Biopsie Liquide Récapitulatif Des Études Cliniques Réalisées

Biopsie Liquide Récapitulatif des études cliniques réalisées

Dr. Tarik MARZOUKI [email protected] Publications et Etudes cliniques réalisées

Dr. Tarik MARZOUKI France Communiqué de presse du CHU DE LYON Communiqué du CHU DE LYON Hospices Civils de Lyon - mardi 03 mai 2016

Cancer du poumon : une simple prise sang remplace la biopsie Etablir un diagnostic, affiner le traitement, accéder à des thérapies ciblées plus efficaces, la biopsie de la tumeur ou d’une de ses métastases a longtemps été considérée comme une pièce maîtresse du traitement. Mais ce geste restait complexe, invasif et douloureux surtout quand il était pratiqué de manière répétée, particulièrement chez des patients fragiles et/ou âgés. De plus, l’échantillon prélevé était parfois de trop petite taille pour permettre des analyses moléculaires approfondies. Grâce aux nouvelles techniques de biologie moléculaire, élaborées par le CHU de Lyon, il est désormais possible de retrouver de petites quantités de fragments d’ADN de la tumeur dans le sang. Pour certains cancers du poumon, les équipes du CHU de Lyon repèrent désormais dans une simple prise de sang certaines mutations et sont capables d’analyser les caractéristiques du cancer en évitant au patient une nouvelle biopsie : c’est la biopsie liquide. Pour que chaque patient de la région bénéficie de cette innovation, un réseau de prescripteurs a été déployé en Auvergne-Rhône- Alpes. Depuis novembre 2015, près de 120 prélèvements issus de cancer du poumon ont été pris en charge par les HCL dont la moitié proviennent des établissements publics et privés hors HCL. Ce projet a reçu le financement Innov’RA 2016 du Cancéropôle Régional CLARA. Outre le cancer du poumon, cette biopsie du futur devrait bientôt être indiquée dans la plupart des cancers. Actuellement, elle est testée dans lest umeurs neuroendocrines et les cancers du côlon. La biopsie liquide a été conçue dans le cadre du projet CIRCAN (CIRculating CANcer) Portée par le CHU de Lyon, CIRCAN veut accélérer le transfert entre la recherche sur les biomarqueurs circulant du cancer et leur utilisation en pratique. Ce programme innovant dispose de moyens de séquençage de nouvelle génération, de techniques de PCR digitale, d’un personnel spécialement formé. Il s’appuie sur les compétences d’un conseil scientifique multidisciplinaire. L’initiative est soutenue par l’Institut de Cancérologie des Hospices Civils de Lyon. CIRCAN a récemment conclu un partenariat exclusif en France avec une société américaine pour la mise à disposition de la méthode de biologie moléculaire appelée PCR (Réaction en Chaîne par Polymérase) digitale optimisée (BEAMing). La livraison de l’automate est prévue fin avril 2016.

Contact : Hospices civils de Lyon Mme Marion Chereul, Directeur de Cabinet et de la Communication . Tel : 0825 0825 69 Email : [email protected] Site : http://www.chu-lyon.fr Standard : 0820 0820 69 France Institut Curie Pr Jean Yves Pierga Chef de département de l’oncologie médical Pr Jean-Yves Pierga. Chef de département de l’oncologie médicale Institut Curie-France

La détection de ’ADN tumoral circulant dans le sang "l’équivalent d’une biopsie liquide", prend également de plus en plus d’importance. Les cellules cancéreuses, comme n’importe quelle cellule, se dégradent naturellement au fil du temps. Ce faisant, elles laissent s’échapper une partie de leur matériel génétique dans le sang. Si l’on parvient à identifier cet ADN tumoral circulant, on peut affirmer qu’une tumeur cancéreuse se trouve quelque part dans l’organisme. Cela pourrait aussi permettre de suivre l’évolution de la masse tumorale ou de rechercher l’apparition de marqueurs de résistance au traitement. Toute la difficulté consiste à identifier cet ADN circulant comme tumoral. Pour ce faire, il faut repérer les anomalies génétiques typiques de la tumeur.

Pour le Pr. Jean-Yves Pierga, "Au-delà d’être moins invasive, moins douloureuse et plus économique, la recherche des anomalies dans l’ADN tumoral circulant est une alternative fiable aux biopsies classiques parfois peu réalisables (surtout au niveau pulmonaire ou de certaines métastases).

