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ESTABLISHING A NATIONAL NETWORK OF LABORATORIES USING NEXT GENERATION AMPLICON DEEP SEQUENCING FOR BCR-ABL1 KINASE DOMAIN MUTATION SCREENING: THE 'NEXT-IN-CML' STUDY
Author(s):
Simona Soverini
,
Simona Soverini
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Caterina De Benedittis
,
Caterina De Benedittis
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Luana Bavaro
,
Luana Bavaro
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Margherita Martelli
,
Margherita Martelli
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Stefania Stella
,
Stefania Stella
Affiliations:
Department of Clinical and Experimental Medicine,University of Catania,Catania,Italy
Alessandra Iurlo
,
Alessandra Iurlo
Affiliations:
Oncohematology Division,IRCCS - Ca' Granda - Ospedale Maggiore,Milan,Italy
Nicola Orofino
,
Nicola Orofino
Affiliations:
Oncohematology Division,IRCCS Cà Granda - Ospedale Maggiore,Milan,Italy
Claudia Baratè
,
Claudia Baratè
Affiliations:
Dept of Clinical and Experimental Medicine,University of Pisa,Pisa,Italy
Sara Galimberti
,
Sara Galimberti
Affiliations:
Dept of Clinical and Experimental Medicine,University of Pisa,Pisa,Italy
Simona Sica
,
Simona Sica
Affiliations:
Institute of Hematology,Università Cattolica Sacro Cuore,Rome,Italy
Federica Sorà
,
Federica Sorà
Affiliations:
Institute of Hematology,Università Cattolica Sacro Cuore,Rome,Italy
Antonella Russo Rossi
,
Antonella Russo Rossi
Affiliations:
Hematology and Transplants Unit,University of Bari,Bari,Italy
Francesco Albano
,
Francesco Albano
Affiliations:
Hematology and Transplants Unit,University of Bari,Bari,Italy
Fabio Ciceri
,
Fabio Ciceri
Affiliations:
Haematology and BMT Unit,San Raffaele Hospital,Milan,Italy
Francesca Lunghi
,
Francesca Lunghi
Affiliations:
Haematology and BMT Unit,San Raffaele Hospital,Milan,Italy
Fausto Castagnetti
,
Fausto Castagnetti
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Gabriele Gugliotta
,
Gabriele Gugliotta
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Elena Tenti
,
Elena Tenti
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Gianantonio Rosti
,
Gianantonio Rosti
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Cristina Papayannidis
,
Cristina Papayannidis
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Fabio Stagno
,
Fabio Stagno
Affiliations:
Hematology Unit,Ferrarotto Hospital,Catania,Italy
Paolo Vigneri
,
Paolo Vigneri
Affiliations:
Department of Clinical and Experimental Medicine,University of Catania,Catania,Italy
Anna Serra
,
Anna Serra
Affiliations:
Department of Clinical and Biological Sciences,University of Turin,Orbassano,Italy
Giuseppe Saglio
,
Giuseppe Saglio
Affiliations:
University of Turin,Turin,Italy
Francesca Carnuccio
,
Francesca Carnuccio
Affiliations:
Department of Clinical and Biological Sciences,University of Turin,Orbassano,Italy
Fabrizio Pane
,
Fabrizio Pane
Affiliations:
CEINGE,University of Naples Federico II,Naples,Italy
Santa Errichiello
,
Santa Errichiello
Affiliations:
CEINGE,University of Naples Federico II,Naples,Italy
Mario Annunziata
,
Mario Annunziata
Affiliations:
Hematology Unit,Cardarelli Hospital,Naples,Italy
Massimo Breccia
,
Massimo Breccia
Affiliations:
Chair of Hematology,La Sapienza University,Rome,Italy
Elisabetta Abruzzese
,
Elisabetta Abruzzese
Affiliations:
Hematology Unit,Sant'Eugenio Hospital,Rome,Italy
Massimiliano Bonifacio
,
Massimiliano Bonifacio
Affiliations:
Department of Medicine,University of Verona,Verona,Italy
Elisabetta Novella
,
Elisabetta Novella
Affiliations:
Division of Hematology,Vicenza,Italy
Eros Di Bona
,
Eros Di Bona
Affiliations:
Division of Hematology,Vicenza,Italy
Rosaria Sancetta
,
Rosaria Sancetta
Affiliations:
Ospedale Dell'Angelo,Mestre,Italy
Elisabetta Calistri
,
Elisabetta Calistri
Affiliations:
Ospedale Ca' Foncello,Treviso,Italy
Giuseppina Spinosa
,
Giuseppina Spinosa
Affiliations:
Hematology Unit,Foggia,Italy
Mariella D'Adda
,
Mariella D'Adda
Affiliations:
Hematology Unit,Spedali Civili,Brescia,Italy
Isabella Capodanno
,
Isabella Capodanno
Affiliations:
Hematology Unit, Reggio Emilia Hospital,Reggio Emilia,Italy
Michele Baccarani
,
Michele Baccarani
Affiliations:
University of Bologna,Bologna,Italy
Michele Cavo
,
Michele Cavo
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
Giovanni Martinelli
Giovanni Martinelli
Affiliations:
Department of Experimental, Diagnostic and Specialty Medicine (DIMES),Hematology/Oncology 'L. e A. Seràgnoli',Bologna,Italy
(Abstract release date: 05/18/17) EHA Library. SOVERINI S. 06/24/17; 181772; S485
Dr. Simona SOVERINI
Dr. Simona SOVERINI
Contributions
PDF Abstract
Abstract

Abstract: S485

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 17:00 - 17:15

Location: Room N101

Background
Benchtop next generation sequencers are gradually replacing Sanger sequencers in diagnostics labs because of greater throughput, better sensitivity and increasing cost-effectiveness. In chronic myeloid leukemia (CML) patients (pts) on tyrosine kinase inhibitor (TKI) therapy, BCR-ABL1 kinase domain (KD) mutation screening is a precious tool for timely and rational therapeutic reassessment and is recommended in case of Failure and Warning. A multicenter, multilaboratory prospective study (‘NEXT-IN-CML’) has been conducted to assess the feasibility, cost, turnaround times and clinical utility of a next generation amplicon deep sequencing (Deep Seq) strategy for routine BCR-ABL1 KD mutation screening.

Aims

The first phase of the study was aimed to i) create a network of 4 labs sharing a common protocol, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and reporting, and ii) verify accuracy and inter-laboratory reproducibility of results. 
The second phase of the study, involving 39 Italian Hematology Units, was meant to prospectively assess the frequency of low burden mutations in CML pts with Failure or Warning to any TKI.

Methods

In the first phase, centrally prepared identical batches of 32 blinded samples (24 clinical samples with known mutation status/load as assessed by Sanger Seq plus 8 T315I+ BaF3 cell line dilutions simulating mutation loads between 20% and 1%) were distributed and analyzed in parallel by each of the 4 participating labs.
In the second phase, 159 consecutive CML pts were prospectively studied in parallel by Sanger Seq and by Deep Seq: 101 Failures (57 pts on 1st-line TKI [IM, n=38; DAS, n=12; NIL, n=7] therapy; 35 pts on 2nd-line TKI [DAS, n=14; NIL, n=17; IM, n=2; BOS, n=1; PON, n=1] therapy; 5 pts on 3rd-line TKI [DAS, n=4; NIL, n=1] therapy and 4 pts on 4th-line TKI [BOS, n=1; PON, n=3] therapy) and 58 Warnings (38 on 1st-line TKI [IM, n=28; DAS, n=4; NIL, n=5; BOS, n=1] therapy and 20 on 2nd-line TKI [NIL, n=10; DAS, n=9; PON, n=1] therapy).

Results

In the first phase, 504/512 amplicons were successfully generated and sequenced, with a median number of forward and reverse reads of 1,757 (range 544-5,838). In the 128 samples analyzed, 51/52 expected mutations were consistently detected by all 4 labs and quantitation of mutation load was highly reproducible across a wide range of frequencies (2%>100%). Three out of 4 labs failed to detect the 1% T315I+ dilution. In clinical samples, additional low burden mutations <3% were occasionally called by one or two labs only, suggesting that this value should be taken as a threshold below which mutation detection is not reproducible and sequencing artifacts and errors cannot be ruled out.
In the second phase of the study, pts positive for mutations were 25/159 (16%; 23 Failures and 2 Warnings) by Sanger Seq and 52/159 (33%; 44 Failures and 8 Warnings) by Deep Seq. Among the pts with low burden mutations detectable by Deep Seq, 4 had a T315I; 34 had other known TKI-resistant mutations; 14 had only mutations with unknown clinical significance. Pts positive for mutations by Deep Seq were more frequent in the High and Intermediate Sokal risk group. The number of positive pts and the number of mutations per pt were not significantly higher in those receiving 2nd- or subsequent-line TKI therapy than in those receiving 1st-line TKI therapy. Compound mutations were found only in 2 out of 52 mutated pts (both in blastic phase).

Conclusion

1) Results of the ‘NEXT-IN-CML’, the first prospective study evaluating the routine diagnostic use of Deep Seq of BCR-ABL1, show that this technology can successfully be implemented in national lab networks and is feasible, robust and reproducible; 2) in a relatively large, nonselected cohort of CML pts analyzed for mutations because of a Failure or Warning response, Deep Seq confirmed that enhancing sensitivity enables to detect BCR-ABL1 KD mutations in twice as many pts as compared to Sanger Seq (33% vs 16%); 3) all the pts who need to be switched to another TKI would benefit from sensitive BCR-ABL1 KD mutation screening by Deep Seq.

Session topic: 8. Chronic myeloid leukemia - Clinical

Keyword(s): Resistance, mutation analysis, BCR-ABL

Abstract: S485

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 17:00 - 17:15

Location: Room N101

Background
Benchtop next generation sequencers are gradually replacing Sanger sequencers in diagnostics labs because of greater throughput, better sensitivity and increasing cost-effectiveness. In chronic myeloid leukemia (CML) patients (pts) on tyrosine kinase inhibitor (TKI) therapy, BCR-ABL1 kinase domain (KD) mutation screening is a precious tool for timely and rational therapeutic reassessment and is recommended in case of Failure and Warning. A multicenter, multilaboratory prospective study (‘NEXT-IN-CML’) has been conducted to assess the feasibility, cost, turnaround times and clinical utility of a next generation amplicon deep sequencing (Deep Seq) strategy for routine BCR-ABL1 KD mutation screening.

Aims

The first phase of the study was aimed to i) create a network of 4 labs sharing a common protocol, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and reporting, and ii) verify accuracy and inter-laboratory reproducibility of results. 
The second phase of the study, involving 39 Italian Hematology Units, was meant to prospectively assess the frequency of low burden mutations in CML pts with Failure or Warning to any TKI.

Methods

In the first phase, centrally prepared identical batches of 32 blinded samples (24 clinical samples with known mutation status/load as assessed by Sanger Seq plus 8 T315I+ BaF3 cell line dilutions simulating mutation loads between 20% and 1%) were distributed and analyzed in parallel by each of the 4 participating labs.
In the second phase, 159 consecutive CML pts were prospectively studied in parallel by Sanger Seq and by Deep Seq: 101 Failures (57 pts on 1st-line TKI [IM, n=38; DAS, n=12; NIL, n=7] therapy; 35 pts on 2nd-line TKI [DAS, n=14; NIL, n=17; IM, n=2; BOS, n=1; PON, n=1] therapy; 5 pts on 3rd-line TKI [DAS, n=4; NIL, n=1] therapy and 4 pts on 4th-line TKI [BOS, n=1; PON, n=3] therapy) and 58 Warnings (38 on 1st-line TKI [IM, n=28; DAS, n=4; NIL, n=5; BOS, n=1] therapy and 20 on 2nd-line TKI [NIL, n=10; DAS, n=9; PON, n=1] therapy).

Results

In the first phase, 504/512 amplicons were successfully generated and sequenced, with a median number of forward and reverse reads of 1,757 (range 544-5,838). In the 128 samples analyzed, 51/52 expected mutations were consistently detected by all 4 labs and quantitation of mutation load was highly reproducible across a wide range of frequencies (2%>100%). Three out of 4 labs failed to detect the 1% T315I+ dilution. In clinical samples, additional low burden mutations <3% were occasionally called by one or two labs only, suggesting that this value should be taken as a threshold below which mutation detection is not reproducible and sequencing artifacts and errors cannot be ruled out.
In the second phase of the study, pts positive for mutations were 25/159 (16%; 23 Failures and 2 Warnings) by Sanger Seq and 52/159 (33%; 44 Failures and 8 Warnings) by Deep Seq. Among the pts with low burden mutations detectable by Deep Seq, 4 had a T315I; 34 had other known TKI-resistant mutations; 14 had only mutations with unknown clinical significance. Pts positive for mutations by Deep Seq were more frequent in the High and Intermediate Sokal risk group. The number of positive pts and the number of mutations per pt were not significantly higher in those receiving 2nd- or subsequent-line TKI therapy than in those receiving 1st-line TKI therapy. Compound mutations were found only in 2 out of 52 mutated pts (both in blastic phase).

Conclusion

1) Results of the ‘NEXT-IN-CML’, the first prospective study evaluating the routine diagnostic use of Deep Seq of BCR-ABL1, show that this technology can successfully be implemented in national lab networks and is feasible, robust and reproducible; 2) in a relatively large, nonselected cohort of CML pts analyzed for mutations because of a Failure or Warning response, Deep Seq confirmed that enhancing sensitivity enables to detect BCR-ABL1 KD mutations in twice as many pts as compared to Sanger Seq (33% vs 16%); 3) all the pts who need to be switched to another TKI would benefit from sensitive BCR-ABL1 KD mutation screening by Deep Seq.

Session topic: 8. Chronic myeloid leukemia - Clinical

Keyword(s): Resistance, mutation analysis, BCR-ABL

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