Research concept paper

Title: An approach in using differential circulatory microRNA expression in breast cancer patients for prediction of chemotherapeutic response and differentiation of subtypes.

Yonas Mulugeta
Jan 10, 2012

0|Page

Introduction:
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% of all cancers. This disease is not restricted to women, as approximately 1% of all cases occur in men. The etiology of breast cancer is complex, and genetic background and environmental factors are believed to contribute to the complexities1. It is now perceived as a heterogeneous group of different diseases characterised by distinct molecular aberrations, rather than one disease with varying histological features and clinical behaviour. If breast cancer is detected and treated at an early stage, successful outcomes can be achieved in some patients. Most patients whose cancer has not spread to the lymph nodes are cured by surgery, chemotherapy and/or radiotherapy tamoxifen; others will go on to develop distant metastases and die. Chemotherapy is given as adjuvant treatment to reduce this risk, preventing distant metastases or as palliation to treat patients with metastatic disease13. It is a systemic treatment given after local treatment to reduce systemic recurrences and overall mortality from breast cancer14. There are multiple combinations of cytotoxic drugs currently accepted as standard protocols care. But, due to frequent severe side effects, including heart failure, leukemia and life threatening infections, selection of most effective chemotherapy agents for individual treatment is a great challenge15. Currently, for prediction of treatment response, first cycle chemotherapy will be administered. But, Such timing would have a notable clinical impact because patients receiving ineffective treatment could be switched to alternative therapies and responding patients could receive more aggressive chemotherapy16.Therefore, a need for specific biomarkers which predict sensitivity and response to a specific panel of chemotherapy is inevitable. Literatures demonstrated that gene expression profile may be a useful tool in defining the signature of cancer and predict the prognosis and response to treatment. Gene expression profiling studies have shown that estrogen-receptor (ER)-positive and ER-negative breast cancers are distinct diseases at the transcriptomic level that additional molecular subtypes might exist within these groups 2. So, based on mRNA profiling breast tumors can be classified into four stable molecular subtypes, with the most prominent discriminators being estrogen receptor (ER), HER2 and tumor differentiation3. The clinical classification of tumors based on gene expression has obvious beneficial implications in the development of multigene prognostic and predictive tests which have been commercialized and have become established as tools in breast cancer diagnostics4. Even though extensive research on molecular mechanisms involved in breast cancer has been done over the decades, challenges still prevail in the early diagnosis and management of breast cancer patients, such as unpredictable response and development of resistance to adjuvant therapies5. As with many cancers, progress in early breast cancer detection has

1|Page

been inadequate, and methods for determining diagnosis and prognosis of breast cancer are still limited to invasive procedures, such as tissue biopsies for histological examination3. Recently promising results are obtained in using miRNAs as potent diagnostic and prognostic biomarkers 6. MicroRNAs (miRNAs) are an evolutionarily conserved class of small non-coding RNAs of approximately 22-nucleotides that decrease gene expression post-transcriptionally by complementary binding to the mRNA 3-untranslated region in a sequence-specific manner, resulting in cleavage or translational repression of the target mRNA7. Mature miRNA molecules, ranging in size from 19 to 23 nt, are cleaved from 70110 nt hairpin precursors. miRNAs have unique expression patterns in various cell types and have been implicated in a number of biological processes including cell proliferation, cell death, fat metabolism, insulin secretion, and hematopoiesis8. Many miRNAs have been correlated with various kinds of cancers and function as oncogenes or tumor suppressor genes. Generally, genes encoding miRNAs located in chromosomal regions that are amplified in cancers function as oncogenes while those deleted in cancers may act as tumor suppressors5. The association of miRNA with breast cancer pathogenesis is supported by the studies examining expression of miRNAs in breast cancer cell lines and clinical samples 1.There is also increasing evidence to suggest that miRNAs may be responsible for a large proportion of breast cancer heterogeneity4. miRNA profiling studies have led to the identification of miRNAs that are aberrantly expressed in human breast cancer, with miR-10b, miR-125b and miR-145 being down-regulated and miR21, and miR-155 being up-regulated5. Recently, significant amounts of miRNA have been found in extracellular human body fluids including blood plasma, urine, saliva and semen 9. Moreover, some circulating miRNAs in the blood have been successfully revealed as biomarkers for several cancers, cardiovascular disease, brain injury and liver injury9. Even if the diagnostic tools which are currently used for diagnosis of breast cancer are important and necessary; lack of sensitivity and specificity for early detection for protein markers, varied interpretation of results on using mammography, adequacy and generality of candidate biomarkers for receptor analysis necessitates an improved and more effective method3. Therefore, recent studies focus on identifying biomarkers that may help in early diagnosis, determination of prognosis and prediction of treatment response, in due course contributing to more favorable patient outcomes. Studies are now continually reporting the presence of miRNAs in circulation system and body fluid, and hypotheses that their potential for use as novel biomarkers for disease and physiological states including invasive cancers, diabetes mellitus and even pregnancy. Their exceptional stability in the visceral tissue formulates them to be easily detectable and quantifiable in the circulation system 5. There is

2|Page

also increasing evidence in the presence of type specific miRNAs at higher level in serum from patients with age-and sex matched healthy volunteers3.

