Integrative multi-region molecular profiling of primary prostate cancer with synchronous lymph node metastasis to characterize the biologically dominant clone

Localized prostate cancer is composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing subclones that may drive the biological trajectory of the disease. Previous large scale sequencing efforts have focused on the evolutionary process of metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we performed multi-region, targeted DNA/RNA next generation sequencing (NGS) and constructed phylogenetic trees from 14 patients with LN metastasis (88 primary prostate cancer foci with 23 synchronous LN metastases) to better define the molecular features of primary disease most likely to spread to the LNs. Of eight primary prostate cancer cases with evidence of extra-prostatic extension (EPE), phylogenetic analysis supported this region as the likely source of LN metastasis in four cases. In two patients with organ-confined disease and LN metastasis, sub-clonal seeding and clonal evolution was observed, with LN metastasis likely arising from a Gleason Grade Group 5 focus. Cribriform pattern was observed in seven patients in both LNs and the primary tumor foci most clonally related. Driver alterations, either oncogenic gene fusions or somatic mutations (e.g., CDK12 , FOXA1 ), were shared among primary tumor and LN metastatic foci. Collectively, we found that a combination of histopathologic and molecular factors, including tumor grade, EPE, cellular morphology (e.g., cribriform pattern), and oncogenic genomic alterations were associated with synchronous LN metastasis. More work is needed to better define the molecular features of primary prostate cancer foci most likely to give rise to metastasis to improve risk stratification, guide treatment allocation, and inform novel therapeutic strategies.