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WO1994003470A1 - Procede et appareil de depistage et de diagnostic de cancers - Google Patents

Procede et appareil de depistage et de diagnostic de cancers Download PDF

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WO1994003470A1
WO1994003470A1 PCT/US1993/006831 US9306831W WO9403470A1 WO 1994003470 A1 WO1994003470 A1 WO 1994003470A1 US 9306831 W US9306831 W US 9306831W WO 9403470 A1 WO9403470 A1 WO 9403470A1
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intracellular
cancer
cells
tissue
risk
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Richard J. Davies
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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 Ca 2+ . Intracellular Ca 2 * regulation is thought to be important in growth regulation and cell division.
  • 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.
  • 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 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.
  • endometrial cancer 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.
  • pancreatic cancer 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.
  • 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.
  • liver cancer 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 cervix consists of squamous epithelium and dysplasia and hyperplasia are well recognized antecedent conditions associated with the development of cervical cancer.
  • Thyroid consists of glandular epithelium and stroma. Thyroid cancer is associated with increased hyperplasia of the surrounding thyroid gland.
  • 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.
  • 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 Ca 2+ 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 Ca 2 * concentration by deriving a signal from the probe-loaded tissue sample. Procedures are described for determining intracellular Ca 2 * concentration for tissues placed in a Ca 2 *-free bath and Ca 2 *-containing bath.
  • FIGURE 1 is a chart comparing intracellular Ca 2 * (Ca 2 *,-) 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) .
  • 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 Ca 2 *,- 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) .
  • 0 CA refers to measure ⁇ ments made in Ca 2 *-free Ringers Solution at 37°C
  • PEAK refers to the highest intracellular Ca 2 * measurement when the bathing solution was changed from a Ca 2 *-free to a Ca 2 *- containing Ringers Solution at 37°C
  • FINAL refers to the plateau intracellular Ca 2 * when the bathing solution was changed from Ca 2 *-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.
  • the inventor conducted a series of tests to provide data showing the difference, if any, in intracellular Ca 2 * for normal, precancerous ("at risk”), and cancerous tissue. The tests are described below.
  • 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.
  • saline as a control
  • DMH 1,2- dimethylhydrazine
  • 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 Ca 2 *-free saline at 24°C to form a cell suspension.
  • Cells were loaded with 5 ⁇ M 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 Ca 2 * 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 Ca 2 *-free Ringer Solution. Then, CaCl 2 , solution was added to the bath of Ringer Solution, until ImM Ca 2 * concentration was reached. The fluorescence measurement was then repeated.
  • ARCM DM3000 spectrofluorometer SPEX Industries, Edison, NJ.
  • 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 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.
  • the control group comprised 22 tissue samples.
  • the intracellular Ca 2 * concentration observed in the Ca 2 *-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 Ca 2 * bath was 421 nM ⁇ 46nM.
  • the difference in measured values of intracellular Ca 2 * between Ca*-free and 1 mM Ca 2+ bath was 178 nM ⁇ 21 nM.
  • the tumor-cell group comprised 10 tissue samples.
  • the intracellular Ca 2 * were 101 nM ⁇ 8 nM for Ca 2 *-free bath, and 233 nM ⁇ 12 nM for 1 mM Ca 2 * bath.
  • the difference in measured values of intracellular Ca 2 * 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 Ca 2 * values were 289 nM ⁇ 21 nM for the Ca 2 *-free bath and 613 nM ⁇ 12 nM for Ca 2 *-containing bath.
  • the difference in measured values of intracellular Ca 2 * was 324 nM ⁇ 27 nM.
  • the intracellular Ca 2 * for mouse colon tissue under Ca 2+ -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 Ca 2 * in control and tumor- free (“at-risk”) cells were not significantly different, when measured in Ca 2 *-free bath.
  • intracellular Ca 2 * measurements were repeated using a 1 mM Ca 2 * bath, a significant increase in intracellular Ca 2 * 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 Ca 2 *, relative to the prior measurement, that was approximately twice the difference measurement for the other two groups.
  • the absolute values for intracellular Ca 2 * in the 1 mM Ca 2 * bath were also significantly different, displaying relative concentration ratios of approximately 1:2:3 for tumor, control, and "at risk" tissue, respectively.
  • 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 1 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 Ca 2 * concentration observed in the Ca 2 *-free Ringer Solution bath was 199 nM ⁇ 22 nM.
  • the value in the 1 mM Ca 2 * bath was 363 nM ⁇ 35 nM.
  • the difference in the measured values of intracellular Ca 2 * between Ca*-free and 1 mM Ca 2 * 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 Ca 2 * concentration values were 329 nM ⁇ 20 nM for Ca 2 *-free bath and 695 nM ⁇ 69 nM for 1 mM Ca 2 *- containing bath.
