Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

• the present invention relates to the use of at least one homeodomain containing transcription factor, such as a HOX peptide or EN-2 peptide, as a biomarker for prostate cancer.
• at least one homeodomain containing transcription factor such as a HOX peptide or EN-2 peptide
• Prostate cancer is currently the leading cause of cancer related death in men, killing 10,000 men each year in the UK alone.
• the current availability of biomarkers for the non-invasive detection of prostate cancer is essentially limited to Prostate Specific Antigen (PSA).
• PSA Prostate Specific Antigen
• Serum levels of PSA are significantly raised in prostate cancer patients, and the PSA test is currently used to follow the progress of the disease and its response to treatment.
• PSA levels are also significantly raised in other, non-cancerous conditions of the prostate, such as prostatitis and benign hypertrophy, it is not always certain that detecting an increase in PSA levels is actually indicative of prostate cancer in a patient. Furthermore, PSA levels do not necessarily correlate with the volume of the disease or to the development of nodal or bone metastasis.
• PSA levels are thought to rise only very slowly in prostate cancer, so that the disease is often far advanced before a positive result is obtained, when measuring PSA levels. Furthermore, PSA levels have to be normalised, as they can vary with age and prostate size.
• HOXC4 a homeotic transcription factor
• HOXA3 and H0XD9 are also considered to be useful as biomarkers.
• EN-2 homeodomain-containing transcription factor Engrailed 2
• the EN-2 protein can be secreted into the surrounding medium of the cells.
• the present invention provides the use of at least one homeodomain- containing transcription factor, or a fragment thereof, as a biomarker for prostate cancer.
• homeodomain-containing transcription factors include HOX and EN-2 peptides.
• the invention also relates to a method for diagnosing prostate cancer disease comprising detecting at least one homeodomain-containing transcription factor in a sample from a patient.
• the at least one HOX or EN-2 peptide, or a fragment thereof is detectable in a body fluid.
• Such combinations include HOXC4 and H0XB5; HOXC4 and HOXB6; HOXB5 and HOXB6; HOXC4, H0XB5 and H0XB6; EN- 2 and HOXC4; EN-2 and H0XB5; EN-2 and H0XB6; EN-2, HOXC4 and HOXB5; EN-2, HOXC4 and HOXB6; EN-2, HOXB5 and HOXB6; EN-2, HOXC4, H0XB5 and H0XB6.
• Such combinations may also further comprise one or both of HOXA3 and/or H0XD9.
• H0XA3 and H0XD9 include H0XA3 and H0XD9; H0XA3 and HOXC4; HOXA3 and H0XB5; H0XA3 and H0XB6; HOXA3 and EN-2; H0XD9 and HOXC4; HOXD9 and H0XB5; H0XD9 and HOXB6; H0XD9 and EN-2.
• a particularly preferred combination of markers comprises one or more of, preferably all of, HOXC4, HOXB5, H0XB6 and EN-2.
• the HOX genes are a family of homeodomain-containing transcription factors that determine the identity of cells and tissues during early development (reviewed by Morgan, 2006). Recent publications, including Miller et al 2003, have effectively identified every single protein that is expressed, and retained intracellularly, in a prostate cancer tumour cell. Of course, this means that a total of around 10,000 to 12,000 proteins, including the HOX peptides, were identified. However, we have surprisingly discovered that, from amongst these thousands of proteins expressed in prostate cancer cells, the HOXC4 protein, for instance, is found extracellularly and is an excellent biomarker for prostate cancer.
• this protein has not been identified as a biomarker for prostate cancer before although it is known to be expressed in prostate cancer cells, along with many thousands of other proteins, many of which could not be described as biomarkers for prostate cancer. This also applies to other HOX peptides including HOXB5 and HOXB6.
• EN proteins In addition to transcriptional regulation, EN proteins have a number of other, apparently unrelated properties that are functionally important. The first of these is the ability to be secreted from cells, and to be internalized by others (Cosgaya et al, 1998; Joliot et al., 1998, reviewed by Morgan, 2006). The exact mechanistic basis for this phenomenon is unknown, although it is clear that it depends upon a conserved nuclear export sequence located in the homeodomain (Maizel et al., 1999; Chatelin et al., 1996; Derossi et al., 1996; Joliot et al., 1997).
• EN-2 has recently been shown to be a potential oncogene in breast cancer (Martin et al., 2005).
• Non-tumorogenic murine mammary cell lines forced to express EN-2 subsequently exhibit a number of malignant characteristics including a shortening of the cell cycle and a loss of cell to cell contact.
• RNAi silencing of EN-2 indicates that it is required for the maintenance of the transformed phenotype in a human breast cancer cell line.
• EN-2 is overexpressed and secreted from the prostate cancer derived cell line LNCaP and in human tissue samples from prostatic adenocarcinoma. Furthermore, EN- 2 is secreted from ductal cells and adenocarcinoma tumours and is present in the urine of prostate cancer patients. This is the first time that EN-2 has been suggested as a marker of prostate cancer.
• the at least one homeodomain-containing transcription factor such as a HOX peptide or EN-2 peptide, or fragment thereof, is detected.
• the at least one HOX peptide is selected from any HOX peptide as listed below, provided that the HOX peptide, or a fragment thereof, is detectable in a body fluid such as serum or urine.
• the at least one HOX peptide, or a fragment thereof is from the HOXA, the HOXB, the HOXC or the HOXD subfamily. If more than one HOX peptide is used, then these may be from the same family or from a combination of families. Combinations of fragments are also envisaged, either fragments of the same protein or fragments of different proteins. Combinations of different peptides can provide more accurate diagnoses. It is preferred that the HOX peptide is not H0XA4, HOXAl, H0XA7, HOXB3, or H0XB9.
• the HOX peptide is the H0XC4 peptide, and/or the H0XB6 peptide and/or the H0XB5 peptide, and/or the H0XA3 peptide, and/or the H0XD9 peptide or fragments thereof.
• the H0XC4 peptide is that provided in Sequence ID NO. 1 (NCBI accession no. P09017, gi: 123279), or a fragment thereof.
• the HOXB6 peptide is that provided in Sequence ID NO. 2 (NCBI accession no. P17509, gi: 116242515), or a fragment thereof.
• the H0XB5 peptide is that provided in Sequence ID NO. 3 (NCBI accession no. P09067, gi:400000), or a fragment thereof.
• the EN-2 peptide is that provided in Sequence ID NO. 4 (NCBI accession no.
• H0XC4 the HOXC4 peptide or a fragment thereof, it will be understood that these terms can be used interchangeably, unless otherwise apparent. The same follows for other HOX peptides and EN-2.
• H0XC4, H0XB6, H0XB5, H0XA3 or H0XD9 this also applies to other HOX peptides, or combinations thereof, unless otherwise apparent.
• reference is made herein to a particular sequence, such as a reference sequence or a Sequence ID NO this applies to all Sequence ID NOs. and any of the HOX sequences provided below, unless otherwise apparent.
• the fragment comprises at least 80% sequence homology to the reference sequence (e.g. HOXC4, HOXB6, HOXB5, HOXA3, H0XD9 or EN-2), more preferably 85%, more preferably 90%, more preferably 95%, more preferably 97%, more preferably 99% and most preferably 99.9% sequence homology to the reference sequence, or as close thereto as appropriate.
• sequence homology include the BLAST programme.
• the fragment comprises at least 80% sequence identity to the reference sequence (e.g. HOXC4, HOXB6, HOXB5, HOXA3, HOXD9 or EN-2), more preferably 85%, more preferably 90%, more preferably 95%, more preferably 97%, more preferably 99% and most preferably 99.9% sequence identity to the reference sequence, or as close thereto as appropriate.
• Suitable methods for establishing sequence identity include the BLAST programme.
• Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing.
• the fragment comprises at least four consecutive amino acids from the reference sequence, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8 consecutive amino acids from the reference sequence, such as Sequence ID NO. 1, 2, 3, 4, 5 or 6, although longer fragments at least 10, 15, 20, 25, 30, 50, 75, 100, 150, 200, 225 and up to at least 250 amino acids are also preferred.
• Fragments also include truncated peptides that have x amino acids deleted from the N-terminus and/or C-terminus. In such truncations, x may be 1 or more (ie. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more), but preferably less than 150.
• HOXC4 is secreted via a non-classical route, with no signal sequence required.
• the sequence given in Sequence ID NO. 1 is, therefore, thought to be the same as the protein found in serum. This is corroborated by the observation that the detected band is of a corresponding size (Mw) to the predicted size based on the gene sequence.
• HOX proteins may also leave cells through other mechanisms e.g. cell lysis.
• biomarker is used throughout the art and means a distinctive biological or biologically-derived indicator of a process, event or condition.
• a biomarker is indicative of a certain biological state, such as the presence of cancerous tissue.
• different forms of biomarkers can be indicative of certain disease states but, without being bound by theory, it is thought that merely the presence of elevated levels of a HOXC4 peptide, or fragment thereof, in body fluids such as serum, is indicative of prostate cancer.
• body fluids such as serum
• glycoforms such as altered glycoform structure or sugar content
• HOXC4 may yet be determined for HOXC4 but these are encompassed and may even also be indicative of the progress of the prostate cancer.
• the principal discovery underpinning the present invention is that there is a significant increase in the level of HOXC4 detectable in body fluids, such as the serum. It is thought that HOXC4 is secreted, but it is also envisaged that it may be due to HOXC4 protein leaking out of dying cells. Similarly, EN-2 may be secreted or may be detectable in body fluids due to leaking from damaged or dead cells. Such increased levels are indicative of both early stage and late stage prostate cancer. Whilst there is a significant rise between control or normal levels and early stage prostate cancer, there is also a very significant increase between early and late stage prostate cancer. Broadly, it is an advantage of the present invention that the substance and also the state of the prostate cancer can be detected. This aids in the prognosis and provision of suitable therapies.
• the present invention is able to distinguish over conditions such as benign prostatic hyperplasia BTH (BPH). Such conditions, which lead to an enlargement of the prostatic gland, can be misinterpreted as prostate cancer using established techniques for determining the presence of prostate cancer, such as Digital Rectal Examination (DRE). It is another advantage of the present invention that an accurate diagnosis can be provided without resorting to unpleasant and potentially harmful invasive procedures, which may also be inaccurate. Furthermore, the present invention is particularly sensitive. Preferably the methods of the present invention may detect the onset of prostate cancer prior to any other detection method and prior to the onset of the overt symptoms of prostate cancer. Thus, the cancer may be treated at an early stage when it is more susceptible to such treatment and less likely to have entered the metastatic stage.
• BPH benign prostatic hyperplasia BTH
• DRE Digital Rectal Examination
• prostate cancer Different types are known. The most common starts in the prostate gland cells and is known as prostate adenocarcinoma. However, other forms of prostate cancer exist, such as, sarcomas, small cell carcinomas, and transitional cell carcinomas. The methods of the invention may be used to detect the onset of any of these types of cancer, although the detection of adenocarcinoma is preferred.
• the progression of cancer is monitored by a staging process. This indicates how well developed the cancer is and if it has spread. The score runs from one to four, with the prognosis becoming progressively worse at each stage.
• Stage Two Gleason scores are five or higher, or over five percent of the gland shows abnormal growth; the cancer is still restricted to the prostate.
• Stage Three Malignant cells have spread to the seminal vesicles, but not to the lymph nodes or other organs.
• Stage Four The lymph nodes, pelvic tissue or more distant organs are affected.
• the methods of the invention detect the onset of prostate cancer prior to, or during stage one or stage two, more preferably stage one.
• the peptide biomarkers of the present invention can be used in methods of diagnosis, for instance clinical screening, and in methods of prognosis assessment, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Furthermore, the biomarkers of the present invention and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.
• the invention provides a method of diagnosing or monitoring the progression of prostate cancer, comprising detecting and/or quantifying at least one homeodomain-containing transcription factor such as a HOX peptide or EN-2 peptide or a fragment thereof.
• the peptide comprises or consists of the amino acid sequence according to SEQ ID NO 1, 2, 3, 4, 5 or 6.
• the at least one HOX peptide, the EN-2 peptide, or a fragment thereof is present in a biological sample from a test subject.
• Said biological sample preferably being a body fluid sample.
• the body fluid may be any human body fluid, including cellular fluid, cerebrospinal fluid (CSF), semen, urine, blood, lymph or saliva.
• CSF cerebrospinal fluid
• the body fluid is whole blood, in particular blood serum, or urine.
• the body fluid is substantially or completely free of whole/intact cells.
• the body fluid is free of platelets and cell debris (such as that produced upon the lysis of cells).
• the body fluid is free of both prokaryotic and eukaryotic cells.
• Such fluids can be obtained by any number of means known in the art, such as will be apparent to the skilled person.
• a urine sample is easily attainable, whilst blood or serum samples can be obtained parenterally by using a needle and syringe, for instance.
• Cell free or substantially cell free samples can be obtained by subjecting the body fluid to various techniques known to those of skill in the art which include, but are not limited to, centrifugation and filtration.
