Hematologic Neoplasm Next Generation Sequencing Panel

Technical Brief:

Hematologic Neoplasm Next-Generation Sequencing Panel


Test Name

Hematologic Neoplasm Next Generation Sequencing Panel – 63 genes
Bone Marrow: HNMNGS   Peripheral Blood: HNPNGS

Subpanels

Myeloid Panel – 50 genes
Bone Marrow: MYNGSM   Peripheral Blood: MYNGSP

Acute Lymphoblastic Leukemia (ALL) Panel – 26 genes
Bone Marrow: ALLBM   Peripheral Blood: ALLPB

Chronic Lymphoproliferative Disorders (LPD) Panel – 7 genes
Bone Marrow: LPMNGS   Peripheral Blood: LPPNGS

Myeloproliferative Neoplasms Panel – 3 genes
Bone Marrow: MPNM   Peripheral Blood: MPNP

CPT Codes

81455

Methodology

Next-Generation Sequencing

Turnaround Time

10 days (upon specimen receipt)

Specimen Requirements

Type:
Bone marrow aspirate

Volume:
2 mL

Type:
Peripheral blood

Volume:
4 mL

Do not freeze specimens.

Stability

Ambient:
48 hours

Refrigerated:
7 days

Frozen:
Unacceptable

Background Information

Recurrent mutations are found in numerous hematologic neoplasms including myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, acute lymphoblastic leukemia, and selected mature lymphoid leukemias.[1-4] The identification of such mutations provides pathologists and clinicians with useful data that may assist in the diagnosis, classification, prognostic evaluation, and therapeutic management of these malignancies. Mutational data in these disorders has been incorporated into the current diagnostic criteria of the World Health Organization Classification of Hematopoietic and Lymphoid Tissues, and into practice guidelines from the National Comprehensive Cancer Network.[5,6]

Cleveland Clinic Laboratories offers a next-generation sequencing panel that analyzes the clinically relevant regions of 62 genes known to be mutated in hematologic neoplasms. This test, performed on peripheral blood or bone marrow aspirate, identifies single nucleotide variants, insertions, and deletions in the targeted genes. Whole-genome copy number analysis may also be obtained by concurrently ordering Cancer Chromosome Microarray + SNP testing.

Smaller subpanels are available for focused disease testing.

Refer to the CC-SIGN® Hematologic Neoplasm Next Generation Sequencing Panel overview for more information.

Panel Highlights & Available Subpanels

Hematologic Neoplasm Next Generation Sequencing Panel – 63 genes

Analyzes the clinically relevant regions of 63 genes known to be mutated in hematologic neoplasms.

  • ABL1
  • ASXL1
  • BCOR
  • BCORL1
  • BRAF
  • CALR
  • CBL
  • CDKN2A
  • CEBPA
  • CSF3R
  • CUX1
  • DDX41
  • DNMT3A
  • EED
  • ETNK1
  • ETV6
  • EZH2
  • FBXW7
  • FLT3
  • GATA1
  • GATA2
  • GNAS
  • IDH1
  • IDH2
  • IKZF1
  • JAK2
  • JAK3
  • KDM6A
  • KIT
  • KMT2A
  • KRAS
  • LUC7L2 (C7orf55)
  • MPL
  • MYD88
  • NF1
  • NOTCH1
  • NPM1
  • NRAS
  • PAX5
  • PHF6
  • PIGA
  • PPM1D
  • PRPF8
  • PTEN
  • PTPN11
  • RAD21
  • RIT1
  • RUNX1
  • SETBP1
  • SF3B1
  • SH2B3
  • SMC1A
  • SMC3
  • SRSF2
  • STAG2
  • STAT3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • U2AF1
  • WT1
  • ZRSR2

Subpanel: Myeloid Neoplasm Next Generation Sequencing Panel – 50 genes

Examines 50 genes mutated in myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia.  This panel includes all 34 genes recommended by the Association for Molecular Pathology for analysis of chronic myeloid neoplasms.[7]

  • ABL1
  • ASXL1
  • BCOR
  • BCORL1
  • CALR
  • CBL
  • CEBPA
  • CSF3R
  • CUX1
  • DDX41
  • DNMT3A
  • EED
  • ETNK1
  • ETV6
  • EZH2
  • FLT3
  • GATA1
  • GATA2
  • IDH1
  • IDH2
  • JAK2
  • KIT
  • KMT2A
  • KRAS
  • MPL
  • NF1
  • NPM1
  • NRAS
  • PHF6
  • PIGA
  • PPMID
  • PTEN
  • PTPN11
  • RAD2
  • RUNX1
  • SETBP1
  • SF3B1
  • SH2B3
  • SMC1A
  • SMC3
  • SRSF2
  • STAG2
  • STAT3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • U2AF1
  • WT1
  • ZRSR2

Subpanel: Acute Lymphoblastic Leukemia Panel – 26 genes

Includes 26 genes recurrently mutated in lymphoblastic leukemias.

  • ABL1
  • CBL
  • CDKN2A
  • EED
  • ETV6
  • EZH2
  • FBXW7
  • FLT3
  • IKZF1
  • JAK2
  • JAK3
  • KDM6A
  • KMT2A
  • KRAS
  • NOTCH1
  • NRAS
  • PAX5
  • PHF6
  • PTEN
  • RUNX1
  • SH2B3
  • STAT5B
  • SUZ12
  • TET2
  • TP53
  • WT1

Subpanel: Chronic Lymphoproliferative Disorders Panel – 7 genes

Targets seven genes mutated in mature lymphoid leukemias, including chronic lymphocytic leukemia, lymphoplasmacytic leukemia, hairy cell leukemia, and large granular lymphocyte leukemias.

  • BRAF
  • MYD88
  • NOTCH1
  • SF3B1
  • STAT3
  • STST5B
  • TP53

Subpanel: Myeloproliferative Neoplasms Panel – 3 genes

Detects mutations associated with myeloproliferative neoplasms.

  • CALR
  • JAK2
  • MPL

Targeted Gene Regions

Genes interrogated, including relevant transcripts and exons, are listed in alphabetical order.

A

Gene, Transcript, Exons

ABL1
NM_005157.5
Exons 4-6

ASXL1
NM_15338.5
Exons 10-13

B

Gene, Transcript, Exons

BCOR
NM_17745.5
Exons 2-15

BCORL1
NM_021946.4
Exons 1-12

BRAF
NM_004333.4
Exon 15

C

Gene, Transcript, Exons

CALR
NM_004343.3
Exon 9

CBL
NM_005188.3
Exons 8, 9

CDKN2A
NM_000077.4
Exons 1, 2

CDKN2A
NM_058195.3
Exon 1

CEBPA
NM_004364.4
Exon 1

CSF3R
NM_000760.3
Exons 14-17

CUX1
NM_001202543.1
Exons 15-24

CUX1
NM_001913.4
Exons 1-23

D

Gene, Transcript, Exons

DDX41
NM_016222.3
Exons 1-17

DNMT3A
NM_022552.4
Exons 2-23

E

Gene, Transcript, Exons

EED
NM_003797.4
Exons 1-12

ETNK1
NM_018638.4
Exon 3

ETV6
NM_001987.4
Exons 1-8

EZH2
NM_004456.4
Exons 2-20

F

Gene, Transcript, Exons

FBXW7
NM_018315.4
Exons 7-11

FLT3
NM_004119.2
Exons 14-17, 19-20

G

Gene, Transcript, Exons

GATA1
NM_002049.3
Exons 2, 4

GATA2
NM_032638.4
Exons 2-6

GNAS
NM_000516.5
Exons 8-11

I

Gene, Transcript, Exons

IDH1
NM_005896.3
Exon 4

IDH2
NM_002168.3
Exon 4

IKZF1
NM_006060.5
Exons 2, 3, 5-7

J

Gene, Transcript, Exons

JAK2
NM_004972.3
Exons 12-16

JAK3
NM_000215.3
Exons 11-18

K

Gene, Transcript, Exons

KDM6A
NM_021140.3
Exons 1-29

KIT
NM_000222.2
Exons 2, 8-11, 13, 17

KMT2A
NM_005933.3
Exons 1-36

KRAS
NM_004985.4
Exons 2-4

L

Gene, Transcript, Exons

LUC7L2 (C7orf55)
NM_001244585.1
Exons 2-11

M

Gene, Transcript, Exons

MPL
NM_005373.2
Exons 10-11

MYD88
NM_002468.4
Exon 5

N

Gene, Transcript, Exons

NF1
NM_000267.3
Exons 1-57

NF1
NM_001042492.2
Exon 31

NOTCH1
NM_17617.4
Exons 26, 27, 34

NPM1
NM_002520.6
Exons 8-11

NRAS
NM_002524.4
Exons 2-4

P

Gene, Transcript, Exons

PAX5
NM_016734.2
Exons 1-10

PHF6
NM_001015877.1
Exons 2-10

PIGA
NM_002641.3
Exons 2-6

PPM1D
NM_003620.3
Exons 1-6

PRPF8
NM_006445.3
Exons 2-43

PTEN
NM_000314.6
Exons 1-9

PTPN11
NM_002834.3
Exons 3, 4, 12, 13

R

Gene, Transcript, Exons

RAD21
NM_006265.2
Exons 2-14

RIT1
NM_006912.5
Exon 5

RUNX1
NM_001754.4
Exons 2-9

RUNX1
NM_001122607.1
Exon 5

S

Gene, Transcript, Exons

SETBP1
NM_015559.2
Exon 4*

* Exon is only partially analyzed from genomic coordinates chr18:42531679-42532175.

SF3B1
NM_012433.3
Exons 13-16

SH2B3
NM_005475.2
Exon 2

SMC1A
NM_006306.3
Exons 1-25

SMC3
NM_005445.3
Exons 1-29

SRSF2
NM_003016.4
Exons 1, 2

STAG2
NM_00104279.2
Exons 3-35

STAT3
NM_003150.3
Exons 20, 21

STAT5B
NM_012448.3
Exons 16-18

SUZ12
NM_015355.3
Exons 1-16

T

Gene, Transcript, Exons

TET2
NM_001127208.2
Exons 3-11

TP53
NM_000546.5
Exons 2-11

U

Gene, Transcript, Exons

U2AF1
NM_006758.2
Exons 2, 6

W

Gene, Transcript, Exons

WT1
NM_000378.4
Exons 1-9

Z

Gene, Transcript, Exons

ZRSR2
NM_005089.3
Exons 1-11

Clinical Indications

This assay is intended for patients with known or suspected hematologic neoplasms including myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, acute lymphoblastic leukemia, and selected mature lymphoid leukemias.

Interpretation

All variants are classified using Association for Molecular Pathology guidelines for interpretation of somatic variants in cancer.[8]

Detailed interpretations are provided for each variant, and an overall interpretation of the entire mutational profile summarizes the case findings.

Reported variants include those of strong or potential clinical significance as well as variants of unclear clinical significance.

Known benign polymorphisms are not reported.

Methodology

Nucleic acid extracted from the specimen is subjected to nested multiplex PCR-based target enrichment.

Coding and non-coding regions of targeted genes are amplified and sequenced on an Illumina instrument (San Diego, CA) with paired-end, 150×2 cycle reads.

A customized bioinformatic analytical pipeline is used to map reads to the reference human genome (Genomic Build GRCh37/hg19).

Limitations

This test does not detect structural variants or copy number changes and does not distinguish between variants that are inherited versus acquired.

During internal validation, this test delivered an average of >500X coverage and >98% of targeted regions showed over 100X coverage. The test demonstrated 95.2% sensitivity and 99.9% specificity in identifying single nucleotide variants, small insertions and deletions (indels) (≤10bp) of >5% variant allele fraction (VAF). For the identification of large indels (>10bp) at >5% VAF the test demonstrated 87.5% sensitivity and 99.9% specificity.

Due to the limitations of next-generation sequencing technology, some large insertions may not be detected.

References

1. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013 Nov 21; 122(22):3616-27.

2. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011 Jun 30;364(26):2496-506.

3. Döhner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015 Sep 17;373(12):1136-52.

4. Nazha A, Zarzour A, Al-Issa K, et al. The complexity of interpreting genomic data in patients with acute myeloid leukemia. Blood Cancer J. 2016 Dec 16;6(12):e510.

5. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016 May 19;127(20):2391-405.

6. “NCCN Guidelines for Treatment of Cancer by Site,” National Comprehensive Cancer Network: https://www.nccn.org/professionals/physician_gls/default.aspx#site.

7. McClure RF, Ewalt MD, Crow J, et al. Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology. J Mol Diagn. 2018 Nov;20(6):717-737. DOI: 10.1016/j.jmoldx.2018.07.002. Epub 2018 Aug 20.

8. Li MM, Datto M, Duncavage EJ, et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017 Jan;19(1):4-23.