US20120040338A1 - RBM3 in Testicular Cancer Diagnostics and Prognostics - Google Patents

RBM3 in Testicular Cancer Diagnostics and Prognostics Download PDF

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US20120040338A1
US20120040338A1 US13/210,809 US201113210809A US2012040338A1 US 20120040338 A1 US20120040338 A1 US 20120040338A1 US 201113210809 A US201113210809 A US 201113210809A US 2012040338 A1 US2012040338 A1 US 2012040338A1
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sample
rbm3
protein
affinity ligand
seq
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Karin Jirström
Jakob Eberhard
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Atlas Antibodies AB
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Atlas Antibodies AB
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Priority claimed from PCT/SE2009/000091 external-priority patent/WO2009102261A1/fr
Priority claimed from EP09158084A external-priority patent/EP2241889A1/fr
Priority claimed from EP09167847A external-priority patent/EP2293070A1/fr
Priority claimed from PCT/EP2009/067419 external-priority patent/WO2010091763A1/fr
Application filed by Atlas Antibodies AB filed Critical Atlas Antibodies AB
Priority to US13/210,809 priority Critical patent/US20120040338A1/en
Assigned to ATLAS ANTIBODIES AB reassignment ATLAS ANTIBODIES AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: EBERHARD, JAKOB, JIRSTROM, KARIN
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    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to the field of testicular cancer. Further, it relates to detection of testicular disorders, including pre-malignant stages of testicular cancer, as well as the establishment of a prognosis for testicular cancer patients.
  • Cancer is one of the most common diseases, and a major cause of death, in the western world. In general, incidence rates increase with age for most forms of cancer. As human populations continue to live longer, due to an increase of the general health status, cancer may affect an increasing number of individuals. The cause of most common cancer types is still largely unknown, although there is an increasing body of knowledge providing a link between environmental factors (dietary, tobacco smoke, UV radiation etc) as well as genetic factors (germ line mutations in “cancer genes” such as p53, APC, BRCA1, XP etc) and the risk for development of cancer.
  • cancer is essentially a cellular disease and defined as a transformed cell population with net cell growth and anti-social behavior.
  • Malignant transformation represents the transition to a malignant phenotype based on irreversible genetic alterations. Although this has not been formally proven, malignant transformation is believed to take place in one cell, from which a subsequently developed tumor originates (the “clonality of cancer” dogma).
  • Carcinogenesis is the process by which cancer is generated and is generally accepted to include multiple events that ultimately lead to growth of a malignant tumor. This multi-step process includes several rate-limiting steps, such as addition of mutations and possibly also epigenetic events, leading to formation of cancer following stages of precancerous proliferation.
  • the stepwise changes involve accumulation of errors (mutations) in vital regulatory pathways that determine cell division, asocial behavior and cell death.
  • Each of these changes may provide a selective Darwinian growth advantage compared to surrounding cells, resulting in a net growth of the tumor cell population.
  • a malignant tumor does not only necessarily consist of the transformed tumor cells themselves but also surrounding normal cells which act as a supportive stroma.
  • This recruited cancer stroma consists of connective tissue, blood vessels and various other normal cells, e.g., inflammatory cells, which act in concert to supply the transformed tumor cells with signals necessary for continued tumor growth.
  • cancers arise in somatic cells and are predominantly of epithelial origin, e.g., prostate, breast, colon, urothelium and skin, followed by cancers originating from the hematopoietic lineage, e.g., leukemia and lymphoma, neuroectoderm, e.g., malignant gliomas, and soft tissue tumors, e.g., sarcomas.
  • epithelial origin e.g., prostate, breast, colon, urothelium and skin
  • cancers originating from the hematopoietic lineage e.g., leukemia and lymphoma
  • neuroectoderm e.g., malignant gliomas
  • soft tissue tumors e.g., sarcomas.
  • mice Microscopic evaluation of biopsy material from suspected tumors remains the golden standard for cancer diagnostics.
  • the tumor tissue is fixated in formalin, histo-processed and paraffin embedded.
  • tissue sections can be produced and stained using both histochemical, i.e., hematoxylin-eosin staining, and immunohistochemical (IHC) methods.
  • IHC immunohistochemical
  • the surgical specimen is then evaluated with pathology techniques, including gross and microscopic analysis. This analysis often forms the basis for assigning a specific diagnosis, i.e., classifying the tumor type and grading the degree of malignancy, of a tumor.
  • TMM tumor-node-metastasis
  • NMM tumor-node-metastasis
  • N stage describes the local extent of the primary tumor, i.e., how far the tumor has invaded and imposed growth into surrounding tissues
  • N stage and M stage describe how the tumor has developed metastases, with the N stage describing spread of tumor to lymph nodes and the M stage describing growth of tumor in other distant organs.
  • Early stages include: T0-1, N0, M0, representing localized tumors with negative lymph nodes.
  • More advanced stages include: T2-4, N0, M0, localized tumors with more widespread growth and T1-4, N1-3, M0, tumors that have metastasized to lymph nodes and T1-4, N1-3, M1, tumors with a metastasis detected in a distant organ.
  • Staging of tumors is often based on several forms of examination, including surgical, radiological and histopathological analyses.
  • a classification system to grade the level of malignancy.
  • the grading systems rely on morphological assessment of a tumor tissue sample and are based on the microscopic features found in a given tumor. These grading systems may be based on the degree of differentiation, proliferation and atypical appearance of the tumor cells. Examples of generally employed grading systems include Gleason grading for prostatic carcinomas and the Nottingham Histological Grade (NHG) grading for breast carcinomas.
  • IHC immunohistochemical staining
  • IHC allows for the detection of protein expression patterns in tissues and cells using specific antibodies.
  • the use of IHC in clinical diagnostics allows for the detection of immunoreactivity in different cell populations, in addition to the information regarding tissue architecture and cellular morphology that is assessed from the histochemically stained tumor tissue section.
  • IHC can be involved in supporting the accurate diagnosis, including staging and grading, of a primary tumor as well as in the diagnostics of metastases of unknown origin.
  • the most commonly used antibodies in clinical practice today include antibodies against cell type “specific” proteins, e.g., PSA (prostate), MelanA (melanocytes) and Thyroglobulin (thyroid gland), and antibodies recognizing intermediate filaments (epithelial, mesenchymal, glial), cluster of differentiation (CD) antigens (hematopoietic, sub-classification of lymphoid cells) and markers of malignant potential, e.g., Ki67 (proliferation), p53 (commonly mutated tumor suppressor gene) and HER-2 (growth factor receptor).
  • testicular cancer only accounts for approximately 1% of all male cancers, it is the most common cancer in men between 20 and 40 years of age.
  • the incidence rate varies between regions: In Asia and Africa the disease is uncommon, with an age standardized incidence of approximately 1/100,000, but in the Nordic countries the incidence is relatively high compared to the rest of the world, with Denmark, Norway, and Sweden all in the top-ten in world incidence rates. Denmark has the highest incidence rate of approximately 10/100,000, Norway is close behind with approximately 9/100,000, and in Sweden the corresponding ratio is approximately 5/100,000.
  • testicular cancers are germ cell tumors, and there are two main systems for classification of these tumors: The British Testicular Tumor Panel System (BTTP) and the WHO classification system. Most pathologists use the WHO system, since it is somewhat more comprehensive.
  • the two main classes, according to both systems, are seminomas and non-seminomatous germ cell tumors (NSGCT). Seminomas are the most common pure germ cell tumors accounting for approximately 50% of germ cell tumors. Since these tumors are generally very sensitive to both radiation and chemotherapy the cure rate is high; for localized disease more than 95%, and for metastatic disease between 85 and 95%.
  • NSGCT can be divided into Embryonal carcinomas, Yolk sac tumors, Choriocarcinoma, and Teratomas. NSGCT are generally not as sensitive to radiation as seminomas, but with the exception of teratomas, most NSGCT are highly sensitive to platinum-based chemotherapy. The tumor specific 10-year survival is approximately 90% for NSGCT.
  • HCG human chorionic gonadotropin
  • AFP ⁇ -fetoprotein
  • LD lactate dehydrogenase
  • HCG is a glucoprotein that is produced in the placenta, and it is present only in very small amounts in adults. It shares some similarity with other hormones, and false positives may therefore occur. Raised HCG-levels can be seen in 60-70% of patients with NSGCT and in 15-20% of patients with seminoma. Lightly raised levels can also be found in some other cancers.
  • AFP is another glucoprotein that is only present in low amounts in adults, it is normally produced in certain foetal tissues. In tumor tissue it is produced by yolk sac cells in NSGCT, but these cells are not present in seminomas. Raised AFP-levels in serum can be seen in 60-70% of patients with NSGCT. Somewhat raised levels can also be found in patients with other cancers.
  • Raised levels of either (or both) marker(s) can be seen in 60-80% of NSGCT patients, and both markers have prognostic value in advanced disease.
  • LD is an enzyme that is present in all human cells, and is thus a less specific marker than HCG and AFP. However, it may be of clinical value, especially in patients without other measurable tumor markers.
  • Diagnostic markers of pre-stages of testicular cancer include Placental-Like Alkaline Phosphatase (FLAP), Octamer-3/4 (OCT3/4) and RNA Binding Motif protein Y (RBMY).
  • FLAP Placental-Like Alkaline Phosphatase
  • OCT3/4 Octamer-3/4
  • RBMY RNA Binding Motif protein Y
  • Testicular cancer is normally diagnosed with ultrasound. If cancer cannot be excluded, orchiectomy (surgical removal of the testicle) is normally performed and the biopsy material is analyzed. A biopsy may also be taken from the contralateral testicle. Often, the level of tumor markers is also measured.
  • stage I represents localized disease
  • stage I Mk+ represents raised levels of tumor markers
  • stage II(A-D) abdominal lymph nodes are involved.
  • stage II(A-D) abdominal lymph nodes are involved.
  • Stage II(A-D) abdominal lymph nodes are involved.
  • Stage II(A-D) abdominal lymph nodes are involved.
  • Stage II(A-D) abdominal lymph nodes are involved.
  • Stage II(A-D) abdominal lymph nodes are involved.
  • stage II(A-D) abdominal lymph nodes are involved.
  • stage II(A-D) abdominal lymph nodes are involved.
  • Patients can be stratified into three prognostic groups, good-risk, intermediate-risk, and poor-risk, based on a classification scheme developed by the International Germ Cell Cancer Collaborative Group (IGCCCG).
  • IGCCCG International Germ Cell Cancer Collaborative Group
  • the 5-year overall survival for the good-prognosis group can be more than 90% in some parts of the world, for the intermediate-prognosis group it can be around 75%, and for the poor-prognosis group it can be around 48%.
  • seminomas there is no poor-prognosis group, and the good-prognosis group covers 90% of patients with a 5-year overall survival of approximately 90%.
  • the seminoma intermediate-prognosis group has a 5-year overall survival of 72%.
  • a number of risk factors have been identified, such as tumor marker increase (see above), size and number of metastases, number and type of metastatic sites, histopathology of the primary tumor, and age of the patient.
  • stage I seminomas radiation therapy may be given as adjuvant.
  • chemotherapy is normally applied.
  • NSGCT Patients with NSGCT are usually given chemotherapy as primary treatment.
  • a frequently applied treatment is a combination treatment consisting of bleomycin, etoposide and cisplatin (BEP). With this treatment, 80% of patients with advanced NSGCT are cured, but the remaining 20% fail curative treatment.
  • Method for determining whether a mammalian subject belongs to a first or a second group, wherein subjects of the first group have a higher risk of having a testicular disorder than subjects of the second group comprising the steps of:
  • step a) is limited to cells other than sertoli cells.
  • step a) is limited to the nuclei of cells of said sample.
  • testicular disorder is selected from testicular cancer in situ, atrophy, and infertility.
  • Method for determining whether a mammalian subject having a testicular cancer belongs to a first or a second group, wherein the prognosis of subjects of the first group is better than the prognosis of subjects of the second group comprising the steps of:
  • Method for determining the level of intensity of a treatment of a mammalian subject having a testicular cancer comprising the steps of:
  • prognosis is a probability of survival, such as overall survival, progression free survival or testicular cancer specific survival.
  • testicular cancer is a testicular germ-cell cancer.
  • testicular cancer is non-seminomatous.
  • Method according to item 14 wherein said body fluid sample is selected from the group consisting of blood, plasma, serum, cerebral fluid, urine, seminal fluid, semen and exudate.
  • step a) is limited to the nuclei and/or cytoplasms of tumor cells of said sample.
  • said reference value is a value corresponding to a predetermined amount of RBM3 protein or RBM3 mRNA in a reference sample.
  • step a) is determined as being either 1, corresponding to detectable RBM3 protein in the sample, or 0, corresponding to no detectable RBM3 protein in the sample.
  • step b) corresponds to a reference sample having no detectable RBM3 protein.
  • step b) is 0.
  • amino acid sequence of the RBM3 protein comprises a sequence selected from:
  • amino acid sequence of the RBM3 protein comprises or consists of a sequence selected from:
  • step a) comprises:
  • step a) comprises:
  • Method according to item 28 or 29, wherein the quantifiable affinity ligand is selected from the group consisting of antibodies, fragments thereof and derivatives thereof.
  • Method according to item 30 wherein said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:6-19.
  • Method according to item 32 wherein said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:8, 16 and 17.
