METHOD OF DIAGNOSING OR PROGNOSING EPITHELIAL OVARIAN CANCER

Abstract

The present invention provides a binding moiety which selectively binds to Sox11 protein and/or mRNA for imaging, diagnosis or prognosis of epithelial ovarian cancer (EOC). Optionally, the moiety is an antibody or antigen-binding fragment thereof. Advantageously, moiety comprises a further, readily detectable moiety. The invention also provides methods of imaging EOC cells as well as methods of diagnosing or prognosing EOC in an individual. A further aspect of the present invention provides a method of identifying cells associated with EOC, the method comprising analysing the pattern of gene expression in a sample of cells to be tested and comparing it to the pattern of gene expression in a sample of known lymphomas cells. Preferably, the cells to be tested are identified as EOC cells if the expression of Sox11 is up-regulated compared to normal B-cells. Preferably EOC cells are identified as improved recurrence-free survival-associated if expression of Sox11 is up-regulated compared with non-cancerous epithelial ovarian cells. Preferably, EOC cells are identified as diminished recurrence-free survival-associated if expression of Sox11 is similar to, or down-regulated, compared with non-cancerous epithelial ovarian cells.

Claims

1. A binding moiety which is capable of binding selectively to Sox11 protein, or to a nucleic acid molecule encoding the same, for use in diagnosing and/or prognosing epithelial ovarian cancer (EOC).

2. A binding moiety which is capable of binding selectively to Sox11 protein, or to a nucleic acid molecule encoding the same, for detecting epithelial ovarian cancer (EOC) cells.

3. A binding moiety according to claim 1 for the diagnosis of epithelial ovarian cancer (EOC).

4. A binding moiety according to claim 1 for the prognosis of epithelial ovarian cancer (EOC).

5. A binding moiety according to any one of the preceding claims wherein the EOC belongs to a histological subtype selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

6. A binding moiety according to claim 5 wherein the EOC is serous EOC.

7. A binding moiety according to claim 5 wherein the EOC is mucinous EOC.

8. A binding moiety according to claim 5 wherein the EOC is endometrioid EOC.

9. A binding moiety according to claim 5 wherein the EOC is clear cell EOC.

10. A binding moiety according to claim 5 wherein the EOC is undifferentiated or unclassifiable EOC.

11. A binding moiety according to any one of the preceding claims for use in vivo.

12. A binding moiety according to any one of the preceding claims for use in vitro.

13. A binding moiety according to any one of the preceding claims for use in the detection of Sox11 expression as a sole biomarker for diagnosing or prognosing epithelial ovarian cancer (EOC).

14. A binding moiety according to any one of the preceding claims for use in combination with one or more additional binding moieties for detecting one or more additional biomarkers for diagnosing or prognosing epithelial ovarian cancer (EOC).

15. A binding moiety according to claim 14 for use in combination with fewer than 20 additional binding moieties, for example fewer than 15, 10, 8, 6, 5, 4, 3, 2 or 1 additional binding moieties.

16. A binding moiety according to any one of the preceding claims for detecting nuclear and/or cytoplasmic expression of Sox11.

17. A binding moiety according to any one of the preceding claims wherein the binding moiety is capable of binding selectively to Sox11 protein.

18. A binding moiety according to claim 17 wherein the binding moiety is capable of binding selectively to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 and/or a natural variant thereof.

19. A binding moiety according to any one of the preceding claims wherein the binding moiety comprises or consists of a polypeptide.

20. A binding moiety according to claim 19 wherein the binding moiety comprises or consists of an antibody, or an antigen-binding fragment or variant thereof.

21. A binding moiety according to claim 20 wherein the antibody is a monoclonal antibody.

22. A binding moiety according to claim 20 or 21 wherein the antibody or antigen-binding fragment or variant thereof is selected from the group consisting of Fv fragments, Fab-like fragments, single variable domains and domain antibodies.

23. A binding moiety according to any one of claims 20 to 22 wherein the antibody or an antigen-binding fragment or variant thereof is humanised.

24. A binding moiety according to any one of claims 1 to 16 wherein the binding moiety is capable of binding selectively to a nucleic acid molecule encoding Sox11 protein.

25. A binding moiety according to claim 24 wherein the binding moiety is capable of binding selectively to a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 and/or natural variants thereof.

26. A binding moiety according to claim 24 or 25 wherein the binding moiety comprises or consists of a nucleic acid molecule.

27. A binding moiety according to claim 24 wherein the binding moiety comprises or consists of a DNA molecule.

28. A binding moiety according to claims 24 to 27 wherein the binding moiety comprises or consists of a fragment of the nucleotide sequence of SEQ ID NO:2, or the complementary sequence thereof, or a variant of the same.

29. A binding moiety according to any one of claims 26 to 28 wherein the nucleic acid molecule is 5 to 100 nucleotides in length.

30. A binding moiety according to claim 29 wherein the nucleic acid molecule is 15 to 35 nucleotides in length

31. A binding moiety according to any one of the preceding claims wherein the binding moiety comprises a detectable moiety.

32. A binding moiety according to claim 31 wherein the detectable moiety comprises or consists of a radioactive atom.

33. A binding moiety according to claim 32 wherein the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

34. A method of diagnosing epithelial ovarian cancer (EOC) in an individual, the method comprising: (a) providing a sample of epithelial ovarian cells from the individual; and (b) determining the amount of Sox11 protein and/or mRNA in the sample of cells. wherein the levels of Sox11 protein and/or mRNA are indicative of the individual having epithelial ovarian cancer (EOC).

35. A method according to claim 34 wherein high levels of Sox11 protein and/or mRNA are indicative of the individual having epithelial ovarian cancer (EOC).

36. A method according to claim 34 or 35 wherein the EOC belongs to a histological subtype selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

37. A method according to claim 36 wherein the EOC is serous EOC.

38. A method according to claim 36 wherein the EOC is mucinous EOC.

39. A method according to claim 36 wherein the EOC is endometrioid EOC.

40. A method according to claim 36 wherein the EOC is clear cell EOC.

41. A method according to claim 36 wherein the EOC is undifferentiated or unclassifiable EOC.

42. A method of prognosing epithelial ovarian cancer (EOC) in an individual, the method comprising: (a) providing a sample of epithelial ovarian cancer cells from the individual; and (b) determining the amount of Sox11 protein and/or mRNA in the sample of cells. wherein the levels of Sox11 protein and/or mRNA are indicative the individual having improved recurrence-free survival (RFS).

43. A method according to claim 42 wherein the EOC belongs to a histological subtype selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

44. A method according to claim 43 wherein the EOC is serous EOC.

45. A method according to claim 43 wherein the EOC is mucinous EOC.

46. A method according to claim 43 wherein the EOC is endometrioid EOC.

47. A method according to claim 43 wherein the EOC is clear cell EOC.

48. A method according to claim 43 wherein the EOC is undifferentiated or unclassifiable EOC.

49. A method according to any one of claims 42 to 48 wherein high levels of Sox11 protein and/or mRNA is indicative of the individual having improved recurrence-free survival (RFS).

50. A method according to any one of claims 42 to 48 wherein low levels of Sox11 protein and/or mRNA is indicative of the individual having diminished recurrence-free survival (RFS).

51. A method of detecting epithelial ovarian cancer (EOC) cells in an individual, the method comprising: (a) providing a sample of epithelial ovarian cells from the individual; and (b) determining the amount of Sox11 protein and/or mRNA in the sample of cells. wherein the levels of Sox11 protein and/or mRNA are indicative of the individual having epithelial ovarian cancer (EOC) cells.

52. A method according to claim 51 wherein high levels of Sox11 protein and/or mRNA are indicative of the cells being epithelial ovarian cancer (EOC) cells.

53. A method according to claims 51 to 52 wherein the EOC belongs to a histological subtype selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

54. A method according to claim 53 wherein the EOC is serous EOC.

55. A method according to claim 53 wherein the EOC is mucinous EOC.

56. A method according to claim 53 wherein the EOC is endometrioid EOC.

57. A method according to claim 53 wherein the EOC is clear cell EOC.

58. A method according to claim 53 wherein the EOC is undifferentiated or unclassifiable EOC.

59. A method according to any one of claims 34 to 58 wherein the method is performed in vivo.

60. A method according to any one of claims 34 to 58 wherein the method is performed in vitro.

61. A method according to any one of Claims 34 to 60 wherein Sox11 is used as a sole biomarker.

62. A method according to any one of Claims 34 to 61 wherein Sox11 is used in combination with one or more additional biomarkers for diagnosing or prognosing EOC.

