Early relapse and increased biological aggressiveness have been observed clinically following radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). We therefore assessed serologic and histopathologic markers of tumorigenesis in distant untreated HCC post-ablation to determine whether these were associated with outcome.The study cohort comprised 13 patients from a prospective single arm study. All patients had 2 ablation sessions of multifocal HCC nodules 14d apart. Core biopsies of untreated tumors were acquired at baseline and at the time of the second ablation. Samples were stained immunohistochemically with Ki-67 (proliferation) and CD34 (microvasculature). Blood plasma was obtained at baseline and 2d post initial ablation and analyzed for HGF, VEGF-C, and Angiopoietin-2 by ELISA. Clinical follow-up ranged 7-25 mo. Patients were stratified as responders (complete remission or limited, delayed recurrence6mo; n=6) or as non-responders (any recurrence within 6mo or3 new tumors or any new tumor3cm thereafter ; n=7).In 3/7 non-responders, the Ki-67-index markedly increased in untreated tumors, whereas all responders were stable. Microvascular density strongly increased in a single non-responder only. HGF and Angiopoeitin-2 increased by30 % in 3/7 and 4/7 non-responders, respectively, whereas they were stable or decreased in responders. Overall, ≥ 2 biomarkers were elevated in 6/7 non-responders (85.7 %), whereas 4/6 responders demonstrated no increased biomarker and 2 patients 1 only (p = 0.002).RFA of HCC can produce pro-tumorigenic factors that induce effects in distant non-treated tumors. These may potentially function as biomarkers of clinical outcome.