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Supplementary Figure from Temozolomide Treatment Alters Mismatch Repair and Boosts Mutational Burden in Tumor and Blood of Colorectal Cancer Patients

Supplementary Figure S4 - PDF file 19K, Ectopic expression of MET in cetuximab sensitive DIFI and LIM1215 cell lines

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRAS.Experimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer.Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03).Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657–66. ©2017 AACR.

Supplementary Legends and Figures

Supplementary Table S1

The file lists Supplementary Materials and Methods and relative References.

Supplementary Table S3

Supplementary Legends and Figures

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

Supplementary Tables S1-S3 - List of drug concentrations to generate resistant cell lines (S1); Drug concentrations applied in the screening depicted in Figure 6 (S2); Primers for gene amplification and sequencing (S3). Supplementary Figure S1. Synergistic activity of targeted therapy combinations in BRAF mutant colorectal cancer cells. Supplementary Figure S2. Amplification of mutant BRAF V600E confers resistance to combined EGFR and MEK targeting in colorectal cancer cells. Supplemental Figure S3. Cytotoxicity induced by ERK inhibition in VACO432 resistant to BRAF combination therapies.

Supplementary methods. Supplementary Table S1. Verification of the analytical sensitivity (lower limit of detection) of the KRASG12/G13 mutation-enrichment NGS assay. Supplementary Table S2. KRASG12/G13 mutations in archival tumor tissue, urine cell-free DNA (cfDNA) and plasma cfDNA. Supplementary Table S3. Systemic therapies in patients with serial urine and/or plasma cell-free DNA (cfDNA) collection. Supplementary Figure S1. Workflow and characteristics of the platform used to analyze cell-free DNA in urine and plasma. Supplementary Figure S2. Schematic of the mutation-enrichment next-generatin sequencing (NGS) assay for the detection of KRASG12/13 mutations in cell-free DNA (cfDNA). Supplementary Figure S2. Schematic of the mutation-enrichment next-generatin sequencing (NGS) assay for the detection of KRASG12/13 mutations in cell-free DNA (cfDNA). Supplementary Figure S3. Kaplan-Meier curves of overall survival (OS) based on the number of KRASG12/13 copies in cell-free DNA (cfDNA). Supplementary Figure S4. Kaplan-Meier curves of overall survival (OS) based on the concentration of cell-free DNA (cfDNA).

PDF file - 53K, Supplementary table 1 showing toxicities

Purpose:Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available.Experimental Design:Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach.Results:XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion.Conclusions:The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

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Purpose: 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 (CRC) display MGMT deficiency due to the 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).Experimental Design: All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m2 intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status.Results: Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1–12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors.Conclusion: Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT. Clin Cancer Res; 19(8); 2265–72. ©2013 AACR.

Purpose:Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need.Experimental Design:We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response.Results:Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice.Conclusions:These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, “maintenance” treatment with PARP inhibitors warrants further clinical investigation.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)–deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment. Significance: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599

Background Patients with solid tumours have high COVID-19 mortality. Limited and heterogeneous data are available regarding the immunogenicity of SARS-CoV-2 mRNA vaccines in this population. Methods and Findings This is a prospective, single-center, cohort study aiming at evaluating seroconversion in terms of anti-spike antibodies in a population of patients with solid tumours undergoing cancer therapy within 2 months before the second vaccine dose, as compared with a cohort of controls. Subjects who were not SARS-CoV-2 naïve were excluded. 171 patients were included in the final study population (150 vaccinated with BNT162b2, 87.7%; 21 with mRNA-1273, 12.3%) and compared with 2406 controls. Median follow-up time from the second dose of vaccination was 30 days (12-42; IQR: 26-34). Most patients had metastatic disease (138, 80.7%). Seroconversion rate was significantly lower in cancer patients than in controls (94.2% vs 99.8%, p70 years). A multivariate logistic model confirmed cancer as the only significant variable in impairing seroconversion (OR 0.03, p2, as the only one of impact (OR 0.07, p=0.012). Conclusions There is a fraction of 6% of patients with solid tumours undergoing cancer treatment, mainly with poorer performance status, who fail to obtain seroconversion after SARS-CoV-2 mRNA vaccines. These patients should be considered for enhanced vaccination strategies and carefully monitored for SARS-CoV-2 infection during cancer treatment.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient (MMRd) tumors often respond to immune checkpoint blockade (ICB). We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB) and, sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR proficient and KRAS mutant mCRC patients receive priming therapy with TMZ. Analysis of solid tissue biopsies and circulating tumor DNA (ctDNA) obtained after TMZ treatment revealed the emergence of TMZ mutational signature, alterations in MMR genes and increased TMB in 14 out of 16 patients. Genetic mutations induced by TMZ were dose-dependent and multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes such as the MSH6 T1219I variant which was detected in ctDNA and tissue of 13/14 (93%) of the cases. A subset of the patients whose tumors after TMZ priming displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment. Overall, we provide proof-of-concept that treatment of MGMT deficient/MMR proficient KRAS mutant mCRCs with TMZ can be tracked by mutational signature analysis and lead to inactivation of the MMR pathway, emergence of the TMZ mutational signature, TMB increase, and, in some cases, to disease stabilization during ICB. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personemi, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide [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 6262.

The definition of oligometastatic colorectal cancer (CRC) identifies a peculiar subpopulation of patients characterized by a limited metastatic spread of disease.1 Oligometastatic disease is defined as the involvement of up to two or occasionally three sites with five or sometimes more metastases that for their anatomic localization is amenable to local ablative therapies, thus rendering the patient free of disease.1-3 Thus, this subgroup is wide and has a significant cohort of patients with CRC. Among patients with oligometastatic CRC, those with liver-limited disease represent a more refined subset and should always be discussed in multidisciplinary teams since they appear to more likely benefit from multimodal approaches with curative intent.2,4-6 A perioperative systemic treatment integrated with surgical liver metastasectomy should be regarded as the best multimodal approach.2,7 However, the best drug regimen to be adopted for this subset of patients with CRC is still debatable and should be tailored case-by-case. Considering left-sided microsatellite stable, RAS and BRAF wild-type CRC, doublet cytotoxic regimens (FOLFIRI or FOLFOX) plus an anti-epidermal growth factor receptor (EGFR) drug can represent the best option on the basis of significant response rate (RR).8-10 However, triplet cytotoxic regimens FOLFOXIRI plus bevacizumab or anti-EGFR drug demonstrate an impressive RR up to 87%, which are increasingly regarded as potential novel neoadjuvant standard strategies.5,11 However, severe treatment-related toxicities have been reported in up to 80% of patients.11,12 Hence, both doublet or triplet combinations and anti-EGFR or antivascular endothelial growth factor are feasible options in this subset of patients. The generation of preclinical models such as patient-derived organoids (PDOs), recapitulating patient tumor histology and genetics, is emerging as a tool to predict treatment efficacy in oncology.13,14 Although genomics has already improved treatment choice in patients with CRC, especially for those carrying RAS/BRAF wild type, coclinical trials are becoming more and more important to directly test different treatment options in patient-derived tumors.15 Here, we present a proof-of-concept case report about the potential role of drug sensitivity testing in PDOs in the clinical decision making of oligometastatic CRC.

Purpose: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need. Experimental Design: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response. Results: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice. Conclusions: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, “maintenance” treatment with PARP inhibitors warrants further clinical investigation.

ObjectiveMutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC).DesignGenome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy.ResultsMethylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival.ConclusionThis five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available. Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach. Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion. Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1. These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504–15. ©2016 AACR.

Background: The analysis of circulating free tumor DNA (ctDNA) in blood, commonly referred to as liquid biopsy, is being used to characterize patients with solid cancers. Tumor-specific genetic variants can also be present in DNA isolated from other body fluids such as urine. Unlike blood, urine sampling is noninvasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumor DNA that clears from the glomerular filtration barrier (transrenal tumor DNA, hereafter trtDNA) are largely unexplored. Patients and Methods: Specimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole-exome sequencing (WES) was performed in DNA isolated from tissue, plasma, and urine. Results: Out of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumor alterations reside primarily in low-molecular-weight fragments. In patients where trtDNA was more than 2.69% of the urine-derived DNA, cancer-specific molecular alterations, mutational signatures, and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA. Conclusions: With current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumor-related genetic information is mainly present in shorter DNA fragments. Although the limited amounts of trtDNA pose analytical challenges, enrichment of low-molecular-weight DNA and optimized computational tools improve detection of tumor-specific genetic information in urine. Citation Format: Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Monica Montone, Alice Bartolini, Ludovic Barault, Antonio Martinetti, Federica Morano, Filippo Pietrantonio, Andrea Sartore-Bianchi, Salvatore Siena, Federica Di Nicolatonio, Silvia Marsoni, Alberto Bardelli, Giulia Siravegna. Whole-exome sequencing analysis of urine transrenal tumor DNA in metastatic colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B11.

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Item does not contain fulltext BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

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.

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRAS. Experimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer. Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03). Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657–66. ©2017 AACR.

Preclinical models that accurately reflect patients’ tumor with regards to histopathology, genomics, and therapeutic response represent a compelling need to fuel the progress of effective cancer treatments. Patient-derived tumor xenografts (PDXs) have significantly accelerated this process, but, although they closely mirror structural and molecular features of the tumor of origin, they still retain important restrictions related to maintenance costs and large-scale screening. To overcome this issue, we have established a novel platform of 2D-cell lines (xeno-cell lines, XL) derived from PDXs of colorectal cancer (CRC) from which patient’s germline gDNA was available. We have characterized XL-cells at multiple levels to confirm their proximity to the PDXs of origin and to assess their suitability as patient avatars in vitro to interrogate functional networks in colorectal cancer. All XL-cells showed an epithelial-like morphology and phenotype, as also confirmed by EMT biomarker transcriptomic analysis. Whole exome and RNA-seq analyses showed that genomic features were consistently preserved between PDXs and matched cell models. Expression analysis revealed the XL-line collection as a significant representative of all CRC subtypes (CMS and CRIS subgroups). Furthermore, genomic analysis allowed the identification of molecular biomarkers of response and resistance to targeted therapies, including EGFR and HER2 blockade. In particular, molecular determinants of resistance to dual-HER2 blockade previously reported in the Heracles-A trial were validated in XL-cells. In conclusion, the XL-cell line and PDX platform represents a unique and comprehensive preclinical tool to validate gene function e to identify novel pharmacological vulnerabilities in colorectal cancer. Citation Format: Luca Lazzari, Giorgio Corti, Claudio Isella, Monica Montone, Pamela Arcella, Eugenia Zanella, Luca Novara, Fabiane Barbosa, Andrea Cassingena, Carlotta Cancelliere, Enzo Medico, Andrea Sartore-Bianchi, Salvatore Siena, Andrea Bertotti, Livio Trusolino, Federica Di Nicolantonio, Michael Linnebacher, Alberto Bardelli, Sabrina Arena. A comprehensive platform of patient-derived xenografts and matched cell lines mirrors the genomic landscape of 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 LB-299.

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.

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.

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.

