Publications by authors named "Alice Bartolini"

24 Publications

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Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients.

ESMO Open 2019 11;4(6)

University of Turin, Department of Oncology, Candiolo (TO), Italy

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 or 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.
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http://dx.doi.org/10.1136/esmoopen-2019-000572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001107PMC
November 2019

A Subset of Colorectal Cancers with Cross-Sensitivity to Olaparib and Oxaliplatin.

Clin Cancer Res 2020 03 12;26(6):1372-1384. Epub 2019 Dec 12.

Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy.

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 and 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 and 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2409DOI Listing
March 2020

Adaptive mutability of colorectal cancers in response to targeted therapies.

Science 2019 12 7;366(6472):1473-1480. Epub 2019 Nov 7.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo (TO) 10060, Italy.

The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.
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http://dx.doi.org/10.1126/science.aav4474DOI Listing
December 2019

Evolving neoantigen profiles in colorectal cancers with DNA repair defects.

Genome Med 2019 06 28;11(1):42. Epub 2019 Jun 28.

Candiolo Cancer Institute, FPO-IRCCS, 10060, Candiolo (TO), Italy.

Background: Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor 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.

Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens.

Results: The 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.

Conclusions: These results indicate that CRCs 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.
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http://dx.doi.org/10.1186/s13073-019-0654-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599263PMC
June 2019

A Genomic Analysis Workflow for Colorectal Cancer Precision Oncology.

Clin Colorectal Cancer 2019 06 7;18(2):91-101.e3. Epub 2019 Mar 7.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo (TO), Italy; University of Turin, Department of Oncology, Candiolo (TO), Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.clcc.2019.02.008DOI Listing
June 2019

Trabectedin and olaparib in patients with advanced and non-resectable bone and soft-tissue sarcomas (TOMAS): an open-label, phase 1b study from the Italian Sarcoma Group.

Lancet Oncol 2018 10 11;19(10):1360-1371. Epub 2018 Sep 11.

Medical Oncology-Sarcoma Unit, Istituto di Candiolo-Fondazione del Piemonte per l'Oncologia, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Candiolo, Italy; Department of Oncology, University of Torino, Regione Gonzole, Orbassano, Italy.

Background: Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma.

Methods: We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675-1·3 mg/m every 3 weeks; olaparib 100-300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058.

Findings: Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2-6; range 1-17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5-23). Considering all dose levels, the most common grade 3-4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6-27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1.

Interpretation: Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas.

Funding: Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.
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http://dx.doi.org/10.1016/S1470-2045(18)30438-8DOI Listing
October 2018

Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer.

Cancer Cell 2018 07;34(1):148-162.e7

Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan 20162, Italy.

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.
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http://dx.doi.org/10.1016/j.ccell.2018.06.004DOI Listing
July 2018

Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth.

Nature 2017 12 29;552(7683):116-120. Epub 2017 Nov 29.

Candiolo Cancer Institute - FPO, IRCCS, Candiolo 10060, Turin, Italy.

Molecular alterations in genes involved in DNA mismatch repair (MMR) promote cancer initiation and foster tumour progression. Cancers deficient in MMR frequently show favourable prognosis and indolent progression. The functional basis of the clinical outcome of patients with tumours that are deficient in MMR is not clear. Here we genetically inactivate MutL homologue 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 on transplantation in immunocompromised mice. By contrast, MMR-deficient cancer cells grew poorly when transplanted in syngeneic mice. The inactivation of MMR increased the mutational burden and led to dynamic mutational profiles, which resulted in the persistent renewal of neoantigens in vitro and in vivo, whereas MMR-proficient cells exhibited stable mutational load and neoantigen profiles over time. Immune surveillance improved when cancer cells, in which MLH1 had been inactivated, accumulated neoantigens for several generations. When restricted to a clonal population, the dynamic generation of neoantigens driven by MMR further increased immune surveillance. Inactivation of MMR, driven by acquired resistance to the clinical agent temozolomide, increased mutational load, promoted continuous renewal of neoantigens in human colorectal cancers and triggered immune surveillance in mouse models. These results suggest that targeting DNA repair processes can increase the burden of neoantigens in tumour cells; this has the potential to be exploited in therapeutic approaches.
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http://dx.doi.org/10.1038/nature24673DOI Listing
December 2017

Genotyping tumour DNA in cerebrospinal fluid and plasma of a HER2-positive breast cancer patient with brain metastases.

ESMO Open 2017 9;2(4):e000253. Epub 2017 Oct 9.

Investigative Clinical Oncology (INCO), Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy.

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 and mutations as well as and c 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.
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http://dx.doi.org/10.1136/esmoopen-2017-000253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640139PMC
October 2017

Emergence of MET hyper-amplification at progression to MET and BRAF inhibition in colorectal cancer.

Br J Cancer 2017 Jul 27;117(3):347-352. Epub 2017 Jun 27.

Department of Oncology, University of Torino, Candiolo (TO) 10060, Italy.

Background: Combined MET and BRAF inhibition showed clinical benefit in a patient with rectal cancer carrying BRAF 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.
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http://dx.doi.org/10.1038/bjc.2017.196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537500PMC
July 2017

Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer.

Nat Commun 2016 12 8;7:13665. Epub 2016 Dec 8.

Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Torino, Italy.

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.
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http://dx.doi.org/10.1038/ncomms13665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155160PMC
December 2016

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer.

Cancer Res 2016 08 16;76(15):4504-15. Epub 2016 Jun 16.

Massachusetts General Hospital Cancer Center, Boston, Massachusetts. Department of Medicine, Harvard Medical School, Boston, Massachusetts.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970882PMC
http://dx.doi.org/10.1158/0008-5472.CAN-16-0396DOI Listing
August 2016

BCAM and LAMA5 Mediate the Recognition between Tumor Cells and the Endothelium in the Metastatic Spreading of KRAS-Mutant Colorectal Cancer.

Clin Cancer Res 2016 Oct 3;22(19):4923-4933. Epub 2016 May 3.

Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin), Italy. Department of Oncology, University of Turin, Candiolo (Turin), Italy. University of New Mexico Comprehensive Cancer Center. Albuquerque, New Mexico. Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2664DOI Listing
October 2016

A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

Cell 2016 Apr;165(2):317-30

Department of Oncology, University of Torino, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy; Candiolo Cancer Institute-FPO, IRCCS, SP142, Km 3.95, 10060 Candiolo, Torino, Italy.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.
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http://dx.doi.org/10.1016/j.cell.2016.02.059DOI Listing
April 2016

Acquired Resistance to the TRK Inhibitor Entrectinib in Colorectal Cancer.

Cancer Discov 2016 Jan 6;6(1):36-44. Epub 2015 Nov 6.

Department of Oncology, University of Torino, Torino, Italy. Candiolo Cancer Institute, FPO, IRCCS, Torino, Italy.

Unlabelled: 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.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0940DOI Listing
January 2016

The Neuronal Pentraxin-2 Pathway Is an Unrecognized Target in Human Neuroblastoma, Which Also Offers Prognostic Value in Patients.

Cancer Res 2015 Oct 20;75(20):4265-71. Epub 2015 Aug 20.

Laboratory of Tumor Microenvironment, Candiolo Cancer Institute-IRCCS, Candiolo, Italy. Department of Oncology, University of Torino, Candiolo, Italy.

Neuronal pentraxins (NPTX) and their corresponding receptors (NPTXR) have been studied as synapse-associated proteins in the nervous system, but their role in cancer is largely unknown. By applying a multidisciplinary, high-throughput proteomic approach, we have recently identified a peptide ligand motif for targeted drug delivery to neuroblastoma. Here, we report the sequence similarity between this peptide and a conserved portion of the pentraxin domain that is involved in the homo- and hetero-oligomerization of NPTX2 and NPTXR. We show that, in comparison with normal tissues, NPTX2 and NPTXR are overexpressed in vivo in mouse models, as well as in human Schwannian stroma-poor, stage IV neuroblastoma. Both proteins are concentrated in the vicinity of tumor blood vessels, with NPTXR also present on neuroblastic tumor cells. In vivo targeting of NPTX2 and NPTXR with the selected peptide or with specific antibodies reduces tumor burden in orthotopic mouse models of human neuroblastoma. In vitro interference with this ligand/receptor system inhibits the organization of neuroblastoma cells in tumor-like masses in close contact with vascular cells, as well as their adhesion to normal microenvironment-derived cells, suggesting a role in the cross-talk between tumor and normal cells in the early steps of neuroblastoma development. Finally, we show that NPTX2 is a marker of poor prognosis for neuroblastoma patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0649DOI Listing
October 2015

Neuroblastoma-targeted nanocarriers improve drug delivery and penetration, delay tumor growth and abrogate metastatic diffusion.

Biomaterials 2015 Nov 1;68:89-99. Epub 2015 Aug 1.

Laboratorio di Oncologia, Istituto G. Gaslini, Genoa, Italy. Electronic address:

Selective tumor targeting is expected to enhance drug delivery and to decrease toxicity, resulting in an improved therapeutic index. We have recently identified the HSYWLRS peptide sequence as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current therapies. Here we validated the specific binding of HSYWLRS to neuroblastoma cell suspensions obtained either from cell lines, animal models, or Schwannian-stroma poor, stage IV neuroblastoma patients. Binding of the biotinylated peptide and of HSYWLRS-functionalized fluorescent quantum dots or liposomal nanoparticles was dose-dependent and inhibited by an excess of free peptide. In animal models obtained by the orthotopic implant of either MYCN-amplified or MYCN single copy human neuroblastoma cell lines, treatment with HSYWLRS-targeted, doxorubicin-loaded Stealth Liposomes increased tumor vascular permeability and perfusion, enhancing tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy, as evaluated by bioluminescence imaging and micro-PET, leading to (i) delay of tumor growth paralleled by decreased tumor glucose consumption, and (ii) abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span. Our findings are functional to the design of targeted nanocarriers with potentiated therapeutic efficacy towards the clinical translation.
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http://dx.doi.org/10.1016/j.biomaterials.2015.07.054DOI Listing
November 2015

BRAF V600E is a determinant of sensitivity to proteasome inhibitors.

Mol Cancer Ther 2013 Dec 9;12(12):2950-61. Epub 2013 Oct 9.

Corresponding Authors: Federica Di Nicolantonio, Department of Oncology, University of Torino, Institute for Cancer Research and Treatment at Candiolo, Strada Provinciale 142 Km 3.95, Candiolo, I-10060, Turin, Italy.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0243DOI Listing
December 2013

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

J Control Release 2013 Sep 25;170(2):233-41. Epub 2013 May 25.

Experimental Therapy Unit, Laboratory of Oncology, Istituto Giannina Gaslini, Genoa, Italy.

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 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 NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.
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http://dx.doi.org/10.1016/j.jconrel.2013.04.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317336PMC
September 2013

A complex of α6 integrin and E-cadherin drives liver metastasis of colorectal cancer cells through hepatic angiopoietin-like 6.

EMBO Mol Med 2012 Nov 16;4(11):1156-75. Epub 2012 Oct 16.

Department of Oncology, University of Turin, Candiolo, Italy.

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.
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http://dx.doi.org/10.1002/emmm.201101164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494873PMC
November 2012

Modulation of cell-cycle dynamics is required to regulate the number of cerebellar GABAergic interneurons and their rhythm of maturation.

Development 2011 Aug 19;138(16):3463-72. Epub 2011 Jul 19.

Neuroscience Institute of Turin (NIT), Department of Neuroscience, University of Turin, I-10125 Turin, Italy.

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2(-/-) interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.
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http://dx.doi.org/10.1242/dev.064378DOI Listing
August 2011

G-CSF administration to adult mice stimulates the proliferation of microglia but does not modify the outcome of ischemic injury.

Neurobiol Dis 2011 Mar 24;41(3):640-9. Epub 2010 Nov 24.

Neuroscience Institute of Turin, Department of Neuroscience, Section of Physiology, University of Turin, I-10125 Turin, Italy.

Recent evidence suggests that adult bone marrow stem cells reduce tissue damage and promote repair following CNS ischemic injury. Since granulocyte-colony stimulating factor (G-CSF) mobilizes hematopoietic stem cells to the circulating compartment, here we tested whether administration of this drug modifies the outcome of a permanent occlusion of the middle cerebral artery in adult mice. To elucidate the behavior and fate of blood-borne cells in the ischemic brain, we produced chimeric animals, in which hematopoietic derivatives are genetically tagged. G-CSF administration enhances the proliferation of microglia in the uninjured CNS but has no effect on the amount of hematopoietic cells that infiltrate the ischemic tissue and on the size of the lesion. The blood-borne elements acquire different mesodermal identities but fail to adopt neural phenotypes, even though they occasionally fuse with Purkinje neurons. These results indicate that G-CSF treatment does not exert a significant beneficial effect on the ischemic injury.
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http://dx.doi.org/10.1016/j.nbd.2010.11.013DOI Listing
March 2011

Laminar fate and phenotype specification of cerebellar GABAergic interneurons.

J Neurosci 2009 May;29(21):7079-91

Department of Neuroscience and Rita Levi-Montalcini Center for Brain Repair, Neuroscience Institute of Turin, University of Turin, I-10125 Turin, Italy.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.0957-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6665607PMC
May 2009

Development of cerebellar GABAergic interneurons: origin and shaping of the "minibrain" local connections.

Cerebellum 2008 ;7(4):523-9

Department of Neuroscience and Rita Levi Montalcini Centre for Brain Repair, National Institute of Neuroscience, University of Turin, Turin, Italy.

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.
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http://dx.doi.org/10.1007/s12311-008-0079-zDOI Listing
May 2009