الفهرس 
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Abstract
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy (30%). The effective treatment of pediatric ALL is one of the great successes of Clinical Oncology, achieving survival rates of 80-85%. However, disease relapse remains the most common cause of treatment failure (15-20%).
The term ”minimal residual disease, MRD” describes a disease that is detected only by laboratory techniques more sensitive than morphology, such as flow cytometry (FCM) for immunologic MRD, or polymerase chain reaction (PCR) for molecular MRD. This residual disease is believed to be minimal, being found in the absence of clinical signs or symptoms. The MRD testing in acute leukemia is used for measuring early treatment response, and identifying patients who achieved morphologic remission, but still harbor considerable levels of disease. The ultimate goal of MRD assays is to guide therapeutic decisions by distinguishing patients in whom therapy must be continued or intensified to minimize the likelihood of clinical relapse.
The quantification of MRD by FCM is based on identification of markers defining a leukemia-associated phenotype (LAP), which is expressed on leukemic B- lymphoblasts, absent on hematogones, and ideally stable during therapy and at relapse. As phenotypic switch is common during treatment, multiple LAPs must be available and used for MRD detection over time. In MRD measurement, the FCM is frequently used being sensitive, rapid, widely applicable and relatively inexpensive.
The CD304 is a non-tyrosine kinase co-receptor for semaphorins and vascular endothelial growth factor (VEGF), implicated in neuronal guidance and angiogenesis. The CD304 is expressed on endothelial cells, blood plasmacytoid dendritic calls, and diverse human solid tumors. Levels of CD304 were reported to be increased in BM specimens of acute myeloid leukemia (AML) and ALL patients.
To study the role of CD304 as a marker for MRD in childhood ALL, thirty-five (35) newly-diagnosed pre-B ALL Egyptian children were included in this study, aiming to (1) detect the frequency of CD304 expression in BM specimens by FCM, (2) evaluate the differential expression of CD304 on B-ALL blasts versus normal hematogones, (3) evaluate CD304, as a marker for detection of MRD, (4) correlate CD304 expression with TEL-AML1, BCR-ABL fusion genes, and with other known prognostic factors in childhood B-ALL (clinical, demographic, laboratory data, immunophenotyping (IPT), risk stratification and clinical outcome). Twenty (20) normal, age and sex-matched, children were enrolled as controls.
All studied patients were subjected to full history taking, with special emphasis on age, gender, presence of leukemia-associated symptoms (fever, easy fatigability, bleeding tendency and bone aches) and thorough clinical examination, laying stress on the presence and extent of leukemia involvement, including: pallor, purpuric eruptions, hepatomegaly, splenomegaly, lymphadenopathy and testicular involvement. Complete blood count (CBC) and examination of Leishman-stained peripheral blood (PB) film, bone marrow (BM) examination, biochemistry profile (serum LDH, renal and liver function tests), IPT, FCM-CD304 at diagnosis and after morphological remission, and FISH analysis for the detection of TEL-AML1 and BCR-ABL (if not present in patient files) were done to all patients.
The FCM-CD304 threshold was adjusted, using a mean fluorescence intensity (MFI) threshold, based on the upper limit of CD304 by normal immature B-cell precursors (hematogones) (MFI cutoff = 1.2). At diagnosis, patients with CD304 >1.2 were designated “group A” (CD304+) and those with CD304 at diagnosis ≤ 1.2 were designated “group B” (CD304-). group A was further classified, according to the CD304 after morphological complete remission at day 28, into “group A1” (CD304+ or MRD+) and “group A2” (CD304- or MRD-).
The present study found that the CD304 expression on normal immature B-cells, in control BM samples, was low [MFI ranged from 0.8-1.2, with a mean of 0.9 ± 0.1] and statistically significantly lower than leukemic B lymphoblasts of pre-B ALL samples [p= 0.0108] which showed CD304 over-expression [MFI ranged from 0.8 to 6.7, with a mean of 1.6 ± 1.1].
The CD304 was over-expressed in 40% of pediatric pre-B ALL patients. The potential usefulness of CD304 as a MRD marker in pediatric ALL, was confirmed owing to its specificity (different expression between hematogones and leukemic lymphoblasts), and stability (in 42% of CD304+ cases).
The CD304+ ALL cases are characterized by higher incidence of lymphadenopathy [p = 0.012], higher uric acid levels [p = 0.012], presence of TEL-AML1 [p = 0.039], and higher blasts % expressing CD304 [p = ˂0.001]. On the other hand, there was no statistically significant difference regarding age, gender, presence of hepatomegaly, splenomegaly, fever, testicular enlargement, TLC, Hb, platelet count, PB or BM blasts %, serum LDH, calcium, creatinine, ALT, albumin, other IPT surface markers (CD10, CD34, CD20, myeloid markers and T-cell markers), presence of BCR-ABL, risk stratification or response to therapy [p = 0.222, 0.739, 0.694, 0.636, 0.685, 1.0, 0.445, 0.08, 0.893, 0.4, 0.126, 0.637, 0.127, 0.892, 0.827, 1.0, 0.506, 0.863, 0.685, 0.259, 0.7, 0.752, and 0.4, respectively].
The MRD+ pre-B ALL patients are characterized by absence of TEL-AML1 [p = 0.01] and belonged more to the high risk patients’ group [p = 0.015]. However, there was no statistical difference regarding age, gender, presence of hepatomegaly, splenomegaly, lymphadenopathy, fever, TLC, Hb level, platelet count, PB blasts, BM blasts %, serum LDH, uric acid, calcium, creatinine, albumin, ALT, other IPT surface markers (CD10, CD20, myeloid markers), BM blasts % expressing CD304 at diagnosis and after morphological CR, CD304 MFI at diagnosis, presence of BCR-ABL, or response to therapy [p = 0.138, 1.0, 0.825, 0.369, 1.0, 0.733, 0.796, 0.302, 0.561, 0.559, 0.074, 1.0, 0.599, 0.948, 0.09, 0.296, 0.897, 0.429, 0.209, 0.733, 0.447, 0.059, 0.235, 0.429, and 0.429, respectively].
Finally, the results of the present study confirmed the potential usefulness of CD304, as a MRD marker at day 28, in pediatric ALL, and determined the characteristic features of CD304+ pediatric pre-B ALL patients at diagnosis. It can be used for risk stratification, relapse prediction, therapy escalation (minimization or intensification) and predicting TEL-AML+ pre-B ALL.
The present work recommends including CD304 in the primary FCM diagnostic panel for pediatric pre-B ALL. The patients who are CD304+ should be re-evaluated for the presence of MRD at day 28. The use CD304 must be in combination with other markers to increase the sensitivity of FCM-based MRD detection, and to avoid false negative MRD results due to phenotypic shift, which is common with treatment. The use of CD304 also can predict TEL-AML1+ ALL. The results of MRD analysis can define prognosis and predict relapse, thus, helps planning treatment. Finally, the present work recommends to study the CD304 marker as a potential target for anti-leukemic and anti-angiogenic treatment strategies in acute leukemia.