Immunophenotypic shifts during minimal residual evaluation in a case of leukemic form of anaplastic large cell lymphoma ALK+

Abstract Background This study aims to describe immunophenotypic explorations at diagnosis and follow up of a pediatric patient with leukemic phase of ALK+ anaplastic large cell lymphoma (ALCL) by multiparametric flow cytometry (MFC). Case An 8‐color MFC combination of antibodies allowed to identify neoplastic cells in concentrations until 0.02% during minimal residual disease (MRD) monitoring. Immunophenotypic shifts occurred in key markers as CD30, CD7, CD2, and CD5, however neoplastic cells were clearly discriminated from normal populations. Conclusion MFC can be a useful tool for ALCL diagnosis and MRD monitoring and may support therapeutic decisions.

F I G U R E 1 Legend on next page. with leukemic presentation. Further, an 8-color panel permitted to identify the neoplastic cells, despite some immunophenotypic shifts during the treatment.

| Samples
All samples were obtained after receiving written informed consent by the donor's legal representative according to the Declaration of Helsinki.
The study was approved by the local ethics committee. BM aspirates were sequentially obtained for MFC analysis at day 0, +22, +48, and +64 of therapy; PB samples were collected at day 0 and +99; and samples from a cervical lymph node and cerebrospinal fluid (CSF) at day 0.

| Multiparameter flow cytometry (MFC) studies
EuroFlow SOPs were used for sample preparation, instrument setup, data acquisition and data analysis, as previously described. 5 More details are described in supplemental material. Monoclonal antibodies panels are available at Table S1.
The disease was treated according to ALCL 99 (arm IV) protocol, the international protocol for the treatment of childhood ALCL, 6 being high dose methotrexate (HD-MTX) omitted from the 1st cycle due to ascites and pleural effusions. Before the 2nd cycle of chemotherapy, MRD evaluation in BM by MCF showed 21.3% of neoplastic cells with a phenotypic profile similar to the one found at diagnosis. MRD decreased to 0.34% at the evaluation performed prior to the 3rd cycle, followed by a reduction to 0.02% prior to the 4th cycle of chemotherapy. In Figure 1, one can note that CD30, CD7, CD5, and CD2 suffered some immunophenotypic shifts during the treatment but neoplastic cells were easily identified using the combination of markers and the principal components analysis (PCA) plot. The population with higher expression of CD30 displayed chemoresistance and this fact can be better observed by comparing dotplot D1 to dotplot F I G U R E 1 Diagnosis and minimal residual disease monitoring of a patient with ALCL. Flow cytometric immunophenotypic analysis of peripheral blood samples (A1-F1) shows ALCL tumor cells (red) with a high sideward scatter (SSC-A), high levels of CD45 and CD30 expression, heterogeneous expression of cyCD3, CD2 and CD7 and the absence of surface CD3 and CD56. This phenotype differs from normal T cells (blue), which show: low SSC-A, CD45+, CD2+, cyCD3+, CD3+, CD7+, and CD30À. NK cells CD56 positive are colored green. Histopathological analysis (G) of the cervical lymph node sample was performed. Paraffin-embedded tissue sections were immuno-stained with monoclonal antibodies against CD30, ALK, EMA, Ki67, CD20, CD3, CD4, EBV, CD68 and CD1a; and sections were counterstained in Harris hematoxylin. Image G shows 100Â and 400Â magnification and ALK staining by immunohistochemistry. FISH analysis (H) of a tumor infiltrated bone marrow sample using dual color ALK break apart probe showing normal and abnormal ALK (2p23) gene arrangement. Fused red/green signals (overlap as a yellow signal) represent the normal ALK gene locus and the separated red and green signals represent an underlying ALK rearrangement (i.e., translocation -arrowhead). for BM staging in ALCL patients as even with the association of HP and IHC less than half of patients with infiltrated BM were identified. 7 Thus, MFC appears as a highly sensitive tool for ALCL diagnosis and follow up. In addition, ALCL MRD detection by MFC was less sensitive (10 -5 ), but less expensive and time consuming compared with the MRD study by reverse-transcriptase polymerase chain reaction (RT-PCR) technique. 8 In Table 1, a summary of the MFC studies performed to characterize ALCL cells, mostly at diagnosis, can be found. 2,3,[9][10][11] One of these studies 8

CONFLICT OF INTEREST
We would like to declare that we have no conflicts of interest on publishing this manuscript.

ETHICS STATEMENT
The present study was approved by the institutional board review and patient consent was obtained for publication of this report.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.