Expression of both estrogen receptor-beta 1 (ER-ß1) and its co-regulator steroid receptor RNA activator protein (SRAP) are predictive for benefit from tamoxifen therapy in patients with estrogen receptor-alpha (ER-α)-negative early breast cancer (EBC).

BACKGROUND: Roles of Estrogen Receptor-beta 1 (ER-ß1) and its co-regulator Steroid Receptor RNA Activator Protein (SRAP) in breast cancer remain unclear. Previously, ER-ß1 and SRAP expression were found positively correlated in breast cancer and, therefore, expression of these two molecules could characterize cancers with a distinct clinical outcome. PATIENTS AND METHODS: ER-ß1 and SRAP expression was determined by immunohistochemistry (IHC) in tissue microarrays from a randomized, placebo-controlled trial (NCIC-CTG-MA12), designed to determine the benefit of tamoxifen following chemotherapy in premenopausal early breast cancer (EBC). Expression was dichotomized into low and high using median IHC scores. Relationships with survival used Cox modeling. RESULTS: In the whole cohort, ER-ß1 and SRAP were not prognostic. However, high ER-ß1 and SRAP significantly predicted tamoxifen responsiveness [overall survival, interaction test, P = 0.03; relapse-free survival (RFS), interaction test, P = 0.01]. Stratification by ER-α-status found predictive benefit only in ER-α-negative cases. The difference in RFS between tamoxifen and placebo was greater in patients whose tumors expressed both high SRAP and ER-ß1[hazard ratio = 0.07; 95% confidence interval (CI) 0.01-0.41; P = 0.003] versus those with low SRAP or ER-ß1 (interaction test, P = 0.02). The interaction test was not significant in ER-α-positive cohorts. CONCLUSIONS: This study provides evidence that both ER-ß1 and SRAP could be predictive biomarkers of tamoxifen benefit in ER-α-negative premenopausal EBC.

Expression of small breast epithelial mucin (SBEM) protein in tissue microarrays (TMAs) of primary invasive breast cancers.

AIMS: Small breast epithelial mucin (SBEM) is a recently described gene product that shows promise as a new breast biomarker. The aim was to investigate for the first time SBEM protein expression in a large cohort (n = 300) of invasive breast cancers, its relationship to established clinical variables and its association with clinical outcome. METHODS AND RESULTS: Immunohistochemical analysis was performed on tissue microarrays consisting of 149 oestrogen receptor (ER) alpha- and 151 ERalpha+ breast cancers. Overall, 18% of tumours were SBEM+ (n = 53/300). However, SBEM protein was more frequently observed in ER- (22%) than in ER+ cancers (13%; P = 0.049). A significant association with psoriasin/S100A7 expression (P < or = 0.0001) was observed in the entire cohort. SBEM was also positively associated with HER-2 (P = 0.046) in ER- cancers, and increased levels of SBEM were strongly associated with higher tumour grade (P = 0.0015). Furthermore, SBEM expression showed a trend towards an association with reduced overall survival and relapse-free survival in the ER+ cohort (P = 0.063 and P = 0.072, respectively). CONCLUSIONS: Our results suggest that SBEM may identify a unique subset of breast cancers with poor prognosis and may have future implications for therapeutic management of this disease.

Expression of oestrogen receptor-beta in oestrogen receptor-alpha negative human breast tumours.

To analyse the phenotype of breast tumours that express oestrogen receptor-beta (ERbeta) alone tissue microarrays were used to investigate if ERbeta isoforms are associated with specific prognostic markers and gene expression phenotypes in ERalpha-negative tumours. ERalpha-negative tumours were positive for ERbeta1 in 58% of cases (n=122/210), total ERbeta in 60% (n=115/192) and ERbeta2/cx in 57% of cases (n=114/199). Oestrogen receptor-beta1 and total ERbeta were significantly correlated with Ki67 (r=0.28, P<0.0001, n=209; r=0.29, P<0.0001, n=191) and with CK5/6, a marker of the basal phenotype (r=0.20, P=0.0106, n=170; r=0.18, P=0.0223, n=158). ERbeta2/cx was strongly associated with p-c-Jun and NF-kappaBp65 (r=0.53, P<0.0001, n=93; r=0.35, P<0.0001, n=176). This study shows that a range of ERbeta isoform expression occurs in ERalpha-negative breast tumours. While expression of ERbeta1, total and ERbeta2/cx are correlated, individual forms show associations with certain phenotypes that suggest different roles in subsets of ERalpha-negative cancers. Based on our in vivo observations, ERbeta may have the potential to become a therapeutic target in the specific subcohort of ERalpha-negative breast cancers.

Inducible upregulation of oestrogen receptor-beta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells.

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.

The steroid receptor RNA activator is the first functional RNA encoding a protein.

The steroid receptor RNA activator (SRA) has previously been characterized as belonging to the growing family of functional non-coding RNAs. However, we recently reported the Western blot detection of a putative endogenous SRA protein (SRAP) in breast cancer cells. Herein, we successfully suppressed the expression of this protein through specific RNA interference assay, unequivocally confirming its existence. Moreover, using database searches and Western blot analysis, we also showed that SRAP is highly conserved among chordata. Overall, our results suggest that SRA is the first example of a new class of functional RNAs also able to encode a protein.

Human small breast epithelial mucin: the promise of a new breast tumor biomarker.

Breast cancer remains one of the most frequently diagnosed cancers today. In developed countries, one in eight women is expected to present with breast cancer within her lifetime and an estimated 1,000,000 cases are detected each year worldwide (Canadian Cancer Statistics, http://www.cancer.ca/vgn/images/ portal/cit_86751114/14/33/1959864 11niw_stats2004_en.pdf). For women with recurrent disease, the median time of survival is about 2 years. Despite optimal surgery, adjuvant irradiation, hormonal treatment, and chemotherapy, approximately 30% of patients with localized breast cancer finally develop distant metastases. Early detection, which enables intervention at a localized and potentially curable stage, remains a central goal in breast cancer treatment. Indeed, the 5-year survival rate for women with breast cancer has been shown to increase dramatically when the disease is diagnosed at an early stage: from less than 25% in women with disseminated cancer to about 75% in patients with regional disease and over 95% in women with a localized tumor (Breast Cancer Facts and Figures, 2001-2002, http://www.cancer.org/downloads/STT/BrCaFF 2001.pdf). Unfortunately, only 60% of all breast cancers are diagnosed at a local stage. Any improvement in early detection through identification of tumor biomarkers would have a significant impact on reducing overall breast cancer mortality.

Putative functional characteristics of human estrogen receptor-beta isoforms.

Estrogen receptors (ERalpha and ERbeta) are clearly multifaceted in terms of structure and function. Several relatively abundant ERbeta isoforms have been identified, which can be differentially expressed in various tIssues. In order to provide insight into the possible role of the ERbeta family in breast tIssue a study of the putative functions of the human (h) ERbeta1, hERbeta2 and hERbeta5 isoforms was undertaken. Only hERbeta1 was found to bind ligand, which induced conformational changes as determined by protease digestion assays. All ERbeta isoforms could bind to and bend DNA although the relative efficiency with which they bound DNA differed with hERalpha>hERbeta1>hERbeta2>>hERbeta5. All ERbeta isoforms inhibited ERalpha transcriptional activity on an estrogen-response element (ERE)-reporter gene. The relative activities were hERbeta1>hERbeta2>hERbeta5; however, only hERbeta1 had transcriptional activity of its own. Both LY117018-hERalpha and LY117018-hERbeta1 complexes alone could activate transcription on a TGF-beta3-CAT gene. Although hERbeta2 and hERbeta5 had no activity alone, they inhibited ERalpha but not hERbeta1 transcriptional activity of transforming growth factor (TGF)-beta3-CAT. In marked contrast to activity on an ERE-CAT reporter gene, hERbeta1 did not modulate ERalpha transcriptional activity on a TGF-beta3-CAT reporter gene. These data support promoter-specific differential activities of hERbeta isoforms with respect to models of ERalpha regulated gene expression, and suggest that they may have a role in differentially modulating estrogen action.

Relationship of coregulator and oestrogen receptor isoform expression to de novo tamoxifen resistance in human breast cancer.

This study addresses the hypothesis that altered expression of oestrogen receptor-beta and/or altered relative expression of coactivators and corepressors of oestrogen receptors are associated with and may be mechanisms of de novo tamoxifen resistance in oestrogen receptor positive breast cancer. All cases were oestrogen receptor +, node negative, primary breast tumours from patients who later had no disease progression (tamoxifen sensitive) or whose disease progressed while on tamoxifen (tamoxifen resistant). Using an antibody to oestrogen receptor-beta that detects multiple forms of this protein (total) but not an antibody that detects only full-length oestrogen receptor-beta 1, it was found that high total oestrogen receptor beta protein expressors were more frequently observed in tamoxifen sensitive tumours than resistant tumours (Fisher's exact test, P=0.046). However, no significant differences in the relative expression of oestrogen receptor beta2, oestrogen receptor beta5 and full-length oestrogen receptor beta1 RNA in the tamoxifen sensitive and resistant groups were found. Also, when the relative expression of two known coactivators, steroid receptor RNA activator and amplified in breast cancer 1 RNA to the known corepressor, repressor of oestrogen receptor activity RNA, was examined, no significant differences between the tamoxifen sensitive and resistant groups were found. Altogether, there is little evidence for altered coregulators expression in breast tumours that are de novo tamoxifen resistant. However, our data provide preliminary evidence that the expression of oestrogen receptor beta protein isoforms may differ in primary tumours of breast cancer patients who prove to have differential sensitivity to tamoxifen therapy.

Altered expression of estrogen receptor coregulators during human breast tumorigenesis.

The hypothesis that altered expression of specific coactivators/repressors of the estrogen receptor occurs during human breast tumorigenesis in vivo is examined in this study. Using in situ hybridization and reverse transcription-PCR assays, the expression of two coactivators (SRA and AIB1) and one repressor (REA) of the estrogen receptor was compared between matched breast tumors and adjacent normal human breast tissue. The levels of SRA and AIB1 mRNA were increased in tumors compared with normal tissues (n = 19; Wilcoxon matched pairs test; P < 0.01). In contrast, the expression of REA mRNA was not different between tumors and normal tissues (n = 19; Wilcoxon; P = 0.110). The ratios of AIB1:REA and SRA:REA were higher (Wilcoxon; P < 0.05) in tumors compared with normal tissues. Furthermore, SRA:AIB1 was higher (Wilcoxon; P = 0.0058) in tumors compared with normal tissues. Although our study is small, these data are consistent with the above hypothesis and suggest that such alterations may have a role in the altered estrogen action occurring during breast tumorigenesis.

Functional characteristics of a novel murine estrogen receptor-beta isoform, estrogen receptor-beta 2.

We have isolated a highly expressed splice variant mRNA of murine estrogen receptor-beta (ERbeta), mERbeta2, containing an in-frame 54 nucleotide insertion between exons 5 and 6 of wild-type mERbeta1. The predicted ERbeta2 protein contains 18 amino acids inserted in the ligand binding domain of mERbeta1. Recombinant protein generated by in vitro transcription/translation showed that mERbeta2 had markedly reduced ligand binding (K(D)=17.7+/-4.7 nM, mean+/-s.e.m., n=3) compared with mERbeta1-bound (3)H-estradiol (K(D)=0.56+/- 0.19 nM, mean+/-s.e.m., n=3). Both receptors bound similarly to palindromic estrogen responsive elements (EREs) in vitro and in vivo, and similarly bent DNA. Transcriptional activity was assessed using transient transfection analysis into a homologous murine cell line, NIH 3T3 cells. mERbeta1 transactivated ERE-tk-CAT reporter genes similarly to mERalpha, whereas mERbeta2 had little activity except at high ligand concentrations. However, under conditions in which mERbeta2 is unlikely to be ligand saturated, co-transfected mERbeta2 inhibited activity of mERalpha and possibly mERbeta1 on ERE-tk-CAT genes. Using a 'novel raloxifene responsive' gene reporter system (TGF-beta3-CAT), we found the ability of estradiol and LY117018 to activate both mERalpha and mERbeta1 on this promoter was identical, and mERbeta2 activity in the presence of either estradiol or LY117018 was only slightly less than that observed with either mERbeta1 or mERalpha. Both mERbeta1 and mERbeta2 when liganded with LY117018 inhibited transcription at a classical ERE-regulated promoter under these transfection conditions, which was in marked contrast to their stimulatory effect at the transforming growth factor-beta3 promoter. These data suggest that responsiveness of gene expression to a relatively highly expressed variant murine ERbeta isoform, mERbeta2, is both ligand and promoter specific. Determination of the relative level of expression of mERbeta1 mRNA and mERbeta2 mRNA in mouse tissues indicated predominance of mERbeta2 mRNA in some but not all tissues. These data suggest that the mERbeta2 may have some tissue-specific and promoter-specific modulatory effects.

Lumican and decorin are differentially expressed in human breast carcinoma.

Previous studies have shown that lumican is expressed and increased in the stroma of breast tumours. Lumican expression has now been examined relative to other members of the small leucine-rich proteoglycan gene family in normal and neoplastic breast tissues, to begin to determine its role in breast tumour progression. Western blot study showed that lumican protein is highly abundant relative to decorin, while biglycan and fibromodulin are only detected occasionally in breast tissues (n=15 cases). Further analysis of lumican and decorin expression performed in matched normal and tumour tissues by in situ hybridization showed that both mRNAs were expressed by similar fibroblast-like cells adjacent to epithelium. However, lumican mRNA expression was significantly increased in tumours (n=34, p<0.0001), while decorin mRNA was decreased (p=0.0002) in neoplastic relative to adjacent normal stroma. This was accompanied by a significant increase in lumican protein (n=12, p=0.0122), but not decorin. Further evidence of altered lumican expression in breast cancer was manifested by discordance between lumican mRNA and protein localization in some regions of tumours but not in adjacent morphologically normal tissues. It is concluded that lumican is the most abundant of these proteoglycans in breast tumours and that lumican and decorin are inversely regulated in association with breast tumourigenesis.

Expression of a repressor of estrogen receptor activity in human breast tumors: relationship to some known prognostic markers.

The expression of a specific repressor of estrogen receptor activity (REA) was investigated by a semiquantitative reverse transcription-PCR assay in 40 human breast tumor biopsy samples with respect to steroid hormone receptor status and other known prognostic variables. The data showed that REA expression was positively correlated with estrogen receptor (ER) levels as defined by ligand-binding assays (Spearman r = 03231; P = 0.042) and that the median level of REA mRNA was significantly (Mann-Whitney two-tailed test, P = 0.0424) higher in ER+ tumors (median = 94.5; n = 30) compared with ER- tumors (median = 645; n = 10), with no significant differences (P = 0.4988) associated with progesterone receptor status alone. In addition, REA expression was inversely correlated with tumor grade (Spearman r = -0.4375; P = 0.0054). When the tumors were divided into two groups based on grade, REA expression was significantly (Mann-Whitney two-tailed test, P = 0.0024) higher in low-grade (median = 97; n = 16) compared with high-grade (median = 76; n = 23) tumors. These results provide preliminary data suggesting that the expression of REA varies among breast tumors and is correlated with known treatment response markers and inversely correlated with a marker of breast cancer progression. REA together with ER status may be an improved marker of endocrine therapy responsiveness in human breast cancer.

Altered expression of estrogen receptor-alpha variant messenger RNAs between adjacent normal breast and breast tumor tissues.

Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-alpha messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-alpha (ERC-4) messenger RNA and a lower exon 3 deleted er-alpha variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-alpha variant (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-alpha variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast carcinogenesis.

Psoriasin (S100A7) expression and invasive breast cancer.

Alteration of psoriasin (S100A7) expression has previously been identified in association with the transition from preinvasive to invasive breast cancer. In this study we have examined persistence of psoriasin mRNA and protein expression in relation to prognostic factors in a cohort of 57 invasive breast tumors, comprising 34 invasive ductal carcinomas and 23 other invasive tumor types (lobular, mucinous, medullary, tubular). We first developed an IgY polyclonal chicken antibody and confirmed specificity for psoriasin by Western blot in transfected cells and tumors. The protein was localized by immunohistochemistry predominantly to epithelial cells, with both nuclear and cytoplasmic staining, as well as occasional stromal cells in psoriatic skin and breast tumors; however, in situ hybridization showed that psoriasin mRNA expression was restricted to epithelial cells. In breast tumors, higher levels of psoriasin measured by reverse transcriptase-polymerase chain reaction and Western blot (93% concordance) were significantly associated with estrogen and progesterone receptor-negative status (P < 0.0001, P = 0.0003), and with nodal metastasis in invasive ductal tumors (P = 0. 035), but not with tumor type or grade. Psoriasin expression also correlated with inflammatory infiltrates (all tumors excluding medullary, P = 0.0022). These results suggest that psoriasin may be a marker of aggressive behavior in invasive tumors and are consistent with a function as a chemotactic factor.

Variant estrogen receptor-alpha messenger RNA expression in hormone-independent human breast cancer cells.

T5-PRF cells are insensitive to the growth-stimulatory effects of estrogen while still retaining expression of estrogen receptor-alpha (ER-alpha). In the apparent absence of ligand, T5-PRF cells have a 3. 6+/-0.5 (s.e.m.)-fold increased basal ER-alpha activity and elevated basal progesterone receptor levels compared with the parent, estrogen-sensitive, T5 cells. Long-range ER-alpha reverse transcription-PCR was performed to characterize variant ER-alpha mRNA expression in the two cell lines. An increased relative expression of an exon 3/4-deleted ER-alpha mRNA variant was found in T5-PRF. Recombinant expression of this ER-alpha variant resulted in significantly increased estrogen responsiveness, as well as a trend to increased basal ligand-independent activity when expressed with wild-type ER-alpha in ER-negative cell lines, as well as significantly increasing both ligand-independent and estrogen-induced ER-alpha transcriptional activity when expressed in parental T5 cells. These results suggest a role for altered variant ER-alpha in ligand-independent activation of ER-alpha which may contribute to hormone independence in breast tumors.

The human orphan receptor PXR messenger RNA is expressed in both normal and neoplastic breast tissue.

The expression of PXR mRNA and a variant PXR mRNA, deleted in 111 nucleotides in the ligand-binding domain, was detected by reverse transcription-PCR amplification in both normal and neoplastic human breast tissues. The level of PXR mRNA did not differ between breast tumors and their adjacent matched normal breast tissues. However, the expression of PXR mRNA did vary among breast tumors. A statistically significant inverse relationship was found between the level of PXR mRNA expression and estrogen receptor (ER) status, as defined by ligand binding analysis. The level of PXR mRNA expression in ER+ tumors (median = 22.4, n = 15) was significantly lower (P = 0.04) than the level of PXR mRNA expression in ER-tumors (median = 46.7, n = 15). No relationship with progesterone receptor status was found. These data raise the possibility that PXR has a role in human breast tissues.

Expression of the steroid receptor RNA activator in human breast tumors.

The expression of the recently described steroid receptor RNA activator (SRA) was measured by semiquantitative reverse transcription-PCR within 27 independent breast tumors, spanning a wide spectrum of grade and estrogen receptor (ER) and progesterone receptor (PR) levels. Subgroup analysis showed that SRA expression was similar in ER+/PR+ (median = 65.5, n = 8) and in ER-/PR- (median = 94.6, n = 5) tumors. Interestingly, SRA expression in these two subgroups was significantly (Mann-Whitney rank-sum test, P < 0.05) lower than that observed in ER+/PR- (median = 156.4, n = 6) and ER-/PR+ (median = 144.8, n = 8) tumors. A variant form of SRA, presenting a deletion of 203 bp within the SRA core sequence, was also observed in breast tumor tissues. The relative expression of this new SRA isoform correlated with tumor grade (Spearman coefficient r = 0.53, n = 27, P = 0.004). These data suggest that changes in the expression of SRA-related molecules occur during breast tumor progression.

Mammaglobin, a potential marker of breast cancer nodal metastasis.

The Mammaglobin gene, a breast-specific member of the uteroglobin gene family, has been previously identified as being overexpressed in some breast tumours, but the cellular origin and relationship to tumour progression are unknown. Using a subtractive hybridization approach, mammaglobin mRNA has also been found to be overexpressed in the in situ compared to the invasive element within an individual breast tumour. Further study by in situ hybridization performed in 13 breast tumours, selected to include normal, in situ, and invasive primary tumour elements, and in most cases axillary lymph node metastases, revealed that mammaglobin expression occurs in all elements, is restricted to epithelial cells, and is significantly increased in tumour cells compared with normal cells ( p< 0.04). Analysis of mammaglobin expression within 20 independent primary breast tumours and their corresponding axillary lymph nodes revealed that all 13 lymph nodes positive and none of the seven nodes negative for metastatic breast carcinoma by histology were mammaglobin-positive by reverse transcription-polymerase chain reaction (RT-PCR) ( p=0.0001). These results suggest that mammaglobin could be a marker of axillary lymph node breast metastases.

Altered expression of exon 6 deleted progesterone receptor variant mRNA between normal human breast and breast tumour tissues.

The progesterone receptor (PR) is an important prognostic marker in breast cancer as well as a marker of responsiveness to endocrine therapies. The presence of several exon-deleted PR variant mRNAs in both normal and neoplastic breast samples has recently been reported. Amongst them, a variant mRNA deleted in exon 6 (D6-PR mRNA) that if translated would encode a truncated PR-like protein missing the hormone binding domain and one of the transactivating domains of the wild-type PR protein. In order to determine whether changes in D6-PR variant expression could occur during tumorigenesis, its expression was investigated by reverse transcription and polymerase chain reaction in ten normal reduction mammoplasty samples, nine breast tumours with high PR levels (> 100 fmol mg(-1) protein) and eight breast tumours with low PR levels (< 15 fmol mg(-1) protein), as determined by ligand binding assay. The relative expression of D6-PR to wild-type PR mRNA was lower (P< 0.01 ) in normal than in all tumour breast samples. Moreover, a trend to lower (P < 0.1) relative D6-PR expression was observed in high PR tumours, compared to low PR tumours. These data suggest that increased expression of D6-PR occurs during tumorigenesis.

Oestrogen receptor-alpha variant mRNA expression in primary human breast tumours and matched lymph node metastases.

We have shown previously that the relative expression of a truncated oestrogen receptor-alpha variant mRNA (ER clone 4) is significantly increased in axillary node-positive primary breast tumours compared with node-negative tumours. In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-alpha variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-alpha variant mRNAs appeared conserved between any primary and its matched secondary tumour.