UCSF Juvenile Myelomonocytic Leukemia Associated Exon Panel (JMML)

Performing Lab:

UCSF Clinical Cancer Genomics Lab

CPT Code:

81450

Specimen Type:

Tumor sample – EDTA (purple top) whole blood or bone marrow aspirate

Germline sample - Buccal swab or cultured fibroblasts

Note: Buccal swabs are available upon request. (CCGL lab 415-502-3252)

Methodology: Next generation sequencing

Turnaround time: 14 days

TEST INDICATIONS:

Designed for patients with suspicion of Juvenile myelomonocytic leukemia (JMML). JMML is characterized by excessive proliferation of myelomonocytes that infiltrate hematopoietic and non-hematopoietic tissues. While the suspicion of JMML is aided by clinical and hematological criteria, the additional finding of a pathogenic DNA variant previously known to be associated with JMML is often the tipping point in establishing a JMML diagnosis.

This assay is quantitative and may be used for recurrence monitoring of the allele burden of one or more previously uncovered mutations in a patient. The sensitivity of detecting a known somatic variant is 1-2%.

HOW THE TEST WORKS:

Genomic DNA is extracted from the sample for library preparation. Target enrichment is performed by hybrid capture using custom oligonucleotides (IDT xgen custom probes). Sequencing of captured libraries is performed on an Illumina NovaSeq 6000 on the SP or S1 flow cell (2 X 101 bp read length). Sequence reads are de-duplicated to allow for accurate allele frequency determination and copy number calling. The analysis uses open source or licensed software for alignment to the human reference sequence UCSC build hg19 (NCBI build 37) and variant calling. Common germline polymorphisms were eliminated from analysis using the complete list of germline mutations from dbSNP. Additional filtering to eliminate technology specific sequencing artifacts was performed before analyzing the data.

GENES ANALYZED:

A characteristic feature of JMML is the presence of a somatic or germline mutation in RAS pathway genes such as NRAS, KRAS, PTPN11, CBL and NF1, which altogether are mutated in about 70% of JMML cases. Mutations outside the RAS pathway have been suggested to constitute secondary mutations that could also lead to the development of JMML and impact on its progression. For example, SETBP1 and JAK3 were found to constitute common targets for secondary mutations. In addition, deleterious mutations in SH2B3 have been associated with the promotion of leukemogenesis, particularly ALL, and are considered as leukemia predisposing variants. Furthermore, heterozygous somatic mutations in ASXL1 may act as tumor suppressor in myeloid malignancies such as AML and CMML, thus assisting in the differential diagnosis of JMML.

Detected variants of known clinical significance in the genes list below will be reported. In cases when one or two genes of interest are requested, the clinically significant variants of only these genes will be reported.

References
Gelsi-Boyer et al. Br. J. Haem. 145:788-800, 2009

Loh ML. Br J Haematol 152: 677-87, 2011
Sakaguchi et al. Nature Genetics 45:937-941, 2013
Perez-Garcia et al. Blood 122:2425-2432, 2013
Stieglitz E, et al. Nat Genet. 48(1):101, 2016

JMML Gene List
DNMT3A	JAK3	PTPN11	RUNX1	SOS1
ETV6	KRAS	RAF1	SAMD9	ZRSR2
EZH2	MAP2K1	RIT1	SAMD9L
FLT3	NF1	RRAS	SETBP1
GATA2	NRAS	RRAS2	SH2B3

Structural Rearrangement (Fusion) List
ALK	PDGFRB	ROS1

Fusions may be detected in FLT3, NUP98, and RARA, depending on the partner and fusion breakpoint.

LIMITATIONS OF THE TEST:

This assay is designed to detect single nucleotide variants and small to medium insertion/deletions. Large insertions/deletions, copy number variants, and structural variants (gene rearrangements) may also be detected by the assay. The test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation.

This test was developed and its performance characteristics determined by the UCSF Clinical Cancer Genomics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is for clinical purposes and should not be considered as investigational or for research purposes.

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST:

Collect

Tumor sample - lavender top (EDTA) Blood or bone marrow

Germline samples - Buccal swab or T25 flask cultured cells

Amount to Collect

Tumor sample
EDTA whole blood: 5 mL
Bone Marrow aspirate: 3 mL

Germline sample
Cultured fibroblasts: Confluent T25 flasks x2
Buccal swab: Collect 4-6 cytobrush on each side of the mouth, according to provided instructions.

Preferred Volume

Minimum Volume

Tumor sample
EDTA whole blood: 2 mL
Bone Marrow aspirate: 1 mL

Germline sample
Cultured fibroblasts: Confluent T25 flasks x2
Buccal swab: Collect 4-6 cytobrush on each side of the mouth, according to provided instructions.

HOW TO ORDER THE TEST:

UCSF Clinicians — Inpatient and Ambulatory Providers are able to place orders electronically in APeX.

30000287 - HC JMML EXON ASSOCIATED PANEL. (APeX search terms include: JMML, JMML Panel, CBL exons 8/9, KRAS exons 2/3, NRAS exons 2/3, PTPN11 exons 3/4/13)

Non-UCSF Clinicians