Series Of Primary Breast Cancers Research Articles

NTHL1 is a recessive cancer susceptibility gene

In search of novel breast cancer (BC) risk variants, we performed a whole-exome sequencing and variant analysis of 69 Finnish BC patients as well as analysed loss-of-function variants identified in DNA repair genes in the Finns from the Genome Aggregation Database. Additionally, we carried out a validation study of SERPINA3 c.918-1G>C, recently suggested for BC predisposition. We estimated the frequencies of 41 rare candidate variants in 38 genes by genotyping them in 2482–4101 BC patients and in 1273–3985 controls. We further evaluated all coding variants in the candidate genes in a dataset of 18,786 BC patients and 182,927 controls from FinnGen. None of the variants associated significantly with cancer risk in the primary BC series; however, in the FinnGen data, NTHL1 c.244C>T p.(Gln82Ter) associated with BC with a high risk for homozygous (OR = 44.7 [95% CI 6.90–290], P = 6.7 × 10–5) and a low risk for heterozygous women (OR = 1.39 [1.18–1.64], P = 7.8 × 10–5). Furthermore, the results suggested a high risk of colorectal, urinary tract, and basal-cell skin cancer for homozygous individuals, supporting NTHL1 as a recessive multi-tumour susceptibility gene. No significant association with BC risk was detected for SERPINA3 or any other evaluated gene.

Abstract 5405: Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value

Abstract Purpose: Cathepsin D is a proteolytic enzyme that is normally localized in the lysosomes and is involved in the malignant progression of breast cancer. The expression of Cathepsin D is regulated by steroid hormones including oestrogen and growth factors. There are conflicting results between different studies regarding Cathepsin D significance as prognostic and predictor marker in breast cancer. Furthermore, Cathepsin D has not been recommended for clinical use by the American Society of Clinical Oncology because of insufficient evidence. This study was aimed to fill the gap in the lack of recent and large studies on prognostic significance of Cathepsin D expression by studying a large well-characterized series of primary breast cancer for cathepsin D expression using immunohistochemistry. Methods: Expression of Cathepsin D was assessed by immunohistochemical staining of tissue microarrays, in a large well-characterized series of early-stage operable breast cancer (n=954) from Nottingham Primary Breast Carcinoma Series between the period of 1988 and 1998 who underwent primary surgery. Correlation of cathepsin D expression with clinicopathological parameters and prognosis was evaluated. Results: Cathepsin D expression was positive in 28.8% (275/954) of breast cancer tumors. Positive expression of cathepsin D was significantly associated with high histological grade (p=0.007), pleomorphism (p=0.002), poor Nottingham Prognostic Index score (NPI) (p<0.002), recurrence (p=0.005) and distant metastasis (p<0.0001). Kaplan-Meier analysis showed that Cathepsin D expression was significantly associated with shorter breast cancer specific survival (BCSS) (p=0.001), higher risk of recurrence (p=0.001) and distant metastasis (p<0.0001). Cathepsin D positive tumors with ER positive staining who were treated with tamoxifen were associated with lower BCSS and higher risk of developing distant metastasis (p=0.001). Conclusion: This study shows Cathepsin D expression as a promising prognostic biomarker for early operable invasive breast cancer. Our results indicate that Cathepsin D expressing tumors are associated with shorter breast cancer specific survival and higher risk of recurrence and metastasis. Cathepsin D could be clinically useful to predict response to tamoxifen treatment. Targeting Cathepsin D either through inhibition of action or interference with the proteolytic pathway could be used to treat metastatic breast cancers expressing Cathepsin D. Citation Format: Inas M. Alhudiri, Christopher C. Nolan, Ian O. Ellis, Emad A. Rakha, Adam Elzagheid, Andrew R. Green, Caroline J. Chapman. Expression of cathepsin D in early-stage breast cancer and its prognostic and predictive value [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5405.

Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes

BackgroundMediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance.MethodsMED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated.ResultsHigh MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001).ConclusionsMED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

Abstract P5-08-17: A study of c-Jun N-terminal kinase (JNK) and c-Jun as biomarkers in early breast cancer

