HER2-amplified Colorectal Cancer Research Articles

Abstract 1923: Cytoplasmic localization of KRAS in Her2-amplified colorectal cancer induces an altered dependency of the oncogenic signaling pathway from RAS/MAPK to PI3K/AKT

Abstract Nowadays, KRAS-G12C specific inhibitor has been used for the treatment of patients with KRAS mutated non-small cell lung cancer, but not for colorectal cancer (CRC) patients. Recent clinical trials have shown that the efficacy of a single KRAS inhibitor treatment was limited for CRC due to multiple primary/adaptive resistance mechanisms. In the CRC, clinical trial with dual inhibition of KRAS-G12C and EGFR has demonstrated that combination therapy had improved partial response rate, suggesting that primary resistance to KRAS inhibitor is mainly caused by EGFR-mediated adaptation. Meanwhile, more than half of the primary resistance mechanisms are still unknown. To understand the intrinsic resistance mechanisms to KRAS inhibitors in CRC, KRAS G12C- or G12D-positive CRC patient-derived cells (PDCs) were established from surgically resected tumor specimens. Analysis of cell proliferation under KRAS inhibitor treatment or KRAS knockdown revealed these PDCs were not solely dependent on KRAS mediated growth signaling. To clarify the compensatory activated pathways in KRAS inhibitor, inhibitor library screening and sequencing analysis were performed among 8 KRAS-mutated PDCs. As a result, PDCs were categorized into EGFR inhibitor with KRAS inhibitor sensitive group and PI3K pathway activating group harboring PIK3CA mutation or/and ERBB2 amplification with KRAS mutation. To explore the novel intrinsic resistance mechanisms, we deeply analyzed one of the PDC named JC261, harboring PIK3CA, ERBB2, and KRAS alterations. We observed that PI3K pathway was highly activated in JC261 cells, and cell proliferation was mainly dependent on PI3K/AKT/mTOR pathway regardless the existence of KRAS-G12C mutation. To examine whether Her2 amplification contributes to the PI3K pathway dependency, we established Her2 knockout JC261 cells. Notably, Her2 knockout suppressed Her2-Her3-PI3K signaling and restored KRAS-MAPK dependency. To analyze the mechanism of how KRAS-MAPK pathway was activated in Her2 knockout cells, we focused on KRAS localization, since the MAPK activation is known to initiate from plasma membrane localization of KRAS after appropriate modifications. Remarkably, immunofluorescence staining of KRAS revealed that KRAS was aberrantly localized in the cytoplasm in JC261, while KRAS localization was changed to the plasm membrane in Her2 knockout JC261 cells. In addition, the cytoplasmic KRAS localization was also observed in Her2-amplified, KRAS-WT PDC (JC69). These results suggest that Her2 amplification may induce aberrant KRAS localization, resulting in low dependence on the KRAS-MAPK pathway. Thus, KRAS localization may regulate the balance between MAPK and PI3K signaling intensity. Citation Format: Kohei Maruyama, Satoshi Nagayama, Ryohei Katayama. Cytoplasmic localization of KRAS in Her2-amplified colorectal cancer induces an altered dependency of the oncogenic signaling pathway from RAS/MAPK to PI3K/AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1923.

Identification of MAPK and mTOR pathway alterations in HER2-amplified colorectal cancer.

185 Background: Human epidermal growth factor receptor 2 (HER2) amplification is now a highly actionable alteration for patients with colorectal cancer (CRC) with distinct combination therapies. Recent studies have shown that oncogenic KRAS, BRAF, and PIK3CA mutations, may serve as biomarkers of response to HER-directed treatments such as Trastuzumab and Tucatinib/Pertuzumab. In this study, we investigated the incidence of MAP kinase and mTOR pathway alternations in patients with HER2-amplified CRC. Methods: The cBioPortal for Cancer Genomics was utilized to obtain data and graphics on HER2 amplification in CRC. cBioPortal was queried to select 21 published studies that contain CRC specimens (assessed April 2023). Clinical, specimen, copy number alteration (CNA), and somatic mutation data of each study were downloaded and aggregated across all studies, followed by filtering, standardization, and harmonization to generate ~30 analysis-ready fields encompassing demographic variables, HER2 amplification, and KRAS/NRAS/PIK3CA/BRAF mutations. Results: Among a pool of 4822 CRC patients with known gender, 44.4% (2139) were female and 55.6% (2683) were male. The incidence of HER2 amplification in the overall cohort was 2.6% (123/4822) and was noted to be higher in males (3.1%, 82/2683) than in females (1.9%, 41/2139). The incidence of HER2 amplification was highest in Asian CRC patients (3.9%, 9/228), followed by Black CRC patients (3.0%, 9/298), and then Caucasian CRC patients (2.7%, 57/2118). Rates of HER2 amplification were similar in the colon and the rectum at 2.6% (61/2360 and 26/1015, respectively). After analyzing rates of concurrent MAPK and mTOR pathway mutations in patients with HER2-amplified CRC, KRAS mutations were noted to be the most common mutation in HER2-amplified colon cancer (27.9%, 17/61), but were less common in HER2-amplified rectal cancer (7.7%, 2/26). PIK3CA mutations were also common, with an incidence of 13.1% (8/61) in HER2-amplified colon cancer and 7.7% (2/26) in HER2-amplified rectal cancer. NRAS mutations occurred at comparable, low rates in HER2-amplified colon and rectal cancer, at 3.3% (2/61) and 3.8% (1/26) respectively. No BRAF mutations were reported in either disease group. Conclusions: In this study, we identified that MAPK and mTOR pathway alterations are relatively common among patients with HER2-amplified CRC. These somatic events appear to be more common in HER2-amplified colon cancer than rectal cancer. Next generation based molecular profiling is recommended to identify potential resistance mechanisms before initiation of HER2-directed therapy.

Somatic mutation profiling, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein expression in HER2-amplified colorectal cancer.

The status of human epidermal growth factor receptor 2 (HER2) for the prognosis in colorectal cancer (CRC) is controversial, and the characteristics of the somatic mutation spectrum, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein are unknown in HER2-amplified colorectal cancer (HACC). In order to explore these characteristics along with their correlation with clinicopathological factors and prognosis in HACC. Samples of 812 CRC patients was collected. After immunohistochemistry (IHC), 59 of 812 were found to be HER2-positive, then 26 of 59 samples were further determined to be HER2 amplification by fluorescence in situ hybridization (FISH). Somatic mutation profiling of HACC was analysed using whole exome sequencing (WES). Multiplex fluorescence immunohistochemistry (mIHC) was used for tumor-infiltrating leukocytes and tertiary lymphoid structures (TLSs), while PD-L1 protein was detected by IHC. Our results indicate that the detection rates of HER2 positivity by IHC and FISH were 7.3% and 3.2% respectively, and HER2 amplification is correlated with distant tumour metastasis. The somatic mutation profiling revealed no differences between HACC and HER2-negative CRC. However, TP 53 strongly correlated with poor prognosis in HACC. Furthermore, tumor-infiltrating T cells and TLSs in the tumor immune microenvironment, as well as PD-L1 expression, were higher in HACC than in HER2-negative controls. However, none of them were associated with the prognosis of HACC. In all, HER2 amplification is correlated with distant metastasis and TP53 gene mutation may be a potential protective mechanism of HACC.

Updates in the Treatment of Metastatic Colorectal Cancer

Molecular biomarker testing for all patients with metastatic colorectal cancer (CRC) has become increasingly important because identifying targetable alterations can lead to meaningful clinical benefits. At a minimum, testing should include RAS, BRAF mutational status, microsatellite instability status, HER2 expression, NTRK, and RET mutations. For HER2-amplified cancer, the NCCN Guidelines offer multiple treatment options, including trastuzumab in combination with tucatinib or pertuzumab, and trastuzumab-deruxtecan. Combination trastuzumab + tucatinib has recently received approval by the FDA for refractory RAS wild-type, HER2-amplified CRC. The addition of bevacizumab to trifluridine/tipiracil treatment has significantly prolonged median overall survival compared with trifluridine/tipiracil alone, regardless of molecular subtypes. KRAS G12C–targeted therapies are on the horizon, with several agents in ongoing studies. Furthermore, bilevel blockade is important when addressing MAP kinase pathway alterations.

Abstract B022: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Abstract Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induces tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a drop-out loss-of-function synthetic lethality screening with a shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at various levels could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2-amplified CRC and found marked sensitivity to pevonedistat in a CRC cell line carrying HER2 amplification. In these cells, long-term in vitro treatment with a combination of lapatinib and trastuzumab induces the emergence of colonies of long-term persisters (i.e., cells surviving to several weeks of HER2/EGFR blockade). These persisters display extremely slow growth and senescence features, however they fully recover upon removal of the blockade, mimicking disease relapse in patients after treatment suspension. Interestingly, persister colonies maintained sensitivity to pevonedistat, displaying a marked decrease when pevonedistat was added after six weeks of HER2/EGFR blockade, independently of the continuation of the blockade. These results unveil the possibility of employing pevonedistat in HER2-amplified CRC patients subsequently to tumor stabilization or regression by HER-2/EGFR blockade. Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Giulio Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B022.

