Table 1

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

Evaluation	Considerations	Diagnostic approach
For bone marrow or peripheral blood involvement
Bone marrow aspirate and biopsy	Biopsy 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 therapy	Obtain 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
Immunophenotype (peripheral blood and bone marrow)	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
Cytogenetics	Evaluate 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
Chimerism	In 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 detection	Consider 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 feasible	In 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 evaluation	Perform as per routine to evaluate for disease	Consider 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.