• samples such as tissue homogenates, tissue sections and biopsy specimens.
• Monitoring methods of the present invention can be used to detect the onset, progression, stabilisation, amelioration and/or remission of prostate cancer.
• the detection of the at least one HOX peptide, EN-2 peptide or a fragment thereof can be used to determine the progression of the disease, in order to direct a physician towards a particular treatment.
• At least two detection and/or quantification steps are provided, preferably spaced apart temporally, for instance by a few days, weeks, years or, more preferably, months, to determine whether the levels of the at least one HOX or EN-2 peptide, or a fragment thereof, have changed, thus indicating whether there has been a change in the progression of the cancer.
• This enables comparisons to be made between a level of the biomarker in samples taken on two or more occasions, as an increase in the level of the peptide over time is indicative of the onset or progression of the cancer, whereas a decrease in the level of the peptide may indicate amelioration and/or remission of the cancer.
• a method of diagnosis or of monitoring comprises comparing the level of the peptide present in a test sample, with the level in one or more controls.
• the controls may be from the same patient from a previous sample, to thus monitor onset or progression.
• the controls may be normalised for a population, particularly a healthy or normal population, where there is no prostate cancer.
• the control may consist of the level of a biomarker found in a normal control sample from a normal subject.
• a method of diagnosing prostate cancer comprising quantifying or detecting the amount of at least one HOX peptide, the EN-2 peptide or a fragment thereof, and comparing the amount of said peptide in said test sample with the amount present in a normal control biological sample from a normal subject or from the same subject well prior to the incidence of disease. If a significant increase in the level of the at least one HOX peptide, the EN-2 peptide or a fragment thereof, is found in the test sample, then it is indicative that the disease is either progressing or has initiated.
• this method of detection of at least one HOX peptide, the EN-2 peptide or a fragment thereof is particularly useful in detecting early stage prostate cancer and is more sensitive than the traditional test of detecting PSA levels.
• the methods of the invention are particularly useful for confirming cancer when a patient has tested negative for cancer using conventional methods, such as the detection of PSA.
• the level of the at least one HOX peptide or EN-2 peptide secretion into the body fluids is in the ranges described below.
• Table 1 and Figure 1 show a very sizeable increase in H0XC4 serum protein concentrations.
• Table 2 and Figure 2 show similar results for HOXB6.
• H0XB6 is a biomarker for late-stage prostate cancer (data not shown).
• H0XB5 is also a biomarker, but is less preferred than H0XB6 or H0XC4 (Table 3 and Figure 7).
• Other alternative biomarkers include H0XA3 and H0XD9 (see Figure 8).
• the increase is statistically significant, determined by using standard methods, such as a "t-test" providing confidence intervals of preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.95%, and most preferably at least 99.99%.
• a "t-test" providing confidence intervals of preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.95%, and most preferably at least 99.99%.
• the peptide may be released into the body fluid, either by cell break-up or leakage, or by secretion.
• Detection and/or quantification of the at least one HOX peptide or the EN-2 peptide, or fragments thereof may be by any method suitable to detect the presence and/or amount of a specific protein or peptide in a biological sample. This may be by direct detection of the biomarker, by instance by MALDI-TOF or SELDI.
• the at least one HOX peptide or EN-2 peptide, or fragment thereof is detected directly or indirectly via interaction with a ligand or ligands.
• ligands may include an anti-HOX or anti-EN-2 antibody or a binding fragment thereof, an aptamer, or an oligonucleotide. It will be appreciated that these ligands must be capable of specifically binding the HOX peptide or EN-2 peptide, or a fragment thereof. It may be suitably labelled and comprise an affinity tag. Suitable labels may be fluorescent, radioactive or luminescent. The labels may be attached to the antibody directly, or via a linker.
• Liquid chromatographic techniques may include HPLC (high pressure liquid chromatography) or even low pressure liquid chromatography (LPLC). Furthermore, methods such as thin-layer chromatography, NMR spectroscopy and any other method described herein, are also preferred.
• the peptide, or a fragment thereof may be detected and/or quantified using immunological methods, such as sandwich immunoassays, for instance an enzyme linked immunosorbent assay (ELISA), radioimmunoassays (RAI), enzyme immunoassays (EIA), Western Blotting, immunoprecipitation.
