WO2014133467A1

WO2014133467A1 - Methods and biomarkers for the detection of circulating tumor cells

Info

Publication number: WO2014133467A1
Application number: PCT/SG2014/000099
Authority: WO
Inventors: Siew-Chuan KOAY; Sai Mun LEONG; Mo-Huang Li
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2013-02-28
Filing date: 2014-02-28
Publication date: 2014-09-04
Anticipated expiration: 2015-08-28

Abstract

The invention relates to methods and biomarkers for the detection of circulating cancer cells in a sample. The methods comprise: a) collecting the circulating tumor cells using a paper; b) extracting the genetic material of the CTCs; and c) analysing the extracted genetic material for the presence of one or more specific biomarkers. Also encompassed are biomarker panels and their use for the diagnosis of cancer.

Description

Methods and biomarkers for the detection of circulating tumor cells

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of United States of America Provisional Patent Application No. 61/770,347 filed February 28, 2013, the contents of which being hereby incorporated by reference in its entirety for all purposes. - Field of the invention

[0002] The invention relates to methods and biomarkers for the detection of circulating cancer cells in a sample.

Background of the invention

[0003] For some specific applications, such as cancer diagnostics via detection of circulating tumor cells (CTCs), the target analytes (CTCs) are within the patients' whole blood. These cells, which are primarily responsible for metastasis from the primary tumors, are normally very rare. Thus, a biomedical device is often applied to isolate or to enrich these cells from the patients' whole blood. The enriched sample solution normally contains the CTCs, with co-captured white blood cells and red blood cells. The red blood cells include an iron-ion-rich heme group which can inhibit PCR amplification.

[0004] To analyse or compare CTC-specific nucleic acid expression profiles, one has to overcome the twin problems of the extreme rarity of the cells and the complex milieu they are embedded in, with an overwhelming presence of red and white blood cells. The current isolation or extraction techniques for CTCs such as by ferrofluid-based or size exclusion methods are thus often contaminated with a high background of blood cells. Although there are several approaches for CTC enrichment from patients whole blood either by size-based separation or antibody-based capturing, no method can completely get rid of white blood cells. Generally, even after separation there are always co-existent white blood cells within an eluted CTC sample solution. Due to the rarity of CTCs in comparison with white blood cells, the eluate often contains outnumbered white blood cells which cause a background containment, and make it challenging to detect genetic material from CTCs.

[0005] MicroRNAs (or miRNAs) are small non-coding R A molecules with important functions in regulating RNA stability and gene expression. Deregulation of their expression has been implicated in cancer development and tumor progression. MiRNA expression fingerprints correlate with clinical and biological characteristics of tumours, and microRNA expression profiling of tumor biopsies has led to identification of signatures for diagnosis, staging, progression, prognosis and treatment prediction. MicroRNA cancer signatures have also been uncovered from bodily fluids such as blood plasma or serum of cancer patients. This is being explored as a non-invasive source of molecular information for diagnosis, staging, progression, prognosis and treatment prediction for better cancer management. There is no reference of microRNAs within a cell. Either a cell will have a constant expression level or abundance independent of cell types.

[0006] It is an object of the present invention to provide methods that address at least some of the problems connected to CTC detection listed above.

Summary of the invention

[0007] The inventors have surprisingly found that some of the existing problems with CTC isolation and detection can be overcome by using a paper-based collection, extraction and analysis approach.

[0008] Accordingly, a first aspect of the invention relates to a method for the detection of circulating tumor cells (CTCs) in a biological sample, comprising: a) collecting the circulating tumor cells using a paper; b) extracting the genetic material of the CTCs; and c) analysing the extracted genetic material for the presence of one or more specific biomarkers.

[0009] Another aspect of the invention relates to a biomarker panel for the detection of CTCs in a biological sample, the panel comprising: a) miR-16, miR-21, miR-93, miR-141 and miR-200c; and/or b) miR-28-3p, miR-28-5-p, miR-106, miR-125b, miR-183, miR-194, miR-206, miR-221 , miR- 222, miR-328, miR-451 , and miR-486.

[0010] Still another aspect of the invention relates to use of the biomarker panel disclosed herein for the diagnosis of cancer, for monitoring the progression of cancer and/or for determining the prognosis of a patient afflicted with cancer.

[0011] A still further aspect of the invention is directed to a method for the diagnosis of cancer or monitoring the progression of cancer, the method comprising determining the expression level of the biomarker panel as described herein.

[0012] Other aspects of the invention will be apparent to a person skilled in the art with reference to the following drawings and description of various non-limiting embodiments.

Brief description of the drawings

[0013] The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following exemplary drawings.

[0014] Figure 1 : Comparison of paper-based method with existing microRNA purification technologies.

[0015] Figure 2: The schematic of one embodiment on using a paper pre-loaded with chemicals for collection of genetic material from cells immobilized on a device surface, (a) A paper is preloaded with chemicals for cell lysis and protein denaturation. Cells within a biological sample are filtrated and captured on a device surface (for example, the device surface is a surface of the membrane filter or a microsieve). (b) The paper is brought in contact with captured cells, (c) Cells are lysed by the pre-loaded chemical, and the lysis solution wets the paper, (d) Genetic material (such as DNA, and RNA) within cells are transferred to the paper by the capillary force of the paper.

[0016] Figure 3: The schematic of one embodiment on using a paper pre-loaded with chemicals for collection of genetic material from cells within microwells. (a) Cells were dispensed into a microwell plate. The volume of each microwell ranges from 10 nL to 10 μί. (b) Side view of cells within microwells. (c) A point collector with a paper tip, pre-loaded with chemicals for cell lysis and protein denaturation. The point collector draws up all analytes within the microwell-solutioh by the capillary force of the dry paper, and then lyses cells within the absorbed solution.

[0017] Figure 4: Split-well approach on using patient's own blood cells as reference for the detection of CTC microRNA biomarkers. Circulating tumor cells were isolated and eluted out from capturing devices. The eluted samples were aliquotted into a mini-well plate with each well containing 0.5-1 μί, of sample solution. The aliquotting process randomly distributes CTCs and WBCs into mini-wells which generates a well-population containing either no CTCs (CTC-negative wells), or CTCs with background WBCs (CTC-positive wells). Solutions within CTC-positive and CTC-negative wells were collected using the paper-based approach respectively. Genetic material such as miRNA, mRNA, and DNA were extracted and retained by the paper. Contaminants within solution were wash off, and the genetic material was eluted out, followed by RT-PCR detection to measure the abundance of specific biomarkers through the cycle threshold values (CT). In another approach, the extraction paper (with entrapped miRNA, mRNA and DNA) was inserted directly into the PCR reagents, skipping the elution step. To remove the background C_T from the contaminating, white blood cells coexisting within the CTC-positive wells, the C_T value difference (AC_T) of CTC- positive and CTC-negative experiments was calculated, instead of the absolute C_T value. The fold difference in expression of each analyzed biomarker was also calculated.

[0018] Figure 5: The schematic of another embodiment for microRNA profiling from CTCs. Circulating tumor cells are isolated from whole blood using either size separation or antibody- capturing methods. The eluted solution is collected with a tube, and often contains both CTCs and other blood cells. The eluted sample is treated with anti-CD45 conjugated magnetic beads which bind to the white blood cells. The magnetic beads with the blood cells are pulled to the tube-bottom using a permanent magnet. The surfactant containing CTCs are transferred to second collection tube. The cells still in solution within these two samples are collected by the paper-based approach, and the genetic materials within these two samples analyzed using PCR detection.

[0019] Figure 6: The relationship of C_T value and microRNA concentration.

[0020] Figure 7: Real-time fluorescence curves of RT-PCRs with samples prepared by a FTA elute paper. Various quantity ( 10⁴— 10¹⁰ copies) of miR-141 was spiked into 1 piL health donor plasma and processed by a FT A paper. The insert shows the relationship of CT value and miR-141 quantity. NT: non-template control.

[0021] Figure 8: The C_T values of paper-based and Cell-to-CT methods from 1 -μΐ. samples containing 10 MCF-7 cells in a background of 1%, 50%, and 95% of blood. C_T values were obtained for miR-16, miR-21 , miR-141 and miR-200c.

[0022] Figure 9: Comparison of the amplification fold difference of paper-based approach and Cell-to-CT method for miR16, miR-21, miR141,,and miR200c. Fold difference (2AC_T) was calculated based on the difference in CT values of experiments with and without blood background (AC_T = C_T of blood-sample - C_T of blood-free). The positive fold difference of blood-sample in paper- based approach is contributed by the microRNAs within blood cells.

[0023] Figure 10: MicroRNA expression profiles of various amounts (3-100 cells) of MCF-7, HT-29, and DU145. The expression profiles vary among cancer cell lines.

[0024] Figure 11 : MicroRNA expression profile from 19 breast and colorectal cancer patients and 2 healthy subjects serving as controls, (a) Expression profiles from microRNA Panel 1 (MiR-16, miR-21 , miR-93, miR-141 , and miR-200c) (b) Expression profiles from microRNA Panel 2 for a subset of patients (Patients 14-19, and 2 healthy subjects). MicroRNA Panel 2: (miR-28-3p, miR-28-5p, miR-106, miR-183, miR-206, miR-221 , miR-222, miR-194, miR-328, miR-451 , miR-486, miR-194, and miR-125b). The circulating tumor cells number was in 7.5 mL whole blood.

[0025] Figure 12: (a) Number of circulating tumor cells detected within 7.5 ml of patient whole blood, (b-d) The microRNA expression profiles corresponding to the cancer progression and treatment, (e) The microRNA expression profiles corresponding to the cancer progression after stopping treatment.

Detailed description

[0026] Described herein are methods to collect genetic material from CTCs and biomarkers that allow detection of the presence of CTCs in a sample.

[0027] In a first aspect, the present invention relates to a method for the detection of circulating tumor cells (CTCs) in a biological sample, comprising: a) collecting the circulating tumor cells using a paper; b) extracting the genetic material of the CTCs; and c) analysing the extracted genetic material for the presence of one or more specific biomarkers.

[0028] The term 'circulating tumor cells (CTCs)', as used herein, refers to tumor cells that are released from a preferably solid tumor and freely circulate in a patients' circulation of blood. As they can freely migrate in an organism, they can initiate metastasis, i.e. form secondary tumors at sites distant from the primary tumor. CTCs are very rare in the blood, with a ratio of 1 : 10⁵ _.(CTC:

common cells) or less. Nevertheless, these cells represent an important population within the circulation with respect to cancer diagnosis and can provide a unique view into disease evolution. [0029] The term 'biological sample', as used herein, includes any sample that is derived from an organism, including tissue and body fluid samples. Preferably, the biological sample is blood, in particular whole blood. . Where the biological sample is blood, the methods described herein include the step of collecting cells including CTCs from blood and extracting their genetic material for subsequent analysis.

[0030] In the methods described herein, the CTCs are collected by a paper. In various embodiments, the paper is porous and fibrous. The porous nature of the paper facilitates

immobilization of genetic material and the capillary force within the paper fibres is used to pick up the liquid sample or solution. The paper approach provides the advantages that it can absorb solutions and biological samples from irregular supporting substrate/surface, which overcomes the limitation of pipetting approaches and automatically adsorbs cells in the solution.

