Paired Immunophenotype Comparison Of Diagnosis and Relapse Samples In B-Lineage Acute Lymphoblastic Leukemia

Although great progress has been made in the treatment of acute lymphoblastic leukemia (ALL), relapse remains a major issue in the follow-up of these patients. Recent data about the emergence of subclones during haematological malignancies suggest that relapses could result from resistant cells initially in minority or from cells driven to resistance by previous treatments. Among the tools allowing for the characterization of leukemic cells, flow cytometry (FCM) is an essential approach. Increasingly used to evaluate minimal residual disease (MRD) based on the immunophenotypic features of the blasts at diagnosis, it can also allow to identify immunophenotypic shifts related to clonal evolution. Such an approach would be best studied by comparing follow-up samples from the same patients. In order to be thorough, this would however require that conditions as similar as possible are applied to both types of cells.

This work was designed 1) to compare the immunophenotypic features of B-ALL blast cells with those of normal hematogones, 2)to assess potential immunophenotypic shifts at relapse 3)to determine the stability of markers not classically used at diagnosis during follow-up and their potential utility for MRD.

FCM was performed simultaneously on thawed paired samples from 15 patients (9 children aged between 1 and 12 years old and 6 adults aged between 23 and 71 years old) with B-lineage ALL. With a three-tubes 8 colours panel comprising a backbone of CD45, CD34, CD22 and CD10, the expression of 8 markers was examined and compared to that observed on normal hematogones contained in 29 bone marrow samples from healthy donors. These 8 markers were CD7, CD19, CD20, CD24, CD38, CD58, CD81, CD123. Moreover, an additional four colours panel was used to examine the more recently described antigens CD44, CD200, CD304 and Her2Neu. The presence of leukemia associated immunophenotypes (LAIP) was defined as a difference in mean fluorescence intensity (MFI) between hematogones and blasts of at least 2 standard deviations.

At diagnosis, the expression of each marker was at variance from that on hematogones yielding LAIP in all patients, with at least 4 aberrant markers (up to 11). Antigens with the most abnormal expression were CD10 (100%), CD24 (93.3%), CD81 (80%), CD38 (60%) and CD58 (53.3%). Antigens with the least aberrant expression were CD19 (20%), CD22 (20%), CD123 (20%), CD34 and CD20 (46,7%).

CD44 which is at a low level on hematogones, was present for 80% of the patients at diagnosis and overexpressed in ALL with MLL rearrangement (3/15 cases). CD200 was overexpressed in 73% of the patients while CD304 was present in only 40% of the patients. A single patient was positive for Her2Neu, which remained present at relapse.

All patients retained at relapse the same global immunophenotype without any change in the EGIL classification (3 B-I, 8 B-II, 4 B-III) and the difference with hematogones remained. The expression of most markers was similar at diagnosis and relapse. There was no change at all for the expression of CD38 which therefore appears as the most interesting marker for follow-up and MRD in ALL. Only one patient each showed a change in the expression of CD44, CD58 or CD123. As a whole, stable markers were CD58, CD44, CD200, CD81 and CD24 in contrast with CD19, CD22, CD123, CD304, CD24 and CD20 which changed in 27 to 67% of the patients.

Four patients displayed no immunophenotypic change at relapse while 3 showed a modification of a single marker. For 5 patients, with respectively 6 and 7 LAIP, two markers were modified at relapse. Three markers changed for the patient with Her2Neu expression. Finally, only two patients (13%) showed major changes possibly associated with the emergence of a new clone.

This study confirms that B-ALL blast cells differ immunophenotypically from hematogones, although the latter have been reported to possibly be their normal counterpart. These data moreover comfort the interest of using LAIP in the detection of MRD in multiparameter FCM. Finally, since molecular targets of therapeutic monoclonal antibodies do not shift sensibly, their use can also be considered at relapse.