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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

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.

BackgroundNeoantigens that arise as a consequence of tumour-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumour types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.MethodsWe performed Whole Exome Sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines,in vitroandvivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures and predicted neoantigens.ResultsThe majority of CRC models showed remarkably stable mutational and neoantigen profiles, however those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures, and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly-evolving CRC.ConclusionsThese results indicate that CRC carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

Background The analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (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 tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose 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. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.

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.

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.

BACKGROUND: Combined MET and BRAF inhibition showed clinical benefit in a patient with rectal cancer carrying BRAF(V600E) and MET amplification. However after 4 months, acquired resistance emerged and the patient deceased shortly after disease progression. The mechanism of resistance to this drug combination is unknown.METHODS: We analysed plasma circulating tumour DNA obtained at progression by exome sequencing and digital PCR. MET gene and mRNA in situ hybridisation analyses in two bioptic specimens obtained at progression were used to confirm the plasma data.RESULTS: We identified in plasma MET gene hyper-amplification as a potential mechanism underlying therapy resistance. Increased MET gene copy and transcript levels were detected in liver and lymph node metastatic biopsies. Finally, transduction of MET in BRAF mutant colorectal cancer cells conferred refractoriness to BRAF and MET inhibition.CONCLUSIONS: We identified in a rectal cancer patient MET gene hyper-amplification as mechanism of resistance to dual BRAF and MET inhibition.

Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies., Some colorectal cancer patients respond well to treatment with anti-EGFR antibodies, however response is almost invariably followed by acquired resistance. Here, the authors show that patients with shorter responses acquire RAS mutations, while those relapsing later preferentially develop mutations in the extracellular domain of EGFR.

Background The diagnosis of colorectal cancer (CRC) is routinely accomplished through histopathologic examination. Prognostic information and treatment decisions are mainly determined by TNM classification, first defined in 1968. In the last decade, patient-specific CRC genomic landscapes were shown to provide important prognostic and predictive information. Therefore, there is a need for developing next generation sequencing (NGS) and bioinformatic workflows that can be routinely used for the assessment of prognostic and predictive biomarkers. Materials and Methods To foster the application of genomics in the clinical management of CRCs, the IDEA workflow has been built to easily adapt to the availability of patient specimens and the clinical question that is being asked. Initially, IDEA deploys ad-hoc NGS assays to interrogate predefined genomic target sequences (from 600 kb to 30 Mb) with optimal detection sensitivity. Next, sequencing data are processed through an integrated bioinformatic pipeline to assess single nucleotide variants, insertions and deletions, gene copy-number alterations, and chromosomal rearrangements. The overall results are gathered into a user-friendly report. Results We provide evidence that IDEA is capable of identifying clinically relevant molecular alterations. When optimized to analyze circulating tumor DNA, IDEA can be used to monitor response and relapse in the blood of patients with metastatic CRC receiving targeted agents. IDEA detected primary and secondary resistance mechanisms to ERBB2 blockade including sub-clonal RAS and BRAF mutations. Conclusions The IDEA workflow provides a flexible platform to integrate NGS and bioinformatic tools for refined diagnosis and management of patients with advanced CRC.

A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair—or boost—the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug–genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E–mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E–mutant colorectal tumors. Mol Cancer Ther; 12(12); 2950–61. ©2013 AACR.

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.

Recent clinical trials have reported that alkylating agents, such as dacarbazine and temozolomide (TMZ) are effective in 10-15% metastatic colorectal cancer (mCRC) patients. In a retrospective analysis of 105 mCRC patients enrolled in clinical trials with alkylating agents, we saw that O6-methylguanine-DNA- methyltransferase (MGMT, the enzyme responsible for repairing drug induced DNA adducts), promoter methylation and protein status could identify a patient subgroup more likely to benefit from these drugs. However, the time to progression of patients treated with alkylating agents was limited by the onset of resistance. In order to identify determinants of response to these agents, we tested TMZ in a collection of 47 molecularly annotated CRC cell lines. We found that only cells displaying mismatch repair (MMR) proficiency as well as promoter methylation and transcriptional silencing of MGMT were sensitive to the treatment. Sensitive cells were chronically exposed to TMZ until the emergence of resistance occurred in seven out of ten models. We identified the mechanisms of acquired drug resistance to be either re-expression of MGMT and/or mutations in MMR genes. Biopsies of eight patients, relapsing upon TMZ based regimen after a long lasting clinical benefit (>3 months), were analyzed for MGMT re-expression and for exome or target panel next generation sequencing. These analyses confirmed the results found in preclinical models: five biopsies showed MGMT re-expression, two demonstrated mutations in the MMR genes (i.e. MSH6 or MSH2), and one displayed a mutation in BRCA2. Interestingly, biopsies and cell lines in which resistance was associated to alterations in DNA repair genes displayed increased mutational loads compared to pre-treatment samples. Despite the absence of the microsatellite switch commonly found in MMR deficient driven cancer, follow up over time in absence of drug of a subset of resistant cell line models demonstrated that those which displayed a mutation in the MMR had an accumulation of predicted neo-antigens, suggesting an increased immunogenicity. This work has implications for the design of future trials incorporating TMZ as part of a multi-modality strategy for treating MGMT deficient and MMR proficient CRC. Citation Format: Ludovic Barault, Giovanni Germano, Simona Lamba, Giuseppe Rospo, Alessandro Magri, Federica Maione, Mariangela Russo, Giovanni Crisafulli, Chiara Falcomatà, Carlotta Cancelliere, Alice Bartolini, Giulia Lerda, Giulia Siravegna, Benedetta Mussolin, Roberta Frapolli, Monica Montone, Giovanni Randon, Filippo de Braud, Nabil Amirouchene-Angelozzi, Silvia Marsoni, Maurizio D'Incalci, Armando Orlandi, Enrico Giraudo, Andrea Sartore-Bianchi, Salvatore Siena, Filippo Pietrantonio, Alberto Bardelli, Federica Di Nicolantonio. Accumulation of predicted neoantigens by MMR deficiency triggered by temozolomide treatment of human 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 2743.

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

Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.

The introduction of biodrugs, e.g. the anti-EGFR antibodies, was initially seen as a promise to radically change the landscape for patients with metastatic colorectal cancer. However, although EGFR-targeted therapies, combined with chemotherapy, have prolonged survival expectancy to 24 months, cure remains anecdotal. Target therapies suffer of high costs, important side effects, and low response rates: it is now clear that this approach as it was originally conceived failed to meet the majority of its expectations. Importantly, because of novel prescription guidelines, patients with KRAS-mutated tumors are excluded from EGFR-targeted therapies: for these patients alternative treatments are urgently needed. We here propose an innovative strategy based on the identification of molecular targets specifically associated with the microenvironment of metastatic colorectal cancer in patients carrying oncogenic KRAS. For this purpose, we set up mice models of metastatic colorectal cancer by intrasplenic (to evaluate liver homing) and intrahepatic (to investigate liver colonization) implantation of human colorectal cancer cell lines (SW48 and LIM1215) in which oncogenic KRAS (G12D, G12V, G13D) variants have been somatically knocked-in. We based our “target fishing” strategy on high-throughput, phage display-mediated proteomic screenings of deriving tumor samples ex vivo. Proteome signatures from the cognate cell lines in vitro served as a subtractive reference for the ex vivo biopanning, aimed at the identification of peptide ligands specific for non-epithelial components. By this combined biological-genetic-bioinformatics approach, we identified peptide/protein signatures selectively associated to metastasis microenvironments with controlled genetic backgrounds in vitro and in vivo. We selected 2 target proteins and 2 targeting peptides, which were exploited for diagnostic and therapeutic purposes. First, we evaluated by IHC the presence and localization of the target proteins in samples (tumor, healthy liver) from a panel of human biopsies and from the described in vivo models. This analysis confirmed a specific enrichment in KRAS-mutated microenvironments. Second, we tested the capability of dye-conjugated targeting peptides to home to these same tumor microenvironments, observing a specific accumulation in target tissues, compared to their scrambled versions and to control tissues. Third, we investigated a potential anti-metastatic effect of these peptides when orthotopically co-injected with colorectal cancer cell lines; preliminary experiments revealed that the targeting peptides, but not the scrambled variants, inhibit the onset of liver metastases from KRAS-mutated cell lines. In summary, we obtained proof-of-concept results in preclinical studies and produced prototype compounds to provide innovative tools that can be translated into the clinical practice with a mid-term perspective. Citation Format: Serena Marchio, Alice Bartolini, Sabrina Cardaci, Marco Soster, Giorgio Corti, Simona Lamba, Federico Bussolino, Davide Cora', Federica Di Nicolantonio. Microenvironment targets in KRAS-mutated metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1163. doi:10.1158/1538-7445.AM2014-1163

A cross-kingdom tale of drug resistance Physicians who treat bacterial infections and those who treat cancer often face a common challenge: the development of drug resistance. It is well known that when bacteria are exposed to antibiotics, they temporarily increase their mutation rate, thus increasing the chance that a descendant antibiotic-resistant cell will arise. Russo et al. now provide evidence that cancer cells exploit a similar mechanism to ensure their survival after drug exposure (see the Perspective by Gerlinger). They found that human colorectal cancer cells treated with certain targeted therapies display a transient up-regulation of errorprone DNA polymerases and a reduction in their ability to repair DNA damage. Thus, like bacteria, cancer cells can adapt to therapeutic pressure by enhancing their mutability. Science , this issue p. 1473 ; see also p. 1458

Background Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. Patients and methods We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying aCAD-ALK translocation during treatment with an ALK inhibitor. Results Using a custom next generation sequencing panel we identified the genomicCAD-ALK rearrangement and aTP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of theCAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient’s clinical course. Detection of theCAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identifiedALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. Conclusion We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.