[0031] In various embodiments, the paper is formed of cellulose comprising parallel D-glucose chains. The structure is stabilized by hydrogen bonds giving it fibrous properties. The cellulose paper sheet is prepared by either mechanical or chemical disintegration of alpha-cellulose, hard or soft wood pulp, purified wood pulp, cotton linter sheet, cotton pulp, or the like. Other sources of cellulose include low crystallinity celluloses and commercially available cellulose excipients, such as micro fibrillated cellulose, powdered cellulose, regenerated cellulose, and microcrystalline cellulose. In certain embodiments the paper may include nitrocellulose or carboxymethylcellulose. It is preferred that the paper be of a porous nature to facilitate collection and immobilization of genetic material when the paper comes into contact with cells. While in principle any type of paper that can absorb a significant quantity of liquids is suitable, it is preferred that the paper has been manufactured with the object of liquid sample collection, in particular in view of potential contamination. Suitable papers for this purpose are known in the art and are commercially available, for example as FTA Elute Sample Collection Cards (GE Healthcare).

[0032] The collecting step thus may include using a paper for soaking up a liquid sample or an aliquot of a liquid sample, including cells.

[0033] In various embodiments, the paper is preloaded with one or more chemicals for lysis of any cells present in the sample and/or protein denaturation. Examples of such paper that are known and commercially available include fast technology for analysis of nucleic acids (FTA) paper, a cellulose-based matrix impregnated with chemicals that lyse cells and preserve nucleic acid. The chemicals are activated when a biological fluid contacts the surface. The paper automatically adsorbs cells in the solution and brings the cells into contact with the pre-loaded cell-lysis chemicals. The cells are lysed on the paper surface and the genetic material within the cells are absorbed on the paper, which provides maximum efficiency in collecting rare CTCs or tiny amounts of genetic materials within CTCs. Additional features of the chemical treatment are bacterial and viral inactivation. This protects the biosample from microbial growth contamination and may also protect the user from potential biohazards present in the biosample. As such FTA paper is a preferred medium that protects and stabilizes DNA for collection, transport, storage, and archival from a variety of biological samples.

[0034] The paper-based approach for cell collection can be advantageously used to remove any sample components that interfere with the subsequent analysis of the genetic material, for example the iron-containing heme groups. To achieve this, use can be made of the interaction of the paper with the extracted genetic material to allow washing of the genetic material adsorbed to the paper to remove or dilute interfering components that originate from the sample.

[0035] The denaturing agents preloaded onto the paper include surfactants or (anionic) detergents that will denature proteins and lyse pathogenic organisms in the genetic sample. The denaturing agents may similarly help to lyse the cells, release the genetic material and and allow the genetic material to be immobilized and preserved. The chemicals preloaded onto the paper may further include buffering chemicals, such as a weak base, chelating agents, and an anionic surfactant or detergent. Uric acids and urate salts may also be used. A weak base may be a Tris

(trishydroxymethyl methane), either as a free base or as the carbonate, and the chelating agent may be EDTA. The anionic detergent may be sodium dodecyl sulfate or sodium lauryl sulfate.

[0036] In other embodiments, chemicals may be used that are capable of lysing the cells but do not denature proteins. In still other embodiments, the chemicals may act to deactivate enzymes without denaturing for instance, by chelating metals that act as cofactors for enzymes.

[0037] Generally, the chemicals may be loaded onto the paper in form of a coating or the paper may be impregnated with them. In certain embodiments, the coating solutions may be applied to the substrate in such a matter that the coatings are disposed, sorbed, or otherwise associated with the cellulose. In certain embodiments the coatings may adhere to the substrate through chemical bonding while in other embodiments, adherence may be physical such as through impregnation.

[0038] The term 'genetic material' as used herein includes nucleic acids that are present in living cells, such as DNA and RNA, including microRNA, ribosomal RNA and messenger RNA. Of particular interest in the context of the present invention is the analysis of the gene expression levels via mRNA or the detection and/or quantitation of specific microRNAs.

[0039] In various embodiments prior to the collecting step, the CTCs are isolated and/or enriched. Methods of isolating and/or enriching CTC from common blood cells are known in the art. Any suitable method of separating CTCs may be used. In various embodiments the CTCs isolation and/or enrichment may be carried out by size separation or antibody capturing methods. In various embodiments the size separation may be carried out using a filter or microsieve. In other

embodiments the isolation and/or enrichment may be carried out by antibody-based capturing in combination with a sorting or filtering technology such as magnetic heads with anti-CD45 conjugated magnetic beads. Generally, antibody-based capturing relies on the recognition of surface molecules that are either specific for CTCs or for the contaminating white and red blood cells and thus allow separation of the different cell types.

[0040] In various embodiments the isolated and/or enriched CTCs are, prior to the collecting step, dispensed into collection tubes or wells of a mini- or microwell plate. This may serve the purpose to dilute the isolated and/or enriched cells to such an extent that the CTCs are further enriched compared to other cells types that may still be present in the sample. As it is common in all existing isolation/enrichment techniques for CTCs that there remains a high background of other cells, such dispensing of the isolated and/or enriched cells into separate compartments may thus subdivide the cells into populations that include CTCs and which are therefore enriched for CTCs and populations that include no CTCs. The CTC containing fractions may then be combined again and collected according to the described methods.

[0041] The method further comprises a step of extracting the genetic material of the CTCs and optionally all other cells inadvertently collected together with the CTCs (background). The extraction can be made by lysing the cells, for example by using a paper that is preloaded with checmicals for cell lysis. Lysis of the cells leads to release of the genetic material, which is then preferably absorbed onto the paper. The genetic material may then be subjected to one or more washing steps that help to get rid of contaminants that are present in the sample and may interfere with subsequent analysis..

[0042] In various embodiments, the extraction step further comprises eluting the genetic material from the paper. Any medium capable of removing nucleic acid molecules from the paper, preferably into solution, is generally suitable for eluting the genetic material collected on the paper. Commonly, the mediums used are either aqueous (e.g. buffers) or non-aqueous liquids (e.g. solvents) or even supercritical gases such as carbon dioxide. However, independent from the type of extraction technique used, the extraction should be such that the structural integrity of the genetic material is ensured and it can be subjected to subsequent analysis.

[0043] In various embodiments the method also comprises a step of drying the paper containing the collected CTCs prior to extracting the nucleic acid. The paper with the dried sample may be stored for at least 24 h, or for at least one week prior to extracting the nucleic acid. The drying can be active, e.g. by application of a stream of dry air to the substrate or by subjecting the substrate to drying solvents such as ethanol or subjecting the substrate to moderately elevated temperatures, e.g. 25-40°C. It can also be passive, where the substrate with the sample is left to dry on e.g. a bench surface. The storage can be performed under dry conditions, such as in the presence of a desiccant, and can be at room temperature (e.g. 20-35°C), under refrigeration (e.g. 0-10°C) or under freezing conditions.

[0044] The method further comprises a step of analysing the extracted genetic material for the presence of one or more specific biomarkers. The term 'specific biomarkers' as used herein refers to at least one biomarker that allows a distinction between CTCs and other cells or a sample containing CTCs and a sample free of CTCs. The specific biomarker is preferably a nucleic acid marker, i.e. a specific gene or expression product, i.e. RNA, such as mRNA or other RNA types, such as microRNA. Alternatively, the specific biomarker may also be an altered expression level of a gene or gene product. The detection and/or quantitation of such a biomarker may occur by use of oligonucleotide probes or primers. In case of using oligonucleotide primers, the analysis commonly occurs by making use of nucleic acid amplification techniques, preferably PCR. Generally, the probes or primers have to be substantially complementary to a nucleic acid biomarker. By "substantially complementary" is meant that the subject probe or primer has a base sequence containing an at least 10 contiguous base region that is at least 70% complementary, preferably at least 80%

complementaty, more preferably at least 90% complementary, and most preferably 100%

complementary to an at least 10 contiguous base region present in the biomarker nucleic acid sequence. The degree of complementarity is determined by comparing the order of nucleobases making up the two sequences and does not take into consideration other structural differences which may exist : between the two sequences, provided the structural differences do not prevent hydrogen bonding with complementary bases. The degree of complementarity between two sequences can also be expressed in terms of the number of base mismatches present in each set of at least 10 contiguous bases being compared, which may range from 0-2 base mismatches.

[0045] In various embodiments the analysing step comprises comparing the expression levels of one or more specific biomarkers with a reference standard. In various embodiments the reference standard is obtained by subjecting white blood cells also contained in the sample to the same collecting, extracting and analysing steps as the CTCs. For example the analysis may include a differential analysis where the determined expression levels of a given biomarker panel of a reference standard are subtracted from the biomarker expression levels determined in the actual sample. This allows very efficiently to remove the background caused by other cells that are present in the sample and more particularly can effectively minimize the contamination and influence of the white blood cells and overcome the limitations of not being able to completely separate CTCs from white blood cells.

[0046] In various embodiments the one or more specific biomarkers are selected from the group of microRNAs. The presence or amount of the biomarkers may be determined by any method known in the art. In various embodiments the presence and an amount of a panel of biomarkers are determined using nucleic acid amplification techniques such as PCR. PCR may include deep sequencing or quantitative PCR, real-time PCR and/or reverse transcriptase PCR. In various enbodiment the cycle threshold of the PCR is measured. 'Cycle threshold' (C,) or, according to the MIQE guidelines, 'quantification cycle' (C_q) refers to the number of cycles at which the detected signal (for example fluorescence by a fluorescently labelled probe) exceeds the threshold (background level).

[0047] The presence of the CTCs in the biological sample can for example be determined by detecting the presence or absence of one or more specific microRNAs. In various embodiments, said microRNAs are selected from the group consisting of miR-16, miR-21 , miR-28-3p, miR-28-5-p, miR- 93, miR-106, miR-125b, miR-141, miR-183, miR-194, miR-200c, miR-206, miR-221, miR-222, miR- 328, miR-451, miR-486, and miR-496. The nucleotide sequences of these marker microRNAs are set forth in SEQ ID Nos. 1-17.

[0048] In various embodiments, the CTCs are selected from the group consisting of breast cancer cells, colon cancer cells, prostate cancer cells, lung cancer cells, bladder cancer cells, bone cancer cells, cervical cancer cells, glioma cells, astrocytoma cells, liver cancer cells, melanoma cells, nasopharyngeal carcinoma cells, ovarian cancer cells, pancreatic cancer cells, renal cancer cells, or cells from any other cancer type in which circulating tumour cells are released into the blood.

Preferably, the cancer is breast cancer, gastric cancer, or rectal cancer .

[0049] Another aspect of the invention relates to a biomarker panel for the detection of CTCs in a biological sample, the panel comprising: a) miR-16, miR-21 , miR-93, miR-141 and miR-200c; and/or b) miR-28-3p, miR-28-5-p, miR-106, miR-125b, miR-183, miR-194, miR-206, miR-221 , miR- 222, miR-328, miR-451 , and miR-486. "Biomarker panel", as used herein, relates to a panel of gene expression products which are either due to their presence or absence or due to altered expression levels indicative for CTCs and allow a distinction between CTCs and normal white blood

cells. Another aspect of the invention relates to use of the biomarker panel comprising: a) miR-16, miR-21 , miR-93, miR-141 and miR-200c; and/or b) miR-28-3p, miR-28-5-p, miR-106, miR-125b, miR-183, miR-194, miR-206, miR-221 , miR-222, miR-328, miR-451, and miR-486; for the diagnosis of cancer, for monitoring the progression of cancer and/or for determining the prognosis of a patient afflicted with cancer. Such a use may include the use of reagents that can detect and optionally also quantify the biomarkers. Examples for such reagents include oligonucleotide probes, such as labelled probes, or oligonucleotide primers, including those described above.

[0050] A still further aspect of the invention relates to a method for the diagnosis of cancer or monitoring the progression of cancer, the method comprising determining the expression level of the biomarker panel comprising: a) miR-16, miR-21 , miR-93, miR-141 and miR-200c; and/or b) miR- 28-3p, miR-28-5-p, miR-106, miR-125b, miR-183, miR-194, miR-206, miR-221 , miR-222, miR-328, miR-451 , and miR-486.

[0051] In the described uses and methods, the presence of or the exceeding of a threshold expression level value of a at least one, at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 or at least 12 of the given biomarkers may be indicative for the presence of CTCs in the sample. This in turn may be indicative for cancer in the patient from which the sample has been obtained. Depending on the specific biomarkers detected, a characterisation of the cancer with respect to cancer type, tumor stage, susceptibility to a given treatment regimen, prognosis of the patent, and the like may be possible..

[0052] By "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0053] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0054] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0055] By "about" in relation to a given numerical value, such as for temperature and period of time, it is meant to include numerical values within 10% of the specified value.

[0056] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0057] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the group.

Examples

Cell separation

[0058] Circulating tumor cells (CTCs) are tumor cells released from the primary tumor cells. The number of CTCs are very rare in comparison with red blood cells (10⁹ cells/ml) and white blood cells (10⁶ cells/ml). Although there are several approaches for CTCs enrichment from patients whole blood either by size-based separation or antibody-based capturing, no method can completely get rid of white blood cells. Figure 5 illustrates an approach for CTC enrichment prior to analyzing the genetic materials within the CTCs using PCR detection. This approach combines further depletion / removal of the contaminating blood cells by use of anti-CD45-labelled magnetic beads, and paper- based genetic material collection.

Extraction from cell separation devices

[0059] This proposed approach overcomes the limitation of transfer via pipetting which is incapable for full / complete collection / pick-up of cells or biological materials from irregular, flat, or perforated surfaces such as filter membranes or biomedical devices. The pipetting method requires that a pipette tip is immersed into sample solution, in order to pick up solution. In addition, the pipetting method utilizes a mechanical aspiratory force to pick up solution which is known to be more suitable for sample volumes larger than 5 μΤ. Moreover, it is known that cells can adsorb onto the surface of the pipette tip, which makes it unsuitable for collecting rare cells from flat surfaces. In Figure 2, the capillary force of a dry paper to collect biological samples from a flat, irregular substrate surface was used. In various embodiments, the dry paper may be preloaded chemicals for cell lysis and protein denaturation. For example, such chemicals may include but are not limited to different detergents with ionic, non-ionic and/or zwitterionic moieties, and/or a surfactant. Detergents may, for example, include sodium dodecyl sulphate (SDS), Triton X-100, 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulphonate. In one example, the dry paper may be a FTA Elute card. In another example, the dry paper may be a standard (normal) cellulose paper.

Extraction from micro-wells

[0060] Another embodiment is to use a paper-based approach to collect samples from the micro- wells (Fig. 3). For example, a paper is rolled on a needle tip which forms a very sharp paper-tip area. The paper may be the same as the dry paper used in Fig. 2. The paper-tip can be used to collect the sample solution (<1 μ ) from mini-microwells with microvolume capacities. The solution within the microwell is automatically withdrawn by the capillary force of a dry paper. Biological materials (such as cells, DNAs, R As, proteins) within the solution are also transported from the microwell to the paper-tip. One can use a paper-tip to collect samples from multi-wells, as long as the paper is not completely wet. Like a normal paper, the volume collection capability of the paper-based approach depends on the paper area and its water absorption properties.

Method proceedure 1

[0061] Figure 4 illustrates a method for accurate microR A or mRNA detection from CTCs using patient's own blood cells as reference. This method overcomes the limitations of: 1) the lack of reference microRNAs which have constant expression level or abundance independent of cell types; 2) the co-existent white blood cells within an eluted CTC sample solution. Unlike in mRNA detection, where several reference mRNAs (such as GAPDH, beta-actin) are able to facilitate the comparison of cycle threshold values of different experiments, there is a lack of widely-accepted reference microRNAs. Using the patient's own white blood cells provides a solution to overcome this limitation. In addition, CTCs are known to be very rare in comparison with white blood cells. Thus, the eluted CTCs sample solutions almost always contain white blood cells, which can cause problems for RT-PCR based CTC detection. The genetic materials within CTCs and WBCs would be mixed together when both of them are lysed in a solution. This makes it very challenging to interpret the biomarker abundance from the cycle threshold value of RT-PCR. By using patients' own blood cells as a reference, the background signals of the contaminating WBCs can be subtracted.

Genetic material extraction using a paper

[0062] A FTA (Whatman, Clifton, NJ) or normal cellulose paper was utilized to absorb solutions and to collect genetic material within samples. Briefly, the paper was brought in contact with sample solutions to transfer solution from a supporting substrate to the paper. The paper was then dried at room temperature for 1 hr, and washed in 70% ethanol for 5 min and air dried again. After that, the absorbed genetic materials was eluted by incubating the dried paper in 12-20 μΐ, of

Diethylpyrocarbonate (DEPC) water at 95°C for 30 min.

Reverse Transcription and SYBR Green Real-time PCR

[0063] Multiplex microRNA to cDNA reverse transcription was carried out in 7.5 reaction volume containing 100 nM stem-loop primers, 1.3mM dNTPs (Applied Biosystems, USA), 1 ^χ reverse transcription buffer (Applied Biosystems, USA), 1.07 U Rnase Inhibitor (Applied Biosystems, USA), 1 x Superscript VILO enzyme (Invitrogen, USA) and 4 μΐ, of paper-eluent. The reaction was conducted at 42 °C for 1 hr using a thermal cycler (Eppendorf, Germany).

[0064] RT-PCR for microRNA experiments was conducted with 10 min of initial denaturation at

95°C, followed by 40 cycles of 30 s denaturation at 95°C and 30 s annealing/extension at 60°C using

ABI 7900HT (Applied Biosystems, USA). Briefly, 2 ih of 10* diluted cDNA sample was added with

8 of l x XtensaMix-SG master mix (BioWORKS, Singapore), containing 2.5 mM MgC12, 200 nM of each primer, and 1.25 U KlearTaq DNA polymerase (KBiosciences, UK). Raw cycle threshold

(CT) values were calculated using the 7500 software v2.0.5 (Applied Biosystems) with automatic baseline and threshold settings. Each cDNA sample was run in duplicates.

Example 1: Collection of microRNAs from 1-jiL solution using a chemical-loaded paper

[0065] The capability of using a FTA paper (Whatman, USA) and normal cellulose paper for collection of biological material from Ι-μί sample solution was examined. The FTA paper is a cellulose paper which is chemically treated, capable to lyse cells and to extract nucleic acids from cells. Briefly, synthetic microRNAs (miR-16, miR-93, miR-141) were serially diluted using

Diethylpyrocarbonate (DEPC) water to concentrations of 102 to 108 per μΕ. Then, Ι-μΙ. microRNA sample was spotted on a FTA paper or a cellulose paper. After that, both papers were dried for 1 hr at room temperature, and washed with 70% ethanol. Then the microRNAs were eluted at 95 °C for 30 min, and the eluted microRNAs were analyzed using RT-PCR. As positive control, serially diluted synthetic microRNAs were used directly for RT-PCR.

[0066] Figure 6 shows the cycle threshold (C_T) values of the RT-PCR detections. Notably, the FTA paper displays almost perfect linearity for the C_T value versus the microRNA concentration (in log scale), with R² values of around 0.990 for all microRNAs tested. The cycle threshold (C_T) values of FTA card and positive control coincided almost perfectly, indicating that the FTA paper collected the microRNAs within solution completely, with minimum loss during wash and elution. Meanwhile, the experiments using normal cellulose paper showed much higher C_T values for all parallel experiments, indicating the requirement of chemical treatment.

Example 2: Collection of microRNAs from Ι-μ , plasma sample

[0067] Synthetic miR-141 was spiked into 1 -μΐ. of plasma from a healthy donor with final concentrations ranging from 10⁴ -10¹⁰ copies^L. A paper pre-loaded cell-lysis chemicals (e.g., FTA paper) was then used to collect microRNA from plasma sample. After that, the microRNA was extracted and RT-PCR amplified.

[0068] Fig. 7 shows the real-time fluorescence curves of the RT-PCR amplification. The C_T value versus the log-scale microRNA concentration is almost perfectly linear (R²= 0.9945) across 7- order microRNA concentrations indicating a perfect RT-PCR amplification. These results demonstrated the suitability of using a paper-based approach to pickup RNA material from plasma. Example 3: Comparisons of paper-based method with Cell-to-CT kits

[0069] The performance of the paper-based approach is further compared with the Cell-to-CT™ kit (Life technologies, USA). The latter uses a chemical for cell lysis followed by a direct PCR amplification without microRNA extraction and purification. Briefly, 10 MCF-7 cells were spiked into 1 \iL of 1 %, 50% and 95% health donors' blood. For paper-based approach, the sample solutions were picked up using a FTA eluted paper (Whatman) and the extracted genetic materials were analyzed using RT-PCR for four microRNAs (miR-16, miR-21 , miR-141 , and miR-200c). For Cell- to-CT experiment, 1 -//L sample solution was directly lysed and RT-PCR analyzed, following the manufacturer's protocol.

[0070] Figure 8 indicates that both methods have comparable performance for samples of blood- free MCF-7 cells, where the CT values were about the same for both methods. However, for samples containing background blood, the paper-based approach greatly outperformed the Cell-to-CT™ kit, as indicated by, the difference in C_T values. The C_T values of Cell-to-CT™ kit are markedly much higher than that of paper-based method (shifted to the upper-left in Fig. 8). Figure 9 shows the fold difference (2AC_T) of four microRNAs derived from the AC_T values of blood and blood-free experiments (control). The Cell-to-CT™ kit has negative fold difference, which indicated the Cell-to- CT kits has lower RT-PCR amplification performance, with samples containing blood. The positive fold difference of paper-based approach indicates that the paper-based method is capable of handling blood samples and preserve the RT-PCR amplification efficiency. The whole blood is known to contain chemicals which may inhibit the enzymatic PGR amplification. In the paper-based approach, the FTA paper selectively captures the genetic materials from biological samples, and gets rid of the PCR-inhibiting chemicals during the wash step.

Example 4: Cancer cells spiked into health donors' whole blood

[0071] The paper-based approach was applied for whole blood experiments. Briefly, 3-100 cells of MCF-7 (breast cancer), HT-29 (colon cancer), arid DU145 (prostate cancer) were spiked into 1 mL of health donors' whole blood, and then filtered using a microsieve device (CellSievo Pte Ltd, Singapore) following the manufacture's protocol. Peripheral blood samples were collected in spray- coated K2 EDTA Vacutainers (BD, USA) from healthy volunteers. The CellSievo's microsieve device is a biomedical device which is designed to capture circulating tumor cells from cancer patients' whole blood.

[0072] A FTA paper was applied directly on the microsieve device surface to collect the biological samples captured on the CellSievo's microsieve surface. After that, the FTA paper was dried at room temperature for 1 hr, and washed with 70% ethanol for 5 min and dried again. The dried paper was then soaked in 12-20 μί of DEPC water for 30 min at 95°C to elute absorbed genetic materials. Finally, the microRNA profiles (miR-16, miR-21 , miR-141 , and miR-200c) of eluted genetic materials using RT-PCR were analyzed.

[0073] Using the paper-based approach, the microRNA profiles with very high sensitivity even from only 3 spiked cancer cells were obtained (Fig. 10). This indicated the paper-based approach can directly pick up biological sample from irregular substrate for RT-PCR analysis. The results also indicated that the microRNA expression level varies among cancer cell lines. Notably, the HT-29 and MCF-7 contains relative high quality of miR-141 , where a fold difference of up to 97.1 ^χ was observed for a mere 3 cells spiked.

Example 5: CTCs from patients' whole blood

[0074] . Venous blood (7.5 mL) were collected in spray-coated K2 EDTA tubes (BD Vacutainer) and diluted with 7.5 mL of 1 * PBS (containing 5 g/L BSA and 2 mM EDTA). The blood samples were then filtered tlirough a 40-μΜ cell strainer (BD,USA), and applied to the CellSievo's microsieve system at a flow rate of 0.5 mL/min (CellSievo Pte Ltd, Singapore), followed by 4 time washes with 1 mL of 1 x PBS. After that, captured cells were incubated for 30 min with 100 μL antibody mix (containing 20 of 100 mg/ mL DAPI (Invitrogen), 20 μΐ, of AlexaFluor-488 labeled anti-CD45 antibody, 20 iL of PE-labeled anti-EpCAM and 20 μΐ, of PBS buffer), followed by 4 time washes with 1 mL of 1 x PBS. CTCs were eluted out into 500 μΐ, of 1 x PBS and collected in a fresh 1.5 mL tubes. [0075] CTCs are very rare in comparison with white blood cells, thus the eluate often contains outnumbered white blood cells which cause a background containment, and make it challenging to detect genetic material from solely CTCs. To overcome this problem, the split-well method as described in Figure 4 was utilized. Briefly, the eluted CTC sample was centrifuged at 2,000 rpm for 5 min. After that, the supernatant was removed, and the cell pellet was re-suspended in 5 μί, of 1 * PBS and randomly aliquot onto a mini-well plate (Greiner HLA Terasaki multiwell plate, Sigma-Aldrich, USA) with each well containing 0.2-0.5 μΐ, of sample solution. Cells within each well was then examined under a BX61 Olympus florescence microscope to record wells with (CTC-positive;

containing CTCs and WBCs) and without (CTC-negative; containing WBCs) CTCs, according to the fluorescent colour of cells. After that, solutions within 10 CTC-positive wells were collected and pooled together, using the paper-based approach with a FTA paper. Same approach was performed to collect solutions from 10 CTC-negative wells which were checked visually to ensure containing only white blood cells. The collected genetic materials were then amplified by RT-PCR detections which generated the cycle threshold values (C_T) for CTC-positive (C_T(CTC)) and CTC-negative (C_T(NC)) samples. Fold difference (2AC_T) was then calculated based on C_T value difference (AC_T = C_T(CTC) - C_T(NC)) of CTC-positive and CTC-negative samples.

[0076] The developed method was used to profile the microRNA expression from CTCs harvested from 19 breast and colorectal cancer patients, with 17 healthy subjects serving as controls. A panel of microRNAs developed from literatures and from the CTC-microRNA array study were utilized, including miR-16, MiR-21 , miR-28-3p, miR-28-5p, miR-93, MiR-106, miR-125b, miR-141 , miR-183, miR-194, miR-206, miR-200c, miR-221 , miR-222, miR-328, and miR-496. Figure 11 shows the detected CTCs number of the microRNAs expression profiles of 19 patients. These results indicated that 11 of the CTC-positive cancer patients showed significant readouts for microRNA Panel 1 (Fig. 1 1a), which includes miR-200c and miR-141 (both involve in suppression of epithelial - mesenchymal transition (EMT)), miR-93 (blocks tumor development and metastasis by inducing mesenchymal-epithelial transition (MET)) and miR-21 (promotes breast tumor invasion and metastasis). Moreover, patients with no detectable CTCs or healthy donor show no observable expression of Panel 1 microRNAs, which indicates these microRNAs are very specific for CTCs detection.

[0077] For the remaining CTC-positive patients with no or low readouts for Panel I , it was found that they can be detected by microRNA Panel 2, which includes microRNAs involved in regulating sensitivity against chemotherapeutic drugs such as Tamoxifen (miR-221 /-222),

Mitoxantrone (miR-328), doxorubicin (miR-28/-106a/-206), Paclitaxel (miR-125b) and Trastuzumab (miR-194). These results demonstrated the applicability on the described panel microRNAs and the split-well approach for CTCs detection, including miR-16, MiR-21, miR-28-3p, miR-28-5p, miR-93, MiR-106, miR-125, miR-141, miR-183, miR-194, miR-206, miR-200c, miR-221 , miR-222, miR-328, and miR-496 (Fig 10b).