Abstract Background The AP-1 transcription factor c-Jun is a key downstream target of c-Jun N-terminal kinase (JNK) which mediates intracellular signalling associated with a variety of cellular functions. The JNK pathway in breast cancer (BC) can be attenuated via loss of function mutations in MAPK kinases as well as via PIK3CA mutations; however, there is contradictory information about the role of JNK pathway and its clinical implications in BC. Methods In the current study, the clinicopathological implications of JNK and JUN mRNA expressions were evaluated in multiple independent BC datasets: a) Training-set (Uppsala cohort; n=249), b) Test-set (human genome atlas database; n=540), c) External validation-set (METABRIC cohort; n=1952) and d) Multicentre pooled databases (n=5530). The clinicopathological associations of their phosphorylated proteins (p-Jnk and p-c-Jun) were assessed in the Nottingham Tenovus Primary BC Series (n= 1650) and in an ER negative cohort (n=450). Results Both JNK and c-JUN mRNA high expressions were significantly associated with PAM50-Luminal A and ER+/HER2-/low proliferation molecular BC subtypes, tubular/lobular types, and integrative molecular clusters 4 (IntClust.4), ps&amp;lt;0.001. Whereas BC that had both low JNK and c-JUN mRNA, were significantly associated with large tumour size, high grade, absence of hormonal receptors (HR), HER2 overexpression, PAM50 HER2 and PAM50 Basal molecular subtypes, and IntClust.1, 9 and 10 BCs; ps&amp;lt;0.001. There was a significant positive correlations between p-Jnk and p-c-Jun protein levels (p&amp;lt;0.0001), however; our data suggested that differential p-Jnk/p-c-Jun expression may influence BC phenotypes. BC with p-Jnk-ve/p-c-Jun-ve were associated with the most aggressive phenotypes including largest tumour size, highest grade, lympho-vascular invasion, absence of HR, basal-like-phenotype, HER2 overexpression, and loss of double strand , single stand and base excision DNA repair proteins (ps&amp;lt;0.0001). In addition p-Jnk-ve/ pc-Jun-ve phenotype was associated with the lowest levels of p-38, ATF2, and p-ATF2; ps&amp;lt;0.001. Interestingly, low levels of either c-JUN-mRNA or pc-Jun protein, was associated with, PAM50-luminal B, epithelial mesenchymal transition and TP53 mutation and loss of its downstream proteins such as MDM2, MDM4, Bcle2 and p21; ps&amp;lt;0.05. JNK+ (mRNA and p-Jnk) and c-JUN+ (mRNA and p-c-Jun) individually were associated with prolonged BC specific survival (ps&amp;lt;0.001). Multivariate cox regression models that included other validated prognostic factors and therapies revealed that c-JUN-mRNA (Uppsala: p=0.005 and METABIRIC: p=0.036) and p-c-Jun (HR: 0.69; 95% CI = 0.55-0.88; p=0.002) were independently associated with clinical outcome. Furthermore, in ER+ high risk BC, exposure to tamoxifen was associated with decreased risk of death from BC in those patients with p-c-Jun-ve BC (HR: 0.65; 95% CI: 0.45-0.95; p=0.025). Conclusion JNK and c-JUN mRNA as well as p-Jnk and p-c-Jun protein levels are associated with luminal BC, with p-c-Jun being found to be an independent prognostic factor. The interaction between p-Jnk, p-c-Jun and TP53 mutation could predict response to endocrine therapy in ER+ BC. The role of the transcriptionally active form of c-JUN warrants further investigation with regard to its role in BC. Citation Format: Palmieri C, Rudraraju B, Giannoudis A, Moore D, Shaw J, Chan S, Ellis IO, Caldas C, Coombes RC, Carroll JS, Ali S, Abdel-Fatah TMA. A study of c-Jun N-terminal kinase (JNK) and c-Jun as biomarkers in early breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-17.

Abstract P3-06-01: Nuclear β-catenin negativity predicts for late relapse in ER+, tamoxifen-treated breast cancer

