Ronald Lebofsky

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.

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.

Time-Dependent Prognostic Impact of Circulating Tumor Cells Detection in Non-Metastatic Breast Cancer: 70-Month Analysis of the REMAGUS02 Study

Introduction. In non-metastatic breast cancer patients, the REMAGUS02 neoadjuvant study was the first to report a significant impact of circulating tumor cells (CTCs) detection by the CellSearch system on the distant metastasis-free survival (DMFS) and overall survival (OS) endpoints. However, these results were only reported after a short follow-up. Here, we present the updated data, with a longer follow-up. Material and Methods. CTC count was performed before and after neoadjuvant chemotherapy in 118 patients and correlated to survival. Results. CTC count results were available before and/or after neoadjuvant chemotherapy in 115 patients. After a median follow-up of 70 months, detection of ≥1 CTC/7.5 mL before chemotherapy (N = 95) was significantly associated with DMFS (P = 0.04) and OS (P = 0.03), whereas postchemotherapy CTC detection (N = 85) had no significant impact. In multivariable analysis, prechemotherapy CTC and triple negative phenotype were the two independent prognostic factors for survival. We observed that the CTC impact is most significant during the first three years of follow-up. Discussion. We confirm that the detection of CTC is independently associated with a significantly worse outcome, but mainly during the first 3-4 years of follow-up. No prognostic impact is seen in patients who are still relapse-free at this moment.

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.

Abstract LB-76: Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool

Circulating tumor DNA (ctDNA) analysis requires absolute single molecule sensitivity, since only a few copies of the mutation may be present per blood draw. Recent developments in next-generation sequencing (NGS) sample preparation have improved the relative sensitivity to detect Citation Format: Ronald Lebofsky, Jordan Madic, Adrien Saliou, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Jean-Yves Pierga. Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool. [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 LB-76. doi:10.1158/1538-7445.AM2014-LB-76

1571PDDE NOVO MUTATION DETECTION FROM CTDNA CORRELATES WITH VARIANTS DETECTED ON METASTASIS OF PATIENTS WITH ANY KIND OF REFRACTORY CANCER FROM THE SHIVA TRIAL

ABSTRACT Aim: The ability to perform molecular analyses on circulating tumor DNA (ctDNA) is a promising non-invasive tool to determine tumor genomic alterations. Liquid biopsies may provide information when the biopsy is not available, and address tumor heterogeneity. Multiple sampling and ctDNA analysis over time allows identification of emerging predictive biomarkers and collection of pharmacodynamic data. The aim of this study was to assess whether targeted sequencing on ctDNA permits the detection of the same mutations observed on solid tumor from the same patient. Methods: SHIVA is a multicentric randomized proof-of-concept phase II trial comparing molecularly targeted therapy based on tumor molecular profiling of a mandatory biopsy of a metastatic site versus conventional therapy in patients with any type of refractory cancer (Le Tourneau et al., BJC 2014). The molecular profile within SHIVA is performed on a mandatory biopsy and includes mutation analyses using AmpliSeq cancer panel (Life Technologies®). Blood samples for ctDNA analyses are taken before starting treatment, between day 7 and day 15, at first tumor evaluation, and at disease progression. The same approach was applied here to ctDNA before treatment and mutational profiles were compared to those from tumor biopsies. The bioinformatics analysis was done in a blinded manner. Results: ctDNA analysis was done on 19 patients including 3 patients with tumor biopsy not eligible for analysis. The mutation(s) identified from the solid tumor biopsy was confirmed in all corresponding blood samples (16/16) and 1 mutation was identified de novo in a patient whose tumor biopsy was uninformative. Tumor types included breast (5), lung (3), ovary (2), cervix (2) among others. Median ctDNA amount was 37 ng/mL [range: 7- 423]. Conclusions: Our results suggest that targeted sequencing of ctDNA across a panel of genes can reliably detect tumor sample mutations de novo without any a priori information from the tumor biopsy. Further analyses on ctDNA from different time points of the patients treatment will give more insight into the potential of liquid biopsies to follow up disease progression and response to treatment. Disclosure: All authors have declared no conflicts of interest.

Abstract OT1-1-10: CirCe T-DM1 phase II trial: Assessing the relevance of HER2-amplified circulating tumor cells as a tool to select HER2-negative metastatic breast cancer for treatment with T-DM1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: T-DM1 has demonstrated its efficacy in the second line of HER2- positive metastatic breast cancer patients. Several studies reported that some breast cancers considered as HER2-negative do have HER2-positive circulating tumor cells (CTC). Our previous report (Lightart & Bidard, Ann Oncol 2013) showed that the reliability of such discrepancy between primary tumor and CTC is directly related to the number of CTC analyzed. This study aims at studying the efficacy of T-DM1 in this setting. Trial design: CirCe T-DM1 is a single arm two-step phase II multicenter study with adaptive design. Patients with HER2-negative measurable metastatic breast cancer will be screened by the FDA-approved CellSearch system, before the start of a second line treatment. HER2/CEP17 ratio will be quantified by FISH on CTC (Janssen Diagnostics). Patients with non HER2-amplified CTC or no CTC will be excluded from the study Patients with HER2-amplified CTC will be treated by T-DM1 single agent q3w, in either cohort “L” (low count: 1 to 4 HER2-amplified CTC) or “H” (high count: 5 or more HER2-amplified CTC). 14 patients (7L+7H) will be included in the first step. If needed, the second step will include 14 additional patients (7L+7H). Tumor response (per RECIST criteria) is the main objective of the trial. Eligibility criteria: main criteria are HER2-negative metastatic breast cancer; measurable disease progressing after a first line treatment; PS 0-1; criteria related to T-DM1 treatment and ethics. Only patients with HER2-positive CTC will be treated by T-DM1. Specific aim: this study will assess the response rate to T-DM1 in patients with HER2-amplified CTC and HER2-negative metastatic breast cancer, taking into account the number of HER2-amplified CTC detected (1-4 or 5+). Statistical methods: To design this adaptive study, response rates were estimated to be H0=5% (no efficacy of T-DM1) and H1=25% (efficacy of T-DM1). After the first step (N=7L and 7H patients): if no response is seen, the trial will be stopped, and considered as negative; if 3 or more responses are seen, we will conclude that the CTC FISH test is relevant to select patient for T-DM1 treatment, in either the L or/and H population, according to the observed pattern of responses; if 1 or 2 responses are observed, 14 more patients will be enrolled (7L+7H) in the second step of the trial. At the end of the second step, if 3 or less responses are observed, the trial will be considered as negative; if 4 or more responses are seen, we will conclude that the CTC FISH test is relevant to select patient for T-DM1 treatment, in either the L or/and H population, according to the observed pattern of responses. Based on the above-mentioned hypotheses and number of patients, the alpha risk was simulated to be of 6%, with a power of 94%. Accrual: the trial started in november 2013 in France. Up to 28 patients will be treated in this study. Citation Format: Francois-Clement Bidard, Yann de Rycke, Bernard Asselain, Paul Cottu, Manuel Rodrigues, Ronald Lebofsky, Jean-Yves Pierga. CirCe T-DM1 phase II trial: Assessing the relevance of HER2-amplified circulating tumor cells as a tool to select HER2-negative metastatic breast cancer for treatment with TDM1. [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 CT317. doi:10.1158/1538-7445.AM2014-CT317

Abstract PD6-5: Pooled analysis of circulating tumor cells in metastatic breast cancer: Findings from 1944 individual patients data

Background: Clinical validity of CTCs (CellSearch®) in metastatic breast cancer (MBC) patients has previously been assessed in studies with limited statistical power. We aimed to pool all European studies to obtain high-level evidence on the prognostic value of CTCs, to investigate their effects across different clinico-pathological characteristics and therapies and to further validate the MD Anderson/Institut Curie/Fox Chase CTC-based prognostic nomogram established in first-line treated MBC patients (Giordano et al, Clin Cancer Res 2013). Material and methods: Methods were predefined in a written protocol. In December 2012, we searched for eligible studies that accrued patients in 2003-2012. We contacted all European laboratories using CellSearch®. We used likelihood ratio tests (LR) in Cox regression models stratified by study to assess the independent prognostic value of CTC when added to a clinicopathological (CP) model for progression-free (PFS) and overall survival (OS). Landmark analyses were used to assess the prognostic effect of early changes in CTC. The CTC-based nomogram (http://cancernomograms.com/CTCOnline.html) score was retrieved for every patient; we calculated C-indices, drew calibration plots and Kaplan-Meier curves according to quintiles of the nomogram score. Results: We collected individual data of 1944 MBC patients, from 20 different studies (some unpublished), from 17 centers in 7 European countries. We observed 1507 PFS events and 929 deaths. Baseline CTC count was significantly associated with several patient characteristics, such as performance status (PS, p Conclusions: This pooled analysis is the largest study ever reported on CTC in MBC, with a previously unreached statistical power. It provides a clear level-of-evidence 1 on the independent prognostic value of CTCs before and during treatment in MBC. Also, the CTC-based prognostic nomogram is independently validated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD6-5.