WO1996012037A1

WO1996012037A1 - Method for analysing a cell cluster

Info

Publication number: WO1996012037A1
Application number: PCT/IB1995/000873
Authority: WO
Inventors: Nicolas Cong Nguyen
Original assignee: Nicolas Cong Nguyen
Priority date: 1994-10-17
Filing date: 1995-10-13
Publication date: 1996-04-25
Also published as: FR2725728B1; FR2725728A1; IL115636A0

Abstract

A method for analysing a cell cluster from a human or animal sample by determining the mitotic index of hormone-dependency of breast or prostate biopsies in cell culture. For this purpose, the cell cluster is divided into n samples of equal mass, the samples are placed in vessels containing a suitable culture medium, n-1 specific hormones and a predetermined amount of a radioactive nucleotide are added to n-1 of the vessels, and the residual radioactivity in each sample is measured by directly counting radioactive particles by means of a counter.

Description

O 96/12037 PCMB95 / 00873

METHOD FOR THE ANALYSIS OF A CELL CLUSTER

The present invention relates to a method for analyzing a cell mass obtained from a sample of human or animal origin by measuring the residual radioactivity of this cell mass after its treatment with at least one radioactive nucleotide.

One of the applications of an analysis of this type is the determination of an index of hormone dependence of breast or prostatic biopsies.

The discovery of a breast nodule, perceived on palpation or during a systematic mammography, in a woman over forty-five years of age, biopsy is generally necessary in the end because histopathology alone allows '' affirm or not the diagnosis of breast cancer. This is the case for one in eleven women in this age group. But for more than three in ten women, it will happily be a benign mastopathy.

So there is the problem of whether or not to treat these patients. P. Mauvais-Jarvis and his collaborators explained this dilemma, in "Hormones and Breasts", P. Scali and R. Villet, Masson Ed. 1991, on "Hormonal treatment of benign mastopathies".

According to this document, it can be admitted that only two methods allow the healing of benign mastopathies at the same time as they prevent cancer.

On the one hand castration which suppresses the production of erative proliferation hormones, on the other hand, mastectomy which suppresses the receptors of erative proliferative hormones. This somewhat simplistic schematization has at least the advantage of advancing the pathophysiological bases of hormonal treatment which are in fact based on two kinds of actions, namely the inhibition of the secretion of gonadotropins which results in varying degrees in a biochemical castration, either the use of steroidal or nonsteroidal antioestrogens preventing hormones from binding to their nuclear receptors at the mammary level.

On a practical level, we have to recognize the difficulties encountered as soon as we approach in concrete terms the medical treatment of benign mastopathies.

There are currently certain methods available for collecting information on the mitotic index observed before and after a given hormone treatment.

These methods of analysis are practiced from a biopsy and consist in taking a sample of a cellular mass, for example of mammary or prostatic origin, which is crushed to make a paste in order to dissociate the receptors. This paste is separated into two parts, one of which is placed in the presence of radioactive estrogens and the other in the presence of radioactive progesterones. "

Another known method of analysis consists in putting the sample of the cell mass in culture to release the cells and in adding enzymes.

The article "Cancer biology for individualized therapy: Correlation of growth fraction index in native-state histoculture with tumor grade and stage" published in the journal "Proceedings of the National Academy of Sciences of the USA" describes a method of cell analysis constituted by an autoradiography process. After three to eleven days, a cell culture obtained from tissues taken by biopsy is subjected to tritriated thymidine intended to mark the cells.

After appropriate treatment, the cells are placed in a dark room for five days to impress a support carrying a sensitive emulsion. Analysis of the support impressed by the radioactive cells makes it possible to determine whether the cells are cancerous.

This process is long and requires a complicated implementation, which makes it difficult to use in practice, especially in the as part of an early detection campaign.

Other methods of analysis are described by publications WO 92/19722 in the name of THE SALK INSTITUTE FOR BIOLOG1CAL STUDIES and WO 92/19722 in the name of MORGAN Lee Roy. However, none of these methods provides a rapid and effective means allowing the safe selection of the most appropriate hormone treatment for resorbing cancerous tumors by allowing rapid and effective analysis of the effects of the results obtained on tissues taken by biopsy, following various hormonal treatments.

These currently commonly used methods have several drawbacks: they are long and unreliable because they do not allow infallible determination of which hormones are favorable for cell division, and therefore for uncontrolled proliferation of cells. , in particular to the formation of mastoses in the breasts, and what are the hormones unfavorable to this cell division.

