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Background: Adding immunotherapy to first-line chemotherapy might improve outcomes for patients with advanced or recurrent endometrial cancer. We aimed to compare carboplatin and paclitaxel versus avelumab plus carboplatin and paclitaxel as first-line treatment with avelumab given concurrent to chemotherapy and as maintenance after the end of chemotherapy. Methods: MITO END-3 is an open-label, randomised, controlled, phase 2 trial conducted at 31 cancer institutes, hospitals, and universities in Italy. Eligible patients were aged 18 years or older with histologically confirmed advanced (FIGO stage III–IV) or recurrent endometrial cancer, an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1, and no previous systemic anticancer therapy as primary treatment for advanced or metastatic disease. Participants were randomly assigned (1:1) using a computerised minimisation procedure stratified by centre, histology, and stage at study entry, to either receive carboplatin (area under the curve [AUC] 5 mg/mL × min) and paclitaxel (175 mg/m2; standard group) intravenously every 3 weeks for six to eight cycles or avelumab (10 mg/kg intravenously) added to carboplatin and paclitaxel (experimental group) every 3 weeks and then every 2 weeks as a single maintenance treatment after the end of chemotherapy until disease progression or unacceptable toxicity. Patients, treating clinicians, and those assessing radiological examinations were not masked to study treatment. The primary endpoint was investigator-assessed progression-free survival, measured in the intention-to-treat (ITT) population. Patients who received at least one dose of study drug were included in the safety analysis. Experimental group superiority was tested with 80% power and one-tailed α 0·20. This trial is registered with ClinicalTrials.gov (NCT03503786) and EudraCT (2016–004403–31). Findings: From April 9, 2018, to May 13, 2021, 166 women were assessed for eligibility and 39 were excluded. 125 eligible patients were randomly assigned to receive carboplatin and paclitaxel (n=62) or avelumab plus carboplatin and paclitaxel (n=63) and included in the ITT population. The median follow-up was 23·3 months (IQR 13·2–29·6) and was similar between the two groups. 91 progression-free survival events were reported, with 49 events in 62 patients in the standard group and 42 events in 63 patients in the experimental group. The median progression-free survival was 9·9 months (95% CI 6·7–12·1) in the standard group and 9·6 months (7·2–17·7) in the experimental group (HR of progression or death 0·78 [60% CI 0·65–0·93]; one-tailed p=0·085). Serious adverse events were reported more frequently in the experimental group (24 vs seven events in the standard group); neutrophil count decrease was the most frequent grade 3–4 adverse event (19 [31%] of 61 patients in the experimental group vs 26 [43%] of 61 patients in the standard group). Two deaths occurred in the experimental group during treatment (one respiratory failure following severe myositis [possibly related to treatment] and one cardiac arrest [not related to treatment]). Interpretation: Adding avelumab to first-line chemotherapy deserves further testing in patients with advanced or recurrent endometrial cancer, although consideration of mismatch repair status is warranted.

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Supplementary Figure Legends

Supplementary Figure 1. "Phase 2-Eligible" Population. Supplementary Figure 2. ALKA-372-001 and STARTRK-1 Dosing Regimens.

Background: Information about the adherence to scientific societies guidelines in the ‘real-world’ therapeutic management of oncological patients are lacking. This multicenter, prospective survey was aimed to improve the knowledge relative to 2017-2018 recommendations of the Italian Association of Medical Oncology (AIOM). Patients and methods: Treatment-naive adult patients with pancreatic adenocarcinoma were enrolled. Group A received adjuvant therapy, group B received primary chemotherapy, and group C had metastatic disease. The results on patients accrued until 31 October 2019 with a mature follow-up were presented. Results: Since July 2017, 833 eligible patients of 923 (90%) were enrolled in 44 Italian centers. The median age was 69 years (range 36-89 years; 24% >75 years); 48% were female; 93% had Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 or 1; group A: 16%, group B: 30%; group C: 54%; 72% Nord, 13% Center, 15% South. In group A, guidelines adherence was 68% [95% confidence interval (CI) 59% to 76%]; 53% of patients received gemcitabine and 15% gemcitabine þ capecitabine; median CA19.9 was 29 (range 0-7300; not reported 15%); median survival was 36.4 months (95% CI 27.5-47.3 months). In group B, guidelines adherence was 96% (95% CI 92% to 98%); 55% of patients received nab-paclitaxel þ gemcitabine, 27% FOLFIRINOX, 12% gemcitabine, and 3% clinical trial; median CA19.9 was 337 (range 0-20220; not reported 9%); median survival was 18.1 months (95% CI 15.6-19.9 months). In group C, guidelines adherence was 96% (95% CI 94% to 98%); 71% of patients received nabpaclitaxel þ gemcitabine, 16% gemcitabine, 8% FOLFIRINOX, and 4% clinical trial; liver and lung metastases were reported in 76% and 23% of patients, respectively; median CA19.9 value was 760 (range 0-1374500; not reported 9%); median survival was 10.0 months (95% CI 9.1-11.1 months). Conclusions: The GARIBALDI survey shows a very high rate of adherence to guidelines and survival outcome in line with the literature. CA19.9 testing should be enhanced; nutritional and psychological counseling represent an unmet need. Enrollment to assess adherence to updated AIOM guidelines is ongoing.

