WO1994003470A1

WO1994003470A1 - Method and apparatus for screening and diagnosing for cancers

Info

Publication number: WO1994003470A1
Application number: PCT/US1993/006831
Authority: WO
Inventors: Richard J. Davies
Original assignee: Davies Richard J
Priority date: 1992-08-10
Filing date: 1993-07-23
Publication date: 1994-02-17

Abstract

An in vitro method of screening and diagnosing patients for cancers at an early stage for the identification of premalignant tissue changes is described. In this in vitro method, a cell preparation is prepared from a tissue sample; a calcium probe, e.g., a fluorescent dye, is added to the sample; an electronic test signal is provided that is representative of intracellular CA2+ concentration in the probed sample; and the test signal is compared with the reference signal representative of intracellular CA2+ concentration in normal tissue. Apparatus for carrying out this method is also described.

Description

Description Method and Apparatus For Screening and Diagnosing For Cancers

Technical Field

The present invention is a method and apparatus for screening and diagnosing patients for cancers at an early stage, or the identification of premalignant changes, which may eventually lead to cancer development.

Background Art

Cancer will become the leading cause of death in the United States and most industrial countries before the end of the decade. This will occur because of, among other things, an aging population, increased incidence of cancers, and decreasing incidence of cardiovascular disease. Most efforts to cure cancers, once they are present with advanced symptoms, have been disappointing. This experience has directed increasing attention to identification of high-risk patients and earlier diagnosis of cancerous and pre- cancerous conditions. It is now recognized that cancer is a multi-step process in which the development of malignancy takes many years or decades. Accordingly, a considerable demand now exists for screening tests to identify early-can¬ cer, pre-cancerous, and "at-risk of cancer" conditions, so that appropriate countermeasures and regimens can be instituted while they may still be effective in preventing death from cancer.

The Larσe Bowel

Cancer Epidemiology. Cancer of the large bowel (colon and rectum) is the second most common cause of cancer in the United States. It is estimated that 160,000 new cases occur annually and that about one half of these patients die within five years. See "Cancer Statistics, 1992." It is estimated that most cancers take years, if not decades, to develop. It may, therefore, be inferred that the identification of early pre-cancerous changes in colonic epithelium would result in the identi ication of a high-risk population in which intensive screening could be directed and in which dietary or chemopreventative measures could be aimed.

Structure. The colon and rectum, known collectively as the large bowel, comprises a hollow muscular tube lined with a mucosa consisting of epithelial and other cells. The mucosa is not a flat sheet of cells. Rather, its structure is like that of an egg crate, with open ends of elongated tubes joined at the mucosal surface by bridges of epithelial cells. These tubes, known as "crypts," are blind-ended deep in the supporting stroma of the mucosa, and open-ended on the surface of the bowel lumen. Normal crypt organoids are the basic functional units of the large bowel. Shaped like a test tube, a crypt is comprised of columnar epithelial cells and goblet mucus cells, which respectively comprise approximately 85 percent and 15 percent of the crypt's total cell population. Cells are replaced from dividing stem cells deep within the lower recesses of the crypts. Stem cells divide to produce proliferating cells, which migrate up the length of the crypt and undergo differentiation and, ultimately, cell death, which occurs shortly before the cells are extruded into the bowel lumen. The epithelial cells within a crypt are completely replaced every three days. The foregoing is the normal cellular process. That process of cell division, differentiation, and cell death becomes drastically altered, however, during the process of cancer development.

Mucosal Proliferation in Colorectal Cancer. It had been shown that colonic mucosa from patients at increased risk for colorectal cancer undergo an increase in cellular proliferation. Hyperplasia occurs attended by an expansion of the proliferative compartment of the intestinal crypts. This phenomenon has also been recognized in rodent models of large bowel cancer. See Deschner, "The relationship of proliferative defects to the colon cancer," in Colorectal Tu ors (eds. Beahrs, Higgins & einstein, 1986) , at 81-86; Lipkin, "Biomarkers of increased susceptibility to gastro¬ intestinal cancer: New applications to studies of cancer prevention in human subjects," Cancer Res. 48:235 (1988).

In a normal person, the major zone of DNA synthesis of crypt cells is the lower third of each crypt. A proliferative compartment occupies the lower two-thirds of each crypt, and DNA synthesis occurs in the lower half of this compartment. One characterization of abnormal crypts (see Deschner, "Significance of the labeling index and labeling distribution as kinetic parameters in colo-rectal mucosa of cancer patients and DMH treated animals." Cancer 50:1136, 1982) divides them into three groups, designated stages I, II, III. In a stage-I abnormal crypt, the proliferative compartment extends all the way along the crypt to the luminal surface, but DNA synthesis continues to occur only in the lower third of the crypt. In a stage- II abnormality, there is a shift of DNA synthesis to the middle third or upper third of the crypt, while the pro¬ liferative compartment still extends from the base of the crypt to the luminal surface. In a stage-Ill abnormality, the labeling index (a measure of DNA synthesis which uses the uptake of radioisotope-labeled thy idine) of the crypt, that has already reached stage-II abnormality reaches 15 percent or higher. A similar increase in cell prolifera¬ tion, associated with the development of cancer has been observed for breast, esophageal, gastric, liver, prostate, cervical, lung, and bladder cancers. The upregulated and abnormal proliferation which occurs throughout an epithelium, which is at increased risk for cancer development, appears to be associated with alterations in intracellular Ca²⁺. Intracellular Ca²* regulation is thought to be important in growth regulation and cell division.

Breast Tissue

Epidemiology. The incidence and mortality from breast cancer is rapidly increasing in the United States. It is estimated that in 1992 there will be 181,000 new cases and 46,600 deaths. It is believed that breast screening programs can significantly reduce mortality from breast cancer. As in the case of colon cancer, it is believed that identification of early pre-cancerous changes in breast tissue would result in the identification of a high-risk population in which intensive screening could be directed and in which dietary and chemopreventative measures could be aimed.

Structure. Breast tissue is comprised of branching ducts which end in lobules. Ductal and lobular tissue are arranged in glands, which are supported by stromal glandular tissue comprised of fat and fibrous tissue. Smaller branching ducts which end in a blind-ended lobule form the principal functional unit of the breast; these organoids are designated as "terminal ductal lobular units." Terminal ductal lobular units undergo cell proliferation, differentiation, and death, in a manner comparable to that of the colonic crypt. Terminal ductal lobular units are also the seat of cancer development in the breast, as crypts are in the large bowel.

Ductal Proliferation in Breast Tissue. Cellular proliferation in normal, non-lactating breast tissue has not been well documented. Increases in labeling index based on thymidine uptake have been observed for patients having malignant breast disease, but the data are highly variable.

The Lung

There will be 168,000 cases of lung cancer in the United States and 146,000 deaths in 1992. The majority of lung cancers develop from bronchoepithelial cells. The nor¬ mal bronchoepithelium is pseudostratified and the development of lung cancer is associated with basal cell, and mucous cell hyperplasia, and squamous metaplasia.

The Prostate

Cancer of the prostate is the second most common cause of cancer amongst males in the United States. It is estimated that in 1992 there will be 132,000 new cases and 34,000 deaths. The prostate consists of glandular epithelium muscle and fibrous stroma. The glandular tissue is arranged into ducts and acini. Prostatic cancer is associated with epithelial hyperplasia.

The Upper Aerodigestive Tract There will be 53,900 cases of upper aerodigestive tract cancer (including esophagus, larynx, lip, mouth, tongue, and pharynx) in the United States and 21,600 deaths in 1992. Most of the upper aerodigestive tract is lined with squamous epithelium and cancers, which develop in this part of the body, are frequently associated with leukoplakia, dysplasia and abnormal proliferation.

The Urinary Bladder

Cancer of the urinary bladder is the fourth most common cause of cancer in the United States. It is estimated that in 1992 there will be 51,600 new cases and 9,500 deaths.

The bladder does not appear to contain organoids analogous to crypts or terminal ductal lobular units, in which cancers develop. Rather, bladder cancer development appears to be associated with the entire bladder lining. An increase in cellular proliferation has been observed as associated with development of bladder cancer.

