Abstract Background: HER2 amplification is found in 5% of RAS wild type (RASWT) metastatic colorectal cancer (mCRC). Dual HER2 blockade with trastuzumab (T) and lapatinib (L), but not treatment with either drug alone, led to remarkable inhibition of tumor growth in patient-derived tumorgrafts of HER2-amplified mCRC. CRC-specific criteria for HER2 positivity by immunohistochemistry (IHC) and in situ hybridization (ISH) were defined retrospectively in 256 CRC paraffin embedded samples (HERACLES DGX criteria). The ensuing diagnostic algorithm was utilised to screen 1299 HER2-positive tumors for therapeutic targeting in patients in the HERACLES phase 2 trial. Methods: HERACLES was conducted at 4 Italian centres. Eligibility criteria were: RASWT exon 2, HER2 positivity, refractoriness to standard of care (including cetuximab or panitumumab), PS-ECOG < 1, and measurable. HER2 positivity was defined by IHC and ISH according to HERACLES DGX criteria. Patients (PTS) received T i.v. at 4 mg/kg loading dose followed by 2 mg/kg weekly, and L p.o. at 1000 mg daily, until progression. The primary endpoint was the objective response rate, assessed by independent central review. Secondary endpoints were progression-free survival and safety. The study was 85% powered to detect an objective response rate of >30% with a one-sided alpha level of 0.05 (EudraCT, number 2012-002128-33). Findings: Between August 27, 2012, and December 31, 2016, 69/1299 (5.3%) RASWT PTS were found HER2-positive. Of these, 33 were enrolled in HERACLES, and evaluable for response. At data cut-off (December 31, 2016), 10 (30.3%, 95% CI 17-47) of 33 heavily refractory PTS (median 5 prior regimens), achieved an objective response (2 complete and 8 a partial responses), 13 (39.3%, 95% CI 24-56) of 33 PTS had stable disease (SD) for a disease control rate of 70% (95% CI 52-82). Toxicity was mild with six (18%) of 33 PTS experiencing grade-3 side effects: fatigue (4), skin rash (1) elevated bilirubin (1). No drug-related SAEs were observed. Interpretation: HERACLES results prove that targeting HER2 is a successful therapeutic strategy in treatment-refractory HER2-positive mCRC patients, which can be easily implemented in clinical practice. Funding: Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus and Roche. Citation Format: Salvatore Siena, Andrea Sartore-Bianchi, Livio Trusolino, Cosimo Martino, Katia Bencardino, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Erika Martinelli, Fortunato Ciardiello, Patrizia Racca, Alessio Amatu, Laura Palmeri, Emanuele Valtorta, Silvia Ghezzi, Angelo Vanzulli, Daniele Regge, Silvio Veronese, Alberto Bardelli, Silvia Marsoni. Final results of the HERACLES trial in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT005. doi:10.1158/1538-7445.AM2017-CT005