63. A method according to claim 62 wherein fewer than 20 additional biomarkers are used in the method, for example fewer than 15, 10, 8, 6, 5, 4, 3, 2 or 1 additional biomarkers.

64. A method according to any one of claims 34 to 63 wherein the method comprises detecting nuclear and/or cytoplasmic expression of Sox11.

65. A method according to any one of 34 32 to 64 wherein the sample of cells to be tested is in the form of a tissue sample.

66. A method according to any one of claims 34 to 65 wherein determining the amount of Sox11 protein and/or mRNA in the sample is performed using a binding moiety according to any one of claims 1 to 31.

67. A method according to any one of claims 34 to 66 further comprising comparing the amount of Sox11 protein and/or mRNA in the sample of cells to be tested with the amount of Sox11 protein and/or mRNA in a control sample.

68. A method according to claim 67 wherein the control sample is a negative control sample comprising or consisting of non-cancerous epithelial ovarian cells.

69. A method according to claim 67 wherein the control sample is a positive control sample comprising or consisting of epithelial ovarian cancer (EOC) cells.

70. A method according to claim 67 wherein the epithelial ovarian cancer (EOC) cells are high recurrence-free survival (RFS)-associated EOC cells.

71. A method according to claim 67 wherein the epithelial ovarian cancer (EOC) cells are low recurrence free survival (RFS)-associated EOC cells.

72. A method according to any one of claims 34 to 71 wherein step (b) is performed using a method selected from the group consisting of macroarray screening, microarray screening, nanoarray screening, reverse transcription PCR, real-time PCR or in situ PCR.

73. A method of imaging epithelial ovarian cancer (EOC) cells in the body of an individual, the method comprising administering to the individual an effective amount of a binding moiety as defined in any one of claims 1 to 33.

74. A method according to claim 73 further comprising the step of detecting the location of the binding moiety in the individual.

75. Use of a binding moiety as defined in any one of claims 1 to 33 in the preparation of a medicament for diagnosing epithelial ovarian cancer (EOC).

76. Use of a binding moiety as defined in any one of claims 1 to 33 in the preparation of a medicament for prognosing epithelial ovarian cancer (EOC).

77. Use of Sox11 protein and/or mRNA encoding the same as a biomarker for diagnosing epithelial ovarian cancer (EOC) cells.

78. Use of Sox11 protein and/or mRNA encoding the same as a biomarker for prognosing epithelial ovarian cancer (EOC) cells.

79. The use according to any one of claims 75 to 78 wherein the EOC belongs to a histological subtype selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

80. The use according to claim 79 wherein the EOC is serous EOC.

81. The use according to claim 79 wherein the EOC us mucinous EOC.

82. The use according to claim 79 wherein the EOC is endometrioid EOC.

83. The use according to claim 79 wherein the EOC is clear cell EOC.

84. The use according to claim 79 wherein the EOC is undifferentiated or unclassifiable EOC.

85. The use according to any one of claims 77 to 84 wherein Sox11 is used as a sole biomarker.

86. The use according to any one of claims 77 to 84 wherein Sox11 is used in combination with one or more additional biomarkers.

87. The use according to claim 86 wherein fewer than 20 additional biomarkers are used in the method, for example fewer than 15, 10, 8, 6, 5, 4, 3, 2 or 1 additional biomarkers.

88. A method of screening for a molecule with efficacy in the diagnosis and/or prognosis of epithelial ovarian cancer (EOC), the method comprising the steps of: (a) contacting a molecule to be tested with Sox11 protein and/or mRNA encoding the same (or with a fragment of said protein or mRNA); and (b) detecting the presence of a complex containing the protein and/or mRNA (or fragment thereof) and the molecule to be tested.

89. A method according to claim 88 wherein the EOC belongs to a histological group selected from the group consisting of serous, mucinous, endometrioid, clear cell and undifferentiated or unclassifiable.

90. The method according to claim 89 wherein the EOC is serous EOC.

91. The method according to claim 89 wherein the EOC us mucinous EOC.

92. The method according to claim 89 wherein the EOC is endometrioid EOC.

93. A method according to claim 89 wherein the EOC is clear cell EOC.

94. A method according to claim 89 wherein the EOC is undifferentiated or unclassifiable EOC.

95. A binding moiety for diagnosing or prognosing epithelial ovarian cancer (EOC) substantially as herein described with reference to the description.

96. A binding moiety for detecting epithelial ovarian cancer (EOC) cells in a sample substantially as herein described with reference to the description.

97. A method of diagnosing or prognosing epithelial ovarian cancer (EOC) in an individual substantially as herein described with reference to the description.

98. A method of imaging epithelial ovarian cancer (EOC) cells in the body of an individual substantially as herein described with reference to the description.

99. Use of a binding moiety in the preparation of a medicament for diagnosing or prognosing epithelial ovarian cancer (EOC) substantially as herein described with reference to the description.

100. Use of Sox11 protein and/or mRNA encoding the same as a marker for epithelial ovarian cancer (EOC) cells substantially as herein described with reference to the description.

101. A method of screening for a molecule with efficacy in the diagnosis, prognosis and/or treatment of epithelial ovarian cancer (EOC) substantially as herein described with reference to the description.

Description

[0113] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

[0114] FIGS. 1A-1B. Sox11 protein expression in ovarian cancer.

[0115] Immunohistochemical staining of Sox 11 showing just cytoplasmic expression (FIG. 1A) and cytoplasmic and nuclear expression (FIG. 1B). Corresponding markup image of the deconvolution algorithm showing stroma in blue, cytoplasmic staining in yellow and orange and nuclear expression in red.

[0116] FIGS. 2A-2C. Sox11 protein expression and survival in ovarian cancer.

[0117] Sox11 expression classified into low, medium and high based on the histogram (FIG. 2A). Kaplan Meier estimate of RFS based on the three Sox11 groups (FIG. 2B). Kaplan Meier estimate of RFS based on comparison of high and medium levels of Sox11 to low levels of Sox11 (FIG. 2C).

[0118] FIG. 3. Amino acid sequence of Homo sapiens Sox11 protein.

[0119] FIG. 4. Nucleic acid sequence of Homo sapiens SOX11 mRNA.

[0120] FIG. 5. Overall survival (25 years) in endometroid ovarian cancer

[0121] The 25-year overall survival of patients with more or less than 10% Sox11 positive tumor cells are compared. When a patient is censored (removed from the study for other reason than death) this is indicated with a tick-mark on the line.

[0122] FIG. 6. Overall survival (5 years) in endometroid ovarian cancer

[0123] The 5-year overall survival of patients with more or less than 10% Sox11 positive tumor cells are compared. When a patient is censored (removed from the study for other reason than death) this is indicated with a tick-mark on the line.

[0124] FIG. 7. Cancer specific survival (5 years) in endometroid ovarian cancer

[0125] The 5-year cancer-specific survival of patients with more or less than 10% Sox11 positive tumor cells are compared. When a patient is censored (removed from the study for other reason than death) this is indicated with a tick-mark on the line.

[0126] FIG. 8. Overall survival (25 years) for high grade EOC

[0127] The 25-year overall survival of high-grade patients with more or less than 10% Sox11 positive tumor cells are compared. When a patient is censored (removed from the study for other reason than death) this is indicated with a tick-mark on the line.

[0128] FIG. 9. Cancer specific (25 years) survival for high grade EOC

[0129] The 25-year cancer-specific survival of high-grade patients with more or less than 10% Sox11 positive tumor cells are compared. When a patient is censored (removed from the study for other reason than death) this is indicated with a tick-mark on the line.