The Endometrium

There will be 32,000 cases of endometrial cancer in the United States and 5,600 deaths in 1992. The endometrium consists of glandular columnar epithelium and it is unknown whether there is upregulated proliferation associated with cancer development.

The Pancreas

There will be 28,300 cases of pancreatic cancer in the United States and 25,000 deaths in 1992. Pancreas consists of ductal and glandular epithelium arranged in acini similar to breast. Although pancreatic cancer is frequently associated with pancreatitis it is unknown whether there is a generalized upregulation in proliferation associated with the development of this malignancy.

The Stomach

Gastric cancer has decreased in incidence in the United States over the past century. In Japan and other Far Eastern countries, however, gastric cancer is a major cause of death. There will be 24,400 cases of gastric cancer in the United States and 13,300 deaths in 1992.

The stomach is lined with gastric pits, which are organoids similar to colorectal crypts. Increase in cellular proliferation has been observed in association with gastric cancer.

The Liver

There will be 15,400 cases of liver or biliary cancer in the United States and 12,300 deaths in 1992. However, worldwide the incidence of liver cancer is extremely high, particularly in countries where hepatitis is endemic. The liver consists of hepatocytes arranged in hepatic lobules with bile canaliculi at the center of each lobule. Primary hepatic carcinoma has been found to be associated with hyperplastic liver nodules and upregulated proliferation of hepatocytes.

The Uterine Cervix

There will be 13,500 cases of cervical cancer in the United States and 4,400 deaths in 1992. The cervix consists of squamous epithelium and dysplasia and hyperplasia are well recognized antecedent conditions associated with the development of cervical cancer.

The Thyroid

There will be 12,500 cases of thyroid cancer in the United States and 1,000 deaths in 1992. Thyroid consists of glandular epithelium and stroma. Thyroid cancer is associated with increased hyperplasia of the surrounding thyroid gland.

Other Tumors Other types of tumors are quite rare such as ovarian, brain, and testicular cancer and it has not been determined as to whether an upregulation in proliferation is associated with the development of cancer. However it is likely that tumors of epithelial or glandular origin (the majority) develop in a background of upregulated proliferation.

Disclosure of the Invention

The inventor had developed technology for making measurements of intracellular calcium (intracellular Ca⁺) levels in order to employ them as diagnostic markers for assessing cancer risks in patients. Altered levels of intracellular Ca²⁺ may be associated with the type of generalized upregulated proliferation which is found in tissues which are at increased risk for cancer development. Such measurements detect or screen for patients who have developed abnormal cell proliferation, but are still years away from developing detectable cancers in the organs with such cell proliferation. Methodology is described for testing isolated cell suspensions, and for testing intact organoids, such as colorectal crypts and terminal ductal lobular units obtained from biopsies.

The basic procedure is to prepare the tissue sample (obtained from biopsies and homogenized into isolated cells or intact organoids) ; load the cells or organoids with a calcium probe, such as a fluorescent dye; and measure intracellular Ca²* concentration by deriving a signal from the probe-loaded tissue sample. Procedures are described for determining intracellular Ca²* concentration for tissues placed in a Ca²*-free bath and Ca²*-containing bath.

Brief Description of the Drawings

FIGURE 1 is a chart comparing intracellular Ca²* (Ca²*,-) concentrations in mouse colon tissue, for mice dosed with the carcinogen 1,2 dimethylhydrazine (DMH) for 20 weeks. 22 tissue samples were examined in the "control" group, 10 tissue samples were examined in the "tumor" group, and 12 samples were examined in the "at-risk" group. Data points refer to the arithmetic mean of the observations, and error bars are the standard error of the mean (SEM) . The y-axis scale is in nanoMoles (nM) . On the x-axis; "0 mM Ca²* Bath" refers to measurements made in Ca²*-free Ringers Solution at 24°C; "1 mM Ca²* Bath" refers to measurements made in Ca²*- containing Ringers Solution at 24°C; "Difference" refers to the change in intracellular Ca²* when the bathing solution was changed from Ca²*-free to Ca*-containing Ringers Solution.

FIGURE 2 is another chart comparing Ca^2*,- in mouse colon tissue, for mice dosed with carcinogen, DMH for 6 weeks. 10 tissue samples were examined in the "control" group, and 10 samples were examined in the "at-risk" group. Otherwise, the legends are as in figure 1.

FIGURE 3 is a chart comparing Ca²*,- in human colon tissue. 11 tissue samples were examined in the "control" group, 9 tissue samples were examined in the "tumor" group, and 10 samples were examined in the "at-risk" group. Otherwise, the legends are as in Figure 1.

FIGURE 4 is a chart comparing Ca*_i in isolated human colonic crypts. The symbol "n" in the legend refers to the number of isolated crypt specimens. Data points refer to the arithmetic mean of the observations, and error bars are the standard error of the mean (SEM) . The y-axis scale is in nanoMoles (nM) . On the x-axis; "0 CA" refers to measure¬ ments made in Ca²*-free Ringers Solution at 37°C; "PEAK" refers to the highest intracellular Ca²* measurement when the bathing solution was changed from a Ca²*-free to a Ca²*- containing Ringers Solution at 37°C; "FINAL" refers to the plateau intracellular Ca²* when the bathing solution was changed from Ca²*-free to Ca^*-containing Ringers Solution. The symbol "#" refers to a p value of < 0.02 using a unpaired Student t-test comparing "at-risk" crypt mouths under 0 CA conditions with controls, and "at-risk" crypt mouths under FINAL conditions compared with controls. The symbol "*" refers to a p value of < 0.001 using an unpaired Student t-test comparing "at-risk" crypt bases under PEAK conditions with controls, and "at-risk" crypt bases under FINAL conditions compared with controls.

Best Mode for Carrying Out the Invention

The inventor conducted a series of tests to provide data showing the difference, if any, in intracellular Ca²* for normal, precancerous ("at risk"), and cancerous tissue. The tests are described below.

EXAMPLE 1- MOUSE COLON CANCER (ISOLATED CELLS)

Measurements of intracellular Ca²* were made in colon cells from normal and pre-malignant mouse colon tissue as follows: CF, mice were given 20 weekly subcutaneous injections of either saline, as a control, or 1,2- dimethylhydrazine(DMH) , 20 mg/kg, a drug used to induce colorectal cancer in rodents.

The animals were sacrificed one week after their last injection. The distal colon was resected and placed in oxygenated physiological saline. Colons from the DMH group were further dissected, to separate gross tumors from the adjacent normal-appearing, tumor-free colon. (The latter group is referred to hereinafter at times as the "at risk" group. These tissues are considered to be precancerous, because mucosal samples were obtained from animals known to have undergone initiation with the carcinogen DMH and with eventual development of colorectal cancer in 96-100% of the animals) .

Tissues from the three groups were minced and treated with collagenase, 2 mg/ml, in a Ca²*-free saline at 24°C to form a cell suspension. Cells were loaded with 5μM Fura/2 AM (Cal Biochem, San Diego, Ca.), a fluorescent probe for calcium, and placed in a cuvette. The cells were alternately excited at 340 n and 380 nM. Intracellular Ca²* was then measured by measuring intensity of light re- emitted at 505 nm. The measurement was made at 24°C, using an ARCM DM3000 spectrofluorometer (SPEX Industries, Edison, NJ.), first in a bath of Ca²*-free Ringer Solution. Then, CaCl₂, solution was added to the bath of Ringer Solution, until ImM Ca²* concentration was reached. The fluorescence measurement was then repeated.

At the completion of the experiment 60 μlΛ digitonin (Cal Biochem) was added to the Ringers Solution to permeablize the cell membrane and permit free movement of Ca^2* across the membrane while still leaving FURA/2 trapped within the cells. In the presence of 1 mM extracellular Ca²*, this step causes maximal saturation of the FURA/2 molecules (i.e., a shift from the free to the calcium-bound form) resulting in a maximal emission intensity at the 340nm excitation wavelength. 60mM EGTA (Sigma Chemical Co.) was then added to chelate cell Ca²* and caused the free-bound FURA/2-Ca²* equilibrium to be shifted so that all FURA/2 was unbound. This results in minimizing the intensity at 340nm excitation while maximizing that at 380nm. Scanning was continued and additional EGTA was added until the emission patterns were consistent with maximal levels of unsaturated FURA/2.