176 (PB056) - The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Background: Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies.

Challenges in the Management of Patients With HER2-Amplified Colorectal Cancer.

Challenges in the Management of Patients With HER2-Amplified Colorectal Cancer.

Abstract 2611: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Abstract Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a "drop-out" loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2 amplified CRC cells and we observed cooperation between pevonedistat and HER2/EGFR blockade by the trastuzumab + lapatinib combination -the current standard treatment for HER2-amplified CRC. In addition, to confirm cooperation in reducing colony formation in vitro, we explored the effect of pevonedistat on long-term persisters, i.e., cell surviving to several weeks of HER2/EGFR blockade. Interestingly, we observed a marked decrease of cell colonies when pevonedistat was added to persisters survived to three weeks of trastuzumab + lapatinib treatment. These results unveil the possibility of testing in vivo the addition of pevonedistat subsequently to tumor stabilization or reduction by HER2/EGFR blockade, to promote further tumor regression. Citation Format: Federica Invrea, Alessandro Carugo, Sabrina Arena, Alberto Bardelli, Giulio F. Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2611.

Sensitivity of HER2-amplified colorectal organotypic cancer spheroids at ex vivo resistance to panitumumab and trastuzumab.

68 Background: HER2 amplification is an emerging biomarker in colorectal cancer (CRC) with increased copy number associated with improved clinical outcomes to HER2 targeting. RAS/RAF wildtype CRC also benefit from use of epidermal growth factor receptor inhibition (EGFRi). The sequencing of EGFRi versus HER2 inhibition in low copy number HER2 amplified CRC remains uncertain. Patient-derived cancer organoids (PDCOs) allow an ex vivo method to assess treatment sensitivity. We examined treatment sensitivity of a HER2 amplified PDCO at baseline and following resistance to panitumumab and trastuzumab. Methods: Following IRB-approval, fresh CRC tissue was cultured to maturation. After expansion, subcultures were treated with stepwise (20%) increase to physiologic Cmax of panitumumab (230ug/mL) and trastuzumab (180ug/mL). Threshold for escalation was median relative growth of +20% at 96h. Sensitivity was assessed on primary culture (RC1), panitumumab resistance (RC1-P) and trastuzumab resistance (RC1-T) using 96h of physiologic Cmax panitumumab, trastuzumab, and combination trastuzumab/pertuzumab. Individual sphere response was assessed for change in mean NADH autofluorescence intensity and ratio of NADH/FAD signal. Response was assessed at 96h in comparison to control using effect size of Glass’s Delta (GΔ). Results: Molecular profiling revealed HER2 copy number of 14 with no concurrent alterations in RAS, RAF, or PIK3CA. Time to resistance was similar between panitumumab (55 days) and trastuzumab (51 days). RC1 had baseline growth (+116%) which was reduced with single agent panitumumab (+17%, GΔ=1.40) with intermediate sensitivity to trastuzumab (+48%, GΔ=0.95) and trastuzumab/pertuzumab (46%, GΔ=0.99). Normalized NADH/FAD ratio revealed significant metabolic response to panitumumab (-20%, GΔ=0.66) and trastuzumab/pertuzumab (-35%, GΔ=1.16) with insignificant effect of single agent trastuzumab (-14%, GΔ=0.46). Following resistance to panitumumab, RC1-P had persistent growth with trastuzumab (+68%) which improved in combination trastuzumab/pertuzumab (+34%, GΔ=1.16). Following resistance to trastuzumab, RC1-T was insensitive to EGFRi with panitumumab including persistent growth (+58%, GΔ=0.70) and unchanged metabolism (+2%, GΔ=-0.10). Conclusions: Therapeutic dose escalation in a single PDCO of HER2 amplified CRC suggests improved sensitivity to EGFRi and dual HER2 targeting with trastuzumab/pertuzumab. Resistance to EGFRi resulted in persistent sensitivity to dual HER2 inhibition using trastuzumab/pertuzumab, however resistance to single agent trastuzumab. Resistance to trastuzumab resulted in future insensitivity to EGFRi. Molecular profiling at resistance revealed no pathologic alterations in EGFR or ERBB2 signaling, with ongoing analysis of transcriptional changes by RNAseq.

