TUMOR MALIGNANCY

Field of the Invention

The present invention relates to in vitro methods and products for the diagnosis and prognosis of ovarian tumor malignancy .

Background of the Invention

Cancer is one of the main health problems in the world. According to the GLOBOCAN database of the International Agency for Research on Cancer of the World Health Organization, more than 12 million cancer cases were diagnosed worldwide in 2008, and the number of deaths due to cancer in 2008 was more than 7 million people.

During the process of malignization, tumor cells change their protein expression pattern, which impairs cellular processes, such as the maintenance of cell architecture, cell proliferation, adhesion and death. These modifications do not only generate changes in the tumor cells themselves, but also cause some cells in the stroma surrounding the tumor to receive impaired molecular signals and change their pattern of behavior and protein expression pattern, acquiring a morphology typical of myofibroblasts. The molecules secreted by these myofibroblasts in response to adjacent tumor can in turn contribute to promoting tumor growth and invasion in a different way, such that a paracrine loop is established between the tumor and stroma. In recent years, scientific evidence pointing with increasing precision to peritumoral stroma as one of the main promoters of tumor invasiveness, as well as of the phenomena of therapy resistance, have been generated.

The methods and products for the diagnosis and prognosis of ovarian tumor malignancy object of the present invention are encompassed in this novel research framework, since they are based on the use of proCOLHAl protein present in the stromal cells of malignant carcinomas as an ovarian tumor diagnosis and malignancy marker.

Ovarian cancer is the gynecological disease causing the highest number of deaths in developed countries. With an incidence of 204,000 cases a year, it causes 125,000 deaths worldwide. The 5-year mortality is about 70-80%, in most cases being due to tumor progression and metastasis.

The most common type of ovarian cancer is ovarian epithelial cancer (90%) . Epithelial tumors include benign tumors, low malignant potential tumors or borderline tumors and malignant tumors, the prognosis of which depends fundamentally on the stage (I-III) (Ovarian Cancer Detailed Guideline, ACS 2013) . Like in other tumors, surgical and pharmacological treatment suitable for each type of tumor depends on the spread of the tumor and on its risk of progression; but in ovarian cancer surgery decision must be the most conservative possible in order to prevent loss of fertility in patients. For this reason, it is of vital importance to correctly classify the tumors and evaluate the tumor progression and metastasis capacity at the time of diagnosis, which would allow selecting the correct surgical procedure for each patient.

Low malignant potential tumors or borderline tumors are benign tumors which can progress into malignant and invasive tumors. These tumors tend to spread outside the ovaries generating implants which can be invasive or non-invasive. Attempt has been made to associate the invasiveness of these implants with the risk of malignization of borderline tumors, but it is very difficult to evaluate the same, particularly in biopsy samples and there are no criteria today that allow classifying borderline tumors according to their progression capacity. Furthermore, these tumors may be mistaken for stage I malignant tumors, given that they share many morphological characteristics. These low malignant potential tumors, as well as benign tumors, are often present together with malignant tumors and can mask said tumors, making the detection thereof difficult, particularly in small biopsies.

Due to the foregoing, a diagnostic system which allows accurately distinguishing benign lesions from malignant ovarian tumors, as well as predicting the risk of malignization of borderline tumors, allowing selecting the most suitable treatment for each patient, assuring disease elimination and preventing excessive or unnecessary treatments which may interfere with patient fertility, is necessary.

Collagen is the main component of the extracellular matrix (ECM) . The correct expression of the genes encoding the different types of collagen is necessary for correct ECM assembly during embryonic development and for the maintenance thereof in the adult body. Collagen XI (COL11) is a little- studied type of collagen which, however, plays a fundamental role in the regulation of fibrillar networks in cartilaginous and non-cartilaginous matrices; these fiber networks are involved in different morphogenesis processes during embryonic development in vertebrate animals. Transcripts of the alpha chain 1 ( l) of collagen XI (COL11A1) have been found during fetal development in cartilaginous tissues and also in other tissues such as the bone, kidney, skin, muscle, tongue, intestine, liver, ear, brain and lung. The extracellular matrix also has an important role in specific biological processes, such as cell differentiation, proliferation and migration; therefore, the deregulation of the expression of genes encoding the proteins forming same, is associated with carcinogenesis and metastasis processes. In the particular case of COL11A1, it has been demonstrated that stromal fibroblasts have high expression levels of the colllal gene in sporadic colorectal carcinomas, whereas this gene is not expressed in healthy colon. The expression of the colllal gene has also been associated with pancreatic cancer, breast cancer, colon cancer, lung cancer, head and neck cancer and bladder cancer and the expression of the COL11A1 protein has been associated with pancreatic cancer and colon cancer.

Recent studies have detected high expression of messenger RNA (mRNA) transcript of the colllal gene in advanced stage ovarian tumors (Kim, H et al. BMC Med Genomics. 2010 Nov 3;3:51), recurrent ovarian tumors or ovarian tumors with a poor prognosis (Wu, YH et al ; Oncogene. 2013 Aug 12), suggesting the possible role of this molecule in tumor progression, although the presence of the protein it encodes, COL11A1, or the precursor protein thereof, proCOLHAl protein, in tumors of patients with ovarian cancer has yet to be described. The high expression of the mRNA transcript of a gene does not necessarily lead to the high expression of the protein encoded by this gene (Gigy et al . , Mol. Cel. Biol., 1999, 9:1720-1733; Greenbaum et al., Genome Biology, 2003, 4:117) .

The authors of the present invention have developed methods and products for detecting the proCOLHAl protein in ovarian tumors and accurately distinguishing benign lesions from malignant ovarian tumors, as well as making a prognosis of the risk of malignization of borderline tumors.

Brief Description of the Invention

The authors of the present invention have discovered, by means of immunohistochemical analysis of ovarian tissue biopsies using the monoclonal antibody 1E8.33 described in International Patent Application WO 2013/021088 A2, that the proCOLHAl protein is expressed in malignant ovarian tumors (carcinomas) , but is not expressed in non-tumoral ovarian tissue, or in in situ or benign or borderline ovarian tumors.

This evidence turns proCOLHAl-specific antibodies into ideal antibodies for developing new in vitro methods and products for the diagnosis of ovarian cancer as well as for differentiating malignant ovarian tumors from benign or in situ tumors in diagnostic biopsy, or in surgical specimens, or in fluids from the patient, including but not being limited to, blood, serum, plasma, ascites fluid or urine, for making a prognosis of ovarian tumor malignancy and for predicting borderline tumor malignancy, therefore guiding the surgery decision and follow-up. Specifically, the invention provides methods and products for identifying, with high sensitivity and specificity, malignant ovarian tumors in vitro, even in the cases in which the morphology of the tumor and the current markers are not detected.

In one aspect, the invention relates to an in vitro method for detecting a malignant ovarian tumor, selected from Method (A) and Method (B) , wherein

A) Method (A) comprises:

detecting the presence of proCOLllAl protein in a sample from a subject having an ovarian tumor or suspected of having an ovarian tumor;

wherein

the detection of the presence of proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor is indicative that said tumor is malignant, and

B) Method (B) comprises:

comparing the expression level of the proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor, with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor greater than the expression level of said proCOLllAl protein in a control sample is indicative that said tumor is a malignant tumor.

In another aspect, the invention relates to an in vitro method for the differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion in a subject, selected from Method (A) and Method (B) , wherein

A) Method (A) comprises:

detecting the presence of proCOLllAl protein in a sample from said subject;

wherein the detection of the presence of proCOLllAl protein in said sample is indicative that the ovarian tumor is malignant; or, alternatively

the non-detection of the presence of proCOLllAl protein in said sample is indicative that the ovarian tumor is benign or in situ, or that the subject has a benign non-tumoral ovarian lesion; and

B) Method (B) comprises:

comparing the expression level of the proCOLllAl protein in said sample with the expression level of said proCOLllAl protein in a control sample; wherein

an expression level of the proCOLllAl protein in said sample greater than the expression level of said proCOLllAl protein in a control sample is indicative that the ovarian tumor is a malignant tumor, or, alternatively

an expression level of the proCOLllAl protein in said sample equal to or less than the expression level of said proCOLllAl protein in a control sample is indicative that the ovarian tumor is benign or in situ, or that the subject has a benign non-tumoral ovarian lesion.

In another aspect, the invention relates to an in vitro method for the diagnosis or prognosis of ovarian tumor malignancy in a subject, wherein said subject is a subject suspected of having a malignant ovarian tumor or is a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, selected from Method (A) and Method (B) , wherein

A) Method (A) comprises

detecting the presence of proCOLllAl protein in a sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; wherein

the detection of the presence of proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that said subject has a malignant ovarian tumor or that said low malignant potential or borderline tumor, with or without extraovarian implantations, is capable of progressing into a malignant ovarian tumor, or, alternatively

the non-detection of the presence of proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that said subject does not have a malignant ovarian tumor, or that said subject has a benign or in situ ovarian tumor, or that said low malignant potential or borderline tumor is not capable of progressing into a malignant ovarian tumor; and

B) Method (B) comprises

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, greater than the expression level of said proCOLllAl protein in a control sample is indicative that said subject has a malignant ovarian tumor or that said low malignant potential or borderline tumor is capable of progressing into a malignant ovarian tumor, or, alternatively

an expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, equal to or less than the expression level of said proCOLllAl protein in a control sample is indicative that the subject does not have a malignant ovarian tumor, or that said subject has a benign or in situ ovarian tumor, or that said low malignant potential or borderline tumor is not capable of progressing into a malignant ovarian tumor.

In another aspect, the invention relates to an in vitro method for determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, in a subject diagnosed with said tumor, selected from Method (A) and Method (B) , wherein

A) Method (A) comprises

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations;

wherein

the detection of the presence of proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that the probability of the tumor progressing into a malignant ovarian tumor is high, or, alternatively the non-detection of the presence of proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that the probability of the tumor progressing into a malignant ovarian tumor is low; and

B) Method (B) comprises

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, greater than the expression level of said proCOLllAl protein in a control sample is indicative that the probability of the tumor progressing into a malignant ovarian tumor is high; or, alternatively

an expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, equal to or less than the expression level of said proCOLllAl protein in a control sample is indicative that the probability of the tumor progressing into a malignant ovarian tumor is low.

In another aspect, the invention relates to a method for selecting a subject suspected of having a malignant ovarian tumor for treatment, wherein said treatment comprises surgically removing said ovarian tumor, performing additional analyses to evaluate tumor infiltration and metastasis, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up, said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject suspected of having a malignant ovarian tumor; or

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor with the expression level of said proCOLllAl protein in a control sample; wherein said subject is selected for said treatment:

if the presence of proCOLllAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in the control sample;

or, alternatively

wherein said subject is not selected for said treatment: if the presence of proCOLllAl protein is not detected in said sample from said subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is equal to or less than the expression level of said proCOLllAl protein in the control sample .

In another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment, wherein said treatment comprises surgically removing said ovarian tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up, said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein said subject is selected for said treatment:

if the presence of proCOLllAl protein is detected in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, is greater than the expression level of said proCOLllAl protein in the control sample;

or, alternatively,

wherein said subject is not selected for said treatment: if the presence of proCOLllAl protein is not detected in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in the control sample.

In another aspect, the invention relates to a method for selecting a subject suspected of having a malignant ovarian tumor for follow-up, wherein said follow-up comprises performing additional analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment, said method comprising: detecting the presence of proCOLllAl protein in a sample from said subject suspected of having a malignant ovarian tumor; or

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor with the expression level of said proCOLllAl protein in a control sample; wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in the control sample .

In another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for follow-up, wherein said follow-up comprises surgically resectioning said tumor and monitoring tumor recurrence, said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is detected in said sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is greater than the expression level of said proCOLllAl protein in the control sample.

In another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for follow-up, wherein said follow-up comprises monitoring tumor recurrence, said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is not detected in said sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in the control sample.

In another aspect, the invention relates to the use of the proCOLllAl protein as a marker for:

a) detecting a malignant ovarian tumor; or for b) performing differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion; or for

c) making a diagnosis or prognosis of ovarian tumor malignancy, or for

d) determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or for

e) selecting a subject suspected of having a malignant ovarian tumor for treatment which comprises surgically removing the tumor, performing additional analyses to evaluate the degree of tumor invasion, applying a chemotherapy and/or radiotherapy treatment, and a subsequent follow-up; or for

f) selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment which comprises surgically removing said tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up; or for

g) selecting a subject suspected of having a malignant ovarian tumor, wherein said follow-up comprises performing complementary analyses for assessing the degree of tumor infiltration for the purpose of determining the subsequent treatment; or for

h) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises surgically resectioning the tumor and monitoring tumor recurrence; or for

i) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises monitoring tumor recurrence.

In another aspect, the invention relates to the use of a specific antibody recognizing the proCOLllAl protein for any of the uses indicated above in relation to said proCOLHAl protein.

In another aspect, the invention relates to the use of a kit comprising a reagent recognizing the proCOLHAl protein, or a reagent for the detection and/or quantification of the expression of the proCOLHAl protein for any of the uses indicated above in relation to said proCOLHAl protein.

Brief Description of the Drawings

Figure 1 shows the results of the immunostain for proCOLHAl in a sample of a benign ovarian tumor (A) , a serous malignant ovarian tumor (carcinoma) (B) and the metastasis of a colon tumor into an ovarian tumor (C) .

Figure 2 is a summary table showing the number of cases classified according to the diagnosis of the biopsy and according to the immunolabeling for proCOLHAl. Additional information can be found in Example 1.

Detailed Description of the Invention

Definitions

To make it easier to understand the present patent application, the meaning of some terms and expressions as they are used within the context of the present invention is provided below .

The term "antibody" refers to a glucoprotein exhibiting a specific binding activity with a particular protein referred to as an "antigen". The term "antibody" comprises whole monoclonal antibodies or polyclonal antibodies, or fragments thereof; and includes human antibodies, humanized antibodies and antibodies of non-human origin. "Monoclonal antibodies" are homogeneous populations of highly specific antibodies targeting a unique site or antigenic "determinant".

The term "cancer" or "carcinoma" refers to the disease which is characterized by an uncontrolled proliferation of abnormal cells capable of invading adjacent tissues and spreading to distant organs.

The term "ovarian cancer", "ovarian carcinoma" or "ovarian tumor" refers to any proliferative disorder of ovary cells, being able to be benign, malignant, in situ, or low malignant potential or borderline, being specified in each case.

As it is used herein, the term "capable of malignization" or "capable of progressing into a malignant tumor" refers to a benign tumor or a low malignant potential tumor being capable of becoming a malignant tumor.

As it is used herein, the term "diagnosis" applied to a malignant ovarian tumor, or to a low malignant potential or borderline ovarian tumor being capable of progressing into a malignant tumor, includes determining the malignancy of said ovarian tumors or whether or not the ovarian tumors are capable of malignization.

As it is used herein, the term "epitope" refers to an antigenic determinant of a protein which is the amino acid sequence of the protein recognized by a specific antibody.

The term "specificity" refers to the capacity to detect true negatives. A specificity of 100% means that there are no false positive (non-affected subjects that obtain positive result) .

The term "infiltration" refers to the presence of tumor cells outside the primary tumor due to tumor invasion.

The term "gene" refers to a molecular deoxyribonucleotide chain encoding a protein.

The term "colllal gene" refers to the gene encoding a collagen XI component referred to as "pro- l(XI) chain" which combines with other two collagen chains (pro- 2(XI) and pro- l(II)) to form a procollagen molecule (proCOLHAl) which is enzymatically processed in cells to form collagen XI fibers. Human colllal gene (also referred to as COLL6 or STL2 ) , the reference gene sequence of which is NG 008033.1, spans over 150 kilobases ( kb ) , contains 68 exons, is located in chromosome 1 (lp21) between the base pairs 103342023 and 103574052, and encodes a protein of 1,806 amino acids (according to the isoform) and 181 KDa containing a signal peptide (amino acids 1-36) . This gene is conserved in humans, chimpanzee, cow, chicken, mouse, rat and zebrafish. Mutations in this gene have been associated with Stickler syndrome type II and Marshall syndrome. Polymorphisms of a nucleotide in this gene have been associated with the susceptibility of having spinal disc herniation. Several transcripts differing from one another mainly in the transcription of the variants of exon 6 have been described, for example, a transcript of 7.2 kb encoding isoform A (NM_001854.3 ( GI : 98985806 ) , NCBI database as of July 21, 2011) of 1,806 amino acids, another transcript of 7,3 kb encoding isoform B (NM_080629.2 NCBI database as of July 21, 2011) of 1, 806 amino acids (GI : 98985810) , another transcript of 6.9 kb encoding isoform C (NM_080630.3 (GI : 299523252 ) , NCBI database as of July 21, 2011) of 1,690 amino acids (GI : 299523253 ) , another transcript of 7.2 kb encoding isoform E (NM_001190709.1 , NCBI database as of August 1, 2011) of 1, 767 amino acids (GI : 299523257 ) , etc. As it is used herein, the term "colllal" does not refer only to the human gene but also to the orthologs of other species. Low and homogeneous expression of the colllal gene is observed in all analyzed tissues except in adipocytes where it is significantly higher (BioGPS: Gene Atlas U133A) . In cell lines, high expression is detected in UASMC cells (umbilical artery smooth muscle cells), HN NP cells (neuronal precursors), HN_Os (osteoblasts), Panc-1 (pancreatic cancer cells), H522 (lung cancer cells), U251 (glioma cells) (RefExA) , A-204 (heart rhabdomyosarcoma cells), SAOS-2 (bone marrow osteosarcoma cells) and SK-MEL28 (skin cancer cells) (GeneCards) .

The terms "equal" or "less" applied to the expression level of a protein, specifically the proCOLllA protein, mean that the amount or concentration of said protein in a specific sample is substantially the same as (equal to) , or lower than (less than) , that found in another sample considered as a control sample or a reference value; therefore the expression level of the proCOLHAl protein in a sample from a subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline tumor, is equal (substantially the same) to or less (lower) than the expression level of said proCOLllAl protein in a control sample obtained from a control population of subjects without history of ovarian tumors. In the context of the present invention, the expression level of a protein, such as the proCOLllAl protein, in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, is considered "equal" to the expression level of said protein in the control sample when the expression level of said protein in the sample from the subject is substantially the same as the expression level of said protein in the control sample, i.e., when it is comprised, for example, between the reference value (expression level of the proCOLllAl protein) plus/minus an amount lower than 3% of said reference value. Likewise, in the context of the present invention, the expression level of a protein, such as the proCOLllAl protein, in the sample from the subject suspected of having a malignant ovarian tumor, or diagnosed with a low malignant potential or borderline ovarian tumor, is considered "less" (or "lower") than the expression level of said protein in the control sample when the expression level of said protein in the sample from the subject decreases, for example, by 3%, 5%, 10%, 25%, 50%, or even 100%, with respect to the reference value.

As it is used herein, the term "malignancy" refers to tumor capacity to spread to or invade the tissue in which it is located, the adjacent or distal tissues, being life-threatening to the subject having the tumor.

As it is used herein, the term "probability" measures the frequency whereby a result (or a group of results) is obtained when carrying out a random experiment, from which all the possible results are known, under sufficiently stable conditions. The probability that a specific result is detected can be "high", i.e., the frequency whereby a result is obtained is greater than 50%, or "low", i.e., the frequency whereby such result is obtained is less than 50%. According to the present invention, the probability that the proCOLllAl protein is detected in a sample from a subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, or the probability that the expression level of the proCOLHAl protein in a sample from a subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor is greater than the expression level of the proCOLHAl protein in a control sample, is higher in those cases in which said subject has a malignant ovarian tumor, or in which said low malignant potential or borderline tumor is capable of progressing into a malignant tumor. As will be understood by the persons skilled in the art, the probability does not have to be 100% for all the evaluated subjects, although it must preferably be so. However, within the context of the present invention, said term requires being able to identify a statistically significant part of the subjects suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor as subjects having a higher probability of obtaining a specific result. The person skilled in the art can determine whether or not an event is statistically significant without major complications using different known statistical evaluation tools, for example, by means of determining the confidence intervals, determining the p-value, the Student's t-test, the Mann-Whitney test, etc. Additional information about these statistical tools can be found in Dowdy and Wearden, Statistics for Research. John Wiley & Sons, New York, 1983. The preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-values are preferably 0.1, 0.05, 0.02, 0.01 or less .

The term "proCOLHAl" refers to the precursor of collagen XI oil chain, the processing of which gives rise to collagen XI oil (COL11A1) . proCOLHAl has a central triple-helix domain with a rod-shaped structure, which is flanked by non-collagenous propeptides at the N- and C-terminal ends; these propeptides can be eliminated by specific peptidases as they are secreted out of the cell generating mature COL11A1, where collagen XI l chains are assembled with one another and with 2 and/or 3 chains to form collagen XI fibers. proCOLllAl protein is encoded by the colllal gene. Several isoforms of human proCOLllAl have been described, for example, isoform A [NM_001854.3 (GI : 98985806) , NCBI database as of July 21, 2011] of 1,806 amino acids, isoform B [NP_542196.2 (GI : 98985810) , NCBI database as of July 21, 2011], isoform C [NM_080630.3 (GI : 299523252 ) , NCBI database as of July 21, 2011], etc. As it is used herein, the term "proCOLllAl" does not refer only to human proCOLllAl protein but also to the orthologs of other species.

The term "protein" refers to a molecular chain of amino acids with biological activity. The term includes all forms of post-translation modifications, for example, glycosylation, phosphorylation or acetylation. The terms "protein", "peptide" and "polypeptide" are used interchangeably throughout this description .

As it is used herein, the term "follow-up" refers to the group of measures to be applied and assessed once the nature of an ovarian tumor in a subject is diagnosed and once a decision on whether or not to use surgical approach on the tumor is made; said measures include, among others, depending on the nature of the ovarian tumor, analyzing the spread of the tumor outside the ovary including the location of possible points of metastasis, applying surgical or pharmacological treatment suitable for the diagnosis of said tests, performing periodic analyses along with or after treatment, which allow detecting tumor relapses, etc. Therefore, the term "follow-up" applied to a subject suspected of having a malignant ovarian tumor who is verified as having a malignant ovarian tumor (either because the presence of proCOLllAl protein is detected in a sample from said subject, or because the expression level of the proCOLllAl protein in a sample from said subject is greater than the expression level of proCOLllAl in a control sample) , and from whom the tumor has been eliminated by means of surgery, refers to the analysis of the spread of the tumor outside the ovary by means of lymph nodes sampling to search for tumor presence, image analysis by suitable techniques such as ultrasound or CAT scan to search for the points of metastasis, application of surgical or pharmacological treatment suitable for the diagnosis of said tests, and the performance of periodic analyses along with or after treatment, which allow detecting tumor relapses, for example, by means of using imaging techniques or analyzing serum markers, such as tumor marker CA-125 (also referred to as "mucin 16" or "MUC16"), for example. Likewise, the term "follow-up" applied to a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, diagnosed with a tumor with the risk of malignization (either because the presence of proCOLllAl protein is detected in a sample from said subject, or because the expression level of the proCOLllAl protein in a sample from said subject is greater than the expression level of proCOLllAl in a control sample) , and, optionally, if the specialist deemed it suitable, subjected to a surgical resection of the tumor, includes monitoring possible tumor recurrence by means of performing periodic tests by imaging techniques or by analyzing serum markers, such as CA125. In this case, surgical resection of the tumor is carried out before monitoring possible tumor recurrence by means of performing periodic tests by imaging techniques or by analyzing serum markers, such as CA-125.

The term "sensitivity" refers to the detection of true positives (for example, positive diagnosis of a pathology when the patient affected by said pathology) ; 100% sensitivity means that there are no false negatives (negative diagnosis in affected patients) .

The term "subject", "individual" or "patient" refers to a member of a mammalian species and includes, but is not limited to, domestic animals, primates and humans; the subject is preferably a male or female human being of any age or race.

The term "greater" applied to the expression level of a protein, specifically the proCOLUA protein, means that the amount or concentration of said protein in a specific sample is higher than in another sample considered as a control sample or a reference value; therefore the expression level of the proCOLllAl protein in a sample from a subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, is higher (greater) than the expression level of said proCOLllAl protein in a control sample obtained from a control population of subjects without history of ovarian tumors, in case that said subject has a malignant ovarian tumor or in case that said low malignant potential or borderline tumor is capable of progressing into a malignant tumor. In the context of the present invention, the expression level of a protein, such as the proCOLllAl protein, in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor is considered "greater" (or "higher") than the expression level of said protein in a control sample (reference value) when the expression level of said protein in the sample from the subject increases, for example, by 3%, 5%, 10%, 25%, 50%, 100%, or even more, compared with the reference value for said protein in the control sample, or when it increases, for example, at least by 1.1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or even more, when compared with the reference value for said protein .

As it is used herein, the term "treatment" generally refers to the application of a therapy to alleviate or eliminate a pathology or to reduce or eliminate one or more symptoms associated with said pathology. Said therapy can include a surgical intervention, a pharmacological treatment, a radiotherapy treatment, etc.

The term "tumor" refers to any abnormal tissue mass resulting from a benign (non-cancerous) or malignant (cancerous) neoplastic process. A benign tumor is a tumor that is not capable of spreading to or invading neither the tissue in which it is located nor the adjacent or distal tissues, such that it is not life-threatening to the subject having the tumor. In contrast, a malignant tumor is a tumor capable of spreading to or invading the tissue in which it is located, the adjacent or distal tissues, such that it is life-threatening to the subject having the tumor. Malignant tumors in ovaries can be serous, mucinous, endometrioid or clear cell tumor, or undifferentiated tumor. Likewise, a low malignant potential or borderline ovarian tumor is an ovarian tumor which, while not being benign, has no clear malignancy characteristics, but can progress in some cases into malignant tumor and grow and spread throughout the abdominal cavity.

As it is used herein, the term "variant" applied to the heavy and light chain sequences of an antibody refers to substantially similar sequences. Generally, from the qualitative viewpoint, variants have the same biological activity as the native sequence. A variant of a polypeptide sequence can be a derivative of a polypeptide sequence comprising addition, deletion or substitution of one or more amino acids present in the native sequence. The variants of the heavy and light chain sequences of an antibody can differ from the sequences described within the frame regions or within the complementarity determining regions or CDRs of any of the heavy or light chains. By way of illustration, the term "variant" applied to the monoclonal antibody 1E8.33 (Garcia-Ocana, et al . , Int J Oncol. 2012; 40 (5) : 1447-54) includes amino acid sequences at least having about at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity with any of the amino acid sequences of the CDRs comprised within the variable region of the heavy chain or the variable region of the light chain of said monoclonal antibody 1E8.33, provided that the resulting variant maintains the biological activity of the native sequence, i.e., the variant of the antibody maintains the capacity of specifically recognizing the proCOLllAl protein. Methods of the Invention

The present invention is based on the discovery that the expression of proCOLllAl protein increases in malignant ovarian tumors or in tumors capable of progressing into a malignant tumor, but not in in situ tumors, benign tumors, low malignant potential or borderline tumors that are not capable of progressing into a malignant tumor, or in benign lesions. The expression of proCOLllAl occurs in fibroblasts and in the extracellular matrix surrounding the tumor, unlike other tumor markers which are expressed in the cancerous cells. Therefore, diagnosis based on proCOLllAl allows detecting malignant ovarian tumors although the infiltration is not morphologically obvious in the primary tumor present in the biopsy, and allows assessing if a low malignant potential or borderline ovarian tumor are capable of malignization at the time of diagnosis.

1. Method for detecting a malignant ovarian tumor

In one aspect, the invention relates to an in vitro method for detecting a malignant ovarian tumor, hereinafter "first method of the invention", selected from Method (A) and Method (B) , wherein

A) Method (A) comprises:

detecting the presence of proCOLllAl protein in a sample from a subject having an ovarian tumor or suspected of having an ovarian tumor;

wherein

the detection of the presence of proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor is indicative that said tumor is malignant, and

B) Method (B) comprises:

comparing the expression level of the proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor, with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor, greater than the expression level of said proCOLllAl protein in a control sample is indicative that said tumor is a malignant tumor.

The first method of the invention is a high sensitivity and specificity method, and is based on the fact that a subject having an ovarian tumor or suspected of having an ovarian tumor expresses and shows the proCOLllAl protein or has a high expression level of the proCOLllAl protein in absolute terms or in comparison with the corresponding levels in control samples from subjects without clinical history of ovarian tumors, or with benign ovarian lesions or benign ovarian tumors.

For putting the first method of the invention into practice in any of the alternatives thereof [Method (A) or (Method (B) ] , a sample, such as a biological sample, is obtained from the individual to be studied. Non-limiting illustrative examples of said sample include a core of tissue obtained by means of biopsy, a tissue specimen obtained by surgery, as well as a biological fluid, such as blood, serum, ascites fluid, urine, etc. In a particular embodiment, said sample comprises tissue obtained by means of biopsy or surgery; in a more particular embodiment, said sample comprising tissue comprises tumor- associated stromal cells, preferably, cancer-associated fibroblasts (also referred to as CAF) , expressing proCOLllAl. In another particular embodiment, said sample comprises a biological fluid (e.g., blood, serum, ascites fluid, urine, etc.) . Depending on the type of samples, to simplify their preservation and handling, these can be fixed in formalin and embedded in paraffin, or, alternatively, they can be first frozen and then embedded in a medium that can be cryogenically solidified.

The samples to be analyzed can be obtained from subjects after an ovarian tumor has been diagnosed, or has not been diagnosed, treated or non-treated.

Method (A)

In a particular embodiment, the first method of the invention comprises detecting the presence of proCOLllAl protein in a sample from a subject having an ovarian tumor or suspected of having an ovarian tumor [Method (A) ] , wherein the detection of the presence of proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor is indicative that the subject has a malignant ovarian tumor (or carcinoma) .

The presence of proCOLllAl protein in the sample to be analyzed can be detected by means of any conventional method which allows detecting the presence of a protein, specifically proCOLllAl, in a sample. In a particular embodiment, said sample is an ovarian tissue sample or abdominal cavity sample or a body fluid sample. Virtually any conventional method for detecting the presence of a protein in a tissue sample or a body fluid sample can be used within the scope of the invention for detecting the presence of proCOLllAl. Non-limiting illustrative examples of said methods include methods based on the use of antibodies, affinity chromatography techniques, ligand binding assays, etc.

In a particular embodiment, the detection of the presence of proCOLllAl protein is performed by means of an immunoassay based on the formation of an antigen-antibody type complex by means of using one (or more) antibodies recognizing one (or more) epitopes of proCOLllAl, and the viewing the complexes formed by any suitable technique, including both radioctive and non-radioctive techniques such as, for example, colorimetric techniques, fluorometric techniques, luminiscent (e.g., bioluminiscent, chemiluminiscent, etc.) techniques, etc., using in turn secondary antibodies labeled with the suitable markers to that end.

The antibodies to be used for putting this particular embodiment of the first method of the invention [Method (A) ] into practice are specific antibodies recognizing the proCOLllAl protein, or an antigenic fragment of said proCOLllAl protein, i.e., antibodies recognizing an epitope of the proCOLllAl protein. The antibodies which can be used in this type of assays can be polyclonal antibodies or sera, monoclonal antibodies, antibody fragments, such as Fv, Fab, Fab', F(ab')2, scFv (single chain Fv) , diabodies, triabodies, tetrabodies, combibodies, etc., capable of recognizing and binding to proCOLllAl, or to an antigenic fragment thereof; said antibodies can be human antibodies, humanized antibodies or antibodies of non-human origin. Advantageously, the antibody recognizing the proCOLllAl protein is an antibody specifically recognizing the VAR subdomain in the N-terminal propeptide of proCOLllAl. Illustrative examples of antibodies specifically detecting proCOLllAl are mentioned in International Patent Application WO 2013/021088 A2. In a particular embodiment, said antibody is the monoclonal antibody 1E8.33 (Garcia-Ocana, et al . , Int J Oncol. 2012; 40 (5 ) : 1447-54 ) , an antibody specifically recognizing the proCOLllAl protein, without detecting other proteins with high sequence homology such as the COL5A1 protein, for example.

The antibody recognizing the proCOLllAl protein can be, optionally, conjugated to a carrier. Likewise, said antibody recognizing the proCOLllAl protein may be labeled or non- labeled; non-limiting illustrative examples of markers which can be used include radioctive isotopes, enzymes, fluorophores , chemiluminiscent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, dyes, etc. The non-labeled antibodies can be used in agglutination assays, whereas labeled antibodies can be used in various assays.

There is a large variety of assays well known by the persons skilled in the art which can be used for putting this particular embodiment of the first method of the invention [Method (A) ] into practice, using non-labeled antibodies (primary antibody) recognizing the proCOLllAl protein and labeled antibodies (secondary antibodies); these techniques include Western-blot, ELISA (enzyme-linked immunosorbent assay) , RIA (radioimmunoassay) , competitive EIA (enzyme immunoassay) , DAS-ELISA (Double-antibody sandwich ELISA) , immunohistochemical assays, multiplex detection techniques based on the use of protein microarrays, microspheres or biochips including specific antibodies, or assays based on colloidal precipitation, etc.

In a particular preferred embodiment, the detection of the presence of proCOLHAl according to Method (A) is carried out by means of an immunohistochemical assay, a known histopathology method based on the use of a specific antibody, usually labeled with a marker (for example, an enzyme) , which can transform a substrate into a visible compound without affecting the capacity of the antibody to form a complex with the antigen (proCOLHAl, in this case), applied to an organic tissue sample. With the use of some of the specific techniques (peroxidase, antiperoxidase, fluorescein, etc.), the antigen-antibody complex formed can be located and identified in the tissue or cell samples to be studied, whereby the antigenic markers characteristics of different cells are identified and the type of cell involved can be determined in the sample.

Briefly, in a particular embodiment of Method (A) , the sample comprising ovary tissue to be analyzed for the purpose of detecting the presence of proCOLHAl protein by means of an immunohistochemical assay, which can be a fresh sample, a frozen sample or a sample embedded in paraffin and fixed using a protecting agent (e.g., formalin or the like), is stained with a proCOLHAl-specific antibody (e.g., the monoclonal antibody 1E8.33 or a variant thereof) and the frequency of cells which have been stained and the intensity of the staining are determined. If necessary, the sample to be analyzed is subjected to a conditioning treatment which can include one or more of the following processes: fixing the tissue, obtaining sections having a suitable thickness, recovering the antigen, blocking molecules that may interfere in the indirect reaction, preventing non-specific bindings, etc. Advantageously, the detection of proCOLHAl by means of said immunohistochemical assay in the sample to be analyzed is carried out in parallel with tissue or cell samples serving as positive marker and as negative marker, and, if desired, as reference, healthy tissues of the same origin as that the of the ovarian tumor or carcinoma being analyzed can be used. It is also common to use a background control. Typically, a value indicative of the total expression which is calculated depending on the frequency of stained cells and on the intensity in each of the stained cells is assigned to the sample. The typical criteria for assigning expression values to the samples were described in the Handbook of Immunohistochemistry and In situ Hybridization in Human Carcinomas, M. Hayat Ed., 2004, Academic Press, for example.

