Page 208 - Haematologica March 2020

M. Bill et al.
of LSC, in 2011, Eppert et al.4 described a LSC-related gene-expression signature comprising 44 genes that were deregulated in LSC. The derived stem cell-like signature was shown to associate with inferior outcome in adult patients with cytogenetically normal AML.4,16 Recently, Ng et al.7 used a similar approach to generate a LSC-derived gene-expression signature consisting of 17 genes that also associated with inferior outcome. However, it is still not fully determined whether this signature is associated with clinical characteristics and gene mutations. Moreover, the prognostic value of the 17-gene LSC score in the context of other, well-established risk classifications, for example the one by the European LeukemiaNet (ELN)1, has not, to our knowledge, been assessed.
We, therefore, derived the 17-gene score from a set of samples from adults with AML and determined associa- tions between the signature and known prognostica- tors1,17,18 as well as mutational data of 81 cancer- and leukemia-associated genes.19 Moreover, we validated the prognostic impact of the 17-gene signature alone and in the context of the 2017 ELN genetic-risk classification.1
Methods
Patients and treatment
We investigated 934 adult patients with de novo AML (other than acute promyelocytic leukemia), for whom material for molecular analyses was available. Availability of material for analysis was the only criterion for inclusion in our study – we did not select AML patients based on their age, ELN risk group, specific clinical trial they were enrolled onto, etc. Because of differences in the treatment protocols between younger and older patients, we performed outcome analyses separately for these two groups of patients. Within each age group, patients were treated similarly, receiving a cytarabine/anthracycline- based induction on Cancer and Leukemia Group B (CALGB) tri- als.20-34 No patient received an allogeneic stem cell transplant in first CR. Details of CALGB treatment protocols are provided in the Online Supplementary Appendix and Online Supplementary Table S1. There were no significant differences in CR rates, disease-free survival (DFS) or overall survival (OS) for younger patients enrolled onto CALGB 8525, 9222, 9621, 10503, 10603 and 19808 treatment trials (Online Supplementary Table S2) nor were there any significant differences in CR rates, DFS or OS among older patients enrolled onto CALBG 9420, 9720, 10201 and 10502 trials (Online Supplementary Table S3). CALGB is now part of the Alliance for Clinical Trials in Oncology (Alliance). All patients were enrolled on CALGB 8461 (cytogenetic studies), CALGB 9665 (leukemia tissue bank) and CALGB 20202 (molecular stud- ies) companion protocols. Patients provided written informed consent, and study protocols were in accordance with the Declaration of Helsinki and approved by Institutional Review Boards.
Transcriptome analyses and calculation of the 17-gene leukemia stem cell score
Pretreatment bone marrow and/or blood samples containing ≥20% leukemic blasts were obtained from all patients and mononuclear cells were enriched through Ficoll-Hypaque gradi- ent centrifugation and cryopreserved until use. Total RNA was extracted from patients’ samples using the TRIzol method according to the manufacturer’s protocol and used for RNA- sequencing analyses (see also the Online Supplementary Appendix). RNA-sequencing libraries were prepared using the
Illumina (San Diego, CA, USA) TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (n. RS1222201) according to the manufacturer's instructions. Sequencing was performed with Illumina HiSeq systems using the HiSeq version 3 sequenc- ing reagents to an approximate cluster density of 800,000/mm2. Image analysis, base calling, error estimation, and quality thresholds were performed using HiSeq Controller software (version 2.2.38) and Real Time Analyzer software (version 1.18.64). Transcript abundance was quantified from the RNA- sequencing data using kallisto,35 with a reference transcriptome consisting of Homo sapiens GRCh38 protein-encoding and non- coding transcripts except rRNA; the strand-specific option of “first read reverse” was chosen. Abundance values are represent- ed in transcripts per million.
The 17-gene LSC score was derived similarly to that in the publication by Ng et al.7 using RNA-sequencing data and the same weights that were published initially for a microarray plat- form.7 Briefly, the 17-gene LSC score was calculated as the weighted sum of the normalized expression values of the 17 genes included in the signature: 17-gene LSC score = (DNMT3B × 0.0874) + (ZBTB46 × −0.0347) + (NYNRIN × 0.00865) + (ARHGAP22 × −0.0138) + (LAPTM4B × 0.00582) + (MMRN1 × 0.0258) + (DPYSL3 × 0.0284) + (KIAA0125 × 0.0196) + (CDK6 × −0.0704) + (CPXM1 × −0.0258) + (SOCS2 × 0.0271) + (SMIM24 × −0.0226) + (EMP1 × 0.0146) + (NGFRAP1 × 0.0465) + (CD34 × 0.0338) + (AKR1C3 × −0.0402) + (GPR56 × 0.0501).7 The derived scores were used to divide patients into two groups using the median as the cutoff: a group with a high score (17- genehigh) and a group with a low score (17-genelow).
Cytogenetic and molecular analyses
Details of the cytogenetic and molecular analyses are provided in the Online Supplementary Appendix.
Results
Clinical and cytogenetic characteristics associated with the 17-gene leukemia stem cell score
Pretreatment characteristics of the 934 patients are shown in Table 1. For all patients, we determined the 17- gene LSC score, which indicates a stem cell-like gene- expression profile, and separated them into 17-genelow and 17-genehigh groups using the median. Comparison between patients with a 17-genelow and 17-genehigh score showed that the former were younger at diagnosis (median: 46 vs. 53 years; P<0.001) and had lower platelet counts (median: 50 vs. 63x109/L; P<0.001). Cytogenetically, there was no difference in the frequency of the presence of cytogeneti- cally normal AML between the groups. Among cytogenet- ically abnormal patients, those with a 17-genelow score more frequently had core-binding factor AML (CBF-AML; P<0.001), including all patients with t(8;21)(q22;q22) and 88% with inv(16)(p13q22) or t(16;16)(p13;q22). On the other hand, the group with a 17-genehigh score included all patients with inv(3)(q21q26) or t(3;3)(q21;q26) and con- tained more patients with a complex karyotype than in the 17-genelow group (P<0.001). Most patients with a com- plex karyotype in the 17-genehigh group had a typical com- plex karyotype (i.e., complex karyotype with unbalanced chromosome abnormalities leading to loss of material from 5q, 7q and/or 17p), whereas an atypical complex karyotype (i.e., complex karyotype without 5q, 7q and/or 17p abnormalities)36 was found with a higher frequency among 17-genelow patients.
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