EP3215636A1 - Method for identifying in a subject tumour cells bearing a t315i mutation - Google Patents

Method for identifying in a subject tumour cells bearing a t315i mutation

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Publication number
EP3215636A1
EP3215636A1 EP15790561.3A EP15790561A EP3215636A1 EP 3215636 A1 EP3215636 A1 EP 3215636A1 EP 15790561 A EP15790561 A EP 15790561A EP 3215636 A1 EP3215636 A1 EP 3215636A1
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European Patent Office
Prior art keywords
cells
vibrational spectrum
mutation
abl
bcr
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EP15790561.3A
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German (de)
French (fr)
Inventor
Ali Turhan
Annelise BENNACEUR GRISCELLI
Christophe SANDT
Olivier FERAUD
Frank Griscelli
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Sud
Universite de Poitiers
Synchrotron Soleil
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Sud
Universite de Poitiers
Synchrotron Soleil
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Publication of EP3215636A1 publication Critical patent/EP3215636A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N2021/3595—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using FTIR

Definitions

  • the present invention relates to a method for identifying in a subject tumour cells bearing a T315I mutation and applications of this method.
  • Chronic myelogenous leukaemia is a malignant myeloproliferative stem cell disorder which is characterized by the occurrence of Philadelphia chromosome in a very primitive hematopoietic stem cell (Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood. 2008 Dec 15;112(13):4808-1713):4808-17).
  • the malignant chronic myelogenous leukaemia clone is responsible for the initiation and maintenance of a myeloproliferative syndrome leading inexorably towards a genetic instability and appearance of an accelerated phase which can foretell an acute lethal leukemia ("blast crisis").
  • Philadelphia chromosome in leukemic cells is the BCR-ABL fusion gene, generating a 210-kd protein with deregulated tyrosine kinase activity.
  • BCR-ABL is the oncogene at the origin of chronic myelogenous leukaemia, as it is universally present in all patients with chronic myelogenous leukaemia and its enforced expression in murine hematopoietic stem cells induces a leukaemia- like disease in mice.
  • the available murine models do not reproduce accurately the human disease as they lead rapidly to a lethal leukaemia.
  • TK deregulated BCR-ABL tyrosine kinase activity
  • small TK inhibitors such as imatinib mesylate (IM) which interacts with the ATP- binding site of the ABL-kinase domain and leads to the inhibition of the downstream signalling pathways induced by BCR-ABL.
  • IM imatinib mesylate
  • the introduction of imatinib mesylate to clinical practice has been one of the major successes in the targeted therapies approaches of human malignancies and currently imatinib mesylate has become the first line therapy in the majority of patients with chronic myelogenous leukaemia.
  • Imatinib mesylate has therefore changed drastically the therapeutic strategies but since its introduction in 1999, a major problem appeared with regard to its ability to eradicate the chronic myelogenous leukaemia clone which is the phenomenon of resistance to tyrosine kinase inhibitors therapy, due essentially but not always, to the occurrence of ABL kinase domain mutations rendering the cells resistant to imatinib mesylate (O'Hare T, Eide CA, Deininger MW. Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood. 2007 Oct 1;110(7) : 2242-9.
  • T315I mutation has attracted particular attention as this mutation renders leukemic cells resistant to all three tyrosine kinase inhibitors currently in clinical use, i.e. Imatinib, Dasatinib and Nilotinib.
  • T315I mutation can be identified by direct sequencing (O'hare et al, Blood), dHPLC (O'hare et al, Blood), or double gradient gel electrophoresis of the PCR products amplified from the BCR- ABL kinase domain (Sorel N, Chazelas F, Brizard A, Chomel JC). Double-gradient- denaturing-gradient gel electrophoresis for mutation screening of the BCR-ABL tyrosine kinase domain in chronic myeloid leukemia patients. Clin Chem. 2005 Jul; 51 (7): 1263-6.
  • T315I mutated cells co-exist with non-mutated CML cells (from which they probably arise). This mutation is also present at the level of leukemic stem cells which currently represent a major therapeutic challenge in the field of CML as they are resistant to currently available therapies. (Chomel and Turhan Oncotarget 2012)
  • Parental cells (not bcr-abl), p210 cells, V299 cells (resistant to dasatinib and imatinib- sensitive) and T315I cells (resistant to both TKIs) are used in the experiments. Spectral changes attributed by the authors to apoptosis in non-resistant treated cells are observed. The authors correlate the IR spectral changes with biochemical markers and protein phosphorylation.
  • An aspect of the present application is therefore a method for identifying in a human subject sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation, comprising the steps consisting in
  • FTIR micro Fourier Transformed infrared microspectroscopy
  • sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed.
  • the spectrum obtained by micro infrared spectroscopy (FTIR) on reference cells from chronic myeloid leukaemia without T315I mutation may be a reference spectrum previously obtained, for example obtained weeks, months or years earlier.
  • a preferred way to compare the sample to the reference is sequentially: first analysing the reference, then replace the reference by the sample and analysing the sample. Errors are possible because each measurement depends not only of the light-absorption properties of the sample, but also of the properties of the device used for the provision of the spectra. More elaborate methods, such as a "two-beam" setup, are used to correct possible errors to give accurate results. The method named "standard addition method” can be used to statistically correct errors.
  • a cell sample used in the present method may be a bone marrow cell sample.
  • a cell sample may be obtained by bone marrow biopsy.
  • the sample is preferably taken from the pelvic or breast bone.
  • a cell sample used in the present method may also be a cell sample comprising nonnucleated blood cells.
  • a sample may be obtained preferably using Ficoll-Paque to separate blood to its components and centrifugation.
  • Infrared Microspectroscopy combines the high brilliance and high collimation of a synchrotron beam with a Fourier transform infrared (FTIR) spectrometer and an IR microscope. High signal-to-noise ratios are obtained at diffraction limited spatial resolutions, usually between 3-8 ⁇ . Infrared Microspectroscopy allows the analysis of microscopic samples. The FTIR microscope allows users to collect large area overview IR images from the samples prior to high resolution mapping using the beamline instrument.
  • FTIR Fourier transform infrared
  • the position, shape and intensity of peaks in the spectra obtained from cells from chronic myeloid leukaemia with and without T315I mutation reveals differences between the molecular structures of both samples, more particularly about the T315I mutation.
  • the wavelength ranges used for the analysis in the present method are preferably in the range of 10000 nm to 5555.56 nm, 3571.43 nm to 3333.33 nm.
  • the range of 10000 to 5555.56 nm is particularly preferred because important differences may be observed for the corresponding peaks between cells from chronic myeloid leukemia with and without T315I mutation.
  • the reference cells from chronic myeloid leukaemia without T315I mutation used to establish the reference spectrum to which the spectrum of the samples analysed by the present method will be compared are preferably similar cells. In other terms, if the method of the invention is implemented on bone marrow cells, the reference spectrum will also be obtained from bone marrow cells.
  • comparison of the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T315I mutation is preferably performed using a multivariate pattern recognition technique.
  • the data obtained by FTIR particularly those obtained after analysis by multivariate pattern recognition technique, are treated by Principal Component Analysis (PCA).
  • PCA is a well-known mathematical procedure that uses orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components.
  • PCA has successfully found linear combinations that separate out different clusters corresponding to tumour cells from chronic myeloid leukaemia bearing a T315I mutation and "normal" cells. By separating out different clusters according to the genetic material contained in the cells, PCA allows to easily determining whether sample cells are tumour cells from chronic myeloid leukaemia bearing a T3151 mutation or not.
  • the methods according to the invention have advantageous properties because they allow identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T3151 mutation.
  • an aspect of the present invention is also a method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) to a treatment with a tyrosine kinase inhibitor (TKI), comprising the steps of:
  • FTIR micro Fourier Transformed infrared microspectroscopy
  • sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed,
  • sample cells are tumour cells from chronic myeloid leukaemia bearing a T315I mutation.
  • the above method allows sorting patients between - a group of patients wherein administration of tyrosine kinase inhibitors would be useless because the said patients would not respond to the treatment, and
  • a further aspect of the present invention is a tyrosine kinase inhibitor compound for use in a method of treatment of a patient affected with CML, wherein the patient is selected by the use of the above method.
  • tyrosine kinase inhibitors are bosutinib, crizotinib, dabrafenib, dasatinib, erlotinib, imatinib, lestaurtinib, nilotinib, ponatinib, regorafenib, le ruxolitinib, sorafenib, sunitinib, tofacitinib and vemurafenib.
  • Preferred tyrosine kinase inhibitors are imatinib, dasatinib, nilotinib, bosutinib and ponatinib.
  • Preferred conditions for implementing the method for identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T315I mutation described above also apply to the other subjects of the invention envisaged above.
  • the present invention relates to a method for treating a patient affected with CML comprising a step of administrating to said patient a tyrosine kinase inhibitor compound, wherein the patient is classified as responder to a TKI treatment by the method according to the above method.
  • FIGURES
  • Figure 1 represents a PC A analysis of parental UT7 (UT7-P), UT7 expressing wild type BCR-ABL (UT7-9), UT7-expressing mutated T315I (UT7-315).
  • FIG. 2 shows a PCA analysis of T315I-mutated BCR-ABL in murine embryonic stem cells.
  • mES GS2 p210 mES cells expressing transduced with BCR-ABL;
  • mES GS2 T3151 T3151-mutated BCR-ABL;
  • mES GS2 WT Parental mES GS2 cells.
  • Figures 3 A, B, C and D are graphs illustrating the PCA analysis of T3151-mutated cells in a doxycycline-inducible system.
  • Figure 4 represents a Western analysis of BCR-ABL expression in inducible UT7 cells.
  • UT7 cells transduced with inducible BCR-ABL vectors express high amounts of BCR- ABL protein in the absence of doxycycline in cell medium.
  • DOX Upon addition of DOX, BCR- ABL expression is reduced steadily at day +3, +5 and +7.
  • Parental UT7 cells transduced with the empty inducible vectors were used as control.
  • FTIR micro Fourier transform infrared spectroscopy
  • Spectra were recorded at the SOLEIL synchrotron facility on the SMIS beamline which exploits the edge and bending radiations of a bending magnet.
  • the synchrotron delivers 430mA current in the 4/4 filling mode, in top-up mode for injection.
  • Spectra were recorded on a Nicolet Continuum XL microscope (Thermo Fischer, Courtaboeuf, France) equipped with a 50x50 ⁇ 2 liquid nitrogen cooled MCT/A detector, a 32X/NA 0.65 Schwarzschild objective, XYZ motorised stage, and coupled to a Nicolet 5700 spectrometer (Thermo Fischer, Courtaboeuf, France) equipped with a Michelson interferometer, and a KBr beamsplitter.
  • the confocal aperture was set at 12x12 ⁇ 2 (stem cells) or 6x6 ⁇ 2 (UT7 cells) which allowed measuring each individual cell with an infrared spot matching their size.
  • Spectra of individual cells were analyzed by multivariate pattern recognition techniques in The Unscrambler 10.2 (Camo Software AS, Oslo, Norway). Spectra were preprocessed before analysis: Extended Multiplicative Scattering Correction (EMSC) was applied between 800-1800 cm “1 , and spectra were baseline corrected by subtracting a linear baseline between 1800 and 800 cm “1 to eliminate baseline shifts after EMSC correction.
