Testing | Overview
The Boston Children’s Hospital Laboratory for Molecular Pediatric Pathology (BCH LaMPP) provides highly innovative tissue-based diagnostic testing, uniquely tailored to serve pediatric patients, particularly those with cancer and developmental anomalies associated with tumor-like tissue overgrowths. Tests include targeted RNA sequencing for gene fusions and internal tandem duplications, and droplet digital PCR assays.
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Nucleic Acid Extraction
DNA
- Overview
- DNA extraction from various sources using the Promega Maxwell automated purification platform, followed by fluorescence-based quantification using the Promega Quantus
- Specimen
- Acceptable sample sources:
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- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
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- Acceptable sample sources:
- Overview
- RNA extraction from various sources using the Promega Maxwell automated purification platform, followed by fluorescence-based quantification using the Promega Quantus
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
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- Acceptable sample sources:
- Overview
- TNA (DNA and RNA) extraction from various sources using the Promega Maxwell automated purification platform, followed by fluorescence-based quantification using the Promega Quantus
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
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- Acceptable sample sources:
Droplet Digital PCR
MYOD1 L122R ddPCR
- Overview
- MYOD1 is protein coding gene for transcription factors in the myogenic factors family that is associated with Rhabomyosarcomas. Rhabomyosarcomas (RMS) are a group of skeletal muscle tumors that include embryonal, alveolar, pleomorphic, and spindle cell/sclerosing subtypes (SC/SRMS). A recurrent hotspot variant in MYOD1, p.L122R (NM_002478.4 c.365G>T), has been described in SC/SRMS. Droplet digital polymerase chain reaction (ddPCR) is performed on DNA using fluorescently labeled allele-specific primer-probe pairs (Bio-Rad) for the MYOD1 p.L122R and wild-type codon 122 (NM_002478.4 c.365G>T). The reaction mix is partitioned into droplets using oil emulsion to a limiting dilution (Bio-Rad QX200 Droplet Generator). End point PCR is performed and the quantity of droplets positive for variant and wild-type alleles is counted using an optical detection system (Bio-Rad QX200 Droplet Reader). Based on internal validation experiments, variant alleles are considered "detected" if they demonstrate variant fractional abundance ≥1%, "indeterminate" if <1% but ≥0.5%, and "not detected" if <0.5%. Therefore this assay may not detect variants with a very low allele fraction or in samples with a very low tumor percentage. For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology.
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
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- Acceptable sample sources:
- Overview
- BRAF is a proto-oncogene with roles in cellular proliferation and differentiation. Somatic activating variants are present in numerous malignancies and may contribute to tumorigenesis. Over 90% of BRAF variants occur at a single site and result in a valine-to-glutamate substitution at amino acid 600 (V600E). Droplet digital polymerase chain reaction (ddPCR) is performed on DNA using fluorescently labeled allele-specific primer-probe pairs (Bio-Rad) for the BRAF p.V600E and wild-type codon 600 (c.1799T>A, transcript NM_001354609.1). The reaction mix is partitioned into droplets using oil emulsion to a limiting dilution (Bio-Rad QX200 Droplet Generator). End point PCR is performed and the quantity of droplets positive for variant and wild-type alleles is counted using an optical detection system (Bio-Rad QX200 Droplet Reader). Based on internal validation experiments, variant alleles are considered "detected" if they demonstrate variant fractional abundance ≥1%, "indeterminate" if <1% but ≥0.5%, and "not detected" if <0.5%. Therefore this assay may not detect variants with a very low allele fraction or in samples with a very low tumor percentage. For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology.
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
-
- Acceptable sample sources:
- Overview
- Somatic activating variants in phosphatidylinositol-3-kinase (PIK3CA) underlie a wide spectrum of partially overlapping, clinically distinct, segmental overgrowth disorders, including CLOVES (congenital lipomatous overgrowth, epidermal nevi, spinal/skeletal anomalies and/or scoliosis) syndrome, Klippel Trenaunay syndrome, FAVAs (fibroadipose vascular anomalies), macrodactyly and muscle hemihypertrophy, megalencephaly syndromes, such as MCAP (megalencephaly-capillary malformation), DMEG (dysplastic megalencephaly) syndromes, and isolated vascular malformations, collectively known as PIK3CA overgrowth spectrum (PROS). Activating PIK3CA variants have also been reported as somatic alterations in a wide variety of tumor types. This assay is performed on extracted DNA using fluorescently labeled probes (Bio-Rad) for detection of five common PIK3CA variants: c.1258T>C (C420R), c.1624G>A (E542K), c.1633G>A (E545K), c.3140A>T (H1047L), and c.3140A>G (H1047R). One probe detects the wild-type (WT) allele (HEX-labeled) and the other detects the variant allele (FAM-labeled). The reaction mix is partitioned into droplets using oil emulsion to a limiting dilution (Bio-Rad QX200 Droplet Generator). End point PCR is performed and the quantity of droplets positive for variant and wild-type alleles is counted using an optical detection system (Bio-Rad QX200 Droplet Reader). Based on internal validation experiments, variant alleles are considered "detected" if they demonstrate variant fractional abundance ≥1%, "indeterminate" if <1% but ≥0.5%, and "not detected" if <0.5%. Therefore this assay may not detect variants with a very low allele fraction or in samples with a very low tumor percentage. For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology. Multiple additional variants in PIK3CA have also been reported and are not evaluated by this assay.
