Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

Accepted Manuscript Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

Crescenzio F. Minervini, Cosimo Cumbo, Paola Orsini, Luisa Anelli, Antonella Zagaria, Luciana Impera, Nicoletta Coccaro, Claudia Brunetti, Angela Minervini, Paola Casieri, Giuseppina Tota, Antonella Russo Rossi, Giorgina Specchia, Francesco Albano PII: DOI: Reference:

S0014-4800(17)30199-5 doi: 10.1016/j.yexmp.2017.06.007 YEXMP 4060

To appear in:

Experimental and Molecular Pathology

Received date: Revised date: Accepted date:

11 April 2017 22 May 2017 25 June 2017

Please cite this article as: Crescenzio F. Minervini, Cosimo Cumbo, Paola Orsini, Luisa Anelli, Antonella Zagaria, Luciana Impera, Nicoletta Coccaro, Claudia Brunetti, Angela Minervini, Paola Casieri, Giuseppina Tota, Antonella Russo Rossi, Giorgina Specchia, Francesco Albano , Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology, Experimental and Molecular Pathology (2017), doi: 10.1016/j.yexmp.2017.06.007

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ACCEPTED MANUSCRIPT Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

Crescenzio F. Minervini, Cosimo Cumbo, Paola Orsini, Luisa Anelli, Antonella Zagaria, Luciana Impera, Nicoletta Coccaro, Claudia Brunetti, Angela Minervini, Paola Casieri, Giuseppina Tota,

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Antonella Russo Rossi, Giorgina Specchia, Francesco Albano

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Affiliation

Department of Emergency and Organ Transplantation (D.E.T.O.) - Hematology Section -

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Corresponding Author:

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Francesco Albano, MD, PhD e-mail: [email protected]

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University of Bari - 70124 - Bari, Italy

Department of Emergency and Organ Transplantation (D.E.T.O.) - Hematology Section -

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University of Bari

P.zza G. Cesare, 11 70124 Bari - Italy

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Tel +39 (0)80-5478031

Fax +39 (0)80-5508369

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ACCEPTED MANUSCRIPT Abstract We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the

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clonal configuration of multiple mutations. We demonstrate that MinION is suitable for

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employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Keywords: chronic myeloid leukemia; acute lymphoblastic leukemia; ABL1 mutation; MinION;

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next generation sequencing.

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ACCEPTED MANUSCRIPT Introduction In newly-diagnosed chronic phase (CP)- chronic myeloid leukemia (CML) patients, 15–30% of those who start first-line tyrosine kinase inhibitors (TKIs) therapy will not reach an optimal response, and a BCR-ABL1 kinase domain (KD) mutation will be detectable in 25–50% of patients

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with treatment failure (Soverini et al., 2011) with an increased frequency of these mutations is observed in accelerated phase (AP) and blast crisis (BC) patients (Jabbour et al., 2006). Compared

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to CML, the frequency of these mutations is much higher in patients with Philadelphia positive

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(Ph+) acute lymphoblastic leukemia (ALL) at the time of relapse (Jones et al., 2008). Currently, the Sanger sequencing (SS) technique analyzing BCR-ABL1 is considered the gold standard for

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mutation detection but this assay has a sensitivity of around 20% (Soverini et al., 2011), and is

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therefore is unsuitable for identifying low-level variants (<20 % variant frequency). Moreover, using SS it is generally not possible to differentiate between multiple mutations occurring in the

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same clone (compound mutants) as opposed to separate clones (polyclonal mutants). Recently, next

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generation sequencing (NGS)-based assays have been reported for detecting BCR-ABL1 KD mutations (Machova Polakova et al., 2015; Soverini et al., 2013; Szankasi et al., 2016). Although these NGS strategies are more accurate and precise than SS, they are burdened by costs related to

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the initial investment, namely purchasing the sequencer, the preparation of specific targets libraries,

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and the required reagents. MinION is a single molecule sequencer connected to a laptop through a USB3.0 interface, based on nanopore technology; it works by connecting two strands of DNA molecules by a hairpin, and sequencing them consecutively (Lu et al., 2016). During sequencing, the single strand of DNA passes through biologic nanopores on a chip, where an electric field is applied and electrical signal variations of consecutive 5-bases DNA in length are recorded. Template and complement sequences obtained are then used to generate the information from both strands (2D), which results in higher base quality. This approach has already been successfully used to sequence the TP53 gene mutations (Minervini et al., 2016). Here, we describe a third-generation 4

ACCEPTED MANUSCRIPT sequencing assay on MinION for detecting BCR-ABL1 KD mutations and compare the results to a SS-based test in 24 Ph+ leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage, in certain circumstances,

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of determining the clonal configuration of multiple mutations.

Material and methods

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Patients

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Overall, 24 patients were included in the study (Supplentary table); they were subdivided in two

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groups and for the purposes of the cDNA barcoding process each group had to be formed by no more than 12 patients; therefore, Group 1 included 11 patients (10 CML and 1 ALL) that developed

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treatment resistance during the TKI treatment course and 1 newly diagnosed ALL, whereas group 2 was exclusively formed by newly diagnosed cases (8 CML and 4 ALL cases). In all cases included

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in this study the presence of t(9;22) rearrangement was verified by conventional and molecular

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cytogenetics, as previously reported (Albano et al., 2010, 2007).

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Library preparation and sequencing

For ABL1 amplification and barcoding the total cellular RNA was extracted from peripheral blood

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cells using the RNeasy Mini Kit (Qiagen) and quantified with Qubit 2.0 Fluorometer (Life Technologies). RNA (1 μg) was reverse-transcribed to complementary DNA (cDNA) using 200U of the SuperScript II Reverse Transcriptase and 2.5μmol/L random hexamer primers (Invitrogen). According

to

the

Oxford

Nanopore

Barcoding

protocol

for

amplicons

(Version

DK003_1148_revB_12Feb2015), for each patient, we performed a Long-PCR using the PrimeSTAR GXL DNA Polymearse (Takara Bio Inc.), three primers, as previously reported6, with a

specific

tail

at

5’end

TTTCTGTTGGTGCTGATATTGCCAACAGTCCTTCGACAGCAG-3’;

(prebar190 prebar210

5’5’5

ACCEPTED MANUSCRIPT TTTCTGTTGGTGCTGATATTGCGAGCAGCAGAAGAAGTGTTTCAGA-3’; prebarABL 5’ACTTGCCTGTCGCTCTATCTTCCTTGGAGTGAGGCATCTCAG-3’), 200 ng of cDNA, in a final volume of 50µl. Thermal-cycling conditions were 98°C for 10 seconds, 60°C for 15 seconds, 68°C for 2 minutes (30 cycles) and 4°C hold. The PCR products, visualized on an agarose-gel (1%), were purified using the QIAquick PCR Purification Kit (Qiagen), and used as templates (30ng) for

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the Barcoding PCR. Barcoding was performed with the same Polymerase and 12 different pairs of

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Barcoding primers from ONT, BC01-BC12, in a final volume of 50 µl. Thermal-cycling conditions

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were 98°C for 10 seconds, 62°C for 15 seconds, 68°C for 2 minutes (15 cycles) and 4°C hold. Before starting library preparation, we purified, quantified and estimated the purity of samples

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(Nanodrop). Barcoded amplicons were then pulled to an equal weight ratio and 1μg of the pool was diluted to 45 µl in nuclease-free water and prepared for sequencing. According to the ONT

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Sequencing protocol (SQK-MAP006), DNA was end-prepared with the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs Inc.), prior to ligation of nanopore-specific

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adapters with Blunt/TA Ligase Master Mix (New England Biolabs Inc.). All purifications were

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performed with AgencourtAMPure XP beads (Beckman Coulter Inc.). Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) were used to elute the library in the pre-sequencing Mix.

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After the Platform QC run, the sequencing mix (75 µl of Running Buffer, 66 µl of nuclease-free water, 4 µl of Fuel Mix and 6 µl of the Pre-sequencing Mix) was loaded and the

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MAP_48Hr_Sequencing_Run.py protocol was started (MinIONflowcell: FLO-MAP105). The sequence data from this study have been submitted to the NCBI Short Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession n° SRP101705. Data Analysis Poretools toolkit was used to extract FASTQ files from FAST5 files. Galaxy, a web-based platform for processing NGS data, was employed for variant analysis (https://usegalaxy.org/). Reads were aligned on GRCh37 human reference genome with the BWA6

ACCEPTED MANUSCRIPT MEM method using specific Nanopore platform parameters and visualized with the Integrative Genomics Viewer (IGV) browser. Single nucleotide variants (SNV) and insertions/deletions (indels) detection was separately performed with the Varscan software, and VCF files were filtered and annotated with Annovar. In particular, for SNV and indels detection, the minimum read depth and supporting reads parameters were set according to the lowest mean coverage (minimum read

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depth of 20, minimum supporting reads of 5). The minimum variant allele frequency threshold

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threshold for variant calling was 0.05, in all Varscan analyses. The complete workflow is available

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at https://usegalaxy.org/u/ematlab/w/varabl1.

Moreover, the BAM files (that is a binary format for storing sequence data) from alignment were

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used for coverage, depth of sequencing and error rate analysis. Coverage and depth were assessed by Gviz R package (Hahne and Ivanek, 2016) using as input bedgraph files obtained from BAM

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files converted with Bedtools (Quinlan and Hall, 2010). Error rate analysis was performed using the Qualimap 2.2.1 java tool (Garcia-Alcalde et al., 2012). SS was performed and electropherograms

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were then analyzed by visual inspection, glass free software for Sanger analysis data

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(http://shiny.bat.infspire.org/igcllglass/) and GeneScreen (Carr et al., 2011). All cases included in

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Results

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the study were analyzed by SS and MinION sequencing in a blinded manner.

Two sequencing runs were performed with the two different pools of patients: the first lasted eight hours and was carried out on the Group 1, whereas the second run included Group 2 and lasted 24 hours to achieve a deeper sequencing. Sequencing results showed that 100% of ABL1 from exon 2 to 10 was covered and that the mean sequencing depth was around 150x and 1000x for Group 1 and 2, respectively. In any case, the depth of sequencing was never below 50X (Fig 1A). A total of 25,345 and 71,331 fast5 files containing raw electric signals were produced from the first and the 7

ACCEPTED MANUSCRIPT second experiment. Fast5 files were then used as input in Metrichor for basecalling and demultiplexing. Each run generated 26 Mbs and 157Mbs, respectively. On the total reads produced in the first run, 3068 passed 2D filters and 3062 had a recognizable barcode. Instead, the second run produced 17,762 reads after 2D filtering, 17,741 of which had a recognizable barcode. Plotting the distribution of read lengths, all passed 2D reads were distributed around a 1.7Kb peak in agreement

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with our amplicon size (Supplementary File1). BAM files were used to calculate the general error

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rate which resulted around 8%. We found 10 BCR-ABL1 KD mutations in 9 patients belonging to

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Group 1 (Case#4 showed compound mutations - supplementary File 2). Notably, almost all mutations had a high allelic ratio and were well detectable both from MinION data and from SS

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(supplementary File 3), apart from Case#2 and Case#8. Moreover, Case#10 presented a single nucleotide polymorphism (SNP) in the BCR-ABL1 KD (Table 1).

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Results from MinION and SS showed 92% concordance in all cases included in this study. Indeed, mutations in Case#2 and Case#8 were initially undetected in SS by the usual electropherograms

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software analysis. These mutations became evident thanks to the indications obtained with MinION

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Discussion

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analysis (Fig 1B).

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BCR-ABL1 KD sequence analysis has become an essential management tool for Ph+ leukemia patients on TKIs therapy and SS is the technique commonly utilized for this purpose. We compared a laboratory-developed nanopore-based BCR-ABL1 mutation test to the traditional SS method. Our findings demonstrate multiple advantages in using MinION approach, first of all the greater sensitivity: our comparison of MinION to SS identified mutations below the detection limit of SS (generally stimated at around <20%) in 2 (22%) among the mutated cases, including mutations known to be clinically important. Moreover, it has been demonstrated that low-level mutations, below the detection limits of SS, may persist after a positive TKIs response and cause resistance 8

ACCEPTED MANUSCRIPT even after long periods of time (Parker et al., 2013; Soverini et al., 2014). Therefore, the possibility of routinely detecting lower level mutations highlights a significant advantage of MinION technology over SS. It is noteworthy that Case #10 showed the K247R polymorphism (Ernst et al., 2008), which in our case could contribute to the failure to respond to TKIs treatment experienced by this patient during his treatment history. Also, our results demonstrated that MinION analysis of

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Ph+ leukemia at diagnosis is potentially able to detect a BCR-ABL1 KD mutation using much lower

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sequencing coverage than the one employed in the experiment performed on Group 2. Another

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point of interest is the compound mutations. Case #4 showed two mutations occurring in the same clone (in cis); SS cannot distinguish between polyclonal (in trans) and compound mutations, unless

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the combined mutant allele burden clearly exceeds 100%, whereas NGS, and particularly the MinION approach, is able to identify compound mutations as long as the average read length

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exceeds the distance between the 2 single nucleotide variations (Supplementary file 2). In fact, with an average read length of 1.7 kbs, our assay is expected to detect all the BCR-ABL1 compound

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mutations located in the KD. The presence of BCR-ABL1 KD compound mutations describe poor-

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risk group of CP-CML patients, with increased chances of disease progression and acquiring resistance to second generation TKIs (Parker et al., 2016). This event may be overcome by using a

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more sensitive mutation analysis able to promptly guide therapy adjustment after TKI. Another advantage of the nanopore technology is the costs profile; in fact, excluding the device cost, that is

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estimated at around USD1000, we calculated that, if 24 samples are run simultaneously in a flowcell (providing a coverage of not less than 500X), the cost per sample is around USD40, a price comparable to that of SS for detecting BCR-ABL1 KD mutations. Moreover, since a very high coverage depth was reached in our analyses, we believe that the overall costs could be further reduced by running more samples per flowcell. Therefore, the main advantage of this technology is that a more efficient and sensitive analysis can be obtained than using SS, at very competitive costs.