  • EMC Extended Multiplicative Scattering Correction
  • PCA Principal component analysis
  • the human UT7 cell lines expressing T315I-mutated BCR-ABL exhibited a spectral signature different from that of Parent wild type BCR-ABL.
  • micro FTIR was able to separate out clearly BCR-ABL T315I expressing UT-7 cells from the parental BCR-ABL cell lines, although for some cells with BCR-ABL, expression spotted along with the parental BCR-ABL cells.
  • Murine embryonic stem cells GS2 stably transduced with BCR-ABL and BCR-ABL T315I viruses were used in the same experimental conditions as in example 1. The results are reported on Figure 2.
  • murine GS2 cells expressing T315I- mutated BCR-ABL exhibited an infrared spectrum clearly different from that induced by wild type BCR-ABL.
  • the spectra could be clearly separated by PCA and the loadings of the principal component 4 show signal from the protein at 1660, 1630, 1558 cm “1 , from the sugar at 1400, 1120 cm “1 and from the nucleic acids at 1240, 1083, 960 cm "1 associated with the T315I mutant transfected cells.
  • Fourier transform infrared spectroscopy allows segregating ES GS2 cell lines expressing T315I-mutated BCR-ABL from wild BCR-ABL cells.
  • T3151 mutation provides a spectral signature in different cell contexts.
  • BCR-ABL-expressing UT-7 cell lines have been generated by retrovirus-mediated gene transfer.
  • UT-7 cell line is a human hematopoietic cell line which is dependent on GM-CSF for its growth and survival.
  • BCR-ABL expression renders the UT-7 cell line independent of GM- CSF for its growth.
  • a MIGR-T315I BCR-ABL retroviral vector was transfected in UT-7 cells and BCR-ABL-expressing clones were isolated and characterized by Western blotting and sequencing.
  • BCR-ABL vectors expressing wild type and T315-mutated BCR-ABL were constructed by first cloning BCR-ABL by long-range PCR and inducing T315I mutation by site directed mutagenesis in the kinase domain of WT BCR-ABL.
  • the presence of 2A peptide sequences between EGFP sequences and BCR-ABL ensured a correlation between the expressions of both genes ( Figure 4).
  • the inducible nature of the vector is due to the TET-Op sequences and tTA sequences, the latter being placed under the control of a CML promoter.
  • BCR-ABL-lentiviruses were generated using 293T cells and lentiviral supematants were titrated using HT1080 cells by measuring EGFP. Due to the size of BCR-ABL inserts, the titres of the BCR-ABL vectors were low ( ⁇ 10e 4 / ml) whereas higher titres were obtained by the use of empty EGFP-expressing vectors.
  • UT7 cells were infected with a MOI of 5 using all three lentiviral supematants and the EGFP+ cells were cell sorted, cloned and individually amplified for further characterization, including Western blots and the presence of T315I mutation by sequencing.
  • BCR-ABL transduced cells express high amounts of BCr-ABL protein in the Absence of DOX in cell medium.
  • GS2 cells expressing T315I-mutated BCR-ABL and wild type BCR-ABL were obtained as follows:
  • Murine embryonic GS2 cell line was previously described (Coppo et al, Oncogene
  • MIGR-BCR-ABL and MIGR-T315I vectors were used for transduction of wild GS2 cell lines of a) above, which grows in the presence of LIF (1000 UI / ml) without the need of MEF stromal layers.
  • Three cell lines, expressing either control (Wild type), or BCR-ABL p210, or BCR-ABL T315I were amplified and characterized with regard to their phenotype and BCR-ABL expression.
  • UT-7 cells expressing BCR-ABL either constitutively or by the use of a TET-sensitive promoter were cultured for XXXX days, harvested after trypsinization and deposited on BaF2 windows prior to analysis by FTIR microspectroscopy. Slides were dried in air.
  • Murine ES GS2 cells were grown on low-e MirrlR slides (Kevley Technologies, OH) and after generation of an embryonic stem cell colony, they were washed with distilled water and fixed for FTIR microspectroscopy analysis using 4% paraformaldehyde.
  • Specificity of the spectral signature conferred by the expression of T3151 protein conferred by the expression of T3151 protein
  • the first strategy was the use of a TET-inducible UT7 cell line expressing mutated T3151 protein.
  • this cell line able to express BCR-ABL according to the presence or absence of DOX in the cell culture medium, several time points were then chosen for FTIR analysis, cells being analyzed at day 0 (Absence of DOX, BCR-ABL ON) and at day + 2, day +4 and day+ 7 after adding DOX in the cell culture medium (BCR-ABL expression OFF or highly reduced).
  • the spectral signatures of the UT7-T315I, UT7-p210 and the UT7P were undistinguishable.
  • TKI tyrosine kinase inhibitor
  • T315I-mutated BCR-ABL is related to the substitution of Isoleucine to Threonine, which normally forms a hydrogen bond with Imatinib, this bond being absolutely required for binding of the drugs to the catalytic site.
  • T315I mutation can be identified by direct sequencing, D-HPLC, allele specific PCR or double gradient gel electrophoresis of the PCR products amplified from the BCR-ABL kinase domain. However these techniques cannot identify directly the leukemic cells expressing T315I-mutated BCR-ABL which are known to co-exist with native BCR-ABL-expressing cells in bone marrow.
  • Infrared microspectroscopy allows the identification of metabolic features of mammalian cells, based on their protein, lipid, amid and nucleic acid contents, generating a spectral signature.
  • infrared microspectroscopy can be used to identify metabolic changes occurring in single cells bearing BCR-ABL T315I mutation.
