Diagnosing and treating breast cancer

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for determining a response to an anti-oestrogen therapy in the individuals suffering diagnosed breast cancer. Substance of the method for determining the response to the anti-oestrogen therapy in the individual suffering breast cancer consists in calculating an endocrine therapeutic index of circulating immune complexes (ETI-COC). If the ETI-COC falls within the range of 0-3, the favourable response to the anti-oestrogen therapy is stated, while the value of 4-6 shows the moderate response, and the value of 7-14 indicates the weak response.

EFFECT: using declared method enables determining the individual's response to the anti-oestrogen therapy effectively.

10 cl, 7 tbl, 8 dwg, 1 ex

This application claims the priority of provisional applications 61/362021, filed July 7, 2010 and 61/469890, filed March 31, 2011, each of which is included here by reference in its entirety.

AREA of TECHNOLOGY

The present invention relates to compositions and methods for diagnosis, research and treatment of cancer, including, but not limited to, cancer markers. In particular, the present invention relates to compositions and methods of the forecast of the entity's response to cancer therapy.

The LEVEL of TECHNOLOGY

Breast cancer is the second most common form of cancer among women in the United States and the second leading cause of cancer deaths among women. While in the 80 years there has been a sharp jump in the number of new cases of breast cancer, at present, it appears that this number has stabilized. Reduction of death from breast cancer, apparently, is due to the fact that more women are getting mammograms. If detected early the chances of successful breast cancer treatment is much better.

Breast cancer that is highly curable by surgery, radiotherapy, chemotherapy and hormonal therapy, most often cured if detected early. Mammography is the most important way of screening for the early detection of breast cancer. Breast cancer is classified into different subtypes, but only few of them affect the prognosis or choice of therapy. Maintaining the patient after initial suspicion of breast cancer includes confirmation of the diagnosis, the assessment of the stage of disease and the choice of therapy. The diagnosis can be confirmed by aspiration Cytology, trephine-biopsy under stereotactic or ultrasound guidance for non-palpable lesions or incisional or excisional biopsy. During the surgical removal of tumor tissue, the part is processed to determine the levels of re and RP.

The prognosis and choice of therapy is influenced by patient age, disease stage, pathologic characteristics of the primary tumor, including tumor necrosis, levels of receptors for estrogen (ER) or to progesterone receptors (PR) in the tumor tissue, overexpression of HER2 status and indicators of proliferative capacity, along with menopausal status and General health status. Patients who are overweight may have a worse prognosis (Bastarrachea et al., Annals of Internal Medicine, 120: 18 [1994]). The prognosis may also vary depending on race, with African Americans and, to a lesser extent, Latin Americans have a worse prognosis compared with white race (Elledge et al., Journal of the National Cancer Institute 86: 705 [1994]; Edwards et al., Journal of Clinical Oncology 16: 2693 [1998]).

Three �main method of treatment for breast cancer - it's surgery, radiation, and drug therapy. No such treatment that would suit each patient, and often requires two or more types of treatment. The choice is determined by many factors, including patient age and menopausal status, type of cancer (e.g., ductal vs. lobular), its stage, whether the tumor has receptors for the hormone or not, and level of invasiveness.

Ways to treat river breast cancer is defined as local or systemic. Surgery and radiation are considered local therapies because they directly affect the tumor, mammary gland, lymph nodes or other specific parts. Drug treatment is called systemic therapy because its effects are widely distributed. Drug therapy include classical chemotherapeutic drugs, treatment, blocking hormones (e.g., aromatase inhibitors, selective modulators of estrogen receptors and inhibitors of estrogen receptors) and treatment with monoclonal antibodies (e.g., against HER-2). They can be used separately or, more often, in various combinations.

There is a need for additional methods of treating, in particular, in the methods of treatment adapted to the tumor of the patient.

Summary of the INVENTION

The present invention relates to compositions and �the means of diagnosis, research and cancer therapy, including, but not limited to, cancer markers. In particular, the present invention relates to compositions and methods of the forecast of the entity's response to cancer therapy.

In some embodiments embodiment the compositions and methods of the present invention find use in determining the prognosis of a subject (e.g., a subject diagnosed as having metastatic breast cancer) in relation to survival or response to treatment (e.g., anti-estrogen treatment). Such methods find application both in research and in clinical applications.

For example, in some embodiments embodiment the present invention provides a method of determining the course of treatment, comprising detecting the level of circulating tumor cells (CTCS) in a sample from the subject diagnosed as having metastatic breast cancer; and determining the course of treatment based on the level of CTCS in the sample. In some embodiments embodiment the treatment contains anti-estrogen therapy (such as tamoxifen or an aromatase inhibitor such as letrozole, anastrozole or exemestane). In other embodiments, embodiments the treatment includes chemotherapy. In some embodiments embodiment the method further includes a step of characterization of one or more tumor markers, associer�bathrooms with CTCS (e.g., estrogen receptor, the receptor of human growth factor-2 - HER-2, regulator of apoptosis bcl-2, markers of apoptosis, receptor insulin-like growth factor-1 - IGFR1, vimentin or proliferation factor ki-67). In some embodiments embodiment, the marker of apoptosis is detected using the monoclonal antibody M30. In some embodiments, embodiments of metastatic breast cancer is positive for the receptor to estrogen. In some embodiments, embodiments, one or more tumor markers are detected using multiple technologies (e.g., multiple PCR) or immunomagnetic analysis. In some embodiments, embodiments of the automated tests. In some embodiments embodiment the method further includes the step of determining the CSC-endocrine therapy index (CSC)-IEP). In some embodiments embodiment the CSC-IEP is calculated by assigning a score to the levels of CTCS and tumor markers. In some embodiments of the incarnation low CTCS-IEP is an indication that the subject who is likely to respond to endocrine (e.g., anti-estrogen) therapy. In some embodiments embodiment the high rate of CTCS-IEP is an indication that the subject probably does not respond to endocrine therapy, and that it is better to chemotherapy.

BRIEF DESCRIPTION of FIGURES

Figure 1 demonstrates�should the expression of estrogen receptor on the cancer cell lines.

Figure 2 shows the expression of (a) Bcl-2, b) Ki-67 and HER-2 in cancer cell lines.

Figure 3 demonstrates the calculation of scores CSC-IET in support of cancer cell lines.

Figure 4 demonstrates the calculation of scores CSC-IEP for three patients.

Figure 5 shows a diagram of an exemplary clinical studies points CTCS-IEP and clinical outcome in breast cancer.

Figure 6 shows a diagram of an exemplary clinical studies points CTCS-IEP and clinical outcome in breast cancer.

Figure 7 shows a diagram of an exemplary clinical studies points CTCS-IEP and clinical outcome in breast cancer.

Figure 8 shows the results of a clinical study of 8 patients who were monitored points (CSC)-IEP.

DEFINITION

To facilitate understanding of the present invention, the following defines a number of terms and phrases.

In this context, the terms "detecting" or "detection" can be described as the overall effect of the recognition or recognition and specific observation of the composition.

In this context, the term "nucleic acid molecule" refers to any molecule containing nucleic acid, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known analogues based�th DNA and RNA including but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridination, pseudoisocyanine, 5-(carboxylatomethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5 - carboxymethylaminomethyl, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouridine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosidosis, 5'-methoxycarbonylmethyl, 5-metacircular, 2-methylthio-N6-isopentenyladenine, uracil-5-oxiana acid methylether, uracil-5-exucuse acid, oxibutinina, pseudorutile, kvasina, 2-thioridazine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxiana acid methylether, uracil-5-exucuse acid, pseudorutile, kvasina, 2-thioridazine and 2,6-diaminopurine.

The term "gene" refers to nucleic acid sequences (e.g. DNA), which contains coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence�activities or part of the coding sequence, assuming that the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full length or a fragment preserved. The term also encompasses the coding phase of the structural gene and the sequences adjacent to codereuse site as at the 5' and 3' ends for a distance of about 1 KB or more from either end, so that the gene belongs to the full-length mRNA.

Sequences located at the 5' coding of the site and present on the mRNA are 5' non-broadcast sequences Sequence located at the 3' or in the course of transcription of the coding of the site and present the mRNAs are 3' non-broadcast sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding phase, interruptible not-coderedii sequences, called "nitrones" or "interveniously sites" or "interveniously sequences". Introns are segments of a gene that are transcribed into nuclear RNA (Garc); introns may contain regulatory elements such as enhancers. Introns are removed or "spiceroads" from the nuclear or primary transcript; introns therefore are absent in transcr�Pte messenger RNA (mRNA). The functions of mRNA during translation are to determine the sequence or order of amino acids in the formed polypeptide.

In this context, the term "heterologous gene" refers to a gene that is not located in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been modified in some fashion (for example, subject to mutation, added in multiple copies, linked non-natural regulatory sequence, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous sequence of the gene, typically joined to DNA sequences that are not found in nature-related gene sequences in the chromosome, or they are associated with parts of a chromosome that is not found in nature (e.g., genes expressed in loci where the gene is normally expressed).

In this context, the term "oligonucleotide" refers to a short section agentcity polynucleotide chain. The oligonucleotides, typically have less than 200 residues in length (e.g., between 15 and 100), however, in this context, the term also, as implied, covers a longer polynucleotide chains. Oligonucleotides are often imenuid� along its length. For example, the oligonucleotide with 24 residues referred to as "24-Mer". The oligonucleotides can form secondary and tertiary structures by samogorodskaia or hybridization with other polynucleotides. Such structures may include, but are not limited to, duplexes, studs, crosses, bends and triplexes.

In this context, the terms "complementary" or "complementarity" are used as applied to polynucleotides (i.e., a sequence of nucleotides) related by the rules of base pairs. For example, the sequence "5'-A-G-T-3' complementary sequence "3'-T-C-A-5'". Complementarity may be "partial" in which only some of the nucleic acid bases are matched according to the rules of base pairs. Or can ever be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between strands of nucleic acids has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend upon binding between nucleic acids.

The term "homology" refers to the degree of complementarity. It may be partial homology or complete homology (i.e. identity). Partial�about complementary sequence - a nucleic acid molecule that at least partially inhibits hybridization between fully complementary nucleic acid molecule with the target nucleic acid is "substantially homologous". The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using the hybridization assay (southern or Northern blotting, hybridization in solution, and the like) under conditions of reduced stiffness. Substantially homologous sequence or probe will compete for binding and inhibit the binding (i.e., hybridization) of a completely homologous nucleic acid molecule with the target under conditions of low stringency. This is not to say that conditions of low stringency are such that allow you to be nonspecific binding; conditions of reduced stiffness require the binding of two sequences to one another was specific (i.e., selective) interaction. The absence of nonspecific binding can be checked by using a second target, which is largely complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not gibridizatsiya with� second complementary target.

When used in relation to donacute nucleic acid sequences, such as cDNA or genomic clone, the term "substantially homologous" refers to any probe that can gibridizatsiya with either or both threads donacute nucleic acid sequence under conditions of low stringency as described above.

A gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript. the cDNAs that are variants of splicing of the same gene will contain areas of identity or complete sequence homology (representing the presence of the same exon on both cDNAs) and areas of complete non-identity (for example, representing the presence of exon "A" on cDNA 1, while cDNA 2 contains instead exon "B"). Due to the fact that the two cDNAs contain the areas of sequence identity, they would both be gibridizatsiya with a probe derived from whole gene or portions of the gene containing sequences found on both cDNAs; two splicing variants therefore substantially homologous to such a probe and to one another.

When used in relation to agentcity nucleic acid sequence, the term "substantially homologous" �tositsa to any probe, which can gibridizatsiya (i.e., complements) sequence agentcity nucleic acid under low stringency as described above.

In this context, the term "hybridization" is used in relation to the formation of pairs of complementary nucleic acids. On hybridization and the strength of hybridization (i.e., the strength of the Association between the nucleic acids) is influenced by such factors as the degree of complementarity between the nucleic acids, the rigidity of the conditions involved, the Tm of the formed hybrid, and the ratio of G:C nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure, is called "samovyrivnyuyucha".

In this context, the term "stiffness" is used in relation to the conditions of temperature, ionic strength and the presence of other compounds such as organic solvents, under which nucleic acid hybridization. Under "low stringency" of the desired nucleic acid sequence will gibridizatsiya with its exact complement, sequences with single mismatches of bases, with closely related sequences (e.g., sequences with 90% or greater homology), and sequences having only partial homology (e.g., �posledovatelnostei with 50-90% homology). Under "conditions of medium hardness" of the desired nucleic acid sequence will gibridizatsiya only with its exact complement, sequences with single mismatches of bases and with closely related sequences (e.g., 90% or more homology). Under "stringent conditions" required sequence of nucleic acid will gibridizatsiya only with its exact complement, and (depending on conditions such as temperature) with sequences with single mismatches of bases. In other words, under conditions of high stringency, the temperature may be raised so as to exclude hybridization with sequences with single mismatches of bases.

"Stringent conditions" when used in relation to nucleic acid hybridization include conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (to 43.8 g/l NaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH brought to 7.4 with NaOH), 0.5% of LTOs, 5X reagent of Deinhard and 100 ág/ml denatured DNA salmon sperm, washed in a solution containing 0.1 X SSPE, 1.0% of LTOs at 42°C when using a probe of about 500 nucleotides in length.

"Medium stringency" when used in relation to nucleic acid hybridization include conditions, e�vigalantee binding or hybridization at 42°C in a solution consisting of 5X SSPE (to 43.8 g/l NaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH brought to 7.4 with NaOH), 0.5% of LTOs, 5X reagent of Deinhard and 100 ág/ml denatured DNA salmon sperm, washed in a solution containing 1.0 X SSPE, 1.0% of LTOs at 42°C when using a probe of about 500 nucleotides in length.

"Conditions of reduced stringency" include conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (to 43.8 g/l NaCl, 6.9 g/l NaH₂PO4 H₂O and 1.85 g/l EDTA, pH brought to 7.4 with NaOH), 0.1% of LTOs, 5X reagent of Deinhard [reagent 50X Deinhard designed for 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ug/ml denatured DNA salmon sperm, washed in a solution containing 5X SSPE, 0.1% of LTOs at 42°C when using a probe of about 500 nucleotides in length.

In engineering it is well known that can be applied to numerous equivalent conditions of low stringency; factors such as the length and nature (DNA, RNA, the composition of the bases) of the probe and nature of the target (DNA, RNA, the composition of the bases, presence in solution or in immobilizerpower, etc.), and takes into account the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), and the hybridization solution may be varied to generate conditions g�britishly in low hardness, than those listed above, however, equivalent. In addition, in the technique known conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of hybridization and/or stages of the laundering, the use of formamide in the hybridization solution, etc.) (see above definition for "stiffness").

In this context, the term "oligonucleotide amplification" refers to an oligonucleotide that's hybrid with the target nucleic acid or its complementary nucleic acid and is involved in the reaction nucleic acid amplification. An example of oligonucleotide amplification is a "primer" that's hybrid with the matrix nucleic acid and contains a 3' HE is the end, which is extended by a polymerase in the amplification process. Another example of an oligonucleotide amplification is an oligonucleotide that is not extended by a polymerase (e.g., due to the fact that he has a blocked 3' end), but participates in amplification or she contributes. The amplification oligonucleotides may optionally include modified oligonucleotides or analogues, or additional nucleotides that participate in the amplification reaction, but is not complementary to them or contained in the target nucleic acid. The amplification oligonucleotides can�to keep the sequence which is not complementary to the target or matrix sequence. For example, the 5' portion of the primer may include a promoter sequence that is not complementary to the target nucleic acid (referred to as "promoter-primer"). Specialist in the field of technology it will be understood that the oligonucleotide amplification, which functions as a primer, can be modified with the inclusion of a 5' promoter sequence, and thus function as a promoter-primer. Similarly, a promoter-primer can be modified by the removal of, or synthesis without promoter sequence and still function as a primer. Blocked 3' oligonucleotide amplification can provide the promotor sequence and serve as template for polymerization (referred to as "promoter-provider").

In this context, the term "primer" refers to an oligonucleotide, as found in nature in a purified restriction cleavage and synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis is induced by the product of the elongation of the primer, complementary strands of nucleic acid {e.g., in the presence of nucleotides and an induction agent such as DNA polymerase and at a suitable tempera�ur and pH). The primer preferably is admonitory for maximum efficiency in amplification, but may alternatively be dunacity. If he donachy, the primer is first treated to separate the strands before being used for cooking elongation. Preferably, the primer is oligodeoxyribonucleotides. The primer must be of a sufficient length for priming the synthesis of the products of the elongation in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method.

In this context, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), as found in nature in a purified restriction cleavage and synthetically, by recombinant or by PCR amplification, which is capable of gibridizatsiya with at least part of another of the desired oligonucleotide. The probe can be admonitory or dunacity. Probes suitable for the detection, identification and isolation of particular gene sequences. It is assumed that any probe used in the present invention, will be labeled with any "reporter molecule," so that it can be detected by any detection system, including, but n� limited to, enzyme (e.g., ELISA, along with based on enzyme histochemical assays), fluorescent, radioactive, and luminescent systems. Not intended that the present invention be limited to any particular detection system or label.

The term "isolated" when used in relation to nucleic acid, as "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is normally associated in its natural source. Thus, the isolated nucleic acid is present in the form or in environments that are different from those in which it is found in nature. In contrast, non-isolated nucleic acid, such nucleic acids, like DNA and RNA, found in the state they exist in nature. For example, a given DNA sequence (e.g. a gene) is detected in the chromosome of the host cell close to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein found in the cell as a mixture with numerous other mRNAs that encode a large number of proteins. However, isolated nuclein�Wai acid, encoding a given protein includes, as an example, such nucleic acid in cells normally expressing the given protein where the nucleic acid is in the localization of chromosomes that is different from the localization in natural cells, or it is otherwise bordered by nucleic acid sequence different from that which is found in nature. An isolated nucleic acid, oligonucleotide or polynucleotide may be present in agentcity or donacute form. When an isolated nucleic acid, oligonucleotide or polynucleotide is used for expression of the protein, the oligonucleotide or polynucleotide will contain at least the semantic encoding or thread (i.e., the oligonucleotide or polynucleotide may be agentcity), however, can contain both semantic and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be dunacity).

In this context, the term "purified" or "to purify" refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind with the target molecule. The removal of non-immunoglobulin proteins and/or the removal of immunoglobulins that do not bind to the target molecule, leads � a result of the increase in sample per cent of immunoglobulins, reacting with the target. In another example, recombinant polypeptides expressed in bacterial host cells and the polypeptides are purified by removing the proteins from the host cells; thus in the sample increases the percent of recombinant polypeptides.

In this context, the term "sample" is used in its broadest sense. In one sense he means, includes a specimen or culture obtained from any source, along with biological samples and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum and the like. Samples of the environment include environment material, such as surface material, soil, water, crystals and industrial designs. Such examples, however, should not be interpreted as limiting the sample types applicable to the present invention.

DETAILED description of the INVENTION

The present invention relates to compositions and methods for diagnosis, research and treatment of cancer, including, but not limited to, cancer markers. In particular, the present invention relates to compositions and methods of the forecast of the entity's response to cancer therapy.

In the past 30 years cm�rtnet from breast cancer in the Western world declined, partly due to the wide use of adjuvant systemic therapy. However, in the United States in 2008, more than 40,000 women will die from metastatic breast cancer. While occasionally a patient with metastatic breast cancer is cured, most eventually die from their disease. Despite this, patients with this condition was introduced a number of new therapies that result led to a moderate prolongation of survival and a significant improvement in the application of palliation.

Thus, the goal of therapy for most patients with metastatic breast cancer is the choice of therapy with a higher likelihood of response and the lowest possible toxicity, thereby counterbalancing the symptoms of cancer side effects of treatment. Indeed, to treat these patients now available with a broad set of strategies and agents. These include chemotherapy (there are now over 10 different agents approved for the treatment of metastatic breast cancer), along with anti-R2 therapy (trastuzumab, lapatinib), and antiangiogenic therapy, although not approved by FDA food and drug administration (FDA), also seem to be quite promising. However, one of the pillars of the treatment of patie�tov with metastatic breast cancer is anti-estrogen therapy.

Breast cancer can be treated using various anti-estrogens approaches, which mainly can be divided into additive and ablative. The most widely used additive therapy is a selective modulator of estrogen receptor (SMRA), tamoxifen, although effective, are also androgen therapy, such as fluoxymesterone and progestogenic agents, such as megestrol acetate. Ablative therapy were in the past, mostly surgical, however more recently been replicated in women in premenopause using agonists and antagonists of the hormone, releasing luteinizing hormone such as goserelin or leuprolide, and postmenopausal women - using agents that inhibit induced aromatase conversion predecessors dehydroepiandrosterone (DHEA) and testosterone to estradiol and estrone. In the U.S. there are three available aromatase inhibitor (AI): letrozole, anastrozole and exemestane.

The choice of appropriate treatment for patients with metastatic breast cancer is based on two factors: prognosis and prediction. In this respect, anti-estrogen therapy may be considered from the earliest targeted therapies. Subsequent studies demonstrated that tamoxifen inactive in patients with ER-negative breast cancers. One�to anti-estrogen therapies are effective in only 30-50% of women with ER-positive (or "rich") tumors, both metastatic and early. It is assumed that these patients with ER-positive, but endocrine-refractory metastatic breast cancer, it would be best treated with immediate chemotherapy, despite its higher toxicity profile than delaying chemotherapy for a few months because of ineffective attempts, though less toxic endocrine treatment.

Once the selected mode of palliative treatment for patients with metastatic breast cancer, it is stopped, and the patient is prescribed a new therapy for one of two reasons: 1) the selected therapy is so toxic that even when it works, it is unlikely to cause relief; or 2) it doesn't work and the tumor progresses. Current methods of determining these phenomena, in particular, the latter include medical history, physical examination, serologic testing and radiographic evaluation. Medical history and physical examination is known to be unreliable due to subjective issues and the fact that more than 50% of patients have only metastatic lesions in the internal organs such as the bones, liver and lungs. Non-specific serological tests, such as enzymes, originating from the bone (alkaline phosphatase) and liver (alkaline phosphatase, serum glutamate oxalate transferase of it.n.) do not have sufficient sensitivity, and specificity to be useful for determining the progression. To monitor these patients used at least three categories of tumor-associated tumor marker. They include tests for protein MUC-1 (CA15-3, CA.29), carcinoembryonic antigen (CEA) and the extracellular domain of HER2. The Commission on the guide to tumor markers American society of Clinical Oncology recommended that for monitoring patients with metastatic breast cancer would be used, at least, CA15-3 and CEA. However, in all three cases, early detection of treatment response or failure is distorted by the phenomenon of "peak tumor marker, wherein the marker actually increases over the course of several weeks to months before the decrease to the original level or lower by up to 25% of patients who ultimately supposed to respond to therapy. Thus, the results of circulating tumor markers currently, on the merits, have no value for determining progression within one or two months from the beginning of the new therapy.

Radiographic imaging is the most highly respected way to determine the clinical course in a patient with metastatic breast cancer. To determine the response, stability, and progression of RA were�developed strict criteria for the review of radiographs, computed tomography and magnetic resonance imaging. However, radiographic imaging has several drawbacks. Sensitivity to the progression of all these methods is low for most patients within the first one or two cycles of therapy, and in particular, the last two methods are inconvenient, expensive and associated with discomfort for the patient. Therefore, these tests are generally not performed until the patient is already on the course for several months after initiation of new therapy. Perhaps more importantly, only approximately 50% of patients with metastatic breast cancer will have a "measurable" disease - rather, they will have "immeasurable" lesions in the skin and/or the pleura or lesions on the skin and in the lungs, the so-called biological weapons, which are too small for accurate quantification. Attempts to monitor patients with the disease "only bones" were particularly disappointing. Bone scintigraphy using technetium pyrophosphate is the most widely used method for monitoring these patients, however, it is limited to relatively weak sensitivity to detect progression and the so-called "scintigraphic reflection", in which patients responding to therapy, in particular on endocrine therapy, nah�with a period of increased tracer uptake in lesions known and even the emergence of new zones of absorption as a function of increased osteoblastic bone regenerating activity.

There are certain patients in whom rapid progression easily determined by clinical, serological and/or radiological means. However, many patients with metastatic breast cancer represents a clinical challenge in determining the likelihood that a newly started therapy will be successful in the next few months, or that it will be useless mode. If so, patients are best treated potentially more effective, even if more toxic treatment, such as chemotherapy.

Accordingly, in some embodiments embodiment the present invention provides methods of identifying patients with ER-positive metastatic breast cancer that probably will not respond to antiestrogen therapy. In these patients with traditional chemotherapy can be started immediately or after a short test antiestrogen therapy. In some embodiments embodiment the methods of the present invention allow for more early monitoring of the effectiveness of the current treatment. This can be selected situation, alternative treatment for subjects who do not meet their current treatment.

In some embodiments embodiment the present invention provides methods, analysis of circulating tumor cells (CTCS. For example, in some embodiments embodiment the present invention provides methods for determining the effectiveness of anti-estrogen therapy by quantifying the level of CTCS. In some embodiments embodiment, the level of CTCS is used to determine prognosis and to guide treatment.

In some embodiments embodiment the levels of CTCS compared with levels of a subject not having cancer, or to the mean value of a population of subjects diagnosed as having cancer. In some embodiments, embodiments of CTCS compared with the average population of subjects diagnosed as having metastatic breast cancer, including subjects who responded to anti-estrogen therapy, and subjects who did not respond. In other embodiments embodiment, the monitoring in time of the levels of CTCS in a subject diagnosed as having non-metastatic breast cancer.

In other embodiments, the embodiment kharakterizovyvatsya one or more tumor markers associated with CSCS, with the aim to distinguish women who fit anti-estrogen therapy, women that are refractory to hormone, and thus to determine the prognosis or course of treatment. In some embodiments of the incarnation of tumor markers include, but are not limited to ER, HER-2, bcl-2, apoptosis (okra�ivanie monoclonal antibody M30), IGRFR1, vimentin and Ki-67. Can be used with additional tumor markers are known to those skilled in the art. The present invention considers both known and unknown (e.g., not yet open) tumor markers. Preferred tumor markers are those that are present on CTCS, and which point (by themselves or in combination) in response to anti-estrogen therapy.

In some embodiments embodiment, the status of tumor markers in CTCS correlates with the status of the same markers in primary and/or metastatic tissue obtained from the same patients.

I. Circulating tumor cells

Tumor cells were first detected in the circulatory system of a human, though, and after his death, more than 150 years ago. Recent advances in technology have allowed the development of highly automated and standardized system for separation of circulating tumor cells (CTCS) from whole blood using immunomagnetic approach. In some embodiments embodiment the methods of the present invention using the CellSearch system™ (Immunicon Corporation, Huntingdon Valley, PA) to quantify CTCS (Allard et al., Clin Cancer Res 2004;10(20):6897-904; Cristofanilli et al., N Engl J Med 2004;351(8):781-91; each of which is included here by reference in its entirety). System CELL SEARCH identifies "events", to�which is then characterized as epithelial in origin by immunofluorescence staining using antiretaliation antibodies and is determined by their cellular nature because of DAPI staining. Contaminating leukocytes are identified by immunofluorescent staining with monoclonal antibody against CD45, and the results are presented graphically in digital format.

The present invention is not limited to the use of the system CELL SEARCH. Can be used with any method of isolating and/or quantifying CTCS.

II. Tumor markers

In some embodiments embodiment the present invention provides methods of identifying tumor markers associated with CSCS. Exemplary tumor markers are described in more detail below.

The estrogen receptor (ER). Among others, selected markers that indicate independence from estrogen therapy include the relative levels of ER, HER-2, bcl-2 and indicators of apoptosis. Several studies indicate that the Association between the expression of ER and benefit from endocrine therapy is not dichotomous, but rather, it refers to quantitative protein level re. Accordingly, we used various limits that have been set to separate "positive" and "negative" for a variety of tests on re. In some embodiments embodiment the relative levels of PE, which can be measured in CTCS, are used to aid in determining the likelihood of response to endocrine therapy.

HER2. Various polinices�s and clinical studies indicate that that over-expression of HER-2 and/or amplification reduces the sensitivity sensitivity to estrogenic treatment in ER-positive cancers of the breast. The present invention is not limited to a particular mechanism. Indeed, the understanding of the mechanism is not necessary for the application in practice of the present invention. However, it is anticipated that patients with ER-positive, but also HER-2 positive breast cancer is likely to get less benefit from antiestrogen treatment.

BCL-2. BCL-2 is an antiapoptotic protein that protects the cell from entering the path of programmed cell death. BCL-2 is normally expressed in breast cancer and is associated with worse prognosis. The expression of BCL-2 is more often expressed in ER-positive than ER-negative breast cancer. Preclinical and some clinical studies indicate that the expression of BCL-2, appears to be associated with relative resistance to anti-estrogen therapy. The present invention is not limited to a particular mechanism. Indeed, the understanding of the mechanism is not necessary for the application in practice of the present invention. However, it is assumed that ER-positive, BCL-2 positive breast cancers are more likely refractory to endocrine treatment.

Apoptosis. One mechanism of action of anti-estrogen therapy is mediated through programmed cell death by apoptosis. Therefore, consistent monitoring of apoptosis provides information about the death of breast cancer cells. Preoperative studies, as with chemotherapy, and hormone therapy, have documented that early induction of apoptosis associated with subsequent clinical response. The present invention is not limited to a particular mechanism. Indeed, the understanding of the mechanism is not necessary for the application in practice of the present invention. However, it is assumed that serial assessment of apoptosis provides an indication of the effectiveness of anti-estrogen therapy.

Ki-67. Ki-67 is an antigen proliferation, which reflects the turnover of cells. Immunohistochemical staining of Ki-67 in primary tissues is associated with prognosis in early stage breast cancer. In neoadjuvant studies, the lower levels of Ki-67 is associated with the apparent benefit from anti-estrogen therapies such as tamoxifen and aromatase inhibitors. The present invention is not limited to a particular mechanism. Indeed, the understanding of the mechanism is not necessary for the application in practice of the present invention. However, it is assumed that serial assessment of Ki-67-associated CTCS p�ed and during endocrine treatment provides an indication of the probability of receiving benefits.

In some embodiments, embodiments, one or more of the above markers is detected in a composite or panel format. For example, in some embodiments, the embodiment uses a multiparametric analysis based on PCR with reverse transcriptase (RT-PCR), such as OncotypeDX™ (Genomic Health Inc, CA). The relative values of expression of ER, BCL-2, HER2 and Ki67 guide the algorithm used to obtain the OncotypeDx "the Rate of relapse (PR)". Well established correlation studies have demonstrated that patients with negative lymph nodes and positive lymph nodes with low PR, reflecting high re, low HER2, low BCL-2 and low Ki-67, have the prognosis of treatment with tamoxifen alone, while patients with a high rate of relapse, reflecting low re, high HER-2, high BCL-2 and high Ki-67 have a significantly worse prognosis when treated with tamoxifen alone, however, is much more likely to benefit from chemotherapy (Paik et al.N Engl J Med 2004;351:2817-26; Paik et al., J Clin Oncol 2006;24:3726-34).

III. Detection methods

Below is the approximate methods to detect markers associated with CSCS. However, there may be used any suitable method of detection of nucleic acids or proteins tumor markers.

A. Detection of DNA and RNA

In some embodiments of the incarnation �Pokolenie markers are detected as mRNA using a variety of methods of nucleic acid known to the ordinary expert in the field of technology, including but not limited to: nucleic acid sequencing, nucleic acid hybridization and amplification of nucleic acids.

1. Sequencing

Illustrative, non-exhaustive examples of methods of sequencing nucleic acids include, but are not limited to, dideoxyadenosine method (Sanger) sequencing method sequencing with dye as a terminator. One of ordinary skill in the field of engineering recognizes that because RNA is less stable in the cell and are more prone to nuclease attack, experimentally RNA usually before sequencing undergoes reverse transcription into DNA.

Dideoxyadenosine sequencing method uses a specific sequence for the termination reaction of DNA synthesis using modified nucleotide substrates. Elongation begins at a specific site on the DNA matrix by using a short oligonucleotide primer labeled radioactively or otherwise, complementary matrix in this phase. Oligonucleotide primer is extended using DNA polymerase, the standard four deoxynucleotide bases and low concentrations of nucleotide that terminates one circuit, most often di-desoxyn�of cleotide. This reaction is repeated in four separate test tubes with each of the bases, alternating di-deoxynucleotides. Limited incorporation of the chain terminating nucleotide DNA polymerase results in a series of related DNA fragments that are terminated only at positions where using a specific di-deoxynucleotide. For each reaction tube fragments are separated by size by electrophoresis in a polyacrylamide gel plate or in capillary tubes filled with a viscous polymer. The sequence is determined by reading what the line gives visualized the mark from the labeled primer as the scan of the gel from top to bottom.

When sequencing with dye as an alternative terminator terminator is marked. Full sequencing can be performed in a single reaction by tagging each of di-deoxynucleotide - chain terminators a different fluorescent dye, which fluoresces at a different wavelength.

2. Hybridization

Illustrative, non-exhaustive examples of the techniques of hybridization of nucleic acids include, but are not limited to in situ hybridization (ISH), micromatrix analysis and southern - or Northern-blotting.

The in situ hybridization (ISH) is a type of hybridization that uses�should of labeled complementary strand of DNA or RNA as a probe to localize a specific DNA sequence or RNA in a part or slice of tissue (in situ), or, if the tissue is small enough, in the entire tissue (ISH in total drug). ISH DNA can be used to determine the structure of chromosomes. ISH RNA is used to measure and localize mRNAs and other transcripts in tissue sections or in total preparations. Cells and tissues are usually treated sample for fixation of the target transcripts in place and to increase access of the probe. Probe hybrid with the target sequence at elevated temperature and then the excess probe is washed. A probe labeled with a radioactive, fluorescent or labeled enzymes on the grounds legalizovyvatsya and quantified in the tissue, respectively, using autoradiography, fluorescence microscopy or immunohistochemistry. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels for the simultaneous determination of two or more transcripts.

2.1 FISH

In some embodiments embodiment the sequence of tumor markers are determined using fluorescent in situ hybridization (FISH). The preferred FISH tests for the present invention using a bacterial artificial chromosome (IIR). They were used extensively in the project to sequence the human genome (see Nature 409: 953-958 (2001) and clones containing specific IIR, available carestation, which can be determined by using many sources, such as the National center for biotechnology information (NCBI). Each clone IIR of the human genome has a name that uniquely identifies. These names can be used to nd the corresponding sequence GenBank and to order copies of the clone distributor.

Specific protocols for performing FISH are well known in the field of technology and can be easily adapted for the present invention. Guidance on methodology can be obtained from many references, including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ Hybridization: A Practical Approach (ed. D. G. Wilkinson), Oxford University Press Inc., England (1992)); Kuo, et al., Am. J. Hum. Genet. 49:112-119 (1991); Klinger, et al., Am. J. Hum. Genet. 57:55-65 (1992); and Ward, et al., Am. J. Hum. Genet. 52:854-865 (1993)). There are also commercially available kits, and they provide protocols to run tests FISH (available from, for example, Oncor, Inc., Gaithersburg, MD). Patents, providing guidance on the methodology include U. S. 5225326; 5545524; 6121489 and 6573043. All these links here by reference in its entirety and can be used together with similar links in the field of technology and with the information provided here in R�account, see the Examples for determining the stages of the procedure, convenient for the specific laboratory.

2.2 Micromatrix tests

Different kinds of biological assays are called micromatrix analyses, including but not limited to: micromatrix DNA analysis (e.g., micromatrix analyses of cDNA and oligonucleotide micromatrix analysis); micromatrix protein analyses; micromatrix tissue tests; transfection or cellular micromatrix analyses; micromatrix analyses of the chemical component; and antibody micromatrix tests. Micromatrix DNA, commonly called gene chip, DNA chip or biochip is a collection of microscopic DNA smears deposited on a solid surface (e.g. glass, plastic or silicon chip) to form a matrix for the purpose of expression profiling or monitoring of expression levels for thousands of genes simultaneously. Fixed DNA segments are known as probes, thousands of which can be used in the micromatrix analysis of isolated DNA. Micromatrix analyses can be used to identify genes associated with diseases, by comparing gene expression in diseased and normal cells. Micromatrix analyses can be performed using a variety of technologies, including, but not limited to: printing by sharp in STI�tov on glass slides; photographically using pre-made stencils; photolithographically using dynamic Micromirror devices; printing ink; or electrochemically on microelectrode arrays.

Southern - and Northern-blotting used to detect, respectively, of certain sequences of DNA or RNA. DNA or RNA extracted from the sample, fragment, separated by electrophoresis on the gel with the substrate and transferred onto a membrane filter. DNA or RNA bound to the filter was subjected to hybridization with a labeled probe complementary to the desired sequence. Conduct detection hybridization probe associated with the filter. Option procedure is the reverse Northern blotting, in which the substrate nucleic acid is attached to the membrane, is a set of isolated DNA fragments and the probe RNA is extracted from tissue and labeled.

3. Amplification

Genomic DNA and mRNA can be amplified before detection, or simultaneously with it. Illustrative, non-exhaustive examples of methods of nucleic acid amplification include, but are not limited to, polymerase chain reaction (PCR), polymerase chain reaction with reverse transcription (RT-PCR), transcription-oborudovaniyeniye (TMA), ligase chain reaction (LCR), the displacement amplification circuit (SDA), and amplification-based nucleic acid sequence (NASBA). One of ordinary skill in the field of engineering recognizes that certain amplification techniques (e.g. PCR) require RNA was transcribed back into DNA prior to amplification (e.g., RT-PCR), while other methods of amplification directly amplificateur RNA (e.g., TMA and NASBA).

Polymerase chain reaction (U.S. patent numbers 4683195; 4683202; 4800159 and 4965188, each of which is included here by reference in its entirety), commonly called PCR, uses multiple cycles of denaturation, annealing pairs of primer on the various circuits and the elongation of the primer for exponential increase in the number of copies of the target nucleic acid sequence. In a variation called RT-PCR, to obtain complementary DNA (cDNA) from mRNA using a reverse transcriptase (FROM), and then cDNA was amplified by PCR to obtain multiple copies of DNA. Other changes in PCR, see, e.g., U.S. patents numbered 4683195; 4683202 and 4800159; Mullis et al., Meth. Enzymol. 155: 335 (1987); and Murakawa et al., DNA 7: 287 (1988), each of which is included here by reference in its entirety.

Transcription-mediated amplification (U.S. patent numbers 5480784 and 5399491, each of which� included here by reference in its entirety), usually called TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytic under conditions of substantially constant temperature, ionic strength and pH, in which multiple RNA copies of the target sequence autocatalytic generate additional copies. See, for example, U.S. patents numbered 5399491 and 5824518, each of which is included here by reference in its entirety. In the embodiment described in the publication. U.S. No. 20060046265 (here incorporated by reference in its entirety), optional TMA includes the use of the blocking of functional groups, functional groups termination and other modifying functional groups to increase the sensitivity and accuracy of the process TMA.

Ligase chain reaction (Weiss, R., Science 254: 1292 (1991), here incorporated by reference in its entirety), commonly referred to LCR, uses two sets of complementary DNA oligonucleotides that gibridizatsiya with adjacent portions of the target nucleic acid. DNA oligonucleotides are covalently joined DNA ligase in repeated cycles of thermal denaturation, hybridization and binding to obtain the detected dontcache linked oligonucleotide product.

Amplification with the moving chain (Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. patent n�d rooms 5270184 and 5455166, each of which is included here by reference in its entirety), commonly called SDA, uses cycles of annealing pairs primerih sequences on opposite chains of the target sequence, extension of the primer in the presence dNTPaS for producing the product of the elongation of duplex hemivariational primer-mediated endonuclease admonitory gap getimagefilename site recognition restriction endonuclease, and mediated polymerase elongation of the primer from the 3' end of the gap to move the existing circuit and receiving circuit for the next round of primer annealing, admonisheth rupture and displacement of the chain that results in geometric amplification of product. Thermophilic SDA (tSDA) uses a thermophilic restriction endonuclease and polymerases at higher temperatures in essentially the same way (European patent application No. 0 684 315).

Other methods of amplification include, for example: amplification-based nucleic acid sequence (U.S. patent number 5130238, here incorporated by reference in its entirety), commonly referred to NASBA; one that uses the RNA replicase for amplification of the probe molecules (Lizardi et al., BioTechnol. 6: 1197 (1988),here incorporated by reference in its entirety), commonly referred QP of replicase; a method of amplification based on Tran�criple (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and self-sustained replication sequence (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is included here by reference in its entirety). For further discussion of known amplification methods see Persing, David N., "In Vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical Microbiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, DC (1993)).

4. Detection methods

Non-amplified or amplified oncomarkers nucleic acid can be detected by any of the traditional methods. For example, in some embodiments of the incarnation oncomarkers nucleic acids are detected by hybridization with-detectable-labeled probe and measurement of the resulting hybrids. Illustrative, non-exhaustive examples of detection methods are described below.

One illustrative method of detecting and analyzing the hybridization protection (HPA), includes chemiluminescent hybridization oligonucleotide probe (e.g., probe labeled with an acridine ester (AE)) with the target sequence, the selective hydrolysis of chemiluminescent labels available on legibilities the probe and measuring the chemiluminescence produced by the remaining probe in a luminometer. See, for example, U.S. patent number 5283174 and Norman C. Nelson et al., Nonisotopic Probing, Blotting, and Sequencing,ch. 17 (Larry J. Kricka ed., 2^nded. 1995, each of which is included here by reference in its entirety).

Another illustrative method of detection is to quantify the amplification process in real time". Evaluation of the amplification process in real time" involves a determination of the amount of amplicon in the reaction mixture, continuously or periodically, during the amplification reaction, and the use of specific values to calculate the amount of target sequence initially present in the sample. In the field of technology is well known various methods for determining the amount of initial target sequence present in the sample, on the basis of amplification in real time. They include the methods disclosed in U.S. patents numbered 6303305 and 6541205, each of which is included here by reference in its entirety. Another way to determine the quantity of target sequence initially present in the sample, however, which is not based on amplification in real time are disclosed in U.S. patent number 5710029, included here by reference in its entirety.

The amplification products can be detected in real time by using different imageregionwidget probes, most of which they�em structure of the stem-loop. Such imageregionwidget probes are labeled so that they emit signals with different detection, depending on whether the probes in imagebrowserurl state or in an altered state due to hybridization with the target sequence. By way of non-exhaustive example, "molecular torches" are a type imageregionwidget the probe, which includes the different parts of semiconcentrated (called the "target binding domain" and "the target closing domain") which are connected by a connecting portion (for example, a non-nucleotide linker) and hybridizers themselves in predefined conditions of a restriction analysis. In a preferred embodiment, molecular torches contain agentcities areas of the bases in the target binding domain, which have a length of from 1 to about 20 bases and available for hybridization with a target sequence present in the amplification reaction in terms of moving the chains. In terms of moving the chains preferred hybridization of two complementary areas of molecular beacon that can be fully or partially complementary, except in cases of presence of the target sequence that will mess with admonitory phase present in �arget binding site, and move all or part of the target closing domain. The target binding domain and the target closing domain of a molecular torch include detectivea label or a pair of interactive labels (e.g., luminescing/dampening), arranged in such a way that it generates various signals when the molecular beacon imagebrowserurl and when the molecular beacon hybrid with the target sequence, thereby allowing the detection of pairs of probe:target in the sample in the presence dehybridization molecular lights. Molecular torches and various types of interacting pairs of labels are disclosed in U.S. patents numbered 6534274, here incorporated by reference in its entirety.

Another example of the detection probe having semicompetent, is a "molecular beacon". Molecular beacons comprise nucleic acid molecules having complementary target sequence, an affinity pair (or shoulders nucleic acid), the carrier of the probe in a closed conformation in the absence of the amplification reaction of the target sequence, and a couple of labels that interact in the case when the probe is in a closed conformation. Hybridization of the target sequence and the complementary target sequence separates the members of AF�Inna pairs, thereby shifting the probe to an open conformation. The transition to the open conformation is determined due to the reduced interaction of labeled pairs, which can represent, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beacons are disclosed in the U.S. patents under nomerami and 6150097, which are here incorporated by reference in its entirety.

Other samovyrivnyuyucha probes are well known to the ordinary expert in the field of technology. By way of non-exhaustive example, binding probe pairs having interacting labels, such as those¹which are disclosed in U.S. patent number 5928862 (¹here incorporated by reference in its entirety) can be adapted for use in the present invention. Additional detection systems include "molecular switches", as is disclosed in the publication U.S. No. 20050042638, included here by reference in its entirety. Other probes, such as those that include intercalating dyes and/or fluorochromes, are also suitable for detection of amplification products in the present invention. See, for example, U.S. patent number 5814447 (here incorporated by reference in its entirety).

B. Detection of protein

In some embodiments embodiment the present invention provides a method� detection oncomarkers protein and/or levels oncomarkers protein. Proteins are detected by applying a variety of protein techniques known to the ordinary expert in the field of technology, including but not limited to: protein sequencing; and immunoassays.

1. Sequencing

Illustrative, non-exhaustive examples of techniques for protein sequencing include, but are not limited to, mass spectrometry and splitting Edman.

Mass spectroscopy can, in principle, to sequence a protein of any size, however, the calculation becomes difficult with increasing size of the protein. Protein is broken down by endoproteases. and the resulting solution is passed through a column of a liquid chromatograph high pressure. At the end of this column from a narrow nozzle, charged to a high positive potential, the solution is sprayed into the mass spectrometer. The charge drops causing their fragmentation to obtain in the residue only single ions. The peptides are then fragmented and the fragments are measured by the ratio of the mass-charge. The mass spectrum is analyzed by the computer and is often compared with a database of previously sequenced proteins with the goal of determining the sequence of the fragments. The process is then repeated with another by the digestive enzyme or overlaps in the sequences used to construct the sequence for protein.

In spallation reactions �about Edman peptide, subject to sequencing, is adsorbed on a solid surface (e.g. glass fibre, covered polybrene). To the adsorbed peptide is added to the Edman reagent, phenylisothiocyanate (FITZ) with a slightly alkaline buffer solution for 12% of trimethylamine, and the reaction occurs with amine group of the N-terminal amino acids. Then the derived terminal amino acid can be selectively separated by the addition of anhydrous acid. Derived isomerized with obtaining substituted phenylthiohydantoin that can be washed and identified chromatographically, and the cycle may be repeated. The efficiency of each stage is approximately 98%, which allows to reliably determine approximately 50 amino acids.

2. The immunoassays

Illustrative, non-exhaustive examples of immunoassays include, but are not limited to: immunoprecipitation; Western-blotting; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR. For use in immunoassays suitable polyclonal or monoclonal antibody-detectable labeled using a variety of methods known to the ordinary expert in the field of technology (e.g., colorimetric, fluorescent, chemiluminescent or radioactive).

Immunoprecipitate is techniques� deposition of antigen out of solution using an antibody, specific to this antigen. This process can be used to identify protein complexes present in cell extracts, with focus on protein, which, presumably, is present in the complex. Complexes stand out from the solution using insoluble antielastase proteins, originally isolated from bacteria such as Protein A and Protein G. the Antibody can also be attached to separatim balls that can be easily separated from the solution. After washing the precipitate can be analyzed using mass spectrometry, Western blotting, or any number of other methods to identify components of the complex.

Western blotting or immunoblotting is a method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. Proteins are then transferred from the gel to the membrane, as a rule, polivinilhloridnyi or nitrocellulose, where they are analyzed by using antibodies specific against the desired protein. As a result, researchers can study the number of proteins in the sample and to compare levels in multiple groups.

ELISA, short for enzyme-linked immunosorbent assay is a biochemical technique�ICA to detect the presence of antibody or antigen in the sample. It uses at least two antibodies, one specific for the antigen, and is connected with the second enzyme. The second antibody will be detected as chromogenic or fluorogenic substrate. Variations include ELISA "sandwich"ELISA, competitive ELISA method ELISA, ELISPOT spots. Due to the fact that ELISA can be performed to assess the presence of antigen or the presence of antibody in the sample, it is a useful tool for determining serum concentrations of antibodies and also to detect the presence of antigen.

Immunohistochemistry and immunocytochemistry refer to the process of localizing proteins, respectively, in tissue sections or cells, based on the principle of binding antigens in tissues or cells with their specific antibodies. Visualization is made possible by the attachment of labels giving the dyeing or fluorescent - antibody. Typical examples of colored labels include, but are not limited to, horseradish peroxidase and alkaline phosphatase. Typical examples fluorophoric labels include, but are not limited to, fluorescein isothiocyanate (FITZ) or phycoerythrin (PE).

Flow cytometry is a technique for counting, research, and sorting microscopic particles suspended in the fluid stream. It allow one�belt multiparametric analysis of the physical and/or chemical characteristics of single cells, passing through the opto-electronic detector device. A beam of light (e.g., laser) of a single frequency or color aims to hydrodynamically focused stream of fluid. To the point where the stream passes through a beam of light aimed a number of detectors, one in accordance with the flow of light (stray light in the direction of propagation or FSC) and several perpendicular to it (side scattering (SSC) and one or more fluorescence detectors). Each particle in the suspension passing through the beam scatters somehow light, and fluorescent chemicals in the particle may be excited by emission light of lower frequency than the light source. The combination of scattered and fluorescent light is picked up by detectors, and by analyzing the deviations in brightness at each detector, one for each peak of fluorescence emission, it is possible to deduce various facts about the physical and chemical structure of each particle. FSC correlates with the cell volume and SSC correlates with the density or inner complexity of the particle (for example, the shape of the nucleus, the amount and type of cytoplasmic granules or the membrane stiffness).

Immuno-polymerase chain reaction (IPCR) uses methods of nucleic acid amplification to intensify the signal in immunoassays based on antibodies. Since�if there is no equivalent of PCR for proteins that is, whites can't be replicated in the same way that the nucleic acid is replicated during PCR, the only way to increase the sensitivity of detection is the amplification of the signal. Target proteins bind to the antibodies, which anywhereman direct or indirect way with the oligonucleotides. Unbound antibodies are washed away, and the remaining antibody-bound amplificateurs their oligonucleotides. Detection of the protein occurs by detection of amplified oligonucleotides using standard methods for detecting polynucleotides, including how real-time.

In some embodiments, the embodiment uses the imunno-magnetic detection. In some embodiments embodiment, the detection is automated. Approximate methods the technique of immune magnetic detection include, but are not limited to, methods, commercially available from Veridex (Raritan, NJ).

V. data Analysis

In some embodiments, the embodiment uses a computer analysis program to translate the raw data generated in the tests of detection (for example, the presence, absence or amount of expression of tumor markers and/or level CSC), data have prognostic value for the Clinician (for example, the choice of cancer therapy). The Clinician can access the predictive data with the ISP�Lovanium any suitable means. Thus, in some preferred embodiments embodiment the present invention provides the further benefit that the Clinician, who may not have received training in genetics or molecular biology, need not understand the raw data. The data is provided directly to the Clinician in its most useful form. The Clinician is then able to immediately use the information to optimize the treatment of the subject.

The present invention considers any method of obtaining, processing and transmission of information in the laboratory and from laboratories that produce the tests, from the information providers, medical staff and actors, and them. For example, in some embodiments, embodiments of the present invention, a sample (e.g., biopsy, or a sample of blood or serum) obtained from the subject and provide the service profiling (e.g., clinical lab at a medical facility, institution genomic profiling, etc.) located in any part of the world (for example, in a country different from that where the subject or where, ultimately, information is used) to obtain the raw data. Where the sample contains tissue or other biological sample, the subject may visit a medical center to sample and sending to the profiling center, or �objecty may collect the sample themselves (for example, a urine sample) and send it directly to the profiling center. Where the sample includes a pre-determined biological information, the information may be directly sent to profiling service by the subject (e.g., an information card containing information, you can scan the computer and transfer data to the computer profiling center using an electronic communication). When receiving the profiling service, the sample is processed and a profile is created (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.

Profile data is then prepared in a format suitable for interpretation by a treating Clinician. For example, instead of providing raw data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of success of cancer treatment) for the subject, along with recommendations for particular treatment options. These data can be presented to the Clinician by any suitable method. For example, in some embodiments embodiment the profiling service generates a report that can be printed for the Clinician (e.g., in place of medical care) or provided to the Clinician on a computer monitor.

In some Varian�Ah embodiments, the information is first analyzed in the place of medical care or in a regional institution. The raw data is then sent to a Central processing facility for further analysis and/or convert raw data into actionable information for the Clinician or the patient. The Central processing facility is an advantage for privacy (all data is stored in a Central facility with standard security protocols), speed and consistency of data analysis. The Central processing facility can control the fate of the data after treatment of the subject. For example, using the electronic communication system, the Central facility can provide data to the Clinician, the subject, or researchers.

In some embodiments embodiment the subject is able to directly access the data, using the electronic communication system. The subject on the basis of the results may elect further intervention or counseling. In some embodiments embodiment the data is used for research applications. For example, the data can be used to further optimize the inclusion or exclusion of markers as useful indicators of a particular condition or stage of the disease.

G. Compositions and kits

Compositions for use in the diagnostic methods of the present invention include, but are not limi�cuautla probes, amplification oligonucleotides and antibodies. Particularly preferred compositions detect the presence of the expression level of tumor markers of CTCS in the sample.

Any of these compositions, alone or in combination with other compositions of the present invention may be provided in the form of a kit. For example, the single labeled probe and pair of amplification oligonucleotides may be provided in the kit for the amplification and detection of tumor markers. The kits can further include appropriate controls and/or reagents for detection.

Composition of probes and antibodies of the present invention can also be provided in the form of a matrix or panel test.

IV. The definition of treatment

In some embodiments embodiment the present invention provides systems, kits and methods for determining the course of treatment.

In some embodiments of the incarnation is determined by the CSC-endocrine therapy index (CSC)-IEP). In some embodiments embodiment the CSC-IEP is calculated by assigning a score to the levels of CTCS and tumor markers (see e.g., Example 1). In some embodiments of the incarnation low CTCS-IEP is an indication that the subject who is likely to respond to endocrine (e.g., anti-estrogen) therapy. In some embodiments, embodiments of the high C rate�To-IEP is an indicator of the subject probably does not respond to endocrine therapy and that it is better to chemotherapy.

EXPERIMENTAL PART

The following examples are provided to demonstrate and further illustrate certain embodiments of the present invention and they should not be construed as limiting its scope.

Example 1

The development of CSC-endocrine therapy index (CSC)-IEP)

CTCS can be reproducibly and reliably estimated using commercially available automated the imunno-magnetic system (for example, the CellSearch system®; Veridex LLC). High levels of CTCS predict rapid progression in patients with metastatic breast cancer (with MBC) (Cristofanilli, et al. NEJM 2004). Only ~ 50% of patients with ER-positive with MBC will benefit from endocrine therapy (ET). Patients with endocrine-refractory with MBC get more relief with the help of chemotherapy. This example describes the multiparametric analysis using CellSearch®, which identifies patients with ER-positive with MBC who are unlikely to benefit from ET, and which are best treated with chemotherapy.

Ways. The CellSearch system® has four fluorescent channels. Three of them are used in order to distinguish CTCS from leukocytes (DAPI, anti-cytokeratin, anti-CD45). The fourth "empty" channel was used to �of serenia expression re, Bcl-2, HER-2 and Ki67 using antigen-specific antibodies with a fluorescent tag. These four markers were chosen because of their Association with sensitivity (ER, Bcl-2) or resistance (HER-2, Ki-67) in FL. Cultivated breast cancer cells (MCF-7: ER+, BCL-2+, HER2-, Ki67+; MDA-MB-231: ER-, Bcl-2-, HER2-, Ki67+; W-474: ER+, Bcl-2+, HER2+, Ki67+) were introduced into 7.5 ml of human whole blood from normal donors and characterized using a set of SHS CellSearch®.

Table 1 shows the Association of different markers of response to FL.

Table 2 shows the antibody and experimental parameters.

Results. Each cell line is colored accordingly markers with corresponding negative controls, although the staining was heterogeneous even within a single cell line (Figures 1-3).

Using these data, was developed by the CSC-IEP, in which points are assigned to certain categories, consisting of a number of cells connected with the relative percentage and degree of positivity of cells for each marker, with lower scores (CTCS few or none, or CTCS with high % and the intensity of ER and Bcl-2; low HER-2 and Ki-67)=predicted favorable response to ET (Tables 3 and 4 (a-b)).

CSC-IEP was determined for 3 patients with metastatic breast cancer using the methods described above. The results are shown in Figure 4.

In further studies was studied 21 patients. Study design and results are shown in Figures 7-8. One patient was ineligible. Five of 20 patients had a low number of CTCS (<5 CTCS/7.5 ml whole blood), and is expected to have a relatively favorable prognosis. CSC-IEP was identified in 10 patients (50%): 2 patients had low, while 3 had intermediate and 5 CTCS had a high-IEP. Technical difficulties prevented the accurate establishment of the CSC-IEP in the remaining 4 patients. It should be noted that the expression of the biomarkers among CTCS in individual patients was heterogeneous.

The lower scores CSC-IEP (a low indicator of CTCS or the lack of, or CTCS with a high rate of re and BCL-2 CTCS and low HER2 and Ki-67 CTCS) were considered as associated with a favorable response to FL. CSC-IEP was calculated in patients with MBC with to determine, predict whether high CSC-IEP resistance and rapid progression of TH.

All publications, �utenti, patents and access numbers mentioned in the foregoing description, here incorporated by reference in its entirety. Although the invention has been described in connection with specific embodiments of the embodiment, it should be understood that the invention as it is claimed, should not properly be limited to such specific choices of implementation. In fact, various modifications and variations of the described compositions and methods of the invention will be apparent to the ordinary skilled in the art and, as implied, they fall within the following claims.

1. The method for determining the response of a subject diagnosed with breast cancer, antiestrogen therapy, including
(a) detecting the level of circulating tumor cells (CTCS) in a sample of whole blood from the subject, representing the person with breast cancer;
b) determining the number of CTCS positive for expression of estrogen receptor, HER-2, bcl-2, and ki67;
b) calculating quantitative CSC-endocrine therapy index (CSC)-IEP) based on the specified number of CTCS positive for expression of the indicated estrogen receptor, HER-2, bcl-2, and ki67, and the total number of CTCS using the allocation of points to specified levels and number of CTCS CTCS positive for expression of estrogen receptor, HER-2, bcl-2, IK, moreover, the specified CSC-IEP is calculated as follows:
(i) in the case where the number of CTCS is 0-4 7.5 ml, is assigned 0 points, in the case where the number of CTCS is 5-10 7.5 ml, is assigned 1 point, and in the case when the number of CTCS is more than 10 7.5 ml, is assigned 2 points;
(ii) in the case where the number of CTCS positive for the estrogen receptor, is more than 10%, is assigned 0 points, in the case where the number of CTCS positive for the estrogen receptor, is 1-10%, is assigned 2 points, and in the case when the number of CTCS positive for the estrogen receptor, is 0, is assigned 6 points;
(iii) in the case where the number of CTCS positive for HER-2 is 0%, is assigned 0 points, in the case where the number of CTCS positive for HER-2, is 1-10%, is assigned 1 point, and in the case when the number of CTCS positive for HER-2 is over 10%, is assigned 2 points;
(iv) in the case where the number of CTCS positive for bcl-2, is more than 10%, is assigned 0 points, in the case where the number of CTCS positive for bcl-2, is 1-10%, is assigned 1 point, and in the case when the number of CTCS positive for bcl-2, is 0%, is assigned 2 points;
v) in the case when the number of CTCS positive for Ki-67, is 0%, is assigned 0 points, in the case where the number of CTCS, positively�based on data for Ki-67, is 1-10%, is assigned 1 point, and in the case when the number of CTCS positive for Ki-67 is more than 10%, is assigned 2 points;
vi) and adding these points to obtain the specified CSC-IEP; and
g) and CSC-IEP gap of 0-3 indicates a favorable response to
anti-estrogen therapy, CSCS-IEP gap of 4-6 indicates moderate response to
anti-estrogen therapy, and the LO-IEP gap of 7-14 indicates a weak response to
anti-estrogen therapy.

2. A method according to claim 1, characterized in that the specified anti-estrogen therapy is selected from the group consisting of a selective modulator of estrogen receptor (SMRA) and aromatase inhibitor.

3. A method according to claim 2, characterized in that the aromatase inhibitor is selected from the group consisting of letrozole, anastrozole and ackzemestana.

4. A method according to claim 1, further comprising the stage of introduction of chemotherapy.

5. A method according to claim 1, characterized in that said determination of the number of CTCS positive for expression of estrogen receptor, HER-2, bcl-2, and ki67 method includes multiple PCR.

6. A method according to claim 1, characterized in that said determination of the number of CTCS positive for expression of estrogen receptor, HER-2, bcl-2, and ki67 includes the use immunomagnetic or immunofluorescent analysis.

7. A method according to claim 1, characterized in that decree�tion breast cancer is metastatic.

8. A method according to claim 1, characterized in that the breast cancer is positive for estrogen receptor.

9. A method according to claim 2, characterized in that the specified SMRA is a tamoxifen.

10. A method according to claim 1, further comprising the step of detecting a marker of apoptosis, and this marker of apoptosis detected using the monoclonal antibody M30.