Feasibility of Targeted RNA Sequencing for Routine Diagnostics of B-Cell Precursor ALL

Introduction: In approximately one third of B-cell precursor acute lymphoblastic leukemia (ALL) major pathogenic or driver cytogenetic abnormalities are missing and they are grouped as "B-other" ALL. Recently, the new provisional entity " BCR-ABL1 -like B-lymphoblastic leukemia/lymphoma ( BCR-ABL1 -likeALL)" has been listed as provisional entity in the 2016 revision to the World Health Organization(WHO) classification. However, a certain portion of "B-other" ALL cases still remain genetically unclassified.

Aims: Evaluating the feasibility of targeted RNA sequencing for identification and characterization of BCR-ABL1 -like and other "B-other" ALL cases in a routine diagnostic workup.

Patients and Methods: The study cohort consisted of 85 consecutive adult precursor B-ALL patients lacking the recurrent aberrations BCR-ABL1, KMT2A-AFF1, TCF3-PBX1 and ETV6-RUNX1. The cohort comprised 34 females and 51 males with a median age of 58 years (range:19-93 years). Precursor B-ALL was diagnosed by immunophenotyping in all cases. All cases were initially analyzed by cytomorphology, cytogenetics based on chromosome banding analysis (CBA) and FISH for exclusion of the above listed recurrent aberrations and retrospectively by molecular screening using TruSight RNA Fusion panel (Illumina, San Diego, CA) covering 507 fusion-associated genes and their expression.

Results: Overall, in 12/85 cases (14%) fusion genes were detected by molecular screening. All fusions were confirmed by specific amplification of the breakpoints using reverse transcription polymerase chain reaction. 5 cases were identified as BCR-ABL1 -like ALL with abnormal ABL pathway activation and comprised 4 cases with previously described ABL-class fusions (ABL1=2, PDGFRB=2) and one JAK2 fusion. ABL1 5' fusion partners were ETV6 and SNX1, PDGFRB 5' fusion partner was EBF1 . JAK2 5' fusion partner was BCR . ETV6-ABL1 could not be detected by CBA as it is cytogenetically cryptic whereas in all other cases the respective chromosomal translocation was initially identified by CBA. ABL1 and PDGFRB fusions are reported to be sensitive to treatment with the multi-tyrosine kinase inhibitors (TKI) imatinib and dasatinib, JAK2 fusions to treatment with the JAK2 inhibitor ruxolitinib.

For the non BCR-ABL1 -like ALL cases, the following fusions were detected: In one case, a CEP110-FGFR1 fusion, which has been previously reported to respond to treatment with dasatinib in 8p11 myeloid neoplasia. The corresponding t(8;9)(p11;q33) chromosomal translocation was identified by CBA.

Additionally one yet undescribed fusion involving the genes NRIP1 located on chromosome 21q and BCL2L1 located on chromosome 20q was detected. The corresponding chromosomal translocation (t(20;21)(q11;q11)) was detected by CBA. Rare cases of NRIP1 fusions have been described in different types of leukemia, including ALL.

Furthermore, in 5 cases, fusion genes involving the zinc-finger protein 384 (ZNF384) were found. 5' fusion partners were the previously described EP300 (n=4) and EWSR1 (n=1). EP300-ZNF384 was not detected by CBA, whereas ZNF384-EWSR1 showed the correlating translocation t(12;22)(p13;q12). EP300-ZNF384 cases are reported to show a good response to prednisone as well as to conventional chemotherapy.

To further classify the remaining 73/85 B-other cases without detectable gene fusion in RNA sequencing analysis, we analyzed CRLF2 expression to identify BCR-ABL1 -like ALL cases with CRLF2 overexpression. 12/73 cases showed elevated CRLF2 expression levels and therefore could be further classified as high-risk B-ALL. In 2/12 cases a t(X;14)(p22;q32) was identified by CBA, which corresponds to the fusion IGH-CRLF2 which is a promoter fusion and therefore not detectable by RNA sequencing. In the remaining 10 cases no corresponding cytogenetic alteration was identified by CBA.

Conclusion: In conclusion, 24/85 B-other ALL cases (28%) could be further genetically and prognostically classified by targeted RNA sequencing. We demonstrate that targeted RNA sequencing 1) improves diagnosis and classification of "B-other" ALL, 2) guides therapeutic decisions allowing targeted treatments and 3) provides the basis for developing sensitive markers for patient specific MRD monitoring. Targeted RNA sequencing therefore may be considered as a standard procedure in a routine diagnostic workup for "B-other" ALL cases.