Next-Generation Sequencing for Myeloid Malignancies · Next-generation sequencing (NGS) technology allows evaluation of numerous abnormalities/variants in one analysis. This is of

Newsletter

Next-Generation Sequencing for Myeloid Malignancies

Van Rensburg

IntroductionThere is increasing awareness of the genetic complexity of malignancy. Haematological neoplasms are no exception. The assessment of genetic lesions has become critical for the correct diagnosis and selection of therapy, as well as more accurate prognostication. Multiple different mutations can be present in a tumour and these can have a modifying effect on one another.

Next-generation sequencing (NGS) technology allows evaluation of numerous abnormalities/variants in one analysis. This is of particular value in the setting of myeloid malignancies as many of the conditions can have overlapping clinical and morphological features, and genetic analysis is frequently critical for classification, potentially requiring extensive genetic testing.

The Molecular Pathology Department at Lancet Laboratories has performed myeloid NGS for over two years, gaining considerable experience with the technique in a range of haematological conditions.

Why use myeloid NGS rather than existing genetic investigations? The diagnosis of many haematological malignancies requires not only confirmation of

the presence of certain abnormalities but exclusion of other mutations. An array of traditional tests (e.g. individual PCR and FISH investigations) may be necessary to make the diagnosis. NGS allows for these to be replaced with a single assay leading to an overall reduction in cost.

Certain abnormalities cannot be currently tested as stand-alone investigations in the South African market, so the panel allows for local testing and comprehensive coverage of myeloid variants.

Compiled by Dr N Holland st 1 Quarter 2020

The actual diagnosis may not have originally been considered by the involved clinician or pathologist due to atypical presentation. Myeloid NGS testing is non-directed and thus facilitates accurate diagnosis.

Myeloid NGS provides a more sensitive assessment of clonality. Haematological disorders such as Myelodysplastic Syndromes require differentiation from reactive conditions which can cause low blood counts and dysplastic change. In 80% � 90% of cases, clonality can be confirmed on NGS, in contrast to the 40% � 50% yield with previous methodologies.

Myeloid NGS does not currently replace cytogenetic analysis. However cytogenetics detects only gross structural chromosomal abnormalities and can only be performed on very fresh samples. It frequently fails due to insufficient material or due to the absence of dividing cells.

Myeloid NGS can detect translocations which may be cryptic on cytogenetic analysis and can define novel translocations involving different gene partners that would be missed on directed FISH studies.

Which conditions are tested for in the myeloid panel performed at Lancet Laboratories?The panel of mutations investigated includes comprehensive assessment of the well described recurrent genetic abnormalities seen in Acute Myeloid Leukaemia (AML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndromes (MDS) and overlap syndromes such as Chronic Myelomonocytic Leukaemia, as well as the condition-defining abnormalities seen, for example, in myeloid/lymphoid neoplasms with eosinophilia and gene rearrangement.

The internationally standardised Oncomine� Myeloid Assay (with sensitivity down to 5%) is utilised to ensure quality results. Please see Table 1 for the full list of variants included in the panel.

NGS can also be useful in the evaluation of unexplained cytopaenias or in certain haematological abnormalities which have a germline predisposition.

HOTSPOT FULL GENE FUSION EXPRESSION

ABL1

BRAF

CBL

CSF3R

DNMT3A

FLT3

GATA2

HRAS

IDH1

IDH2

JAK2

KIT

KRAS

MPL

MYD88

NPM1

NRAS

PTPN11

SETBP1

SF3B1

SRSF2

U2AF1

WT1

WT1

ASXL1

BCOR

CALR

CEBPA

ETV6

EZH2

IKZF1

NF1

PHF6

PRPF8

RB1

RUNX1

SH2B3

STAG2

TET2

TP53

ZRSR2

ABL1 BAALC

ALK MECOM

BCL2 MYC

BRAF SMC1A

CCND1 WT1

CREBBP

ETV6

EGFR

FGFR1

FGFR2

FUS

HMGA2

JAK2

KMT2A (MILL)

MECOM

MET

MLLT10

MLLT3

MYBL1

MYH11

NTRK3

NUP214

PDGFRA

PDGFRB

RARA

RBM15

RUNX1

TCF3

TFE3

Table 1. Variants included in the Oncomine� Myeloid Assay

Dys

pla

sia

or

inef

fect

ive

haem

atro

po

iese

s

Increased proliferation

Normal

Loss of diffl

erentiatio

n

MDS MDP/MPN

02 AML

MPN

AML

Myeloid Disorder Development

Practical pointsWhich clinicians request myeloid NGS?The test is frequently requested by the treating clinical haematologist or oncologist. In certain cases, the haematological pathologist will recommend testing to the treating physician based on the patient�s bone marrow or peripheral blood findings. This could be a newly diagnosed or a suspected haematological malignancy in a patient who has not as yet been transferred to a super-specialist.

What conditions would benefit from Myeloid NGS testing?The analysis is of benefit in the setting of clonal myeloid disorders such as Acute Myeloid Leukaemia, Myeloproliferative Neoplasms, Myelodysplastic Syndromes and Myelodysplastic/Myeloproliferative Neoplasms (e.g. Chronic Myelomonocytic Leukaemia). Repeat testing may be necessary if the haematological malignancy shows findings suggestive of disease progression/evolution. Testing may also be requested in the context of unexplained low or high blood counts to investigate clonality.

When is myeloid NGS performed?The test will most frequently be performed at the time of the initial diagnostic bone marrow investigation. If disease progression or relapse is suspected, the analysis may be repeated to reassess the clonal signature. In certain cases, the analysis will be used for evaluation of residual disease, however the current assay (with a sensitivity of 5%) is not suitable for minimal residual disease analysis.

What samples are required?The test is performed on a peripheral blood or bone marrow aspirate sample drawn in an EDTA tube. If it is uncertain whether the investigation is necessary, a myeloid extract can be performed and the sample stored for up to a month. Processing can be done within that period if the analysis is required.

Where is the test done and how is it requested?The specimens are processed by the Lancet Laboratories Molecular Pathology Department at Richmond, Johannesburg. The test can be requested on a bone marrow or peripheral blood requisition form as Myeloid NGS. If there are any queries about the test, please discuss with the haematologist on call (via Lancet Switchboard � 011 358 0800) or the Molecular Pathology Department (via [email protected]).

AML with mutated NPM1 and FLT3ITD (cytogenetically normal)

What is the turnaround time on the test?TAT: 10 to 14 days

What is the report format?The report describes the gene variants and fusions detected and discusses the diagnostic and prognostic significance of the abnormalities in relation to the provisional diagnosis. In addition, a list of the clinical trials and relevant FDA, NCCN, EMA and ESMO recommended therapies (in the setting of a particular mutation) are supplied. This is the Oncomine Knowledgebase Reporter (OKR) report. The reports are available on the Lancet Pathportal and Oculus, and, in the case of bone marrow samples, as a consolidated diagnostic report.

ConclusionThere is increasing emphasis on precision medicine. As our understanding of the complexity of the genetic landscape of cancer increases, the requirement to map the pattern in individual malignancies becomes apparent. The data informs diagnostic, prognostic and therapeutic decision making. Myeloid next-generation sequencing allows extensive analysis of the make-up of myeloid malignancies such as AML, MDS and MPN. As full assessment of malignancy increasingly requires evaluation of multiple genes, NGS provides a comprehensively informative, cost-effective and time-saving approach.

References1. Yang F & Press RD. Next-Generation Sequencing multi-gene mutation panels in

myeloid malignancies. The Hematologist 2016; 13(3): 6 � 7. 2. Valent P, et al. Proposed minimal diagnostic criteria for myelodysplastic syndromes

(MDS) and potential pre-MDS conditions. Oncotarget 2017; 8(43): 73483 � 73500. 3. Grinfeld J, et al. Classification and personalized prognosis in Myeloproliferative

Neoplasms. N Engl J Med 2018; 379(15): 1416 � 1430. 4. Gotlib J. How I treat atypical chronic myeloid leukemia. Blood 2017; 129(7): 838 �

845. 5. Solary E & Itzykson R. How I treat chronic myelomonocytic leukemia. Blood 2017;

130(2): 126 � 136.6. Leisch M, et al. Next-Generation Sequencing in AML � on the way to becoming a

new treatment standard for treatment initiation and/or modulation? Cancers (Basel) 2019; 11(2): pii. E252.

7. Bacher U, et al. Challenges in the introduction of next-generation sequencing (NGS) for diagnostics of myeloid malignancies into routine clinical use. Blood Cancer J 2018; 8(11): 113.

8. Kim B, et al. Targeted next generation sequencing can serve as an alternative to conventional tests in myeloid neoplasms. PLoS One 2019; 14(3): e0212228.

9. Swerdlow SH, et al. WHO classification of tumours of haematopoietic and lymphoid tissues, revised fourth edition. Lyon, France: IARC; 2017.

10. Deininger MWN et al. Turning the tide in myelodysplastic/myeloproliferative neoplasms. Nat Rev Cancer 2017; 17(7): 425-440.

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