Surface Expression of CRLF2 Protein Is Associated with Lower Minimal Residual Disease (MRD) Among Children with IKZF1-deleted Acute Lymphoblastic Leukemia (ALL)

* AP and LS contributed equally

^ TS and WM contributed equally as senior authors

on behalf of the Polish Pediatric Leukemia/Lymphoma Study Group

Background High MRD levels as well as adverse outcome characterize ‘’BCR-ABL 1-like’’ subtype of ALL. Nevertheless, from genetic abnormalities associated with this subtype of ALL, only IKZF1 deletions were unequivocally associated with high MRD and increased ALL relapse risk across the published studies. Prognostic significance of CRLF2 alterations still remains with discordant conclusions. To date, CRLF2 status has been determined based on CRLF2 mRNA expression or CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2F232C) but not regarding protein expression.

Aim The goal of this project was to investigate early response to treatment in patients with childhood ALL with respect to CRLF2 protein expression on leukemic cells at diagnosis.

Methods Between August 2011 and March 2014, 384 consecutive children (median age 4.4 yrs; median follow-up 15±8.7 months), with newly diagnosed BCP-ALL treated according to ALL-IC BFM09 protocol in 15 centers of the Polish Pediatric Leukemia/Lymphoma Study Group were prospectively enrolled into the study. Targeted copy number screening of selected 23 loci was performed on available DNA samples (n=359) by using the P335-B2 and P202-A2 SALSA MLPA kits (MRC-Holland, Netherlands). CRLF2 protein expression on leukemic cells collected at diagnosis (n=384) was determined by flow cytometry (FCM) using anti-TSLP-R antibody (Biolegend, USA). CRLF2-P2RY8fusion was identified using RT-PCR with specific primers followed by direct sequencing. MRD was measured at day 15 of induction therapy using FCM with EuroFlow 8-color antibody panels (n=377).

Results CRLF2 expression was present at diagnosis in 21 cases (21/384=5.4%), in 10 out of the 21 CRLF2-P2RY8 fusion was found. Among 359 patients, 54 (15%) harbored IKZF1 deletions which were significantly associated with CRLF2 expression (47/341=13.7% vs. 7/18= 38.8%, p=0.01). The presence of CRLF2 expression was also linked with Down syndrome (5/12=41.6% vs. 3/258=11.6, p<10^-5). Median MRD levels at day 15 were significantly higher among patients with IKZF1 deletion, delIKZF1+=2.65(0.1-12)% vs. delIKZF1-=0.37(0.05-2.99)%, p=0.001; and in patients negative for CRLF2 expression, CRLF2-=0.45(0.06-3.8)% vs. CRLF2+= 0.01 (0.00-1.12)%, p=0.007]. Moreover, in conditional analysis MRD levels were lower among ALL cases with delIKZF1+CRLF2+ compared to delIKZF1+CRLF2-, 0.3 (0.02-5.6) vs. 2.7 (0.2-12) respectively, p=0.001; and patients double negative delIKZF1-CRLF2- showed even higher MRD level than patients delIKZF1-CRLF2+, 0.4 (0.05-3.1) vs 0.01 (0.00-1.75), p=0.001.

Conclusions We reported for the first time clinical relevance of the CRLF2 protein surface expression with specific impact on MRD levels, which did not worsen induction response in patients with IKZF1 deletions. Considering relatively short follow-up, further prospective observation of events in the study cohort is indispensable to assess prognostic value of CRLF2 protein expression.