Fluorescence In Situ Hybridisation (FISH)

Fluorescence in situ hybridisation (FISH) uses fluorescently labelled DNA probes that bind to complementary sequences on chromosomes in metaphase spreads, interphase nuclei, or tissue sections, and visualises specific loci under a fluorescence microscope.

It detects copy number changes (deletion, amplification, aneuploidy), translocations (break apart, dual fusion and extra-signal probes), and locates genes or markers. Resolution is about 100 to 200 kilobases, higher than karyotype (5 to 10 megabases), and it works on non-dividing cells, so results are available in 24 to 72 hours with no culture.

Sensitivity for a defined target is over 98% in more than 200 scored nuclei, but it only looks at the regions targeted by the probes.

Rapid prenatal aneuploidy FISH (chromosomes 13, 18, 21, X, Y) in uncultured amniocytes gives answers in 24 to 48 hours.

In oncology, FISH detects BCR-ABL1, PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, KMT2A rearrangement, TP53 and ATM deletion, 13q deletion, trisomy 12 and IGH translocations in chronic lymphocytic leukaemia and myeloma, MYC, BCL2 and BCL6 rearrangements in lymphomas (double hit lymphoma), HER2 (ERBB2) amplification in breast and gastric cancer, ALK, ROS1 and RET rearrangements in lung cancer,

MYCN amplification in neuroblastoma, 1p/19q co-deletion in oligodendroglioma, and EWSR1 rearrangements in sarcomas.

In constitutional work it detects microdeletion syndromes (22q11.2, Williams 7q11.23, Prader-Willi and Angelman 15q11-q13, Smith-Magenis 17p11.2, Wolf-Hirschhorn 4p16.3, cri du chat 5p15) and subtelomeric rearrangements.

Indications. Rapid prenatal diagnosis of common aneuploidies. Suspected microdeletion syndrome with clinical features. Confirming and characterising a rearrangement or marker chromosome seen on karyotype. Haematological malignancy for diagnosis, risk stratification and treatment selection, and monitoring of minimal residual disease or chimerism after sex mismatched transplant.

HER2 status in breast and gastric cancer with equivocal immunohistochemistry (score 2+). Lung cancer biomarkers (ALK, ROS1, RET) and sarcoma translocations. Neuroblastoma (MYCN) and glioma (1p/19q). Failed or uninformative karyotype. Detection of mosaicism in interphase cells (Turner, Klinefelter).

Specimen. Peripheral blood (sodium heparin, 2 to 5 mL), bone marrow (sodium heparin, 1 to 2 mL), amniotic fluid, chorionic villi, cultured cells, fixed cell pellets (methanol:acetic acid 3 to 1) from cytogenetic cultures, buccal smears, or formalin fixed paraffin embedded tissue (4 micrometre sections, fixed in neutral buffered formalin for 6 to 72 hours, tumour area marked by a pathologist), fresh tumour touch preparations, and cytology smears.

Do not use acid decalcified bone biopsies. Request forms state the probe required or the clinical question.

Reference values. Normal pattern for the probe set: for example, two signals for each locus (2R2G), and no fusion signal or break apart separation in at least 200 cells (cut offs for each probe, usually 1 to 5% for deletions and 0.5 to 3% for fusions in interphase, set by validated normal controls).

Interpretation.

• Fusion signal (dual colour dual fusion): BCR-ABL1 in chronic myeloid leukaemia, PML-RARA in acute promyelocytic leukaemia, RUNX1-RUNX1T1, and EWSR1 partner genes in sarcomas, with percentages of positive nuclei recorded.
• Break apart pattern (separation of 5' and 3' signals): KMT2A, MYC, BCL6, ALK, ROS1, RET and EWSR1 rearrangements without identification of the partner.
• Loss of a signal (deletion): 13q14, 17p13 (TP53), 11q22 (ATM) in CLL, 1p/19q co-deletion, 22q11.2 deletion (DiGeorge syndrome, found in over 90% of cases), and 7q11.23 in Williams syndrome.
• Extra signals (gain, amplification): trisomy 12, trisomy 21 (three signals), MYCN and HER2 amplification (ratio of HER2 to chromosome 17 centromere of 2.0 or more, or average HER2 copy number of 6 or more, positive), and EGFR or MET amplification.
• Aneuploidy: three signals for chromosomes 13, 18, 21 or two X and one Y: confirmed by karyotype or quantitative fluorescent PCR and clinical context before irreversible decisions.
• Mosaic signals in interphase cells: sex chromosome mosaicism (45,X/46,XX) are sought by counting 200 to 500 nuclei.
• Atypical patterns (variant fusions, deletions at breakpoints, cryptic insertions) need further studies, such as RT-PCR or sequencing.
• Negative FISH does not exclude other genetic abnormalities outside the probe region; karyotype, microarray, RT-PCR, next generation sequencing or immunohistochemistry may be required.