  • the difference in the measured values of intracellular Ca 2 * 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.
  • EXAMPLE 3 - HUMAN COLON CANCER Measurements of intracellular Ca 2 * 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) .
  • the control group comprised 11 tissue samples.
  • the intracellular Ca 2 * concentration observed in the Ca 2 *-free Ringers Solution bath was 151 nM ⁇ lOnM.
  • the intracellular Ca 2 * concentration observed in the 1 mM Ca 2 * bath was 256 nM ⁇ 17nM.
  • the difference figure was 104 nM ⁇ 8nM.
  • the corresponding figures for the tumor-cell group (9 tissue samples) were: Ca 2 *-free, 130 nM ⁇ llnM; 1 mM Ca 2 *, 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 Ca 2 *, 1116 nM ⁇ 321 nM; difference, 421 nM ⁇ 155nM.
  • the human test data shows that there is a marked difference between intracellular Ca 2 * concentration for "at risk” tissue samples, on the one hand, and tumor- or control-tissue samples, on the other hand.
  • the intracellular Ca 2 * 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 Ca 2 *-free and Ca 2 *- containing solutions for "at-risk" tissue is approximately 4 to 8 times the difference for control (or tumor) cells.
  • the inventor also concluded that the foregoing Ca 2 * 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 Ca 2 * 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.
  • Intracellular Ca 2 * concentration was measured in Ca*-free Ringer Solution and then 1 mM Ca 2 *- 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 Ca 2 * 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 Ca 2 * concentration observed in the Ca 2 *-free Ringers Solution bath was 100-200 nM.
  • the intracellular Ca 2 * concentration observed in the 1 mM Ca 2* bath was 200-300 nM. Similar to the observations in human colonocytes, intracellular Ca 2 * 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: Ca 2 *-free, 300-800 nM; 1 mM Ca 2 *, 500-1400 nM. Intracellular Ca 2 * was 2-10 fold higher in isolated breast ductal epithelial cells, compared with clumps of cells.
  • the human test data demonstrated that there was a marked difference between intracellular Ca 2 * concentration for "at risk” tissue samples, on the one hand, and control-tissue samples, on the other hand.
  • the intracellular Ca 2 * 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 Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2* concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated bronchial epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk” cells, compared with controls.
  • EXAMPLE 6 - HUMAN PROSTATE CANCER (ISOLATED CELLS) Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated prostate cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * 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 Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca 2 *-free Ringer Solution and then 1 mM Ca 2 *- containing Ringer Solution, as before, using a SPEX ARCM DM3000 spectrofluorometer, flow cyto eter, or digital imaging system. The measurements of intracellular Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated upper aerodigestive tract epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk" cells, compared with controls.
  • EXAMPLE 8 - URINARY BLADDER CANCER (ISOLATED CELLS) Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated bladder epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2* in "at risk” cells, compared with controls.
  • Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca*-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated endometrial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * in "at risk" cells, compared with controls.
  • pancreatic epithelial cells Measurements of intracellular Ca 2 * 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.
  • ERCP endoscopic retrograde cholangio- pancreaticography
  • 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 Ca 2 * concentration is measured in Ca*-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated pancreatic epithelial cells compared with clumps of cells. Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk" cells, compared with controls.
  • EXAMPLE 11 - HUMAN GASTRIC CANCER (ISOLATED CELLS) Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated gastric epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk” cells, compared with controls.
  • EXAMPLE 12 - LIVER CANCER (ISOLATED CELLS) Measurements of intracellular Ca 2 * 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 Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger in ⁇ crease in intracellular Ca 2 * in "at risk” cells, compared with controls.
  • EXAMPLE 13 - CANCER OF THE UTERINE CERVIX Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2* concentration is measured in Ca 2* -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 Ca 2 * 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.
  • intracellular Ca 2* is 2-3 fold higher in isolated cervical epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk” cells, compared with controls.
  • Measurements of intracellular Ca 2 * 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 Ca 2 *) . Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * 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.
  • intracellular Ca 2 * is 2-3 fold higher in isolated thyroid epithelial cells compared with clumps of cells.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at risk” cells, compared with controls.
  • EXAMPLE 15 - OTHER TUMORS 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).
  • the procedures of Examples 3 and 4 for instance, use measurements of intracellular Ca 2 * 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.
  • isolated organoids such as crypts or terminal ductal lobular units
  • 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).
  • 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 Ca 2 *-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 cm 3 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.
  • 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 ⁇ M 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.
  • 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.