BackgroundHER2 amplification is detected in 3% of patients with colorectal cancer (CRC), making tumors in the metastatic setting vulnerable to double pharmacological HER2 blockade. Preclinical findings show that it also might impair response to anti-epidermal growth factor receptor (EGFR) treatment.Subjects and MethodsPatients with KRAS exon 2 wild-type metastatic CRC underwent molecular screening of HER2 positivity by HERACLES criteria (immunohistochemistry 3+ or 2+ in ≥50% of cells, confirmed by fluorescence in situ hybridization). A sample of consecutive HER2-negative patients was selected as control. A regression modeling strategy was applied to identify predictors explaining the bulk of HER2 positivity and the association with response to previous anti-EGFR treatment.ResultsFrom August 2012 to April 2018, a total of 100 HER2-positive metastatic CRC tumors were detected out of 1,485 KRAS exon 2 wild-type screened patients (6.7%). HER2-positive patients show more frequently lung metastases (odds ratio [OR], 2.04; 95% confidence interval [CI], 1.15–3.61; p = .014) and higher tumor burden (OR, 1.48; 95% CI, 1.10–2.01; p = .011), and tumors were more likely to be left sided (OR, 0.50; 95% CI, 0.22–1.11; p = .088). HER2-positive patients who received treatment with anti-EGFR agents (n = 79) showed poorer outcome (objective response rate, 31.2% vs. 46.9%, p = .031; progression-free survival, 5.7 months vs. 7 months, p = .087).ConclusionTesting for HER2 should be offered to all patients with metastatic CRC because the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Patients with HER2-amplified metastatic CRC are less likely to respond to anti-EGFR therapy.Implications for PracticePatients with HER2-amplified/overexpressed metastatic colorectal cancer (mCRC) harbor a driver actionable molecular alteration that has been shown in preclinical models to hamper efficacy of the anti-epidermal growth factor receptor (EGFR) targeted therapies. The present study confirmed that this molecular feature was associated with worse objective tumor response and shorter progression-free survival in response to previous anti-EGFR therapies. Moreover, it was found that the occurrence of this biomarker is unlikely to be predicted based on main clinicopathological features. Therefore, HER2 status assessment should be included in the molecular diagnostic workup of all mCRC for speedy referral to clinical trials encompassing HER2-targeted double blockade independently of previous anti-EGFR treatment.

Introduction: Gene fusions are strong driver alterations in various cancers, increasingly diagnosed with multiple testing techniques. ROS1 fusions can be found in 1-2% of non-small cell lung cancer...

AimsNeurotrophic Tropomyosin Kinase Receptor 1 (NTRK1) gene encodes for the protein Tropomyosin-related kinase A (TRKA). Deregulated activity of TRKA has been shown to have oncogenic potential. We present here the results of an immunohistochemical (IHC) observational cohort study of TRKA expression together with gene copy number (GCN) assessment in various solid tumours.MethodsFormalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess NTRK1 gene rearrangements and/or individual GCN gain. All patients underwent this molecular assessment within the phase I ALKA-001 clinical trial.Results1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayed NTRK1 gene rearrangement and 15/17 NTRK1 GCN gain by FISH. No correlation was found between intensity of TRKA IHC staining and number of copies of NTRK1.ConclusionsTRKA expression can be found in 1.6% of solid tumours and can be paralleled by NTRK1 gene rearrangements or mostly GCN gain. The prognostic and translational therapeutic impact of the latter remains to be established.

