Pathology

• Specimens: Minimum five FFPE tissue sections or scrolls (non-decalcified), 150 mg of Fresh/frozen tissue, 2 mL Whole blood (EDTA tube), 2 mL Bone marrow (EDTA tube), Two Air-dried bone marrow aspirate smears, RNA, Fluids

Contact information

Please contact us via email at Path_Lampp-dl@childrens.harvard.edu.

Solid and Brain Tumor Fusion Panel | CPT:81479

• Clinical utility: The Boston Children’s Hospital Solid and Brain Tumor Fusion Panel targets genes with known diagnostic, prognostic and/or therapeutic significance in pediatric solid tumors and brain tumors. The assay is validated to detect fusions of 64 target genes, either between the targeted genes, or between a targeted gene and a non-targeted partner.
• Methodology: Massively parallel sequencing using Archer FusionPlex™ library kit on an Illumina sequencers. Sequences are analyzed using Archer Analysis bioinformatics software system (ArcherDX, Boulder, Colo.). For detailed methodology, please contact the laboratory.
• Sample types: This assay is validated on formalin-fixed paraffin-embedded tissue (FFPE; block or unstained slides acceptable), fresh or frozen tissue, and air-dried smears.
• References:
  • Church, Alanna J., et al. "Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer." Nature medicine (2022): 1-9. PMID: 35739269
  • Wachter, Franziska, et al. "Molecular Characterization of Inflammatory Tumors Facilitates Initiation of Effective Therapy." Pediatrics 148.6 (2021). PMID: 34814185
  • Al-Ibraheemi, Alyaa, et al. "Assessment of BCOR Internal Tandem Duplications in Pediatric Cancers by Targeted RNA Sequencing." The Journal of Molecular Diagnostics 23.10 (2021): 1269-1278. PMID: 34325058
  • Slack, Jonathan C., and Alanna J. Church. "Molecular Alterations in Pediatric Solid Tumors." Surgical Pathology Clinics 14.3 (2021): 473-492
  • Paulson, Vera A., et al. "Recurrent and novel USP6 fusions in cranial fasciitis identified by targeted RNA sequencing." Modern Pathology 33.5 (2020): 775-780. PMID: 31827231
  • Church, Alanna J., et al. "Recurrent EML4–NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy." Modern Pathology 31.3 (2018): 463-473. PMID: 29099503
• Target genes: 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 and tyrosine kinase domain duplications in FGFR1. All genomic alterations are reported in hg19/GRCh37 coordinates.

Hematologic Malignancy Fusion Panel |  CPT:81479

• Clinical utility: 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.
• Methodology: Massively parallel sequencing using Archer FusionPlex™ library kit on an Illumina sequencers. Sequences are analyzed using Archer Analysis bioinformatics software system (ArcherDX, Boulder, Colo.). For detailed methodology, please contact the laboratory.
• Sample types: This assay is validated on formalin-fixed paraffin-embedded tissue (FFPE; block or unstained slides acceptable), fresh or frozen tissue, and air-dried smears. Decalcified bone marrow core biopsy tissue/slides are not accepted.
• References:
  • Mueller SB, Pikman Y, Tasian SK, Silverman LB, Harris MH, Tsai HK. ETV6 fusions from insertions of exons 3-5 in pediatric hematologic malignancies. Haematologica. 2023 Dec 1;108(12):3471-3476. PMID: 37381775
  • Paolino J,et al. Integration of genomic sequencing drives therapeutic targeting of PDGFRA in T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma. Clin Cancer Res. 2023 Sep 19. PMID: 37725576
  • Tsai HK, et al. Outlier Expression of Isoforms by Targeted or Total RNA Sequencing Identifies Clinically Significant Genomic Variants in Hematolymphoid Tumors. J Mol Diagn. 2023 Sep;25(9):665-681. PMID: 37419244
  • Pikman, Yana, et al. "Matched targeted therapy for pediatric patients with relapsed, refractory, or high-risk leukemias: a report from the LEAP Consortium." Cancer discovery 11.6 (2021): 1424-1439.PMID: 33563661
  • Harris, Marian H. "Gene rearrangement detection in pediatric leukemia." Clinics in Laboratory Medicine 41.3 (2021): 551-561.PMID: 34304782
• Target genes: ABL1, ABL2, ALK, BCL11B, BCL2, BCL6, BCR, BIRC3, CBFB, CCND1, CCND3, CDK6, CHD1, CHIC2, CIITA, CREBBP, CRLF2, CSF1R, DEK, DUSP22, EBF1, EIF4A1, EPOR, ERG, ETV6, FGFR1, GLIS2, IKZF1, IKZF2, IKZF3, JAK2, KAT6A, KLF2, KMT2A, MALT1, MECOM, MKL1, MLF1, MLLT10, MLLT4, MYC, MYH11, NF1, NFKB2, NOTCH1, NTRK3, NUP214, NUP98, P2RY8, PAG1, PAX5, PBX1, PDCD1LG2, PDGFRA, PDGFRB, PICALM, PML, PRDM16, PTK2B, RARA, RBM15, ROS1, RUNX1, RUNX1T1, SEMA6A, SETD2, STIL, TAL1, TCF3, TFG, TP63, ZCCHC7. [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.]