  • human (UT7) and murine embryonic stem cells (GS2) expressing either native (N) or T315I-mutated BCR-ABL.
  • UT7 human
  • GS2 murine embryonic stem cells
  • N-BCR- ABL we were able to clearly distinguish at the single cell level, cells expressing BCR-ABL from wild type cells using principal component analysis.
  • T315I mutation induced a clearly different signature, allowing a highly significant separation by the use of spectral signature.
  • UT7 cells engineered to express either native or T315I-mutated BCR-ABL under the control of Doxycycline (DOX) -sensitive promoters In this TET-OFF system, the addition of doxycycline to the culture medium inhibits BCR-ABL expression as monitored by Western blots, in approximately 6 days. Using this system, at day 0, UT7 cells expressing N-BCR- ABL and T315I mutated BCR-ABL were clearly distinguishable from parental UT7 cells.

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Abstract

The present invention relates to a method for identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T315I mutation, comprising the steps consisting in: - passing a beam of infrared light through a bone marrow cell sample or a blood nonnucleated cell sample in vitro according to micro Fourier Transformed infrared microspectroscopy (FTIR) which produces a vibrational spectrum in the wavelength range of 22222.2 nm to 2500 nm, and therefore obtaining a first vibrational spectrum, - comparing the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T3151 mutation, - identifying the sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed.

Description

METHOD FOR IDENTIFYING IN A SUBJECT
TUMOUR CELLS BEARING A T315I MUTATION
FIELD OF THE INVENTION:
The present invention relates to a method for identifying in a subject tumour cells bearing a T315I mutation and applications of this method.
BACKGROUND OF THE INVENTION:
Chronic myelogenous leukaemia (CML) is a malignant myeloproliferative stem cell disorder which is characterized by the occurrence of Philadelphia chromosome in a very primitive hematopoietic stem cell (Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood. 2008 Dec 15;112(13):4808-1713):4808-17).
The malignant chronic myelogenous leukaemia clone is responsible for the initiation and maintenance of a myeloproliferative syndrome leading inexorably towards a genetic instability and appearance of an accelerated phase which can foretell an acute lethal leukemia ("blast crisis"). The molecular counterpart of Philadelphia chromosome in leukemic cells is the BCR-ABL fusion gene, generating a 210-kd protein with deregulated tyrosine kinase activity. BCR-ABL is the oncogene at the origin of chronic myelogenous leukaemia, as it is universally present in all patients with chronic myelogenous leukaemia and its enforced expression in murine hematopoietic stem cells induces a leukaemia- like disease in mice. However, the available murine models do not reproduce accurately the human disease as they lead rapidly to a lethal leukaemia.
The deregulated BCR-ABL tyrosine kinase (TK) activity can be successfully targeted by small TK inhibitors (TKI) such as imatinib mesylate (IM) which interacts with the ATP- binding site of the ABL-kinase domain and leads to the inhibition of the downstream signalling pathways induced by BCR-ABL. The introduction of imatinib mesylate to clinical practice has been one of the major successes in the targeted therapies approaches of human malignancies and currently imatinib mesylate has become the first line therapy in the majority of patients with chronic myelogenous leukaemia. Imatinib mesylate has therefore changed drastically the therapeutic strategies but since its introduction in 1999, a major problem appeared with regard to its ability to eradicate the chronic myelogenous leukaemia clone which is the phenomenon of resistance to tyrosine kinase inhibitors therapy, due essentially but not always, to the occurrence of ABL kinase domain mutations rendering the cells resistant to imatinib mesylate (O'Hare T, Eide CA, Deininger MW. Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood. 2007 Oct 1;110(7) : 2242-9.
Amongst the ABL-kinase mutations, T315I mutation has attracted particular attention as this mutation renders leukemic cells resistant to all three tyrosine kinase inhibitors currently in clinical use, i.e. Imatinib, Dasatinib and Nilotinib.
It would therefore be particularly interesting to identify T315I mutation. T315I mutation can be identified by direct sequencing (O'hare et al, Blood), dHPLC (O'hare et al, Blood), or double gradient gel electrophoresis of the PCR products amplified from the BCR- ABL kinase domain (Sorel N, Chazelas F, Brizard A, Chomel JC). Double-gradient- denaturing-gradient gel electrophoresis for mutation screening of the BCR-ABL tyrosine kinase domain in chronic myeloid leukemia patients. Clin Chem. 2005 Jul; 51 (7): 1263-6.
However these techniques cannot directly identify cells expressing the BCR-ABL oncogene with T315I mutation. In addition, even in advances stages of the disease, T315I mutated cells co-exist with non-mutated CML cells (from which they probably arise). This mutation is also present at the level of leukemic stem cells which currently represent a major therapeutic challenge in the field of CML as they are resistant to currently available therapies. (Chomel and Turhan Oncotarget 2012)
Bellisola et al. Analyst, 2013, 138, 3934 tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) in combination with unsupervised Hierarchical Cluster Analysis (HCA) in identifying drug-resistance/sensitivity in murine leukemic cells exposed to tyrosine kinase inhibitors. Experiments were carried out in a well-established mouse model of human Chronic Myelogenous Leukemia (CML). The authors compare murine BaF3 cells treated and non-treated with TKIs dasatinib and imatinib. Parental cells (not bcr-abl), p210 cells, V299 cells (resistant to dasatinib and imatinib- sensitive) and T315I cells (resistant to both TKIs) are used in the experiments. Spectral changes attributed by the authors to apoptosis in non-resistant treated cells are observed. The authors correlate the IR spectral changes with biochemical markers and protein phosphorylation.
It would therefore be desirable to have a technique for analysis of leukemic stem cells in leukemia patients, especially to lead to appropriate therapeutic decisions at diagnosis. These decisions may be based in particular on the heterogeneity of the original tumour and the clonal evolution of the disease.
It would also be desirable that these analytical techniques allow the rapid delivery of results, and are preferably further automatizable and quantifiable. Advantageously, it could be used for analyzing very large number of stem cells, for example, several thousand, and this quickly.
In both wild type or T315I-mutated BCR-ABL-expressing cells, there is no specific cell surface marker allowing a direct identification of leukemic cells as compared to non- leukemic cells.
SUMMARY OF THE INVENTION:
It is an aspect of the present invention to eliminate or at least to substantially mitigate said drawbacks of existing methods of identifying T3151 mutation.
Now the applicant has discovered a new satisfactory technique of identifying T315I mutation.
It has been noted in a surprising and unexpected fashion that the use of Fourier Transformed infrared (FTIR) microspectroscopy allows identifying T315I mutation, with the result that a very good compromise is obtained regarding the speed, the simplicity of the technique and the quality of results obtained.
DETAILED DESCRIPTION OF THE INVENTION:
An aspect of the present application is therefore a method for identifying in a human subject sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation, comprising the steps consisting in
- passing a beam of infrared light through a bone marrow cell sample or a blood nonnucleated cell sample in vitro according to micro Fourier Transformed infrared microspectroscopy (FTIR) which produces a vibrational spectrum in the wavelength range of 22222.2 nm to 2500 nm, and therefore obtaining a first vibrational spectrum,
- comparing the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T3151 mutation,
- identifying the sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed.
The spectrum obtained by micro infrared spectroscopy (FTIR) on reference cells from chronic myeloid leukaemia without T315I mutation may be a reference spectrum previously obtained, for example obtained weeks, months or years earlier.
A preferred way to compare the sample to the reference is sequentially: first analysing the reference, then replace the reference by the sample and analysing the sample. Errors are possible because each measurement depends not only of the light-absorption properties of the sample, but also of the properties of the device used for the provision of the spectra. More elaborate methods, such as a "two-beam" setup, are used to correct possible errors to give accurate results. The method named "standard addition method" can be used to statistically correct errors.
A cell sample used in the present method may be a bone marrow cell sample. Such a cell sample may be obtained by bone marrow biopsy. The sample is preferably taken from the pelvic or breast bone.
A cell sample used in the present method may also be a cell sample comprising nonnucleated blood cells. Such a sample may be obtained preferably using Ficoll-Paque to separate blood to its components and centrifugation.
Infrared Microspectroscopy combines the high brilliance and high collimation of a synchrotron beam with a Fourier transform infrared (FTIR) spectrometer and an IR microscope. High signal-to-noise ratios are obtained at diffraction limited spatial resolutions, usually between 3-8 μιη. Infrared Microspectroscopy allows the analysis of microscopic samples. The FTIR microscope allows users to collect large area overview IR images from the samples prior to high resolution mapping using the beamline instrument.
The position, shape and intensity of peaks in the spectra obtained from cells from chronic myeloid leukaemia with and without T315I mutation reveals differences between the molecular structures of both samples, more particularly about the T315I mutation. The wavelength ranges used for the analysis in the present method are preferably in the range of 10000 nm to 5555.56 nm, 3571.43 nm to 3333.33 nm. The range of 10000 to 5555.56 nm is particularly preferred because important differences may be observed for the corresponding peaks between cells from chronic myeloid leukemia with and without T315I mutation.
The reference cells from chronic myeloid leukaemia without T315I mutation used to establish the reference spectrum to which the spectrum of the samples analysed by the present method will be compared are preferably similar cells. In other terms, if the method of the invention is implemented on bone marrow cells, the reference spectrum will also be obtained from bone marrow cells.
Under preferred conditions for implementing the invention, comparison of the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T315I mutation is preferably performed using a multivariate pattern recognition technique. Under preferred conditions for implementing the invention, the data obtained by FTIR, particularly those obtained after analysis by multivariate pattern recognition technique, are treated by Principal Component Analysis (PCA).
PCA is a well-known mathematical procedure that uses orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. In the present invention, PCA has successfully found linear combinations that separate out different clusters corresponding to tumour cells from chronic myeloid leukaemia bearing a T315I mutation and "normal" cells. By separating out different clusters according to the genetic material contained in the cells, PCA allows to easily determining whether sample cells are tumour cells from chronic myeloid leukaemia bearing a T3151 mutation or not.
The methods according to the invention have advantageous properties because they allow identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T3151 mutation.
This is why an aspect of the present invention is also a method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) to a treatment with a tyrosine kinase inhibitor (TKI), comprising the steps of:
- obtaining a bone marrow cell sample or a blood nonnucleated cell sample from the patient,
- passing a beam of infrared light through the bone marrow cell sample or blood nonnucleated cell sample in vitro according to micro Fourier Transformed infrared microspectroscopy (FTIR) which produces a vibrational spectrum in the wavelength range of 22222.2 nm to 2500 nm, and therefore obtaining a first vibrational spectrum,
- comparing the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T3151 mutation,
- identifying the sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed,
- concluding that the patient would not advantageously receive a tyrosine kinase inhibitor treatment, if the sample cells are tumour cells from chronic myeloid leukaemia bearing a T315I mutation.
Therefore, the above method allows sorting patients between - a group of patients wherein administration of tyrosine kinase inhibitors would be useless because the said patients would not respond to the treatment, and
- a group of patients who should be responsive to a tyrosine kinase inhibitor treatment. Accordingly, a further aspect of the present invention is a tyrosine kinase inhibitor compound for use in a method of treatment of a patient affected with CML, wherein the patient is selected by the use of the above method.
Examples of tyrosine kinase inhibitors are bosutinib, crizotinib, dabrafenib, dasatinib, erlotinib, imatinib, lestaurtinib, nilotinib, ponatinib, regorafenib, le ruxolitinib, sorafenib, sunitinib, tofacitinib and vemurafenib. Preferred tyrosine kinase inhibitors are imatinib, dasatinib, nilotinib, bosutinib and ponatinib.
Preferred conditions for implementing the method for identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T315I mutation described above also apply to the other subjects of the invention envisaged above.
In a further embodiment, the present invention relates to a method for treating a patient affected with CML comprising a step of administrating to said patient a tyrosine kinase inhibitor compound, wherein the patient is classified as responder to a TKI treatment by the method according to the above method.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:
Figure 1 represents a PC A analysis of parental UT7 (UT7-P), UT7 expressing wild type BCR-ABL (UT7-9), UT7-expressing mutated T315I (UT7-315).
Figure 2 shows a PCA analysis of T315I-mutated BCR-ABL in murine embryonic stem cells. mES GS2 p210: mES cells expressing transduced with BCR-ABL; mES GS2 T3151: T3151-mutated BCR-ABL; mES GS2 WT: Parental mES GS2 cells.
Figures 3 A, B, C and D are graphs illustrating the PCA analysis of T3151-mutated cells in a doxycycline-inducible system.
Figure 4 represents a Western analysis of BCR-ABL expression in inducible UT7 cells. UT7 cells transduced with inducible BCR-ABL vectors express high amounts of BCR- ABL protein in the absence of doxycycline in cell medium. Upon addition of DOX, BCR- ABL expression is reduced steadily at day +3, +5 and +7. Parental UT7 cells transduced with the empty inducible vectors were used as control. EXAMPLE:
In examples 1 and 2, cell lines were analysed using micro Fourier transform infrared spectroscopy (FTIR) by taking an average of 100 spectra per sample using a spectral frequency from 800 cm 1 (λ=12500 nm) to 4000 cm"1 (λ=2500 nm). Data obtained were then used in principal component analysis (PC A).
Spectra were recorded at the SOLEIL synchrotron facility on the SMIS beamline which exploits the edge and bending radiations of a bending magnet. The synchrotron delivers 430mA current in the 4/4 filling mode, in top-up mode for injection. Spectra were recorded on a Nicolet Continuum XL microscope (Thermo Fischer, Courtaboeuf, France) equipped with a 50x50 μιη2 liquid nitrogen cooled MCT/A detector, a 32X/NA 0.65 Schwarzschild objective, XYZ motorised stage, and coupled to a Nicolet 5700 spectrometer (Thermo Fischer, Courtaboeuf, France) equipped with a Michelson interferometer, and a KBr beamsplitter.
For individual cells, 128 to 256 scans per spectrum were coadded at 4 cm"1 resolution with Happ-Genzel apodization, and Mertz phase correction. The confocal aperture was set at 12x12 μιη2 (stem cells) or 6x6 μιη2 (UT7 cells) which allowed measuring each individual cell with an infrared spot matching their size.
Spectra of individual cells were analyzed by multivariate pattern recognition techniques in The Unscrambler 10.2 (Camo Software AS, Oslo, Norway). Spectra were preprocessed before analysis: Extended Multiplicative Scattering Correction (EMSC) was applied between 800-1800 cm"1, and spectra were baseline corrected by subtracting a linear baseline between 1800 and 800 cm"1 to eliminate baseline shifts after EMSC correction.
Principal component analysis (PCA) was performed on mean centered data using the NIPALS algorithm with leverage correction and 5 PCs were computed. Outliers were detected using the influence (residual versus leverage) plot and Hotelling T2 ellipse. Where separation between clusters was not obvious in PCA, a Partial Least Square-Discriminant Analysis (PLS- DA) identification model was created and the efficiency of the identification was evaluated. PLS-DA was performed in The Unscrambler 10.2 using mean centered data, and the NIPALS algorithm with random validation (20 segments). The initial dataset was split in 2 sets, one for calibration and one for validation with half of the data in each. The calibration and prediction steps were performed alternatively on the calibration and validation set to verify that the identification was not depending on the calibration dataset. During the identification step spectrum identities are predicted as 0 or 1, the threshold for identification was set at 0.5. Spectra predicted with deviation above 0.5 were considered prediction outliers.
Example 1: UT7 Cells
Principal components analysis scatterplots of the tested cells calculated from repeat- count values were obtained.
PCA has successfully found linear combinations that separate out different clusters corresponding to different lines cell populations. Results for Human BCR-ABL and BCR- ABL T315I cell lines are reported in Figure 1.
Indeed, the human UT7 cell lines expressing T315I-mutated BCR-ABL exhibited a spectral signature different from that of Parent wild type BCR-ABL.
As can be seen on Figure 1 , micro FTIR was able to separate out clearly BCR-ABL T315I expressing UT-7 cells from the parental BCR-ABL cell lines, although for some cells with BCR-ABL, expression spotted along with the parental BCR-ABL cells.
Example 2: GS2 Cells
Murine embryonic stem cells GS2 stably transduced with BCR-ABL and BCR-ABL T315I viruses were used in the same experimental conditions as in example 1. The results are reported on Figure 2.
As can be seen on Figure 2, in this cell context, murine GS2 cells expressing T315I- mutated BCR-ABL exhibited an infrared spectrum clearly different from that induced by wild type BCR-ABL. The spectra could be clearly separated by PCA and the loadings of the principal component 4 show signal from the protein at 1660, 1630, 1558 cm"1, from the sugar at 1400, 1120 cm"1 and from the nucleic acids at 1240, 1083, 960 cm"1 associated with the T315I mutant transfected cells.
Thus, Fourier transform infrared spectroscopy allows segregating ES GS2 cell lines expressing T315I-mutated BCR-ABL from wild BCR-ABL cells.
Accordingly, T3151 mutation provides a spectral signature in different cell contexts.
Preparations
1. Preparation of UT-7 cells a) wild UT-7 cells
BCR-ABL-expressing UT-7 cell lines have been generated by retrovirus-mediated gene transfer.
UT-7 cell line is a human hematopoietic cell line which is dependent on GM-CSF for its growth and survival. BCR-ABL expression renders the UT-7 cell line independent of GM- CSF for its growth.
b) mutant UT-7 cells
To generate a T315I-expressing UT-7 cell line, a MIGR-T315I BCR-ABL retroviral vector was transfected in UT-7 cells and BCR-ABL-expressing clones were isolated and characterized by Western blotting and sequencing.
c) Inducible UT-7 cells
BCR-ABL vectors expressing wild type and T315-mutated BCR-ABL were constructed by first cloning BCR-ABL by long-range PCR and inducing T315I mutation by site directed mutagenesis in the kinase domain of WT BCR-ABL. The presence of 2A peptide sequences between EGFP sequences and BCR-ABL ensured a correlation between the expressions of both genes (Figure 4).
The inducible nature of the vector is due to the TET-Op sequences and tTA sequences, the latter being placed under the control of a CML promoter.
BCR-ABL-lentiviruses were generated using 293T cells and lentiviral supematants were titrated using HT1080 cells by measuring EGFP. Due to the size of BCR-ABL inserts, the titres of the BCR-ABL vectors were low (< 10e4 / ml) whereas higher titres were obtained by the use of empty EGFP-expressing vectors. UT7 cells were infected with a MOI of 5 using all three lentiviral supematants and the EGFP+ cells were cell sorted, cloned and individually amplified for further characterization, including Western blots and the presence of T315I mutation by sequencing.
BCR-ABL transduced cells express high amounts of BCr-ABL protein in the Absence of DOX in cell medium.
2. Preparation of GS2 cells
GS2 cells expressing T315I-mutated BCR-ABL and wild type BCR-ABL were obtained as follows:
a) wild GS2 cells
Murine embryonic GS2 cell line was previously described (Coppo et al, Oncogene
2003). b) mutant GS2 cells
MIGR-BCR-ABL and MIGR-T315I vectors were used for transduction of wild GS2 cell lines of a) above, which grows in the presence of LIF (1000 UI / ml) without the need of MEF stromal layers. Three cell lines, expressing either control (Wild type), or BCR-ABL p210, or BCR-ABL T315I were amplified and characterized with regard to their phenotype and BCR-ABL expression.
3. Preparation of slides for FTIR microspectroscopy
UT-7 cells expressing BCR-ABL either constitutively or by the use of a TET-sensitive promoter were cultured for XXXX days, harvested after trypsinization and deposited on BaF2 windows prior to analysis by FTIR microspectroscopy. Slides were dried in air.
In experiments with the TET-inducible UT7 cell lines, cells growing in the absence of doxycycline (DOX) were spotted on slides at day 0. In Cultures in which the DOX was added, aliquots were then taken at day +2 and day +4 after DOX treatment, these two samples were labeled day+2 and day+4.
Murine ES GS2 cells were grown on low-e MirrlR slides (Kevley Technologies, OH) and after generation of an embryonic stem cell colony, they were washed with distilled water and fixed for FTIR microspectroscopy analysis using 4% paraformaldehyde. Experiment: Specificity of the spectral signature conferred by the expression of T3151 protein
In order to determine the specificity of the spectral signature conferred by the expression of T315I protein, two strategies were used.
The first strategy was the use of a TET-inducible UT7 cell line expressing mutated T3151 protein. In this cell line able to express BCR-ABL according to the presence or absence of DOX in the cell culture medium, several time points were then chosen for FTIR analysis, cells being analyzed at day 0 (Absence of DOX, BCR-ABL ON) and at day + 2, day +4 and day+ 7 after adding DOX in the cell culture medium (BCR-ABL expression OFF or highly reduced).
In this TET-OFF system, the expression of BCR-ABL in - DOX conditions generated a spectrum which was clearly different from that of cells expressing wild type (p210) BCR- ABL or parental control cells (P) as shown on Figure 3.
Upon addition of DOX to the cell culture medium, BCR-ABL T315I expression declined and in the same time the UT7-T315I cells adopted a spectral signature which approached the signature observed in UT-7 cells expressing wild type p210 BCR-ABL at day +2 and at day +4, with however, a clear distinction from parental UT-7 cells.
At day+7, the spectral signatures of the UT7-T315I, UT7-p210 and the UT7P were undistinguishable.
This experiment confirms that the spectral signature of the UT7-T315I is caused by the expression of the transgene, and not by random alteration of the cell metabolism caused by the transfection process.
Indeed, in the above experiments, the clear signature observed between BCR-ABL and BCR-ABL-T315I-mutated cells, tended to disappear when the cells were treated with Doxycycline, leading to the inhibition of mutated BCR-ABL expression and adopted features similar to those observed in parental UT7 cells not expressing wild-type BCR-ABL.
Infrared Microspectroscopy Allows Direct Identification of Leukemic Cells Expressing T315I-Mutated BCR-ABL Via a Unique Spectral Signature:
The use of tyrosine kinase inhibitor (TKI) therapies has dramatically changed the prognosis of chronic myeloid leukemia (CML) and modified the natural history of the disease. However, the generation of ABL-kinase domain mutations due to the genetic instability of leukemic cells continues to be very significant challenge, especially in advanced phases of the disease. Amongst these mutations, T315I is the most problematic as it reduces the binding of Imatinib, Dasatinib or Nilotinib to their target, leading to a total resistance to all three TKIs. Although the mechanism of the appearance of this mutation is not completely known, it is possible that it induces a signalling pathway not entirely similar to that induced by classical wild-type BCR-ABL. From the mechanistic point of view, the mechanism of resistance of T315I-mutated BCR-ABL to Imatinib is related to the substitution of Isoleucine to Threonine, which normally forms a hydrogen bond with Imatinib, this bond being absolutely required for binding of the drugs to the catalytic site. T315I mutation can be identified by direct sequencing, D-HPLC, allele specific PCR or double gradient gel electrophoresis of the PCR products amplified from the BCR-ABL kinase domain. However these techniques cannot identify directly the leukemic cells expressing T315I-mutated BCR-ABL which are known to co-exist with native BCR-ABL-expressing cells in bone marrow. Infrared microspectroscopy allows the identification of metabolic features of mammalian cells, based on their protein, lipid, amid and nucleic acid contents, generating a spectral signature. In this work we asked whether infrared microspectroscopy can be used to identify metabolic changes occurring in single cells bearing BCR-ABL T315I mutation. For this purpose we have used human (UT7) and murine embryonic stem cells (GS2) expressing either native (N) or T315I-mutated BCR-ABL. In the human UT7 cells expressing N-BCR- ABL, we were able to clearly distinguish at the single cell level, cells expressing BCR-ABL from wild type cells using principal component analysis. However, the presence of T315I mutation induced a clearly different signature, allowing a highly significant separation by the use of spectral signature. To confirm the specificity of this signature, we have used UT7 cells engineered to express either native or T315I-mutated BCR-ABL under the control of Doxycycline (DOX) -sensitive promoters. In this TET-OFF system, the addition of doxycycline to the culture medium inhibits BCR-ABL expression as monitored by Western blots, in approximately 6 days. Using this system, at day 0, UT7 cells expressing N-BCR- ABL and T315I mutated BCR-ABL were clearly distinguishable from parental UT7 cells. Upon inhibition of BCR-ABL expression by addition of Doxycycline, the spectral signature of N-BCR-ABL and T315I-mutated BCR-ABL cells became similar at day 2 and indistinguishable at day 4, but still distinguishable from wild type UT7 cells expressing DOX- inducible GFP. To confirm these results in a different cell context we have used a murine embryonic stem (ES) cell line (GS2) which was transduced with either N-BCR-ABL or T315I-mutated BCR-ABL-expressing lentiviral vectors. BCR-ABL-expressing individual clones grown were analyzed to analyze the spectral signature. In this cell line also, infrared microspectroscopy was able to distinguish ES cells expressing BCR-ABL T315I as compared to N-BCR-ABL. Thus, these results suggest that T315I mutation clearly induces metabolic changes different from N-BCR-ABL in leukemic cells, rendering them identifiable by the analysis of nucleic acid, lipid, amid and sugar contents. This new methodology can now be applied to the identification of primary CML leukemic cells harbouring T315I at the single cell level and could be of interest for rapid identification of leukemic cells in a single step. This technique can also be used for rapid screening of novel compounds active against T315I mutation using microfiuidic technologies leading to novel drug discoveries.

Claims

CLAIMS:
A method for identifying in a human subject sample a cell as being a tumour cell from chronic myeloid leukaemia bearing a T315I mutation, comprising the steps consisting in:
- passing a beam of infrared light through a bone marrow cell sample or a blood nonnucleated cell sample in vitro according to micro Fourier Transformed infrared microspectroscopy (FTIR) which produces a vibrational spectrum in the wavelength range of 22222.2 nm to 2500 nm, and therefore obtaining a first vibrational spectrum,
- comparing the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T3151 mutation,
- identifying the sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed.
The method according to claim 1 , characterized in that the vibrational spectrum is in the wavelength range of 10000 to 5555.56 nm
The method according to claim 1 or 2, wherein FTIR is combined with Principal Component Analysis (PCA) analysis of the results of FTIR.
A method for predicting the responsiveness of a patient affected by a chronic myeloid leukemia (CML) to a treatment with a tyrosine kinase inhibitor (TKI), comprising the steps of
- obtaining a bone marrow cell sample or a blood nonnucleated cell sample from the patient,
- passing a beam of infrared light through the bone marrow cell sample or blood nonnucleated cell sample in vitro according to micro Fourier Transformed infrared microspectroscopy (FTIR) which produces a vibrational spectrum in the wavelength range of 22222.2 nm to 2500 nm, and therefore obtaining a first vibrational spectrum, - comparing the first vibrational spectrum to a reference vibrational spectrum obtained by FTIR on reference cells from chronic myeloid leukaemia without T3151 mutation,
- identifying the sample cells as being tumour cells from chronic myeloid leukaemia bearing a T315I mutation if a difference in composition or band shape between the reference vibrational spectrum and the first vibrational spectrum is noticed,
- concluding that the patient would not advantageously receive a tyrosine kinase inhibitor treatment, if the sample cells are tumour cells from chronic myeloid leukaemia bearing a T315I mutation.
5. A method for treating a patient affected with CML comprising a step of administrating to said patient a tyrosine kinase inhibitor compound, wherein the patient is classified as responder to a TKI treatment by the method according to claim 4.
EP15790561.3A 2014-11-06 2015-11-05 Method for identifying in a subject tumour cells bearing a t315i mutation Withdrawn EP3215636A1 (en)

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