Entrectinib, a potent oral inhibitor of the tyrosine kinases TRKA/B/C, ROS1, and ALK, was evaluated in two phase I studies in patients with advanced or metastatic solid tumors, including patients with active central nervous system (CNS) disease. Here, we summarize the overall safety and report the antitumor activity of entrectinib in a cohort of patients with tumors harboring NTRK1/2/3, ROS1, or ALK gene fusions, naïve to prior TKI treatment targeting the specific gene, and who were treated at doses that achieved therapeutic exposures consistent with the recommended phase II dose. Entrectinib was well tolerated, with predominantly Grades 1/2 adverse events that were reversible with dose modification. Responses were observed in non–small cell lung cancer, colorectal cancer, mammary analogue secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as >2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1–NTRK1-rearranged lung cancer.Significance: Gene fusions of NTRK1/2/3, ROS1, and ALK (encoding TRKA/B/C, ROS1, and ALK, respectively) lead to constitutive activation of oncogenic pathways. Entrectinib was shown to be well tolerated and active against those gene fusions in solid tumors, including in patients with primary or secondary CNS disease. Cancer Discov; 7(4); 400–9. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 339

Neurotrophic Tropomyosin Kinase Receptor 1 (Formalin-fixed, paraffin-embedded consecutive samples of different tumour types were tested for TRKA expression. Samples showing TRKA IHC staining in at least 10% of cells were analysed by fluorescence in situ hybridisation to assess1043 samples were tested and annotation for histology was available in 1023. Most of the samples were colorectal adenocarcinoma (CRC) (n=550, 52.7%) and lung adenocarcinoma (n=312, 29.9%). 24 samples (2.3%) were biliary tract carcinoma (BTC). Overall, 17 (1.6%) samples were characterised by TRKA IHC expression (four weak, eight moderate, five strong): 9/17 lung adenocarcinoma, 3/17 CRC, 3/17 BTC, 1/17 thyroid cancer and 1/17 cancer of unknown primary. Of these, 1/17 with strong TRKA IHC staining displayedTRKA expression can be found in 1.6% of solid tumours and can be paralleled by

Patients with metastatic colorectal cancer (mCRC) refractory to standard therapies have a poor prognosis. In this setting, recruitment into clinical trials is warranted, and studies driven by selection according to individual tumor molecular characteristics are expected to provide added value. We retrospectively analyzed data from patients with mCRC refractory to or following failure of standard therapies who were enrolled into phase I/II clinical studies at the Niguarda Cancer Center based on the presence of a specific molecular profile expected to represent the target of susceptibility to the experimental drug(s). From June 2011 to May 2016, 2044 patients with mCRC underwent molecular screening. Eighty patients (3.9%) were enrolled in ad hoc studies; the median age was 60 years (range 36–86) and the median number of previous treatment lines was five (range 2–8). Molecular characteristics exploited within these studies were MGMT promoter hypermethylation (48.7%), HER2 amplification (28.8%), BRAF V600E mutation (20%), and novel gene fusions involving ALK or NTRK (2.5%). One patient (1%) had RECIST (Response Evaluation Criteria In Solid Tumors) complete response (CR), 13 patients (16.5%) experienced a partial response (PR), and 28 (35%) stable disease (SD). Median progression-free survival (PFS) was 2.8 months (range 2.63–3.83), with 24% of patients displaying PFS >5 months. Median growth modulation index (GMI) was 0.85 (range 0–15.61) and 32.5% of patients had GMI >1.33. KRAS exon 2 mutations were found in 38.5% of patients, and among the 78 patients with known KRAS status, those with wild-type tumors had longer PFS than those with mutated tumors (3.80 [95% CI 2.80–5.03] vs. 2.13 months [95% CI 1.77–2.87], respectively, p = 0.001). Median overall survival (OS) was 7.83 months (range 7.17–9.33) for all patients, and patients with KRAS wild-type tumors had longer OS than those with mutated tumors (7.83 [95% CI 7.33–10.80] vs. 7.18 months [95% CI 5.63–9.33], respectively, p = 0.06). This single-institution retrospective study indicates that in a heavily pretreated population approximately 4% of mCRC tumors display a potential actionable molecular context suitable for therapeutic intervention. Application of molecular selection is challenging but improves clinical outcome even in later lines of treatment.

