Clincial and Genetic Landscapes Differ Between IGH-CRLF2 and P2RY8-CRLF2 Acute Lymphoblastic Leukaemia

Deregulated expression of the type I cytokine receptor, cytokine receptor-like factor 2 (CRLF2-d), is observed in 5% of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It occurs via three known aberrations; a cryptic reciprocal translocation with the immunoglobulin heavy chain locus (IGH); an interstitial deletion within PAR1, resulting in the P2RY8-CRLF2 fusion; rare but recurrent CRLF2 mutation (F323C). All three result in overexpression of CRLF2, however alone it is insufficient to cause overt leukaemia. Mutations of the Janus kinase (JAK) family genes and the IL7 receptor, are recurrent (50% of CRLF2-d) and together with CRLF2-d result in transformation of mouse BaF3 cells. We noted that outcome data from different study groups were inconsistent, with patients being classified as either high or intermediate risk. Thus, in this largest study to date, our aim was to acertain whether differences in clinical and/or genetic features between patients with CRLF2-d and involvement of either IGH or P2RY8 may define them as different disease entities and partly explain the outcome heterogeneity.

Among 160 CRLF2-d patients, chromosomal analysis confirmed a high incidence of additional somatic copies of chromosomes X (39/88, 44%) and 21 (23/88, 26%) and identified that aneuploidy of chromosomes 9 (5/88, 6%) and 17 (7/88, 8%)  were recurrent in both groups. From comparison of patients with IGH-CRLF2 and P2RY8-CRLF2, we noted a higher frequency of IKZF1 (79% v 37% p<0.001) and BTG1 (50% v 8%, p<0.001) deletions and greater complexity (>3 additional copy number alterations (CNA)) by MLPA (SALSA MLPA P335 kit, MRC Holland, The Netherlands) (p=0.037) among IGH-CRLF2 patients. We verified that (48/160) 30% of CRLF2-d patients were Down syndrome (DS).

In addition to CNA within the immunoglobulin loci and the genes investigated by MLPA, SNP6.0 arrays (Affymetrix, Santa Clara, USA)  (n=25) and subsequent FISH screening identified recurrent CNA in the histone cluster at 6p22.2 (7/25, 28%), VPREB1 (6/25, 24%), ADD3 (14/58, 24%), BTLA (4/25, 16%), SLX4IP (12/43, 28%), SERP2 and TSC22D1 (6/53, 11%), PBX3 (16/54, 30%). A novel finding was an interstitial deletion at Xp11.4 fusing USP9X to DDX3X(9/50, 18%).

A total of 137 somatic structural variants (SV) were detected by whole genome sequencing of 11 patients [tandem duplication- (n=6), deletion- (n=104), inversion- (n=14) and translocation-type (n=13)] creating an average of 12.7 validated SV per patient. Mutations were identified at an incidence of 12.8 per patient, including mutations in JAK1 and JAK2 (40%) and IL7R, CACNA1D and USH2A (n=2). Mutations in kinase genes were common and unique to all patients, a number have not been previously reported (e.g. ERBB4, TTBK1 and STK38L). Of interest, 7 patients had ≥1 point mutation in gene(s) involved in cell adhesion (e.g. NPNT, ADAM29, ITGB7, COL3A1 and USH2A).

There was a significant difference in the median age and white blood cell count (WBC) between P2RY8-CRLF2 and IGH-CRLF2 (4yrs v 14yrs, p<0.001 and WBC >50x10⁶/L - 25% v 44%, p=0.021) . Overall IGH-CRLF2 patients had a worse outcome compared to P2RY8-CRLF2 (p=0.191) with outcome heterogeneity in relation to gender (IGH females, HR 3.75, p=0.003), number of CNA (IGH <3 genes deleted, HR 3.79, p=0.047) and treatment trial (IGHhave worse outcome on ALL97, HR 3.15, p=0.014) being observed. There was no heterogeneity for age (p=0.458), WBC (p=0.331), cytogenetic risk (p=1.0), MRD (p=1.0) or DS-ALL (p=0.877).

In summary we have confirmed differences in age and WBC, involvement of kinase gene mutations and chromosomal gains in CRLF2-d ALL. Other findings included recurrent deletions involving TSC22D1 and PBX3, transcription factors involved in other human cancers; SLX4IP, an uncharacterized gene, frequently deleted in childhood ALL; the novel involvement of USP9X, a deubiquitinase involved in cancer development. We also identified recurrent mutations in cell adhesion genes. We showed that IGH-CRLF2 patients had a worse outcome, with heterogeneity seen between gender, number of gene deletions and treatment trial. We have shown that there are genetic and clinical differences dependent on whether CRLF2 is deregulated by IGH or P2RY8, suggesting that they should be regarded as different clincial entities in future studies.