Patent Publication Number: US-4317877-A

Title: Process for detecting the presence of malignant and pre-malignant cells in humans

Description:
The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare. 
    
    
     BACKGROUND 
     This invention relates to a process for detecting the presence of malignant and pre-malignant cells in humans which utilizes an antibody to oncofoetal deoxythymidine kinase (TK). 
     The expression of oncodevelopmental by transformed cells antigens is well-documented (Ibsen et al, Biochim. Biophys. Acta, 560, 243-280 (1979); Rees et al, Methods Can. Res., 18, 99-133 (1979) and may well be a characteristic feature of many malignancies. The pyrimidine nucleoside recycling enzyme closely associated with proliferation, TK, is particularly interesting in this regard since quantitative and/or qualitative changes in TK activity invariably accompany neoplastic transformation. Progressively increasing levels of TK activity and shifts toward enzyme variants with foetal-like characteristics were present in preneoplastic and neoplastic intestinal lesions induced in rats by 1,2-dimethylhydrazine (Salser et al, Cancer Res., 36, 3495-3498 (1976); Ball, et al, Cancer Res., 36, 2686-2689 (1976)). There were also increases in the levels of the more thermolabile foetal-like TK variant(s) in the surface and the crypt of the colonic mucosa from these animals, i.e., surface vs crypt=6:1, reminiscent of the expansion of the proliferative zone to the upper two-thirds of the colon seen in carcinogen-treated mice and in patients with predilection to or prior history of colon cancer (Maskens et al, J. Nat&#39;l. Cancer Inst., 58, 1221-1224 (1977); Deschner, Z. Krebsforsch., 91, 205-216 (1978). These data suggest that during carcinogenesis TK variant(s), qualitatively distinguishable from normal TK, begin to appear in the premalignant intestines. 
     In man, oncofoetal TK&#39;s have been identified (a) in several different types of cancer on the basis of electrophoretic mobilities and various biochemical parameters similar to those of foetal liver enzyme (Taylor et al, J. Biol. Chem., 247, 1930-1935 (1972); Stafford et al, Biochim. Biophys. Acta, 277, 439-442 (1972)) and (b) in colonic flat mucosa (but not in colonic crypt), several colorectal tumors and a variety of other unrelated solid tumors by using antisera produced against 2100-fold purified colonic mucosal TK to demonstrate the antigenic cross-reactivities between term-placental TK and cytosolic TK&#39;s from the above mentioned tissues (Salser et al, Nature, 260, 261-263 (1976)). 
     SUMMARY 
     The present invention provides a process for detecting the presence of malignant and pre-malignant cells of humans which comprises: 
     (1) contacting an antibody to oncofoetal deoxythymidine kinase with human cells; and 
     (2) detecting the presence of cells containing oncofoetal deoxythymidine kinase antigen bound to said antibody. The antibody can be in an antiserum and can be contacted (incubated). with human cells grown in culture, human cell smears and human tissue sections. The cell-bound antibody can be detected by indirect immunofluoresence and by coupled immuno peroxidase assay. The antiserum can be prepared against placental or tumor deoxythymidine kinase. 
     DESCRIPTION 
     The present invention in one embodiment, uses an antiserum prepared against an oncodevelopmental enzyme, deoxythymidine kinase (TK), purified from human term placenta that can discriminate between normal and various malignant human cells. An indirect immunofluorescence technique is used with primary cells in culture, cervical cell smears, malignant effusion cell smears and both cryostat and formalin-fixed paraffin-embedded tissue sections. The positive cytoplasmic fluorescence of primary tumor cells in culture exhibit a perinuclear concentration; normal cells in explant cultures do not stain. In tissue sections, malignant cells within the tumors show a heterogeneous positive cytoplasmic fluorescence whereas adjacent normal cells do not. Leukaemic leucocytes and both normal and mitogen-stimulated peripheral lymphocytes do not show any reactivity to this antiserum. 
     To identify the TK variants and to delineate the specific TK-associated antigenic relationship between normal and malignant tissues, antisera are produced in rabbits against 4000-fold purified TK from normal colonic mucosa (pooled autopsy specimens) and 3400-fold purified TK from term-placentae. The purification procedure is as previously described (Salser et al, Nature, 260, 261-263 (1976)). The smaller molecular weight component initially resolved by gel filtration serves as the antigen. This particular placental TK, like its colonic counterpart, is comprised of a major TK variant (M. Wt., about 85,000 daltons, determined by sedimentation in a linear glycerol gradient and by gel filtration) and at least three other minor TK variants (revealed by PAGE analysis). The electrophoretically identical variants from the two sources, however, are not antigenically identical. This lack of complete identity between the two TK preparations is also borne out by the differences in the precipitin patterns (Balis et al, in &#34;Biological Markers of Neoplasia: Basic and Applied Aspects&#34; (edited by Ruddon, R. W.), 517-534 (Elsevier North Holland, Inc., New York, (1978)) and in the inhibition of TK activity when identical antigens are assayed with the antisera produced against these two enzymes (Table 1). The lack of complete neutralization by these antisera of their respective homologous enzymes is not unexpected since only the small molecular weight component is used as antigen. The more effective inhibition of tumor and colonic surface cells by anti-placental TK when compared to its colonic counterpart reaffirms the antigenic relatedness between these tissues reported previously (Salser et al, Cancer Res., 36, 3495-3498 (1976); Ball et al Cancer Res., 36, 2686-2689 (1976) and Salser et al, Nature, 260, 261-263 ( 1976). The positive cytoplasmic fluorescence obtained with anti-placental TK, but not with anti-colonic TK, when assayed in indirect immunofluorescence tests against established human tumor cell lines in culture (Table 1) can be used to differentiate malignant and pre-malignant cells from normal cells according to the invention. 
     Explants of human tissues are examined (Table 2) to demonstrate that anti-placental TK can discriminate between normal and malignant cells. Cells derived from some of these explants exhibit positive cytoplasmic fluorescence with a perinuclear concentration. An examination of cryostat and of formalin-fixed paraffin-embedded tissue sections (Table 3) confirms the finding with the explant cultures (Table 2). Thirteen out of 16 solid tumors of diverse origins exhibit positive cytoplasmic fluorescence, e.g., in an infiltrating squamous cell carcinoma of the epiglottis, numerous malignant cells show a markedly heterogeneous granular positive cytoplasmic fluorescence. Except for term-placentae, normal cells do not react with anti-placental TK whether they are located among other normal cells in a tissue or are adjacent to the malignant cells in a tumor. The absence of staining in cell preparations of leucophoresed leukaemic leucocytes and both normal and phytohemagglutinin-stimulated peripheral lymphocytes is consistent with earlier neutralization and immunodiffusion studies (Salser et al, Nature, 260, 261-263 (1976). Cervical and malignant effusion cell smears illustrate the feasibility of using such specimens in screening routines. 
    
