Provisional entity: B-lymphoblastic leukemia/lymphoma, BCR-ABL1-like (Arber et al., 2016)

Among positive patients the PR2Y8/CRLF2 rearrangement has been detected in different proportions of cells, in principal clones and in minor subclones. Original major clones may persist, disappear, or revert to a minor clone; the minor clones may evolve into a major clone or loss, continue or substitute by a clone with different breakpoint. Based on this, it has been proposed that PR2Y8/CRLF2 is not a driver abnormality in the process of leukemogenesis. Instead, it is a secondary lesion and requires concomitant mutations to become functionally important. The presence of acquire CRLF2 rearrangements in constitutional trisomy 21, and the coexistence of the PR2Y8/CRLF2 and iAMP21 in non-Down syndrome B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), also suggest that CRLF2 rearrangements requires cooperating mutations to activate JAK/STAT and PI3K/mTOR pathways (Morak et al., 2012).

PR2Y8/CRLF2 positive patients are included in the Ph-like BCP-ALL subgroup, based on its characteristic gene expression signature (Arber et al., 2016). The prevalence of this subgroup is associated with age, high-risk stratification and ethnicity (Harvey et al., 2010). It presents in about 10% of standard-risk childhood ALL, and in >25% of young adults; CRLF2 is the most frequent deregulated gene in the Ph-like subgroup. Overexpression of CRLF2 results in constitutive JAK/STAT activation and cytokine-independent growth (Roberts et al., 2015). In the total of BCP-ALL patients 6% present over-expression of CRLF2, this deregulation is more prevalent among Down syndrome patients (54%). In an unselected cohort of pediatric patients, the Medical Research Council (MRC) found predominance of PR2Y8/CRLF2 (5%) in contrast to IGH/CRLF2 (1%). In comparison, the Childrens Oncology Group (COG) observed that IGH/CRLF2 is twice as prevalent as PR2Y8/CRLF2 (Ensor et al., 2011); this variability could be explained by the selection of high risk ALL patients analysed in this study, including a high proportion of older patients and Hispanic patients, who are 5 times more possible to have CRLF2 abnormalities compared with patients with other ethnicities. In children with BCP-ALL and Down syndrome PR2Y8/CRLF2 was identified in 54.5% of cases (Mullighan et al., 2009). In adults with BCP-ALL the prevalence of Ph-like is >20%, of this group, the IGH/CRLF2 (57.6%) is more prevalent than PR2Y8/CRLF2 (21.2%) (Roberts et al., 2017). In general, IGH/CRLF2 patients are older than PR2Y8/CRLF2 patients.

ALL with BCP immunophenotype with rearrangement involving a cytokine receptor, included in the BCR-ABL1-like ALL subgroup.

The PR2Y8/CRLF2 rearrangement results in kinase-activating lesion that causes the constitutive activation of the JAK/STAT signalling pathway. Therefore, patients with PR2Y8/CRLF2 ALL could benefit from the treatment with clinically approved tyrosine kinase inhibitors such as Ruxolitinib, which target these pathways. The treatment of these patients with the relevant tyrosine kinase inhibitor could potentially improve their survival (Harrison, 2013).

In different cohorts of children with high risk ALL, the presence of CRLF2 alterations have been associated with poor clinical outcome. However, not in all the study groups have been found positive correlations. The different treatment protocols, not standardized methods for diagnosis of CRLF2 alterations or over-expression, and limited prospective studies for analysing prognostic value are source of bias. A meta-analysis study including 5945 patients of seven different studies analysed the association between CRLF2 alterations and survival of children with ALL, the results showed that CRLF2 deregulation predicts poor prognosis. In adults, the outcome of patients with Ph-like ALL is inferior compared with the outcome of patients with non-Ph-like ALL, excluding BCR/ABL1 and MLL positive cases (Roberts et al., 2017).
It is expected that PR2Y8/CRLF2 positive clones contribute to disease progression. However, in children it has been found heterogeneity in PR2Y8/CRLF2 clone sizes, and no participation in relapses if the size is very small at diagnosis. In part, these may be the basis of the controversies about the prognostic value of this rearrangement and the over-expression of CRLF2 as risk markers. A revision of these parameters should be done.

T-lymphoblastic leukemia/lymphoma (T-ALL)

: It has been reported CRLF2 over-expression in T-ALL patients: however, PR2Y8/CRLF2 has not been found in these cases. Deregulation of CRLF2 in this entity is considered a poor prognosis marker (Palmi et al., 2016).

The PR2Y8/CRLF2 rearrangement results from an interstitial microdeletion involving the PAR1, which is located in the short arm of both sex chromosomes. This deletion causes the CRLF2 overexpression through the juxtaposition of the entire coding sequence of CRLF2 with the first non-coding exon of P2RY8, and their transcriptional control elements that are highly active in lymphoid cells (Morak et al., 2016; Vesely et al., 2016; Ensor et al; 2016; Mullighan et al; 2009).

The PAR1 deletion measures approximately 320 Kb and deletes the complete coding sequence of P2RY8 along with ASMTL , SLC25A6, IL3RA and CSF2RA genes, bringing together the first exon of P2RY8 to exon 1 of CRLF2. The breakpoints of the deletion are highly conserved and are located 3.4 Kb upstream of the CRLF2 exon 1 and 0.3-1 Kb distal to P2RY8 exon 1 (Mullighan et al., 2009). Two new alternative breakpoints have been described, 2.1 Kb and 0.1 Kb upstream of CRLF2 exon 1, but in all cases the deletion place the full CRLF2 open reading frame under transcriptional control of the P2RY8 promoter (Morak et al., 2016). The presence of recombination signal sequences immediately internal to the deletion breakpoints suggests that the PAR1 deletion may arise as a result of aberrant activity of the RAG recombinases, this phenomenon has been described for other chromosomal rearrangements in B- ALL (Mullighan et al; 2009).

The chimeric transcript contains the 5-UTR region of P2RY8 joined to exon 1 of CRLF2, prior to the transcription start codon (Hertzberg et al., 2016). The PR2Y8 5-UTR region measures approximately 226 bp, and the open reading frame of CRLF2, which is approximately of 1116 bp, remains complete; therefore the chimeric transcript size is 1342 bp (Yap et al., 2016).

The PR2Y8/CRLF2 positive cases can be detected at genomic level by long range PCR or MLPA techniques using genomic DNA extracted from leukemic cells, however, these assays only reveal the presence of the rearrangement. On the other hand, FISH assay informs about the proportion of abnormal cells, it has been designed deletion probes for covering the telomeric end of P2RY8 and a region distal to the gene, and a control probe that covers a region of the proximal side of P2RY8 (Morak et al., 2016). At RNA level, RT-PCR can confirm the presence of chimeric transcripts. The overexpression of CRLF2 can be measured by qRT-PCR using different methods of absolute and relative measurement (Mullighan et al; 2009).

The overexpression of CRLF2 produced by the PR2Y8/CRLF2 rearrangement causes the constitutive activation of JAK/SAT signaling and the PI3K/mTOR, Ras, MAPK and Bcl-2 downstream related pathways in the leukemic blast. These signaling pathways have a central role in the regulation of the cell proliferation, survival, differentiation and immune response; hence its malfunction contributes to the leukemic transformation (Roberts KG et al., 2015).

P2RY8/CRLF2