Radiomics predicts response of individual HER2-amplified colorectal cancer liver metastases in patients treated with HER2-targeted therapy.

The aim of our study was to develop and validate a machine learning algorithm to predict response of individual HER2-amplified colorectal cancer liver metastases (lmCRC) undergoing dual HER2-targeted therapy. Twenty-four radiomics features were extracted after 3D manual segmentation of 141 lmCRC on pretreatment portal CT scans of a cohort including 38 HER2-amplified patients; feature selection was then performed using genetic algorithms. lmCRC were classified as nonresponders (R-), if their largest diameter increased more than 10% at a CT scan performed after 3 months of treatment, responders (R+) otherwise. Sensitivity, specificity, negative (NPV) and positive (PPV) predictive values in correctly classifying individual lesion and overall patient response were assessed on a training dataset and then validated on a second dataset using a Gaussian naïve Bayesian classifier. Per-lesion sensitivity, specificity, NPV and PPV were 89%, 85%, 93%, 78% and 90%, 42%, 73%, 71% respectively in the testing and validation datasets. Per-patient sensitivity and specificity were 92% and 86%. Heterogeneous response was observed in 9 of 38 patients (24%). Five of nine patients were carriers of nonresponder lesions correctly classified as such by our radiomics signature, including four of seven harboring only one nonresponder lesion. The developed method has been proven effective in predicting behavior of individual metastases to targeted treatment in a cohort of HER2 amplified patients. The model accurately detects responder lesions and identifies nonresponder lesions in patients with heterogeneous response, potentially paving the way to multimodal treatment in selected patients. Further validation will be needed to confirm our findings.

Abstract 1414: Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer

Abstract Background: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality with limited precision strategies. The human epidermal growth factor receptor (HER2) is amplified in 5% of patients with metastatic CRC. Therapeutic studies have demonstrated activity of HER2 targeting regimens in the late-line treatment setting though little characterization has been done in the setting of not having prior anti-epidermal growth factor receptor antibody therapy or in the first-line treatment setting. Methods: Patient with metastatic colorectal cancer underwent next-generation sequencing (NGS) panel testing by Strata Oncology through the University of Wisconsin Precision Medicine Molecular Tumor Board. Patients were treated per multi-disciplinary recommendation across multiple lines of therapy and tracked by prospective registry (IRB#UW15068). HER2 copy numbers >/= 10 were used to determine amplification. A subset of patients with HER2-amplified CRC were treated with single agent trastuzumab. Results: HER2 amplifications were identified in 4 of 78 cases of metastatic CRC (5.1%). All biopsies were performed prior to exposure to anti-epidermal growth factor receptor inhibitors. Median estimated copy number (CN) was 55.5 [range 13-178]. There was no concurrent alterations in extended RAS, BRAF, PIK3CA, AKT or MTOR. All cases included concurrent mutations in TP53 at R175H (2), C135W, or C135F. Single agent trastuzumab was administered to two patients at standard dosing (trastuzumab 8mg/kg intravenous day 1 cycle 1 followed by 6 mg/kg intravenous day 1 cycle 2+ on a 21 day cycle). One patient was treated in the treatment refractory setting. They had resolution of bowel obstructive symptoms and a minor response on imaging. Additionally, a patient with a HER2 amplification (CN 178) was treated in the first-line setting with single agent trastuzumab. The patient had grade 1 fever with the first cycle and no other treatment related adverse events thereafter. The patient’s CEA decreased from 7,506 to 2,525 ng/mL with the first cycle of therapy and overall reduced to 6.5 ng/mL after 12 cycles of therapy. The patient has had a partial response per RECIST v1.1 response criteria with a reduction of cross-sectional diameter of their disease by 60%. The patient currently continues on this therapy without evidence of disease progression. Conclusions: HER2 amplified colorectal cancers are an important subtype of colorectal cancer. Here we describe the potential benefit of HER2 targeting with single agent trastuzumab and even demonstrate single agent activity in the first-line setting. Further studies should be done to further characterize the benefit of this low toxicity treatment option for patients with metastatic HER2 amplified colorectal cancer. Citation Format: Jeremy Kratz, Nataliya Uboha, Kayla Lemmon, Hannah Houtler, Mark Burkard, Dustin Deming. Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1414.

Combined blockade of MEK and PI3KCA as an effective antitumor strategy in HER2 gene amplified human colorectal cancer models

BackgroundTargeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy.MethodsWe have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer.ResultsLIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients.ConclusionsThese results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer.

Abstract CT005: Final results of the HERACLES trial in HER2-amplified colorectal cancer

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