Thomas Rio Frio

Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types

Cell‐free tumor DNA (ctDNA) has the potential to enable non‐invasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The histology‐independent phase II SHIVA trial matches patients with targeted therapeutics based on previous screening of multiple somatic mutations using metastatic biopsies. To evaluate the utility of ctDNA in this trial, as an ancillary study we performed de novo detection of somatic mutations using plasma DNA compared to metastasis biopsies in 34 patients covering 18 different tumor types, scanning 46 genes and more than 6800 COSMIC mutations with a multiplexed next‐generation sequencing panel. In 27 patients, 28 of 29 mutations identified in metastasis biopsies (97%) were detected in matched ctDNA. Among these 27 patients, one additional mutation was found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. These results suggest that ctDNA analysis is a potential alternative and/or replacement to analyses using costly, harmful and lengthy tissue biopsies of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patients tumor genetic content.

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Emergence of New ALK Mutations at Relapse of Neuroblastoma

PURPOSE In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. METHODS We analyzed ALK mutations in 54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. RESULTS All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). CONCLUSION In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.

Designs and challenges for personalized medicine studies in oncology: focus on the SHIVA trial

Personalized medicine is defined by the National Cancer Institute as “a form of medicine that uses information about a person’s genes, proteins, and environment to prevent, diagnose, and treat disease.” In oncology, the term “personalized medicine” arose with the emergence of molecularly targeted agents. The prescription of approved molecularly targeted agents to cancer patients currently relies on the primary tumor location and histological subtype. Predictive biomarkers of efficacy of these modern agents have been exclusively validated in specific tumor types. A major concern today is to determine whether the prescription of molecularly targeted therapies based on tumor molecular abnormalities, independently of primary tumor location and histology, would improve the outcome of cancer patients. This new paradigm requires prospective validation before being implemented in clinical practice. In this paper, we will first review different designs, including observational cohorts, as well as nonrandomized and randomized clinical trials, that have been recently proposed to evaluate the relevance of this approach, and further discuss their advantages and drawbacks. The design of the SHIVA trial, a randomized proof-of-concept phase II trial comparing therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer will be detailed. Finally, we will discuss the multiple challenges associated with the implementation of personalized medicine in oncology, as well as perspectives for the future.

Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1–dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

Breakpoint Features of Genomic Rearrangements in Neuroblastoma with Unbalanced Translocations and Chromothripsis

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.

Frequent Somatic Mutation in Adult Intestinal Stem Cells Drives Neoplasia and Genetic Mosaicism during Aging

Summary Adult stem cells may acquire mutations that modify cellular behavior, leading to functional declines in homeostasis or providing a competitive advantage resulting in premalignancy. However, the frequency, phenotypic impact, and mechanisms underlying spontaneous mutagenesis during aging are unclear. Here, we report two mechanisms of genome instability in adult Drosophila intestinal stem cells (ISCs) that cause phenotypic alterations in the aging intestine. First, we found frequent loss of heterozygosity arising from mitotic homologous recombination in ISCs that results in genetic mosaicism. Second, somatic deletion of DNA sequences and large structural rearrangements, resembling those described in cancers and congenital diseases, frequently result in gene inactivation. Such modifications induced somatic inactivation of the X-linked tumor suppressor Notch in ISCs, leading to spontaneous neoplasias in wild-type males. Together, our findings reveal frequent genomic modification in adult stem cells and show that somatic genetic mosaicism has important functional consequences on aging tissues.

Deep Sequencing Reveals Occurrence of Subclonal ALK Mutations in Neuroblastoma at Diagnosis

Purpose: In neuroblastoma, activating ALK receptor tyrosine kinase point mutations play a major role in oncogenesis. We explored the potential occurrence of ALK mutations at a subclonal level using targeted deep sequencing. Experimental Design: In a clinically representative series of 276 diagnostic neuroblastoma samples, exons 23 and 25 of the ALK gene, containing the F1174 and R1275 mutation hotspots, respectively, were resequenced with an extremely high depth of coverage. Results: At the F1174 hotspot (exon 23), mutations were observed in 15 of 277 samples (range of fraction of mutated allele per sample: 0.562%–40.409%). At the R1275 hotspot (exon 25), ALK mutations were detected in 12 of 276 samples (range of fraction of mutated allele: 0.811%–73.001%). Altogether, subclonal events with a mutated allele fraction below 20% were observed in 15/27 ALK-mutated samples. The presence of an ALK mutation was associated with poorer 5-year overall survival (OS: 75% vs. 57%, P = 0.0212 log-rank test), with a strong correlation between F1174 ALK mutations and MYCN amplification being observed. Conclusions: In this series, deep sequencing allows the detection of F1174 and R1275 ALK mutational events at diagnosis in 10% of cases, with subclonal events in more than half of these, which would have gone undetected by Sanger sequencing. These findings are of clinical importance given the potential role of ALK mutations in clonal evolution and relapse. These findings also demonstrate the importance of deep sequencing techniques for the identification of patients especially when considering targeted therapy. Clin Cancer Res; 21(21); 4913–21. ©2015 AACR. See related commentary by George, p. 4747

Abstract 2400: Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for targeted sequencing in a prospective randomized trial for personalized treatment in all tumor type: The SHIVA study

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rationale: The noninvasive biomarker circulating cell-free tumor DNA (ctDNA) has the potential to enable noninvasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The ongoing large multicentric randomized histology-independent phase II trial (SHIVA, [NCT01771458][1]) compares molecularly targeted therapy based on tumor molecular profiling versus conventional therapy in patients with any type of refractory cancer. The screening phase consists of the invasive biopsy of metastatic tumor tissue and downstream analysis using Ion Torrents Ampliseq hotspot cancer panel. We evaluated whether ctDNA analysis would identify the same mutations as those obtained through invasive biopsy. Patients and methods: de novo detection of somatic mutations using ctDNA in 34 patients covering 18 different tumor types, scanning ∼50 genes and more than 2500 mutations with a multiplexed next generation sequencing panel. Results: In 27 patients, 27 of 28 actionable mutations identified in metastasis biopsies (96%) were detected in matched ctDNA. Among these 27 patients, two additional mutations were found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. Conclusion; These results suggest that ctDNA analysis is a cheaper, safer and quicker alternative to invasive biopsy of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patients tumor genetic content. Citation Format: Jean-Yves Pierga, Charles Decraene, Virginie Bernard, Maud Kamal, Anthony Blin, Quentin Leroy, Thomas Rio Frio, Gaelle Pierron, Celine Callens, Ivan Bieche, Adrien Saliou, Jordan Madic, Etienne Rouleau, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Ronald Lebofsky, Christophe Le Tourneau. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for targeted sequencing in a prospective randomized trial for personalized treatment in all tumor type: The SHIVA study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2015-2400 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01771458&atom=%2Fcanres%2F75%2F15_Supplement%2F2400.atom

Abstract PD3-8: Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients

Background Preliminary reports suggested that circulating tumor DNA (ctDNA) can be used as a prognostic marker in a way akin to circulating tumor cells (CTC) in metastatic breast cancer patients. However ctDNA detection is often performed on multiple mutations, combining heterogeneous techniques. Here we used the high prevalence of TP53 mutations in triple-negative metastatic breast cancer (TNMBC) to compare CTC and ctDNA detection rates and prognostic value. Methods A cohort of 40 patients treated at the Institut Curie (Paris, France) was enrolled before starting a new line of treatment for TNMBC. CTC were detected by the CellSearch system (in 7.5 mL of blood). Using massively parallel sequencing (NGS), TP53 mutations were first characterized in tumor tissue, then in plasma DNA extracted from fresh frozen plasma samples (from 15-20 mL of blood). ctDNA detection was performed using high depth targeted sequencing using two platforms in parallel (Illumina HiSeq 2500 and Roche 454). Libraries for Illumina were prepared following the TAm-Seq procedure (Forshew et al, Sci Transl Med 2012), with preamplification of all coding TP53 exons and flanking untranslated regions followed by both paired-end 150bp Illumina and 454/Roche sequencing. CTC, ctDNA and usual patient characteristics were correlated with time to progression (TTP) and overall survival (OS). Results Archived tumor (FFPE or frozen) tissue was available for 36 patients, and 31 were successfully sequenced: TP53 mutations were found in 27 patients. As measured on the Illumina platform, ctDNA was detected in 21/27 patients (81%), ranging from 48 to 648,000 copies/mL of plasma (median 1620). Mutant allele fraction in circulating cell-free DNA ranged from 2 to 70% (median 5%). Comparison between ctDNA levels measured by Illumina and 454/Roche platforms in plasma displayed a good correlation (R2 = 0.903), with a single discordance. ≥1 CTC were detected in 19 of these 27 patients (70%). Strikingly, high ctDNA levels had prognostic impact neither on OS, nor on TTP, whatever the dataset used (Illumina or 454) whereas CTC≥5/7.5 mL were correlated with OS (p=0.04) and marginally with TTP (p=0.06). Other known usual factors, such as poor performance status, elevated LDH and number of previous treatment lines had also significant prognostic factors in this cohort. CTC and ctDNA early changes during treatment were available for 12 patients and changes (increase/decrease) of the two biomarkers were globally similar. Conclusion Demonstrating a good sensitivity (81%), ctDNA by the TAm-Seq is more frequently detected than CTCs in the 27 TNMBC with TP53 mutations. The observed correlation between the 2 massively parallel sequencing approaches suggested that ctDNA levels data were quantitative. In contrast to other usual prognostic factors, baseline ctDNA level did not demonstrate a prognostic impact,in this proof-of-principle study, suggesting that mechanisms of ctDNA release in TNMBC rely on biological features that do not dramatically impact patient’s outcome. Citation Format: Francois-Clement Bidard, Jordan Madic, Anna Kiialainen, Fabian Birzele, Guillemette Ramey, Quentin Leroy, Thomas Rio Frio, Virginie Raynal, Virginie Bernard, Alban Lermine, Inga Clausen, Nicolas Giroud, Roland Schmucki, Carsten Horn, Olivia Spleiss, Olivier Lantz, Marc-Henri Stern, Martin Weisser, Ronald Lebofsky, Jean-Yves Pierga. Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-8.