GeneVa (Targeted DNA Sequencing for Vascular Anomalies) | CPT:81479

• Clinical utility: Vascular anomalies include a group of disorders of vascular malformations and overgrowths due to genetic alterations. Somatic mosaic genetic alterations underlie groups of disorders associated with capillary malformations, venous malformations, lymphatic malformations, arteriovenous malformations, and complex syndromes associated with various vascular anomalies. GeneVa, a targeted DNA sequencing panel, detects single nucleotide variants and small insertions/deletions (≤30 bp) in 35 genes with known diagnostic, prognostic, and/or therapeutic significance in vascular anomalies.
• Methodology: Massively parallel sequencing using Archer VariantPlex™ library kit on an Illumina sequencers. Sequences are analyzed using Archer Analysis bioinformatics software system (ArcherDX, Boulder, Colo.) as well as custom analysis. For detailed methodology, please contact the laboratory.
• Sample types: This assay is validated on formalin-fixed paraffin-embedded tissue (FFPE; block or unstained slides acceptable), fresh or frozen tissue, and air-dried smears.
• References:
  • Kurek, Kyle C., et al. "Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome." The American Journal of Human Genetics 90.6 (2012): 1108-1115
• Target genes: AKT1, AKT2, AKT3, BRAF, EPBH4, FGFR1, FGFR2, FGFR3, FLT4, FOXC2, GDF2, GLMN, GNA11, GNA14, GNAQ, GNAS, HRAS, KRAS, KRIT1, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K3, MAPK1, NRAS, PDCD10, PIK3CA, PIK3R1, PIK3R2, PTEN, RASA1, SMAD4, SOS1, SOX18, TEK (TIE2)
• Billing: Patients must be financially cleared through verification of coverage from the insurance company or prior authorization.

