WO2015124607A1 - Method to predict risk of recurrence in endometrial carcinoma - Google Patents

Method to predict risk of recurrence in endometrial carcinoma Download PDF

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WO2015124607A1
WO2015124607A1 PCT/EP2015/053387 EP2015053387W WO2015124607A1 WO 2015124607 A1 WO2015124607 A1 WO 2015124607A1 EP 2015053387 W EP2015053387 W EP 2015053387W WO 2015124607 A1 WO2015124607 A1 WO 2015124607A1
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Prior art keywords
annexin
level
expression
recurrence
endometrial carcinoma
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PCT/EP2015/053387
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French (fr)
Inventor
Francisco Javier MATIAS-GUIU GUIA
Francisco Javier DOLCET ROCA
Jaume REVENTÓS PUIGJANER
Eva COLÁS ORTEGA
Jaime PRAT DÍAZ DE LOSADA
José PALACIOS CALVO
Miguel Abal Posada
Rafael LÓPEZ LÓPEZ
Gema Moreno Bueno
Antonio Gil Moreno
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Institut De Recerca Biomèdica De Lleida Fundació Doctor Pifarré
Universitat De Lleida
Fundació Hospital Universitari Vall D'hebron - Institut De Recerca
Fundació Institut De Recerca De L'hospital De La Santa Creu I Sant Pau
Servicio Andaluz De Salud
Servizo Galego De Saúde
Fundación Md Anderson International España
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Publication of WO2015124607A1 publication Critical patent/WO2015124607A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present invention provides an in vitro method for predicting risk to develop recurrence in a patient suffering from endometrial carcinoma.
  • the method allows to readily detect patients with a high risk to develop recurrence independently of the FIGO stage and/or histology grade.
  • the method has potential applications in optimizing the clinical management of endometrial carcinoma, with a special interest in the early stages of disease.
  • Endometrial carcinoma is the most common gynaecologic malignancy in the first world. There are several types of classifications to stratify EC.
  • endometrioid endometrial carcinoma EEC or type I
  • NEEC or type II non- endometrioid endometrial carcinomas
  • UPSC uterine papillary serous carcinoma
  • CCC clear cell carcinomas
  • a second classification criterion is based on histologic grade, which is the degree of similarity of the tumour tissue in comparison with normal tissue (the higher the area with loss of glandular morphology, the higher the grade).
  • Grades I, II and III are assigned if the histology sample has an area of 5% or less, 5-50%, and more than 50% of non-glandular morphology, respectively.
  • a third classification is the so-called FIGO (International Federation of Gynaecology and Obstetrics) stage, which is derived from a pathologic assessment of a hysterectomy specimen, and which classifies EC into stage 0 (the cancer cells are found only in the surface layer of the endometrium), stage I (the cancer is found in the body of the uterus), stage II (the cancer has spread from the uterus to the supportive connective tissue) and stage III (the cancer has spread outside the uterus).
  • FIGO International Federation of Gynaecology and Obstetrics
  • biomarker discovery is one of the most rapidly evolving fields of cancer research. Biomarkers are being discovered to determine risk to develop disease as well as to assist in the diagnosis, prognosis and response to treatment. This field is now being propelled by the recent advances in the "- omics" technologies. With the aim of identifying candidate proteins that could potentially be used as biomarkers, a series of proteomics techniques are currently applied. All of them rely on the comparison of protein profiles between two states (normal vs. disease, untreated vs. treated, etc.).
  • proteomics-derived hypotheses are often misleading and many of the markers discovered do not turn out to be robust, validated and applicable tools (Meehan KL. et.al. "Proteomics and the search for
  • tumour tissue samples are a heterogeneous mixture of multiple cell types (both malignant and normal), all of which contribute to the proteomic profile derived from the 2D gels that proteomics studies are based on.
  • further validation by other techniques is always necessary in order to validate identified markers.
  • IHC immunohistochemistry
  • Endometrial Carcinoma there are currently no tissue biomarkers to be used in the prediction of recurrence for EC patients (Zaino R. et.al, "Epithelial tumours and Precursor Lesions” 2014, Chapter 5 "Uterine Corpus", World Health Organization). Consequently, there is a significant unmet need in the clinics for tools to assess risk of recurrence for EC patients and specifically for the early stage EC patient subset (as recognized by Lu K.H., in the publication "Management of early-stage endometrial cancer” Seminars in Oncology 2009, vol. 36, pp. 137-144).
  • aneuploidy i.e. an abnormal number of chromosomes in a cell
  • aneuploidy is nowadays not regarded as a crucial feature in the assessment of risk of recurrence.
  • aneuploidy is nowadays not regarded as a crucial feature in the assessment of risk of recurrence.
  • many EEC patients can still show recurrence even in the absence of aneuploidy, and conversely some aneuploid tumors turn out to have low risk of recurrence (Pradhan M., et.al.
  • Annexin A2 (ANXA2) levels in EC patients can reliably be used to assess risk to develop recurrence
  • Annexin A2 when is used as a biomarker, in the evaluation of developing recurrence even in the early FIGO stage I EEC patient subpopulation.
  • the invention is especially relevant because many of the markers that are highlighted in prospective "omics" studies of cancer in general (and of endometrial cancer in particular) are later on found not to be suitable for applied clinical applications.
  • inventors have discovered in the past that a wide variety of cancer-related gene products identified by "omics” approaches do not perform as reliable markers in the assessment of risk to develop recurrence when immunohistochemistry techniques are used. As it has been cited before (Matias-Guiu X.
  • the invention fills a long felt need in the art for markers that can be incorporated in routine clinical practice in the proper diagnosis of EC, and especially EEC.
  • the invention relies in the detection of this marker in tumor tissue and in easy-to-access body fluids (i.e. exosomes, soluble and pellet fraction of uterine aspirates, and circulating tumour cells).
  • the detection of ANXA2 in these samples has many advantages, namely: i) it is applicable both in fresh and fixed samples; and ii) its determination can be combined with the determination of other markers in an effort to find patterns to aid in the prognosis.
  • a first aspect of the invention is an in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma, the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient.
  • the invention provides a new diagnostic tool that can be used without resorting to any further classification methods available (and in use in the clinics) such as the determination of the ploidy status of the tumor, the histological grade and others.
  • the application of this method will allow to take the proper measures with the different subpopulation of patients that, independently of their clinical prognosis, are found to be at high risk of relapse; the latter can be monitored more strictly so that relapses can be detected as early as possible.
  • EC FIGO Stage la patients are usually not given radiotherapy, because for most of them the risk/benefit ratio does not advice to do so.
  • EC FIGO Stage la patients could be divided into two groups, those with high risk of relapse and those with low risk. The former could be given radiotherapy in order to diminish the potential recurrence of disease.
  • a second aspect of the present invention is the use of Annexin A2 as an in vitro marker for predicting the risk to develop recurrence in a patient diagnosed with endometrial carcinoma.
  • a third aspect aspect of the present invention is the use of means for determining the level of expression of Annexin A2 in an isolated tumor tissue sample in the method of the first aspect of the invention.
  • FIG. 1 Representative examples of ANXA2 staining in endometrioid carcinomas. ANXA2-negative endometrioid carcinomas samples that did not recur (right) and ANXA2-positive endometrioid carcinomas samples that did recur (left).
  • FIG. 2. Retrospective study demonstrating an increased probability of endometrioid carcinoma recurrence with ANXA2 expression. A multicentric study including 121 patients was conducted by analyzing histoscore ANXA2 expression and the probability of a primary endometrioid carcinoma to recur.
  • FIG. 3 Expression of ANXA2 in uterine aspirates of EC patients in both supernatant and pellet fractions.
  • the level of expression of this marker in recurrent cases is increased when compared to non recurrent cases for both supernatant and pellet fractions (R means Recurrence, NR means No- Recurrence, IA means endometrioid cancer diagnosed in FIGO stage IA, IB means endometrioid cancer diagnosed in FIGO stage IB).
  • FIG. 4 Expression of ANXA2 in circulating tumour cells (CTCs).
  • CTCs circulating tumour cells
  • the level of expression of ANXA2 is increased in patients with recurrent disease (R) when compared to healthy controls (C), and patients with non-recurrent EC (NR).
  • recurrence in a patient diagnosed with endometrial carcinoma refers to a situation where a patient who has been diagnosed of endometrial carcinoma and has received chemical and/or surgical treatment later on suffers from a relapse.
  • the term "ploidy” as used herein refers to the number of sets of chromosomes in the nucleus of a cell. In humans the cells in all non-reproductive tissues are of the diploid type, meaning that they have two sets of chromosomes (2n).
  • the term "aneuploidy” is an abnormal number of chromosomes in a cell. It can be the result of a defective segregation of chromosomes during mitosis, and usually leads to multiple genetic disfunctions, some of them potentially associated to cancer.
  • isolated tumor tissue sample as used herein can refer to both a histological cut of the tumor tissue as well as a cell or cells derived from such tumor by any extraction means (such as uterine aspirates or any other procedures). In particular, it also includes exosomes, secreted proteins, cell fragments, genetic material, and vesicles derived from cells of the tumor tissue, as well as circulating tumour cells.
  • risk to develop recurrence refers to the risk to develop a further cancer once the previous cancer has been treated and/or removed from the body. This recurrence can be either local, wherein the cancer appears after treatment in a tissue adjacent to the removed tissue, or remote, wherein the cancer appears in a remote tissue via lymphatic or haematic trasport.
  • level of expression of Annexin A2 refers to the quantity of Annexin A2 mRNA and/or Annexin A2 protein produced by the Annexin A2 gene present in the cells of the isolated tumor tissue sample of the patient.
  • immunochemistry refers to a variety of techniques for detecting antigens (usually proteins and peptides, and in the present case Annexin A2) in a sample by exploiting the principle of antibodies binding specifically to said antigens.
  • antigens usually proteins and peptides, and in the present case Annexin A2
  • immunocytochemistry and
  • immunohistochemistry as used herein are basically interchangeable, and refer to the application of immunochemistry techniques in the context of a cell or a group of cells present in a histology sample. Visualising an antibody- antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine.
  • the immunohistochemistry technique can be direct or indirect. The direct method is a one-step staining method and involves a labeled antibody (e.g.
  • FITC-conjugated antiserum reacting directly with the antigen. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, such as with indirect methods, and is less commonly used than indirect methods.
  • the indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the sample and a labeled secondary antibody (second layer) that reacts with the primary antibody. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.
  • the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or Neutravidin-enzyme.
  • the indirect method aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be
  • Annexin A2 RNA could be performed by using a tagged nucleic acid designed to bind a specific sequence of Annexin A2 RNA, and then detecting said tagged nucleic acid with a labelled antibody which selectively binds to the tag.
  • An example of a putative antibody that could be used in the method of the invention is Abeam ab1803 (other examples of antibodies that could be used are those found in
  • FIGO stage refers to the International Federation of Gynaecology and Obstetrics classification of endometrial carcinoma (EC).
  • the current classification has the following stages:
  • Stage la No or less than half myometrial invasion
  • Stage lb Invasion equal to or more than half the
  • Tumour invades the serosa of the body of the uterus and adnexae.
  • Stage 111 Pelvic or para-aortic lymphadenopathy.
  • Stage IV Involvement of rectum and or bladder mucosa and or distant metastasis.
  • reference expression level referred to in the methods of the invention is to be understood as a predefined value of a given molecular marker, Annexin A2 in the present case, which is derived from the values of said molecular marker in a tumour sample or group of samples. If the level of expression of Annexin A2 is determined at the protein level, then the
  • reference expression level is a predefined value of Annexin A2 protein quantity, whereas if the level of expression of Annexin A2 is determined at the mRNA level, then the “reference expression level” is a predefined value of Annexin A2 mRNA quantity.
  • the samples are taken from a subject or group of subjects wherein the presence, absence, stage, or course of the disease has previously been determined. The subject or subjects from whom the
  • reference expression level is derived may include subject(s) wherein the condition is absent, subject(s) wherein the condition is present, or both.
  • the person skilled in the art, making use of the general knowledge, is able to choose the subject or group of subjects more adequate for obtaining the reference expression levels for each of the methods of the present invention.
  • Methods for obtaining the reference expression level from the group of subjects selected are well-known in the state of the art (Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values”).
  • the "reference expression level” for Annexin A2 is a cut-off value defined by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). As the skilled person will appreciate, optimal cut-off values will be defined according to the particular application of the method: target population for the prediction, balance between specificity and sensitivity of the immunochemistry technique, and others.
  • antibody or a fragment thereof able to bind to Annexin A2 is to be understood as any immunoglobulin or fragment thereof able to selectively bind the Annexin A2. It includes monoclonal and polyclonal antibodies.
  • fragment thereof encompasses any part of an antibody having the size and conformation suitable to bind an epitope of Annexin A2. Suitable fragments include F(ab), F(ab') and Fv.
  • An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies).
  • Human Annexin A2 (also known as ANXA2, ANX2, ANX2L4, CAL1 H and LPC2D) is found under the code P07355 in the UniProt database (last modified October 16, 2013). It belongs to the Annexin familiy of proteins, and is involved in cell motility and cell-matrix interactions.
  • a first aspect of the present invention is an in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma, the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient.
  • the determination of the level of expression of Annexin A2 in the isolated tissue sample of the patient is carried out by contacting the test sample with a reagent that binds Annexin A2.
  • the in vitro method comprises the steps of: a) determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient; and b) comparing the level of expression of Annexin A2 determined in step a) with a reference expression level, wherein if the level of expression determined in step (a) is higher than the reference expression level, it is indicative that the patient is in risk to develop recurrence, or alternatively, if the level of expression determined in step (a) is lower than the reference expression level, it is indicative that the patient is not in risk to develop recurrence.
  • the in vitro method according to the first aspect of the invention comprising the steps of:
  • step (c) comparing the level of expression of Annexin A2 determined in step a) with the reference expression level of step b). wherein if the level of expression determined in step (a) is higher than the reference expression level of step (b), it is indicative that the patient is in risk to develop recurrence, or alternatively, if the level of expression determined in step (a) is lower than the reference expression level of step (b), it is indicative that the patient is not in risk to develop recurrence.
  • the in vitro method according to the first aspect of the invention, wherein the isolated tumor tissue sample of the patient is selected from the group consisting of a histological cut of the tumor tissue, circulating tumor cells, exosomes, secreted proteins, cell fragments, genetic material and vesicles derived from cells of the tumor tissue, or combinations thereof.
  • the endometrial carcinoma is of the endometrioid type.
  • the endometrial carcinoma is a FIGO stage I endometrial carcinoma.
  • the endometrial carcinoma is a FIGO stage la endometrial carcinoma.
  • the level of expression of Annexin A2 is determined at the protein level.
  • the level of expression of Annexin A2 is determined at the mRNA level. In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 determined at the mRNA level is based on a nucleic acid hybridization technique.
  • the level of expression of Annexin A2 protein is determined by immunohistochemistry.
  • the level of expression of Annexin A2 protein is determined using an antibody or a fragment thereof able to bind to Annexin A2.
  • the antibody or fragment thereof forms part of a kit.
  • a third aspect of the present invention is the use of means for determining the level of expression of Annexin A2 in an isolated tumour tissue sample in the method of the first aspect of the invention.
  • the means to carry out the invention comprise an antibody or a fragment thereof able to bind to Annexin A2.
  • the antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies are made by injecting animals with the peptide antigen and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mixture of antibodies that recognize several epitopes.
  • Monoclonal antibodies show specificity for a single epitope and are therefore considered more specific to the target antigen than polyclonal antibodies.
  • the means to carry out the invention form part of a kit.
  • the antibody or fragment thereof for detecting Annexin A2 can be included in a kit.
  • the kit may additionally comprise means (additives, solvents) to visualize the antigen-antibody interactions.
  • Endometrial cancer samples were collected from patients who underwent surgery for endometrial carcinoma at Vail d'Hebron University Hospital (Barcelona, Spain), University Hospital of Santiago de Compostela (Santiago de Compostela, Spain), Arnau de Vilanova Hospital (Lleida, Spain), MD-
  • Table 1 Clinicopathology characteristics of the patients included in the 2DIGE proteomic analysis.
  • Tissue microarrays were constructed from paraffin-embedded endometrial cancer tissue blocks, including a first series of endometrial carcinomas of 1 15 primary lesions and 25 post radiation recurrences, and a second cohort of 121 patients including 59 primary carcinomas that did not relapse and 62 primary carcinomas that progressed to recurrent disease.
  • uterine aspirates were collected by aspiration of the endometrial fluid with a Cornier Pipelle®. Then, the fluid was mixed in 1 :1 ration with PBS1 x and centrifuged in order to separate the supernatant, which
  • ANXA2 was evaluated by ELISA in both fractions obtained from a uterine aspirate, supernatant and pellet, following manufacturer's
  • CTC circulating tumour cells
  • ANXA2 expression was determined by RT-q-PCR upon EpCAM based immunoisolation of CTC, using the CELLectionTM Epithelial Enrich kit (Invitrogen, Dynal, Oslo, Norway) as described (Alonso-Alconada et al., "Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer” Mol. Cancer 2014, 13:223).
  • Total RNA from CTC was extracted with the QIAmp viral RNA mini kit (Qiagen, Valencia, CA) and cDNA was synthesized using SuperScriptlll (Invitrogen, Carlsbad, CA) following manufacturer's protocol. To further optimize the sensibility of detection, a preamplification step was performed using TaqMan PreAmp Master Mix kit (Applied Biosystems, Foster City, CA) with 14 reaction cycles.
  • ANXA2 expression level was determined by RT-q-PCR with TaqManH
  • ANXA2 expression was assessed in the group of endometrioid endometrial carcinomas (EECs). ANXA2 expression was found to be a 24.76% superior in those Type I endometrioid endometrial carcinomas that ended up in recurrent disease compared to those that did not recur (p ⁇ 0.0001 ; mean 217.87 versus 174.62 histoscore, respectively), evidencing the potential of ANXA2 as biomarker in intermediate-risk endometrial carcinomas. Further, ANXA2 expression was evaluated in stages I and II, Type I endometrioid endometrial carcinomas regarding development of recurrence. This was important since stages I and II of Type I endometrioid carcinomas represent the subset of endometrial carcinomas that are less prone to develop recurrences. From a total of 70 stage I tumors (39
  • stage I and stage II endometrioid carcinomas 86 cases; 38 associated with recurrence and 48 unassociated with recurrence
  • the tumors that recurred had 25.22% higher ANXA2 levels in comparison with those that did not recur.
  • Annexin A2 could actually be used to discriminate the endometrioid carcinomas with high likelihood of recurrence from those with low likelihood of recurrence.
  • Uterine aspirates are in direct contact with the endometrium and its collection is performed in routinely basis to patients suspected to suffer of endometrial cancer. It has been reported that there is a very high correlation at RNA and DNA level between endometrial tissues and uterine aspirates, making this biofluid an interesting source of biomarkers for the screening of gynecological diseases such as endometrial cancer.
  • the identification of ANXA2 as marker of recurrence in uterine aspirates would represent an improvement in the process of treatment decision-making.
  • CTC Circulating Tumor Cells
  • Meehan KL. et.al. Proteomics and the search for biomarkers of female reproductive diseases" Reproduction 2010, vol. 140, pp. 505-519 Wulfkuhule J.D., et.al. "Proteomic applications for the early detection of cancer” Nat. Reviews Cancer 2003, vol. 3, pp. 267-275.

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Abstract

There is provided an in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient. Additionally, the use of Annexin A2 as an in vitro marker for predicting the risk to develop recurrence in a patient diagnosed with endometrial carcinoma and the use of means for determining the amount of Annexin A2 in said method are also provided.

Description

Method to predict risk of recurrence in endometrial carcinoma
The present invention provides an in vitro method for predicting risk to develop recurrence in a patient suffering from endometrial carcinoma. In particular, the method allows to readily detect patients with a high risk to develop recurrence independently of the FIGO stage and/or histology grade. The method has potential applications in optimizing the clinical management of endometrial carcinoma, with a special interest in the early stages of disease.
BACKGROUND ART
Endometrial carcinoma (EC) is the most common gynaecologic malignancy in the first world. There are several types of classifications to stratify EC.
Depending on the histological type, it can be classified into endometrioid endometrial carcinoma (EEC or type I), which comprises approximately 85% of all cases and is usually associated to good prognosis, and non- endometrioid endometrial carcinomas (NEEC or type II - being either uterine papillary serous carcinoma [UPSC], clear cell carcinomas [CCC] or other minor subtypes) which account for 15% of cases and are usually associated with bad prognosis. A second classification criterion is based on histologic grade, which is the degree of similarity of the tumour tissue in comparison with normal tissue (the higher the area with loss of glandular morphology, the higher the grade). Grades I, II and III are assigned if the histology sample has an area of 5% or less, 5-50%, and more than 50% of non-glandular morphology, respectively. Yet, a third classification is the so-called FIGO (International Federation of Gynaecology and Obstetrics) stage, which is derived from a pathologic assessment of a hysterectomy specimen, and which classifies EC into stage 0 (the cancer cells are found only in the surface layer of the endometrium), stage I (the cancer is found in the body of the uterus), stage II (the cancer has spread from the uterus to the supportive connective tissue) and stage III (the cancer has spread outside the uterus). In order to properly stratify an endometrial carcinoma, the physician must determine type, grade and stage in an attempt to give an accurate prognosis that guarantees the best treatment. Nowadays, biomarker discovery is one of the most rapidly evolving fields of cancer research. Biomarkers are being discovered to determine risk to develop disease as well as to assist in the diagnosis, prognosis and response to treatment. This field is now being propelled by the recent advances in the "- omics" technologies. With the aim of identifying candidate proteins that could potentially be used as biomarkers, a series of proteomics techniques are currently applied. All of them rely on the comparison of protein profiles between two states (normal vs. disease, untreated vs. treated, etc.). Although it was anticipated that proteomic technologies would easily lead to the unveiling of a whole new range of cancer biomarkers, it is now widely recognized that proteomics-derived hypotheses are often misleading and many of the markers discovered do not turn out to be robust, validated and applicable tools (Meehan KL. et.al. "Proteomics and the search for
biomarkers of female reproductive diseases" Reproduction 2010, vol. 140, pp. 505-519). In particular, it has been known that tumour tissue samples are a heterogeneous mixture of multiple cell types (both malignant and normal), all of which contribute to the proteomic profile derived from the 2D gels that proteomics studies are based on. This represents a significant hindrance for the search of biomarkers, especially in early stage cancers because the lesions are often very small and contamination from the surrounding stromal tissue confounds the detection of tumour-specific markers (Wulfkuhule J.D., et.al. "Proteomic applications for the early detection of cancer" Nat. Reviews Cancer 2003, vol. 3, pp. 267-275). Hence, further validation by other techniques is always necessary in order to validate identified markers. In relation to this, more conventional and reliable techniques in the prognosis of
EC are used in the clinical setting, such as those based on histopathology. Among these techniques, immunohistochemistry (IHC) plays an essential role in tumor identification and classification due mainly to its high specificity and convenience, which renders it one of the core techniques exploited for a reliable determination in all pathology departments.
Even though most patients with early stage disease (type I, EEC) can be cured by surgery alone, there is a subset of 15-20% of EEC patients that suffer from recurrence which can be manifested locally, distantly or both, and is usually associated with bad prognosis. This EEC patient subset cannot be easily pointed out with current tissue and clinical biomarkers. A similar scenario occurs with other stages and histologies of EC regarding the utility of current prognostic factors. Current prognostic factors are mostly based on clinical findings; being histological type, histological grade, ploidy, depth of myometrial infiltration, lymphovascular space invasion, presence of atypical endometrial hyperplasia, cervical involvement and hormone receptor status the most important factors. Those factors allow the classification of patients into low, medium or high risk of recurrence. Despite the general consensus among clinicians to apply this classification for treatment decisionmaking, a percentage of low risk patients develop recurrences, and
conversely a percentage of high risk patients do not develop recurrences.
Hence, identification of new independent biomarkers which can predict risk of recurrence independently of the most common prognostic factors is badly needed. A number of disclosures have probed the relationship between some oncogenes and tumor suppressor genes (such as p53, PTEN, pi3K, β-catenin and others) and risk of recurrence for EC patients (see for instance Gadduci A., et.al., "Tissue and serum biomarkers as prognostic variables in
endometioid-type endometrial cancer" Critical Rev, in Oncology 201 1 , vol 80, pp. 181 -192, and references therein). However, the usability of such molecules as IHC biomarkers for the assessment of risk to develop
recurrence is questionable at best. (Matias-Guiu X. et. al. "Prognostic biomarkers in endometrial and ovarian carcinoma" Virchows Archiv. in press). As is seen in the World Health Organization (WHO) classification for
Endometrial Carcinoma, there are currently no tissue biomarkers to be used in the prediction of recurrence for EC patients (Zaino R. et.al, "Epithelial tumours and Precursor Lesions" 2014, Chapter 5 "Uterine Corpus", World Health Organization). Consequently, there is a significant unmet need in the clinics for tools to assess risk of recurrence for EC patients and specifically for the early stage EC patient subset (as recognized by Lu K.H., in the publication "Management of early-stage endometrial cancer" Seminars in Oncology 2009, vol. 36, pp. 137-144).
Recent disclosures (such as Gemoll et.al. "Protein profiling of genomic instability in endometrial cancer" Cell. Mol. Life Sci. 2012, vol. 69, pp. 325- 333) have attempted to link aneuploidy-related risk of recurrence in endometrial cancer with a number biomarkers in a small series of patients. However, in addition to being based on non-conclusive proteomics
techniques, they rest upon the questionable hypothesis that aneuploidy, i.e. an abnormal number of chromosomes in a cell, is synonymous to recurrence. In spite of being used in the past as a risk assessment variable, aneuploidy is nowadays not regarded as a crucial feature in the assessment of risk of recurrence. As it has been noted in ample statistical studies, many EEC patients can still show recurrence even in the absence of aneuploidy, and conversely some aneuploid tumors turn out to have low risk of recurrence (Pradhan M., et.al. "Prognostic importance of DNA ploidy and DNA index in stage I and II endometrioid adenocarcinoma of the endometrium" Annals of Oncology 201 1 , vol. 23, pp. 1 178-1 184). In view of the above, there is a need in the art to discover biomarkers which can reliably be used in pathology tests for the assessment of risk to develop recurrence in EC patients, especially those at the early stages of disease.
SUMMARY OF THE INVENTION
Inventors have remarkably found that Annexin A2 (ANXA2) levels in EC patients can reliably be used to assess risk to develop recurrence
independently of the FIGO stage and/or histology in which the patient was diagnosed. Especially interesting is the role of Annexin A2, when is used as a biomarker, in the evaluation of developing recurrence even in the early FIGO stage I EEC patient subpopulation.
The invention is especially relevant because many of the markers that are highlighted in prospective "omics" studies of cancer in general (and of endometrial cancer in particular) are later on found not to be suitable for applied clinical applications. In this respect, inventors have discovered in the past that a wide variety of cancer-related gene products identified by "omics" approaches do not perform as reliable markers in the assessment of risk to develop recurrence when immunohistochemistry techniques are used. As it has been cited before (Matias-Guiu X. et.al., ibid), in this regard, inventors have formerly evaluated a number of gene products related to cell signalling pathways (such as FGFR2, PTEN, beta-catenin, SPRY, RASF1A, estrogen receptors), myometrial invasion (RUNX, ETV5), DNA mismatch repair (MLH- 1 , MSH-2, MSH-6, PMS-2), apoptosis (NfkB, FLIP, BAX, Bcl-xL), cell cycle (cyclins) and others. Although many of them could have been considered as promising biomarker candidates to predict risk of relapse, none of them were found to be of value in immunochemistry tests for a proper assessment of risk of relapse in endometrial carcinoma. Therefore, the invention fills a long felt need in the art for markers that can be incorporated in routine clinical practice in the proper diagnosis of EC, and especially EEC. Of note, the invention relies in the detection of this marker in tumor tissue and in easy-to-access body fluids (i.e. exosomes, soluble and pellet fraction of uterine aspirates, and circulating tumour cells). The detection of ANXA2 in these samples has many advantages, namely: i) it is applicable both in fresh and fixed samples; and ii) its determination can be combined with the determination of other markers in an effort to find patterns to aid in the prognosis. Specific advantages of tumor tissue are: iii) it is a direct measure of the expression of this marker in the tumour, and not an indirect measure subject to many variable factors such as those taken from plasma or blood, and iv) not only the quantity but also the morphology of expression within the tumour tissue can be assessed which aids in the prognosis. Specific advantages of easy-to-access body fluids are: v) ANXA2 could be detected prior to treatment and therefore taken into account in treatment decisionmaking. Thus, a first aspect of the invention is an in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma, the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient. Advantageously, the invention provides a new diagnostic tool that can be used without resorting to any further classification methods available (and in use in the clinics) such as the determination of the ploidy status of the tumor, the histological grade and others. The application of this method will allow to take the proper measures with the different subpopulation of patients that, independently of their clinical prognosis, are found to be at high risk of relapse; the latter can be monitored more strictly so that relapses can be detected as early as possible.
Specifically, it is known that EC FIGO Stage la patients are usually not given radiotherapy, because for most of them the risk/benefit ratio does not advice to do so. With the application of the method of the invention, EC FIGO Stage la patients could be divided into two groups, those with high risk of relapse and those with low risk. The former could be given radiotherapy in order to diminish the potential recurrence of disease. A second aspect of the present invention is the use of Annexin A2 as an in vitro marker for predicting the risk to develop recurrence in a patient diagnosed with endometrial carcinoma.
A third aspect aspect of the present invention is the use of means for determining the level of expression of Annexin A2 in an isolated tumor tissue sample in the method of the first aspect of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 . Representative examples of ANXA2 staining in endometrioid carcinomas. ANXA2-negative endometrioid carcinomas samples that did not recur (right) and ANXA2-positive endometrioid carcinomas samples that did recur (left). FIG. 2. Retrospective study demonstrating an increased probability of endometrioid carcinoma recurrence with ANXA2 expression. A multicentric study including 121 patients was conducted by analyzing histoscore ANXA2 expression and the probability of a primary endometrioid carcinoma to recur. Mean+sd ANXA2 immunoexpression levels (1 ) in stage I EC samples depending on recurrence (R), (2) in cases with no recurrences (NR) comparing stages I and II and (3) in cases with recurrences comparing stages I and II.
FIG. 3. Expression of ANXA2 in uterine aspirates of EC patients in both supernatant and pellet fractions. The level of expression of this marker in recurrent cases is increased when compared to non recurrent cases for both supernatant and pellet fractions (R means Recurrence, NR means No- Recurrence, IA means endometrioid cancer diagnosed in FIGO stage IA, IB means endometrioid cancer diagnosed in FIGO stage IB).
FIG. 4. Expression of ANXA2 in circulating tumour cells (CTCs). The level of expression of ANXA2 is increased in patients with recurrent disease (R) when compared to healthy controls (C), and patients with non-recurrent EC (NR).
DETAILED DESCRIPTION OF THE INVENTION For the sake of understanding, the following definitions are included.
The term "recurrence in a patient diagnosed with endometrial carcinoma" as used herein refers to a situation where a patient who has been diagnosed of endometrial carcinoma and has received chemical and/or surgical treatment later on suffers from a relapse.
The term "ploidy" as used herein refers to the number of sets of chromosomes in the nucleus of a cell. In humans the cells in all non-reproductive tissues are of the diploid type, meaning that they have two sets of chromosomes (2n). The term "aneuploidy" is an abnormal number of chromosomes in a cell. It can be the result of a defective segregation of chromosomes during mitosis, and usually leads to multiple genetic disfunctions, some of them potentially associated to cancer. The term "isolated tumor tissue sample" as used herein can refer to both a histological cut of the tumor tissue as well as a cell or cells derived from such tumor by any extraction means (such as uterine aspirates or any other procedures). In particular, it also includes exosomes, secreted proteins, cell fragments, genetic material, and vesicles derived from cells of the tumor tissue, as well as circulating tumour cells.
The term "risk to develop recurrence" as used herein refers to the risk to develop a further cancer once the previous cancer has been treated and/or removed from the body. This recurrence can be either local, wherein the cancer appears after treatment in a tissue adjacent to the removed tissue, or remote, wherein the cancer appears in a remote tissue via lymphatic or haematic trasport. The term "level of expression of Annexin A2" as used herein refers to the quantity of Annexin A2 mRNA and/or Annexin A2 protein produced by the Annexin A2 gene present in the cells of the isolated tumor tissue sample of the patient.
The term "immunochemistry" as used herein refers to a variety of techniques for detecting antigens (usually proteins and peptides, and in the present case Annexin A2) in a sample by exploiting the principle of antibodies binding specifically to said antigens. The terms "immunocytochemistry" and
"immunohistochemistry" as used herein are basically interchangeable, and refer to the application of immunochemistry techniques in the context of a cell or a group of cells present in a histology sample. Visualising an antibody- antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine. The immunohistochemistry technique can be direct or indirect. The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, such as with indirect methods, and is less commonly used than indirect methods. The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the sample and a labeled secondary antibody (second layer) that reacts with the primary antibody. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.
Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or Neutravidin-enzyme. The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be
generated. With the direct method, it would be necessary to label each primary antibody for every antigen of interest. It must be borne in mind that immunochemistry techniques can also be used to detect certain nucleic acid sequences if a tagged nucleic acid probe (designed to specifically bind to a certain target nucleic acid sequence) can later on be detected with a labelled antibody. Thus, the detection of Annexin A2 RNA could be performed by using a tagged nucleic acid designed to bind a specific sequence of Annexin A2 RNA, and then detecting said tagged nucleic acid with a labelled antibody which selectively binds to the tag. An example of a putative antibody that could be used in the method of the invention is Abeam ab1803 (other examples of antibodies that could be used are those found in
http://www.antibodyresource.com/search/Antibodies/17f2c95b-6bbb-4cab- 3121 -7509bd571476/anxa2 as of November 20th, 2013)
The term "FIGO stage" as used herein refers to the International Federation of Gynaecology and Obstetrics classification of endometrial carcinoma (EC). The current classification has the following stages:
• Stage 0. Carcinoma in situ.
• Stage I. Carcinoma limited to the body of the uterus.
o Stage la. No or less than half myometrial invasion, o Stage lb. Invasion equal to or more than half the
myometrium
• Stage II. Cervical stromal involvement.
• Stage III. Local or regional spread of the tumour.
o Stage Ilia. Tumour invades the serosa of the body of the uterus and adnexae.
o Stage 1Mb. Vaginal or parametria! involvement,
o Stage 111 . Pelvic or para-aortic lymphadenopathy.
• Stage IV. Involvement of rectum and or bladder mucosa and or distant metastasis.
The term "reference expression level" referred to in the methods of the invention is to be understood as a predefined value of a given molecular marker, Annexin A2 in the present case, which is derived from the values of said molecular marker in a tumour sample or group of samples. If the level of expression of Annexin A2 is determined at the protein level, then the
"reference expression level" is a predefined value of Annexin A2 protein quantity, whereas if the level of expression of Annexin A2 is determined at the mRNA level, then the "reference expression level" is a predefined value of Annexin A2 mRNA quantity. The samples are taken from a subject or group of subjects wherein the presence, absence, stage, or course of the disease has previously been determined. The subject or subjects from whom the
"reference expression level" is derived may include subject(s) wherein the condition is absent, subject(s) wherein the condition is present, or both. The person skilled in the art, making use of the general knowledge, is able to choose the subject or group of subjects more adequate for obtaining the reference expression levels for each of the methods of the present invention. Methods for obtaining the reference expression level from the group of subjects selected are well-known in the state of the art (Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values"). In a particular case the "reference expression level" for Annexin A2 is a cut-off value defined by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). As the skilled person will appreciate, optimal cut-off values will be defined according to the particular application of the method: target population for the prediction, balance between specificity and sensitivity of the immunochemistry technique, and others.
The term "antibody or a fragment thereof able to bind to Annexin A2" is to be understood as any immunoglobulin or fragment thereof able to selectively bind the Annexin A2. It includes monoclonal and polyclonal antibodies. The term "fragment thereof encompasses any part of an antibody having the size and conformation suitable to bind an epitope of Annexin A2. Suitable fragments include F(ab), F(ab') and Fv. An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies).
Human Annexin A2 (also known as ANXA2, ANX2, ANX2L4, CAL1 H and LPC2D) is found under the code P07355 in the UniProt database (last modified October 16, 2013). It belongs to the Annexin familiy of proteins, and is involved in cell motility and cell-matrix interactions.
As mentioned above, a first aspect of the present invention is an in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma, the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient.
In a particular embodiment of the invention, the determination of the level of expression of Annexin A2 in the isolated tissue sample of the patient is carried out by contacting the test sample with a reagent that binds Annexin A2. In a particular embodiment of the first aspect of the invention, the in vitro method comprises the steps of: a) determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient; and b) comparing the level of expression of Annexin A2 determined in step a) with a reference expression level, wherein if the level of expression determined in step (a) is higher than the reference expression level, it is indicative that the patient is in risk to develop recurrence, or alternatively, if the level of expression determined in step (a) is lower than the reference expression level, it is indicative that the patient is not in risk to develop recurrence. It also forms part of the invention the in vitro method according to the first aspect of the invention, comprising the steps of:
(a) determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient;
(b) determining a reference expression level of Annexin A2, wherein the reference expression level is derived from: a group of samples taken from one or more subjects where the condition is absent, or
alternatively a group of samples taken from one or more subjects where the condition is present, or alternatively a group of samples taken from subjects where the condition is either present or absent; and
(c) comparing the level of expression of Annexin A2 determined in step a) with the reference expression level of step b). wherein if the level of expression determined in step (a) is higher than the reference expression level of step (b), it is indicative that the patient is in risk to develop recurrence, or alternatively, if the level of expression determined in step (a) is lower than the reference expression level of step (b), it is indicative that the patient is not in risk to develop recurrence. It also forms part of the invention the in vitro method according to the first aspect of the invention, wherein the isolated tumor tissue sample of the patient is selected from the group consisting of a histological cut of the tumor tissue, circulating tumor cells, exosomes, secreted proteins, cell fragments, genetic material and vesicles derived from cells of the tumor tissue, or combinations thereof. In a particular embodiment of the first aspect of the invention, the endometrial carcinoma is of the endometrioid type.
In a particular embodiment of the first aspect of the invention, the endometrial carcinoma is a FIGO stage I endometrial carcinoma.
In a particular embodiment of the first aspect of the invention, the endometrial carcinoma is a FIGO stage la endometrial carcinoma.
In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 is determined at the protein level.
In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 is determined at the mRNA level. In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 determined at the mRNA level is based on a nucleic acid hybridization technique.
In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 protein is determined by immunohistochemistry.
In another particular embodiment of the first aspect of the invention, the level of expression of Annexin A2 protein is determined using an antibody or a fragment thereof able to bind to Annexin A2.
In another particular embodiment of the first aspect of the invention, the antibody or fragment thereof forms part of a kit.
As mentioned above, a third aspect of the present invention is the use of means for determining the level of expression of Annexin A2 in an isolated tumour tissue sample in the method of the first aspect of the invention. In a particular embodiment of the third aspect, the means to carry out the invention comprise an antibody or a fragment thereof able to bind to Annexin A2. The antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies are made by injecting animals with the peptide antigen and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mixture of antibodies that recognize several epitopes.
Monoclonal antibodies show specificity for a single epitope and are therefore considered more specific to the target antigen than polyclonal antibodies.
In another particular embodiment of the third aspect, the means to carry out the invention form part of a kit. The antibody or fragment thereof for detecting Annexin A2 can be included in a kit. The kit may additionally comprise means (additives, solvents) to visualize the antigen-antibody interactions.
Throughout the description and claims the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" and its variations encompasses the term "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
EXAMPLES
A) Materials and Methods
Patients
Endometrial cancer samples were collected from patients who underwent surgery for endometrial carcinoma at Vail d'Hebron University Hospital (Barcelona, Spain), University Hospital of Santiago de Compostela (Santiago de Compostela, Spain), Arnau de Vilanova Hospital (Lleida, Spain), MD-
Anderson Cancer Center Madrid (Madrid, Spain), Hospital de la Santa Creu i Sant Pau (Barcelona, Spain), and Hospital Virgen del Rocio (Sevilla, Spain). All participants signed an informed consent and the protocols were approved by the Institutional Review Boards.
2D-DIGE Proteomic analysis
Proteomic comparison of primary endometrial carcinomas versus EC recurrences was carried out in 12 samples (Table 1 ), presenting similar percentages of epithelial tumor glands. Samples were processed as described (Monge et al., "Substractive proteomic approach to the endometrial carcinoma invasion front" J. Proteome Res. 2009; vol 8, pp. 4676-84). Briefly, DIGE lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) was used for protein extraction and was labeled with Cy3 and Cy5 cyanine dyes. A pool consisting of equal amounts of each of the samples was used as internal standard and was labeled with Cy2 dye. Samples were combined according to the experimental design, at 50 g of protein per Cy dye per gel, and diluted 2-fold with IEF sample buffer (7 Murea, 2Mthiourea, 4% wt/vol CHAPS, 2% dithiothreitol, 2% pharmalytes pH 3-10 and 0.002%
bromophenol blue). The 2-dimension electrophoresis was performed using GE Healthcare reagents and equipment. Fluorescence images of the gels were acquired on a Typhoon 9400 scanner (GE Healthcare). Cy2, Cy3 and Cy5 images were scanned at 488/520, 532/580 and 633/670 nm
excitation/emission wavelengths, respectively, at a 100 μιτι resolution. Image analysis and statistical quantification of relative protein abundances were performed using Progenesis SameSpots v3.2 (Nonlinear Dynamics,
Newcastle, UK). Differentially expressed proteins between primary tumors and recurrences fulfilled the restrictive selection criteria of fold -1 .5 > x > 1 .5 and Anova p<0,05 were selected.
Table 1 . Clinicopathology characteristics of the patients included in the 2DIGE proteomic analysis.
Primary tumor
Diagnosis
Diagnosis Grade Stage
Recurrence Endometrioid EC 3 IA
Primary tumor Endometrioid EC 3 IA
Recurrence Serous EC <<< IA
Primary tumor Serous EC 3 IA
Recurrence Endometrioid EC 1 MB
Primary tumor Endometrioid EC 1 IIA
Recurrence Endometrioid EC 1 IC Primary tumor Endometrioid EC 1 IC
Recurrence Endometrioid EC 1 IB
Primary tumor Endometrioid EC 1 IB
Recurrence Endometrioid EC 2 MB
Primary tumor Endometrioid EC 2 MB
Immunohistochemistry
Tissue microarrays were constructed from paraffin-embedded endometrial cancer tissue blocks, including a first series of endometrial carcinomas of 1 15 primary lesions and 25 post radiation recurrences, and a second cohort of 121 patients including 59 primary carcinomas that did not relapse and 62 primary carcinomas that progressed to recurrent disease.
Immunohistochemistry was performed in two different tumor cylinders per case, with anti-Anxa2 monoclonal antibody (diluted 1 :100 ; ab41803 Abeam). Immunohistochemical staining was semi-quantitatively graded based on the percentage and on the intensity of the labelling. Histological scores ranged from 0 (no immunoreaction) to 300 (maximum immunoreactivity) by applying the formula: Histoscore = 1 X (% light staining) + 2X (% moderate staining) + 3X (% strong staining).
Uterine aspirates
In this study, uterine aspirates were collected by aspiration of the endometrial fluid with a Cornier Pipelle®. Then, the fluid was mixed in 1 :1 ration with PBS1 x and centrifuged in order to separate the supernatant, which
corresponds to a soluble fraction containing proteins mainly secreted from the endometrium and plasma proteins; and the pellet, which corresponds to a solid fraction containing mainly red blood cells and cells coming from the endometrium. ANXA2 was evaluated by ELISA in both fractions obtained from a uterine aspirate, supernatant and pellet, following manufacturer's
instructions (cat. No. SEB944Hu; Uscn Science Inc.). This study enrolled a total of 15 patients with endometrial cancer. A description of the
clinicopathological characteristics of each tumor as well as the clinical management of each patient is detailed in Table 2.
Figure imgf000017_0001
Table 2
ANXA2 expression in circulating tumour cells (CTC) from endometrial cancer patients
ANXA2 expression was determined by RT-q-PCR upon EpCAM based immunoisolation of CTC, using the CELLectionTM Epithelial Enrich kit (Invitrogen, Dynal, Oslo, Norway) as described (Alonso-Alconada et al., "Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer" Mol. Cancer 2014, 13:223). Total RNA from CTC was extracted with the QIAmp viral RNA mini kit (Qiagen, Valencia, CA) and cDNA was synthesized using SuperScriptlll (Invitrogen, Carlsbad, CA) following manufacturer's protocol. To further optimize the sensibility of detection, a preamplification step was performed using TaqMan PreAmp Master Mix kit (Applied Biosystems, Foster City, CA) with 14 reaction cycles. ANXA2 expression level was determined by RT-q-PCR with TaqManH
PreAmp Master Mix kit (Applied Biosystems, Foster City, CA) and a 7.500 quantitative Real-time PCR Machine. Data were analyzed with StepOne Software v.2.1 (Applied Biosystems, Foster City, CA) and normalized to protein tyrosine phosphatase receptor type C (PTPRC, CD45), as a marker of non-specific isolation. Data were represented as (40 - DCt), whereby DCt=duplicate mean (CtANXA2 - CtCD45). Kruskal-Wallis non-parametric test with Dunn's post-test was used to determine the differences in ANXA2 expression level from CTC.
B) Results B1
As it is routinely done in this type of studies, we first performed a 2D-DIGE proteomic analysis to narrow down the number of biomarkers handled. For this, we macroscopically dissected endometrial primary lesions and matched endometrial recurrences (n=12; see Table 1 for clinicopathology
characteristics), presenting similar percentages of epithelial tumor glands.
Samples were processed as described (Monge et al 2009, ibid.), and differentially expressed proteins between primary tumors and recurrences fulfilled the restrictive selection criteria of fold -1 .5 > x > 1 .5 and Anova p<0,05.
B2
We evaluated ANXA2 by immunohistochemistry in tissue microarrays including 1 15 primary endometrioid carcinomas of endometrium and 25 post radiation recurrences. Two different tumor cylinders per case were evaluated and immunohistochemical staining was semi-quantitatively graded based on the percentage and on the intensity of the labelling. Histological scores ranged from 0 (no immunoreaction) to 300 (maximum immunoreactivity) by applying the formula: Histoscore = 1 X (% light staining) + 2X (% moderate staining) + 3X (% strong staining). Representative examples of ANXA2 staining are presented in FIG. 1 . ANXA2 over-expression in postradiation recurrences (mean Histoscore 221 .6; range 90-295) was found to be statistically significant compared to primary carcinomas (mean Histoscore
155.4; range 0-295) (p-value <0.0001 ).
B3 Once demonstrated that ANXA2 expression was up-regulated in recurrent disease compared to primary lesions, we assessed the clinical utility of ANXA2 as a predictor of endometrial cancer recurrences. For this, we conducted a retrospective multi-centric study to evaluate by
immunohistochemistry the expression of ANXA2 in a cohort of 121 patients including 59 primary carcinomas that did not relapse and 62 primary carcinomas that progressed to recurrent disease. Globally, we found a 25.12% increased levels in those carcinomas that finally recurred when compared to primary tumors that did not recur (p<0.00001 ; mean 218.81 versus 174.87 histoscore, respectively). When we adjusted the levels of ANXA2 expression to a logistic regression model and distributed them into intervals, we found a significant probability of a primary carcinoma to recur associated with increasing ANXA2 expression (Table 3 and FIG. 2).
Moreover, when we set up a threshold of 253 histoscore for ANXA2
expression, we could identify those primary carcinomas with a significant statistical probability to end up in recurrent disease (96.55% versus 40% of carcinomas that did not recur; p<0.00001 ).
Figure imgf000019_0001
Table 3.
Histologically, non-endometrioid Type II endometrial carcinomas presented a 17% elevated ANXA2 expression compared to endometrioid Type I endometrial carcinomas (p<0.005; mean 231 .35 versus 197.42 histoscore, respectively).
Finally and more interestingly, ANXA2 expression was assessed in the group of endometrioid endometrial carcinomas (EECs). ANXA2 expression was found to be a 24.76% superior in those Type I endometrioid endometrial carcinomas that ended up in recurrent disease compared to those that did not recur (p<0.0001 ; mean 217.87 versus 174.62 histoscore, respectively), evidencing the potential of ANXA2 as biomarker in intermediate-risk endometrial carcinomas. Further, ANXA2 expression was evaluated in stages I and II, Type I endometrioid endometrial carcinomas regarding development of recurrence. This was important since stages I and II of Type I endometrioid carcinomas represent the subset of endometrial carcinomas that are less prone to develop recurrences. From a total of 70 stage I tumors (39
associated with recurrences, and 31 unassociated with recurrences), the tumors that recurred had 18.29% higher ANXA2 levels in comparison with those that did not recur (p=0.01 ). When evaluating stage I and stage II endometrioid carcinomas together (86 cases; 38 associated with recurrence and 48 unassociated with recurrence), the tumors that recurred had 25.22% higher ANXA2 levels in comparison with those that did not recur.
Thus, our studies demonstrated that Annexin A2 could actually be used to discriminate the endometrioid carcinomas with high likelihood of recurrence from those with low likelihood of recurrence.
B4
Uterine aspirates are in direct contact with the endometrium and its collection is performed in routinely basis to patients suspected to suffer of endometrial cancer. It has been reported that there is a very high correlation at RNA and DNA level between endometrial tissues and uterine aspirates, making this biofluid an interesting source of biomarkers for the screening of gynecological diseases such as endometrial cancer. The identification of ANXA2 as marker of recurrence in uterine aspirates would represent an improvement in the process of treatment decision-making.
As depictured in Figure 3A, expression of ANXA2 was increased in uterine aspirates that recurred compared to those that did not recur, both when the expression was analyzed in supernatant and in pellet fractions. If results were analyzed independently of stage (Figure 3B and 3C), we again confirmed that expression of ANXA2 was increased in supernatant and pellet fractions of uterine aspirates from patients that finally recurred. Altogether, our results indicated that ANXA2 has great potential as marker of recurrence in uterine aspirates.
B5 We explored ANXA2 expression in Circulating Tumor Cells (CTC) isolated from 34 high-risk endometrial cancer patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls. Samples were subjected to EpCAM-based immunoisolation using CELLectionTM
Epithelial Enrich kit (Invitrogen, Dynal, Oslo, Norway) followed by quantitative Real-Time PCR analysis as described above. We could confirm a gradual increase from healthy controls to patients with no recurrent disease, and a further increase in patients presenting recurrent disease (Figure 4). These data translate into clinically relevant samples the proposed role of ANXA2 during metastatic dissemination, and suggest its potential as surrogate marker of disseminated CTC for the management of high-risk endometrial cancer patients. REFERENCES CITED IN THE APPLICATION
Meehan KL. et.al. "Proteomics and the search for biomarkers of female reproductive diseases" Reproduction 2010, vol. 140, pp. 505-519 Wulfkuhule J.D., et.al. "Proteomic applications for the early detection of cancer" Nat. Reviews Cancer 2003, vol. 3, pp. 267-275.
Lu K.H., "Management of early-stage endometrial cancer" Seminars in Oncology 2009, vol. 36, pp. 137-144.
Gadduci A., et.al., "Tissue and serum biomarkers as prognostic variables in endometioid-type endometrial cancer" Critical Rev. in Oncology 201 1 , vol 80, pp. 181 -192. Gemoll et.al. "Protein profiling of genomic instability in endometrial cancer"
Cell. Mol. Life Sci. 2012, vol. 69, pp. 325-333.
Pradhan M., et.al. "Prognostic importance of DNA ploidy and DNA index in stage I and II endometrioid adenocarcinoma of the endometrium" Annals of Oncology 201 1 , vol. 23, pp. 1 178-1 184.
Burtis C. A. et al., 2008, Chapter 14, section "Statistical Treatment of Reference Values"
Matias-Guiu X. et. al. "Prognostic biomarkers in endometrial and ovarian carcinoma" Virchows Archiv. in press
Zaino R. et.al, "Epithelial tumours and Precursor Lesions" 2014, Chapter 5 "Uterine Corpus", World Health Organization
Monge et al., "Substractive proteomic approach to the endometrial carcinoma invasion front" J. Proteome Res. 2009; vol 8, pp. 4676-84

Claims

1 . An in vitro method for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma, the method comprising the step of determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient.
2. The in vitro method according to claim 1 , comprising the steps of: a) determining the level of expression of Annexin A2 in an isolated tumor tissue sample of the patient;
b) determining a reference expression level of Annexin A2, wherein the reference expression level is derived from: a group of samples taken from one or more subjects where the condition is absent, or alternatively a group of samples taken from one or more subjects where the condition is present, or alternatively a group of samples taken from subjects where the condition is either present or absent; and
c) comparing the level of expression of Annexin A2 determined in step a) with the reference expression level of step b). wherein if the level of expression determined in step (a) is higher than the reference expression level of step (b), it is indicative that the patient is in risk to develop recurrence, or alternatively, if the level of expression determined in step (a) is lower than the reference expression level of step (b), it is indicative that the patient is not in risk to develop recurrence.
3. The in vitro method according to any one of claims 1 -2, wherein the endometrial carcinoma is of the endometrioid type.
4. The in vitro method according to any one of claims 1 -3, wherein the endometrial carcinoma is FIGO stage I endometrial carcinoma.
5. The in vitro method according to claim 4, wherein the endometrial carcinoma is FIGO stage la endometrial carcinoma.
6. The in vitro method according to anyone of claims 1 -5, wherein the level of expression of Annexin A2 is determined at the protein level.
7. The in vitro method according to anyone of claims 1 -6, wherein the level of expression of Annexin A2 is determined by immunohistochemistry.
5 8. The in vitro method according to claim 7, wherein the level of expression of Annexin A2 protein is determined using an antibody or a fragment thereof able to bind to Annexin A2.
9. The in vitro method according to claim 8, wherein said antibody or fragment o thereof forms part of a kit.
10. Use of Annexin A2 as an in vitro marker for predicting the risk to develop recurrence in a patient diagnosed with endometrial carcinoma. 5
1 1 . Use of means for determining the level of expression of Annexin A2 in an isolated tumor tissue sample for predicting risk to develop recurrence in a patient diagnosed with endometrial carcinoma in the method of any one of the claims 1 -9. 0
12. The use according to claim 1 1 , wherein said means comprise an antibody or a fragment thereof able to bind to Annexin A2.
13. The use according to any one of claims 1 1 -12, wherein said means form part of a kit.
5
14. The in vitro method according to claim 1 , wherein the isolated tumor tissue sample of the patient is selected from the group consisting of a histological cut of the tumor tissue, circulating tumor cells, exosomes, secreted proteins, cell fragments, genetic material and vesicles derived from 0 cells of the tumor tissue, or combinations thereof
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