Van’t Veer et al, Nature 415:530-536 (2002) Primary breast carcinomas Can identify an expression profile that correlates with incidence of metastases Suggests bulk primary tumor already has properties that predispose to metastasis That is, not (only) rare variant metastatic cells

Ramaswamy et al, Nature Genetics 33: 49-54 (2003) Miscellaneous collection of 12 metastases and 64 primary tumors of same histological types - all adenocarcinomas Can identify an expression profile of 128 genes that distinguishes primaries from metastases Some primaries show the “metastasis pattern” Analyzed available data sets and found that the 128 gene set could split primaries into two sets, one of which showed the “metastasis pattern” and had poor prognosis - same result with a 17 gene set Suggests bulk primary tumors already have properties that predispose to metastasis That is, not (only) rare variant metastatic cells

128 gene signature 17 gene signature Clustered by all genes

17 gene signature

Kang/Massague et al Preprint Breast cancer cell line Select variants highly metastatic to bone They “breed true” They have a characteristic expression profile Transfection of 2/3 of the overexpressed genes -> increased metastasis Random isolation and screening of clones from parent line identifies clones with the “metastatic signature” These unselected clones ARE metastatic Therefore there ARE preexisting variant cells in the parent population The “metastatic signature” is overlaid on the “poor prognosis signature” of van’t Veer DIFFERENCE - this was done with cell lines

In vivo selection of metastatic variants

Overexpression enhancing metastasis

In vitro isolation of preexisting metastatic variant clones

Conclusion There IS a global difference between tumors with a predisposition to metastasize Within such tumors there ARE preexisting variant cells that are more metastatic than the bulk population Can isolate these metastatic variants and they are ‘stably’ different Poor prognosis with variants Good prognosis Poor prognosis