Background. Metastatic colorectal cancer (CRC) remains mostly incurable, with a survival of about two years only. It has been recently proved that CRCs with genetic defects in the mismatch-repair pathway (MMRd), occurring in 15% of early CRC but only in 5% of metastatic CRC, present with a high tumor mutational burden (TMB), which results in an increased number of neoantigens that can be recognized by the immune system. Indeed, treatment with the anti-programmed cell death protein 1 (PD-1) immune checkpoint inhibitor pembrolizumab or nivolumab is effective in inducing durable objective responses in metastatic CRC MMRd cases. These results are quite remarkable considering that the clinical efficacy was independent from RAS mutations, which constrain the use of targeted treatments and negatively affect prognosis. We recently showed in preclinical models that the pharmacological treatment with temozolomide (TMZ) can induce the inactivation of MMR genes and consequently trigger an increase in immunogenic neoantigens. This suggests that TMZ could be used to prime MMR proficient (MMRp) tumors for response to checkpoint inhibitors. Accordingly, mCRC patients recruited in previous clinical trials where TMZ was administered, acquired alterations of MMR genes upon treatment and showed remarkable increase in TMB at disease progression (PD). We thus designed the ARETHUSA clinical trial to test whether a priming course with TMZ in patients can sensitize mCRC to the anti-PD1 inhibitor pembrolizumab. Methods. Arethusa is a 2-cohorts, phase II trial consisting of three different phases (NCT03519412). During screening-phase, 344 mCRC patients with RAS-extended mutations who failed standard therapies will be tested for MMR status. MMRd CRC patients will proceed directly to trial-phase for immediate pembrolizumab treatment (expected N=14). MMR-proficient (MMRp) patients will be further tested for TMZ sensitivity via assessment of expression of O6-methylguanine-DNA methyltransferase (MGMT) by immunohistochemistry and by promoter methylation analysis. Expected 67 IHC-negative, promoter methylation-positive MMRp patients will thus be eligible for priming-phase and will receive TMZ until PD; TMB will then be assessed on tumor biopsies at resistance. Those patients that will have >20 mutations/megabase (expected N=20) will proceed to that trial-phase and will be treated with pembrolizumab. Overall response rate (primary outcome), Progression Free, and Overall Survival, and treatment related toxicities (secondary outcomes) in MMRp pembrolizumab-treated patients will be estimated. Treatment efficacy and toxicity within pembrolizumab-treated MMRd cohort will be used for comparison. Pre- and post-TMZ biopsies and longitudinal blood and stool collection during priming and trial phases will allow for discovery of predictive molecular markers and for the assessment of integrated tumor and (immune)environment evolution in response to therapy. Citation Format: Silvia Marsoni, Giovanni Germano, Andrea Sartore Bianchi, Filippo Pietrantonio, Nicola Personeni, Alessio Amatu, Emanuela Bonoldi, Emanuele Valtorta, Ludovic Barault, Federica Di Nicolantonio, Filippo de Braud, Lorenza Rimassa, Armando Santoro, Silvia Ghezzi, Andrea Cassingena, Giovanna Marrapese, Loredana Lupica, Giulia Siravegna, Giuseppe Rospo, Cosimo Martino, Luca Lazzari, Paolo Luraghi, Nabil Amirouchene-Angelozzi, Alberto Bardelli, Salvatore Siena. Pharmacological inactivation of DNA repair to improve response to immunotherapy: The Arethusa trial in metastatic 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 CT215.

The introduction into clinical practice of KRAS mutational status for selection of patients has dramatically improved the results from use of anti-EGFR monoclonal antibodies cetuximab or panitumumab for metastatic colorectal cancer. More refined selection of patients by means of other molecular alterations, for example BRAF, PIK3CA, and NRAS has enabled further increases in responses to first-line and other therapy for metastatic disease. Elucidation of differences among specific subtypes of KRAS mutations affecting sensitivity, and identification of other mechanisms by which tumor cell resistance is acquired, revealing “druggable” molecular targets to overcome resistance, are clearly a priority of clinical research. Recent data have revealed potentially different activity of the G13D KRAS mutation in conferring resistance to cetuximab. This review examines the most recent evidence available on codon 13 mutation in metastatic colorectal cancer, including both preclinical and available clinical data, indicating differences between codon 13 and other KRAS mutations and analyzing its prognostic and predictive use in EGFR-targeted therapy.

