Page 148 - Haematologica May 2020

B. Depreter et al.
Table 1. Characteristics of de novo acute myeloid leukemia (AML) patients used for sorting CD34+CD38+ and CD34+CD38– cell fractions and qual- itative polymerase chain reaction evaluation.
Age, years
WBC count, x 109/L Morphological blast count
BM, % PB, %
Pediatric AML (n=13) Median (Range)
10 (2-16)
66 (2.7-336)
81 (34-96) 67 (1-95)
Adult AML (n=17) Median (Range)
48 (20 - 76)
15 (6-274)†
77 (28-90)† 73 (7-93)||
N%N%
Gender
F 7 53.8% 9 M 6 46.2% 8
Sample
BM 8 61.5% 11
PB 5 38.5% 6
52.9% 47.1%
64.7%
35.3% 88.2% 18.8% 11.8% 71.4%
35.7% 60.0%
423.5% 741.2% 317.6% 317.6%
CD34 positivity 13 Fusion transcript 6 CBF leukemia 4 WT1 overexpression 10 Mutation status
NPM1 0
FLT3-ITD 8 Risk classification
SR 7 HR 5 Unknown 1
100.0% 15 46.2% 3* 30.8% 2 76.9% 10‡
0.0% 5‡ 61.5% 9†
53.8% Favorable 38.5% Intermediate-I/II
7.7% Adverse
Unknown
Pediatric acute myeloid leukemia (pedAML) patients were diagnosed in Belgium and treated according to the DB AML-01 (n=9, 69%) or NOPHO-DBH AML 2012 (n=4, 31%) pro- tocol. Pediatric patients were risk stratified as previously published8 and categorized according to the French-American-British (FAB) classification into M0 (n=1), M1 (n=1), M2 (n=4), M3 (n=1), M4 (n=3), M5 (n=2), and M7 (n=1). Adult AML samples were from patients treated at the Ghent University Hospital, Ghent, Belgium (n=12, 71%) or VUmc, Amsterdam, the Netherlands (n=5, 29%). Belgian patients were treated according to local and international guidelines, whereas Dutch patients were included in the HOVON 102 (n=3) or HOVON 132 (n=2) study. Adults were risk stratified according to the European LeukemiaNet 2010 guidelines1 and categorized according to the FAB classification into M1 (n=6),M2 (n=6),and M3 (n=2).WT1 overexpression was interpreted according to in-house or published cut-offs.Core binding factor (CBF)-positive leukemias comprised AML with t(8;21)(q22;q22) (pedAML=3) and inv(16)(p13q22) (pedAML=1, adult AML=2). Other fusion transcripts detected were DEK-NUP214 (pedAML=1) and PML-RARA (pedAML=1, adult AML=1). *One, †two, ‡three or ||five missing data. BM: bone marrow; F: female; M: male; NPM1: nucleophosmin; PB: peripheral blood; WBC: white blood cell; WT1:Wilms' tumor 1.
ed synthetic TARP peptide TARP(P5L).4-13 Regarding RV transduced TARP-TCR CTL, mock CTL were used to correct for non-TARP mediated lysis, and CMV-TCR transduced CTL to evaluate aspecific killing.
Results
Discovery of T-cell receptor γ chain alternate reading frame protein transcript expression in acute myeloid leukemia
In order to identify LSC-specific antigens, we re-ana- lyzed the gene set enrichment (GSE) 17054 micro-array dataset from Majeti et al.,31 which included gene expres- sion profiles of CD34+CD38– sorted fractions of four healthy adults (HSC) and nine adult AML patients (LSC). TARP ranked first amongst the top differentially expressed genes, with all four probes in the top 20 (range log2-FC 5.13-6.92), showing a significantly higher expression in LSC compared to HSC (P<0.01) (Online Supplementary Figure S1). TARP had previously been identified as a trun- cated TCR transcript expressed in androgen-sensitive
prostate and breast adenocarcinoma (Online Supplementary Figure S2).41,42 We further explored TARP expression in pedAML by micro-array profiling CD34+CD38+ (n=4, leukemic blast) and CD34+CD38– (n=3, LSC) sorted cell populations from four pedAML patients (2 FLT3-ITD and 2 FLT3 WT) (Online Supplementary Table S1). In addition, sorted CD34+CD38+ (n=3) and CD34+CD38- (n=2) cells from CB were profiled to examine the expression in their normal counterparts (Online Supplementary Figure S3). TARP appeared to be more highly expressed in leukemic blasts and LSC from FLT3-ITD patients compared to FLT3 WT patients and CB (Figure 1A). This finding suggested that TARP might represent a LSC-associated target within HR pedAML patients harboring FLT3-ITD.
To validate these data in a larger patient group, we sort- ed CD34+CD38+ and CD34+CD38– cell populations from nine additional pedAML (resulting in a total of 13 pedAML patients), 17 adult AML (Table 1) and 15 control samples (n=7 CB, n=6 NBM, n=2 mPBSC). qPCR analysis using TARP short primers (Online Supplementary Table S3 and Online Supplementary Figure S2) showed that TARP transcripts were consistently low in HSC and myeloblasts
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