BRIGHTseq Beacon | CPT:81479

• Clinical utility: The BRIGHTseq Beacon assay is designed for children, adolescents, and young adults (including those eligible for Medicare) with known or suspected cancers, including both solid tumors and hematolymphoid malignancies. This test utilizes whole-exome sequencing (WES) of tumor and matched germline DNA to accurately distinguish somatic variants from background variants. Reporting focuses on a curated subset of genes relevant to pediatric and adolescent and young adult (AYA) cancers. The assay evaluates single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations (CNAs), providing comprehensive genomic characterization to refine diagnosis, inform prognostic risk stratification, and identify therapeutic targets.
• Methodology: Massively parallel sequencing using custom TWIST library kits on Illumina sequencers. Sequences are analyzed using DRAGEN analysis pipeline. For detailed methodology, please contact the laboratory.
• Sample types: This assay is validated on formalin-fixed paraffin-embedded tissue (FFPE; block or unstained slides acceptable) paired with a germline comparator sample (blood).
• References:
  • Church AJ et al. "Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer." Nature medicine (2022): 1-9. PMID: 35739269
  • Slack JC and Church AJ. Molecular Alterations in Pediatric Solid Tumors Clinics in Laboratory Medicine, Volume 44, Issue 2, 277 – 304
  • Villani, A., et al., The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations. Nat Cancer, 2023. 4(2): p. 203-221.
  • Simon, J., et al., Clinical impact of large genomic explorations at diagnosis in 198 pediatric solid tumors: a monocentric study aiming practical feasibility of precision oncology. BMC Cancer, 2024. 24(1): p. 1296.
  • Ramkissoon, S.H., et al., Clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors. Neuro Oncol, 2017. 19(7): p. 986-996
  • Lau, L.M.S., et al., Precision-guided treatment in high-risk pediatric cancers. Nat Med, 2024. 30(7): p. 1913-1922.
• Target genes: ABL1, ACVR1, AKT1, AKT2, AKT3, ALK, AMER1, ANKRD26, APC, ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXL, B2M, BAP1, BARD1, BAX, BCL2, BCOR, BCORL1, BLM, BRAF, BRCA1, BRCA2, BRCC3, BTG1, BTK, CABLES1, CALR, CARD11, CBL, CCND1, CCND2, CCND3, CCNE1, CD79B, CDK2, CDK4, CDK5, CDK6, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIITA, CREBBP, CRLF2, CSF3R, CSNK1A1, CSNK2B, CTCF, CTDNEP1, CTNNA1, CTNNB1, CUX1, CXCR4, CYSLTR2, DDR2, DDX3X, DDX41, DGCR8, DICER1, DIS3, DIS3L2, DKC1, DNAJC21, DNMT3A, DROSHA, EFL1, EGFR, EIF1AX, EIF6, ELANE, ELP1, EP300, EPCAM, EPOR, ERBB2, ERBB3, ERBB4, ERCC4, ERCC6L2, ERG, ETNK1, ETV6, EZH2, EZHIP, FANCC, FAS, FAT1, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FLT4, GATA1, GATA2, GFI1B, GLI1, GLI2, GLI3, GNA11, GNA13, GNAQ, GNAS, GNB1, H1-4, H3-3A, H3-3B, H3C2, H3C3, H3C4, HNF1A, HRAS, ID2, IDH1, IDH2, IGF1R, IGF2, IKZF1, IKZF2, IKZF3, IL7R, JAK1, JAK2, JAK3, KBTBD4, KDM6A, KDM6B, KDR, KIAA1549, KIT, KLF2, KLF4, KMT2A, KMT2C, KMT2D, KRAS, LIG4, LZTR1, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAPK1, MBD4, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MGA, MLH1, MPL, MSH2, MSH6, MTAP, MTOR, MYB, MYC, MYCN, MYD88, MYOD1, NBN, NF1, NF2, NFE2, NFE2L2, NFKBIA, NFKBIE, NFKBIZ, NOTCH1, NOTCH2, NOTCH3, NPM1, NR3C1, NRAS, NSD2, NT5C2, NTRK1, NTRK2, NTRK3, OTX2, PALB2, PAX5, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIGA, PIK3C2B, PIK3CA, PIK3CB, PIK3CG, PIK3R1, PIK3R2, PIM1, PLCB4, PLCG2, PMS1, PMS2, POLD1, POLE, POLH, POLQ, POT1, PPM1D, PRDM1, PRDM6, PRKAR1A, PRKCA, PRPF8, PTCH1, PTEN, PTPN11, PTPN14, PVALB, RAD21, RAD50, RAD51, RAD51C, RAD51D, RAD52, RAD54B, RAF1, RASA1, RASA2, RB1, RECQL4, REL, RET, RHOA, RICTOR, RIT1, RNU1-1, ROS1, RPTOR, RRAS, RRAS2, RTEL1, RUNX1, SAMD9, SAMD9L, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SERPINA1, SETBP1, SETD2, SF3B1, SH2B3, SMAD4, SMARCA4, SMARCAL1, SMARCB1, SMARCE1, SMC1A, SMC3, SMO, SOCS1, SRP72, SRSF2, SS18, STAG1, STAG2, STAT3, STAT5B, STAT6, SUFU, TBR1, TCF3, TCF4, TERC, TERT, TET2, TNFAIP3, TNFRSF14, TOP3A, TP53, TP53BP1, TRAF1, TRAF3, TRPS1, TSC1, TSC2, U2AF1, UBA1, UBR5, UBTF, USP48, USP8, VHL, WRN, WT1, XPC, XPO1, YAP1, ZEB2, ZIC1, ZMYM3, ZRSR2.

BRAF p.V600E | CPT 81210

• Clinical utility: 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. BRAF p.V600E (NM_001354609.1, c.1799T>A) occurs in over 90% of BRAF variants. 

PIK3CA hotspots | CPT 81309

• Clinical utility: 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) found in 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). 

MYOD1 p.L122R | CPT 81479

• Clinical utility: A recurrent hotspot variant in MYOD1, p.L122R (NM_002478.4 c.365G>T), has been described in rhabomyosarcomas (RMS), a group of skeletal muscle tumors that include embryonal, alveolar, pleomorphic, and spindle cell/sclerosing subtypes (SC/SRMS) 

Methodology for all ddPCR assays

Droplet digital polymerase chain reaction (ddPCR) is performed on DNA using fluorescently labeled allele-specific primer-probe pairs to target specific nucleotide mutations. 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). For detailed methodology, please contact the Laboratory for Molecular Pediatric Pathology.

The laboratory can also perform DNA, RNA, and/or TNA (total nucleic acid) extractions on a variety of sample types.

These tests were developed, and their 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 U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary.