WO2014108707A1

WO2014108707A1 - Identification of patients for breast cancer theraphy using t128 family members as markers

Info

Publication number: WO2014108707A1
Application number: PCT/GB2014/050090
Authority: WO
Inventors: Robert Charles Rees; Amanda Kathleen Miles; Stephen Yan Tat CHAN; Tarek Mohamed Ahmed ABDEL-FATAH
Original assignee: Nottingham Trent University
Current assignee: Nottingham Trent University
Priority date: 2013-01-14
Filing date: 2014-01-14
Publication date: 2014-07-17
Anticipated expiration: 2015-07-14
Also published as: GB201300563D0

Abstract

The invention provides a method of detecting or monitoring breast cancer in a subject, or identifying the prognosis of a subject with breast cancer, comprising determining the level of and/or position of expression of a T128 family member, in a sample of tissue from the subject. Assay kits and methods of treating patients are also provided.

Description

IDENTIFICATION OF PATIENTS FOR BREAST CANCER THERAPHY USING T128 FAMILY MEMBERS AS MARKERS

The invention relates to methods of detecting or monitoring breast cancer in a subject, or identifying the prognosis of a subject with breast cancer comprising determining the level and/or position of the expression of a T128 family, and optionally one or both of ER and HER2 in a sample.

In the UK in the past 25 years the incidence of breast cancer has grown by 50 % making breast cancer the United Kingdom's most common form of cancer. In 2008 almost 48,000 women were diagnosed with breast cancer and although the number of diagnosed cases has increased so has the 10-year survival rate for patients, with almost two thirds of women alive 10 years after diagnosis (CRUK website).

The promotion of cell growth and proliferation via growth signalling pathways contributes to the generation and progression of breast cancer (Hung MC, 2006, novel signalling pathways in breast cancer). These growth signalling pathways are promoted by a number of membrane-bound and intracellular receptors including oestrogen receptor, progesterone receptor, rearranged during transfection and human epidermal growth factor receptor 2 (HER2) and the expression and biological activities of these receptors may have a great impact on tumour initiation, progression, relapse and prevention or treatment (Shamabadi, 2011, target points in Trastuzumab resistance).

HER2 belongs to the family of epidermal growth factor receptors and is also known as Neu and Erb-2. Amplification or overexpression of HER2 occurs in approximately 20-30 % of human breast cancers (Slaman DJ, 1989, studies of the her-2/neu proto-oncogene). HER2 amplification is generally associated with poor prognosis and levels correlate strongly with carcinogenesis (ref 3-5 from review 1). Furthermore HER2 overexpression is found at metastatic sites as well as at the primary tumour site suggesting that anti-HER2 therapy may be effective for both localised and metastatic disease (ref review 1). HER2 does not have a natural ligand and this allows it to be distinguished from other family members and makes the molecule a suitable therapeutic candidate (ref 2 review 1). Trastuzumab was the first such candidate which was registered for use in patients with over-expressing HER2 breast cancer. Trastuzumab is a humanised monoclonal antibody targeting the extracellular domain of HER2 and it has been used both as a single agent (review 1 ref 7) as well as in combination with chemotherapy, endocrine therapy and other HER2 targeting agents producing encouraging results in patients with early stage HER2-positive breast cancer (ref review 2). However, not all HER2-overexpressing cancers respond clinically to Trastuzumab and in some cases resistance develops after the initial response. Trastuzumab is also associated with significant drug acquisition costs that should be examined within the context of all its associated benefits (review 2 ref 5). In addition, a number of Trastuzumab-related side effects have been described including infusion reactions, cardiac toxicity and pulmonary toxicity (review 2 ref 3). Given the costs, side effects and resistance to therapy associated with Trastuzumab treatment a niche has developed whereby patient stratification would be a valuable tool in determining those patients that would do well clinically following Trastuzumab therapy.

In the current application, the applicants used a well characterised series of patients with primary early stage breast cancer to investigate the expression of a novel candidate antigen identified in their laboratory designated T128. T128 was identified in the laboratory using a modified serological screening approach (SEREX). In brief, a normal testicular cDNA library was plated out and screened with pooled allogeneic sera from patients with prostate cancer. Following rapid amplification of the cDNA ends on the library cDNA insert a putative gene of 1050 nucleotides long was obtained. Upon three phase translation of this nucleotide sequence a protein of 349 amino acids was predicted. This protein sequence was then searched against entries submitted to the NCBI GenBank database. The search revealed 100 % sequence homology to a gene named PRPF38B, which is a pre-mRNA splicing factor. PRPF38B is a 546 amino acid, 64 kDa protein that has been implicated in RNA splicing and mRNA processing but there are no publications on this gene and its relationship to cancer. An isoform of the protein exists that generates a 21 kDa protein and we believe that T128 is a further isoform of PRPF38 generating a 43 kDa protein. In addition a further partial protein sequence belonging to sarcoma antigen NY-SAR-27 shares 41 % sequence similarity to T128 but this protein does not have a reported translational initiation site therefore the size of the protein remains uncharacterised.

WO 2005/108420 describes the association of T128 with prostate, kidney and gastrointestinal cancers. There was no suggestion at the time of the relationship of the marker with breast cancer. Here the applicant reports on data associated with potential clinical and economical benefits of screening ER-/HER2+ patients for T128 prior to administration of Trastuzumab. They demonstrate that screening of ER-/HER2+ breast cancer patients for T128 would enable clinicians to stratify more accurately a cohort of patients who would gain maximum clinical benefit from being treated with Trastuzumab, thus reducing the costs associated with this treatment while concurrently increasing the success rate of a well known and established breast cancer treatment.

The invention provides a method of detecting or monitoring breast cancer in a subject, or identifying the prognosis of a subject with breast cancer, comprising determining the level of and/or position of expression of a T128 family member.

There are a number of members of the T128 family showing high levels of homology or identity to T 128.

Figure 1 shows the nucleotide sequence and the amino acid sequence of T128 (SEQID Nos: 1 and 2). This shows 100% identity with the overlapping sequences of GenBank accession number BAA91546.1, P_060531.2 and CAH72070.1. The latter two proteins are pre- mRNA splicing factor 38B from Homo sapiens and yeast. It is also similar to NY-SAR-27 (Lee S.Y. et al PNAS (2003) 100(5) 2651-2656). These proteins are capable of being specifically bound by an antibody raised against the protein shown in Figure 1 and are therefore considered to be members of the T128 family.

Typically the family member assayed is T128.

The applicant has found that the protein detected by anti-T128 specific antibodies is differentially expressed in breast cancer tissue.

The T128 family member may be assayed for the presence or absence of the expressed protein product or mRNA transcript, or the concentration of the product of mRNA transcript. The concentration may be compared to predetermined normal levels for normal tissues to indicate whether the level is significantly different. T128 has been found to be significantly associated with breast cancer grade. In normal tissue the product is normally found in the nucleus of the cells, with some cytoplasmic staining, but no substantial membrane staining. In breast cancer the localisation of T 128 varied depending on the clinical features of the tumour. Membranous staining of the product was an independent prognostic factor of breast cancer.

The sample may also have the presence of or level of expression of estrogen receptor 2 (ER) and/or human epidermal growth factor receptor 2 (HER2) may also be measured. Whether the cell stains ER^" or HER2⁺ with anti-ER or anti-HER2 antibodies has been shown by the applicant to have an effect on the prognosis of the breast cancer. Significant correlation has been found between ER7HER2 positive breast cancers.

The presence or absence of, or levels of, the T128 family, HER2 or ER may be determined using techniques generally known in the art, such as Western blotting or immunological assays such as immunohistological staining or FACS staining.

Typically the sample is a sample of a breast tumour.

The sample may be fixed or freeze dried.

Cells in the sample may be stained, for example, in the form of a section of tissue on a slide. Flow cytometry, such as FACS, may be used to count stained cells.

Antibodies or fragments thereof raised against T128, ER and/or HER2 may be used to stain or detect the expression of the protein. Fab or F(ab')₂ antibodies may be used which still have specificity to the protein to be detected. The antibodies may be specific for T128 family proteins, typically specific for T128, or ER or HER2 as appropriate.

They may be labelled with levels well know in the art. These include FITC (Fluorescein isothiocyanate) optionally with a fluorescent tissue counterstain such as Pontamine Sky Blue. Other labels include fluorescein, rhodamine, phycocrythrin, AMCA, Oregan Green™ and CyDyes™. Enzyme labels include alkaline phosphatase (with naphthol phosphate as a substrate) and horse radish peroxidase (with 3,3'-diaminabenzidine substrate). β-D-galactosidase and glucose oxidase. Particulate labels such as colloidal gold or radioisotope labels may be used.

The antibody may be detected indirectly. For example, a second antibody raised against the immunoglobulin from the host species of the first antibody. In indirect labelling the primary unconjugated antibody from a first animal type, or fragment thereof, is allowed to bind to the antigen on the cell. A second tracer-conjugated antibody, raised in another animal type and specific for the animal and immunoglobulin class of the primary antibody, is applied to the cell and allowed to bind to the primary antibody. The complex which forms can then be visualised. Labels for the second tracer-conjugated antibody may be as defined above for direct visualisation techniques. That is, the second tracer-conjugated antibody may be attached to one or more of the labels described above. The advantage of the indirect technique is that it is more sensitive than the direct technique and is still relatively rapid and inexpensive. A further advantage of this method is that provided that the primary antibody host species remains the same, any number of tests can be performed using the common conjugated second antibody.

Antibodies may also be attached to, for example, avidin which is then used to attach labelled biotin to avidin to form an avidin/biotin complex.

The expression of mRNA may be determined using, for example, reverse transcription polymerase chain reaction and real time qualitative PCR.

The applicant has found that T128 membrane positive, ER^", HER2⁺ tumours are especially suitable to be treated with anti HER2 antibodies or fragments thereof. Trastuzumab (marketed under the trade name Herceptin), is an antibody which targets HER2 and may be used to treat cancer. The presence of T128 on the membrane of breast cancer cells significantly improved the ability of Trastuzumab to treat the cancer.

This allows patients to be better treated. If T128 as a membrane protein is not found, then it indicates that alternative treatments should be used. It also suggests that T128 membrane proteins could be targeted by anti-T128 family antibodies or fragments thereof as a therapeutic target, in a similar manner to Trastuzumab.

Assay kits comprising optionally labelled anti-T128 family antibodies or fragments thereof in combination with one or both of anti-HER2 and/or anti-ER antibodies or fragments thereof, are also provided.

Methods of treating breast cancer patients by determining if the patient is suitable to be treated with anti-HER2 antibodies by a method of the invention and then treating the patient with anti-HER2 antibodies or fragments are also provided.

The invention also provide anti-HER-2 antibodies or fragments thereof for use in the treatment of breast cancer in a patient who is T128 membrane positive, ER^" of HER2⁺.

The invention also provides a computer implemented method comprising receiving a signal indicating a level of and/or position of expression of a T128 family member in a sample of tissue from a subject, processing the signal and producing a read out showing the level of and/or position of expression of said T128 family member.

Assay devices are also provided comprising a computer readable memory adapted to be used in a method of the invention. This may be used in combination with a kit according to the invention or as described above.

The computer readable memory may control the assay device or comprise a predetermined threshold or calibration curve to indicate the presence, level or position of the T128 family member.

The invention will now be described by way of example only with reference to the following figures:

Figure 1. (A) PRPF38B-T128 encodes a putative protein of 349 amino acids and when this sequence was submitted for Blast alignment against the Homo sapiens database on the NCBI website, 100 % sequence homology was identified to the PRPF38B gene. (B) The nucleotide sequence of PRPF38B, its related predicted iso forms (that could potential cross- react with the antibody used) and PRPF38B-T128 were aligned against the genomic sequence from Homo sapiens chromosome 1, GRCh37.p9 Primary Assembly (gi_224589800) using the Spidey alignment tool.

Figure 2. PRPF38B mRNA expression levels in a panel of normal tissues, paired breast cancer and adjacent normal tissues and breast cancer cell lines. The quantification of PRPF38B mRNA expression was performed by qPCR. Each PCR run was performed in triplicate and replicates for each sample were included in each run. HPRT1 and TBP were used as housekeeping genes for normalising the ct values.

Figure 3. Expression of PRPF38B protein in breast cancer cell lines and breast tissues. (A) Immunofluorescent staining on 5 breast cancer cell lines using antibody to PRPF38B (green). Cell nuclei are stained with DAPI (blue). PRPF38B protein was expressed in a variety of subcellular locations in all the cell lines tested. Scale bars represent 60 μπι. (B) Immunoblotting of the 5 breast cancer cell lines using antibody against PRPF38B. β-actin was measured as a loading control. IB analysis reveals 3 distinct bands at 64 kDa, 43 kDa and 34 kDa corresponding to the full length of PRPF38B, the putative PRPF38B-T128 isoform and the predicted PRPF38B 34 kDa isoform respectively. These immunoblots are representative of three independent experiments. (C) Photomicrographs of PRPF38B immunohistochemistry. (i) In normal breast tissue PRPF38B demonstrated homogenously intense nuclear staining, (ii) In ductal carcinoma in situ PRPF38B was localised in the nucleus of peripheral cells and tumour epithelial cells had membranous staining, (iii) High grade invasive breast cancer demonstrated staining of PRPF38B in the cytoplasm and nucleus of cells with intense membranous staining also observed, (iv) Enlarged image of (iii) demonstrating intense membranous staining.

Figure 4. Membranous T128 expression was significantly associated with poor breast cancer specific survival (BCSS) and disease free survival (DFS) in patients in both the discovery data set [(A) p=0.003 and (B) p=0.003] and the test data set [(C) p=0.00002 and (D) p=0.004). Materials and Methodology

Cell Lines and Growth Conditions

The breast cancer cell lines used were: MDA468 (Dr Li Li, University of Nottingham, Nottingham, UK), MDA231 (Dr Anne Vessieres, Ecole Nationale Superieure de Chimie de Paris, Paris, France), MCF7 (HTB-22, ATCC), T47D (HTB-133, ATCC) and SkBr3 (HTB- 30, ATCC). MDA468, MDA231 and T47D cells were maintained in RPMI 1640 media (Lonza) supplemented with 10 % fetal calf serum (FCS) (Perbio) and 1 % (w/v) L-glutamine (Lonza). MCF7 cells were maintained in DMEM media (Lonza) supplemented with 10 % FCS and SkBr3 cells were maintained in Mc Coy's 5 A media (Lonza) supplemented with 10 % FCS. All cells were incubated in a humid chamber at 37°C with 5 % C0₂.

Semi-quantitative reverse transcription-polymerase chain reaction

The breast cancer cell lines were grown to 80 % confluency as described previously before the extraction of total RNA using RNA-STAT 60 reagent (AMS Biotechnology). Total RNA (2 μg) was reverse transcribed into cDNA using Moloney Murine Leukemia Virus reverse transcriptase (M-MLV) (Promega) and oligo-dT primers (Promega) following the manufacturer's protocol. Real-time qPCR was then carried out using the following primers specific for T128 (Forward 5 ' -GACGTTC AAGGTCTCC AAGG-3 ' ; Reverse 5'-TAGTCGCTGGCGTTCTTTCT-3') and the housekeeping genes HPRT1 (Forward 5 ' -TGAC ACTGGC AAAAC AATGC A-3 ' ;

Reverse 5'-GGTCCTTTTCACCAGCAAGCT-3') and TBP (Forward 5 ' -TGC AC AGGAGCC AAGAGTGAA-3 ' ;

Reverse 5'-CACATCACAGCTCCCCACCA-3'). All primers were supplied by Eurofins MWG Operon. Cycling was performed using a SYBR™ Green master mix (BioRad, UK) and a hot start procedure in a Rotagene real time PCR cycler (Qiagen) as follows: initial denaturation step at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at primer-specific Tm for 30 seconds and extension at 72°C for 10 seconds. Following each PCR the melt curves were examined prior to data analysis. 2ACT calculations were performed to calculate the expression of T128 relative to that of the averaged housekeeping genes.

Antibodies

For this study we used a monospecific T128 antibody (1 :500 for immunoblotting (IB); 1 :650 for immunofluorescence (IF), 1 : 1000 immunohistochemistry (IHC), Pacific Immunology), anti-human erbB2/HER-2 (1 :2000 for IF, 24D2, Biolegend), anti-human Neu (1 : 1000 For IB, H-200, Santa Cruz Biotechnology, Inc), anti-human c-erbB2 (1 : 100 for IHC, Dako), β-actin (1 :5000 for IB, A2066, Sigma). Alexa Fluor 488- and Alexa Fluor 568-conjugated antibodies were used as secondary antibodies for IF (1 : 1500, Invitrogen). Horse Radish peroxidase (HRP) -conjugated antibodies were used as secondary antibodies for IB (1 : 1000, Dako). Need Tareks secondary antibody.

Antibody validation on the monospecific T128 antibody was validated by preabsorbtion of the antibody with its respective blocking peptide. Specific T128 staining (IHC) and bands (IB) were completely abrogated by preabsorbtion with the relevant peptide but not with an irrelevant peptide (data not shown).

Immunobloting (IB)

For IB, breast cancer cells were harvested, washed with lxPBS and total cell protein was extracted using a modified RIPA buffer (150 nM MaCl, 50 mM Tris-base pH 8.0, 5 mM EDTA, 1 % v/v IGEPAL, 0.5 % w/v sodium deoxycholate, 0.1 % w/v SDS, 1 mM benzamidine, 0.1 mM PMSF, 1 mM sodium ortho-vanadate, 1 mM sodium azide) followed by a 30 minute incubation on ice and centrifugation at 12000 rpm for 30 minutes at 4°C to pellet insoluble protein. Supernatant was then collected, aliquoted and stored at -20°C. Protein concentration was determined using a DC Protein Assay Kit (Biorad) following the manufacturer's protocol and using BSA diluted from 0 to 1 μg/ml to determine the standard curve. For IB analysis, 30 μg of total protein was subjected to Tris/glycine SDS-polyacrylamide gel electrophoresis. Proteins were separated alongside a molecular weight marker (Geneflow). Protein bands were transferred onto Amersham Hybond-P PVDF membranes (GE Healthcare). Membranes were blocked for 1 hour with 10 % marvel milk/tris buffered saline (TBS) solution with 0.05 % Tween-20 (TBST). Following blocking, membranes were incubated with primary antibody (in blocking solution) at 4°C overnight followed by washing and incubation with secondary antibodies for one hour at room temperature. The IB's were washed ahead of visualisation with Rapid Step ECL reagent (Calbiochem) and viewed using a CCD camera (Fujifilm).

Immunofluorescence

Breast cancer cells were seeded at 10000 cells/well in 8-well chamber slides and allowed to adhere down for 48 hours prior to the start of assay. The cells were then treated as follows: fixed in 4 % (w/v) paraformaldehyde followed by washing three times in lx phosphate-buffered saline (PBS) for 10 minutes each, blocked and permeabilised in 10 % (w/v) bovine serum albumin (BSA) in 0.1 % (v/v) PBS-Tween, one hour incubation at room temperature with primary antibody (in blocking solution), washed 3 times in lx PBS for 10 minutes each, one hour incubation at room temperature with Alexa fluor-conjugated secondary antibody (in blocking solution) and three lx PBS washes for 10 minutes. Breast cancer cells were counterstained and mounted with DAPI fluorescent medium (Vector Laboratories) for visualisation using IF microscopy.

Tritiated thymidine proliferation assays

Cells were plated out at 5x10³ cells per well in 24-well plates (Sarstedt) and each treatment was performed in quadruplicate. Assessment of the effect of herceptin treatment on the proliferation of breast cancer cells was assessed 24 hours and 72 hours after addition of herceptin (n=3 independent experiments). After plating out the cells were rested overnight to allow them to re-adhere down. The following morning herceptin was added to quadruplicate wells at the following concentrations: 0, 0,2, 0.4, 0.6, 0.8, 1.0 and 10 ug/ml. The cells were then left for 8 hours before removal of media and addition of complete media containing both the test concentration of herceptin and tritiated thymidine. The cells were then incubated for 16 hours to allow absorption of the isotope. On the day of assay, media was removed from the cells and they were washed with PBS before the addition of trypsin. Following 30 minutes incubation the cells were harvested onto a Unifilter 96-well plate (Sarstedt) and allowed to dry at room temperature. Each test well was treated with 40 μΐ of Microscint-0 media (Perkin-Elmer) before the plate was read using a Top-Count NXT microplate scintillation counter (Packard). Results

T128 identification and characterisation

In the preliminary mRNA screen of T128 we were able to detect significantly elevated levels of T128 in normal breast and prostate tissues but lower levels of T128 mRNA were detected in the other normal tissues tested (kidney, adrenal, colon, liver, PBMC, tonsil, small intestine, muscle, lung, kidney, brain, trachea, heart and testis). In a more comprehensive study of breast tissues we found that T128 had significantly higher levels of expression in the breast cancer tissue compared to the normal adjacent tissue (p<0.0103). T128 mRNA transcripts could also be detected in the 5 well characterised breast cancer cell lines routinely used in our lab (MDA468, MDA231, MCF7, T47D and SkBr3) [Figure ] Western blotting of T128 protein in 5 well characterised breast cancer cell lines revealed bands of varying intensity [Figure ]. In two of the breast cancer cell lines (MDA231 and MCF7) an extremely faint band corresponding to the molecular mass (64 kDa) of PRPF38B was detected, however all of the breast cancer cell lines tested had T128 protein present (43 kDa) in variable amounts ranging from the highest level of expression detected in MDA468>MDA231>MCF7>T47D>SkBr3. In addition another potential splice variant of the T128/PRPF38B protein was detected at a molecular weight of approximately 38 kDa and specificity of this band was confirmed by peptide blocking experiments (data not shown).

Immuno fluorescent staining of T128 and HER2 in the breast cancer cell lines revealed heterogeneous T128 expression across the five cell lines. HER2 was detected in four out of the five cell lines (SkBr3, T47D, MCF7 and T47D) with significant over-expression only detectable in the SkBr3 cell line which is in line with the current literature. The only cell line that was truly negative for HER2 protein expression as demonstrated by both IF and FACS was the MDA468 cell line (include FACS as supplementary data) More over the dual IF staining of T128 with HER2 strongly suggested that a small population of cells in four of the breast cancer cell lines (MDA468, MCF7, T47D and SkBr3) expressed T128 on the cell surface and this was confirmed by FACS analysis performed on the un-permeabilised breast cancer cell lines followed by probing with T128 antibody (supplementary data to be included) (need to include % positive cells as well) Immunofluorescent staining of T128 in the breast cancer cell lines revealed heterogeneous T128 expression across the five cell lines. More over the IF staining of T128 strongly suggested that a small population of cells in four of the breast cancer cell lines (MDA468, MCF7, T47D and SkBr3) expressed T128 on the cell surface and this was confirmed by FACS analysis performed on un-permeabilised breast cancer cell lines followed by probing with T128 antibody (supplementary data to be included) (need to include % positive cells as well)

T128 cellular localisation as validated by IHC

The cellular localisation of T128 varies depending on the tissue origin (normal or cancerous) and also on the clinico-pathological features of the tumour. In the normal tissue TMA screened T128 was predominately located within the nucleus of cells with some cytoplasmic staining but no membranous staining observed. In ductal carcinoma in situ nuclear staining of T128 was observed in the peripheral cells, however tumour epithelial cells had membranous staining. In breast cancer the localisation of T 128 varied depending on the clinical features of the tumour.