Objectives Evaluation of cardiotoxicity of novel targeted therapies tyrosine-kinase inhibitors. Cardiotoxicity is an important side effect of drugs used to treat cancer patients. First data about cardiotoxicity concern anthracyclines, 5-fluorouracil and recently trastuzumab. We have less information about toxicity induced by a new category of drugs, the tyrosine kinase inhibitors (TKIs), which have modified the natural history of many diseases. It is possible that the cardiac damage resulting from treatment with TKIs is an understimated phenomenon than its real incidence. The predictor factors of cardiotoxicity are prior history of hypertension and coronary artery disease. The most relevant side-effects in clinical practice are hypertension and left ventricular dysfunction. Conclusions Collaboration between oncologists and cardiologists is necessary to identify patients who could manifest cardiac dysfunction, to control and prevent cardiotoxicity and to decide which is the best diagnostic and therapeutic algorithm for patients of this kind.

Summary Background We previously found that dual HER2 blockade with trastuzumab and lapatinib led to inhibition of tumour growth in patient-derived xenografts of HER2 -amplified metastatic colorectal cancer. In this study, we aimed to assess the antitumour activity of trastuzumab and lapatinib in patients with HER2-positive colorectal cancer. Methods HERACLES was a proof-of-concept, multicentre, open-label, phase 2 trial done at four Italian academic cancer centres. We enrolled adult patients with KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive metastatic colorectal cancer refractory to standard of care (including cetuximab or panitumumab), an Eastern Cooperative Oncology Group performance status of 0 or 1, and at least one measurable lesion. We defined HER2 positivity in tumour samples by use of immunohistochemistry and fluorescence in-situ hybridisation in accordance with our previously validated colorectal cancer-specific diagnostic criteria. Eligible patients received intravenous trastuzumab at 4 mg/kg loading dose followed by 2 mg/kg once per week, and oral lapatinib at 1000 mg per day until evidence of disease progression. The primary endpoint was the proportion of patients achieving an objective response (defined as complete response or partial response), which was assessed by independent central review in the intention-to-treat population. This trial is registered with EudraCT, number 2012-002128-33. Findings Between Aug 27, 2012, and May 15, 2015, we screened 914 patients with KRAS exon 2 (codons 12 and 13) wild-type metastatic colorectal cancer and identified 48 (5%) patients with HER2-positive tumours, although two died before enrolment. Of these patients, 27 were eligible for the trial. All were evaluable for response. At the time of data cutoff on Oct 15, 2015, with a median follow-up of 94 weeks (IQR 51–127), eight (30%, 95% CI 14–50) of 27 patients had achieved an objective response, with one patient (4%, 95% CI −3 to 11) achieving a complete response, and seven (26%, 95% CI 9–43) achieving partial responses; 12 (44%, 95% CI 25–63) patients had stable disease. Six (22%) of 27 patients had grade 3 adverse events, which consisted of fatigue in four patients, skin rash in one patient, and increased bilirubin concentration in one patient. No grade 4 or 5 adverse events were reported. We detected no drug-related serious adverse events. Interpretation The combination of trastuzumab and lapatinib is active and well tolerated in treatment-refractory patients with HER2-positive metastatic colorectal cancer. Funding Associazione Italiana Ricerca Cancro (AIRC), Fondazione Oncologia Niguarda Onlus, and Roche.

Background Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). Patients and methods We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m2 days 1–5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error=0.059 (one-sided), power=0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. Results The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9–24.6]. Objective response rate was 3.4% (90% CI 0.2–15.3), disease control rate 48.3% (90% CI 32.0–64.8), median OS 6.2 months (95% CI 3.8–7.6), and median PFS 2.6 months (95% CI 1.4–2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. Conclusion Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.

Background: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. Methods: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. Results: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. Conclusions: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Background KRAS mutations occur in 35–45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance. Methodology/Principal Findings We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with ≥2 alterations (p

2517 Background: Entrectinib (formerly RXDX-101) is a potent and selective oral small molecule inhibitor of the TrkA/B/C, ROS1, and ALK kinases. Schedule A (fasted, 4d on/3d off for 3wks, 1 wk rest...