The aim of the invention is to develop a rapid, effective and reliable method for determining the hormone (s) which are the most effective in halting cell division, in order to allow the infallible development of a hormone treatment suitable for benign mastopathies corresponding to a cellular hyperplasia predominantly epithelial, in particular adenomas, f ibroadenomas, polyadenastoses, or conjunctives such as ibrocystic disease.

This object is achieved by the method according to the invention characterized in that the said cell mass is divided into a predetermined number n of samples of substantially equal mass, in that each of these samples is placed in an appropriate container containing a suitable culture medium, in that one introduces into each of these containers an identical amount of said radioactive nucleotide, in that one introduces into at most n-1 of said containers respecti¬ vely at most n-1 hormones and in that said measurement of the residual radioactivity of the samples is carried out thoroughly washed and centrifuged after a predetermined time of bringing the samples and the radioactive nucleotide into contact, by direct counting of the radioactive particles by means of a counter.

The advantage of direct counting is that it is fast, precise and does not lend itself to any misinterpretation.

According to a preferred embodiment, said predetermined time for bringing together the samples and the radioactive nucleotide is between twelve and thirty-six hours and preferably substantially equal to twenty-four hours.

Furthermore, said radioactive nucleotide is preferably tritrium-labeled thymidine, or tritiated thymidine.

Said n-1 specific hormones are either of the estrogen type or of the progesterone type.

Preferably, said cell mass is removed by surgical excision or by biopsy puncture and deposited sterile in bottles containing isotonic saline serum at least during the time of transport to a laboratory.

Advantageously, this cell mass is then cut into thin strips of 0.1 to 0.2 mm thick in a Petri dish containing a solution of Earle.

Depending on the case, before being cut up, said cell mass can be sterilized on its external zone in a substance such as 70% ethanol and transferred to a petri dish containing an Earle solution without calcium or magnesium.

After each cut the cells are released into the solution and this solution is preferably filtered through a gauze filter to retain the large pieces and the fibers. Then, the filtered solution is centrifuged. Preferably, part of the cells are stored at a temperature between - 80 ° C and - 100 ° C, and preferably at around - 90 ° C in a Me Coy 5A medium modified with:

20% patient (breast) or patient (prostate) serum,

100 Units / ml of penicillin,

100 micrograms / ml of streptomycin; thereafter called medium A.

In this context, part of the cells are then cultured by dissolving approximately 10 ⁷ to 10 ⁹ cells in the medium of Me Coy 5A modified with:

20% patient (breast) or patient (prostate) serum, 100 Units / ml of penicillin,

100 micrograms / ml of streptomycin, or in a medium of Me Coy 5A modified with: 20% serum ^- from bovine fetus, 100 Units / ml of penicillin, 100 micrograms / ml of streptomycin, hereinafter called medium B, and tritiated thymidine is added at a dose of 1 to 2 microcuries per ml to constitute a culture mixture.

Preferably, a part of said culture mixture is taken which is deposited in cell culture tubes and a part which is deposited in reference tubes.

The reference tubes preferably receive 0.025 ml of isotonic serum.

The culture tubes are advantageously placed in an incubation study for forty-eight hours at approximately +37 ° C. in a controlled atmosphere which preferably contains 95% air and 5% CO ₂ .

In the advantageous embodiment of the method, the counter used to determine the number of radioactive particles is a liquid scintillation counter.

In general, the method consists in determining the mitotic index and the hormone dependence of breast or prostate biopsies in tissue culture, in the presence of nucleotide markers, such as tritiated thymidine, necessary for the synthesis of DNA.

In practice, this method of analysis measures mitotic activity much more effectively than the measurement of hormone receptors on homogenates of tissue samples, because it better reflects the effect of hormones on the cell rather than the presence or absence of such and such a receptor on the cell membrane.

It makes it possible to precisely determine the rate of incorporation of tritiated thymidine into the DNA synthesis of the tissue samples studied, this incorporation being more or less high compared to a reference culture in the presence of specific hormones. fiques. It is thus possible to determine which is the hormone whose presence promotes or inhibits the multiplication of cells, which makes it possible to recommend the treatment which could reduce the lesion especially when the response of the pathological anatomy is negative, that is ie when the lesion is non-cancerous.

The invention will be better understood with reference to the attached drawing in which:

FIG. 1 represents a schematic view illustrating the main phases of the method and its equipment for implementation.

With reference to FIG. 1, the general principle of the method comprises the following essential steps A to F.

