Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the commonest childhood tumor and remains a leading cause of cancer death in the young. In the last decade, microarray and sequencing analysis of large ALL cohorts has revolutionized our understanding of the genetic basis of this disease. These studies have identified new ALL subtypes, each characterized by constellations of structural and sequence alterations that perturb key cellular pathways, including lymphoid development, cell-cycle regulation, and tumor suppression; cytokine receptor, kinase, and Ras signaling; and chromatin modifications. Several of these pathways, particularly kinase-activating lesions and epigenetic alterations, are logical targets for new precision medicine therapies. Genomic profiling has also identified important interactions between inherited genetic variants that influence the risk of leukemia development and the somatic genetic alterations that are required to establish the leukemic clone. Moreover, sequential sequencing studies at diagnosis, remission, and relapse have provided important insights into the relationship among genetic variants, clonal heterogeneity, and the risk of relapse. Ongoing studies are extending our understanding of coding and noncoding genetic alterations in B-progenitor and T-lineage ALL and using these insights to inform the development of faithful experimental models to test the efficacy of new treatment approaches. (Blood. 2015;125(26):3977-3987)

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“…This is important given the increasing application of nextgeneration sequencing approaches to MRD detection. 85 …”

Section: Epigenetic Alterations In Allmentioning

confidence: 99%

“…Finally, sequencing is likely to have an important role in monitoring response to therapy, by deeply sequencing antigen receptor loci 85 or other targets of genetic alteration, particularly those known to facilitate resistance to therapy (for example, IKZF1, CREBBP, and NT5C2).…”

Section: Prospects For Precision Medicine In Allmentioning

confidence: 99%

Redefining ALL classification: toward detecting high-risk ALL and implementing precision medicine

Hunger

Mullighan

2015

Blood

252

198

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“…In extreme cases, such bias can result in undetectable levels of specific under-amplifying target templates. A bias-free assay is critical for studies aiming to quantitatively measure the frequency of specific immune receptor rearrangements, such as minimal residual disease (MRD) monitoring in leukaemia [9][10][11][12] , following exposure-specific immune repertoires over time 13,14 and research to study basic B-and T-cell biology 15,16 .…”

mentioning

confidence: 99%

Using synthetic templates to design an unbiased multiplex PCR assay

et al. 2013

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T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

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“…Others have previously identified clonal IGH and TCR transcripts from massively parallel sequencing data (5,(26)(27)(28) via targeted or enriched sequencing with ad hoc experiments for this specific purpose, and specifically for the detection of MRD. Early techniques required patient-specific amplification, whereas latter methods have been refined to work with consensus primers; at least one group has described the use of degenerate IGH targeted primers (28).…”

Section: Discussionmentioning

confidence: 99%

Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia

Blachly

Ruppert

Zhao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (>1 × 10 12 ) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition. Cancers arising from mature B cells are characterized by clonal production of Ig heavy (IGH@) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@ V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was outof-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.RNA sequencing | immunoglobulin | somatic hypermutation | B cells | CLL I mmunoglobulins (Igs) are proteins produced by mature B-lymphocytes that recognize foreign antigens, both as soluble antibody molecules and as part of the B-cell receptor. The generation of Ig diversity through gene recombination and hypermutation of the heavy chain (H) variable region (V) is essential to adaptive immunity. The extent of this process is strongly associated with both pathology and prognosis in chronic lymphocytic leukemia (CLL), wherein CLL that expresses an unmutated IGHV tends to be more aggressive than CLL using unmutated IGHV (1, 2). The accurate assessment of this IGHV mutation status is thus of a high clinical priority. As each patient's leukemia generally expresses only a single IGH@, the mutation status of IGHV is determined by amplifying the expressed transcript via RT-PCR, sequencing the gene via the Sanger technique, and then comparing this sequence with known inherited IGHV sequences. However, there are limitations to such methods, including variation in technique across institutions. RNA-sequencing is a powerful technology that can simultaneously yield information about gene and isoform expression as well as underlying DNA sequence (3, 4). Motivated by the notion that a single RNA sequencing experiment could replace many other discrete tests (qPCR, genotyping, microarray, I...

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Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia

Cited by 369 publications

References 42 publications

Redefining ALL classification: toward detecting high-risk ALL and implementing precision medicine

Redefining ALL classification: toward detecting high-risk ALL and implementing precision medicine

Using synthetic templates to design an unbiased multiplex PCR assay

Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia

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