Identification of an Ultra High-Risk and Targetable Molecular Signature in Relapsed Pediatric T-ALL
Authors: Richter-Pechanska Paulina , Kunz Joachim B. , Hof Jana , Zimmermann Martin , Rausch Tobias , Bandapalli Obul Reddy , Orlova Elena , Scapinello Greta , Sagi Judit , Stanulla Martin , Schrappe Martin , Kirschner-Schwabe Renate , Eckert Cornelia , Benes Vladimir , Korbel Jan O. , Muckenthaler Martina U. , Kulozik Andreas E 
Published: November 2019

Abstract

Relapsed T-cell acute lymphoblastic leukemia (T-ALL) represents one of the major challenges of pediatric oncology as it is often resistant to treatment and fatal. In the search for genes that define critical steps of relapse and can serve as prognostic markers we compared 67 relapses (REL) and 147 samples collected at initial diagnosis (INI) by targeted sequencing of 313 leukemia-related genes. In addition to the analysis of single nucleotide variants (SNV) and small insertions and deletions (InDels), we made use of the available coverage data for profiling of copy number alterations (CNA).
Of the 147 INI patients, 31 were treated according to the ALL-BFM 2000 and 116 according to the AIEOP-BFM ALL 2009 protocol. All REL patients were recruited from the ALL-REZ BFM 2002 trial. We analyzed bone marrow DNA by targeted capture of 313 genes (5964 exons) using the Haloplex Target Enrichment Kit (Agilent). We used Varscan to detect both SNV and InDels. In the absence of available remission samples, we subtracted known SNPs (dbSNP, 1000 gp) and variants present in at least one of 20 non-leukemic samples that we sequenced in parallel to the patients' samples. Only mutations with an AF >10% were considered. Copy number analysis based on read-depth data was validated by MLPA analysis of 14 genes showing a sensitivity of 99% and by low coverage WGS. In total, we have SNV data on all 147 INI and 67 REL patients and CNA data on 144 and 58 patients, respectively.
Altogether, we identified relapse specific genetic events in 32 of 67 RELs. Our results confirm that NT5C2 mutations are highly enriched in relapse (REL: 17/67 vs. INI: 1/147; p=0.0001). Although activation of NT5C2 was associated with the occurrence of early first relapse (p=0.02), it did not correlate with induction failure and therefore has no prognostic impact. Similarly, amplifications of chromosome 17 q11.2-24.3, a region that contains genes of the STAT and ABCA families were significantly more frequent in REL than in INI (REL 7/58 vs. INI 3/144, p=0.0068), but also had no prognostic implications. By contrast, TP53 mutations were highly predictive of a second event: all 8 patients who carried a total of 9 TP53 mutations and deletions died within 9 months after first relapse (p=0.002), whereas 17 of the other 58 (29%) survived. Inactivation of TP53 was significantly correlated with higher mutation rates in other genes compared to those with wild-type TP53 (ttest= p<0.0001). TP53 alterations combined with other recurrent mutations in genes responsible for surveillance of DNA integrity (MSH6 and USP7) recognize 15 of the 49 patients with a fatal post-relapse outcome. This group appeared to be particularly resistant to induction therapy as only 2/14 (no data available for 1 pt) patients compared to 17/52 without TP53/MSH6/USP7 mutations achieved second remission and could receive stem cell transplantation (p=0.0006). Activating mutations of KRAS or NRAS thus activating the Ras/Raf/MEK/ERK pathway were found in 8 of these 49 REL patients, 6 of whom died within 3 months, indicating that these mutations cause treatment resistance (p<0.05). Further, likely inactivating mutations of CNOT3 and known activating insertions of IL7R have been exclusively found in 7 REL patients who failed induction. Four of these 7 RELs also carried TP53 or RAS mutations. This combination of mutations predicts an exquisitely poor outcome and results in an ultra-high risk prognostic signature that identifies 24/49 REL patients, who failed induction treatment and died.
In conclusion, targeted sequencing of relapsed T-ALL identified a molecular signature predicting an exquisitely poor outcome in 50% of relapses, who failed salvage treatment. This group of patients does not benefit from current treatment strategies thus identifying a subgroup with a dire clinical need for experimental therapy. Notably, approx. 25% REL patients who failed induction carried RAS and IL7R mutations. These patients may be considered for personalized treatment with either MEK- or JAK/STAT-inhibitors. Another 4 patients carry TP53 missense mutations and may benefit from treatment with p53 refolding compounds.

Disclosures
No relevant conflicts of interest to declare.