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
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- Acceptable sample sources:
Gene Fusion Sequencing: Solid and Brain Tumor
- Overview
- The Solid Tumor and Brain Tumor Fusion Panel is designed to include genes known to be somatically altered in pediatric tumors that may be used to support the diagnosis, prognosis or therapy for patients. The assay uses total nucleic acid (TNA), which is a combination of DNA and RNA, or RNA alone, to interrogate 64 genes. TNA is isolated from tissue containing at least 20% tumor nuclei and analyzed by massively parallel sequencing using Archer FusionPlexTM library kit with BCH custom Gene Specific Primers from ArcherDX (Boulder, CO) is used for library preparation and libraries are sequenced on an Illumina MiSeq sequencer. Sequences are analyzed using ArcherAnalysis bioinformatics software system (ArcherDX, Boulder, CO).
- Genes tested for select rearrangements:
- ALK, BCOR, BRAF, BRD3, BRD4, C11orf95, CAMTA1, CCNB3, CIC, CDH11, DNAJB1, EGFR, EPC1, ERG, ETV1, ETV4, ETV5, ETV6, EWSR1, FGFR1, FGFR3, FOSB, FOXO1, FOXO4, FUS, GLI1, HMGA2, MAML2, MEAF6, MET, MKL2, MYB, MYBL1, NCOA1, NCOA2, NTRK1, NTRK2, NTRK3, NUTM1, PDGFB, PHF1, PLAG1, PPARG, PRKACA, PRKCA, PRKCB, PRKCD, QKI, RAF1, RELA, RET, ROS1, SMARCB1, SS18, SS18L1, STAT6, TAF15, TCF12, TFE3, TFEB, TFG, USP6, VGLL2, YAP1, YWHAE
- The assay is also validated to detect internal tandem duplications in exon 15 of BCOR. All genomic alterations are reported in hg19/GRCh37 coordinates.
- Genetic alterations are classified into four tiers based on their level of clinical significance in cancer diagnosis, prognosis and/or therapeutics, according the standards and guidelines recommended by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists
- For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology
- References:
- Dupain C, Harttrampf AC, Urbinati G, Geoerger B, Massaad-Massade L. Relevance of Fusion Genes in Pediatric Cancers: Toward Precision Medicine. Mol Ther Nucleic Acids. 2017 Mar 17;6:315-26.
- Wong MK, Ng CCY, Kuick CH, Aw SJ, Rajasegaran V, Lim JQ, et al. Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion. Histopathology. 2018 Jan;72(2):320-9.
- Zheng Z, Liebers M, Zhelyazkova B, Cao Y, Panditi D, Lynch KD, et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med. 2014 Dec;20(12):1479-84.
- Li MM, Datto, M, Duncavage EJ, Kulkarni S, Lindeman NI, Roy S, Tsimneridou AM, Vnencak-Jones CL, Wolff DJ, Younes A, Nikiforova MN. 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 Diag, 2017 Jan;19(1):4-23.
- Specimen
- Acceptable sample sources:
-
- FFPE tissue sections or scrolls
- Fresh/frozen tissue
- Whole blood
- Bone marrow
- Bone marrow aspirate smears
-
- Acceptable sample sources:
Gene Fusion Sequencing: Hematologic Malignancy
- Overview
- The Boston Children’s Hospital Heme Malignancy Fusion Panel assay is based on the ArcherDX FusionPlex® Heme v2 panel. The assay targets genes with known diagnostic, prognostic and/or therapeutic significance in hematologic malignancies, and is validated to detect fusions of 72 target genes, either between the targeted genes, or between a targeted gene and a non-targeted partner. [Note that this assay is based on the detection of fusion transcripts. Rearrangements involving B-cell receptor or T-cell receptor loci (including IGH, IGL, IGK, TRA, TRB, TRD, and TRG) may not be detected because these often do not result in chimeric transcripts. Examples of these types of rearrangements include IGH-MYC rearrangements in Burkitt lymphoma or TRA-TLX1 rearrangements in T-ALL.]
- Genes tested for select rearrangements:
- ABL1 (NM_005157, exons 1-5), ABL2 (NM_005158, exons 2-8), ALK (NM_004304, exons 2, 4, 6, 10, 16- 23), BCL11B (NM_138576, exons 2-4), BCL2 (NM_000633, exons 2-3), BCL6 (NM_001706, exons 2-3), BCR (NM_004327, exons 1-3, 8, 12-16), BIRC3 (NM_001165, exons 4-7), CBFB (NM_022845, exons 4-5), CCND1 (NM_053056, exon 5), CCND3 (NM_001760, exon 2), CDK6 (NM_001259, exons 1-4), CHD1 (NM_001270, exons 1-2), CHIC2 (NM_012110, exons 1-3), CIITA (NM_000246, exons 1-2), CREBBP (NM_004380, exons 2-6), CRLF2 (NM_022148, exon 1), CSF1R (NM_005211, exons 9-14), DEK (NM_003472, exons 2-3), DUSP22 (NM_020185, exons 1-2), EBF1 (NM_024007, exons 10-14), EIF4A1 (NM_001416, exons 2-3), EPOR (NM_000121, exons 7-8), ERG (NM_004449, exons 7-11), ETV6 (NM_001987, exons 1-6), FGFR1 (NM_023110, exons 2-12, 17), GLIS2 (NM_032575, exons 2-3), IKZF1 (NM_006060, exons 1-3, 7-8), IKZF2 (NM_016260, exons 3-4), IKZF3 (NM_012481, exons 2-7), JAK2 (NM_004972, exons 6-13, 15-20), KAT6A (NM_006766, exons 13-16), KLF2 (NM_016270, exons 2-3), KMT2A(MLL) (NM_005933, exons 4-35), MALT1 (NM_006785, exon 9), MECOM (NM_004991, exons 1-4), MRTFA(MKL1) (NM_020831, exons 4-6), MLF1 (NM_022443, exons 2-4), MLLT10 (NM_004641, exons 2-18), MLLT4 (NM_001040000, exon 2), MYC (NM_002467, exons 1-2), MYH11 (NM_002474, exons 7-11, 14-16), NF1 (NM_000267, exons 14 and 36), NFKB2 (NM_002502, exons 14-21), NOTCH1 (NM_017617, exons 24-29, 34), NTRK3 (NM_002530, exons 13-15), NUP214 (NM_005085, exons 17-19), NUP98 (NM_016320, exons 8-17), P2RY8 (NM_178129, exon 1), PAG1 (NM_018440, exon 2), PAX5 (NM_016734, exons 1, 4-8), PDCD1LG2 (NM_025239, exons 1-3, 5-6), PDGFRA (NM_006206, exons 9-14), PDGFRB (NM_002609, exons 8-14), PICALM (NM_007166, exons 16-19), PML (NM_002675, exons 2-7), PRDM16 (NM_022114, exons 1-4), PTK2B (NM_173176, exons 2-8), RARA (NM_000964, exons 2-5), RBM15 (NM_022768, exon 1), ROS1 (NM_002944, exons 31-36), RUNX1 (NM_001754, exons 2-9), RUNX1T1 (NM_001198679, exons 2-3), SEMA6A (NM_020796, exons 1-2), SETD2 (NM_014159, exons 1-12), STIL (NM_003035, exons 1-2), TAL1 (NM_003189, exons 2-4), TCF3 (NM_003200, 11-18), TFG (NM_006070, exons 2-4), TP63 (NM_003722, exons 3-5), TYK2 (NM_003331, exons 16 and 18), ZCCHC7 (NM_032226, exons 1-4).
- Genetic alterations are classified into four tiers based on their level of clinical significance in cancer diagnosis, prognosis and/or therapeutics, according the standards and guidelines recommended by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists
- For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology
- Acceptable sample types include:
-
- Air-dried bone marrow aspirate smears
- Fresh blood or bone marrow (EDTA tube)
- Fresh/frozen tissue
- FFPE tissue sections or scrolls (non-decalcified)
-
These tests were developed and its performance characteristics determined by the Laboratory for Molecular Pediatric Pathology (LaMPP) in the Department of Pathology at Boston Children’s Hospital under the supervision of Marian H. Harris, MD, PhD, LaMPP Director. This laboratory is regulated under the Clinical Laboratory Improvement Amendments of 1988 as qualified to perform high-complexity clinical testing. This test has not been cleared of approved by the US Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary.