Patent Application
20200041513
The present invention lies in the field of diagnosing or assisting diagnosing and monitoring minimal residual disease in malignant hemopathies and the subject thereof is a method for detecting tumoral plasma cells and a method for diagnosing or assisting diagnosing and monitoring monoclonal gammopathies.
Multiple myeloma (MM) or Kahler's disease is a malignant hemopathy characterized by the accumulation of tumoral plasma cells (B lymphocytes) in the bone marrow, and the accumulation of a whole monoclonal immunoglobulin or of a monoclonal light chain. This pathological condition affects 2000 new patients per year in France, 19 000 in Europe and 19 000 in the United States. The median survival expectancy of patients 65 years old or younger is 6-7 years. The diagnosis of MM includes bone signs, an increased sedimentation rate, the presence of monoclonal immunoglobulin and the use of a myelogram showing:
MM is in all cases preceded by a benign monoclonal gammopathy (MGUS from Monoclonal Gammopathy of Undeterminated Significance). MGUSs are detected in 2 to 4% of subjects 60 years old, 10% of subjects 70 years old, 20% of subjects 80 years old. MGUSs progress to an MM with a degree of transformation of 1% per year, independent of the date of diagnosis of the MGUS.
MM represents 1% of new cases of cancer per year, 10 to 15% of malignant hemopathies and is the second most common malignant hemopathy after non-Hodgkin's lymphoma. Furthermore, the costs associated with its treatment are among the highest.
Recent data from the literature show that the identification and characterization of tumoral and normal plasma cells in patients suffering from MM provide a reliable marker for the response to treatment. The group of Prof. San Miguel (Paiva et al., 2014) has shown that, when there exists fewer than 1 tumoral plasma cell per 10 000 total leukocyte cells (with a sensitivity of 10−4) three months after a high-dose chemotherapy followed by a hematopoietic stem cell autograft, the patients have a significantly increased survival. The tumoral plasma cell/normal plasma cell ratio is a prognostic factor independent of the usual prognostic markers (Mateo et al., 2008). The proliferation index of tumoral plasma cells, calculated by flow cytometry, is also a powerful prognostic factor. It can be decisive for evaluating whether or not a patient is in active relapse. The median level of S-phase cells is 1% in an individual who is ill (Paiva et al., 2012) and is increased in the patients who are in relapse.
Moreover, minimal residual disease (MRD) in multiple myeloma denotes the persistence of residual tumor cells after treatment. Plasma cells represent up to 5% of the cells in the bone marrow, and the presence of MRD is defined by the presence of at least 50 tumoral plasma cells per 500 000 total leukocyte cells, i.e. 0.01% of the cells (Rawstron et al., 2013).
The median survival time of a patient is 7 months without treatment, from 3 to 4 years with conventional chemotherapy treatment and from 6 to 7 years with a treatment with high-dose chemotherapy and autograft. Thus, the monitoring of MRD after treatment is also very important in order to detect relapses as early as possible and to improve the therapeutic treatment.
At the current time, there are various methods for detecting MRD, for example “Deep Sequencing”, quantitative PCR (Polymerase Chain Reaction) with specific primers and multiparametric flow cytometry (MFC) Martinez Lopez et al., 2014; Paiva et al., 2008). The first two methods have a high sensitivity; however, they remain very expensive, and lengthy, and are difficult to automate. Thus, multiple color flow cytometry is at the current time the most suitable method for evaluating patient response, for detecting relapse early on and for improving the therapeutic treatment in order to maximize the treatment benefit/cost ratio.
Given the very small amount of normal plasma cells present in the bone marrow, i.e. a median value of 0.44% mononuclear cells in a healthy donor, this means that a large number of cell events must be acquired in order to minimize statistical errors.
Currently, a method set up by the STI laboratory (Caraux et al., 2010, 2012) takes 2 days, i.e. 16 working hours, in order to provide clinicians with a result. Furthermore, such a method uses at least seven colors, seven detectors, two lasers and five tubes, and therefore a large and expensive number of devices and reagents. In addition, the use of such a method requires the knowhow of an expert in cytometry for the preparation, acquisition, compensation and analysis of the data.
There is therefore a real need to develop new methods and compositions for the diagnosis and monitoring of the progression of this disease, and in particular to improve the sensitivity and the reliability of the tests for detecting tumoral plasma cells, while at the same time reducing the costs and the complexity of the various experimental steps.
The inventors have developed a method for detecting tumoral plasma cells which has very high sensitivity, reproducibility and reliability.
The method according to the invention makes it possible, through the use of a particular combination of tumoral and normal plasma cell markers, to obtain results from detection of normal and tumoral plasma cells that are at least equivalent to those obtained using the detection methods already known in the prior art.
Compared with the prior art methods, the method according to the invention has the advantage of being able to be carried out in a single container, such as a tube or a well of a plate with wells, comprising the sample to be analyzed and the various markers. This has in particular the advantage of being less expensive, of reducing the time and the complexity of the various experimental steps, and of reducing the delay in obtaining the results. Thus, the method according to the invention makes it possible to decrease the requirements for qualified personnel and the associated risks of errors, thus at the same time increasing the reliability of the method. Furthermore, the decrease in complexity for various experimental steps of the method according to the invention makes it possible to improve the reproducibility and to increase the productivity.
The subject of the invention is a method for detecting, by flow cytometry, the presence of normal plasma cells and of tumoral plasma cells in a sample of cells from a patient, said method comprising the following steps:
a) bringing said sample of cells into contact with:
According to other embodiments, use may be made, as an alternative to the respective CD38 and CD138 markers, of one or more markers chosen from the group consisting of: a CD229 marker, a CD54 marker, a CD319 marker (Pojero et al., Utility of CD54, CD229, and CD319 for the identification of Plasma Cells in Patients with Clonal Plasma Cell Diseases), a CD319 marker, or a CD269 marker (Frigyesi et al., Blood. 2014 Feb. 27; 123(9):1336-40. doi: 10.1182/blood-2013-09-529800). Mention may also be made of a MUM1/IRF4 marker which is an intracytoplasmid marker.
The abovementioned markers can be used individually, as a replacement for the respective CD38 and CD138 markers, or in combination with them and/or in particular with CD38.
A κ chain marker, a λ chain marker and a CD229 marker may thus for example be used.
It is understood from the remainder of the description and the examples illustrating the invention which are described hereinafter that the CD marker(s) according to the invention, for one and the same group of markers having a common expression differential (decrease or loss versus increase or gain), are coupled or conjugated to one and the same fluorochrome, or chromophore.
According to the present invention, firstly the tumoral plasma cell marker(s) in step a) emit a (fourth) signal different than the first three signals, in the sense that it is one and the same signal different than the first three signals, in other words a common signal or a “single detectable signal” different than the first three signals.
According to the invention, the term “one and the same signal” or “single detectable signal” is intended to mean that the tumoral plasma cell marker(s) are characterized in that they emit a common signal recognized by the detector.
According to one preferred embodiment, said tumoral plasma cell marker(s) are coupled or conjugated to the same fluorochrome so as to emit one and the same signal otherwise known as “single detectable signal”, different than the first three signals.
According to one alternative, said tumoral plasma cell marker(s) are coupled conjugated to fluorochromes which emit signals that can be detected, in frequency bands close to, equivalent to or that can be likened to one and the same signal otherwise known as “single detectable signal”, by a detector of suitable frequency, said single detectable signal being different than the first three signals.
According to the present invention, secondly, the normal plasma cell marker(s) in step a) emit a (fifth) signal different than the first four signals, in the sense that it is one and the same signal different from the first four signals, in other words a common signal or a “single detectable signal” different that the first four signals.
According to the invention, the term “one and the same signal” or “single detectable signal” is intended to mean that the normal plasma cell marker(s) are characterized in that they emit a common signal recognized by the detector.
According to one preferred embodiment, the normal plasma cell marker(s) are coupled or conjugated to the same fluorochrome so as to emit one and the same signal otherwise known as “single detectable signal”, different than the first four signals.
According to one alternative, said normal plasma cell marker(s) are coupled or conjugated to fluorochromes which emit signals that are detectable, in frequency bands close to, equivalent to or that can be likened to one and the same signal otherwise known as “single detectable signal”, by a detector of suitable frequency, said single detectable signal being different than the first four signals.
The detection of one and the same signal for one and the same group of markers having a common expression differential (decrease or loss versus increase or gain), advantageously makes it possible to reduce the number of fluorochromes and/or of detectable signals, and therefore the number of detectors and of sources in the detection method according to the invention, for a rapid (smaller data files) and simplified analysis.
Preferentially, the plasma cell marker according to the invention is a plasma cell antigen marker, preferentially a plasma cell cluster of differentiation (CD) marker.
The term “marker” is intended to mean any molecule(s) enabling the recognition of the target and the detection thereof by the emission of a detectable signal. Advantageously, for a peptide sequence labeling, the markers according to the invention are aptamers or antibodies conjugated to fluorochromes, preferentially the fluorochromes are selected from PE-Cy5.5, PE-CF594, PE-Cy7, PE, APC and FITC. Alternatively, for a DNA labeling, the markers according to the invention can also be base analogs, autofluorescent molecules when they bind to or intercalate into DNA (for example DAPI; blue fluorescence at approximately 456 nm) or molecules conjugated to fluorochromes (for example antibodies) which can bind to or intercalate into DNA. Advantageously, according to the invention, the tumoral plasma cell marker(s) are conjugated to one and the same fluorochrome (chromophore) for the emission of one and the same signal, distinct from the other signals. In addition, the normal plasma cell marker(s) are conjugated to another fluorochrome (chromophore) for the emission of one and the same signal, distinct from the other signals.
This makes it possible to obtain one and the same signal per group of markers having a common expression differential (decrease or loss versus increase or gain), simplifying the method and advantageously allowing implementation in a single container.
Thus, the amount of signal emitted by a marker according invention is linked to the amount of marker/target complexes, and therefore to the level of expression of peptide sequences and/or to the amount of target DNA.
The term “signal” is intended to mean any signal emitted with properties which make it possible to recover this signal after transmission. These properties can for example be frequencies, modulations, encodings. Preferentially, the detectable signals are emitted in frequency bands which they are assigned, more preferentially colors of the visible spectrum in an optical transmission. Thus, various markers which emit different detectable signals in close frequency bands can be detected by one and the same detectors, or different markers which emit one and the same single detectable signal can be detected by one and the same detector.
According to one particular preferred embodiment, use will be made, according to the invention, of one or more markers which emit one and same signal otherwise known as “single detectable signal” or one and the same group of markers having a common expression differential (decrease or loss versus increase or gain), distinct from the other signals.
One or the same fluorochrome (chromophore) thus corresponds to each group of markers having a common expression differential (decrease or loss versus increase or gain), so as to give the emission of a single signal.
The term “detector” is intended to mean any system/device which makes it possible to recover one of the detectable signals among the others, the detection thereof and/or the quantification thereof. Preferentially, the detector uses a filter to isolate the signal that it detects. More preferentially, this filter is of band-pass type. Preferentially, the detection and/or quantification is carried out by a photonic/electronic converter, for example photodiodes, avalanche photodiodes, photomultipliers (PMTs).
The term “normal plasma cell” is intended to mean plasma cells, plasmatic cells or plasmatocytes having the (cluster of differentiation; CD) antigens chosen from CD19, CD27, CD38, CD45, CD81, CD5L, CD11a, CD16b, CD24, CD36, CD52, CD68, CD79a, CD80, CD82, CD84, CD99, CD138, CD163, and not having the CD20, CD28, CD52, CD56, CD117 and CD200 antigens.
Normal plasma cells exhibit the kappa or lambda intra-cytoplasmic chains with respective proportions of ⅔ and ⅓.
The term “tumoral plasma cell” is intended to mean plasma cells, plasmatic cells or plasmatocytes having at least one modification relative to the normal profile:
The expression “positive signal” signifies according to the invention that the signal is said to be positive when the plasma-cell-specific markers used bind to their targets and emit a significantly detectable signal. The “positive signal” thus shows that the plasma cell(s) detected in step b) of the method according to the invention has(have) a significantly increased level of expression of the molecule, recognized by the marker(s) used, by the plasma cell(s) detected. A positive signal for a “CD” marker is commonly expressed by “CD−” or by the terminologies “CDlow or dim”, “CDmid or intermediate” or “CDhigh or bright” depending on the level of expression of said marker detected. Reference will also be made to a low or dim, mid or intermediate, or high or bright positive signal.