Clinico-pathological significance of T128 expression

A total of 1215 tumours were suitable for analysis of T128 expression. When these tumours were subdivided into non-membranous (nuclear and cytoplasmic) and membranous T128 staining and scored accordingly, 11 % of the total cohort demonstrated membranous expression of T128. Associations between T128 membranous/non-membranous expression and a range of standard clinico-pathological parameters were tested, but only those showing statistical significance are shown in Table 1. T128 expression was significantly associated with grade, p53 and Bcl2 but more interestingly the membranous expression of T128 was significantly associated with ER-negative (p=5.9xl0^"19) and HER2-positive (p=4.9xl0^"57) status. Furthermore there was a significant association between the membranous expression of T 128 in a more selective ER-negative/HER2-positive patient cohort (p=1.2xl0^"50).

In the Cox-multivariate regression analysis, membranous expression of T128 was an independent prognostic factor for breast cancer [RR (95 % CI); 1.48 (1.1-1.9)] and was better than tumour size [RR (95 % CI); 1.30 (1.2-1.4)] and grade [RR (95 % (CI); 0.59 (0.4-0.9)] (Table 3).

Membranous T128 expression and patient outcome

Kaplan-Meier survival analysis was used to determine disease free survival with respect to the cellular localisation of T128. When the patients were further subdivided according to their ER hormone receptor status, the cellular localisation of T 128 again varied and was predictive of disease free survival. In ER-positive patients, T128 expression was predominantly nuclear and did not associate with disease free survival (p=0.285). However, in the ER-negative cohort of patients that were treated with CMF between 1989-1998, membranous expression of T128 was associated with poor prognosis (p=0.008). This was also true for the ER-negative cohort of patients that were treated post 2000 with anthracycline (p=0.00005).

T128 expression and patient outcome in ER-negative/HER2-positive patients treated with Trastuzumab

Due to the significant correlation found between T128 membranous expression with ER- negative cases and HER2-positive cases, these associations were investigated by further stratifying the patients based on the routine treatment regime that these patients would classically receive, namely Trastuzumab therapy. Kaplan-Meier survival analysis was used to determine disease free survival (DFS) with respect to cellular expression of T128 (membranous vs. non-membranous) and Trastuzumab therapy (treatment vs. no treatment). In those patients that were ER-negative/HER2-positive and did not receive Trastuzumab therapy (patients treated prior to 2006), membranous T128 expression was associated with poor prognosis (p=0.01). In the ER-negative/HER2-positive cohort of patients that did receive Trastuzumab therapy it was the cellular localisation of T128 that was a significant factor in the DFS of these patients; the non-membranous expression of T128 had no significant impact on DFS (p=0.56729) regardless of whether or not the patients received Trastuzumab therapy. However, those patients that had membranous expression of T 128 and received Trastuzumab therapy had a significantly better DFS (p=0.00058) compared to patients that did not receive Trastuzumab therapy. Table 1: Significant associations between T128 expression and other clinico-pathological variables

Table 2: Multivariate analysis demonstrating significant predictive associations between membranous T128 expression and other clinically significant markers

Table 3: Cox-multivariate regression analysis of the association between T128 expression with other cancer relevant markers

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Glu Arg Arg Arg Ser Arg Ser Arg Glu Arg His Arg Ser Arg Ser Arg

