IDENTIFICATION OF BIOMARKERS FOR EARLY DIAGNOSIS OF BREAST CANCER"

Transcription

1 IDENTIFICATION OF BIOMARKERS FOR EARLY DIAGNOSIS OF BREAST CANCER" Edmond Marzbani, MD December 2, 2008

2 Early diagnosis of cancer Many solid tumors are potentially curable if diagnosed at an early stage when the cancer can be surgically removed. It follows then that new methods to aid in early diagnosis of cancer are needed to increase cure rates.

3 Early diagnosis of breast cancer Breast cancer represents the most commonly diagnosed cancer in women. However, despite availability of routine screening with mammography, approximately 40% of breast cancers when diagnosed are not localized. In addition, the sensitivity of mammography itself declines significantly in younger, premenopausal women due to increased breast density.

4 Early diagnosis of breast cancer The identification of new biomarkers may facilitate development of a novel serum-based assay that would permit early detection of breast cancer. This would greatly enhance clinical management of disease.

5 Tumor shed proteins as assay target Tumor shed proteins represent one focus for serum based assays. Many challenges for development including abundance of nonspecific serum proteins such as albumin. May require larger tumor bulk for detection, reducing benefit of assay. May be complicated by transient shedding, rapid degradation, or clearance.

6 Serum antibodies as assay target Serum autoantibodies generated in response to tumor-associated antigens (TAAs) are promising alternative. Tumors express aberrant levels of mutated or modified forms of proteins associated with malignant growth. TAAs can be immunogenic and stimulate cellular and humoral immune responses.

7 Tumor associated antigens Image from Finn. (2008) Tumor Immunology. N Engl J Med 358:2704,

8 Benefits of serum autoantibodies Multiple benefits associated with using antibody responses to TAAs in serum assay. Serum antibody is stable in serum samples and can readily be detected with secondary antibodies. B-cells produce specific antibodies in large amounts after stimulation by small amount of tumor antigen, amplifying response. TAA-specific serum antibodies can be detected at high titer in patients with early stages of cancer.

9 Serum antibodies as assay target Most autoantibodies discovered thus far have had limited diagnostic value alone, requiring development of antibody panels in order to achieve predictive ability. Multiple reasons for this.

10 Serum antibodies as assay target 1. Frequencies of autoantibodies specific for a particular TAA in a cancer population are often low, ranging 10-30%. 2. May discriminate presence of malignancy but not between cancer types. 3. May arise as consequence of molecular events associated with cancer or other diseases.

11 Serum antibodies as assay target That said, promising models are being developed. A panel of four tumor antigens can discriminate sera from cancer patients vs. healthy donors with 74.8% power. IGFBP2, P53, HER2, Topoisomerase 2a.

12 Serum antibodies as assay target Current goal is to find candidates that will increase sensitivity and specificity of panel assay.

13 SEREX One powerful method for identification of targets of humoral immunity is the serological screening of cdna expression library, or SEREX.

14 SEREX illustration Membrane blocked. Incubated with serum. GAM-AP 2 Ab. Color development.

15 Experimental design Neu-transgenic mice harboring nonmutated neu were bred. Mice develop spontaneous breast cancers that are her positive and ER negative, mimicking premenopausal human breast cancer. Serum was drawn at regular intervals and mice were concurrently monitored for spontaneous development of breast cancer. Neu-tg mouse Tumor development

16 Experimental design Using SEREX, we probed a cdna library constructed from mouse mammary carcinoma cells with serum from Neu-tg mice to evaluate for autoantibody responses to TAAs.

17 Experimental design 1º screen + - 2º screen No further tests + in post-tumor serum, - in pre-tumor serum SCE and sequence. + in post-tumor serum, + in pre-tumor serum No further tests. - in post-tumor serum, - in pre-tumor serum.

18 Examples 1 screen 2 screen Isolation

19 Results A total of 3 x 10 4 phage clones were each screened with serum from 16 tumor bearing mice. Total of 23 positive clones were identified with primary screen. Total of 6 positive clones were identified with secondary screen. Later, 3 of these were found to be false positives and 3 were true positives.

20 Results 1 screen 2 screen Isolation #3 #13

21 Results 1 screen 2 screen Isolation #16 Pending

22 Results Single clone excision was performed and samples were sent to outside facility for sequencing.

23 Sequencing results Gene Symbol # of clones Full Gene Name RPL5 1 Ribosome protein L5. TNFAIP3/ 2 Tumor necrosis factor alpha-induced protein 3 A20

24 Time course of antibody development ** **

25 Discussion 1. SEREX is useful in identifying autoantibodies to tumor associated antigens. 2. Serum antibody response was detectable prior to clinical tumor development based on preliminary time course studies. 3. Next question: what is the biological relevance of the tumor associated antigens identified?

26 TNFaip3/A20 Expression is rapidly induced by TNF. Protein encoded by gene is zinc finger protein. Inhibits NF-kappa B activation as well as TNFmediated apoptosis. Knockout studies of gene in mice suggests that it is critical for limiting inflammation by terminating TNFinduced NK-kappa B responses.

27 TNFaip3/A20 A20 expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma. A20 expression closely associated with resistance to certain alkylating agents in glioblastoma cells and proposed as predictive marker associated with patient survival. A20 overexpression associated with development of resistance to TNFa-mediated apoptosis in breast tumor cellline MCF-7.

28 TNFaip3/A20 A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells. Vendrell, Ghayad, Ben-Larbi, Dumonet, Mechti, Cohen. Oncogene (2007) 26, A20 found to be negatively regulated by E2 treatment in MCF-7 cells. MCF-7 cells overexpressing A20 protein developed resistance to cytotoxic and proapoptotic effects of OH-Tam not due to loss or diminution of ER expression. A20 expression is increased in tumors with poor prognostic markers, that is, ER- status, PR- status, and high histiologic grade. Data suggest increased expression of A20 in breast carcinoma is associated with aggressive phenotype.

29 TNFaip3/A20 NF-kappa B genes have a major role in inflammatory breast cancer. Lerebours et al. BMC Cancer (2008) 8:41 Evaluated role of NF-κB-related genes in series of IBC patients due to involvement in cell proliferation, invasiveness, angiogenesis and inflammation with RT- PCR. TNFAIP3/A20 was one of five of the most strongly deregulated genes (overexpressed) in series out of total of 60.

30 RPL5 RPL5 is a component of the 60S ribosomal subunit (4 RNA species and 80 structurally distinct proteins). Located in the cytoplasm where it binds 5S rrnp to form complex involved in transport of other ribosomal components to nucleolus.

31 RPL5 MDM2 (double minute 2) is an oncogene which binds to and inhibits p53 but also has p53 independent negative effects on DNA double strand break repair. MDM2 gene amplification is found in sporadic breast cancers and overexpression is associated with accelerated tumor development. Recent paper demonstrated that RPL5 was found to bind MDM2 and inhibit it, permitting activation of p53. Dai and Lu. Journal of Biological Chemistry (2004) 279,

32 Conclusions These findings are promising candidates for use in a serum-based assay. Further studies in mice must be performed to prioritize antigens and potentially inform future human trials.

33 Future studies Future studies in mouse model should evaluate the frequency of autoantibody development in normal and tumor-bearing mice in a population. Tissue expression patterns should be examined to elucidate how the identified antigens are immunogenic and to determine whether there is restricted expression.

34 Translation to humans Future translation of these findings would involve: 1. Evaluating immunogenicity of TNFaip3 and RPL5 as tumor antigens in humans. Promising information regarding immunogenicity in humans was found. a. Cancer Immunome database revealed autoantibodies have been detected to RPL5 in one human patient (KY-CC-18). b. Autoantibodies have been detected against TNFIP1 and 9, suggesting this class can be immunogenic in humans as well.

35 Translation to humans 2. Evaluating whether autoantibody responses are present in those with breast cancer compared to those without breast cancer. 3. Evaluation of the frequency of these autoantibodies within the breast cancer population and whether they are present in other types of cancer.

36 Other directions Ultimately, the development of uniquely designed TAA arrays for autoantibody profiling in cancer may give rise to diagnostic chips with high sensitivity and specificity. These TAA arrays may also be useful for monitoring treatment response and predicting recurrence. Finally, TAA arrays specific for particular cancer type could be exploited for the development of cancer vaccines designed to boost immune system to suppress tumor.

37 Thanks Nora Disis Hailing Lu Amy Chang Ekram Gad Mei Wu Yifeng Zhou The entire Tumor Vaccine Group.

38 References Anderson and LaBaer. (2005) The Sentinel Within: Exploiting the Immune System from Cancer Biomarkers. Journal of Proteome Research. 4: Casiano et al. (2006) Tumor-associated Antigen Arrays for the Serological Diagnosis of Cancer. Molecular and Cellular Proteomics. 5: Mu-Shui Dai and Hua Lu. (2004) Inhibition of MDM2-mediated p53 Ubiquitination and Degradation by Ribosomal Protein L5. Journal of Biological Chemistry. 279 (43): Finn. (2008) Cancer Immunology. NEJM 358 (25): Heyninck and Beyaert. (2005) A20 inhibits NF-K-B activation by dual ubiquitin-editing functions. Trends in Biochemical Sciences 30:1-4. Lerebours et al. (2008) NF-kappa B genes have a major role in inflammatory breast cancer. BMC Cancer 8:41. Lu et al. (2006) The Tumor Antigen Repertoire Identified in Tumor-Bearing Neu Transgenic Mice Predicts Human Tumor Antigens. Cancer Res. 66 (19): Lu et al. (2008) Humoral Immunity Directed against Tumor-Associated Antigens As Potential Biomarkers for Early Diagnosis of Cancer. Journal of Proteome Research. 7: Vendrell et al. (2007) A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells. Oncogene. 26: