Molecular tools for personalised treatment of breast cancer
- A multidisciplinary research project in basic breast cancer research within cell and molecular biology which may improve the treatment of patients suffering from breast cancer.
The project represents an innovative approach to translational science that aims at identifying, developing, evaluating and clinically validating new predictive breast cancer biomarkers by genomic, proteomic and molecular pathology strategies, to be used to optimize current adjuvant treatment and metastatic treatment of breast cancer. In particular, the project focuses on the huge problems related to resistance towards chemotherapy and endocrine treatment.
The Consortium consists of a unique, multidisciplinary combination of internationally recognized Danish and Chinese scientists with a strong track record within basic clinical cancer research, clinical genetics, genomics, molecular and cellular biology, proteomics, and bioinformatics. The Consortium thus possesses the necessary competences to successfully carry out the research project.
Background of breast cancer research
Breast cancer is the second most frequent cancer type and the second most frequent cause of cancer related death among women. In spite of improvements over the last two decades, there is still an urgent need for development of improved treatment strategies.
We will approach this important challenge by identification of new and improved biomarkers, enabling improved treatment efficacy prediction, which furthermore will create the foundation for a personalised approach in which each patient receives the most optimal cancer treatment based on a comprehensive sensitivity/resistance signature of the tumour.
Objectives in the translational breast cancer research project
Resistance to chemotherapeutic drugs and/or endocrine therapy is considered the greatest obstacle to the successful clinical management of cancer patients and drug resistance is considered the main contributor to cancer related deaths.
Despite many efforts over the last decades, there has been no significant progress in solving this fundamental problem. The primary objective of this translational cancer research project is the identification, evaluation and clinical validation of candidate biomarkers for the prediction of treatment benefit/response to routine chemotherapy and routine endocrine treatment in breast cancer - thereby allowing for individualized treatment of breast cancer patients. We will design one or more in vitro diagnostic tests that can be used on paraffin sections required to meet the needs of predictive biomarker assays. The anticipated features of such a new test are: high predictive value, reproducibility, low cost and easy implementation into the health care system.
The importance and novelty of the breast cancer research projectWith approximately 230,000 new cases and 90,000 deaths/year, breast cancer is the second most frequent cancer and the second most common cause of cancer related death in Europe. The incidence of breast cancer has been continuously growing over the last years with a 38 percent increase in China during the last two years. It is thus clear that there is an urgent need for the development of more effective treatment strategies.
Systemic chemotherapy is the treatment of choice in metastatic breast cancer patients with hormone receptor negative tumors and in hormone receptor positive tumors following failure to endocrine therapy. So far, chemotherapy has been the only treatment option in these situations. However, recently biological therapy has been added for patients with HER2 positive disease. Survival following chemotherapy of metastatic breast cancer has improved over the last years. The latest studies with anthracycline, cyclophosphamide and/or taxane-based chemotherapy reported a median overall survival of more than 20 months.
The medical problem in breast cancer
In the adjuvant setting, routine treatment of all high-risk breast cancer patients under 60 years of age and estrogen receptor negative high-risk patients over 60 years, consists of a combination of anthracyclines, cyclophosphamide and taxanes.
Antracyclines and taxanes are among the most efficient drugs used in breast cancer therapy. As described, both drugs are used in adjuvant treatment and as the first-line treatment of metastatic breast cancer as single agent or in combination with other drugs. However, other drugs or combinations without taxanes or antracyclines are also used.
The choice among the different treatment options is primarily driven by the type of prior adjuvant chemotherapy and by the time interval between its discontinuation and the development of metastatic breast cancer. One unresolved problem is that only up to 50% of patients with metastatic breast cancer obtain an objective response or benefit from the chosen chemotherapy. Thus, with the current clinical approach there is a large group of patients who do not obtain any benefit and they may even suffer from the side effects induced by the chemotherapy. These patients are unnecessarily exposed to treatment toxicity, and they experience disease progression which affects the performance status and the capability to tolerate further chemotherapy.
The estrogen receptor (ER) status, which is routinely analysed by immunohistochemistry, is an important factor for selecting the therapeutic approach in breast cancer. ER is expressed by approximately 80% of all primary breast carcinomas and in these patients an anti-estrogen therapy with the drugs tamoxifen or aromatase inhibitors significantly reduces the risk of recurrence and death in all age groups studied. However, a significant number of ER postive patients will not respond to endocrine therapy and presently we do not have any method to identify these ER positive but endocrine therapy resistant patients.
The challenges in breast cancer treatment
Identification, validation and clinical implementation of new biomarkers for treatment sensitivity/resistance require a work intensive phase of research and development. Needless to say, such an effort requires a strong and intensive scientific collaboration to reach the final goal as represented by the present breast cancer Consortium combining the strengths of the Chinese and Danish centres.
Our efforts are expected to result in improved patient survival, improved quality of life, and saved economical resources.
