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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

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.

PDF file - 635K, Supplementary Figure S1. Genotypes included in the combinatorial genetic matrix. PTEN or RB1 were silenced by shRNA in isogenic KI cells.

XLSX file - 144K, Supplementary table S1. List of compounds employed in the present work. Supplementary Table S2. List of t-tests p values comparing the responses of the different mutant cell lines to a given drug concentration with the response of the WT Scramble control cells. Supplementary Table S3. List of the drugs included in the library and tested on HME-1 cell lines.

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.

Purpose: KRAS mutations confer adverse prognosis to colorectal cancer, and no targeted therapies have shown efficacy in this patient subset. Paracrine, nongenetic events induced by KRAS-mutant tumor cells are expected to result in specific deregulation and/or relocation of tumor microenvironment (TME) proteins, which in principle can be exploited as alternative therapeutic targets.Experimental Design: A multimodal strategy combining ex vivo/in vitro phage display screens with deep-sequencing and bioinformatics was applied to uncover TME-specific targets in KRAS-mutant hepatic metastasis from colorectal cancer. Expression and localization of BCAM and LAMA5 were validated by immunohistochemistry in preclinical models of human hepatic metastasis and in a panel of human specimens (n = 71). The antimetastatic efficacy of two BCAM-mimic peptides was evaluated in mouse models. The role of BCAM in the interaction of KRAS-mutant colorectal cancer cells with TME cells was investigated by adhesion assays.Results: BCAM and LAMA5 were identified as molecular targets within both tumor cells and TME of KRAS-mutant hepatic metastasis from colorectal cancer, where they were specifically overexpressed. Two BCAM-mimic peptides inhibited KRAS-mutant hepatic metastasis in preclinical models. Genetic suppression and biochemical inhibition of either BCAM or LAMA5 impaired adhesion of KRAS-mutant colorectal cancer cells specifically to endothelial cells, whereas adhesion to pericytes and hepatocytes was unaffected.Conclusions: These data show that the BCAM/LAMA5 system plays a functional role in the metastatic spreading of KRAS-mutant colorectal cancer by mediating tumor–TME interactions and as such represents a valuable therapeutic candidate for this large, currently untreatable patient group. Clin Cancer Res; 22(19); 4923–33. ©2016 AACR.

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.

The supplementary material includes Supplementary Methods, two Supplementary Tables, and six Supplementary Figures Table S1. Complete set of BCAM-mimic peptides. Table S2. Characterization of human CRC cell lines in animal models. Figure S1. Quality control of the phage display experiments: sequencing readout. Figure S2. BCAM protein expression in the complete panel of human hepatic metastases. Figure S3. BCAM protein expression in human primary CRCs. Figure S4. BCAM protein and mRNA expression in all cell lines. Figure S5. Photographic documentation of the experimental hepatic metastasis models. Figure S6. HT-55, DLD-1, LIM1215 and SW-48 cells cannot be reduced to adhesion-functional monocellular suspensions.

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 Table S2

Supplementary Table S1

Supplementary Table S4

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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.

Supplemental Figure 4: neuroblastoma cells migrate towards microenvironment-derived cells

Supplemental Methods and Figure Legends

Supplemental Figure 1: ephrin A1 and α11 integrin are barely detectable in mouse models of neuroblastoma

Supplemental Figure 2: expression levels of NPTX2 and NPTXR in vitro and in vivo

Supplemental Figure 5: NPTX2 and NPTXR are barely or not expressed in normal human tissues

Supplemental Figure 3: NPTX2 and NPTXR are barely or not expressed in normal mouse tissues

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

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.

Patients affected by colorectal cancer (CRC) with DNA mismatch repair deficiency (MMRd), often respond to immune checkpoint blockade therapies, while those with mismatch repair-proficient (MMRp) tumors generally do not. Interestingly, a subset of MMRp CRCs contains variable fractions of MMRd cells, but it is unknown how their presence impacts immune surveillance. We asked whether modulation of the MMRd fraction in MMR heterogeneous tumors acts as an endogenous cancer vaccine by promoting immune surveillance. To test this hypothesis, we use isogenic MMRp (Mlh1+/+) and MMRd (Mlh1-/-) mouse CRC cells. MMRp/MMRd cells mixed at different ratios are injected in immunocompetent mice and tumor rejection is observed when at least 50% of cells are MMRd. To enrich the MMRd fraction, MMRp/MMRd tumors are treated with 6-thioguanine, which leads to tumor rejection. These results suggest that genetic and pharmacological modulation of the DNA mismatch repair machinery potentiate the immunogenicity of MMR heterogeneous tumors.

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.

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.

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.

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

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.

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.

The tumor mutational burden affects immune surveillance and is associated with response to immune checkpoint blockade. We recently reported that inactivation of the DNA mismatch repair (MMR) pathway in cancer cells increases the mutational burden and modifies the neoantigen landscapes of cancer cells leading to their increased recognition by the immune system. We designed a pharmacologic screening to identify FDA-approved drugs capable of differentially affecting cancer cells MMR proficient and deficient. MMR-deficienT-cells displayed lower sensitivity to the alkylating agent Temozolomide (TMZ) and to the antimetabolite 6-Thioguanine (6-TG). Cells lacking key elements of the MMR pathway such as MutL homolog1, MutS homolog2 (MSH2) or MutS homolog 6 (MSH6), displayed an increased resistance to both TMZ and 6-TG. Next we treated two mouse colorectal cancer cell lines (MC38 and CT26) with TMZ until resistant populations emerged. MC38 cells acquired TMZ resistance through inactivation of the MMR pathway. Bioinformatic analysis revealed that these cells had higher numbers of neoantigens compared to parental cells. Importantly, when MC38 MMRd cells were injected in syngeneic mice, they were unable to form tumors. On the contrary, CT26 cells that acquired TMZ-resistance through other mechanisms, efficiently formed tumors in mice. Therefore, TMZ-induced MMR inactivation, and not TMZ treatment per se, triggered immune surveillance. To assess whether results obtained in mouse cancer models might translate to human disease, we tested TMZ in 47 molecularly annotated colorectal cancer (CRC) cancer cell lines. Only MMR-proficienT-cells and cells in which O6-methylguanine-DNA- methyltransferase (MGMT, the enzyme responsible for repairing the DNA adducts formed by TMZ) was not expressed were sensible to TMZ. Ten sensitive cell lines were chronically treated with TMZ until resistant populations emerged; we found that MGMT re-expression and loss of MMR genes were the main mechanisms of acquired resistance. In agreement with in vitro observations, analysis of biopsies from eight patients relapsing upon TMZ-based therapeutic regimens revealed MGMT re-expression (5 patients) and MMR genes mutations (i.e., MSH2 or MSH6) as main resistance mechanism. In both cell lines and biopsies, MMR inactivation led to increased mutational load and, consequently, to higher levels of predicted neo-antigens, suggesting an augmented immunogenicity. These preclinical data led to the clinical trial Arethusa (NCT03519412; https://clinicaltrials.gov/ct2/show/NCT03519412). Within Arethusa MMR-proficient patients will be tested for (MGMT) expression (IHC) and then for MGMT promoter methylation. MGMT negative patients will be treated with temozolomide (TMZ). Patients progressing under temozolomide will be tested for tumor mutational burden (TMB) and proceed to pembrolizumab if TMB is > 20 mutations/Mb. The primary study hypothesis is that tumors with acquired resistance to temozolomide might become hypermutated and could be sensitive to the anti PD-1 antibody, pembrolizumab. Citation Format: Vito Amodio, Giovanni Grmano, Ludovic Barault, Simona Lamba, Giuseppe Rospo, Alessandro Magrì, Federica Maione, Giovanni Crisafulli, Carlotta Cancelliere, Giulia Lerda, Alice Bartolini, Giulia Siravegna, Benedetta Mussolin, Roberta Frappolli, Monica Montone, Giovanni Randon, Filippo de Braud, Nabil Amirouchene Angelozzi, Silvia Marsoni, Maurizio D'Incalci, Armando Orlandi, Enrico Giraudo, Andrea Satore-Bianchi, Salvatore Siena, Filippo Pietrantonio, Federica Di Nicolantonio, Alberto Bardelli. Temozolomide drives mismatch repair deficiency and fosters neoantigen generation in tumor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B069.

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.

Colorectal, ovarian, endometrial and other tumors carrying defects in DNA mismatch repair often show favorable prognosis and indolent progression. The genomes of these tumors bear hundreds of thousands of somatic mutations, a feature which fosters cancer progression and might lead to rapid evolution of resistance to targeted therapies. Recent evidences that a subset of MSI (microsatellite instable) tumors respond prominently to immune checkpoint blockade led to the hypothesis that the presence of high number of somatic mutations may be responsible for effective immune-surveillance. However, several reports indicate that a relevant fraction of hyper-mutated tumors have unfavorable prognosis and do not respond to immune-modulators. To understand the molecular and functional bases of response to immune checkpoint inhibitors, we genetically inactivated MutL homolog 1 (MLH1) in colorectal, breast and pancreatic mouse cancer cells. The growth of MMR deficient cells was comparable to their proficient counterparts in vitro and upon transplantation in immune-compromised mice. However, isogenic MMR deficient cancer cells, acquiring alterations over time, were unable to form tumors when injected subcutaneously or orthotopically in syngeneic mouse models according to cell-passage number. Moreover, we found that MMR-driven dynamic generation of neoantigens, when restricted to a clonal population, further increases immune detection. Mechanistically, MLH1 inactivation increased the mutational burden and led to dynamic mutational profiles, resulting in persistent renewal of neoantigens in vitro and in vivo, while control cells exhibited stable mutational loads and neoantigen profiles over time. These results led us hypothesize that enforced increase of the number of mutations in cancer cells could restrict cancer growth and might be beneficial for therapeutic purposes. We therefore performed a pharmacological screen to identify agents capable of permanent inactivation of MMR in colorectal, breast and PDAC cancer cells. We found that temozolomide triggers MLH1 inactivation in cancer cells that -as a result- are unable to form tumors in syngeneic animals. Genomic analysis of temozolomide resistant cells revealed that fluctuating levels of neoantigens, rather than the absolute number of mutations, might be critical to provoke immune surveillance. Overall, these results provide the rationale for developing innovative anticancer therapies based on inhibition of DNA repair mechanisms. Citation Format: Giovanni Germano, Simona Lamba, Giuseppe Rospo, Ludovic Barault, Alessandro Magri', Federica Maione, Mariangela Russo, Giovanni Crisafulli, Alice Bartolini, Giulia Lerda, Giulia Siravegna, Benedetta Mussolin, Roberta Frapolli, Monica Montone, Federica Morano, Filippo de Braud, Nabil Amirouchene-Angelozzi, Silvia Marsoni, Maurizio D'Incalci, Armando Orlandi, Enrico Giraudo, Andrea Sartore-Bianchi, Salvatore Siena, Filippo Pietrantonio, Federica DiNicolantonio, Alberto Bardelli. Inactivation of DNA repair triggers neoantigen generation and impairs tumor growth [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 5723.

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.

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

Cetuximab and panitumumab are monoclonal anti-EGFR antibodies (moAbs) currently used for the treatment of advanced RAS wild type colorectal cancers (CRC). Emergence of acquired resistance invariably limits the efficacy of these agents, and the dynamics of clonal evolution during anti-EGFR blockade are poorly understood. At progression, RAS mutations represent the most common genetic alterations, while EGFR extracellular domain (ECD) mutations are acquired by a smaller cohort of patients. We found that the mutation profile correlates with the clinical outcome of patients; in particular those who develop RAS mutations upon EGFR blockade achieve reduced tumor shrinkage and shorter duration of response respect to patients in which EGFR ECD mutations emerge during therapy. We investigated in preclinical models the potential role of RAS and EGFR ECD mutations during the emergence of acquired resistance, by tracking the evolution of clones in a genetically barcoded population of CRC cells chronically treated with cetuximab. We observed that therapeutic (target therapy, chemotherapy) and environmental (reduced nutrient condition) pressures differentially shape the clonal composition of CRC cell populations, leading to the emergence of clones with the highest fitness in presence of the external pressure. In conclusion, a multistep clonal evolution process characterizes the development of drug resistance and is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies. Citation Format: Sabrina Arena, Beth Van Emburgh, Giulia Siravegna, Luca Lazzari, Giovanni Crisafulli, Giorgio Corti, Benedetta Mussolin, Federica Baldi, Michela Buscarino, Alice Bartolini, Emanuele Valtorta, Joana Vidal, Beatriz Bellosillo, Giovanni Germano, Filippo Pietrantonio, Agostino Ponzetti, Joan Albanell, Salvatore Siena, Andrea Sartore-Bianchi, Federica Di Nicolantonio, Clara Montagut, Alberto Bardelli. Emergence of RAS or EGFR mutant clones affects duration of response to EGFR blockade in colorectal cancers [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 2913. doi:10.1158/1538-7445.AM2017-2913

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

Background Central nervous system (CNS) involvement contributes to significant morbidity and mortality in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (mBC) and represents a major challenge for clinicians. Liquid biopsy of cerebrospinal fluid (CSF)-derived circulating tumour DNA (ctDNA) harbours clinically relevant genomic alterations in patients with CNS metastases and could be effective in tracking tumour evolution. Methods In a HER2-positive mBC patient with brain metastases, we applied droplet digital PCR (ddPCR) and next-generation whole exome sequencing (WES) analysis to measure ctDNA dynamic changes in CSF and plasma collected during treatment. Results Baseline CSF-derived ctDNA analysis revealed TP53 and PIK3CA mutations as well as ERBB2 and c MYC amplification. Post-treatment ctDNA analysis showed decreased markers level in plasma, consistent with extra-CNS disease control, while increased in the CSF, confirming poor treatment benefit in the CNS. Discussion Analysis of ctDNA in the CSF of HER2-positive mBC is feasible and could represent a useful companion for clinical management of brain metastases.

The cerebellar circuits comprise a limited number of neuronal phenotypes embedded in a defined cytoarchitecture and generated according to specific spatio-temporal patterns. The local GABAergic network is composed of several interneuron phenotypes that play essential roles in information processing by modulating the activity of cerebellar cortical inputs and outputs. A major issue in the study of cerebellar development is to understand the mechanisms that underlie the generation of different interneuron classes and regulate their placement in the cerebellar architecture and integration in the cortico-nuclear network. Recent findings indicate that the variety of cerebellar interneurons derives from a single population of multipotent progenitors whose fate choices are determined by instructive environmental information. Such a strategy, which is unique for the cerebellum along the neuraxis, allows great flexibility in the control of the quality and quantity of GABAergic interneurons that are produced, thus facilitating the adaptive shaping of the cerebellar network to specific functional demands.

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

Purpose: Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. The identification of novel peptide ligands specific for cells present in high-risk neuroblastoma, a childhood tumor mostly refractory to current therapies, is needed. Experimental design: We performed combined in vitro/ex-vivo phage display screenings on human neuroblastoma cell lines and on tumors derived from orthotopic mouse models of human neuroblastoma. Binding validation and homing in vivo of selected phage clones were tested by immunohistochemistry/immunofluorescent analyses. Cell association experiments in vitro with the corresponding synthetic biotin-labeled peptides were performed. In vitro cytotoxicity and in vivo tumor accumulation and therapeutic experiments were performed using peptide-targeted, doxorubicin-loaded, nanocarriers. Results: By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for the stromal components. Globally, we isolated 121 phage-displayed neuroblastoma-binding peptides; of these, 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV neuroblastoma patients and to neuroblastoma tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into neuroblastoma-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded nanocarriers led to a significant inhibition in tumor volume and enhanced survival in preclinical neuroblastoma models. Conclusions: Our findings demonstrate that novel ligands of neuroblastoma-associated markers are functional in the design of nanocarriers with therapeutic efficacy paving the way to their clinical development. Citation Format: Monica Loi, Daniela Di Paolo, Marco Soster, Chiara Brignole, Alice Bartolini, Laura Emionite, Jessica Sun, Pamela Becherini, Flavio Curnis, Andrea Petretto, Monica Sani, Alessandro Gori, Claudio Gambini, Renato Longhi, Michele Cilli, Theresa M. Allen, Federico Bussolino, Wadih Arap, Renata Pasqualini, Angelo Corti, Mirco Ponzoni, Serena Marchiò, Fabio Pastorino. Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5620. doi:10.1158/1538-7445.AM2013-5620