Table 1

Diagnostic Approach to Evaluation of Leukemia Detection Following B-cell Targeted Immunotherapy

EvaluationConsiderationsDiagnostic approach
For bone marrow or peripheral blood involvement
Bone marrow aspirate and biopsyBiopsy will provide essential information about bone marrow cellularity which will be helpful in the determination of potential myelodysplasia or cytopenias related to CAR T-cell therapyObtain both aspirate and biopsy
Immunophenotype (peripheral blood and bone marrow)What antigen was previously targeted?Select a flow cytometry panel to assess for the possibility of antigen loss/diminution
Is there any history of a myeloid malignancy
  • Select a flow panel which will assess for myeloid markers

  • Obtain prior diagnostic flow cytometry report to select an appropriate panel to identify relapse

CytogeneticsEvaluate prior cytogenetics, and if there is KMT2A/MLLr, consider possibility of lineage switch
  • Send cytogenetics on all samples (karyotyping, FISH)

  • Obtain prior reports

In patients with constitutional trisomy 21 and a predisposition to MDS/AML, consider the possibility of lineage switch
  • Send cytogenetics on all samples

  • Obtain prior reports

Consider impact of prior therapy in heavily pretreated patients
  • Evaluate for treatment-related malignancy

Genomic analysis
  • Evaluate for novel therapeutic approaches in patients with multiply relapsed/refractory disease

  • Evaluate for clonal evolution

  • Consider DNA-based deep sequencing or RNAseq to identify targetable mutations

  • If there is potential for leukemic evolution, consider repeating sequencing

ChimerismIn patients with history of HSCT, chimerism studies will help elucidate origin of disease
  • Consider XY-based or STR-based chimerism as available to evaluate for etiology of new/relapsed disease or donor-derived malignancy

CAR T cell detectionConsider the potential for CAR T cell-associated malignancy
  • Evaluate for CAR T cell persistence and clonal expansion, including ddPCR, vector integration site studies, and TCR sequencing studies

  • Evaluate for RCL

For extramedullary (EM) disease
Biopsy of any extramedullary disease as feasibleIn addition to the above, there is the possibility of discrepant results between EM disease and blood/marrow
  • Consider biopsy of EM in any patient with newly diagnosed EM disease

  • Flow cytometry for EM disease to look for immunophenotype

  • Consider PET/CT or PET/MRI scan to assess both extent of disease and treatment response

CSF evaluationPerform as per routine to evaluate for diseaseConsider additional flow cytometry
General considerations
  • For the evaluation of new disease detection following immunotherapy, always consider the possibility of lineage switch, antigen loss and/or secondary/treatment-related malignancies

  • In patients with prolonged cytopenias following immunotherapy, consider the possibility of MDS

  • Report findings of secondary malignancies to the appropriate regulatory authorities and industry sponsors

  • AML, acute myeloid leukemia; CAR, chimeric antigen receptor; CSF, cerebrospinal fluid; ddPCR, droplet digital PCR; FISH, fluorescence in situ hybridization; HSCT, hematopoietic stem cell transplantation; MDS, myelodysplastic syndrome; PET, positron emission tomography; RCL, replication competent lentivirus; STR, short-tandem repeats; TCR, T-cell receptor.