BOX BINDING PROTEIN (YB-1)

Field of the invention

The present invention is in the field of medicine, in particular to the field diagnostics and prognosis of cancer, particularly ovarian cancer. Furthermore, it relates to methods, means and kits for diagnosis of cancer and for the detection of YB-1 antibodies in samples of patients.

Background of the invention

According to the American Cancer Society ovarian cancer is expected to account for over 22,000 new cancer diagnoses and more than 14,000 deaths in 2013 in the US alone. Of the gynaecologic malignancies, ovarian cancer has the highest mortality rate. In early stages of the disease, ovarian cancer is nearly asymptomatic. Hence, a large portion of the patients present with clinically advanced stages of ovarian cancer. However, the 5-year survival rate for patients diagnosed with early-stage disease is often >90%, but it is <20% for advanced- stage disease, underscoring the importance of early detection.

Current diagnosis of ovarian cancer relies on pelvic exam, transvaginal ultrasonography, (TVS), abdominal ultrasonography, and exploratory or diagnostic laparoscopy. The most commonly used biomarker for clinical screening and prognosis in patients with ovarian cancer is ovarian cancer antigen 125 (CA125) (Coticchia et al. (2008), J. Natl. Compr. Cane. Netw. 6(8):795-802). Serum CA125 levels are elevated in ¾80% of patients with advanced- stage epithelial ovarian cancer but are increased in only 50—60% of patients with early-stage disease. Serum CA125 levels may be falsely elevated in women with any i.p. pathology resulting in irritation of the serosa of the peritoneum or pericardium, uterine fibroids, renal disorders, and normal menses. Moreover, serum CA125 levels do not predict the outcome of cytoreductive surgery in patients with advanced epithelial ovarian cancer. Further biomarkers include, for example, Human Epidymis Protein 4 (HE4) and Mesothelin (Sarojini et al. (2012), Journal of Oncology 102, Article ID 709049). Severeness of ovarian cancer is categorized by the grade and stage of tumorization. This nowadays can only be performed by evaluation of the tumors under or after surgical treatment or by combining marker evaluation and (histological) evaluation of tissue. Staging may be very important because ovarian cancers have different prognosis at different stages and may be treated differently. The accuracy of the staging may determine whether or not a patient will be cured. If the cancer isn't accurately staged, then cancer that has spread outside the ovary might be missed and not treated. Once a stage has been given it does not change, even when the cancer comes back or spreads to new locations in the body.

Ovarian cancer staging is by FIGO staging system uses information obtained after surgery, which can include a total abdominal hysterectomy, removal of (usually) both ovaries and fallopian tubes, (usually) the omentum, and pelvic (peritoneal) washings for cytopathology. The AJCC stage is the same as the FIGO stage. The AJCC staging system describes the extent of the primary Tumor (T), the absence or presence of metastasis to nearby lymph Nodes (N), and the absence or presence of distant Metastasis (M).

Stage I limited to one or both ovaries

LA involves one ovary; capsule intact; no tumor on ovarian surface; no malignant cells in ascites or peritoneal washings

LB involves both ovaries; capsule intact; no tumor on ovarian surface; negative washings

LC tumor limited to ovaries with any of the following: capsule ruptured, tumor on ovarian surface, positive washings

Stage Π pelvic extension or implants

LLA extension or implants onto uterus or fallopian tube; negative washings

LLB extension or implants onto other pelvic structures; negative washings

LLC pelvic extension or implants with positive peritoneal washings

Stage ΠΙ peritoneal implants outside of the pelvis; or limited to the pelvis with extension

to the small bowel or omentum ΠΙΑ microscopic peritoneal metastases beyond pelvis

IIIB macroscopic peritoneal metastases beyond pelvis less than 2 cm in size

IIIC peritoneal metastases beyond pelvis > 2 cm or lymph node metastases

Stage IV distant metastases to the liver or outside the peritoneal cavity

Para-aortic lymph node metastases are considered regional lymph nodes (Stage IIIC). As there is only one para-aortic lymph node intervening before the thoracic duct on the right side of the body, the ovarian cancer can rapidly spread to distant sites such as the lung. The AJCC/TNM staging system includes three categories for ovarian cancer, T, N and M. The T category contains three other subcategories, Tl , T2 and T3, each of them being classified according to the place where the tumor has developed (in one or both ovaries, inside or outside the ovary). The Tl category of ovarian cancer describes ovarian tumors that are confined to the ovaries, and which may affect one or both of them. The sub-subcategory Tl a is used to stage cancer that is found in only one ovary, which has left the capsule intact and which cannot be found in the fluid taken from the pelvis. Cancer that has not affected the capsule, is confined to the inside of the ovaries and cannot be found in the fluid taken from the pelvis but has affected both ovaries is staged as Tib. Tic category describes a type of tumor that can affect one or both ovaries, and which has grown through the capsule of an ovary or it is present in the fluid taken from the pelvis. T2 is a more advanced stage of cancer. In this case, the tumor has grown in one or both ovaries and is spread to the uterus, fallopian tubes or other pelvic tissues. Stage T2a is used to describe a cancerous tumor that has spread to the uterus or the fallopian tubes (or both) but which is not present in the fluid taken from the pelvis. Stages T2b and T2c indicate cancer that metastasized to other pelvic tissues than the uterus and fallopian tubes and which cannot be seen in the fluid taken from the pelvis, respectively tumors that spread to any of the pelvic tissues (including uterus and fallopian tubes) but which can also be found in the fluid taken from the pelvis. T3 is the stage used to describe cancer that has spread to the peritoneum. This stage provides information on the size of the metastatic tumors (tumors that are located in other areas of the body, but are caused by ovarian cancer). These tumors can be very small, visible only under the microscope (T3a), visible but not larger than 2 centimeters (T3b) and bigger than 2 centimeters (T3c). This staging system also uses N categories to describe cancers that have or not spread to nearby lymph nodes. There are only two N categories, NO which indicates that the cancerous tumors have not affected the lymph nodes, and Nl which indicates the involvement of lymph nodes close to the tumor. The M categories in the AJCC/TNM staging system provide information on whether the ovarian cancer has metastasized to distant organs such as liver or lungs. MO indicates that the cancer did not spread to distant organs and Ml category is used for cancer that has spread to other organs of the body.

The AJCC/TNM staging system also contains a Tx and a Nx sub-category which indicates that the extent of the tumor cannot be described because of insufficient data, respectively the involvement of the lymph nodes cannot be described because of the same reason. The ovarian cancer stages are made up by combining the TNM categories in the following manner:

Stage I: T1+N0+M0; Li: Tl a+N0+M0; IB: Tlb+N0+M0; IC: Tl c+N0+M0;

Stage Π: T2+N0+M0; Ila: T2a+N0+M0; IIB: T2b+N0+M0; IIC: T2c+N0+M0;

Stage 111: T3+ N0+M0; IIIA : T3a+ N0+M0; MB: T3b+ N0+M0; IIIC: T3c+ N0+M0 or any T+N1+M0;

Stage IV: Any T+ Any N+Ml

In addition to being staged, like all cancers ovarian cancer is also graded. The histologic grade of a tumor measures how abnormal or malignant its cells look under the microscope. There are four grades indicating the likelihood of the cancer to spread and the higher the grade, the more likely for this to occur. Grade 0 is used to describe non-invasive tumors. Grade 0 cancers are also referred to as borderline tumors. Grade 1 tumors have cells that are well differentiated (look very similar to the normal tissue) and are the ones with the best prognosis. Grade 2 tumors are also called moderately well differentiated and they are made up by cells that resemble the normal tissue. Grade 3 tumors have the worst prognosis and their cells are abnormal, referred to as poorly differentiated. However, there is a need for improved tools for the early detection; staging, grading and prognosis of ovarian cancer. In particular there is a need for predicting response to a cancer treatment.

The pi 8 fragment of Y binding protein (YB-1) has been shown to have a high specific prevalence in malignancies; see Tacke et al.: High prevalence of Y-boxprotein-l/pl 8 fragment in plasma of patients with malignancies of different origin, BMC Cancer 2014, 14:33.

Summary of the invention

The inventors now found that auto-antibodies directed against the Y binding protein (YB-1) exist. Furthermore, it has been observed that levels of such antibodies are lower in samples of patients suffering from cancer, in particular ovarian cancer. Furthermore, as shown in the Examples provided herein, the higher the level of antibodies against YB-1 in patients suffering from cancer the higher the chance that the patient will respond to chemotherapy. The data presented herein demonstrate that auto-antibodies directed against YB-1 provide an advantageous tool for diagnosis and treatment of cancer.

Hence, the present application relates to a method for diagnosis of cancer, comprising the steps of (i) determining the level of antibodies against Y box binding protein 1 (YB-1) in a sample from a subject to be diagnosed, and (ii) comparing the determined level in the sample to a control level of YB-1 antibodies derived from subjects without cancer; wherein a decreased level in the sample from the subject to be diagnosed as compared to the control level is indicative of cancer in the subject to be diagnosed

The invention further pertains to a method for diagnosis of cancer, wherein a level of anti- YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.8 fold as compared to the control level is indicative of the presence of cancer in the subject to be diagnosed, preferably a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.7 fold as compared to the control level is indicative of the presence of cancer in the subject to be diagnosed, more preferably a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.65 fold as compared to the control level is indicative of the presence of cancer in the subject to be diagnosed; yet further preferred a level of anti-YB- 1 antibodies in the sample of the patient to be diagnosed of less than 0.6 fold as compared to the control level is indicative of the presence of cancer in the subject to be diagnosed.

The invention also pertains to a method for diagnosis of a cancer, wherein the level of antibodies against YB-1 is determined in a sample from a subject to be diagnosed, and wherein a level of anti-YB-1 antibodies below 5 units/ml is indicative of cancer, preferably below 4 units/ml, more preferably below 3.5 units/ml, further preferred below 3 units/ml.

The inventors for the first time show the presence of antibodies in samples of subjects. The antibodies in said samples may be detected through there ability to bind YB-1 or an antigenic fragment thereof. Hence, the present invention also relates to a method for detecting an anti-YB-1 antibody in a sample from a subject, comprising the steps of

(a) contacting the sample suspected of comprising an anti-YB-1 antibody with Y box binding protein 1 (YB-1) or an antigenic peptide fragment thereof under conditions allowing for the formation of a complex between the anti-YB-1 antibody with YB-1 or the peptide fragment thereof,

(b) detecting the complex. The assay is preferably an immunoassay.

In the context of the present invention YB-1 or an antigenic peptide fragment thereof can thus be used for the diagnosis of cancer.

The present invention further relates to research and/or diagnostic kit for the diagnosis of cancer or for the prediction of response or non-response in a patient, wherein the kit comprises Y box binding protein 1 (YB-1) or an antigenic (immunogenic) peptide fragment thereof. The inventors also found that the level of antibodies against Y box binding protein 1 (YB-1) correlates with the risk of relapse in subjects treated with a chemotherapeutic drug. Decreased levels of anti-YB-1 antibodies in samples correlated with a higher risk of relapse in patients treated with said chemotherapeutic drug. Hence, levels of anti-YB-1 antibodies in samples of patients to be treated with a chemotherapeutic drug are an indicator for response or non-response of a patient, i.e. whether amelioration of the disease is achieved in a patient (responder) or not (non-responder). If a patient responds to a treatment the disease is ameliorated. It might be the case that a patient responds to a treatment at first but suffers from relapse of the disease at a later stage. Also this is a form of non-response. However, it is difficult to predict whether a patient will respond or not to a treatment, as it may be determined only at later stages with the known methods, e.g. when relapse. This problem is solved by the present invention as it provides a method to predict whether a subject will respond or not to a certain treatment, e.g. a treatment with a chemotherapeutic drug. Hence, the present invention also relates to method for predicting whether a subject being treated or to be treated for cancer with a drug will respond to said treatment comprising the steps of

(i) determining the level of antibodies against Y box binding protein 1 (YB-1) in a sample from said subject to be treated,

(ii) comparing the determined level in the sample to either one or both of a first and second anti-YB-1 antibody control level,

a) wherein the first anti-YB-1 antibody control level is derived from one or more subjects responding to said treatment, and

b) wherein the second anti-YB-1 antibody control level is derived from one or more subjects not responding to said treatment,

wherein a decreased level in the sample from the subject to be treated as compared to the first anti-YB-1 antibody control level and/or an equal level as compared to the second anti- YB-1 antibody control level is indicative of a non-response of said subject to said treatment, and wherein an increased level in the sample from the subject to be treated as compared to the second anti-YB-1 antibody control level and/or an equal level as compared to the first anti-YB-1 antibody control level is indicative of a response of said subject to said treatment. In a preferred embodiment of the invention the subject is to be treated, i.e. the method to determine response of a subject is performed before the onset of treatment. In a preferred embodiment the treatment comprises the administration of a drug, preferably a chemotherapeutic drug. Preferred chemotherapeutic drugs are platinum-based antineoplastic drugs (e.g. platinum analogues), e.g. as further defined herein. Hence, the present invention also relates to method for predicting whether a subject being treated or to be treated for cancer with a platinum-based antineoplastic will respond to said treatment comprising the steps of

(i) determining the level of antibodies against Y box binding protein 1 (YB-1) in a sample from said subject to be treated,

(ii) comparing the determined level in the sample to either one or both of a first and second anti-YB-1 antibody control level,

a) wherein the first anti-YB-1 antibody control level is derived from one or more subjects responding to said treatment, and

b) wherein the second anti-YB-1 antibody control level is derived from one or more subjects not responding to said treatment,

wherein a decreased level in the sample from the subject to be treated as compared to the first anti-YB-1 antibody control level and/or an equal level as compared to the second anti- YB-1 antibody control level is indicative of a non-response of said subject to said treatment, and wherein an increased level in the sample from the subject to be treated as compared to the second anti-YB-1 antibody control level and/or an equal level as compared to the first anti-YB-1 antibody control level is indicative of a response of said subject to said treatment.

Furthermore, ratios may be used in order to determine response or non-response to a treatment with a platinum-based antineoplastic. In such embodiment a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of less than 0.9 fold as compared to the first anti-YB-1 antibody control level is indicative of a non-response of said subject to said treatment, preferably a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of less than 0.8 fold as compared to the first anti-YB-1 antibody control level is indicative of a non-response of said subject to said treatment, further preferred a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of less than 0.75 fold as compared to the first anti-YB- 1 antibody control level is indicative of a non-response of said subject to said treatment. Furthermore, ratios may alternatively or additionally be determined to the second control level, in such embodiment a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.1 fold as compared to the second anti- YB-1 antibody control level is indicative of a response of said subject to said treatment; preferably a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.2 fold as compared to the second anti-YB-1 antibody control level is indicative of a response of said subject to said treatment; further preferred a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.3 fold as compared to the second anti-YB-1 antibody control level is indicative of a response of said subject to said treatment; yet further preferred a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.35 fold as compared to the second anti-YB-1 antibody control level is indicative of a response of said subject to said treatment.

As will be readily understood by the skilled person, these methods of the present invention may be performed as methods for monitoring cancer treatment efficiency. In this embodiment the levels of anti-YB-1 antibodies in said subject is determined during treatment, i.e. in a subject being treated with said drug.

The present invention also relates to a method of treating cancer with a chemotherapeutic drug, e.g. a platinum-based antineoplastic in a subject, comprising determining the level of antibodies against Y box binding protein 1 (YB-1) in a sample from the subject, wherein when the level of anti-YB-1 antibodies in a sample from the subject is above 1.9 units/ml, said chemotherapeutic drug analogue is administered to the subject, preferably above 2.0 units/ml, more preferably above 2.3 units/ml, also preferred above 2.5 units/ml. However, the threshold may also be determined as outlined above, hence, the method of treating cancer in a subject may also comprise the method for determining whether a subject being treated or to be treated for cancer with a chemotherapeutic drug will respond to said treatment, wherein the chemotherapeutic drug is administered if the determined levels of anti-YB-1 antibodies in said subject is indicative of response to the chemotherapeutic drug, as outlined above. The chemotherapeutic drug in accordance with the present invention is preferably a platinum-based anti-neoplastic. As outlined herein, results of non-response of a patient to a treatment may be relapse or progression of cancer of the patient. Hence, in a preferred embodiment of the method for the prediction of risk stratification for relapse in a patient to be treated or being treated for cancer with a drug, the method comprising the steps of

(i) determining the level of antibodies against YB-1 in a sample from said subject to be treated,

(ii) comparing the determined level in the sample to either one or both of a first and second anti-YB-1 antibody control level, wherein

a) the first anti-YB-1 antibody control level is derived from one or more subjects not showing relapse of cancer after treatment with said drug, and b) the second anti-YB-1 antibody control level is derived from one or more subjects showing relapse of cancer after treatment with said drug, wherein a decreased level in the sample from the subject being treated as compared to the first anti-YB-1 antibody control level and/or an equal level as compared to the second anti- YB-1 antibody control level is indicative of relapse in the subject, and wherein an increased level in the sample from the subject being treated as compared to the second anti-YB-1 antibody control level and/or an equal level as compared to the first anti-YB-1 antibody control level is indicative of no relapse in the subject. Preferably the level in said patient is determined before the onset of treatment. The preferred subject is therefore in this context a subject to be treated with said drug. In a preferred embodiment of the present invention first YB-1 antibody control level is derived from one or more subjects that did not show relapse or progression of cancer within 12 months after onset of treatment with said drug and the second YB-1 antibody control level is derived from one or more subjects that did show relapse or progression of cancer within 12 months after onset of treatment with said drug.

The present invention also relates to a method prediction of risk stratification for relapse of cancer in a patient being treated or to be treated with a chemotherapeutic drug, e.g. a platinum-based antineoplastic, comprising determining the level of antibodies against YB-1 in a sample from the subject, wherein when the level of anti-YB-1 antibodies in a sample from the subject is below 5 units/ml, is indicative of relapse of cancer in the subject.

Description of drawings Fig. 1: Comparison of the mean level of anti-YB-1 antibodies (In of units/ml) in serum samples of ovarian cancer patients (OvCA; In of mean = 0.89; n = 198) to the mean level of anti-YB-1 antibodies in serum samples of a healthy control group (Control, In of mean = 1.634 units/ml; n = 132). The assay used was the YB-l-AB-ELISA. The p-value is indicated on top. Bars indicate standard error of mean.

Fig. 2: ROC-analysis of the sensitivity of the diagnosis of ovarian cancer is plotted against the specificity. The assay used was the YB-l-AB-ELISA. The graph displays the relation between sensitivity and specificity for an exemplary cutoff value (2.77 units/ml). Fig. 3: Comparison of the mean level of anti-YB-1 antibodies (In of units/ml) in serum samples of ovarian cancer patients responding to treatment with Carboplatin (responder; In of mean = 0.966; n = 1 1 1); mean level of anti-YB-1 antibodies (In of units/ml) in serum samples ovarian cancer patients not responding to the treatment with Carboplatin (non- responder; In of mean = 0.632 units/ml; n = 52); and the mean level of anti-YB-1 antibodies in serum samples of a healthy control group (Control, In of mean = 1.634 units/ml; n = 132). The assay used was the YB-l-AB-ELISA of Example 1. The p-value is indicated on top. Bars indicate standard error of mean.

Fig. 4: Comparison of the mean level of anti-YB-1 antibodies (In of units/ml) in serum samples of ovarian cancer patients showing relapse after treatment by surgery and carboplatin (relapse; In of mean = 0.833; n = 123); mean level of anti-YB-1 antibodies (In of units/ml) in serum samples ovarian cancer patients not showing relapse after said treatment (no relapse; In of mean = 0.985 units/ml; n = 75); and the mean level of anti-YB-1 antibodies in serum samples of a healthy control group (Control, In of mean = 1.634 units/ml; n = 132). The assay used was the YB-l -AB-ELISA of Example 1. The p-value is indicated on top. Bars indicate standard error of mean.

Fig. 5: ROC-analysis of the sensitivity of the prediction of relapse is plotted against the specificity. The graph displays the relation between sensitivity and specificity for an exemplary cutoff value (ln=1.74 units/ml (i.e. 5.7 units/ml)).

Fig. 6: Standard curve of the YB-1 -Auto- Antibody ELISA Detailed description of the invention The present invention is based on the surprising finding of the inventors that in samples of patients with cancer decreased levels of anti-YB-1 antibodies can be found as compared to subjects without cancer. In other words the inventors have found that patients with cancer have little or no detectable antibodies against Y box binding protein 1 (YB-1) in the blood (e.g. determined in serum) whereas in control groups anti-YB-1 auto-antibodies can be detected at higher levels.

The present invention is based on the finding of that levels of the autoimmune-antibodies in subjects have diagnostic and predictive properties. The antibodies to be detected in connection with the present invention are therefore autoantibodies, i.e. those produced by immune system of the subject to be diagnosed, or being or to be treated.

The invention relates to a method for the diagnosis of a cancer, comprising the steps of (i) determining the level of antibodies against YB-1 in a sample from a subject to be diagnosed; and (ii) comparing the determined level in the sample to a control level of antibodies against YB-1 in samples derived from one or more subjects without cancer; wherein a decreased level in the sample from the subject to be diagnosed as compared to the control level is indicative of cancer in the subject. Preferably, the cancer is an ovarian cancer. It will be understood by those of ordinary skills in the art, that if a preferred cancer is chose to be diagnosed, the control level is preferably derived from subjects without that specific cancer, i.e. if ovarian cancer is to be diagnosed, the control level shall be derived from subjects without ovarian cancer.

However, the skilled person will also understand that the "control" level may be implicated in the used assay for detecting said auto antibodies. The skilled person hence may chose particulars of the assay so that the test is positive for the presence of the antibody in the sample if levels above a certain level is reached and vice versa be negative for the presence of said autoantibody if levels are determined that are below the control value.

Hence, in one embodiment of the present invention the method for diagnosis of cancer in a subject, comprising the steps of determining the absence or presence of antibodies against Y box binding protein 1 (YB-1) in a sample from a subject to be diagnosed, wherein absence of YB-1 antibodies in the sample from the subject to be diagnosed are indicative of the presence of cancer in said patient, preferably for the presence of ovarian cancer.

The terms "anti-YB-1 antibody", "YB-1 antibody" and YB-1 auto-antibody" are used synonymously herein and refer to the antibodies present in subjects that specifically bind to YB-1 or an immunogenic fragment thereof. In this context the term "specific binding" refers to antibodies raised against peptides derived from YB-1. Such peptides can comprise additional or less N- or C-terminal amino acids. An antibody is considered to be specific to YB-1 or an immunogenic peptide thereof, if its affinity towards the variant it is at least 50- fold higher, preferably 100-fold higher, more preferably at least 1000-fold higher than towards other proteins or peptides. It is well known in the art how to determine binding of antibodies to a specific antigen.

Auto-antibodies directed against YB-1 are not known until today. The inventors of the present application for the first time demonstrate the presence of such antibodies as well as the diagnostic and predictive value. It was found that a decrease in the level of antibodies directed against YB-1 in samples of a subject to be diagnosed as compared samples from subjects with proven absence of cancer is indicative of the presence of cancer as well as for the prediction of response or non-response to a treatment of the cancer with a drug.

"Cancer" in connection with the present invention is to be understood as any diseases involving unregulated cell growth. Cancer in this regard is a disease where cells divide and grow uncontrollably resulting in the formation of malignant tumors.

However, in a preferred embodiment of the present invention "cancer" refers to an ovarian cancer. In a particularly preferred embodiment the cancer according to the present invention, including all embodiments, is an ovarian cancer. Ovarian cancer often derives from the epithelium of the ovary, but may also be derived from fallopian tube. However, it was found that in both cases the method of the present invention is predictive for the presence of cancer or the response to a certain treatment. Hence, in one embodiment of the present invention cancer is an ovarian cancer, the ovarian cancer being epithelial ovarian cancer or cancer derived from the fallopian tube.

Hence, the present application relates to a method for diagnosis of ovarian cancer, comprising the steps of (i) determining the level of antibodies against Y box binding protein 1 (YB-1) in a sample from a subject to be diagnosed, and (ii) comparing the determined level in the sample to a control level of YB-1 antibodies derived from one or more subjects without cancer, preferably without ovarian cancer; wherein a decreased level in the sample from the subject to be diagnosed as compared to the control level is indicative of the presence of ovarian cancer in the subject to be diagnosed The invention further pertains to a method for diagnosis of a ovarian cancer, wherein a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.8 fold as compared to the control level is indicative of the presence of ovarian cancer in the subject to be diagnosed, preferably a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.7 fold as compared to the control level is indicative of the presence of ovarian cancer in the subject to be diagnosed, more preferably a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.65 fold as compared to the control level is indicative of the presence of ovarian cancer in the subject to be diagnosed; yet further preferred a level of anti-YB-1 antibodies in the sample of the patient to be diagnosed of less than 0.6 fold as compared to the control level is indicative of the presence of ovarian cancer in the subject to be diagnosed.

The invention also pertains to a method for diagnosis of ovarian cancer, wherein the level of antibodies against YB-1 is determined in a sample from a subject to be diagnosed and wherein a level of anti-YB-1 antibodies below 5 units/ml is indicative of ovarian cancer in the patient to be diagnosed, preferably below 4 units/ml, more preferably below 3.5 units/ml, further preferred below 3 units/ml.

The skilled person knows that depending on the subject, different cancers may be diagnosed. He is aware that he also may have to consider further parameters to diagnose the subject, e.g. when diagnosing ovarian cancer, the subject has to be female. In the context of the present invention the subject to be diagnosed is a mammal, preferably a human. The subject is preferably a human suspected to have cancer. In a further preferred embodiment the subject is a female mammal, preferably a female human subject suspected of having ovarian cancer or a female mammal, preferably a female human subject to be screened for the presence of ovarian cancer, preferably a female human subject to be treated or being treated for ovarian cancer with a drug, preferably a chemotherapeutic drug, e.g. a platinum-based antineoplastic drug.

In the context of the present invention the terms "YB" or "YBX1" or "YB-1" are interchangeably used herein and refer to "Y box binding protein 1", "Y-box transcription factor" or "nuclease-sensitive element-binding protein 1", and are also known as BP-8; CSDB; DBPB; CSDA2; NSEP1 ; NSEP-1 ; or MDR-NF1. This protein is a protein that in humans is encoded by the YBX1 gene (Entrez Gene ID: 4904). In context with the present invention the subject is preferably a human. YB-1 is a cold shock protein. YB-1 lacks an N- terminal signal peptide motif and therefore it s secretion is regulated similar to that of other leaderless proteins. Further to the full-length YB-1 protein which is 324 amino acids in length, a truncated fragment could be detected (Frye et al.; Ybox-protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen. EMBO reports 2009, 10(7):783-789).

In the context of the immunoassays of the present invention the "YB-1" may be present in its natural cellular environment and can be used together with the material associated with YB-1 in its natural state as well as in isolated form with respect to its primary, secondary and tertiary structures. The YB-1 is well known to those skilled in the art. The protein or its immunogenic fragment is preferably used in isolated form, i.e. essentially free of other proteins, lipids, carbohydrates or other substances naturally associated with YB-1. "Essentially free of means that the protein or its immunogenic fragment is at least 75%, preferably at least 85%, more preferably at least 95% and especially preferably at least 99% free of other proteins, lipids, carbohydrates or other substances naturally associated with YB-1. In connection with the present invention, the naturally occurring protein as well as all modifications, mutants or derivatives of the YB-1 can be used. This includes all naturally present modifications as known to the killed person. Similarly, a YB-1 produced by means of recombinant techniques, which includes amino acid modifications, such as inversions, deletions, insertions, additions etc. can be used according to the invention provided that this part of the essential function of the YB-1 is present, namely the capability of binding antibodies. The YB-1 being used may also comprise exceptional amino acids and/or modifications of such as alkylation, oxidation, thiol-modification, denaturation, oligomerization and the like. The YB-1 can also be synthesized by chemical means. According to the invention the YB-1 particularly can be a protein and/or peptide or a fusion protein, which in addition to other proteins, peptides or fragments thereof, includes the YB- 1 as a whole or in part. Using conventional methods, peptides or polypeptides of the YB-1 which have functionally analogs, analogous properties can be determined by those skilled in the art. For example such polypeptides or peptides have 50-60%, 70% or 80%, preferably 90%, more preferably 95%, and most preferably 98% sequence homology to peptides identified as YB-1, and said homology can be determined, e.g. by means of Smith- Waterman homology search algorithm, using the MPFRCH program (Oxford Molecular), for example.

The term "peptide" or "polypeptide" of an YB-1 used in the present invention, comprises also molecules differing from the original sequence by deletion(s), insertion(s), substitution(s) and/or other modifications well known in the prior art and/or comprising a fragment of the original amino acid molecule, the YB-1 still exhibiting the properties mentioned above. Such a peptide has preferably at least a length of 57 amino acid residues but may also be shorter, e.g. at least 12, 15, 20 or 25 amino acid residues in length. Also included are allele variants and modifications. Methods of producing the above changes in the amino acid sequence are well known to those skilled in the art and have been described in the standard textbooks of molecular biology, e.g. Sambrook et al, "Molecular cloning" (2012) Cold Spring Habor Laboratory Press, ISBN 978-1 -9361 13-42-2. Those skilled in the art will also be able to determine whether a YB-1 , thus, modified still has the properties mentioned above. The amino acid sequence of YB-1 is known. Database entries exist in several well known Databases. When referring to the amino acid sequence of YB-1 any amino acid sequence known is meant, particularly those disclosed in common databases, preferably of human origin. This protein in humans is encoded by the YBX1 gene (Entrez Gene ID: 4904). One preferred sequence of YB-1 are given herein, i.e. SEQ ID NO: l for full-length YB-1. The YB-1 may be glycosylated in vivo. In the present specification all of the above illustrated modifications of the YB-1 will be referred to as "functionally analogous peptides or proteins" or "immunogenic peptides" or "antigenic peptides" in brief.

The antibodies to be detected or determined according to the present invention are directed against YB-1. This means that the antibodies specifically bind YB-1. Specific binding of an antibody normally occurs via binding of a binding site of the antigen. The antibodies of the present invention are those specifically binding to YB-1 or immunogenic fragments thereof. This binding may occur via recognition of sequence or structural epitopes. The skilled person is aware of methods of how determining specific epitopes, e.g. fragments of the antigen YB-1, which are recognized and bound by the antibodies to be determined. Fragments of YB-1 binding to the autoantibodies are called immunogenic or antigenic fragments. Methods for determining fragments of an antigen binding the antibody are described in several publications which are incorporated herein by reference (e.g. Gershoni, JM; Roitburd-Berman, A; Siman-Tov, DD; Tarnovitski Freund, N; Weiss, Y (2007). "Epitope mapping: The first step in developing epitope-based vaccines". BioDrugs 21 (3): 145-56; Westwood, MR; Hay, FC (2001). Epitope Mapping: a practical approach. Oxford, Oxfordshire: Oxford University Press. ISBN 0-19-963652-4; Flanagan et al. (201 1), "Mapping Epitopes with H/D-Ex Mass Spec". Genetic Engineering and Biotechnology news; 31(1); Gaseitsiwe, S.; Valentini, D.; Mahdavifar, S.; Reilly, M.; Ehrnst, A.; Maeurer, M. (2009) "Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1 *0101 , DRB1 *1501 , and DRB1 *0401 ". Clinical and Vaccine Immunology 17 (1): 168-75; Linnebacher, Michael; Lorenz, Peter; Koy, Cornelia; Jahnke, Annika; Born, Nadine; Steinbeck, Felix; Wollbold, Johannes; Latzkow, Tobias et al. (2012). "Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase Ha by peptide chip and proteome analysis: A pilot study with colorectal carcinoma patient samples" Analytical and Bioanalytical Chemistry 403 (1): 227- 38; Cragg, M. S. (201 1). "CD20 antibodies: Doing the time warp". Blood 1 18 (2): 219-20; Banik, Soma S. R.; Doranz, Benjamin J. (2010). "Mapping Complex Antibody Epitopes". Genetic Engineering and Biotechnology News 3 (2): 25-8; and Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; otelnikov, Valery; Greene, Tiffani A. et al. (2009). "Atomic-Level Mapping of Antibody Epitopes on a GPCR". Journal of the American Chemical Society 131 (20): 6952-4). In context with the present invention anti-YB-1 antibodies are understood as any immunoglobulin specifically recognizing/binding to YB-1. The antibody to be detected in a preferred embodiment binds any of YB- 1 , preferably to a sequence comprising or consisting of SEQ ID NO: 1.

In the context of the present invention the anti-YB-1 antibody may particularly be selected from the group of IgA-antibody, IgG-antibody and IgM-antibody, preferably an IgG antibody, e.g. IgGl, IgG2, IgG3 and IgG4.

The control levels as disclosed herein refer to control levels of YB-1 antibodies. It will be readily understood by the skilled person that the control levels from subjects having the desired disease or response as defined in the methods and to which the determined levels are compared to, are not necessarily determined in parallel but may be represented by previously determined levels. Nevertheless, control levels may be determined in parallel. The skilled person with the disclosure of the present invention and his knowledge is able to determine such levels, as outlined herein. Hence, the control levels of the present invention may be previously defined thresholds or cut off values. Preferred thresholds are disclosed herein. Furthermore, it will be acknowledged by the skilled person that control levels are, like the levels to be determined in the subject to be diagnosed or treated, determined in samples of the recited subjects having the desired disease or response or being healthy. Preferably, the sample is the same kind of sample as the sample of the person to be diagnosed or to be treated, e.g. when the sample of the latter is serum, the control levels are preferably derived from serum samples of the control subjects.

As outlined herein, the levels of YB-1 antibodies in samples of the patient to be diagnosed and treated or to be treated are compared with the control groups as defined herein. However, in one embodiment the levels are compared to fixed values, i.e. thresholds under or over which a certain diagnosis, or prognosis of response is given. To this end, unit- standards may be applied. The inventors of the present invention set out such standard for the YB-1 antibody using a serum sample from a systemic sclerosis patient. Systemic sclerosis patients are known to have high levels of autoimmune antibodies in general. Hence, the inventors took a serum sample of a systemic sclerosis patient for the unit standard met herein. However, it will be acknowledged by the skilled person that also other samples may be taken to set a different standard, e.g. samples of healthy subjects, samples of cancer patients. Nevertheless the principle of generating a standard (units) is the same in any case and are exemplified herein using serum samples of systemic sclerosis patients. In the context of the present invention "units/ml", unless specified otherwise, refers to the concentration of antibodies standardised as exemplified herein. Hence, in one embodiment of the present invention 20 units/ml refers to a dilution of 1 :200 of a serum sample of systemic sclerosis patients. The serum sample may be derived from a single patient. The inventors of the present invention found that the concentration of YB-1 antibodies in samples of systemic sclerosis do not differ by more than about 10 %, showing such standard being reproducible. In one preferred embodiment the standard for the concentrations of the autoimmune antibodies is generated in the following way: a serum sample of a systemic sclerosis patient (or a larger cohort) is diluted (a) 1 :200 for standard point 20 Units/ml, (b) 1 :400 for standard point 10 Units/ml, (c) 1 :800 for standard point 5 Units/ml, (d) 1 : 1600 for standard point 2.5 Units/ml, (e) 1 :3200 for standard point 1.25 Units/ml and (f) 1 :6400 for standard point 0.63 Units/ml . These standards are then used for the immunoassay chosen, e.g. ELISA, and then correlated with the respective read-out value, e.g. for ELISA ratio of optical density at 450 nm/ optical density at 620 nm. A typical standard curve of YB-1 autoantibody ELISA is shown in Figure 6. Nevertheless, the skilled person will readily understand that it may also be possible to standardize the levels of YB-1 -autoantibodies using different samples, e.g. samples of healthy subjects or cancer patients.

"Equal" level in context with the present invention means that the levels differ by not more than ± 10 %, preferably by not more than ± 5 %, more preferably by not more than ± 2 %."Decreased" or "increased" level in the context of the present invention mean that the levels differ by more than 10 %, preferably by more than 15 %, preferably more than 20 %.

Herein, the sample of the subject to be diagnosed in which the level of anti-YB-1 antibodies is to be determined is preferably a bodily fluid such as whole blood or lymph or fractions of blood such as serum or plasma. Preferably in the context of the present invention the sample is plasma or serum.

Where appropriate, the sample may need to be homogenized, or extracted with a solvent prior to use in the present invention in order to obtain a liquid sample. A liquid sample hereby may be a solution or suspension. Liquid samples may be subjected to one or more pre-treatments prior to use in the present invention. Such pre-treatments include, but are not limited to dilution, filtration, centrifugation, concentration, sedimentation, precipitation, and dialysis. Pre-treatments may also include the addition of chemical or biochemical substances to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, chelators. "Plasma" in the context of the present invention is the virtually cell-free supernatant of blood containing anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparinates or hirudin. Cell-free plasma can be obtained by centrifugation of the anticoagulated blood (e.g. citrated, EDTA or heparinized blood) for at least 15 minutes at 2000 to 3000 g.

"Serum" is the liquid fraction of whole blood that is collected after the blood is allowed to clot. When coagulated blood (clotted blood) is centrifuged serum can be obtained as supernatant. It does not contain fibrinogen, although some clotting factors remain. In the methods of the present invention, the anti-YB-1 antibody is preferably detected in an immunoassay. Suitable immunoassays may be selected from the group of immunoprecipitation, enzyme immunoassay (EIA)), enzyme-linked immunosorbenassys (ELISA), radioimmunoassay (RIA), fluorescent immunoassay, a chemiluminescent assay, an agglutination assay, nephelometric assay, turbidimetric assay, a Western Blot, a competitive immunoassay, a noncompetitive immunoassay, a homogeneous immunoassay a heterogeneous immunoassay, a bioassay and a reporter assay such as a luciferase assay. Preferably herein the immunoassay is an enzyme linked immunosorbent assay (ELISA).

The immunoassays can be homogenous or heterogeneous assays, competitive and non- competitive assays. In a particularly preferred embodiment, the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the anti-YB-1 antibody (i.e. the "analyte") to be detected and/or quantified is allowed to bind to an immobilized YB-1 protein or immunogenic peptide fragment thereof and to a secondary antibody. The YB-1 or fragment thereof (i.e. a peptide), may e.g., be bound to a solid phase, e.g. a bead, a surface of a well or other container, a chip or a strip, and the secondary antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety such as a peroxidase, e.g. horseradish peroxidase. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with "sandwich assays" are well-established and known to the skilled person (The Immunoassay Handbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005), ISBN-13: 978-0080445267; Hultschig C et al, Curr Opin Chem Biol. 2006 Feb;10(l):4-10. PMID: 16376134, incorporated herein by reference). Sandwich immunoassays can for example be designed as one-step assays or as two-step assays. The detectable label may for example be based on fluorescence or chemiluminescence. The labelling system comprises rare earth cryptates or rare earth chelates in combination with a fluorescence dye or chemiluminescence dye, in particular a dye of the cyanine type. In the context of the present invention, fluorescence based assays comprise the use of dyes, which may for instance be selected from the group comprising FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyanine dyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen, 6-Carboxy-2',4',7',4,7- hexachlorofluorescein (HEX), TET, 6-Carboxy-4',5'-dichloro-2',7'-dimethodyfluorescein (JOE), N,N,N',N'-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy-X-rhodamine (ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes, such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone, Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red, Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes, Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, and the like.

In the context of the present invention, chemiluminescence based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in Kirk- Othmer, Encyclopedia of chemical technology, 4 ^th ed., executive editor, J. I. Kroschwitz; editor, M. Howe-Grant, John Wiley & Sons, 1993, vol.15, p. 518-562, incorporated herein by reference, including citations on pages 551 -562. Preferred chemiluminescent dyes are acridiniumesters.

The "sensitivity" of an assay relates to the proportion of actual positives which are correctly identified as such, i.e. the ability to identify positive results (true positives positive results / number of positives). Hence, the lower the concentrations of the analyte that can be detected with an assay, the more sensitive the immunoassay is. The "specificity" of an assay relates to the proportion of negatives which are correctly identified as such, i.e. the ability to identify negative results (true negatives / negative results). For an antibody the "specificity" is defined as the ability of an individual antigen binding site to react with only one antigenic epitope. The binding behaviour of an antibody can also be characterized in terms of its "affinity" and its "avidity". The "affinity" of an antibody is a measure for the strength of the reaction between a single antigenic epitope and a single antigen binding site. The "avidity" of an antibody is a measure for the overall strength of binding between an antigen with many epitopes and multivalent antibodies.

An "immunogenic peptide" or "antigenic peptide" as used herein is a portion of an YB-1 protein that is recognized (i.e., specifically bound) by the anti-YB-1 antibody. Such immunogenic peptides generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of YB-1. However, they may also comprise at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acid residues.

For the purposes of the immunoassays and diagnostic methods of the invention YB-1 by expression in cells, preferably eukaryotic cells or in cell free, preferably eukaryotic cell free systems. Hence, in the assays and methods of the invention YB-1 may be present in its natural cellular environment and can be used together with the material associated with the protein in its natural state as well as in isolated form. Suitable expression systems include prokaryotic cells (e.g. E. coli) or eukaryotic cells (e.g. mammalian cells such asChinese hamster ovary (CHO) cells) overexpressing the human YB-1. Cell extracts or purified protein can be used to detect anti-YB-1 antibodies. Based on the weight of the whole protein or its immunogenic fragment in the preparation (e.g. the "extract") to be used according to the invention, the isolated protein should account for at least 0.5%, preferably at least 5% more preferably at least 25%, and in a particular preferred embodiment at least 50%. The protein is preferably used in isolated form, i.e. essentially free of other proteins, lipids, carbohydrates or other substances naturally associated with the YB-1. "Essentially free of means that the protein is at least 75%, preferably at least 85%, more preferably at least 95% and especially preferably at least 99% free of other proteins, lipids, carbohydrates or other substances naturally associated with the protein. In particular, the determining steps of the methods according to the present invention comprise the steps of

(a) contacting the sample with Y box binding protein 1 (YB-1) or an antigenic peptide fragment thereof under conditions allowing for the formation of a complex between anti-YB-1 antibodies with YB-1 or a peptide fragment thereof,

(b) detecting the complex.

Hence, the invention relates to a method for detecting an anti-YB-1 antibody in a sample from a subject, comprising the steps of

(a) contacting the sample suspected of comprising an anti-YB-1 antibody with YB-1 or an antigenic peptide fragment thereof under conditions allowing for the formation of a complex between the anti-YB-1 antibody with YB-1 or the antigenic peptide fragment thereof,

(b) detecting the complex.

The YB-1 or the antigenic peptide fragment thereof may preferably be immobilized on a surface. In a preferred embodiment the YB-1 or the antigenic peptide fragment thereof is immobilized directly on a surface. "Direct" immobilization in context with the present invention relates to an immobilization without using tags fused to the YB-1, e.g. without YB-1 being fused to a GST-Tag. A direct immobilization to the surface means that the protein is not fused to a tag. Such tag may interfere with the conformation of the protein that could cause the autoantibodies to be detected no longer binding to the protein. Hence, conformation stabilizing means are preferred in context with the methods according to the invention. Preferably the YB-1 is immobilized directly on a surface or stabilized by a buffer covering the surface, the buffer comprising Calcium chloride, further preferred the YB-1 is immobilized directly on a surface and stabilized by a buffer covering the surface, the buffer comprising Calcium chloride. The Calcium chloride concentration of the buffer may vary, however it shall stabilize the protein to allow proper binding of antibodies to the YB-1. In a preferred embodiment the Calcium chloride concentration is from 0.1 mM to 10 mM, further preferred from 0.5 to 5 mM, particularly preferred 1 mM. The Calcium chloride may in a preferred embodiment be added to any buffer of the methods of the present invention that come into contact with the YB-1 protein, e.g. also washing and/or detection buffers.

The complex may for example be detected using a secondary antibody against the Fc portion of the anti-YB-1 antibody. When the anti-YB-1 antibody is an IgG-antibody, the secondary antibody may be an anti-IgG antibody. Hence, in one embodiment the anti-YB-1 antibody to be detected is an IgG-antibody and the secondary antibody is an anti-IgG antibody, particularly preferred the subject is a human and the second antibody is an anti-human-IgG antibody. The skilled person will understand that it is possible to detect total IgG, i.e. the method does not distinguish between the subtypes of IgG antibodies. Hence, in one embodiment the secondary antibody is an anti-human-total IgG antibody. Nevertheless, in some embodiment it may be preferred that the subtypes are differentially detected. Hence, in a particular embodiment, the subject is a human and

(i) the anti-YB-1 antibody is an IgG 1 -antibody and the secondary antibody is an anti- human-IgGl antibody; or

(ii) the anti-YB-1 antibody is an IgG2-antibody and the secondary antibody is an anti- human-IgG2 antibody; or

(iii) the anti-YB-1 antibody is an IgG3 -antibody and the secondary antibody is an anti- human-IgG3 antibody; or

(iv) the anti-YB-1 antibody is an IgG4-antibody and the secondary antibody is an anti- human-IgG4 antibody.

The secondary antibody may for example be labeled with a detectable marker, e.g. a peroxidase.

Furthermore, in the methods of the present invention further parameters of the subject may be considered as well for diagnosis, differential diagnosis, prognosis of response etc. Such parameters in a multivariate model may include gender, age, histological evaluation, Figo or histopathological staging, grading of the tumor and other markers. Dependent variables for determining survival may also be time till death, time till first relapse, time till death or first relapse (shorter interval if both events occurred). A Cox-Proportional-Hazard regression predicts the dependent variable based on one or more independent variables. These predictors can either be measures (as e.g. level of a biomarker) or categorical data (as e.g. response to a previous treatment). The skilled person is aware of the fact that diagnostic markers only give a certain degree of sensitivity and specificity, as also outlined herein. He knows that different further parameters might be considered in order to increase both. For example, when detecting levels of a marker indicative of epithelial cancer, inter alia ovarian cancer, the skilled person would not diagnose ovarian cancer in a male human subject. Nevertheless, the present invention provides a new and superior marker for diagnosis, prognosis of cancer, particularly for ovarian cancer. In the context of the methods of the invention and particularly the immunoassays of the invention, the presence of one or more further diagnostic markers for ovarian cancer is detected in the sample. For example, in a diagnostic method of the present invention levels of CA125, Human Epidymis Protein 4 (HE4) and/or Mesothelin are detected in addition. The invention also relates to the use of YB-1 or an antigenic peptide fragment thereof, preferably as set out herein above, for the diagnosis of cancer, preferably for the diagnosis of an YB-1 or YB-1 associated cancer, more preferably for the diagnosis of ovarian cancer. As outlined above for the methods according to the invention, the YB-1 or the antigenic peptide fragment thereof may preferably be immobilized on a surface. In a preferred embodiment the YB-1 or the antigenic peptide fragment thereof is immobilized directly on a surface. "Direct" immobilization in context with the present invention relates to an immobilization without using tags fused to the YB-1 , e.g. without YB-1 being fused to a GST-Tag. A direct immobilization to the surface means that the protein is not fused to a tag. Such tag may interfere with the conformation of the protein that could cause the autoantibodies to be detected no longer binding to the protein. Hence, conformation stabilizing means are preferred in context with the methods according to the invention. Preferably the YB-1 is immobilized directly on a surface or stabilized by a buffer covering the surface, the buffer comprising Calcium chloride, further preferred the YB-1 is immobilized directly on a surface and stabilized by a buffer covering the surface, the buffer comprising Calcium chloride. The Calcium chloride concentration of the buffer may vary, however it shall stabilize the protein to allow proper binding of antibodies to the YB-1. In a preferred embodiment the Calcium chloride concentration is from 0.1 mM to 10 mM, further preferred from 0.5 to 5 mM, particularly preferred 1 mM. The Calcium chloride may in a preferred embodiment be added to any buffer of the methods of the present invention that come into contact with the YB-1 protein, e.g. also washing and/or detection buffers.

In the context of the present invention, the levels of the anti-YB-1 antibodies a may be analyzed in a number of fashions well known to a person skilled in the art. For example, each assay result obtained may be compared to a "normal" value, or a value indicating a particular disease or outcome. A particular diagnosis/prognosis may depend upon the comparison of each assay result to such a value, which may be referred to as a diagnostic or prognostic "threshold". In certain embodiments, assays for one or more diagnostic or prognostic indicators are correlated to a condition or disease by merely the presence or absence of the indicator(s) in the assay. For example, an assay can be designed so that a positive signal only occurs above a particular threshold concentration of interest, and below which concentration the assay provides no signal above background.

The sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "normal" (i.e. apparently healthy individuals not having ovarian cancer) and "disease" populations. For any particular marker, a distribution of marker levels for subjects with and without a disease will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease. A threshold is selected, below which the test is considered to be abnormal and above which the test is considered to be normal. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results don't necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. l=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art. See, e.g., Hartley et al. 1982. Radiology 143: 29-36. Preferably, a threshold is selected to provide a ROC curve area of greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to +/- 5% of a given measurement.

The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1- specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

In other embodiments, a positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease. In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. Γη the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group.

In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.

In the case of a hazard ratio, a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and "control" groups; a value greater than 1 indicates that the risk is greater in the diseased group; and a value less than 1 indicates that the risk is greater in the control group. The skilled artisan will understand that associating a diagnostic or prognostic indicator, with a diagnosis or with a prognostic risk of a future clinical outcome is a statistical analysis. For example, a marker level of lower than X may signal that a patient is more likely to suffer from an adverse outcome than patients with a level more than or equal to X, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels may be reflective of patient prognosis, and the degree of change in marker level may be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1 , 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 , and 0.0001.

Suitable threshold levels for the stratification of subjects into different groups (categories) have to be determined for each particular combination of YB-1 -antibodies, disease and/or medication. This can e.g. be done by grouping a reference population of patients according to their level of YB-1 -antibodies into certain quantiles, e.g. quartiles, quintiles or even according to suitable percentiles. For each of the quantiles or groups above and below certain percentiles, hazard ratios can be calculated comparing the risk for an adverse outcome, i.e. an "cancer" or a "non response", e.g. in terms of survival rate/mortality, between those patients who have received a certain medication and those who did not, or in terms of presence and absence of cancer in patients. In such a scenario, a hazard ratio (HR) above 1 indicates a higher risk for an adverse outcome for the patients who have received a treatment than for patients who did not. A FIR below 1 indicates beneficial effects of a certain treatment in the group of patients. A HR around 1 (e.g. +/- 0.1) indicates no elevated risk but also no benefit from medication for the particular group of patients. By comparison of the FIR between certain quantiles of patients with each other and with the HR of the overall population of patients, it is possible to identify those quantiles of patients who have an elevated risk and those who benefit from medication and thereby stratify subjects according to the present invention. In some cases presence of cancer, relapse and/or mortality upon treatment with an platinum- based antineoplastic will affect patients with high levels (e.g. in the fifth quintile) of YB-1- antibodies, while in other cases only patients with low levels of YB-1 -antibodies will be affected (e.g. in the first quintile). However, with the above explanations, a skilled person is able to identify those groups of patients having cancer, those groups that do respond to a medication and those groups that do not respond to the medication. The gist of the invention is the correlation of reduced antibody levels against YB-1 with a certain outcome as outlined herein. Exemplarily, some combinations of medications are listed for several diseases in the appended examples. In another embodiment of the invention, the diagnosis, risk for relapse of cancer and/or outcome for a patient are determined by relating the patient's individual level of marker antibody to certain percentiles (e.g. 20 ^th , 10 ^th , 5 ^th or 2.5 ^th percentile) of a healthy population or responders, respectively.

Kaplan-Meier estimators may be used for the assessment or prediction of the outcome or risk (e.g. diagnosis, relapse, progression or morbidity) of a patient.

The term "drug" in connection with the present invention is to be understood as any substance, pharmaceutical composition or the like which are intended for the treatment of cancer, preferably ovarian cancer. Different drugs are known. Preferred drugs are chemotherapeutic agents or chemotherapeutic drugs, preferably platinum analogues used for treating cancer. Such platinum analogues are known by the skilled person and are preferably platinum-based antineoplastic agents, preferably selected from the group consisting of cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin. Also encompassed by the invention is a method of treating cancer, e.g. ovarian cancer, in a subject, comprising determining the level of antibodies against YB-1 in a sample from the subject, wherein when the level of anti-YB-1 antibodies in a sample from the subject is above a level determined as the control level for no-response to the treatment with a method according to the present invention as disclosed herein above. Preferably the invention encompasses a method of treating ovarian cancer in a subject, comprising determining the level of antibodies against YB-1 in a sample from the subject, wherein when the level of anti-YB-1 antibodies in a sample from the subject is above 1.9 units/ml, said chemotherapeutic drug analogue is administered to the subject, preferably above 2.0 units/ml, more preferably above 2.3 units/ml, also preferred above 2.5 units/ml. Drugs used in the treatment of cancer include platinum analogues, e.g. an platinum-based antineoplastic, preferably selected from the group consisting of cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin.

The invention, thus, also relates a drug for use in the treatment of cancer, preferably ovarian cancer, in a subject, wherein the drug is administered to the subject when the level anti-YB- 1 antibodies in a sample from the subject is above a level determined as the control level for non-response to the treatment with a method according to the present invention as disclosed herein above. Particularly the invention relates to a drug for use in the treatment of cancer in a subject, wherein the drug is administered to the subject when the level anti-YB-1 antibodies in a sample from the subject is above 1.9 units/ml, said chemotherapeutic drug analogue is administered to the subject, preferably above 2.0 units/ml, more preferably above 2.3 units/ml, also preferred above 2.5 units/ml.

Furthermore, the invention relates to a drug, preferably a platinum-based antineoplastic, for use in the treatment of cancer, preferably ovarian cancer, in a subject, wherein the drug is administered to the subject if a level of antibodies against Y box binding protein 1 in a sample from the subject to be treated of more than 1.1 fold as compared to anti-YB-1 antibody control levels derived from samples of patients not responding to said treatment is determined; preferably if a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.2 fold as compared to the anti-YB-1 antibody control level is determined; further preferred if a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.3 fold as compared to the anti-YB-1 antibody control level is determined; yet further preferred if a level of antibodies against Y box binding protein 1 in the sample from the subject to be treated of more than 1.35 fold as compared to the anti-YB-1 antibody control level is determined. The embodiments as outlined herein above for the methods according to the present invention, particularly for the methods for determining the response, apply also to the drug for use in the treatment of cancer, e.g. the cancer preferably being an ovarian cancer, the subject being a human, preferably a female human.

In a preferred embodiment the drug is for use in the treatment of ovarian cancer. The drug is preferably a platinum analogue, such as a platinum-based antineoplastic, preferably selected from the group consisting of cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin. The invention, thus, also relates to platinum-based antineoplastic for use in the treatment of ovarian cancer in a subject, wherein bevacizumab is administered to the subject when the level anti-YB-1 antibodies in a sample from the subject is above 1.9 units/ml, said chemotherapeutic drug analogue is administered to the subject, preferably above 2.0 units/ml, more preferably above 2.3 units/ml, also preferred above 2.5 units/ml.

"Antineoplastic" in connection with the present invention relates to substances acting to prevent, inhibit or halt the development of a neoplasm (a tumor), i.e. an agent with antineoplastic properties.

The invention also pertains to a research and/or diagnostic kit for the diagnosis of cancer, e.g. ovarian cancer, or for the prediction or risk stratification for relapse of cancer in a patient, wherein the kit comprises YB-1 or an antigenic peptide fragment thereof. The kit may further comprise an antibody directed to the Fc portion of the anti-YB-1 antibody to be detected, i.e. an anti-human IgG antibody.

Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific therapeutic or non- therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert which is included with the kit.

The invention hence also relates to a kit for diagnosing cancer, e.g. ovarian cancer, as outlined above, or predicting the response of a cancer patient to the treatment for cancer, e.g. ovarian cancer by e.g. a platinum-based antineoplastic, said kit comprising the YB-1 protein or an antigenic peptide thereof, and means to detect antibodies binding to said YB-1 or antigenic peptide thereof. As outlined herein, conformation stabilization of the YB-1 is preferred, hence, in a preferred embodiment the kit of the comprises YB-1 or an antigenic peptide fragment thereof immobilized on a surface; preferably immobilized directly on a surface. Preferably the YB-1 is immobilized directly on a surface or stabilized by a buffer covering the surface, the buffer comprising Calcium chloride, further preferred the YB-1 is immobilized directly on a surface and stabilized by a buffer covering the surface, the buffer comprising Calcium chloride. The Calcium chloride concentration of the buffer may vary, however it shall stabilize the protein to allow proper binding of antibodies to the YB-1. In a preferred embodiment the Calcium chloride concentration is from 0.1 mM to 10 mM, further preferred from 0.5 to 5 mM, and particularly preferred 1 mM. The Calcium chloride may in a preferred embodiment be added to any buffer of the kit coming into contact with the immobilized YB-1 protein, e.g. also washing and/or detection buffers. Preferably the kit is designed for a method of the present invention. It will be understood that the embodiments disclosed herein above for YB-1 or an antigenic peptide thereof also apply to the kit. The kit is preferably designed to detect autoimmune antibodies in samples of subject and hence comprises means to detect such antibodies, particularly antibodies binding to said YB-1 or peptide thereof. Such means are outlined herein above, e.g. for immunoassays, and applying likewise to the kit. The embodiments set out for the immunoassays, proteins and methods apply also to the kit of the invention. The kits of the present invention are meant for the detection of autoimmune antibodies in samples, e.g. blood samples, like serum or plasma. Hence, in one embodiment they comprise means for the preparation of blood, e.g. for gaining serum thereof. The kit may also comprise means for detecting antibodies, e.g. secondary antibodies, as outlined herein above. Furthermore, the kit may comprise control composition and/or standards. The control composition preferably comprises YB-1 antibodies as positive control. Furthermore, the kit may comprise one or a plurality of standard compositions. A standard composition comprises YB-1 antibodies at a defined concentration. As outlined herein, determination of concentration of autoimmune-antibodies may be performed using standard curves. These curves set out which concentration of antibodies in a sample or solution corresponds to what read-out value of the assay used, e.g. optical density or proportion of optical density at different wavelengths (e.g. 450nm/620nm). To this end the kits of the present invention may comprise one or more standard compositions having a defined concentration of YB-1 antibodies, preferably of the kind to be detected in the method. A standard composition of the kit according to the present invention may for instance comprise YB-1 antibodies at concentrations selected from the group consisting of 20 units/ml, 10 units/ml, 5 units/ml, 2.5 units/ml, 1.25 units/ml, and 0.63 units/ml. In one embodiment the kit comprises six standard compositions with the recited concentration. In another embodiment the kit comprises one standard composition with the highest concentration of the standard curve, e.g. 20 units/ml or 10 units/ml. The other concentrations may be produced at the side of the end user by further dilutions, e.g. in PBS. A dilution buffer may therefore also be comprised in the kits according to the invention.

It will be readily understood that the embodiments outlined above shall apply to the invention as a whole and not be limited to a specific method, unless stated otherwise. It will for example be understood the embodiments for the type of cancer shall be applied to every method, kit or the like disclosed herein. The invention is further illustrated by the following non-limiting examples and figures.

Sequences SEQ ID NO: 1:

Amino acid sequence of YB-1 [SEQ ID NO:l] MSSEAETQQP PAAPPAAPAL SAADTKPGTT GSGAGSGGPG GLTSAAPAGG

DKKV I ATKVL GTVKWFNVRN GYGFINRNDT KEDVFVHQTA IKKNNPRKYL

RSVGDGETVE FDWEGEKGA EAANVTGPGG VPVQGSKYAA DRNHYRRYPR

RRGPPRNYQQ NYQNSESGEK NEGSESAPEG QAQQRRPYRR RRFPPYYMRR

PYGRRPQYSN PPVQGEVMEG ADNQGAGEQG RPVRQNMYRG YRPRFRRGPP

RQRQPREDGN EEDKENQGDE TQGQQPPQRR YRRNFNYRRR RPENPKPQDG

KETKAADPPA ENSSAPEAEQ GGAE

EXAMPLES

Example 1 : YB-1 autoantibody levels in serum samples were measured using two different sandwich ELISA kit (CellTrend GmbH Luckenwalde, Germany). For the first kit the microtiter 96- well polystyrene plates were directly coated with human full length YB-1 of the sequence of SEQ ID NO:l . This kit is referred to herein as YB- 1 -AB-ELIS A. To maintain the conformational epitopes of the protein/fragment, 1 mM Calcium chloride was added to every buffer. Duplicate samples of a 1 : 800 serum dilution were incubated at 4°C for 2 hours. After washing steps, plates were incubated for 60 minutes with a 1 :20.000 dilution of horseradish-peroxidase-labeled goat anti-human IgG (Jackson, USA) used for detection. In order to obtain a standard curve, plates were incubated with test sera from an anti-YB-1 autoantibody positive index patient suffering from systemic sclerosis. The ELISA was validated according to the FDA's "Guidance for industry: Bioanalytical method validation".

YB-1 -autoantibodies are not commercially available; a serum sample from a patient with a systemic sclerosis is used for the standard curve. A 1 :200 dilution of the serum sample is defined as 20 units/ml YB-1 -antibodies. To set a standard for the concentrations of the autoimmune antibodies, a standard curve was generated. In detail, a serum sample of systemic sclerosis serum sample was diluted (a) 1 : 200 for standard point 20 units/ml, (b) 1 : 400 for standard point 10 units/ml, (c) 1 : 800 for standard point 5 units/ml, (d) 1 : 1600 for standard point 2.5 units/ml, (e) 1 : 3200 for standard point 1.25 units/ml and (f) 1 : 6400 for standard point 0.63 units/ml. Then the optical density was determined using the kit and method of above. Each standard point was performed in duplicates. Results are shown in Figure 6. Example 2:

YB-1 antibody levels in serum samples from 132 healthy donors ("Control") and 198 patients with ovarian cancer ("OvCA") were measured using the kit and method of example 1. The levels were determined in units/mL.

Figure 1 shows the mean values of the natural logarithm (In) of YB-1 antibody levels for OvCA and Control subjects determined using the YB-l-AB- ELISA. Patient suffering from ovarian cancer had significantly lower levels (p<0.0001) of anti-YB-1 antibodies as compared to healthy controls.

ROC-analysis was performed. The results are shown if Fig. 2 for YB-1-AB-ELISA. The results for YB-l-AB-ELISA clearly show that the use of YB-1 full length protein allows for a good detection of anti-YB-1 antibodies and delivers a diagnostic assay with high sensitivity and specificity.

Example 3: Levels of YB-1 antibodies were determined in samples from patients suffering from ovarian cancer taken after surgical removal of the cancer and before onset of treatment with a platinum-based antineoplastic. The patients were treated with a chemotherapeutic agent (platinum-based antineoplastic) after surgical removal of ovarian cancer. YB-1 antibody levels in serum samples from 132 healthy donors ("control"; see Example 2), 1 1 1 patients with ovarian cancer showing response to the treatment with the platinum-based antineoplastic carboplatin ("responder") and 52 patients not showing response to the platinum-based antineoplastic ("non-responder") were measured using the kit and method of Example 1. The levels were determined in units/mL. Figure 3 shows the mean values of the natural logarithm of the YB-1 antibody levels for control, responder and non-responder subjects. Patients not responding to the treatment with the platinum-based antineoplastic had significantly lower levels (p<0.0372) of YB-1 antibodies as compared to patients responding to said treatment. Example 4: Serum samples of ovarian cancer patients were taken before treatment with carboplatin. The treatment was conducted by physicians. The patients were categorized into relapse free ("no relapse") and patients who received a relapse ("relapse"). The levels of YB-1 antibodies were determined as outlined in Example 1. The results are shown in Figure 4. Levels of anti-YB-1 antibodies were lower in patients of the "relapse" group (In mean: 0.985 units/ml) compared to the "no relapse" group (In mean: 0.833 units/ml).

Example 5:

The sensitivity and specificity for levels of YB-1 antibodies as predictor of relapse was calculated using ROC-analysis and Kaplan-Meier estimation. Relapse were determined after treatment by surgery and a platinum-based antineoplastic. The results for an exemplary cut off value of 1.74 units/ml are given are given in Figure 5. The results show that the levels of YB-1 antibodies are a good predictor for relapse after treatment of cancer patients, giving a sensitivity of 70 % by maintaining a specificity of 50 %.

Summary

The results of the present Examples show that YB antibody levels are significant lower in patients with cancer compared to healthy controls. This allows diagnosis of cancer. Furthermore, the levels are significantly higher in patients in which show no relapse after treatment as compared to patients showing relapse of cancer after treatment. Levels of YB-1 antibodies in samples are a well suited predictor for the response to the treatment with a platinum-based antineoplastic.