CA1340595C - Method for early detection of lung cancer - Google Patents
Method for early detection of lung cancerInfo
- Publication number
- CA1340595C CA1340595C CA000595508A CA595508A CA1340595C CA 1340595 C CA1340595 C CA 1340595C CA 000595508 A CA000595508 A CA 000595508A CA 595508 A CA595508 A CA 595508A CA 1340595 C CA1340595 C CA 1340595C
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- Prior art keywords
- lung cancer
- chest
- ray
- sputum
- cells
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Abstract
A method for early detection of lung cancer comprising: obtaining samples from the body, especially the respiratory tract material including sputum or bronchial fluid or any other pulmonary tissue or regional lymphnodes or thoracic cells and assaying said samples with monoclonal antibodies for the presence of antigens whose enhanced presence correlates with the development of lung cancer. The method of the present invention permits identification of lung cancer up to two years prior to the development of clinical lung cancer, and thus enables early treatment of the lung cancer.
Description
13:0595 METHOD FOR EARLY DETECTION OF LUNG CANCER
Field of the Invention The ;present invention relates to the early diagnosis of cancer; and more particularly, to a method and assay kit for early detection of lung cancer.
Background of the Invention Lung cancer remains the major cause of cancer death among both males and females. Recognition of the expression of one or more neoplastic antigens in advance of clinical cancer opens several potential therapeutic alternatives.
Four types of lung cancer are found in humans:
squamous, adeno, small cell, and large cell. Each tumor expresses specific differentiation features or surface phenotype determinants, a11 of which distinguish these cells from normal cells. The development of monoclonal antibody diagnostic techniques has greatly enhanced the production of reagents capable of differentiating normal cells from cancer cells and differentiating types of cancer cells from other cancer cells.
Sputum cytology screening of bronchial epithelial-cell morphologic atypia has not so far led to frequent, early-stage lung cancer detection and cure.
This was most convincingly demonstrated in a large three-institution study sponsored by the National Cancer ~.340~.~5 Institute, as reported in ~ ev Resgir Dis Q:545-549, 555-560, 561-565, and 565-570, 1984. As part of this study to determine whether the addition of sputum cytology screening could significantly enhance lung cancer detection and reduce lung cancer mortality when compared to radiographic screening alone, the Johns Hopkins Lung Project. obtained expectorated sputum cytology specimens amd chest radiographs serially over a period of from five to eight years from male smokers, 45 years of age and oldler. From 1973 to l977, l0,384 of these high risk individuals were recruited. Half were randomized to receive cytology screening plus chest radiography, and the: other half were screened by radiography alone. Cytology screening was found to be insufficiently sensitive, there being too many false negatives.
Several mouse monoclonal antibodies produced against antigens on small cell and non-small cell human lung cancer have been used in immunohistochemical assays to study tumor biology, lung cancer immunolocalization, and to give clues to tumor ancestry. The antigens recognized by these antibodies are expressed on a variety of tumors a~: well as normal fetal tissue. As summarized in the proceedings of the First International Workshop on Antigens: of Small Cell Lung Cancer, Souhami et al., Lancet, 2 8554): 325-6, 1987, there are nearly 100 monoclonal antibodies being investigated to study small cell and non-s:mall cell cancer of the lung. This workshop supported central registry coding of antibodies followed by blinded staining of a variety of normal and neoplastic tissues. Statistical analyses of the results led to a definition of clusters of reactivity which suggested similar antigenic determinants were being recognized by two or more monoclonal antibody reagents.
None of the an.tigen:: studied were either specific for small cell lung cancer or were universally present on a11 small cell lung cancer specimens studied.
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Field of the Invention The ;present invention relates to the early diagnosis of cancer; and more particularly, to a method and assay kit for early detection of lung cancer.
Background of the Invention Lung cancer remains the major cause of cancer death among both males and females. Recognition of the expression of one or more neoplastic antigens in advance of clinical cancer opens several potential therapeutic alternatives.
Four types of lung cancer are found in humans:
squamous, adeno, small cell, and large cell. Each tumor expresses specific differentiation features or surface phenotype determinants, a11 of which distinguish these cells from normal cells. The development of monoclonal antibody diagnostic techniques has greatly enhanced the production of reagents capable of differentiating normal cells from cancer cells and differentiating types of cancer cells from other cancer cells.
Sputum cytology screening of bronchial epithelial-cell morphologic atypia has not so far led to frequent, early-stage lung cancer detection and cure.
This was most convincingly demonstrated in a large three-institution study sponsored by the National Cancer ~.340~.~5 Institute, as reported in ~ ev Resgir Dis Q:545-549, 555-560, 561-565, and 565-570, 1984. As part of this study to determine whether the addition of sputum cytology screening could significantly enhance lung cancer detection and reduce lung cancer mortality when compared to radiographic screening alone, the Johns Hopkins Lung Project. obtained expectorated sputum cytology specimens amd chest radiographs serially over a period of from five to eight years from male smokers, 45 years of age and oldler. From 1973 to l977, l0,384 of these high risk individuals were recruited. Half were randomized to receive cytology screening plus chest radiography, and the: other half were screened by radiography alone. Cytology screening was found to be insufficiently sensitive, there being too many false negatives.
Several mouse monoclonal antibodies produced against antigens on small cell and non-small cell human lung cancer have been used in immunohistochemical assays to study tumor biology, lung cancer immunolocalization, and to give clues to tumor ancestry. The antigens recognized by these antibodies are expressed on a variety of tumors a~: well as normal fetal tissue. As summarized in the proceedings of the First International Workshop on Antigens: of Small Cell Lung Cancer, Souhami et al., Lancet, 2 8554): 325-6, 1987, there are nearly 100 monoclonal antibodies being investigated to study small cell and non-s:mall cell cancer of the lung. This workshop supported central registry coding of antibodies followed by blinded staining of a variety of normal and neoplastic tissues. Statistical analyses of the results led to a definition of clusters of reactivity which suggested similar antigenic determinants were being recognized by two or more monoclonal antibody reagents.
None of the an.tigen:: studied were either specific for small cell lung cancer or were universally present on a11 small cell lung cancer specimens studied.
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Rather than strict tumor markers, these antigenic determinants may be markers of differentiation. Progressive neoplastic differentiation in carcinogen exposed individuals may lead to an increased expression of these markers in the bronchial epithelium before overt development of a pulmonary neoplasm.
Mulshine et al, in U.S. Patent No. 4,569,788, disclose mon~~clonal antibodies which can be used to detect human non-small cell lung cancer and distinguish this type of cancer from all other types of lung cancer and normal tissue cells. These two antibodies may be utilized in :kit form to distinguish non-small cell lung cancer from ether forms of lung cancer by testing the tumor tissue.
Among other monoclonal antibodies used to determine cancer in humans is a monoclonal antibody of the IgM class, U.S. Patent No. 4,683,200, to Hirohashi et al. The monoclonal antibody disclosed in this patent is reactive with human cancers of the lung, and can be used for ser~ua diagnosis of a patient suffering from cancer.
Loor et .al, U.S. Patent No. 4,690,890, disclose a p~:ocess for detecting at. least two antigens using an immunomet:ric dual sandwich assay containing an effective amount of at least one monoclonal antibody against each antigen. This technique is particularly useful for assaying for prostatic acid phosphatase and prostate antigen.
Tanswell et al, U.S. Patent No. 4,624,930, disclose a process for determining the presence of polyvalent antigens by incubation with three receptors wherein the first receptor is a complete antibody or an antibody covalently bound to hapten, the second receptor is an antibody whit;h is capable of binding with only a part of the first rec;eptor,, and the third receptor, which must not cross-react wii~h the second receptor, can be an ~~~o~~
Mulshine et al, in U.S. Patent No. 4,569,788, disclose mon~~clonal antibodies which can be used to detect human non-small cell lung cancer and distinguish this type of cancer from all other types of lung cancer and normal tissue cells. These two antibodies may be utilized in :kit form to distinguish non-small cell lung cancer from ether forms of lung cancer by testing the tumor tissue.
Among other monoclonal antibodies used to determine cancer in humans is a monoclonal antibody of the IgM class, U.S. Patent No. 4,683,200, to Hirohashi et al. The monoclonal antibody disclosed in this patent is reactive with human cancers of the lung, and can be used for ser~ua diagnosis of a patient suffering from cancer.
Loor et .al, U.S. Patent No. 4,690,890, disclose a p~:ocess for detecting at. least two antigens using an immunomet:ric dual sandwich assay containing an effective amount of at least one monoclonal antibody against each antigen. This technique is particularly useful for assaying for prostatic acid phosphatase and prostate antigen.
Tanswell et al, U.S. Patent No. 4,624,930, disclose a process for determining the presence of polyvalent antigens by incubation with three receptors wherein the first receptor is a complete antibody or an antibody covalently bound to hapten, the second receptor is an antibody whit;h is capable of binding with only a part of the first rec;eptor,, and the third receptor, which must not cross-react wii~h the second receptor, can be an ~~~o~~
antibody caF~able of binding with the antigen, which is obtained from an animal species different from that from which the first receptor is obtained.
Summary of the Invention It is an object of the present invention to overcome deficiencies in the prior art, such as indicated above.
It is another object of the present invention to provide for early diagnosis of lung cancer.
It is yea another object of the present invention to provide a kit and a technique for use in early diagnosis of lung cancer.
According to the present invention, lung cancer can be detected at a much earlier stage than has previously been possible, in some cases more than two years earlier than with any other assay method, by using monoclonal antibodies to detect cells that express antigens whose enhanced presence correlates with the development of lung cancer. The process of the present invention comprises assaying for the presence of antigens in :bronchial fluid or sputum, using antibodies which recognize tumor associated antigens, differentiation antigens, Class I or II antigens, or any antigen differentially expressed on normal bronchial cells versus dysplastic bronchial cells versus neoplastic bronchial cells. This method has been found to be far more sensitive in detecting lung cancer than any previous assaying methods, including the John Hopkins study reported, _sy~ra .
.. ~.~40~9 - 4a -z'he present invention, according to one aspect, provides. a process for screening for the presence of lung cancer which comprises:
a) obtaining sputum or bronchial fluid samples from the respiratory tract of an individual who appears to be free of lung cancer by sputum cytology and chest x-ray;
b) assaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplast:ic bronchial cells or neoplastic bronchial cells;
ii) are specific for antigens whose differential expressionleas been shown to distinguish between samples which contain cancer cells and those which do not by evaluation against control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, some but not a11 of whom went on to develop lung cancer evident by chest x-ray and sputum cytology, thereby yielding both positive and negative control samples; and iii) s~~reening for the binding of the monoclonal antibodies to antigens in the sample.
Tlhe present invention also provides a process for screening fo:r the presence of lung cancer in an individual comprising:
1) olbtaining a sputum or bronchial fluid sample from said individual;
2) contacting said sample with at least one pre-selected monoclonal antibody under conditions such that said antibody imm~unogenically binds with a binding site present in said sample for which binding site said antibody is specific, whereby a complex is formed; and 3 ) d~atecti:ng the presence of said complex;
~~~t~59 a 4b wherein said antibocLy is pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites or neoplastic bronchial cells under dysplastic versus neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently deve:Lop lung cancer detectable by chest x-ray or clinical symptoms.
The present invention, in another aspect, resides in 134Q5~~
4c a diagnostic kit for screening for the presence of early lung cancer in an individual, comprising:
means for sputu~,m or bronchial fluid or other pulmonary cells induction, a means of detection selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels, a.nd a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently d~welop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals wh~c subsequently develop lung cancer k, .~t.
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4d detectable by chest x-ray or clinical symptoms compared to preserved samples. from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
The present invention in a further aspect, resides in:
a diagnostic kit for early diagnosis of lung cancer comprlslng:
a means of detection employing labels selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels;
a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or newplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) dete~~ting the presence or absence of said complex resulting from step (ii);
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4e iv) monitoring said test individuals and determining which of~ said test individulas subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptons;
v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and a sputum inducing means;
the above elements of the kit to be used in conjunction with target cells from sputum bronchial fluid or other pulmonary cells for testing for said antigens, said kit including container means for combining said target cell with said monoclonal antibodies, and a packaging means for combining said container means, said combining means and said means of detection.
The present invention further provides a method for assaying for antigens whose enhanced presence correlates with the development of lung cancer in an individual before it is detectable by chest x-ray or sputum cytology, said antigens being components of a sputum or bronchial fluid sample obtained from the body, .$ ..:
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4f characterised by asaaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples with contain cancer cells and those which do not by comparison with both. positive and negative control samples, said control samples having been taken from individuals having n.o evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, the positive control sarr~ples having been collected from individuals who went on to develop lung cancer within the next 24 months as evidenced by chest x-ray and sputum cytology, the negative control samples having been collected from individuals who did not develop lung cancer within 24 months.
The detection systems that can be used in the process according to the present invention include standard immunometric detection systems, including ELISA, cell sorting or fluorescence Activated Cell sorting assays, Western blotting assays, immunoprecipitation assays, colorimetric or densitometry based assays, and the like.
~,~4~595 Using the technique of the present invention, murine monoclonal antibodies to a glycolipid antigen of small cell and. a protein antigen of non-small cell lung cancer were applied to preserved sputum specimens from individuals wh.o participated in the Johns Hopkins Lung Project, which. projsact is described supra.
In that study, which was originally undertaken to evaluate th.e efficacy of sputum cytology screening, half of the high rissk participants (5226 males of at least 45 years of ache, currently smoking at least one pack of cigarettes per day) were randomly assigned to produce specimens far cytopathological analysis. During regular screenings over the next five to eight years, 626 (12%) showed moderate or greater atypia. Sixty-nine of these (26 who progressed to cancer, 43 who did not) were randomly selected for a blind improved monoclonal antibody immunostaining protocol. Satisfactory specimens with morphologic atypia immunostained positively in 14 of the 22 patients who eventually progressed to cancer (sensitivity 64%j, and were non-reactive in 35 of the 40 patients who did not progress to lung cancer (spec:ificity 88%). Review of the false negative atypias, failure to stain, showed that they were collected an average of 57 months preceding a diagnosis of cancer. In contrast, the true positive specimens had been collected 24 months prior to diagnosis. Later specimens, an average of 26 months prior to cancer, from those which were originally false negative, did stain positively, improving the sensitivity to 91%. The specificity among truly negative specimens collected from individuals who had not developed lung cancer during seven to eight years of follow-up screening was 88%.
Thus, the assay method of the present invention, using samples collected in a previous three-institute study, identified lung cancer in patients up to two years before these patients developed clinical 1~44~95 symptoms of lung cancer. This assay method was far more sensitive than the methods used in the previous study in detecting lung cancer in the very early stages.
Recognition of neopl,astic antigen expression two years in advance of clinical cancer may thus be a valuable intermediate endpoint in studies of lung cancer prevention, dei:ection, and therapy.
Detai7led Description of the Invention According ito the present invention, antibodies are used to detect cells in bronchial fluid or sputum that express antigens whose enhanced presence correlates with the development of lung cancer. The assay is particularly uf:eful for patients who are at risk for lung cancer, including smokers, workers who have been exposed to asbestos, and the like. The assays may be of any convention2~l typ~a of immunoassay, including ELISA, radioimmunoassay, fluorescence immunoassay, chemiluminescence imzaunoassay, cell sorting or fluorescence acaivated cell sorting assays, Western blotting techniques, immunoprecipitation assays, colorimetric or densitometry based assay, and the like.
The cytology screening as described above consisted of a sputum induction with either a 25-minute inhalation of ultrasonically aerosolized balanced salt solution (Hanks. BSS) or a 15-minute inhalation of hypertonic saline. Aliquots of the resulting sputum were smeared on. glass. slides for Papanicolaou staining and interpretation. The remaining material was homogenized, concentrated, and placed in Saccomanno's preservative solution (SPS, 2~ polyethylene glycol in 50~ ethanol) using standard methods, as described in Saccomanno et al., g~~ ~rtQl i:305-10, 1963. Slides were also prepared for Papanicolaou staining from the SPS-preserved material. At four months and again at eight months following induction, a jar of SPS was mailed to these participants. For the next three ,.,~
_,_ mornings, sputum was expectorated into the jar containing the SPS, and this combined specimen was mailed back to the laboratory for cytologic analysis.
As lzas been previously described, 5226 men had been allocated to receive cytologic screening.
During the course of the project, 626 (12%) of these participants had moderate or greater atypic on one or more of their specimens, excluding upper airways cancers and metastases from extra-thoracic primaries.
Individuals with at least moderate atypic in their sputum cytolocty underwent a second sputum induction.
A11 such specimens were placed in SPS and stored for future investigation.
The first atypical cytology specimens of these 626 participants were divided into four groups, as shown in Table 1. Z'wo of the groups consisted of participants whose sputum demonstrated moderate atypic on two screenings: 537(86%) of these never developed lung cancer, Group I, and 40 (6.4%) progressed to lung cancer, Group II. All four major lung cancer cell types were represented in Group II: 12 squamous, 9 small cell, 7 adeno, 8 large cell, and 4 others or mixed.
Groups III: and IV consisted of those participants with marked atypic on at least two occasions. Group III consisted of three individuals (0.5%) who never progressed to cancer. Group IV
consisted of 46 individuals (7.4%) who progressed to non-small cell cancer: 41 developed squamous, three adenocarcinoma, and two developed large cell cancer.
There were no amall cell cancers in this category. In fact, a11 of tlhe small cell cancers that were detected in the John Ho~pkins Lung Project study were either detected by radiography or came to clinical attention between screenings.
_8_ Allocation of JHLP Participants with Stored Sputum Specimens by Severity of atypia and Development of Lung Cancer 626 (100%) Moderate Atypia 537 (86%) Group I At:ypia < Marked ( x 2) No Lung Cancer 40 (6.4%) Grc~up II Atypia < Marked ( x 2) 12 Squamous 9 Small Cell 7 Adeno 8 Large Cell 4 Other, mixed 3 (0.5%) Group II7C Atypia >= Marked (x 2) No Lung Cancer 46 (7.4) Group IV Atypia >= Marked (x 2) 41 Squamous 3 Adeno 2 Large Cell From the total of 626 specimens, samples from these four groups weare randomly selected to be immunostained. The sampling fractions are shown in Table 2. Of t:he 537 who did not go on to cancer, Group I, 40, with an aver<ige of 96.8 months of follow-up, range 34 to 19:2 monl:hs, were randomly selected. Of those who progressed to cancer, 40 in Group II, 46 in Group IV, subjects were randomly selected from each cell type stratum, 15 and 11 from Groups II and IV, respectively. All of the three cases form Group III
(marked atypia~ that did not progress to cancer) were selected. These procedures resulted in the selection of a total of 69 cases which were then coded for the blinded.monocl.onal antibody immunostaining protocol described below.
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- g -TABL$ Z
Sampling Fractions n Selected %
GroupI' 537 40 7.4 GroupII 40 15 37.5 GroupIII 3 3 100.0 .
GroupIV 46 11 23.9 The characteristics of the NCI-Navy Medical Oncology Branch monoclonal antibodies with specificity for a glycolip:~d antigen of small cell cancer (code numbers 534F8, 600A6, and 624H12) and a protein antigen of non-small call cancer (code numbers 702A6, 703D4, and 704A1) have been previously reported, cf. Fargion et al., Cancer $g=~ ~:2~633-2638, 1986: Mulshine et al., Immunol ~,:49;~-502, 1983: Rosen et al., Cancer $~
i:2052-2061, 7L984; Spitalnik et al., Cancer $~ ~¢:
4751-4755, 198Ei. One SCC Mab (624H12) and one NSCC Mab (703D4) were selected for this study; the monoclonal antibodies are the subaect of U.S. Patent No. 4,569,788, Biotinylated anti-rat IgM was purchased from Zymed Laboratories (South San Francisco,, CA); biotinylated anti-mouse IgG, -horse IgG, -rabbit :CgG, and Avidin-Biotin-Peroxidase Complex (ABC) :-eageni~s were all purchased from Vector Laboratories (FSUrlinc~ame, CA) .
Small. cell cancer (SCC) and non-small cell cancer (NSCC) ~.ung tumor cells from cell lines maintained at t:he Johns Hopkins Oncology Center were preserved in SPS and used as positive controls. Sputum specimens and control cells, each stored in SPS, were resuspended by brief vortexing, then deposited on glass 1340~9~
slides using a'~Cytospin"centrifugation apparatus purchased from Shandon Southern Instruments (Sewickley, PA ) .
Since the. variability from one "run" to the next could potentially affect the overall results of the study, multiple slides of each sputum specimen were stained to minimize the effects of staining variability.
In many cases, ten slides were stained; five with each of the two (S~CC and NSCC) antibodies. Individual specimens were also evaluated for potential cross-reactivity with SCC and NSCC antibodies by staining at least one slide of each specimen with each of the two antibodies separately.
A c~~mplete description of the staining protocol used in this procedure is contained in Gupta et al., 'a n ~topath~ i:133-136, 1985. Throughout the procedure, the slides were washed in phosphate buffered saline, 0.01M at pH 7.4, using a magnetic stirrer at its lower setting. All incubations took place in a sealed, humidified chamber .at room temperature (25'C). Briefly, the immunosta:ining ;procedure involved application of a marker-specific primary antibody solution, followed successively by a biotinylated secondary antibody solution (dir~acted against the species/subclass of immunoglobulin in which the primary antibody was raised), a biotinyl~ated tertiary antibody solution (directed aga:Lnst the second antibody), the Avidin-Biotin-Complex (ABC) reagent, and finally the substrate-chromogen solution (0.01% hydrogen peroxide and 0.05%
diaminobenzidine in PBS). Specimens were then counterstained with 0.l25% methylene blue and mounted by routine methods.
The completed preparations were independently evaluated by t:wo observers who were unaware of whether or not the spE~cimen had been produced by a participant who subsequently de~reloped lung cancer. The results were recorded on specially designed report forms which * Trademark 13~Q~95 contained the coded identification and a means for recording data pertinent to the technical aspects of the protocol, such as the antibody and control specimen used; the numr~er and type of epithelial (atypical and neoplastic) arid inf7Lammatory cells; and the qualitative and quantitative features of positively-stained cells.
Staining intensity was graded independently by the two obser~~ers using a scale which ranged from negative to equivocal (~) to strongly positive (4+), compared to the positive control. The immunocytochemical reactivity of each lung cancer case with either sea of monoclonal antibodies (anti-SCC and anti-NSCC) was. determined by dividing the sum of the scores given for each preparation by the total number of preparations for each specimen. Only staining intensities of 2+ or greater were considered unequivocably positive. Negative specimens were defined as those which contained atypical cells that did not stain with a 2;+ intE:nsity. The complete absence of tumor or atypical cells, the presence of obscuring quantities of inflammatory cells, the presence of excessive levels of nonspecific "background" staining or nonspecific staining of the entire slide were considered grounds for scoring a specimen as unsatisfactory, cf.
Table 3.
Staining Definitions I. Unsatisfactory Specimen Quality (preserved 4-8 yr) 1. Contains only non-pulmonary material 2. Excessive inflammation 3. Excessive cellular degeneration 4. Pulmonary material without atypical cells Technical 1. Excessive "background" staining 2. Complete lack of any staining II. Negative!
Atypical. cell: present in specimen but not staining III. Positive!
Stain >_= 2+/4+
Of t:he 69 specimens selected, 26 specimens had been obtained from participants who progressed to lung cancer. Four of these (15%) were unsatisfactory, not significantly different from the proportion (7%) of unsatisfactory staining specimens which was found among the 43 participants that did not progress to lung cancer, as shown in Table 4. The Participants with unsatisfactory staining were excluded from analysis. Of the satisfactory specimens from participants who progressed to lung cancer, two thirds (14 of 22) showed positive reactivity with the antibody (sensitivity 64%).
In contrast, of tho;~e that did not progress to lung cancer, 35 of 40 (specificity 88%) did not stain. The likelihood the~t a premalignant specimen from a participant who would ultimately develop lung cancer would stain with eii~her the SCC or NSCC monoclonal antibodies wa:c highly significant (OR=12.25, 95% C.I. -134~~9~
2.94-55.20, p==0.000:L). Even if the unsatisfactory specimens are considered as non-staining (negative) in the analysis, the si=aining response remains significant (OR=8.87, 95% C.I.=:?.32-36.08, p=0.0004).
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Applied to the First Atypical. Sputum Specimen Stored by the JHLP
Lung Cancer No Lung Cancer Total Satisfactory Stain + 14 5 19 Subtotal 22 40 62 Unsatisfactory 4 3 7 Total 26 43 69 The staining patterns were then examined separately fot- the i~wo (SCC,NSCC antibody cell types.
Of the five participants whose Papanicolaou smear results showed less than marked atypic but who developed small cell lung cancer, shown in Table 5, all five stained with t:he smell cell antibody. Although not shown in this table" a11 of the small cell pre-malignant specimens also stained with the non-small cell antibody (100% positives cross-staining).
13~Q~9~
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Specific for Cell Type Pap Smear Small Cell Ab < Marked (x2) _> Marked (x2) Total with Small Cell Ca 5 0 Stained 5 -% 1o0 -Non Small Cell Ab Total with for Small Cell Ca 9 8 Stained 2 7 % 22 88 Of the nine participants whose sputum cytology Papanicolaou smear results showed less than marked atypic but went on t:o non-small cell cancer, as shown in Table 5, only two (22%) stained with the non-small cell antibody. In contrast, when the atypic was marked on two occasions or showed frank cancer, a11 but one (7 of 8, or 88%) of the non-small cell premalignant specimens stained with the non-small cell antibody. Among these 17 NSCC specimens, only one (a marked atypic) stained with the small. cell antibody (6% positive cross-staining).
Review of the eight of 22 false negative atypias (failure to stain) showed that they were collected on a.n average of 57 months preceding a diagnosis of cancer, as shown in Table 6. In contrast, the true positive specimens (14 of 22 atypias) had been collected approximat:ely 24 months in advance of diagnosis. Those participants who did not develop cancer had been followed for seven to eight years. The duration of this cancer-free period confirms these latter specimens as true negatives.
~3~~59~
The hypothesis that Stage I lung cancer could be detected by morphologic changes in sputum cytology, lead. to successful resection, and result in a lowered lung cancer mortality was not borne out by the results of the: John: Hopkins Lung Project study. The dual-screen group that received sputum cytology screening plus chest: radiography and the radiographically screened group had identical survival and mortality rates. Failures of both detection and intervention contributed to those results.
Unfortunately, 51% of the new cancers that arose during the screening period were "interval": or clinical cases neither detected by regular cytologic nor radiographic screening. Furthermore, of the half which were detected, only 57% were sufficiently well localized for surgical intervention, stage I. The survival in the project was also compared to that of earlier studies.
The overall age- and smoking-adjusted lung cancer mortality in x~oth dual-screen and control groups was not less than that. of unscreened populations. It was concluded, the:refors~, that there was no mortality benefit associated with the addition of the cyto-morphologic screening of sputum to chest films.
Almost two-thirds (147 of 233, or 63%) of the lung cancers i.n the dual-screen group occurred in those without positive cyt:omorphology. This observation indicates that. compared to the absence of atypia, the presence of morphologic atypia is not a sufficiently sensitive intermediate indicator for the subsequent development of lung cancer. Yet when present, atypia has been shown to bs~ reasonably predictive in reflecting an enhanced risk of developing cancer. Over eight to ten years of screening, 86 of 626 (14%) with at least moderate atypi.a progressed to lung cancer, compared with 147 cases of lung cancer (3%) which developed among 4600 participants without: atypia. Further, a11 of the different cell. type:a are represented in these atypical ~3~4595 specimens. The eventual lung cancer cell types could not be disting~uisheci by separate morphologic characteristics in t:he pre-malignant atypias.
Nevertheless, for the majority of lung cancer cases, pre-malignant morphologic change in sputum cytology was not apparent. This suggests that if examination of exfoliated epithelial cells can provide an intermediate endpoint for early detection, morphologic criteria must be supplemented by other indices.
By design,, the first available specimen for each subject which :showed at least moderately atypical morphology was. stained. Among those with positive immunostaining who Eventually developed cancer, the sputum specimen had been collected approximately two years (23.8 months) before the development of clinical lung cancer, ass shown in Table 6. In reviewing the data of those who developed cancer but whose specimen failed to stain (farce negatives), it was postulated that the average interval of more than four years (57.2 months) between the time of sputum collection and development of lung cancer might h<ive adversely affected the likelihood of antibody binding. It was possible that cell differentiation associated with tumor progression might lead to substantial changes in the membrane glycolipid antigen. It is also possible that the antigen may not havE; been expressed'as early as four years in advance of clinical cancer, but may well have been expressed two years in advance of clinical manifestation of thE~ cancer.
~~t~0o9fi Average Duration in Months from First Atypical Sputum Specimen Collection to Development of Cancer or Last Follow-up :Gong Cancer No Lung Cancer Stain + 23.8 83.8 Stain - 57.2 96.7 Staining Results of Sputum Specimens with ~: Marked (x2) Pap Smear Morphology of Individual:~ Who :Progressed to Non Small Cell Cancer First ~ Last Atypical Available Specimen Specimen Total with Non Small Cell Ca 9 9 Sta~Lned 2 8 Average: 40 months prior to cancer * Average: 2ti months prior to cancer .13~~J:o9~
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Applied to the Most Recent Atypical Sputum Specimens Stored by the JHLP
Lw~g Cancer No Lung Cancer Total Satisfactory Stain ~~ 20 5 25 -_ 2 35 37 Subtotal 22 40 62 Unsatisfactory 4 3 7 Total 26 43 69 Sensitivity = 91% O.R. = 70 Specificity = 88% 95% C.I. = l0.46 - 297.8 Chi-square = 35..63, p < 1 x 10'6 Of the nine participants with less than marked atypic on at least t:wo occasions who went on to develop non-small cell cancer, only 2 (22%) had positive immunostaining, as shown in Table 5. For these participants, the average interval between sputum specimen collection and lung cancer development was 40 months, as shown in Table 7. More recent specimens from these individuals (average 26 months prior to cancer) were chosen for immunostaining as well. In Contrast to the earlier result, eight of nine (89%) of these latter specimens took up the immunostain. The single specimen that did not stain was technically unsatisfactory. A
reconstitution of the sensitivity table, Table 8, shows that a11 but two of those that progressed to lung cancer within two yeaz~s stained, i.e., 91% sensitivity. Of those that did not progress to lung cancer, the ~.3~~ a9'~
specificity remained at 88%. Therefore, in addition to the morphologic epithelial cell changes which occur early in only a third of those who develop lung cancer, there appears to be a cell surface marker on exfoliated sputum cells from more than 90% of lung cancer patients that is expressed at least two years in advance of the cancer.
Three participants with marked atypic were followed for .a prolonged interval without developing lung cancer, i~roup III. One of the. three remains alive 98 months after his atypical sputum. His atypical sputum specimen failed to take up either the NSCC or SCC
immunostains, and has since reverted to normal. A
second individual is dead from cardiovascular disease 89 months after lzis atypical sputum. His sputum specimen also failed to take up the immunostains, and had reverted to normal. The third individual died of colon cancer 86 moni~hs after his atypical sputum. His sputum specimen took up the NSCC but not the SCC stain, and had remained moderately atypical at his last examination .
These results are consistent, therefore, with the high Mab specificii:y described above.
The small cell antibodies used in the assays described above did not show binding affinities defined by the (SCC-Antigen Workshop) Cluster 5 antigens, supra.
These antigen sites are preserved after tissue fixation and appear to be more specific than other SCC antigens.
The antigen sEaems to be expressed on a 100 kD
glycoprotein present to some degree on other neuroendocrins! tumors and only rarely on NSCC tumors.
The common pathogenesis of SCC and NSCC from a common pleuripotential stem cell may explain some of the cross-reactivity observed between the different tumor types in the assays performed.
It should be noted that most specimens used remained in storage for from five to eight years. Thus, it is possiblE~ that the cellular material could have c 134~~9~
deteriorated during this period of time. Secondly, the rigor of the .specimen homogenization process could have destroyed or altered the antigens on the cell's surface, resulting in complete lack of any staining.
Automated staining procedures will reduce variability and reduce the frequency of technically unsatisfactor~r slid~as. Automated quantitation of staining will minimize the possibility of subjectivity in interpretal;ion.
The assays of the present invention are suitable for rise in diagnostic kits consisting of antibodies, the bronchial or sputum cells to be tested, and any suitable screening technique, such as immunoassay, :Lmmunoprecipitation assay, or immunohistocheamistr:y assays. An outside source of target cells are added to the kit's ingredients. The kit includes a source of antibody for use in the assay and the screening means for the assay.
A typical kit comprises a container means for the monoclona7L antibodies, plate or slide means for combining said targ~at cells with the monoclonal antibodies, and a packaging means for combining said container means, said plate or slide means, and said means of detecaion.
Recognition of the expression of one or more neoplastic ani;igens in advance of clinical cancer opens several poteni~ial therapeutic alternatives. Aggressive selective bronchoscopy and either earlier surgery or laser-photothE:rapy rnay be considered for newly detected in-situ or mic:roinvasive cancers. early detection and local therapy might also be appropriate during therapeutic monitoring of treated lung cancers. The greatest impacts upon lung cancer survival might result, however, if nutritional interventions were effective in halting or reversing tumor progression from the pre-malignant stages potentially detectable by monoclonal antibodies.
While teh invention is described above in relation to certain sepecific embodiments, it will be understood that many variations are possible, and that alternative materials and reagents can be used without departing from the invention. In some cases such variations and substitutions may require some experimentation, but such will only involve routine testing.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.
Summary of the Invention It is an object of the present invention to overcome deficiencies in the prior art, such as indicated above.
It is another object of the present invention to provide for early diagnosis of lung cancer.
It is yea another object of the present invention to provide a kit and a technique for use in early diagnosis of lung cancer.
According to the present invention, lung cancer can be detected at a much earlier stage than has previously been possible, in some cases more than two years earlier than with any other assay method, by using monoclonal antibodies to detect cells that express antigens whose enhanced presence correlates with the development of lung cancer. The process of the present invention comprises assaying for the presence of antigens in :bronchial fluid or sputum, using antibodies which recognize tumor associated antigens, differentiation antigens, Class I or II antigens, or any antigen differentially expressed on normal bronchial cells versus dysplastic bronchial cells versus neoplastic bronchial cells. This method has been found to be far more sensitive in detecting lung cancer than any previous assaying methods, including the John Hopkins study reported, _sy~ra .
.. ~.~40~9 - 4a -z'he present invention, according to one aspect, provides. a process for screening for the presence of lung cancer which comprises:
a) obtaining sputum or bronchial fluid samples from the respiratory tract of an individual who appears to be free of lung cancer by sputum cytology and chest x-ray;
b) assaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplast:ic bronchial cells or neoplastic bronchial cells;
ii) are specific for antigens whose differential expressionleas been shown to distinguish between samples which contain cancer cells and those which do not by evaluation against control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, some but not a11 of whom went on to develop lung cancer evident by chest x-ray and sputum cytology, thereby yielding both positive and negative control samples; and iii) s~~reening for the binding of the monoclonal antibodies to antigens in the sample.
Tlhe present invention also provides a process for screening fo:r the presence of lung cancer in an individual comprising:
1) olbtaining a sputum or bronchial fluid sample from said individual;
2) contacting said sample with at least one pre-selected monoclonal antibody under conditions such that said antibody imm~unogenically binds with a binding site present in said sample for which binding site said antibody is specific, whereby a complex is formed; and 3 ) d~atecti:ng the presence of said complex;
~~~t~59 a 4b wherein said antibocLy is pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites or neoplastic bronchial cells under dysplastic versus neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently deve:Lop lung cancer detectable by chest x-ray or clinical symptoms.
The present invention, in another aspect, resides in 134Q5~~
4c a diagnostic kit for screening for the presence of early lung cancer in an individual, comprising:
means for sputu~,m or bronchial fluid or other pulmonary cells induction, a means of detection selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels, a.nd a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently d~welop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals wh~c subsequently develop lung cancer k, .~t.
13~059~
4d detectable by chest x-ray or clinical symptoms compared to preserved samples. from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
The present invention in a further aspect, resides in:
a diagnostic kit for early diagnosis of lung cancer comprlslng:
a means of detection employing labels selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels;
a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or newplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) dete~~ting the presence or absence of said complex resulting from step (ii);
1~4059~
4e iv) monitoring said test individuals and determining which of~ said test individulas subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptons;
v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and a sputum inducing means;
the above elements of the kit to be used in conjunction with target cells from sputum bronchial fluid or other pulmonary cells for testing for said antigens, said kit including container means for combining said target cell with said monoclonal antibodies, and a packaging means for combining said container means, said combining means and said means of detection.
The present invention further provides a method for assaying for antigens whose enhanced presence correlates with the development of lung cancer in an individual before it is detectable by chest x-ray or sputum cytology, said antigens being components of a sputum or bronchial fluid sample obtained from the body, .$ ..:
~3~0~9~
4f characterised by asaaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples with contain cancer cells and those which do not by comparison with both. positive and negative control samples, said control samples having been taken from individuals having n.o evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, the positive control sarr~ples having been collected from individuals who went on to develop lung cancer within the next 24 months as evidenced by chest x-ray and sputum cytology, the negative control samples having been collected from individuals who did not develop lung cancer within 24 months.
The detection systems that can be used in the process according to the present invention include standard immunometric detection systems, including ELISA, cell sorting or fluorescence Activated Cell sorting assays, Western blotting assays, immunoprecipitation assays, colorimetric or densitometry based assays, and the like.
~,~4~595 Using the technique of the present invention, murine monoclonal antibodies to a glycolipid antigen of small cell and. a protein antigen of non-small cell lung cancer were applied to preserved sputum specimens from individuals wh.o participated in the Johns Hopkins Lung Project, which. projsact is described supra.
In that study, which was originally undertaken to evaluate th.e efficacy of sputum cytology screening, half of the high rissk participants (5226 males of at least 45 years of ache, currently smoking at least one pack of cigarettes per day) were randomly assigned to produce specimens far cytopathological analysis. During regular screenings over the next five to eight years, 626 (12%) showed moderate or greater atypia. Sixty-nine of these (26 who progressed to cancer, 43 who did not) were randomly selected for a blind improved monoclonal antibody immunostaining protocol. Satisfactory specimens with morphologic atypia immunostained positively in 14 of the 22 patients who eventually progressed to cancer (sensitivity 64%j, and were non-reactive in 35 of the 40 patients who did not progress to lung cancer (spec:ificity 88%). Review of the false negative atypias, failure to stain, showed that they were collected an average of 57 months preceding a diagnosis of cancer. In contrast, the true positive specimens had been collected 24 months prior to diagnosis. Later specimens, an average of 26 months prior to cancer, from those which were originally false negative, did stain positively, improving the sensitivity to 91%. The specificity among truly negative specimens collected from individuals who had not developed lung cancer during seven to eight years of follow-up screening was 88%.
Thus, the assay method of the present invention, using samples collected in a previous three-institute study, identified lung cancer in patients up to two years before these patients developed clinical 1~44~95 symptoms of lung cancer. This assay method was far more sensitive than the methods used in the previous study in detecting lung cancer in the very early stages.
Recognition of neopl,astic antigen expression two years in advance of clinical cancer may thus be a valuable intermediate endpoint in studies of lung cancer prevention, dei:ection, and therapy.
Detai7led Description of the Invention According ito the present invention, antibodies are used to detect cells in bronchial fluid or sputum that express antigens whose enhanced presence correlates with the development of lung cancer. The assay is particularly uf:eful for patients who are at risk for lung cancer, including smokers, workers who have been exposed to asbestos, and the like. The assays may be of any convention2~l typ~a of immunoassay, including ELISA, radioimmunoassay, fluorescence immunoassay, chemiluminescence imzaunoassay, cell sorting or fluorescence acaivated cell sorting assays, Western blotting techniques, immunoprecipitation assays, colorimetric or densitometry based assay, and the like.
The cytology screening as described above consisted of a sputum induction with either a 25-minute inhalation of ultrasonically aerosolized balanced salt solution (Hanks. BSS) or a 15-minute inhalation of hypertonic saline. Aliquots of the resulting sputum were smeared on. glass. slides for Papanicolaou staining and interpretation. The remaining material was homogenized, concentrated, and placed in Saccomanno's preservative solution (SPS, 2~ polyethylene glycol in 50~ ethanol) using standard methods, as described in Saccomanno et al., g~~ ~rtQl i:305-10, 1963. Slides were also prepared for Papanicolaou staining from the SPS-preserved material. At four months and again at eight months following induction, a jar of SPS was mailed to these participants. For the next three ,.,~
_,_ mornings, sputum was expectorated into the jar containing the SPS, and this combined specimen was mailed back to the laboratory for cytologic analysis.
As lzas been previously described, 5226 men had been allocated to receive cytologic screening.
During the course of the project, 626 (12%) of these participants had moderate or greater atypic on one or more of their specimens, excluding upper airways cancers and metastases from extra-thoracic primaries.
Individuals with at least moderate atypic in their sputum cytolocty underwent a second sputum induction.
A11 such specimens were placed in SPS and stored for future investigation.
The first atypical cytology specimens of these 626 participants were divided into four groups, as shown in Table 1. Z'wo of the groups consisted of participants whose sputum demonstrated moderate atypic on two screenings: 537(86%) of these never developed lung cancer, Group I, and 40 (6.4%) progressed to lung cancer, Group II. All four major lung cancer cell types were represented in Group II: 12 squamous, 9 small cell, 7 adeno, 8 large cell, and 4 others or mixed.
Groups III: and IV consisted of those participants with marked atypic on at least two occasions. Group III consisted of three individuals (0.5%) who never progressed to cancer. Group IV
consisted of 46 individuals (7.4%) who progressed to non-small cell cancer: 41 developed squamous, three adenocarcinoma, and two developed large cell cancer.
There were no amall cell cancers in this category. In fact, a11 of tlhe small cell cancers that were detected in the John Ho~pkins Lung Project study were either detected by radiography or came to clinical attention between screenings.
_8_ Allocation of JHLP Participants with Stored Sputum Specimens by Severity of atypia and Development of Lung Cancer 626 (100%) Moderate Atypia 537 (86%) Group I At:ypia < Marked ( x 2) No Lung Cancer 40 (6.4%) Grc~up II Atypia < Marked ( x 2) 12 Squamous 9 Small Cell 7 Adeno 8 Large Cell 4 Other, mixed 3 (0.5%) Group II7C Atypia >= Marked (x 2) No Lung Cancer 46 (7.4) Group IV Atypia >= Marked (x 2) 41 Squamous 3 Adeno 2 Large Cell From the total of 626 specimens, samples from these four groups weare randomly selected to be immunostained. The sampling fractions are shown in Table 2. Of t:he 537 who did not go on to cancer, Group I, 40, with an aver<ige of 96.8 months of follow-up, range 34 to 19:2 monl:hs, were randomly selected. Of those who progressed to cancer, 40 in Group II, 46 in Group IV, subjects were randomly selected from each cell type stratum, 15 and 11 from Groups II and IV, respectively. All of the three cases form Group III
(marked atypia~ that did not progress to cancer) were selected. These procedures resulted in the selection of a total of 69 cases which were then coded for the blinded.monocl.onal antibody immunostaining protocol described below.
13~0~9~
- g -TABL$ Z
Sampling Fractions n Selected %
GroupI' 537 40 7.4 GroupII 40 15 37.5 GroupIII 3 3 100.0 .
GroupIV 46 11 23.9 The characteristics of the NCI-Navy Medical Oncology Branch monoclonal antibodies with specificity for a glycolip:~d antigen of small cell cancer (code numbers 534F8, 600A6, and 624H12) and a protein antigen of non-small call cancer (code numbers 702A6, 703D4, and 704A1) have been previously reported, cf. Fargion et al., Cancer $g=~ ~:2~633-2638, 1986: Mulshine et al., Immunol ~,:49;~-502, 1983: Rosen et al., Cancer $~
i:2052-2061, 7L984; Spitalnik et al., Cancer $~ ~¢:
4751-4755, 198Ei. One SCC Mab (624H12) and one NSCC Mab (703D4) were selected for this study; the monoclonal antibodies are the subaect of U.S. Patent No. 4,569,788, Biotinylated anti-rat IgM was purchased from Zymed Laboratories (South San Francisco,, CA); biotinylated anti-mouse IgG, -horse IgG, -rabbit :CgG, and Avidin-Biotin-Peroxidase Complex (ABC) :-eageni~s were all purchased from Vector Laboratories (FSUrlinc~ame, CA) .
