Characterization of Philadelphia-like Pre-B Acute Lymphoblastic Leukemia: Experiences in Mexican Pediatric Patients

Abstract

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano-Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

Keywords: CRLF2 overexpression; IGH::CRLF2; Mexican; P2RY8::CRLF2; Ph-like; children; iAMP21; pCrkl; pre-B ALL.

Figure 2

CRLF2 genetic lesions. (A) FISH assay showing cell clones with hyperdiploidy (left) and cell clones with IGH::CRLF2 translocation and hyperdiploidy coexistence (right). White arrows represent the IGH and CRLF2 loci and orange triangles represent the IGH::CRLF2 translocation (fused signals green/red represent IGH or CRLF2 normal loci, and separate green and red signals represent broken IGH or CRLF2 loci). (B) aCGH shows a copy number gain at Xp22.33 (ChrX: 403328-2865335) involving the CRLF2 gene. (C) Sanger sequencing shows the CRLF2 point mutation.

Figure 5

Adapted strategy for Ph-like diagnosis. Proposed testing strategy for the Ph-like subgroup. The analysis comprises the pre-B ALL patients without recurrent gene fusions. ✔ = no further evaluation since a final result was obtained.

Figure 1

Genetic landscape of pre-B ALL cases clustered according to the CRLF2 expression. Each column represents a patient, and the rows represent genetic or biochemical features. Gene mutations are shown in parenthesis as follows: patients harboring the mutation/different mutations identified. CNG: copy number gain.

Figure 3

CRLF2 expression distribution between the different GEP coefficients. Different GEP coefficients were compared between patients with and low/null CRLF2 overexpression, statistic differences are presented (* p < 0.05 chi-square test).

Figure 4

Two patients with signaling pathway activation. (A) Patient ALL 036 showing positivity to the three surrogate markers pStat5, pCrkl, and pErk. (B) Patient ALL 046 showing positivity to pCrkl and reversion produced by imatinib, and interphase nuclei hybridized with ABL2 break-apart FISH probe with a normal pattern (two large green signals in the left nucleus) and with an ABL2 breakage (one large green signal representing the normal gene, and two small green signals representing the broken gene in the rightright nucleus). White arrows represent the normal ABL2 loci and orange triangles represent the breakage of ABL2.The Abl pathway was assessed in 29 patients and the surrogate marker pCrkl was positive in 10 (Figure 1 and Figure 4A); of these, 7 patients presented a genetic abnormality involving CRLF2, interestingly, 4 out of 7 cases were also positive for pStat5. On the other hand, three of the seven cases showed no evidence of abnormalities in CRLF2 and the GEPs were 3.3, 6.7, and 1.0. It was possible to analyze two patients positive to pCrkl (ALL 039 and ALL 046) with ABL1 and ABL2 break-apart FISH probes, positive results were obtained for ABL2 breakage in 16% and 6% of cells analyzed, respectively. Interestingly, patient ALL 046 was also found to be positive for IGH::CRLF2 and showed the presence of both surrogate markers pStat5 and pCrkl, revealing the coexistence of both abnormalities in the same patient. Remarkably, this case showed pCrkl reversion using imatinib (Figure 1 and Figure 4B).