The detection of breast cancer in humans by evaluating antigen
induced leukocyte adherence inhibition caused by tumor specific
cell mediated immunity by incubating a measured amount of a patients
blood leukocytes with aqueous basic extracts of human breast tumor,
then determining whether the leukocyte adherence inhibition measured
by the non-adherence index as compared to a control of a non-specific
antigen is high, indicating the presence of breast cancer, or low,
indicating the absence of breast cancer.
1. A method for detecting the presence of breast cancer in humans
which comprises incubating a measured amount of a patient's blood
leukocytes with aqueous basic extracts of human breast tumor, then
determining the leukocyte non-adherence index by comparison to a
non-specific antigen control, and concluding that breast cancer
is present if the non-adherence index of the patient being tested
is higher than the control non-adherence index.
2. The method of claim 1 wherein the breast tumor extract is from
a breast tumor of the patient being tested.
3. The method of claim 2 wherein the breast tumor extract is from
a breast tumor of a patient not being tested.
4. The process of claim 1 wherein the breast tumor extract is an
aqueous extract at pH 7.3.
BACKGROUND OF THE INVENTION
Cell mediated tumor immunity has been demonstrated in experiments
with transplanted tumors in mice and rats. For example, Halliday
et al. Int. J. Cancer, Vol. 9, pages 477-483 (1972) established
a simple assay of cell mediated immunity called Leukocyte Adherence
Inhibition. This assay was based on the findings that the adherence
of peritoneal leukocytes to glass is specifically inhibited by the
addition of tumor extracts if the peritoneal leukocytes are obtained
from mice which have been presensitized against the specific tumor.
Holan et al., Cellular Immunology, Vol. 13, pages 107-116 (1974),
using transplanted tumors in rats, mice and guinea pigs also demonstrated
the same phenomenon with macrophage cells. There has been much research
in attempts to utilize immunological principles for the detection
and possible cure for various human cancers. While success has been
met in animal models having transplanted tumors, immunological methods
developed for the detection of breast cancers have not been successful
for mass screening. While it has been generally known that an immunological
defense mechanism exists in the early stages of malignant diseases,
while such defense mechanism has been demonstrated with breast cancer
in humans with macrophage migration inhibition and by interdermal
injections of breast antigens which show a delayed hypersensitivity
response, such assays are unsuitable for large scale screening.
In recent years a great many studies have been made in the medical
diagnostics field to achieve detection of breast cancer in humans,
however, no blood tests have been developed which can be used in
mass screening programs for breast cancer detection. The diagnostic
tests available to the clinician are based on visual observation
of cells, x-rays, or thermography. These tests, however, are time
consuming, complex and subjective. Ideally, a desirable test for
human breast carcinoma would be a blood test which is simple, specific
and reproducible and which detects the disease at a sufficiently
early stage so that the disease is still curable.
DESCRIPTION OF THE INVENTION
We have discovered that the product of the immunological defense
mechanism of the human body to breast cancer can be detected at
a stage of the disease which is sufficiently early so the disease
is still curable. The method used to detect the product of the immunological
defense mechanism is a blood test which is simple to perform, is
specific to breast cancer and gives reproducible results. As used
herein, "breast cancer" means carcinomas of the breast
of humans. Carcinomas include adenocarcinoma.
The process of this invention involves collecting the leukocytes
from heparinized venous blood of the patient, then suspending a
measured amount of the leukocytes in a buffered solution followed
by mixing the leukocytes with extracts of breast cancer tumors,
and using as a control extracts of normal breast tissue, or extracts
of other tissues or malignant tumors not being detected. After suitable
incubation procedures, the number of cells in each case not adhering
to the sides of the test tube are counted and the leukocyte adherence
inhibition is measured as the non-adherence index (NAI). This determines
the presence or absence of breast cancer. ##EQU1##
The leukocytes are collected and counted by conventional methods.
The heparinized blood sample is incubated at about 37.degree. C.
for about one hour and the plasma fraction is collected. The plasma
fraction is rich in leukocytes. The leukocytes are separated by
centrifugation and suspended in an isotonic buffer. Any erythrocytes
remaining are lysed from the leukocyte suspension. The leukocytes
are then separated by centrifugation and suspended in an appropriate
aqueous medium at a cell concentration of 1 .times. 10.sup.7 /ml.
The tumor extracts are made by homogenizing the tumor samples (or
normal tissue samples), in an ice-cold buffered saline at pH about
7.3, centrifuging the homogenate and collecting the resulting supernatant.
The buffer used is preferably a phosphate buffered saline (PBS).
It is necessary to use a basic pH since acid pH will destroy the
antigenic activity. In order to use in the assay, the extracts are
diluted with an appropriate aqueous medium, preferably Medium 199
(Microbiological Associates, Bethesda, Md.). The optimum dilution
for use in the assay is established for each extract by determining
the concentration of tumor antigen which produces the largest increment
between the specific and non-specific inhibition of leukocyte adherence.
The breast tumor extract is the antigenic material which gives
rise to the cell mediated immunity. For purposes of illustrating
this invention, breast adenocarcinoma extracts are used. The antigens
which are suitable for use as the non-specific controls are extracts
of normal breast tissue, melanoma tumor, adenocarcinoma of the bowel,
ovary and bladder and lung cancer.
The assay is carried out by mixing aliquots of a small amount of
a blood leukocyte suspension containing about 1 .times. 10.sup.7
leukocytes/ml. with separate small amounts of buffer, breast tumor
antigen, or unrelated antigen in separate glass containers. Each
mixture is then brought to a predetermined volume. Each mixture
is then incubated in the glass container which is placed so that
the contents cover at least three-fourths of the inner surfaces
of the container. The incubation is carried out at about 37.degree.
C. for a sufficient time to complete the reaction, if any, under
a humidified atmosphere of 5% CO.sub.2 -air. When the incubation
is completed, the contents are agitated and the number of non-adherent
cells are counted.
The aliquots of leukocyte suspension are conveniently very small,
usually about 0.1 ml. The concentration of leukocytes is such that
counting of the cells gives a meaningful result and the reaction
with the antigen material can be readily assayed. The buffer used
for dilution is Medium 199 which has a pH of 7.2-7.4.
The amount of antigen extract used is conveniently about 0.1 ml.
of the diluted extract. The extract is diluted generally with Medium
199. The amount of dilution of the concentrated antigen extract
on a volume basis can be between 1 part antigen extract per 4 to
16 parts of diluent. Preferably a 1 to 4 dilution is used.
The final volume of the test mixture of leukocytes and antigen
is conveniently about 0.5 ml. The diluent used to achieve this volume
is Medium 199.
Suitable glass containers used are any convenient size and shape.
For convenience of use and availability, 20 ml. Pyrex test tubes
are preferred. The incubation is carried out with the test tubes
in a horizontal position in a holder which prevents their movement.
This is critical since movement during incubation can adversely
affect the test results. The incubation is carried out preferably
for about two hours. Longer times can be used but do not increase
the accuracy of the assay or the completeness of the reaction. Shorter
times can also be used but are not optimum. Thus incubation times
of from about 1 to 3 hours are operable.
The non-adherent cells are counted in any convenient manner, for
example, by the use of a hemocytometer.
It has been found according to the process used in this invention
that the response of the leukocytes of a patient to breast cancer
tumor extracts does not depend upon the source of the extract. Extracts
from the patient who is being tested or extracts from other patient's
breast cancers give equivalent results. This indicates that the
leukocytes of a patient with breast cancer exhibit cell mediated
immunity which is specific to all breast cancers of the same type
and is not affected by the source of the breast cancer antigen.
The following Examples illustrate the invention.
Breast tumor samples were received at operation and placed in a
sterile container. Fat and fibrous tissue were dissected away and
the specimen was finely minced with sharp scissors in ice-cold phosphate-buffered
saline (PBS), pH 7.3. The resulting material was homogenized for
10-15 minutes in four volumes of PBS at 40,000 rpm in a VirTis 45
homogenizer. The homogenate was centrifuged at 20,000 .times. gravity
for 30 minutes and the supernatants were collected and stored at
-40.degree. C. in small aliquots. Extracts of normal breast tissue,
malignant melanoma and other tumors were prepared in an identical
manner. These extracts were used as the controls for the process
of this invention. For use in the assay, the extracts were thawed
and diluted to 1:4 by volume with Medium 199. Medium 199 is marketed
by Microbiological Associates, Bethesda, Maryland and is composed
of Components mg./liter ______________________________________ Amino
Acids L-Alanine 25.0 L-Aginine HCl 70.0 L-Aspartic Acid 30.0 L-cysteine
HCl 0.1 L-Cystine 20.0 L-Glutamic Acid 67.0 L-Glutamine 100.0 Glycine
50.0 L-Histidine HCl-H.sub.2 O 22.0 Hydroxy-L-proline 10.0 L-Isoleucine
20.0 L-Leucine 60.0 L-Lysine HCl 70.0 L-Methionine 15.0 L-Phenylalanine
25.0 L-Proline 40.0 L-Serine 25.0 L-Threonine 30.0 L-Tryptophan
10.0 L-Tyrosine 40.0 L-Valine 25.0 Vitamins P-Aminobenzoic Acid
0.050 Ascorbic Acid 0.050 D-Biotin 0.010 Calciferol 0.100 D-Ca-Pantothenate
0.010 Cholesterol 0.200 Choline Chloride 0.500 Folic Acid 0.010
i-Inositol 0.050 Menadione 0.010 Nicotinamide 0.025 Nicotinic Acid
0.025 Pyridoxal HCl 0.025 Pyridoxine HCl 0.025 Riboflavin 0.010
Thiamine HCl 0.010 DL-.alpha.-Tocopherolphosphate (Na.sub.2) 0.010
Tween 80* 5.000 Vitamin A.Acetate 0.140 Other Components Adenine
HCl.2H.sub.2 O 12.10 Adenosine-5'-Monophosphoric acid, dihydrate
(AMP) (Muscle Adenylic Acid) 0.20 Adenosine-5'-Triphosphate disodium,
tetrahydrate (ATP) 1.08 Deoxyribose 0.50 Dextrose 1000.00 Glutathione
(Reduced) 0.05 Guanine HCl.H.sub.2 O 0.33 Hypoxanthine 0.30 Phenol
Red 20.00 Ribose 0.50 Sodium Acetate.3H.sub.2 O 83.00 Thymine 0.30
Uracil 0.30 Xanthine 0.34 Inorganic Salts CaCl.sub.2.2H.sub.2 O
186.0 Fe(NO.sub.3).sub.3.9H.sub.2 O 0.7 KCl 400.0 KH.sub.2 PO.sub.4
60.0 MgSO.sub.4.7H.sub.2 O 200.0 NaCl 8000.0 NaHCO.sub.3 1400.0
Na.sub.2 HPO.sub.4.7H.sub.2 O 90.0 Vialed at pH 7.2 to 7.4 ______________________________________
*Trademark of Atlas Powder Company - Polyoxyethylene (20) sorbitan
Preparation of the Leukocyte Reagent
Blood was taken from patients with breast cancer and control subjects
and immediately stored at 4.degree. C. After retraction of the clot
overnight, the serum was separated and stored at -40.degree.C. These
blood samples were heparinized blood samples obtained from patients
with adenocarcinoma of the breast and controls with benign breast
disease and a variety of non-malignant diseases or unrelated malignancies.
The diagnosis of breast cancer in the so designated patients was
confirmed histologically. The clinical diagnosis of all apparently
benign breast lesions was confirmed by histological examinations
of the surgical specimen. 20 Ml. samples of the heparinized blood,
i.e., venous blood, was placed vertically in a glass universal bottle
at 37.degree. C. for 1 hour. The resulting leukocyte rich plasma
fraction was aspirated and centrifuged at 200 .times. gravity for
15 minutes. The plasma was removed and discarded. The remaining
cell button was suspended in an ice cold isotonic Tris-buffered
ammonium chloride solution by repeated pipetting and left for 15
minutes at 4.degree. C. in order to lyse any remaining erythrocytes.
This procedure was terminated by the addition of 3 ml. of Medium
199 and the cells were centrifuged at 200 .times. gravity for 15
minutes. The resulting supernatant was removed and discarded and
the remaining cells were then washed twice in 20 volumes of Medium
199. Aliquots of the leukocytes were made at a concentration of
1 .times. 10.sup.7 cells per ml. using Medium 199 as the diluent.
Aliquots of 0.1 ml. of blood leukocytes suspensions containing
1 .times. 10.sup.7 cells per ml. were placed in glass test tubes.
Added to these leukocytes were 0.1 ml. of extracts of either breast
tissue or other unrelated tumor cell derived preparations or 0.1
ml. of extracts of cancer tumors from the breast. Various concentrations
of tumor extracts were used to determine the optimum concentration.
The mixture of leukocytes and test cells were brought to a final
volume of 0.5 ml. by the addition of Medium 199. The mixture was
then stirred and the tubes were put in a horizontal position so
that the contents of the tubes covered three-fourths of the surface
of the tubes. The horizontal tubes were then incubated at 37.degree.
C. in a humidified atmosphere of 5% CO.sub.2 -air for 2 hours. The
tubes were not disturbed or moved during the incubation period.
After the incubation the tubes were placed vertically and the contents
were agitated with a pasteur pipette. The number of non-adherent
cells/ml. was counted in a hemocytometer. All assays were done in
triplicate. The results expressed as the non-adherence index (NAI)
is shown in the following table:
The results indicate that the patients with breast cancer have
a significantly higher NAI when compared to other patients.
When blood leukocytes from breast cancer patients or control subjects
were incubated in glass tubes without added antigen for 2 hours,
about 10 percent non-adherent cells were found. The addition of
any tumor extract or normal breast tissue extract to non-sensitized
blood leukocytes inhibited adherence of 15-40 percent of the leukocytes.
Incubation of blood leukocytes from patients with breast cancer,
with breast tumor extracts, caused a 40-65 percent non-adherence
of leukocytes to glass, whereas incubating the same cells with normal
breast tissue, melanoma extract or other unrelated tumor extracts
produced a 15-40 percent non-adherence of leukocytes. Leukocyte
adherence-inhibition was the same when the breast carcinoma patient's
leukocytes were exposed to extracts of their own tumor as when the
extract was from breast carcinoma of another individual.
In order to achieve the optimum assay conditions, that is, the
maximum specific inhibition of leukocyte adherence with the least
non-specific inhibition, the antigen extracts were tested at different
concentrations with leukocytes from patients with malignant melanoma,
breast cancer, and control subject. As shown in Table 2, the NAI
falls with increasing dilutions. In this instance, however, a dilution
of one-fourth of the breast cancer extract and melanoma extract
gave a high NAI value for the patient with breast cancer and a low
NAI to breast cancer for both the control and patient with malignant
melanoma. Therefore, for the purpose of this invention, a dilution
of one-fourth of the antigen, that is, one part antigen extract
to 4 parts of diluent are preferred. Good results are obtained,
however, when the dilution is as high as 1:16.
The results shown in Tables 3 and 4 indicate that significant inhibition
of leukocyte adherence to glass occurred with breast cancer extracts
with cells from 40 of 47 breast cancer patients. The breast cancer
patients showed a mean NAI of 58 and 189 to the breast cancer extract
when the non-specific antigens were melanoma and normal breast tissue,
respectively. There is a striking difference in the NAI between
breast cancer patients who have clinically localized disease to
breast or breast and lymph nodes compared with individuals with
cancer spread to multiple organs. Six patients showed a NAI below
the arbitrary level of 18 when the melanoma extract was employed
as a control. Three of the six patients had disseminated disease,
one patient had axillary node involvement but no clinical evidence
of metastasis and the remaining two patients had small breast lumps
which histologically showed invasive adenocarcinoma. This is apparent
from Table 3. When normal breast tissue was used as the non-specific
control antigen, only one breast cancer patient had a NAI below
the arbitrary level of 60. This is shown in Table 4. This patient
had disseminated cancer.
Thirty-two controls had no clinical evidence of breast cancer.
In general, the controls failed to demonstrate breast tumor antigen-induced
inhibition of leukocyte glass adherence as a group and as individuals.
The mean Non-Adherence Index of the breast cancer patients is significantly
different from the corresponding control group (P<.001).
Tumor antigen-induced inhibition of leukocyte adherence to glass
in patients with breast cancer and controls. The Non-Adherence Index
was calculated using breast cancer antigen as specific antigen and
melanoma extract as unrelated antigen. NAI = % non-adherent cells
with specific antigen - % non-adherent cells with non-specific antigen
divided by % non-adherent cells with non-specific antigen. A NAI
of 18 or greater was considered significant. The breast cancer group
consisted of 23 patients with active disease. The control group
consisted of 19 patients with variety of diagnosis, 7 of whom had
histologically proven benign breast disease. The mean NAI of the
breast cancer group of 58 is significantly different from the control
group (-18). (P<.001 by unpaired t-test).
Tumor antigen induced inhibition of leukocyte adherence to glass
in patients with breast cancer and controls. The Non-Adherence Index
was calculated using breast cancer as specific antigen and normal
breast tissue as unrelated antigen. A NAI of 60 and greater was
considered significant. The breast cancer group consisted of 24
patients with active disease. The control consisted of 20 patients,
7 of whom were the same patients with the benign breast disease.
The mean NAI of the breast cancer group is 158 and is significantly
different from the controup, 30. (P<.001 by unpaired t-test).
Other tumor extracts have been employed as the non-specific antigens.
The results have been exactly similar to those obtained with the
melanoma extract as the non-specific control. No evidence of cross-reaction
has been observed.