EXAMPLE A

Introduction

[0130] The transcription factor Sox11 is a member of the Sox gene family and has been mapped to chromosome 2p25.3 (Azuma T, Ao S, Saito Y, et al. Human SOX11, an upregulated gene during the neural differentiation, has a long 3′ untranslated region. DNA research 1999; 6:357-60). Sox proteins are identified as proteins that contain a DNA-binding high mobility group (HMG) domain with strong amino acid homology (usually >50%) to the HMG domain of the male sex determination gene, Sry (Wegner M. From head to toes: the multiple facets of Sox proteins. Nucleic acids research 1999; 27:1409-20). More than 20 orthologous Sox genes have been identified in the human and mouse genomes, and family members are divided into eight subgroups according to the degree of homology within and outside the HMG-domain (Schepers G E, Teasdale R D, and Koopman P. Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families. Developmental cell 2002; 3:167-70). Sox proteins act as transcription factors by binding to the minor groove of DNA and inducing a sharp bend of DNA allowing them to play a key architectural role in the assembly of transcriptional enhancer complexes (Dy P, Penzo-Mendez A, Wang H, et al. The three SoxC proteins Sox4, Sox11 and Sox12 exhibit overlapping expression patterns and molecular properties. Nucleic acids research 2008; 36:3101-17; van de Wetering M and Clevers H. Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix. The EMBO journal 1992; 11:3039-44). In addition to protein-DNA interactions, Sox proteins also interact with various other transcription factors to increase their efficiency and specificity of action (Dy P, Penzo-Mendez A, Wang H, et al. The three SoxC proteins Sox4, Sox11 and Sox12 exhibit overlapping expression patterns and molecular properties. Nucleic acids research 2008; 36:3101-17).

[0131] Sox11 belongs to the C subgroup, along with Sox4 and Sox12 (Schepers G E, Teasdale R D, and Koopman P. Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families. Developmental cell 2002; 3:167-70), and all three proteins demonstrate a high degree of homology within both the C-terminal transactivation domain and the HMG domain (Dy P, Penzo-Mendez A, Wang H, et al. The three SoxC proteins Sox4, Sox11 and Sox12 exhibit overlapping expression patterns and molecular properties. Nucleic acids research 2008; 36:3101-17; Jay P, Goze C, Marsollier C, et al. The human SOX11 gene: cloning, chromosomal assignment and tissue expression. Genomics 1995; 29:541-5). Sox11 and Sox 4 play major roles in cardiac, neuronal and other major embryonic processes, whilst less is known about Sox12 (Dy P, Penzo-Mendez A, Wang H, et al. The three SoxC proteins Sox4, Sox11 and Sox12 exhibit overlapping expression patterns and molecular properties. Nucleic acids research 2008; 36:3101-17).

[0132] We have recently demonstrated the nuclear Sox11 is specifically up-regulated in mantle cell lymphoma (MCL) and distinguishes MCL from other B-cell lymphomas (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5). Sox4 is a prominent transcription factor in lymphocytes of both the B and T-cell lineage (Wegner M. From head to toes: the multiple facets of Sox proteins. Nucleic acids research 1999; 27:1409-20; van de Wetering M, Oosterwegel M, van Norren K, and Clevers H. Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. The EMBO journal 1993; 12:3847-54) and is crucial for B lymphopoiesis (Smith E and Sigvardsson M. The roles of transcription factors in B lymphocyte commitment, development, and transformation. Journal of leukocyte biology 2004; 75:973-81), whilst Sox11 has no known lymphopoietic function and is not expressed in B cells (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5). Both Sox4 and Sox11 are expressed in medulloblastoma (Lee C J, Appleby V J, Orme A T, Chan W I, and Scotting P J. Differential expression of SOX4 and SOX11 in medulloblastoma. Journal of neuro-oncology 2002; 57:201-14) and Sox11 is also overexpressed in malignant glioma (Weigle B, Ebner R, Temme A, et al. Highly specific overexpression of the transcription factor SOX11 in human malignant gliomas. Oncology reports 2005; 13:139-44). Additionally Sox4 is expressed in bladder cancer with increased levels of expression associated with improved patient outcome (Aaboe M, Birkenkamp-Demtroder K, Wiuf C, et al. SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization. Cancer research 2006; 66:3434-42).

[0133] This study outlines the expression of Sox11 mRNA across a large number of normal tissues and tumours, and reveals Sox11 mRNA to be overexpressed in a large number of malignant tissues. In addition, we specifically examined Sox11 protein expression in EOC and demonstrated that increased levels of Sox11 protein, as determined by image analysis, were associated with an improve recurrence free survival (RFS).

Materials and Methods

Transcriptional Profiling

[0134] SOX11 gene mRNA expression levels across a large number of human tissues were retrieved from the In Silico Transcriptomics (IST) database, containing data from a meta-analysis of 14,095 samples analyzed using the Affymetrix gene expression microarrays.

Patients and Tumour Samples

[0135] The TMA, used in this study, was constructed from a consecutive cohort of 76 patients diagnosed with primary invasive epithelial ovarian cancer at the National Maternity Hospital, Dublin, with a median follow-up of of 4.3 years. The patient cohort is summarised in table 1. The standard surgical approach was a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy with cytological evaluation of peritonea fluid or washings. Residual disease was resected to less than 2 cm where possible. Stage and volume of residual disease (no residual disease, residual disease greater or less than 2 cm) was recorded in all cases. Adjuvant chemotherapy consisted of cisplatin or carboplatin prior to 1992 and combined with paclitaxel from 1992 to 2002. No patient received neo-adjuvant chemotherapy. Benign or borderline ovarian cancers, non-epithelial ovarian cancer and cases with histological features typical of secondary ovarian cancer were excluded from the study. Diagnostic specimens were all formalin fixed and paraffin embedded in the Department of Pathology at the National Maternity Hospital, Dublin, Ireland. All tissue blocks were stored in this department prior to construction of the TMA. Full ethical approval was obtained from the ethics committee of the National Maternity Hospital, Dublin.

Tissue Microarrays and Immunohistochemistry

[0136] Seventy six paraffin-embedded tumour specimens were used for tissue microarray (TMA) construction. Areas representative of invasive cancer were marked on haematoxylin and eosin-stained slides and the TMA was constructed, using a manual tissue arrayer (MTA-1, Beecher Inc, WI). The array consisted of four cores per patient. Two 1.0 mm cores were extracted from each donor block and assembled in a recipient block. Recipient blocks were limited to approximately 100 cores each. In general, cores were taken from the peripheral part of the tumour in cases where the tumour had well-defined borders. In more diffusely growing tumours, areas with the highest tumour cell density were primarily targeted. Necrotic tissue was avoided.

[0137] TMA sections (4 μm) were dried, deparaffinized, rehydrated and put through descending concentrations of ethanol. Heat mediated antigen retrieval was performed in a BORGdecloaker (Biocare, Concord, Calif., USA) at pH 9.0 and sections were then stained with the primary rabbit anti-human Sox11 antibody (1:100) at room temperature for 25 minutes. This specific antibody was raised, as previously described (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5) and targeted the following protein sequence:

TABLE-US-00001 [SEQ ID NO: 3] FMVWSKIERRKIMEQSPDMHNAEISKRLGKRWKMLKDSEKIPFIREAER LRLKHMADYPDYKYRPRKKPKMDPSAKPSASQSPEKSAAGGGGGSAGGG AGGAKTSKGSSKK

[0138] Signal was detected, using the Dako REAL Detection system, containing the secondary biotinylated goat anti-rabbit/mouse antibody, the strepavidin/horseradish peroxidase complex and 3,3′-diaminobenzidine, according to the manufacturer's protocol. Slides were counterstained with Mayers hematoxylin (Sigma-Aldrich, St Louis, Mo.).

Image Acquisition, Management and Automated Analysis

[0139] The Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, Calif.) system was used to capture whole slide digital images with a 20× objective. Slides were de-arrayed to visualise individual cores, using TMA Lab (Aperio). A color deconvolution algorithm (Aperio) was used to develop a quantitative scoring model for Sox11 expression.

Statistical Analysis

[0140] Spearman's Rho correlation was used estimate the relationship between cores from individual tumours, Differences in distribution of clinical data and tumour characteristics between samples with a high and low Sox11 expression (described below) were evaluated using the χ.sup.2 test. Kaplan-Meier analysis and the log rank test were used to illustrate differences between RFS. Cox regression proportional hazards models were used to estimate the relationship to RFS and Sox11, stage and grade. All calculations were performed, using SPSS version 11.0 (SPSS Inc, Chicago, Ill.). P value <0.05 was considered statistically significant.

Results

[0141] Sox11 mRNA Expression in Normal and Tumour Tissues

[0142] A metanalysis of Sox11 mRNA expression levels was performed in 14 095 samples analyzed using the Affymetrix gene expression microarrays.

[0143] Increased levels of Sox11 mRNA expression were evident in epithelial ovarian carcinoma samples (data not shown).

Sox11 Protein Expression in Ovarian Cancer

[0144] Having identified Sox11 as a gene that was overexpressed in epithelial ovarian carcinoma cells, Sox11 protein expression was examined using IHC in EOC as illustrated in FIG. 1. Sox11 expression was seen exclusively in tumour epithelium and IHC signal was evident in both the nucleus and the cytoplasm. Nuclear expression of Sox11 was present only when accompanied by cytoplasmic signal, whereas a proportion (49%) of tumours did demonstrate cytoplasmic expression in the absence of nuclear signal (FIG. 1).

Quantitative Determination of Sox11 Expression as Determined by Image Analysis

[0145] Quantitative determination of Sox11 expression was then ascertained, using an image analysis approach, in particular via the use of a commercial colour deconvolution algorithm (Aperio). A pseudo-colour “mark-up” image was generated as an algorithm result, thus allowing confirmation that the algorithm was accurately identifying epithelial and stromal pixels (FIG. 1). A full description of the algorithm was recently published (Brennan D J, Rexhepaj E, O'Brien S L, et al. Altered Cytoplasmic-to-Nuclear Ratio of Survivin Is a Prognostic Indicator in Breast Cancer. Clinical cancer research 2008; 14:2681-9).

[0146] The algorithm was used to calculate a total intensity (TI) for Sox11 for the each core. There was a strong correlation between quadruplicate cores from individual tumours for TI (Spearman's Rho=0.858, p<0.001), indicating that Sox11 has a homogenous pattern of expression in ovarian cancer and is suitable for TMA based analysis. As tumours were arrayed in quadruplicate, the median value for each tumour was used for further analysis. The algorithm accurately distinguished between nuclear and cytoplasmic staining in all cores, as confirmed by a histopathologist.

[0147] A histogram of Sox11 image analysis data for the entire cohort is shown in FIG. 2a. Using this histogram, the tumours were placed into three categories—high, medium and low level of Sox11 expression, as determined by image analysis. Based on image analysis categorization, 20% (n=17) of tumours were classified as having high levels 43% (n=35) medium levels and 29% (n=24) low/negative levels of Sox11 expression, as determined by image analysis. Tumours in the high expression group all showed expression of nuclear and cytoplasmic Sox11, whilst those in the medium group generally exhibited only cytoplasmic Sox11. No association was found between Sox11 expression and age, grade or stage of disease.

Associations Between Sox11 Expression as Determined by Automated Image Analysis and Survival

[0148] Kaplan Meier analysis of RFS based on the expression of Sox11 revealed a stepwise decrease in RFS between the high, medium and low groups (p=0.033) (FIG. 2b). Further subset analysis revealed a markedly reduced RFS in patients with low levels of Sox11 expression, as determined by image analysis compared to high and medium Sox11 expressers (p=0.02) (FIG. 2c). We proceeded to perform a multivariate Cox regression analysis of RFS, which revealed that Sox11 expression was an independent predictor of RFS when compared to stage and grade (HR=0.56, 95% Cl=0.319-0.997, p=0.049).

Discussion

[0149] In this study we combined a meta-analysis of transcriptomic data, TMAs and automated image analysis to identify and validate Sox11 as a prognostic biomarker in epithelial ovarian carcinoma (EOC). This study is the first to describe the relationship between Sox11 expression and prognosis in EOC. Having previously identified Sox11 as a new diagnostic marker in MCL (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5), we proceeded to use the IST database containing data from a meta-analysis of 14 095 samples analyzed, using the Affymetrix gene expression microarrays to profile the expression of Sox11 mRNA in EOC cells.

[0150] We then proceeded to use TMAs and a quantitative automated analysis of IHC to evaluate Sox11 protein expression in EOC in relation to recurrence free survival. This revealed that epithelial-specific Sox11 expression in both the nuclear and cytoplasmic compartments. Increased levels of Sox11, particularly nuclear Sox11, was associated with an increased RFS and Cox regression multivariate analysis revealed Sox11 was an independent predictor of RFS when controlling for grade and stage (see Table 2).