Targeting HER2 with trastuzumab and lapatinib is effective in ERBB2 amplified metastatic colorectal cancer (mCRC). Although at least 30% of the patients initially respond, secondary resistance occurs in most of the cases. Since the drivers of secondary resistance to trastuzumab and lapatinib in ERBB2 amplified mCRC are unknown, we exploited longitudinal plasma collections and patient-derived cell models to define the molecular bases of resistance to HER2 blockade. Levels of ERBB2 amplification in plasma circulating tumor DNA (ctDNA) paralleled response and relapse. The emergence of EGFR, ERBB2, RAS, BRAF and PIK3CA variants in ctDNA was associated with resistance. Radiographic measurements of individual metastases coupled with longitudinal liquid biopsies unveiled lesion-specific patterns of heterogeneous response in several patients. Phylogenetic tracking and functional analyses on tissue samples and patient-derived cell models established from eight metastases of a single case revealed new druggable oncogenic dependencies and genomic evolution associated with resistance. These data highlight the relevance of coupling imaging and liquid biopsies analyses in precision oncology and provide the rationale for additional lines of therapies in HER2 positive mCRC relapsing upon HER2 blockade. Citation Format: Giulia Siravegna, Luca Lazzari, Andrea Sartore-Bianchi, Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Cosimo Martino, Richard Lanman, Rebecca Nagy, Giorgio Corti, Alice Bartolini, Pamela Arcella, Monica Montone, Francesca Lodi, Alice Vanzati, Emanuele Valtorta, Giovanni Cappello, Andrea Bertotti, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Mariangela Russo, Antonella Balsamo, Mauro Truini, Federica Di Nicolantonio, Alessio Amatu, Erica Bonazzina, Silvia Ghezzi, Daniele Regge, Angelo Vanzulli, Livio Trusolino, Salvatore Siena, Silvia Marsoni, Alberto Bardelli. Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2848.

3506Background: The rate of HER2 (ERBB2) copy number amplification (CNA) in metastatic colorectal cancer (mCRC) ranges from 2-13%. HERACLES, a phase II trial of trastuzumab and lapatinib (T+L) in H...

The introduction of KRAS testing as a diagnostic tool to select patients for epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized cancer medicine. Oncologists are now facing emerging issues in the treatment of metastatic colorectal cancer, including: (i) the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients; (ii) the explanation of rare cases of patients carrying KRAS-mutated tumors who have been reported to respond to either cetuximab or panitumumab and (iii) the discovery of mechanisms of secondary resistance to anti-EGFR antibody therapies. Here we discuss the potential role of comprehensive dissection of the key oncogenic nodes in the EGFR signaling cascade to predict resistance and sensitivity to EGFR monoclonal antibodies in metastatic colorectal cancer. Current data suggest that, together with KRAS mutations, the evaluation of BRAF and PIK3CA/PTEN alterations could also be useful for selecting patients with reduced chance to benefit from EGFR-targeted therapy. Furthermore, measuring EGFR gene copy number also appears relevant to positively identify responders. Up until now, each of these markers has been mainly assessed as a single event, often in retrospective analyses and patients' series. As these molecular alterations display overlapping patterns of occurrence, this adds considerable complexity to the drawing of an algorithm suitable for clinical decision-making. We suggest that in the near future comprehensive molecular analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of individual markers.