Example 1 describes an immunohistochemical assay for detecting the presence of proCOLllAl in which the previously conditioned sample comprising ovary tissue to be analyzed is contacted with the monoclonal antibody targeting proCOLllAl and, after the incubation period, it is developed with diaminobenzidine (DAB) using a suitable detection system.

The detection of the presence of proCOLllAl by means of an immunohistochemical assay has several advantages since it is widely used in pathological anatomy laboratories, which enables the immediate applicability both of the first method of the invention and of the rest of the methods provided by the present invention by means of said technique, and, furthermore, it is highly automated, which makes it easier to perform same under homogeneous and reproducible conditions .

Based on the detection of the presence of proCOLllAl protein in a sample from a subject having an ovarian tumor or suspected of having an ovarian tumor, it is possible to link the detection of the presence (or the non-detection of the presence- absence) of proCOLllAl in said sample with tumor malignancy.

Therefore, in a subsequent step, Method (A) involves correlating the detection of the presence (or the non-detection of the presence-absence) of proCOLllAl protein with the diagnosis of tumor malignancy.

This correlation allows indicating that the ovarian tumor is malignant when the presence of proCOLllAl protein is detected in the sample from the subject having an ovarian tumor or suspected of having an ovarian tumor.

This information can be used effectively by the specialist for selecting the most suitable treatment to be administered to the subject having an ovarian tumor or suspected of having an ovarian tumor depending on whether or not the tumor is malignant .

In the sense used in this description, the expression "detection of the presence of proCOLllAl protein", or the like, refers to the capacity of detecting proCOLllAl protein in the analyzed sample using one of the methods referred to above.

Method (B)

In another particular embodiment, the first method of the invention comprises comparing the expression level of the proCOLllAl protein in the sample from a subject having an ovarian tumor or suspected of having an ovarian tumor with the expression level of said proCOLllAl protein in a control sample, wherein the expression level of the proCOLllAl protein in said sample from said subject having an ovarian tumor or suspected of having an ovarian tumor greater than the expression level of said proCOLllAl protein in a control sample is indicative that the ovarian tumor is malignant.

The characteristics of the sample to be analyzed have already been mentioned above.

Method (B) comprises determining the expression level of the proCOLllAl protein in a sample from a subject having an ovarian tumor or suspected of having an ovarian tumor and comparing it with the expression level of said proCOLllAl protein in a control sample.

The determination of the expression level of the proCOLllAl protein is carried out by means of the quantification or determination of the amount or concentration of said proCOLllAl protein in said sample.

The amount or concentration of the proCOLllAl protein present in said samples can be quantified by means of any conventional method which allows detecting and quantifying said protein in the samples to be analyzed. In this case, Method (B) comprises performing an extraction step to obtain a protein extract containing said protein. The protein extracts can be obtained by means of conventional methods (Chomczynski et al . , Anal. Biochem., 1987, 162:156; Chomczynski P., Biotechniques , 1993, 15:532) .

Virtually any duly adapted conventional method for determining the amount or concentration of proteins in a sample can be used within the scope of the present invention for quantifying the amount or concentration of proCOLllAl protein present in a sample. By way of non-limiting illustration, the amount or concentration of said proCOLllAl protein can be determined by means of conventional methods, for example, by means of using antibodies capable of binding with proCOLllAl and subsequently quantifying the complexes formed. In a particular embodiment, Method (B) comprises contacting the protein extract of the sample with a composition comprising one or more specific antibodies targeting one or more epitopes of the proCOLllAl protein, under conditions allowing the formation of antibody :proCOLHAl complexes and determining the amount or concentration of proCOLllAl protein present in the sample. Non- limiting illustrative examples of said methods include methods based on the use of antibodies, affinity chromatography techniques, ligand binding assays, etc.

In a particular embodiment, the detection of the presence of proCOLllAl is performed by means of an immunoassay based on the formation of an antigen-antibody type complex by means of using one (or more) antibodies recognizing one (or more) epitopes of proCOLllAl, and then viewing and quantifying the complexes formed by any suitable technique, including both radioctive and non-radioctive techniques such as, for example, colorimetric techniques, fluorometric techniques, luminiscent (e.g., bioluminiscent, chemiluminiscent, etc.) techniques, etc., using in turn secondary antibodies labeled with the suitable markers to that end.

The antibodies to be used for putting this particular embodiment of the first method of the invention [Method (B) ] into practice are specific antibodies recognizing the proCOLllAl protein, or an antigenic fragment of proCOLllAl, i.e., antibodies recognizing an epitope of proCOLllAl. The antibodies which can be used in this type of assays can be polyclonal sera, monoclonal antibodies, antibody fragments, such as Fv, Fab, Fab', F(ab')2, scFv (single chain Fv) , diabodies, triabodies, tetrabodies, combibodies, etc., capable of recognizing and binding to proCOLHAl, or to an antigenic fragment thereof; said antibodies can be human antibodies, humanized antibodies or antibodies of non-human origin. Advantageously, the antibody recognizing the proCOLHAl protein is an antibody specifically recognizing the VAR subdomain in the N-terminal propeptide of proCOLHAl. Illustrative examples of antibodies specifically detecting proCOLHAl have been mentioned in relation to Method (A), the content of which is incorporated herein by reference. International Patent Application WO 2013/021088 A2 describes antibodies specifically recognizing the VAR subdomain in the N- terminal propeptide of proCOLHAl. In a particular embodiment, said antibody is the monoclonal antibody, clone 1E8.33, (Garcia- Ocana, et al . , Int J Oncol. 2012; 40 (5) : 1447-54 ) .

The antibody recognizing proCOLHAl can be, optionally, conjugated to a carrier. Likewise, said antibody recognizing proCOLHAl can be labeled or non-labeled; non-limiting illustrative examples of markers which can be used include radioctive isotopes, enzymes, fluorophores , chemiluminiscent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, dyes, etc. The non-labeled antibodies can be used in agglutination assays, whereas labeled antibodies can be used in various assays.

There is a large variety of assays well known by the persons skilled in the art which can be used for putting this particular embodiment [Method (B) ] of the first method of the invention into practice, using non-labeled antibodies (primary antibody) recognizing proCOLHAl and labeled antibodies (secondary antibodies); these techniques include Western-blot, ELI SA, RIA, competitive EIA, DAS-ELI SA, immunohistochemical assays, multiplex detection techniques based on the use of protein microarrays, microspheres or biochips including specific antibodies, or assays based on colloidal precipitation, etc. In a particular preferred embodiment, the detection and quantification of the proCOLllAl protein according to Method (B) is carried out by means of an enzyme-linked immunosorbent assay (ELISA) , an known immunodetection method based on the use of one or two specific antibodies, one of them usually labeled with an enzyme, which can transform a substrate into a visible compound without affecting the capacity of the antibody to form a complex with the antigen (proCOLllAl, in this case), applied to a protein extract generally originating from a biological fluid (e.g., blood, serum, urine, ascites fluid, etc.) or from a tissue portion, or applied to the biological fluid itself. With the use of some of the specific techniques (peroxidase, antiperoxidase, fluorescein, etc.), the antigen-antibody complex formed can be detected and quantified in the samples to be studied, the levels of protein to be analyzed in samples of different origin being able to be easily quantified. In a specific embodiment, monoclonal antibody 1E8.33 or a variant thereof such as the antibody, or one of the antibodies, recognizing the proCOLllAl protein is used.

Briefly, in a particular embodiment of Method (B) , the sample comprising a biological fluid for the purpose of detecting the presence of proCOLllAl by means of an immunohistochemical assay, which can be a fresh sample or a frozen sample comprising blood, serum, urine, ascites fluid, etc., is contacted with labeled anti-proCOLl 1A1 antibody/antibodies (e.g., monoclonal antibody 1E8.33 or a variant thereof) and the concentration of the protein is quantified depending on the intensity of the enzymatic reaction associated with the antibody label. If necessary, the sample to be analyzed is subjected to a protein extraction process by means of conventional methods to concentrate the proteins, making detection thereof easier (Chomczynski et al . , Anal. Biochem., 1987, 162:156; Chomczynski P., Biotechniques , 1993, 15:532) . Advantageously, detection by means of said immunohistochemical assay of proCOLllAl in the sample to be analyzed is carried out in parallel with samples containing known levels of proCOLllAl as reference (standard curve), formed by human samples of known concentration, proCOLllAl recombinant protein or extracts from cell lines expressing the protein, which will allow calculating the concentration of proCOLUA in the samples to be analyzed by comparing the intensities obtained.

The quantification of the proCOLllAl protein by means of an ELISA-type immunodetection assay has several advantages since it is widely used in biochemical laboratories, which enables the immediate applicability not only of the first method of the invention, particularly Method (B) , but also any of the other methods provided by this invention, by means of said technique, and, furthermore, it is highly automated, which makes it easier to perform same under the same conditions .

Method (B) comprises the step of comparing the amount or concentration of proCOLllAl protein determined in the sample from the subject having an ovarian tumor or suspected of having an ovarian tumor object of study with the amount or concentration of proCOLll protein of the control sample (reference value) . In a particular embodiment, the control sample is a sample from subjects without clinical history of ovarian tumors .

Therefore, once the reference value has been established, the expression level of the proCOLllAl protein in the sample from the subject having an ovarian tumor or suspected of having an ovarian tumor under study is compared with the reference value. As a result of this comparison, the expression level of the proCOLllAl protein in the sample from the subject can be "greater than" (higher than) , "less than" ("lower than") or "equal to" said reference value for said protein in the control sample. As described above, in the context of the present invention, the expression level of the protein of interest (proCOLllAl) in the sample from the subject under study is considered "greater" than the reference value for said protein when the expression level of said protein in the sample from the subject under study increases, for example, by 3%, 5%, 10%, 25%, 50%, 100% or even more when compared with the reference value for said protein, or when it increases, for example, at least by 1.1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more when compared with the reference value for said protein. Likewise, in the context of the present invention, the expression level of the gene of interest in the sample from the subject is considered "less" than the reference value for said protein when the expression level of said protein in the sample from the subject under study decreases, for example, by 3%, 5%, 10%, 25%, 50%, 75%, or even 100% when compared with the reference value for said protein. Additionally, in the context of the present invention, the expression level of the gene of interest in the sample from the subject is considered "equal" to the reference value for said protein when the expression level of said protein in the sample from the subject under study is substantially the same as the reference value for said protein (i.e., the reference value + 3%) .

Additionally, Method (B) comprises in a subsequent step the step of correlating the result obtained after comparing the amount or concentration of proCOLllAl protein in the sample from a subject having an ovarian tumor or suspected of having an ovarian tumor with the amount or concentration of said proCOLllAl protein in a control sample with the diagnosis of malignancy of said ovarian tumor.

This correlation allows indicating that the ovarian tumor of the subject under study (subject having an ovarian tumor or suspected of having an ovarian tumor) is a malignant tumor in the event that the amount or concentration of the proCOLllAl protein in the sample from said subject under study is greater than the amount or concentration of said proCOLllAl protein in the control sample.

This information can be used effectively by the specialist for selecting the most suitable treatment to be administered to the subject having an ovarian tumor or suspected of having an ovarian tumor depending on the diagnosis of malignancy of said ovarian tumor.

2. Method for the differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion

In another aspect, the invention relates to an in vitro method for the differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion in a subject, hereinafter "second method of the invention", selected from Method (A) and Method (B) , wherein

A) Method (A) comprises:

detecting the presence of proCOLllAl protein in a sample from said subject;

wherein

the detection of the presence of proCOLllAl protein in said sample is indicative that the ovarian tumor is malignant; or, alternatively

the non-detection of the presence of proCOLllAl protein in said sample is indicative that the ovarian tumor is a benign or in situ tumor, or that the subject has a benign non-tumoral ovarian lesion; and

B) Method (B) comprises:

comparing the expression level of the proCOLllAl protein in said sample with the expression level of said proCOLllAl protein in a control sample; wherein

an expression level of the proCOLllAl protein in said sample greater than the expression level of said proCOLllAl protein in a control sample is indicative that the ovarian tumor is a malignant tumor, or, alternatively

an expression level of the proCOLllAl protein in said sample equal to or less than the expression level of said proCOLllAl protein in a control sample is indicative that the ovarian tumor is a benign or in situ tumor, or that the subject has a benign non- tumoral ovarian lesion.

Therefore according to the second method of the invention, the presence of proCOLHAl protein is detected and/or the expression level of said proCOLHAl protein is determined in a sample from the subject under study and the obtained result is correlated with possible ovarian tumor malignancy or with the existence of a benign non-tumoral ovarian lesion.

The characteristics of the sample and the proCOLHAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference .

The subject under study can be a subject having a tumoral or non-tumoral lesion in the ovary, such as, for example, a subject with an ovarian tumor, a subject suspected of having a ovarian tumor, a subject suspected of having a malignant ovarian tumor, a subject diagnosed with a low malignant potential or borderline ovarian tumor, a subject with a benign ovarian tumor, a subject with an in situ ovarian tumor, or a subject with a benign non-tumoral ovarian lesion.

Based on the detection of the presence of proCOLHAl protein or of its expression level in a sample from a subject under study, it is possible to link the detection of the presence (or the non-detection of the presence-absence) of said proCOLHAl protein in said sample, or the expression level of said proCOL-llAl protein in said sample from said subject, with possible ovarian tumor malignancy or with the existence of a benign non-tumoral ovarian lesion.

Therefore, in a subsequent step, the second method of the invention involves correlating either the detection of the presence (or the non-detection of the presence-absence) of the proCOLHAl protein in the sample from the subject under study with possible ovarian tumor malignancy or with the existence of a benign non-tumoral ovarian lesion [Method (A) ] or correlating the expression level of the proCOLHAl protein in the sample from the subject under study in comparison with the expression level of the proCOLllAl protein in a control sample with possible ovarian tumor malignancy or with the existence of a benign non-tumoral ovarian lesion [Method (B) ] .

This correlation allows establishing that the ovarian tumor is a malignant ovarian tumor when:

the presence of proCOLllAl protein is detected in the sample from the subject under study, or when the expression level of the proCOLllAl protein in the sample from the subject under study is greater than the expression level of said proCOLllAl protein in a control sample.

Said correlation also allows establishing that the ovarian tumor is a benign ovarian tumor or an in situ ovarian tumor when :

the presence of proCOLllAl protein is not detected in the sample from the subject under study, or when the expression level of the proCOLllAl protein in the sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in a control sample.

Likewise, said correlation also allows establishing that the subject has a benign non-tumoral ovarian lesion when:

the presence of proCOLllAl protein is not detected in the sample from the subject under study, or when the expression level of the proCOLllAl protein in the sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in a control sample.

Therefore, in a particular embodiment, the second method of the invention allows differentiating between a malignant ovarian tumor and a benign or in situ ovarian tumor; i.e., it allows distinguishing a benign or in situ ovarian tumor from a malignant ovarian tumor. In another particular embodiment, the second method of the invention allows differentiating between a malignant ovarian tumor and a benign non-tumoral ovarian lesion; i.e., it allows distinguishing a benign non-tumoral ovarian lesion from a malignant ovarian tumor, or in other words, it allows excluding a benign non-tumoral ovarian lesion from the ovarian tumor or carcinoma diagnosis.

The meaning of the expression "detection of the presence of proCOLHAl protein" or the like, as well as the meaning of an expression level of the proCOLHAl protein "greater" than, "equal" to or "less" than that of the proCOLHAl protein in a control sample, as it is used herein, has already been indicated above and is incorporated herein by reference. Likewise, in the sense used in this description, the expression "non-detection of the presence of proCOLHAl protein" or the like refers to the incapability to detect the proCOLHAl protein in the analyzed sample using any of the methods referenced to above.

The information provided by the second method of the invention can be used effectively by the specialist for selecting the most suitable treatment to be administered to the subject under study depending on the nature of the tumoral (benign/malignant) or non-tumoral lesion of the subject under study .

3. Method for the diagnosis or prognosis of ovarian tumor malignancy

In another aspect, the invention relates to an in vitro method for the diagnosis or prognosis of ovarian tumor malignancy in a subject, wherein said subject is a subject suspected of having a malignant ovarian tumor or is a subject diagnosed with a low malignant potential or borderline ovarian tumor, hereinafter "third method of the invention", selected from Method (A) and Method (B) , wherein

A) Method (A) comprises

detecting the presence of proCOLHAl protein in a sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor;

wherein the detection of the presence of proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor is indicative that said subject has a malignant ovarian tumor or that said low malignant potential or borderline tumor is capable of progressing into a malignant ovarian tumor, or, alternatively

the non-detection of the presence of proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor is indicative that said subject does not have a malignant ovarian tumor, or that said subject has a benign ovarian tumor, or that said subject has an in situ ovarian tumor, or that said low malignant potential or borderline tumor is not capable of progressing into a malignant ovarian tumor; and

B) Method (B) comprises

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor greater than the expression level of said proCOLllAl protein in a control sample is indicative that said subject has a malignant ovarian tumor or that said low malignant potential or borderline tumor is capable of progressing into a malignant ovarian tumor, or, alternatively an expression level of the proCOLHAl protein in said sample from said subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, equal to or less than the expression level of said proCOLHAl protein in a control sample is indicative that the subject does not have a malignant ovarian tumor, or that said subject has a benign ovarian tumor, or that said subject has an in situ ovarian tumor, or that said low malignant potential or borderline tumor is not capable of progressing into a malignant ovarian tumor.

The third method of the invention is a method of high sensitivity and specificity and is based on the fact that a subject with a malignant ovarian tumor or with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, being capable of progressing into a malignant tumor, expresses and shows the proCOLHAl protein or has high levels of proCOLHAl protein in absolute terms or in comparison with the corresponding levels in control samples from subjects without clinical history of ovarian tumors, or with benign ovarian lesions or benign ovarian tumors.

Therefore according to the third method of the invention, the presence of proCOLHAl protein is detected and/or the expression level of said proCOLHAl protein is determined in a sample from the subject under study, and the obtained result is correlated with ovarian tumor malignancy or with whether or not a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is capable of progressing into a malignant ovarian tumor.

The characteristics of the sample and the proCOLHAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first and second methods of the invention and are incorporated herein by reference.

According to the third method of the invention, the subject under study is a subject with a malignant ovarian tumor or a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations.

Based on the detection/non-detection of the presence of proCOLHAl or of its expression level in a sample from a subject under study, i.e., a subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, it is possible to link the detection of the presence (or the non-detection of the presence-absence) of the proCOLHAl protein in said sample with ovarian tumor malignancy or with whether or not the ovarian tumor is capable of malignization (progression of the tumor into a malignant tumor) .

Therefore, in a subsequent step, the third method of the invention involves correlating either the detection/non- detection (absence) of the presence of proCOLHAl protein [Method (A) ] , or correlating its expression level [Method (B) ] with a diagnosis of malignancy or of whether or not the ovarian tumor is capable of malignization.

This correlation can indicate that:

the ovarian tumor is malignant or is capable of progressing into a malignant tumor when the presence of proCOLHAl protein is detected in the sample from the subject suspected of having a malignant ovarian carcinoma or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, or when the expression level of said proCOLHAl protein in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is greater than the expression level of said proCOLHAl protein in a control sample, or

the ovarian tumor is a benign or in situ tumor, or there is no tumor whatsoever in the ovary, or the low malignant potential or borderline ovarian tumor is not capable of progressing into a malignant tumor, when the presence of proCOLllAl protein is not detected in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, under study, or when the expression level of said proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in a control sample .

In other words, this correlation allows establishing that the ovarian tumor in a subject suspected of having a malignant ovarian tumor is a malignant ovarian tumor when:

the presence of proCOLllAl protein is detected in the sample from the subject suspected of having a malignant ovarian tumor, or when

the expression level of the proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in a control sample .

Said correlation also allows establishing that the low malignant potential or borderline ovarian tumor is capable of progressing into a malignant tumor when:

the presence of proCOLllAl protein is detected in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, or when the expression level of the proCOLllAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is greater than the expression level of said proCOLllAl protein in a control sample.

Likewise, said correlation also allows establishing that the ovarian tumor in a subject suspected of having a malignant ovarian tumor is not a malignant ovarian tumor when:

the presence of proCOLllAl protein is not detected in the sample from the subject suspected of having a malignant ovarian tumor, or when

the expression level of the proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor is equal to or less than the expression level of said proCOLllAl protein in a control sample .

Furthermore, said correlation also allows establishing that the low malignant potential or borderline ovarian tumor is not capable of progressing into a malignant tumor when:

the presence of proCOLllAl protein is not detected in the sample from the subject suspected of having a malignant ovarian tumor, or when

the expression level of the proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor is equal to or less than the expression level of said proCOLllAl protein in a control sample .

Finally, this correlation together with other diagnostic tests also allows establishing that the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, does not have a malignant ovarian tumor but rather a non-tumoral ovarian lesion, a benign ovarian tumor, an in situ tumor or a borderline tumor without malignization probability, when:

the presence of proCOLllAl protein is not detected in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, or when the expression level of the proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in a control sample.

Therefore, the third method of the invention allows establishing if a subject suspected of having a malignant ovarian tumor, or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, has a malignant ovarian tumor, or a benign ovarian tumor, or an in situ ovarian tumor, or whether or not the low malignant potential or borderline ovarian tumor is capable of progressing into a malignant tumor, or if said subject does not have any tumoral ovarian lesion but rather a non-tumoral ovarian lesion .

This information can be used effectively by the specialist for selecting the most suitable treatment to be administered to the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, depending on whether or not the tumor is malignant, or whether or not it is capable of progressing into a malignant tumor, or if the subject has a non-tumoral ovarian lesion.

4. Method for determining the malignization probability of a low malignant potential or borderline ovarian tumor

In another aspect, the invention relates to an in vitro method for determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, in a subject diagnosed with said tumor, hereinafter "fourth method of the invention", selected from Method (A) and Method (B) , wherein A) Method (A) comprises

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations;

wherein

the detection of the presence of proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that the probability of the tumor progressing into a malignant ovarian tumor is high, or, alternatively the non-detection of the presence of proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is indicative that the probability of the tumor progressing into a malignant ovarian tumor is low; and

B) Method (B) comprises

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein

an expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, greater than the expression level of said proCOLllAl protein in a control sample is indicative that the probability of the tumor progressing into a malignant ovarian tumor is high; or, alternatively

an expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, equal to or less than the expression level of said proCOLHAl protein in a control sample is indicative that the probability of the tumor progressing into a malignant ovarian tumor is low.

Therefore according to the fourth method of the invention, the presence of proCOLHAl protein is detected and/or the expression level of said proCOLHAl protein is determined in a sample from the subject under study, such as a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, and the obtained result is correlated with the malignization probability of the tumor, i.e., with the probability of said tumor progressing into a malignant ovarian tumor.

The characteristics of the sample and the proCOLHAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference .

In this case, the subject under study is a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations.

Based on the detection of the presence of proCOLHAl protein or of its expression level in a sample from said subject, it is possible to link the detection of the presence (or the non-detection of the presence-absence) of said proCOLHAl protein in said sample, or the expression level of said proCOL-llAl protein in said sample from said subject, with the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor.

Therefore, in a subsequent step, the fourth method of the invention involves correlating either the detection of the presence (or the non-detection of the presence-absence) of the proCOLllAl protein in the sample from the subject under study, with the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor [Method (A) ] or correlating the expression level of the proCOLllAl protein in the sample from the subject under study in comparison with the expression level of the proCOLllAl protein in a control sample, with the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor [Method (B) ] .

This correlation allows establishing that the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor is high when:

the presence of proCOLllAl protein is detected in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, or when the expression level of the proCOLllAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is greater than the expression level of said proCOLllAl protein in a control sample.

This correlation also allows establishing that the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor is low when:

the presence of proCOLllAl protein is not detected in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, or when the expression level of the proCOLllAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in a control sample.

Generally, when the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is high, said tumor will progress into a malignant ovarian tumor; likewise, generally, when the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is low, said tumor will not progress into a malignant ovarian tumor.

Therefore, in a particular embodiment, the fourth method of the invention allows establishing the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor .

The meaning of the terms "probability", "malignization", as well as the meaning of the terms "high" and "low" applied to probability, as it is used herein, has already been indicated above and is incorporated herein by reference. The expressions "detection of the presence of proCOLllAl protein", or the like, and "non-detection of the presence of proCOLllAl protein" or the like, have also been defined above.

The information provided by the fourth method of the invention can be used effectively by the specialist for selecting the most suitable treatment to be administered to the subject under study depending on the probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, progressing into a malignant ovarian tumor .

5. Methods for selecting treatments

There are different treatments which can be applied on the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, depending on whether or not said subject has a malignant tumor or on whether or not the tumor is capable of progressing into a malignant tumor. Generally, patients having malignant tumors are subjected to a radical surgery to eliminate the affected tissue entirely, and are also subjected to additional tests for assessing the tumor infiltration level and the presence of metastasis. The degree of tumor infiltration determines the chemotherapy or radiotherapy treatments necessary for each patient, as well as the follow-up to which the patient will be subjected. In the case of benign and in situ tumors, the tumor resection can be carried out by means of conservative surgery, and additional tests for detecting metastasis are not indicated. In the case of low malignant potential or borderline tumors, if the latter are not capable of progressing into malignant tumors, they may be treated as a benign tumor, but if they are capable of progressing, they must be treated as a malignant tumor at least as regards the surgery, completely eliminating the tumor and the extraovarian implants, if any.

It is sometimes complicated to differentiate benign and in situ tumors from malignant tumors with current tumor classification methods due to the morphological and structural similarities of the tumors and to the fact that different types of tumor often appear in the same sample. In the case of low malignant potential or borderline tumors, there is no tool available today that allows predicting the progress of said tumors into malignant carcinomas.

Now it has been observed that, because it is possible to correlate the detection of the presence of, and/or the expression level of, the proCOLllAl protein in a sample from a subject suspected of having a malignant ovarian tumor with the diagnosis of tumor malignancy in the tissue extracted by means of biopsy, the specialist can correctly classify the malignant tumors. Furthermore, the detection of the proCOLllAl protein has also been linked with whether or not low malignant potential or borderline tumors are capable of progressing into a malignant tumor, being useful for classifying said tumors according to the risk of progression thereof. Therapeutic care for the subject is thus optimized and sped up and drawbacks associated with the application of insufficient or excessive surgery or post-surgery treatment are prevented, and additional surgical interventions are prevented, with the subsequent economical repercussion that it entails.

Therefore in another aspect, the invention relates to a method for selecting a subject suspected of having a malignant ovarian tumor for treatment, wherein said treatment comprises surgically removing said ovarian tumor, performing additional analyses to evaluate tumor infiltration and metastasis and choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up, hereinafter "fifth method of the invention", said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject suspected of having a malignant ovarian tumor; or

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor with the expression level of said proCOLllAl protein in a control sample; wherein said subject is selected for said treatment:

if the presence of proCOLllAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in the control sample; or, alternatively,

wherein said subject is not selected for said treatment: if the presence of proCOLllAl protein is not detected in said sample from said subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is equal to or less than the expression level of said proCOLHAl protein in the control sample .

Therefore according to the fifth method of the invention, the presence/absence of the proCOLHAl protein is detected or the expression level of said proCOLHAl protein is determined in a sample from the subject suspected of having a malignant ovarian tumor which is being evaluated and the obtained result is correlated with the possibility of selecting said subject to receive a treatment which comprises completely removing the tumor, performing additional analyses to evaluate the degree of tumor invasion, i.e., of tumor infiltration and possible metastasis that may exist; choosing the chemotherapy and/or radiotherapy treatment to be applied to the subject; and the subsequent follow-up of the subject.

The characteristics of the sample and the proCOLHAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference. In this case, the subject under study is a subject suspected of having a malignant ovarian tumor.

Once the presence/absence or the amount of proCOLHAl protein in the sample from the subject suspected of having a malignant ovarian tumor under study is analyzed, it is possible to evaluate the possibility of selecting said subject to receive a treatment, hereinafter "Treatment 1", which comprises completely removing the tumor, performing additional analyses to evaluate tumor infiltration and metastasis, applying the suitable chemotherapy and/or radiotherapy treatment and the subsequent follow-up of the patient (subject) .

The ovarian tumor can be completely removed by means of any method which allows assuring the complete removal of the tumor and/or of the affected area; non-limiting illustrative examples of such methods include cytoreduction, hysterectomy, adnexectomy, colostomy, lymph node removal, etc.

The analyses to be performed for the purpose of evaluating the degree of ovarian tumor invasion and determining tumor infiltration and metastasis include, among others, lymph nodes sampling to search for tumor presence, and image analysis by suitable techniques such as, for example, ultrasound, CAT scan, etc., to search for the points of metastasis.

In view of the obtained results, the specialist may choose a chemotherapy and/or radiotherapy treatment. In a particular embodiment, said treatment comprises a chemotherapy treatment. In a specific embodiment, said chemotherapy treatment comprises the administration of a suitable anti-tumor drug; non-limiting illustrative examples of such anti-tumor drugs include paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, doxorubicin, irinotecan, topotecan, etc. In another particular embodiment, said treatment comprises a radiotherapy treatment. In another particular embodiment, said treatment comprises a chemotherapy treatment followed by a subsequent radiotherapy treatment.

The treatment further comprises the subsequent follow-up of the patient for which the necessary diagnostic tests will be performed periodically to search for tumor relapses or the onset of metastasis, such as using imaging techniques or analyzing serum markers, such as tumor marker CA-125 (also referred to as "mucin 16" or "MUC16"), for example.

The possibility of selecting a subject to receive said Treatment 1 is established based on the detection of the presence/absence of the proCOLHAl protein or on the expression level of said protein in a sample from the subject suspected of having a malignant ovarian tumor, such that if the presence of proCOLHAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor or if the expression level of the proCOLHAl protein in said sample from the subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLHAl protein in the control sample, then said subject is selected for said Treatment 1. In contrast, i.e., if the presence of proCOLHAl protein is not detected in the sample from the subject under study (subject suspected of having a malignant ovarian tumor) or if the expression level of the proCOLllAl protein m said sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in the control sample, then said subject is not selected for said Treatment 1.

The fifth method of the invention therefore allows selecting a subject suspected of having a malignant ovarian tumor to receive said Treatment 1, a treatment which comprises completely removing the tumor, performing additional analyses to evaluate tumor infiltration and metastasis, applying the chosen chemotherapy and/or radiotherapy treatment and the subsequent follow-up of the subject. Therefore, if the presence of proCOLllAl protein is detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is greater than the expression level of said proCOLllAl protein in a control sample, then said subject is a suitable candidate a priori for receiving said Treatment 1, whereas if the presence of proCOLllAl protein is not detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is equal to or less than the expression level of said proCOLllAl protein in a control sample, then said subject no is a suitable candidate a priori for receiving said Treatment 1.

This fifth method of the invention is a valuable tool for the treatment of ovarian tumors since, in view of the results provided by evaluating ovarian tumor malignancy by means of a method such as any of the first to fourth methods provided by this invention, the specialist can select the subject who is being evaluated to receive said Treatment 1 which comprises completely removing the tumor, performing additional analyses to evaluate tumor infiltration and metastasis, applying the chosen chemotherapy and/or radiotherapy treatment and the subsequent follow-up, depending on the detection of the presence/absence of the proCOLllAl protein, or on the expression level of the proCOLllAl protein in the sample from said subject. Therefore, the methods and means provided by the present invention can aid the specialists in selecting the most suitable treatment to be administered to a subject having a malignant ovarian tumor and preventing adverse effects associated with delay in applying the suitable treatment as a result of an incorrect first diagnosis, or associated with the application of an overly aggressive treatment in subjects with benign or in situ tumors, or in low malignant potential or borderline tumors that are not capable of progressing into malignant tumors.

The fact that the subject under study is not selected to receive Treatment 1 can mean that the ovarian lesion is not a malignant tumor but rather, for example, a non-tumoral benign lesion, a benign tumor, an in situ tumor or a borderline tumor, with or without extraovarian implantations, without probability of progressing into a malignant tumor, which can be treated by means of a treatment different from Treatment 1, which may or may not consist of the surgical resection of the lesion, an optional pharmacological treatment and the absence of a subsequent follow-up for detecting relapses or progression of the lesion.

In another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment, wherein said treatment comprises surgically removing said ovarian tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up, hereinafter "sixth method of the invention", said method comprising :

detecting the presence of proCOLHAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLHAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLHAl protein in a control sample; wherein said subject is selected for said treatment:

if the presence of proCOLllAl protein is detected in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is greater than the expression level of said proCOLllAl protein in the control sample; or, alternatively

wherein said subject is not selected for said treatment: if the presence of proCOLllAl protein is not detected in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in the control sample.

Therefore according to the sixth method of the invention, the presence/absence of the proCOLllAl protein is detected or the expression level of said proCOLllAl protein is determined in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, which is being evaluated and the obtained result is correlated with the possibility of selecting said subject to receive a treatment which comprises completely removing the tumor, choosing the chemotherapy and/or radiotherapy treatment to be applied to the subject; and the subsequent follow-up of the subject.

The characteristics of the sample and the proCOLllAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference. In this case, the subject under study is a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations.

Once the presence/absence or the amount of proCOLHAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, under study is analyzed, it is possible to evaluate the possibility of selecting said subject to receive a treatment, hereinafter "Treatment 2", which comprises completely removing the tumor, applying the suitable chemotherapy and/or radiotherapy treatment and the subsequent follow-up of the subject.

The ovarian tumor can be completely removed by means of any method which allows assuring the complete removal of the tumor and/or of the affected area, such as the methods indicated in relation to the fifth method of the invention, for example.

In view of the obtained results, the specialist may choose a chemotherapy and/or radiotherapy treatment, as indicated above in relation to the fifth method of the invention. Therefore, in a particular embodiment, said treatment comprises a chemotherapy treatment based on the administration of a suitable anti-tumor drug, such as, for example, paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide, gemcitabine, doxorubicin, irinotecan, topotecan, etc., optionally followed by a subsequent radiotherapy treatment. In another particular embodiment, said treatment comprises a radiotherapy treatment.

Treatment 2 further comprises the subsequent follow-up of the patient, for which the necessary diagnostic tests will be performed periodically to search for tumor relapses or the onset of metastasis, which can include, for example, analyzing images by means of ultrasound, CAT scan, etc., analyzing serum markers such as CA-125 ( "MUC16" ) .

The possibility of selecting a subject to receive said Treatment 2 is established based on the detection of the presence/absence of the proCOLHAl protein or on the expression level of said protein in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, under study such that if the presence of proCOLllAl protein is detected in said sample from said subject or if the expression level of the proCOLllAl protein in said sample from said subject is greater than the expression level of said proCOLllAl protein in the control sample, then said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is selected for said Treatment 2. In contrast, i.e., if the presence of proCOLllAl protein is not detected in the sample from the subject under study or if the expression level of the proCOLllAl protein in said sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in the control sample, then said subject is not selected for said Treatment 2.

The sixth method of the invention therefore allows selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, to receive said Treatment 2, a treatment which comprises completely removing the tumor, applying the chosen chemotherapy and/or radiotherapy treatment and the subsequent follow-up of the subject. Therefore, if the presence of proCOLllAl protein is detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is greater than the expression level of said proCOLllAl protein in a control sample, then said subject is a suitable candidate a priori for receiving said Treatment 2, whereas if the presence of proCOLllAl protein is not detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is equal to or less than the expression level of said proCOLllAl protein in a control sample, then said subject is not a suitable candidate a priori for receiving said Treatment 2.

This sixth method of the invention is a valuable tool for the treatment of ovarian tumors since, in view of the results provided by evaluating ovarian tumor malignancy by means of a method such as any of the first to fourth methods provided by this invention, the specialist can select the subject who is being evaluated to receive said Treatment 2 which comprises completely removing the tumor, applying the chosen chemotherapy and/or radiotherapy treatment and the subsequent follow-up, depending on the detection of the presence/absence of the proCOLllAl protein, or on the expression level of the proCOLllAl protein in the sample from said subject. Therefore, the methods and means provided by the present invention can aid the specialists in selecting the most suitable treatment to be administered to a subject having a malignant ovarian tumor and preventing adverse effects associated with delay in applying the suitable treatment as a result of an incorrect first diagnosis, or associated with the application of an overly aggressive treatment in subjects with benign or in situ tumors, or in low malignant potential or borderline tumors, with or without extraovarian implantations, that are not capable of progressing into malignant tumors .

The fact that the subject under study is not selected to receive Treatment 2 can mean that the borderline tumor has no probability of progressing into a malignant tumor, and can be treated by means of a treatment different from Treatment 2, which may or may not consist of the surgical resection of the tumor, an optional pharmacological treatment and the absence of a subsequent follow-up for detecting relapses or progression of the lesion.

6. Methods for selecting follow-ups

There are different follow-ups which can be applied on the subject suspected of having a malignant ovarian tumor or diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, depending on whether or not said subject has a malignant tumor or on whether or not the tumor is capable of progressing into a malignant tumor. As mentioned above, the patients having malignant tumors are usually subjected to a radical surgery to eliminate the affected tissue entirely, and also to additional tests for assessing the tumor infiltration level and the presence of metastasis, the result of which determines the chemotherapy and/or radiotherapy treatment to be administered to the patient, as well as the follow-up up to which the patient will be subjected. In the case of benign or in situ tumors, the tumor resection can be carried out by means of conservative surgery and additional tests for detecting metastasis are generally not indicated, although performing a follow-up of the disease is advisable. In the case of low malignant potential or borderline tumors, if the latter are not capable of progressing into malignant tumors, they may be treated as benign tumors, but if they are capable of progressing into malignant tumors, they must be treated as malignant tumors at least as regards the surgery, completely eliminating the tumor and the extraovarian implants, if any; in any case, performing a follow-up of the subject is advisable to prevent or treat possible relapses as soon as possible .

Now it has been observed that it is possible to correlate the detection of the presence of, and/or the expression level of, the proCOLHAl protein in a sample from a subject suspected of having a malignant ovarian tumor with the diagnosis of tumor malignancy in the tissue extracted by means of biopsy, as well as with whether or not a low malignant potential or borderline tumor is capable of progressing into a malignant tumor, so the specialist can correctly classify the tumoral or non-tumoral ovarian lesions, establish the treatment to be applied and design the corresponding follow-up.

Therefore in another aspect, the invention relates to a method for selecting a subject suspected of having a malignant ovarian tumor for follow-up, wherein said follow-up comprises performing additional analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment, hereinafter "seventh method of the invention", said method comprising: detecting the presence of proCOLllAl protein in a sample from said subject suspected of having a malignant ovarian tumor; or

comparing the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor with the expression level of said proCOLllAl protein in a control sample; wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor; or

if the expression level of the proCOLllAl protein in said sample from said subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in the control sample .

Therefore according to the seventh method of the invention, the presence/absence of the proCOLllAl protein is detected or the expression level of said proCOLllAl protein is determined in a sample from the subject suspected of having a malignant ovarian tumor which is being evaluated and the obtained result is correlated with the possibility of selecting said subject to be subjected to a follow-up which comprises performing additional analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment.

The characteristics of the sample and the proCOLllAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference. In this case, the subject under study is a subject suspected of having a malignant ovarian tumor.

Once the presence/absence or the amount of proCOLllAl protein in the sample from the subject suspected of having a malignant ovarian tumor under study is analyzed, it is possible to evaluate the possibility of selecting said subject to be subjected to a follow-up, hereinafter "Follow-up 1", which comprises performing additional analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment.

In the event that the presence of proCOLllAl protein is detected in a sample from the subject under study (subject suspected of having a malignant ovarian tumor) , or in the event that the expression level of the proCOLllAl protein in a sample from said subject is greater than the expression level of proCOLllAl in a control sample, then the subject can be selected to be subjected to Follow-up 1, once the ovarian tumor has been surgically eliminated. In this case, the subject is subjected to analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment; said analyses include, among others, analysis of the spread of the tumor outside the ovary by means of lymph nodes sampling to search for tumor presence, image analysis by suitable techniques such as, for example, ultrasound, CAT scan, etc., to search for the points of metastasis, application of surgical or pharmacological treatment suitable for the diagnosis of said tests, and the performance of periodic analyses along with or after treatment, which allow detecting tumor relapses, for example, by means of using imaging techniques or analyzing serum markers, such as tumor marker CA-125 (also referred to as "mucin 16" or "MUC16"), for example.

In view of the results obtained by means of the follow-up, the specialist can choose the suitable treatment, for example, a chemotherapy and/or radiotherapy treatment, as mentioned above in relation to the fifth method of the invention.

The possibility of selecting a subject to be subjected to said Follow-up 1 is established based on the detection of the presence/absence of the proCOLllAl protein or on the expression level of said protein in a sample from the subject suspected of having a malignant ovarian tumor, such that if the presence of proCOLllAl protein is detected in said sample from the subject suspected of having a malignant ovarian tumor, or if the expression level of the proCOLllAl protein in said sample from the subject suspected of having a malignant ovarian tumor is greater than the expression level of said proCOLllAl protein in the control sample, then said subject is selected to be subjected to said Follow-up 1. In contrast, i.e., if the presence of proCOLllAl protein is not detected in the sample from the subject under study (subject suspected of having a malignant ovarian tumor) or if the expression level of the proCOLllAl protein in said sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in the control sample, then said subject is not selected to be subjected to said Follow-up 1.

The seventh method of the invention therefore allows selecting a subject suspected of having a malignant ovarian tumor to be subjected to said Follow-up 1, a follow-up which comprises performing additional analyses to evaluate the degree of tumor infiltration for the purpose of determining the subsequent treatment. Therefore, if the presence of proCOLllAl protein is detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is greater than the expression level of said proCOLllAl protein in a control sample, then said subject is a suitable candidate a priori to be subjected to said Follow-up 1.

This seventh method of the invention is also a valuable tool for the treatment of ovarian tumors since, in view of the results provided by evaluating ovarian tumor malignancy by means of a method such as any of the first to fourth methods provided by this invention, the specialist can select the subject who is being evaluated to be subjected to said Follow-up 1 depending on the detection of the presence/absence of the proCOLllAl protein, or on the expression level of the proCOLllAl protein in the sample from said subject. Therefore, the methods and means provided by the present invention can aid the specialists in selecting the most suitable follow-up to which a subject having a malignant ovarian tumor is subjected with the advantages that it entails.

Additionally in another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for follow-up, wherein said follow- up comprises surgically resectioning said tumor and monitoring tumor recurrence, hereinafter "eight method of the invention", said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is detected in said sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor; with or without extraovarian implantations, is greater than the expression level of said proCOLllAl protein in the control sample.

Therefore according to the eight method of the invention, the presence/absence of the proCOLllAl protein is detected or the expression level of said proCOLllAl protein is determined in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, which is being evaluated and the obtained result is correlated with the possibility of selecting said subject to be subjected to a follow-up which comprises surgically resectioning said tumor and monitoring tumor recurrence .

The characteristics of the sample and the proCOLHAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference. In this case, the subject under study is a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations.

Once the presence/absence or the amount of proCOLHAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, under study is analyzed, it is possible to evaluate the possibility of selecting said subject to be subjected to a follow-up, hereinafter "Follow-up 2", which comprises surgically resectioning the tumor and monitoring tumor recurrence .

In the event that the presence of proCOLHAl protein is detected in a sample from the subject under study, or in the event that the expression level of the proCOLHAl protein in a sample from said subject is greater than the expression level of proCOLHAl in a control sample, then the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, can be selected to be subjected to Follow-up 2. In this case, since the tumor would have a high probability of progressing into a malignant ovarian tumor, then the follow-up comprises surgically resectioning the tumor by means of any suitable surgical method which allows completely eliminating or removing the tumor (for example, by means of any of the methods indicated in relation to the fifth method of the invention) , and monitoring tumor recurrence, for which the necessary periodic tests would be performed either by means of image analysis by suitable techniques such as ultrasound, CAT scan, etc., or by means of analyzing serum markers, such as, tumor marker CA-125 ("MUC16"), for example, which allows detecting tumor relapses.

In view of the results obtained by means of Follow-up 2, the specialist can therefore in turn select the suitable treatment .

The possibility of selecting a subject to be subjected to said Follow-up 2 is established based on the detection of the presence/absence of the proCOLllAl protein or on the expression level of said protein in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, such that if the presence of proCOLllAl protein is detected in said sample from the subject, or if the expression level of the proCOLllAl protein in said sample from the subject is greater than the expression level of said proCOLllAl protein in the control sample, then said subject is selected to be subjected to said Follow-up 2. In contrast, i.e., if the presence of proCOLllAl protein is not detected in the sample from the subject under study or if the expression level of the proCOLllAl protein in said sample from the subject under study is equal to or less than the expression level of said proCOLllAl protein in the control sample, then said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is not selected to be subjected to said Follow-up 2.

The eight method of the invention therefore allows selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, to be subjected to said Follow-up 2, a follow-up which comprises surgically resectioning the tumor and monitoring tumor recurrence. Therefore, if the presence of proCOLllAl protein is detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is greater than the expression level of said proCOLllAl protein in a control sample, then said subject is a suitable candidate a priori to be subjected to said Follow-up 2.

This eight method of the invention is also a valuable tool for the treatment of ovarian tumors since, in view of the results provided by evaluating ovarian tumor malignancy by means of a method such as any of the first to fourth methods provided by this invention, the specialist can select the subject who is being evaluated to be subjected to said Follow-up 2 depending on the detection of the presence/absence of the proCOLllAl protein, or on the expression level of the proCOLllAl protein in the sample from said subject. Therefore, the methods and means provided by the present invention can aid the specialists in selecting the most suitable follow-up to which a subject having a malignant ovarian tumor is subjected with the advantages that it entails.

Likewise in another aspect, the invention relates to a method for selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for follow-up, wherein said follow- up comprises monitoring tumor recurrence, hereinafter "ninth method of the invention", said method comprising:

detecting the presence of proCOLllAl protein in a sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

comparing the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, with the expression level of said proCOLllAl protein in a control sample;

wherein said subject is selected for said follow-up:

if the presence of proCOLllAl protein is not detected in said sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or

if the expression level of the proCOLllAl protein in said sample from said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is equal to or less than the expression level of said proCOLllAl protein in the control sample.

Therefore according to the ninth method of the invention, the presence/absence of the proCOLllAl protein is detected or the expression level of said proCOLllAl protein is determined in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, which is being evaluated and the obtained result is correlated with the possibility of selecting said subject to be subjected to a follow-up which comprises monitoring tumor recurrence.

The characteristics of the sample and the proCOLllAl protein as well as the methods for detecting and/or quantifying said protein have already been mentioned above in relation to the first method of the invention and are incorporated herein by reference. In this case, the subject under study is a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations.

Once the presence/absence or the amount of proCOLllAl protein in the sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, under study is analyzed, it is possible to evaluate the possibility of selecting said subject to be subjected to a follow-up, hereinafter "Follow-up 3", which comprises monitoring tumor recurrence.

In the event that the presence of proCOLllAl protein is not detected in a sample from the subject under study, or in the event that the expression level of the proCOLllAl protein in a sample from said subject is equal to or less than the expression level of proCOLllAl in a control sample, then the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, can be selected to be subjected to Follow-up 3. In this case, since the tumor would have a low probability of progressing into a malignant ovarian tumor, then the follow-up may not require surgically resectioning the tumor, rather monitoring possible tumor progression would be enough, for which the necessary periodic tests would be performed either by means of image analysis by suitable techniques such as ultrasound, CAT scan, etc., or by means of analyzing markers, such as, for example, proCOLllAl, CA-125 ("MUC16"), etc., which allow detecting any change in whether or not the tumor is capable of malignization.

In view of the results obtained by means of Follow-up 3, the specialist can therefore in turn select the suitable treatment .

The possibility of selecting a subject to be subjected to said Follow-up 3 is established based on the detection of the presence/absence of the proCOLllAl protein or on the expression level of said protein in a sample from the subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, such that if the presence of proCOLllAl protein is not detected in said sample from the subject, or if the expression level of the proCOLllAl protein in said sample from the subject is equal to or less than the expression level of said proCOLllAl protein in the control sample, then said subject is selected to be subjected to said Follow-up 3. In contrast, i.e., if the presence of proCOLllAl protein is detected in the sample from the subject under study or if the expression level of the proCOLllAl protein in said sample from the subject under study is greater than the expression level of said proCOLllAl protein in the control sample, then said subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, is not selected to be subjected to said Follow-up 3.

The ninth method of the invention therefore allows selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, to be subjected to said Follow-up 3, a follow-up which comprises monitoring tumor recurrence. Therefore, if the presence of proCOLllAl protein is not detected in the analyzed sample from the subject, or if the expression level of the proCOLllAl protein in said sample is equal to or less than the expression level of said proCOLllAl protein in a control sample, then said subject is a suitable candidate a priori to be subjected to said Follow-up 3.

This ninth method of the invention is also a valuable tool for the treatment of ovarian tumors since, in view of the results provided by evaluating ovarian tumor malignancy by means of a method such as any of the first to fourth methods provided by this invention, the specialist can select the subject who is being evaluated to be subjected to said Follow-up 3 depending on the detection of the presence/absence of the proCOLllAl protein, or on the expression level of the proCOLllAl protein in the sample from said subject. Therefore, the methods and means provided by the present invention can aid the specialists in selecting the most suitable follow-up to which a subject having a malignant ovarian tumor is subjected with the advantages that it entails.

Uses

In another aspect, the invention relates to the use of the proCOLllAl protein as a marker for:

a) detecting a malignant ovarian tumor; or for

b) performing differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion; or for c) making a diagnosis or prognosis of ovarian tumor malignancy, or for

d) determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or for e) selecting a subject suspected of having a malignant ovarian tumor for treatment which comprises surgically removing the tumor, performing additional analyses to evaluate the degree of tumor invasion, applying a chemotherapy and/or radiotherapy treatment, and a subsequent follow-up; or for

f) selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment which comprises surgically removing said tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up; or for

g) selecting a subject suspected of having a malignant ovarian tumor, wherein said follow-up comprises performing complementary analyses for assessing the degree of tumor infiltration for the purpose of determining the subsequent treatment; or for h) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises surgically resectioning the tumor and monitoring tumor recurrence; or for

i) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises monitoring tumor recurrence.

In another aspect, the invention relates to the use of a specific antibody recognizing the proCOLllAl protein for:

a) detecting a malignant ovarian tumor; or for

b) performing differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion; or for c) making a diagnosis or prognosis of ovarian tumor malignancy, or for

d) determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or for e) selecting a subject suspected of having a malignant ovarian tumor for treatment which comprises surgically removing the tumor, performing additional analyses to evaluate the degree of tumor invasion, applying a chemotherapy and/or radiotherapy treatment, and a subsequent follow-up; or for

f) selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment which comprises surgically removing said tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up; or for

g) selecting a subject suspected of having a malignant ovarian tumor, wherein said follow-up comprises performing complementary analyses for assessing the degree of tumor infiltration for the purpose of determining the subsequent treatment; or for h) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises surgically resectioning the tumor and monitoring tumor recurrence; or for

i) selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises monitoring tumor recurrence.

The characteristics of the specific antibody recognizing the proCOLllAl protein, or a fragment thereof recognizing the proCOLllAl protein, have been mentioned in relation to the first method of the invention, particularly in relation to Method (A) , and are incorporated herein by reference. Advantageously, the antibody recognizing the proCOLllAl protein is an antibody specifically recognizing the VAR subdomain in the N-terminal propeptide of proCOLllAl. Illustrative examples of antibodies specifically detecting proCOLllAl are mentioned in International Patent Application WO 2013/021088 A2. In a particular embodiment, said antibody is the monoclonal antibody 1E8.33 (Garcia-Ocana, et al . , Int J Oncol. 2012; 40 (5 ) : 1447-54 ) , an antibody specifically recognizing the proCOLllAl protein, without detecting other proteins with high sequence homology such as the COL5A1 protein.

In another aspect, the invention relates to the use of a kit comprising a reagent recognizing the proCOLllAl protein for: detecting a malignant ovarian tumor; or for

performing differential diagnosis of a malignant ovarian tumor from a benign or in situ ovarian tumor or from a benign non-tumoral ovarian lesion; or for making a diagnosis or prognosis of ovarian tumor malignancy, or for

determining the malignization probability of a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations; or for

selecting a subject suspected of having a malignant ovarian tumor for treatment which comprises surgically removing the tumor, performing additional analyses to evaluate the degree of tumor invasion, applying a chemotherapy and/or radiotherapy treatment, and a subsequent follow-up; or for

selecting a subject diagnosed with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, for treatment which comprises surgically removing said tumor, choosing a chemotherapy and/or radiotherapy treatment, and the subsequent follow-up; or for

selecting a subject suspected of having a malignant ovarian tumor, wherein said follow-up comprises performing complementary analyses for assessing the degree of tumor infiltration for the purpose of determining the subsequent treatment; or for

selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises surgically resectioning the tumor and monitoring tumor recurrence; or for

selecting a subject with a low malignant potential or borderline ovarian tumor, with or without extraovarian implantations, wherein said follow-up comprises monitoring tumor recurrence,

a particular embodiment, said reagent recognizing the proCOLHAl protein is a specific antibody recognizing the proCOLHAl protein, or a fragment thereof recognizing the proCOLHAl protein. The characteristics of said specific antibody recognizing the proCOLHAl protein, or a fragment thereof recognizing the proCOLHAl protein, have been mentioned in relation to the first method of the invention, particularly in relation to Method (A) and are incorporated herein by reference. Advantageously, the antibody recognizing the proCOLHAl protein is an antibody specifically recognizing the VAR subdomain in the N-terminal propeptide of proCOLHAl. Illustrative examples of antibodies specifically detecting proCOLHAl are mentioned in International Patent Application WO 2013/021088 A2. In a particular embodiment, said antibody is the monoclonal antibody 1E8.33 (Garcia-Ocana, et al . , Int J Oncol. 2012; 40 (5 ) : 1447-54 ) , an antibody specifically recognizing the proCOLHAl protein, without detecting other proteins with high sequence homology such as the COL5A1 protein.

The following example illustrates the invention and must not be considered as limiting same.

Example 1

Differential expression of proCOLHAl in ovarian tumors and the relation thereof with tumor malignancy

1.1 Materials and Methods

Immunohistochemistry

Sections of the biopsies fixed in formalin and embedded in paraffin from patients suspected of having an ovarian tumor were made with microtome. The sections of 3-4 μιτι were dried throughout the night at 54-56°C in an oven. A pre-treatment with CC2 [Cell Conditioning 2] buffer [Ventana Medical Systems, Inc.; catalog no. 950-123] was applied at 98°C for 32 minutes. After titrating to adjust the suitable antibody concentration of 65, 26 and 13 g/ml, the preparations were incubated with the proCOLHAl-specific monoclonal antibody 1E8.33 (WO 2013/021088) at a concentration of 26 g/ml in an antibody diluent (Ventana- Roche, Tucson, Arizona) for 32 minutes at room temperature. The Optiview detection system (Ventana) was used and it was developed with diaminobenzidine (DAB) (Ventana) .

Thirty-one cases of patients suspected of having ovarian tumor were analyzed with the proCOLHAl-specific monoclonal antibody 1E8.33. The immunostaining was evaluated with a double- blind assay by 2 different observers. It was considered positive when at least one cell with fibroblastic morphology had immunoprecipitate .

1.2 Analysis of the statistical data

Fisher's exact test was used to calculate frequency distribution. p<0.05 was considered significant.

1.3 Results

The expression of the proCOLllAl protein in the histology sections of biopsies of the ovary from patients suspected of having an ovarian tumor was studied by means of using a proCOLllAl protein-specific antibody (1E8.33) . The objective of these experiments was to determine if the presence of proCOLllAl immunolabeling could be associated with the presence of a malignant ovarian tumor. From the 31 analyzed cases, 21 were positive for immunolabeling with the proCOLllAl protein-specific antibody, i.e., they showed the expression of the proCOLllAl protein, and 10 were negative (Figure 1) .

From the 21 cases which were positive for proCOLllAl immunolabeling, 20 patients were diagnosed based on the biopsy with malignant ovarian tumor: 2 endometrioid adenocarcinomas, 3 mucinous adenocarcinomas, 2 mixed or unclassified carcinomas, 10 papillary serous carcinomas, and 3 colon metastasis to ovary. One of the samples which were positive for proCOLllAl immunolabeling was clinically diagnosed in the biopsy with a benign tumor (Figure 2) .

From the 10 negative cases without the expression of proCOLllAl, 7 cases corresponded to malignant ovarian tumor and 3 cases corresponded to benign or in situ ovarian tumors.

According to the Fischer's test, the frequency of positive staining for proCOLllAl had statistically significant differences (p<0.05) between the group of patients with malignant ovarian tumors and the group of patients with benign and in situ ovarian tumors.

Furthermore, the sensitivity for detecting malignant ovarian tumors in biopsy tissues (74%), and the specificity for detecting tumor infiltration in biopsy cores (75%) as well as the positive predictive values (PPVs) and negative predictive values (NPVs) for diagnosing infiltration (PPV 95% and NPV 30%), were calculated. These results demonstrate that this marker (proCOLHAl) has a high sensitivity and specificity for detecting malignant ovarian tumors.

1.4 Conclusions

Immunostaining with the proCOLHAl-specific monoclonal antibody 1E8.33 shows high sensitivity and specificity in the detection of malignant ovarian tumors.