Residency · Residency · Pathology

Fluorescence In Situ Hybridization (FISH) in Hematopathology and Solid Tumors

Introduction

Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescently labeled DNA probes to detect specific chromosomal abnormalities in interphase or metaphase cells. FISH bridges the gap between conventional cytogenetics and molecular diagnostics, providing rapid, sensitive, and spatially resolved detection of translocations, amplifications, and deletions critical to cancer diagnosis and management.

Principles and Technique

Basic Methodology

A probe is designed as a single-stranded, fluorescently labeled DNA sequence complementary to the target chromosomal region. During hybridization, the probe binds to denatured target DNA on fixed tissue sections, cytology preparations, or cytogenetic preparations. Visualization uses fluorescence microscopy, with signals counted in individual cells (typically 50-200 cells scored). Results are expressed as the percentage of cells with the abnormality and compared against laboratory-established cutoff values.

Probe Types

Centromeric (CEP) probes target alpha-satellite DNA to identify specific chromosomes and are used for ploidy assessment such as trisomy and monosomy. Locus-specific identifier (LSI) probes target specific genes or chromosomal loci. Break-apart probes use two differently colored probes flanking a breakpoint, where separation of signals indicates translocation. Dual-fusion probes label two genes involved in a translocation, with fusion signals confirming the rearrangement. Dual-color, dual-fusion (DCDF) probes are the most specific for reciprocal translocations and reduce false positives from random signal juxtaposition.

Specimen Types

FISH works on touch preparations, cytospins, bone marrow aspirate smears, FFPE tissue sections (which require deparaffinization and protease digestion), peripheral blood smears (for example in CLL monitoring), and cytogenetic pellets from cultured cells.

Applications in Hematopathology

Acute Myeloid Leukemia (AML)

PML::RARA [t(15;17)] defines acute promyelocytic leukemia, which is ATRA-responsive; FISH provides rapid confirmation. RUNX1::RUNX1T1 [t(8;21)] and CBFB::MYH11 [inv(16)/t(16;16)] identify core binding factor AML with favorable prognosis. KMT2A (MLL) rearrangements at 11q23 involve multiple partners and generally carry adverse prognosis. DEK::NUP214 [t(6;9)] also confers adverse prognosis.

Acute Lymphoblastic Leukemia (ALL)

ETV6::RUNX1 [t(12;21)] is the most common pediatric ALL translocation with favorable prognosis and is cryptic on conventional karyotype, requiring FISH. BCR::ABL1 [t(9;22)] identifies the Philadelphia chromosome and tyrosine kinase inhibitor target. KMT2A rearrangements confer adverse prognosis, especially in infant ALL. Hyperdiploidy with more than 50 chromosomes is favorable and detected by CEP probes. Intrachromosomal amplification of chromosome 21 (iAMP21) is adverse.

Chronic Lymphocytic Leukemia (CLL)

del(13q14) is the most common abnormality, with isolated del(13q) carrying favorable prognosis. Trisomy 12 is intermediate. del(11q) at the ATM locus is adverse. del(17p) at the TP53 locus is the most adverse finding and predicts poor response to chemoimmunotherapy. The FISH panel is standard for CLL prognostication and treatment planning.

Multiple Myeloma

IGH rearrangements include t(4;14) [FGFR3/MMSET], t(14;16) [MAF], and t(14;20) [MAFB], all high-risk. t(11;14) [CCND1] is standard risk and venetoclax-sensitive. del(17p) is high-risk. 1q gain/amplification (CKS1B) is adverse. FISH is performed on CD138-selected plasma cells for optimal sensitivity.

Lymphoma

BCL2 rearrangement [t(14;18)] is the hallmark of follicular lymphoma. MYC rearrangement defines Burkitt lymphoma and is also tested in DLBCL for high-grade B-cell lymphoma classification. CCND1 [t(11;14)] identifies mantle cell lymphoma. ALK rearrangement is diagnostic of ALK-positive anaplastic large cell lymphoma. Double-hit or triple-hit lymphoma with concurrent MYC and BCL2 and/or BCL6 rearrangements is aggressive and requires FISH confirmation.

