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THE VALUE OF NGS PANEL SEQUENCING TO MOLECULARLY DEFINE MYELOID MALIGNANCIES AND CLARIFY BORDERLINE CASES: A STUDY ON 39 GENES IN 1143 PATIENTS
Author(s): ,
Constance Baer
Affiliations:
MLL Munich Leukemia Laboratory,Munich,Germany
,
Karolína Perglerová
Affiliations:
MLL2 s.r.o.,Prague,Czech Republic
,
Wolfgang Kern
Affiliations:
MLL Munich Leukemia Laboratory,Munich,Germany
,
Claudia Haferlach
Affiliations:
MLL Munich Leukemia Laboratory,Munich,Germany
Torsten Haferlach
Affiliations:
MLL Munich Leukemia Laboratory,Munich,Germany
(Abstract release date: 05/18/17) EHA Library. Baer C. 06/23/17; 181408; S121
Dr. Constance Baer
Dr. Constance Baer
Contributions
Abstract

Abstract: S121

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:00 - 12:15

Location: Hall E

Background

The 2016 revision of the WHO classification for myeloid malignancies includes numerous molecular markers for classification and prognostication. Next generation sequencing allows analyzing relevant genes in one panel.

Aims

Exploit clinical usefulness of panel sequencing in routine diagnostics in order to describe genetic changes and use respective patterns in cases with undefinitive morphology.

Methods

According to WHO 2016, 1143 patients were morphologically categorized as AML (n=261), MDS (n=176), MPN (n=19), CMML (n=51) or AML/MDS (n=21) and MDS/MPN overlap (n=28). Patients, who did not fulfill all characteristic criteria or had insufficient sample quality, were classified as “possible” AML (n=28), MDS (n=211), MPN (n=5), CMML (n=14) and as reactive (n=193) or unclear (n=136). DNA was isolated from BM (n=958) or PB (n=185) for NextSeq or MiSeq sequencing after TruSeq library preparation (all Illumina, San Diego, CA). Data was analyzed with SeqNext 4.3 (JSI Medical Systems, Kippenheim, Germany). FLT3-ITD and KMT2A-PTD data was obtained according to standard protocols.

Results

Analyzing 39 genes, we found ≥1 molecular change in 90% of patients (500/556) with a definite morphologic diagnosis (median: 2 genes; max: 7).
In de novo AML, 212/229 (93%) patients showed ≥1 molecular hit, of which 211 (92%) had aberrations that define WHO categories or have prognostic (according to ELN/MRC) or predictive value. More than one mutation was found in 166/229 patients (72%), including information of adverse impact (e.g. of 68 NPM1 positive patients, 17 had DNMT3A mutations and 20 FLT3-ITD). Following NPM1, RUNX1 was the second most frequently mutated gene (46/225; 20%) and mutations were significantly more common in patients with ≥3 aberrations (38/104; 37% vs. 8/96; 8%; p<0.001). A similar RUNX1 pattern was found in s-AML and t-AML.
In the cohort of “possible AML” (including MDS overlap), 45/48 (94%) patients had ≥1 hit. Most frequently mutated were ASXL1 (16/48; 33%), TET2 (32%; 14/44) and SRSF2 (29%; 14/48); 16% had all three mutated. This combination is also the most frequent three-way interaction in CMML (10/44; 23%).
In MDS, 124/157 (79%) cases showed mutations, of which 108 had ≥1 prognostic change (according to Bejar, 2015). The prognostically favorable SF3B1 mutation was present in 31/157 (20%) and significantly enriched among cases with ring sideroblasts (p<0.001). Overall, TET2 showed the highest mutation rate (25%) and was also the most commonly mutated gene in cases with “possible” MDS (36/190; 19%), reactive morphologic changes (17/201; 8%) or even unclear morphology (19/116; 17%). Of these three subsets, five patients had only the TET2 mutation with <10% burden, which is observed in clonal hematopoiesis of indeterminate potential (CHIP), too. However, using panel sequencing in cases with possible MDS, unclear or reactive morphology revealed at least one molecular marker for clonal disease in 47% (91/199), 36% (43/118) or 17% (36/211) of cases, respectively (excluding sole ASXL1, DNMT3A, TET2 mutations with <10% burden).

Conclusion
WHO 2016 requires information on numerous genes for diagnosis, prognosis and therapeutic decisions. This challenges conventional laboratory approaches and suggests panel sequencing. We demonstrate the feasibility in routine settings for a broad spectrum of myeloid malignancies and identify 1) relevant patterns and mutation interactions; 2) genetic aberrations supporting diagnosis for samples with borderline morphology or poor quality and 3) patient-specific clonality useful for follow-up.

Session topic: 9. Myelodysplastic syndromes - Biology

Keyword(s): Myeloid malignancies, mutation analysis, MDS/AML

Abstract: S121

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:00 - 12:15

Location: Hall E

Background

The 2016 revision of the WHO classification for myeloid malignancies includes numerous molecular markers for classification and prognostication. Next generation sequencing allows analyzing relevant genes in one panel.

Aims

Exploit clinical usefulness of panel sequencing in routine diagnostics in order to describe genetic changes and use respective patterns in cases with undefinitive morphology.

Methods

According to WHO 2016, 1143 patients were morphologically categorized as AML (n=261), MDS (n=176), MPN (n=19), CMML (n=51) or AML/MDS (n=21) and MDS/MPN overlap (n=28). Patients, who did not fulfill all characteristic criteria or had insufficient sample quality, were classified as “possible” AML (n=28), MDS (n=211), MPN (n=5), CMML (n=14) and as reactive (n=193) or unclear (n=136). DNA was isolated from BM (n=958) or PB (n=185) for NextSeq or MiSeq sequencing after TruSeq library preparation (all Illumina, San Diego, CA). Data was analyzed with SeqNext 4.3 (JSI Medical Systems, Kippenheim, Germany). FLT3-ITD and KMT2A-PTD data was obtained according to standard protocols.

Results

Analyzing 39 genes, we found ≥1 molecular change in 90% of patients (500/556) with a definite morphologic diagnosis (median: 2 genes; max: 7).
In de novo AML, 212/229 (93%) patients showed ≥1 molecular hit, of which 211 (92%) had aberrations that define WHO categories or have prognostic (according to ELN/MRC) or predictive value. More than one mutation was found in 166/229 patients (72%), including information of adverse impact (e.g. of 68 NPM1 positive patients, 17 had DNMT3A mutations and 20 FLT3-ITD). Following NPM1, RUNX1 was the second most frequently mutated gene (46/225; 20%) and mutations were significantly more common in patients with ≥3 aberrations (38/104; 37% vs. 8/96; 8%; p<0.001). A similar RUNX1 pattern was found in s-AML and t-AML.
In the cohort of “possible AML” (including MDS overlap), 45/48 (94%) patients had ≥1 hit. Most frequently mutated were ASXL1 (16/48; 33%), TET2 (32%; 14/44) and SRSF2 (29%; 14/48); 16% had all three mutated. This combination is also the most frequent three-way interaction in CMML (10/44; 23%).
In MDS, 124/157 (79%) cases showed mutations, of which 108 had ≥1 prognostic change (according to Bejar, 2015). The prognostically favorable SF3B1 mutation was present in 31/157 (20%) and significantly enriched among cases with ring sideroblasts (p<0.001). Overall, TET2 showed the highest mutation rate (25%) and was also the most commonly mutated gene in cases with “possible” MDS (36/190; 19%), reactive morphologic changes (17/201; 8%) or even unclear morphology (19/116; 17%). Of these three subsets, five patients had only the TET2 mutation with <10% burden, which is observed in clonal hematopoiesis of indeterminate potential (CHIP), too. However, using panel sequencing in cases with possible MDS, unclear or reactive morphology revealed at least one molecular marker for clonal disease in 47% (91/199), 36% (43/118) or 17% (36/211) of cases, respectively (excluding sole ASXL1, DNMT3A, TET2 mutations with <10% burden).

Conclusion
WHO 2016 requires information on numerous genes for diagnosis, prognosis and therapeutic decisions. This challenges conventional laboratory approaches and suggests panel sequencing. We demonstrate the feasibility in routine settings for a broad spectrum of myeloid malignancies and identify 1) relevant patterns and mutation interactions; 2) genetic aberrations supporting diagnosis for samples with borderline morphology or poor quality and 3) patient-specific clonality useful for follow-up.

Session topic: 9. Myelodysplastic syndromes - Biology

Keyword(s): Myeloid malignancies, mutation analysis, MDS/AML

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