[0151] Sox11 plays an important role in embryogenesis and tissue remodeling, and consequently is present during gastrulation and early post-gastrulation development throughout the embryo (Hargrave M, Wright E, Kun J, et al. Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Developmental dynamics 1997; 210:79-86; Sock E, Rettig S D, Enderich J, et al. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Sox11 in tissue remodeling. Molecular and cellular biology 2004; 24:6635-44). Later during development, Sox11 is prominently expressed in the developing nervous system and at many sites throughout the embryo where epithelial-mesenchymal interactions occur (Hargrave M, Wright E, Kun J, et al. Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Developmental dynamics 1997; 210:79-86). At sites of such epithelial-mesenchymal interactions, Sox11 can be found in the mesenchymal or epithelial compartment, and it has been postulated to be involved in inductive remodelling (Hargrave M, et al., 1997supra.). Sox11 expression in most tissues is transient and as a consequence, little Sox11 expression has been found in terminally differentiated adult tissues, in contrast to its widespread expression during embryogenesis (Sock E, Rettig S D, Enderich J, et al. Gene targeting reveals a widespread role for the high-mobility-group transcription factor Sox11 in tissue remodeling. Molecular and cellular biology 2004; 24:6635-44). Our findings complement these data, whereby Sox11 expression was absent in normal tissue.

[0152] The role played by Sox11 in tumourogenesis remains to be fully elucidated. As mentioned previously, a marked upregulation of Sox11 mRNA was evident in EOC cells. The exact functional role of Sox11 in adult tissues is not fully understood, although the Sox proteins appear to play a dual role (i) DNA binding and (ii) transcriptional partner selection, which may permit selective recruitment of individual Sox proteins to specific genes (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5). Whilst a number of studies have described Sox11 expression in gliomas (Weigle B, Ebner R, Temme A, et al. Highly specific overexpression of the transcription factor SOX11 in human malignant gliomas. Oncology reports 2005; 13:139-44), neuroblastomas (Lee C J, Appleby V J, Orme A T, Chan W I, and Scotting P J. Differential expression of SOX4 and SOX11 in medulloblastoma. Journal of neuro-oncology 2002; 57:201-14) and MCL (Ek S, Dictor M, Jerkeman M, Jirstrom K, and Borrebaeck C A. Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma. Blood 2008; 111:800-5), its functional role in these tumours is not completely understood.

[0153] In summary, this is the first description of the differential expression of Sox11 in EOC. Our findings demonstrate that Sox11 is an independent predictor of improved RFS in EOC.

TABLE-US-00002 TABLE 1 Patient and tumour characteristics Age Median (Range) 52 (31-77) Histology Serous 50 Mucinous 4 Endometroid 17 Clear cell 1 Other 4 Grade Well Differentiated 12 Moderately Differentiated 29 Poorly Differentiated 35 Stage 1 0 2 21 3 54 4 1

TABLE-US-00003 TABLE 2 Multivariate* Cox regression analysis of RFS HR 95.0% CI P value Sox11 (high/medium v's low) 0.56 0.319-0.997 0.049 Stage (continuous) 1.92 0.971-3.800 0.061 Grade (Well and moderately diff 1.08 0.740-1.562 0.702 v's poorly Diff) *Adjusted for all other variables in the table Abbreviations: HR = Hazard ratio, 95% CI = 95% Confidence intervals

Example B

Introduction

[0154] Epithelial ovarian cancer (EOC) comprises three major histological subtypes (serous, mucinous and endometrioid) and can also be subgrouped based on stage and grade. Endometrioid tumors make up about 2 to 4 percent of all ovarian tumors and most of them (about 80 percent) are malignant, representing 10 to 20 percent of all ovarian carcinomas.

Material & Methods

[0155] Sections of high grade EOC and endometrioid EOC were stained for Sox11 and analyzed as previously described (Brennan et al., 2009, European Journal of Cancer, 45(8):1510-1517).

Results & Discussion

[0156] As shown in FIGS. 5, 6 and 7, overall and cancer specific survival can be predicted using Sox11 for endometrioid EOCs. Also, as shown in FIGS. 8 and 9, overall and cancer specific survival can be predicted using Sox11 for high grade EOCs.

[0157] When calculating cancer-specific survival probability only data for cancer-related deaths are used, in contrast to when calculating overall survival.

[0158] These data indicate that Sox11 can be used in both high grade EOC and endometrioid ovarian cancer to stratify patients into clinically relevant groups based on overall and cancer specific survival.

[0159] In conclusion, Sox11 is not only a useful biomarker for EOC as a group, but can be used for subgroups of patients with different clinical and/or histological features.