What pediatric conditions is the FISH (Fluorescent In Situ Hybridization) test a diagnostic and preferred test for?

FISH Testing in Pediatric Conditions: Diagnostic and Preferred Applications

Primary Hematologic Malignancies

FISH is the diagnostic and preferred test for detecting specific genetic abnormalities in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), particularly when these abnormalities are cryptic or require rapid diagnosis for risk stratification and treatment decisions. 1

Pediatric B-Cell Acute Lymphoblastic Leukemia (B-ALL)

The American College of Medical Genetics (ACMG) recommends the following initial FISH panel for all pediatric B-ALL cases: 1

• BCR::ABL1 fusion probes - Detects Philadelphia chromosome-positive ALL, which requires specific tyrosine kinase inhibitor therapy 1
• KMT2A (MLL) rearrangement probes - Identifies MLL-rearranged ALL, accounting for up to 25% of pediatric cases and associated with poor prognosis 1
• ETV6::RUNX1 fusion probes - Detects the most common fusion in pediatric B-ALL (15% of cases), associated with favorable prognosis; this probe also identifies ETV6 deletion and iAMP21 (intrachromosomal amplification of chromosome 21) 1, 2
• Centromeric probes for chromosomes 4 and 10 - Detects high hyperdiploidy (trisomies 4 and 10), which carries favorable prognosis 1

Critical advantage: FISH detects cryptic abnormalities that conventional cytogenetics misses - studies show that 32% of cytogenetically normal pediatric ALL cases harbor cryptic abnormalities detectable only by FISH, particularly t(12;21) and del(9p) 2

Pediatric Acute Myeloid Leukemia (AML)

For pediatric AML, FISH panels should include: 1

• RUNX1::RUNX1T1 fusion probes for t(8;21) - Defines core binding factor (CBF)-AML with favorable prognosis 1
• CBFB rearrangement probes for inv(16) or t(16;16) - FISH confirmation is specifically advised because these abnormalities can be subtle on conventional cytogenetics, particularly with suboptimal chromosome morphology 1, 3
• KMT2A (MLL) rearrangement probes - Detects 11q23 abnormalities, which are frequently complex and/or cryptic 1
• PML::RARA fusion probes for t(15;17) - Diagnostic of acute promyelocytic leukemia; FISH may be initiated expeditiously when APL is suspected clinically 1
• NUP98 rearrangement probes - Particularly important for pediatric cases where NUP98 abnormalities are more common and often cryptic, associated with unfavorable outcome 1
• -5/5q- and -7/7q- probes - Detects monosomy 7 and 5q deletions, which occur in 3-5% of pediatric AML and carry adverse prognosis 1

Key clinical context: These genetic abnormalities together account for 50% of pediatric AML cases, a much higher frequency than in adults, making FISH particularly valuable in the pediatric population 1

Constitutional Genetic Disorders

Microdeletion/Microduplication Syndromes

FISH is the primary diagnostic test for microdeletion syndromes where deletions are frequent mutational mechanisms: 1, 4

• DiGeorge syndrome (22q11.2 deletion) - FISH is the preferred initial test when clinical diagnosis is highly suggestive 1, 4
• Prader-Willi and Angelman syndromes (15q11-q13 deletion) - Deletions account for approximately 70% of cases, making FISH highly clinically sensitive 1, 4
• Williams syndrome (7q11.23 deletion) - FISH detects the characteristic elastin gene deletion 1, 4

Important caveat: For syndromes where microdeletion is a minor cause of mutation (e.g., Rubinstein-Taybi syndrome with only 11-12% deletion rate), FISH has reduced clinical utility and should be considered only after other etiologies are excluded 1

Prenatal Diagnosis

FISH is a preferred rapid screening test for common aneuploidies in prenatal diagnosis, particularly in advanced maternal age (>35 years): 4, 5, 6

• Chromosomes 13,18,21, X, and Y - FISH using probes for these chromosomes detects approximately 80% of clinically significant abnormalities in advanced maternal age patients 4
• Trisomy 21 detection - Studies of 5,049 consecutive amniotic fluid samples demonstrate 100% sensitivity and 100% specificity for trisomy 21 detection by FISH 5
• Overall detection rate - FISH detects 65-70% of significant cytogenetic abnormalities across all prenatal indications 4

Technical advantage: FISH provides results in 24-48 hours compared to 7-14 days for conventional cytogenetics, allowing earlier clinical decision-making 5, 6

Clinical Algorithm for FISH Testing in Pediatric Hematologic Malignancies

Step 1: Initial Diagnostic Workup

• Perform bone marrow aspiration with morphology, immunophenotyping, and flow cytometry 1
• Initiate G-banded chromosome analysis and FISH panel simultaneously for rapid turnaround 1

Step 2: FISH Panel Selection

• For B-ALL: Use BCR::ABL1, KMT2A, ETV6::RUNX1, and centromeric probes for chromosomes 4 and 10 1
• For AML: Use RUNX1::RUNX1T1, CBFB, KMT2A, PML::RARA, NUP98, -5/5q-, and -7/7q- probes 1

Step 3: Interpretation and Additional Testing

• If initial FISH panel is negative in B-ALL, proceed with Ph-like ALL testing (CRLF2, PDGFRB, ABL1, ABL2, JAK2 rearrangement probes) 1
• If cytogenetics fails or shows poor morphology, FISH becomes the primary diagnostic modality 1
• For cryptic abnormalities suspected clinically but not confirmed by cytogenetics, targeted FISH is mandatory 1

Step 4: Risk Stratification

• Favorable risk: t(8;21), inv(16), t(15;17), ETV6::RUNX1, high hyperdiploidy 1, 3
• Adverse risk: KMT2A rearrangements (except t(9;11)), -7, -5/5q-, complex karyotype 1
• Use FISH results to guide intensity of therapy and consideration for stem cell transplantation 1, 3

Critical Pitfalls and Limitations

Maternal cell contamination: Occurs in 1.27% of prenatal samples, rendering 0.26% uninformative; always correlate FISH results with clinical context 5

False-negative risk: FISH only detects abnormalities for which specific probes are used; normal FISH does not exclude all chromosomal abnormalities 1, 4

Metaphase analysis requirement: When amplification or complex rearrangements are detected, conventional chromosome analysis should follow to detect homogeneously staining regions or double minutes 4

Probe limitations: FISH probes target specific loci and do not substitute for complete karyotypic analysis; very small deletions or point mutations may yield false-negative results 1, 4