The fluorescence intensity observed on matrices such as those illustrated in the examples hereinafter, with, on the x-axis, the fluorescence intensity measured, is usually classified according to the following orders of magnitude (decades), by way of illustration:
The term “negative signal” is intended to mean that the signal is said to be negative when the plasma-cell-specific markers used do not bind, or bind weakly, to their targets and therefore do not emit a significantly detectable signal. The “negative signal” of these specific markers thus shows that the plasma cell(s) detected in step b) of the method according to the invention have a negative or significantly reduced level of expression of the molecule recognized by the marker(s) used. A negative signal for a “CD” marker is commonly expressed “CD”.
Preferentially, the cell sample used for the method according to the invention is chosen from, or obtained from, bone marrow, blood, serum, a blood extract, PBMCs (Peripheral Blood Mononuclear Cells), cerebrospinal fluid, pleural fluid and lymph nodes, preferentially the bone marrow of a patient.
According to one particular embodiment, the cell sample of the method according to the invention comes from a patient suffering from monoclonal gammopathy, or from an individual who may be suffering from a monoclonal gammopathy.
According to one preferred embodiment, the CDs specific for normal plasma cells of the method according to the invention are chosen from: CD19, CD27, CD45, CD81, CD5L, CD11a, CD16b, CD24, CD36, CD52, CD68, CD79a, CD80, CD82, CD84, CD99 and CD163, preferentially CD19, CD27, CD45 and CD81, more preferentially CD19, CD27.
According to one preferred embodiment, the CDs specific for tumurol plasma cells of the method according to the invention are chosen from: CD56, CD117, CD200, CD20, CD28, CD1D, CD2BP2, CD32c, CD47, CD59, CD109, CD229, CD300a and CD320, preferentially CD56, CD117, CD200, CD20, and CD28, more preferentially CD56, CD117, CD200.
Preferentially, the method according to the invention also comprises analyzing the FSC (Forward Scatter) and SSC (Side Scatter) signals. FSC and SSC correspond to simultaneous measurements carried out by flow cytometry making it possible to determine the physical and biological characteristics of isolated cells. By definition, the FSC corresponds to the intensity of the light, from the excitation source scattered in the axis (angle <12°), which is proportional to the size of the cell, while the SSC corresponds to the intensity of the light that is orthogonally scattered, termed “wide angle” (close to 90°), relative to the incident light source, which is representative of the intracellular complexity.
According to one preferred embodiment, the sample of the method according to the invention is brought into contact with:
Preferentially, the CD38 or CD138 marker is respectively an anti-CD38 or an anti-CD138 antibody conjugated to the PE-Cy5.5 fluorochrome, the κ chain marker is an anti-κ antibody conjugated to the PE-CF594 fluorochrome and the λ chain marker is an anti-λ chain antibody conjugated to the FITC fluorochrome; the detection being carried out on the appropriate flow cytometry detectors according to the prior art.
Advantageously, the CD19 and CD27 markers are anti-CD19 and anti-CD27 antibodies conjugated to the PE-Cy7 fluorochrome, and the CD56, CD117 and CD200 markers are anti-CD56, anti-CD117 and anti-CD200 antibodies conjugated to the PE fluorochrome; the detection being carried out on the appropriate flow cytometry detectors according to the prior art.
Furthermore, as previously indicated, the proliferation index of the tumoral plasma cells corresponds to the percentage of tumor cells in the S phase, calculated by flow cytometry, and is a powerful prognostic factor, making it possible to evaluate the speed of development of the tumor mass and to determine whether or not a patient is in active relapse.
Thus, according to one preferred embodiment, the method according to the invention also comprises determining the proliferation index of the tumoral plasma cells, comprising the following steps:
The term “proliferation markers” is intended to mean any markers which make it possible to determine whether a cell is in the G0/G1, G2/M or S cell cycle phase. The proliferation markers are for example chosen from:
According to one particular and preferred embodiment, use will be made of at least one proliferation marker which makes it possible to identify the S-phase cells, such as base analogs of BrdU or EdU type, and preferably of BrdU type.
According to one particular embodiment, the markers for tumoral plasma cell proliferation of the method according to the invention are selected from:
The term “BrdU” (BrdU FlowKit; BD Pharmingen) is defined as a synthetic nucleoside, which is a structural analog of thymidine, which can be incorporated into DNA and which can be detected by a specific antibody for determining the phase of the cell cycle for a cell and in particular whether said cell is in the DNA replication phase (S phase). The term “EdU” (Click-iT® EdU; Thermofisher) is defined as a synthetic nucleoside (structural analog of thymidine) which is incorporated into DNA during its replication. The bonding between an alkyne of the ethynyl fragment of EdU and an azide coupled to a fluorochrome is carried out by the cycloaddition reaction, one of the most popular ligation reactions in click-chemistry. The EdU can then be detected when it is incorporated into the DNA in order to determine whether the cells of interest are in the DNA replication phase (S phase).
Advantageously, the method for determining the proliferation index of the tumoral plasma cells according to the invention comprises the following steps:
Preferentially, the method for determining the proliferation index of the tumoral plasma cells according to the invention comprises the following steps:
Preferentially, the method for determining the proliferation index of the tumoral plasma cells according to the invention also comprises an additional step of evaluating the diploidy consisting in adding DAPI between steps C1 and D1 or B2 and C2.
Preferentially, the method for determining the proliferation index of the tumoral plasma cells according to the invention comprises the following steps:
Preferentially, the method for determining the proliferation index of the tumoral plasma cells according to the invention comprises the following steps:
The proportion of tumoral plasma cells relative to the other leukocytes gives a picture of the cell environment, but this proportion is influenced by the variations in the other leukocyte populations.
Quantification of the tumor cells gives, for its parts, an evaluation of the tumor mass. This quantification can be carried out by analyzing a known volume by flow cytometry, using counting particles, such as fluorescent polystyrene beads (for example sold by Coulter, FlowCyto Standard Corporation, Polysciences, Sperotech, BD TrueCounts, DakoCytomation, etc.), introduced in a known amount into a known volume of sample, or else indirectly by the proportion of tumoral plasma cells relative to an otherwise quantified population (for example a number of total leukocytes of the sample, resulting from an automated counting device).
Thus, according to one particular embodiment, the method for detecting, by flow cytometry, the presence of normal plasma cells and of tumoral plasma cells in a sample of cells from a patient according to the invention also comprises:
The expression “receiving the amount of a population” is intended to mean an action which makes it possible to provide the system with information regarding the amount of cells present in the sample to be analyzed or already analyzed (for example: quantification of the leukocytes). This action can for example consist of a computer transmission, or an input by the operator.
According to one particular embodiment, said patient is considered to be at risk of developing a monoclonal gammopathy when the number of tumoral plasma cells per unit of volume is greater than a predefined threshold value. This “predefined threshold value” is determined from results obtained by means of the method according to the invention from healthy and/or unwell control subjects. According to one particular embodiment of the present invention, the predefined threshold values according to the invention are parametrizable.
A subject of the present invention is also a method for diagnosing or assisting in diagnosing, in vitro, or monitoring monoclonal gammopathy, using a sample of cells from a patient, comprising the following steps:
A subject of the present invention is also a method for diagnosing or assisting in diagnosing, in vitro, monoclonal gammopathy using a sample of cells of a patient, wherein steps 1) and 2) according to the invention are automated.
A subject of the present invention is also a method for monitoring, in vitro, the progression of the disease of a patient suffering from a monoclonal gammopathy using a first and a second sample of cells from the patient, comprising the following steps:
A subject of the present invention is also a method for monitoring, in vitro, the progression of the disease of a patient suffering from a monoclonal gammopathy, using a first and a second sample of cells from the patient, characterized in that steps i), ii), iii) and iv) are automated.
The present invention also relates to a composition comprising at least:
Advantageously, the composition according to the invention comprises at least:
According to one advantageous embodiment, the composition according to the invention also comprises one or more marker(s) for tumoral plasma cells proliferation according to the invention, emitting a signal different than each of the signals of the other markers present in said composition.
The subject of the present invention is also the use of a composition comprising at least:
for carrying out the method according to the invention.
Advantageously, the subject of the present invention is the use of a composition comprising at least:
According to one particular embodiment, the use of a composition according to the invention also comprises one or more markers for tumoral plasma cell proliferation according to the invention, emitting a signal different than the signals of each of the other markers present in said composition.
A subject of the present invention is also a kit for carrying out the method according to the invention, comprising at least:
According to one preferred embodiment, the kit for carrying out the method according to the invention comprises at least:
According to one particular embodiment, the kit according to the invention also comprises one or more markers for tumoral plasma cell proliferation according to the invention, emitting a signal different than each of the other markers present in said kit.
Advantageously, the kit according to the invention also comprises at least one cell proliferation marker chosen from:
According to one preferential embodiment, the kit according to the invention is characterized in that it comprises at least one cell proliferation marker chosen from:
According to one preferential embodiment, the kit according to the invention is characterized in that it comprises at least one cell proliferation marker chosen from:
Other characteristics and advantages of the invention emerge in the following examples and figures:
The principle of this strategy is the discrimination of the cells of interest by means of their size, their structural complexity and the presence of cell markers, or antigens, recognized by antibodies coupled to fluorochromes. There are 8 antigens characteristic of plasma cells (CD38, κ, λ, CD19, CD27, CD56, CD117 and CD200) of which the specific antibodies are coupled to 5 different fluorochromes in a single tube (CD38/PeCy5.5, κ/PECF594, λ/FITC, (CD19, CD27)/PeCy7, (CD56, CD117, CD200)/PE). This strategy can be carried out in half a day. The anti-CD38, anti-kappa and anti-lambda antibodies are used to discriminate the total plasma cells from the other leukocytes and two groups of antibodies make it possible to identify the tumor cells. The negative group contains the anti-CD19 and anti-CD27 antibodies and the positive group contains the anti-CD56, anti-CD117 and anti-CD200 antibodies.
Following the taking of the bone marrow sample, said bone marrow must be filtered in order to remove any bone debris. The red blood cells are then lysed with an NH4Cl lysis (ratio) and the debris from this is to a large extent eliminated by centrifugation. The membrane antibodies coupled to their respective fluorochrome CD56-PE (Beckman Coulter; Cat.: A07788), CD117-PE (Beckman Coulter; Cat.: IM2732), CD200-PE (BD Pharmingen; Cat.: 552475) (positive group), CD19-PeCy7 (Beckman Coulter; Cat.: IM3628), CD27-PeCy7 (Beckman Coulter; Cat.: A54823) (negative group), CD38-PeCy5.5 (Beckman Coulter; Cat.: A70205) are directly deposited in the cell preparation according to the producer's recommendations. After incubation for 20 minutes at 4° C. and washing with PBS, the cells are fixed (Intraprep kit; Beckman Coulter; Cat.: A07803). Said cells are then washed with PBS in order to remove all of the fixing solution. Finally, the cells are permeabilized (Intraprep kit; Beckman Coulter; Cat.: A07803) and labeled with the pairs consisting of intracytoplasmic antibodies/fluorochromes κ-PE-CF594 (BD Pharmingen; Cat.: 562620) and λ-FITC (BD Pharmingen; Cat.: 555796) according to the producer's recommendations. After incubation for 20 minutes at 4° C. and washing with PBS, the sample is analyzed by means of the flow cytometry device (BD LSRFORTESSA X-20, BD Biosciences).
Firstly, the cells which pass through the measurement window at the same time are eliminated using the FSC and SSC parameters which correspond, respectively, to the size and to the structural complexity of the cell. For these two parameters, the matrix of height and area of the measurement peak is used and the events departing from the diagonal of this matrix are eliminated. This is because the height and area values are proportional and the absence of this proportionality (points outside the diagonal) reflects the presence of doublets or even multiplets.
The cell debris due to the lysis, mainly the red blood cell debris, is then eliminated. The FSC/SSC matrix is used to select all of the leukocytes and to eliminate the cell debris having a small size and a structure which is not very complex.
The selection of the total plasma cells is carried out by means of three markers, making it possible to detect CD38 and the two intracytoplasmic proteins κ and λ. The plasma cells are cells strongly expressing the CD38 marker on the surface of their membrane. These cells are capable of producing antibodies composed of (κ or λ) light chains and of heavy chains. Each plasma cell can only produce one type of light chain and the proportion of plasma cells termed “kappa” or “Lambda” plasma cells is ⅔ and ⅓, respectively. In the patients suffering from multiple myeloma, the proliferation of a κ or λ clone will induce an imbalance of the κ/λ ratio. The plasma cells are selected on the CD38/κ and CD38/λ matrices independently.
The events which are on the diagonal of the κ and λ dimensions are eliminated. Indeed, plasma cells can secrete in the κ light chain or the λ light chain throughout their life, but never both.
Once the total plasma cells have been selected, the tumoral plasma cells must be discriminated with respect to the normal plasma cells by selecting the cells expressing a marker of the positive group (CD56/CD117/CD200) measured on the PE channel and/or having a loss of expression of one of the markers of the negative group (CD19/CD27) measured on the PE-Cy7 channel.
The strategy developed and carried out by the “Suivi des thérapie innovant” [“Innovative therapy monitoring” ] laboratory and described in the prior art (Caraux et al.; 2012), that will be referred to herein as “STI strategy”, calls for 10 antibodies directed against antigens characteristic of plasma cells (CD38, CD45, CD19, CD20, κ, λ, CD27, CD56, CD117 and CD200) coupled to 7 different fluorochromes distributed into 4 independent tubes.
The BrdU analysis makes it possible to determine the percentage of S-phase (synthesis phase) tumor cells. DAPI (4′,6′-diamidino-2-phenylindole, a fluorescent molecule capable of strongly binding to the adenine (A) and thymidine (T) bases of DNA) is used to quantify the DNA (2N/4N).
In order to monitor cell proliferation, the APC BrdU Flow Kit was used according to the recommendations of the supplier (BD Pharmingen, Cat.: 557892).
Subsequent to the protocol described in example 1, 350 μl of a DAPI/PermWash mixture at 2 μl/ml were added to the cell solution and then incubated for 20 minutes at 4° C. The cell solution was then washed with PBS and then taken up in PBS, before being analyzed by flow cytometry (CyAn™ ADP cytometer—Beckman Coulter).
Patient suffering from multiple myeloma with a normal level of S-phase plasma cells (0.60%) (
Patient suffering from multiple myeloma with a high level of S-phase plasma cells (4.89%) (
Ki67 makes it possible to quantify the percentage of cells engaged in the cell cycle (non-quiescent cells). DAPI (4′,6′-diamidino-2-phenylindole, a fluorescent molecule capable of binding strongly to the adenine (A) and thymidine (T) bases of DNA) is used to quantify the DNA (2N/4N).
Two tubes are used, a negative control with the APC isotype (Beckman Coulter, Ref: IM2475U) added to the cell solution at the time of the surface antibody labeling, and a Ki67 tube positive with the anti-Ki67 coupled to Alexa Fluor 647 (BD Pharmingen, Cat.: 558615) added to the cell solution at the time of the intracytoplasmic antibody labeling.
Subsequent to the preceding protocol, 350 μl of a DAPI/PermWash mixture at 2 μl/ml were added to the cell solution and then incubated for 20 minutes at 4° C. The cell solution is then washed with PBS then taken up in PBS, before being analyzed by flow cytometry (CyAn™ ADP cytometer—Beckman Coulter).
Patient suffering from multiple myeloma with a level of cells engaged in the cell cycle (non-quiescent cells) (21%) (
Patient suffering from multiple myeloma with a level of cells engaged in the cell cycle (non-quiescent cells) (39.5%) (
To monitor the cell proliferation, the Click It® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit was used according to the recommendations of the supplier (Life Technologies, Cat.: C10635).
Subsequent to the preceding protocol, 350 μl of a DAPI/PermWash mixture at 2 μl/ml were added to the cell solution and then incubated for 20 minutes at 4° C. The cell solution is then washed with PBS then taken up in PBS, before being analyzed by flow cytometry (BD LSRFortessa™ X-20 cytometer—BD Biosciences).
The monitoring of the plasma cell proliferation can be carried out in two dimensions (DAPI vs EdU) or in one dimension (EdU histogram).
The results presented in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1654514 | May 2016 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/062307 | 5/22/2017 | WO | 00 |
