive idiotype families exist.

Antibody response of BALB/c mice to dextran B1355S: alterations in the expression of an idiotype associated with the depletion of idiotype-binding cells.

In this study, we established that BALB/c mice recognize and respond to the idiotype (M104E IdI) of a major dextran-specific clonotype within the BALB/c mouse repertoire. This idiotype recognition is established by demonstrating the presence of idiotype-binding cells and by the production of antibodies specific for the private M104E idiotype. To determine whether or not the idiotype-recognizing cells play a regulatory role during an immune response to dextran, the idiotype-binding cells were selectively removed either by panning or by radiation-induced killing. Two significant effects are observed when the depleted spleen cells are immunized with dextran. First, there is a substantial increase in the proportion of anti-dextran antibodies that are M104E IdI+. The second effect of the idiotype-specific cell depletion is the production of significant amounts of M104E IdI+ immunoglobulin molecules which do not bind dextran. The depletion experiments produced no alteration in the concentration of anti-dextran antibodies found in the serum or in the number of dextran-specific PFC in the spleen. The data indicate that idiotype-reactive cells can play a role in regulating the level of individual clonotype expression (i.e., the M104E clonotype), but that an alternative mechanism must exist for regulating the absolute amount of anti-dextran antibody produced after immunization.

Expression in L cells of transfected class I genes from the mouse major histocompatibility complex.

One of the major surprises of the molecular analysis of major histocompatibility complex (MHC) genes is the large number of class I (K/D)-related sequences in the genome. Both restriction fragment length polymorphisms and cosmid cloning experiments showed them all to be closely linked to the MHC. Until now little information was available concerning either their expression or recognition by the immune system. Here we report that these non-K/D genes can provoke antibody responses and be recognized by cytolytic T cells. Immunization of C3H mice with L cells transfected with class I genomic clones resulted in antisera that reacted preferentially with cells from strain B10.P (the gene donor). Thus, these genes can be expressed by L cells. These products were recognized by cytolytic T cells produced by mixed lymphocyte culture with B10.P stimulators. One gene, represented in clone lambda 3a, was chosen for further analysis. A restriction fragment length polymorphism, detected between B10.P (KpDp) and B10.F(14R) (KbDp) and between B10 (KbDb) and B10.F(13R) (KpDb), has enabled us to map the lambda 3a sequence to the D or Tla region. Restriction endonuclease mapping of the lambda 3a clone shows that the gene is intact and that, although many restriction sites are conserved, the gene in lambda 3a differs from other class I genes. When the lambda 3a clone was transfected into mouse L cells, a new product was expressed. Cells expressing this product (designated L3a cells) were killed by primary D-end-reactive, allospecific cytolytic T lymphocytes. The L3a cells were unreactive with monoclonal antibodies specific for the Kp,Dp,Qa-2, Tla.3, and Tla.5 molecules.

Monoclonal antibodies specific for two different histological types of human lung carcinoma.

Hybridomas secreting monoclonal antibodies specific for human lung cancer were produced by fusing immunized mouse spleen cells with mouse myeloma line X63-Ag8.653. Prior to fusion, BALB/c mice were immunized with two different histological types of human lung cancer (Squamous cell carcinoma and adenocarcinoma) obtained from surgery. An immunocytoadherence test was used to select hybridomas secreting antibodies that bound the patient's lung tumor, but did not bind to a B-lymphoblastoid cell line derived from the same patient. Five stable antibody-producing hybrids have been established and cloned. The antibodies produced by these clones have been characterized according to their light and heavy chain isotypes and for their specificity. In addition to binding to the tumor used for immunization, the antibodies bound to other lung tumors of the same histological type (i.e., squamous cell or adenocarcinoma). This reactivity was observed with both established lung tumor cell lines and with fresh tumors obtained from biopsy of patients in our clinic. Some significant reactivity was also observed with large cell carcinoma but the antibodies did not react with small cell carcinomas of the lung, bronchiolo-alveolar cell carcinoma, cancer of the esophagus and stomach, melanomas, several types of leukemias, normal human lung tissue, fibroblasts, or erythrocytes of type A, B, or O. Two of the five antibodies, 5C7 and 5E8 cross-reacted with one breast cancer obtained from surgery, and 5C7 also cross-reacted with one melanoma biopsy specimen. These results suggest that we have generated monoclonal antibodies that recognize a set of antigenic determinants that are commonly expressed on a portion of human lung tumors that are not detectable on a variety of other human tumors or normal human tissue.

Monoclonal antibodies and a heterobifunctional reagent: a novel approach to the vectorial labeling of selected membrane proteins.

Many applications exist and others are envisioned for the chemical coupling of macromolecules to membrane proteins on the surface of mammalian cells. The ability to use antibody as a means to label and subsequently to follow the distribution of cell surface proteins is reported here. A new procedure is outlined for covalently coupling monoclonal antibodies to thiol-containing membrane proteins. The key reagent in the coupling reaction is the commercially available heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The coupling proceeds in a simple two-step reaction in aqueous medium under very mild conditions. This results in a very efficient and stable attachment of anti-hapten antibodies to a selected set of cell surface proteins without any loss in cell viability and without denaturing the antibody molecule. The hapten-binding activity of the antibody is exploited to monitor the re-distribution of the antibody-labeled cell surface proteins periodically after the coupling reaction. The hapten binding activity can also be utilized to isolate membrane macromolecules via affinity chromatography.

Studies on osteoporosis III. Effect of estrogens and fluoride.

Total skeletal calcium levels were determined in female mice with the aid of whole body neutron activation analysis. Three months treatment with heparin produced significant osteoporosis in C3-H/St(Ha) mice but not in C57/BL6 (J) mice. Treatment with a conjugated natural estrogen preparation (Premarin) prevented heparin accelerated osteoporosis, but high level fluoride in the drinking water had no preventive effect. In some experiments there was a suggestion of a deleterious effect of fluoride.

Studies on osteoporosis. II. Effect of estrogens and fluoride on experimental osteoporosis. A preliminary report.

Total skeletal calcium level was determined in female mice with the aid of whole body neutron activation analysis. Three months treatment with heparin produced significant osteoporosis in C3H/St(Ha) mice of 3--5 months of age. Treatment with a conjugated natural estrogen preparation (Premarin) prevented this phenomenon but high level fluoride in the drinking water failed to show preventive activity.

Dupuytren's disease presenting as palmar pits.

A 37 year old man with a non-Hodgkin's lymphoma and a history of heavy alcohol use presented with bilateral palmar pits. Similar lesions were noted in several family members, some of whom had limited use of the hands. Although the patient was referred for evaluation of the palmar pits of the basal cell nevus syndrome, close evaluation revealed the diagnosis of Dupuytren's disease.