Massive Parallel Sequencing and Mutations Analysis of Mismatch Repair Genes in Recurrent Childhood Acute Lymphoblastic Leukemia Cases-Identification of Possible Molecular Signature of Poor Clinical Outcome

[Introduction] Mechanism of recurrence is fully characterized and is significant to improve prognosis in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and T lymphoblastic leukemia (T-ALL).. We have analyzed recurrent cases of childhood BCP-ALL and T-ALL. [Procedure] We analyzed clinical samples of 18 cases with BCP-ALL and 3 cases with T-ALL, who had been treated in our institution. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B, JAKs, IL7RA, and TP53 using RT-PCR and MLPA methods and IGH/TCR on genomic PCR. Whole exome analysis was done using Ion AmpliSeq Exome in seven cases. [Result] We analyzed clinical samples of 21 cases with ALL, who had been treated in our institution. The cases were 18 cases with BCP-ALL and 3 cases with T-ALL. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B,JAKs,IL7RA, NOTCH (for T-ALL), and TP53. Whole exome analysis was done in two cases, particularly for a case from which we have established cell line. < Result > Eight BCP-ALL cases with P2RY8-CRLF2 chimeric transcript: four cases of which had P2RY8-CRLF2 chimeric transcript only at initial diagnosis; two cases obtained transcript at recurrence. This was confirmed employing LD-PCR genomic analysis. Ten BCP-ALL cases had deletion of IKZF1; two showed deletion at relapse and a case demonstrated deletion at diagnosis only. Two BCP-ALL cases showed mutation of IL7RA. One of T-ALL cases from which we have established cell line showed mutation of MSH2 and more than 200 non-synonymous mutations on whole exome analysis. One T-ALL cases showed mutation of JAK3 at diagnosis. One BCP-ALL case had MLH1 mutation < Discussion > The present study has suggested acquisition of complex genetic change at different point of evolution may work in recurrence of ALL. The present study has indicated P2RY8-CRLF2 works not as simple growth advantage but rather as manifestation of genomic instability. This may be also illustrated by recurrent cases with T-ALL having mutation of MSH2 and BCP-ALL having mutation of MLH1.

Mutation of mismatch repair gene may be driver of gene mutation acquisition and consequently alterations of CREBBP and RAS or IKZF1, TP53, mismatch repair genes, or emergence of P2RY-CRLF2 chimeric transcript may be prognostically relevant in childhood acute lymphoblastic leukemia.