Biological Features and Prognostic Impact of CRLF2 Overexpression In Childhood ALL
Authors: Cazzaniga Giovanni , Vendramini Elena , Palmi Chiara , Silvestri Daniela , Frison Daniela , Longinotti Giulia , Leszl Anna , Fazio Grazia , Giarin Emanuela , Rossi Vincenzo , Bungaro Silvia , Basso Giuseppe , Biondi Andrea , Valsecchi Maria Grazia , Conter Valentino , Kronnie Truus te 
Published: October 2019

Abstract
Abstract 271

Introduction.
Although in AIEOP-ALL 2000 protocol, early MRD assessment allowed to better recognize high risk (HR) cases, still about 20% of children relapse, with more than half of relapses occurring in the largest subgroup of non-HR patients. Therefore, new prognostic markers are useful to adapt treatment. Recently, high-level expression of the CRLF2 gene was recognized to characterize a novel subgroup of BCP-ALL. Over-expression of CRLF2 can be the consequence of a translocation at the IGH@ locus, and/or an interstitial deletion, juxtaposing CRLF2 with the promoter of the P2RY8 gene. CRLF2 over-expression appeared to be the driver of STAT5 activation, either alone or in combination with gain-of-function mutations in Janus Kinases (JAKs), mainly JAK2 R683. Aim. In order to estimate the incidence of this abnormality, its prognostic value and the association with patients' biological features, we analyzed CRLF2 gene expression in 515 non-Down Syndrome BCP-ALL children, treated according to the ALL-AIEOP 2000 protocol.

Methods.
Ig/TcR PCR-MRD and RT-PCR screening for known chromosomal translocation were routinely performed. Gene expression profiling by Affymetrix HG U133 Plus 2.0 arrays; RQ-PCR for CRLF2 expression; PCR detection of P2RY8-CRLF2 fusion, FISH for CRLF2 translocations, High Resolution Melting (HRM) and sequencing for JAK2 mutations, gene Copy Number Analysis by Affymetrix Cyto 2.7M array were performed. CRLF2 expression was reported as a fold increase to the median of all values detected in the complete series.

Results.
Out of 515 samples, CRLF2 expression was 10 times higher than the overall median in 46 cases (8.9%), and 20 times more in 24 (4.7%). The P2RY8-CRLF2 rearrangement was detected in 15/168 cases overall (8.9%), in 13/43 tested as CRLF2-high(>10) samples (30%) and in 11/22 considering only the CRLF2-highest(>20) expression group (50%). JAK2 mutations were tested in the first 121 cases highly expressing CRLF2 (>3 times), and were observed in 4 cases (3.3%). All JAK2 mutated cases were over-expressing CRLF2 (19, 31, 43 and 58 times), and all were P2RY8-CRLF2 positive; 2 were R683G substitutions, and 2 cases presented 6 and 12 inserted nucleotides, respectively. Comparing CRLF2-highest(>20) and low expressing patients, none of the CRLF2-highest cases had recurrent chromosomal translocations (while 1 BCR/ABL and 3 TEL/AML1 positive cases were in the >10 times, and <20, overexpressing group). CRLF2-highest patients tend to have a lower probability for high WBC count at diagnosis (>20,000/μ l: 25% vs 35%) and a higher age at diagnosis (median age 67 months vs 56). CRLF2-highest and low had a similar incidence of prednisone poor response (9.2% vs 12.5%). Interestingly, among CRLF2-highest patients only 2 were in the PCR-MRD HR group. Remarkably, all 15 cases P2RY8-CRLF2-positive were non-HR by MRD (4 were SR, one of them in the CRLF2>20 group). Patients CRLF2-highest had a statistically significant higher 5-year Cumulative Incidence of Relapse (CIR) compared to CRLF2-low patients (37.5±9.8% vs. 17.7±1.7%; relapses=9/24 vs 93/491, p=0.00296). This difference was due to a higher CIR for P2RY8-CRLF2 positive cases; in fact, among CRLF2 >20 times cases, CIR for deleted cases was 60±15.5% compared to 16.6±10.7% for non-deleted (relapses=6/10 versus 3/14). The difference in CIR between CRLF2>20 times and CRLF2-low remains statistically different within the hyperdiploid subgroup (interestingly, none of the P2RY8-CRLF2 positive was hyperdiploid). Only 2 of the 4 JAK2 mutated (and CRLF2 deleted) cases relapsed.

Conclusions.
High levels of CRLF2 expression (>20 times the median) define a subgroup of childhood ALL (around 5%) without known high risk features, associated with a higher incidence of relapse if treated according to the AIEOP-ALL 2000 protocol. This effect is mainly related to the presence of the P2RY8-CRLF2 rearrangement. Once prospectively confirmed in large series, the assessment of CRLF2 status may serve as a new stratification marker.

Disclosures:
No relevant conflicts of interest to declare.