Page 188 - Williams Hematology ( PDFDrive )

162 Part IV: Molecular and Cellular Hematology Chapter 11: Genomics 163

NEXT-GENERATION SEQUENCING AS A also may reveal the clonal architecture of persistent disease from the
CLINICAL ASSAY: IMPLICATIONS FOR THE mutations detected. Finally, clinicians could use knowledge gained by
sequencing an individual’s genome to determine the choice of therapy,
PRACTICING HEMATOLOGIST whether for a malignant or non-malignant hematologic disease. In one
Using NGS as a clinical assay platform offers many opportunities for such example, a therapy choice could be optimized based on pharma-
new clinical tests and potential therapeutic interventions. Clinical cogenomic studies in which the response or toxicity of a given drug is
64
sequencing requires the same high standards regarding sample bank- associated with underlying inherited genetic variation in the patient.
ing, nucleic acid isolation and proper informed consent. Moreover, In a second example, a genomic assay might identify a somatic altera-
clinical sequencing must be done in an appropriately certified clinical tion corresponding to a targeted therapy that might help a patient with
laboratory environment. As the depth of coverage increases for NGS- residual MRD to achieve remission. These examples are illustrative of
based platforms, the statistical power to detect variation will increase the translational potential of NGS from research tool into clinical care.
up until the point that it is outweighed by the intrinsic error associated
with the sequencing platform. For clinical NGS-based diagnostic tests, REFERENCES
these error metrics must be predetermined for each protocol, whether
it be whole-genome sequencing, a specific exome reagent, or a specific 1. International Human Genome Sequencing Consortium: Finishing the euchromatic
gene panel test. sequence of the human genome. Nature 431(7011):931–945, 2004.
NGS-based diagnostics have specific potential applications for 2. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci U S A 74(12):5463–5467, 1977.
both hematologic malignancies and nonmalignant hematologic condi- 3. Prober JM, Trainor GL, Dam RJ, et al: A system for rapid DNA sequencing with fluo-
tions. For leukemias, lymphomas, myelodysplastic syndromes (MDS), rescent chain-terminating dideoxynucleotides. Science 238(4825):336–341, 1987.
myeloproliferative neoplasms and other hematologic malignancies 4. Smith LM, Sanders JZ, Kaiser RJ, et al: Fluorescence detection in automated DNA
sequence analysis. Nature 321(6071):674–679, 1986.
(or premalignant conditions), the use of clinical sequencing can be 5. Tabor S, Huber HE, Richardson CC: Escherichia coli thioredoxin confers processivity
employed to determine the spectrum of mutations that are driving the on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem
particular malignancy. For any given tumor, comprehensive sequencing 262(33):16212–16223, 1987.
may identify an “actionable” mutation that could lead to the use of a 6. Huber HE, Tabor S, Richardson CC: Escherichia coli thioredoxin stabilizes complexes
of bacteriophage T7 DNA polymerase and primed templates. J Biol Chem 262(33):
targeted therapy. 16224–16232, 1987.
In the past, a clinician may have ordered a single gene test to deter- 7. Heller C: Principles of DNA separation with capillary electrophoresis. Electrophoresis
22(4):629–643, 2001.
mine if a particular molecular abnormality were present in a tumor 8. Metzker ML: Sequencing technologies-the next generation. Nat Rev Genet 11(1):31–46,
sample, such as testing for FLT3 internal tandem duplications (FLT3- 2010.
ITD) in patients with acute myeloid leukemia (AML). The presence of 9. Mardis ER: Next-generation sequencing platforms. Annu Rev Anal Chem (Palo Alto
Calif) 6:287–303, 2013.
such an alteration has prognostic implications and may have therapeu- 10. Mardis ER: A decade’s perspective on DNA sequencing technology. Nature 470
tic significance pending the results of ongoing clinical studies with FLT3 (7333):198–203, 2011.
inhibitors. A single-gene or single “hot spot” assay that is designed to 11. Bentley DR, Balasubramanian S, Swerdlow HP, et al: Accurate whole human genome
detect a specific alteration has several limitations that are now leading to sequencing using reversible terminator chemistry. Nature 456(7218):53–59, 2008.
wider use of NGS-based approaches to clinical diagnostics. To continue 12. Dohm JC, Lottaz C, Borodina T, Himmelbauer H: Substantial biases in ultra-short read
data sets from high-throughput DNA sequencing. Nucleic Acids Res 36(16):e105, 2008.
using FLT3-ITD alterations in AML as an example, the use of a more 13. Flicek, P, Birney E: Sense from sequence reads: Methods for alignment and assembly.
comprehensive sequencing platform could be used to discern the sub- Nat Methods 6(11 Suppl):S6-S12, 2009.
clonal architecture of the cancer tissue. If the FLT3-ITD mutation was 14. Fullwood MJ, Wei CL, Liu ET, Ruan Y: Next-generation DNA sequencing of paired-end
tags (PET) for transcriptome and genome analyses. Genome Res 2009;19(4):521–532,
present only in a subclone but not in the founding clone, one would pre- 2008.
dict that a FLT3 inhibitor would only be active in eradicating the sub- 15. Rothberg JM, Hinz W, Rearick TM, et al: An integrated semiconductor device enabling
clone. Therefore, clinicians would need to incorporate another therapy non-optical genome sequencing. Nature 475(7356):348–352, 2011.
to eradicate the founding clone, so as to achieve remission or prevent 16. Bragg LM, Stone G, Butler MK, et al: Shining a light on dark sequencing: Characterising
errors in Ion Torrent PGM data. PLoS Comput Biol 9(4):e1003031, 2013.
disease relapse. Ideally, the choice of this therapy would be determined 17. Eid J, Fehr A, Gray J, et al: Real-time DNA sequencing from single polymerase mole-
based on the other mutations identified in the founding clone. cules. Science 323(5910):133–138, 2009.
As clinicians better understand the mutational drivers of any par- 18. Ross MG, Russ C, Costello M, et al: Characterizing and measuring bias in sequence
data. Genome Biol 14(5):R51, 2013.
ticular tumor, they may be able to use targeted therapies directed at par- 19. Branton D, Deamer DW, Marziali A, et al: The potential and challenges of nanopore
ticular pathways rather than individual gene mutations. For example, sequencing. Nat Biotechnol 26(10):1146–1153, 2008.
researchers may be able to develop a drug that is effective if a patient 20. Wheeler DA, Srinivasan M, Egholm M, et al: The complete genome of an individual by
massively parallel DNA sequencing. Nature 452(7189):872–876, 2008.
with AML or MDS harbors a mutation in any of the genes involved 21. Ley TJ, Mardis ER, Ding L, et al: DNA sequencing of a cytogenetically normal acute
in the spliceosome complex or associated proteins. A similar scenario myeloid leukaemia genome. Nature 456(7218):66–72, 2008.
could be envisioned targeting mutations in genes in the cohesin com- 22. Ng SB, Turner EH, Robertson PD, et al: Targeted capture and massively parallel
sequencing of 12 human exomes. Nature 461(7261):272–276, 2009.
plex (protein complex that regulates the separation of sister chromatids 23. Mamanova L, Coffey AJ, Scott CE, et al: Target-enrichment strategies for next-genera-
in cell division) or genes that alter the hydroxymethylation of cytosine tion sequencing. Nat Methods 7(2):111–118, 2010.
in DNA. Clinical trials built around these concepts will be necessary 24. Altmuller J, Budde BS, Nurnberg P: Enrichment of target sequences for next-generation
sequencing applications in research and diagnostics. Biol Chem 395(2):231–237, 2014.
to establish their validity. Additionally, comprehensive sequencing can 25. Koboldt DC, Steinberg KM, Larson DE, et al: The next-generation sequencing revolu-
identify somatic mutations that result in the formation of neo-antigens tion and its impact on genomics. Cell 155(1):27–38, 2013.
expressed on the tumor cells. Clinicians could then use tumor-specific 26. Alkan C, Coe BP, Eichler EE: Genome structural variation discovery and genotyping.
immunotherapy to target the malignancy. 59,60 Nat Rev Genet 12(5):363–376, 2011.
Researchers are using NGS-based technology to detect minimal 27. Koboldt DC, Larson DE, Chen K, et al: Massively parallel sequencing approaches for
characterization of structural variation. Methods Mol Biol 838:369–384, 2012.
residual disease (MRD) in hematologic malignancies. 61–63 An advan- 28. Mitelman F, Johansson B, Mertens F: The impact of translocations and gene fusions on
tage of NGS-based methods of MRD detection is that the data not only cancer causation. Nat Rev Cancer 7(4):233–245, 2007.
provide information regarding the presence or absence of MRD but 29. Landau DA, Carter SL, Getz G, Wu CJ: Clonal evolution in hematological malignancies
and therapeutic implications. Leukemia 28(1):34–43, 2014.

Kaushansky_chapter 11_p0155-0164.indd 163 9/18/15 11:48 PM