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Chapter 45: Cytogenetic & Molecular Pathology of Paediatric Cancer – Comprehensive Bullet Notes

Introduction

  • Pediatric cancers often harbor pathognomonic cytogenetic / molecular lesions that (1) establish diagnosis, (2) drive biology, (3) stratify risk, (4) uncover drug-gable targets.
  • Classic examples: t(11;22) EWS-FLI1 (Ewing sarcoma), t(8;21) RUNX1–RUNX1T1 (AML-M2), t(15;17) PML–RARA (APL) and MYCN amplification (neuroblastoma).
  • Core laboratory armamentarium now spans low-resolution karyotyping to single–base, single-cell next-generation sequencing (NGS).

Cytogenetic & Molecular Methodologies

• Karyotyping

  • Arrest metaphases → G/T-banding → global 5–10 Mb resolution.
  • Pros: genome-wide, detects novel/complex changes.
  • Cons: requires fresh, viable cells; culture failure common in solid tumours; low resolution; slow.
    • Fluorescence / Chromogenic in-situ hybridization (FISH / CISH)
  • Break-apart, dual-fusion, centromeric & copy-number probes.
  • Detect cryptic, masked, non-dividing nuclei; works on paraffin sections, touch imprints, smears.
  • Break-apart EWSR1, FOXO1, MLL, ALK, MYCN widely used.
    • Highly combinatorial FISH
  • SKY / M-FISH → each chromosome painted unique colour.
  • Fiber-FISH & DIRVISH → 5 kb–700 kb resolution.
    • Comparative genomic hybridisation
  • Conventional metaphase CGH (obsolete) vs Array-CGH (10–50 kb) vs SNP-array (copy-neutral LOH).
  • Detect gains/losses >1–10 kb; balanced rearrangements missed.
    • PCR-based assays
  • Genomic PCR → LOH, ITDs (eg FLT3).
  • RT-PCR → fusion transcripts; sensitivity ≈ (1/10^4) cells; used for MRD.
  • qPCR / digital PCR → copy number, expression, allelic ratio (eg FLT3-ITD AR > 0.5).
  • Panhandle / LDI-PCR → partner-unknown breakpoint cloning (MLL, NUP98, EWSR1, NTRK3).
    • Next-generation sequencing
  • Whole genome, exome, RNA-seq; or targeted "hemo-oncology" panels (100–600 genes); detects SNV, indel, CNV & fusions in 1 assay.
  • Challenges: DNA quality/quantity, turnaround, data analysis, cost.
  • Emerging multiplex non-PCR platforms (NanoString nCounter, Ab-FISH).
    • Method comparison (Table 45-4 key points)
  • Cost: Sequencing > CGH/SNP > Karyotype > FISH > PCR.
  • Fresh material mandatory only for karyotype/CGH; others work on fixed.
  • Genome-wide only by karyotype/CGH/SNP/NGS.

Genomic Mechanisms in Pediatric Tumours

• Primary classes of aberration

  1. Balanced translocation → fusion oncoprotein (TF, kinase, epigenetic).
  2. Translocation → promoter-swap/up-regulation (MYC-Ig in Burkitt).
  3. High-level amplification (MYCN, FOXO1, MDM2, CDK4).
  4. Deletion / LOH (1p/11q neuroblastoma; 22q SMARCB1 rhabdoid).
  5. Chromothripsis / chromoplexy (i(17q) medulloblastoma; osteosarcoma).
    • Etiology & predisposition
  6. DNA double-strand break stimuli: V(D)J, meiotic recombination, TOP2 poisons, IR/UV, ROS.
  7. Chromatin proximity determines partner choice (RET–H4, MYC–Ig).
  8. Hereditary repair defects: NF1, ATM, NBN, BLM, RECQL4 (Rothmund–Thomson), BRCA2 (Fanconi D1), TP53 (Li-Fraumeni).
  9. Pharmacologic links: Alkylators → −5/−7; TOP2 poisons → MLLr.

Transcription-Factor Fusion Oncoproteins

• RUNX1-RUNX1T1 t(8;21) → block myeloid differentiation; good risk if high-dose Ara-C.
• CBFB-MYH11 inv(16)/t(16;16) → myelomonocytic + eosinophilia; good risk.
• PAX3/7-FOXO1 t(2;13)/(1;13) → alveolar RMS; PAX7 variant highly amplified → better OS.
• EWSR1-ETS family (FLI1 > ERG > others) ext{t}(11;22) etc → Ewing sarcoma; Type 1 (EWS7-FLI1 6) trend to superior outcome.
• EWSR1-WT1 t(11;22) → desmoplastic small round cell tumour; drives PDGFA & IGF1R.
• CIC-DUX4 t(4;19)/t(10;19) → Ewing-like SRBCT.
• SS18-SSX 1/2 t(X;18) → synovial sarcoma; SSX2 fusion & reduced SNAIL recruitment → better DFS.

Tyrosine-Kinase Fusion Oncoproteins

• ABL1 fusions

  • t(9;22) BCR-ABL1 in 5 % childhood ALL (P190).
  • ABL1, ABL2, PDGFRB, JAK2 fusions in Ph-like ALL.
    • Receptor kinases in solid tumours
  • TPM3/4-ALK, CLTC-ALK, RANBP2-ALK in IMT (ALK IHC pattern useful).
  • ASPSCR1-TFE3 t(X;17) alveolar soft part sarcoma & pediatric RCC (Xp11).
  • COL1A1-PDGFB t(17;22) / ring 17-22 DFSP → imatinib responses.
    • Internal tandem duplication
  • FLT3-ITD in 12–15 \% paediatric AML; allelic ratio > 0.5 → poor EFS.

Promoter-Swap / Up-regulation Translocations

• MYC-Ig t(8;14), t(2;8), t(8;22) in Burkitt lymphoma.
• LMO1/2, TAL1 (SIL), HOX11 to TCR loci \sim 40 % T-ALL.
• JAZF1-SUZ12 t(7;17) low-grade endometrial stromal tumour.
• PLAG1 activation by 8q12 rearrangements in lipoblastoma, pleomorphic adenoma.

Tumour-Type Highlights

Soft Tissue & Bone

• Ewing sarcoma

  • CD99+, EWSR1-ETS fusion (95 %); secondary TP53 mut & CDKN2A del worsen prognosis.
  • 1q gain (DTL) & 16q loss → relapse risk.
    • Rhabdomyosarcoma
  • Fusion-positive alveolar (PAX3/7-FOXO1) vs fusion-negative embryonal.
  • PAX7 fusion, <10 y, extremity location → favourable.
  • CTNNB1 mut / APC loss in desmoid tumours; nuclear β-catenin IHC.
    • Synovial sarcoma
  • TLE1 & SOX2 IHC; SS18-SSX fusion; HDAC, EZH2 & BRD4 dependency.
    • Inflammatory myofibroblastic tumour – ALK fusions (50 %); crizotinib.
    • GIST children – SDH-deficient (SDHB IHC–), KIT/PDGFRA WT; respond poorly to imatinib.

Non-mesenchymal paediatric solids

• Wilms tumour

  • WT1 loss (11p13), 11p15 LOH (IGF2 up), 1p/16q LOH & 1q gain → relapse.
  • Nephrogenic rests: PLNR (11p15) vs ILNR (WT1/CTNNB1).
    • TFE3/TFEB-RCC in children (Xp11 & 6p21 translocations).
    • NUT carcinoma t(15;19) BRD4-NUT; NUT IHC & BET inhibitors.

Neuroblastoma

• Risk genetics:

  • MYCN amp (>10 copies), 1p36 del, 11q del, 17q gain → poor.
  • Hyperdiploidy without 1p/11q loss → good.
  • ALK mut/F1174L & germline R1275Q; ALK IHC vs sequencing.
  • SNP 6p22 (FLJ44180) & 2q35 (BARD1) susceptibility loci.

CNS

• Medulloblastoma four molecular groups:

  • WNT (CTNNB1, i(17q), >90 \% OS).
  • SHH (PTCH1, SUFU, SMO).
  • Group 3 (MYC amp, isochr 17q, worst).
  • Group 4 (CDK6, SNCAIP, i17q).
    • ATRT/rhabdoid: SMARCB1 (INI1) or SMARCA4 loss → cyclin D1 up; INI1 IHC–.

Germ Cell Tumours

• Isochromosome 12p pathognomonic (FISH or karyotype) except paediatric yolk-sac.

Hematologic Neoplasms

• Hodgkin lymphoma – 2p/9p/12q gains; L&H (BCL6) vs H-RS (REL, NF-κB activation); EBV + subset.
• Burkitt – MYC–Ig translocation, Ki-67 ≈ 100 \%.
• Anaplastic large cell – ALK fusions, CD30+, EMA+; ALK IHC pattern mirrors partner.
• B-ALL key lesions –

  • TEL-AML1 t(12;21) good.
  • Ph-like ALL (ABL1/JAK fusions, IKZF1 del) poor; TKI/JAKi sensitive.
  • iAMP21, CRLF2-P2RY8, FLT3-ITD.
    • T-ALL – NOTCH1 mut (50 %), PTEN del (8 %), ETP-ALL (MEF2C, JAK/STAT) worst.
    • AML – Core binding factor (t8;21, inv16) good; FLT3-ITD, WT1, NUP98 poor.
    • JMML – PTPN11 (35 %), NRAS/KRAS (25 %), NF1 (10 %); monosomy 7; RAS pathway inhibitors.
    • Myeloid leukaemia Down syndrome – GATA1 mut + trisomy 21; high Ara-C sensitivity.
    • Therapy-related (t-MDS/AML) –
  • Alkylator: −5/−7, complex, TP53 mut; latency ~6 yrs.
  • TOP2 poison: MLLr, t(8;21), inv(16); latency 1–3 yrs.

Practical Diagnostic Algorithms

  1. Undifferentiated round cell tumour → CD99, desmin, keratin ±FISH (EWSR1 break-apart, FOXO1, CIC, BCOR).
  2. Paediatric renal mass < 10 y → TFE3 FISH ± WT1 IHC, look for t(1;22) in infants.
  3. Leukaemia with \ge15 % blasts post-topo II chemo → MLL FISH, RUNX1-RUNX1T1 PCR, inv16 FISH.
  4. Child with persistent monocytosis, splenomegaly → karyotype + RAS/NF1/PTPN11 sequencing; if monosomy 7 present consider HSCT.

Equations / Cut-offs (clinical)

• FLT3-ITD allelic ratio = \frac{\text{mutant peak}}{\text{mutant+wt peaks}}; AR >0.5 denotes high risk.
• MYCN amplification = >10 copies by FISH or >4 fold by qPCR.
• iAMP21 defined by \ge 3 extra RUNX1 signals on a single abnormal 21.
• High hyperdiploid ALL = >50 chromosomes; hypodiploid = <44.

Ethical / Practical Considerations

  • Need for rapid (< 7 d) assays to influence front-line therapy (e.g.
    ATRA in APL, imatinib in Ph+ ALL, crizotinib in ALK+ IMT).
  • Importance of sample triage: fresh tissue for karyotype/NGS; reserve touch imprints / FFPE for FISH.
  • Informed consent for germline findings (TP53, ALK, DICER1).
  • Long-term surveillance of childhood cancer survivors (t-MDS/AML risk rises \sim1 % per yr after epipodophyllotoxin).

Take-Home Bullet Summary

• Combine morphology + immunophenotype + cytogenetics for definitive paediatric cancer diagnosis.
• Balanced translocations dominate sarcomas, leukaemias; CNVs dominate neuroblastoma, osteosarcoma.
• Core binding factor AML, high hyperdiploid B-ALL, WNT-medulloblastoma = excellent outcomes.
• FLT3-ITD, iAMP21, IKZF1-del, CRLF2-rearranged ALL; MLL-r infant leukaemia; ETP-ALL = very high risk but offer novel targeted therapy windows.
• SDH-deficient GIST, ALK-IMT, COL1A1-PDGFB DFSP, NTRK3-fibrosarcoma = prototypic kinase-addicted paediatric solids responding to small-molecule inhibitors.
• Therapy-related myeloid neoplasms segregate by agent: alkylator → −5/−7/TP53; TOP2 → MLL.
• NGS panels increasingly frontline (fusion, SNV, CNV) but FISH/qPCR remain indispensable for speed and MRD.