JP7836038B2 - Method for determining the effectiveness of treatment in melanoma patients - Google Patents

Description

This invention relates to a method for determining the effectiveness of treatment for melanoma patients. In particular, this invention relates to a method for determining the effectiveness of treatment for melanoma patients using tumor cells derived from biological samples other than primary melanoma tissue and melanoma metastasis tissue (e.g., blood samples).

Determining the effectiveness of treatments and predicting prognosis in cancer patients is crucial for providing physicians with information on the risks and survival probabilities associated with the patient's condition, enabling them to select the optimal therapy. This reduces the risk of unnecessary treatments and associated costs, and contributes to improving the patient's prognosis.

To assist in the treatment of cancer patients, tests using soluble tumor antigens (CHTs) are being conducted. Soluble tumor antigens are secreted by tumor cells and can be detected in blood samples, leading to attempts at applications such as monitoring treatment effectiveness. For example, CEA (Carcinoembryonic Antigen) and CA19-9 (Carbohydrate Antigen 19-9) are used as tumor markers for gastrointestinal cancers. However, since soluble tumor antigens are also released upon the destruction of tumor cells, their presence does not necessarily fully reflect the prognosis of cancer patients.

On the other hand, circulating tumor cells (CTCs) in the blood are attracting attention as an alternative detection target to primary cancer tissue or metastatic cancer tissue. By monitoring the number of CTCs in blood samples collected from patients, it is possible to evaluate the patient's overall condition, including prognosis and treatment effectiveness.

For example, Patent Document 1 discloses a method for predicting the patient's prognosis by using an epithelial marker expressed on the surface of CTCs to immunomagnetically concentrate CTCs from a patient's blood sample, then detecting the CTCs in the concentrate using immunofluorescence staining with the epithelial marker, measuring the number of cells, and using the change in the number of these cells as an indicator. However, in the case of melanoma, since the cells are not epithelial, they cannot be detected with an epithelial marker. Therefore, the method described in Patent Document 1 has the problem of not being able to predict the therapeutic effect or prognosis of melanoma patients with high accuracy.

Furthermore, Patent Document 2 discloses a method for monitoring disease progression in melanoma patients by immunomagnetically concentrating CTCs expressing CD146, a melanoma cell marker, from blood samples collected from melanoma patients, and then detecting the CTCs contained in the concentrate using immunofluorescence staining with Ki-67, a cell proliferation-related marker, and using the change in the number of these cells as an indicator. However, the method disclosed in Patent Document 2, like that in Patent Document 1, has problems in terms of accuracy.

Special Publication No. 2008-533487 Special Publication No. 2011-505012

This invention has been made in view of these circumstances, and its objective is to provide a method for determining the effectiveness of treatment in melanoma patients with minimal invasiveness and high accuracy.

To solve the above problems, the inventors conducted extensive research and discovered that it is possible to determine the therapeutic effect on melanoma patients by using changes in the number of circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes in biological samples collected from melanoma patients as indicators. This led to the present invention.

In other words, the first aspect of the present invention is a method for determining the therapeutic effect of treatment on a melanoma patient, comprising the steps of detecting circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes in a biological sample collected from the patient, and measuring the number of such cells over time, with the change in the number of such cells being used as an indicator of the therapeutic effect on the patient.

Furthermore, a second aspect of the present invention is the method according to the first aspect, comprising the step of concentrating circulating tumor cells contained in a biological sample collected from the patient to obtain a concentrate, prior to detecting circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes.

Furthermore, a third aspect of the present invention is the method according to the first aspect, wherein the melanoma cell marker gene is at least one gene selected from the group consisting of (i) to (iii) below.
(i) A gene encoding a polypeptide comprising at least one of the amino acid sequences described in Sequence ID No. 1 to 128,
(ii) A gene encoding a polypeptide having at least one amino acid sequence having 70% or more homology to the amino acid sequence described in any of Sequence IDs 1 to 128.
(iii) A gene encoding a polypeptide comprising at least one amino acid sequence in which one or more amino acids are substituted, deleted, inserted and/or added, as described in any of Sequence IDs 1 to 128.

Furthermore, a fourth aspect of the present invention is the method according to the first to third aspects, wherein the disease state determination marker gene is the ICAM-1 gene.

This invention is characterized by evaluating the effectiveness of treatment for melanoma (malignant melanoma) patients using changes in the number of circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes contained in the patient's biological sample as indicators. This invention makes it possible to determine the effectiveness of treatment for melanoma patients with minimal invasiveness and high accuracy. This allows physicians to provide optimal drug dosage indicators for the patient's condition, as well as information on the risk of the disease and the patient's survival probability, enabling them to select the most appropriate treatment. As a result, the risk of administering unnecessary treatments (unnecessary anticancer drug administration) to patients can be reduced, contributing not only to saving unnecessary treatment costs but also to improving the patient's prognosis. Furthermore, this invention can also be applied to the early detection of disease progression in melanoma diagnosis.

This figure (exploded view) shows an example of a cell detection device capable of retaining circulating tumor cells (CTCs) that can be used in the process of detecting CTCs in the blood according to the present invention. Figure 1 is a front view of the device shown. This figure shows an example of a process for detecting CTC using the apparatus shown in Figure 1.

This invention provides a method for determining the effectiveness of treatment in melanoma patients. The method of this invention is characterized by detecting circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes in a biological sample collected from a melanoma patient, and measuring the number of such cells over time.

In this invention, "melanoma patients" include patients who have received treatment for melanoma (malignant melanoma), patients undergoing treatment for melanoma, and patients before treatment for melanoma. While there are no particular limitations on "biological samples" as long as they contain circulating blood cells (CTCs), examples include blood samples such as whole blood, diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and component blood samples, as well as samples that may contain blood-derived components such as urine, saliva, semen, feces, sputum, amniotic fluid, and ascites.

In this invention, "circulating tumor cells" refers to tumor cells that have infiltrated the blood vessels or lymphatic vessels from a primary tumor (primary lesion) or metastatic tumor (metastatic lesion). Of these, circulating tumor cells that circulate in the blood are specifically referred to as "circulating tumor cells" (CTCs). Circulating tumor cells exist, for example, as tumor cells circulating in the peripheral blood flow of cancer patients.

In the present invention, it is preferable to include a step of concentrating CTCs contained in a biological sample collected from the patient to obtain a concentrate, prior to detecting circulating tumor cells in the blood that express melanoma cell marker genes and disease state determination marker genes. The concentration of CTCs can be performed by known methods, such as density gradient centrifugation (Japanese Patent Publication No. 2015-006169) or filtering (Japanese Patent Publication No. 2014-233267).

When obtaining a fraction (concentrate) containing CTCs by density gradient centrifugation, the sample is layered in a density gradient solution and then centrifuged. During this centrifugation, contaminating cells (red blood cells, white blood cells, etc.) in the sample move to the lower layer (density gradient solution side), while the CTCs remain in the upper layer (sample side). Therefore, by recovering this upper layer, a fraction containing CTCs (hereinafter also referred to as "CTC concentrate") can be obtained. It is preferable to obtain the CTC concentrate using a container (Japanese Patent Publication No. 2015-006169) that allows for the separation of the upper and lower layers, as this facilitates the acquisition of the CTC concentrate. Furthermore, if the biological sample collected from the patient is a blood sample or a sample containing blood-derived components, it is preferable to perform a hemolysis step (hemolysis operation) of the sample before layering it in the density gradient solution. This reduces the number of contaminating cells, such as red blood cells, and thus reduces the number of red blood cells mixed into the upper layer. Note that the hemolysis operation may also be performed after density gradient centrifugation, in which case further removal of contaminating cells by centrifugation or other methods may be performed.

The CTC concentrate obtained by the method described above may be preserved by cryopreservation or chemical fixation. For example, in the case of cryopreservation, the solution should be replaced with a cell preservation solution and then stored at a temperature of 0°C or below, preferably -20°C or below, and more preferably -80°C or below. In the case of chemical fixation, a stabilizer should be added to the cell suspension, and a cell fixation treatment should be performed to insolubilize and/or inactivate proteins, thereby suppressing the deterioration of the cells for a long period of time. Examples of stabilizers used in chemical fixation include solutions containing cell fixatives such as aldehydes, acids, dehydrating agents/organic solvents, and metal salts.

In the method of the present invention, CTCs expressing melanoma cell marker genes and disease state determination marker genes are detected in the aforementioned biological sample. In one preferred embodiment for detecting CTCs, first, a sample containing CTCs (e.g., a CTC concentrate) is applied to a slide or introduced into a device capable of holding CTCs to retain them. Then, the retained CTCs expressing the melanoma cell markers and disease state determination markers can be detected using a microscope or optical detector. In another preferred embodiment for detecting CTCs, the sample containing CTCs may be introduced into a flow cytometer to detect CTCs expressing the melanoma cell markers and disease state determination markers.

An example of a cell detection device capable of detecting CTCs is shown in Figure 1, and its front view is shown in Figure 2.

The cell detection device 100 shown in Figures 1 and 2 is
A flat plate-shaped light-shielding member 11 having a through hole 11a,
A flat plate-shaped insulator 12 having a through hole 12a,
A flat plate-shaped spacer 20 having an inlet 21, an outlet 22 and a through-hole 23,
Electrode substrates 31 and 32 are provided so as to be in close contact with the lower part of the light-shielding member 11 and the upper part of the spacer 20,
A conductor 40 connects electrode substrates 31 and 32,
A signal generator 50 that applies a signal to electrode substrates 31 and 32,
It is equipped with.

The through-holes 11a in the light-shielding member 11 and 12a in the insulator 12 are identical in size and shape, and the light-shielding member 11 and the insulator 12 are positioned so that the locations of their respective through-holes coincide. The through-holes 11a and 12a, along with the electrode substrate 31 which is in close contact with the lower part of the light-shielding member 11, constitute a holding section 60 within the cell holding means 10. When a liquid containing cells is introduced through the inlet 21, the cells are introduced into the holding section 60 through the through-holes 23. The electrode substrate 32 is in close contact with the upper part of the spacer 20, preventing the scattering and evaporation of the liquid containing cells introduced through the inlet 21. To facilitate the retrieval of cells held in the holding section 60, the electrode substrate 32 is detachable from the spacer 20. Furthermore, using transparent electrodes such as ITO (indium tin oxide) for the electrode substrates 31 and 32 is preferable, as it allows the cells held in the holding section 60 to be detected using a microscope or optical detector.

When holding CTCs in the cell detection device 100 shown in Figures 1 and 2, it is preferable to introduce the sample containing CTCs through the inlet 21 provided in the spacer 20, and then generate dielectrophoretic force by applying an AC voltage from the signal generator 50 to the electrode substrates 31 and 32 via the conductor 40 to hold the CTCs. When introducing a sample containing CTCs (for example, a concentrated CTC solution) into the cell detection device 100, it is preferable to first obtain a pellet containing CTCs by centrifuging the sample, and then suspend the pellet in a solution containing sugars such as mannitol, glucose, and sucrose before introducing it into the cell detection device 100, as this reduces damage to the CTCs. In addition to the sugars mentioned above, the suspension of the pellet may further contain proteins such as BSA (bovine serum albumin) and casein, or proteins to which hydrophilic polymers are bound. The concentration of sugar in the pellet suspension should be isotonic with respect to the CTC. When using mannitol as the sugar, the final concentration should be between 250 mM and 350 mM. Examples of AC voltages applied to the electrode substrates 31 and 32 include sine waves, square waves, triangular waves, and trapezoidal waves, with peak voltages of approximately 1V to 20V and frequencies of approximately 10kHz to 10MHz. For example, if you want to hold each live CTC individually in the holding section, it is preferable to use a square wave with a frequency of 100kHz to 3MHz.

In this invention, the "melanoma cell marker gene" whose expression is detected in CTCs is a gene that has higher specificity to melanoma cells than to contaminating cells contained in a biological sample (e.g., a blood sample or a sample containing blood components). "Contaminating cells" refer to cells other than melanoma cells, such as red blood cells, white blood cells, and platelets. "High specificity" means that melanoma cells express the gene at a higher level at the transcriptional or translational level than at least one contaminating cell. Preferably, melanoma cells express the melanoma cell-specific gene at 1.5 times or more, more preferably at 2 times or more, even more preferably at 3 times or more, and particularly preferably at 5 times or more (e.g., 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more) than contaminating cells.

In the method of the present invention, a preferred embodiment of the "melanoma cell marker gene" is, if of human origin, typically a gene encoding a polypeptide containing at least one of the amino acid sequences described in SEQ ID NOs: 1 to 128. However, in the present invention, homologs of these genes (e.g., counterpart genes in organisms other than humans) can also be targeted (detected). Furthermore, since the DNA sequence of a gene can mutate naturally (i.e., unartificially) due to mutations, such naturally occurring mutants can also be targeted in the present invention.

The homologous amino acid sequence may be an amino acid sequence having 70% or more homology, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (for example, 96%, 97%, 98%, 99% or more) to the entire amino acid sequence described in any of Sequence IDs 1 to 128. Sequence homology can be determined, for example, using the BLASTP program (Altschul et al., J. Mol. Biol., 215:403-410, 1990).

Furthermore, the amino acid sequence of the mutant may be an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added in any of the amino acid sequences described in (iii) SEQ ID NOs: 1 to 128. The number of amino acid mutations in nature is generally within a few. Here, "a few" means an integer of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. One to several amino acid residues preferably consist of 1 to 10 amino acid residues, more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 2 or fewer.

Tables 1 to 11 below show the sequence number, the name of the protein to which the polypeptide composed of each amino acid sequence corresponds, its official name (full name) and alias, and the GenBank accession number (GenBank No.) for each amino acid sequence, for each sequence from sequence number 1 to 128.

These polypeptides are markers that can distinguish and detect melanoma cells contained in a biological sample from leukocytes contained in the same sample, and were discovered by the inventors as transmembrane polypeptides. Among these, the genes encoding gp100 (also known as PMEL, SEQ ID NO: 1) and MART-1 (Melanoma Antigen Recognized by T cells-1, also known as MLANA, SEQ ID NO: 3), which have a high gene expression ratio of melanoma cells to leukocytes, are particularly preferred as the melanoma cell marker genes.

In the present invention, the "pathological condition marker gene" whose expression is detected in CTC is a gene that serves as an indicator for evaluating the invasiveness and metastatic potential of tumor cells. Examples of such genes include the ICAM-1 gene, MMPs (Matrix metalloproteinases, e.g., MMP-2, MMP-7, MMP-9, etc.) genes, TIMPs (Tissue inhibitor of metalloproteinases, e.g., TIMP-1, TIMP-2, etc.) genes, and ADAMs (A disintegrin and metalloproteinases, e.g., ADAM28, etc.) genes. Among these, the ICAM-1 (Intercellular Adhesion Molecule-1) gene, which is more highly expressed in melanoma metastatic tissue than in primary melanoma tissue and has been reported to be involved in metastasis (Oncotargetet, 2017, pp. 99580-99586), is preferred as the disease state determination marker gene. Similar to the melanoma cell marker genes mentioned above, the disease state determination marker gene can target not only genes encoding typical human polypeptides, but also homologs of these genes (e.g., counterpart genes in non-human organisms) or natural mutants.

In this invention, "gene expression" includes both gene transcription and translation. Therefore, detection of gene expression in CTC includes detection at the transcriptional level (mRNA level) and detection at the translational level (protein level) (i.e., detection of the polypeptide encoded by the gene).

Furthermore, in eukaryotic cells, during gene transcription, a reaction called splicing occurs in which introns are removed from mRNA precursors and exons are rejoined. However, diversity can arise in this exon rejoining, resulting in the production of various mature mRNAs. Ultimately, these mature mRNAs are translated into various proteins. These diverse mRNAs and proteins resulting from differences in splicing are called "splicing variants." Therefore, the detection of gene expression in this invention includes the detection of these splicing variants.

For the detection of gene expression in the present invention, known methods or methods similar thereto can be used as appropriate. For detection at the transcription level, for example, a probe corresponding to an appropriate position in the base sequence (polynucleotide) of the transcript (mRNA) of the target gene may be designed and then detected by Northern blotting, dot blotting, RNase protection assay, DNA microarray analysis, in situ hybridization, etc. Alternatively, a primer set corresponding to an appropriate position in the polynucleotide may be designed and then detected by, for example, PCR, RT-PCR, TRC (Transscription Reverse transcription Concerted), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transscription-Mediated) The polynucleotides may be amplified and detected using methods such as amplification, or the sample containing the polynucleotides may be directly subjected to a sequencer for detection.

Furthermore, detection at the translational level can be achieved using methods such as immunohistochemistry, imaging cytometry, flow cytometry, ELISA, radioimmunoassay, immunoprecipitation, immunoblotting (Western blotting, etc.), antibody arrays, and in vivo imaging (immunological methods); or by using aptamers instead of antibodies. These immunological methods may also be performed automatically using enzyme immunoassay instruments such as the AIA-900 or AIA-CL2400 (both manufactured by Tosoh Corporation) after preparing the necessary reagents.

In the present invention, from the viewpoint of simplicity, detection of gene expression is preferably performed at the translational level (detection of the polypeptide encoded by the target gene). In particular, a method of detecting the polypeptide using an antibody that specifically recognizes the polypeptide (hereinafter, optionally referred to as "anti-polypeptide antibody") or an aptamer that specifically recognizes the polypeptide (hereinafter, optionally referred to simply as "aptamer") is preferred, and a method of detecting the polypeptide using the anti-polypeptide antibody is more preferred.

In the present invention, "antibody" may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. Furthermore, "antibody" includes all classes and subclasses of immunoglobulins. A "functional fragment" of an antibody is a part (partial fragment) of an antibody that specifically recognizes the polypeptide encoded by the target gene. Specifically, examples include Fab, Fab', F(ab') ₂ , variable region fragment (Fv), disulfide bond Fv, single-stranded Fv (scFv), sc(Fv) ₂ , diabodies, polyspecific antibodies, and polymers thereof.

The anti-polypeptide antibodies according to the present invention, if polyclonal antibodies, can be obtained by immunizing an immunized animal with an antigen (a polypeptide encoded by a target gene, a partial peptide thereof, or cells expressing these, etc.), and purifying the antiserum using conventional methods (salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be produced by hybridoma or recombinant DNA methods. Examples of hybridoma methods include the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)), and examples of recombinant DNA methods include cloning DNA encoding the anti-polypeptide antibody from hybridomas or B cells, incorporating it into a suitable vector, introducing it into host cells (mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the anti-polypeptide antibody as a recombinant antibody (e.g., P.J. Delves, Antibody Production: Essential Techniques, 1997; WILEY, P. Shepherd and C. Dean, Monoclonal Antibodies, 2000; Oxford University). PRESS, Vandamme A. M. et al. , Eur. J. Biochem. 192:767-775 (1990)).

In this invention, molecules that bind to the expression product of a target gene, such as anti-polypeptide antibodies and aptamers, can be those to which a labeling substance has been attached. By using the molecule to which the labeling substance has been attached and detecting the labeling substance, the amount of the molecule bound to the expression product encoded by the target gene can be directly measured. The labeling substance is not particularly limited as long as it can bind to the molecule and can be detected by chemical or optical methods. Examples include fluorescent substances such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), and Alexa Fluor (trade name), as well as enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), and alkaline phosphatase, and radioactive substances.

Furthermore, in this invention, when detecting the expression of a target gene is the detection of the polypeptide encoded by that gene, and when using the anti-polypeptide antibody, indirect detection methods such as using a secondary antibody conjugated with the labeling substance, or using a polymer conjugated with the secondary antibody and the labeling substance, can also be used. Here, "secondary antibody" refers to an antibody that exhibits specific binding affinity to the anti-polypeptide antibody. For example, if the anti-polypeptide antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies usable for antibodies derived from various biological species such as rabbits, goats, and mice are commercially available, and an appropriate secondary antibody can be selected and used depending on the biological species from which the anti-polypeptide antibody originates. Alternatively, it is also possible to use protein G or protein A conjugated with a labeling substance instead of a secondary antibody.

When melanoma cell marker genes or disease-determining marker genes encode polypeptides expressed on the cell membrane, such as transmembrane polypeptides, CTCs can be captured and/or recovered via these polypeptides expressed on the cell membrane, and then the expression of target genes can be detected by targeting these polypeptides. For this capture and/or recovery, a carrier-immobilized molecule comprising a carrier and a molecule that binds to the polypeptide supported on the carrier can be used.

Examples of carriers for the carrier-immobilized molecule include plates, fibers, membranes, particles, and microfluidic chips. Preferably, the molecule that binds to the polypeptide is an antibody that binds to the polypeptide. Such carrier-immobilized molecules can be conventionally known molecules or commercially available molecules.

The method for recovering the CTCs captured by the carrier-immobilized molecules is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, if the carrier is a plate, a method of removing the liquid phase (supernatant) from the plate after the capture step can be used, or if the carrier is particles, a method of recovering the particles by centrifugation or magnetic collection after the capture step and then removing the liquid phase (supernatant) can be used. A preferred example of a recovery means is a means comprising a substrate with a holding part capable of holding the CTCs and a recovery part that recovers the CTCs by suction discharge using a nozzle. A specific example is the recovery device disclosed in Japanese Patent Application Publication No. 2016-142616.

In a preferred embodiment of CTC detection in the present invention, the presence of a cell nucleus and/or low expression of a leukocyte marker gene are further indicators. Examples of the "leukocyte marker gene" include, but are not limited to, the CD45 gene. Furthermore, "low expression of a leukocyte marker gene" means that the expression level of the leukocyte marker gene in the target cell is less than half, preferably less than 1/3, more preferably less than 1/5, and even more preferably less than 1/10, the expression level in leukocytes. Additionally, if the expression level of the leukocyte marker gene in the target cell is equivalent to that of negative control cells known not to express the leukocyte marker gene (e.g., vascular endothelial cells, mesenchymal stem cells), it can also be evaluated as "low expression of a leukocyte marker gene."

When detecting CTCs using optical instruments such as microscopes and optical detectors, cell nuclei can be detected by staining with a cell nucleus staining reagent such as 4',6-diamidino-2-phenyllindole (DAPI) or Hoechst 33342 (trade name). Furthermore, the detection of leukocyte marker gene expression can be performed using the same method as described above for detecting melanoma cell marker genes and disease state determination marker genes.

CTC detection using optical instruments can be performed by, for example, capturing images (bright-field images, fluorescence images, emission images, etc.) using an imaging device such as a camera, importing the images into a computer, and then using software to determine whether or not a CTC is present. Alternatively, CTC can be determined visually without the use of software.

In the method of the present invention, based on the detection described above, the number of CTCs expressing melanoma cell marker genes and disease state determination marker genes is measured over time, and the resulting change in cell count is used as an indicator of the therapeutic effect on melanoma patients. There are no particular limitations on the "treatment of melanoma patients" to be evaluated for therapeutic effect; examples include excision of melanoma tissue, molecular targeted therapy, immunotherapy, cytotoxic anticancer drug therapy, and radiation therapy. Furthermore, "measurement over time" means measuring the number of CTCs in at least two biological samples collected from melanoma patients at least once before and once during treatment. This may include at least once before and once during a specific treatment. It may also include all stages before, during, and after a specific treatment. By evaluating using biological samples collected at more time points, more detailed information for determining therapeutic effect can be obtained.

Specifically, the changes in the number of CTCs expressing melanoma cell marker genes and disease state determination marker genes, detected by the method described above, can be correlated with the treatment effectiveness of the patient, using identification tables and graphs that show the relationship between the progression of the disease in the patient and the number of CTCs in that patient. Such identification tables and graphs can be determined by first examining the number of CTCs in a patient group and then tracking the course of treatment.

As shown in the embodiments of this application, a higher number of detected CTC cells is considered to indicate disease progression. Therefore, generally, a decrease in the measured CTC cell count over time indicates a good therapeutic effect, and a stable disease state can be predicted when the cell count remains low with little fluctuation. Conversely, an increase in the cell count over time, or a sustained high cell count, indicates a poor therapeutic effect. Here, "low value" refers to the cell count generally observed in patients with a good or stable disease state; for example, when measured using the method described in Embodiment 2 of this application, this is 0 to 4 cells per 4 mL of blood. On the other hand, "high value" refers to the cell count generally observed in patients with a progressing disease state; for example, when measured using the method described in Embodiment 2 of this application, this is 5 or more cells per 4 mL of blood.

Based on the above evaluation, the therapeutic effect determined by the present invention can be classified into, for example, complete response/partial response groups (e.g., curative group, disease improvement group, disease stabilization group), progression group (e.g., relapse group, disease worsening group), etc. This classification may be performed by a medical assistant or other person, rather than by a physician, or it may be automatically classified by the device and software. Therefore, the method of the present invention can also be considered a preliminary diagnostic method (a method for obtaining indicators for determining therapeutic effect).

The evaluation of treatment effectiveness according to the present invention provides highly accurate information for determining treatment strategies and monitoring treatment effectiveness. Specifically, it provides physicians with information regarding the risks and survival probabilities of the patient's condition, enabling them to select the optimal treatment. Therefore, it contributes not only to reducing the risk of unnecessary treatment and saving on unnecessary treatment costs, but also to improving the patient's prognosis.

Furthermore, the method of the present invention can be applied not only to determining the effectiveness of treatment in melanoma patients, but also to the early detection of melanoma and the prediction of the prognosis of melanoma patients. Based on the method of the present invention, for example, patients judged to have a low treatment response can be given further appropriate medical treatment (such as administration of anticancer drugs, application of radiation therapy, or surgery).

The following describes a method for determining the therapeutic effect on melanoma patients using the cell detection device 100 shown in Figures 1 and 2, as one aspect of the present invention. However, the present invention is not limited to this aspect.

(1) Collect blood from a melanoma patient. Anticoagulants such as citrate, heparin, or ethylenediaminetetraacetic acid (EDTA) may be added during blood collection. If necessary, the collected blood may be diluted with physiological saline solution.

(2) The collected blood (or diluted blood) is subjected to density gradient centrifugation to remove contaminating cells (red blood cells, white blood cells, etc.) contained in the blood. Density gradient centrifugation is a method of separating substances based on their specific gravity. By layering the collected blood (or diluted blood) onto a medium that forms a density gradient (density gradient solution) and then centrifuging it, contaminating cells and debris can be removed, and the fraction (upper layer) containing CTCs can be recovered. Before performing the centrifugation, a binder capable of binding to contaminating cells (red blood cells, white blood cells, etc.) (for example, RosetteSep (manufactured by StemCell Technologies)) may be added to the collected blood (or diluted blood). The binder forms cell aggregates by binding to red blood cells, white blood cells and/or surface antigens of these cells, thereby increasing the density of these cells and facilitating the separation of CTCs by density gradient centrifugation. While it is preferable to promptly perform subsequent operations on the CTC-containing fraction from which contaminating cells and debris have been removed by density gradient centrifugation, if prompt operation is not possible, cryopreservation may be performed. For cryopreservation, the CTC-containing fraction should be suspended in a cell preservation solution such as CELLBANKER2 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), and then cryopreserved at -80°C.

(3) Adding a solution containing ammonium chloride to the fraction containing CTC obtained in (2) and stirring will lyse the red blood cells mixed in the fraction. This procedure improves the observation of the separated and recovered CTC.

(4) The solution containing the hemolyzed CTC obtained in (3) is centrifuged to remove blood components, and the CTC is formed into pellets. The CTC is then suspended in an appropriate solution.

(5) The suspension containing CTC prepared in (4) is centrifuged again to recover the pellets containing CTC. If necessary, an additional step may be added to resuspend the recovered pellets in the solution and centrifuge again.

The CTCs obtained in (6) and (5) are spread onto the cell holding means 10 provided on the cell detection device 100 shown in Figure 1, and then the cells 70 containing the CTCs are held in the holding unit 60 by the dielectrophoretic force 80 (Figure 3(1)).

(7) The adhesive substance 90 is introduced into the cell detection device 100, and the cells 70 are adhered to the holding part 60 (Figure 3(2)). For example, poly-L-lysine can be used as the adhesive substance 90, and its concentration is preferably 0.01% (w/v) or less.

(8) The preservation agent and cell membrane permeabilization agent are introduced into the cell detection device 100 to preserve and permeabilize the CTCs. Examples of preservation agents include aldehydes such as formaldehyde, formaldehyde donor compounds (compounds capable of releasing formaldehyde upon hydrolysis), glutaraldehyde, alcohols such as methanol and ethanol, and solutions containing heavy metals. Examples of cell membrane permeabilization agents include alcohols such as methanol and ethanol, and surfactants such as saponins.

(9) To prevent nonspecific reactions by antibodies, the retention region containing the target cells after storage and membrane permeabilization is subjected to protein blocking treatment.

(10) After blocking, the cells are labeled with a fluorescently labeled antibody against proteins expressed by leukocytes (leukocyte markers), a fluorescently labeled antibody against proteins expressed by melanoma cells (melanoma cell markers), a fluorescently labeled antibody against disease state determination markers, and a reagent for fluorescently staining the cell nucleus (Figure 3 (3)). After washing, the fluorescence and bright-field images of the cells are observed using a fluorescence microscope 200 or similar (Figure 3 (4)). Anti-CD45 antibody can be used as the antibody against proteins expressed by leukocytes. Anti-gp100 antibody or anti-MART-1 antibody can be used as the antibody against melanoma cell markers, and anti-ICAM-1 antibody can be used as the antibody against disease state determination markers. DAPI or Hoechst 33342 (trade name) can be used as the reagent for fluorescently staining the cell nucleus.

(11) Detect CTC71 based on the observed fluorescence and bright-field images (Figure 3(4)). CTCs can be detected by, for example, by identifying cells whose nuclei are stained, are not labeled with anti-CD45 antibody, and are labeled with antibodies against melanoma cell markers (e.g., anti-gp100 antibody or anti-MART-1 antibody) and disease state determination markers (e.g., anti-ICAM-1 antibody).

(12) Measure the number of detected CTC cells and determine the therapeutic effect on the patient based on the fluctuations in the number of CTC cells.

The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

(Example 1) Identification of melanoma cell marker genes Three melanoma cell lines, 501 mel, 888 mel, and 928 mel, were selected, and the differences in gene expression levels between these melanoma cell lines and healthy leukocytes were analyzed using a next-generation sequencer in the following manner.

(1) Melanoma cell lines were cultured in RPMI-1640 medium containing 10% (v/v) FBS (fetal bovine serum) at 37°C under a 5% _CO2 environment, and then melanoma cells were harvested by detaching the cells from the medium using 0.25% trypsin/1 mM EDTA (n=3).

(2) After recovering the total RNA from the melanoma cells obtained in (1) using the RNeasy Mini Kit (QIAGEN), cDNA synthesis and amplification were performed from 10 ng of total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). Similarly, RNA was recovered from leukocytes collected from the blood of four healthy individuals, and cDNA synthesis and amplification were performed from 10 ng of RNA.

(3) Using 1 ng of cDNA obtained in (2) above, libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina) and Nextera XT v2 Index Kit Set A (Illumina). Sequence analysis was performed using Next-seq500 (Illumina) under the conditions of "read length 75 bp, single-end read," thereby decoding more than 10 million reads per sample.

(4) The nucleotide sequences (sequence data) decoded in (3) above were mapped to the human genome sequence using TopHat2 (Johns Hopkins University) and Bowtie2 (Johns Hopkins University). The human genome sequence and human gene information used were BUILD GRCh38, publicly available from NCBI (National Center for Biological Information). The mapped nucleotide sequences were then used to determine the gene expression values for each gene, expressed in units of FPKM (Fragments Per Kilobase of exon per Million reads mapped), based on the number of reads for each gene decoded, using Cufflinks (University of Washington).

(5) Expression comparisons were performed on four samples of healthy leukocytes and nine samples of three melanoma cell lines. The average expression levels (FPKM values) in melanoma cells (three types, nine samples) were 10.00 times or more higher than the average expression levels (FPKM values) in healthy leukocytes (four samples), and the genes encoding transmembrane proteins are shown in Tables 1 to 11 above.

(Example 2) Correlation between the number of circulating tumor cells (CTCs) expressing melanoma cell marker genes and disease state determination marker genes and changes in disease state due to treatment The gp100 gene was selected as an example of a melanoma cell marker gene, and the ICAM-1 gene was selected as an example of a disease state determination marker gene, and the following experiment was conducted.

(1) From three melanoma patients who had given informed consent, 10 mL of blood was collected from each patient and centrifuged at 200 × g for 10 minutes at room temperature. After removing the supernatant, the blood was suspended in 20 mL of PBS (Phosphatate-buffered saline) to prepare diluted blood samples. The treatment and medical information of the patients are shown below.

Patient A: Liver metastases were found after surgery (disease progression).
Patient B: Liver metastases shrank with molecular targeted therapy using dabrafenib and trametinib, demonstrating a response to the drugs (partial response).
Patient C: Undergoing targeted therapy with dabrafenib and trametinib (condition stable).

(2) The diluted blood sample was overlaid in a density gradient solution with a density of 1.077 g/mL, centrifuged at 800 × g for 20 minutes at room temperature, and the fraction containing CTCs in the supernatant was collected.

(3) Add 30 mL of PBS to the fraction containing CTC recovered in (2), and centrifuge at 600 × g for 10 minutes at room temperature. Remove the supernatant to obtain a pellet containing CTC.

(4) The pellet containing CTC was resuspended in 20 mL of PBS and centrifuged at 300 × g for 8 minutes at room temperature to remove the supernatant and obtain a pellet containing CTC.

(5) The pellet containing CTC was resuspended in 20 mL of PBS, then centrifuged at 300 × g for 8 minutes at room temperature, and the supernatant was removed. Note that steps (3), (4), and this procedure are for removing blood components and concentrating the desired CTC.

(6) For the purpose of long-term storage of CTCs, the pellet containing CTCs was resuspended in 2 mL of cell cryopreservation solution (CELLBANKER 2, manufactured by Nippon Zenyaku Kogyo Co., Ltd.) and cryopreserved at -80°C.

(7) The suspension containing CTC, which had been frozen and stored in (6), was thawed. A portion of this suspension was then suspended in 10 mL of a solution containing 300 mM mannitol, and the supernatant was removed by centrifugation at 300 × g for 5 minutes at room temperature.

(8) The cells were again suspended in 10 mL of a solution containing 300 mM mannitol, then centrifuged at 300 × g for 5 minutes at room temperature, and the supernatant was removed. Note that (7) and this procedure are for removing the cell cryopreservation solution and concentrating the CTCs.

The suspension containing CTCs, from which the supernatant was removed in (9) and (8), was introduced into the cell holding means 10 provided in the cell detection device 100 shown in Figures 1 and 2. An AC voltage (frequency 1 MHz) was applied from the signal generator 50 to the electrode substrates 31 and 32 for 3 minutes, causing the cells 70 containing CTCs to be held in the holding portion 60 of the means. The cell detection device 100 used in this embodiment has a structure in which a 1 mm thick spacer 20 and electrode substrate 32 are in close contact with the upper surface of the holding portion 60 provided in the cell holding means 10, which consists of an insulator 12 having multiple micropores with a diameter of 30 μm and a depth of 40 μm, a light-shielding chromium film (light-shielding member 11) provided between the insulator 12 and the electrode substrate 31, and the electrode substrate 31.

Under the conditions of (10) and (9), an AC voltage was applied while introducing a 300 mM mannitol aqueous solution containing 0.01% (w/v) poly-L-lysine. After standing for 3 minutes, the application of the AC voltage was stopped, and the aqueous solution was removed by aspirate.

(11) An aqueous solution containing 50% (v/v) ethanol and 2% (w/v) formaldehyde (hereinafter referred to as the "cell membrane permeability reagent") was introduced and allowed to stand for 10 minutes to permeate the cell membrane, and cells containing CTCs in the retention area were prepared as specimens.

(12) The cell membrane permeability reagent was aspirated and removed, and any remaining reagent was washed away by introducing PBS.

(13) An aqueous solution containing a fluorescently labeled antibody capable of specifically binding to proteins inside and outside the cell membrane, and a fluorescent reagent (DAPI: 4',6-diamidino-2-phenyllindole) that labels the cell nucleus (hereinafter referred to as "labeling reagent A") was introduced and allowed to stand for 30 minutes. The labeling antibodies used were those labeled against CD45 expressed on the surface of leukocytes, and those labeled against gp100 and ICAM-1 expressed in the cytoplasm of melanoma cells.

(14) Labeled reagent A was removed by aspirate, and any remaining labeled reagent A was removed by introducing PBS.

After placing the cell retention means containing CTCs labeled in (15) and (14) onto the stage of a fluorescence microscope, imaging of the entire retention unit was performed to observe all cells captured in the multiple retention pores. A fluorescence microscope (IX83; Olympus Corporation) equipped with a computer-controlled motorized stage and a CMOS camera (ORCA-Flash 4.0; Hamamatsu Photonics Corporation) was used. LabVIEW (National Instruments Corporation) was used for image acquisition and analysis software.

From the cells imaged in (16) and (15), CTCs were detected that were stained with DAPI (DAPI positive), indicating the presence of a cell nucleus; not stained with antibodies against CD45 expressed on leukocytes (CD45 negative); and stained with antibodies against gp100, a melanoma cell marker, or ICAM-1, a disease state determination marker (gp100/ICAM-1 positive).

Table 12 shows the results of counting the number of CTCs detected at each blood sampling in this embodiment. In Table 12, "1st" and "2nd" indicate chronological order (2nd being later).

In patients with progressive disease (Patient A), the number of cytoplasmic tumor cells (CTCs) that possessed a cell nucleus and were positive for gp100 (a melanoma cell marker) and ICAM-1 (a disease progression marker) showed a significant increase (from 1 cell per 4 mL of blood (1st test) to 19 cells per 4 mL of blood (2nd test)). On the other hand, in patients with a partial response to treatment (Patient B), the CTCs disappeared (from 2 cells per 4 mL of blood (1st test) to 0 cells per 4 mL of blood (2nd test)), and in patients with stable disease (Patient C), the levels remained low (0 cells per 4 mL of blood (1st test) and 1 cell per 4 mL of blood (2nd test)).

The above results indicate that the number of cytoplasmic tumor cells (CTCs) possessing a nucleus and positive for melanoma cell markers and disease state assessment markers correlates with the patient's condition, and that the effectiveness of treatment for melanoma patients can be determined with high accuracy based on the fluctuations in the CTC count.

(Comparative Example 1)
Except for the fact that the patients from whom blood was collected were five Stage I melanoma patients different from those in Example 1, who had given informed consent, CTCs were counted in the same manner as in Example 1.

The results are shown in Table 13. gp100-positive CTCs were detected in all blood samples taken before surgery from five patients with stage I melanoma (8 to 28 CTCs per 4 mL of blood). On the other hand, gp100 and ICAM-1-positive CTCs were not present in any of the patients' blood samples.

(Example 3) Correlation between the number of CTCs expressing melanoma cell marker genes and disease state determination marker genes and changes in disease state during long-term treatment. In melanoma patient C (Example 2) and a newly diagnosed melanoma patient (Patient D) who gave informed consent, the number of gp100 and ICAM-1 positive CTCs was counted during a long-term treatment period, including changes in treatment. The counting of CTCs at each blood sampling was performed in the same manner as in Example 2.

The results for patient C are shown in Table 14, and the results for patient D are shown in Table 15. Note that in Tables 14 and 15, the higher the number of trials, the later the results in the time series.

As mentioned earlier, Patient C's condition was stable with molecular targeted therapy, but the metastases did not shrink (3rd time). Therefore, the treatment was changed to immunotherapy, which resulted in a reduction in metastases (4th time). Subsequently, new metastases appeared (5th time), so the treatment was changed back to molecular targeted therapy (6th time). The metastases then shrank again (9th time), and the patient has maintained that condition (10th time).

The number of gp100 and ICAM-1 positive CTCs was almost undetectable (1 per 4 mL of blood) at the time the disease state was stable after the start of molecular targeted therapy (second dose), but increased to 18 per 4 mL of blood by the third dose. Subsequently, when the patient switched to immunotherapy and the metastases shrank (fourth dose), the number of CTCs decreased significantly to 2 per 4 mL of blood. After switching back to molecular targeted therapy due to the appearance of new metastases (sixth dose onward), the metastases shrank, but during that time, gp100 and ICAM-1 positive CTCs were almost undetectable (less than 1 per 4 mL of blood).

Patient D is a patient who, after primary tumor resection, experienced lung metastasis (second time), underwent molecular targeted therapy, achieved complete remission (fourth time), and has maintained that state (fifth time and beyond).

Following the appearance of lung metastases (second time), the number of gp100 and ICAM-1 positive CTCs increased significantly (43 cells per 4 mL of blood). However, with the initiation of molecular targeted therapy (third time), the number decreased significantly (17 cells per 4 mL of blood), and at the time of complete remission (fourth time), it was 4 cells per 4 mL of blood. Since then, complete remission has been maintained (from the fifth to the seventh time), during which time the number of gp100 and ICAM-1 positive CTCs remained below 2 cells per 4 mL of blood.

The above results indicate that even with long-term treatment for melanoma patients, the correlation between the number of cytoplasmic tumor cells (CTCs) possessing a nucleus and positive for melanoma cell markers and disease state assessment markers and the patient's disease state is maintained. This suggests that the effectiveness of long-term treatment for melanoma patients can be assessed with high accuracy based on the fluctuations in the CTC count.

100: Cell detection device 10: Cell holding means 11: Light-shielding member 12: Insulator 11a, 12a: Through hole 20: Spacer 21: Inlet 22: Outlet 23: Through section 31, 32: Electrode substrate 40: Conductor 50: Signal generator 60: Holding section 70: Cell 71: Target cell (circulating tumor cell [CTC] in the blood)
80: Dielectrophoretic force 90: Adhesive material 200: Fluorescence microscope

Claims (2)

A method for obtaining an index for determining the effectiveness of treatment in melanoma patients, comprising the steps of detecting circulating tumor cells expressing melanoma cell marker genes and disease state determination marker genes in a biological sample taken from the patient, and measuring the number of such cells over time.
The melanoma cell marker gene is either (i) or (ii) below,
(i) A gene encoding a polypeptide containing at least one amino acid sequence described in any of Sequence IDs 1 to 128 (ii) A gene encoding a polypeptide containing at least one amino acid sequence having 90 % or more homology to the amino acid sequence described in any of Sequence IDs 1 to 128 The disease state determination marker gene is the ICAM-1 gene,
A method for using the aforementioned fluctuation in cell count as an indicator for determining the therapeutic effect on the patient. The method according to claim 1, comprising the step of concentrating circulating tumor cells in a biological sample collected from the patient to obtain a concentrate, prior to detecting circulating tumor cells in the blood expressing melanoma cell marker genes and disease state determination marker genes.