Three Steps to the Diagnosis of Adult Ph-like ALL

Introduction: Philadelphia chromosome-like acute lymphoblasic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to BCR-ABL-positive ALL and is associated with particularly poor prognosis. Aberrant gene-expression, point mutations or fusion translocations cause activation of either the ABL1 or JAK signaling pathways. The diagnosis of Ph-like ALL is challenging but of vital importance as it is likely to respond to targeted therapy with ABL or JAK inhibitors.

Aims: To develop a robust and efficient stepwise diagnostic work-up to identify Ph-like ALL in routine diagnostics.

Patients and Methods: We studied 132 adult precursor B-ALL patients (pts) lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 (age 18-85 years, median 54 years). Precursor B-ALL was diagnosed by immunophenotyping in all cases. The following methods were performed in parallel:  Microarray gene-expression profiling (GEP) according to Roberts et al, NEJM 2014; fluorescence in situ hybridization (FISH) with customized probes for ABL2, TSLP, CSF1R, PDGFRB, PTK2B, JAK2, ETV6, IGH, NTRK3, EPOR, TYK2, and IL2RB; and RT-PCR for detection of ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2; quantitative real-time PCR for evaluation of CRLF2-expression, fragment analysis for detection of IKZF1 deletions;  next generation sequencing for mutations in JAK1, JAK2,  JAK3, KRAS, and NRAS.

Results: Based on GEP, we identified 13/132 adult precursor B-ALL pts (10%) as Ph-like ALL. As an alternative approach, we applied a combination of FISH, RT-PCR, fragment analysis and next generation sequencing for diagnosis of Ph-like ALL and simultaneous identification of  therapy sensitive targets. Quantitative real time PCR identified 11/132 cases with CRLF2 overexpression (8%), of which 9 cases were also classified as Ph-like ALL according to GEP. 7/11 (64%) cases with elevated CRLF2 expression harbored IGH-CRLF2 rearrangement detected by our FISH assay. Additionally, RT-PCR and FISH analysis identified 4 pts with rearrangements involving TKI sensitive genes ABL1 (SNX2-ABL1; n=1), and PDGFRB (EBF1-PDGFRB; n=3). Rearrangements of EPOR and JAK2 were not detected. Taken together, the combination of quantitative RT-PCR, FISH and fusion specific RT-PCR identified 15 cases (12%) compared to 13 cases (10%) identified by GEP with Ph-like ALL in a cohort of 132 preselected adult precursor B-ALL cases: 13 pts were assigned to the Ph-like subset by both approaches, while no patient was identified by GEP only and two were identified by the alternative approach only.

Mutations in JAK2 were detected in 8/15 Ph-like ALL cases, all of which were characterized by CRLF2 rearrangements and/or overexpression of CRLF2. In comparison, none of the 117/132 non Ph-like ALL cases harbored JAK2 mutations. NRAS mutations were found in two pts with CRLF2 rearrangement. Mutations in JAK1, JAK3 and KRAS were not detected. Furthermore, we found a high frequency of IKZF1 deletions in Ph-like ALL pts (n=12/16; 75%) compared to 19% (22/117) in the remaining 117 cases with non Ph-like ALL (p<0.001), which is in line with previous published data.

Regarding age, 5/15 Ph-like pts were young adults (age: 31-39 years), 3/15 pts adults (age: 40-55 years) and 7/15 pts were older adults (age: 56-76 years), indicating an incidence of Ph-like ALL of 8% in adults age ≥40 years.

Conclusions: In adult precursor B-ALL lacking the recurrent aberrations BCR-ABL1, MLL-AF4 and E2A-PBX1 Ph-like ALL was identified in 10% of pts based on GEP and in 12% by an alternative approach based on RT-PCR and FISH, respectively. As GEP per se is challenging in routine diagnostics and does not provide therapeutic information regarding potential susceptibility to ABL or JAK inhibitors, we postulate the following diagnostic algorithm to identify Ph-like ALL:

1. Analysis of CRLF2 expression.
2. FISH targeting ABL and JAK pathway activating fusions involving the genes ABL1, ABL2, CSF1R, PDGFRB and JAK2.
3. Fusion specific RT-PCR for the identification of the respective ABL and JAK fusion partner and sensitive MRD monitoring.

We show that the alternative approach is efficient for identification of Ph-like ALL and is feasible for routine diagnostics.