Small. cell cancer (SCC) and non-small cell cancer (NSCC) ~.ung tumor cells from cell lines maintained at t:he Johns Hopkins Oncology Center were preserved in SPS and used as positive controls. Sputum specimens and control cells, each stored in SPS, were resuspended by brief vortexing, then deposited on glass 1340~9~
slides using a'~Cytospin"centrifugation apparatus purchased from Shandon Southern Instruments (Sewickley, PA ) .
Since the. variability from one "run" to the next could potentially affect the overall results of the study, multiple slides of each sputum specimen were stained to minimize the effects of staining variability.
In many cases, ten slides were stained; five with each of the two (S~CC and NSCC) antibodies. Individual specimens were also evaluated for potential cross-reactivity with SCC and NSCC antibodies by staining at least one slide of each specimen with each of the two antibodies separately.
A c~~mplete description of the staining protocol used in this procedure is contained in Gupta et al., 'a n ~topath~ i:133-136, 1985. Throughout the procedure, the slides were washed in phosphate buffered saline, 0.01M at pH 7.4, using a magnetic stirrer at its lower setting. All incubations took place in a sealed, humidified chamber .at room temperature (25'C). Briefly, the immunosta:ining ;procedure involved application of a marker-specific primary antibody solution, followed successively by a biotinylated secondary antibody solution (dir~acted against the species/subclass of immunoglobulin in which the primary antibody was raised), a biotinyl~ated tertiary antibody solution (directed aga:Lnst the second antibody), the Avidin-Biotin-Complex (ABC) reagent, and finally the substrate-chromogen solution (0.01% hydrogen peroxide and 0.05%
diaminobenzidine in PBS). Specimens were then counterstained with 0.l25% methylene blue and mounted by routine methods.
The completed preparations were independently evaluated by t:wo observers who were unaware of whether or not the spE~cimen had been produced by a participant who subsequently de~reloped lung cancer. The results were recorded on specially designed report forms which * Trademark 13~Q~95 contained the coded identification and a means for recording data pertinent to the technical aspects of the protocol, such as the antibody and control specimen used; the numr~er and type of epithelial (atypical and neoplastic) arid inf7Lammatory cells; and the qualitative and quantitative features of positively-stained cells.
Staining intensity was graded independently by the two obser~~ers using a scale which ranged from negative to equivocal (~) to strongly positive (4+), compared to the positive control. The immunocytochemical reactivity of each lung cancer case with either sea of monoclonal antibodies (anti-SCC and anti-NSCC) was. determined by dividing the sum of the scores given for each preparation by the total number of preparations for each specimen. Only staining intensities of 2+ or greater were considered unequivocably positive. Negative specimens were defined as those which contained atypical cells that did not stain with a 2;+ intE:nsity. The complete absence of tumor or atypical cells, the presence of obscuring quantities of inflammatory cells, the presence of excessive levels of nonspecific "background" staining or nonspecific staining of the entire slide were considered grounds for scoring a specimen as unsatisfactory, cf.
Table 3.
Staining Definitions I. Unsatisfactory Specimen Quality (preserved 4-8 yr) 1. Contains only non-pulmonary material 2. Excessive inflammation 3. Excessive cellular degeneration 4. Pulmonary material without atypical cells Technical 1. Excessive "background" staining 2. Complete lack of any staining II. Negative!
Atypical. cell: present in specimen but not staining III. Positive!
Stain >_= 2+/4+
Of t:he 69 specimens selected, 26 specimens had been obtained from participants who progressed to lung cancer. Four of these (15%) were unsatisfactory, not significantly different from the proportion (7%) of unsatisfactory staining specimens which was found among the 43 participants that did not progress to lung cancer, as shown in Table 4. The Participants with unsatisfactory staining were excluded from analysis. Of the satisfactory specimens from participants who progressed to lung cancer, two thirds (14 of 22) showed positive reactivity with the antibody (sensitivity 64%).
In contrast, of tho;~e that did not progress to lung cancer, 35 of 40 (specificity 88%) did not stain. The likelihood the~t a premalignant specimen from a participant who would ultimately develop lung cancer would stain with eii~her the SCC or NSCC monoclonal antibodies wa:c highly significant (OR=12.25, 95% C.I. -134~~9~
2.94-55.20, p==0.000:L). Even if the unsatisfactory specimens are considered as non-staining (negative) in the analysis, the si=aining response remains significant (OR=8.87, 95% C.I.=:?.32-36.08, p=0.0004).
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Applied to the First Atypical. Sputum Specimen Stored by the JHLP
Lung Cancer No Lung Cancer Total Satisfactory Stain + 14 5 19 Subtotal 22 40 62 Unsatisfactory 4 3 7 Total 26 43 69 The staining patterns were then examined separately fot- the i~wo (SCC,NSCC antibody cell types.
Of the five participants whose Papanicolaou smear results showed less than marked atypic but who developed small cell lung cancer, shown in Table 5, all five stained with t:he smell cell antibody. Although not shown in this table" a11 of the small cell pre-malignant specimens also stained with the non-small cell antibody (100% positives cross-staining).
13~Q~9~
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Specific for Cell Type Pap Smear Small Cell Ab < Marked (x2) _> Marked (x2) Total with Small Cell Ca 5 0 Stained 5 -% 1o0 -Non Small Cell Ab Total with for Small Cell Ca 9 8 Stained 2 7 % 22 88 Of the nine participants whose sputum cytology Papanicolaou smear results showed less than marked atypic but went on t:o non-small cell cancer, as shown in Table 5, only two (22%) stained with the non-small cell antibody. In contrast, when the atypic was marked on two occasions or showed frank cancer, a11 but one (7 of 8, or 88%) of the non-small cell premalignant specimens stained with the non-small cell antibody. Among these 17 NSCC specimens, only one (a marked atypic) stained with the small. cell antibody (6% positive cross-staining).
Review of the eight of 22 false negative atypias (failure to stain) showed that they were collected on a.n average of 57 months preceding a diagnosis of cancer, as shown in Table 6. In contrast, the true positive specimens (14 of 22 atypias) had been collected approximat:ely 24 months in advance of diagnosis. Those participants who did not develop cancer had been followed for seven to eight years. The duration of this cancer-free period confirms these latter specimens as true negatives.
~3~~59~
The hypothesis that Stage I lung cancer could be detected by morphologic changes in sputum cytology, lead. to successful resection, and result in a lowered lung cancer mortality was not borne out by the results of the: John: Hopkins Lung Project study. The dual-screen group that received sputum cytology screening plus chest: radiography and the radiographically screened group had identical survival and mortality rates. Failures of both detection and intervention contributed to those results.
Unfortunately, 51% of the new cancers that arose during the screening period were "interval": or clinical cases neither detected by regular cytologic nor radiographic screening. Furthermore, of the half which were detected, only 57% were sufficiently well localized for surgical intervention, stage I. The survival in the project was also compared to that of earlier studies.
The overall age- and smoking-adjusted lung cancer mortality in x~oth dual-screen and control groups was not less than that. of unscreened populations. It was concluded, the:refors~, that there was no mortality benefit associated with the addition of the cyto-morphologic screening of sputum to chest films.
Almost two-thirds (147 of 233, or 63%) of the lung cancers i.n the dual-screen group occurred in those without positive cyt:omorphology. This observation indicates that. compared to the absence of atypia, the presence of morphologic atypia is not a sufficiently sensitive intermediate indicator for the subsequent development of lung cancer. Yet when present, atypia has been shown to bs~ reasonably predictive in reflecting an enhanced risk of developing cancer. Over eight to ten years of screening, 86 of 626 (14%) with at least moderate atypi.a progressed to lung cancer, compared with 147 cases of lung cancer (3%) which developed among 4600 participants without: atypia. Further, a11 of the different cell. type:a are represented in these atypical ~3~4595 specimens. The eventual lung cancer cell types could not be disting~uisheci by separate morphologic characteristics in t:he pre-malignant atypias.
Nevertheless, for the majority of lung cancer cases, pre-malignant morphologic change in sputum cytology was not apparent. This suggests that if examination of exfoliated epithelial cells can provide an intermediate endpoint for early detection, morphologic criteria must be supplemented by other indices.
By design,, the first available specimen for each subject which :showed at least moderately atypical morphology was. stained. Among those with positive immunostaining who Eventually developed cancer, the sputum specimen had been collected approximately two years (23.8 months) before the development of clinical lung cancer, ass shown in Table 6. In reviewing the data of those who developed cancer but whose specimen failed to stain (farce negatives), it was postulated that the average interval of more than four years (57.2 months) between the time of sputum collection and development of lung cancer might h<ive adversely affected the likelihood of antibody binding. It was possible that cell differentiation associated with tumor progression might lead to substantial changes in the membrane glycolipid antigen. It is also possible that the antigen may not havE; been expressed'as early as four years in advance of clinical cancer, but may well have been expressed two years in advance of clinical manifestation of thE~ cancer.