[0078] 6 patients overexpressed the combination of mir-16 and mir-21 either alone or with one or both over expression of mir-200c and mir-93. 4 patients overexpressed the combination of mir-21, mir-200c and mir-93 either alone or with addittion of overexpression of mir-141. 4 of the 6 patients tested with panel 2 had a combination of over expressed mir-221, mir 328 and mir-486

Example 6: Serial microRNA profile from a patient

[0079] The developed protocol and the microRNA biomarkers described in Example 5 were used to monitor the cancer progression of a breast cancer patient under neoadjuvant treatment (chemotherapy) (Fig. 12). Patient's whole blood was collected and analyzed at patient's initial and three follow-up visits. Briefly, 7.5 mL of venous blood was filtered using a microsieve device (CellSievo Pte Ltd), and the captured cells are eluted out into mini-96-wells for cell counting. After that, the CTCs and WBCs were collected using the paper-based approach for microRNA analysis. A panel of microRNA biomarkers was analyzed including miR-28-3p, miR-28-5p, miR-106, miR-183, miR-206, miR-221 , miR-222, miR-328, miR 451 , and miR-486.

[0080] The CTCs number varied during the cancer treatment, where the CTCs number was 55 cells/7.5 ml blood at initial visit, decreased to only 1 and 2 cells/7.5 ml blood at days 33 and 47 respectively during the neoadjuvant treatment, and increased to 41 cells/7.5 ml blood at days 63 again when the chemotherapy was stopped. In addition, the microRNAs expression profiles changed among a therapy treatment. The miR-28-5p, miR-221 , mir-328, and mir-486 were over-expression at the initial visit (before chemotherapy, CTCs number: 55 cells/7.5 mL), while no significant microRNA (except miR-183 which also exists in some healthy people at low level) was found during the first visit and the second follow-up visit (CTC numbers were very low). However, at the third visit (day 63; after chemotherapy has stopped), the expression of mir-28-5p, mir-328 and mir-451 disapeared compared to the expression level at the initial visit; miR-221 and mir-486, over-expressed at the initial visit, was diminished, while a new miR-106 emerged. These results demonstrated the applicability on using the paper-based method, and the split-well approach for detection of CTCs and microRNA biomarkers from cancer patients' whole blood.

[0081] Based on the results in Figures 11 and 12 it can be concluded that and increased expression of miR-138 is an indication of a reduced deseased state where either cancer is not present as in seen in healthy 1 sample or the circulating cancer cells are reduced as seen in figure 12 (c) and (d).

Claims

Claims:

1. Method for the detection of circulating tumor cells (CTCs) in a biological sample, comprising: a) collecting the circulating tumor cells using a paper;

b) extracting the genetic material of the CTCs; and

c) analysing the extracted genetic material for the presence of one or more specific biomarkers.

2. The method of claim 1 , wherein prior to the collecting step, the CTCs are isolated and/or enriched.

3. The method of claim 2, wherein the isolation and/or enrichment is carried out by size separation or antibody capturing methods.

4. The method of claim 3, wherein the isolation and/or enrichment is carried out using a microsieve or anti-CD45 conjugated magnetic beads.

5. The method of any one of claims 2-4, wherein the isolated or enriched CTCs are, prior to the collecting step, dispensed into collection tubes or wells of a mini- or microwell plate.

6. The method of any one of claims 1 to 5, wherein the paper is preloaded with one or more chemicals for cell lysis and/or protein denaturation.

7. · The method of claim 6, wherein the one or more chemicals comprise at least one detergent and/or surfactant.

8. The method of any one of the preceding claims, wherein the collecting step further comprises one or more washing steps.

9. The method of any one of the preceding claims, wherein the extraction step comprises eluting the genetic material from the paper.

10. The method of any one of the preceding claims, wherein the genetic material comprises DNA, mRNA and/or microRNA.

1 1. The method of claim 10, wherein the genetic material comprises microRNA.

12. The method of any one of the preceding steps, wherein the analysing step comprises amplifying the extracted genetic material.

13. The method of claim 12, wherein the amplification is done by PCR.

14. The method of claim 13, wherein the PCR comprises quantitative PCR, real time PCR and/or reverse transcriptase PCR.

15. The method of any one of claims 1 to 14, wherein the analysing step comprises comparing the expression levels of one or more specific biomarkers with a reference standard.

16. The method of claim 15, wherein the reference standard is obtained by subjecting white blood cells also contained in the sample to the same collecting, extracting and analysing steps as the CTCs.

17. The method of any one of claims 1 to 16, wherein the one or more specific biomarkers are selected from microRNAs.

18. The method of claim 17, wherein said microRNAs are selected from the group consisting of miR-16, miR-21, miR-28-3p, miR-28-5-p, miR-93, miR-106, miR-125b, miR-141 , miR-183, miR- 194, miR-200c, miR-206, miR-221 , miR-222, miR-328, miR-451 , miR-486, and miR-496.

19. The method of any one of the preceding claims, wherein the biological sample is whole blood.

20. The method of any one of the preceding claims, wherein the CTCs are selected from the group consisting of breast cancer cells, colon cancer cells, prostate cancer cells, lung cancer cells, bladder cancer cells, bone cancer cells, cervical cancer cells, glioma cells, astrocytoma cells, liver cancer cells, melanoma cells, nasopharyngeal carcinoma cells, ovarian cancer cells, pancreatic cancer cells, renal cancer cells, gastric cancer cells or rectal cancer cells.

21. Biomarker panel for the detection of CTCs in a biological sample, the panel comprising a) miR- 16, miR-21 , miR-93 , miR- 141 and miR-200c; and/or

b) miR-28-3p, miR-28-5-p, miR-106, miR-125b, miR-183, miR-194, miR-206, miR-221, miR-222, miR-328, miR-451 , and miR-486.

22. Use of the biomarker panel of claim 21 for the diagnosis of cancer, for monitoring the progression of cancer and/or for determining the prognosis of a patient afflicted with cancer.

23. Method for the diagnosis of cancer or monitoring the progression of cancer, the method comprising determining the expression level of the biomarker panel of claim 21.