ES2528493B1 - TUMOR BIOMARCATOR OF THE STATE OF AFFECTION OF NON-CENTINELA LYMPHATIC GANGLES - Google Patents

Abstract

The present invention identifies matrix metalloprotease (MMP) -1 as a tumor biomarker to predict tumor involvement of the non-sentinel nodes, from the positive sentinel node in cases of cancer. In cases of positive sentinel node, the non-expression of this factor in a particular cell type, predicts non-sentinel node involvement by tumor cells. The specificity and sensitivity of this prognostic / diagnostic test is 61.5% and 100%, respectively. However, the most important thing is that the negative predictive value is 100%. Which means that all cases with a negative expression of MMP-1 by peritumoral inflammatory mononuclear cells, in the positive sentinel node, have no tumor involvement of the other nodes. This implies that this invention can prevent at least 60% of unnecessary complete axillary dissections.

Description

. Tumor biomarker of non-sentinel lymph node involvement status.

SECTOR OF THE TECHNIQUE

The present invention focuses on the medical sector, specifically in the medical sector.

5 oncology, pathology and surgery. The main applications of the invention are as support in making surgical decisions in oncology, pathology and molecular diagnosis.

STATE OF THE PREVIOUS TECHNIQUE

The present invention is based on a tumor biomarker of the state of affectation

10 metastatic non-sentinel lymph nodes in patients with a positive sentinel node, preferably in tumors with lymph node involvement, more preferably in patients with breast cancer.

In the nineteenth century, Virchow formulated the theory that lymph nodes filter particulate substances in lymph. This important statement made us aware that the cancer could be cured at an early stage with proper surgery. Halsted introduced at the end of the 19th century, the next logical step of Virchow's theory, radical mastectomy (Halsted WS, 1894). Almost a century later, Kett et al. Administered a contrast substance in the lymphatic vessels of the breast, which were visualized with the help of an injection with a blue dye in the breast areola 20 (Kett K, et al., 1970). Thus they observed the flow to an isolated lymph node, called the "sorgius ~ node, and then a drain through many vessels and lymph nodes into the collecting system around the axillary vein. Using lymphoscintigraphy in 1980, Christensen and collaborators observed" ganglia of primary drainage "(Christensen B, et al., 1980). Haagensen studied the route of metastasis through the filter

25 of the axillary lymph nodes, and noted that the central group nodes are not only more frequently affected, but also more frequently exclusively affected (Haagensen CD, 1972). However, the concept of lymphatic mapping was not introduced until the end of the 20th century.

The concept of sentinel node biopsy is based on two basic principles:

The existence of an orderly and predictable pattern of lymphatic drainage to a regional lymph node chain, and the functioning of a first lymph node as an effective filter for tumor cells. The development of the sentinel lymph node biopsy in breast cancer began at the "John Wayne Caneer Institute ~ in 1991. Previously, this intraoperative lymphatic mapping technique was presented at the" 2 <1 International Conference on Melanoma of the World Organization for Health 'in 1989. However, shortly thereafter an intraoperative screening test of the sentinel node by gamma rays was introduced using an injection of colloidal radiomarkers (Krag DN, aL, 1993). Currently, different methodologies based on this

5 lymphatic mapping technique are applied worldwide.

Current methods for assessing sentinel node involvement during surgery employ frozen sections, imprinting or scraping cytology, rapid immunocytochemistry and combinations thereof. Sections on frozen tissue is the most used technique. The protocols for intraoperative analysis of sections of frozen tissue and the making of 105 cuts of formalin-fixed and paraffin-embedded tissue vary considerably in each institution. In addition, frozen sections are of lower quality for morphological evaluation than paraffin sections, and small metastases of 105 lymph nodes may be lost. Moreover, the process of cutting the frozen sections produces an irreversible loss of tissue.

15 Cytological techniques, such as imprinting or scraping, consist of pressing or scraping the cut surface of the sentinel node on a glass slide, staining and examining it. The preparation time and the cost of 105 cytological specimens are less than 105 of the frozen sections, there being, in addition, tissue loss.

Intraoperative standard histological sample analysis protocols examine

20 only a small part of the total sentinel node volume. This introduces the probability of significant sampling errors in these techniques: a negative result may simply be due to a failure to not examine the part of the node that contains the metastasis. Therefore, these techniques require a subsequent and complete examination with specific tumor markers, such as cytokeratins. The techniques

25 molecular have the potential to eliminate sampling error. The tissue to be analyzed is homogenized, and the presence of genetic markers is examined, so that the entire ganglion is analyzed. Viable techniques for this intraoperative analysis of the sentinel node would be both the quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the amplification of nucleic acids in a single

30 step (OSNA). These techniques are based on the detection of messenger RNA (mRNA) of marker genes that are overexpressed in tumor cells but not in normal tissue. The value of these molecular assays in the detection of lymph node metastasis was limited by the high sensitivity of these techniques: tumor mRNA markers, although expressed in neoplastic cells, are also

35 present in normal tissues, although to a lesser extent (Gillanders WE, et al, 2004). Therefore, the specificity was too low to be used in the detection of

metastasis by qualitative techniques (Manzotti M, et al., 2001; Sakaguchi M, et aL, 2003).

The first commercial trial available for intraoperative analysis of sentinel node material using qRT-PCR was '' GeneSearch ™ Breast lymph Nade Assay "5 (Veridex, Warren, New Jersey, USA), based on the use of mamaglobin (MGB1) and cytokeratin -19 (CK-19) in a double marker test A positive test would be one in which the expression of either of the two markers exceeds a threshold level, calibrated to correlate with the presence of metastases larger than 0 , 2 mm in diameter Unfortunately, because the qRT-PCR technique

10 requires tissue homogenization, a direct comparison by histological examination of the same tissue is not possible.

The aSNA technique, like qRT-PCR, is a molecular diagnostic technique used to detect the target mRNA. This technique also uses reverse transcriptase to convert mRNA into its complementary DNA (cDNA), but by means of a loop-mediated isothermal amplification (LAMP): this variation in the PCR uses 6 specific primers for the same target cDNA. These primers are designed so that the DNA loop occurs during the amplification phase, resulting in a release of pyrophosphate as a reaction byproduct, which binds with magnesium and precipitates. The precipitation or turbidity rate of the solution is used

20 to quantify the amount of target gene present (Godey F, et al., 2012). Unfortunately, as in qRT-PCR, aSNA also requires tissue homogenization, and therefore a direct comparison by histological examination of the same tissue is not possible. In addition, tissue homogenization causes irreversible tissue loss, and even data if a technical failure occurs.

25 Dissection of axillary nodes in breast cancer is a procedure originally designed to maximize survival and regional control, and to determine the classification of nodes. Axillary lymph node dissection (DGLA) with removal and histopathological examination of at least 10 nodes has been an inherent part of the surgical treatment of breast cancer for a long period.

30 In the past decade, DGLA was replaced by sentinel lymph node biopsy (BGC). Sentinel node resection was designed to minimize the side effects of lymph node surgery, but still offers equivalent results to axillary node dissection, when the sentinel node is affected. In case the sentinel lymph node (GLC) is positive, DGLA is completed

35 (DGLAc) routinely due to the possibility that non-sentinel nodes (GLnC) are affected by tumor. However, between 50 and 68% of patients with

GLC positive, it has been seen that GLC was the only lymph node affected by tumor cells (Domenech A, et aL, 2009; Hung WK, aL, 2005).

To avoid unnecessary DGLA, different tools have been developed to predict the probability of metastasis in GlnC in breast cancer patients with 5 GLC positive (Barranger E, aL, 2005; Degnim AC, aL, 2005; Hwang RF, et aL , 2003; Kohrt HE, al., 2008; Pal A, et al., 2008; Saidi RF, al., 2004; Van Zee KJ, al., 2003). These tools, called nomograms, are based on clinical parameters and histopathological characteristics of the primary tumor and its metastases, which correlate with the risk of developing metastasis in 105 GLnC. The most relevant parameters are

10 the size of the primary tumor and GLC metastasis. However, the majority of 105 nomograms have been created based on data from a single institution with a population of patients and specific protocols (for example, specialties in surgical procedures, intraoperative examination of GLCs, pathological analysis of GLCs, etc.).

15 The most validated predictive tool was created by van Zee et al., Of the Memorial Sloan Kettering Caneer Center (MSKCC) (Van Zee KJ, et al., 2003), so that, to avoid inter-institutional differences in the intraoperative examination of the GLC, they created two nomograms, one with and one without the information of the frozen sections. As a result, a nomogram can be used in institutions where

20 the frozen sections are made, and the other in those in which they are not made, which expands the applicability of its tool. Specifically, it is a multivariate logistic regression analysis of a long series of histopathological variables (tumor size, lymphovascular invasion in the primary tumor, size of the metastasis in the GLC, detection of micrometastases in the GLC). However, 105 nomograms

25 currently available are not yet reliable enough to allow therapeutic stratification during surgery to avoid extensive DGLA in patients with a low risk of axillary recurrence.

Sauer et al. Have used "multiplex sandwich immunoassays" to find molecular indicators of the status of GLnC, but this technique does not yet have clinical relevance (Sauer G, et al, 2011).

