A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Abstract

Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD < 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

Regulation and Functions of α6-Integrin (CD49f) in Cancer Biology

“…Aside from motility, PI3K inhibition reduces cell surface expression of ITGA6 in acute lymphoblastic leukemia cells and limits CNS metastasis [ 56 , 59 , 117 ]. ITGA6 is strongly expressed in most acute lymphoblastic leukemia patients, and its interaction with laminin enhances the development of CNS disease [ 5 , 59 , 94 , 118 , 119 ] and the maintenance of minimal residual disease (MRD) after chemotherapy [ 15 ].…”

“…The condition under which clinical remission is established and remaining leukemia cells are detected using flow cytometry or polymerase chain reaction (PCR) testing is referred to as minimum residual disease [ 15 ]. In precursor B-acute lymphoblastic leukemia, the persistence of minimum residual disease at the completion of induction treatment (day 29) is a well-established prognostic predictor [ 15 , 119 , 120 , 123 , 148 , 149 ]. Although some mutations are linked to a bad prognosis, their presence at diagnosis does not always indicate relapse: some mutations can be lost at relapse, and others can be found in diagnostic samples that never relapse [ 68 ].…”

“…One of the top five elevated genes in CD34-positive leukemia cells is itga6 , and it is linked to poor therapeutic response [ 150 ]. In addition, itga6 is one of several genes overexpressed in B-acute lymphoblastic leukemia upon diagnosis, and it has been linked to the discovery of minimal residual disease later on [ 15 , 119 , 120 , 123 , 148 , 149 ]. The expression of ITGA6 may also be upregulated during chemotherapeutic agent treatment [ 60 , 123 , 148 ], or alternatively, the therapy may select a subset of ITGA6 blasts that are not visible at diagnosis [ 148 ].…”

“…The proportion of ITGA6+ blasts in hypodiploid and hyperdiploid patients appears to be more varied than in BCR-ABL1+ or ETV6-RUNX1+ instances [ 123 ]. ITGA6 expression has been found to be higher among (t12,21) and chromosome 21 amplification (iAMP21) patients [ 119 ]. It has also been reported that ITGA6+ is linked to a greater prevalence of ETV6-RUNX1 cytogenetic abnormalities and a lower frequency, although not statistically significant, of KMT2A rearrangement and hypodiploidy, while none of the ITGA6+ patients have iAMP21 or TCF3-PBX1 (t1,19).…”

“…Notably, ITGA6 expression is negatively associated with white blood cell count, as is characteristic of the B-other cytogenetic leukemia category, which cannot be categorized into any of the known cytogenetic groups [ 119 ]. In the case of strict bone marrow adherence, lesser amounts of leukemic blasts in circulation may be predicted, resulting in a high minimum residual disease but low white blood cell counts [ 119 , 149 ].…”