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Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC), but the emergence of resistance mutations restricts their efficacy. We previously showed that RAS, BRAF and EGFR mutant alleles, which appear in circulating tumor DNA (ctDNA) during EGFR blockade, decline upon therapy withdrawal. We hypothesized that monitoring resistance mutations in blood could rationally guide subsequent therapy with anti-EGFR antibodies. We report here the results of CHRONOS, an open-label, single-arm phase 2 clinical trial exploiting blood-based identification of RAS/BRAF/EGFR mutations levels to tailor a chemotherapy-free anti-EGFR rechallenge with panitumumab (ClinicalTrials.gov: NCT03227926; EudraCT 2016-002597-12). The primary endpoint was objective response rate. Secondary endpoints were progression-free survival, overall survival, safety and tolerability of this strategy. In CHRONOS, patients with tissue-RAS WT tumors after a previous treatment with anti-EGFR-based regimens underwent an interventional ctDNA-based screening. Of 52 patients, 16 (31%) carried at least one mutation conferring resistance to anti-EGFR therapy and were excluded. The primary endpoint of the trial was met; and, of 27 enrolled patients, eight (30%) achieved partial response and 17 (63%) disease control, including two unconfirmed responses. These clinical results favorably compare with standard third-line treatments and show that interventional liquid biopsies can be effectively and safely exploited in a timely manner to guide anti-EGFR rechallenge therapy with panitumumab in patients with mCRC. Further larger and randomized trials are warranted to formally compare panitumumab rechallenge with standard-of-care therapies in this patient setting.

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

The Cell Model Network-UK group author list

Purpose:Current glioma diagnostic guidelines call for molecular profiling to stratify patients into prognostic and treatment subgroups. In case the tumor tissue is inaccessible, cerebrospinal fluid (CSF) has been proposed as a reliable tumor DNA source for liquid biopsy. We prospectively investigated the use of CSF for molecular characterization of newly diagnosed gliomas.Experimental design:We recruited two cohorts of newly diagnosed patients with glioma, one (n = 45) providing CSF collected in proximity of the tumor, the other (n = 39) CSF collected by lumbar puncture (LP). Both cohorts provided tumor tissues by surgery concomitant with CSF sampling. DNA samples retrieved from CSF and matched tumors were systematically characterized and compared by comprehensive (NGS, next-generation sequencing) or targeted (ddPCR, droplet digital PCR) methodologies. Conventional and molecular diagnosis outcomes were compared.Results:We report that tumor DNA is abundant in CSF close to the tumor, but scanty and mostly below NGS sensitivity threshold in CSF from LP. Indeed, tumor DNA is mostly released by cells invading liquoral spaces, generating a gradient that attenuates by departing from the tumor. Nevertheless, in >60% of LP CSF samples, tumor DNA is sufficient to assess a selected panel of genetic alterations (IDH and TERT promoter mutations, EGFR amplification, CDKN2A/B deletion: ITEC protocol) and MGMT methylation that, combined with imaging, enable tissue-agnostic identification of main glioma molecular subtypes.Conclusions:This study shows potentialities and limitations of CSF liquid biopsy in achieving molecular characterization of gliomas at first clinical presentation and proposes a protocol to maximize diagnostic information retrievable from CSF DNA.

Supplementary Table 1

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Supplementary Figures 1-4, Supplementary Tables S1-S13, Description of Supplementary Data 1-3, Supplementary Materials and Methods, Supplementary References.

Supplementary Figure 2

Supplementary Figure S1. LMNA-NTRK1 genetic rearrangement detected in patient's plasma and xeno. Supplementary Figure S2. Acquisition of mutations in the TRKA kinase domain drives secondary resistance to entrectinib in Ba/F3 TRKA WT cells. Supplementary Figure S3. NTRK1 mutations confer resistance to TRKA inhibition. Supplementary Figure S4. Homology alignment of NTRK1 p.G595 and p.G667 variants. Supplementary Table S1. NGS analysis of NTRK1 gene in patient's plasma and xeno. Supplementary Table S2. NGS analysis of patient's derived samples. Supplementary Table S3. Summary of serial ctDNA analyses. Supplementary Table S4. The p.G595R and p.G667C mutations confer resistance to multiple TRK inhibitors. Supplementary Table S5. NTRK1 p. G595R, p.G667C and LMNA-NTRK1 fusion probes for ddPCR.

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Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair–deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer. The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of patients receiving immunotherapy have disease that is refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR colorectal cancer preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in patients with dMMR/MSI-H colorectal cancer and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies.Significance:We found that a large, diverse set of dMMR/MSI-H colorectal cancer preclinical models, including models of treatment-refractory disease, are WRN-dependent. Our results support WRN as a promising synthetic-lethal target in dMMR/MSI-H colorectal cancer tumors as a monotherapy or in combination with targeted agents, chemotherapy, or immunotherapy.This article is highlighted in the In This Issue feature, p. 1861

Supplementary Figure S1. MEK1 K57 mutation confers resistance to anti-EGFR antibodies in CRC preclinical models. Supplementary Figure S2. Dual blockade of EGFR and MEK restores sensitivity to CRC cells expressing MEK1 K57N or K57T. Supplementary Figure S3. Longitudinal analysis of founder mutations in patient plasma during panitumumab and trametinib treatment. Supplementary Table S1. 1000 gene sequencing panel. Supplementary Table S2. 40 gene targeted sequencing panel. Supplementary Table S3. Summary of targeted sequencing and ddPCR data on tissue specimens. Supplementary Table S4. Next generation sequencing data from plasma ctDNA. Supplementary Table S5. Summary of serial ctDNA analyses. Supplementary Table S6. Ratio of resistance-associated genetic alterations and founder mutations in serial plasma ctDNA timepoints.

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Primers and probes.

SNP ID.

Purpose: Patients with colorectal cancer who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment, the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signaling axis on the acquisition of resistance to cetuximab in patients and cellular models.Experimental Design: Tissue samples were obtained from 37 patients with colorectal cancer who became refractory to cetuximab. Colorectal cancer cells sensitive to cetuximab were treated until resistant derivatives emerged. Mutational profiling of biopsies and cell lines was performed. Structural modeling and functional analyses were performed to causally associate the alleles to resistance.Results: The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF, and PIK3CA genes, including two novel EGFR ectodomain mutations (R451C and K467T). Mutational profiling of cetuximab-resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR alleles: S464L, G465R, and I491M. Structurally, these mutations are located in the cetuximab-binding region, except for the R451C mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab.Conclusions: Colorectal tumors evade EGFR blockade by constitutive activation of downstream signaling effectors and through mutations affecting receptor–antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for patients with colorectal cancer who relapse upon treatment with anti-EGFR antibodies. Clin Cancer Res; 21(9); 2157–66. ©2015 AACR.

Supplementary Table S3. Prevalence of EGFR mutations in resistant cell lines.

The file lists Supplementary Materials and Methods and relative References.

The file contains Supplementary Table and Figure Legends

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 Figure S3. Nucleotide sequence of the EGFR gene in cetuximab resistant cell lines.

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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

The number of effective therapies approved for metastatic colorectal cancer (mCRC) is still limited and multiple strategies are continuously explored to expand the drug target repertoire. Among these, the identification of overexpressed genes has prompted the discovery of actionable oncogenic dependencies in multiple tumour types. Starting from RNA sequencing data, we identified transcriptome-wide gene expression outliers, defined as samples showing abnormal expression for a particular gene, across 226 CRC cell lines, considering both overexpression and underexpression events as positive or negative outliers. Then, the distance of each outlier from gene-specific reference points, absolute expression values and differential expression values were considered in a multi-filter strategy to select extreme gene expression outliers, with the hypothesis that they are more likely to be functionally relevant in cancer cells. We also profiled genetic and epigenetic features of CRC cell lines based on whole exome sequencing and DNA methylation microarray data. Extreme positive and negative gene expression outliers were found for 3,533 and 965 genes, respectively, and only some of them were associated with underlying genetic and epigenetic alterations. Gene expression alterations with known therapeutic or diagnostic value in CRC were pinpointed as extreme positive and negative outliers thus confirming the validity of the approach. Annotation of overexpressed enzyme genes according to the Target Development Level (TDL) classification revealed numerous enzymes for which inhibitors are already available. We next explored underexpression events to identify potential synthetic lethal targets. Intriguingly, we found that CRC models lacking expression of the MTAP gene were sensitive to treatment with an inhibitor of the PRMT5:MTA complex currently under clinical development. We found that mapping extreme and transcriptome-wide positive and negative gene expression outliers in CRC cell lines is an effective strategy to identify putative drug targets and biomarkers, independently from the underlying genetic or epigenetic alterations. We indeed present a comprehensive atlas of CRC extreme gene expression outliers which includes events with diagnostic or therapeutic relevance. This resource could also serve as a reference for further discoveries in CRC and other tumour types. Citation Format: Gaia Grasso, Elisa Marriella, Martina Miotto, Kristi Buzo, Nicole M. Reilly, Pietro Andrei, Pietro P. Vitiello, Giovanni Crisafulli, Sabrina Arena, Giuseppe Rospo, Giorgio Corti, Annalisa Lorenzato, Carlotta Cancelliere, Ludovic Barault, Giulia Gionfriddo, Michael Linnebacher, Mariangela Russo, Federica Di Nicolantonio, Alberto Bardelli. A transcriptome-wide gene expression outlier analysis pinpoints therapeutic vulnerabilities in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB058.