Abstract Background: The annual recurrence rates of post-menopausal ER-positive breast cancers persist beyond the first 5 years of diagnosis and treatment and the mortality rates in this period are higher versus ER-negative cancers. Extended endocrine therapy past 5 years has been shown to be of benefit but is associated with increased toxicity and cost thus the ability to predict patients who are at highest risk of late relapse would be of significant benefit in clinical decision making in this context. β-catenin is an intracellular protein that undergoes Wnt-mediated nuclear translocation where it transactivates genes implicated in tumour development and progression. We have also previously reported that β-catenin can play a role in aggressive resistance that accompanies prolonged endocrine treatment in vitro. In this study, we thus investigated whether β-catenin expression in ER+ breast cancer was predictive of recurrence beyond 5 years in an analysis of three separate endocrine-treated cohorts. Methods: Associations between β-catenin gene expression and relapse free survival (RFS) were performed using the online KMplotter tool. Immunostaining for total β-catenin was performed on tissue samples from 3 ER+ primary breast cancer series with long-term follow-up data: Nottingham 2000 (n=384, tamoxifen-only); ABC (n=570; tamoxifen only); transATAC (n=743; tamoxifen or ansatrozole). The association between subcellular (nuclear or cytoplasmic) β-catenin expression and RFS was determined in (i) the entire cohort, (ii) the first 5 years of tamoxifen treatment versus post-5 years. Results: KMplotter analysis of β-catenin in ER+, tamoxifen-treated patient samples (n=665) revealed a significant relationship with improved RFS in the post-five year cohort [HR: 0.48 (0.34-0.68); p=0.000019) versus the first 5 years [HR: 0.91 (0.61-1.36; p=0.64)]. No significant association was observed in untreated ER+ patients. In the Nottingham series, nuclear β-catenin positivity was significantly associated with improved survival in the 20-year follow-up for tamoxifen-treated patients (p=0.047) but not in the first five years (p=0.239). Cytoplasmic β-catenin did not associate with survival. Further analysis in the ABC series revealed an association between nuclear β-catenin positivity and improved survival for tamoxifen-treated patients in the entire cohort (HR: 0.52 (0.18, 0.55); p=0.00005). However, nuclear β-catenin was more strongly associated with improved outcome in the post 5-year treatment group (HR: 0.30 (0.14, 0.66); p=0.01) versus the first five years of treatment (HR: 0.33 (0.15,0.73); p=0.06). No significant associations were seen with cytoplasmic β-catenin. The transATAC trial material again revealed an association between presence of nuclear β-catenin and reduced distant recurrence in the post 5-years endocrine-treated cohort (HR: 0.54 (0.33, 0.91): x2=5.52, p=0.018) versus years 1-5 (HR: 1.16 (0.67, 2.01); x2=0.29, p=0.59). Conclusions: This is the first study to demonstrate that nuclear β-catenin may represent a predictive biomarker for late relapse following tamoxifen treatment in ER+ breast cancer where, contrary to traditional hypotheses and pre-clinical data, its nuclear expression is strongly associated with good outcome post-five years of treatment. Citation Format: Stephen Hiscox, Chris Smith, Robert I Nicholson, Julia Gee, Adrian Harris, Judith Bliss, Eleftheria Kalaizak, Ivana Sestak, Mitch Dowsett, Jack Cuzick, Ian Ellis, Peter Barrett-Lee. Nuclear β-catenin negativity predicts for late relapse in ER+, tamoxifen-treated breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-01.

Abstract P5-09-01: Nottingham prognostic index plus (NPI+): Validation of the modern clinical decision making tool in breast cancer

Abstract Introduction Current management of breast cancer (BC) relies on risk stratification based on well-defined clinicopathologic factors. The Nottingham Prognostic Index Plus (NPI+) is based on the assessment of biological class combined with established clinicopathologic prognostic variables providing improved patient outcome stratification superior to the traditional NPI*. This study aimed to validate the NPI+ in an independent series of BC. Methods A Validation series of 469 primary early-stage BC cases treated in Edinburgh, UK were matched for size, stage and grade to cases from Nottingham, UK used to develop the NPI+ (Training series). Adjuvant therapy was similar in both series except that 143 Edinburgh cases received endocrine therapy whilst the matched Nottingham cases had no adjuvant therapy. However, there was no significant difference in 10 year BC specific survival (BCSS) between the Training and Validation series. Cases, prepared as TMAs, were immunohistochemically assessed for Cytokeratin (Ck)5/6, Ck18, EGFR, Estrogen Receptor (ER), Progesterone Receptor (PgR), HER2, HER3, HER4, Mucin 1 and p53 expression. NPI+ biological class based on the expression of the 10 biomarkers was determined. Subsequent NPI+ prognostic scores were assigned using individual algorithms for each biological class developed using the Training series incorporating clinicopathologic parameters: positive nodes (including nodal stage), tumour size, tumour grade (including mitotic index) and PgR. NPI+ biological classes, prognostic scores and prognostic groups were compared between the Validation and Training series and their role in prediction of patient outcome. A p-value of &amp;lt;0.01 was considered significant. Results As anticipated, there was a comparable distribution of NPI+ biological classes between Training and Validation series: Luminal A, n=143 (31%) vs n=115 (25%); Luminal N, n=99 (21%) vs n=89 (19%); Luminal B, n=75 (16%) vs n=85 (18%); Basal p53 altered, n=54 (12%) vs n=72 (15%); Basal p53 normal, n=37 (8%) vs n=53 (11%); HER2+/ER+, n=31 (7%) vs 18 (4%); HER2+/ER-, n=30 (6%) vs n=37 (8%; X2=13.792, p=0.032). BCSS was analogous between the Validation and Training series in each of the NPI+ biological classes except Luminal B (p=0.042). Similar BCSS was observed in the NPI+ Biological classes of the Training versus Validation series when taking into consideration adjuvant treatment modalities. The mean NPI+ score was similar between the Validation and Training series (2.30 vs 1.89, Pearson’s Regression p=0.079). The NPI+ prognostic groups significantly predicted patient outcome in each molecular class (BCSS, p&amp;lt;0.0001) in the Validation series irrespective of adjuvant treatment. Comparing the BCSS in each of the NPI+ prognostic groups demonstrated there were no significant differences between patient outcome in each of the NPI+ prognostic groups between the Validation and Training series. Conclusion This study validates the NPI+ in an independent series of primary BC confirming its’ reproducibility. The NPI+ provides improved individualised clinical decision making for breast cancer for both prediction of clinical outcome and relevant therapeutic options. Acknowledgements Funded by the MRC References *Rakha EA et al Br J Cancer. 2014 110:1688-97. Citation Format: Andrew R Green, Daniel Soria, Jacqueline Stephen, Desmond G Powe, Christopher C Nolan, Ian Kunkler, Jeremy Thomas, Gill Kerr, Wilma Jack, David Camreron, Tammy Piper, Graham R Ball, Jonathan M Garibaldi, Emad A Rakha, John MS Bartlett, Ian O Ellis. Nottingham prognostic index plus (NPI+): Validation of the modern clinical decision making tool in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-09-01.