~~t~0o9fi Average Duration in Months from First Atypical Sputum Specimen Collection to Development of Cancer or Last Follow-up :Gong Cancer No Lung Cancer Stain + 23.8 83.8 Stain - 57.2 96.7 Staining Results of Sputum Specimens with ~: Marked (x2) Pap Smear Morphology of Individual:~ Who :Progressed to Non Small Cell Cancer First ~ Last Atypical Available Specimen Specimen Total with Non Small Cell Ca 9 9 Sta~Lned 2 8 Average: 40 months prior to cancer * Average: 2ti months prior to cancer .13~~J:o9~
Result of Double-Bridge Immuno Peroxidase Staining of Monoclonal Ab Surface Markers Applied to the Most Recent Atypical Sputum Specimens Stored by the JHLP
Lw~g Cancer No Lung Cancer Total Satisfactory Stain ~~ 20 5 25 -_ 2 35 37 Subtotal 22 40 62 Unsatisfactory 4 3 7 Total 26 43 69 Sensitivity = 91% O.R. = 70 Specificity = 88% 95% C.I. = l0.46 - 297.8 Chi-square = 35..63, p < 1 x 10'6 Of the nine participants with less than marked atypic on at least t:wo occasions who went on to develop non-small cell cancer, only 2 (22%) had positive immunostaining, as shown in Table 5. For these participants, the average interval between sputum specimen collection and lung cancer development was 40 months, as shown in Table 7. More recent specimens from these individuals (average 26 months prior to cancer) were chosen for immunostaining as well. In Contrast to the earlier result, eight of nine (89%) of these latter specimens took up the immunostain. The single specimen that did not stain was technically unsatisfactory. A
reconstitution of the sensitivity table, Table 8, shows that a11 but two of those that progressed to lung cancer within two yeaz~s stained, i.e., 91% sensitivity. Of those that did not progress to lung cancer, the ~.3~~ a9'~
specificity remained at 88%. Therefore, in addition to the morphologic epithelial cell changes which occur early in only a third of those who develop lung cancer, there appears to be a cell surface marker on exfoliated sputum cells from more than 90% of lung cancer patients that is expressed at least two years in advance of the cancer.
Three participants with marked atypic were followed for .a prolonged interval without developing lung cancer, i~roup III. One of the. three remains alive 98 months after his atypical sputum. His atypical sputum specimen failed to take up either the NSCC or SCC
immunostains, and has since reverted to normal. A
second individual is dead from cardiovascular disease 89 months after lzis atypical sputum. His sputum specimen also failed to take up the immunostains, and had reverted to normal. The third individual died of colon cancer 86 moni~hs after his atypical sputum. His sputum specimen took up the NSCC but not the SCC stain, and had remained moderately atypical at his last examination .
These results are consistent, therefore, with the high Mab specificii:y described above.
The small cell antibodies used in the assays described above did not show binding affinities defined by the (SCC-Antigen Workshop) Cluster 5 antigens, supra.
These antigen sites are preserved after tissue fixation and appear to be more specific than other SCC antigens.
The antigen sEaems to be expressed on a 100 kD
glycoprotein present to some degree on other neuroendocrins! tumors and only rarely on NSCC tumors.
The common pathogenesis of SCC and NSCC from a common pleuripotential stem cell may explain some of the cross-reactivity observed between the different tumor types in the assays performed.
It should be noted that most specimens used remained in storage for from five to eight years. Thus, it is possiblE~ that the cellular material could have c 134~~9~
deteriorated during this period of time. Secondly, the rigor of the .specimen homogenization process could have destroyed or altered the antigens on the cell's surface, resulting in complete lack of any staining.
Automated staining procedures will reduce variability and reduce the frequency of technically unsatisfactor~r slid~as. Automated quantitation of staining will minimize the possibility of subjectivity in interpretal;ion.
The assays of the present invention are suitable for rise in diagnostic kits consisting of antibodies, the bronchial or sputum cells to be tested, and any suitable screening technique, such as immunoassay, :Lmmunoprecipitation assay, or immunohistocheamistr:y assays. An outside source of target cells are added to the kit's ingredients. The kit includes a source of antibody for use in the assay and the screening means for the assay.
A typical kit comprises a container means for the monoclona7L antibodies, plate or slide means for combining said targ~at cells with the monoclonal antibodies, and a packaging means for combining said container means, said plate or slide means, and said means of detecaion.
Recognition of the expression of one or more neoplastic ani;igens in advance of clinical cancer opens several poteni~ial therapeutic alternatives. Aggressive selective bronchoscopy and either earlier surgery or laser-photothE:rapy rnay be considered for newly detected in-situ or mic:roinvasive cancers. early detection and local therapy might also be appropriate during therapeutic monitoring of treated lung cancers. The greatest impacts upon lung cancer survival might result, however, if nutritional interventions were effective in halting or reversing tumor progression from the pre-malignant stages potentially detectable by monoclonal antibodies.
While teh invention is described above in relation to certain sepecific embodiments, it will be understood that many variations are possible, and that alternative materials and reagents can be used without departing from the invention. In some cases such variations and substitutions may require some experimentation, but such will only involve routine testing.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.
Claims (11)
1. A process for screening for the presence of lung cancer in an individual comprising:
1) obtaining a sputum or bronchial fluid sample from said individual;
1) obtaining a sputum or bronchial fluid sample from said individual;
2) contacting said sample with at least one pre-selected monoclonal antibody under conditions such that said antibody immunogenically binds with a binding site present in said sample for which binding site said antibody is specific, whereby a complex is formed; and
3) detecting the presence of said complex;
wherein said antibody is pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal versus dysplastic or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms.
2. A process for screening for the presence of lung cancer which comprises:
a) obtaining sputum or bronchial fluid samples from the respiratory tract of an individual who appears to be free of lung cancer by sputum cytology and chest x-ray;
b) assaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples which contain cancer cells and those which do not by evaluation against control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray and sputum cytology, thereby yielding both positive and negative control samplers; and c) screening for the binding of the monoclonal antibodies to antigens in the sample.
3. The method of claim 1 or 2 wherein the monoclonal antibody is selected from the group consisting of monoclonal antibody designated 704A1 having ATCC
Accession No. HB8302, monoclonal antibody designated 703D4 having ATCC Accession No. HB8301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
wherein said antibody is pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal versus dysplastic or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms.
2. A process for screening for the presence of lung cancer which comprises:
a) obtaining sputum or bronchial fluid samples from the respiratory tract of an individual who appears to be free of lung cancer by sputum cytology and chest x-ray;
b) assaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples which contain cancer cells and those which do not by evaluation against control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray and sputum cytology, thereby yielding both positive and negative control samplers; and c) screening for the binding of the monoclonal antibodies to antigens in the sample.
3. The method of claim 1 or 2 wherein the monoclonal antibody is selected from the group consisting of monoclonal antibody designated 704A1 having ATCC
Accession No. HB8302, monoclonal antibody designated 703D4 having ATCC Accession No. HB8301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
4. A diagnostic kit for screening for the presence of early lung cancer in an individual, comprising:
means for sputum or bronchial fluid or other pulmonary cells induction, a means of detection selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels, and a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
means for sputum or bronchial fluid or other pulmonary cells induction, a means of detection selected from the group consisting of radioisotopic labels, chromophoric labels, and enzyme labels, and a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
5. The diagnostic kit of claim 4, wherein said monoclonal antibody is selected from the group consisting of monoclonal antibody designated 704A1 having ATCC
Accession No. HB8302, monoclonal antibody designated 703B4 having ATCC Accession No. HB8301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
Accession No. HB8302, monoclonal antibody designated 703B4 having ATCC Accession No. HB8301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
6. A diagnostic kit for screening for the presence of early lung cancer in an individual, comprising:
means for sputum or bronchial fluid induction, a means for detection comprising a biotinylated secondary antibody and a biotinylated tertiary antibody, and a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detestably by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
means for sputum or bronchial fluid induction, a means for detection comprising a biotinylated secondary antibody and a biotinylated tertiary antibody, and a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detestably by chest x-ray or sputum cytology;
ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells under conditions such that immunological binding to said binding sites can occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individuals subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms, the above elements of the kit to be used in conjunction with target cells from sputum, bronchial fluid or other pulmonary cells for testing for binding sites present on target cells, said kit including container means for combining said target cells with said monoclonal antibody.
7. The diagnostic kit of claim 6, wherein said monoclonal antibody is selected from the group consisting of monoclonal antibody designated 704A1 having ATCC
Accession No. HB8302, monoclonal antibody designated 703D4 having ATCC Accession No. HB301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
Accession No. HB8302, monoclonal antibody designated 703D4 having ATCC Accession No. HB301, and monoclonal antibody 624H12 having ATCC Accession No. HB10479.
8. A method for assaying for antigens whose enhanced presence correlates with the development of lung cancer in an individual before it is detectable by chest x-ray or sputum cytology, said antigens being components of a sputum or bronchial fluid sample obtained from the body, characterised by assaying said samples with monoclonal antibodies which:
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples which contain cancer cells and those which do not by comparison with both positive and negative control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, the positive control samples having been collected from individuals who went on to develop lung cancer within the next 24 months as evidenced by chest x-ray and sputum cytology, the negative control samples having been collected from individuals who did not develop lung cancer with 24 months;
said assaying step being selected from the group consisting of reading immunostained slides, ELISA, immunofluorescence, Western blotting, immunoprecipitation, and densitometric assays.
i) are specific for antigens which are differentially expressed on normal bronchial cells versus dysplastic bronchial cells or neoplastic bronchial cells;
and ii) are specific for antigens whose differential expression has been shown to distinguish between samples which contain cancer cells and those which do not by comparison with both positive and negative control samples, said control samples having been taken from individuals having no evidence of lung cancer by sputum cytology or chest x-ray at the time of sampling, the positive control samples having been collected from individuals who went on to develop lung cancer within the next 24 months as evidenced by chest x-ray and sputum cytology, the negative control samples having been collected from individuals who did not develop lung cancer with 24 months;
said assaying step being selected from the group consisting of reading immunostained slides, ELISA, immunofluorescence, Western blotting, immunoprecipitation, and densitometric assays.
9. The method of claim 8 wherein the monoclonal antibody is selected from IgG2Ak monoclonal antibody designated 704A1, ATCC Accession No. HB8302, and IgG2Ak monoclonal antibody designated 703D4, ATCC Accession No.
HB8301.
HB8301.
10. A diagnostic kit for early diagnosis of lung cancer comprising:
a means of detection employing labels selected from the group consisting of radioisotopic labels, chromophric labels, and enzyme labels;
a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray pt d[iyi, vuyp;phu' ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or newplastic bronchial cells under conditions such that immunological binding to said binding sites pan occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individulas subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptons;
v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and a sputum inducing means;
the above elements of the kit to be used in conjunction with target cells from sputum bronchial fluid or other pulmonary cells for testing for said antigens, said kit including container means for combining said target cell with said monoclonal antibodies, and a packaging means for conbining said container means, said combining means and said means of detection.
a means of detection employing labels selected from the group consisting of radioisotopic labels, chromophric labels, and enzyme labels;
a monoclonal antibody wherein said antibody is immunogenically pre-selected by the process of:
i) collecting and preserving sputum or bronchial fluid samples from test individuals in which lung cancer is not detectable by chest x-ray pt d[iyi, vuyp;phu' ii) contacting said preserved samples with monoclonal antibodies that are specific for binding sites differentially expressed on normal bronchial cells versus dysplastic bronchial cells or newplastic bronchial cells under conditions such that immunological binding to said binding sites pan occur, whereby a complex is formed;
iii) detecting the presence or absence of said complex resulting from step (ii);
iv) monitoring said test individuals and determining which of said test individulas subsequently develop lung cancer detectable by chest x-ray or clinical symptoms and which of said test individuals do not subsequently develop lung cancer detectable by chest x-ray or clinical symptons;
v) selecting from said antibodies of step (ii) those antibodies that preferentially bind to binding sites present in preserved samples from said test individuals who subsequently develop lung cancer detectable by chest x-ray or clinical symptoms compared to preserved samples from said test individuals who do not subsequently develop lung cancer detectable by chest x-ray or clinical symptoms; and a sputum inducing means;
the above elements of the kit to be used in conjunction with target cells from sputum bronchial fluid or other pulmonary cells for testing for said antigens, said kit including container means for combining said target cell with said monoclonal antibodies, and a packaging means for conbining said container means, said combining means and said means of detection.
11. The kit according to claim 10 wherein said monoclonal antibody is selected from 703D4, ATCC
Accession No. HB8301 and 704A1, ATCC Accession No.
HB8302.
Accession No. HB8301 and 704A1, ATCC Accession No.
HB8302.
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| EP0544898A1 (en) * | 1991-06-26 | 1993-06-09 | The Biomembrane Institute | Early diagnosis of lung cancer using anti-carbohydrate antibody combinations |
| US5790761A (en) * | 1992-12-11 | 1998-08-04 | Heseltine; Gary L. | Method and apparatus for the diagnosis of colorectal cancer |
| US6251586B1 (en) | 1995-10-02 | 2001-06-26 | The United States Of America As Represented By The Department Of Health And Human Services | Epithelial protein and DNA thereof for use in early cancer detection |
| US5994062A (en) | 1995-10-02 | 1999-11-30 | The United States Of America As Represented By The Department Of Health And Human Services | Epithelial protein and DNA thereof for use in early cancer detection |
| US5955287A (en) * | 1995-12-29 | 1999-09-21 | Fernandez-Pol; Jose Alberto | Method of determining level of biological substances elevated in the presence of cancer and other neoplasms |
| WO1998015835A1 (en) * | 1996-10-08 | 1998-04-16 | Inspire Pharmaceuticals, Inc. | Method of early lung cancer detection via sputum induction and analysis of sputum to detect cancer associated substances |
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| US6471663B1 (en) | 1999-08-31 | 2002-10-29 | American Biosystems, Inc. | Chest compression vest with connecting belt |
| US6916298B2 (en) * | 1999-08-31 | 2005-07-12 | Advanced Respiratory, Inc. | Pneumatic chest compression vest with front panel air bladder |
| US6379316B1 (en) * | 1999-08-31 | 2002-04-30 | Advanced Respiratory, Inc. | Method and apparatus for inducing sputum samples for diagnostic evaluation |
| US20040158177A1 (en) * | 1999-08-31 | 2004-08-12 | Van Brunt Nicholas P. | Pneumatic chest compression vest with front panel bib |
| US6910479B1 (en) | 1999-10-04 | 2005-06-28 | Advanced Respiratory, Inc. | Airway treatment apparatus with bias line cancellation |
| US7316658B2 (en) * | 2003-09-08 | 2008-01-08 | Hill-Rom Services, Inc. | Single patient use vest |
| JP5855326B2 (en) | 2005-01-06 | 2016-02-09 | ノヴォ ノルディスク アー／エス | Anti-KIR combination therapy and method |
| US7785280B2 (en) | 2005-10-14 | 2010-08-31 | Hill-Rom Services, Inc. | Variable stroke air pulse generator |
| JP2009516178A (en) * | 2005-11-10 | 2009-04-16 | ユニバーシティー オブ ケンタッキー | Lung cancer diagnostic assay |
| US8460223B2 (en) | 2006-03-15 | 2013-06-11 | Hill-Rom Services Pte. Ltd. | High frequency chest wall oscillation system |
| US20080113350A1 (en) * | 2006-11-09 | 2008-05-15 | Terstappen Leon W M M | Blood test to monitor the genetic changes of progressive cancer using immunomagnetic enrichment and fluorescence in situ hybridization (FISH) |
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| US11471366B2 (en) | 2016-08-22 | 2022-10-18 | Hill-Rom Services Pte. Ltd. | Percussion therapy apparatus and methods thereof |
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| US4865998A (en) * | 1983-03-11 | 1989-09-12 | Sloan-Kettering Institute For Cancer Research | Monoclonal antibodies to human lung cancers and method |
| US4569788A (en) * | 1983-05-18 | 1986-02-11 | The United States Of America As Represented By The Department Of Health And Human Services | Monoclonal antibodies against non small cell lung cancer |
| US4687733A (en) * | 1984-01-30 | 1987-08-18 | Ohio State University | Early cancer detection method |
| US4886745A (en) * | 1984-03-30 | 1989-12-12 | Syntex Inc. | Monoclonal antibody specific for human basal cell surface antigen |
| US4782015A (en) * | 1984-03-30 | 1988-11-01 | Syntex (U.S.A.) Inc. | Method for determining the presence of malignant cells |
| US4690890A (en) * | 1984-04-04 | 1987-09-01 | Cetus Corporation | Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay |
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| US4873188A (en) * | 1985-05-28 | 1989-10-10 | Oncogen | Method, monoclonal antibody, and monoclonal antibody fragments for detecting human non-small cell lung carcinomas and cell line for producing such antibodies |
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- 1989-04-03 JP JP1081664A patent/JP2988630B2/en not_active Expired - Lifetime
- 1989-04-03 EP EP89303267A patent/EP0336677B1/en not_active Expired - Lifetime
- 1989-04-03 AT AT89303267T patent/ATE131620T1/en not_active IP Right Cessation
- 1989-04-03 CA CA000595508A patent/CA1340595C/en not_active Expired - Fee Related
-
1993
- 1993-11-16 US US08/152,881 patent/US5455159A/en not_active Expired - Lifetime
-
1998
- 1998-03-31 JP JP10087822A patent/JPH116836A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE68925058D1 (en) | 1996-01-25 |
| ATE131620T1 (en) | 1995-12-15 |
| KR0141608B1 (en) | 1998-06-15 |
| KR890015714A (en) | 1989-11-25 |
| EP0336677A1 (en) | 1989-10-11 |
| EP0336677B1 (en) | 1995-12-13 |
| AU3117289A (en) | 1989-10-05 |
| JPH116836A (en) | 1999-01-12 |
| JP2988630B2 (en) | 1999-12-13 |
| DE68925058T2 (en) | 1996-05-09 |
| AU632469B2 (en) | 1993-01-07 |
| US5455159A (en) | 1995-10-03 |
| JPH0296658A (en) | 1990-04-09 |
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