ES2356738T3 - CIRCULATING TUMOR CELLS (CTC): EARLY EVALUATION OF EVOLUTION TIME, SURVIVAL AND THERAPY RESPONSE IN PATIENTS WITH METASTASIC CANCER. - Google Patents

Abstract

A method for the prognosis of the survival of a patient comprising: a) immunologically enriching a fraction of a blood sample of said patient, said sample comprising a population of mixed cells suspected of containing circulating tumor cells (CTC), wherein said enrichment step comprises mixing said sample with magnetic particles coupled to an antibody that specifically binds to EpCAM and subjecting said sample-magnetic particle mixture to a magnetic field to produce a CTC-enriched cell suspension bound to magnetic particles, b) confirm the Structural integrity of cytokeratin-positive CTCs that are intact, where cytokeratin-positive cells are identified by labeling with a monoclonal antibody to cytokeratin, and where said structural integrity is determined by an analysis of morphological characteristics based in fluorescence, and c) measuring the number of said intact CTCs to determine patient survival, where a CTC number above or equal to five CTC per 7.5 ml of blood is indicative of a lower survival prognosis.

Description

Background

Field of the Invention

 The present invention relates generally to the prognosis of cancer and survival in metastatic cancer patients, based on the presence of circulating cancer cells (CTC) morphologically intact in blood. More specifically, diagnostic procedures, reagents and apparatus that correlate the presence of circulating cancer cells in 7.5 ml of blood of patients with metastatic breast cancer with the time of disease progression and survival are described. Circulating tumor cells are determined by very sensitive methodologies capable of isolating and taking images of 1 or 2 cancer cells in approximately 5 to 50 ml of peripheral blood, the level of tumor cell counts and the increase in the number of tumor cells during treatment they correlate with the time of evolution, the time until death and the response to therapy.

Prior art

 Despite efforts to improve the treatment and management of cancer patients, survival in cancer patients has not increased in the last two decades for many types of cancer. Consequently, the majority of cancer patients do not die from their primary tumor, but instead succumb to metastasis: multiple widespread tumor colonies established by malignant cells that shed from the original tumor and travel through the body, often to sites distant. The most successful therapeutic strategy in cancer is the early detection and surgical removal of the tumor while it is still confined to the organ. Early detection of cancer has proven feasible for some cancers, particularly where there are appropriate diagnostic tests such as PAP smears in cervical cancer, mammography in breast cancer, and prostate specific serum antigen (PSA) in prostate cancer. However, many cancers detected in early stages have already established micrometastases before surgical resection. Thus, it is important the early and precise determination of the malignant potential of cancer for the selection of the appropriate therapy.

 Optimal therapy will be based on a combination of diagnostic and prognostic information. An accurate and reproducible diagnostic test is necessary to provide specific information regarding the metastatic nature of a particular cancer, along with an evaluation of the prognosis that will provide specific information regarding survival.

 A well-designed prognostic test will provide doctors with information about the risk and likelihood of survival, which in turn benefits the patient by not having to endure unnecessary treatment. 30 The mood of the patient will also be stimulated by knowing that a selected therapy will be effective based on a prognostic test. The savings associated with this test can be significant since it provides the doctor with a justification for the substitution of ineffective therapies. Obviously, a well-developed diagnostic and prognostic database in the treatment and detection of survival-focused metastatic cancer would be a huge benefit to medicine (US 6,063,586). 35

 If a primary tumor is detected early enough, it can often be removed by surgery, radiation, or chemotherapy or some combination of these treatments. Unfortunately, metastatic colonies are difficult to detect and eliminate and it is often impossible to treat them all successfully. Therefore, from a clinical point of view, metastasis can be considered the conclusive event in the natural progression of cancer. In addition, the ability to metastasize is a property that uniquely characterizes a malignant tumor. 40

Soluble Tumor Antigen:

 Based on the complexity of cancer and the metastasis of cancer and frustration in the treatment of cancer patients over the years, numerous attempts have been made to develop diagnostic tests that guide the treatment and control the effects of that treatment. about metastasis or recurrence.

 One of the first attempts to develop a useful test for diagnostic oncology was formulation 45 of an immunoassay for the carcinoembryonic antigen (CEA). This antigen appears in fetal cells and reappears in tumor cells in certain cancers. Extensive efforts have been made to evaluate the usefulness of tests for CEA, as well as many other "tumor" antigens, such as prostate specific antigen (PSA), CA 15.3, CA 125, membrane specific antigen Prostate (PSMA), CA 27.29, p27 found in tissue samples or in blood as soluble cell debris. It is believed that these and other cell debris antigens proceed from the destruction of circulating and non-circulating tumor cells, and therefore their presence does not always reflect the metastatic potential, especially if the cell rupture occurs while in a state of apoptosis

 Additional tests used to predict tumor progression in cancer patients have focused on the correlation of enzymatic indices such as telomerase activity in tumor samples obtained by biopsy with an indication of a favorable or unfavorable prognosis (US document 5,693,474; U.S. document 5,639,613). The evaluation of enzymatic activity in this type of analysis may involve the use of time-consuming laboratory procedures, such as gel electrophoresis and Western blot analysis. In addition, there are variations in the signal to noise ratio and in the sensitivity in the analysis of samples based on the origin of the tumor. Despite these deficiencies, specific blood-soluble tumor markers can provide a quick and effective approach to the development of a therapeutic strategy at the beginning of treatment. For example, the detection of serum HER-2 / neu and serum CA 15-3 in breast cancer patients has been shown to be prognostic factors for metastatic breast cancer (Ali SM, Leitzel K., Chinchilli 10 VM, Engle L ., Demers L., Harvey HA, Carney W., Allard JW and Lipton A., Relationship of Serum HER-2 / neu and Serum CA 15-3 in Patients with Metastatic Breast Cancer, Clinical Chemistry, 48 (8): 1314 -1320 (2002)). The increase in HER-2 / neu results in a decrease in the response to hormonal therapy, and is a significant prognostic factor in the prediction of responses to positive metastatic breast cancer for the hormone receptor. Thus, in the malignant neoplasms where the product of the HER-2 / neu oncogene is associated, procedures have been described to monitor the therapy or assess the risks based on high levels (US document 5,876,712). However, in both cases, the initial levels during remission, or even in healthy individuals, are relatively high and can overlap with the concentrations found in patients, so they require multiple tests and surveillance to establish initial levels and levels. limit that depend on the patient.

 In prostate cancer, serum PSA levels have proven useful in early detection. 20 When used with a physical follow-up exam (rectal examination) and biopsy, the PSA test has improved the detection of prostate cancer at an early stage, when treated better.

 However, the PSA or related PSMA tests leave much to be desired. For example, elevated PSA levels correlate weakly with the stage of the disease and do not appear to be a reliable indicator of the metastatic potential of the tumor. This may be due in part to the fact that PSA is a component of normal prostate tissue and benign prostatic hyperplasia (BHP) tissue. On the other hand, approximately 30% of patients with suspected localized prostate cancer and corresponding low concentrations of serum PSA may present with metastatic disease (Moreno et al., Cancer Research, 52: 6110 (1992)).

Genetic markers:

 An approach to determine the presence of malignant cells of the prostate tumor has been test 30 for the expression of PSA messenger RNA in blood. This is done through a laborious isolation procedure of the whole mRNA from the blood sample and the performance of reverse transcriptase PCR. No significant correlation has been reported between the presence of disseminated tumor cells in blood and the ability to identify which patients would benefit from a more vigorous treatment (Gomella LG., J of Urology, 158: 326-337 (1997)). In addition, false positives are often observed with this technique. There is an added drawback, and there is a practical and finite limit to the sensitivity of this technique based on the sample size. Typically, the test is performed on 105 to 106 cells separated from the interfering red blood cells, which corresponds to a practical lower sensitivity limit of a tumor cell / 0.1 ml of blood (about 10 tumor cells in one ml of blood) before a signal is detected. Greater sensitivity has been suggested to detect hK2 RNA in tumor cells isolated from blood (US 6,479,263; US 6,235,486). 40

 Studies based on qualitative RT-PCR with nucleotide markers in the blood have been used to indicate that the disease-free survival potential for patients with positive CEA mRNA in blood before the operation is worse than that of mRNA-negative patients. from CEA (Hardingham JE, Hewett PJ, Sage RE, Finch JL, Nuttal JD, Kotasel D. and Dovrovic A., Molecular detection of blood-borne epithelial cells in colorectal cancer patients and in patients with benign bowel disease, Int. J. Cancer 89: 8-13 (2000): Taniguchi T., Makino 45 M., Suzuki K., Kaibara N., Prognostic significance of reverse transcriptase-polymerase chain reaction measurement of carcinoembryonic antigen mRNA levels in tumor drainage blood and peripheral blood of patients with colorectal carcinoma, Cancer 89: 970-976 (2000)). The prognostic value of this parameter depends on CEA mRNA levels, which are also induced in healthy individuals by G-CSF, cytokines, steroids, or environmental factors. Therefore, the CEA mRNA marker lacks specificity and is clearly not exclusive to circulating colorectal cancer cells. Other reports have involved tyrosinase mRNA in peripheral blood and bone marrow as a marker for malignant melanoma in phase II-IV patients (Ghossein RA, Coit D., Brennan M., Zhang ZF, Wang Y., Bhattacharya S., Houghton A., and Rosai J., Prognostic significance of peripheral blood and bone marrow tyrosinase messenger RNA in malignant melanoma, Clin Cancer Res., 4 (2): 419-428 (1998)). Again, the use of tyrosinase mRNA as a soluble tumor marker is subject to the aforementioned limitations of soluble tumor antigens as indicators of metastatic potential and patient survival.

 The aforementioned and other studies, although in principle seem prognostic in experimental conditions, do not provide data consistent with a prolonged follow-up period or with

satisfactory specificity. Consequently, these efforts have proved somewhat useless since the appearance of mRNA for blood antigens in general has not been predictive for most cancers and is often detected when there is little hope for the patient.

 Despite this, the polymerase chain reaction with real-time reverse transcriptase (RT-PCR) has been the only published procedure that correlates the quantitative detection of circulating tumor cells with the patient's prognosis. Real-time RT-PCR has been used to quantify CEA mRNA in peripheral blood of patients with colorectal cancer (Ito S., Nakanishi H., Hirai T., Kato T., Kodera Y., Feng Z., Kasai Y., Ito K., Akiyama S., Nakao A., and Tatematsu M., Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a Light Cycler, Cancer Letters, 183: 195-203 (2002)). Using Kaplan-Meier-type analysis, the disease-free survival of patients with positive blood CEA mRNA after surgery was significantly shorter than in cases that were negative for CEA mRNA. These results suggest that the tumor cells spread in the bloodstream (possibly during surgical procedures or micrometastases already existing at the time of the operation), and resulted in poor results in patients with colorectal cancer. The sensitivity of this assay provides a similar detectable reproducible range in sensitivity to conventional RT-PCR. As mentioned, these detection ranges are based on unreliable conversions of amplified product in number of tumor cells. Extrapolated cell count may include damaged CTCs unable to produce metastatic proliferation. In addition, PCR-based analyzes are limited by the possible contamination of the sample, along with the inability to quantify tumor cells. More importantly, procedures based on PCR, flow cytometry, cytoplasmic enzymes and circulating tumor antigens cannot provide essential morphological information that confirms the metastatic potential underlying the structural integrity of the alleged CTC and therefore constitutes substitute tests functionally less reliable than the high sensitivity imaging methods incorporated, in part, in this invention.

Evaluation of intact tumor cells in the detection and prognosis of cancer:

 The detection of intact tumor cells in the blood provides a direct connection to recurrent metastatic disease in cancer patients who have undergone resection of the primary tumor. Unfortunately, the diffusion of malignant cells itself is still overlooked in conventional tumor staging procedures. Recent studies have shown that the presence of a single carcinoma cell in the bone marrow of cancer patients is an independent prognostic factor for metastatic recurrence (Dial IJ, Kaufman M, Goerner R, Costa SD, Kaul S, Bastert G. Detection of tumor cells in bone marrow of patients with primary breast 30 cancer: a prognostic factor for distant metastasis. J Clin Oncol, 10: 1534-1539, 1992). But these invasive techniques are considered undesirable or unacceptable for routine or multiple clinical trials compared to the detection of disseminated tumor epithelial cells in the blood.

 An alternative approach incorporates immunomagnetic separation technology and provides greater sensitivity and specificity in the unequivocal detection of intact circulating cancer cells. This simple and sensitive diagnostic tool, as described (US 6,365,362; U.S. 6,551,843; U.S. 6,623,982; U.S. 6,620,627; U.S. 6,645,731, WO 02/077604; WO03 / 065042; and WO 03/019141) is used in the present invention to correlate the statistical survival of an individual patient based on a threshold level greater than or equal to 5 tumor cells in 7 , 5 to 30 ml of blood (1 to 2 tumor cells correspond to about 3000 to 4000 total tumor cells in circulation for a given individual). 40

 With this diagnostic tool, a blood sample from a cancer patient (WO 03/018757) is incubated with magnetic beads, coated with antibodies directed against an antigen on the surface of an epithelial cell, such as EpCAM, as described in WO99 / 41613, US 2002/0172987, Kagan et al., J. Clin. Ligand Assay 25: 104-110 (2002) and Witzig et al., Clin. Canc. Res. 8: 1085-1091 (2002). After labeling with magnetic nanoparticles coated with anti-EpCAM, the magnetically labeled cells are isolated using a magnetic separator. The immunomagnetically enriched fraction is processed for downstream immunocytochemical analysis or for image cytometry, for example, in the Cell Spotter® System (Immunicon Corp., PA). The magnetic fraction can also be used for downstream immunocytochemical analysis, RT-PCR, PCR, FISH, flow cytometry, or other types of image cytometry.

 The Cell Spotter® System uses immunomagnetic selection and separation to enrich and concentrate all epithelial cells present in whole blood samples. Captured cells are detectably labeled with a specific leukocyte marker and with one or more fluorescent monoclonal antibodies specific to tumor cells to allow identification and counting of the captured CTCs, as well as the unambiguous instrumental or visual differentiation of contamination of non-target cells With an extraordinary sensitivity of 1 or 2 epithelial cells per 7.5 to 30 ml of blood, this assay allows the detection of 55 tumor cells, even in the early stages of low tumor mass. The embodiment of the invention is not limited to the Cell Spotter® System, but includes all isolation and imaging protocols of comparable sensitivity and specificity.

 The prognostic protocols currently available have not proven to be a consistently reliable means of correlating CTC to predict progression-free survival or overall survival in patients with cancers such as metastatic breast cancer (MBC). Therefore, there is a clear need for accurate detection of cancer cells with metastatic potential, not only in the MBC, but also in metastatic cancers in general. On the other hand, this need is accentuated by the need to select the most effective therapy for a given patient.

Summary of the Invention

 The present invention is a method for the prognosis of cancer, as defined in claim 1. It uses diagnostic tools, such as the Cell Spotter® System, to evaluate the time of disease progression, survival, and Therapy response based on the absolute number of circulating cancer cells (CTC) in patients with metastatic cancer. The system immunomagnetically concentrates the epithelial cells, marks the cells with fluorescence, identifies and quantifies the CTCs for the positive count. Statistical analysis of cell count predicts survival. Survival prediction is based on a comparison of the threshold of the number of circulating tumor cells in blood with the time of disease progression and death. The statistical analysis of long-term follow-up studies 15 of patients diagnosed with cancer sets a threshold for the number of CTCs found in the blood and the prediction of survival. The absence of CTC is defined as less than 5 morphologically intact CTC. The presence or absence of CTC to predict survival is useful in the choice of treatment. For example, the absence of CTC in a woman not previously treated for metastatic breast cancer could be used to select hormonal therapy versus chemotherapy with fewer side effects and a better quality of life. 20 By contrast, the presence of CTC could be used to select chemotherapy, which has greater side effects, but may prolong survival more effectively in a high-risk population. Thus, the invention has a prognostic role in the detection of CTC in women with metastatic breast cancer.

Brief description of the drawings

 Figure 1: Cell Spotter® fluorescence analysis profile used to confirm captured objects such as 25 tumor cells. Check marks mean a positive tumor cell based on the composite image. The composite images come from the positive selection of the epithelial cell marker (EC-PE) and the nuclear dye (NADYE). A negative selection for the leukocyte marker (L-APC) and for the control (CNTL) is also necessary.

 Figure 2: Comparison of diagnostic procedures to measure changes in tumor status. The current pattern of the physical measurement of discrete lesions is shown using radiographic imaging (Group A). A model is shown that illustrates the ability to assess changes in the burden of metastatic cancer by counting the number of CTC in blood (Group B).

 Figure 3: Lack of correlation between the number of CTC and tumor size in 69 patients.

 Figure 4: Changes in the number of CTC in patients with partial response to therapy or with a stable disease status. CTCs were reduced or remained undetectable in all cases.

 Figure 5: Changes in the number of CTC in patients with disease progression. CTCs increased or remained undetectable with the progression of the disease.

 Figure 6: Evolution in patients of the CTC number. Group A shows a type patient with less than 5 CTC per 7.5 ml of blood. Group B shows a type patient with a decrease in CTC over the course of therapy. Group C shows a type patient with a decrease in CTC, followed by an increase. Group D shows a type patient with an increase in CTC.

 Figure 7: Determination of an optimal CTC limit to distinguish MBC patients with rapid progression. The analysis was performed using the CTC numbers obtained at the start of the study of the 102 patients included in the training group. The median SLP of patients with greater than or equal to the selected number of CTC 45 in 7.5 ml of blood is indicated by the solid line and the median SLP of patients below the selected CTC level is indicated by the line. discontinuous The median of the SLP was reduced as the CTCs increased and reached a level that stabilized at 5 CTC (indicated by the vertical line). The black dot indicates the median PFS of ~ 5.9 months for the 102 patients. The selected limit of ≥ 5 CTC / 7.5 ml was used in all subsequent analyzes. fifty

 Figure 8: The predictive value of the CTC at the beginning for the SLP and the SG. The probabilities of PFS and OS of patients with MBC with <5 (black line) and ≥ 5 (gray line) of CTC in 7.5 ml of blood are shown using the initial blood draw before the start of a new line of therapy. The SLP and the SG were calculated from the moment of blood collection at the beginning. Group A: the probability of SLP using the initial CTC count (n =

177, log-rank p = 0.0001, CoxHR = 1.95, chi2 = 15.33, p = 0.0001). Group B: the probability of SG using the initial CTC count (n = 102, log-rank p = 0.0003, CoxHR = 3.98, chi2 = 12.64, p = 0.0004).

 Figure 9: The predictive value of the CTC in the first follow-up for the SLP and the SG. The probabilities of PFS and OS of patients with MBC with <5 (black line) and ≥ 5 (gray line) of CTC in 7.5 ml of blood at the first follow-up after the start of a new therapy line are shown . The SLP and the SG were calculated 5 from the moment of blood collection at the beginning. Group A: the probability of PFS using the first blood collection follow-up (n = 163, log-rank p <0.0001, CoxHR = 2.73, chi2 = 25.25, p <0.0001). Group B: the probability of OS using the first blood collection follow-up (n = 68, log-rank p = 0.0001, CoxHR = 6.12, chi2 = 13.24, p = 0.0003).

 Figure 10: A reduction in the CTC count below 5 in the first follow-up of blood collection after the start of therapy (4 to 5 weeks) predicts an improved median of PFS and OS. For both groups, the black line shows <5 CTC in both initial extraction and blood collection for the first follow-up. The black dotted line shows ≥ 5 CTC initially at the start of the study, but decreases below 5 CTC at the first follow-up. The gray dotted line shows ≥ 5 CTC, both at the beginning and after the follow-up, and the solid gray line shows an increase in the CTC compared to the initial level with ≥ 5 CTC 15 at the follow-up. Group A shows the probability of PFS in patients, while Group B shows the probability of OS.

Detailed description of the invention

 The objective of the present invention provides the detection of circulating tumor cells as an indicator of the early prognosis of patient survival. The procedure combines immunomagnetic enrichment technology 20, and immunofluorescence labeling technology with a suitable analysis platform after initial blood collection. The associated test has the sensitivity and specificity to detect these rare cells in a whole blood sample and to investigate their role in the clinical evolution of the disease in malignant tumors of epithelial origin. From a whole blood sample, rare cells are detected with a sensitivity and specificity that allow them to be collected and used in the diagnostic tests of the invention, that is, to predict the clinical evolution of the disease in malignant tumors. .

 With this technology, it has been shown that there are circulating tumor cells (CTC) in the blood in detectable amounts. This has generated a tool to investigate the importance of cells of epithelial origin in the peripheral circulation of cancer patients (Racila E., Euhus D., Weiss AJ, Rao C., McConnell J., Terstappen LWMM and Uhr JW, Detection and characterization of carcinoma cells in the blood, Proc. Natl. Acad. Sci. 30 USA, 95: 4589-4594 (1998)). This study demonstrates that these blood-borne cells could play an important role in the pathophysiology of cancer. By having a detection sensitivity of 1 epithelial cell per 5 ml of blood, the analysis incorporates the immunomagnetic enrichment of the sample and the staining of fluorescent monoclonal antibodies, followed by flow cytometry for a rapid and sensitive analysis of a sample. The results show that the number of epithelial cells in peripheral blood of eight patients treated for metastatic carcinoma of the breast correlate with the progression of the disease and the response to treatment. In 13 of 14 patients with localized disease, 5 of 5 patients with lymph node involvement and 11 of 11 patients with distant metastases, epithelial cells were found in peripheral blood. The number of epithelial cells was significantly higher in patients with extended disease.

 The assay has been further configured for cytometric image analysis such that the immunomagnetically enriched sample is analyzed by the Cell Spotter® System (see Example 1). This is an image analysis using a fluorescence-based microscope, which unlike flow cytometry analysis allows the visualization of events and the evaluation of morphological characteristics to better identify objects.

 The term Cell Spotter® System refers to an automated microscopic fluorescence system for automated counting of cells isolated from blood. The system contains an integrated computer-controlled fluorescence microscope and an automated phase with a magnetic fork that will house a disposable sample cartridge. The magnetic fork is designed to allow candidate tumor cells marked with an iron fluid inside the sample chamber to be magnetically located on the top viewing surface of the sample cartridge for microscopic viewing. The software presents the operator with 50 suspicious cancer cells, labeled with antibodies to the cytokeratin and having an epithelial origin, for final selection.

 Although the isolation of tumor cells for the Cell Spotter® System can be achieved by any means known in the art, one embodiment uses the Immunicon CellPrep ™ System to isolate tumor cells using 7.5 ml of whole blood. Specific magnetic particles of epithelial cells are added and incubated for 20 minutes. After magnetic separation, the antibody bound cells

Immunomagnetic junction is magnetically held to the tube wall. The unbound components are then aspirated, and an isotonic solution is added to resuspend the sample. A sample of nucleic acids, monoclonal antibodies for cytokeratin (a marker of epithelial cells) and CD 45 (a marker of broad-spectrum leukocytes) is incubated with the sample. After magnetic separation, the free fraction is aspirated again and the labeled and bound cells are resuspended in 0.2 ml of an isotonic solution. The sample is suspended in a cell presentation chamber and placed in a magnetic device whose field orients the magnetically labeled cells for examination under a fluorescence microscope in the Cell Spotter® System. Cells are automatically identified in the Cell Spotter® System and candidate circulating tumor cells are presented to the operator for verification with a list. A checklist consists of predetermined morphological criteria that constitute a complete cell (see Example 1). 10

 The diagnostic potential of the Cell Spotter® System, together with the use of intact circulating tumor cells as a prognostic factor in cancer survival, can provide a rapid and sensitive procedure to determine the appropriate treatment. Accordingly, in the present invention there is provided a method for rapid counting and characterization of disseminated tumor cells in the blood of patients with metastatic and primary disease for the evaluation of the prognosis of survival potential. fifteen

 The methods of the invention are useful in the evaluation of a favorable or unfavorable survival, and even in the prevention of unnecessary therapy that could lead to harmful side effects when the prognosis is favorable. Therefore, the present invention can be used for the prognosis of any of a wide variety of cancers, including, without limitation, solid tumors and leukemia, including prominent cancers: apudoma, choristoma, branquioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (i.e., 20 Walker, basal cell, basoscamosus, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinosa, non-small cell lung, small cell, papillary, scirro , bronchiolar, bronchogenic, squamous cell and transition cell), histiocytic disorders, leukemia (i.e., B-cell, mixed cell, null cell, T-cell, chronic T-cell, associated with HTLV-II, acute lymphocytic or chronic lymphocytic, mast cell, and myeloid), malignant histiocytosis, Hodgkin's disease, immunoproliferative small intestine, non-Hodgkin's lymphoma, plasmacytoma, reticuloendothelium sis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolinphoma, carcinosararingi, chordoma , dysgerminoma, hamartoma, mesenchygoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, 30 cylromaroma, cystoadenocarcinoma, cystadenoma, gland tumor, granuloasstoma cells pancreatic islet cell tumor, icydig cell tumor, papilloma, Sertoli cell tumor, teak cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paragan glioma, non-chromaffin paraganglioma, 35 angiokeratoma, angiolinfoid hyperplasia with eosinophilia, sclerosing angioma, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyarcomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomacomalarcoma , leukosarcoma, liposarcoma, lymphangiosarcoma, myoswarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (i.e., experimental Ewing, Kaposi and mastocytic cells), neoplasms (i.e., bone, breast, digestive, colorectal , liver, pancreas, pituitary gland, testis, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory and urogenital tract, neurofibromatosis, and cervical dysplasia.

 The following examples illustrate the predictive value and prognosis of CTCs in the blood of patients. Note that the following examples are offered by way of illustration and are not intended in any way to limit the scope of the invention.

Example 1

Circulating cytokeratin positive cell count with CellPrep ™

 Cells positive for cytokeratin were isolated with the CellPrep ™ System using a 7.5 ml sample of whole blood. Specific immunomagnetic fluid from epithelial cells was added and incubated for 20 minutes. After magnetic separation for 20 minutes, the cells bound to the immunomagnetic binding antibodies were magnetically attached to the tube wall. The unbound components are then aspirated, and an isotonic solution is added to resuspend the sample. A sample of nucleic acids, monoclonal antibodies to cytokeratin (a marker of epithelial cells) and CD 45 (a marker of broad-spectrum leukocytes) is incubated with the sample for 15 minutes. After magnetic separation, the free fraction is aspirated again and the labeled and bound cells are resuspended in 0.2 ml of an isotonic solution. The sample is suspended in a cell presentation chamber and placed in a magnetic device whose field orients the magnetically labeled cells for examination under a fluorescence microscope in the Cell

Spotter® System. Cells are automatically identified in the Cell Spotter® System; Control cells are counted by the system, while candidate circulating tumor cells are presented to the operator for counting by a checklist as shown (Figure 1).

Example 2

Tumor load assessment: Comparison between the analysis of radiographic images and the absolute number 5 of CTC.

 Radiological measurements of metastatic lesions are currently used to assess tumor burden in cancer patients with metastases. In general, the largest lesions are measured and added together to obtain a tumor burden. An example of a two-dimensional measure of a liver metastasis in a patient with breast cancer is illustrated in Figure 2A. Figure 2B illustrates a model that represents the need to measure the tumor burden 10 in the blood as a measure of the actual active tumor burden, and therefore a better measure of the overall activity of the disease. To determine whether the absolute number of CTC is correlated or not with the size of the tumor measured by the image, a prospective study was conducted in patients with MBC.

 The Cell Spotter® System was used for the CTC count in 7.5 ml of blood from 69 patients with measurable MBC. Tumor load was assessed by two-dimensional radiographic measurements of up to 15 8 measurable lesions before the start of therapy. Tumor load was determined by the addition of individual measurements (mm²). CTCs are counted in blood collection before the start of therapy.

 Figure 3 shows the number of CTC in 7.5 ml compared to the two-dimensional sums of tumor measurements in the 69 patients. In Figure 3, there is no correlation between the size of the tumor and the absolute number of tumor cells in the blood. Some patients with large tumors, measured by imaging, have a low CTC number, and vice versa.

 Therefore, tumor burden, measured by radiographic imaging, does not correlate with the absolute number of tumor cells in the blood.

Example 3

Evaluation of tumor burden: Comparison between changes in radiographic image and changes in the absolute number of CTC.

 Radiographic imaging is the current standard to assess whether a certain disease is responding, is stabilized, or is progressing with treatment. The interval between radiographic measurements must be at least 3 months to give significant results. Therefore, a test that could predict an earlier treatment response during the treatment cycle could improve the management of 30 patients treated for metastatic diseases, which could increase their quality of life and possibly improve their survival. In this study, patients who initiate a new line of treatment for MBC were evaluated to determine if a change in the number of CTC is correlated with a change in the patient's condition, measured by imaging.

 The Cell Spotter® System was used for the CTC count in 7.5 ml of blood in patients with MBC at the point of starting a new treatment, and at different times during the treatment cycle. Radiographic measurements were made before the start of therapy, 10-12 weeks after the start of therapy and after the end of the treatment cycle (approximately 6 months after the start of therapy), or at the time when the patient evolved in therapy, whichever came first.

 From the image analysis, a partial response was found in 14 patients (17 data segments). 40 CTCs were reduced or remained undetectable in all cases (see Figure 4). The image showed the stabilization of the disease in 30 patients (37 data segments). CTCs were reduced or remained undetectable in all cases (see Figure 4). The image showed the progression of the disease in 14 patients (15 data segments). CTCs increased in 7 of 15 cases. No CTC was detected at any time in the other 8 cases. Four. Five

 Only an increase in CTC was observed in patients with disease progression (100%). Only a decrease in CTC was observed in patients with partial response or stable disease (100%). In patients with partial response or stable disease, no CTC was detected at both times in 54 of 61 cases (89%).

Example 4

Evolution of the number of CTC in patients treated by MBC as a guide for treatment.

 A study was conducted in patients with MBC to determine whether clear trends can be observed or not in changes in the number of CTC in patients treated by MBC, and whether simple rules can be applied to these trends or not in order to guide to the doctor who assists in the optimization of the treatment of patients with MBC.

 The Cell Spotter® System was used for the CTC count in 7.5 ml of blood. In the study 5 81 patients were included, starting a new line of treatment for MBC. CTCs are counted in blood collection before the start of therapy and approximately every month thereafter.

 Clear trends were observed in the number of CTC in 76 of 81 (94%) patients. During the course of treatment, the number of CTC was not detectable or remained below 5 CTC per 7.5 ml of blood in 50% of patients. A typical example is shown in Figure 6A. The number of CTC was reduced during the course of therapy in 22% of patients. A typical example is shown in Figure 6B. A decrease in the number of CTC was observed followed by an increase in the course of treatment in 6% of patients. A typical example is shown in Figure 6C. The number of CTC increased during the course of therapy in 16% of patients. A typical example is shown in Figure 6D. In 42 cases, 2 blood samples were prepared and analyzed at the time of each blood draw. The results with the first tubes removed at the initial moment 15 and the first tube removed at the follow-up point were compared with the results of the second tubes removed at each point. In only one of those cases, the change in the number of CTCs was different between the first tubes removed and the second (or duplicate) tubes removed (98% concordance). In this case, the two tubes of the first blood collection had 0 CTC, while for the second blood collection, one tube had 5 CTC (below the limit) and the second tube had 6 CTC (above the limit). Compared to the reproducibility of the CTC measurements, the inter-radiological variability of radiographic images, when the same films were read by two different expert radiologists, resulted in a concordance of only 81%. In addition, the concordance between the two radiologists in a set of 146 image segments was 85% when Progression was measured against non-progression, and reduced to only 58% when Progression, stable disease and partial response were measured. . In contrast, the CTC measurement analysis was performed on the same set of data 25 by two different technicians, which results in a 100% match.

 Therefore, the detection and monitoring of CTC in patients treated by MBC is a more reproducible procedure to measure the response to therapy than radiographic images.

Example 5

Prediction of PFS and OS before the start of therapy. 30

 A study was carried out to correlate CTC levels before the start of therapy with progression-free survival (PFS) and overall survival (OS), so a threshold value of ≥ 5 CTC / 7.5 ml was used. .

 177 patients with measurable MBC were tested for CTC in 7.5 ml of blood before starting a new line of treatment and at subsequent monthly intervals for a period of up to six months. In the 35 trial, patients entering any type of therapy or any line of treatment were included. The progression or response to the disease was evaluated by doctors at the locations of each patient.

 As shown in Figure 7, the median PFS decreased as CTC levels increased and reached a level that stabilized at 5 CTC (vertical line). The median PFS was approximately 5.9 months for all patients (black dot). Based on the change in the average SLP 40 for positive patients and the Cox risk ratio, a limit of ≥ 5 CTC was used for all subsequent analyzes.

 Figure 8 shows a Kaplan-Meier analysis of Progression-free Survival (SLP) and Global Survival (SG) using the number of CTC measured at the start of blood draws. In the 177 patients, the average time of PFS was approximately 5.0 months. Patients with ≥ 5 CTC / 7.5 ml of blood at baseline had a significantly lower PFS than patients with <5 CTC (approximately 2.7 45 months versus 7.0 months, respectively). Overall survival (OS) reflects the same trend with a median OS of 10.1 months versus> 18 months for patients with ≥ 5 CTC vs <5 CTC, respectively.

 The measurement of the number of CTC before the start of a new treatment line predicts time until patients progress in their therapy, and predicts survival time. Due to this prediction capability, the detection and measurement of CTCs at the beginning provides information to physicians that will be useful in the selection of an appropriate treatment. In addition, the ability to stratify patients into high and low risk groups in terms of PFS and OS can be very useful in selecting suitable patients for inclusion in

therapeutic trials To obtain new drugs with potentially high toxicity, patients with poor prognostic factors may be the most appropriate target population. In contrast, drugs with minimal toxicity and promising therapeutic efficacy can be more appropriately directed to patients with favorable prognostic factors.

Example 6 5

Prediction of PFS and OS after the start of treatment.

 A study was conducted to correlate CTC levels after the start of therapy with progression-free survival (PFS) and overall survival (OS) using the CTC number at the first follow-up to predict PFS and OS .

 For this analysis, 163 patients with measurable MBC were evaluated. Blood was drawn an average of 4 weeks 10 after the start of a new treatment line. The progression or response to the disease was evaluated by the doctors in the places for each patient in an average time of 12 weeks after the start of therapy.

 As shown in Figure 9, of the 49 patients with ≥ 5 CTC per 7.5 ml at first follow-up had a significantly lower SLP median compared to 114 patients with <5 CTC per 7.5 ml, 15 approximately 2.1 months versus 7.0 months, respectively. The same trend was observed for the median overall survival, approximately 8.2 months for ≥ 5 CTC and> 18 months for <5 CTC.

 In a separate analysis, we compared two groups with a shorter or longer known SLP and OS to that of patients with decreased CTC. Specifically, patients whose CTC were <5 at baseline and at the first follow-up are known to have a relatively long PFS and OS; that is, it was a population with a relatively good performance. On the contrary, patients whose CTC increased from the beginning to the first follow-up with a CTC level> 5 at the first follow-up are known to have a relatively short PFS and OS, that is, it was a population of patients with relatively poor results. Next, two additional groups of patients were compared with these first two groups: first, patients whose CTC decreased from baseline to first follow-up at a level <5. Second, patients 25 whose CTC decreased from the beginning until the first follow-up, but the number of CTC at the first follow-up was ≥ 5. The results are shown in Figure 10. For the first control groups with a CTC <5 at the start and at the first follow-up, the SLP and the SG are relatively long, as expected. For the second control group with cell augmentation, the SLP and the SG are relatively much shorter, again as expected. For patients whose CTC was reduced to <5 at the first follow-up, the SLP and OS approached that of patients 30 who had <5 CTC at both times. On the contrary, for patients whose CTC decreased, but did not decrease to <5, their prognosis was as bad as that of patients with increasing CTC.

 Consequently, CTCs should fall below 5 at the first follow-up (approximately 4 weeks) to maximize PFS and OS, and to maximize the benefit associated with therapy.

Example 7 35

Unifactorial and multifactorial analysis of predictors of the SLP and the SG.

 In order to compare CTC levels with known parameters associated with PFS and OS, single-factor and multifactorial regression analysis of Cox proportional risk was carried out. To predict PFS, only the therapy line, the type of therapy and CTC levels at the beginning and at the first follow-up were uniformly significant. For OS, the ER / PR status was also unifactorially significant, where ER / PR is considered positive if the estrogen receptor, the progesterone receptor, or both are positive. The status of the patient measured using the ECOG guidelines was also unifactorially significant for OS, where ECOG is the status of the European Cooperative Oncology Group, which ranges from 0 to 5 (Table 2).

Table 2. Cox unifactorial regression analysis of independent parameters for the prediction of the SLP and the SG.

 Categories SLP SG

 Parameter

 Pos Neg No. pac HR p-val chi2 HR p-val chi2

 Age (years)

 Age at start 175 0.99 0.1099 2.56 0.99 0.1992 1.65

 ECOG

 2 vs 1 vs 0 172 1.10 0.4465 0.58 1.63 0.0075 7.16

 Stage

 4 vs 3 vs 2 vs 0 164 0.94 0.5591 0.34 1.10 0.4746 0.51

 ER / PR status

 + - 175 0.85 0.3827 0.76 0.57 0.0253 5.00

 HER2 state

 3 vs 2 vs 1 148 0.90 0.1895 1.72 0.90 0.3557 0.85

 Time for metastasis

 Years 175 0.97 0.0252 5.01 0.92 0.0028 8.92

 Therapy Line

 ≥ 2nd 1st 175 1.68 0.0025 9.14 2.06 0.0042 8.19

 Therapy Type

 Hormonal Chyme 172 1.81 0.0016 9.97 3.46 0.0001 15.61

 CTC at startup

 ≥ 5 <5 177 1.95 0.0001 15.33 4.39 0.0000 31.73

 CTC in the 1st follow-up

 ≥ 5 <5 163 2.73 0.0000 25.25 5.54 0.0000 38.02

Stage = stage of the disease at the time of primary diagnosis, Pos = positive, Neg = negative, Chemo = chemotherapy with or without other treatments, Horm. = Hormone therapy and / or immunotherapy, HR = Cox risk ratio. Age and time for metastasis were evaluated as continuous variables.

 Cox regression in stages at a severity level of p <0.05 to include and exclude parameters is used separately for the initial CTC levels and the first follow-up to predict the SLP and the SG. Although some of the clinical factors remained important in the multivariate analysis, persistent initial CTC and positive CTC levels at the first follow-up appeared as the strongest predictors of PFS and OS (Table 3).

Table 3. Cox multifactorial regression analysis for the prediction of PFS and OS by means of 10-stage selection at a severity level of p <0.06.

 Categories SLP SG

 Parameter

 Pos Neg HR p-val chi2 HR p-val chi2

 Analysis using the initial CTC count

 (n = 172) (n = 170)

 CTC at startup

 ≥ 5 <5 1.76 0.001 10.58 4.26 0.000 22.35

 Therapy Line

 ≥ 2nd 1st 1.73 0.002 9.75 2.38 0.001 10.32

 Therapy Type

 Hormonal Chyme 1.61 0.016 5.85 2.54 0.015 5.90

 ECOG

 2 vs 1 vs 0 ns ns ns 1.48 0.024 5.10

 Time for metastasis

 Years ns ns ns 0.92 0.028 4.82

 Analysis using the 1st CTC Tracking count

 (n = 162) (n = 160)

 1st CTC Tracking

 ≥ 5 <5 2.52 0.000 23.56 6.49 0.000 38.34

 ER / PR status

 + - ns ns ns 0.35 0.001 11.19

 Therapy Line

 ≥ 2nd 1st 1.58 0.013 6.22 2.29 0.006 7.67

 ECOG

 2 vs 1 vs 0 ns ns ns 1.53 0.025 5.05

 Cox multifactorial regression analysis was used with a step selection process to evaluate the association with the SLP and the GS. A level of rigor (p value) of 0.05 was used to include and exclude parameters in the multifactor analysis. The results for each parameter that has demonstrated a statistically significant correlation for the SLP and the SG are summarized in the table. The CTC number was the strongest predictor of the SLP and the SG. 5

Stage = stage of the disease at the time of the main diagnosis, Pos = positive

Neg = negative, HR = Cox risk ratio, ns = not significant in the multifactor analysis

Example 8

Evaluation of the response to therapy based on CTC after the first follow-up.

 A study was conducted in patients with MBC to determine if the number of CTC after the first follow-up provides a relevant index, or not, to assess the response to treatment.

 The Cell Spotter® System was used to count the CTC in 7.5 ml of blood. 163 patients diagnosed clinically with metastatic breast cancer were compared for CTC in blood collection at the first follow-up with an average of 4.5 ± 2.4 weeks (median 4.0 weeks, varying between 1.4 and 16 , 9 weeks) from the moment of initial blood collection. CTCs were counted in blood collection before 15 the start of therapy and approximately every month thereafter. Using a threshold value of less than 5 CTC per 7.5 ml of blood, the CTC count at the first follow-up was compared with the patient's clinical status, so that patients with stable or responding disease were classified as No progression , and patients with clinical disease progression based on the determination of two-dimensional images from the beginning and the first follow-up were classified as Progression (See Table 4). twenty

Table 4: Count of patients with metastatic breast cancer at the time of blood collection from the first follow-up.

 Determination of imaging

 Patients with <5 CTC after 1st follow-up Patients with ≥ 5 CTC after 1st follow-up Total

 Stable or responsive (no progression)

 94 14 108

 Progression

 20 35 55

 Total

 114 49 163

 When 94 patients with less than 5 CTC were tested at the first follow-up, they did not show disease progression, which shows a concordance between the CTC counts and the response to treatment. When 35 patients with 5 or more CTCs were tested at the first follow-up, they showed disease progression, which again demonstrates a concordance between the number of CTC at the time of the first follow-up and the lack of response to treatment. 20 patients had less than 5 CTC with disease progression, which represents false negative results. These results would not be clinically counterproductive since these patients continued to receive treatment, in the same way that they would receive 30 if the CTC analysis was not used. However, 14 patients presented ≥ 5 CTC at the first follow-up without radiographic evidence of progression, indicating false positive responses. Although these responses could lead to a change in therapy in a patient who could benefit from such therapy, it is expected that the new therapy will be useful in these patients, and that the number of false positives will be acceptably low. Therefore, in general, the CTC count at the first follow-up gave an indication of the response to treatment in 129 of the 163 35 patients evaluated.

Claims (3)

 1. A procedure for the prognosis of the survival of a patient comprising: a) immunologically enriching a fraction of a blood sample of said patient, said sample comprising a population of mixed cells suspected of containing circulating tumor cells (CTC), wherein said enrichment step comprises mixing said sample with magnetic particles coupled to an antibody which specifically binds to EpCAM and subject said sample-magnetic particle mixture to a magnetic field to produce a CTC-enriched cell suspension bound to magnetic particles, b) confirm the structural integrity of cytokeratin-positive CTCs that are intact, where cytokeratin-positive cells are identified by labeling with a monoclonal antibody to cytokeratin, and where said structural integrity is determined by an analysis of the morphological characteristics based on fluorescence, and 10 c) measuring the number of said intact CTCs to determine patient survival, where a number of CTCs greater than or equal to five CTCs per 7.5 ml of blood is indicative of a lower survival prognosis.  2. A method according to claim 1, wherein said survival prognosis is determined in patients with metastatic breast cancer, patients with metastatic prostate cancer and / or patients with metastatic colon cancer. fifteen