Background: Metastatic colorectal cancer still has a dismal 5-yr survival of less than 10%. Chemotherapy, targeted therapies, and immune-checkpoint inhibitors are effective in only a fraction of patients, and even in responders, long-term remission are rare. To harness insight into the evolving heterogeneity of mCRC, and to combat it therapeutically, we have established the logistic research matrix FUNNEL that integrates the topographic acquisition of patient samples with the clinical validation of hypothesis-driven trials in primary or secondary resistance disease. Methods: FUNNEL will follow 1000 sequential cases from diagnosis to death, acquiring biologic specimens, imaging, and clinical data at critical steps along the disease continuum. At diagnosis of metastatic disease, patients are genotyped with a CRC-specific panel designed to map the landscape of primary resistance to anti-EGFR therapies (Bertotti A. et al,, Nature 2015). Paraffin samples are centrally processed and tested using Sequenom® (with a 5% detection sensitivity for 168 mutations in KRAS, BRAF, NRAS, PIK3CA, EGFR, HER2, IDH1, MEK1) and NanoString® technologies [measuring gene copy number variation (CNV) for RAF1, EGFR, FGRF1/2/3, HER2, IGF1/2, IGF1R, KRAS, MET, and NF1]. Genes are considered amplified with a CNV ≥ 10 cut-off validated by FISH. Dynamic data of centralized disease assessments imaging coupled with longitudinal liquid biopsies are also collected at significant clinical events (baseline, response to therapy, and progression of sequential treatment’s lines). Proteomics and neoantigenic profile of cancer cells in parallel with function and tumor specificity of infiltrating T cells will also be determined in retrospectively selected patients. Results: From 7/2015 to 6/2017, 428 patients have been successfully genotyped. Overall we found 52% mutated and 6% amplified tumors (2% both amplified and mutated). Of these, 81% (N 180) had a single gene mutation in KRAS (67%), BRAF (13%), PIK3CA (11%), or NRAS (9%). Remaining cases had a double gene alteration in PIK3CA (N 38) and KRAS (81%) or BRAF (7%) or NRAS (5%), or BRAF (N3: KRAS, NRAS, IDH1 ). 29% of the 24 amplified tumors showed concomitant mutations, while 16% had multiple amplifications. Interestingly, 2 out of 13 HER2-amplified tumors also harbored KRAS (G12D) and PIK3CA mutations (E542K and E545A). Conclusion: Upfront genotyping detected 44% multiple wild type and 9% patients with potentially actionable drivers including HER2, FGFR1 and 3, EGFR, MET, IGF1 and 2, and IGF1R. Two embedded trials for HER2-amplified (HERACLES - EudraCT 2012-002128-33, NCT03225937) and multiple wild type patients (CHRONOS EudraCT 2016-002597-12, NCT03227926) are ongoing. Beyond "molecular triage" purposes, FUNNEL is also building an integrated repository of lifetime clinical and molecular information, that all together will allow a comprehensive insight of mCRC clonal evolution, laying the groundwork to design further innovative trials, and, eventually, inform patient care. Funded by AIRC Grant 9970; Italian Ministry of Health grant NET-2011-02352137. Citation Format: Cosimo Martino, Enrico Berrino, Carmine Dell'Aglio, Laura Casorzo, Mara Panero, Salvatore Siena, Andrea Sartore-Bianchi, Andrea Cassingena, Vittorina Zagonel, Sara Lonardi, Fotios Loupakis, Giuseppe Tonini, Patrizia Racca, Livio Trusolino, Andrea Bertotti, Ilaria Depetris, Antonella Balsamo, Anna Sapino, Silvia Marsoni. Empowering precision medicine in metastatic colorectal cancer: preliminary results from the FUNNEL platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A087.

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

A metastatic colorectal cancer (mCRC) patient carrying CAD-ALK translocation achieved partial response to an experimental ALK inhibitor and then progressed after 5 months. We studied whether urine cell-free, trans-renal DNA (tr-DNA) could be used to monitor tumor burden and patient’s response. A NGS panel was developed to interrogate 52 common cancer gene rearrangements and 14 frequently mutated genes in cancer patients. A TP53 p.R248W mutation and the CAD-ALK genomic breakpoint (rearrangement) were identified in the tumor tissue and matched plasma circulating tumor DNA (ctDNA) Urine samples were longitudinally obtained from the patient during ALK inhibitor treatment in parallel with blood. To detect the CAD-ALK translocation in urine tr-DNA we designed ultra-short (51 bp amplicon) primer pairs spanning the genomic breakpoint as a unique tumor marker. This approach allowed the non-invasive monitoring of the gene fusion in urine with amounts paralleling tumor burden. Of note, CAD-ALK gene fusion was apparent in urine tr-DNA before radiological confirmation of disease progression. The same strategy was applied to plasma ctDNA and the results were compared. To detect point mutations in urine tr-DNA, we exploited a peptide nucleic acids (PNA)-CLAMP PCR coupled with droplet digital PCR (ddPCR) analysis to specifically suppress amplification of wild-type DNA fragments. Custom PNA probes were designed for TP53 codon 248, and a ddPCR assay was optimized to detect the TP53 p.R248W mutation, which was then identified in all urine tr-DNA samples, with absolute copies correlating with tumor burden throughout ALK inhibitor treatment. In conclusion, we find that urine tr-DNA can be exploited to non-invasively monitor tumor burden by detecting tumor-specific gene fusions as well as point mutations. Citation Format: Giulia Siravegna, Andrea Sartore-Bianchi, Benedetta Mussolin, Laura Palmieri, Federica Tosi, Andrea Cassingena, Luca Novara, Michela Buscarino, Giorgio Corti, Giovanni Crisafulli, Alice Bartolini, Mark Erlander, Federica Di Nicolantonio, Salvatore Siena, Alberto Bardelli. Tracking CAD-ALK gene translocation in urine and plasma of a colorectal cancer patient treated with ALK blockade [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 3834. doi:10.1158/1538-7445.AM2017-3834

602 Background: Molecular testing of cell-free (cf) DNA from urine is a completely non-invasive approach for detection of actionable mutations in cancer. Methods: A quantitative mutation enrichment next-generation sequencing (NGS) urine cell-free (cf) DNA KRASG12/G13mutation test was developed and results compared to clinical testing of archival tumor tissue and research testing of plasma cfDNA from patients with advanced colorectal (n=56, 79%) and other advanced cancers (n=15, 21%). Results: The analytical sensitivity of the KRASG12/G13 cfDNA test was 0.002%-0.006% mutant copies in wild-type background. In 71 patients, the agreement between urine cfDNA and tumor was 73% (sensitivity 63%; specificity 96%); the agreement increased to 89% for patients with recommended 90-110mL of urine. In 33 patients with available plasma samples, the agreement with tumor was 94% (sensitivity 92%; specificity 100%). In patients treated with systemic therapy there was lower number of KRASG12/G13 copies in urine and plasma cfDNA on therapy compared to baseline and progression ( PG12/G13 copies on therapy compared to no change/increase was associated with longer median time to treatment failure ( PG12/G13 mutations in urine cfDNA has good concordance with archival tumor tissue. Changes in urine cfDNA correspond with time to treatment failure.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification. Significance: Amplification of the MET proto-oncogene is responsible for de novo and acquired resistance to anti-EGFR therapy in a subset of colorectal cancers. As multiple anti-MET therapeutic strategies are available, these findings offer immediate novel opportunities to design clinical studies. Cancer Discov; 3(6); 658–73. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 591

Purpose: 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 (CRC) display MGMT deficiency due to the 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). Experimental Design: All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m2 intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status. Results: Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1–12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Conclusion: Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT. Clin Cancer Res; 19(8); 2265–72. ©2013 AACR.

Metastatic colorectal cancer (mCRC) patients carrying KRAS mutated tumors do not benefit from epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies. Indeed, the mutational status of KRAS is currently a validated predictive biomarker employed to select mCRC patients for EGFR targeted drugs. When patients fail standard 5-fluorouracil-, oxaliplatin-, irinotecan- and bevacizumab-based therapies, EGFR-targeted salvage therapy can be prescribed only for those individuals with KRAS wild-type cancer. Thus, clinicians are now facing the urgent issue of better understanding the biology of KRAS mutant disease, in order to devise novel effective therapies in such defined genetic setting. In addition to KRAS, recent data point out that BRAF and PIK3CA exon 20 mutations hamper response to EGFR-targeted treatment in mCRC, potentially excluding from treatment also patients with these molecular alterations in their tumor. This review will focus on current knowledge regarding the molecular landscape of mCRC including and beyond KRAS, and will summarize novel rationally-developed combinatorial regimens that are being evaluated in early clinical trials.

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.