Summary Background The antiepidermal growth factor receptor (antiEGFR) monoclonal antibodies cetuximab and panitumumab have good clinical activity in about 10% of patients with metastatic colorectal cancer that is resistant to chemotherapy. The molecular mechanisms underlying clinical response or resistance to these agents are unknown. Methods Tumours from 31 patients with metastatic colorectal cancer who had either an objective response (n=10) or stable disease or progressive disease (n=21) after treatment with cetuximab or panitumumab were screened for genetic changes in EGFR or its immediate intracellular effectors. Specifically, we assessed the EGFR copy number and the mutation profile of the EGFR catalytic domain and of selected exons in KRAS , BRAF , and PIK3CA . Results Eight of nine of patients with objective responses who were assessable by fluorescence in-situ hybridisation (FISH) had an increased EGFR copy number. By contrast, one of 21 non-responders assessable by FISH had an increased EGFR copy number (p vs non-responders, Fisher's exact test). The mutation status of the EGFR catalytic domain and its immediate downstream effectors PIK3CA , KRAS , and BRAF did not correlate with disease response. In colorectal-cancer cell lines, the concentration of cetuximab that completely inhibited proliferation of cells with amplified EGFR copy number did not affect proliferation of cells with unamplified EGFR . Interpretation We propose that the response to antiEGFR treatment has a genetic basis and suggest that patients might be selected for treatment on the basis of EGFR copy number. Published online April 14, 2005 DOI 10.1016/S1470-2045(05)70102-9

583 Background: O(6)-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein. About 40% of mCRC display MGMT deficiency due to promoter hypermethylation, leading to susceptibility to treatment with cytotoxic alkylating agents. We previously reported that objective clinical response to dacarbazine is confined to tumors harboring MGMT promoter hypermethylation (Amatu et al. Clin Cancer Res 2013). Based on these findings, we conducted a phase II study with TMZ in MGMT-deficient mCRCs after failure of standard therapies (EudraCT 2012-003338-17). Methods: We screened mCRC patients for MGMT promoter hypermethylation on archival FFPE specimens by methylation-specific PCR (MSP). All eligible patients underwent tumor biopsy prior to start of treatment. Patients received TMZ 200 mg/m2 po qd days 1-5 q28 until progression or intolerable toxicity. We used a Fleming single-stage design to determine whether PFS rate at 12 week would be ≥35% (H0≤15%, type I error=0.059 (1-sided) and power=0.849). Secondary endpoints included response rate (RR), disease control rate (DCR), overall survival (OS), and association of clinical outcomes with quantitative MGMT evaluation in serum and fresh tissue. Results: 150 patients were screened: 54 (36%) presented MGMT promoter hypermethylation and 29 were eligible and enrolled from December 2012 to May 2014 (M 69%, median age 60y [38-77], mean number of previous treatment lines 5.6). A median of 2 cycles of TMZ were administered (range 1-5). Primary endpoint was not achieved, since PFS rate at 12 weeks was 10.3% (90% CI: 2.9-24.6). DCR was 48.3% (90% CI: 32.0-64.8), median OS 6.2 months (95% CI: 3.7-7.6), and median PFS 2.6 months (95% CI: 1.4-2.7). G3/4 toxicities occurring in ≥10% of patients included: thrombocytopenia (10.3/13.8%), neutropenia (0/10.3%) and increased bilirubin (11.1/0%). Exploratory MGMT quantitative analysis is in progress. Conclusions: In mCRC patients treated with TMZ, selection according to MGMT promoter hypermethylation by MSP did not provide meaningful PFS rate at 12 weeks. Biomarkers analyses on serum and fresh tumor biopsy are ongoing to identify the subgroup of mCRCs benefiting from treatment. Clinical trial information: 2012-003338-17.