Applications in Solid Tumors

Breast Cancer

HER2 (ERBB2) amplification by FISH is a standard method when IHC is equivocal (2+). A HER2/CEP17 ratio of 2.0 or greater or HER2 copy number of 6.0 or greater constitutes amplification per ASCO/CAP guidelines. At least 20 cells must be counted in the invasive component using a dual-probe assay. Results guide trastuzumab and other HER2-directed therapy.

Lung Cancer

ALK rearrangement was originally detected by break-apart FISH as the FDA-approved companion diagnostic, though IHC and NGS are now often used instead. ROS1 rearrangement is confirmed by FISH. RET rearrangement is detected by break-apart FISH. MET amplification is assessed by the MET/CEP7 ratio.

Soft Tissue Tumors

EWSR1 rearrangement is diagnostic of Ewing sarcoma (with EWSR1::FLI1 being most common). SYT (SS18) rearrangement identifies synovial sarcoma. MDM2 amplification distinguishes well-differentiated and dedifferentiated liposarcoma from benign lipomatous tumors. DDIT3 rearrangement characterizes myxoid liposarcoma. USP6 rearrangement identifies aneurysmal bone cyst.

Urothelial Carcinoma

UroVysion FISH is a multiprobe assay detecting chromosomes 3, 7, and 17 polysomy along with 9p21 deletion. It is used on urine cytology specimens for surveillance and diagnosis of urothelial carcinoma and has higher sensitivity than cytology alone for low-grade tumors.

Brain Tumors

1p/19q co-deletion is the defining feature of oligodendroglioma per WHO criteria and is detected by FISH or NGS. EGFR amplification characterizes glioblastoma. CDKN2A/B homozygous deletion is an adverse prognostic marker in IDH-mutant astrocytoma and leads to a grade 4 designation.

Technical Considerations and Pitfalls

Signal Interpretation

Truncation artifact occurs because FFPE sections are only 4 micrometers thick, and nuclei may be cut, causing signal loss. This necessitates appropriate cutoff values. Cutoff establishment requires testing 200 or more normal cells to determine the false-positive rate, with the cutoff set at the mean plus 3 standard deviations. Overlapping signals may mimic fusion, and Z-stack imaging helps resolve ambiguity. Probe failure producing no signals or dim signals may indicate poor hybridization, making positive controls essential.

Quality Assurance

Validated protocols must exist for each probe and specimen type. Annual proficiency testing through CAP or equivalent programs is required. Technologist competency is assessed regularly, and results are correlated with morphology and other ancillary testing.

Clinical Pearls

FISH provides cell-level resolution for detecting specific chromosomal abnormalities and works on both fresh and FFPE specimens, making it versatile across hematopathology and surgical pathology. In CLL, the FISH panel directly informs treatment decisions, with del(17p) identifying patients unlikely to respond to standard chemoimmunotherapy. HER2 FISH in breast cancer must follow ASCO/CAP guidelines for scoring, and equivocal IHC results (2+) require FISH confirmation before initiating HER2-directed therapy. Break-apart FISH probes are particularly useful for detecting translocations when the partner gene is variable or unknown.

References

1. Wolff AC, et al. HER2 testing in breast cancer: ASCO/CAP clinical practice guideline focused update. J Clin Oncol. 2018;36(20):2105-2122.
2. Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed. IARC; 2017.
3. Bridge JA. Advantages and limitations of cytogenetic, molecular cytogenetic, and molecular diagnostic testing in mesenchymal neoplasms. J Orthop Sci. 2008;13(3):273-282.
4. Hastings RJ, et al. Guidelines for cytogenetic investigations in tumours. Eur J Hum Genet. 2016;24(1):6-13.

Read this lecture as Markdown