METHOD AND KIT FOR DIAGNOSIS AND/OR PROGNOSIS OF NON-HEMATOLOGICAL TUMORS

Abstract

The present invention relates to an in vitro method for the diagnosis and/or prognosis of non-haematological tumours by analysing circulating tumour cells isolated from a blood sample or a derivative thereof and to a kit for such purpose. The present invention further relates to a specific culture medium particularly effective for use in such method.

Claims

1. An in vitro method for the diagnosis and/or prognosis of non-hematological tumors comprising the following steps: i) Isolating a population of circulating tumor cells from a blood sample, or a derivative thereof, obtained from a patient suffering from, or potentially suffering from tumor by means of the following steps: a) separating said blood sample or derivative thereof on a Ficoll gradient by centrifugation; b) collecting from said separated sample the phase with a density value comprised between 1,080 and 1,090 (kg/m3) and diluting the collected phase c) centrifuging the collected and diluted phase; and d) recovering the obtained pellet ii) cultivating the pellet obtained in step d) in a culture medium containing: Nutrient mixture supplemented with fetal bovine serum Heparin; Epidermal growth factor; Fibroblast growth factor; Bovine serum albumin; D-Glucose, L-ascorbic acid; and one or more antibiotics selected from penicillin, streptomycin and/or amphotericin; and iii) performing a cytological analysis of cells cultured in step ii) so as to provide a diagnosis and/or prognosis of said cancer.

2. The method according to claim 1, wherein said circulating tumor cells derive from a solid tumor or are epithelial cells.

3. The method according to claim 1, wherein said non-hematological tumor is selected from the group consisting of: stromal cancers, cardiac myxomas, intracranial cancers including multiform glioblastoma, thyroid cancers, adrenal cancers, pancreatic cancers, colon cancers, breast cancers, stomach cancers, cholangiocarcinomas, melanoma, spinocellular cancers and basal cancers and lung small cell cancers.

4. The method according to claim 1, wherein the sample of said blood derivative is a sample of whole blood, pleural or ascites fluid.

5. The method according to claim 1, wherein said centrifugation at said step a) is carried out at 700 g for 20 minutes at 4 C. or at 1,840 rpm in a centrifuge swinging bucket for 25 minutes at 22 C.

6. The method according to claim 1, wherein said centrifugation at step d) is carried out at 1850 or 1860 rpm for 10 minutes at room temperature.

7. The method according to claim 1, wherein the D-glucose in said medium is in a concentration between 4 and 10 mM and/or ascorbic acid is in a concentration between 5 and 20 mM.

8. The method according to claim 1, wherein said medium comprises: Nutrient mixture Ham F-12 supplemented with fetal bovine serum at 10%: Heparin 25000 u/5 ml (fc: 0.5 U/ml); epidermal growth factor (EGF) 200 g/ml (fc: 50 ng/ml); fibroblast growth factor (FGF) 25 g/ml (fc: 25 ng/ml); bovine serum albumin (BSA) 1%, D-Glucose 5.55 mM, L-ascorbic acid 14 mm; and penicillin and/or streptomycin and/or amphotericin B.

9. A medium for culturing circulating tumor cells from a blood sample, or a derivative thereof, comprising Nutrient mixture supplemented with fetal bovine serum Heparin; Epidermal growth factor; Fibroblast growth factor; Bovine serum albumin; D-Glucose, L-ascorbic acid; and one or more antibiotics selected from penicillin, streptomycin and/or amphotericin.

10. The medium for culturing circulating tumor cells from a blood sample according to claim 9 wherein the D-glucose in said medium is in a concentration between 4 and 10 mM.

11. The medium for culturing circulating tumor cells from a blood sample according to claim 9 wherein ascorbic acid is in a concentration between 5 and 20 mM.

12. The medium for culturing circulating tumor cells from a blood sample according to claim 9, wherein said medium comprises the following mixture: Nutrient mixture Ham F-12 supplemented with fetal bovine serum at 10%; Heparin 25000 u/5 ml (fc: 0.5 U/ml); epidermal growth factor (EGF) 200 g/ml (fc: 50 ng/ml); fibroblast growth factor (FGF) 25 g/ml (fc: 25 ng/ml); bovine serum albumin (BSA) 1%, D-Glucose 5.55 mM, L-ascorbic acid 14 mm; and penicillin and/or streptomycin and/or amphotericin B.