3|Page

Hypothesis:
Differential expression of circulatory miRNA is associated with molecular heterogeneity and sensitivity to chemotherapeutic treatment in breast cancer patients.

Objectives: General objective:

To investigate predictive potential of circulatory microRNAs in treatment response and in subtype identification among breast cancer patients.

Specific objectives:
Analysis of circulatory miRNAs in breast cancer patients Testing the use of circulatory microRNAs in predicting responsiveness or resistance to a specific panel of chemotherapy. Looking at the possibility of using circulatory microRNAs as diagnostic tool in specifying subtypes of breast cancer. Search for possible target-miRNA in the treatment of breast cancer find out differences in circulatory miRNAs due to polymorphism

Significance of the research:

Breast cancer case fatality rates are highest in economically disadvantaged countries, where survival is worsened due to the advanced stage of disease at initial presentation combined with inadequate resources to provide standard cancer therapy10. In Ethiopia, breast cancer is typically a fatal disease with high mortality unlike the experience of the Western world where breast cancer is frequently treatable, and with lower mortality. In almost every type of cancer, attempts have been made to identify molecular markers that could distinguish between different subtypes of tumors in order to choose the most efficient therapy. The treatment for breast cancer has to also rely on specific, effective and early diagnostic procedures for successful personalized medicine. Complexity of sample taking procedures, lack of precision on repeated analysis, lack of early and specific diagnosis and need for specialized personal in the area of invasive diagnosis necessitates biomarkers that can be measured repeatedly and non-invasively. Such markers would need to be sensitive and specific enough to aid in the detection of breast cancer at an early stage,

4|Page

would monitor progression of the disease, and could predict the individual patients response to treatment. It is well known that, the degree of response to chemotherapy provides important prognostic information, and patients achieving a complete pathologic response show significantly longer diseasefree and overall survival than nonresponders16. However, to increase the efficacy of such treatment, and to reduce its toxic effects on normal tissue, determining specific criteria and biomarkers which predict the selection for responsiveness or resistance to a specific treatment is yet to be established. Gene expression signatures of sensitive and resistant cell lines might help in this aspect14. Specific cancer characteristics, both genetic and epigenetic, are detectable in the plasma and serum of cancer patients and may be useful as a tool for: early detection, diagnosis and follow-up of cancer patients. Over the last decade mRNA arrays have been investigated extensively for this use, and disease-specific and prognostic signatures have been identified. However, so far these techniques have not been implemented in clinical practice, mainly because of the requirement for fresh tumor material, problems with the reproducibility when the method is applied to different platforms, complicated bioinformatics due to massive amounts of data, and extensive costs11. Even if studies on using circulating miRNAs as diagnostic tool is still in its infancy; evidences show a positive correlation between these potentially noninvasive biomarkers and cancer diagnosis3. miRNAs due to their small size are relatively resistant to RNase degradation suggesting they would be eminently suitable candidates for detection in biological fluids11. One of the big advantages of miRNAs is their stability, and it has been shown that cell-free miRNAs in body fluids are stable under harsh conditions such as high temperatures, extreme pH values, repeated freeze-thaw cycles and long-term storage12. Large number of studies has been done on the diagnostic application of microRNAs focusing on comparing normal tissues to tumors. But, due to the need for novel and targeted therapy for breast cancer, it will be more important to correlate miRNA expression with tumor subtypes or clinical parameters. miRNA expression signatures may turn out to be an efficient tool for identifying subgroups of breast cancer patients who will benefit from alternative treatment regimens producing a desired or intended result. The massive polymorphism which is seen in miRNAs might also have a differing effect on gene and protein expression which represent another type of genetic variability that can influence the risk of breast cancer11.

5|Page

Ethical consideration:
This study will be approved by the relevant research ethics committee, and informed written consent will be collected from participants. The confidentiality of the study subject data will be maintained by coding before analysis.

Research methodology:
Tumor specimen collection and analysis of ER, PR, and HER2/neu status Breast tumor specimens will be obtained from patients, and tissue samples will be immediately snapfrozen in liquid nitrogen and will be stored at -800C. The ER, PR, and HER2/neu status of patients will be determined by immunohistochemistry on formalin-fixed paraffin embedded sections as per the routine procedures. State which histological type and stage of tumor tissues will be collected? Blood Collection and serum preparation Venous blood (6mL) will be collected before and after 24 weeks of chemotherapy (chemotherapy will depend upon type/stage of tumor and the duration/cycles of chemotherapy will also vary) and it will be placed in a serum separator tube. The blood will be centrifuged at 1600 rpm for 5 min and the serum aliquot will be transferred into Eppendorf tubes, followed by a 15 min high speed centrifugation at 12,000 rpm to completely remove cell debris, leaving only circulating RNA. Extraction of total RNA Total RNA (including miRNA) will be extracted from 400L of serum by using RNA isolation kit following manufacturer instruction. And the purified RNA will be stored at -800C until analysis. Extraction and quantification of specific miRNAs Real time quantitative reverse transcription (Real-time q-RT-PCR) methodology will be used to evaluate expression level of specific miRNAS from serum samples. The relative miRNA levels in serum from breast cancer patients and controls? will be determined using comparative Ct method. Stratification of miRNA data Patient data will be sorted into different catagories based on ER, PR and HER2/neu status, and specific miRNA expression which will characterize each specific molecular subtype will be identified. The patient

6|Page

data will also be sorted based on the panel of chemotherapy administered, and in each group the type of specific circulatory miRNAs which are expressed by resistant and responsive patients (based on pathologic complete response) will be reported.

7|Page

References:
1. Vivien Wang and Wei Wu1. MicroRNA: A New Player in Breast Cancer Development. Journal of Cancer Molecules 2007; 3(5): 133-138. 2. Jorge S Reis-Filho, Lajos Pusztai. Using DOIGene expression profiling in breast cancer: classification, prognostication, and prediction. The lancet 2011;378(9805): 1812 1823 3. Claire Corcoran, Anne M. Friel, Michael J. Duffy, John Crown, and Lorraine ODriscoll1. Intracellular and Extracellular MicroRNAs in Breast Cancer. Clinical Chemistry 2011; 57(1): 1832 4. Aoife J Lowery, Nicola Miller, Amanda Devaney, Roisin E McNeill, Pamela A Davoren, Christophe Lemetre, Vladimir Benes, Sabine Schmidt, Jonathon Blake, Graham Ball and Michael J Kerin. MicroRNA signatures predict oestrogen receptor, progesterone receptor and HER2/neu receptor status in breast cancer. Breast Cancer Research 2009. 11(3):1-18 5. Sidney W. Fu, Liang Chen and Yan-gao Man. miRNA Biomarkers in Breast Cancer Detection and Management. Journal of Cancer 2011. 2:116-122 6. Thomas Litman, Nana Jacobsen, Mikkel Nrholm, Christian Glue, Nina Stahlberg & Sren Mller. MicroRNA profiling of breast cancer tissue using an LNA-based microarray. METHODS 2007. 1-2 7. Yee-Ming Lee1, Jen-Yi Lee1, Chao-Chi Ho, Qi-Sheng Hong, Sung-Liang Yu, Chii-Ruey Tzeng, Pan-Chyr Yang and Huei-Wen Chen. MicroRNA 34b as a tumor suppressor in estrogendependent growth of breast cancer cells. Breast Cancer Research 2011. 13; 1-30 8. Lisa Wenrich, Mark Landers, Dan Krissinger, Peter Welch, and Christopher Adams. miRNA profiling in breast cancer using the NCode Rapid miRNA Labeling System. Quest 2007. 5;1: 9. Andrey Turchinovich, Ludmila Weiz, Anne Langheinz and Barbara Burwinke. Characterization of extracellular circulating microRNAs. Nucleic acid research 2011; 1-11 10. Benjamin O. Anderson, Cheng-Har Yip, Scott D. Ramsey, Rafael Bengoa, Susan Braun, Margaret Fitch, Martijn Groot, Helene Sancho-Garnier and Vivien D. Tsu. Breast Cancer in LimitedResource Countries: Health Care Systems and Public Policy. The Breast Journal 2006; 12 (1): S54 S69 11. G P George and R D Mittal. MicroRNAs: Potential biomarkers in cancer. Indian Journal of Clinical Biochemistry 2010; 25 (1): 4-14 12. Bianca Mostert, Anieta M. Sieuwerts, John W.M. Martens, Stefan Sleijfer. Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients.2011; 1-30 13. Balazs Gyrffy, Pawel Surowiak and Hermann Lage. Application of Microarrays for the NATURE

rediction of Therapy Response in Breast Cancer. Cancer genomics & proteomics 2005; 2: 255-264

8|Page

14. Charles L. Shapiro, and Abram Recht. Side effects of adjuvant treatment in breast cancer. The New
England Journal of Medicine 2001; 344(26): 1997-2008

15. Kelly H. Salter, Chaitanya R. Acharya, Kelli S. Walters, Richard Redman, Ariel Anguiano, Katherine S. Garman, Carey K. Anders, Sayan Mukherjee, Holly K. Dressman, William T. Barry, Kelly P. Marcom, John Olson, Joseph R. Nevins, Anil Potti1. An Integrated Approach to the Prediction of Chemotherapeutic Response in Patients with Breast Cancer. PLoS ONE 2008.; 3(4): 1-8 16. Norbert Avril, Stefanie Sassen, and Rebecca Roylance. Response to therapy in breast cancer. The journal of nuclear medicine 2009; 50(5): 55S-63S

9|Page