Les équipes de Marc-Henri Stern et Olivier Lantz ont montré pour la première fois qu’il était possible de détecter de l’ADN tumoral circulant dans le sang de patients atteints de mélanome de l’œil métastatique. Sa présence révèle l’existence d’une tumeur et sa quantité reflète sa taille: ceci en fait un nouveau biomarqueur susceptible de repérer très tôt la présence d’une tumeur ou d’une récidive. Bien que réalisée sur un nombre limité de patients atteints d'une maladie rare, cette étude publiée dans Clinical Cancer Research est une preuve de concept de la faisabilité et de l’intérêt clinique de la détection et de la quantification de l'ADN tumoral dans le sang. Récemment les chercheurs ont également démontré la supériorité de la détection de l’ADN tumoral sur celle des CTC dans le cas du mélanome de l’œil. Cette technique va bien au-delà du mélanome de l’œil, puisqu’elle pourrait s’appliquer à tous les cancers chez lesquels une mutation spécifique a été identifiée. La détection de l’ADN circulant apporte des réponses complémentaires à celles des cellules tumorales circulantes aux cliniciens,

Cette recherche s’inscrit également dans les programmes de médecine personnalisée de l’Institut Curie comme l’étude SHIVA dirigé par le Dr Christophe , la détection des anomalies moléculaires guidant le traitement ciblé sur la biopsie d’une métastase est également recherché dans l’ADN tumoral circulant. ASCO 2016 Résumés des études cliniques publiés CANCER/ASCO16 le 4 juin 2016 : les « biopsies liquides » ou comment mieux traiter les cancers grâce à une prise de sang.

Pour diagnostiquer un cancer et en suivre l’évolution il faut recouru à des prélèvements de tissus, des biopsies, qui imposent des gestes pas toujours simples et souvent douloureux. Une avancée technologique importante est en train de changer les choses, la biopsie liquide.

C’est souvent un très mauvais souvenir pour une femme chez laquelle on suspectait un cancer du sein. Les biopsies faites à travers la peau pour aller chercher des fragments de tissus ne laissent généralement pas un excellent souvenir. Et quand il faut les refaire parce que la tumeur a métastasé vers le foie, par exemple, il y a des moments pénibles.

Mais les progrès technologiques font qu’on développe depuis quelques années des outils biologiques qui permettent de mettre en évidence dans le sang des éléments provenant de la tumeur. Des éléments qu’on peut analyser très finement et qui permettent donc de réaliser ce qu’on appelle une « biopsie liquide ». Que va-t-on rechercher dans ces prélèvements sanguins ? Soit des cellules tumorales, soit de l’ADN venu des cellules cancéreuses. Mais grâce aux outils génétiques comme le séquençage de nouvelle génération (Next Generation Sequencing ou NGS) on peut détecter sur cet ADN des modifications sur les gènes de la tumeur et ainsi réagir rapidement concernant le traitement des patients.

Dans un certain type de cancer du poumon par exemple, celui dans lequel il y a une mutation du gène EGFR qui représente 15 % des tumeurs broncho-pulmonaires, Une étude américaine présentée aujourd’hui à l’ASCO et qui a concerné 15191 patients, on a pu montrer une très bonne corrélation entre les prélèvements tissulaires et les prélèvements sanguins pour la mise en évidence de la mutation. Mais l’analyse de l’ADN a aussi pu montrer rapidement la présence d’une mutation particulière appelée T790M et qui entraine une résistance au traitement par les médicaments de thérapie ciblée appelés inhibiteurs de tyrosine kinase. L’apparition de cette mutation sur l’ADN permet donc de modifier le traitement de venu inefficace et d’autant plus toxique. On sait également rechercher sur ce ctADN des anomalies d’un gène, ESR1, impliqué dans la résistance aux traitements hormonaux du cancer du sein.

Si ces analyses permettent de réagir rapidement et de corriger les choix thérapeutiques en cas de mise en évidence de résistance, elles ne remplaceront pas la pratique des biopsies tissulaires classiques, notamment pour le diagnostic initial de cancer, elles sont complémentaires. La biopsie tissulaire a en effet un intérêt majeur puisqu’elle permet d’avoir des informations sur le microenvironnement de la tumeur. Une information essentielle notamment avec le développement de l’immunothérapie. Voir quelles sont les cellules de défenses présentes et en quelle quantité est une information que seule la biopsie classique peut apporter. Etudes cliniques Sensibilité, et seuil de détection de la biopsie liquide Mutation detection in cell free DNA from healthy donors. (PATHWAY GENOMICS laboratory)

Sub-category: Circulating Biomarkers

Category: Tumor Biology

Meeting: 2016 ASCO Annual Meeting

Abstract No: e23054

Citation: J Clin Oncol 34, 2016 (suppl; abstr e23054)

Author(s): Anja Kammesheidt, Aaron Chen, Sean Li, Jair Jaboni, Megan Anselmo, Troy Viloria, Julie A Neidich, Glenn D Braunstein; Pathway Genomics, San Diego, CA

Abstract Disclosures

Abstract: Background: Tumor cells release small DNA fragments into the bloodstream as circulating tumor DNA (ctDNA), a subset of cell-free DNA (cfDNA). CancerIntercept Detect is a CLIA validated, laboratory-developed blood test that can be used by physicians as a screening tool for the detection of biomarkers associated with specific cancer types in high-risk patients without a cancer diagnosis. The assay detects the presence of 96 mutations in nine genes (BRAF, CTNNB1, EGFR, FOXL2, GNAS, KRAS, NRAS, PIK3CA, TP53). To investigate the positive mutation rate in an unselected, general population, we initiated a small IRB-approved study in August 2015. Methods: Subjects were asymptomatic, had no personal history of cancer and filled out a health questionnaire. Ages ranged from 20-71 (mean 40 years); 55 females and 47 males participated. 20 ml of blood was collected in cfDNA blood tubes, plasma was separated, cfDNA isolated from 5 ml of plasma, and ctDNA enriched and then sequenced via next-generation methods. We report on as few as 2 copies of mutant ctDNA. Results: 98/102 (96%) subjects were negative for the 96 mutation panel. A single mutation was detected in 3 subjects at just above the detection threshold, and 2 mutations were detected in one individual. All 4 positive subjects were over 50 years old. We continue to test ctDNA levels every ~2-3 months and levels have remained steady with small copy number variations. Subject 4 underwent a CT scan of the lungs showing multiple lung nodules. Results at ~9 months follow-up will be presented. Conclusions: In our study, 3% of non-selected adults were found to have low levels of ctDNA detected without an underlying neoplasm found, while 1% had moderately elevated levels associated with lung nodules concerning for neoplasm. 2nd Blood Draw (within 1st Blood Draw 1-2 months of first draw)

Subject Mutation Copy# Abundance (%) Copy# Abundance (%)

1 NRAS_G12S 2.19 0.010 3.5 0.032

2 TP53_R273H 3.70 0.037 4.1 0.048

3 GNAS_R201H 4.4 0.053 3.0 0.065

4 KRAS_A146T 76.2 0.457 30.1 0.7

5 TP53_R175H 4.1 0.025 5.5 0.128 Mutation detection in circulating tumor DNA in stage I vs. later stage cancer samples.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting Abstract No: e23046 Citation: J Clin Oncol 34, 2016 (suppl; abstr e23046) Author(s): Aaron Chen, Sean Li, Jair Jaboni, Megan Anselmo, Julie A Neidich, Glenn D Braunstein, Anja Kammesheidt; Pathway Genomics, San Diego, CA

Abstract Disclosures Abstract: Background: Tumor cells release small DNA fragments into the bloodstream as circulating tumor DNA (ctDNA), a subset of cell-free DNA (cfDNA). CancerIntercept is a CLIA validated, laboratory-developed blood test that can be used by physicians as a screening tool for the detection of biomarkers associated with specific cancer types in patients without a cancer diagnosis but at high risk for developing a cancer or as a test to monitor patients who have been diagnosed with cancer. The assay detects the presence of 96 mutations in nine genes (BRAF, CTNNB1, EGFR, FOXL2, GNAS, KRAS, NRAS, PIK3CA, TP53). Since less has been published about ctDNA mutation detection in patients with early stage cancers, we sought to determine the frequency detected in these patients in comparison to those with stage III or IV tumors. Methods: 45 commercial plasma samples from individuals with stage I-II cancer and 36 samples from patients with stage III-IV cancer were analyzed. Cancer types included breast, colorectal, \endometrial, gastric, lung, melanoma, ovarian, pancreatic, thyroid, head and neck, and prostate cancer. Clinical information such as grade, TNM, tumor size, lymph node status was available for most samples. For each sample, 1-2 ml of frozen plasma were processed, cfDNA isolated, and run on the 96 mutation panel for ctDNA enrichment followed by next- generation sequencing. Results: Six of the 42 stage I specimens (14%) had at least one positive mutation above limit of detection (2/6 gastric, 3/7 pancreatic, 1/8 thyroid cancer). One pancreatic cancer sample had two mutations. Notably these mutations were detected in individuals with stage IA, T1N0M0 tumors. 18 (50%) of the stage III-IV samples had mutations detected (3/9 breast, 6/9 colorectal, 3/7 lung, 1/1 endometrial, 3/7 pancreatic, 1/1 gastric, 1/1 ovarian). The mutations found in the stage I cancers include recurrent mutations in TP53, GNAS and KRAS. The later stage samples harbored mutations in all of the genes except BRAF and FOXL2, and several samples had multiple mutations and higher on average copy numbers. Conclusions: Overall the ctDNA mutation rate in the stage I cancers we examined was 14%, while stage III-IV samples had a higher mutation rate (50%) and higher numbers of mutations per sample. Etudes cliniques Cancer de Poumon Evaluation of liquid biopsies for molecular profiling and monitoring in non-small cell lung cancer (NSCLC) patients.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting

Session: Tumor Biology Time: Monday June 6, 1:00 PM to 4:30 PM

Abstract No: 11533 Poster Board Number: Poster Session (Board #230) Citation: J Clin Oncol 34, 2016 (suppl; abstr 11533) Author(s): Jordi Remon, Jean-Charles Soria, Ludovic Lacroix, Karen Howarth, Andrew Lawson, Sarah Smalley, Davina Gale, Vincent Plagnol, Emma Green, Nitzan Rosenfeld, Cecile Jovelet, Chloe Pannet, Benjamin Besse; Gustave Roussy, Université Paris-Saclay, Department of Medicine, Villejuif, France; Gustave Roussy Cancer Center, Villejuif, France; Translational Research, Gustave Roussy, Villejuif, France; INIVATA, Cambridge, United Kingdom; Inivata, Cambridge, United Kingdom; Gustave Roussy, Villejuif, France; Department of Medical Oncology, Gustave Roussy, Villejuif, France Abstract Disclosures Abstract: Background: Effective and non-invasive methods for actionable molecular alterations in NSCLC patients (pts) are urgently needed. Molecular profiling is often limited by access to sufficient tumour tissue for comprehensive analysis and due to tumour heterogeneity, the complete range of tumor DNA abnormalities may not be represented. Circulating tumour DNA (ctDNA) can be used as a minimally-invasive liquid biopsy for the detection and quantification of molecular abnormalitiesMethods: We enrolled a prospective cohort of 70 Stage IIIB/IV NSCLC pts to collect blood (10ml K2-EDTA) prior to treatment (D1) and after treatment (D2, D21, D42 and every imaging assessment until relapse in a subset of 25 patients). Blood samples were processed (double-spin method) and analysed using an enhanced TAm-Seq method, which enables detection of molecular abnormalities across 35 genes. Statistical analyses are in progress to quantify the concordance of ctDNA with tissue biopsy, and to assess the value of ctDNA as a dynamic tool following treatment. Results: As of January 2015, 26 pts were analysed (69% were male, 19% never-smoker, 84% had an adenocarcinoma subtype, and 88% stage IV). Only 46% (n = 12) of tumor biopsies provided sufficient sample tissue for molecular analysis, obtaining a molecular profile for 9 out of 12 pts. ctDNA profiling was possible for all pts, which detected cancer mutations in 20 out of 26 pts. Median number of plasma somatic mutations was 2, predominantly located in KRAS, TP53 and EGFRmutation. Of that, 46% of mutations detected in ctDNA were observed at a frequency below 1%. ctDNA profiling reported the same molecular abnormality than in the tissue in 7 out of 9 patients, with a 78% concordance rate. Indeed, 51% of mutations detected at D1 were also detected at D21. For mutations present at both time points, the reduction in allele fraction was small (median 18%). Conclusions: ctDNA can be used as a ‘liquid biopsy’ for molecular profiling of NSCLC pts to detect actionable molecular alterations when tissue biopsy is unavailable providing real-time information. The full analysis of 70 pts, including dynamic analysis profiles following treatment will be presented during the meeting. Utility of liquid biopsies to assess circulating tumor DNA in patients with lung adenocarcinoma.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting Abstract No: e23097 Citation: J Clin Oncol 34, 2016 (suppl; abstr e23097) Author(s): Maria Clemence Schwaederle, Sandip Pravin Patel, Hatim Husain, Megumi Ikeda, Richard Burnham Lanman, Kimberly C. Banks, AmirAli Talasaz, Lyudmila Bazhenova, Razelle Kurzrock; Center for Personalized Cancer Therapy, UCSD Moores Cancer Center, La Jolla, CA; Center for Personalized Cancer Therapy and Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA; Guardant Health, Inc., Redwood City, CA; University of California, San Diego, La Jolla, CA Abstract Disclosures Abstract: Background: Liquid biopsies were used to assess genomic alterations in plasma circulating tumor DNA (ctDNA) from patients with non-small cell lung adenocarcinoma (NSCLC), and therapeutic outcomes analyzed. Methods: ctDNA testing (digital next-generation sequencing (NGS)) was performed in 88 patients with advanced NSCLC; (54 or 68 gene panel; 40 and 48 patients, respectively (Guardant Health, Inc. (Guardant360 test))) (Clinical Laboratory Improvement Amendment (CLIA) laboratory). Fifty-five patients (62.5%) also had some tissue molecular testing, 37 of whom had a consistent tissue NGS panel ( ≥ 182-genes). Results: Eighty-two percent of patients (72/88) had ≥ 1 ctDNA alteration(s) (median = 2 alterations), most frequently TP53 (44.3% of patients), EGFR (27.3%), MET (14.8%), KRAS (13.6%), and ALK (6.8%). Retrospective analysis showed that 25 of 88 patients (28.4%) had received a therapy matching at least one ctDNA alteration(s) (most frequently an inhibitor of EGFR (N = 18), ALK, BRAF, other). Sixty-five percent of evaluable (N = 5 patients, too early) matched patients (N = 13/20) achieved stable disease (SD) ≥ 6 months or partial response (PR). The median progression-free survival for the 25 matched patients was 13.9 months. For the 37 patients who had both the tissue NGS panel and the ctDNA test (median 1.3 months (range, 0.1 to 20.9) between tissue biopsy and blood draw), the concordance rate for EGFR ctDNA alterations with tissue NGS was 70.3% (k = 0.361), with higher concordance when time interval between tissue and blood tests was ≤ 1.3 months. When we only considered patients in whom ctDNA confirmed present (N = 27), the EGFRconcordance was 77.8% (k = 0.526). Fifty-one patients (58%) did not have the tissue NGS test performed, including 33 (37.5%) who had no other tissue molecular testing, usually because of inadequate tissue or potential morbidity for repeat biopsy. Conclusions: ctDNA frequently revealed genomic alterations in NSCLC adenocarcinoma, including in difficult-to-biopsy patients. Patients who received cognate therapies demonstrated a high rate of SD ≥ 6months/PR, suggesting clinical utility. EFIRM liquid biopsy (eLB) to detect actionable mutations in NSCLC.

Meeting: 2016 ASCO Annual Meeting

Category: Lung Cancer—Non-Small Cell Local-Regional/Small Cell/Other Thoracic Cancers

Subcategory: Adjuvant Therapy

Session Type and Session Title: Poster Session, Lung Cancer—Non-Small Cell Local-Regional/Small Cell/Other Thoracic Cancers

Abstract Number: 8520

Citation: J Clin Oncol 34, 2016 (suppl; abstr 8520)

Author(s): David T. Wong, Fang Wei, Wei Liao, Ziding Feng, David Chia, Yong Kim, Youlin Qiao, Qinghua Zhou; University of California, Los Angeles, Los Angeles, CA; University of California, Los Angeles, North Hills, CA; EZLife BioSciTech Co. Ltd, Guangzhou, China; The University of Texas MD Anderson Cancer Center, Houston, TX; Department of Pathology and Laboratory Medicine, Los Angeles, CA; University of California Los Angeles, Los Angeles, CA; Department of Cancer Epidemiology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; Sichuan Lung Cancer Institute, Chengdu, China

Background: Liquid biopsy to detect actionable mutations in NSCLC is in clinical practice primarily based on digital droplet PCR (ddPCR) and/or next generation sequencing (NGS) technologies with performance at ~70-80% concordance with tissue-based genotyping. Technologies that will permit 100% concordance detection (sensitivity) will provide the ultimate complementation to the tumor-specific fingerprint (specificity) for unambiguous detection of the tumor in a non-invasive setting. The electric field induced released and measurement (EFIRM) fulfilled this translational and clinical goal. Methods: A double blinded study enrolled 37 NSCLC patients at three lung cancer centers in China (SLCI/Sichuan, JCH/Nanjing and HCH/Henan). For each patient, both pre- and post- biopsy/surgery, saliva and plasma were collected. Codes were removed from samples and blinded. Tissues were genotyped for EGFR L858R and exon 19del genotyping by Cobas (Roche Molecular Systems) while saliva and plasma were assayed for same EGFR TKI-sensitive mutation by eLB. Results were sent to MD Anderson Cancer Center for concordance analysis with the tissue-based genotyping data from Cobas. Results: In this blinded study, using tumor tissue (surgery/biopsy) genotyping as gold standard (Cobas), eLB correctly predicted both EGFR exon 19del and L858R status for all 37 pre- and post- surgery/biopsy saliva samples (AUC = 1.0). Plasma from the same 37 patients’ pre- and post-surgery/biopsy were measured by eLB with AUC = 1.0 for exon 19del, and AUC = 0.96 for L858R. Signals in saliva are cleaner than that in plasma for L858R. For Exon 19del, both plasma and saliva have clean separation between mutants and wild types. Correlations between pre- and post- saliva assays are excellent (0.82 and 0.95), a bit lower but still high for pre- and post- plasma (0.70 and 0.93). Conclusions: This study confirmed the concordance performance of eLB for detecting EGFR mutations from pre- and post-biopsy serum and saliva samples for actionable EGFR mutations in NSCLC patients.

Source URL: http://meetinglibrary.asco.org/content/162539-176 Etudes cliniques Cancer de sein Cell-free DNA detected by “liquid biopsy” as a potential prognostic biomarker in patients with different subtypes of breast cancer.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting Abstract No: e23081 Citation: J Clin Oncol 34, 2016 (suppl; abstr e23081) Author(s): Sara Ravaioli, Roberta Maltoni, Valentina Casadio, Maria Maddalena Tumedei, Flavia Foca, Samanta Salvi, Filippo Martignano, Daniele Calistri, Andrea Rocca, Sara Bravaccini; Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy Abstract Disclosures Abstract: Background: Conventional biomarkers used to define breast cancer (BC) subtypes are not always capable of predicting prognosis. Thus, the search for new biomarkers that can be easily detected by liquid biopsy is ongoing. Recent studies have shown that cell-free DNA (cf-DNA) could be a promising diagnostic marker in different tumor types. However, its prognostic value in BC has yet to be confirmed. This retrospective study evaluated the prognostic role of cf-DNA quantity of BCAS1, PI3KCA, HER2 and c-Myc, 4 frequently altered genes in BC, considering different BC subtypes (luminal A, luminal B, triple negative, HER2 positive). Methods: A total of 80 serum samples were collected before surgery from women at first diagnosis of BC operated on in the Breast Unit of Forlì Hospital (Italy) from 2005 to 2008. Twenty relapsed patients and 60 non-relapsed patients (5 relapsed and 15 non-relapsed for each tumor subtype) were paired by subtype and age. Blood samples were also collected from 10 healthy donors. All samples were analyzed by Real Time PCR for cf-DNA quantity of BCAS1, PI3KCA, HER2 and c-Myc. The relationship between cf-DNA quantity values and prognosis was analyzed by a nonparametric ranking statistic test. Accuracy was measured by the area under the ROC curve (AUC). Results: Significantly higher median values for the absolute quantity (ng) of HER2, BCAS1 and PI3KCA cf-DNA were found in BC patients than in healthy controls (HER2 0.238 vs 0.067, p = 0.0160; BCAS1 1.259 vs 0.085, p = 0.0011; PI3KCA 1.619 vs 0.174, p= 0.0015). A difference in the quantity of HER2 cf-DNA was observed between non-relapsed and relapsed patients although non-statistically significant (median values 0.202 vs 0.438, p = 0.057), independently of tumor subtype. Of the 4 analyzed oncogenes, HER2 and PI3KCA showed AUC values of 0.639 and 0.612, respectively, showing the highest accuracy in predicting relapse of disease. Conclusions: The present study supports the rationale for a liquid biopsy approach to detect prognostic biomarkers on which to base the clinical management of patients independently of BC subtype. Our preliminary data now require validation in a larger case series to establish their prognostic value in BC. Detection, frequency and actionability of recurrent copy number gains detected by non- invasive liquid biopsy of 3,942 lung and breast cancer samples.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting Abstract No: 11541 Poster Board Number: Poster Session (Board #238) Citation: J Clin Oncol 34, 2016 (suppl; abstr 11541) Author(s): Diana Abdueva, Darya Chudova, Richard Burnham Lanman, Kimberly C. Banks, Rebecca J Nagy, Stefanie Mortimer, AmirAli Talasaz, Helmy Eltoukhy, Razelle Kurzrock; Guardant Health Inc, Redwood City, CA; Guardant Health, Inc., Redwood City, CA; University of California, San Diego, La Jolla, CA Abstract Disclosures

Abstract: Background: Circulating tumor DNA (ctDNA) testing must include detection of single nucleotide variants (SNVs), indels, fusions, and copy number amplifications (CNAs) to identify all NCCN-recommended genomic targets. We examined CNA landscape in a large clinical cohort of breast and lung cancer ctDNA samples analyzed with Guardant360 (G360) test, classifying recurrent gene-level copy number changes across distinct oncogenic pathways and disease subtypes. Methods:We developed an improved CNA detection pipeline and evaluated its analytical and clinical performance on a set of cell-line spike-ins with known amplifications and 72 clinical plasma samples from patients with stage IV breast cancer with known ERBB2 amplification/overexpression. We then examined 1,935 lung adenocarcinoma and 2,007 breast carcinoma clinical cases: CNA patterns in samples with and without known driver mutations were compared using Fisher’s exact test to identify candidate driver amplification events. Results: Analytical sensitivity, specificity, and precision of the new CNA pipeline improved significantly - average coefficient of variation (CV) of copy number estimates across 18 genes on the panel was 1.2% for spike-in replicates, all spike-ins and copy number neutral samples were detected correctly, and signal-to-noise ratio increased 4.3-fold. We observed high concordance between tissue-verified amplification events in breast carcinoma subset when the mutant allele fraction of the driver mutation exceeded 0.5%. In the lung adenocarcinoma cohort, CNA-driver mutations were identified in ~7% of 1,935 samples, excluding cases known functional driver events. Focal amplification events in MET, CCND1, and ERBB2 were enriched in samples lacking other driver oncogene mutations (p-value < 0.001).Conclusions: Copy number variation is an important mechanism of oncogene activation in lung and breast cancers. Highly accurate quantification of copy number state in ctDNA allows for clinically meaningful identification of amplified therapeutic targets with otherwise wildtype DNA sequence. Etude clinique Cancer de l’Ovaire Correlation of mutation status between tissue and blood-drop liquid biopsies from ovarian cancer patients following chemotherapy.

Meeting: 2016 ASCO Annual Meeting

Category: Cancer Prevention, Hereditary Genetics, and Epidemiology

Subcategory: Genetic Testing

Session Type and Session Title: This abstract will not be presented at the 2016 ASCO Annual Meeting but has been published in conjunction with the meeting.