13. A kit for the diagnosis and/or prognosis of non-hematological tumors comprising the culture medium according to claim 9 and at least one aliquot of a solution for carrying out the Ficoll gradient and/or at least one tube wherein the phase with a gradient value between 1,080 and 1,090 (kg/m3) is highlighted and/or at least one means selected from the group consisting of: a tube, pipette, tube with EDTA, syringe, needle, aliquot with phosphate buffered saline and sterile water aliquot.

14. The method according to claim 7, wherein the D-glucose concentration in said medium is 5.55 mM and/or the ascorbic acid concentration is 14 mM.

15. The medium according to claim 10, wherein the D-glucose in said medium is at a concentration of 5.55 mM.

16. The medium according to claim 11, wherein the ascorbic acid is at a concentration of 14 mM.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGS. 1 and 2 show table 1 and table 2 wherein the estimate of the circulating tumour cells is shown, obtained with the method traditionally used for isolating CTC cells from blood and the number of CTC cells isolated with the herein described method, respectively.

[0015] FIG. 3: (a), (b), (c), (d), (e), (f) represent primary cultures of CTC derived from the blood of patients affected by colon (a), breast (b) and lung (c) cancers, whereas (d), (e), (f) primary cultures derived in equal experimental conditions from the blood of healthy subjects.

[0016] FIG. 4 shows the expression of calpain-2 in lung tumour cells (PT), lung circulating tumour cells (CTC) isolated according to the herein disclosed method, before (C1) and after (C2) the sample surgical removal.

[0017] FIG. 5. Analysis of the pre-cultivation phenotype. The graph relates to the cells collected after the passage in centrifuge and isolated from the phase.

SUMMARY OF THE INVENTION

[0018] For the first time an ameliorating method is herein described in terms of sensitivity and reproducibility for detecting the circulating tumour cells (CTC) in patients affected by cancer. The sensitivity of the illustrated method facilitates experiment repeatability. The routine isolation and the cultivation of malignant epithelial cells according to what herein described provides a better control of diagnosis and prognosis of tumour disease. Moreover, the herein described method provides information about feasibility of specific chemotherapy on the patient and based upon a simple in vitro test it allows to detect the heterogeneity of sensibility to drug. Moreover, the used culture medium has been optimized to increase the CTC yield in vitro both in adhesion and suspension. At last it was observed that the number of spontaneous formations of cell spheres increases as the disease stage progresses and therefore they are considered as a negative prognosis sign.

[0019] In the publication Malara et al. Journal of Biological Regulators & Homeostatic agents, 1 Jan. 2014 717-731 the Protocol for culturing ctc from Non-Small Cell Lung Cancer (NCSLC) is described. With respect to the described method in this publication the use of optimized culture mediums allows to obtain circulating tumour cells for the diagnosis and/or prognosis in patients with non-haematological tumours of different type not only with NCSLC. The culture medium composition was tested in order to avoid qualitative discrepancies between the subset types of CTC found after culture and those observed in the same sample before in vitro passage. The medium type and the time of 14 days modify the absolute number of CTCs per single phenotypic subset not modifying the phenotypic inter subset proportions. In other words, the number of cells is modified but they are not modified qualitatively.

[0020] Firstly, the present invention then relates to an in vitro method for the diagnosis and/or prognosis of a not-haematological tumour by means of analysing the circulating tumour cells isolated from a blood sample or a derivative thereof as defined by claim 1.

[0021] Secondly, the present invention relates to a cell culture medium as defined by claim 9.

[0022] Thirdly, the present invention relates to a kit for the diagnosis and/or prognosis of a not-haematological tumour by means of analysing circulating tumour cells isolated from a blood sample or a derivative thereof as defined by claim 10.

[0023] Preferred features of the present description are set forth in the relative depending claims.

DETAILED DESCRIPTION OF THE INVENTION

[0024] An in vitro method for the diagnosis and/or prognosis of non-haematological tumours by analysing circulating tumour cells is herein disclosed. The method provides a first isolation passage of circulating tumour cells from a blood sample, or a derivative thereof, which will be performed according to what described in the patent application ITRM20120028 shown herein too for sake of completeness.

[0025] Under blood derivative in the present description a derivative is meant obtained from blood, for example, by filtration and/or natural separation such as in case of plasma, pleural or ascites fluid.

[0026] The separation of a blood sample with Ficoll is conventionally performed by a person skilled in the art in his/her daily work and described in any laboratory manual, therefore it does not require additional examination herein. Preferably, such separation is performed by centrifugation. In an embodiment of the present invention, the first passage of the method for isolating CTCs is obtained by centrifugation for about 20 minutes at 4 C.

[0027] As a consequence of the just described procedure the different components of the sample of interest separate along the gradient according to their specific density value. The inventor of the present method has surprisingly detected a phase of Ficoll gradient therealong preferentially CTCs concentrate. Such phase of Ficoll gradient corresponds to the phase with a density value comprised between about 1,080 and 1,090 (kg/m.sup.3), phase which has never been described or suggested in literature as useful to isolate circulating tumour cells. In particular, the state of prior art designates as value useful to the above purpose a value ranging between 1,050 and 1,070 (kg/m.sup.3).

[0028] Then, the second passage of the herein described method consists in collecting from said separated sample on Ficoll, as previously indicated, the phase with density value comprised between 1,080 and 1,090 (kg/m.sup.3). Optionally, the so-collected phase can be diluted with a suitable solution, wherein under suitable a solution is meant which does not induce any type of chemical/physical alteration to the portion of up-to-now separated and collected sample. Examples of such solutions are phosphate-buffered saline or a solution of standard citrate of saline solution (CSD), solution of stringent washing (pH 7.0 at 71 C., 0.06 M of NaCl, 6 mm C.sub.6H.sub.5Na.sub.3O.sub.7.2H.sub.2O) Subsequently, the collected gradient phase is subjected to additional separation, preferably by centrifugation. In an embodiment of the present invention, such centrifugation is performed at about 1,500 rmp preferably for 10 minutes and at room temperature.

[0029] At last, the so-obtained pellet is recovered. Such pellet corresponds to the circulating tumour cells, thus to the CTCs of a patient and circulating.

[0030] In particular, the isolating method as described herein characterizes for a yield relatively to the number of isolated CT which is of about 10.sup.5 tumour circulating cells/ml of blood or a derivative thereof.

[0031] The method provides an additional passage in which the isolated circulating tumour cells as described above are cultured in a medium optimized for such purpose comprising:

[0032] Nutrient mixture supplemented with Foetal bovine serum

[0033] Heparin, in particular Heparin 25000;

[0034] Epidermal growth factor;

[0035] Fibroblast growth factor;

[0036] Bovine serum albumin;

[0037] D-Glucose,

[0038] L-ascorbic acid;

[0039] one or more antibiotics selected from penicillin, streptomycin and/or amphotericin.

[0040] According to an embodiment the medium will include D-glucose in a concentration comprised between 4 and 10 mM, in particular 5.55 mM and ascorbic acid in a concentration comprised between 5 and 20 mM, in particular 14 mM.

[0041] According to an embodiment the nutrient mixture supplemented with Foetal bovine serum is of Ham F-12 type. Hereinafter the composition of a commercial medium of F-12 Ham, commercialized by Sigma-Aldrich, is shown:

TABLE-US-00001 N4888 N6658 N6760 Component [1x] g/L [1x] g/L g/L Inorganic Salts Calcium Chloride 0.0333 0.0333 0.0333 Cupric Sulfate5H.sub.2O 0.0000025 0.0000025 0.0000025 Ferrous Sulfate7H.sub.2O 0.000834 0.000834 0.000834 Magnesium Chloride 0.0576 0.0576 0.0576 Potassium Chloride 0.224 0.224 0.224 Sodium Bicarbonate 1.176 1.176 Sodium Chloride 7.599 7.599 7.599 Sodium Phosphate Dibasic 0.14204 0.14204 0.14204 (anhydrous) Zinc Sulfate7H.sub.2O 0.000863 0.000863 0.000863 Amino Acids L-Alanine 0.009 0.009 0.009 L-ArginineHCl 0.211 0.211 0.211 L-AsparagineH.sub.2O 0.01501 0.01501 0.01501 L-Aspartic Acid 0.0133 0.0133 0.0133 L-CysteineHClH.sub.2O 0.035 0.035 0.035 L-Glulamic Acid 0.0147 0.0147 0.0147 L-Glulamine 0.146 0.146 Glycine 0.00751 0.00751 0.00751 L-Histidine3HClH.sub.2O 0.02096 0.02096 0.02096 L-Isoleucine 0.00394 0.00394 0.00394 L-Leucine 0.0131 0.0131 0.0131 L-LysineHCl 0.0365 0.0365 0.0365 L-Methionine 0.00448 0.00448 0.00448 L-Phenylalanine 0.00496 0.00496 0.00496 L-Proline 0.0345 0.0345 0.345 L-Serine 0.0105 0.0105 0.0105 L-Threonine 0.0119 0.0119 0.0119 L-Tryptophan 0.00204 0.00204 0.00204 L-Tyrosine2Na2H.sub.2O 0.00778 0.00778 0.00778 L-Valine 0.0117 0.0117 0.0117 Vitamins D-Biotin 0.0000073 0.0000073 0.000073 Choline Chloride 0.01396 0.01396 0.01396 Folic Acid 0.00132 0.00132 0.00132 myo-Inositol 0.018 0.018 0.018 Niacinamide 0.000037 0.000037 0.000037 D-Pantothenic Acid (hemicalcium) 0.00048 0.00048 0.00048 PyridoxineHCl 0.000062 0.000062 0.000062 Riboflavin 0.000038 0.000038 0.000038 ThiamineHCl 0.00034 0.00034 0.00034 Vitamin B.sub.12 0.00136 0.00136 0.00136 Other D-Glucose 1.802 1.802 1.802 Hypoxanthine 0.00408 0.00408 0.00408 Linoleic Acid 0.000084 0.000084 0.000084 Phenol RedNa 0.0013 0.0013 0.0013 PutrescineHCl 0.000161 0.000161 0.000161 Pyruvic AcidNa 0.11 0.11 0.11 Thioctic Acid 0.00021 0.00021 0.00021 Thymidine 0.00073 0.00073 0.00073 Add L-Glutamine 0.146 Sodium Bicarbonate 1.176

[0042] According to an additional embodiment the medium will include or will consists of:

[0043] Nutrient mixture supplemented with Foetal bovine serum, in particular Ham F-12 (commercially available for example by Sigma Aldrich catalogue number N3520) supplemented with Foetal bovine serum at 10%:

[0044] Heparin 25000 u/5 ml (fc: 0.5 U/ml);

[0045] epidermal growth factor (EGF) 200 g/ml (fc: 50 ng/ml);

[0046] fibroblast growth factor (FGF) 25 g/ml (fc: 25 ng/ml)

[0047] bovine serum albumin (BSA) 1%,

[0048] D-Glucose 5.55 mM,

[0049] L-ascorbic acid 14 mm;

[0050] Solution of penicillin-streptomycin 1% and/or amphotericin B 0.1%.

[0051] The method provides an additional passage of cytological analysis of the cells cultivated with the purpose of performing a diagnosis and/or prognosis of tumour of the patient therefrom the blood sample was collected. The cytological analysis preferably will be a microscope analysis and/or an analysis by means of cytofluorimetry, for example by means of Fluorescence-activated cell sorting (FACS).

[0052] The method could be used to obtain circulating tumour cells deriving from a tumour of solid type or from epithelial cells. For example stromal cancers, cardiac myxomas, intracranial cancers including the multiform glioblastoma, thyroid cancers, adrenal cancers, pancreatic, colon, breast, stomach cancers, cholangiocarcinomas, melanoma, spinocellular and basal cancers and lung small cell cancers. The present description further relates to a kit for the diagnosis and/or prognosis of non-haematological tumours by analysing circulating tumour cells comprising the culture medium as described herein. The kit could further comprise at least one tube wherein the phase with a density value comprised between about 1.080 and 1.090 (kg/m.sup.3) is highlighted, and at least a solution for carrying out the Ficoll gradient. Preferably such tube is a sterile tube having capacity suitable to contain a quantity of blood sample or a derivative thereof sufficient to perform the separation according to the above method. The tube characterizes in having the area corresponding to the phase of Ficoll gradient comprised between about 1,080 and 1,090 (kg/m.sup.3) limited visibly so as to allow and lead the operator towards a correct isolation of the tumour circulating cells according to the herein claimed method. The absence of highlighting the phase 1,080 and 1,090 (kg/m.sup.3) on the tube would imply the collection of phases characterized by the presence of blood components, such as for example, lymphocytes and monocytes, with consequent contamination of the obtained CTC sample. It appears clear that the quantity of the solution aliquot for carrying out the Ficoll gradient will be selected by taking into account the capacity and the number of tube included in the above kit.

[0053] The kit can further comprise at least an operating means useful to perform the isolation of the circulating tumour cells according to the herein disclosed method. Therefore, such means can be selected in the group comprising a: tube, pipette, tube with EDTA, syringe, needle, phosphate buffered saline and sterile water.

