The origin of metastatic disease: clues from genomics 7/13/2011

Tumor evolution

Metastasis: Current questions When do tumor cells leave the primary tumor? Are metastases clonal? Do metastases evolve at the metastatic site? Do different tumor types have different metastatic origins? Nguyen, D. J Pathol 2011; 223:

Why do we care? Treatment for metastasis is virtually non-existent Some treatments increase the likelihood of metastasis Analyzing the primary tumor may indicate the genetic make-up of metastases Diagnostics do not exist for metastases, but may be required if metastases are genetically dissimilar to the primary tumor CSCs might be a source of metastasis that are refractory to current treatment

Genomic studies of metastases How do we track the differences between primary tumors and mets? Loss of heterozygosity: two distinct alleles exist in normal tissue, one is lost in tumor Copy number: number of alleles or chromosomes increases or decreases Epigenetic markers: methylation states

Loss of heterozygosity Caused by – Non-disjunction – Recombination – Double strand break repair – Deletion Detect with SNPs or microsatelllites

Copy Number Measure of genomic instability Detected by sequencing (single cell) or CGH (tissue)

CGH Comparative Genomic Hybridization Label tumor vs. normal tissue

Metastasis: Current questions When do tumor cells leave the primary tumor? Are metastases clonal? Do metastases evolve at the metastatic site? Do different tumor types have different metastatic origins? Nguyen, D. J Pathol 2011; 223:

Metastasis: Current questions When do tumor cells disseminate? – Early: DTCs or Mets have fewer or different mutations than the primary tumor – Late: DTCs or Mets look like aggressive primary tumor Are metastases clonal? – The entire met or all mets in the body look the same Do metastases evolve at the metastatic site? – No: Mets look like primary tumor – Yes: Mets show significant divergence from the primary tumor Do different tumor types have different metastatic origins? – Mets look like primary tumor in some cancers – Mets look different than primary tumor in other cancers

When do tumor cells disseminate?

Disseminated tumor cells Metastasis Primary tumor

DTCs often do not cluster with metastases or primary tumors

Conclusions and Caveats DTCs exist very early in tumor progression Cytokeratin marker causes bias

Are Metastases Clonal?

Copy Number Analysis Liu et al. Nature Medicine 15, (2009)

Metastases are clonal in prostate cancer Metastases found in different regions of the body are clonal, implying a single cell of origin Liu et al. Nature Medicine 15, (2009)

Metastases are not clonal in breast cancer Wu et al. Clin Cancer Res April 1, ; 1938

Primary tumors are similar to metastases

Copy number analysis of single cells 6% coverage Navin et al. Nature 472, 90–94 (07 April 2011)

Copy number analysis of single cells in breast tumors Stromal cells Tumor cells Navin et al. Nature 472, 90–94 (07 April 2011)

Copy number analysis of single cells

Clinical examples of tracing metastatic origin Breast cancer – Her2-pos – 65 PTs with 42 macrometastases and 18 micrometastases – In HER-2-positive PTs, 26 of 29 metastases were HER-2-positive – HER-2 amplification and ER and PR status were conserved in mets in 71% of cases Strein et al. Pathol Res Pract Apr 15;206(4):253-8

Similarities between primary tumor and metastases in colorectal cancer Jones et al. PNAS March 18, 2008 vol. 105 no

Screened 233 known mutations in PTs and Mets Only seven not found in both Jones et al. PNAS March 18, 2008 vol. 105 no

Implications of this data Mets arise late from aggressive, advanced carcinoma cells Implies that genes required for primary tumor growth might confer metastatic ability

Primary tumors are significantly different than metastases

Clinical examples of tracing metastatic origin Fehm et al. Breast Cancer Research :R59

Clinical examples of tracing metastatic origin Fehm et al. Breast Cancer Research :R59

Significant divergence of Mets from PTs in breast cancer Ramaswamy et al. Nature Genetics 33, (2002)

Methylation states change between primary tumor and metastases in prostate cancer Prostate cancer cell lines Yegnasubramanian et al. Cancer Res March 15, ; 1975

Methylation states change between primary tumor and metastases Yegnasubramanian et al. Cancer Res March 15, ; 1975

Methylation states change between primary tumor and metastases Yegnasubramanian et al. Cancer Res March 15, ; 1975

Hypermethylation occurs in breast cancer metastases Mehrotra et al. Clin Cancer Res. Vol. 10, , 2004.

Interpretations of this data Metastases require additional mutations to successfully grow at a distant site Three possible scenarios: – Metastatic cells leave the primary tumor early on – Metastatic cells leave the primary tumor late but then require additional mutations to colonize a distant organ – Clones exist in primary tumor but are not detected

The best current example of using genomics to understand metastatic origin

Metastases occur late in pancreatic cancer Yachida et al. Nature 467, 1114–1117 (28 October 2010)

Conclusions Some metastases are genetically similar to the primary tumor, while others are not Metastases can be clonal or heterogeneous Information from primary tumor could help to determine characteristics about metastases Primary tumor diagnostics may not be sufficient Origin of metastatic precursor may be early or late in tumor progression These studies imply that it may not be cancer-type dependent Studies are limited…

Stoecklein, N. H. and Klein, C. A. (2010). International Journal of Cancer, 126: 589–598.

Conclusions Seems to be mostly patient or study dependent!

Future directions Next generation sequencing – Can get more accurate data – Single cell resolution Analysis of CTCs and DTCs “Personalized medicine”