Introduction: Immunotherapy based on PD1/PDL1 and/or CTLA4 blocking antibodies has shown efficacy in the molecular subgroup of colorectal cancers (CRCs) characterized by deficient mismatch repair (MMRd). This feature, present in only 4-5% of metastatic CRCs, causes a significant increase of tumor mutational burden (TMB) and tumor neoantigens, ultimately leading to immune rejection. On the other hand, for the majority of MMR proficient (MMRp) CRCs, cytotoxic chemotherapy ± targeted agents still constitute the mainstay of the treatment Interestingly, even though it is known that cytotoxic agents induce mutations, their immunogenic potential is still not fully understood. The aim of this work is to systematically investigate the immunogenicity of chemotherapy-induced mutations in CRC. Methods: We performed a two-step experiment: first, murine CRC models were exposed to chemotherapy agents in an immune-free context (priming phase) and then challenged in immune-deficient and -proficient contexts (editing phase). Chemotherapy agents included the commonly used cytotoxic drugs 5-fluorouracil (5FU), oxaliplatin, SN38 (the active metabolite of irinotecan), cisplatin, and temozolomide, used as single drugs or combinations, in a pulsatile schedule using weekly ‘cycles’ (2 days on/5 days off) in order to mimic drug exposure in clinical settings. Whole exome sequencing (WES) was performed at baseline, and after both the priming and the editing phases. Results and discussion: WES analysis showed that cisplatin-based treatment is associated with the highest increase in TMB and predicted neoantigens when compared to 5FU-based combinations. Multiple mutational processes contribute to the accumulation of mutations upon exposure to chemotherapy, as revealed by analysis of mutational signatures. A statistically significant growth delay and increased survival was reported for tumors primed with cisplatin/temozolomide and 5FU/Oxaliplatin/SN38 (FOLFOXIRI) combinations compared to unprimed tumors in immunocompetent but not in immunodeficient mice, suggesting the active involvement of the immune system in controlling tumor growth. Sequencing data of residual (escaped) tumors at the end of the editing phase revealed a preferential loss of mutations etiologically linked to chemotherapy in the immunocompetent models, pinpointing an active immune-editing of chemotherapy-induced mutations. Conclusions: Cisplatin/temozolomide and FOLFOXIRI treatment effectively prime murine CRC models for subsequent immune rejection. These results suggest that the anticancer effect of chemotherapy treatment may be in part mediated by the immune-editing of chemotherapy-induced mutant cancer subclones. Citation Format: Pietro Paolo Vitiello, Rosaria Chilà, Giuseppe Rospo, Gaia Grasso, Giovanni Crisafulli, Alice Bartolini, Vito Amodio, Federica Di Nicolantonio, Giovanni Germano, Alberto Bardelli. The effects of cytotoxic chemotherapy on colorectal cancer immunogenicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6402.

Compelling evidence shows that cancer persister cells represent a major limit to the long-term efficacy of targeted therapies. However, the phenotype and population dynamics of cancer persister cells remain unclear. We developed a quantitative framework to study persisters by combining experimental characterization and mathematical modeling. We found that, in colorectal cancer, a fraction of persisters slowly replicates. Clinically approved targeted therapies induce a switch to drug-tolerant persisters and a temporary 7- to 50-fold increase of their mutation rate, thus increasing the number of persister-derived resistant cells. These findings reveal that treatment may influence persistence and mutability in cancer cells and pinpoint inhibition of error-prone DNA polymerases as a strategy to restrict tumor recurrence.

Simple Summary Immunotherapy has revolutionized cancer treatment, but not for all tumor types. Indeed, sarcomas are considered “immune-cold” tumors, which are relatively unresponsive to immunotherapy. One strategy to potentiate immunotherapy efficacy is to increase tumor immunogenicity, for instance by boosting the number of candidate targets (neoantigens) to be recognized by the immune system. Tumor mutational burden indicates the number of somatic mutations identified in the tumor and normalized per megabase. Tumor mutational burden is considered as an acceptable, measurable surrogate of tumor neoantigens. Here, we explored whether the combination of two DNA-damaging agents, trabectedin and olaparib, could increase tumor mutational burden in sarcomas, to prime subsequent immunotherapy. We found no variation in tumor mutational burden after trabectedin + olaparib in preclinical and clinical samples. Therefore, other aspects should be considered to increase sarcoma immunogenicity, by exploiting different pathways such as the potential modulation of the tumor microenvironment induced by trabectedin + olaparib. Abstract Drug-induced tumor mutational burden (TMB) may contribute to unleashing the immune response in relatively “immune-cold” tumors, such as sarcomas. We previously showed that PARP1 inhibition perpetuates the DNA damage induced by the chemotherapeutic agent trabectedin in both preclinical models and sarcoma patients. In the present work, we explored acquired genetic changes in DNA repair genes, mutational signatures, and TMB in a translational platform composed of cell lines, xenografts, and tumor samples from patients treated with trabectedin and olaparib combination, compared to cells treated with temozolomide, an alkylating agent that induces hypermutation. Whole-exome and targeted panel sequencing data analyses revealed that three cycles of trabectedin and olaparib combination neither affected the mutational profiles, DNA repair gene status, or copy number alterations, nor increased TMB both in homologous recombinant-defective and proficient cells or in xenografts. Moreover, TMB was not increased in tumor specimens derived from trabectedin- and olaparib-treated patients (5–6 cycles) when compared to pre-treatment biopsies. Conversely, repeated treatments with temozolomide induced a massive TMB increase in the SJSA-1 osteosarcoma model. In conclusion, a trabectedin and olaparib combination did not show mutagenic effects and is unlikely to prime subsequent immune-therapeutic interventions based on TMB increase. On the other hand, these findings are reassuring in the increasing warning of treatment-induced hematologic malignancies correlated to PARP1 inhibitor use.

Colorectal cancer (CRC) is one of the most prevalent and deadly cancers worldwide. Despite recent improvements in treatment and prevention, most of the current therapeutic options are weighted by side effects impacting patients’ quality of life. Better patient selection towards systemic treatments represents an unmet clinical need. The recent multidisciplinary and molecular advancements in the treatment of CRC patients demand the identification of efficient biomarkers allowing to personalise patient care. Currently, core tumour biopsy specimens represent the gold-standard biological tissue to identify such biomarkers. However, technical feasibility, tumour heterogeneity and cancer evolution are major limitations of this single-snapshot approach. Genotyping circulating tumour DNA (ctDNA) has been addressed as potentially overcoming such limitations. Indeed, ctDNA has been retrospectively demonstrated capable of identifying minimal residual disease post-surgery and post-adjuvant treatment, as well as spotting druggable molecular alterations for tailoring treatments in metastatic disease. In this review, we summarise the available evidence on ctDNA applicability in CRC. Then, we review ongoing clinical trials assessing how liquid biopsy can be used interventionally to guide therapeutic choice in localised, locally advanced and metastatic CRC. Finally, we discuss how its widespread could transform CRC patients’ management, dissecting its limitations while suggesting improvement strategies.

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.

When cancer cells are exposed to lethal doses of targeted therapies, the emergence of a subpopulation of drug-tolerant persister cells (DTPs) is often observed. We previously reported that colorectal cancer (CRC) cells exposed to targeted therapies activate an adaptive mutability stress response, involving DNA damage induction and a switch to low-fidelity DNA replication. Therefore, targeted treatment might lead to increased mutation rate in DTPs, but mutation rates of cancer cells during treatment have not been quantitatively assessed. Here, we combined biological experiments and mathematical modelling to characterize emergence and dynamics of DTPs. From this, we extrapolated parameters governing dynamics of cancer cells populations and designed a modified Luria-Delbrück assay on mammalian cells (MC-LD) to quantify mutations rates of CRC cells under standard growth conditions and during exposure to targeted therapy. We selected mismatch repair proficient CRC cell lines sensitive to different clinically used therapeutic agents, and derived clones to be used for experiments. By monitoring cell dynamics in drug-response growth assays, we found that CRC cells exposed to targeted therapy display a biphasic killing curve reaching a stable plateau, a pattern indicative of emergence of DTPs. By fitting model estimation to population growth assays, we predicted that, even if a subgroup of DTPs predated treatment, the majority of them emerged only upon exposure to targeted therapies. We also observed that DTPs slowly replicate under treatment, as shown by Carboxy fluorescein succinimidyl ester (CFSE) analysis and staining with 5-ethynyl-2’-deoxyuridine (EdU). We used these population dynamics parameters to design the MC-LD assay. CRC clones were plated in several 96-multiwell plates each, and after an expansion phase in standard culture conditions, treatment was added. After 3-4 weeks, a minority of wells showed growth of resistant colonies: based on the measured growth rates, we could predict that the resistant cells arose before treatment by spontaneous mutation. The remaining wells contained a homogenous population of DTPs. After several weeks of treatment, when pre-treatment resistant clones would have already emerged, late-emerging resistant colonies appeared in a subset of wells in which DTPs had previously been detected. Using the number of residual DTPs and resistant colonies to infer mutation rates, we found a 7- to 50-fold increase (depending on the cell line) in DTPs’ mutation rate compared to sensitive cells.In conclusion, we developed a new assay which allows quantitative comparisons of spontaneous and drug-induced mutation rates in cancer cells and showed that adaptive mutability in DTPs leads to increased mutation rates. This approach could be used to measure whether and how a wide range of environmental conditions affect DTP phenotype and mutation rates in mammalian cells. Citation Format: Alberto Sogari, Mariangela Russo, Simone Pompei, Mattia Corigliano, Giovanni Crisafulli, Andrea Bertotti, Marco Gherardi, Federica Di Nicolantonio, Marco Cosentino Lagomarsino, Alberto Bardelli. A modified Luria-Delbrück assay allows quantification of colorectal cancer persister cells’ mutation rate [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 2613.

Compelling evidence shows that cancer persister cells limit the efficacy of targeted therapies. However, it is unclear whether persister cells are induced by anticancer drugs, and if their mutation rate quantitatively increases during treatment. Here, combining experimental characterization and mathematical modeling, we show that, in colorectal cancer, persisters are induced by drug treatment and show a 7- to 50-fold increase of mutation rate when exposed to clinically approved targeted therapies. These findings reveal that treatment may influence persistence and mutability in cancer cells and pinpoints new strategies to restrict tumor recurrence.

3589 Background: HER2 amplified mCRC has emerged as a unique clinical subset, characterized by resistance to anti-EGFR therapy and response to anti-HER2 strategies. Accurate identification and quantification of HER2amp has predictive value for efficacy of anti-HER2 therapies and appropriate patient selection. Despite availability and use of various tumor tissue-based and blood-based assays for detecting HER2amp, data on cross-performance of these platforms are lacking. Methods: Leveraging a multicenter international consortium (Italy, Japan and USA), we generated a large cohort (N = 353) of mCRC patients (pts), tested for HER2amp using both tissue and blood. Tissue testing was done using immunohistochemistry (IHC), in-situ hybridization (ISH) and (NGS). ctDNA NGS was performed using CLIA-certified Guardant360 ctDNA assay, capable of detecting HER2 copy number (CN) variations. The primary endpoint was to correlate HER2 gene CNs in tissue (tCN) and plasma (pCN). Descriptive statistics, spearman correlation (r) and Fisher’s exact test were used. Results: Baseline tumors characteristics included right-sided primary in 234 (23%), proficient mismatch repair in 264 (98%) and RAS/BRAF wild type (WT) genotype in 194 (67%) pts. Tissue testing was done by IHC, ISH and NGS in 76%, 64% and 74% pts, respectively. A total of 177 pts had HER2amp detected by at least one test: 116 (66%), 157 (89%) and 96 (54%) of which had tissue +, ctDNA +, and both tissue and ctDNA + disease, respectively. Discordant cases consisted of 20 (6%) with positivity in tumor only and 61 (17%) in ctDNA only. Sensitivity, specificity, positive and negative predictive values of ctDNA assay (vis-à-vis tissue) were 83%, 74%, 61% and 90% respectively. Among HER2amp pts, median (range) HER2/CEP17 (ISH) ratio, tCN and pCN were 5.2 (2–12), 11.6 (2–700) and 3.5 (2–122), respectively. The pCN showed strong correlation with ISH ratio (r = 0.69) and tCN (r = 0.68) (P < 0.001). Median pCN differed significantly between pts with HER2 IHC 3+ (12.0), 2+ (2.2) and 0/1+ (2.0) tumors (P < 0.001). High HER2amp (pCN > 4.0) appeared to be enriched with tissue + cases (69% vs 8% [OR 24.6, P < 0.001]), tumor tissue HER2 + status (IHC3+ [75%] vs IHC2+ISH+ [50%] vs IHC2+/ISH- or IHC0/1+ [12%], P < 0.001), HER2 tCN > 6 (79% vs 31% [OR 8.7, P < 0.001]) and RAS/BRAF WT tumors (41% vs 17% [OR 3.5, P = 0.064) but not left sidedness (41% vs 38%; OR 1.1; P = 0.82). Conclusions: In this large diverse cohort of mCRC, we demonstrated correlation of HER2 tCN and pCN obtained by tissue-based and blood-based ctDNA assay. Further prospective efforts are needed to standardize this cross-platform quantification of HER2amp to facilitate robust clinical application of HER2 therapies. This effort shows the value of strategic international partnership in furthering research for rare cancer subsets.

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.

Entrectinib is a first-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA–NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profiling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1, p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confirmed that either mutation renders the TRKA kinase insensitive to entrectinib. These findings can be immediately exploited to design next-generation TRKA inhibitors. Significance: We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identification of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the first time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1); 36–44. ©2015 AACR. See related commentary by Okimoto and Bivona, p. 14. This article is highlighted in the In This Issue feature, p. 1

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.

Success in eradicating human cancer with targeted therapies is limited by the emergence of secondary resistance. The prevalent view is that resistance is a fait accompli: when treatment is initiated, tumors already contain drug-resistant mutant cells. However, when cancer cells are challenged with targeted agents, the emergence of drug tolerant ‘persister’ cell population is often observed. Persisters survive exposure to targeted therapies through non-genetic, poorly understood mechanisms, and constitute a reservoir from which genetically divergent, drug-resistant derivatives eventually emerge. Analogously, when bacteria are exposed to stress, such as antibiotic treatment, persister cells can survive and, by switching from high-fidelity to low-fidelity DNA replication process, increase transiently their mutation rate (adaptive mutability), thus improving chances of survival. We used colorectal cancer (CRC) as model system to explore the hypothesis that, in addition to pre-existing drug resistant cells, resistance to targeted therapies could be fostered by a transient increase in genomic instability during treatment, leading to de novo genetic alterations. We found that, when exposed to EGFR and/or BRAF inhibition, mismatch repair proficient (MMRp) CRC cell lines exhibited a down-modulation of MMR and Homologous Recombination (HR) DNA repair genes, and a concomitant up-regulation of error-prone polymerases, resulting in reduced MMR and HR proficiency. Therapy-induced modulation of DNA repair genes was transient and returned to initial levels upon removal of the drugs, or when the cells developed permanent resistance to targeted agents. Notably, MLH1 and MSH2 MMR genes were down-regulated in patients-derived xenografts and tissue samples obtained at clinical response (minimal residual disease) compared to pre-treatment samples, confirming the clinical relevance of our findings. Activation of error-prone polymerases was associated with increased ROS production and accumulation of DNA damage marker γ-H2AX, in a time- a dose-dependent manner upon drug administration. The combination of DNA repair down-modulation and error-prone polymerases upregulation increases mutagenic ability and triggers microsatellite instability under drug-induced stress in CRC cells. Our results demonstrate that cancer cells, like unicellular organisms, evade therapeutic pressures by enhancing mutability. The notion that cancer cells exposed to targeted therapies activate a stress-induced adaptive mutability process may prompt the design of novel therapeutic strategies aimed at interfering with clonal evolution, reducing the generation of new variants during therapeutic treatment. Citation Format: MARIANGELA RUSSO, Giovanni Crisafulli, Alberto Sogari, Nicole Megan Reilly, Sabrina Arena, Simona Lamba, Alice Bartolini, Vito Amodio, Alessandro Magrì, Luca Novara, Ivana Sarotto, Zachary Nagel, Cortt Piett, Alessio Amatu, Andrea Sartore-Bianchi, Salvatore Siena, Andrea Bertotti, Livio Trusolino, Federica Di Nicolantonio, Alberto Bardelli. Adaptive mutability of colorectal cancers in response to targeted therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A120. doi:10.1158/1535-7163.TARG-19-A120