Method for Detection of Human Mammary Carcinoma

The method of the invention is illustrated by the following example:

Peripheral blood leucocytes are isolated according to the procedure of McCoy with some modifications. All steps are carried out at room temperature using sterile technique. Two ml of plasma gel (obtained from HTI Corporation) is mixed with 10 ml of heparinized whole blood and the mixture is incubated for 1 hour. The upper plasma phase containing the leucocytes is centrifuged at 220 Xg for 20 minutes, and the supernatant is discarded. The leucocyte pellet is triturated with 2 ml of tris buffered ammonium chloride, pH 7.20, and incubated for 10 minutes. The leucocytes are centrifuged at 220 Xg for 5 minutes and the supernatant is again discarded. The leucocyte pellet is washed twice with 2 ml of medium 199 (Flow Laboratories), pH 7.1-7.2, containing penicillin, streptomycin, and phenol red indicator. The resulting leucocyte pellet is resuspended in 2 ml of medium 199 to make the leucocyte suspension to be tested. Then 1/10 ml of this suspension (which suspension contains 1.78 .times. 10.sup.6 leucocytes per ml) is placed in a capped 1 dram glass vial and is incubated for 18 to 20 hours at 5.degree. C.

Mouse mammary tumor extract is then prepared as follows: CaD.sub.1 subcutaneous primary mammary carcinomas, 1.0 to 1.5 cm in diameter, from DBA/1J mice (Jackson Laboratories) are excised, pooled, and immediately cut into 1-5 millimeter cubes. Ten ml of tumor tissue is pressed through a 16 gauge stainless steel mesh and is then suspended in 10 ml of medium 199. The carcinoma suspension is centrifuged at 10,000 Xg for 30 minutes at 5.degree. C; the resulting supernatant is removed and is then passed through a 0.45 micron millipore filter. The resulting filtered extract is adjusted to 9.6 mg/ml and to a pH of from about 7.1 to about 7.2 and is stored at 5.degree. C.

To perform the test, 1/10 ml of the extract is added to the vial containing the leucocyte suspension and the resulting mixture is incubated, preferably at 37.degree. C. The mixture is examined for color change due to the pH indicator from red (basic) to yellow (acid) over a period of at least 48 hours, whereby the decrease in pH is measured.

The results of this test on 16 subjects clinically diagnosed as having mammary carcinoma, 7 subjects known to have other neoplastic diseases, and 14 ostensibly normal blood bank samples are shown in Table I. Samples from sixteen subjects gave positive results in the above test, as evidenced by a pH decrease of at least 0.55 pH units. Samples from twenty subjects gave a pH decrease of less than 0.55 pH units, a negative result. These results are significant to the 99% confidence level.

These results indicate that the test of the invention is useful for specific detection of mammary carcinoma. It detected 96% of the clinically diagnosed mammary carcinoma hosts and yielded only one false positive - the subject with rectal cancer. No false positives were obtained with ostensably normal subjects.

The results of a double blind study on seventeen mammary carcinoma hosts and twenty-three subjects with non-cancerous breast conditions are shown in Table II. The test was conducted as described in the Example, a pH decrease of at least 0.60 pH units being the criterion for a positive result.

For reasons not clearly understood at the present time, the method of the invention is of greatest utility in the detection of mammary carcinoma in younger hosts. This fact is illustrated in Table II, in which age 61 was chosen as a dividing line for presentation of results. The results for hosts less than 61 years of age are significant to the 99% confidence level. One false positive was obtained, which may indicate that the particular subject has a mammary carcinoma which is as yet clinically undetectable.