The role of BUB and CDC proteins in low-grade breast cancers

BackgroundThe drivers of neoplasia within low-grade luminal breast cancers remain undelineated. The BUB and CDC family are among kinase genes known recently to help to identify luminal breast cancers with poorer prognosis. Additionally, other CDC kinase genes (CDC42) are associated with luminal A breast cancers with good prognosis. We aimed to investigate the role of these kinases at the protein level within low-grade luminal breast cancers. MethodsThe Nottingham Tenovus Primary Breast Cancer Series (n=1858) microarrays were immunostained for BUB (BUB1, BUB1B, BUB3) and CDC proteins (CDC2, CDC42) and expression correlated with clinicopathological and molecular variables and patient outcome (SPSS, version 22). FindingsOn χ2 analysis, cytoplasmic BUB1 and nuclear BUB3 were negatively associated with grade including pleomorphism, mitosis, and Nottingham Prognostic Index, whereas BUB1B was positively associated (p=0·05). BUB1 and BUB3 expression was positively correlated with oestrogen and progesterone receptor expression whereas BUB1B was negatively correlated (p=0·01). CDC42 had strong associations with tumour morphology within the low-grade luminal breast cancers, tubular and lobular (p=0·02). CDC42 nuclear expression revealed negative correlations with basal (CK5) and HER family biomarkers (p=0·02). By contrast, cytoplasmic CDC2 overexpression was associated with high-grade tumours (p=0·01). BUB1, BUB1B, and CDC42 showed significant associations (p=0·04) with breast-cancer-specific survival even at the 15–20-year range, indicating their long-term prognostic potential. InterpretationThese results suggest that BUB1, BUB3, and CDC42 are key kinases for low-grade luminal tumours whereas BUB1B and CDC2 kinases are preferentially expressed in high-grade disease. High protein expression of BUB1, BUB3, and CDC42 in low-grade breast cancers was associated with longer overall survival whereas lower expression resulted in poorer outcome. FundingPathological Society of Great Britain and Northern Ireland, National Institute for Health Research.

E4BP4 is a repressor of epigenetically regulated SOSTDC1 expression in breast cancer cells.

We and others show that SOSTDC1 is down-regulated in breast cancer tissues compared to matched normal tissues. Previously, we found that epigenetic mechanisms underlie the down-regulation of SOSTDC1 in gastric cancer cells. The aim of this study was to assess the putative epigenetic regulation of SOSTDC1 expression in breast cancer cells. Microarray-based expression profiling was performed in a series of primary breast cancers and matched normal tissues. Real-time PCR was performed to assess SOSTDC1 and E4BP4 mRNA levels in MCF7, BT549, MBMDA231, T47D (breast cancer) and HEK293T (normal kidney) cell lines. Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were performed to assess the methylation level of the SOSTDC1 gene promoter, and 5-Aza 2-deoxycytidine (5'-Aza-dC) treatment was used to induce its demethylation. A luciferase assay was used to measure SOSTDC1 promoter activity in vitro. Stable shRNA-mediated knockdown of E4BP4 was carried out in MCF7 cells and confirmed by Western blotting. Finally, MCF7 cell proliferation and survival were measured by MTS assay. We found that SOSTDC1 is frequently down-regulated in primary breast cancers (98.2%) and in all breast cancer cell lines tested. MSP and BSP analyses revealed SOSTC1 promoter hypermethylation at CpG sites. 5'-Aza-dC treatment induced a striking down-regulation of SOSTDC1 gene expression, whereas BSP analysis showed demethylation of its promoter. Subsequent in silico SOSTDC1 promoter analysis indicated the presence of putative transcriptional repressor E4BP4 binding sites, and promoter deletion studies indeed revealed repressor binding regions encompassing these E4BP4 binding sites. Relative quantification of E4BP4 expression showed an inverse correlation to SOSTDC1 expression in the breast cancer cell lines tested. Exogenous over-expression of E4BP4 in HEK-293 and BT549 cells reduced SOSTDC1 expression and its promoter activity, respectively. Stable shRNA-mediated E4BP4 BT549 and MCF7 knock-down cells treated with 5'-Aza-dC exhibited up-regulation of SOSTDC1 expression and a concomitant inhibition of cell proliferation and survival. From our results we conclude that the transcriptional repressor E4BP4 plays a role in repressing epigenetically regulated SOSTDC1 expression in breast cancer cells, which can be reverted by E4BP4 silencing.

Abstract P2-10-22: Phosphorylation of Steroid Receptor Coactivator 3 (SRC3) at Ser543 is a novel independent prognostic marker in breast cancer

Abstract Introduction: Steroid receptor coactivator 3 (SRC3) acts as a coactivator of nuclear receptors including estrogen receptor-alpha (ER). SRC3 has been implicated in the pathogenesis of breast cancer (BC) as well as in resistance to endocrine therapy. SRC3 is phosphorylated at a number of residues following the stimulation of growth factors or hormones. Tyr24 and Ser543 are both phosphorylated upon estrogen stimulation, while Tyr24 is modulated by JNK and Ser543 by both p38 and JNK. To date, the importance and potential role of phosphorylation at these residues has not been explored in BC. In this study we assessed the protein expression of SRC3, pTyr24 and pSer543 and association with clinico-pathological features and outcome in a well defined breast cancer series. Methods: The expressions of SRC3, pTyr24 and pSer543 were assessed in the Nottingham Tenovus Primary Breast Cancer Series which consists of 1650 cases of primary invasive. SRC3, pTyr24 and pSer543 were correlated with clinico-pathological data as well as outcome. Results: SRC3 expression was significantly associated with unfavourable clinicopathological features including ER -ve (p = 0.02), PR –ve (p = .038), HER2 overexpression (p &amp;lt; 0.0001), Triple negative phenotype (p = 0.001), high proliferation (p &amp;lt; 0.0001), high histological grade (p &amp;lt; 0.001), and lympho-vascular invasion. On contrast, the expression of pSer543 was significantly associated with a luminal phenotype, well differentiation, low proliferation (low mitotic index, low Ki67 and low SPAG5; p &amp;lt; 0.0001), hormonal receptors (ERα+ve/PR+ve/AR+ve), absence of both ER-B1 and ER-B5 (p &amp;lt; 0.01), high expression of ER-α associated proteins (cyclin D1 and Bcl2; p &amp;lt; 0.0001), high expression of c-jun (p &amp;lt; 0.0001), JNK (p &amp;lt; 0.0001), SRC3 (p &amp;lt; 0.0001), T24 (p &amp;lt; 0.001) and active p53 transcriptional pathways that regulate cell cycle progression and apoptosis (MDM2+ve, MDM4+ve and Bax+ve; p &amp;lt; 0.01) and absence of basal like phenotypes (p &amp;lt; 0.01). The absence of pSer543 was significantly associated with loss of expression of the key DNA repair proteins including XRCC1 (p &amp;lt; 0.0001), BRCA1 (p &amp;lt; 0.0001), ATM (p = 0.008) and TOP2A (p &amp;lt; 0.0001) reflecting a higher risk of genomic instability. Moreover, absence of pSer543 was more common in BC with a triple negative phenotype (p &amp;lt; 0.001). With regard to outcome, no association with outcome based on the expression of SRC3 either with or without tamoxifen was observed. However, expression of pSer543 was associated with significantly longer disease free survival (DFS) (p &amp;lt; 0.00001) and breast cancer specific survival (BCSS) (p = 0.0001). Furthermore, absence of pSer543 was associated with both a shorter DFS (p = 0.007) and BCSS (p = 0.01) in ER+ ve high risk BC. pSer534 was confirmed as an independent prognostic factor after adjustment for endocrine therapy and other validated prognostic factors and absent of pSer534 was associated with a two-fold increased risk of recurrence (HR = 1.9, CI 95%= 1.2–3.1). Data on Tyr24 will also be presented. Conclusion: Phosphorylation at Ser543 is associated with a luminal phenotype, positive prognostic factors and sensitivity to tamoxifen. Furthermore, it is an independent prognostic factor. pS543 is a novel prognostic marker in BC and warrants further investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-22.

Molecular Alterations Between the Primary Breast Cancer and the Subsequent Locoregional/Metastatic Tumor

Metastatic breast cancers have historically been presumed to have the same predictive biomarkers as the initial primary tumor. We compared the expression of these biomarkers in a large paired tissue microarray (TMA) series of primary and subsequent relapsed tumors. Using the British Columbia Cancer Agency Breast Cancer Outcomes Unit database, patients with biopsy-proven relapses were identified and linked to a large TMA series of primary breast cancers from 1986-1992. Charts were reviewed, and tissue blocks of the metastatic cancer were collected to create a separate TMA. Immunohistochemical assessment with the same antibodies and conditions was performed for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER)-2 on both the primary and relapsed tumors. One hundred sixty cases were received that had tumor adequate for analyses. Of these, 71.9% had no changes in either the ER or PR status or HER-2 status. Of the 45 (28.1%; 95% confidence interval [CI], 21.2%-35.1%) tumors that did have changes in receptor status, 7.5% were in-breast recurrences or new breast primaries, 4.4% had changes in PR status only and were therefore deemed clinically irrelevant, and 19.4% (95% CI, 13.3%-25.5%) had changes in either the ER or HER-2 status from regional or distant relapses. Five percent of tumors had a receptor status change going from ER(+) or PR(+) to ER(-) or PR(-); 9.4% went from ER(-) or PR(-) to ER(+) or PR(+). With regard to HER-2 status, 3.8% of tumors went from positive to negative and 1.3% went from negative to positive. For all discordant cases, biopsies of the relapsed lesion were obtained prior to initiation of first-line treatment for metastatic disease. In the primary tumors that were ER(+), time to relapse was significantly shorter in the discordant relapsed cases than in the concordant ones (p = .0002). Changes in loss or gain of either biomarker were seen across the discordant cases. A significant proportion of relapsed tumors had changes in either ER or HER-2 status, which would dramatically alter treatment recommendations and clinical behavior. This study suggests that biopsies of relapsed and metastatic breast cancers should be performed routinely in clinical practice.

P1-06-14: Topoisomerase II alpha (Top2a) Protein Expression Is a Predictor for Response to Anthracycline-Based Chemotherapy (ATC-CT): Is It Due to Gene Amplification, HER2−Coamplification or a Summation of Pathways Leading to This Highly Proliferative Phenotype?

Abstract Background: The evaluation of Top2a protein may be clinically more useful than gene alterations as a predictive marker for ATC-CT. In this study we assessed the association between gene copy number, gene and protein expressions of both TOP2A and HER2, and their effect on clinicopathological outcomes and management of breast cancer (BC). Methods: 1- To study the response to anthracycline based chemotherapy (ATC-CT): The associations between clinical outcomes and both gene copy number changes (using in-situ hybridization; CISH) and protein expression (using immunohistochemistry) were studied in the neoadjuvant and adjuvant settings: a) 250 locally advanced primary BC treated with Neoadjuvant ATC-CT with or without Taxane followed by surgery (S) + radiotherapy (RT); pathological complete response (pCR) was used as the primary end point (PEP), b) 245 BC in which all patients were treated with S + RT followed by Adjuvant ATC-CT; progression free survival (PFS) was used as PEP ii) 145 primary BC overexpressing HER-2 treated with S+ RT followed by sequential adjuvant ATC-CT+ trastuzumab; PFS was used as PEP. 2- To study the clinic-pathological association of TOP2A alterations, we evaluated TOP2A alterations detected by CISH and IHC in unselected series of 1650 consecutive cases of primary BC who treated with S + RT and received adjuvant CMF and/or endocrine therapies according to Nottingham prognostic index and ER status. 3- To study in details the molecular alterations of TOP2A/HER2, in 171 unselected series of primary BC, we evaluated a) gene copy number changes using both high resolution oligo array CGH and CISH, b) mRNA expression using Agilent gene expression array and c) protein expression using IHC. We analysed 48,000 gene transcripts using Artificial Neural Networks (ANN) and pathway analysis to identify genes and biological pathways that related to TOP2A gene alterations. Results: 1) In the ATC-CT neoadjuvant series, the pCR rate was 32/115 (28%) in tumours expressing high levels of Topo2A, compared to 5/74 (7%) in tumours expressing low levels of Topo2A (p&amp;lt;0.0001). In multivariate analysis, Top2A overexpression was an independent predictor for pCR (HR 5.1, CI 95%; 1.4−18.4, p&amp;lt;0.001). 2) TOP2A overexpression was strongly associated with mitotic index, histological grade, KIF2C, loss of p53 function and the absence of both BRCA1 and ATM inactivation (p&amp;lt;0.0001). 3) ANN and pathway analysis revealed that TOP2A-strongly correlated genes are involved in: mitotic cell cycle regulation especially M phase and cell division (AURKB, KIF2C, BRIC5, ASPM, CCNA2, BUB1, FBXO5, PTTG1, CDCA5, CDCA3 CDCA8), Kinesin and microtubules regulator genes (KIF2C, KIF11, KIF14, KIF20A, KIF23, and KIFC1), and metastases (BRIC5, BUB1B, CCNA2, CCNE, PRRG1, PRM2, STMN1). Conclusions: Top2A protein expression is an independent predictor for pCR after ATC-CT treatment. The high response rate of top2A protein overexpression supports the theory that Top2a protein is a direct target of ATC-CT in these highly proliferative tumour cells. Furthermore, evaluation of Top2A protein may lead to a clinically useful test. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-14.