Experimental Section and Examples

Procedure

Selection of Patients

Cancer Patients

[0054] 1. Caucasic people
2. Age within 18 and 65 years old
3. Cancer diagnosis

Exclusion Criteria:

[0055] 1. The patients with infective active states
2. The subjects not without food
3. The individuals subjected to pharmacological treatment for at least 48 hours Collection of blood samples
1) The blood (5 ml) collected from each donor in tubes for the blood collection including EDTA. In order to avoid contamination of blood samples with epithelial skin cells, collect the second tube once the first blood sample has been collected.
CRITICAL PASSAGE: Overturn gently the blood tube 10 times and keep it at room temperature. The processes within maximum 4 hours after blood collection.
All subsequent passages are at room temperature (20-22) in sterile hood at room temperature
2) mix gently 5 ml of blood with 3 ml of dilution buffer (PBS1) ATTENTION: for point 4) use 10-ml pipettes (Corning cat.n. cc4488) for reducing cell lysis.

CELL SEPARATION BY CENTRIFUGATION IN DENSITY GRADIENT TIME: MORE THAN 50 MIN

[0056] 5) apply carefully 4 ml of cell suspension on 3 ml of Ficoll density gradient. The cell suspension should float above the gradient.
6) centrifuge the gradient at 1,840 rpm in a centrifuge swinging bucket for 25 min at 22. Note: transfer even the tube with PCM medium kept at 4 at room temperature ready for passage 10.
7) Collect the wished fraction with a pipette. In particular, of fraction 2, the most opaque band includes fragments of cells, the circulating not-haematological cells, the haematological cells. The fraction 1 includes debris, the fraction 3 includes mainly monocytes and lymphocytes, the fraction 4 or pellet mainly includes red blood cells. Collect the fraction 3 for the maximum yield of cells.
8) dilute the gradient, mix the cell suspension with a ratio of 1:2 with washing buffer (PBS 1).
9) Centrifuge the cell suspension including the wished fraction for 10 min. at 20, 1860 rpm per minute in a centrifuge swinging bucket. Repeat twice.
10) Eliminate the supernatant including debris. Remove the pellet of cells by moving the tube bottom with a finger. Immediately suspend again the cells in 1 ml of PCM
11) Count the cells under an inverted microscope by using 200-ul of cell suspension staining with trypan blue.
CRITICAL PHASE: cover and put the tube in CO.sub.2 incubator to avoid toxic exhalations of pH in CO.sub.2 environment

Plate Culture

[0057] 2) Plate and cultivate cells.
Follow option A for the cells adhering to the culture system and option B for the spheres (A) In order to favour the adherence to the cell culture plate the cells at wished concentration in COMPLETE PCM are placed in wells or in 60 mm15 mm culture plate. Preferably 24-well plates will be used to ease the management in several points of time or concentrations and the widespread of contaminations is less probable.
13) continue the culture for the subsequent 5-7 days, provide new nutriment and remove waste by removing half of medium on day 4 or 5 and replace it with an equal volume of complete medium. Repeat this medium change every 3 days.
14) after 4-5 days the adherent cells can be collected. Different types of cells can be identified by specific immunostainings or through evaluation by cytometry. Such culture also includes endothelial cells and lymphocytes.

Option (B) for Sphere Cancer

[0058] After 4-5 days the spheres can be collected, disaggregated (by using a 1-ml pipette) for the characterization in cytometry or for sorter of epithelial tumour cells. The selected CTC can be planted again for proliferation or differentiation of additional tumour spheres. The density is 1000 cells/ml.

Composition of the Optimized Culture Medium

[0059] Hereinafter the composition of the culture medium is illustrated which resulted to be the most effective one:

[0060] Nutrient mixture Ham F-12 (available on the market for example by Sigma Aldrich catalogue number N3520) supplemented with Foetal bovine serum at 10%:

[0061] Heparin 25000 u/5 ml (fc: 0.5 U/ml);

[0062] epidermal growth factor (EGF) 200 g/ml (fc: 50 ng/ml);

[0063] fibroblast growth factor (FGF) 25 g/ml (fc: 25 ng/ml)

[0064] bovine serum albumin (BSA) 1%,

[0065] D-Glucose 5.55 mM,

[0066] L-ascorbic acid 14 mm;

[0067] Solution of penicillin-streptomycin 1% and/or amphotericin B 0.1%.