WHOLE EXOME SEQUENCING IDENTIFIES NOVEL MUTATIONS IN RELAPSED OR REFRACTORY ACUTE PROMYELOCYTIC LEUKAEMIA (APL) UNRESPONSIVE TO ORAL ARSENIC TRIOXIDE
(Abstract release date: 05/19/16)
EHA Library. Gill H. 06/09/16; 132436; E887
Dr. Harinder Gill
Contributions
Contributions
Abstract
Abstract: E887
Type: Eposter Presentation
Background
Arsenic trioxide (As2O3)-based regimens induce remissions in >90% of relapsed APL. Further relapses after As2O3-induced remissions portend a poor prognosis. With the use of As2O3 in frontline treatment of APL, relapses in patients previously treated with As2O3 resulting in arsenic resistance is an emerging clinical problem. There is a paucity of information on the molecular mechanisms of As2O3-resistance.
Aims
To determine mutations associated with As2O3-resistance with whole exome sequencing (WES).
Methods
Seven patients with prior As2O3 treatment, who had ≥2 subsequent relapses occurring during or refractory to As2O3 treatment, were assigned to the discovery cohort. Serial bone marrow (BM) samples that were arsenic-sensitive (taken before treatment in a relapse responding to As2O3) and arsenic-resistant (obtained in subsequent relapses refractory to As2O3) underwent WES the illumina HiSeq 1500 platform at an average depth of 100X. Exome data were analysed utilizing bioinformatic pipeline programs designed to identify single nucleotide variants (SNVs) that were present either only or at increased frequencies in the resistant samples. The functional significance of non-synonymous missense mutations was evaluated using SIFT, Polyphen2 and FATHMM programs. Putative SNVs were confirmed by Sanger sequencing. Confirmed SNVs were tested in arsenic-resistant BM samples obtained from a validation cohort of 22 relapsed patients previously salvaged with As2O3, who had subsequent As2O3-refractory relapse again. Clinicopathologic features, karyotype, FLT3 and PML-RARA mutations were determined in the validation cohort.
Results
In the discovery cohort, serial WES showed following confirmed SNVs of potential functional significance showed increased allele frequency during As2O3-resistance: NOTCH2, FOXD4L5, KCNJ11/18, MADCAL1, CBR3, NSD1, FLG1, CCCDC179, SIGLEC11, ROBO4, CISD2, WT1, PTCH1, KMT2D and MED17. PML B2 domain mutations were not seen. The frequencies of the mutations were validated in a cohort of 22 patients with relapsed or refractory APL following oral As2O3-based treatment. The recruited patients comprised 14 men and 8 women with a median age of 44.5 (range:24-76) years at relapse. 1 (5%) patient had microgranular variant of APL while 2 patients (9%) had therapy-related APL. Additional karyotypic abnormalities were seen in 3 (14%) patients.14 patients (64%) had central nervous system (CNS) involvement at relapse. Internal tandem duplication of FLT3 (FLT3-ITD) was detected in 7 (32%) patients. The following recurrent mutations were found in As2O3-refractory relapse BM samples: NOTCH2 (n=17, 77%), FOXD4L5 (n=22, 100%), KCNJ11/18 (n=14, 64%), MADCAM1 (n=15, 68%), CBR3 (n=17, 77%), NSD1 (n=17, 77%), FLG1 (n=20, 91%), CCCDC179 (n=7, 32%), SIGLEC11 (n=5, 23%), ROBO4 (n=3, 14%), CISD2 (n=3, 14%), WT1 (n=1, 5%), PTCH1(n=1, 5%), KMT2D (n=1, 5%) and MED17 (n=6, 27%).
Conclusion
Mechanisms other than PML B2 domain mutations may account for As2O3 resistance. Novel genes regulating the cell signaling and apoptotic pathways, cellular proliferation, histone modification, DNA repair and angiogenesis were frequently mutated in As2O3-refractory APL. Further functional validation on the role of these mutations in conferring As2O3 resistance is required.
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Arsenic trioxide, Resistance
Type: Eposter Presentation
Background
Arsenic trioxide (As2O3)-based regimens induce remissions in >90% of relapsed APL. Further relapses after As2O3-induced remissions portend a poor prognosis. With the use of As2O3 in frontline treatment of APL, relapses in patients previously treated with As2O3 resulting in arsenic resistance is an emerging clinical problem. There is a paucity of information on the molecular mechanisms of As2O3-resistance.
Aims
To determine mutations associated with As2O3-resistance with whole exome sequencing (WES).
Methods
Seven patients with prior As2O3 treatment, who had ≥2 subsequent relapses occurring during or refractory to As2O3 treatment, were assigned to the discovery cohort. Serial bone marrow (BM) samples that were arsenic-sensitive (taken before treatment in a relapse responding to As2O3) and arsenic-resistant (obtained in subsequent relapses refractory to As2O3) underwent WES the illumina HiSeq 1500 platform at an average depth of 100X. Exome data were analysed utilizing bioinformatic pipeline programs designed to identify single nucleotide variants (SNVs) that were present either only or at increased frequencies in the resistant samples. The functional significance of non-synonymous missense mutations was evaluated using SIFT, Polyphen2 and FATHMM programs. Putative SNVs were confirmed by Sanger sequencing. Confirmed SNVs were tested in arsenic-resistant BM samples obtained from a validation cohort of 22 relapsed patients previously salvaged with As2O3, who had subsequent As2O3-refractory relapse again. Clinicopathologic features, karyotype, FLT3 and PML-RARA mutations were determined in the validation cohort.
Results
In the discovery cohort, serial WES showed following confirmed SNVs of potential functional significance showed increased allele frequency during As2O3-resistance: NOTCH2, FOXD4L5, KCNJ11/18, MADCAL1, CBR3, NSD1, FLG1, CCCDC179, SIGLEC11, ROBO4, CISD2, WT1, PTCH1, KMT2D and MED17. PML B2 domain mutations were not seen. The frequencies of the mutations were validated in a cohort of 22 patients with relapsed or refractory APL following oral As2O3-based treatment. The recruited patients comprised 14 men and 8 women with a median age of 44.5 (range:24-76) years at relapse. 1 (5%) patient had microgranular variant of APL while 2 patients (9%) had therapy-related APL. Additional karyotypic abnormalities were seen in 3 (14%) patients.14 patients (64%) had central nervous system (CNS) involvement at relapse. Internal tandem duplication of FLT3 (FLT3-ITD) was detected in 7 (32%) patients. The following recurrent mutations were found in As2O3-refractory relapse BM samples: NOTCH2 (n=17, 77%), FOXD4L5 (n=22, 100%), KCNJ11/18 (n=14, 64%), MADCAM1 (n=15, 68%), CBR3 (n=17, 77%), NSD1 (n=17, 77%), FLG1 (n=20, 91%), CCCDC179 (n=7, 32%), SIGLEC11 (n=5, 23%), ROBO4 (n=3, 14%), CISD2 (n=3, 14%), WT1 (n=1, 5%), PTCH1(n=1, 5%), KMT2D (n=1, 5%) and MED17 (n=6, 27%).
Conclusion
Mechanisms other than PML B2 domain mutations may account for As2O3 resistance. Novel genes regulating the cell signaling and apoptotic pathways, cellular proliferation, histone modification, DNA repair and angiogenesis were frequently mutated in As2O3-refractory APL. Further functional validation on the role of these mutations in conferring As2O3 resistance is required.
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Arsenic trioxide, Resistance
Abstract: E887
Type: Eposter Presentation
Background
Arsenic trioxide (As2O3)-based regimens induce remissions in >90% of relapsed APL. Further relapses after As2O3-induced remissions portend a poor prognosis. With the use of As2O3 in frontline treatment of APL, relapses in patients previously treated with As2O3 resulting in arsenic resistance is an emerging clinical problem. There is a paucity of information on the molecular mechanisms of As2O3-resistance.
Aims
To determine mutations associated with As2O3-resistance with whole exome sequencing (WES).
Methods
Seven patients with prior As2O3 treatment, who had ≥2 subsequent relapses occurring during or refractory to As2O3 treatment, were assigned to the discovery cohort. Serial bone marrow (BM) samples that were arsenic-sensitive (taken before treatment in a relapse responding to As2O3) and arsenic-resistant (obtained in subsequent relapses refractory to As2O3) underwent WES the illumina HiSeq 1500 platform at an average depth of 100X. Exome data were analysed utilizing bioinformatic pipeline programs designed to identify single nucleotide variants (SNVs) that were present either only or at increased frequencies in the resistant samples. The functional significance of non-synonymous missense mutations was evaluated using SIFT, Polyphen2 and FATHMM programs. Putative SNVs were confirmed by Sanger sequencing. Confirmed SNVs were tested in arsenic-resistant BM samples obtained from a validation cohort of 22 relapsed patients previously salvaged with As2O3, who had subsequent As2O3-refractory relapse again. Clinicopathologic features, karyotype, FLT3 and PML-RARA mutations were determined in the validation cohort.
Results
In the discovery cohort, serial WES showed following confirmed SNVs of potential functional significance showed increased allele frequency during As2O3-resistance: NOTCH2, FOXD4L5, KCNJ11/18, MADCAL1, CBR3, NSD1, FLG1, CCCDC179, SIGLEC11, ROBO4, CISD2, WT1, PTCH1, KMT2D and MED17. PML B2 domain mutations were not seen. The frequencies of the mutations were validated in a cohort of 22 patients with relapsed or refractory APL following oral As2O3-based treatment. The recruited patients comprised 14 men and 8 women with a median age of 44.5 (range:24-76) years at relapse. 1 (5%) patient had microgranular variant of APL while 2 patients (9%) had therapy-related APL. Additional karyotypic abnormalities were seen in 3 (14%) patients.14 patients (64%) had central nervous system (CNS) involvement at relapse. Internal tandem duplication of FLT3 (FLT3-ITD) was detected in 7 (32%) patients. The following recurrent mutations were found in As2O3-refractory relapse BM samples: NOTCH2 (n=17, 77%), FOXD4L5 (n=22, 100%), KCNJ11/18 (n=14, 64%), MADCAM1 (n=15, 68%), CBR3 (n=17, 77%), NSD1 (n=17, 77%), FLG1 (n=20, 91%), CCCDC179 (n=7, 32%), SIGLEC11 (n=5, 23%), ROBO4 (n=3, 14%), CISD2 (n=3, 14%), WT1 (n=1, 5%), PTCH1(n=1, 5%), KMT2D (n=1, 5%) and MED17 (n=6, 27%).
Conclusion
Mechanisms other than PML B2 domain mutations may account for As2O3 resistance. Novel genes regulating the cell signaling and apoptotic pathways, cellular proliferation, histone modification, DNA repair and angiogenesis were frequently mutated in As2O3-refractory APL. Further functional validation on the role of these mutations in conferring As2O3 resistance is required.
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Arsenic trioxide, Resistance
Type: Eposter Presentation
Background
Arsenic trioxide (As2O3)-based regimens induce remissions in >90% of relapsed APL. Further relapses after As2O3-induced remissions portend a poor prognosis. With the use of As2O3 in frontline treatment of APL, relapses in patients previously treated with As2O3 resulting in arsenic resistance is an emerging clinical problem. There is a paucity of information on the molecular mechanisms of As2O3-resistance.
Aims
To determine mutations associated with As2O3-resistance with whole exome sequencing (WES).
Methods
Seven patients with prior As2O3 treatment, who had ≥2 subsequent relapses occurring during or refractory to As2O3 treatment, were assigned to the discovery cohort. Serial bone marrow (BM) samples that were arsenic-sensitive (taken before treatment in a relapse responding to As2O3) and arsenic-resistant (obtained in subsequent relapses refractory to As2O3) underwent WES the illumina HiSeq 1500 platform at an average depth of 100X. Exome data were analysed utilizing bioinformatic pipeline programs designed to identify single nucleotide variants (SNVs) that were present either only or at increased frequencies in the resistant samples. The functional significance of non-synonymous missense mutations was evaluated using SIFT, Polyphen2 and FATHMM programs. Putative SNVs were confirmed by Sanger sequencing. Confirmed SNVs were tested in arsenic-resistant BM samples obtained from a validation cohort of 22 relapsed patients previously salvaged with As2O3, who had subsequent As2O3-refractory relapse again. Clinicopathologic features, karyotype, FLT3 and PML-RARA mutations were determined in the validation cohort.
Results
In the discovery cohort, serial WES showed following confirmed SNVs of potential functional significance showed increased allele frequency during As2O3-resistance: NOTCH2, FOXD4L5, KCNJ11/18, MADCAL1, CBR3, NSD1, FLG1, CCCDC179, SIGLEC11, ROBO4, CISD2, WT1, PTCH1, KMT2D and MED17. PML B2 domain mutations were not seen. The frequencies of the mutations were validated in a cohort of 22 patients with relapsed or refractory APL following oral As2O3-based treatment. The recruited patients comprised 14 men and 8 women with a median age of 44.5 (range:24-76) years at relapse. 1 (5%) patient had microgranular variant of APL while 2 patients (9%) had therapy-related APL. Additional karyotypic abnormalities were seen in 3 (14%) patients.14 patients (64%) had central nervous system (CNS) involvement at relapse. Internal tandem duplication of FLT3 (FLT3-ITD) was detected in 7 (32%) patients. The following recurrent mutations were found in As2O3-refractory relapse BM samples: NOTCH2 (n=17, 77%), FOXD4L5 (n=22, 100%), KCNJ11/18 (n=14, 64%), MADCAM1 (n=15, 68%), CBR3 (n=17, 77%), NSD1 (n=17, 77%), FLG1 (n=20, 91%), CCCDC179 (n=7, 32%), SIGLEC11 (n=5, 23%), ROBO4 (n=3, 14%), CISD2 (n=3, 14%), WT1 (n=1, 5%), PTCH1(n=1, 5%), KMT2D (n=1, 5%) and MED17 (n=6, 27%).
Conclusion
Mechanisms other than PML B2 domain mutations may account for As2O3 resistance. Novel genes regulating the cell signaling and apoptotic pathways, cellular proliferation, histone modification, DNA repair and angiogenesis were frequently mutated in As2O3-refractory APL. Further functional validation on the role of these mutations in conferring As2O3 resistance is required.
Session topic: E-poster
Keyword(s): Acute promyelocytic leukemia, Arsenic trioxide, Resistance
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