The Role of CCR9 and Melanoma Metastasis to Small Intestine. Farin Amersi, MD Samuel Oschin Comprehensive Cancer Center Cedar Sinai Medical Center

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1 The Role of CCR9 and Melanoma Metastasis to Small Intestine Farin Amersi, MD Samuel Oschin Comprehensive Cancer Center Cedar Sinai Medical Center

2 BACKGROUND Melanoma is the fifth most common cancer. Estimated that 67,190 cases (5% cancer cases) will be diagnosed this year. (Am. Cancer Soc, 2007) Postmortem specimens 50-60% metastases to GI tract

3 BACKGROUND Melanoma metastatic to GI tract in only % of all pts 1/3 of all metastatic disease of the GI tract is from melanoma Postmortem specimens 50-60% metastases to GI tract

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5 CHEMOKINES -RECEPTORS

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7 Chemokines and their receptors determine the metastatic destination of tumor cells. Final distribution of metastases reflects relative abundance of chemokines in different organs Neutralizing the interaction of chemokines and their receptors impairs metastasis of cancer cells

8 THE MECHANISM DETERMINING THE DIRECTIONAL MIGRATION AND INVASION OF MELANOMA CELLS INTO SPECIFIC ORGANS IN THE GASTROINTESTINAL TRACT NEEDS TO BE ESTABLISHED

9 OVERVIEW BACKGROUND HYPOTHESIS METHODS RESULTS PLANNED STUDIES

10 TECK / CCL25 Thymus expressed chemokine (TECK) initially reported to be expressed by thymic cells TECK expression recently localized to epithelium of small intestine but not colon or stomach CCR9 is the only known receptor for TECK; expressed at high levels by CD4+ and CD8+ lymphocytes in small bowel

11 Zabel et al, JEM 1999, 190(9):

12 The Role of Thymus-Expressed Chemokine and its Receptor CCR9 on Lymphocytes in the Regional Specialization of the Mucosal Immune System K. Papadakis,, J. Prehn,, V. Nelson, A. Cheng, S.W. Binder, P.D. Ponath,, D.P. Andrew, and S.T. Targan Journal of Immunology, 2000; 165(9):

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15 CCR9 and Immune Diseases In PBL, CCR9+ lymphocytes markedly elevated in small bowel Crohn s,, but not in pure colonic Crohn s TECK expression intensely expressed in small bowel but was not expressed in either normal or inflamed colon. Gastroenterology, 2001; 121:

16 QUESTIONS Is CCR9 expressed in melanoma cell lines generated from metastases to the small intestine Are there differences in CCR9 expression in melanoma cell lines derived from small intestinal metastases when compared to cell lines derived from metastases to other GI organs

17 QUESTIONS If CCR9 is expressed only in melanoma cell lines derived from small intestinal metastases, will these findings correlate with our analysis using paraffin embedded tissue. Is CCR9 expressed in the primary or regional lymph nodes

18 QUESTIONS If CCR9 is expressed only in small intestinal metastases, will these findings correlate with overall survival If CCR9 is expressed in the primary tumor or lymph nodes, can it be utilized as a prognostic indicator for future metastatic progression

19 HYPOTHESIS CCR9 is expressed in melanoma cell lines derived from small bowel Melanoma cells that express CCR9 functionally, respond to TECK in a manner that facilitates metastases of these cells from the primary site or lymph nodes to small bowel

20 METHODS: CELL LINES RNA extraction Reverse transcription Real Time PCR Cell migration/invasion assay

21 CELL LINES Eight cell lines from melanoma metastasis to small bowel from JWCI tissue repository Fifteen cell lines from melanoma metastasis to other distant organs Liver- 4 Pancreas-1 Lung- 3 Adrenal- 2 Kidney- 2 Colon- 2 Gastric- 1

22 RESULTS CCR9 expressed in 8/8 (100%) small bowel cell lines CCR9 not expressed in 15/15 metastatic cell lines to other distant organs

23 CCR9/GAPDH mrna CCR9 Expression FL UK PC DM MG HC MP SJ MOLT4 SMALL BOWEL MELANOMA CELL LINES

24 CCR9 Expression CCR9/GAPDH mrna liver adrenal colon kidney pancreas stomach small bowel Cell lines lung MOLT

25 Further Studies Complete analysis of paraffin specimens from small bowel metastasis/distant organs Validate CCR9 Expression by flow cytometry TECK expression in paraffin tissue from small bowel metastasis/distant organs Immunohistochemistry Migration/Invasion Assay in SB cell lines

26 METHODS PARAFFIN EMBEDDED TISSSUE 198 STAGE 4 MELANOMA PATIENTS Small bowel resections - 96 Surgical resection to other distant sites 23 STAGE 1 PRIMARY MELANOMA PATIENTS - 7 matched pairs to stage IV pts with small bowel metastasis - 16 stage 1 primary