Calculation of intracellular Ca²* (Ca²*,.) was performed using the Grynkiewicz Equation:

Ca²*,. = (K_d) (b) (R - R_unsat) / (R_sat - R) where

K_d = dissociation constant (2.24 x 10^"7) . b = ³⁸⁰ _sat/³⁸⁰ _unsat emission ratio for excitation at 380nm for the two indicated conditions. R = 340/380 emission ratio for a given data point _unsat = 340/380 emission ratio for unsaturated FURA/2 dye (all free) . R_sat = 340/380 emission ratio for saturated FURA/2 dye (all bound) . Fluorescence intensity of bound (340nm excitation) to unbound (380nm excitation) emissions were generated separately. Next, points from the maximally saturated (following the addition of digitonin) and unsaturated (following the additions of EGTA) portions of the dual- excitation intensities were used in the Grynkiewicz Equa¬ tion. This generates a curve representing intracellular Ca²* in calcium-free Ringer solution and after the addition of ImM Ca²* that is both qualitative and quantitative. Using SPEX DM3000 Cation Measurement (SPEX Industries) , a ratio was obtained from the calibration curve using the for¬ mula above. Ratio intensities for each excitation wavelength was used to calculate the intracellular Ca²* for each sample.

The control group comprised 22 tissue samples. The intracellular Ca²* concentration observed in the Ca²*-free Ringer Solution bath was 244 nM ±39nM. (The first figure, 244 nM, is a mean, and the second figure, 39 nM, is a standard error.) The value in the 1 mM Ca²* bath was 421 nM ±46nM.

The difference in measured values of intracellular Ca²* between Ca*-free and 1 mM Ca₂₊ bath was 178 nM ±21 nM. The tumor-cell group comprised 10 tissue samples. The intracellular Ca²* were 101 nM ± 8 nM for Ca²*-free bath, and 233 nM ±12 nM for 1 mM Ca²* bath. The difference in measured values of intracellular Ca²* was 132 nM ±19 nM.

The normal-appearing, tumor-free("at risk") tissue group taken from colon tissue adjacent to tumor-containing tissue comprised 12 samples. The intracellular Ca²* values were 289 nM ±21 nM for the Ca²*-free bath and 613 nM ±12 nM for Ca²*-containing bath. The difference in measured values of intracellular Ca²* was 324 nM ±27 nM.

The following conclusions are drawn from this data, which is depicted in FIG. l: The intracellular Ca²* for mouse colon tissue under Ca²⁺-free conditions was significantly lower for the tumor tissue, compared to both the control tissue and the tumor-free ("at-risk") tissue. The p-values, using an unpaired Student t-test, were p < 0.05 for control tissue and p < 0.01 for tumor-free ("at- risk") cells. The intracellular Ca²* in control and tumor- free ("at-risk") cells were not significantly different, when measured in Ca²*-free bath. The difference in intracellular Ca²* in a Ca²*-free solution between tumor cells, on the one hand, and non-tumor(normal or precan¬ cerous), on the other hand, was approximately 2:1.

When the intracellular Ca²* measurements were repeated using a 1 mM Ca²* bath, a significant increase in intracellular Ca²* was observed for each group. However, the increases were different in the groups. The tumor-free (but "at risk") cells, taken from colon tissue adjacent to tumor tissue, showed a difference of intracellular Ca²*, relative to the prior measurement, that was approximately twice the difference measurement for the other two groups. The absolute values for intracellular Ca²* in the 1 mM Ca²* bath were also significantly different, displaying relative concentration ratios of approximately 1:2:3 for tumor, control, and "at risk" tissue, respectively.

It was therefore concluded from the mouse data that the absolute values of Ca²* concentration in the 1 mM Ca²*- containing bath may permit this measurement to be used for diagnostic purposes to distinguish normal, tumor, and precancerous cells. Further, it was concluded that the difference between intracellular Ca²* in Ca²*-free and Ca²*- containing baths may be useful for screening pre-cancerous tissue samples to distinguish them from other samples.

In addition, the mouse data is considered useful, in itself, because of the known utility of mouse models for testing anticancer drugs, chemopreventative, genotoxic, or cancer causing agents. Mouse models are widely used to screen prospective anti-cancer drugs or cancer causing agents. Accordingly, an early biomarker for cancer is useful in connection with such mouse tests.

EXAMPLE 2- MOUSE COLON. EARLIER TEST (ISOLATED CELLS)

The procedure of Example 1 was repeated using a course of six weekly injections of DMH and control, instead of 20. At seven weeks, the mice were sacrificed and the same measurements were made.

The control group comprised 10 tissue samples. The intracellular Ca²* concentration observed in the Ca²*-free Ringer Solution bath was 199 nM ±22 nM. The value in the 1 mM Ca²* bath was 363 nM ±35 nM. The difference in the measured values of intracellular Ca²* between Ca*-free and 1 mM Ca²* bath was 164 nM ±28nM.

The normal-appearing, tumor-free ("at risk") tissue group taken from the colonic mucosa of mice exposed to six weekly injections of DMH comprised 10 samples. (Note: There were no tumors observed after only 6 weekly DMH treatments.) The intracellular Ca²* concentration values were 329 nM ±20 nM for Ca²*-free bath and 695 nM ±69 nM for 1 mM Ca²*- containing bath. The difference in the measured values of intracellular Ca²* concentration was 366 nM ±73 nM. Differences between control and "at-risk" cells were significant at p < 0.05, or less, under all conditions using an unpaired Student t-test.

The following conclusions are drawn from this data, which is depicted in FIG: 2. Since DMH is a known carcinogen for mice, as illustrated by the data of Example 1, it is probable that the colon tissue from at least a very substantial part of the DMH-treated mice was precancerous. Hence, an elevation in intracellular Ca²* occurs in pre¬ cancerous colon before tumors develop, and said elevation is therefore a marker of the "at-risk" or premalignant state.

On the basis of the data from Examples 1 and 2, the inventor concluded that intracellular Ca²* measurements showed promise of utility as an early marker of precancerous conditions in human colon tissue samples. The foregoing measurement procedures were therefore used with human tissue samples, as described in Example 3, below. The procedure and data were published, in somewhat more extensive detail, in the paper, Edelstein, Thompson, and Davies, "Altered intracellular calcium regulation in human colorectal cancers and in ^,normal^, adjacent mucosa," Cancer Res. 51:4492-94 (Aug. 15, 1991) . (Among other things, the data for each of the 30 tissue samples was published in the paper, rather than only summary data as provided in Example 3, below.)

EXAMPLE 3 - HUMAN COLON CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* were made in colon cells from human colon tissue as follows: Samples isolated from surgical specimens or biopsies of human colon tissue which were obtained from patients having either benign disease (used as control samples) or distal adenocarcinomas. In the latter group, the specimens were obtained either directly from the tumor site or else from normal-appearing mucosa located approximately 20 cm proximal to the tumor lesion (tumor-free but "at risk"—that is, considered by the inventor to be precancerous tissue because of the known etiology of bowel cancer) .

Mucosa specimens were minced and treated with collagenase, 2 mg/ml, in Ca²*-free saline at 4°C to form a cell suspension. Cells were then loaded with FURA/2 as a probe.. Intracellular Ca²* concentration was measured in Ca²*-free Ringer Solution and then 1 mM Ca^2*-containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer. The measurements of intracellular Ca²* concentration were made, as in Example l, and the following results were observed:

The control group comprised 11 tissue samples. The intracellular Ca²* concentration observed in the Ca²*-free Ringers Solution bath was 151 nM ± lOnM. The intracellular Ca²* concentration observed in the 1 mM Ca²* bath was 256 nM ± 17nM. The difference figure was 104 nM ± 8nM.

The corresponding figures for the tumor-cell group (9 tissue samples) were: Ca²*-free, 130 nM ± llnM; 1 mM Ca²*, 186 nM ± 17 nM; difference, 56 nM ± 7nM.

The corresponding figures for the "at risk" (believed precancerous) group (10 tissue samples) were: Ca*-free, 695 nM ± 178nM: 1 mM Ca²*, 1116 nM ± 321 nM; difference, 421 nM ± 155nM.

As shown in Figure 3, the human test data shows that there is a marked difference between intracellular Ca²* concentration for "at risk" tissue samples, on the one hand, and tumor- or control-tissue samples, on the other hand. The intracellular Ca²* level for "at risk" tissue is approximately 4 to 6 or more times than the value for control (or tumor) cells. The difference in intracellular Ca^2* when measurements were made in Ca²*-free and Ca²*- containing solutions for "at-risk" tissue is approximately 4 to 8 times the difference for control (or tumor) cells. It is therefore concluded that the differences between "at- risk" values for each of these measurements (or parameters) and the corresponding values for control (and tumor) tissues is so great that the test is useful for distinguishing "at risk" (precancerous) tissue samples from normal tissue (or tumor tissue) in screening tests.

The inventor also concluded that the foregoing Ca²* tests should be used, also, for breast and other cancers. Accordingly, he adapted the preceding procedures as described below.

EXAMPLE 4 - HUMAN BREAST CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* were made in epithelial cells from human breast tissue as follows: Samples isolated from surgical biopsies or fine needle aspiration biopsies of human breast were obtained from patients having either benign disease (used as control sa ples) or carcinoma of the breast. In the latter group, the specimens were obtained away from the known tumor site and were considered tumor-free but "at risk"—that is, considered by the inventor to be precancerous tissue because of the known etiology of breast cancer, which similar to bowel cancer, tends to develop in a background of upregulated epithelial proliferation.

Breast specimens from open biopsies were cut into 0.5- 1.0 cm pieces and incubated in hyaluronidase and collagenase dissolved in RPMI 1640 medium (discussed below, see example 17) . Filtration yielded isolated ductal epithelial cells, clumps of epithelial cells, mesenchymal, and fat cells in the filtrate, separate from the intact terminal ductal lobular units on the filter (see example 17) . Other specimens of single ductal cells or clusters of cells were obtained from breast aspirations using a syringe and a 22- gauge hypodermic needle from patients' breasts. Cell aspirates were placed in either a physiological oxygenated buffer or onto a glass slide coated with polylysine, in order to stick the cells onto the slide, and the slide was then placed in the buffer. Cells were then loaded with FURA/2 as a probe. Intracellular Ca²* concentration was measured in Ca*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer. Similar measurements are made using microfluorimetry and digital imaging (see example 19) , and flow cytometry (see example 26) . The measurements of intracellular Ca²* were made either in the isolated cells or clumps of cells. The measurements of intracellular Ca^2* concentration were made, as in Example 1, and the following results were observed:

The control group comprised 5 samples. The intracellular Ca²* concentration observed in the Ca²*-free Ringers Solution bath was 100-200 nM. The intracellular Ca²* concentration observed in the 1 mM Ca^2* bath was 200-300 nM. Similar to the observations in human colonocytes, intracellular Ca²* was 2-3 fold higher in isolated breast ductal epithelial cells, compared with clumps of cells.

The corresponding values for the "at-risk" group (5 samples obtained from breast tissue known to contain breast cancer elsewhere in the breast) were: Ca²*-free, 300-800 nM; 1 mM Ca²*, 500-1400 nM. Intracellular Ca²* was 2-10 fold higher in isolated breast ductal epithelial cells, compared with clumps of cells.

Therefore, the human test data demonstrated that there was a marked difference between intracellular Ca²* concentration for "at risk" tissue samples, on the one hand, and control-tissue samples, on the other hand. The intracellular Ca²* level for "at risk" tissue was approximately 2 to 8 or more times than the value for control cells. It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control tissues is so great that the test is useful for distinguishing "at risk" (precancerous) tissue samples from normal tissue in screening tests.

EXAMPLE 5 - LUNG CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in bronchial epithelial cells from human lung as follows: Samples isolated from surgical biopsies, fine-needle aspiration biopsies of human lung, bronchial washings, bronchial brushings, or sputum samples are obtained from patients being screened or evaluated for lung cancer or risk of lung cancer development.

Surgical or bronchoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings or brushings from bronchoscopy, and sputum samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca^2* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated bronchial epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in bronchial epithelium of the "at-risk" group of patients, who either have lung cancer or are at high risk of developing lung cancer, compared with controls, who are at average or low risk of lung cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control tissues is so great that the test is useful for distinguishing "at risk" (precancerous) tissue samples from normal tissue in screening tests.

EXAMPLE 6 - HUMAN PROSTATE CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in prostate epithelial and stromal cells from human prostate as follows: Samples isolated from surgical biopsies, or fine needle aspiration biopsies of human prostate are obtained from patients being screened or evaluated for prostate cancer or risk of prostate cancer development.

Surgical biopsies are disaggregated as described in examples 1-4. Aspiration samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated prostate cells compared with clumps of cells.

Intracellular Ca²* is elevated in prostate cells of the "at-risk" group of patients, who either have prostate cancer or are at high risk of developing prostate cancer, compared with controls, who are at average or low risk of prostate cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca^2* in "at risk" cells, compared with controls.

EXAMPLE 7 - UPPER AERODIGESTIVE TRACT CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in epithelial cells from human upper aerodigestive tract as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human upper aerodigestive tract, washings, brushings, saliva or mucus samples are obtained from patients being screened or evaluated for cancer of the upper aerodigestive tract or risk of upper aerodigestive tract cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings, brushings saliva or mucus samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cyto eter, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated upper aerodigestive tract epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in the upper aerodigestive epithelium of the "at-risk" group of patients, who either have upper aerodigestive tract cancer or are at high risk of developing it, compared with controls, who are at average or low risk of this type of cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

EXAMPLE 8 - URINARY BLADDER CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in bladder epithelial cells from human bladder as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human bladder, bladder washings, bladder brushings or urine samples are obtained from patients being screened or evaluated for urinary bladder cancer or risk of urinary bladder cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings or brushings from cystoscopy, and urine samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated bladder epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in bladder epithelium of the "at-risk" group of patients, who either have bladder cancer or are at high risk of developing bladder cancer, compared with controls, who are at average or low risk of bladder cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca^2* in "at risk" cells, compared with controls.

EXAMPLE 9 - ENDOMETRIAL CANCER (ISOLATED CELLS)

Measurements of intracellular Ca²* are made in endometrial cells from human uterus as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human endometrium, washings, endometrial brushings or mucus samples are obtained from patients being screened or evaluated for endometrial cancer or risk of endometrial cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings, brushings, and mucus samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated endometrial cells compared with clumps of cells.

Intracellular Ca²* is elevated in endometrial epithelium of the "at-risk" group of patients, who either have endometrial cancer or are at high risk of developing endometrial cancer, compared with controls, who are at average or low risk of endometrial cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca^2* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

EXAMPLE 10 - PANCREATIC CANCER (ISOLATED CELLS)

Measurements of intracellular Ca²* are made in pancreatic epithelial cells from human pancreas as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human pancreas, washings, brushings obtained at endoscopic retrograde cholangio- pancreaticography (ERCP) , or pancreatico-biliary secretions are obtained from patients being screened or evaluated for pancreatic cancer or risk of pancreatic cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings or brushings from ERCP, and^' pancreatico-biliary secretions samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca^2*) . Intracellular Ca²* concentration is measured in Ca*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated pancreatic epithelial cells compared with clumps of cells. Intracellular Ca²* is elevated in pancreatic epithelium of the "at-risk" group of patients, who either have pancreatic cancer or are at high risk of developing pancreatic cancer, compared with controls, who are at average or low risk of pancreatic cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

EXAMPLE 11 - HUMAN GASTRIC CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in gastric epithelial cells from human stomach as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human stomach, gastric washings, gastric brushings or gastric secretion samples are obtained from patients being screened or evaluated for gastric cancer or risk of gastric cancer development.

Surgical or gastroscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings or brushings from gastroscopy, and gastric secretion samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca^2*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated gastric epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in gastric epithelium of the "at-risk" group of patients, who either have gastric cancer or are at high risk of developing gastric cancer, compared with controls, who are at average or low risk of gastric cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

EXAMPLE 12 - LIVER CANCER (ISOLATED CELLS) Measurements of intracellular Ca²* are made in hepatocytes from human liver as follows: Samples isolated from surgical biopsies, fine needle aspiration biopsies of human liver, biliary tree washings, brushings or choledochoscopically obtained samples are obtained from patients being screened or evaluated for primary liver or biliary cancer or risk of liver or biliary cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings, or brushings are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca^2*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca^2* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated hepatocytes or biliary epithelial cells compared with clumps of cells.

Intracellular Ca^2* is elevated in hepatocytes or biliary epithelial cells of the "at-risk" group of patients, who either have liver or biliary cancer respectively, or are at high risk of developing these particular cancers, compared with controls, who are at average or low risk of development of these types of cancer. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger in¬ crease in intracellular Ca²* in "at risk" cells, compared with controls.

EXAMPLE 13 - CANCER OF THE UTERINE CERVIX (ISOLATED CELLS) Measurements of intracellular Ca²* are made in cervical epithelial cells from human uterine cervix as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies of human lung, cervical smears, washings, brushings or mucus samples are obtained from patients being screened or evaluated for cervical cancer or risk of cervical cancer development.

Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, smears, washings, brushings, and mucus samples are handled as de¬ scribed in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca^2* concentration is measured in Ca^2*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca^2* is 2-3 fold higher in isolated cervical epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in cervical epithelium of the "at-risk" group of patients, who either have cervical cancer or are at high risk of developing cervical cancer, compared with controls, who are at average or low risk of cervical cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control tissues is so great that the test is useful for distinguishing "at risk" (precancerous) tissue samples from normal tissue in screening tests. EXAMPLE 14 - THYROID CANCER (ISOLATED CELLS)

Measurements of intracellular Ca²* are made in thyroid epithelial cells from human thyroid as follows: Samples isolated from surgical biopsies, or fine needle aspiration biopsies of human lung are obtained from patients being screened or evaluated for thyroid cancer or risk of thyroid cancer development.

Surgical biopsies are disaggregated as described in examples 1-4. Aspiration samples are handled as described in example 4 for breast aspirates. Cells are loaded with FURA/2 (or other probe for intracellular Ca²*) . Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cytometer, or digital .imaging system. The measurements of intracellular Ca²* are made either in the isolated cells or in clumps of cells. The measurements of intracellular Ca²* concentration are made, as in Example 1.

Similar to the observations in human colonocytes and breast epithelium, intracellular Ca²* is 2-3 fold higher in isolated thyroid epithelial cells compared with clumps of cells.

Intracellular Ca²* is elevated in thyroid epithelium of the "at-risk" group of patients, who either have thyroid cancer or are at high risk of developing thyroid cancer, compared with controls, who are at average or low risk of thyroid cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at risk" cells, compared with controls.

It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control tissues is so great that the test is useful for distinguishing "at risk" (precancerous) tissue samples from normal tissue

EXAMPLE 15 - OTHER TUMORS (ISOLATED CELLS) Similar approaches may be used in diagnosis or screening for cancer or identifying patients at high risk for other rarer tumors, providing tissue is accessible by surgical or endoscopic biopsy or sampling. This would include tumors of ovarian, small bowel, muscle or neurological origin.

The foregoing data relate to data based on cells, or clumps of cells obtained from minced tissue samples or aspiration biopsies, available from persons known to have cancer, or believed not the have cancer (or a precancerous condition). However, the procedures of Examples 3 and 4, for instance, use measurements of intracellular Ca²* in heterogeneous cell suspensions in which epithelial cells in different states of proliferation or differentiation are mixed together. The inventor believes that measurements of intracellular Ca^2* at different levels within intact organoids is more sensitive and specific in identifying patients who are "at-risk" for developing cancer in the specific organ of question. Therefore, the inventor has devised procedures specifically directed to diagnosis (screening) of tissue samples from patients whose condition is unknown.

The inventor believes that use of isolated organoids, such as crypts or terminal ductal lobular units, better preserves aberrant proliferative characteristics of precancerous cells than does use of isolated cells, clumps of a few cells, or cultured cells derived from biopsies. Thus, in the case of bowel tissue, the inventor considers it preferable to isolate intact crypts from segments of colorectal mucosa. This was done for thousands of intact crypts from 1 cm bowel segments using a modified version of the technique described by Bjerknes and Chang in "Methods for the isolation of intact epithelium from the mouse intestine," Anat. Rec. 199:565 (1981). Also, the inventor has been able to obtain several hundred intact crypts from single pinch biopsies, by the procedure described below.

EXAMPLE 16 - ISOLATION OF CRYPTS FOR SCREENING Mucosa or biopsy specimens were placed in Ca²*-free Ringers Solution with 25 mM EDTA and 0.15 bovine serum albumin. Then, they were incubated for 40-80 minutes at 4°C. Then, they were vibrated vigorously for 20-30 seconds using a vortex mixer, to release the crypts from the underlying submucosa and muscle layers. This resulted in release of isolated crypts, which were then subjected to the procedures described below.

Terminal ductal lobular units were obtained from breast tissue by two methods. One was a collagenase method described in the following example.

EXAMPLE 17 - ISOLATION OF TERMINAL DUCTAL LOBULAR UNITS Biopsied breast tissue was divided into 0.5 cm³ pieces and digested for 48 hours in Type 1 collagenase (250 U/ml) plus hyaluronidase (100 U/ml) dissolved in RPMI 1640 medium containing 10% fetal calf serum, thereby yielding a suspension of intact terminal ductal lobular units and mesenchymal cells. The mesenchymal cells and isolated ductal epithelial cells were then filtered away using 105 micron controlled pore size polyester cloth.

A second method used was to stain the terminal ductal lobular units in a surgical biopsy with 0.1% methylene blue. A dissecting microscope was then used to excise the terminal ductal lobular units for testing. Both methods appeared to be satisfactory, but the first is considered preferable to avoid use of skilled labor.

It is believed that, when FURA/2-AM crosses a cell membrane and enters a cell, the acetoxymethylester bonds of the dye are cleaved by intracellular esterases to generate an impermeable, fluorescent form of the dye. The result of this is to "load" the cells so that, when irradiated with light of an appropriate wavelength, they re-emit light and provide a characteristic scan pattern which can be observed and recorded by the procedures described herein. A loading procedure is now described.

EXAMPLE 18 - PROBE LOADING

Crypts or terminal ductal lobular units, isolated by the procedures of Examples 16 or 17, were incubated for 30 minutes at 4°C and pH 7.4 in oxygenated Ringers Solution with fluorescent dye (8-10 μM FURA/2-AM) . Although it was found that crypts and terminal ductal lobular units can be loaded at temperatures from 4°C to 37°C, it is believed that they are viable longer after loading at 4°C.

Similar fluoroscopy techniques were used for analysis of isolated crypts and terminal ductal lobular units. A laboratory technique was used to provide the data presented herein, and it is described in the following example. A procedure for a stand alone screening system is subsequently described.

EXAMPLE 19 - LAB FLUOROSCOPY TECHNIQUE The crypts or terminal ductal lobular units were attached to a glass coverslip using polylysine or CELLTAK to prevent their movement. The coverslip was placed in a flow-through chamber on the stage of a Nikon inverted fluorescence microscope. The solutions were heated to 37°C and oxygenated.

UV light was filtered from a Xenon source to permit passage of narrow bands about 340 nm and 380 nm. The UV light was passed through the Nikon UV objectives to the tissue. The emitted fluorescence image was filtered at 510 nM and then passed to the input of a Dage Genesis II image intensifier tube and then to a CCD camera. The video image from the camera was digitized, using a frame grabber and commercial software ("UIC", Universal Imaging) , and stored on an optical disk for further use. The image information was then corrected for autofluorescence and background. Then the intracellular Ca²* was calculated, using the same software package, which is marketed specifically for performing quantitative fluorescence ratio imaging.

Calibration of the signal was accomplished on the same cell preparation by adding digitonin (Cal Biochem, San Diego, Ca.), 60 μM for 100 sec to maximize binding of Ca²* to FURA. Then 60 mM ethyleneglycol-bis (β-aminoethyl ether)-N,N,N',N^,-tetracetic acid was added to remove all Ca²* and to determine emission for unbound FURA. Correction for cell autofluorescence was made by scanning the same cells prior to FURA loading. Intracellular Ca^2* concentration was determined by using the Grynkiewicz equation and the ratio of bound 340 nM excitation) emission intensity to unbound (380 nM excitation) emission intensity, and a K_d for FURA/2 of 224 nM.

The foregoing procedures were used to determine intracellular Ca²* concentration in isolated organoid specimens, as described in the following examples.

EXAMPLE 20 - HUMAN COLON CANCER (ISOLATED CRYPTS) Human colorectal crypts were isolated from tissue samples by the procedure of Example 16. The crypts were then loaded with FURA/2 by the procedure of Example 18. The crypts were then subjected to the lab fluoroscopic procedure of example 19. The following data was obtained:

All samples were from distal colonic or rectal biopsies obtained at colonoscopy, proctoscopy or from surgical specimens. The "at-risk" specimens were obtained at least 10 to 20 cm from the polyp or cancer (when present) or from patients with known risk factors for colorectal cancer. The control group comprised 14 samples of isolated human colonic crypts in which intracellular Ca²* was measured in cells at the base of the crypts, and 14 samples of isolated human crypts in which intracellular Ca²* was measured at the mouth of the crypt. The "at-risk" group comprised 23 samples of isolated human colonic crypts in which intracellular Ca²* was measured in cells at the base of the crypts, and 9 samples of isolated human crypts in which intracellular Ca²* was measured at the mouth of the crypt. The results of these studies are illustrated in figure 4. The intracellular Ca^2* concentration in control basal crypt cells observed in the Ca²*-free Ringers Solution bath (0 CA) was 27 nM ± 4nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca²* was 98 nM ± 20nM. The final intracel¬ lular Ca²* concentration (FINAL) was 67 nM ± 16nM after a period of equilibration.

The intracellular Ca²* concentration in control mouth crypt cells observed in the Ca²*-free Ringers Solution bath (0 CA) was 44 nM ± 7nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca²* was 131 nM ± 30nM. The final intracellular Ca^2* concentration (FINAL) was 81 nM ± 14nM after a period of equilibration.

The intracellular Ca²* concentration in "at-risk" basal crypt cells observed in the Ca²*-free Ringers Solution bath (0 CA) was 41 nM ± 5nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca²* was 215 nM ± 22nM. The final intracellular Ca²* concentration (FINAL) was 179 nM ± 22nM after a period of equilibration.

The intracellular Ca²* concentration in "at-risk" mouth crypt cells observed in the Ca²*-free Ringers Solution bath (0 CA) was 91 nM ± 20nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca^2* was 211 nM ± 44nM. The final intracellular Ca^2* concentration (FINAL) was 142 nM ± 16nM after a period of equilibration.

As shown in Figure 4, the human test data show that there is a marked difference between intracellular Ca²* concentration for "at-risk" crypt samples, on the one hand, and control-crypt samples, on the other hand. The intracellular Ca²* level for "at-risk" crypt basal cells is approximately 2 to 3 times more than the value for control basal cells in Ca²*-free or Ca²*-containing Ringers Solution. Furthermore, the human test data show that there is a marked difference between intracellular Ca²* concentration for "at- risk" crypt mouth cells, on the one hand, and control-crypt mouth cells, on the other hand. The intracellular Ca²* level for "at-risk" crypt mouth cells is approximately 2 times more than the value for control mouth cells in Ca²*- free or Ca²*-containing Ringers Solution. In addition the intracellular Ca²* gradient which in control crypts is low at the base and high at the crypt-mouth is lost or reversed in the "at-risk" crypt when bathed in Ca²*-containing Ringers Solution (i.e. the peak intracellular Ca²* is similar in the base and the mouth, and the final intracellu¬ lar Ca²* gradient is reversed between base and mouth crypt cells when the bathing solution is changed to Ca²*- containing Ringers Solution in figure 4) . It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control isolated crypts is so great that the test is useful for distinguishing "at-risk" (precancerous) crypt samples from normal tissue in screening tests.

EXAMPLE 21 - HUMAN BREAST CANCER (ISOLATED TERMINAL DUCTAL LOBULAR UNITS)

Human terminal ductal lobular units were isolated from tissue samples by the procedure of Example 17, The terminal ductal lobular units were then loaded with FURA by the procedure of Example 18. The terminal ductal lobular units were then subjected to the lab fluoroscopy procedure of Example 19. The following data was obtained:

The control group comprised 3 samples. Measurements were made in the terminal ductal epithelial cells of the terminal ductal lobular unit. All samples were from breast tissue away from known cancers in the "at-risk" group or from patients undergoing biopsy for benign disease and without known risk factors for breast cancer; the control group. The intracellular Ca²* concentration in control terminal ductal epithelial cells in the Ca*-free Ringers Solution bath (0 CA) was 5-10 nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca²* was 200-350 nM. The final intracellular Ca²* concentration (FINAL) was 40-80 nM after a period of equilibration.

The "at-risk" group^" comprised 5 samples. The intracellular Ca²* concentration in "at-risk" terminal ductal epithelial cells observed in the Ca²*-free Ringers Solution bath (0 CA) was 5-20 nM. The peak intracellular Ca²* concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca²* was 300-600nM. The final intracellular Ca²* concentration (FINAL) was 70-200 nM after a period of equilibration.

The human test data show that there is a marked difference between intracellular Ca²* concentration for "at- risk" breast terminal ductal lobular units, on the one hand, and control-terminal ductal lobular units, on the other hand. The intracellular Ca²* level for "at-risk" terminal ductal epithelial cells is approximately 2 to 3 times more than the value for control cells in Ca²*-free or Ca²*- containing Ringers Solution. It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control isolated crypts is so great that the test is useful for distinguishing "at-risk" (precancerous) breast terminal ductal lobular units from normal tissue in screening tests.

EXAMPLE 22 - HUMAN PROSTATE CANCER (ISOLATED PROSTATE DUCTAL ACINAR UNITS)

Measurements of intracellular Ca²* are made in prostate ductal, and acinar epithelial and stromal cells from human prostate as follows: Samples isolated from surgical biopsies, or needle biopsies of human prostate are obtained from patients being screened or evaluated for prostate cancer or risk of prostate cancer development.

Human ductal acinar units are isolated from prostate tissue samples, obtained from surgical or needle biopsies, by the procedure of Example 17. The organoids are loaded with FURA/2 (or other probe for intracellular Ca²*) by the procedure of Example 18. The ductal acinar units are then subjected to the lab fluoroscopy procedure of Example 19. Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a digital imaging system and described in Example 19. The measurements of intracellular Ca²* are made in the isolated organoid.

Similar to the observations in human colonic crypts and breast terminal ductal lobular units, intracellular Ca²* is 2-4 fold higher in prostatic organoids and the pattern and distribution of the calcium gradient in these organoids is altered in "at-risk" patients.

Intracellular Ca²* is elevated in prostatic organoids of the "at-risk" group of patients, who either have prostate cancer or are at high risk of developing prostate cancer, compared with controls, who are at average or low risk of prostate cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at-risk" cells, compared with controls.

It is therefore concluded that the differences between "at-risk" values for each of these measurements (or parameters) and the corresponding values for control tissues is so great that the test is useful for distinguishing "at- risk" (precancerous) tissue samples from normal tissue in screening tests.

EXAMPLE 23 - HUMAN PANCREATIC CANCER (ISOLATED PANCREATIC DUCTAL ACINAR UNITS)

Measurements of intracellular Ca²* are made in pancreatic ductal, acinar units from human pancreas as follows: Samples isolated from surgical biopsies, or needle biopsies of human pancreas are obtained from patients being screened or evaluated for pancreatic cancer or risk of pancreatic cancer development.

Human ductal acinar units are isolated from pancreatic tissue samples, obtained from surgical or needle biopsies, by the procedure of Example 17. The organoids are loaded with FURA/2 (or other probe for intracellular Ca²*) by the procedure of Example 18. The ductal acinar units are then subjected to the lab fluoroscopy procedure of Example 19. Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a digital imaging system and described in Example 19. The measurements of intracellular Ca²* are made in the isolated organoid.

Similar to the observations in human colonic crypts and breast terminal ductal lobular units, intracellular Ca²* is 2-4 fold higher in pancreatic organoids and the pattern and distribution of the calcium gradient in these organoids is altered in "at-risk" patients. Intracellular Ca²* is elevated in pancreatic organoids of the "at-risk" group of patients, who either have pancreas cancer or are at high risk of developing pancreatic cancer, compared with controls, who are at average or low risk of pancreatic cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at-risk" cells, compared with controls.

EXAMPLE 24 - HUMAN GASTRIC CANCER (ISOLATED GASTRIC PITS)

Measurements of intracellular Ca²* are made in isolated gastric glands or pits from human stomach as follows: Samples isolated from surgical, endoscopic, or needle biopsies of human stomach are obtained from patients being screened or evaluated for gastric cancer or risk of gastric cancer development.

Human gastric glands are isolated from gastric tissue samples, obtained from surgical, endoscopic or needle biopsies, by the procedure of Example 17. The organoids are loaded with FURA/2 (or other probe for intracellular Ca²*) by the procedure of Example 18. The gastric glands are then subjected to the lab fluoroscopy procedure of Example 19. Intracellular Ca²* concentration is measured in Ca^z*-free Ringer Solution and then 1 mM Ca²*- containing Ringer Solution, as before, using a digital imaging system and described in Example 19. The measurements of intracellular Ca²* are made in the isolated organoid. Similar to the observations in human colonic crypts and breast terminal ductal lobular units, intracellular Ca²* is 2-4 fold higher in gastric organoids and the pattern and distribution of the calcium gradient in these organoids is altered in "at-risk" patients.

Intracellular Ca²* is elevated in gastric organoids of the "at-risk" group of patients, who either have gastric cancer or are at high risk of developing gastric cancer, compared with controls, who are at average or low risk of gastric cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at-risk" cells, compared with controls.

EXAMPLE 25 - HUMAN LIVER CANCER (ISOLATED HEPATIC LOBULES)

Measurements of intracellular Ca^2* are made in isolated hepatic lobules from human liver as follows: Samples isolated from surgical, or needle biopsies of human liver are obtained from patients being screened or evaluated for liver cancer or risk of liver cancer development.

Isolated human hepatic lobules are isolated from liver tissue samples, obtained from surgical, or needle biopsies, by the procedure of Example 17. The organoids are loaded with FURA/2 (or other probe for intracellular Ca^2*) by the procedure of Example 18. The hepatic lobules are then subjected to the lab fluoroscopy procedure of Example 19. Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then 1 mM Ca^2*- containing Ringer Solution, as before, using a digital imaging system and described in Example 19. The measurements of intracellular Ca²* are made in the isolated organoid.

Similar to the observations in human colonic crypts and breast terminal ductal lobular units, intracellular Ca^2* is 2-4 fold higher in hepatic lobules and the pattern and distribution of the calcium gradient in these organoids is altered in "at-risk" patients. Intracellular Ca²* is elevated in hepatic lobules of the "at-risk" group of pa¬ tients, who either have primary liver cancer or are at high risk of developing liver cancer, compared with controls, who are at average or low risk of liver cancer development. Furthermore, similar to large bowel and breast epithelium, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at- risk" cells, compared with controls.

EXAMPLE 26 - MEASUREMENT OF INTRACELLULAR Ca²* USING FLOW CYTOMETRY

Because of the need to automate measurements of intracellular Ca²* in isolated cells, flow cytometry is employed to measure intracellular calcium in isolated cells prepared as described in examples 1-15.

Automated measurements of intracellular Ca²* are made in isolated cells as follows: Samples isolated from surgical or endoscopic biopsies, fine needle aspiration biopsies, endoscopic washings, brushings or body fluid samples are obtained from patients being screened or evaluated for a particular cancer or risk of cancer develop¬ ment as described in examples 1-15. Surgical or endoscopic biopsies are disaggregated as described in examples 1-4. Aspiration samples, washings or brushings from endoscopy, and body fluid samples are handled as described in example 4 for breast aspirates. Cells are loaded with the acetoxy-methyl ester of Indo-1 (Indo-1/AM; Molecular Probes, OR.), or other intracellular Ca²*-probe, as has been previously described (Rabinovitch et.al., "Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3: Simultaneous use of Indo-1 and immunofluorescence with flow cytometry," J. Immunol., 137:952-961 (1986)). 5 x 10⁶ cell are incubated with 5 μM Indo-1 for 1 hour at 37°C in an oxygenated physiological buffer. Intracellular Ca²* concentration is measured in Ca²*-free Ringer Solution and then l mM Ca²*-containing Ringer Solution, using an EPICS 753 flow cytometer (Coulter Corporation, Hialeah, FL, USA) interfaced with a MDADS II computer for subsequent flow cytometric analysis. The cell suspension is placed in a pressurized chamber in the flow cytometer. The cells exit in single file through a small aperture into the flow chamber. The differential pressure between the pressurized chamber and the flow chamber is used to regulate the cell flow rate. Fluorescence is measured using single excitation wavelength (355 nm) to excite the stream of flowing cells, and dual emission (405 nm/485 nm, violet/blue) as previously described (Grynkiewicz et. al., "A new generation of Ca^2* indicators with greatly improved fluorescent properties," J. Biol. Chem., 260:3440-3450 (1985)) to measure the intracellular Ca²*. The ratio of violet/blue fluorescence is measured in intact epithelial cells in Ca²*-free for 60 seconds, and ImM Ca*-containing Ringers Solution for a further 60 seconds. Then 5 μM Ionomycin (Calbiochem, La Jolla, CA) is added to obtain the ratio, R^, at the saturated level of intracellular Ca²*. The cells are then lysed with 0.05% Triton X-100 in the presence of 60 mM ethyleneglycol-bis (β-aminoethyl ether)-N,N,N',N'-tetracetic acid to remove all Ca^2* and to determine the fluorescent ratio, R^, when unbound intracellular Ca²* is zero. The intracellular Ca^2* (Ca²^) is then calculated from the for¬ mula:

Ca 2_{* =} ^'^ ⁾^!

where R is the violet/blue fluorescence ratio of the cells, under equilibrium conditions in Ca²*-free, and then Ca²*- containing Ringers Solution; and S^/S^ is the ratio of the blue fluorescence intensity of Ca*-free to Ca²*-bound dye.

The EPICS 753 flow cytometer uses a 5 W argon laser operating in the UV region (355-361nm) . Emitted forward light scatter, side scatter, linear violet fluorescence, linear blue fluorescence, the violet/blue ratio using the MDADS II function card, and time are collected and analyzed using a commercial analytical package, "ReproMan." Forward scatter is filtered with a 365DF10 band pass filter, and side scatter is collected after refection by a float glass beam splitter. Violet Indo-1 emission is filtered through a 395DF25 band pass after reflection from a 430LP dichroic beam splitter, and blue Indo-1 emission is collected after the 430LP dichroic. Data for each cell suspension is collected at 30,000 events/minute for up to 12 minutes before analysis using ReproMan.

Intracellular Ca²* is elevated in isolated cells of the "at-risk" group of patients, who either have cancer or are at high risk of developing cancer, compared with controls, who are at average or low risk of gastric cancer development. Furthermore, an increase in the bathing solution Ca²* from 0 to 1 mM results in a larger increase in intracellular Ca²* in "at-risk" cells, compared with controls.

