How is multiple clonality managed in patients with hematological malignancies?

Management of Multiple Clonality in Hematological Malignancies

Primary Recommendation

When multiple clonal populations are detected in hematological malignancies, verify their presence through immunophenotypic analysis and manage patients according to the most aggressive clone, particularly treating cases with discordant somatic hypermutation status as unmutated disease. 1

Detection and Verification Strategy

Initial Assessment

• Perform duplicate PCR analyses from the same or separate DNA isolates to confirm reproducibility of clonal peaks or bands, as non-reproducible findings may represent selective amplification from paucicellular specimens rather than true multiple clonality 1
• Analyze a minimum of 20 metaphases at diagnosis when no abnormality is initially found, which excludes chromosomally abnormal clones of ≥14% with 95% confidence 1
• Examine cells of varying quality to maximize detection of neoplastic clones, as normal cells with better chromosome morphology may mask abnormal populations 1

Confirmation of Multiple Clones

• Verify through immunophenotyping to confirm the presence of two or more distinct B-cell or T-cell clonal populations before concluding multiple clonality exists 1
• Consider next-generation sequencing (NGS) to assess the relative frequency of each clonotype and identify the predominant clone when Sanger sequencing shows multiple productive rearrangements 1
• Be aware that co-existing clones and clonal evolution may be present, requiring additional analysis from more than one culture regimen if suspected 1

Disease-Specific Management Approaches

Chronic Lymphocytic Leukemia (CLL)

Double Productive Rearrangements:

• Concordant somatic hypermutation (SHM) status: Manage as standard mutated (M-CLL) or unmutated (U-CLL) based on the shared SHM status 1
• Discordant SHM status: Manage as U-CLL regardless of which clone is dominant, as this approach errs on the side of patient benefit given the adverse prognosis associated with unmutated disease 1
• One productive and one unproductive rearrangement: Define mutational status solely by the productive rearrangement, ignoring the SHM status of the unproductive rearrangement 1

Multiple (≥3) Productive Rearrangements:

• Perform immunophenotyping to verify presence of multiple clonal populations 1
• Use NGS to determine the relative frequency of each clonotype 1
• Manage according to the predominant clonotype if clearly identified, though specific frequency thresholds remain to be established 1
• If SHM status is discordant across clones, manage as U-CLL with close follow-up 1

Multiple Myeloma (MM)

Clonal Evolution Recognition:

• Understand that tumor populations consist of various subpopulations with differing sensitivities to available agents, leading to "clonal tides" where therapy suppresses one clone while another emerges 1
• Disease progression on a specific agent does not preclude its future use, as clonal selection may render previously resistant populations sensitive again 1
• Monitor for clonal evolution during follow-up, as minor IG rearrangements can persist over time and account for 0.2-3% of the total repertoire 1

Treatment Implications:

• Use triple combination therapy with at least one new drug class when relapse occurs, as this addresses potential clonal heterogeneity more effectively than continuing the same therapeutic approach 1
• Consider second autologous stem cell transplant (ASCT) for patients with meaningful initial response (≥18 months without maintenance or >36 months with maintenance), as this can address evolving clonal populations 1

B-Cell Lymphoproliferations

Target Selection Strategy:

• Begin with IGH VH-JH multiplex PCR (3 tubes) as the first-line approach 1
• Add IGK analysis (Vκ-Jκ and Kde rearrangements, 2 tubes) as the second step, preferably in parallel to avoid diagnostic delay 1
• Reserve IGH DH-JH and IGL analyses for cases where preceding PCRs fail to detect monoclonality and show no clear polyclonality 1

T-Cell Lymphoproliferations

Complementary Testing:

• Use TCRB and TCRG PCR in parallel as they provide complementary information, with TCRB generally slightly more informative 1
• Analyze both TCRB Vβ-Jβ tubes and both TCRG V-J tubes in duplicate to ensure no PCR products are missed 1

Follow-Up and Monitoring

Post-Treatment Assessment

• When normal cytogenetics at diagnosis: Further cytogenetic analysis is usually not appropriate 1
• When abnormal cytogenetics at diagnosis and normal result obtained post-treatment: Analyze or score a minimum of 20 metaphases for the diagnostic abnormality 1
• When abnormal clone persists: Limit minimum analysis to 10 metaphases 1
• Prefer quantitative PCR, molecular technologies, or multi-parametric flow cytometry for minimal residual disease detection over cytogenetics when possible 1

Relapse or Transformation Surveillance

• Process samples as diagnostic specimens when clinical information suggests relapse, refractory disease, or secondary hematological neoplasm 1
• Perform bone marrow aspiration to identify and quantify clonal plasma cell percentage and detect high-risk genetic abnormalities in MM 1

Critical Pitfalls to Avoid

Technical Considerations

• Do not dismiss single abnormal metaphases without further screening or complementary testing, particularly single cells with trisomy 8 or monosomy 7 in myeloid neoplasms 1
• Do not exclude polyploid or hypodiploid metaphases from analysis, though cells with loss of >6 chromosomes cannot be fully analyzed unless loss is clonal 1
• Avoid relying solely on scored cells for follow-up reports; qualify that analysis only excludes the specific abnormality scored 1

Biological Considerations

• Recognize that heavily mutated B-cell clones may remain undetected in standard IGH PCR, resulting in false polyclonal or absent profiles 1
• Be aware that monoclonal products in one multiplex PCR may cause lack of specific amplicons in complementary multiplex PCR targeting the same locus due to absence of reactive lymphocytes 1
• Understand that subset #2 CLL membership is associated with adverse prognosis and inferior response to chemoimmunotherapy regardless of SHM status 1

Clinical Management Errors

• Never assume all clones require simultaneous targeting; sequential therapy may be more appropriate as different clones emerge over time 1, 2
• Do not continue the same therapeutic approach when multiple clonality is suspected at relapse; change to at least one new drug class 1
• Avoid underestimating the clinical significance of minor clonotypes detected by NGS, as they may represent biologically relevant populations that persist and expand over time 1