Step A consists of taking a sample of a cell mass 10 which is divided into two parts 11 and 12, one of which 11 is used to carry out conventional histopathology using conventional equipment. 13 schematically represented by a microscope. The other part 12 is divided into n equal parts by weight, during step B. Each of these n equal parts is introduced into a test tube

14 containing a culture medium, in this case physiological serum from the person or animal from which the cell mass was removed.

During step C, each of these test tubes is introduced with a predetermined equal amount of a radioactive nucleotide, such as for example tritium-labeled thymidine, schematically represented by a container 15.

During step D, two test tubes 16 are separated which constitute the reference tubes. The other n-2 tubes, called culture tubes, each receive a specific hormone Hi, H ₂ , H3,. . . .

Hn-2 which is introduced into the test tube.

Stage E is a waiting stage during which the hormones act either to stimulate or to slow down cell division. The duration t of this waiting is between twelve and thirty-six hours and is preferably l 'order of twenty-four hours.

During step F, the samples are centrifuged and washed and their residual radioactivity is measured using equipment 17 of the Geiger counter type or similar, for example a Packard type liquid scintillation counter with display screen 18 This counter allows direct and precise counting of radioactive particles.

Laboratory tests have been performed and are described in detail below.

The biopsy samples are taken either by surgical excision or by biopsy puncture. They are immediately placed in a sterile manner in vials ^" specially prepared for this purpose containing isotonic saline, and kept at + - + ° C during transport to the laboratory. The biopsy is studied macroscopically and microscopically by a few sections after freezing to determine the degree of cellularity. Sometimes it is necessary to sterilize the external area of the sample by placing it for five minutes in ethanol at

70% before being transferred to a Petri dish containing Earle's solution (EBSS) without calcium or magnesium.

In the case of relatively substantial surgical biopsies, the external part and the adipose tissue are resected by dissection and separated from the healthy breast or prostate tissue and the necrotic or haemorrhagic parts. For biopsy punctures, examination under a binocular microscope allows the same operations to be carried out to prepare the material taken.

All dissections are performed under the cover of an EBSS bath.

When the biopsy is well trimmed, it is cut into small very thin strips of 0.1 to 0.2 mm thick in a new petri dish under an EBSS bath and the pieces are cut and not scraped with the blade. scalpel while being held stationary with dissecting forceps. After each cut, cells are released into the medium which will be poured through a gauze filter to retain large lumps and any fibers.

The filtered medium is centrifuged at 500G at + ° C for ten minutes. Part of the cells of the biopsy can be stored for further studies at -90 ° C in a medium of Me Coy 5A modified with:

20% patient (breast) or patient (prostate) serum,

100 Units / ml of penicillin, 100 micrograms / ml of streptomycin; and the other part can be cultivated immediately.

The biopsy cell filtrate is studied by counting figurative elements to determine its cell content, according to the same technique as that of blood studies. 11 must be from 10 ⁷ to 10 ⁹ cells to be diluted in 25 ml of a modified Me Coy medium A or of medium B, to which tritiated thymidine H-thymidine is added at a dose of 1 to 2 microcuries per ml, ie 25 to 50 microcuries in all. Then, ml of aliquot of this suspension are put into 150 x 16 mm Falcon tubes for tissue culture, at the rate of four tubes per hormone or per substance studied.

The reference tubes receive 0.025 ml of isotonic saline.

All the culture tubes are placed in the incubation oven for forty-eight hours at + 37 ° C. in an atmosphere of 95% air and 5% CO ₂ .

At the end of the incubation period, the cells are centrifuged at 500 G for ten minutes at + A ° C, and washed three times with the washing solution.

The small ball of cells obtained is then frozen and thawed twice to break the membranes, then 5 ml of trichloroacetic acid are added to it.

After two hours at + 4 ° C, the tubes are again centrifuged at 750G for fifteen minutes at + 4. ° C. The small cell ball is treated with 1.0 ml of hyamine hydroxide (10X) for twelve hours at +37 ° C to digest the membranes and release the cytoplasm and nuclei.

The culture tubes are transferred to the counting tubes by adding 1.5 ml of methanol and then adding 10 ml of modified Bray solution.

The tubes are then counted in a liquid scintillation counter of the Packard type.

After correction for adjustment (Quelching), the results of the counting of each group of tubes are expressed in disintegration by minutes, or strokes / min. The incorporation rate of tritiated thymidine in the cells of each group tested is expressed as a percentage more or less compared to the average of the control group, or reference group, respective to each series. A difference where p is greater than or equal to 0.05 is required to be significant.

The results from these tests are given in detail below.

On twenty surgical biopsies, the results obtained are as follows: Increase in the rate of incorporation of ³ H-thymidine:

4 cases (N ⁹ 5, 7, 8, 19) in the presence of estradiol, 2 cases (N ⁹ 5, 7) in the presence of testosterone; Decrease in the incorporation rate of ³ H-thymidine:

7 cases (N ⁹ 4, 6, 10, 14, 15, 17, 19) in the presence of testosterone,

5 cases (N ⁹ 10, 14, 15, 17, 19) in the presence of progesterone, 2 cases (N ⁹ 14, 17) in the presence of contisol,

2 cases (N ⁹ 10, 14) in the presence of estradiol; Response to treatment:

- case N ⁹ 19, which is histomologically a carcinoma of the breast, shows an increase under estradiol of + 41% and a decrease under testone of -33% and -23% under progesterone, and responds very favorably with androgen treatment. seven cases of benign estradiol sensitive breast tumor regressed completely after treatment with progesterone in the form of ointment for local applications.

This process has the advantage of allowing very rapid and perfectly exact and precise detection of the tests carried out.

Claims

1. Method for analyzing a cell mass obtained from a sample of human or animal origin by measuring the residual radioactivity of this cell mass after its treatment with at least one radioactive nucleotide, characterized in that dividing said cell mass into a predetermined number n of samples of substantially equal mass, in that each of these samples is placed in a suitable container containing a suitable culture medium, in that one introduces into each of these receptacles an identical quantity of said radioactive nucleotide, in that one introduces into at most n-1 of said receptacles respectively at most n-1 specific hormones, and in that one carries out said measurement of radioactivity residual of the samples previously washed and centrifuged after a predetermined time of bringing the samples and the radioactive nucleotide into contact, by direct counting of the radioactive particles at by means of a counter.

2. Method according to claim 1, characterized in that said predetermined time for bringing the samples and the radioactive nucleotide into play is between twelve and thirty-six hours and preferably substantially equal to twenty-four hours.

3. Method according to claim 1, characterized in that said radioactive nucleotide is thymidine labeled with tritrium, or tritiated thymidine.

4. Method according to claim 1, characterized in that said n-1 specific hormones are of the estrogen or progesterone type.

5. Method according to claim 1, characterized in that said cell mass is removed by surgical excision or by biopsy puncture and sterile deposited in vials containing isotonic saline serum at least during the time of transport to a laboratory.

6. Method according to claim 5, characterized in that said cluster cell is cut into thin strips of 0.1 to 0.2 mm thick in a petri dish containing Earle's solution.

7. Method according to claim 5, characterized in that before its cutting, said cell mass is sterilized on its external area by treatment in a substance such as 70% ethanol and transferred to a petri dish containing a solution of 'Earle without calcium or magnesium.

8. Method according to claim 6, characterized in that after each cut the cells are released into the solution and in that this solution is filtered through a gauze filter to retain large pieces and fibers.

9 "Method according to claim 8, characterized in that the filtered solution is centrifuged.

10. Method according to claim 8, characterized in that a part of the cells is kept at a temperature between - 80 ° C and - 100 ° C, and preferably at about - 90 ° C in a Me Coy medium 5A modified with:

20% patient (breast) or patient (prostate) serum,

100 Units / ml of penicillin, 100 micrograms / ml of streptomycin.

11. Method according to claims 3 and 8, characterized in that part of the cells are cultured by dissolving approximately 10 ⁷ to 10 ⁹ cells in the medium of Me Coy 5A modified with: 20% of the patient's serum (breast) or patient

(prostate),

100 Units / ml of penicillin, 100 micrograms / ml of streptomycin, or in a medium of Me Coy 5A modified with: 20% of fetal bovine serum,

100 Units / ml of penicillin, 100 microgram s / ml of streptomycin, and tritiated thymidine is added at a dose of 1 to 2 microcuries per ml to constitute a culture mixture.

12. Method according to claim 11, characterized in that one takes a part of said culture mixture which is deposited in tubes for cell culture and a part which is deposited in reference tubes.

13. Method according to claim 12, characterized in that the reference tubes receive 0.025 ml of isotonic serum.

14. Method according to claim 12, characterized in that the culture tubes are placed in an incubation study for forty-eight hours at approximately 37 ° C in a controlled atmosphere.

15. The method of claim 14, characterized in that said controlled atmosphere contains 95% air and 5% C0 ₂ .

16. Method according to claim 1, characterized in that the counter is a liquid scintillation counter.