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ACCEPTED MANUSCRIPT In conclusion, we demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

Acknowledgements

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This work was supported by “Associazione Italiana contro le Leucemie (AIL)-BARI”.

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The authors would like to thank Ms. MVC Pragnell, B.A. for language revision of the manuscript.

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The authors declare that there are no conflict of interest.

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Conflict of Interest statement

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Authors’ contributions

CFM and FA conceived and designed the study, and wrote the manuscript. CC, CFM, LA, and AZ performed the Long-PCRs, barcoding and nanopore experiments. PO and CFM performed all bioinformatics

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analysis. PC, NC, GT, AM, CB, and LI conducted molecular analysis and interpreted data. performed

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diagnostic molecular analysis. ARR provided clinical data. GS and FA supervised the manuscript preparation. All authors read and approved the final manuscript.

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Figures and Table Legends

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Table 1. Main characteristics of cases resulting positive by MinION analysis Figure 1. (A) Coverage plot from reads mapping on human genome. Reads are mapped and coverages for each patient are plotted reporting the sequencing depth values (x-axis: the average number times that a nucleotide is sequenced) versus genomic position (y-axis). All exons of ABL1, excluding the first which is not involved in the BCR-ABL1 fusion gene, resulted covered (relative ABL1 exons positions are showed below). (B) Example of mutations detected in two cases (Case#2, Case#8). Aligned reads are visualized by Integrative Genomics Viewer (IGV) browser. Below the genomic position, the depth of sequencing for each base is reported as a gray bar. Variants with an 10

ACCEPTED MANUSCRIPT allelic ratio >10% are reported as colored depth of sequencing bars, where each color represent the specific base fraction. As shown the mutations were barely visible in SS and were detected thanks to indications obtained using MinION. Supplementary Table. Summary of main characteristics of the patients included in this study.

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Supplementary File 1. 2D Sequence Length vs Quality Score. Data from Sequence Length distribution (y-axis) and Mean Quality Score distribution (x-axis) are plotted. 2D reads with the

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higher quality score are distributed around a 1.7kb as expected.

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Supplementary File 2. Case#4 reads aligned on ABL1 cDNA sequence. A group of reads carrying

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the mutations c.1076T>G and c.742C>G (arrows) at same time are detected (blue box). Supplementary File 3. Two mutated cases that were well visible both in MinION analysis than in

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Sanger Sequencing.

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ACCEPTED MANUSCRIPT Table 1 Patients

Diagnosis

Protein Description

Transcript description

Allelic Ratio

SS Detection

Treatment at the time of mutational analysis

Response at the time of mutation detection

Case#1

CML

p.F317L

c.951C>G

95%

Yes

Nilotinib

Failure to achieve Cytogenetic Response

Case#2

CML

p.E459K

c.1375G>A

15%

No

Imatinib

Case#3

CML

p.M351T

c.1052T>C

99%

Yes

Imatinib

Case#4

CML

p.F359C

c.1076T>G

96%

Yes

p.L248V

c.742C>G

75%

Yes

Case#5

CML

p.T315I

c.944C>T

96%

Yes

Case#6

CML

p.H396R

c.1187A>G

94%

Yes

Case#7

CML

p.Q252H

c.756G>T

89%

Case#8

ALL

p.F317L

c.951C>A

14%

Case#9

ALL

p.T315I

c.944C>T

Case#10

CML

p.K247R

C C

I R

N A

Failure to achieve Cytogenetic Response Loss of MMR

Imatinib

Progression to blast crisis

Imatinib

Loss of Cytogenetic Response

Yes

Nilotinib

Loss of Cytogenetic response

No

None

Diagnosis

53%

Yes

Dasatinib

ALL progression

89%

Yes

Imatinib

Failure to achieve MMR

D E

T P E

c.740A>G

Failure to achieve Cytogenetic Response

C S U Nilotinib

T P

M

A

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ACCEPTED MANUSCRIPT Highlights We used MinION nanopore sequencing to detect BCR-ABL1 mutations in Ph+ leukemia. MinION shows greater sensitivity than Sanger sequencing test at competitive costs. MinION approach allows to identify compound mutations at long distance.

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