WO2008085777A2 - A method for early detection of cancer - Google Patents

Abstract

The method is a device or apparatus of unique design, material or a combination thereof, which identifies, separates, does imaging and classifies cells and prognostic bio-markers associated with cancer of epithelial nature. The contrivance uses a chip made of glass, silicon, quartz, a combination of these materials, or a plastic material, fabricated by nanotechnology. The method can be used to diagnose, monitor the disease state, disease progression, drug efficacy and a patient's treatment response.

Description

Patent Application of Ajay Smghal

A METHOD FOREARLYDETECTION OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional patent application Ser No

60/883,236 filed January 3, 2007 by the present inventor

FEDERALLY SPONSORED RESEARCH

[0002] Not Applicable

SEQUENCE LISTING OR PROGRAM

[0003] Not Applicable

BACKGROUND— FIELD OF INVENTION

[0004] This invention relates to early detection of cancer

BACKGROUND OF THE INVENTION— PRIOR ART

[0005] a) US Patent, 6,365,362, " Methods and reagents for the rapid and efficient isolation of circulating cancer cells "

[0006] b) US Patent, 4,654,267, " Magnetic Polymer Particles and Process for the

Preparation Thereof "

[0007] c) Vladimir V Iakovlev, Rashmi S Goswami, Jonathan Vecchiarelh, Nona C

R Arneson and Susan J Done, "Quantitative detection of circulating epithelial cells by Q-

RT-PCR" Breast Cancer Research and Treatment, March 09, 2007 [0008] d) Ahki Stathopoulou, Anna Gizi, Maria Perraki, Stella Apostolaki, Nikos

Malamos, Dimistπs Mavroudis, Vassihs Georgouhas, and Evi S Liamdou, " Rea-Time

Quantification of CK- 19 mRNA- Positive Cells in Peripheral Blood of Breast Cancer Patients

Using the Lightcycler System", VoI 9, 5145- 5151, November 1, 2003, Clinical Cancer

Research

[0009] e) US Patent, 7,156,049, " Optical bio-discs and fluidic circuits for analysis of cells and methods relating thereto"

[0010] f) Automated blood counting machines include the Beckman Coulter LH series, Sysmex XE-2100, Bayer ADVIA 120, and the Abbott Cell-Dyn series

[0011] g) Other prior art includes MRI (magnetic resonance imaging), ELISA

(enzyme-linked immuno-adsorbant assay), flow-cytometry and PET (positron emission tomography), bone-marrow biopsy and lymph node biopsy

[0012] h) US Patent 6,905,827, "Methods and compositions for diagnosing or monitoring auto immune and chronic inflammatory diseases" focuses on diagnosing chronic inflammatory disease and detection

[0013] i) US Patent 6,706,867 reports PCR method of K B Mullis U S Pat Nos

4,683195, 4,683,202, and 4,965,188 and amplifies a single copy of a specific target sequence in genomic DNA to a detectable level

[0014] j) US Patent, 7,026,121, "Methods and compositions for diagnosing and monitoring transplant rejection "

[0015] k) US Patent, 6,974,667, "Gene expression profiles in liver cancer "

[0016] 1) US Patent, 6,310,034, "Agouti polypeptide compositions "

[0017] m) US Patent, 6,514,747, "Agouti polynucleotide compositions and methods of use " [0018] n) US Patent, 6,921,638, "Hydorgel-based microarray signal amplification methods and devices thereof"

[0019] o) US patent, 6,399,365, "Bioarray chip reaction apparatus and its manufacture "

[0020] p) US Patent, 6,986,992, "P450 single nucleotide polymorphism biochip analysis "

[0021] q) US Patent, 7,163,789, "Cancer Diagnosis Method "

r) Mammogram

[0022] Typical methods for detecting CTC (circulating tumor cells) are immunohistocytochemistry (ICCS) or RT-PCR (Reverse Transcription Polymerase Cham Reaction) or flow cytometry, which ranges from $30,000 to $150,000 However, these methods are labor-intensive, have technical and operational complexity, present the need for reliable electricity, are unreliable, and not as accurate, rapid, or reproducible as the invention proposed here Without an enrichment procedure, ICCS is known to require several (up to 10 slides) to accurately detect cancer cells Flow cytometry can take as long as 10 hours for analysis, which is not practical for rapid screening and diagnostic in a clinical setting

[0023] Companies such as Affymetπx have incorporated semiconductor fabrication methods, by use of lithography and creating a micro-array for DNA sequence analysis Their technology, however, does not detect single cells Our technology is expected to use proven semiconductor fabrication methods and to detect single cells

[0024] a) Better Cell-Trapping Our methodology proposes the use of unique device and entrapment structures to better trap cancer cells, as well as solid-phase than does the FDA approved Veπdex's CellSearch™ after the cells are isolated and enriched, based on the preferred bio-markers, for a given cancer [0025] b) Higher Statistics The ability of our solid-phase device structures to separate excess chemicals will allow for better statistical results than that reported by Veπdex, LLC or Immumcon i e , an error of up to 50% for 5 CTC per 7 5-ml blood sample can result from multiple wash steps The results from our technology based capture on a solid-phase substrate, will be more reliable

[0026] The table below lists key differentiators of the present invention and technology

CellSearch and CellSpotter ■ -IΪJ5TJϊlllιW-]ιH[.1ιl

NEED FORPRESENT INVENTION

[0027] Worldwide, there are 25 million cases of cancer About one-third of the cancer cases develop into metastatic cancer, a devastating and often terminal illness These demographics underscore a need for a rapid, near-patient, reliable, low-cost early diagnostic method We expect the cost of the present test to be less than $100 Therefore, the test will not only offer the advantage of low-cost, simple test, but also the ability of increased diagnostic validation [0028] The present invention focuses on an in vitro (i) cancer cell enrichment and (n) a cancer cell separation process and device The present invention will enable large pharmaceutical companies by a offering companion diagnostic modality for therapeutics The solution will also provide a diagnostic modality to large diagnostic device companies and laboratories

[0029] These platform solutions will be targeted for (i) the early detection of such epithelial cancers as prostate, breast, colon, and lung, as well as blood-based cancers (ii) monitoring of patient treatment response, and (in) evaluation of cancer recurrence after surgery

[0030] Our solution will address cellular analysis of blood withm the in vitro cancer diagnostics Current methods to detect and monitor breast cancer, e g , mammography, are not sufficiently sensitive insensitive, are inadequate or too expensive The present invention is non-mvasive, avoids painful biopsy, and can be used as a screening method or to increase diagnostic validation, in combination with other methods such as X-ray

PLATFORM TECHNOLOGY FOR MANY CANCERS

[0031] With the platform approach of the present invention, we propose early diagnosis and monitoring of lung, prostate, colon, and other cancers The low cost and ease of tumor cell detection offered by our platform will help increase diagnostic validation and will complement other tests such as that of X-ray when, for example, a spot is detected in the lungs Likewise, our method will complement sigmoidoscopy for colon cancer

[0032] The high prevalence of new cancer cases in 2006 in the US alone, listed in the table below, indicates a large market opportunity

SUMMARY

[0033] The method is a nanotechnology or microchip device, apparatus, system or contrivance It preferentially identifies, enriches, purifies, separates, marks, detects, images and counts circulating tumor cells (CTC) or leukemic cells or other biological species, associated with prognostic markers, in a known volume of sample of human blood, bone marrow or other fluid Thus, the device helps early detection and monitoring of cancer and other related illnesses The method may or may not be used in combination with other cancer diagnostic methods

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG 1 is a flowchart of the nanotechnology chip in operation to detect circulating tumor cells (CTC) and other illnesses associated with the related biomarkers

[0035] FIG 2 is an array of micro-well and array of micro-structures fabricated by nanotechnology and chip fabrication methods The array carries micro-probes to mark and identify bio-markers, unique to cancer and the cancer cells to be diagnosed

[0036] FIG 3 is a schematic of apparatus for detection of epithelial and blood-based cancers and any other types of cancers, and of diagnostic and prognostic markers in a sample of blood or another fluid from a human or animal

[0037] FIG 4 is a schematic of apparatus for cancer cell enrichment, with example methods, to transfer to the chip, detection, imaging and analysis on the chip of the cells or circulating tumor cells (CTC) for epithelial cancers and other types of cancers, and of diagnostic and prognostic and therapeutic markers in a sample of blood or another fluid from a human or animal

[0038] FIG 5 is a schematic of apparatus for cancer cell enrichment, with removal of

"debris" by micro-plumbmg and incubation of secondary antibodies onto the circulating tumor cells (CTC) deposited on micro-chip assembly

[0039] FIG 6 is a schematic of Dynabeads, showing antibody tagging to protein expression sites on a T-cell

DETAILED DESCRIPTION— FIGS 1 THROUGH 6— PREFERRED EMBODIMENT

[0040] A preferential embodiment of the flow of the present invention is illustrated in

Fig 1 The figure shows taking a known volume (0 lμl to 20ml) of sample of human blood, bone marrow or other fluid such as saliva, sputum (4A) in a vacutamer tube As illustrated in Figure 4, the sample 4A is subjected to removal of red blood cells by lysis buffer step (4B), and removal of "debris" (4C) or of leukocytes or of white blood cells, which lack CD45 [0041] A sample of 10ml blood contains about 5x108 red blood cells, 1x106 white blood cells (WBC or leukocytes) and 4x107 platelets Threshold concentration of the circulating tumor cells (CTC), for example, for metastatic breast cancer has been shown to be 5 cells per 7 5ml of blood Thus, the sensitivity requirement for tumor cell detection is of the order of 1 in 108 to 1 in 109 (or a billion) cells, since 10ml of whole blood contains about a billion cells

[0042] Following the lysis buffer step by centπfugmg for example, at 300g for 1 to 2 minutes, and other conditions shown in Fig 4, and after removal of the WBC (4C), serum consisting of the desired cells, and with 30 to 40% volume (or 3 to 4ml), of the original starting sample volume is left In a separate step, in one of the preferred embodiments, primary antibodies are attached on the surface of the microcavities or micro-wells on the micro-chip device (3E, 4F), by optimizing the surface chemistry of the substrate so as to expose the hydrophobic sites In one variation of the preferred embodiment, PVDF (poly- vmylene difluoπde) substrate is soaked in methanol to expose the hydrophobic sites, and for subsequent preferred binding

[0043] In a preferred embodiment, the cell-enriched serum (4D) is overlaid and incubated on the chip, carrying primary antibodies The tumor cells will bind to the microchip device (3E, 4F, 5D) The kinetics of this binding will be determined by the number of binding sites (6B) per cell and the dissociation constant (kD) After an initial wash-step, secondary fluorescent antibodies (4G, 5C, 6A) are attached onto the cells bound to the microchip device (3E, 4F, 5D) The microchip device is then analyzed for counting and imaging the cells and their simultaneous pathological examination (5E)

[0044] These steps can be carried out in 1 to 8 hours time, in a near-patient setting, in a hospital laboratory or in a clinical laboratory Further, the results will be objective and operator independent Thus, quick clinical decision-making can be made, for patient screening, for patient response to therapeutic treatment, for risk-assessment for cancer recurrence after surgery, for determining if the tumor cells are benign or malignant, and therefore, decision-making for surgery, and for disease prognosis [0045] The cells or biological species are analyzed (3G, 3H, 31) by a microchip assembly system The apparatus uses fluorescent meter and analyte-specific antibody immunohistochemical such that cell species, diagnostic and prognostic biomarkers for epithelial cancers such as lung cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, colon cancer and other forms of cancers

[0046] For example, breast cancer genes, BRCA-I, and BRCA-2, antigens CEA

(carcmoembryomc antigen), PSA (prostate specific antigen), CA 125, CA15 3, CA19 9, breast cancer protein human epidermal growth factor receptor (HER-2), are preferentially stained and identified

[0047] In another variation of the preferred embodiment, the associated tumor cells can be separated by magnetic polymer beads either by positive or by negative enrichment and by applying a magnetic field to the container (4D) These species can be marked with anti-

BRCA-I, anti-BRCA-2, anti-PSA, anti-CA125, anti-CA15 2 or anti-CA19 9 antibodies

Likewise in another variation, DNA, mRNA, protein species or the tumor cells associated with the proteins or biomarkers characterizing the disease are preferentially extracted, enriched, transferred, marked and analyzed onto the nanotechnology chip or device (3E, 4F,

5D)

[0048] The species and its sequence are identified, marked and quantitatively analyzed (Fig 1, 3H, 4H, 5E) If the results are withm the desired range, the patient is not ill with one of the above epithelial cancers For example, the CA125 antigen expressed in epithelial ovarian cancer, at levels higher than 30U/ml of serum will indicate presence of ovarian cancer If the results are outside the range, the patient is diagnosed ill or needing attention

[0049] The static sample in the chip assembly (3C, 3D, 3E) is optically illuminated, and fluoresced In one variation of the preferred embodiment, the species will be electro- magnetically excited The output optical or fluorescence, or luminescent or spectrometer or electrical or electromagnetic signal is detected using a photomultipher, CCD camera, CMOS detector or other microscopy or spectroscopy or electronic or electromagnetic device The detector is connected to a computer assembly (3G) The system also has the software capability of statistical image acquisition, classification and analysis (3G and 31)

[0050] The micro-chip assembly (Fig 2) is fabricated by using the following steps as one of the preferred embodiment The size of the micro-chip device will range from a stamp size (l"x 1") to a post-card-size (4"x5") While the stamp size chip device may be smgle-use, the larger device and its different sections may be used multiple times for diagnosing and monitoring the same patient The geometry of the micro-array on the lithography mask is shown After the mask is laid out and created, micro-well structures and micro-array are fabricated

[0051] The process sequence will include photolithography (contact mask lithography, i-lme-365nm, h-lme-405nm, g-lme-436nm, 248nm, 193nm, another equivalent printing method or a conventional complementary metal-on-semiconductor- CMOS processing sequence of a technology generation between 5μm to 22nanometer) Precise reactive ion etching (RIE) will control the critical dimensions (CDs) withm a reasonable dimensional tolerance (of 10 to 30%) such that the micro-volume of the sample can be controlled The substrates of interest are sapphire (A12O3), silicon, quartz, Si3N4, SiO2/Si, Si3N4/SiO2/Si, or a combination of these film stacks and plastic such as PMMA or other such material

[0052] In one variation, the substrates of the microchip device (Fig 2, 3E, 4F, 5E) will be a composite consisting of micro-spheres of magnetic particles (for example, of permanent magnets- Nd2Fel4B, Sm2Col7) embedded and dispersed throughout the substrate for preferential paramagnetic enrichment of the cell species This variation of the preferred embodiment will help the tumor cell enrichment and separation process Cell enrichment, separation and adhesion of cells to the antibodies on the micro-chip substrate can, therefore, be carried out in a single step

[0053] In another variation, the plastic or polymer or composite polymer substrate,

"negative" trenches or pillars may be formed The desired cell species, characterized by certain biomarkers or proteins or antigens will collect and adhere to the these pillars, which carry the biological probes or analytes or antibodies [0054] Typical thickness of these substrates is between 200μm to lOOOμm (or lmm)

In other variations, the substrate thickness may be larger, for ease of fabrication and sample handling The example embodiment description and critical dimensions (CDs) of the micro- array structures are as follows

1) 5x5 array, 20μm diameter holes, 50μm deep (or 0 2μl sample volume/micro-well)

2) 5x5 array, 50μm diameter holes, 50μm deep (or 1 25μl sample volume/micro-well)

3) 5x5 array, lOOμm diameter holes, 50μm deep (or 5μl sample volume/micro-well)

4) 5x5 array, lmm diameter holes, 500μm deep (or 5000μl sample volume/micro-well)

5) 2x2 array, 10mm diameter holes, 500μm deep (or 50ml sample volume/micro-well)

6) 2x2 array, 15mm diameter holes, 500μm deep (or 112 5ml sample volume/micro-well) [0055] In this preferred embodiment, an optional second photolithography step will be used This step will be used to make holes at the bottom of these microwells (5E) These holes will help better cell entrapment, filter the undesired cell and biological species and help remove excess fluid The antibody conjugated species or cells, will be better retained on the microchip substrate, on top of the surface due to increased surface area, which will then be imaged These cells will be analyzed using the imaging, classification, and by quantitative, statistical analysis Example dimensions for the group of arrays 1) to 3) in the above list is a 2x2 array of 2μm diameter holes For the group 4) to 6) array, the specifications can be a 5x5 array of 2μm diameter holes In another variation, the size of these holes may be made 5μm diameter, which is still smaller than the size of the tumor cells The size of tumor cells is similar to that of leukocytes or slightly larger and ranges between 5 and 15μm

[0056] In another variation of the preferred embodiment, the holes sizes, shapes, depths and array sizes may be modified The volume of these micro-wells is such that they structures can handle varying volumes of the raw or enriched sample carrying the cellular or biological species of interest THEORY OF OPERATION

[0057] The preferred embodiment is the platform illustrated in Fig 1 , which is a combination of device and chemistry (assay) that preferentially enriches, isolates by filtering unwanted cells based on their size (< 6-7μm) and surface treatment and by binding the desired cells to the device substrate, images and analyzes circulating tumor cells (CTC) associated with one or more bio-markers The error in sensitivity of the rare cancer cells will be inversely proportional to their concentration or number (N) in the sample volume In other words, error, expressed as Λ/N/N = 1/Λ/N will improve with rising concentration (N) of the cells in the whole blood sample, as the denominator N increases in the above error term Therefore, the % error will be higher (100%) for N = 1 cell compared to (33%) for N = 10 cells in the same volume of the sample of whole blood

[0058] These bio-markers are molecules or proteins that characterize the surface of

CTC for a certain cancer type Further, depending upon the cancer type and the behavior of the CTC, one or more of these proteins or bio-markers may be more highly or less highly expressed It is desirable that the cancer cells equally and highly expressive such that they can be isolated and imaged with high signal-to-noise ratio or high mean fluorescence intensity (MFI)

[0059] For example, epithelial cancer cells for breast cancer, unlike leukocytes (or white blood cells), consist of cytokeratm, CK+, namely CK8, CK9, CK19, and CD45- (a marker for leukocytes) These bio-markers are FDA approved in 2004 (CellSearch™ and Cell Spotter™) for monitoring treatment response to therapy for breast cancer patients As shown in the FIG 5, cellular debris, i e , leukocytes, red-blood cells, and platelets will be preferentially separated [0060] The present invention for cancer cell detection and imaging utilizes preferential separation labelling, and purification of CD3, CD4 and CD8 with appropriate antibodies Typical volumes of the antibodies requirements are whole blood to the antibody reagent in the ratio of 10 1 Work-flow for another application of HIV testing, by CD4- T cell lymphocyte detection and imaging, which uses much smaller whole blood volume (lμl compared to about 10ml for the present invention), is illustrated in an article published in, PLoS Medicine, July 2005, Volume 2, Issue 7, el82 The CD4- cell testing was carried out using a peripheral blood sample of 1 μl, and antibody-taggmg (Alexafluor-488 and Alexafluor-647 conjugated antibodies, Invitrogen, Carlsbad, CA) of these cells (CD4- T cell lymphocytes) was performed

[0061] Subsequent to mechanical removal of "debris," namely the red-blood cells and platelets and of antibody-conjugated leukocytes (WBC), one is left with an enriched sample containing the tumor cells Alternatively, red-blood cells may be separated by lysis The micro-plumbed, enriched sample (l-200μl) is transferred to the microchip device (3E, 4F, 5E) with microwell or micro-pillar or micro-posts structures In one variation, the microwell structures are loaded with bead-based assay carrying the fluorescent-conjugated antibody tagging agent, e g , Alexafluor-488 (A21335, clone 289-14120, Molecular Probes, Eugene, Oregon, United States )

[0062] Our experimental plan will develop antibodies-based assays for cancer cell enrichment from peripheral whole blood samples In particular, in a 7 5-ml sample of blood, a threshold of 5 or more CTCs has been reported for metastatic breast cancer To ensure high specificity, we will target a family of known bio-markers, e g , CD45-, CK8, CK9, CK19, CK20 (breast cancer), CEA, CK20, EGFR (for colorectal cancer) and PSA, Her-2 (for prostate cancer) The technology is a platform technology, as one will be able investigate CTC for breast cancer, colorectal and prostate cancer and other cancer types Tumor cells for prostate cancer have been reported to be characterized by msulm-hke growth factor (IGF-IR) [CIm Cancer Res 2007,13(12) June 15, 2007]

[0063] The preferred embodiment includes optimizing the cell capture and binding affinity to the device cartridge first with cell lines with known surface density and expression These samples are readily available and can be easily engineered in a laboratory environment to optimize the expression rate, which determines their detectability The known cell lines, which will serve as internal controls, are the CD3 and CD4 cell lines The concentration of these cells relative to circulating tumor cells (CTC) in peripheral blood is 10,000 to 100,000x more abundant Alternatively, CD34 are about 1000 times more abundant compared to CTC in blood and can also serve as internal reference and calibration species

[0064] Another important aspect of the present invention is the performance of a prevalence study Initially, one will use known cell samples and dilute them to certain concentrations such that they can be treated as standards With these pre-determined concentrations, one will be able to demonstrate the higher efficacy of our method over that of the benchmark, namely flow cytometry Flow cytometry, however, takes up to 10 hours for analysis, and therefore, while it can be a good calibration tool, it is slow for clinical application Surface chemistry optimization, namely, hydrophobicity and protem-protem interactions between the surface molecule proteins and the surface of the device, will be optimized, first with the cell lines for highly abundant, well characterized cells for example, with CD3, CD4 and CD34 Subsequently, one can carry out this optimization specific for the desired species of rare cancer cells These optimizations are illustrated in tables at the end of this section

PLATFORM COMPONENTS

[0065] The device will consist of one or more table-top instruments for sample preparation and cell quantification In one of the variations, cancer cell enrichment will be carried out by positive or negative immunomagnetic enrichment, e g by using Dynal Beads CD45-, in combination with magnetic particle separation, for negative separation While positive enrichment tags the specific CTC species, negative enrichment method leaves the cell character prime and untouched Subsequent sample handling and processing will be performed using a disposable, cartridge-based micro-chip device, a consumable containing an array of microwells or micro-pillars The unique structure of the micro-well device and its surface treatment with primary antibodies will promote tight trapping and binding of the CTCs (6B) The fabrication of the device and novel structures will use proven methods The trapped cells will be sandwiched by subsequently treating them with secondary antibodies, for fluorescent imaging and enhanced mean fluorescence intensity (MFI) Cell quantitation will be performed by imaging the micro-array substrate (3E) with fluorescence microscopy and image analysis or other variation of imaging or microscopy or detection method Subsequently, cell imaging and statistical analysis will be carried out by digital image analysis software (3G) customized for the application

CHEMISTRY, CELL ENRICHMENT AND RECOVERY

[0066] Tumor-cell recovery is known to be in the range of 50% to 90% (as published by Veπdex, LLC) However, in other reports,by quadrapole magnetic flow sorter, cancer cell recovery is shown to be 89%, [Nakamura M, Decker K, Chosy J, Cornelia K, Melmk K, Moore L, Lasky LC, Zborowski M, Chalmers JJ The separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter Biotechnol Prog 2001, 17 1145- 1155]

[0067] Off-the-shelf reagents are available, for example, immunofluorescence staining of the enriched cancer cell sample can be carried out with anticytokeratm (CK3- 6H5)-FITC CD45-MACS (Cat No 130-045-801, Miltenyi Biotec, Auburn, CA), for example for breast cancer, biomarkers for epithelial cancer cells are CK8+, CK9+, CK19+, CD45-, or Her2 Positive or negative cell-labelmg can be applied Positive labeling has been reported to limit further labeling, for analysis if specific sites are already bound with antibodies

[0068] In another variation, Dynabeads, used in magnetic cell separation technology, are commercially available from Invitrogen Corporation (Carlsbad, CA) and are shown in Fig 6 The present invention contrasts, with the liquid phase approach used by Veπdex, LLC, which uses ferro-fluids The figure 6 shows Dynabeads (6A) signaling to T cells (6B) and the mechanism of binding of to the cell specific sites

DIFFERENTIATION AND ADVANTAGES

[0069] Our method is based on single cancer cell detection Because of the ability for single-cell detection, the method is expected to assist in the early detection of cancer or metastatic cancer With an X-ray or a mammogram, however, detection requires a tumor comprising at least 1,000,000 cells, below which the sensitivity of the test decays dramatically ( 0 5mm to 5mm tumor size will have 100,000 to 100,000,000 cells, with an average size of about 10mm) Further, the present invention is non-mvasive, avoids painful biopsy, and can be used as a screening method and to increase diagnostic validation, in combination with other methods such as X-ray, and ιn-vιvo biological imaging

[0070] The present invention offers the ability to test multiple (3 or 4 ) biomarkers simultaneously The method offers the ability to format and detect these markers in a single test compared to multiple tests, as well as an ability to carry out pathological examination of the cells The method also offers a tool to test the efficacy of novel drugs or drug combination in the treatment of specific cancer Finally, the method can help surgeons improve decision- making whether or not to perform surgery, depending upon the benign or malignant nature of the cells Finally, the results will be in on a solid-phase substrate, will be objective and operator independent, and will reduce use of expensive chemicals and reagents

CONCLUSION, RAMIFICATIONS, AND SCOPE

[0071] Although the description above contains many specificities, these should not be construed as limitations on the scope of any embodiment, but as merely providing illustrations of some of the presently preferred embodiments thereof Many other ramifications and variations are possible withm the teachings of the various embodiments Thus the scope of the invention should be determined by the appended claims and their legal equivalents, and not by the examples given

Claims

1. A method for early diagnosis or monitoring of cancer comprising: a) a device b) obtaining a sample of blood of a human or animal subject suspected of having early stage cancer or with metastatic disease, a test specimen comprising circulating tumor cells (CTC), a mixed DNA, RNA, gene or protein population suspected of associated cancer tumor cells of epithelial nature; c) enriching said test specimen of the cancer cells, DNA, messenger RNA or genes specific to cancer, to the substantial exclusion of the other test specimen components; d) subjecting the specimen to staining and marking by specific probe species distributed in a micro-array fashion on a micro-chip, fabricated by lithography and other nanotechnology methods, such that if said cancer biomarkers are present in said test specimen; and e) analyzing said biological species on the above nanotechnology device, for the presence of said cancer, the presence of said cancer in said specimen indicating the presence of early stage cancer in said test subject.

2. A method as claimed in claim 1, wherein before analysis said enriched sample is contacted with at least one probe species that labels said cancer biomarkers.

3. A method as claimed in claim 1, wherein after binding of the cell or biological species to the device, it is used for pathological examination of the cell or biological species associated with cancer or the disease.

4. A method as claimed in claim 1, wherein the chemistry, for primary and secondary binding and analysis of the cancer cell or biological species on the device, is used as companion diagnostic with the drug used for therapeutic treatment of the cancer or other disease.

5. A method as claimed in claim 1, which when combined with or independent of other diagnostic methods, will help distinguish benign cell species from malignant, and will help decide surgical procedure on a patient.

6. A method as claimed in claim 1, which comprises analyzing biological species specific to one of the cancers, which is a carcinoma selected from the group consisting of apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, Walker, basal cell, basosquamous, Brow-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non small cell lung, oat cell, papillary, scirhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, tibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxocarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofϊbroma, adenolymphoma, carcinosarcoma, chordoma, mesenchymoma, mesonephroma, myosarcoma, ameloblactoma, cementoma, odontoma, teratoma, throphoplastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulose cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepitholioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, antiokeratoma, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphagioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, lipsarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, myxosarcoma, ovarian carcinoma, rhabomysarcoma, Kaposi's sarcoma, and mast-cell sarcoma.