  • the intracellular Ca 2 * 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 ⁇ M for 100 sec to maximize binding of Ca 2 * to FURA. Then 60 mM ethyleneglycol-bis ( ⁇ -aminoethyl ether)-N,N,N',N , -tetracetic acid was added to remove all Ca 2 * 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.
  • 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 Ca 2 * was measured in cells at the base of the crypts, and 14 samples of isolated human crypts in which intracellular Ca 2 * was measured at the mouth of the crypt.
  • the "at-risk” group comprised 23 samples of isolated human colonic crypts in which intracellular Ca 2 * was measured in cells at the base of the crypts, and 9 samples of isolated human crypts in which intracellular Ca 2 * 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 Ca 2 *-free Ringers Solution bath (0 CA) was 27 nM ⁇ 4nM.
  • the peak intracellular Ca 2 * concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca 2 * was 98 nM ⁇ 20nM.
  • the final intracel ⁇ lular Ca 2 * concentration (FINAL) was 67 nM ⁇ 16nM after a period of equilibration.
  • the intracellular Ca 2 * concentration in control mouth crypt cells observed in the Ca 2 *-free Ringers Solution bath (0 CA) was 44 nM ⁇ 7nM.
  • the peak intracellular Ca 2 * concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca 2 * was 131 nM ⁇ 30nM.
  • the final intracellular Ca 2* concentration (FINAL) was 81 nM ⁇ 14nM after a period of equilibration.
  • the intracellular Ca 2 * concentration in "at-risk" basal crypt cells observed in the Ca 2 *-free Ringers Solution bath (0 CA) was 41 nM ⁇ 5nM.
  • the peak intracellular Ca 2 * concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca 2 * was 215 nM ⁇ 22nM.
  • the final intracellular Ca 2 * concentration (FINAL) was 179 nM ⁇ 22nM after a period of equilibration.
  • the intracellular Ca 2 * concentration in "at-risk" mouth crypt cells observed in the Ca 2 *-free Ringers Solution bath (0 CA) was 91 nM ⁇ 20nM.
  • the peak intracellular Ca 2 * 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.
  • the human test data show that there is a marked difference between intracellular Ca 2 * concentration for "at-risk” crypt samples, on the one hand, and control-crypt samples, on the other hand.
  • the intracellular Ca 2 * level for "at-risk” crypt basal cells is approximately 2 to 3 times more than the value for control basal cells in Ca 2 *-free or Ca 2 *-containing Ringers Solution.
  • the human test data show that there is a marked difference between intracellular Ca 2 * concentration for "at- risk” crypt mouth cells, on the one hand, and control-crypt mouth cells, on the other hand.
  • the intracellular Ca 2 * level for "at-risk" crypt mouth cells is approximately 2 times more than the value for control mouth cells in Ca 2 *- free or Ca 2 *-containing Ringers Solution.
  • the intracellular Ca 2 * 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 Ca 2 *-containing Ringers Solution (i.e. the peak intracellular Ca 2 * is similar in the base and the mouth, and the final intracellu ⁇ lar Ca 2 * gradient is reversed between base and mouth crypt cells when the bathing solution is changed to Ca 2 *- containing Ringers Solution in figure 4) .
  • the inventor also concluded that the foregoing Ca 2 * tests should be used, also, for breast and other cancers. Accordingly, he adapted the preceding procedures as described below.
  • 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 Ca 2 * concentration in control terminal ductal epithelial cells in the Ca*-free Ringers Solution bath (0 CA) was 5-10 nM.
  • the peak intracellular Ca 2 * concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca 2 * was 200-350 nM.
  • the final intracellular Ca 2 * concentration (FINAL) was 40-80 nM after a period of equilibration.
  • the "at-risk” group comprised 5 samples.
  • the intracellular Ca 2 * concentration in "at-risk” terminal ductal epithelial cells observed in the Ca 2 *-free Ringers Solution bath (0 CA) was 5-20 nM.
  • the peak intracellular Ca 2 * concentration (PEAK) observed when the bathing solution was changed to contain 1 mM Ca 2 * was 300-600nM.
  • the final intracellular Ca 2 * concentration (FINAL) was 70-200 nM after a period of equilibration.
  • the human test data show that there is a marked difference between intracellular Ca 2 * 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 Ca 2 * level for "at-risk” terminal ductal epithelial cells is approximately 2 to 3 times more than the value for control cells in Ca 2 *-free or Ca 2 *- 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.
  • Measurements of intracellular Ca 2 * 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 Ca 2 *) by the procedure of Example 18.
  • the ductal acinar units are then subjected to the lab fluoroscopy procedure of Example 19.
  • Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * are made in the isolated organoid.
  • intracellular Ca 2 * 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 Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at-risk" cells, compared with controls.
