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BTK AND PLCG2 MUTATIONS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA RELAPSING ON IBRUTINIB: A EUROPEAN RESEARCH INITIATIVE ON CLL (ERIC) STUDY BASED ON REAL-WORLD EVIDENCE
Author(s): ,
Lydia Scarfò
Affiliations:
Division of Experimental Oncology, Strategic Research Program on CLL and B-cell Neoplasia Unit,Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele,Milan,Italy
,
Silvia Bonfiglio
Affiliations:
Division of Experimental Oncology, Strategic Research Program on CLL and B-cell Neoplasia Unit,Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele,Milan,Italy;Centre for Omics Sciences,IRCCS Ospedale San Raffaele,Milan,Italy
,
Lesley-Ann Sutton
Affiliations:
Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden
,
Viktor Ljungstrom
Affiliations:
Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden;Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Tatjana Pandzic
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Diego Cortese
Affiliations:
Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden
,
Gianluca Gaidano
Affiliations:
Division of Hematology, Department of Translational Medicine,University of Eastern Piedmont,Novara,Italy
,
Livio Trentin
Affiliations:
Hematology and Clinical Immunology, Department of Medicine,University of Padua,Padua,Italy
,
Lisa Bonello
Affiliations:
Pathology Unit,AOU Città della Salute e della Scienza di Torino and Department of Molecular Biotechnologies and Health Sciences, University of Turin,Turin,Italy
,
Gianluigi Reda
Affiliations:
Hematology Department,Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico,Milan,Italy
,
Panayiotis Panayiotidis
Affiliations:
Department of Propaedeutic Internal Medicine,Laiko Hospital, University of Athens,Athens,Greece
,
Francesco Forconi
Affiliations:
Haematology Dept and School of Cancer Sciences,Cancer Research UK and NIHR Experimental Cancer Medicine Centres, University of Southampton Hospital Trust,Southampton,United Kingdom
,
Niki Stavroyianni
Affiliations:
Hematology Department and HCT Unit,G. Papanicolaou Hospital,Thessaloniki,Greece
,
Csaba Bödör
Affiliations:
MTA-SE Momentum Molecular Oncohematology Research Group,Semmelweis University, 1st Department of Pathology and Experimental Cancer Research,Budapest,Hungary
,
Constantine Tam
Affiliations:
Peter MacCallum Cancer Center,Royal Melbourne Hospital and University of Melbourne,Melbourne, Victoria,Australia
,
Sunil Iyengar
Affiliations:
Department of Haemato-Oncology,Royal Marsden Hospital and Institute of Cancer Research Fulham Road,London,United Kingdom
,
Anders Österborg
Affiliations:
Department of Hematology,Karolinska University Hospital,Stockholm,Sweden
,
Marta Coscia
Affiliations:
Division of Hematology,A.O.U. Città della Salute e della Scienza di Torino and Department of Molecular Biotechnology and Health Sciences, University of Turin,Turin,Italy
,
Loïc Ysebaert
Affiliations:
Département d'Hématologie,Institut Universitaire du Cancer-Oncopole de Toulouse,Toulouse,France
,
Ozren Jaksic
Affiliations:
Dubrava University Hospital,Zagreb,Croatia
,
Ingo Ringshausen
Affiliations:
Department of Hematology,University of Cambridge,Cambridge,United Kingdom
,
Stephen Mulligan
Affiliations:
Royal North Shore Hospital, University of Sydney,Sidney,Australia
,
Alessandra Tedeschi
Affiliations:
Department of Hematology,Niguarda Cancer Center, ASST Grande Ospedale Metropolitano Niguarda,Milan,Italy
,
Vladimir Strugov
Affiliations:
Almazov National Medical Research Centre,St Petersburg,Russian Federation
,
Zadie Davis
Affiliations:
Department of Haematology,Royal Bournemouth Hospital,Bournemouth,United Kingdom
,
Carolina Pavlovsky
Affiliations:
Fundaleu,Buenos Aires,Argentina
,
Roberto Marasca
Affiliations:
Hematology Unit, Department of Medical and Surgical Sciences,University of Modena and Reggio Emilia,Modena,Italy
,
Antonella Capasso
Affiliations:
Division of Experimental Oncology, Strategic Research Program on CLL and B-cell Neoplasia Unit,Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele,Milan,Italy
,
Pamela Ranghetti
Affiliations:
Division of Experimental Oncology, Strategic Research Program on CLL and B-cell Neoplasia Unit,Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele,Milan,Italy
,
Kostas Stamatopoulos
Affiliations:
Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
,
Panagiotis Baliakas
Affiliations:
Department of Immunology, Genetics and Pathology, Science for Life Laboratory,Uppsala University,Uppsala,Sweden
,
Richard Rosenquist
Affiliations:
Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden
Paolo Ghia
Affiliations:
Division of Experimental Oncology, Strategic Research Program on CLL and B-cell Neoplasia Unit,Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele,Milan,Italy
(Abstract release date: 05/14/20) EHA Library. Scarfò L. 06/12/20; 294981; S161
Dr. Lydia Scarfò
Dr. Lydia Scarfò
Contributions
Abstract

Abstract: S161

Type: Oral Presentation

Session title: CLL - Targeted therapy II

Background

Acquired mutations within BTK gene at position C481, the binding site for ibrutinib, or the PLCG2 gene, have been reported in relapsing ibrutinib-treated patients (pts) with CLL. However, real-world evidence is lacking.

Aims
We performed a retrospective observational multicenter study aimed at determining the actual prevalence of BTK and PLCG2 mutations in pts with CLL relapsing (R/R) or responding to ibrutinib outside clinical trials.

Methods
Samples from 108 pts with CLL treated with ibrutinib [59 R/R and 49 responders (>1 year from therapy initiation)] from 22 institutions worldwide were included. Paired samples i.e. baseline and relapsed/responding, were available for 54 pts. Libraries were prepared using a custom Agilent HaloPlexHS kit targeting BTK and PLCG2 and sequenced on an Illumina NextSeq instrument. Reads were aligned to the hg19 reference genome using BWA and variants were called with Pisces with a variant allelic frequency (VAF) cutoff of 1% and annotated with SnpEff and SnpSift. Ninety-five samples were analyzed with digital droplet PCR (ddPCR) with a sensitivity of 0.1% at hotspot C481S (c.G1442C and T1441A) to confirm mutations.

Results
59 pts progressed on ibrutinib (median 33 months; range, 5-67); 49 had a sustained response (ibr-responders) at last follow up (median 35 m; range, 12-73) most concerned PR/nPR (37/49). Samples for ibr-responders were obtained ≥1 year from ibrutinib initiation (median time 20 m) while samples of R/R cases were obtained at the time of relapse (median 34 m). Thirty-one of 59 (53%) R/R pts carried at least 1 hotspot BTK mutation (p.C481S/R/Y) (median VAF 23.3%, range, 1.4%>97.3%) (Table 1). Eight of 31 (26%) mutated pts carried ≥2 hotspot BTK mutations with different VAFs. Seven R/R pts harboring BTK mutation(s) also carried PLCG2 mutation(s) (Table 1). PLCG2 mutations were rarely observed in the absence of BTK mutations with only 2 R/R BTK-wildtype pts carrying a hotspot PLCG2 mutation (Table 1). No hotspot mutations in BTK or PLCG2 were found at baseline. Thirty-nine of 59 R/R pts were analyzed with ddPCR, which confirmed BTK mutation(s) in all pts mutated according to NGS. The ddPCR identified low VAF BTK mutations (range: 0.03-1.2%: 3/5 <1%) in 5 wildtype patients by initial NGS analysis. A per base re-analysis of NGS data revealed that 4/5 pts indeed carried the BTK mutations retrieved by ddPCR at similar VAFs. Only 2/49 (4%) ibr-responders carried BTK and PLCG2 mutations by both NGS and ddPCR: 1 progressed 18 m after sampling, the second (sampled at 40 m) carried a minor BTK p.C481S-mutated subclone (VAF 4.36%) with 3 low-frequency variants in PLCG2 (VAF 2.65%>5.53%). Fourty-four of 46 BTK wildtype ibr-responders were analyzed with ddPCR and in 3/44 pts a low VAF C481S mutation was found (0.44-1.18%), again close or below the NGS cutoff.

Conclusion
This study reveals that ~50% of pts relapsing on ibrutinib had a BTK p.C481 mutation by NGS. ddPCR validated NGS analysis with 100% concordance above the pre-set threshold of 1%. Additional low-frequency BTK-mutated clones in 5 R/R cases by ddPCR were indeed present also in the NGS reads though initially disregarded  (<1% VAF in 3/5 cases). PLCG2 mutations were rare (9/59 R/R pts) and rarely isolated (2 R/R pts). There remains to dissect additional mechanisms underlying resistance to BTKi in the ~40% of pts without BTK /PLCG2 mutations. Analysis of additional genes (e.g. TP53, NOTCH1, SF3B1) is currently ongoing and will be presented at the meeting.

Session topic: 06. Chronic lymphocytic leukemia and related disorders - Clinical

Keyword(s): Chronic lymphocytic leukemia, Ibrutinib, Resistance

Abstract: S161

Type: Oral Presentation

Session title: CLL - Targeted therapy II

Background

Acquired mutations within BTK gene at position C481, the binding site for ibrutinib, or the PLCG2 gene, have been reported in relapsing ibrutinib-treated patients (pts) with CLL. However, real-world evidence is lacking.

Aims
We performed a retrospective observational multicenter study aimed at determining the actual prevalence of BTK and PLCG2 mutations in pts with CLL relapsing (R/R) or responding to ibrutinib outside clinical trials.

Methods
Samples from 108 pts with CLL treated with ibrutinib [59 R/R and 49 responders (>1 year from therapy initiation)] from 22 institutions worldwide were included. Paired samples i.e. baseline and relapsed/responding, were available for 54 pts. Libraries were prepared using a custom Agilent HaloPlexHS kit targeting BTK and PLCG2 and sequenced on an Illumina NextSeq instrument. Reads were aligned to the hg19 reference genome using BWA and variants were called with Pisces with a variant allelic frequency (VAF) cutoff of 1% and annotated with SnpEff and SnpSift. Ninety-five samples were analyzed with digital droplet PCR (ddPCR) with a sensitivity of 0.1% at hotspot C481S (c.G1442C and T1441A) to confirm mutations.

Results
59 pts progressed on ibrutinib (median 33 months; range, 5-67); 49 had a sustained response (ibr-responders) at last follow up (median 35 m; range, 12-73) most concerned PR/nPR (37/49). Samples for ibr-responders were obtained ≥1 year from ibrutinib initiation (median time 20 m) while samples of R/R cases were obtained at the time of relapse (median 34 m). Thirty-one of 59 (53%) R/R pts carried at least 1 hotspot BTK mutation (p.C481S/R/Y) (median VAF 23.3%, range, 1.4%>97.3%) (Table 1). Eight of 31 (26%) mutated pts carried ≥2 hotspot BTK mutations with different VAFs. Seven R/R pts harboring BTK mutation(s) also carried PLCG2 mutation(s) (Table 1). PLCG2 mutations were rarely observed in the absence of BTK mutations with only 2 R/R BTK-wildtype pts carrying a hotspot PLCG2 mutation (Table 1). No hotspot mutations in BTK or PLCG2 were found at baseline. Thirty-nine of 59 R/R pts were analyzed with ddPCR, which confirmed BTK mutation(s) in all pts mutated according to NGS. The ddPCR identified low VAF BTK mutations (range: 0.03-1.2%: 3/5 <1%) in 5 wildtype patients by initial NGS analysis. A per base re-analysis of NGS data revealed that 4/5 pts indeed carried the BTK mutations retrieved by ddPCR at similar VAFs. Only 2/49 (4%) ibr-responders carried BTK and PLCG2 mutations by both NGS and ddPCR: 1 progressed 18 m after sampling, the second (sampled at 40 m) carried a minor BTK p.C481S-mutated subclone (VAF 4.36%) with 3 low-frequency variants in PLCG2 (VAF 2.65%>5.53%). Fourty-four of 46 BTK wildtype ibr-responders were analyzed with ddPCR and in 3/44 pts a low VAF C481S mutation was found (0.44-1.18%), again close or below the NGS cutoff.

Conclusion
This study reveals that ~50% of pts relapsing on ibrutinib had a BTK p.C481 mutation by NGS. ddPCR validated NGS analysis with 100% concordance above the pre-set threshold of 1%. Additional low-frequency BTK-mutated clones in 5 R/R cases by ddPCR were indeed present also in the NGS reads though initially disregarded  (<1% VAF in 3/5 cases). PLCG2 mutations were rare (9/59 R/R pts) and rarely isolated (2 R/R pts). There remains to dissect additional mechanisms underlying resistance to BTKi in the ~40% of pts without BTK /PLCG2 mutations. Analysis of additional genes (e.g. TP53, NOTCH1, SF3B1) is currently ongoing and will be presented at the meeting.

Session topic: 06. Chronic lymphocytic leukemia and related disorders - Clinical

Keyword(s): Chronic lymphocytic leukemia, Ibrutinib, Resistance

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