Rascall: Rapid (Ra) screening (Sc) of RNA-seq data for prognostically significant genomic alterations in acute lymphoblastic leukaemia (ALL)

RNA-sequencing (RNA-seq) efforts in acute lymphoblastic leukaemia (ALL) have identified numerous prognostically significant genomic alterations which can guide diagnostic risk stratification and treatment choices when detected early. However, integrating RNA-seq in a clinical setting requires rapid detection and accurate reporting of clinically relevant alterations. Here we present RaScALL, an implementation of the k-mer based variant detection tool km, capable of identifying more than 100 prognostically significant lesions observed in ALL, including gene fusions, single nucleotide variants and focal gene deletions. We compared genomic alterations detected by RaScALL and those reported by alignment-based de novo variant detection tools in a study cohort of 180 Australian patient samples. Results were validated using 100 patient samples from a published North American cohort. RaScALL demonstrated a high degree of accuracy for reporting subtype defining genomic alterations. Gene fusions, including difficult to detect fusions involving EPOR and DUX4, were accurately identified in 98% of reported cases in the study cohort (n = 164) and 95% of samples (n = 63) in the validation cohort. Pathogenic sequence variants were correctly identified in 75% of tested samples, including all cases involving subtype defining variants PAX5 p.P80R (n = 12) and IKZF1 p.N159Y (n = 4). Intragenic IKZF1 deletions resulting in aberrant transcript isoforms were also detectable with 98% accuracy. Importantly, the median analysis time for detection of all targeted alterations averaged 22 minutes per sample, significantly shorter than standard alignment-based approaches. The application of RaScALL enables rapid identification and reporting of previously identified genomic alterations of known clinical relevance.Jacqueline Rehn, Chelsea Mayoh, Susan L Heatley, Barbara J McClure, Laura N Eadie, Caitlin Schutz, David T Yeung, Mark J Cowley, James Breen, Deborah L Whit

Case Report: Precision Medicine Target Revealed by In Vitro Modeling of Relapsed, Refractory Acute Lymphoblastic Leukemia From a Child With Neurofibromatosis

Children with neurofibromatosis have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop B-cell acute lymphoblastic leukemia (B-ALL). Through in-vitro modeling, a novel NF1 p.L2467 frameshift (fs) mutation identified in a relapsed/refractory Ph-like B-ALL patient with neurofibromatosis demonstrated cytokine independence and increased RAS signaling, indicative of leukemic transformation. Furthermore, these cells were sensitive to the MEK inhibitors trametinib and mirdametinib. Bi-allelic NF1 loss of function may be a contributing factor to relapse and with sensitivity to MEK inhibitors, suggests a novel precision medicine target in the setting of neurofibromatosis patients with B-ALL.Susan L. Heatley, Elyse C. Page, Laura N. Eadie, Barbara J. McClure, Jacqueline Rehn, David T. Yeung, Michael Osborn, Tamas Revesz, Maria Kirby, and Deborah L. Whit

Case Report: Rare IKZF1 Gene Fusions Identified in Neonate with Congenital KMT2A-Rearranged Acute Lymphoblastic Leukemia

Published: 19 January 2023Chromosomal rearrangements involving the KMT2A gene occur frequently in acute lymphoblastic leukaemia (ALL). KMT2A-rearranged ALL (KMT2Ar ALL) has poor long-term survival rates and is the most common ALL subtype in infants less than 1 year of age. KMT2Ar ALL frequently occurs with additional chromosomal abnormalities including disruption of the IKZF1 gene, usually by exon deletion. Typically, KMT2Ar ALL in infants is accompanied by a limited number of cooperative le-sions. Here we report a case of aggressive infant KMT2Ar ALL harbouring additional rare IKZF1 gene fusions. Comprehensive genomic and transcriptomic analyses were performed on sequential samples. This report highlights the genomic complexity of this particular disease and describes the novel gene fusions IKZF1::TUT1 and KDM2A::IKZF1.Laura N. Eadie, Jacqueline A. Rehn, James Breen, Michael P. Osborn, Sophie Jessop, Charlotte E. J. Downes, Susan L. Heatley, Barbara J. McClure, David T. Yeung, Tamas Revesz, Benjamin Saxon and Deborah L. Whit

KMT2A rearranged acute lymphoblastic leukaemia: unravelling the genomic complexity and heterogeneity of this high-risk disease

KMT2A rearranged (KMT2Ar) acute lymphoblastic leukaemia (ALL) is a high-risk genomic subtype, with long-term survival rates of less than 60% across all age groups. These cases present a complex clinical challenge, with a high incidence in infants, high-risk clinical features and propensity for aggressive relapse. KMT2A rearrangements are highly pathogenic leukaemic drivers, reflected by the high incidence of KMT2Ar ALL in infants, who carry few leukaemia-associated cooperative mutations. However, transgenic murine models of KMT2Ar ALL typically exhibit long latency and mature or mixed phenotype, and fail to recapitulate the aggressive disease observed clinically. Next-generation sequencing has revealed that KMT2Ar ALL also occurs in adolescents and adults, and potentially cooperative genomic lesions such as PI3K-RAS pathway variants are present in KMT2Ar patients of all ages. This review addresses the aetiology of KMT2Ar ALL, with a focus on the cell of origin and mutational landscape, and how genomic profiling of KMT2Ar ALL patients in the era of next-generation sequencing demonstrates that KMT2Ar ALL is a complex heterogenous disease. Ultimately, understanding the underlying biology of KMT2Ar ALL will be important in improving long-term outcomes for these high-risk patients.Michelle O.Forgione, Barbara J.McClure, Laura N.Eadie, David T.Yeung, Deborah L.Whit

MLLT10 rearranged acute leukemia: incidence, prognosis and possible therapeutic strategies

Rearrangements of the MLLT10 gene occur in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), most commonly T-lineage ALL (T-ALL), in patients of all ages. MLLT10 rearranged (MLLT10r) acute leukemia presents a complex diagnostic and therapeutic challenge due to frequent presentation of immature or mixed phenotype, and a lack of consensus regarding optimal therapy. Cases of MLLT10r AML or T-ALL bearing immature phenotype are at high risk of poor outcome, but the underlying molecular mechanisms and sensitivity to targeted therapies remain poorly characterized. This review addresses the incidence and prognostic significance of MLLT10r in acute leukemia, and how the aberrant gene expression profile of this disease can inform potential targeted therapeutic strategies. Understanding the underlying genomics of MLLT10r acute leukemia, both clinically and molecularly, will improve prognostic stratification and accelerate the development of targeted therapeutic strategies, to improve patient outcomes.Michelle O. Forgione, Barbara J. McClure, David T. Yeung, Laura N. Eadie, Deborah L. Whit

In-vitro modeling of TKI resistance in the high-risk B-cell acute lymphoblastic leukemia fusion gene RANBP2-ABL1 - implications for targeted therapy

.Acute lymphoblastic leukemia remains a leading cause of cancer-related death in children. Furthermore, subtypes such as Ph-like ALL remain at high-risk of relapse, and treatment resistance remains a significant clinical issue. The patient-derived Ph-like ALL RANBP2-ABL1 fusion gene was transduced into Ba/F3 cells and allowed to become resistant to the tyrosine kinase inhibitors (TKIs) imatinib or dasatinib, followed by secondary resistance to ponatinib. RANBP2-ABL1 Ba/F3 cells developed the clinically relevant ABL1 p.T315I mutation and upon secondary resistance to ponatinib, developed compound mutations, including a novel ABL1 p.L302H mutation. Significantly, compound mutations were targetable with a combination of asciminib and ponatinib. In-vitro modeling of Ph-like ALL RANBP2-ABL1 has identified kinase domain mutations in response to TKI treatment, that may have important clinical ramifications. Early detection of mutations is paramount to guide treatment strategies and improve survival in this high-risk group of patients.Susan L. Heatley, Kartini Asari, Caitlin E. Schutz, Tamara M. Leclercq, Barbara J. McClure, Laura N. Eadie , Timothy P. Hughes, David T. Yeung and Deborah L. Whit

HMGN1 plays a significant role in CRLF2 driven Down Syndrome leukemia and provides a potential therapeutic target in this high-risk cohort

Published online: 2 December 2021The genetic basis of the predisposition for Down Syndrome (DS) patients to develop cytokine receptor-like factor 2 rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) is currently unknown. Genes located on chromosome 21 and expressed in hematopoietic cells are likely candidates for investigation of CRLF2r DS-ALL pathogenesis. We explored the high-mobility group nucleosome-binding protein 1 (HMGN1), located in the DS critical region, in an inducible CRISPR/Cas9 knockout (KO) xenograft model to assess the effect of HMGN1 loss of function on the leukemic burden. We demonstrated HMGN1 KO-mitigated leukemic phenotypes including hepatosplenomegaly, thrombocytopenia, and anemia, commonly observed in leukemia patients, and significantly increased survival in vivo. HMGN1 overexpression in murine stem cells and Ba/F3 cells in vitro, in combination with P2RY8-CRLF2, resulted in cytokine-independent transformation and upregulation of cell signaling pathways associated with leukemic development. Finally, in vitro screening demonstrated successful targeting of P2RY8-CRLF2 and HMGN1 co-expressing cell lines and patient samples with fedratinib (JAK2 inhibitor), and GSK-J4 (demethylase inhibitor) in combination. Together, these data provide critical insight into the development and persistence of CRLF2r DS-ALL and identify HMGN1 as a potential therapeutic target to improve outcomes and reduce toxicity in this high-risk cohort of young patients.Elyse C. Page, Susan L. Heatley, Laura N. Eadie, Barbara J. McClure, Charles E. de Bock, Sofia Omari ... et al