  • Measurements of intracellular Ca 2 * 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 Ca 2 *) by the procedure of Example 18.
  • the ductal acinar units are then subjected to the lab fluoroscopy procedure of Example 19.
  • Intracellular Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * are made in the isolated organoid.
  • intracellular Ca 2 * 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 Ca 2 * 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.
  • an increase in the bathing solution Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at-risk” cells, compared with controls.
  • Measurements of intracellular Ca 2 * 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 Ca 2 *) by the procedure of Example 18.
  • the gastric glands are then subjected to the lab fluoroscopy procedure of Example 19.
  • Intracellular Ca 2 * concentration is measured in Ca z *-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 Ca 2 * 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 gastric organoids and the pattern and distribution of the calcium gradient in these organoids is altered in "at-risk" patients.
  • Intracellular Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at-risk" cells, compared with controls.
  • 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 Ca 2 * concentration is measured in Ca 2 *-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 Ca 2 * are made in the isolated organoid.
  • 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 Ca 2 * 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.
  • an increase in the bathing solution Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * in "at- risk” cells, compared with controls.
  • Automated measurements of intracellular Ca 2 * 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.
  • Intracellular Ca 2 * concentration is measured in Ca 2 *-free Ringer Solution and then l mM Ca 2 *-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 Ca 2 *.
  • the ratio of violet/blue fluorescence is measured in intact epithelial cells in Ca 2 *-free for 60 seconds, and ImM Ca*-containing Ringers Solution for a further 60 seconds.
  • R is the violet/blue fluorescence ratio of the cells, under equilibrium conditions in Ca 2 *-free, and then Ca 2 *- containing Ringers Solution; and S ⁇ /S ⁇ is the ratio of the blue fluorescence intensity of Ca*-free to Ca 2 *-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 Ca 2 * 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 Ca 2 * from 0 to 1 mM results in a larger increase in intracellular Ca 2 * 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.
  • 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.
  • DH 1,2 dimethylhydrazine
  • DMBA 7,12 dimethylben- zanthracene
  • 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 Ca 2 *-probe FURA/2, (or other Ca 2 *-probe) , as described in Examples 1-4, or Indo-1, (or other Ca 2 *-probe) , as described in Example 26. Organoids are loaded with FURA/2, (or other Ca 2* -probe) , as described in Example 18. Intracellular Ca 2 * 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 Ca 2 * is measured in Ca 2 *-free and then Ca 2 *-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.
  • the in vitro effect of anticancer agents on intracellular Ca 2 * is determined to assess the efficacy of these drugs or agents in promoting or preventing cancer.
  • Intracellular Ca 2 * is measured in organoids as described in Example 19. Intracellular Ca 2* is measured in Ca 2 *-free and then Ca 2 *-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 organoids, and the altered distribution and gradient of intracellular Ca 2 *, by previous treatment with a . cancer causing, or an anticancer drug or agent.
  • the in vitro effect of cancer causing or anticancer agents on intracellular Ca 2 *, 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.

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Abstract

L'invention se rapporte à une méthode de dépistage et de diagnostic in vitro de cancers à un stade précoce afin d'identifier des changements ayant lieu dans des tissus précancéreux. Selon cette méthode in vitro, une préparation cellulaire est obtenue à partir d'un échantillon de tissu; une sonde calcique, telle qu'un colorant fluorescent, par exemple, est ajoutée à l'échantillon; un signal d'essai électronique, représentatif de la concentration intracellulaire de CA2+ dans l'échantillon sondé, est émis, et le signal d'essai est comparé au signal de référence représentatif de la concentration intracellulaire de CA2+ dans des tissus normaux. Un appareil permettant d'effectuer cette méthode est également décrit.
PCT/US1993/006831 1992-08-10 1993-07-23 Procede et appareil de depistage et de diagnostic de cancers Ceased WO1994003470A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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WO2002079779A1 (fr) * 2001-03-31 2002-10-10 The University Court Of The University Of Dundee Essai de criblage a haut rendement permettant d'identifier des substances capables de moduler la survie et/ou la proliferation cellulaire
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WO2001035078A1 (fr) * 1999-11-09 2001-05-17 Raytheon Company Procede et dispositif servant a executer une analyse de cellules d'apres des emissions simultanees et multiples de marqueurs provenant d'une neoplasie (casmmen)
WO2002079779A1 (fr) * 2001-03-31 2002-10-10 The University Court Of The University Of Dundee Essai de criblage a haut rendement permettant d'identifier des substances capables de moduler la survie et/ou la proliferation cellulaire
KR100496829B1 (ko) * 2002-04-09 2005-06-22 김유만 중간층이 울 섬유로 된 의류용 다층부직포 및 그 제조방법과 제조장치

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