  • the quantifiable affinity ligand is a protein ligand derived from a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, lipocalins, ankyrin repeat domains, cellulose binding domains, ⁇ crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain and zinc fingers.
  • a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, lipocalins, ankyrin repeat domains, cellulose binding domains, ⁇ crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain and zinc fingers.
  • said quantifiable affinity ligand comprises a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radioisotopes, particles and quantum dots.
  • a quantifiable affinity ligand capable of selective interaction with a protein selected from Placental-Like Alkaline Phosphatase (PLAP), Octamer-3/4 (OCT3/4) and (RNA Binding Motif protein Y) RBMY; and
  • a quantifiable affinity ligand capable of selective interaction with a protein selected from human chorionic gonadotropin (HCG), ⁇ -fetoprotein (AFP) and lactate dehydrogenase (LD); and
  • Kit according to item 43 or 44 in which said quantifiable affinity ligand is selected from the group consisting of antibodies, fragments thereof and derivatives thereof.
  • Kit according to item 45 in which said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with a protein whose amino acid sequence consists of SEQ ID NO:1.
  • Kit according to item 45 in which said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with a peptide whose amino acid sequence consists of an amino acid sequence selected from SEQ ID NO:4 and 5.
  • Kit according to item 45 in which said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises an amino acid sequence selected from SEQ ID NO:6-19.
  • Kit according to item 48 in which said quantifiable affinity ligand is obtainable by a process comprising a step of immunizing an animal with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises an amino acid sequence selected from SEQ ID NO:8, 16 and 17.
  • Kit according to item 43 or 44 in which said quantifiable affinity ligand is a protein ligand derived from a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, lipocalins, ankyrin repeat domains, cellulose binding domains, ⁇ crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain and zinc fingers.
  • a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, lipocalins, ankyrin repeat domains, cellulose binding domains, ⁇ crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, PDZ domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain and zinc fingers.
  • Kit according to any one of items 43-51, in which said quantifiable affinity ligand is capable of selective interaction with an RBM3 protein comprising, or consisting of, a sequence selected from:
  • Kit according to any one of items 43-52, in which said quantifiable affinity ligand is capable of selective interaction with an RBM3 protein comprising, or consisting of, a sequence selected from:
  • kits according to any one of items 43-56 in which said quantifiable affinity ligand comprises a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radioisotopes, particles and quantum dots.
  • a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radioisotopes, particles and quantum dots.
  • Kit according to item 58 in which said secondary affinity ligand comprises a label selected from the group consisting of fluorescent dyes or metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radioisotopes, particles and quantum dots.
  • Kit according to item 60 in which at least one reference sample is a sample comprising no detectable RBM3 protein.
  • Kit according to item 65 in which at least one reference sample comprises an amount of RBM3 protein corresponding to a strong nuclear or cytoplasmic intensity.
  • a first reference sample comprising an amount of RBM3 protein corresponding to a value (positive reference value) being higher than a reference value
  • a second reference sample comprising an amount of RBM3 protein corresponding to a value (negative reference value) being lower than or equal to said reference value.
  • RBM3 protein fragment which consists of 50 amino acids or less and comprises an amino acid sequence selected from SEQ ID NO:4-19.
  • RBM3 protein fragment according to item 70 which consists of 29 amino acids or less.
  • RBM3 protein fragment according to item 70 or 71 which consists of 20 amino acids or less and comprises an amino acid sequence selected from SEQ ID NO:6-19.
  • RBM3 protein fragment according to item 72 which consists of 20 amino acids or less and comprises an amino acid sequence selected from SEQ ID NO:8, 16 and 17.
  • RBM3 protein fragment according to item 72 or 73 which consists of 15 amino acids or less.
  • RBM3 protein or an antigenically active fragment thereof, for the production of a diagnostic agent for a testicular disorder, such as testicular cancer in situ, or a prognostic agent for establishing a prognosis for a mammalian subject having a testicular cancer.
  • prognostic agent is an affinity ligand capable of selective interaction with the RBM3 protein or the antigenically active fragment thereof.
  • amino acid sequence of the RBM3 protein comprises a sequence selected from:
  • amino acid sequence of the RBM3 protein comprises or consists of a sequence selected from:
  • Affinity ligand which is obtainable by a process comprising a step of immunizing an animal with a peptide whose amino acid sequence consists of sequence SEQ ID NO:4 or 5 or a RBM3 protein fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises an amino acid sequence selected from SEQ ID NO:6-19.
  • Affinity ligand which is obtainable by a process comprising a step of immunizing an animal with a peptide whose amino acid sequence consists of SEQ ID NO:5 or a RBM3 protein fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:8, 16 and 17.
  • Affinity ligand capable of selective interaction with a peptide whose amino acid sequence consists of SEQ ID NO:4 or 5 or an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises an amino acid sequence selected from SEQ ID NO:6-19.
  • Affinity ligand capable of selective interaction with a peptide whose amino acid sequence consists of SEQ ID NO:5 or an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises an amino acid sequence selected from SEQ ID NO:8, 16 and 17.
  • affinity ligand is an affinity ligand according to any one of items 87-90.
  • FIG. 1 shows Western blot results for Anti-RBM3, 1B5 and 6F11.
  • FIG. 2 shows RBM3 protein expression in testicular cancer patients. Patients were split into four different prognosis groups: poor prognosis, intermediate prognosis, good prognosis, and seminomas.
  • FIG. 3 shows the RBM3 protein expression in testicular cancer patients. Patients were split into two different groups: diseased or survived.
  • FIG. 4 shows Western blot results for, from left to right, 7F5, 10F1, 12A10, 12C9, 14D9 and anti-RBM3.
  • lanes 1 through 5 show the monoclonal antibodies while lane 6 shows the polyclonal anti-RBM3 antibody.
  • FIG. 5 shows alignment of SEQ ID NO:4 as well as the peptides used for epitope mapping of the monoclonal antibodies.
  • a method for determining whether a mammalian subject belongs to a first or a second group, wherein subjects of the first group have a higher risk of having a testicular disorder than subjects of the second group comprising the steps of:
  • This first aspect of the present disclosure is based on the inventors' finding that RBM3 generally is highly expressed in tissue of testicular cancer in situ, whereas the RBM3 expression in normal testicular tissue is generally low.
  • the inventors thus conclude that the presence of RBM3 in biological material from a testicle is a marker of testicular cancer in situ, or of other conditions associated with an elevated risk for subsequent development of testicular cancer.
  • RBM3 is likely to be a marker for testicular disorders in general, not only of pre-stages of testicular cancer. Examples of other testicular disorders are atrophy and sertoli cell-only syndrome, which are associated with infertility or cancer risk.
  • the inventors believe that the RBM3 may be an oncogene that is turned on as a protection against testicular disorders.
  • subjects showing high RBM3 expression in a testicle may be closely monitored and/or further examined (e.g. with ultra sound), and possibly, the testicle in question may be surgically removed to avoid development and spread of cancer. Further, if a subject is diagnosed with cancer in one testicle, an RBM3 measurement may indicate if the other testicle is also at risk of developing cancer. Also, it may be particularly relevant to monitor subjects having cryptorchidism with RBM3 measurements according to the present disclosure, because such subjects already have an elevated risk of developing cancer.
  • the biological material of the sample of step a) of the first aspect may comprise seminal fluid or cells from the testicle.
  • the cells may for example be part of tissue material from the testicle.
  • the tissue material may be obtained from a previously performed testicle biopsy. Such biopsies are often performed when a subject shows symptoms of a testicle disorder or during a fertility examination. Collection of seminal fluid may be a more convenient way of obtaining biological material from the testicle, which also saves the subject the discomfort of the biopsy.
  • the evaluation of step a) may be limited to cells other than sertoli cells.
  • the inventors have found that sertoli cells of normal testicular tissue express some RBM3 protein, and the precision of the method of the first aspect may thus be increased if sertoli cells are excluded from the evaluation of step a).
  • An example of cells other than sertoli cells that may be relevant in the evaluation is spermatogonia or more differentiated forms thereof.
  • step a) may thus be limited to the nuclei of cells of said sample.
  • the difference between the first and the second group may be that subjects of the first group have a higher risk of having testicular cancer in situ than subjects of the second group.
  • the difference between the first and the second group may be that subjects of the first group have a higher risk of developing testicular cancer, such as a testicular germ-cell tumor, than subjects of the second group.
  • a physician assessing the risk of a subject developing cancer may assign to someone else, such as a lab worker, to perform step a), and optionally step b) of the method of the first aspect, while performing step c), and optionally b), himself.
  • the cure rate of subjects diagnosed with testicular cancer is comparatively high. Accordingly, the inventors conclude that there is an interest in even better stratification of patients to avoid over treatment in low-risk groups, and focusing the more intense treatment strategies on the patients that are in most need of them.
  • a method for determining whether a mammalian subject having a testicular cancer belongs to a first or a second group, wherein the prognosis of subjects of the first group is better than the prognosis of subjects of the second group comprising the steps of:
  • This second aspect of the present disclosure is based on the inventors' finding that testicular cancer subjects showing high levels of RBM3 expression generally have a greater chance of survival than such subjects showing low levels of RBM3 expression.
  • RBM3 is thus a prognostic marker for testicular cancer.
  • the suggested protective role of the RBM3 may explain why it is up-regulated in pre-stages of testicular cancer and in testicular cancers of good prognosis. Accordingly, the low RBM3 protein levels in testicular cancers of poor prognosis may be an indication of malfunctioning protection against the cancer.
  • prognostic indication entails a number of benefits.
  • prognostic information may form the basis of a physician's decision regarding the treatment of a subject.
  • the prognosis for a testicular cancer subject normally reflects the level of aggressiveness of the cancer and, if a particularly aggressive form of a cancer is identified, a painful or in any other sense unpleasant treatment which normally is avoided may anyway be considered. Further, if less aggressive forms can be identified, over-treatment may be avoided.
  • the RBM3 protein or the RBM3 mRNA has a great potential for example in a panel for making predictions or prognoses or for the selection of a treatment regimen.
  • testicular cancer subject belongs to a first or a second group, wherein subjects of the first group generally have a better prognosis than subjects of the second group.
  • the division of testicular cancer subjects into the two groups is determined by comparing samples values from the subjects with a reference value.
  • the reference value is thus the determinant for the size of the respective groups; the higher the reference value, the fewer the subjects in the first group and the lower the likelihood that a tested subject belongs to the first group.
  • a high reference value may in some instances be selected to identify subjects with a particularly good prognosis.
  • the person skilled in the art may select relevant reference values without undue burden. This is further discussed below.
  • the first and the second group may consist exclusively of subjects having testicular cancers of the same or similar stage, differentiation grade and/or subtype as the tested subject. Further, the groups may consist only of subjects having the same or similar age, race, geographic residence, genetic characteristics or medical status or history, such as testicular cancer history.
  • a physician may use the method according to the second aspect to obtain additional information regarding the prognosis of an testicular cancer subject, which in turn may help him to select the most appropriate treatment regimen. For example, a subject shown to belong to the first group using the method of the second aspect may be given a less aggressive treatment than what would otherwise have been considered, and vice versa.
  • a method for determining the level of intensity of a treatment of a mammalian subject having a testicular cancer comprising the steps of:
  • the level of intensity may for example be measured as the average daily or weekly dose of a therapeutic agent given to the subject.
  • a treatment of the second intensity may thus be applied more frequently or in higher individual doses than a treatment of the first intensity.
  • the treatment of the second intensity may also comprise application of a more aggressive therapy than the treatment of the first intensity.
  • the treatment of the second intensity may be a combination treatment while the treatment of the first intensity is a mono-therapy. Yet another possibility is that the treatment of the second intensity is applied for a longer period than the treatment of the first intensity.
  • c1) may thus be concluding that said subject should undergo treatment during a first period and c2) may be concluding that said subject should undergo treatment during a second period, wherein the second period is longer than the first period.
  • Platinum-based treatment is frequently applied to testicular cancer subjects.
  • a platinum-based treatment comprises application of a platinum-based therapeutic agent.
  • Carboplatin including paraplatin, oxaliplatin, satraplatin, picoplatin and cisplatin are some platinum-based therapeutic agents tested or used in the clinic today.
  • the platinum-based treatment may thus be application of an agent selected from carboplatin, oxaliplatin, satraplatin, picoplatin and cisplatin.
  • the treatment of the first intensity may for example be application of a combination of bleomycin, etoposide and cisplatin whereas the treatment of the first intensity may be application of only one or two of these agents.
  • prognosis refers to the prediction of the course or outcome of a disease and its treatment.
  • prognosis may also refer to a determination of chance of survival or recovery from a disease, as well as to a prediction of the expected survival time of a subject.
  • a prognosis may specifically involve establishing the likelihood for survival of a subject during a period of time into the future, such as three years, five years, ten years or any other period of time.
  • a prognosis may further be represented by a single value or a range of values.
  • the prognosis may be a probability of survival.
  • the survival of the present disclosure may for example be overall survival, progression free survival or testicular cancer specific survival.
  • the “survival” may be measured over different periods, such as five, ten or 15 years. Accordingly, the survival may be a five-year, ten-year or 15-year survival.
  • testicular germ-cell cancers are from testicular germ-cell cancers.
  • the testicular cancer is thus a testicular germ-cell cancer.
  • testicular cancer In general, subjects having seminomatous testicular cancer have a very good prognosis. It may thus be more relevant to establish a prognosis for a subject having a non-seminomatous testicular cancer. Further, RBM3 is shown to be prognostically relevant for such subjects in Examples below. In embodiments of the second or third aspect, the testicular cancer may thus be non-seminomatous.
  • the sample may be a body fluid sample.
  • the body fluid sample may be selected from the group consisting of blood, plasma, serum, cerebral fluid, urine, seminal fluid, semen, lymph and exudate.
  • the sample may be a cytology sample or a stool sample.
  • the level of expression of RBM3 protein or RBM3 mRNA may preferably be measured intracellularly or in sample derived from cells.
  • the body fluid, cytology or stool sample may for example comprise cells, such as tumor cells derived from a testicle of the subject
  • the sample may be a testicular tumor tissue sample, e.g. from an earlier surgical removal of a testicle.
  • RBM3 protein is particularly relevant for determining prognoses or selecting treatments.
  • step a) of the second or third aspect may be limited to the nuclei of tumor cells of said sample. Consequently, when a tissue sample is examined, only the nuclei of tumor cells may be taken into consideration. Such examination may for example be aided by immunohistochemical staining.
  • an increase in the amount of RBM3 protein or RBM3 mRNA typically results in an increase in the sample value, and not the other way around.
  • the evaluated amount may correspond to any of a predetermined number of discrete sample values.
  • a first amount and a second, increased, amount may correspond to the same sample value.
  • an increase in the amount of RBM3 protein or RBM3 mRNA will not result in a decrease in the sample value in the context of the present disclosure.
  • the evaluated amounts may be inversely related to sample values if the qualification between step b) and c) is inverted.
  • the qualification between step b) and c1) is inverted if the phrase “if the sample value is higher than the reference value” is replaced with “if the sample value is lower than the reference value”.
  • the physician responsible for the treatment may take several parameters into account, such as the result of an immunohistochemical evaluation, patient age, tumor subtype, stage and grade, general condition and medical history, such as testicular cancer history.
  • the physician may perform a test, or order a test performed, according to the second or third aspect. Further, the physician may assign to someone else, such as a lab worker, to perform step a), and optionally step b), while performing step c), and optionally b), himself.
  • “earlier obtained” refers to obtained before the method is performed. Consequently, if a sample earlier obtained from a subject is used in a method, the method does not involve obtaining the sample from the subject, i.e., the sample was previously obtained from the subject in a step separate from the method.
  • a mammalian subject having a testicular cancer refers to a mammalian subject having a testicular tumor or a mammalian subject which has had a testicular tumor removed, wherein the removal of the tumor refers to killing or removing the tumor by any appropriate type of surgery or therapy.
  • a mammalian subject having testicular cancer also includes the cases wherein the mammalian subject is suspected of having a testicular cancer at the time of the performance of the use or method and the testicular cancer diagnosis is established later.
  • the “reference value” refers to a predetermined value found to be relevant for making decisions or drawing conclusions regarding the diagnosis, prognosis or a suitable treatment strategy for the subject.
  • Step a) of the methods of the above aspects involve evaluating an amount of RBM3 protein or RBM3 mRNA present in at least part of the sample, and determining a sample value corresponding to the amount.
  • the “at least part of the sample” refers to a relevant part or relevant parts of the sample for establishing the diagnosis or prognosis or drawing conclusions regarding suitable treatments. The person skilled in the art understands which part or parts that are relevant under the circumstances present when performing the method.
  • step a) an amount is evaluated and a sample value corresponding to the amount is determined. Consequently, an exact measurement of the amount of RBM3 protein or RBM3 mRNA is not required for obtaining the sample value.
  • the amount of RBM3 protein may be evaluated by visual inspection of a stained tissue sample and the sample value may then be categorized as for example “high” or “low” based on the evaluated amount.
  • step a) requires some kind of processing or manipulation of the sample. It is not possible to determine the sample value by mere inspection. Various techniques, of which some are presented below, for such evaluation and determination, are well known to the skilled person. The methods of the present disclosure are therefore not limited to any specific technique or techniques for the performance of step a).
  • step a) it may be convenient to use zero as the reference value, because in such case, it has only to be established in step a) whether RBM3 protein or RBM3 mRNA is present in the sample or not.
  • the sample value of step a) may be either 1, corresponding to detectable RBM3 protein in the sample, or 0, corresponding to no detectable RBM3 protein in the sample. Consequently, in such embodiments, the evaluation of the sample is digital: RBM3 protein is considered to be either present or not.
  • no detectable RBM3 protein refers to an amount of RBM3 protein that is so small that it is not, during normal operational circumstances, detectable by a person or an apparatus performing the step a).
  • the “normal operational circumstances” refer to the laboratory methods and techniques a person skilled in the art would find appropriate for performing the methods of the present disclosure.
  • the reference value of step b) may be 0.
  • the reference value of step b) may correspond to a reference sample having no detectable RBM3 protein or RBM3 mRNA (see below).
  • sample value and “reference value” are to be interpreted broadly.
  • the quantification of RBM3 protein or RBM3 mRNA to obtain these values may be done via automatic means, via a scoring system based on visual or microscopic inspection of samples, or via combinations thereof.
  • a skilled person such as a person skilled in the art of histopathology, to determine the sample and reference values by inspection, e.g., of tissue slides that have been prepared and stained for RBM3 protein expression. Determining that the sample value is higher than the reference value may thus correspond to determining, upon visual or microscopic inspection, that a sample tissue slide is more densely stained than a reference tissue slide.
  • sample value may also be compared to a reference value given by a literal reference, such as a reference value described in wording or by a reference picture. Consequently, the sample and/or reference values may in some cases be mental values that the skilled person determines upon inspection and comparison.
  • step a) and optionally step b) may be performed in an automatic analysis apparatus, and such apparatus may be based on a platform adapted for immunohistochemical analysis.
  • one or more tissue sample(s) from the subject in question may be prepared for immunohistochemical analysis manually and then loaded into the automatic analysis apparatus, which gives the sample value of step a) and optionally also performs the comparison with the reference value of step b).
  • the operator performing the analysis, the physician ordering the analysis or the apparatus itself may then draw the conclusion of step c). Consequently, software adapted for drawing the conclusion of step c) may be implemented on the apparatus.
  • a reference value found to be relevant for establishing a diagnosis or prognosis or making treatment decisions, for use as comparison with the sample value from the subject, may be provided in various ways. With the knowledge of the teachings of the present disclosure, the skilled artisan can, without undue burden, provide relevant reference values for performing the methods of the present disclosure.
  • the person performing the methods of the above aspects may, for example, adapt the reference value to the desired information.
  • the reference value may for example be adapted to yield the most significant information with regard to survival.
  • the reference value may correspond to the amount of RBM3 protein or RBM3 mRNA measured in a reference sample of normal testicular tissue.
  • the amount of protein expression of the reference sample may be previously established.
  • the reference value may correspond to the amount of RBM3 protein or RBM3 mRNA measured in a reference sample comprising or being derived from tumor cells, such as a reference sample of tumor tissue.
  • the amount of protein expression of the reference sample may preferably be previously established.
  • the reference value may be provided by the amount of RBM3 protein or RBM3 mRNA measured in a reference sample comprising (or being derived from) cells expressing a predetermined amount of RBM3 protein or RBM3 mRNA.
  • the reference value may for example be provided by the amount of RBM3 protein or RBM3 mRNA measured in a reference sample comprising cell lines, such as cancer cell lines, expressing a predetermined, or controlled, amount of RBM3 protein or RBM3 mRNA.
  • cell lines such as cancer cell lines
  • expressing a predetermined, or controlled, amount of RBM3 protein or RBM3 mRNA The person skilled in the art understands how to provide such cell lines, for example guided by the disclosure of Rhodes et al. (2006) The biomedical scientist, p 515-520.
  • the reference value may be a predetermined value corresponding to the amount of RBM3 protein or RBM3 mRNA in a reference sample.
  • the amount of RBM3 protein in the reference sample does not have to directly correspond to the reference value.
  • the reference sample may also provide an amount of RBM3 protein that helps a person performing the method to assess various reference values.
  • the reference sample(s) may help in creating a mental image of the reference value by providing a “positive” reference value and/or a “negative” reference value.
  • the fraction may for example be: a “cellular fraction”, wherein the RBM3 protein expression of the whole cells is taken into account; a “cytoplasmic fraction”, wherein the RBM3 protein expression of only the cytoplasms of the cells is taken into account; or a “nuclear fraction”, wherein the RBM3 protein expression of only the nuclei of the cells is taken into account.
  • the nuclear or cytoplasmic fraction may for example be classified as ⁇ 2%, 2-25%, >25-75% or >75% immunoreactive cells of the relevant cell population.
  • the “cytoplasmic fraction” corresponds to the percentage of relevant cells in a sample that exhibits a positive staining in the cytoplasm, wherein a medium or distinct and strong immunoreactivity in the cytoplasm is considered positive and no or faint immunoreactivity in the cytoplasm is considered negative.
  • the “nuclear fraction” corresponds to the percentage of relevant cells in a sample that exhibits a positive staining in the nucleus, wherein a medium or distinct and strong immunoreactivity in the nucleus is considered positive and no or faint immunoreactivity in the nucleus is considered negative.
  • the person skilled in the art of pathology understands which cells that are relevant under the conditions present when performing the method and may determine a cytoplasmic or nuclear fraction based on his general knowledge and the teachings of the present disclosure. Further, the skilled artisan understands how to perform corresponding measurements employing the “cellular fraction”.
  • the intensity may for example be: a “cellular intensity”, wherein the RBM3 protein expression of the whole cells is taken into account; a “cytoplasmic intensity”, wherein the RBM3 protein expression of only the cytoplasms of the cells is taken into account, or a “nuclear intensity”, wherein the RBM3 protein expression of only the nuclei of the cells is taken into account. Cytoplasmic and nuclear intensity is subjectively evaluated in accordance with standards used in clinical histopathological diagnostics.
  • the weak and moderate values may be combined into a weak/moderate value.
  • the reference value may thus be a nuclear fraction, a nuclear intensity or a combination thereof.
  • the sample value may be a nuclear fraction, a nuclear intensity or a combination thereof.
  • the criterion for the conclusion in step c) is that the sample value is higher than a nuclear or cytoplasmic fraction of 0%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95%.
  • the reference value of step b) is a nuclear or cytoplasmic fraction of 95% or lower, such as 90% or lower, such as 85% or lower, such as 80% or lower, such as 75% or lower, such as 70% or lower, such as 65% or lower, such as 60% or lower, such as 55% or lower, such as 50% or lower, such as 45% or lower, such as 40% or lower, such as 35% or lower, such as 30% or lower, such as 25% or lower, such as 20% or lower, such as 15% or lower, such as 10% or lower, such as 5% or lower, such as 2% or lower, such as 1% or lower, such as 0%.
  • 95% or lower such as 90% or lower, such as 85% or lower, such as 80% or lower, such as 75% or lower, such as 70% or lower, such as 65% or lower, such as 60% or lower, such as 55% or lower, such as 50% or lower, such as 45% or lower, such as 40% or lower, such as 35% or lower, such as 30% or lower, such as 25% or
  • the criterion for the conclusion in step c) may be a sample value, which is higher than absent cytoplasmic or nuclear intensity, such as higher than weak cytoplasmic or nuclear intensity, such as higher than moderate cytoplasmic or nuclear intensity.
  • the reference value of step b) may be a moderate cytoplasmic or nuclear intensity of RBM3 protein expression or lower, such as a weak cytoplasmic or nuclear intensity of RBM3 protein expression or lower, such as an absent cytoplasmic or nuclear intensity.
  • the reference value may be a combination or a function of a fraction value and an intensity value.
  • the reference value may thus involve two, and even more, criteria.
  • an intensity value and/or a fraction value as the reference value may depend on the staining procedure, e.g., on the type and amount/concentration of the employed antibody and on the type and concentration of the staining reagents.
  • relative low reference values refer to nuclear or cytoplasmic fractions of 25% or lower, such as 10% or lower, a weak nuclear or cytoplasmic intensity or lower, such as an absent nuclear or cytoplasmic intensity or a staining score (SS, see Examples, section 8a) of 1 or lower, such as 0.
  • a relatively high reference value may also be particularly relevant for making diagnostic or prognostic conclusions according to the above aspects.
  • a “relatively high” reference value refer to nuclear or cytoplasmic fractions of 50% or higher, a moderate nuclear or cytoplasmic intensity or a staining score (SS, see Examples, section 8A) of 2.
  • a person skilled in the art e.g., a pathologist understands how to perform the evaluation yielding a fraction, such as a cellular, cytoplasmic or nuclear fraction, or an intensity, such as a cellular, cytoplasmic or nuclear intensity.
  • a fraction such as a cellular, cytoplasmic or nuclear fraction
  • an intensity such as a cellular, cytoplasmic or nuclear intensity.
  • the skilled artisan may use a reference sample comprising a predetermined amount of RBM3 protein for establishing the appearance of a certain fraction or intensity.
  • a reference sample may not only be used for the provision of the actual reference value, but also for the provision of an example of a sample with an amount of RBM3 protein that is higher than the amount corresponding to the reference value.
  • histochemical staining such as in immunohistochemical staining
  • the skilled artisan may use a reference sample for establishing the appearance of a stained sample having a high amount of RBM3 protein, e.g., a positive reference. Subsequently, the skilled artisan may assess the appearances of samples having lower amounts of RBM3 protein, such as the appearance of a sample with an amount of RBM3 protein corresponding to the reference value.
  • the skilled artisan may use a reference sample to create a mental image of a reference value corresponding to an amount of RBM3 protein which is lower than that of the reference sample.
  • the skilled artisan may use another reference sample having a low amount of RBM3 protein, or lacking detectable RBM3 protein, for establishing the appearance of such sample, e.g., as a “negative reference”.
  • two reference samples may be employed: a first reference sample having no detectable RBM3 protein, and thus corresponding to an absent nuclear intensity, which is lower than the reference value; and a second reference sample having an amount of RBM3 protein corresponding to a strong nuclear intensity, which is higher than the reference value.
  • reference sample for establishing the appearance of a sample with a high amount of RBM3 protein.
  • Such reference sample may be a sample comprising tissue expressing a high amount of RBM3 protein, such as a sample comprising tissue having a pre-established high expression of RBM3 protein.
  • the reference sample may provide an example of a strong nuclear intensity (NI).
  • NI nuclear intensity
  • the skilled artisan may then divide samples into the NI categories absent, weak, moderate and strong. This division may be further assisted by a reference sample lacking detectable RBM3 protein (negative reference), i.e., a reference sample providing an absent nuclear intensity.
  • the reference sample may provide an example of a sample with a nuclear fraction (NF) higher than 75%. With the knowledge of the appearance of a sample with more than 75% positive cells, the skilled artisan may then evaluate the NF of other samples having e.g., a lower percentage of positive cells.
  • This division may be further assisted by a reference sample essentially lacking RBM3 protein (negative reference), i.e., a reference sample providing a low NF (e.g., ⁇ 5%, such as ⁇ 2%), or a NF of 0.
  • cell lines expressing a controlled amount of RBM3 protein may be used as the reference, in particular as a positive reference.
  • One or more pictures may also be provided as the “reference sample”.
  • a picture may show an example of a tissue slide stained with a certain antibody during certain conditions and exhibiting a certain nuclear intensity and/or fraction.
  • the above discussion about the “reference sample” applies mutatis mutandis to pictures.
  • the cell lines or pictures may also form part of the kit according to the present disclosure (see below).
  • the RBM3 protein may comprise a sequence selected from:
  • sequence ii) above is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to SEQ ID NO:1.
  • the RBM3 protein may comprise, or consists of, a sequence selected from:
  • sequence ii) above is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to SEQ ID NO:2.
  • % identical is calculated as follows.
  • the query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson, J. D., Higgins, D. G. and Gibson, T. J., Nucleic Acids Research, 22: 4673-4680 (1994)).
  • the amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identical.
  • the target sequence determines the number of positions that are compared. Consequently, in the context of the present disclosure, a query sequence that is shorter than the target sequence can never be 100% identical to the target sequence. For example, a query sequence of 85 amino acids may at the most be 85% identical to a target sequence of 100 amino acids.
  • step a) of the methods of the above aspects may comprise:
  • Step a) may thus, as an example, comprise obtaining tissue material from the subject, optionally fixating the tissue material in paraffin or formalin, histo-processing the tissue material to obtain a section which constitute said sample and optionally mounting said sample on a transparent slide, such as a glass slide, for microscopy.
  • the RBM3 protein may be detected and/or quantified through the application to the sample of a detectable and/or quantifiable affinity ligand, which is capable of selective interaction with the RBM3 protein.
  • the application of the affinity ligand is performed under conditions that enable binding of the affinity ligand to RBM3 protein in the sample.
  • step a) may comprise:
  • affinity ligand remaining in association with the sample refers to affinity ligand which was not removed in step a2), e.g., the affinity ligand bound to the sample.
  • the binding may for example be the interaction between antibody and antigen.
  • step a) may comprise:
  • “specific” or “selective” interaction of e.g., an affinity ligand with its target or antigen means that the interaction is such that a distinction between specific and non-specific, or between selective and non-selective, interaction becomes meaningful.
  • the interaction between two proteins is sometimes measured by the dissociation constant.
  • the dissociation constant describes the strength of binding (or affinity) between two molecules.
  • the dissociation constant between an antibody and its antigen is from 10 ⁇ 7 to 10 ⁇ 11 M.
  • high specificity/selectivity does not necessarily require high affinity. Molecules with low affinity (in the molar range) for its counterpart have been shown to be as selective/specific as molecules with much higher affinity.
  • a specific or selective interaction refers to the extent to which a particular method can be used to determine the presence and/or amount of a specific protein, the target protein, under given conditions in the presence of other proteins in a tissue sample or fluid sample of a naturally occurring or processed biological fluid.
  • specificity or selectivity is the capacity to distinguish between related proteins.
  • Specific and selective are sometimes used interchangeably in the present description.
  • the specificity or selectivity of an antibody may be determined as in Examples, Section 2, below, wherein analysis is performed using a protein array set-up, a suspension bead array and a multiplexed competition assay, respectively. Specificity and selectivity determinations are also described in Nilsson P et al. (2005) Proteomics 5:4327-4337.
  • affinity ligands that may prove useful, as well as examples of formats and conditions for detection and/or quantification, are given below for the sake of illustration.
  • the affinity ligand may be selected from the group consisting of antibodies, fragments thereof and derivatives thereof, i.e., affinity ligands based on an immunoglobulin scaffold.
  • the antibodies and the fragments or derivatives thereof may be isolated.
  • Antibodies comprise monoclonal and polyclonal antibodies of any origin, including murine, rabbit, human and other antibodies, as well as chimeric antibodies comprising sequences from different species, such as partly humanized antibodies, e.g., partly humanized mouse antibodies.
  • Polyclonal antibodies are produced by immunization of animals with the antigen of choice.
  • the polyclonal antibodies may be mono-specific.
  • Monoclonal antibodies of defined specificity can be produced using the hybridoma technology developed by Köhler and Milstein (Köhler G and Milstein C (1976) Eur. J. Immunol. 6:511-519).
  • the antibody fragments and derivatives of the present disclosure are capable of selective interaction with the same antigen (e.g. RBM3 protein) as the antibody they are fragments or derivatives of.
  • Antibody fragments and derivatives comprise Fab fragments, consisting of the first constant domain of the heavy chain (CH1), the constant domain of the light chain (CL), the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of an intact immunoglobulin protein; Fv fragments, consisting of the two variable antibody domains VH and VL (Skerra A and Plückthun A (1988) Science 240:1038-1041); single chain Fv fragments (scFv), consisting of the two VH and VL domains linked together by a flexible peptide linker (Bird R E and Walker B W (1991) Trends Biotechnol. 9:132-137); Bence Jones dimers (Stevens F J et al.
  • the affinity ligand of the present disclosure is capable of selective interaction with a peptide consisting of the amino acid sequence SEQ ID NO:1.
  • the RBM3 fragment SEQ ID NO:1 was designed to lack transmembrane regions to ensure efficient expression in E. coli, and to lack any signal peptide, since those are cleaved off in the mature protein. SEQ ID NO:1 was thus designed for immunizations.
  • the protein fragment was designed to consist of a unique sequence with low homology with other human proteins, to minimize cross reactivity of generated affinity reagents, and to be of a suitable size to allow the formation of conformational epitopes and still allow efficient cloning and expression in bacterial systems.
  • the affinity ligand may be obtainable by a process comprising a step of immunizing an animal with a peptide whose amino acid sequence consists of the sequence SEQ ID NO:1.
  • the immunization process may comprise primary immunization with the protein in Freund's complete adjuvant.
  • the immunization process may further comprise boosting at least two times, in intervals of 2-6 weeks, with the protein in Freund's incomplete adjuvant. Processes for the production of antibodies or fragments or derivatives thereof against a given target are known in the art.
  • the affinity ligand may thus be obtainable by a process comprising a step of immunizing an animal with a peptide whose amino acid sequence consists of SEQ ID NO:4 or SEQ ID NO:5.
  • the antibody or fragment may be obtainable by a process comprising a step of immunizing an animal with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:6-19.
  • a “mono-specific antibody” is one or a population of polyclonal antibodies which has been affinity purified on its own antigen, thereby separating such mono-specific antibodies from other antiserum proteins and non-specific antibodies. This affinity purification results in antibodies that bind selectively to its antigen.
  • the polyclonal antisera are purified by a two-step immunoaffinity based protocol to obtain mono-specific antibodies selective for the target protein. Antibodies directed against generic affinity tags of antigen fragments are removed in a primary depletion step, using the immobilized tag protein as the capturing agent.
  • the serum is loaded on a second affinity column with the antigen as capturing agent, in order to enrich for antibodies specific for the antigen (see also Nilsson P et al. (2005) Proteomics 5:4327-4337).
  • the biomolecular diversity needed for selection of affinity ligands may be generated by combinatorial engineering of one of a plurality of possible scaffold molecules, and specific/selective affinity ligands are then selected using a suitable selection platform.
  • the scaffold molecule may be of immunoglobulin protein origin (Bradbury A R and Marks J D (2004) J. Immunol. Meths. 290:29-49), of non-immunoglobulin protein origin (Nygren P ⁇ and Skerra A (2004) J. Immunol. Meths. 290:3-28), or of an oligonucleotide origin (Gold L et al. (1995) Annu. Rev. Biochem. 64:763-797).
  • Non-limiting examples of such structures useful for generating affinity ligands against RBM3 protein for use according to the present disclosure, are staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z (Nord K et al. (1997) Nat. Biotechnol. 15:772-777); lipocalins (Beste G et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:1898-1903); ankyrin repeat domains (Binz H K et al. (2003) J. Mol. Biol.
  • CBD cellulose binding domains
  • CBD cellulose binding domains
  • GFP green fluorescent protein
  • CTL-4 human cytotoxic T lymphocyte-associated antigen 4
  • protease inhibitors such as Knottin proteins (Wentzel A et al. (2001) J. Bacteriol. 183:7273-7284; Baggio R et al. (2002) J. Mol. Recognit. 15:126-134) and Kunitz domains (Roberts B L et al. (1992) Gene 121:9-15; Dennis M S and Lazarus R A (1994) J. Biol. Chem.269:22137-22144); PDZ domains (Schneider S et al. (1999) Nat. Biotechnol. 17:170-175); peptide aptamers, such as thioredoxin (Lu Z et al.
  • non-immunoglobulin protein scaffolds include scaffold proteins presenting a single randomized loop used for the generation of novel binding specificities, protein scaffolds with a rigid secondary structure where side chains protruding from the protein surface are randomized for the generation of novel binding specificities, and scaffolds exhibiting a non-contiguous hyper-variable loop region used for the generation of novel binding specificities.
  • oligonucleotides may also be used as affinity ligands.
  • Single stranded nucleic acids called aptamers or decoys, fold into well-defined three-dimensional structures and bind to their target with high affinity and specificity.
  • aptamers or decoys Single stranded nucleic acids
  • the oligonucleotide ligands can be either RNA or DNA and can bind to a wide range of target molecule classes.
  • Selection platforms include, but are not limited to, phage display (Smith G P (1985) Science 228:1315-1317), ribosome display (Hanes J and Plückthun A (1997) Proc. Natl. Acad. Sci. U.S.A.
  • yeast two-hybrid system yeast two-hybrid system
  • yeast display yeast display
  • Gai S A and Wittrup K D (2007) Curr Opin Struct Biol 17:467-473
  • mRNA display Robots R W and Szostak J W (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302
  • bacterial display Daugherty P S (2007) Curr Opin Struct Biol 17:474-480, Kronqvist N et al. (2008) Protein Eng Des Sel 1-9, Harvey B R et al.
  • the affinity ligand may be a non-immunoglobulin affinity ligand derived from any of the protein scaffolds listed above, or an oligonucleotide molecule.
  • the RBM3 protein fragment SEQ ID NO:1 was designed to consist of a unique sequence with low homology with other human proteins and to minimize cross reactivity of generated affinity reagents. Consequently, in embodiments of the present disclosure, the affinity ligand may be capable of selective interaction with a polypeptide consisting of the amino acid sequence SEQ ID NO:1.
  • the affinity ligand of the present disclosure is capable of selective interaction with a peptide consisting of an amino acid sequence selected from SEQ ID NO:4 and 5.
  • the affinity ligand of the present disclosure is capable of selective interaction with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:6-9.
  • the affinity ligand of the present disclosure is capable of selective interaction with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:10-19.
  • Antibodies having selectivity for a single epitope region may provide for increased reproducibility in detection analyses as compared to antibodies generated against a longer peptide sequence (such as a PrEST or a full-length protein).
  • the antibodies selective for a single epitope region may also provide for distinct and strong staining in immunohistochemical analyses. These benefits, independently or jointly, may be valuable when and making treatment predictions or decisions regarding treatments according to the present disclosure.
  • FIG. 1 a benefit (increased selectivity) of monoclonal antibodies according to the present disclosure as compared to a polyclonal antibody is illustrated.
  • the monoclonal antibodies 6F11 and 1 B5 are considered to be particularly beneficial.
  • 6 F11 and 1 B5 are both shown to be more selective than a polyclonal anti-RBM3 antibody. Further, 1 B5 is shown to be more selective than 6F11. 1 B5 is also employed in Examples, Sections 8 and 9 below.
  • the affinity ligand is thus capable of selective interaction with an RBM3 fragment which consists of SEQ ID NO:5, and in particularly preferred embodiments of the present disclosure, the affinity ligand is capable of selective interaction with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises the sequence SEQ ID NO:17.
  • 6F11 is shown to bind to SEQ ID NO:8 and SEQ ID NO:16.
  • the affinity ligand is thus capable of selective interaction with an RBM3 fragment which consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:8 and 16. Note that SEQ ID NO:8 and 16 are overlapping and that such a fragment may comprise the sequences of both SEQ ID NO:8 and 16.
  • the detection and/or quantification of the affinity ligand capable of selective interaction with the RBM3 protein may be accomplished in any way known to the skilled person for detection and/or quantification of binding reagents in assays based on biological interactions. Accordingly, any affinity ligand described above may be used to quantitatively and/or qualitatively detect the presence of the RBM3 protein.
  • These “primary” affinity ligands may be labeled themselves with various markers or may in turn be detected by secondary, labeled affinity ligands to allow detection, visualization and/or quantification.
  • Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole) and bioluminescent proteins (e.g., luciferin, luciferase), haptens (e.g., biotin).
  • fluorescent dyes or metals e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine
  • chromophoric dyes e.g., rhodopsin
  • chemiluminescent compounds e.g., luminal, imidazole
  • bioluminescent proteins e.g., luciferin, luciferase
  • haptens
  • Affinity ligands can also be labeled with enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3 H, 14 C, 32 P, 35 S or 125 I) and particles (e.g., gold).
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase
  • radioisotopes e.g., 3 H, 14 C, 32 P, 35 S or 125 I
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.g., gold
  • particles e.
  • the different types of labels can be conjugated to an affinity ligand using various chemistries, e.g., the amine reaction or the thiol reaction.
  • chemistries e.g., the amine reaction or the thiol reaction.
  • other reactive groups than amines and thiols can be used, e.g., aldehydes, carboxylic acids and glutamine.
  • the detection, localization and/or quantification of a labeled affinity ligand bound to its RBM3 protein target may involve visualizing techniques, such as light microscopy or immunofluorescence microscopy. Other methods may involve the detection via flow cytometry or luminometry.
  • Biological material from the subject may be used for obtaining the sample for detection and/or quantification of RBM3 protein.
  • the sample may thus be an earlier obtained sample. If using an earlier obtained sample in a method, no steps of the method are practiced on the human or animal body.
  • the affinity ligand may be applied to the sample for detection and/or quantification of the RBM3 protein. This procedure enables not only detection of RBM3 protein, but may in addition show the distribution and relative level of expression thereof.
  • the method of visualization of labels on the affinity ligand may include, but is not restricted to, fluorometric, luminometric and/or enzymatic techniques. Fluorescence is detected and/or quantified by exposing fluorescent labels to light of a specific wavelength and thereafter detecting and/or quantifying the emitted light in a specific wavelength region. The presence of a luminescently tagged affinity ligand may be detected and/or quantified by luminescence developed during a chemical reaction. Detection of an enzymatic reaction is due to a color shift in the sample arising from a chemical reaction. Those of skill in the art are aware that a variety of different protocols can be modified in order for proper detection and/or quantification.
  • the sample may be immobilized onto a solid phase support or carrier, such as nitrocellulose or any other solid support matrix capable of immobilizing RBM3 protein present in the biological sample applied to it.
  • solid state support materials useful in the present invention include glass, carbohydrate (e.g., Sepharose), nylon, plastic, wool, polystyrene, polyethene, polypropylene, dextran, amylase, films, resins, cellulose, polyacrylamide, agarose, alumina, gabbros and magnetite.
  • primary affinity ligand selective for RBM3 protein may be applied, e.g., as described in Examples, Sections 8 and 9, of the present disclosure.
  • the supporting matrix may be washed with one or more appropriate buffers known in the art, followed by exposure to a secondary labeled affinity ligand and washed once again with buffers to remove unbound affinity ligands. Thereafter, selective affinity ligands may be detected and/or quantified with conventional methods.
  • the binding properties for an affinity ligand may vary from one solid state support to the other, but those skilled in the art should be able to determine operative and optimal assay conditions for each determination by routine experimentation.
  • the quantifiable affinity ligand of al) or al) may be detected using a secondary affinity ligand capable of recognizing the quantifiable affinity ligand.
  • the quantification of a3) or all) may thus be carried out by means of a secondary affinity ligand with affinity for the quantifiable affinity ligand.
  • the secondary affinity ligand may be an antibody or a fragment or a derivative thereof.
  • one available method for detection and/or quantification of the RBM3 protein is by linking the affinity ligand to an enzyme that can then later be detected and/or quantified in an enzyme immunoassay (such as an EIA or ELISA).
  • an enzyme immunoassay such as an EIA or ELISA.
  • the biological sample is brought into contact with a solid material or with a solid material conjugated to an affinity ligand against the RBM3 protein, which is then detected and/or quantified with an enzymatically labeled secondary affinity ligand.
  • an appropriate substrate is brought to react in appropriate buffers with the enzymatic label to produce a chemical moiety, which for example is detected and/or quantified using a spectrophotometer, fluorometer, luminometer or by visual means.
  • primary and any secondary affinity ligands can be labeled with radioisotopes to enable detection and/or quantification.
  • appropriate radiolabels in the present disclosure are 3 H, 14 C, 32 P, 35 S or 125 I.
  • the specific activity of the labeled affinity ligand is dependent upon the half-life of the radiolabel, isotopic purity, and how the label has been incorporated into the affinity ligand.
  • Affinity ligands are preferably labeled using well-known techniques (Wensel T G and Meares C F (1983) in: Radioimmunoimaging and Radioimmunotherapy (Burchiel S W and Rhodes B A eds.) Elsevier, New York, pp 185-196).
  • a thus radiolabeled affinity ligand can be used to visualize RBM3 protein by detection of radioactivity in vivo or in vitro.
  • Radionuclear scanning with e.g., gamma camera, magnetic resonance spectroscopy or emission tomography function for detection in vivo and in vitro, while gamma/beta counters, scintillation counters and radiographies are also used in vitro.
  • the protein expression of the RBM3 gene is detected and found to correlate with diagnostic and prognostic indications.
  • the present disclosure also encompasses the mRNA expression of the RBM3 gene as the inventors have found the RBM3 mRNA level and the RBM3 protein level to co-vary in other types of cancer tissue in which RBM3 is also of prognostic significance.
  • total cellular RNA is purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are then precipitated, in order to remove DNA by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., the so-called “Northern” blotting technique. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question.
  • the nucleic acid probe may be labeled with, e.g., a radionuclide such as 3 H, 32 P, 33 P, 14 C, or 35 S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin, or an antibody), a fluorescent molecule, a chemiluminescent molecule, an enzyme, or the like.
  • a radionuclide such as 3 H, 32 P, 33 P, 14 C, or 35 S
  • a heavy metal e.g., a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin, or an antibody), a fluorescent molecule, a chemiluminescent molecule, an enzyme, or the like.
  • Probes may be labeled to high specific activity by either the nick translation method (Rigby et al., (1977) J. Mol Biol, 113: 237-251), or by the random priming method (Fienberg, (1983) Anal. Biochem., 132: 6-13).
  • the latter can be a method for synthesizing 32 P-labeled probes of high specific activity from RNA templates. For example, by replacing preexisting nucleotides with highly radioactive nucleotides according to the nick translation method, it is possible to prepare 32 P- labeled nucleic acid probes with a specific activity well in excess of 10 cpm/microgram. Autoradiographic detection of hybridization then can be performed by exposing hybridized filters to photographic film.
  • Biomarker levels can be quantified by computerized imaging systems, such as the Molecular Dynamics 400-B 2D Phosphorimager (Amersham Biosciences, Piscataway, N.J., USA).
  • the random-primer method can be used to incorporate an analogue, for example, the dTTP analogue 5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate, into the probe molecule.
  • analogue for example, the dTTP analogue 5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate
  • the biotinylated probe oligonucleotide can be detected by reaction with biotin-binding proteins, such as avidin, streptavidin, and antibodies (e.g., anti-biotin antibodies) coupled to fluorescent dyes or enzymes that produce color reactions.
  • determining the levels of RNA transcript may be accomplished using the technique of in situ hybridization. This technique requires fewer cells than the Northern blotting technique, and involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled nucleic acid (e.g., cDNA or RNA) probes. This technique is particularly well-suited for analyzing tissue biopsy samples from subjects.
  • a solution containing radioactive or otherwise labeled nucleic acid e.g., cDNA or RNA
  • RNA transcripts in cells can also be determined by reverse transcription of RNA transcripts, followed by amplification of the reverse-transcribed transcripts by polymerase chain reaction (RT-PCR).
  • the levels of RNA transcripts can be quantified in comparison with an internal standard, for example, the level of mRNA from a standard gene present in the same sample.
  • an internal standard for example, the level of mRNA from a standard gene present in the same sample.
  • the person skilled in the art is capable of selecting suitable genes for use as an internal standard.
  • the methods for quantitative RT-PCR and variations thereof are within the skill in the art.
  • primers can be used for the quantitative RT-PCR.
  • the primers are specific to RBM3 It is within the skill in the art to generate primers specific to RBM3 (e.g. starting from SEQ ID NO:3).
  • Primers can be of any suitable length, but are preferably between 19 and 23 (e.g., 19, 20, 21, 22, or 23) nucleotides.
  • amplicon length should be 50 to 150 (up to 250 may be necessary but then optimization of the thermal cycling protocol and reaction components may be necessary) bases for optimal PCR efficiency. Designing primers that generate a very long amplicon may lead to poor amplification efficiency. Information about primer design and optimal amplicon size may for example be found at www.ambion.com.
  • microchip technology it may be desirable to use microchip technology to detect biomarker expression.
  • the microchip can be fabricated by techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5′-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GENEMACHINE OmniGrid 100 Microarrayer and Amersham CODELINK activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates.
  • the labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6 times SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75 times TNT at 37° C. for 40 minutes.
  • hybridizing conditions e.g., 6 times SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75 times TNT at 37° C. for 40 minutes.
  • hybridization occurs at positions on the array, where the immobilized probe DNA recognizes a complementary target cDNA in the sample.
  • the labeled target cDNA marks the exact position on the array where binding occurs, thereby allowing automatic detection and quantification.
  • the output consists of a list of hybridization events, which indicate the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary biomarker, in the subject sample.
  • the labeled cDNA oligomer is a biotin-labeled cDNA prepared from a biotin-labeled primer.
  • the microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding biomarker in the subject sample.
  • the use of the array has one or more advantages for mRNA expression detection.
  • the RBM3 mRNA (as well as the RBM3 protein) may for example be extracted from formalin-fixed, paraffin-embedded tumor tissue. Accordingly, the sample of the methods of the present disclosure may be formalin-fixed and/or paraffin-embedded tissue.
  • RBM3 is a diagnostic marker for testicular cancer in situ.
  • Placental-Like Alkaline Phosphatase (PLAP), Octamer-3/4 (OCT3/4) and (RNA Binding Motif protein Y) RBMY are other markers of pre-stages of testicular cancer.
  • PLAP Placental-Like Alkaline Phosphatase
  • OCT3/4 Octamer-3/4
  • RNA Binding Motif protein Y RNA Binding Motif protein Y
  • kit for carrying out a method according the first aspect which comprises:
  • a quantifiable affinity ligand capable of selective interaction with a protein selected from Placental-Like Alkaline Phosphatase (PLAP), Octamer-3/4 (OCT3/4) and (RNA Binding Motif protein Y) RBMY; and
  • RBM3 is a prognostic marker for testicular cancer.
  • Human chorionic gonadotropin (HCG), a-fetoprotein (AFP) and lactate dehydrogenase (LD) are other prognostic markers in testicular cancer.
  • HCG Human chorionic gonadotropin
  • AFP a-fetoprotein
  • LD lactate dehydrogenase
  • the inventors have realized the value of combining affinity ligands targeting the RBM3 protein and at least one of the other prognostic markers in a single kit.
  • kit for carrying out a method according to the second or third aspect which comprises
  • a quantifiable affinity ligand capable of selective interaction with a protein selected from human chorionic gonadotropin (HCG), a-fetoprotein (AFP) and lactate dehydrogenase (LD); and
  • the same reagents such as the same secondary antibody, may be used for quantifying both the anti-RBM3 protein affinity ligand and the affinity ligand targeting the other marker.
  • kits may be selected and specified as described above in connection with the method aspects of the present disclosure.
  • the kit according to the fourth or fifth aspect comprises an affinity ligand against an RBM3 protein, an affinity ligand against another marker as well as other means that help to quantify the specific and/or selective affinity ligands after they have bound specifically and/or selectively to their respective targets.
  • the kits may contain a secondary affinity ligand for detecting and/or quantifying a complex formed by the targets and the affinity ligands.
  • the kits may also contain various auxiliary substances other than affinity ligands, to enable the kits to be used easily and efficiently.
  • auxiliary substances include solvents for dissolving or reconstituting lyophilized protein components of the kits, wash buffers, substrates for measuring enzyme activity in cases where an enzyme is used as a label, target retrieval solution to enhance the accessibility to antigens in cases where paraffin or formalin-fixed tissue samples are used, and substances such as reaction arresters, e.g., endogenous enzyme block solution to decrease the background staining and/or counterstaining solution to increase staining contrast, that are commonly used in immunoassay reagent kits.
  • reaction arresters e.g., endogenous enzyme block solution to decrease the background staining and/or counterstaining solution to increase staining contrast, that are commonly used in immunoassay reagent kits.
  • the kit according to the kit aspects may also advantageously comprise a reference sample for provision of, or yielding, the reference value to be used for comparison with the sample value.
  • the reference sample may comprise a predetermined amount of RBM3 protein.
  • a reference sample may for example be constituted by a tissue sample containing the predetermined amount of RBM3 protein. The tissue reference sample may then be used in the determination of the RBM3 expression status in the sample being studied, by manual, such as ocular, or automated comparison of expression levels in the reference tissue sample and the subject sample.
  • the reference sample may comprise cell lines, such as cancer cell lines, expressing a predetermined, or controlled, amount of RBM3 protein.
  • the cell lines may be formalin fixed.
  • such formalin fixed cell lines may be paraffin embedded.
  • the wording “reference sample for provision of the reference value” is to be interpreted broadly in the context of the present disclosure.
  • the reference sample may comprise an amount of RBM3 protein actually corresponding to the reference value, but it may also comprise an amount of RBM3 protein corresponding to a value being higher than the reference value.
  • the “high” value may be used by a person performing the method as an upper reference (positive reference) for assessing, e.g., the appearance of, a reference value which is lower than the “high” value.
  • the person skilled in the art of immunohistochemistry understands how to do such an assessment.
  • the skilled person may use another reference sample comprising a low amount of RBM3 protein for provision of a “low” value in such an assessment, e.g., as a negative reference. This is further discussed above in connection with the method aspects.
  • the reference sample may comprise an amount of RBM3 protein corresponding to the reference value.
  • the reference sample may comprise an amount of RBM3 protein corresponding to a nuclear or cytoplasmic fraction of 95% or lower, such as 90% or lower, such as 85% or lower, such as 80% or lower, such as 75% or lower, such as 70% or lower, such as 65% or lower, such as 60% or lower, such as 55% or lower, such as 50% or lower, such as 45% or lower, such as 40% or lower, such as 35% or lower, such as 30% or lower, such as 25% or lower, such as 20% or lower, such as 15% or lower, such as 10% or lower, such as 5% or lower, such as 2% or lower, such as 1% or lower, such as 0%.
  • the reference sample may comprise an amount of RBM3 protein corresponding to a moderate nuclear or cytoplasmic intensity or lower, such as a weak nuclear or cytoplasmic intensity or lower, such as an absent nuclear or cytoplasmic intensity.
  • kits may comprise a reference sample comprising an amount of RBM3 protein corresponding to a value being higher than the reference value.
  • the reference sample may for example comprise an amount of RBM3 protein corresponding to a nuclear or cytoplasmic fraction of 75% or higher and/or a strong nuclear or cytoplasmic intensity.
  • kits may comprise a reference sample comprising an amount of RBM3 protein corresponding to a value being lower than or equal to the reference value, e.g., an absent nuclear or cytoplasmic intensity and/or a nuclear or cytoplasmic fraction of ⁇ 2%, such as 0%.
  • kits may thus comprise: a reference sample comprising an amount of RBM3 protein corresponding to a predetermined reference value; a reference sample comprising an amount of RBM3 protein corresponding to a value being higher than a predetermined reference value; and/or a reference sample comprising an amount of RBM3 protein corresponding to a value being lower than or equal to a predetermined reference value.
  • kits may comprise: a first reference sample comprising an amount of RBM3 protein being higher than a predetermined reference value; and a second reference sample comprising an amount of RBM3 protein being lower than or equal to the predetermined reference value.
  • the reference sample may be a tissue sample, such as a tissue sample adapted to ocular or microscopic evaluation.
  • the tissue reference sample may be fixated in paraffin or buffered formalin and/or histo-processed to sections (e.g., ⁇ m-thin sections) that are mounted on microscopic glass-slides.
  • the tissue reference sample may be further adapted to staining with affinity ligands, such as antibodies, against an RBM3 protein.
  • the reference sample may be adapted to directly, or indirectly, provide any relevant reference value, such as any one of the reference values discussed above.
  • RBM3 may also be detected on the mRNA level. Such detection may for example be an in situ mRNA analysis or a quantitative RT-PCR mRNA analysis. Further, the mRNA of a sample may be copied into cDNA to increase stability prior to detection.
  • kits comprising at least one probe or primer for detection and/or quantification of RBM3 mRNA or RBM3 cDNA.
  • kit of the configuration of the third aspect may further comprise:
  • a probe or primer for detection and/or quantification of the mRNA or cDNA one or more of PLAP, OCT3/4, RBMY, HCG, AFP and LD.
  • a probe or primer according to the configuration of the third aspect may for example be a single or double stranded oligonucleotide that is complementary to a part of the mRNA or cDNA in question.
  • the RBM3 cDNA is represented by SEQ ID NO:3. If the probe is double stranded, it is denaturated prior to detection/hybridization to become single stranded, e.g. by means of heating.
  • the length of the probe(s) or primer(s) may for example be at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 50, such as at least 75, such as at least 100, such as at least 150 consecutive nucleotides.
  • a primer is normally shorter than a probe.
  • the kit may comprise further auxiliary products. Examples of such products are described above in connection with the discussion about mRNA analysis.
  • the kit of the configuration of the third aspect may for example comprise one or more auxiliary products selected from the group consisting of a pre-treatment solution (for preparing the sample), a proteolytic enzyme such as pepsin, a second probe (to be used as a reference), a buffer such as a wash buffer and a fluorescence mounting medium (if fluorescent labels are used).
  • the probes of the configuration of the third aspect may be labeled or conjugated to other chemical moieties. This is also exemplified above in connection with the discussion about mRNA analysis.
  • the probes of the configuration of the third aspect may for example be arranged on a solid phase, optionally together with probes for one of the other targets.
  • examples of such other probes are thus those capable of detecting the mRNA of PLAP, OCT3/4, RBMY, HCG, AFP and LD.
  • the inventors have realized several uses for the RBM3 protein and fragments thereof and the RBM3 mRNA.
  • an RBM3 protein fragment which consists of 50 amino acids or less and comprises a sequence selected from SEQ ID NO:4-19.
  • the fragment consists of 29 amino acids or less.
  • the fragment consists of 20 amino acids or less, such as 15 amino acids or less, and comprises a sequence selected from SEQ ID NO:6-19.
  • an RBM3 protein or an RBM3 mRNA molecule as a diagnostic marker for a testicular disorder, such as testicular cancer in situ.
  • the use of the first configuration may be entirely in vitro, e.g., on previously obtained samples.
  • diagnostic marker refers to something material which presence indicates a medical condition.
  • the marker may thus be a biomarker, such as a human protein. It is to be understood that the presence of the diagnostic marker, or a relatively high level thereof, is indicative of a relatively high likelihood of having the testicular disorder.
  • the “relatively high likelihood” is high in comparison with a subject not expressing such high levels of the diagnostic marker.
  • an RBM3 protein or an antigenically active fragment thereof for the production, selection or purification of a diagnostic agent for a testicular disorder, such as testicular cancer in situ.
  • an “antigenically active fragment” refers to a fragment of sufficient size to be capable of generating an affinity ligand capable of selective interaction with the fragment.
  • the selection and purification may be in vitro, while the production may be in vivo.
  • diagnostic agent refers to an agent having at least one property being valuable in an establishment of a diagnosis.
  • the diagnostic agent may be capable of selective interaction with the diagnostic marker.
  • the diagnostic agent may thus be an affinity ligand capable of selective interaction with the RBM3 protein or the antigenically active fragment thereof. Examples of such affinity ligands are discussed above in connection with the method aspects.
  • the person skilled in the art understands how to use the RBM3 protein or fragment in the production, selection or purification of the diagnostic agent.
  • the use may comprise affinity purification on a solid support onto which the RBM3 protein or fragment thereof has been immobilized.
  • the solid support may for example be arranged in a column.
  • the use may comprise selection of affinity ligands having specificity for the RBM3 protein or fragment thereof using a solid support onto which the RBM3 protein or fragment thereof has been immobilized.
  • Such solid support may be well plates (such as 96 well plates), magnetic beads, agarose beads or sepharose beads.
  • the use may comprise analysis of affinity ligands on a soluble matrix, for example using a dextran matrix, or use in a surface plasmon resonance instrument, such as a BiacoreTM instrument, wherein the analysis may for example comprise monitoring the affinity of a number of potential affinity ligands for the immobilized RBM3 protein or fragment thereof.
  • the RBM3 protein or the antigenically active fragment thereof may be used in an immunization of an animal, such as a rabbit or mouse.
  • an RBM3 protein or an RBM3 mRNA molecule as a prognostic marker for testicular cancer.
  • the use of the first configuration may be entirely in vitro, e.g., on previously obtained samples.
  • prognostic marker refers to something material which presence indicates a prognosis.
  • the marker may thus be a biomarker, such as a human protein. It is to be understood that the presence of the prognostic marker, or a relatively high level thereof, is indicative of a relatively good prognosis.
  • the “relatively good prognosis” is good in comparison with a comparable testicular cancer subject not expressing such high levels of the prognostic marker.
  • an RBM3 protein or an antigenically active fragment thereof for the production, selection or purification of a prognostic agent for testicular cancer.
  • the selection and purification may be in vitro, while the production may be in vivo.
  • prognostic agent refers to an agent having at least one property being valuable in an establishment of a prognosis, e.g., a prognosis for a mammalian subject having a testicular cancer.
  • the prognostic agent may be capable of selective interaction with the prognostic marker.
  • the prognostic agent may thus be an affinity ligand capable of selective interaction with the RBM3 protein or the antigenically active fragment thereof. Examples of such affinity ligands are discussed above in connection with the method aspects.
  • testicular cancer of the eighth aspect may for example be a testicular germ-cell cancer. Further, in alternative or complementary embodiments, it may be non-seminomatous.
  • amino acid sequence of the RBM3 protein may comprise a sequence selected from:
  • sequence ii) is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to SEQ ID NO:1.
  • amino acid sequence of the RBM3 protein may comprise or consist of a sequence selected from:
  • sequence ii) is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to SEQ ID NO:2.
  • the antigenically active fragment of the seventh or eighth aspect may for example be any one of the fragments of the sixth aspect.
  • an affinity ligand capable of selective interaction with an RBM3 protein.
  • an affinity ligand according to the ninth aspect as a diagnostic agent for a testicular disorder, such as testicular cancer in situ.
  • testicular cancer may for example be a testicular germ-cell cancer. Also, it may for example be non-seminomatous.
  • Such uses may for example be performed in vitro, e.g., involving the determination of the amount of RBM3 in at least part of a sample earlier obtained from the subject.
  • an affinity ligand capable of selective interaction with an RBM3 protein in the manufacture of a diagnostic agent for a testicular disorder, such as testicular cancer in situ, or a prognostic agent for testicular cancer.
  • a suitable fragment of the target protein encoded by the EnsEMBL Gene ID ENSG00000102317 was selected using bioinformatic tools with the human genome sequence as template (Lindskog M et al (2005) Biotechniques 38:723-727, EnsEMBL, www.ensembl.org). The fragment was used as template for the production of a 134 amino acid long fragment corresponding to amino acids 18-151 (SEQ ID NO:1) of the RBM3 protein (SEQ ID NO:2; EnsEMBL entry no. ENSP00000365946).
  • downstream primer was biotinylated to allow solid-phase cloning as previously described, and the resulting biotinylated PCR product was immobilized onto Dynabeads M280 Streptavidin (Dynal Biotech) (Larsson M et al (2000) J. Biotechnol. 80:143-157).
  • the fragment was released from the solid support by Notl-Ascl digestion (New England Biolabs), ligated into the pAff8c vector (Larsson M et al, supra) in frame with a dual affinity tag consisting of a hexahistidyl tag for immobilized metal ion chromatography (IMAC) purification and an immunopotentiating albumin binding protein (ABP) from streptococcal protein G (Sjölander A et al (1997) J. Immunol.
  • Notl-Ascl digestion New England Biolabs
  • IMAC immobilized metal ion chromatography
  • ABSP immunopotentiating albumin binding protein
  • BL21(DE3) cells harboring the expression vector were inoculated in 100 ml 30 g/I tryptic soy broth (Merck KGaA) supplemented with 5 g/; yeast extract (Merck KGaA) and 50 mg/l kanamycin (Sigma-Aldrich) by addition of 1 ml of an overnight culture in the same culture medium.
  • the cell culture was incubated in a 1 liter shake flask at 37° C. and 150 rpm until the optical density at 600 nm reached 0.5-1.5.
  • Protein expression was then induced by addition of isopropyl- ⁇ -D-thiogalactopyranoside (Apollo Scientific) to a final concentration of 1 mM, and the incubation was continued overnight at 25° C. and 150 rpm.
  • the His 6 -tagged fusion protein was purified by immobilized metal ion affinity chromatography (IMAC) on columns with 1 ml Talon® metal (Co 2+ ) affinity resin (BD Biosciences Clontech) using an automated protein purification procedure (Steen J et al (2006) Protein Expr. Purif. 46:173-178) on an ASPEC XL4TM (Gilson).
  • the resin was equilibrated with 20 ml denaturing washing buffer (6 M guanidine hydrochloride, 46.6 mM Na 2 HPO 4 , 3.4 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0-8.2). Clarified cell lysates were then added to the column.
  • the resin was washed with a minimum of 31.5 ml washing buffer prior to elution in 2.5 ml elution buffer (6 M urea, 50 mM NaH 2 PO 4 , 100 mM NaCl, 30 mM acetic acid, 70 mM Na-acetate, pH 5.0).
  • the eluted material was fractioned in three pools of 500, 700 and 1300 ⁇ l.
  • the 700 ⁇ l fraction, containing the antigen, and the pooled 500 and 1300 ⁇ l fractions were stored for further use.
  • the antigen fraction was diluted to a final concentration of 1 M urea with phosphate buffered saline (PBS; 1.9 mM NaH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 154 mM NaCl) followed by a concentration step to increase the protein concentration using Vivapore 10/20 ml concentrator with molecular weight cut off at 7500 Da (Vivascience AG).
  • the protein concentration was determined using a bicinchoninic acid (BCA) micro assay protocol (Pierce) with a bovine serum albumin standard according to the manufacturer's recommendations.
  • BCA bicinchoninic acid
  • the protein quality was analyzed on a Bioanalyzer instrument using the Protein 50 or 200 assay (Agilent Technologies).
  • a gene fragment corresponding to nucleotides 281-682 of the full-lengths transcript of RBM3 was successfully isolated by RT-PCR from a human RNA pool using primers specific.
  • the fragment codes for amino acids 18 to 151 of the target protein RBM3 (SEQ ID NO:2).
  • the 134 amino acid fragment (SEQ ID NO:1) of the target protein (SEQ ID NO:2) was designed to lack transmembrane regions to ensure efficient expression in E. coli , and to lack any signal peptide, since those are cleaved off in the mature protein.
  • the protein fragment was designed to consist of a unique sequence with low homology with other human proteins, to minimize cross reactivity of generated affinity reagents, and to be of a suitable size to allow the formation of conformational epitopes and still allow efficient cloning and expression in bacterial systems.
  • a clone encoding the correct amino acid sequence was identified, and, upon expression in E. coli , a single protein of the correct size was produced and subsequently purified using immobilized metal ion chromatography. After dilution of the eluted sample to a final concentration of 1 M urea and concentration of the sample to 1 ml, the concentration of the protein fragment was determined to be 10.4 mg/ml and was 96.0% pure according to purity analysis.
  • the purified RBM3 fragment as obtained above was used as antigen to immunize a rabbit in accordance with the national guidelines (Swedish permit no. A 84-02).
  • the rabbit was immunized intramuscularly with 200 ⁇ g of antigen in Freund's complete adjuvant as the primary immunization, and boosted three times in four week intervals with 100 ⁇ g antigen in Freund's incomplete adjuvant.
  • Antiserum from the immunized animal was purified by a three-step immunoaffinity based protocol (Agaton C et al (2004) J. Chromatogr. A 1043:33-40; Nilsson P et al (2005) Proteomics 5:4327-4337).
  • the first step 7 ml of total antiserum was buffered with 10 ⁇ PBS to a final concentration of 1 ⁇ PBS (1.9 mM NaH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 154 mM NaCl), filtered using a 0.45 ⁇ m pore-size filter (Acrodisc®, Life Science) and applied to an affinity column containing 5 ml N-hydroxysuccinimide-activated SepharoseTM 4 Fast Flow (GE Healthcare) coupled to the dual affinity tag protein His 6 -ABP (a hexahistidyl tag and an albumin binding protein tag) expressed from the pAff8c vector and purified in the same way as described above for the antigen protein fragment.
  • 1 ⁇ PBS 1.9 mM NaH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 154 mM NaCl
  • the flow-through depleted of antibodies against the dual affinity tag His 6 -ABP
  • the His 6 -ABP protein and the protein fragment antigen were coupled to the NHS activated matrix as recommended by the manufacturer. Unbound material was washed away with 1 ⁇ PBST (1 ⁇ PBS, 0.1% Tween20, pH 7.25), and captured antibodies were eluted using a low pH glycine buffer (0.2 M glycine, 1 mM EGTA, pH 2.5).
  • the eluted antibody fraction was collected automatically, and loaded onto two 5 ml HiTrapTM desalting columns (GE Healthcare) connected in series for efficient buffer exchange in the third step.
  • the second and third purification steps were run on the AKTAxpressTM platform (GE Healthcare).
  • the antigen selective (mono-specific) antibodies (msAbs) were eluted with PBS buffer, supplemented with glycerol and NaN 3 to final concentrations of 40% and 0.02%, respectively, for long term storage at ⁇ 20° C. (Nilsson P et al (2005) Proteomics 5:4327-4337).
  • the specificity and selectivity of the affinity purified antibody fraction were analyzed by binding analysis against the antigen itself and against 94 other human protein fragments in a protein array set-up (Nilsson P et al (2005) Proteomics 5:4327-4337).
  • the protein fragments were diluted to 40 ⁇ g/ml in 0.1 M urea and 1 ⁇ PBS (pH 7.4) and 50 ⁇ l of each were transferred to the wells of a 96-well spotting plate.
  • the protein fragments were spotted in duplicate and immobilized onto epoxy slides (SuperEpoxy, TeleChem) using a pin-and-ring arrayer (Affymetrix 427).
  • the slide was washed in 1 ⁇ PBS (5 min) and the surface was then blocked (SuperBlock®, Pierce) for 30 minutes.
  • An adhesive 16-well silicone mask (Schleicher & Schuell) was applied to the glass before the mono-specific antibodies were added (diluted 1:2000 in 1 ⁇ PBST to appr. 50 ng/ml) and incubated on a shaker for 60 min.
  • Affinity tag-specific IgY antibodies were co-incubated with the mono-specific antibodies in order to quantify the amount of protein in each spot.
  • the slide was washed with 1 ⁇ PBST and 1 ⁇ PBS twice for 10 min each.
  • the specificity and selectivity of the affinity-purified antibody were analyzed by Western blot.
  • Western blot was performed by separation of total protein extracts from selected human cell lines on pre-cast 10-20% SDS-PAGE gradient gels (Bio-Rad Laboratories) under reducing conditions, followed by electro-transfer to PVDF membranes (Bio-Rad Laboratories) according to the manufacturer's recommendations. The membranes were blocked (5% dry milk, 1 ⁇ TBST; 0.1 M Tris-HCl, 0.5 M NaCl, 0.1% Tween20) for 1 h at room temperature, incubated with the primary affinity purified antibody (diluted 1:500 in blocking buffer) and washed in TBST.
  • the secondary HRP-conjugated antibody (swine anti-rabbit immunoglobulin/HRP, DakoCytomation) was diluted 1:3000 in blocking buffer and chemiluminescence detection was carried out using a ChemidocTM CCD camera (Bio-Rad Laboratories) and SuperSignal® West Dura Extended Duration substrate (Pierce), according to the manufacturer's protocol.
  • a two-color dye labeling system was used, with a combination of primary and secondary antibodies.
  • Tag-specific IgY antibodies generated in hen were detected with a secondary goat anti-hen antibody labeled with Alexa 555 fluorescent dye.
  • the specific binding of the rabbit msAb to its antigen on the array was detected with a fluorescently Alexa 647 labeled goat anti-rabbit antibody.
  • Each protein fragment was spotted in duplicates.
  • the protein array analysis shows that the affinity purified mono-specific antibody against RBM3 is highly selective to the correct protein fragment and has a very low background to all other protein fragments analyzed on the array.
  • the result of the Western blot analysis shows that the antibody specifically detects a single band of approximately 16 kDa in two breast tumor cell lines, T47D and MCF-7.
  • the theoretical molecular weight of RBM3 is 16 kDa (as calculated from the RBM3 amino acid sequence SEQ ID NO:2), corresponding well to the result obtained.
  • the purified fragment (SEQ ID NO:1) obtained in Section 1 was used as antigen for production of monoclonal antibodies.
  • Antigen was sent to AbSea Biotechnology Ltd (Beijing, China) and briefly, the antigen was injected subcutaneously into BALB/c mice (4-6 weeks old, female) at three week intervals. The antigen was mixed with complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for the following injections. Three days before fusion, the mouse was last challenged with antigen intravenously. Hybridomas were generated by fusion of mouse splenocytes with the Sp2/0 myeloma cell line.
  • Cell-lines were screened by ELISA (at AbSea) to identify lines that produce monoclonal antibodies (mAbs) that recognize the antigen (SEQ ID NO:1), but not the affinity tag His-ABP. Eight cell-lines showed specific binding to the antigen SEQ ID NO:1 in ELISA and were selected for further testing. For each of the selected eight clones 150-300 ⁇ l supernatant was collected, azide was added, and the supernatants were delivered to Atlas Antibodies AB on wet ice. The supernatants were stored at +4° C. upon arrival according to the instructions from AbSea.
  • clones 1 B5, 6F11 and 7G3 that gave positive results in both Western blot and IHC analysis. These clones were selected for subcloning and expansion, performed by AbSea Biotechnology Ltd.
  • RBM3 DNA corresponding to SEQ ID NO:1 was amplified by PCR using vector pAff8c as template.
  • the amplified DNA was fragmentized to various lengths (approximately 50-150 bp) by sonication, followed by ligation into the staphylococcal display vector (pSCEM2) and transformed into S. Carnosus yielding around 100000 transformants. In-frame DNA fragments were displayed as peptides on the staphylococcal surface.
  • a PEPscreen library consisting of 25 biotinylated peptides corresponding to the PrEST HPRR232631 (SEQ ID NO:1) on RBM3 was synthesized by Sigma-Genosys (Sigma-Aldrich).
  • the peptides were 15 amino acids long with a 10 amino acid overlap, together covering the entire PrEST-sequence.
  • the peptides were resolved in 80% DMSO to a final concentration of 10 mg/ml.
  • Neutravidin (Pierce, Rockford, Ill.) was immobilized on carboxylated beads (COON Microspheres, Luminex-Corp., Austin, Tex.) in accordance to the manufacturer's protocol. Coupling of 10 6 beads was performed using a filter membrane bottomed microtiter plate (MultiScreen-HTS, Millipore, Billerica, Mass.) as previously described (Larsson et al (2009) J Immunol Methods 15;34(1-2):20-32, Schwenk et al (2007) Mol Cell Proteomics 6(1) 125:32).
  • a bead mixture containing all 25 bead IDs was prepared and 45 ⁇ l of each antibody diluted to 50 ng/ml in PBS was mixed with 5 ⁇ l of the bead mix and incubated for 60 min at RT.
  • a filter bottomed microtiter plate (Millipore) was utilized for washing and following each incubation all wells were washed with 3 ⁇ 100 ⁇ l PBST.
  • 50 ⁇ l of R-Phycoerythrine labeled anti-rabbit IgG antibody 0.5 ⁇ g/ml, Jackson ImmunoResearch
  • 50 ⁇ l of Alexa Fluor 555 goat anti-mouse IgG were added (0.4 ug/mI) for a final incubation of 60 min at RT.
  • Measurements were performed using the Luminex LX200 instrumentation with Luminex xPONENT software. For each experiment 50 events per bead ID were counted and the median fluorescence intensity (MFI) was used as a measurement of antibody binding to individual bead populations.
  • MFI median fluorescence intensity
  • the specificities of the monospecific polyclonal antibody (anti-RBM3, HPA003624) and the monoclonal antibody 6F11 were tested in an assay using beads coupled with synthetic biotinylated peptides.
  • Anti-RBM3 showed strong binding to 8 of the peptides, namely 6, 7, 8, 14, 15, 16, 24 and 25, corresponding to three distinct regions on the PrEST sequence, consensus sequences SEQ ID NO: 6, 7, 8 and 9.
  • peptide 24 and 25, corresponding to SEQ ID NO:9 generated a strong signal.
  • the monoclonal antibody 6F11 reacted with two peptides: 15 and 16, corresponding to one distinct region on the PrEST sequence, consensus sequence SEQ ID NO: 8.
  • SEQ ID NO:6 is within SEQ ID NO:4 and that SEQ ID NO:8 to some extent overlaps with SEQ ID NO:5.
  • RBM3 DNA corresponding to SEQ ID NO:1 was amplified by PCR using vector pAff8c as template.
  • the amplified DNA was fragmentized to various lengths (approximately 50-150 bp) by sonication, followed by ligation into the staphylococcal display vector (pSCEM2) and transformed into S. Carnosus yielding around 100000 transformants. In-frame DNA fragments were displayed as peptides on the staphylococcal surface.
  • the regions SEQ ID NO:10-15 within SEQ ID NO:1 were identified.
  • the regions SEQ ID NO:11 and SEQ ID NO:12 were of interest, since they were found within the earlier identified region SEQ ID NO:4.
  • the regions SEQ ID NO:13 and 14 were particularly interesting, since they to a large extent overlapped with previously identified SEQ ID NO:6 and 7, respectively.
  • the region SEQ ID NO:16 within SEQ ID NO:1 was identified, and this region (SEQ ID NO:16) is within the earlier identified region SEQ ID NO:5.
  • the epitope region of 6F11 identified here in Section 7 has a one-amino acid overlap with the 6F11 epitope region identified in Section 6.
  • the results of Sections 6 and 7 are, however, not in contrast; one of the peptides found to bind 6F11 in Section 6 (peptide 16) comprises SEQ ID NO:16 (and SEQ ID NO:19).
  • the results of Sections 6 and 7 may thus be considered complementary.
  • the region SEQ ID NO:17 within SEQ ID NO:1 was identified, and this region (SEQ ID NO:17) was also found within the earlier identified region SEQ ID NO:5.
  • the region SEQ ID NO:18 within SEQ ID NO:1 was identified. This region (SEQ ID NO:18) was also found within the earlier identified region SEQ ID NO:5. This region (SEQ ID NO:18) overlaps with the epitope for the 6F11 antibody (SEQ ID NO:16).
  • the region SEQ ID NO:19 within SEQ ID NO:1 was identified.
  • the specificity of the polyclonal antibody (anti-RBM3), and two of the monoclonal antibodies (6F11 and 1 B5) were analyzed by Western Blot.
  • Western blot was performed by separation of total protein extracts from selected human cell lines on 17% SDS-PAGE gels under reducing conditions, followed by electro-transfer to PVDF membranes (Bio-Rad Laboratories) according to the manufacturer's recommendations. The membranes were blocked (5% BSA in 1 ⁇ PBS with 0.1% Tween20) for 1 h at room temperature, incubated with the primary affinity purified antibody (diluted 1:1000 in blocking buffer) and washed in PBST.
  • the secondary HRP-conjugated antibody (sheep anti-mouse immunoglobulin/HRP, GE) was diluted 1:10000 in blocking buffer and chemiluminescence detection was carried out using a ChemidocTM CCD camera (Bio-Rad Laboratories) and Western Blotting Luminol Reagent (Santa Cruz Biotechnologies, Inc), according to the manufacturer's protocol.
  • the results of the Western blot analysis shows that the antibodies specifically detect a band of approximately 16 kDa in the cell lines.
  • the theoretical molecular weight of RBM3 is 16 kDa (as calculated from the RBM3 amino acid sequence SEQ ID NO:2), corresponding well to the result obtained. Additional bands were observed for anti-RBM3 and 6F11.
  • the results show that the monoclonal antibodies selectively interacting with epitopes within SEQ ID NO:5 were more specific to the target protein than the polyclonal antibody raised against a peptide covering almost all of the RBM3 protein, and that the 1 B5 antibody was even more specific than the 6F11 antibody (see FIG. 1 ).
  • Peptide specific antibodies were obtained by affinity purification of anti-RMB3 against peptides to which the polyclonal anti-RBM3 antibody was shown to bind in Examples, section 5.
  • peptide 6 SEQ ID NO:20
  • 600 nmol of biotinylated peptide were diluted with HiTrap TM Streptavidin binding buffer to a final volume of 1100 ⁇ I and applied to 1 ml HiTrapTM Streptavidin HP columns (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for binding.
  • Serum obtained from a New Zeeland white rabbit immunized with the recombinant RBM3 fragment SEQ ID NO: 1 fused to a His6-ABP tag was purified on a AKTAxpressTM (GE Healthcare) liquid chromatography system on eight columns in a serial mode as follows: two 5 ml His6-ABP columns followed by 5 epitope specific peptide columns and at the end a His6-ABP-RBM3 fusion protein column. After sample loading, the columns were washed and eluted in parallel to obtain separate antibody fractions.
  • the eluted antibody fractions were epitope mapped using BioPlex, as described above.
  • alanine scanning of the peptide was performed using the following method: Twenty biotinylated synthetic peptides of the sequence TQRSRGFGFITFTNPEHASV (SEQ ID NO: 21), each with a single alanine mutation introduced at every residue (Sigma-Aldrich) were dissolved in DMSO and diluted to 4 pmoles peptide in 100 ⁇ L PBS 7.4 supplemented with 1 mg/mL BSA. The peptides were coupled to 20 Bioplex neutravidin coated beads with 20 unique reporter dyes as described above.
  • the antibody fraction binding to peptide 6 was incubated for one hour in PBST with a cocktail of the different beads consisting of around 15,000 beads per ID.
  • the antibodies were subsequently labeled with PE-conjugated secondary reagent (Moss Inc., USA) and analyzed using Bioplex 200 Suspension Array instrumentation with Bio-Plex Manager 5.0 software.
  • the fractionated antibody bound its expected peptide.
  • the antibody fraction that bound to peptide 6 was confirmed to bind the full-length RBM3 protein (SEQ ID NO:2) by IHC and Western Blot analysis. Preserved antibody binding for the fraction that bound peptide 6 was observed for all amino acid positions except the alanine-substitutions Phe12Ala, Thrl3Ala, and Asnl4Ala of the epitope.
  • the epitope for the antibody fraction was thus determined to include the sequence FTN (SEQ ID NO:22) within SEQ ID NO:4 (see FIG. 5 ).
  • a synthetic peptide (SEQ ID NO: 23), including peptide 6 (SEQ ID NO: 20) and peptide 7 (SEQ ID NO:24) in Section 5 and having a cystein residue added at its N-terminal to which BSA was coupled according to standard procedure, was used as antigen for production of monoclonal antibodies.
  • the antigen was injected subcutaneously into BALB/c mice (4-6 weeks old, female) at three week intervals. The antigen was mixed with complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for the following injections. Three days before fusion, the mouse was last challenged with antigen intravenously. Hybridomas were generated by fusion of mouse splenocytes with the Sp2/0 myeloma cell line.
  • Cell-lines were screened by ELISA to identify lines that produce monoclonal antibodies (mAbs) that recognize the antigen (SEQ ID NO:1), but not the fusion tag, BSA. There were 37 cell-lines showing specific binding to the antigen SEQ ID NO:1 in ELISA and these were selected for further testing. For each of the selected 37 clones 150-300 ⁇ l supernatant was collected and azide was added. The supernatants were stored at +4° C. Further testing of the cell lines showed that clones denoted 7F5, 10F1, 12A10, 12C9, and 14D9 gave positive results in both Western blot and IHC analysis. These clones were selected for subcloning and expansion.
  • the membranes were blocked (5% milk in 1 ⁇ TBST (0.1% Tween20)) for 1 h at room temperature, incubated with the primary monoclonal antibody (diluted 1:10 in 1% BSA, 1 ⁇ TBST) and washed in PBST.
  • the secondary HRP-conjugated antibody polyclonal goat anti-mouse or polyclonal swine anti-rabbit, both Dako
  • chemiluminescence detection was carried out using a CCD camera (Syngene) and Immobilon Western Chemiluminescent HRP Substrate (Millipore), according to the manufacturer's protocol.
  • the results of the Western blot analysis shows that the antibodies specifically detect a band of approximately 16 kDa in the RT4 cell line.
  • the theoretical molecular weight of RBM3 is 16 kDa (as calculated from the RBM3 amino acid sequence SEQ ID NO:2), corresponding well to the result obtained. Additional bands were observed for anti-RBM3.
  • the results show that the monoclonal antibodies were more specific than the polyclonal antibody, (see FIG. 4 ).
  • the monoclonal antibodies obtained as described in Section 9 were epitope mapped using Bioplex. Synthetic peptide preparation and bead coupling was performed as described in Section 6. A bead mixture containing all 25 bead IDs was prepared and 10 ⁇ l of the monoclonal antibodies, diluted 1:10 in PBS-BN (1% BSA), was mixed with 5 ⁇ l of the bead mix and incubated for 60 min at RT. A filter bottomed microtiter plate (Millipore) was utilized for washing and following each incubation all wells were washed with 3 ⁇ 100 ⁇ l PBST. 25 ⁇ l of PE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) were added (4 ug/ml) for a final incubation of 60 min at RT.
  • PE-conjugated goat anti-mouse IgG Jackson ImmunoResearch
  • Measurements were performed using BioPlex 200 Suspension Array instrumentation with Bio-Plex Manager 5.0 software. For each experiment 50 events per bead ID were counted and the median fluorescence intensity (MFI) was used as a measurement of antibody binding to individual bead populations.
  • MFI median fluorescence intensity
  • TGCT testicular germ-cell tumors
  • UMAS Department of Pathology
  • 5 of the samples were pure seminoma and 25 were non-seminomatous TGCT.
  • Non-seminomatous TGCT were classified according to the International Germ Cell Cancer Collaborative Group (IGCCC).
  • IGCCC International Germ Cell Cancer Collaborative Group
  • Four of the 25 samples were cases classified as IGCCC intermediate prognosis and five samples as IGCCC poor prognosis.
  • the remaining 16 non-seminomatous TGCT cases were classified as IGCCC good prognosis.
  • Permission for this study was obtained from the Ethics Committee at Lund University; ref nr 447-07 and 493-09. Five patients died from their disease, 4 of whom were IGCCC poor and 1 IGCCC intermediate. All patients received standard treatment including cisplatin.
  • Basic annotation parameters included an evaluation of i) subcellular localization (nuclear and/or cytoplasmic/membranous), ii) staining intensity (SI) and iii) fraction of stained cells (FSC).
  • fraction of stained cells was subjectively evaluated in accordance to standards used in clinical histo-pathological diagnostics and outcome was classified as: ⁇ 2%, 2-25%, >25-75% or >75% immunoreactive cells of the relevant cell population.
  • This annotation procedure is similar to a calculation of an Allred score, see e.g. Allred et al (1998) Mod Pathol 11(2), 155.
  • SS staining score
  • RBM3 expression was annotated as 0, 1, 2, or 3, with 3 denoting the highest staining score (SS).
  • SS staining score
  • a patient diagnosed with a RBM3 high TGCT is more likely to have a longer survival than a patient diagnosed with a RBM3 low TGCT.
  • Testis TMA Diagnosis
  • Tissue material was collected from 30 patients diagnosed with testicular germ-cell tumors (TGCT) at the Department of Pathology, UMAS, between 1995 and 2008. From this material 15 samples were collected from areas of unclassified intratubular germ cell neoplasia (ITGCN) and 22 samples were collected from normal testis tissue. Permission for this study was obtained from the Ethics Committee at Lund University; ref nr 447-07 and 493-09.
  • Basic annotation parameters included an evaluation of i) subcellular localization (nuclear and/or cytoplasmic/membranous), ii) staining intensity (SI) and iii) fraction of stained cells (FSC).
  • fraction of stained cells was subjectively evaluated in accordance to standards used in clinical histo-pathological diagnostics and outcome was classified as: ⁇ 2%, 2-25%, >25-75% or >75% immunoreactive cells of the relevant cell population.
  • This annotation procedure is similar to a calculation of an Allred score, see e.g. Allred et al (1998) Mod Pathol 11(2), 155.
  • SS staining score
  • ITGCN is a precursor of testicular germ cell tumors (TGCT) and is also referred to as cancer in situ (CIS). The strong expressions of RBM3 in all ITGCN cells suggest a role for RBM3 in CIS.
  • testicular cancer in situ can be diagnosed by detection of RBM3 expression.

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US13/210,809 2009-02-16 2011-08-16 RBM3 in Testicular Cancer Diagnostics and Prognostics Abandoned US20120040338A1 (en)

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PCT/SE2009/000091 WO2009102261A1 (fr) 2008-02-15 2009-02-16 Rbm3 en tant que marqueur pour un pronostic du cancer du sein
SEPCT/SE2009/000091 2009-02-16
US16996309P 2009-04-16 2009-04-16
EP09158084.5 2009-04-16
EP09158084A EP2241889A1 (fr) 2009-04-16 2009-04-16 Protéine RBM3 dans les pronostics de cancer colorectal
US23376909P 2009-08-13 2009-08-13
EP09167847A EP2293070A1 (fr) 2009-08-13 2009-08-13 Moyens et procédés pour le pronostic du cancer ovarien
EP09167847.4 2009-08-13
PCT/EP2009/067419 WO2010091763A1 (fr) 2009-02-16 2009-12-17 Rbm3 comme marqueur pour le pronostic du mélanome malin
EPPCT/EP2009/067419 2009-12-17
PCT/EP2010/051935 WO2010092187A1 (fr) 2009-02-16 2010-02-16 Protéine rbm3 utilisée dans le diagnostic et le pronostic du cancer des testicules
US201161487341P 2011-05-18 2011-05-18
EP11166558.4 2011-05-18
EP11166558A EP2524928A1 (fr) 2011-05-18 2011-05-18 RBM3 dans le cancer de la vessie
US13/210,809 US20120040338A1 (en) 2009-02-16 2011-08-16 RBM3 in Testicular Cancer Diagnostics and Prognostics

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US14/117,704 Expired - Fee Related US9701741B2 (en) 2011-05-18 2012-05-11 RBM3 in bladder cancer
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CN110195109A (zh) * 2019-07-03 2019-09-03 北京太东生物科技有限公司 Rbm46作为睾丸肿瘤标志物的应用

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US20120034317A1 (en) * 2009-02-16 2012-02-09 Jirstroem Karin Prediction of Response to Platinum-Based Therapy
US8747910B2 (en) * 2009-02-16 2014-06-10 Atlas Antibodies Ab Prediction of response to platinum-based therapy
CN110195109A (zh) * 2019-07-03 2019-09-03 北京太东生物科技有限公司 Rbm46作为睾丸肿瘤标志物的应用

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JP2014517282A (ja) 2014-07-17
EP2710030A1 (fr) 2014-03-26
WO2012156330A1 (fr) 2012-11-22
US20120034218A1 (en) 2012-02-09
US20140295462A1 (en) 2014-10-02
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