    
     The following examples are intended to further illustrate the present invention without limiting same in any manner. 
     
                       TABLE 1                                                     
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Comparison of anti-colonic TK and anti-placental TK                       
Neutralization*  Anti-colonic                                             
                            Anti-placental                                
______________________________________                                    
Normal colon                                                              
Total mucosa     68         35                                            
Surface (flat mucosa)                                                     
                 38         59                                            
Crypt            64         30                                            
Term-placentae   25         64                                            
Tumors                                                                    
Colonic, rectal, gastric,                                                 
                 18-35      34-57                                         
ovarian and breast                                                        
Indirect Immunofluorescent                                                
                 negative   postive cyto-                                 
test.sup.+                  plasmic                                       
                            fluorescence                                  
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 *Values are the percent inhibition of TK activity. Dilutions (1:10) of   
 preimmune and sera of rabbit immunized with colonic TK (4000fold purified
 and of rabbit immunized with placental TK (3400fold purified) are        
 preincubated overnight at 4° with equal volumes (0.2 ml, containin
 250-300 μg protein) of dialyzed preparations of TK&#39;s partially purifie
 by two successive ammonium sulfate precipitations from the cytosol       
 fraction of the tissue extracts. Three aliquots of the resultant         
 supernatant solutions are removed for TK assay as previously described   
 (Salser et al, Cancer Res., 33, 1889-1897 (1973); Cancer, 34, 889-895    
 (1974).                                                                  
 .sup.+ Established human colon carcinoma and breast carcinoma cell lines 
 are grown on glass coverslips using Eagle&#39;s Minimum Essential Medium     
 supplemented with 10-20% fetal calf serum. Culture conditions employed   
 have been described (Higgins et al, Rurop. J. Cancer, 15, 423-431 (1979).
 The growth media is aspirated from the cultures, the cells washed twice i
 phosphatebuffered saline (PBS), pH 7.4 and fixed in 100% methanol for 30 
 min at room temperature and these fixed cell preparations stored at      
 -20°. Rabbit antiplacental TK is diluted 1:40-1:50 in foetal calf 
 serum (or in PBS when tissue sections are processed) and used as the     
 primary antiserum which is layered onto the rehydrated specimens (with   
 PBS) and incubated for 1 hr at 22° in a humid chamber. Controls ar
 preimmune serum or antiplacental TK absorbed with total placental cytosol
 fraction or placental TK preparations of varying degrees of purification.
 Washing of the cells and tissue sections for treatment with secondary    
 fluorescein isothiocyanateconjugated goat antirabbit IgG (Miles          
 Laboratory) and subsequent fluorescence microscopy are previously        
 described (Higgins et al, Europ. J. Cancer, 15, 423-431 (1979)).         
 