Critical values and findings needing immediate action.

• PML-RARA positive: acute promyelocytic leukaemia, start all-trans retinoic acid immediately (even before confirmation by PCR) with arsenic trioxide, coagulopathy correction and intensive care.
• BCR-ABL1 positive in leukaemia: tyrosine kinase inhibitor treatment, and baseline quantitative PCR.
• Double hit lymphoma (MYC with BCL2 or BCL6 rearrangement): intensified therapy and central nervous system prophylaxis.
• HER2 amplification: targeted therapy with trastuzumab and other agents; ALK, ROS1 or RET rearrangement in lung cancer: targeted tyrosine kinase inhibitors.
• Prenatal trisomy 13, 18 or 21 on rapid FISH: urgent genetic counselling, confirmation by karyotype, and decision support within legal and gestational limits.
• TP53 deletion in CLL or myeloma: high risk disease, with selection of targeted agents rather than chemoimmunotherapy.
• MYCN amplification in neuroblastoma: high risk stratification and intensive therapy.

Interferences and pitfalls.

• FISH is a targeted test; abnormalities in other parts of the genome are missed, and a normal FISH result does not equal a normal karyotype.
• Poor fixation (cold ischaemia, over- or under fixation, acid decalcification), thick sections, necrotic tissue and autofluorescence reduce signal quality and may produce failed results (2 to 10% of solid tumours).
• Truncation of nuclei in tissue sections gives false deletions (signal loss); cut offs and scoring of at least 50 to 200 nuclei by two scorers reduce this.
• Overlapping signals can mimic fusions, and split signals in a single nucleus can occur by chance, which is why each probe has laboratory validated normal ranges.
• Cryptic, complex and variant rearrangements may not follow expected patterns; low level mosaicism in blood needs hundreds of cells.
• False negative interpretation from probe design (breakpoints outside the probe region), for example ALK and KMT2A, requires RT-PCR or sequencing in the face of high suspicion.
• FISH is labour intensive and expensive compared with PCR and immunohistochemistry, and it is being supplemented or replaced by next generation sequencing panels and chromosomal microarray in many applications.
• Results must be interpreted with morphology, immunophenotype and clinical findings, and incidental findings need reporting policies.
• Quality assurance requires validation of probes, positive and negative controls, proficiency testing and trained scorers.

Special groups.

• Prenatal patients: rapid FISH has a risk of maternal contamination and mosaicism; results are confirmed by karyotype, with microarray when ultrasound is abnormal.
• Children and infants with congenital heart disease, hypocalcaemia, cleft palate and learning problems: 22q11.2 deletion testing.
• Leukaemia patients: baseline FISH at diagnosis, with repeat tests at relapse and for clonal evolution, and chimerism studies after sex mismatched transplant.
• Patients with solid tumours: ensuring adequate tissue, with tumour content over 20 to 30% and pathologist selection.
• Infertile men and women: Y chromosome and X chromosome studies, and sperm FISH for aneuploidy in selected men.
• Regions: access is limited in low resource settings, where core tests (karyotype, immunohistochemistry, PCR) are prioritised and samples are sent to reference centres.

Follow up tests. Karyotype, chromosomal microarray, RT-PCR and quantitative PCR for fusion transcripts (BCR-ABL1, PML-RARA), next generation sequencing panels, immunohistochemistry (HER2, ALK, ROS1), flow cytometry and morphology for leukaemia, parental studies for microdeletions, echocardiography, calcium and immunological workup in 22q11.2 deletion, repeat FISH for relapse and minimal residual disease, and genetic counselling.