Breast cancer cells - Background
At the cellular level, cancer is a genetic disease and the tumour cells have acquired genetic changes that are responsible for the multi-step process that drives the malignant transformation. The cancer specific genetic changes may lead to altered mRNA and protein levels (including different isoforms) and may represent the most important mechanism by which the tumour can permanently acquire new functionalities. The acquired specific genetic changes in the cancer cells may, however, not be present in the non-malignant cells of the patient. Therefore, it is obvious to exploit the specific genetic changes of the tumour cells as diagnostic, prognostic and especially predictive tools in the management of cancer patients.
The malignant transformation is driven by inactivation of tumour suppressor genes combined with activation of proto-oncogenes. The inactivation of tumour suppressor genes can occur by a variety of mechanisms, including physical deletion, point mutation and/or methylation - all leading to loss of function. Proto-oncogenes, on the other hand, can be activated by amplification, point mutation or structural rearrangements leading to gain of functions. Minor changes include point mutations and smaller intragenic deletions. These changes can be studied by sequencing, LOH (loss of heterozygosity) analysis and/or PCR based techniques combined with blotting techniques.
The major genetic changes include large stretches of DNA, from several thousands to millions of base pairs, and may, depending on methodology, be detected as structural rearrangements or copy number changes or structural rearrangements. The copy number changes can be studied by CGH (comparative genomic hybridisation), array-CGH and/or FISH (fluorescence in situ hybridization). Using the FISH technique, structural rearrangements can also be detected. During the last decade of research a number of novel biomarker molecules/genes of potential interest for prediction of response to chemotherapy or endocrine treatment in breast cancer have emerged. Of particular interest is that many of the genes that seem to confer sensitivity or resistance to therapy are either amplified or deleted in cancer.
In endocrine therapy, recent results have shown that patients with amplification of the PAK1 gene have decreased benefit of Tamoxifen compared to patients without amplification (6). Amplification of the estrogen receptor alpha gene (ESR1) has also recently been observed in approximately 20% of breast cancer and these tumors respond more efficiently to Tamoxifen than tumours without amplification (7,8).
The mechanisms underlying endocrine resistance in breast cancer are not well-defined, but could result from genetic or epigenetic changes within the tumor that activate hormone-independent mitogenic pathways (9). Such changes may be downstream of the ER within receptor-dependent or -independent pathways. There is no evidence that changes in ER function, ligand binding, and ability to induce expression of the progesterone receptor might account for the development of hormone resistance. Methylation of the ER gene has been shown to vary between tumours but the degree of methylation does not correlate with the levels of expression of the receptor protein. Thus, analysis for identification of potential gene amplifications and measurements of gene expression profiles (including alternative splice forms) that reflect the activity of the entire ER and related pathways could provide an important advance in understanding the behaviour of these breast cancers and in predicting treatment response .
One example of a gene where amplifications have been proposed linked to chemosensitivity in breast cancer is the TOP2A gene. This gene codes for topoisomerase II which is the target for the group of widely used chemotherapeutic drugs, the anthracyclines. Amplifications of the TOP2A gene have been shown to correspond to sensitivity to antracyclines (10). A second gene often amplified in breast cancer is the HER2 gene. Amplification of this gene is strongly associated with response to the antibody Herceptin, but some publications have indicated a significant association also to sensitivity to antracyclines (11) explained by frequent co-amplification of TOP2A and HER2. A third relationship between gene copy number and sensitivity/resistance to chemotherapy is the TYMS, DHFR and ECGF1 genes which all are involved in sensitivity to 5FU treatment (12).
We have recently published that the lack of TIMP-1 in experimental cancer models confers sensitivity to chemotherapy (12) while overexpression of TIMP-1 is associated with cellular resistance to chemotherapy in metastatic breast cancer (13). In metastatic breast cancer patients with high tumor tissue content of TIMP-1 protein, the objective response to chemotherapy was 0% while the objective response rate in TIMP-1 low patients approximated 45%.
TIMP-1 has also been shown to be associated with reduced benefit from adjuvant anthracycline containing chemotherapy. One of our results shows that the TIMP-1 gene may be amplified in a number of breast cancers.
There is a good base of knowledge of different molecular markers that potentially can be used to predict individual outcome or, more specifically, resistance to a given treatment. Retrospective analyses of some of these markers identified subgroups of patients who are less likely to respond to one drug regimen while they might benefit from another drug regimen. Thus, it might be possible to obtain knowledge prior to treatment whether a patient would be more likely to respond to one rather than the other of the available drug regimens. Only with this information rational treatment selection can be put into practice for the individual patient.
Predictive biomarkers in the research project
The Sino-Danish translational research project represents a four phase strategy in establishing selected biomarkers as predictive markers in chemotherapeutic or endocrine treatment of breast cancer. Since access to freshly frozen tissue is extremely difficult, we have based our selection and research proposal on markers that ultimately can be evaluated on paraffin sections either by immunohistochemistry or Fluorescence In Situ Hybridization (FISH) technology.
The research will use present knowledge and newly generated information on specific molecular markers to evaluate their potential in selecting the most beneficial treatment choice for the individual patient. An algorithm describing interactions and possible clinical significance of the various markers and clinical information will be constructed in collaboration between bioinformaticists at Beijing Genomics Institute and at the Centre for Biological Sequence Analysis (CBS) at the Technical University of Denmark.
In the first phase of the research programme, we will identify new markers of treatment sensitivity/resistance. In addition, based on the availability and on a literature surveys we will select a number of potential breast cancer predictive biomarkers that all have some degree of pre-validation data available. All markers will be adjusted, if possible, to paraffin embedded breast cancer tissue.
In the second phase of the research programme, we will study the significance of the individual genes that have been identified by the use of breast cancer cell line experiments in which the genes of interest are either up-regulated or suppressed.
In the third phase we will validate the results from phase 1 and 2 in an independent set of paraffin embedded breast cancer tissue samples. These samples will be derived from approximately 1000 primary breast cancer patients who have received chemotherapy and for whom information on time to recurrence and overall survival is available. A similar cohort of breast cancer patients who have received endocrine therapy will be selected. We will make Tissue Micro Arrays (TMAs) from these tumors.
The fourth phase, which will cover years 4-6 of the research program, relates to the identification and validation of the identified molecules as potential new targets for anticancer therapy. These studies will use the established genetically modified breast cancer cell lines and in vivo experiments in tumour-bearing mice. In addition, we will aim at performing prospective clinical validation of the identified biomarkers, resulting in the implementation of these markers into daily clinical routine treatment of breast cancer patients and thereby allowing for individualised treatment.
References - Breast cancer treatment:
1. Knoop AS, Bentzen SM, Nielsen MM, Rasmussen BB, and Rose C, Value of epidermal growth factor receptor, HER2, p53, and steroid receptors in predicting the efficacy of tamoxifen in high-risk postmenopausal breast cancer patients. J Clin Oncol 19: 3376-3384, 2001.
2. Honig S, Diseases of the Breast. Lippencott-Raven, Philidelphia, 1996.
3. West M, Blanchette C, Dressman H, Huang E, Ishida S, Spang R, Zuzan H, Olson JA, Jr., Marks JR, and Nevins JR, Predicting the clinical status of human breast cancer by using gene expression profiles. Proc Natl Acad Sci U S A 98: 11462-11467, 2001.
4. Hayashi S, Prediction of hormone sensitivity by DNA microarray. Biomed.Pharmacother. 58: 1-9, 2004.
5. Bostner J, Ahnström Waltersson M, Fornander T, Skoog L, Nordenskjöld B, Stål O. Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer. Oncogene. 26:6997-7005, 2007.
6. Holst F, Stahl PR, Ruiz C, Hellwinkel O, Jehan Z, Wendland M, Lebeau A, Terracciano L, Al-Kuraya K, Jänicke F, Sauter G, Simon R. Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer. Nat Genet. 2007;39:655-60. 2007.
7. Ejlertsen B, Nielsen KV, Rasmussen BB, Balslev E, Müller S, Møller S, Mouridsen HT. Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer. Breast Cancer Research and Treatment 2007; 106(S1): S34 (abstr 402).
8. Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer. 2:101-12, 2002.
9. Knoop AS, Knudsen H, Balslev E, Rasmussen BB, Overgaard J, Nielsen KV, Schonau A,Gunnarsdóttir K, Olesen KE, Mouridsen HAT, Ejlertsen B. Retrospective analysis of To-poisomerase IIa amplifications and deletions as predictive markers in primary breast can-cer patients randomly assigned to cyclophosphamide, methotrexate and fluorouracil orcyclophosphamide, epirubicin and fluorouracil: Danish Breast Cancer Cooperative Group.JCO 2005;23:7483-90.
10. Pritchard KI, Shepherd LE, O
11. Christensen AJ, Jensen LB, Balslev E, Nielsen KV, Poulsen TS, Nielsen DL, Møller S,Mouridsen H, Ejlertsen B, Rigshospitalet, Copenhagen, Denmark; DakoCytomation, Glo-strup Denmark. Breast Res Treat 2005;94:555 (Abstr 1048).Genet. 39:655-60, 2007.
12. Louise Davidsen, Sidse Würtz, Maria Unni Rømer, Nanna M Sørensen, Simon Johansen, Ib Jarle Christensen, Jørgen K Larsen, Hanne Offenberg, Nils Brünner and Ulrik Lademann. TIMP-1 gene deficiency increases tumor cell sensitivity to chemotherapy-induced apoptosis.Br J Cancer, 2006, 95: 1114-1120
13. Anne Sofie Schrohl , Marion E Meijer-van Gelder, Mads Holten-Andersen, Ib Jarle Christensen, Maxime P Look, Henning T Mouridsen, Nils Brünner and John Foekens. Primary tumor levels of Tissue Inhibitor of Metalloproteinases-1 are predictive of resistance to chemotherapy in patients with metastatic breast cancer. Clin Cancer Res, 2006, 12: 7054-7058
14. O ’Malley FP, Andrulis IL, Tu D, Bramwell VH, Levine MN, for the National Cancer Institute of Canada Clinical Trials Group HER2 and Responsiveness of Breast Cancer to Adjuvant Chemotherapy. N Engl Med 2006;354:2103-11.1. Early Breast Cancer Trialists' Collaborative Group, Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 351: 1451-1467, 1998.