Abstract Number: e13108

Citation: J Clin Oncol 34, 2016 (suppl; abstr e13108)

Author(s): Angelina I Londono, Chen-Hsiung Yeh, Ronald David Alvarez, Charles A. Leath, John Michael Straughn, Rebecca Christian Arend; University of Alabama at Birmingham, Birmingham, AL; Circulogene Theranostics, Homewood, AL

Background: Peripheral circulating cell-free DNA (cfDNA) has great promise for targeted therapy and monitoring tumor clonal evolution during treatment, thus overcoming the limitations of invasive tissue biopsy. However, the requirement of large sample input, low extraction efficiency, and poor yield, coupled with a labor- intensive and time-consuming procedure in order to obtain a liquid biopsy presents a major challenge. Methods: Circulogene has developed a highly efficient and high yield cfDNA enrichment technology from only 20ul of blood. We were able to sequence cfDNA and tumor DNA from 14 de-identified plasma and tumor tissue samples pre- and post- chemotherapy for a total of 56 samples. We ascertained the mutation status of five therapeutically targetable genes implicated in ovarian cancer: TP53, PIK3CA, PTEN, KRAS, and BRAF. These genes were interrogated by deep next-generation sequencing, and concordance was determined between matched tissue and plasma, and pre- and post-treatment samples. Results: 13 out of 14 of the samples, either tissue or plasma, contained at least 1 mutation. Overall, more mutations were detected in plasma than the corresponding tissue samples at baseline, and much less dramatic mutation changes were observed in tissue than plasma following therapy, indicating that a more complete tumor heterogeneity landscape was captured from the blood. The genomic response to chemotherapy was consistent in both tissue and plasma samples. The overall frequency of TP53, PIK3CA, PTEN, KRAS, BRAF mutations in both patient cohorts (pre and post chemotherapy) were 100%, 16.7%, 6.7%, 6.7%, 0% in tissue, and 70%, 30%, 20%, 0%, 0% in plasma, respectively. The concordance of TP53, PIK3CA, PTEN, KRAS, BRAF mutation status between matched tissue and plasma was 33.3%, 86.7%, 73.3%, 93.3%, 100%, respectively. Conclusions: These results illustrate the potential utility of our blood-drop liquid biopsy as a non-invasive, real-time and longitudinal means for monitoring treatment response, outcomes and disease progression, and could become an invaluable tool clinical decision-making.Source URL: http://meetinglibrary.asco.org/content/171741-176

• Etude clinique Cancer Colorectal Practical applications of “liquid biopsy” in the colorectal cancer treatment.

Sub-category: Circulating Biomarkers Category: Tumor Biology Meeting: 2016 ASCO Annual Meeting Abstract No: e23044 Citation: J Clin Oncol 34, 2016 (suppl; abstr e23044) Author(s): Koichi Suzuki, Yuji Takayama, Kosuke Ichida, Taro Fukui, Nao Kakizawa, Tamotsu Obitsu, Yuta Muto, Fumi Hasegawa, Fumiaki Watanabe, Rina Kikugawa, Takaharu Kato, Yuji Kaneda, Masaaki Saito, Shingo Tsujinaka, Kazushige Futsuhara, Yasuyuki Miyakura, Hiroshi Noda, Hirokazu Kiyozaki, Fumio Konishi, Toshiki Rikiyama; Department of Surgery, Saitama Medical Center, Jichi Medical University, Saitama, Japan; Department of Surgery, Nerima Hikarigtaoka Hospital, Tokyo, Japan

Abstract Disclosures Abstract: Background: A blood-based technology platform tracking circulating tumor DNA, “Liquid biopsy” represents an ideal non-invasive tool allowing multiple testing over time, monitoring real time changes occurring within the tumor and evaluation of therapeutic response. Here, we show its practical applications via monitoring of KRAS mutation in detection of circulating tumor DNA (ctDNA) during treatments. Methods: 238 plasma samples were collected from 57 patients (pts) who underwent chemotherapy for metastatic colorectal cancer. KRAS mutant ctDNA (MctDNA) was determined by digital PCR as a tool of Liquid biopsy. Results:KRAS assessment in tumor tissues showed 19 patients with mutation (MT) and 38 without mutation (WT). Among 19 patients with MT, 12 patients displayed mutant ctDNA at the initial assessment and 7 patients showed no MctDNA. Regardless of KRAS status in tumor tissues, poor response was likely seen in patients with MctDNA. While 38 patients with WT showed no MctDNA before chemotherapy except one patient (Case 1), 4 patients exhibited MctDNA after treatments with various regimens. 1) Prediction of the drug resistance: MctDNA was detectable in blood of these 4 patients prior to radiographic detection of disease progression, suggesting monitoring of ctDNA was a promising molecular biomarker to predict drug resistance. 2) Selection of candidates for treatments: These 4 patients displaying MctDNA in blood are unlikely benefit from the anti-EGFR antibody treatment despite of their KRAS wild status in tumor tissues. 3) Choice of optimal interventions: MctDNA was detected before treatment in Case 2 without KRAS mutation in the tissue, indicating metastatic liver tumor consisted of KRAS mutant clones. Optional interventions can be available such as surgery for liver metastasis and chemotherapy for primary tumor in this patient. 4) Re-introduction of the drug: The KRAS mutant clones declined in blood of Case 1 during treatment with regorafenib and TAS-102, suggesting that drug sensitivity to the anti-EGFR antibody could be recovered and re-introduction of this antibody would be effective. Conclusions: Liquid biopsy provides a circulating biomarker not only for treatment response but decision making of a sequential strategy. Etude clinique Cancer de Prostate et cancer de Foie High sensitivity and specificity NGS analysis of prostate and kidney cancer samples using a novel liquid biopsy workflow.

Meeting:

2016 ASCO Annual Meeting

Category: Genitourinary (Prostate) Cancer

Subcategory: Biomarkers/Epidemiology/Outcomes

Session Type and Session Title: This abstract will not be presented at the 2016 ASCO Annual Meeting but has been published in conjunction with the meeting.

Abstract Number: E16607

Citation:

J Clin Oncol 34, 2016 (suppl; abstr e16607)

Author(s): Cristian Ionescu-Zanetti, Reynolds Brobey, Hubert Tseng, Kevin P Rosenblatt, Nicholas Kamps-Hughes, Tony Tran, Robert J. Amato; Fluxion Biosciences, South San Francisco, CA; The University of Texas Health Science Center at Houston, Houston, TX; Nano3D Biosciences, Houston, TX; CompanionDx Reference Lab, LLC, Houston, TX; The University of Texas Health Science Center Houston, Houston, TX

Background: Next generation sequencing (NGS) of blood-derived nucleic acids is an emerging paradigm for determining the mutational status of cancer patients over time. Circulating tumor cells (CTC) and cell-free circulating DNA have been proposed as possible sample types. Here we present data from a CTC enrichment modality that results in tumor cell purities of > 10% and a high sensitivity NGS data analysis workflow that enables the use of standard amplicon panels typically used for primary tissue. This study was aimed at urological cancers of the kidney and prostate). Methods: Blood samples from prostate and kidney cancer patients were enriched for CTCs using the IsoFlux System, from a starting blood volume of 7.5-14ml. Matched samples were enumerated to determine the CTC load, where CTCs were defined as CK+, CD45- nucleated cells (DAPI+). Cells were lysed and DNA was amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion), and quantified via qPCR. Targeted libraries were sequenced using either the PGM or MiSeq instruments; the analysis used a high sensitivity variant calling pipeline and ERASE algorithm (Elimination of Recurring Artifacts and Stochastic Errors) as well as VarSeq (Golden Helix Inc.) for variant filtering and functional interpretation. Results: Multisite analytical validation data, based on spiking of cells into whole blood, and a matched molecular and bioinformatics approach demonstrates a detection limit down to 10 cells from a blood draw with a false positive rate of below 0.1 calls per sample. DNA spike data shows detection down to 0.4% allele frequency without false positives. Clinical data from two different urological cancer pilot studies demonstrates the detection of somatic variants for a majority of samples, and significant overlap between detected mutations and known somatic mutation sites. We also describe concordance between biological replicates, and a comparison of the liquid biopsy samples to germ line DNA derived from white blood cells. Conclusions: This assay makes possible the detection of somatic variants from urological cancer patients without the need for a tissue biopsy.Source URL: http://meetinglibrary.asco.org/content/170350-176 Liste des références de l’ensemble des études cliniques réalisées 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for clinicians. 2015;65:5-29. 2. Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic acids research. 2015;43:D805-11. 3. Shah SP, Kobel M, Senz J, et al. Mutation of FOXL2 in granulosa-cell tumors of the ovary. The New England journal of medicine. 2009;360:2719-29. 4. Schirripa M, Cremolini C, Loupakis F, et al. Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer. International journal of cancer. Journal international du cancer. 2015;136:83-90. 5. Janku F, Lee JJ, Tsimberidou AM, et al. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. PloS one. 2011;6:e22769. 6. Kalfa N, Lumbroso S, Boulle N, et al. Activating mutations of Gsalpha in kidney cancer. The Journal of urology. 2006;176:891-5. 7. Fecteau RE, Lutterbaugh J, Markowitz SD, Willis J, Guda K. GNAS mutations identify a set of right-sided, RAS mutant, villous colon cancers. PloS one. 2014;9:e87966. 8. Sparks AB, Morin PJ, Vogelstein B, Kinzler KW. Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer. Cancer research. 1998;58:1130-4. 9. Heitzer E, Ulz P, Geigl JB. Circulating tumor DNA as a liquid biopsy for cancer. Clinical chemistry. 2015;61:112-23. 10. Lebofsky R, Decraene C, Bernard V, et al. 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. Molecular oncology.2015;9:783-90. 11. Esposito A, Bardelli A, Criscitiello C, et al. Monitoring tumor-derived cell-free DNA in patients with solid tumors:clinical perspectives and research opportunities. Cancer treatment reviews. 2014;40:648-55. 12. Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2014;32:579-86.

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Dr. Tarik MARZOUKI