Aim: RXDX-101 is a small molecule inhibitor of TrkA, TrkB, TrkC, ROS1 and ALK, with high potency and selectivity. RXDX-101 demonstrated potent pharmacological activity in preclinical studies and is potentially first-in-class against the Trk family of kinases. This study aims to determine MTD, PD, PK and anti-tumor activity in patients with advanced cancer and applicable molecular alterations. Methods: Phase 1 dose escalation in patients with advanced solid tumors. RXDX-101 was dosed orally once/day in a 4 day on, 3 day off schedule for 3 weeks, followed by a 7 day rest period, in continuous 28-day cycles. Minimum of 3 patients were enrolled at each dose level. Endpoints include safety, PK, and tumor response by RECIST. Results: 19 patients have been treated at 6 dose levels (100, 200, 400, 800,1200 and 1600 mg/m2). RXDX-101 has been well tolerated to date; the MTD has not been reached. No DLT has been seen at any dose level. The most common AEs (mainly grade 1-2) considered possibly treatment-related included asthenia, paresthesias, nausea and diarrhea. Possibly related grade 3/4 AEs include asthenia and increased lipase. No treatment-related SAEs were observed. A patient with colorectal carcinoma (TrkA+) has a PR and is in cycle 2. Two patients with NSCLC (1 ROS1 + ; 1 ALK+) have also achieved PRs. An additional patient with neuroblastoma (ALK+) has a PR and is currently in cycle 16. Two patients have had prolonged stabilization of their disease and remain on treatment: a patient with NSCLC (ALK+) in cycle 14 and a patient with pancreatic cancer (ROS1+) in cycle 11. PK analysis shows maximum concentrations of RXDX-101 were generally achieved within 2 to 4 hours following dosing and exposure increased in an approximate dose proportional manner up to doses of 800 mg/M2 with minimal accumulation following multiple doses. Mean terminal half-life was 21- 32 hours and steady state reached within 4 days. Conclusions: RXDX-101 has been well tolerated in patients with advanced solid tumors. Early responses in patients with 3 different relevant molecular alterations are promising. PK profile seems suitable for once daily dosing, which is being evaluated. A global phase I/II trial was recently initiated. Disclosure: F.G.M. De Braud: Consultant or Advisory Roles Novartis Compensated Bristol-Myers Squibb; ompensated Roche Compensated Stock Ownership No Honoraria No Research Funding Yes; E. Ardini: Senior Scientist, Nerviano Medical Sciences Employee Compensated; M. Martignoni: CLIOSS Employee, Clinical Leader Compensated; A. Isacchi: Director, Nerviano Medical Sciences Compensated Employee; P. Pearson: Consultant Role Ignyta, Inc. Compensated Stock Ownership Ignyta, Inc.; D. Luo: Ignyta, Inc. Employee Sr. Director, Clinical Operations Compensated; J.L. Freddo: Consultant Role; Member Board of Directors Ignyta, Inc. Compensated Stock Ownership, Ignyta, Inc. Yes; S. Siena: Consultant or Advisory Role Amgen Compensated Bayer Compensated Celgene Compensated Sanofi Compensated Roche Compensated; tock Ownership No Research Funding Bayer. All other authors have declared no conflicts of interest.

2502 Background: RXDX-101 is an oral small molecule inhibitor of TrkA, TrkB and TrkC, as well as ROS1 and ALK, with high potency and selectivity. RXDX-101 has demonstrated potent pharmacological ac...

3581 Background: O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O6-guanine in DNA. Approximately 40% of colorectal cancers (CRCs) display MGMT deficiency due to promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O6–guanine site, inducing base pair mismatch, therefore activity of dacarbazine could be enhanced in CRCs lacking MGMT. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if KRAS wild type). Methods: All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m2 i.v. qd for 4 consecutive days q21 until PD or intolerable toxicity. We employed a Simon two-stage design to determinate if the ORR would be ≥ 10%. Secondary endpoints included association of response, PFS and disease control rate with MGMT status. Results: Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of 3 cycles of dacarbazine [range 1-12]. Grade 3-4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), anemia (18%). Overall, 2 patients (3%) achieved partial response (PR) and 8 patients (12%) had stable disease (SD). Disease control rate (PR+SD) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Conclusions: Objective clinical responses to dacarbazine in metastatic CRC patients are confined to those tumors harbouring epigenetic inactivation of the DNA repair enzyme MGMT. Clinical trial information: 2011-002080-21.

3570 Background: O6-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from O6-guanine in DNA. Roughly 40% of colorectal cancers (CRCs) display MGMT deficiency due to promoter hypermethylation leading to silencing of the gene. Alkylating agents exert their antitumor activity by DNA methylation at the O6–guanine site, inducing base pair mismatch; therefore, activity of these compounds may be enhanced in CRCs lacking MGMT (Esteller, 2004; Pegg, 1990). Despite anecdotal reports about activity of dacarbazine in CRC (Shacham-Shmueli, 2011) no clinical trials assessed monotherapy with dacarbazine for metastatic CRC. Methods: We conducted a phase II study of dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines and cetuximab or panitumumab if KRAS WT), PS=0-1, with adequate organ function, and measurable disease as per RECIST criteria assessed by CT scans at baseline and every 8 weeks. All patients had tumor tissue assessed for MGMT as promoter hypermethylation. Patients received dacarbazine 250 mg/m2 i.v. d1-4, q21 until PD or untolerable toxicity (with a maximum of 6 cycles in case of SD). We employed a Simon, two-stage design to determinate if the ORR of dacarbazine treatment would be ≥ 10%. Secondary endpoints were association of response/resistance with loss of expression of MGMT, PFS and disease control rate. Results: In the first stage, from June 2011 to November 2011 we enrolled 40 pts (male 68%, median age 67 y [range 29-81], PS=0 50%, KRAS mut 55%). Pts received a median of 3 cycles [range 1-11]. Toxicities included (All Grades/Grade 3-4 %): fatigue (58/5), constipation (38/0), nausea/vomiting (35/0), anemia (20/2.5), platelet count decrease (10/5). 36 pts were evaluable for objective response. Overall, 2 pts (5%) achieved PR and 5 pts (12.5%) had SD. All pts with PR had tumors with MGMT promoter hypermethylation. Conclusions: Having exceeded the boundaries of futility (at least 2 objective responses in the first stage), the trial proceeds to the second stage, enrolling 28 more patients. Analysis of MGMT promoter hypermethylation and association with sensitivity/resistance is performed at the end of the second stage.