Therefore, it would be of great importance to identify 105 patients who do not have additional lymph nodes affected by metastases in order to avoid unnecessary DGLA and its consequent morbidity. The key question that needs to be answered is: Is there any possibility of avoiding axillary lymph node surgery in patients with breast cancer? To answer this question, we should be able to identify

potential biomarkers involved in metastatic progression through axillary lymph nodes in the breast caneer. One of the possible markers for this purpose may be human matrix metalloproteases (MMPs).

The family of human MMPs is currently composed of 28 members of

5 zinc-dependent homologous endopeptidases, which can be divided into 8 structural classes, or based on their specificity by the substrate and their primary structure, in the best known subgroups of collagenases (MMP-1, 8 and 13), gelatinases (MMP- 2 and 9), stromalisins (MMP-3, 10 and 11), membrane-associated MMPs (MMP-14, 15, 16, 17, 23, 24 and 25), and other new MMPs. MMPs are synthesized as inactive zymogens

10 which are then predominantly activated pericellularly, either by other MMPs or by serine proteases. The activity of MMPs can be specifically inhibited by tissue inhibitors of MMPs (TIMPs). Currently, 4 different TIMPs are known: TIMP-1, 2, 3 and 4. MMPs are responsible for tissue remodeling and degradation of the extracellular matrix, including collagen, elastin,

15 jellies, matrix glycoproteins and proteoglycans. In addition, these proteins are regulated by hormones, growth factors and cytokines and are produced by a variety of connective tissues and stromal cells, such as fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils and lymphocytes. MMPs are able to modify cell behavior in vivo as a result of their ability to

20 degrade growth factors, cell surface receptors, cell adhesion molecules, and cytokines or chemokines. For these reasons, MMPs can produce metastases through modulation of extracellular matrix remodeling. In addition, some of the authors of the invention have previously observed that the expression of several MMPs and TIMPs by fibroblasts or inflammatory cells

25 mononuclear tumors of the tumor stroma are closely associated with the appearance of distant metastases, by hematogenous route, in patients with breast cancer (Vizoso FJ, et al., 2007; González LO, et al., 2007; Del Casar JM, et al., 2009; González LO, et al., 2009; Gonzalez LO, et al., 2010).

In 2010, the Jove Hospital Foundation published a description of the objectives in the

30 that his research was based to identify biological factors associated with sentinel node and / or non-sentinel node involvement in breast cancer. This research was funded by the Carlos 111 Institute, of the then Ministry of Health, within the health techniques evaluation program. Among the main objectives of this research, the variations of the populations of different

35 lymphocytes (C04, C08 and C01 a) and other inflammatory cells, as well as their expression phenotypes of MMPs and TIMPs, in the sentinel node and their possible relationship with tumor involvement of the non-sentinel nodes (MEMORY FHJ 2010, Hospital Foundation from Jove, page 45).

Despite the different procedures described in the prior art, there is currently a need to identify a reliable method for predicting the

5 tumor involvement of the sentinel lymph nodes. This, in order to avoid unnecessary axillary lymph node dissection and thus avoid the adverse effects of the technique, in patients with a tumor with lymph node involvement, preferably in patients with breast cancer.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a new marker for the detection of positive sentinel node metastasis. Specifically, the present invention relates to the detection of MMP-1 expression in a specific cell type, to predict whether non-sentinel lymph nodes are affected by tumor. That is, it is necessary to identify the cell type where the expression of MMP-1 must be determined to predict whether

15 non-sentinel lymph nodes are affected by tumor. In a positive lymph node, the determination of the expression of MMP-1 and the identification of the cell type that shows said expression, makes it possible to predict the involvement of non-sentinel lymph nodes and therefore avoid complete axillary dissection.

Sentinel node biopsy is accepted as a standard method for patients.

20 with breast cancer and clinically negative nodes. Axillary complete dissection is performed when the sentinel node is affected by the tumor. However, in 50% to 68% of cases, the sentinel node has been shown to be the only node affected by tumor cells. Therefore, in these patients a complete axillary dissection could have been avoided, and thus avoid the associated comorbidities, such as

25 lymphedema, shoulder pain, paraesthesia and a prolonged recovery time. In addition, a second unnecessary surgical intervention could be avoided, when the diagnosis of sentinel node tumor involvement is not reached intraoperatively, as is currently the case in many cases.

The authors of the present invention have identified a new tumor biomarker for

30 Predict tumor involvement of the non-sentinel nodes, from the positive sentinel node in breast cancer. In cases of positive sentinel node, non-expression of that factor, by a specific cell type, predicts non-sentinel node involvement by tumor cells. This invention relates to the non-expression of matrix metalloprotease (MMP) -1, also called collagenase

interstitial or collagenase 1, by peritumoral inflammatory mononuclear cells, in the positive sentinel node.

The specificity and sensitivity of this prognostic / diagnostic test is 61.5% and 100%, respectively. However, the most important thing is that the negative predictive value of the invention is 100%. Which means that all cases with a negative expression of MMP-1 by peritumoral inflammatory mononuclear cells, in the positive sentinel node, have no tumor involvement of the other nodes. This implies that the present invention can prevent at least 60% of unnecessary complete axillary dissections.

Detailed description of the invention

The present invention relates to a new prognostic / diagnostic tumor biomarker, in the positive sentinel node, of the presence of non-sentinel node metastases. Likewise, the invention relates to the detection of MMP-1 expression by a specific cell type, to predict non-sentinel node involvement. In a positive sentinel node, the determination of the expression of MMP-1 and the identification of the cell type that expresses it, allows to predict the involvement of the non-sentinel nodes and thus avoid unnecessary complete axillary dissection. Specifically, if MMP-1 is not expressed by peritumoral inflammatory cells in the sentinel node, then the non-sentinel nodes are not affected by tumor cells.

Specifically, the present invention refers to a method for the in vitro prediction of tumor involvement (neoplastic or metastatic involvement) of non-sentinel lymph nodes (GLnC) in a patient with at least one tumor with lymph node involvement, characterized by comprising the Next steps:

a) from at least one sample already obtained from a positive sentinel lymph node, preferably a fixed sample, and more preferably, a sample fixed and included in paraffin; Y

b) determine the expression of MMP-1 in peritumoral inflammatory mononuclear cells (Mies) of said sample, by means of a cell type identification technique.

As used herein, the term "sample already obtained" refers to a sentinel node sample previously removed by routine surgical procedure for diagnostic use in the usual clinical protocol.

As used herein, the term "positive sentinel node" refers to a sentinel node with micrometastases (metastasis diameter between 0.2mm to 2mm) or macrometastasis (metastasis diameter greater than 2mm).

As used herein, the term "non-sentinel node involvement" refers to the presence of tumor cells in the non-sentinel lymph nodes.

As used herein, the term "determination of MMP-1 expression" refers to antibodies or any other system that can evaluate the expression of said protein.

In a preferred embodiment, the present invention is characterized in that the value

10 negative predictive of such method is 100%. That is, when MMP-1 expression is not detected in peritumoral inflammatory mononuclear cells in the patient's positive sentinel lymph node sample, it means that the non-sentinel lymph nodes will not be affected by the tumor, thus preventing complete dissection of axillary lymph nodes (DGLAc).

15 As used herein, the term "negative predictive value" refers to the percentage of cases that having the negative test turn out to have no disease (involvement of non-sentinel nodes by tumor cells).

In another preferred embodiment, the present invention is characterized in that the patient with at least one tumor with lymph node involvement suffers from a disease selected from the following group: breast cancer, mela noma, prostate cancer, stomach cancer, and cancer of colon Preferably, the patient suffers from breast cancer, and more preferably, the patient has at least one infiltrating ductal carcinoma (CDI).

As used herein, the term "infiltrating ductal carcinoma," also known as carcinoma of no special type (NST), refers to a carcinoma that

25 begins in the milk ducts of the breast and invades or infiltrates the surrounding tissue of the breast.

In another preferred embodiment, the present invention is characterized in that the technique for cell type identification defined in step b) is selected from the following group: immunohistochemistry, immunofluorescence, and flow cytometry.

Preferably, the technique for cell type identification defined in step b) is immunohistochemistry, and more preferably, immunohistochemistry comprises the following steps:

i) sample pretreatment,

ii) immunostaining;

Ii) contratinción; Y

iv) immunostaining analysis.

In another preferred embodiment, the present invention is characterized in that the inflammatory mononuclear cells defined in step b) are selected from the following group: monocytes, macrophages and lymphocytes.

As used herein, the term "inflammatory mononuclear cells" refers to immune system mononuclear cells, such as monocytes, macrophages and lymphocytes.

In another preferred embodiment, the present invention is characterized in that the inflammatory mononuclear cells defined in step b) are selected from the tumor periphery of the positive sentinel node and therefore are peritumoral inflammatory mononuclear cells.

As used herein, the term "peritumoral inflammatory mononuclear cells" refers to inflammatory mononuclear cells in contact or in the immediate vicinity of tumor foci, in positive sentinel nodes.

In another preferred embodiment, the present invention is characterized in that the sample already obtained from a positive sentinel lymph node defined in step a) is selected from the following group: fixed tissue sample, fresh tissue sample, and sample of

20 frozen tissue. Preferably, it is a fixed tissue sample.

In another preferred embodiment, the present invention is characterized in that the patient is a mammal. Preferably, the mammal is selected from the following group: human, primate and non-primate. More preferably, the patient is a human.

As used herein, the term "patient" preferably refers to

25 to a human. However, it can refer to a mammal, including noprimate mammals (eg, horse, dog, cat, rat, mouse, cow or pig) and primate mammals (eg, monkey).

The present invention also relates to an MMP-1 for use as a tumor biomarker in the method described above.

As used herein, the term "molecular marker / tumor biomarker" refers to a biological marker of cancer, that is, a substance (s) produced by the tumor or narrow stromal cells. relationship with their presence,

and that offers information of clinical interest on the status of non-sentinel lymph node involvement by tumor cells.

The present invention also relates to a kit for use in the method described above.

The embodiment of the invention can be carried out in fresh, fixed or frozen sentinel node tissue by flow cytometry, immunohistochemistry, immunofluorescence or any other technique that allows the identification of the cell type known to a person skilled in the art.

Throughout the description and the claims the word "comprises" and its variants

10 are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

15 DESCRIPTION OF THE FIGURES

FIGURE 1 Example of negative immunohistochemical staining of MMP-1 in peritumoral inflammatory mononuclear cells (arrows) in a positive sentinel node (200X).

FIGURE 2. Example of positive immunohistochemical staining of MMP-1 in cells

20 peritumoral inflammatory mononuclear (arrows) in a positive sentinel node (200X)

FIGURE 3. Example of negative immunohistochemical staining of MMP-1 in peritumoral inflammatory mononuclear cells (arrows) in a positive sentinel node (400X).

FIGURE 4. Example of positive immunohistochemical staining of MMP-1 in peritumoral inflammatory mononuclear cells (arrows) in a positive sentinel node (400X).

EXAMPLES

The following specific examples provided in this patent document

30 serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1: Identification of the possible relationship of MMPs or TIMPs with the

metastatic process in axillary lymph nodes.

1.1 Patients

In order to identify the possible relationship of MMPs or TIMPs with tumor involvement 5 (metastatic process) in axillary lymph nodes, two populations of patients with a tumor with lymph node involvement (breast cancer) were studied.

One of them consisted of a population of 50 women who underwent a classic axillary dissection (Group 1), and with the following inclusion criteria: nfiltrating duetal carcinoma, affected axillary lymph nodes and at least 6 axillary nodes

10 histologically evaluated.

The exclusion criteria were as follows: metastatic disease at diagnosis, history of any type of malignant tumor, bilateral breast cancer at diagnosis, having received any type of neoadjuvant treatment, and absence of sufficient sample in the paraffin blocks used for the realization of the meshes

15 of fabric. Regarding the number of axillary nodes affected, a total of 12 patients had only one affected node, and 38 patients had two or more axillary nodes with metastases.

Once the MMPs and TIMPs candidates for predicting lymph node involvement in Group 1 were identified, a population of patients undergoing surgery was subsequently investigated.

20 intraoperative evaluation of the sentinel node (Group 2).

The objective in this second group of patients was to explore the possible role of the expression of MMPs or TIMPs in the positive sentinel node to predict the involvement of the other axillary ganglia (not sentinels). To do this, the database of the "Hospital de Jove Foundation", Gijón (Spain) and the "University Hospital of Santiago de 25 Compostela" (Spain) were reviewed, including all patients undergoing a selective sentinel lymph node biopsy with cancer of infiltrating breast and clinically negative nodes, between January 2007 and January 2012. Patients with positive sentinel lymph node underwent a complete posterior axillary dissection were selected, according to the following inclusion criteria: infiltrating ductal carcinoma, lymph node metastasis

30 sentinel (diameter greater than 0.2mm), only one or two sentinel nodes, at least 6 axillary nodes evaluated histologically. The exclusion criteria were the following: history of any type of malignant tumor, breast cancer bilateral diagnosis, multifocality, having received any type of neoadjuvant treatment. A total of 105 patients who met these criteria were included in the study.

The patients signed an informed consent for the use of their tissue samples for this study. The study adhered to national regulations and was approved by the Research Commission of our institution and by the Regional Ethical Committee of Clinical Trials.

5 1.2 Sample handling and realization of tissue meshes

It is based on tissue samples already obtained (previously obtained, at the time of surgery, by ordinary procedure). Routinely fixed samples (overnight in 10% v / v formalin), included in paraffin and stored in Pathological Anatomy services, were used in this study. The histologically representative areas representative of the lymph node tumor involvement were defined in sections stained with hematoxylin / eosin and marked on the lamella itself. Tissue meshes, from patients in Group 1, were performed as described previously (Parker RL, et al., 2002). Each tissue mesh contains, a cylindrical sample of each patient, with a diameter of 1.5 mm taken through a histological zone.

15 representative of the tumor involvement of the sentinel node. One cylinder of each affected node was analyzed. The samples of the patients of Group 2 were evaluated on complete biopsy cutting, without performing tissue meshes.

1.3 lnmunohistochemistry

The 3-lJm sections of the paraffin blocks already obtained during the biopsies or from

20 the tissue meshes already obtained during the biopsies were consecutively cut with a microtome (Leica Microsystems GmbH, Wetzlar, Germany) and transferred to a lamella. The cuts were dewaxed in xylene and then rehydrated in gradual concentrations of ethyl alcohol (100%, 96%, 70%) and then with water. To perform antigen unmasking, only for some antibodies, the cuts

25 were treated in a PT-Link® (Dako) at 97 "C for 20 min, in citrate buffer pH 6.1 (MMP-1, -14 and TIMP-1. -3) 0 in Tris-EDTA pH 9 ( MMP-13, TIMP-2), and then the cuts were washed in a phosphate buffered saline solution (PBS) .The cuts were stained using a TechMate TM50 autostainer automatic immunotoner (Dako, Glostrup, Denmark). MMPs and TIMPs were obtained

30 from Neomarker (Lab Vision Corporation, Fremont, CA, USA). The dilution of each antibody was as follows: 1/50 for MMP-2, -7, -13, -14; 11100 for MMP-9, TIMP-1, -2 AND -3; 1/200 for MMP-1 and 1/400 for MMP-11. As negative controls, the process described above was used, replacing the primary antibody with the diluted mouse or rabbit DakoCytomation serum at that same concentration. The dilutions were

35 performed in Antibody Diluent (Dako) and incubated between 30 min and 2 hours at

room temperature. Endogenous peroxidase activity was blocked by incubating the slides in peroxidase blocking solution (Dako) for 5 minutes.

The uEnVision Detection Kie (Dako) kit was used as a detection system. The cuts

5 were counterstained with hematoxylin, dehydrated in elanol and mounted. As positive controls, cases were used in which the presence of the proteins evaluated by Western blot was confirmed as described in previous studies (González LO, et al., 2009; González LO, et al., 2007). A single band of the expected molecular weight was observed for each protein (González LO, et al, 2009).

10 1.4 Analysis of immunostaining

The overall expression of the factors (score or score value) was evaluated in the different cuts. In addition, the type of stained cells was determined for each preparation. For each staining studied, the location of immunoreactivity, the percentage of stained area and the intensity of it were determined. All cases were semiquantified for each area of staining. An image analysis system composed of an Olympus BX51 microscope and an analysis program (analySIS®, Soft imaging system, Münster, Germany) were used as follows: the cuts were stained with antibodies, according to the method explained above and a hematoxylin contratinction was performed. There are thus different optical thresholds for both stains. For tissue meshes of Group 1 patients, each cylinder was scanned with a 400X magnification objective in two fields per cylinder. The fields were selected within the tumor areas looking for the dyed areas. The computer program selects and draws a line around the area stained by the antibody (highest optical threshold: red spots), unstained areas (tissue stained 25 by hematoxylin with a lower optical threshold) are excluded and shown as a blue background. Any field has a relationship of dyed area (red) versus unstained area (blue). A final area ratio is obtained after averaging two fields. To assess the intensity of immunostaining, a numerical score of O to 3 was used, which reflects the intensity as follows: O, without reactivity; 1, weak reactivity; 2, reactivity

30 moderate; and 3, intense reactivity. The average score is obtained by multiplying the intensity score (1) by the percentage of stained area (PA), using an Excel spreadsheet. The final score is based on the overall evaluation of the sample that includes the staining areas of both tumor cells and stroma.

In Group 2 of patients, sentinel node samples were scanned in two 35 fields with an objective of 400X magnification. The fields were selected within

of the tumor areas looking for the dyed areas in each cut obtained during the biopsy.

In the present study, immunohistochemical tindon was also assessed separately for each major cell type: tumor cells, fibroblasts and inflammatory mononuclear cells (CMls). Stromal cells were distinguished from cancer cells because the latter are larger. In addition, fibroblasts are fusiform cells, while CMls are round cells. On the other hand, while cancer cells form acinar or trabecular patterns, stromal cells show a diffuse pattern.

When any of these cell types were positive for a given MMP or TIMP, at least 70% of the cells showed a positive immunostaining in each field evaluated in each case, which made the division between "positive" and "negative" reaction, simple.

1.5 Statistical analyzes

The differences between the percentages were calculated using the chi-square test. The comparison of immunostaining values between the groups was performed with the Mann-Whitney test or the Kruskall-Wallis test. The statistical results were corrected by applying the Bonferroni correction. A value of p <0.05 was considered significant. The PASW Statistics 18.0 program (SPSS Inc., Chicago, IL, USA) was used for all calculations.

1.6 Results

A. Group 1 patients

Table 1 shows the overall expression (score values according to the median

or score) of the different MMPs and TIMPs, as well as the different cell types, depending on the number of lymph nodes affected. Cases with more than one lymph node affected have a higher proportion of positive cases for MMP-1 and TIMP-1, in all cell types, than cases with only one affected lymph node. Similarly, cases with more than one lymph node affected have a higher proportion of MMP-13 expression by CMls, than cases with only one lymph node affected.

Table 1: Relationship between the former pressure of MMPs and TIMPs in ganglia

lymph nodes with metastases and the number of nodes affected in cancer

mom. 1 or> 1 lymph node affected by tumor cells.

Factor

1 + N ° patients > 1+ N ° patients P

MMP-l

Score <median vs> median

11/114/24 0.010

Tumor cell (-) vs (+)

8/4 3/35 0.0001

Fibroblast (-) vs (+)

11/1 12/26 0.003

WCC (-) vs (+)

12/0 8/30 0.0001

MMP-2

Score <median vs> median

6/6 20/18 1,000

Tumor cell (-) vs (+)

4/8 19/19 0.476

Fibroblast (-) vs (+)

12/0 30/8 0.160

WCC (-) vs (+)

10/2 33/5 0.862

MMP-7

Score <median vs> median

7/5 18/20 0754

Tumor cell (-) vs (+)

3/9 7/31 0.042

Fibroblast (-) vs (+)

11/1 24/14 0.052

WCC (-) vs (+)

11/1 24/14 0.052

MMP-9

Score <median vs> median

7/5 20/18 0.722

Tumor cell (-) vs (+)

2/10 5/33 0.973

Fibroblaslo (-) vs (+)

8/4 11/27 0.466

WCC (-) vs (+)

10/2 10/28 0.148

MMP-l1

Score <median vs> median

6/6 19/19 1,000

Tumor cell (-) vs (+)

2/10 3/35 0.629

Fibroblast (-) vs (+)

7/5 16/22 0.326

CM I (-) vs (+)

7/5 10/18 0.476

MMP-1 3

Score <median vs> median

4/8 19/19 0.933

Tumor cell (-) vs (+)

2/10 4/34 0.791

Fibroblast (-) vs (+)

8/4 19/19 0.088

WCC (-) vs (+)

8/4 14/24 0.028

MMP-14

Score <median vs> median

7/5 19/19 1,000

Tumor cell (-) vs (+)

4/8 5/33 0.204

Fibroblast (-) vs (+)

8/4 25/13 0.786

WCC (-) vs (+)

8/4 25/13 0.288

TIMP-l

Score <median vs> median

8/4 16/22 0.441

Tumor cell (-) vs (+)

3/9 2/36 0.010

Fibroblast (-) vs (+)

11/1 19/19 0.039

WCC (-) vs (+)

11/1 19/19 0.039

TIMP-2

Score <median vs> median

7/2 12/16 0,150

Tumor cell (-) vs (+)

3/9 2/36 0.010

Fibroblast (-) vs (+)

10/2 24/14 0.571

WCC (-) vs (+)

10/2 24/14 0.571

TIMP-3

Score <median vs> median

8/4 2/17 1 0.291

Tumor cell (-) vs (+)

4/8 7/31 0,327

Fibroblast (-) vs (+)

8/4 22/16 0.144

CM I (-) vs (+)

8/4 25/13 0.288

Abbreviation: MIC, inflammatory mononuclear cell.

B. Group 2 patients

The proteins most significantly associated with the metastatic process through the axillary lymphatic system in Group 1 of patients, were analyzed in the samples of 5 patients in Group 2, where there was less tissue availability due to its use for diagnosis.

Table 2 shows the overall expression (score or score values) of MMP-1, -7, -13 and TIMP-1, as well as the expression by the different cell types, in the positive sentinel node, and depending on the condition of non-sentinel node metastasis. The expression of MMP-1 was significantly associated with non-sentinel node involvement (p <0.0001). It was also found that MMP-1 expression by CMls in the positive sentinel node was significantly and strongly associated with non-sentinel node involvement (p <0.0001). The study of the predictive capacity of this last result has shown that the specificity and sensitivity of the test is

15 61.5% and 100%, respectively. In addition, the negative predictive value of the invention is 100%.