130 135 140

Ser Arg Asp Arg Lys Gly Asp Arg Arg Asp Arg Asp Arg Glu Arg Glu 145 150 155 160

Lys Glu Asn Glu Arg Gly Arg Arg Arg Asp Arg Asp Tyr Asp Lys Glu 165 170 175

Arg Gly Asn Glu Arg Glu Lys Glu Arg Glu Arg Ser Arg Glu Arg Ser

180 185 190

Lys Glu Gin Arg Ser Arg Gly Glu Val Glu Glu Lys Lys His Lys Glu

195 200 205

Asp Lys Asp Asp Arg Arg His Arg Asp Asp Lys Arg Asp Ser Lys Lys

210 215 220

Glu Lys Lys His Ser Arg Ser Arg Ser Arg Glu Arg Lys His Arg Ser 225 230 235 240

Arg Ser Arg Ser Arg Asn Ala Gly Lys Arg Ser Arg Ser Arg Ser Lys

245 250 255

Glu Lys Ser Ser Lys His Lys Asn Glu Ser Lys Glu Lys Ser Asn Lys

260 265 270

Arg Ser Gly Ser Gin Gly Arg Thr Asp Ser Val Glu Lys Ser Lys Lys

275 280 285

Arg Glu His Ser Pro Ser Lys Glu Lys Ser Arg Lys Arg Ser Arg Ser

290 295 300

Lys Glu Arg Ser His Lys Arg Asp His Ser Asp Ser Lys Asp Gin Ser 305 310 315 320

Asp Lys His Asp Arg Arg Arg Ser Gin Ser lie Glu Gin Glu Ser Gin

325 330 335

Lys Gin His Lys Asn Lys Asp Glu Thr Val

340 345 <211> 20

<212> DNA

<213> Homo sapiens

<400> 3

gacgttcaag gtctccaagg 20

<210> 4

<211> 20

<212> DNA

<213> Homo sapiens

<400> 4

tagtcgctgg cgttctttct

<210> 5

<211> 21

<212> DNA

<213> Homo sapiens

<400> 5

tgacactggc aaaacaatgc

<210> 6

<211> 21

<212> DNA

<213> Homo sapiens

<400> 6

ggtccttttc accagcaagc

<210> 7

<211> 21

<212> DNA

<213> Homo sapiens

<400> 7

tgcacaggag ccaagagtga a 21 <211> 20

<212> DNA

<213> Homo sapi ens

<400> 8

cacatcacag ctccccacca 20

Claims

1. Method of detecting or monitoring breast cancer in a subject, or identifying the prognosis of a subject with breast cancer, comprising determining the level of and/or position of expression of a T128 family member, in a sample of tissue from the subject.

2. A method according to claim 1, wherein the T128 family member is selected from T128, PRPF38B or NY-SAR-27.

3. A method according to claims 1 or 2, wherein the level of membrane-associated expression of the T128 family member is determined.

4. A method according to claims 1 to 3, wherein the sample is further tested to identify whether the sample is estogen receptor negative (ER^") and/or human epidermal growth factor 2 positive (HER2⁺).

5. A method according to claim 4, further comprising the step of determining whether the subject is suitable to be treated with an anti-HER2 antibody or fragment thereof.

6. A method according to claims 1 to 5, wherein the sample is a tumour sample.

7. A method according to claims 1 to 6, wherein the expression of the T128 family member and optionally ER and/or HER2, is determined by Western blotting or immunostaining.

8. An assay kit for use in a method according to any preceding claims, comprising an anti-T128 family specific antibody or a fragment thereof and one or both of an anti-ER antibody and/or anti-HER2 antibody, or fragments thereof.

9. A method according to claim 5, wherein if the sample is T128 membrane positive, ER^", HER2⁺, the patient is treated with anti-HER2 antibodies or fragments thereof.

10. A method of treating breast cancer, comprising the use of an anti-T128 antibody to screen the subject to identify an optimum treatment regime for that subject.

11. A method of treating a patient with breast cancer comprising determining identifying that a patient is suitable to be treated with an anti-HER2 antibody by a method according to claims 1 to 7 and treating the patient with an anti-HER2 antibody or fragment thereof.

12. An anti-HER2 antibody or fragment thereof for use in the treatment of breast cancer in a patient who is T128 membrane positive, ER^", HER2⁺.

13. A computer implemented method comprising receiving a signal indicating a level of and/or position of expression of a T128 family member is a sample of tissue from a subject, processing the signal and producing a read out showing the level of and/or position of expression of said T128 family member.

14. An assay device comprising a computer readable memory adapted to be used in a method or assay but according to claims 1 to 9 or 13.