3553 Background: The most relevant recent advance in the treatment of metastatic colorectal cancer is the fact that cure is still possible under very selected conditions. However the goal of chemotherapy remains palliative in the great majority of patients, justifying less toxic innovative approaches in so- called “window of opportunity trials”. We have pursued this idea in a phase II study of cetuximab monotherapy in chemo-naive patients with advanced colorectal cancer beyond any possibility of curative resection. Methods: Patients with non-resectable metastatic colorectal cancer (at least two metastatic sites and/or otherwise inoperable metastatic disease) were treated with cetuximab (400 mg/m2 week 1 and 250 mg/m2 weekly thereafter) until progressive disease (PD) or unacceptable toxicity. The primary end-point was objective response; secondary end-points were: stable disease, time to treatment failure and time to progression. According to Simon’s two-stage design, the number of responses/patients to stop the trial was 0/10 for stage 1 and 3/29 for stage 2. Results: Of the 44 patients screened, 42 (97%) had EGFR-expressing tumors. Thirty-nine patients (median age: 69) initiated the treatment. Two had grade 3 allergic reactions to cetuximab at the first administration leading to treatment discontinuation. The most common adverse event was skin toxicity, which occurred in 90% of the patients ( 31% grade 2, 10 % grade 3). We observed 1 complete response, 3 partial responses, 13 stable diseases (5 of which were minor responses), and 22 PD. The duration of the 4 responses were 12, 9, 9 and 6 months. Median time to progression was 2.0 months. Conclusions: The study is negative because the response rate is low. However, the duration of benefit in the few responding patients is such that it is imperative to find the molecular determinants of cetuximab activity in these cases. [Table: see text]

Background ERBB2 amplification occurs in 5% of RAS wild-type metastatic colorectal cancer (mCRC) and it has been shown to be a target for treatment with 2 HER2-directed combinations of trastuzumab and lapatinib or trastuzumab and pertuzumab. We present long-term clinical results of trastuzumab and lapatinib (HERACLES-A trial) at 6.7 years (82 months) follow-up and focus on central nervous system (CNS) recurrences. Patients and Methods Patients had histologically confirmed KRAS exon 2 (codons 12 and 13) wild-type and HER2-positive mCRC. HER2 positivity was assessed by immunohistochemistry and in situ hybridization HERACLES diagnostic criteria. Patients were treated with intravenous trastuzumab 4 mg/kg loading dose, then 2 mg/kg once per week, and oral lapatinib 1000 mg per day until disease progression or toxicity. Patients who presented with symptoms or signs of CNS disease received brain computed tomography scan or magnetic resonance imaging. Results A total of 35 patients received trastuzumab and lapatinib and 32 were evaluable for response. One patient (3%) achieved complete response (CR), 8 (25%) partial response, and 13 (41%) stable disease. Therefore, response rate was 28%. Median progression-free survival was 4.7 months (95% confidence interval [CI] 3.7–6.1). Median overall survival was 10.0 months (95% CI 7.9–15.8). One patient achieved sustained CR still maintained at 7 years of follow-up. Progression in the central nervous system (CNS) occurred in 6 (19%) of 32 patients. Conclusions Long-term (6.7 years) follow-up analysis of HERACLES-A supports using of trastuzumab and lapatinib as treatment reference for KRAS wild-type, chemorefractory HER2-positive mCRC. In this subset of patients, prolongation of survival is accompanied by CNS recurrences that will require diagnostic and therapeutic attention in future studies. Clinicaltrials. Gov identifier: NCT 03225937.