When should fluorescence in situ hybridization (FISH) testing be performed in multiple myeloma, and which cytogenetic panel should be ordered for risk stratification?

FISH Testing in Multiple Myeloma

FISH testing should be performed at diagnosis in all patients with newly diagnosed multiple myeloma (NDMM) and repeated at relapse, using a standardized panel that includes 17p deletion (TP53), 1p deletion, 1q gain/amplification, and IGH rearrangements with reflex testing for t(4;14), t(14;16), t(14;20), and t(11;14). 1

Timing of FISH Testing

At Diagnosis

• Perform FISH on all newly diagnosed MM patients as part of the initial bone marrow evaluation 2, 1
• Testing should be done on bone marrow aspirate and/or biopsy specimens, ideally with CD138+ plasma cell enrichment to improve detection rates 2, 1
• FISH is essential for risk stratification using the Revised International Staging System (R-ISS and R2-ISS), which directly impacts treatment decisions and prognosis 1, 3

At Relapse

• Repeat FISH testing at relapse/progression in all patients with relapsed/refractory MM (RRMM) 1
• This is critical because cytogenetic abnormalities evolve: del(17p) increases from 7-10% at diagnosis to up to 80% at relapse, and 1q gain increases from 30-45% to 55-70% 1
• The relapse panel must include at least one probe targeting the primary abnormality found at diagnosis as a positive control to confirm you're analyzing the malignant clone 1

Recommended FISH Panel Design

For Newly Diagnosed MM (NDMM)

The 2025 Cancer Genomics Consortium guidelines provide the most current standardized approach 1:

Mandatory probes:

• 17p13 deletion (TP53) - present in 7-10% of NDMM, defines high-risk disease 1
• 1p32 deletion (CDKN2C) - present in 8-12% of NDMM 1
• 1q21 gain/amplification (CKS1B) - present in 30-45% of NDMM; distinguish between gain (3 copies) and amplification (≥4 copies) 1
• IGH break-apart probe (BAP) as initial screen for IGH rearrangements 1

Reflex testing when IGH-BAP is abnormal:

• t(4;14) - present in ~15% of NDMM, high-risk 1
• t(14;16) - present in 3-5% of NDMM, high-risk 1
• t(14;20) - present in 1-2% of NDMM, high-risk 1
• t(11;14) - present in 15-20% of NDMM, standard-risk but predicts BCL2 inhibitor sensitivity 1

For Relapsed/Refractory MM (RRMM)

Mandatory probes: 1

• 17p deletion (TP53)
• 1p deletion
• 1q gain/amplification
• Plus at least one probe targeting the primary abnormality detected at diagnosis (serves as positive control)

Technical Approach

Laboratory Methodology

• Use an IGH break-apart probe as initial screen - this is cost-effective and labor-conscious 1
• When IGH-BAP is abnormal (~80% will have a recurrent translocation), perform reflex testing with dual-color, dual-fusion (DC-DF) probes for the four key translocations 1
• This two-step approach is supported by evidence that nearly all recurrent IGH rearrangements result in an abnormal IGH-BAP result, and primary IGH rearrangements are generally mutually exclusive 1

Plasma Cell Enrichment

• CD138+ plasma cell enrichment is strongly recommended to improve detection sensitivity 2, 1
• Standard metaphase cytogenetics should also be included despite low yield (20%), as it can identify hyperdiploid vs. non-hyperdiploid patients and capture uncommon abnormalities 2

Clinical Significance and Risk Stratification

High-Risk Cytogenetic Abnormalities

The presence and number of high-risk abnormalities directly impacts survival 4, 5:

Ultra high-risk (worst prognosis):

• del(17p) - associated with shortest PFS and OS 4
• "Double-hit" or "triple-hit" profiles (multiple high-risk abnormalities together) 5

High-risk combinations per updated IMS/IMWG criteria:

• 1q gain is high-risk only when co-occurring with monoallelic del(1p) 4
• IGH translocations [t(4;14), t(14;16), t(14;20)] are high-risk only when accompanied by 1q gain or del(1p) 4

Prognostic Impact

• Increasing burden of high-risk cytogenetic abnormalities correlates with shorter PFS and OS 4
• Cytogenetic complexity (multiple abnormalities) is a key determinant of outcome, more so than individual abnormalities alone 5
• Each high-risk abnormality independently confers poorer outcomes compared to no abnormalities 4

Common Pitfalls and Caveats

Testing Gaps

• In real-world practice, FISH data are available in only 57% of patients 4, representing a significant care gap
• Some centers do not routinely perform FISH in elderly patients (>80 years) 3 - this is inappropriate as cytogenetic risk stratification should guide treatment decisions regardless of age

Standardization Issues

• Variability exists in CD138+ cell purification methods and cytogenetic abnormality detection thresholds between laboratories 3
• Reporting of 1q abnormalities has been particularly challenging - ensure your laboratory distinguishes between gain (3 copies) and amplification (≥4 copies) 1

Post-Treatment Monitoring

• FISH testing is NOT recommended for minimum residual disease (MRD) evaluation - use flow cytometry or next-generation sequencing instead 1
• FISH should only be repeated at clinical relapse/progression, not during routine follow-up of responding patients

Interpretation Errors

• Do not rely on del(13q) alone for risk stratification - it was included in older panels but is no longer considered independently prognostic 3
• Hyperdiploidy detection by FISH is inconsistently performed (only 52% of centers) 3 - while useful, it's not part of the minimal required panel