Estrogen receptor-negative breast carcinomas: a review of morphology and immunophenotypical analysis

Estrogen receptor (ER)-negative breast cancers are a group of tumors with poor prognosis and fewer cancer prevention and treatment strategies compared to ER-positive tumors. The aim of this study was to assess the morphological characteristics and immunohistochemical profile of ER-negative tumors and thus to understand the biological behavior and unique nature. In total, 291 consecutive ER-negative cases available from our primary breast cancer series were examined. Hematoxylin- and eosin-stained sections of all the cases were studied for several morphological parameters and their immunophenotype profile. These findings were correlated with patient and tumor characteristics and survival data. ER-negative tumors constituted 30% of the primary operable breast cancer series. The majority of tumors were grade 3 (94%) and the commonest histological types were ductal/no specific type (85%), and atypical medullary carcinoma (8%). High-grade comedo-type necrosis, lymphoid stroma, central necrosis/fibrosis and pushing margins were the most common morphological features. The presence of a pushing margin showed a significant relation to androgen receptor negativity, absence of epidermal growth factor receptor expression and negative lymph nodes. Lymphoid stroma and comedo-necrosis correlated with higher tumor grade. ER-negative breast cancers are a distinct group of tumors with several common morphological features. Grade 3 histology, pushing margin, lymphoid stroma, comedo-type necrosis and central fibrosis/necrosis are the dominant morphological findings. The presence of a pushing margin appears to have a significant correlation with negative lymph node status. ER-negative tumors show a higher expression of p53, CerbB2 and epidermal growth factor receptor compared to ER-positive breast cancer. These unique features support the concept that ER-negative tumors are a morphologically and phenotypically distinct entity and provide a rationale for the study and use of newer promising agents in the treatment of ER-negative breast cancer.

EGF-R and Her2/neu overexpressing tumors: independent collectives for treatment of breast cancer by specific monoclonal antibody-therapy.

In our recent studies, we demonstrated that breast cancer treatment by anti-EGF-R antibody resulted in a significant therapeutic effect in vivo. Furthermore, we were able to elucidate histopathologic parameters with an impact on therapy success. The aim of this study was the evaluation of EGF-R and Her2/neu protein expression on a large series of primary breast cancer, to elucidate if anti-EGF-R antibody therapy is a new therapeutic option for patients who are Her2/neu negative and where, therefore, anti-Her2/neu antibody treatment is not applicable. We analyzed EGF-R and Her2/neu protein expression of 149 consecutive primary breast cancer specimens by fully quantitative enzyme-linked immunosorbent assay. For evaluation of stochastic independence, we used chi(2)-test as goodness-of-fit test. We found EGF-R and Her2/neu expression as stochastically independent in primary breast cancer. Anti-EGF-R antibody treatment is a potential therapeutic option for patients with Her2/neu negative breast cancer.

Subcellular localisation of cyclin B, Cdc2 and p21 WAF1/CIP1 in breast cancer : association with prognosis

The heterodimeric cyclin B/Cdc2 protein kinase governs entry into mitosis, and can be negatively regulated through p53-mediated transcriptional induction of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1 . Ectopic expression of p21 WAF1/CIP1 in cultured cells has been shown previously to influence the subcellular distribution of the cyclin-dependent kinases (CDKs) including Cdc2. In this study, we have examined the subcellular localisation of Cdc2, cyclin B and p21 WAF1/CIP1 by immunohistochemistry in a well characterised series of primary breast cancers. Surprisingly, p21 WAF1/CIP1 was predominantly cytoplasmic in many of the tumours, where it was associated with high p53 levels; cytoplasmic p21 WAF1/CIP1 and high cyclin B levels were also significant predictors of poor prognosis. We conclude that breast tumorigenesis may be characterised by abnormalities in pathways determining not only levels of expression of key regulatory molecules, but also their subcellular localisation. Investigation of the subcellular distribution of cell cycle regulatory proteins, particularly p21 WAF1/CIP1 , could provide valuable prognostic markers in breast cancer.

Glyceraldehyde-3-phosphate dehydrogenase gene expression in human breast cancer

Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) has been widely used as a control RNA in Northern blotting and in reverse transcriptase–polymerase chain reaction (RT–PCR) analyses. We investigated the expression of GAPDH in a large series of primary breast cancers and in MCF7 human mammary epithelial breast cancer cells treated with oestradiol. The expression of GAPDH was quantified by a real-time one-step RT–PCR assay, based upon the 5′ nuclease activity of Taq polymerase using an Abi Prism 7700 Sequence Detector System (Perkin Elmer, France). Using the Spearman test, GAPDH expression was found to correlate inversely with the age of the patients at diagnosis ( P=0.003; r=−0.147), oestradiol receptors (ER) ( P<0.0001; r=−0.327) and progesterone receptors (PgR) ( P<0.0001; r=−0.206). A positive correlation was observed between GAPDH expression and the histo-prognostic grading (HPG) ( P<0.0001; r=0.344). Moreover, the overall survival (OS) and the relapse-free survival (RFS) were significantly reduced in patients whose tumours showed an enhanced level of GAPDH expression (OS, P=0.046; RFS, P=0.021). Multivariate analyses demonstrated that GAPDH was not an independent prognostic factor. Finally, in MCF7 cells treated with oestradiol, a statistically significant dose-dependent increase in GAPDH expression was observed. These results show that GAPDH expression is associated with breast cancer cell proliferation and with the aggressiveness of tumours. The present study demonstrates that, in cancer, the use of GAPDH gene expression should not be used as a control RNA.

Immunohistochemical localization of cytochrome P450 CYP1B1 in breast cancer with monoclonal antibodies specific for CYP1B1.

Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase. (J Histochem Cytochem 47:1457-1464, 1999)

Human estrogen receptor messenger RNA variants in both normal and tumor breast tissues

By using the PCR-SSCP technique we characterized various ER-specific RNA species present in a series of primary breast cancers, as well as in cell lines established from breast carcinomas and in mammary gland tissues from healthy specimens. A series of six truncated messenger RNAs generated by alternative splicing was characterized. These RNAs correspond to specific deletions of one (exons 2–7, except exon 6) or two (exons 3 + 4) exons. All these RNA variants are observed in each one of the analyzed RNAs, regardless of origin. In addition, the relative amount of these different variants in ER + tumors is comparable to that measured in ER — tumors and healthy mammary gland tissues. This data suggests that tumor progression is not related to the emergence of any of the ER mRNA variants.

Glycolytic enzyme activities in breast cancer metastases.

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Estrogen receptors and breast cancer.

Estrogen receptors have been assayed in a series of primary breast cancers from postmenopausal women; 59% of which were estrogen-receptor positive. These patients survived for a significantly longer period of time than those whose tumors were estrogen-receptor negative. The effect of estrogen-receptor status was only seen (and then markedly accentuated) in patients who had lymph-node invasion at the time of mastectomy. Such determinations also appear to be of value in preselecting those patients who, on recurrence, will benefit from tamoxifen therapy.

Estrogen Receptors and Breast Cancer

Estrogen receptors have been assayed in a series of primary breast cancers from postmenopausal women; 59% of which were estrogen-receptor positive. These patients survived for a significantly longer period of time than those whose tumors were estrogen-receptor negative. The effect of estrogen-receptor status was only seen (and then markedly accentuated) in patients who had lymph-node invasion at the time of mastectomy. Such determinations also appear to be of value in preselecting those patients who, on recurrence, will benefit from tamoxifen therapy.