• sandwich immunoassays for instance an enzyme linked immunosorbent assay (ELISA), radioimmunoassays (RAI), enzyme immunoassays (EIA), Western Blotting, immunoprecipitation.
• particle-based immunoassays which may include, for instance, using gold, silver, or latex particles, magnetic particles or Q-dots. Such methods may be performed, for example, in microtitre plate or strip format
• anti-human HOXC4 antibody is available from Abeam, UK, Catalogue No. ab24338. This is rabbit anti-human HOXC4 antibody and, therefore, it may be preferable to use a secondary, anti-rabbit IgG antibody linked to a reporter, such as alkaline phosphatase, to detect the HOXC4 antibody-complex.
• An example of an anti-HOXB6 antibody is from Abeam, cat no. ab26077.
• An example of an anti-HOXB5 antibody is from Abeam, cat no. ab26079.
• An example of an anti-HOXA3 antibody is from Abeam, cat no. ab28771.
• An example of an anti- H0XD9 antibody is from Abeam, cat no. ab60708. Examples of other anti-HOX antibodies are known.
• An example of an anti-EN-2 antibody is from Abeam, cat no. ab45867.
• antibodies may be polyclonal, monoclonal, bispecific, humanised or chimeric antibodies.
• the antibodies may consist of a single chain but would preferably consist of at least a light chain or a heavy chain but it will be appreciated that at least one Complementarity Determining Region (CDR) is required in order to bind a HOX epitope or an EN-2 epotope.
• CDR Complementarity Determining Region
• the epitope or epitopes must be available for binding or recognition by the ligand, such as an antibody.
• Anti-HOX and anti-EN-2 antibodies are already known, but if further antibodies or binding ligands, with different specificities are required then these could be obtained.
• Methods of making antibodies are known in the art. For example, if polyclonal antibodies are desired, then a selected mammal, such as a mouse, rabbit, goat or horse may be immunised with the antigen of choice, such as human HOX or EN-2. The serum from the immunised animal is then collected and treated to obtain the antibody, for instance by immunoaffinity chromatography. Human HOX or EN-2 could, for instance, be injected into rabbits triggering a rabbit immune response, raising anti-HOX or anti-EN-2 antibodies, which can thereby be isolated, using standard methods.
• monoclonal antibodies could be produced by methods known in the art, and are generally preferred.
• the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
• an antibody as referred to herein, must consist of an epitope-binding region, such as CDR.
• the term "antibody” refers to intact molecules as well as to fragments thereof, such as Fab, Fab', F(ab') 2 and Fv, which are capable to binding to an epitope.
• the antibody also comprises an effector portion, such as the Fc region.
• the antibody may of any suitable class, including IgE, IgM, IgD, IgA and, in particular, IgG.
• IgE immunoglobulin A
• IgM immunoglobulin A
• IgD immunoglobulin A
• IgG immunoglobulin A
• the various subclasses of these antibodies are also envisaged.
• a biosensor is used to detect the at least one peptide biomarker, or a fragment thereof.
• the biosensor may incorporate an immunological method, as described above, for instance, for detection of the biomarker, or electrical, thermal, magnetic, optical for instance a hologram, or acoustic means or technologies. Using such biosensors, it is envisaged that the biomarker is detected and/or quantified.
• the present invention provides the use of a biosensor to detect and/or quantify the peptide, or a fragment thereof.
• the at least one HOX or EN-2 biomarker can be detected using a biosensor based on the use of "smart" holograms, or high frequency acoustic systems, particularly as such systems are amenable to array configurations.
• a biosensor based on the use of "smart" holograms, or high frequency acoustic systems, particularly as such systems are amenable to array configurations.
• smart hologram sensors available from Smart Holograms Limited, Cambridge UK
• a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker.
• the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram.
• the test result readout can see a change in the optical brightness, image, colour and/or position of the image. This can even be read by eye.
• a biosensor according to the invention will detect one or more of, preferably two, three, four, five or all six of, HOXC4, HOXB5, HOXB6, H0XA3, HOXD9 and EN-2. Further preferred biosensors may also further detect other HOX biomarkers as referred to above.
• a method may comprise the steps of:
• step (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c);
• Figure 3 (a) RT-QPCR analysis of EN-2 expression in LNCaP and primary tumour cells (transcript level as a proportion of ⁇ -actin). (b,c) Core biopsy of a prostatic adenocarcinoma stained with anti-En2 antibody. En2 positive staining (brown) is present in the cytoplasm of tumour cells, with strongest staining at the luminal border (1). En2 positive blebs are visible attached to the luminal border (2) or free within the lumen (3). The nuclei are stained blue. Magnification: (a) xlOO, (b) x60.
• Figure 7 Detection of HOXB5 in three patients with early stage prostate cancer where tumours are small and confined to the prostate, three patients with late stage prostate cancer and from three age-matched controls.
• Figure 8 Expression levels of various HOXA and HOXD genes in tumour tissue compared to normal adjacent tissue.
• DU145 and PC3 are cell lines derived from prostate tumours.
• Serum was obtained from six patients with advanced, hormone-refractory prostate cancer, six patients with early stage prostate cancer where tumours are small and confined to the prostate, and from six age-matched controls. This was de-salted and fractionated using a 12.5% PAGE/SDS gel.
• Human HOXC4 protein (UniProtKB / Swiss-Prot entry number P09017) was detected using western blotting with a rabbit anti-human HOXC4 antibody (Abeam, UK, catalogue number ab24338), together with a secondary anti-rabbit IgG antibody linked to alkaline phosphatase (Invitrogen, UK). Using this method, we detected high levels of HOXC4 protein in the serum of all the prostate cancer patients, but only very low levels in age-matched control serum (Fig 1).
• H0XC4 could be used as the basis of a diagnostic and / or prognostic test for prostate cancer by using a standard assay technique such as ELISA. This would involve the use of an anti-HOXC4 antibody linked to a reporter such as alkaline phosphatase in order to quantify the amount of H0XC4 protein in patient serum samples. Other methodologies could also be used, including western blotting. The amount of H0XC4 protein would provide an indication as to whether the disease is present and what stage it has reached.
• HOXB6 The detection of HOXB6 in these samples was done exactly as for HOXC4 except that the primaiy antibody was rabbit-anti human HOXB6 (from Abeam, cat no. ab26077).
• the relative values for HOXB6 protein were assessed by densitometry of the X-ray film (using the bands located at its predicted molecular weight). These values are the optical density of the band relative to that of the local background. Therefore, it will be appreciated that a negative comparative densitometry value is possible, as shown for the Age Matched Controls (AMCs), although it will be appreciated that this does not translate to a negative amount of H0XB6 protein.
• AMCs Age Matched Controls
• HOXB5 The detection of HOXB5 in these samples was done exactly as for HOXC4 and HOXB6 except that the primary antibody was rabbit-anti human HOXB5 (from Abeam, cat no. ab26079).
• the relative values for HOXB5 protein were assessed by densitometry of the X-ray film (using the bands located at its predicted molecular weight). These values are the optical density of the band relative to that of the local background.
• RNA Extraction Tumour and normal adjacent tissue RNA was purchased from Ambion, USA. RNA from cell cultures was extracted using the RNeasy Mini Kit (Qiagen, UK). cDNA Synthesis: RNA was first denatured by heating to 65 0 C for five minutes. l-5mg of RNA was incubated in a volume of 50ml at 37 0 C for one hour with final concentrations of 1OmM DTT, ImM dNTPmix, as well as lOOmg/ml polyT primers, 200 units of reverse transcriptase (Invitrogen, USA) and 40 units of RNASEout (Invitrogen, USA). The cDNA synthesis reaction was terminated by placing tubes at 65 0 C for five minutes.
• Real-Time PCR Semi-quantitative RT-PCR was performed using the Stratagene MX4000 Real Time PCR machine.
• the Stratagene MX4000 measures PCR product accumulation during the exponential phase of the reaction, prior to the amplification becoming vulnerable to limited reagents and cycling variability. Fluorescence increases in accordance with increasing levels of PCR product.
• the relative amount of each transcript was determined as a ratio with the amount of Beta-actin transcript in each sample.
• the primers used are listed in table 4 below.
• GIGAATGTGGSS EPSACSDHPIPGCSLKEEEKQHSQPQQQQLDPNNPAANWIHARSTR
• EN-2 is a candidate oncogene in human breast cancer. Oncogene 24, 6890-6901.

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• 2007
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  • 2007-12-19 PL PL07848631T patent/PL2115472T3/pl unknown
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  • 2007-12-19 DK DK07848631.3T patent/DK2115472T3/da active
  • 2007-12-19 ES ES07848631.3T patent/ES2445185T3/es active Active
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  • 2007-12-19 US US12/518,708 patent/US8460882B2/en active Active
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• 2009
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• 2014
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