Predictive Value of Next-Generation Sequencing (NGS) Following Tisagenlecleucel in Pediatric and Young Adult B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias, and features different outcomes depending on the age of onset. Improvements in ALL genomic analysis achieved thanks to the implementation of next-generation sequencing (NGS) have led to the recent discovery of several novel molecular entities and to a deeper understanding of the existing ones. The purpose of our review is to report the most recent discoveries obtained by NGS studies for ALL diagnosis, risk stratification, and treatment planning. We also report the first efforts at NGS use for minimal residual disease (MRD) assessment, and early studies on the application of third generation sequencing in cancer research. Lastly, we consider the need for the integration of NGS analyses in clinical practice for genomic patients profiling from the personalized medicine perspective.

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Next Generation Sequencing Identifies Novel Oncogene Alterations In CRLF2-rearranged Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.472.472 ◽

2010 ◽

Vol 116 (21) ◽

pp. 472-472 ◽

Cited By ~ 1

Author(s):

Akinori Yoda ◽

Terry Haley ◽

Oliver B. Weigert ◽

Jeffery Kutok ◽

Adalis Maisonet ◽

...

Keyword(s):

Next Generation Sequencing ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Exome Sequencing ◽

Rna Editing ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Next Generation ◽

High Confidence ◽

Generation Sequencing

Abstract Abstract 472 We and others recently identified locus rearrangements involving the type I cytokine receptor subunit CRLF2 (also known as thymic stromal lymphopoietin receptor (TSLPR)) in 5–7% of all adult and pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a CRLF2-IGH translocation. Prognosis for CRLF2-rearranged B-ALL is particularly poor, suggesting that a sizable fraction of relapsed and ultimately fatal B-ALL harbor CRLF2 rearrangements. Approximately 50% of CRLF2-rearranged B-ALL harbor mutations in JAK2 (and rarely JAK1) that cluster around JAK2 Arg683. A separate 15% have a CRLF2 F232C mutation that promotes constitutive homodimerization and signaling. In the remaining cases, the driver of CRLF2 signaling is not known. To identify additional genetic alterations that contribute to CRLF2-mediated leukemogenesis, we performed next-generation sequencing on 3 CRLF2-rearranged B-ALL specimens, including one with CRLF2 F232C (#536). Exome libraries were assembled from bone marrow specimens with greater than 90% B-ALL involvement and from paired remission bone marrows. Transcriptome sequencing was also performed on cDNA isolated from the involved marrow of patient #536 to: 1) confirm the findings from exome sequencing, 2) focus on transcribed genes and 3) identify possible RNA editing events. Sequencing utilized the SOLiD (Sequencing by Oligonucleotide Ligation and Detection; Applied BioSystems) platform, with a target recovery of greater than 40 million (transcriptome) and 25 million (exome) uniquely mapping reads per sample. Mutations were considered high confidence if a minimum of 3 individual reads identified the same mutation within the involved specimen and the mutation was not identified in any reads from the remission specimen (minimum 10 reads at that base-pair). Paired exome sequencing of germline and involved marrow specimens identified 129, 297 and 630 high-confidence, non-synonymous, somatic mutations in the three samples. Of the total 1056 mutations, 1023 (96.9%) were missense and the remaining 33 (3.1%) were nonsense mutations. The expected mutation in CRLF2 F232C (#536) was recovered, as was a novel JAK2 mutation from a sample previously thought to be JAK2 wild-type (#002). Comparison between transcriptome and exome sequence markedly reduced the number of potential “driver” alterations, i.e., nonsynonymous coding mutations expressed in the tumor but not present in the remission exome. For example, from the 129 somatic alterations in the involved exome from #536, only 15 were recovered from the involved transcriptome. Sequence alterations affected genes known to be involved in histone modification, oxygen metabolism, and steroid responsiveness. To our knowledge, none of the identified mutations have previously been described. One mutation in a gene involved in E2F transcription was observed in two of three cases. In addition, 2,428 coding sequence mutations were identified from the involved marrow transcriptome of #536 that were not present in the involved marrow exome, possibly indicating a high rate of RNA editing. Additional sequence analysis and molecular epidemiology of the novel sequence alterations in CRLF2-rearranged and —unrearranged B-ALL is underway. Disclosures: No relevant conflicts of interest to declare.

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Exome Sequencing Identifies Highly Recurrent Somatic GATA2 and CEBPA Mutations in Acute Erythroid Leukemia

Blood ◽

10.1182/blood.v126.23.1394.1394 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1394-1394

Author(s):

Suning Chen ◽

Nana Ping ◽

Jia Yin ◽

Wenxiu Cheng ◽

Qinrong Wang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Acute Lymphoblastic Leukemia ◽

Exome Sequencing ◽

Myeloid Leukemia ◽

Transcriptional Activity ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Next Generation ◽

Cell Acute Lymphoblastic Leukemia ◽

Generation Sequencing

Abstract Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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