Table 2: Relationship between the expression of MMP-1, 7, -13, and TlMP-1, in the positive sentinel node, and non-sentinel node status in 10 5 patients with infiltrating ductal carcinoma of the breast.

Factors

Cases Ganglios no Sentinel negative N ('lo) Cases Ganglios no Sentinel positive N ('lo) P value

Total Cases

65 (100) 40 (100)

MMP-l

Score <median vs.> median

42 (64.6) / 23 (35.4) 11 (27.5) / 29 (72.5) 0.0001

Tumor cell (vs. +

19 (29.2) / 46 (70.8 7 17.5 / 33 82.5 0.263

Fibroblasts (-) vs. +

57 (87.7 / 8 12.3 36 90.0 / 4 (10.0 0.984

WCC (-) vs. (+)

40 (61 5) / 25 (38 5) O (O) / 40 (100) 00001

MMP-7

Score <median vs.> median

33 (50.8) / 31 (47.7) 19 (47.5) / 20 (50.0) 0.939

Tumor cell (vs. +

12 (185/52 (800 5 12.5 / 34 (85.0 0608

Fibroblasts (-) vs. +

27 (4 1.5 / 37 (56.9 21 52.5 / 18 45.0 0,344

WCC (-) vs. (+)

23 (35.4) / 41 (63.1) 19 (47.5) / 20 (50.0) 0.283

MMP-1 3

Score <median vs.> median

36 (55.4) / 28 (43.1) 16 (40.0) / 24 (60.0) 0.158

Tumor cell (-) vs. (+)

20 (30.8) / 44 (67.7) 13 (32.5) / 27 (67.5) 1,000

Fibroblasts (-) vs. (+)

39 (60.0) / 25 (38.5) 28 (70.0) / 12 (30.0) 0.466

WCC (-) vs. (+)

36 (55.4) / 28 (43.1) 21 (52.5) / 19 (47.5) 0.864

TlMP-1

Score <median vS.> median

33 (50.8) / 30 (46.2) 19 (47.5) / 21 (52.5) 0.779

Tumor cell (-) vs. (+)

24 (36.9) / 39 (60.0) 18 (45.0) / 22 (55.0) 0.625

Fibroblasts (-) vs. (+)

35 (58.3) / 28 (43.1) 22 (55.0) / 18 (45.0) 1,000

WCC (-) vs. (+)

50 (76.9) / 13 (20.0) 27 (67.5) / 13 (32.5) 0.263

Abbreviations: MIC, inflammatory mononuclear cell.

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Claims (14)

1. Method for in vitro prediction of non-sentinel lymph node tumor involvement of a patient with at least one tumor with lymph node involvement, characterized by comprising the following steps: a) from at least one sample already obtained from a positive sentinel lymph node; Y b) determine the absence of MMP-1 expression in the inflammatory mononuclear cells of said sample, associated with the non-involvement of the non-sentinel nodes, by means of a cell type identification technique.

2.

Method according to claim 1, characterized in that the patient with at least one tumor with lymph node involvement suffers from a disease selected from the following group: breast cancer, melanoma, prostate cancer, stomach cancer, and colon cancer.

3.

Method according to claim 2, characterized in that the patient suffers from breast cancer.

Four.

Method according to claim 3, characterized in that the patient has at least one infiltrating ductal carcinoma.

5.

Method according to claims 1-4, characterized in that the technique for the identification of the cell type defined in step b) is selected from the following group: immunohistochemistry, immunofluorescence, and flow cytometry.

6.

Method according to claim 5, characterized in that the technique for the identification of cellular type defined in step b) is immunohistochemistry.

7.

Method according to claim 6, characterized in that the immunohistochemistry

It comprises the following steps: i) sample pretreatment, ii) immunostaining; iii) contratinción; and iv) immunostaining analysis.

8.

Method according to claims 1-7, characterized in that the inflammatory mononuclear cells defined in step b) are selected from the following group: monocytes, macrophages and lymphocytes.

9.

Method according to claims 1-8, characterized in that the inflammatory mononuclear cells defined in step b) are selected from the tumor periphery of the positive sentinel node and therefore are peritumoral inflammatory mononuclear cells.

10.

Method according to claims 1-9, characterized in that the sample already obtained from a positive sentinel lymph node defined in step a) is selected from the following group: fixed tissue sample, fresh tissue sample, and frozen tissue sample .

eleven . Method according to claim 10, characterized in that it is a fixed tissue sample.

12.

Method according to claims 1-11, characterized in that the patient is a mammal.

13.

Method according to claim 12, characterized in that the patient is a human.

14.

Use of MMP-1 as a tumor biomarker for in vitro prediction of non-sentinel lymph node tumor involvement of a cancer patient in the method described in claims 1-13.