Examples 3-15 involve measurements made in human tissue samples to diagnose existing cancer, or to determine if a patient is at-risk for cancer development. Animal models of human cancer are used in research and by the pharmaceutical industry to test anticancer drugs, chemopreventative, genotoxic, or cancer causing agents. Rodent or larger animal models are widely used to screen prospective anti-cancer drugs or cancer causing agents. Accordingly, an early biomarker for cancer is useful in connection with such animal tests.

EXAMPLE 27 - MEASUREMENTS OF INTRACELLULAR Ca²* IN ANIMAL MODELS OF CANCER

Cancer is induced in animal models of human cancer including colon cancer in rodents with 1,2 dimethylhydrazine (DMH), breast cancer in rodents with 7,12 dimethylben- zanthracene (DMBA) , bladder cancer in rodents with N-methyl- N-nitrosourea.

Isolated cells suspensions are prepared using the methodology outlined in Examples 1-4, organoids are prepared for colon as in Example 16, or organoids for breast are prepared as in Example 17. Isolated cells are loaded with the Ca²*-probe FURA/2, (or other Ca²*-probe) , as described in Examples 1-4, or Indo-1, (or other Ca²*-probe) , as described in Example 26. Organoids are loaded with FURA/2, (or other Ca^2*-probe) , as described in Example 18. Intracellular Ca²* is then measured in isolated cells as in Examples 1-4 using an ARCM DM3000 spectrofluorometer, or digital imaging microfluorimetry as in Example 19, or flow cytometry as in Example 26.

Intracellular Ca²* is measured in Ca²*-free and then Ca²*-containing Ringers Solutions. The efficacy of cancer causing, chemopreventative agents and anticancer drugs is determined by evaluating the abrogation or stimulation of elevated intracellular Ca^2* in these cells by previous treatment with a cancer causing, or an anticancer drug or agent. Similarly the in vitro effect of anticancer agents on intracellular Ca²* is determined to assess the efficacy of these drugs or agents in promoting or preventing cancer.

Intracellular Ca²* is measured in organoids as described in Example 19. Intracellular Ca^2* is measured in Ca²*-free and then Ca²*-containing Ringers Solutions. The efficacy of cancer causing, chemopreventative agents and anticancer drugs is determined by evaluating the abrogation or stimulation of elevated intracellular Ca²* in these organoids, and the altered distribution and gradient of intracellular Ca²*, by previous treatment with a . cancer causing, or an anticancer drug or agent. Similarly the in vitro effect of cancer causing or anticancer agents on intracellular Ca²*, and its altered distribution and gradient in these organoids is determined to assess the efficacy of these drugs or agents in promoting or preventing cancer.

The foregoing procedures provide an entirely new diagnostic method, hitherto unknown to medical science. These tests employ measurements of intracellular Ca²* as a biomarker of tissue that is "at risk" for cancer (precancerous) . These measurements consist of altered levels of intracellular Ca²* in isolated cells or altered distributions of intracellular Ca²* in intact organoids derived from biopsies. The inventor believes that this bio¬ marker is of considerable utility in diagnosing patients at risk for different types of cancer, and for assessing the effects of various drugs or agents in preventing or causing cancer in various animal models of cancer.

While the invention has been described in connection with specific and preferred embodiments thereof, it is capable of further modifications without departure from the spirit and scope of the invention. This application is intended to cover all variations, used, or adaptions of the invention, following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains, or as are obvious to persons skilled in the art, at the time the departure is made. It should be appreciated that the scope of this invention is not limited to the detailed description of the invention hereinabove, but rather comprehends the subject matter defined by the following claims.

Claims

Clai s

1. An in vitro method of screening a tissue sample for precancerous, or early cancerous, condition, said method comprising:

(1) making a cell preparation from the tissue sample, wherein said tissue sample essentially consists of isolated organoids;

(2) adding a calcium probe to the cell preparation, providing a probed sample;

(3) providing a electronic test signal representative of intracellular Ca²* concentration in the probed sample; and

(4) comparing the test signal with a reference signal representative of intracellular Ca²* concentration in normal tissue.

2. A method according to claim 1, wherein a first electronic test signal is provided after placing said probed sample in a Ca²*-free bath; a second electronic test signal is provided after placing said probed sample in a Ca²*- containing bath; and said signals are processed to provide a difference test signal which is representative of a difference between intracellular Ca²* concentrations of which said first and second electronic test signals are representative.

3. A method according to claim 1, wherein said calcium probe is a fluorescent dye, and said test signal is provided by exciting said probed sample with light at a first wave¬ length and then filtering and electronically registering light re-emitted from said probed sample at a second wavelength.

4. A method according to claim 1, directed to screening for: colorectal cancer, wherein said isolated organoids are colorectal crypts; breast cancer, wherein said isolated organoids are terminal ductal lobular units; prostate cancer, wherein said isolated organoids are prostate ductal acinar units; pancreatic cancer, wherein said isolated organoids are pancreatic ductal acinar units; gastric cancer, wherein said isolated organoids are gastric pits; or liver cancer, wherein said isolated organoids are hepatic lobules.

5. A method according to claim 1, for screening human colon tissue or human breast tissue for precancerous, or early cancerous, condition, wherein:

(a) said step (1) comprises isolating colorectal crypts from colon tissue samples or terminal ductal lobular units from breast tissue samples;

(b) said step (2) comprises loading said crypts or units with a fluorescent dye, providing a probed sample;

(c) said step (3) comprises passing ultraviolet light through said probed sample, and collecting and measuring re-emitted light from said probed sample, providing a signal representative of intracellular Ca²* concentration in said crypts or units; and

(d) said step (4) comprises:

(A) passing said re-emited light to a CCD camera and means for light amplifica¬ tion, providing a video image;

(B) digitizing said video image, thereby providing a digitized video image; and

(C) electronically processing said digitized image, providing a signal repre¬ sentative of intracellular Ca²* concentration in said colorectal crypts or said terminal ductal lobular units.

6. A method for screening human tissue for precancerous, or early cancerous, condition, said method comprising:

(1) preparing a cell suspension from a tissue sample; (2) loading said suspension with a fluorescent dye, providing a probed cell suspension;

(3) placing said probed cell suspension into a pressurized chamber of a flow cytometer, said chamber exiting via a narrow aperture to a flow chamber, whereby a narrow stream of flowing cells is provided that flows from said pressurized chamber to said flow chamber;

(4) exciting said stream of cells with UV light;

(5) filtering and measuring re-emitted light from said stream of cells, providing a test signal;

(6) electronically processing said signal, providing a first signal representative of intracellular Ca²* concentration; and

(7) comparing said first signal with a. second signal representative of intracellular Ca²* concentration in normal cells of the type being tested. ^~~

7. An apparatus for screening tissue for precancerous condition, said apparatus comprising: a container permitting passage of UV light and containing a tissue cell preparation treated with a fluorescent- dye calcium probe; a source of light for illuminating said container and exciting said fluorescent-dye calcium probe; means for receiving and filtering light re-emitted from said container, and for providing a signal representative of intensity of said light; a spectrofluorometer or CCD camera for registering said signal; and signal-processing means for deriving from said a signal a further representative of intracellular Ca²*-concen- tration in said tissue cell preparation.

8. An apparatus for automatically testing tissue samples for cancer, comprising: a source of narrow-band UV light; a pressurizable first chamber for receiving a cell suspension, said chamber having a narrow exit conduit slightly more than one cell diameter in width, said conduit exiting to a second chamber; means for illuminating said cell suspension with said UV light as said cell suspension passes from said first chamber to said second chamber via said conduit; means for collecting re-emitted light from said cell suspension; means for filtering and measuring said re-emitted light, providing a test signal; and signal-processing means for processing said test signal, providing a further signal representative of intracellular Ca²* concentration in the cells of said tissue.

9. An apparatus according to claim 8, further comprising means for comparing said further signal with a reference signal representative of intracellular Ca²* concentration in normal cells of said tissue.

10. An apparatus according to claim 8, wherein said means for collecting re-emitted light from said cell suspension comprises a CCD camera that provides a signal to a light amplifier.