    
     
                       TABLE 2                                                     
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Immunofluorescent reactivity of methanol-fixed                            
explant cultures of human tissues.                                        
              Individual    Reactivity to                                 
Explant       samples       antiplacental TK                              
______________________________________                                    
Normal colon  1             -                                             
Adenomatous polyps                                                        
              3             +                                             
Normal mammary tissue                                                     
              3             -                                             
Mammary carcinoma                                                         
Epithelioid   3             +                                             
              1             -                                             
Fibroblasts   3             +                                             
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     Explant cultures are established from human tissues by seeding minced tissue fragments onto 60 mm petri dish cultures containing coverslips and 5 ml of foetal calf serum-supplemented growth medium according to a method described for the establishment of a mouse hepatoma cell line (Higgins, et al, Europ. J. Cancer, 15, 423-431 (1979). After 2-4 weeks of culture, the coverslips are removed and the adherent cells washed in PBS prior to fixation in 100% methanol for 30 min at room temperature. These fixed cell preparations are stored at -20° C. and are rehydrated with PBS for immunofluorescence testing as described in Table 1. 
     
                       TABLE 3                                                     
______________________________________                                    
Reactivity of anti-placental TK in tissue preparation.                    
                      Number of                                           
Tissue                positive/Total                                      
______________________________________                                    
Tissue sections*                                                          
Normal                                                                    
Colon                 0/6                                                 
Salivary gland        0/2                                                 
Thymus                0/2                                                 
Term placentae        2/2                                                 
Tumor                                                                     
Colon adenoma         2/2                                                 
Colon adenocarcinoma  5/6                                                 
Gastric adenocarcinoma                                                    
                      2/2                                                 
Small intestinal tumor                                                    
                      1/1                                                 
Squamous carcinoma of the head and neck                                   
                      2/2                                                 
Lung epidermoid carcinoma                                                 
                      1/1                                                 
Uterine choriocarcinoma                                                   
                      0/1                                                 
Auxillary melanoma    0/1                                                 
Cell smears.sup.+                                                         
Cervical cells                                                            
Normal                0/3                                                 
Abnormal              14/46                                               
Peripheral lymphocytes                                                    
Normal control        0/5                                                 
phytohemagglutinin-stimulated                                             
                      0/2                                                 
Leukaemic (CLL and AML)                                                   
                      0/6                                                 
Malignant effusions, e.g., ascitic fluids                                 
                      4/4                                                 
and pleural fluids                                                        
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 *Routine 4-5 μ sections of normal and malignant human tissues are     
 prepared by cryostat sectioning of unfixed specimens or by microtome     
 sectioning of 100% methanol or 10% formalinfixed paraffinembedded tissues
 Cryostat sectioned specimens are fixed in 100% methanol for 30 min at roo
 temperature, immediately after cutting. Prior to use in the indirect     
 immunofluorescence test (cf. Table 1), paraffinembedded are cleared in   
 xylene and taken through graded dilutions of ethanol.                    
 .sup.+ Cervical cell smears are samples taken for routine PAP tests. Cell
 from malignant effusions were collected by centrifugation and washed with
 PBS. Normal lymphocytes are examined either as the total buffy coat or   
 after purification with FicollPaque; leukaemic leucocytes from           
 leucophoresis samples are also purified with FicollPaque. These cell     
 preparations are resuspended in Eagle&#39;s Minimal Essential Medium         
 supplemented with 10% foetal calf serum and slides are prepared like     
 standard blood smears.                                                   
 
    
     The present invention can be used to diagnose human cancers other than leukemia by detecting the presence of malignant and pre-malignant cells as described above. The oncofoetal deoxythymidine kinase antigen described herein can also be used to produce monoclonal antibodies for example as described by Barnstable et al, Cell, 14, 9-20 (1978).