VLA-4 and CXCR4 overexpression in bone marrow of an aleukemic B-cell acute lymphoblastic leukemia presenting with osteolytic bone lesions

Acute B-lymphoblastic leukemia (B-ALL) is usually characterized by a hyperleukocytosis together with infiltration of the bone marrow (BM) and lymphoid organs. Occasionally, extralymphatic B-ALL manifestation can occur, and a rare atypical B-ALL can develop with osteolytic lesions in the absence of any peripheral blasts [1]. The extensive bone involvement in these exceptional cases may be caused by deregulated tumor–bone interactions with increased recruitment of cell types responsible for bone destruction, particularly osteoclasts. Recent data suggested that vascular cell adhesion protein-1 (VCAM-1) serves to recruit and activate osteoclasts, resulting in bone destruction and enhanced tumor growth [2]. The receptor for VCAM-1, the integrin very late antigen-4 (VLA-4), is a key molecule of B cell homing, retention and early B cell development in the BM environment [3–5]. A second key regulator of these processes is the chemokine receptor CXCR4, in concert with its ligand stromal cell-derived factor-1 (SDF-1, CXCL12) [5,6]. CXCL12/CXCR4 signals activate VLA-4 [5] and deregulation of CXCL12/CXCR4 signaling can contribute to osteolysis [7].

Here, we present a case of an atypical presentation of B-ALL with initial manifestation of disseminated osteolytic bone lesions accompanied by significantly up-regulated expression of VLA-4 and CXCR4 and the respective ligands VCAM-1 and CXCL12.

In September 2012, a 23-year-old woman was referred to the 3rd Medical Department, Paracelsus Medical University, Salzburg, because of fatigue, night sweats and diffuse skeletal pain. A consequential X-ray showed disseminated osteolyses of the pelvis and proximal femora. Bone scintigraphy revealed multiple pathological accumulations in the skeleton [Figure 1(A)]. For detection of a supposed solid tumor, computed tomography (CT) was performed confirming lytic lesions in the pelvis, femur and proximal humerus on both sides [Figure 1(B)]. Nevertheless, a subsequent extended clinical work-up including gastroscopy, colonoscopy and mammography as well as dermatological and gynecological examination showed no evidence of a primary solid tumor. Blood count measurements showed a modest thrombocytopenia (platelet count 129 × 10⁹/L; normal range 150–400 × 10⁹/L) as well as slight normocytic, normochromic anemia (11.2 g/dL; normal range 11.5–16 g/dL). The leukocyte count was 5.06 × 10⁹/L (normal range 4–10 × 10⁹/L) with a normal distribution of leukocyte subsets. Notably, lactate dehydrogenase and alkaline phosphatase, expected to be elevated in cases of bone involvement of solid cancers or hematological neoplasia, were normal.

Figure 1. Radiological and immunohistochemical assessment of BM osteolysis in atypical c-ALL characterized by increased VCAM-1/VLA-4 and CXCL12/CXCR4 expression. (A) Bone scintigraphy anterior (left) and posterior (right) of the female patient with atypical c-ALL demonstrated multiple pathological accumulations in the skeleton with increased symmetric signals in the pelvis, femur and proximal humerus on both sides as indicated by arrows. (B) Computed tomography scan of the bone reveals osteolytic lesions in the pelvis and femurs on both sides (arrows). (C) Immunostainings (DAKO EnVision™ FLEX detection system; all antibodies ready to use; incubation for 30 min at 24°C) of TdT (pAB; Dako), CD34 (Clone QBEnd 10; Dako) and CD68 (Clone KP1; Dako) (brown) and hematoxylin counterstains (blue). First column, expression of TdT illustrates infiltration by precursor B-ALL cells; second column, CD34 expression for identification of hematopoietic stem/progenitor cells and distinct endothelial cells; third column, CD68 staining indicates the presence of macrophages and monocytes. First row, osteolytic BM from the patient with atypical c-ALL; second row, BM without osteolyses (atypical c-ALL); third row, representative BM from a case of typical c-ALL (n = 4). (D) Immunostaining (DAKO EnVision™ FLEX detection system; all antibodies incubated for 30 min at 24°C) of VCAM-1 (1:800; R&D), VLA-4 (1:30; AbD Serotec), CXCL12 (1:75, Clone 79018; R&D), CXCR4 (1:100, Clone UMB2; Epitomics) (brown) and hematoxylin counterstains (blue). First column, VCAM-1; second column, VLA-4; third column, CXCL12; fourth column, CXCR4. First row, osteolytic BM (atypical c-ALL); second row, BM region without osteolysis (atypical c-ALL); third row, representative BM from a case of typical c-ALL (n = 4); fourth row, representative BM from a healthy donor (n = 2). All immunostaining images were captured by the Olympus IX81 Xcellence System (UPLSAPO20 × O/0.85 objective lens; DP71 digital camera; cell^R software for Windows). Original magnification × 20 for all panels. Formalin-fixed, paraffin-embedded BM samples were obtained upon written informed consent (Salzburg Ethics Committee statement 415-EP/73/375-2014). (E) Immune reactive score (IRS) (0–1 negative, 2–3 mild, 4–8 moderate, 9–12 strong expression) of cells positive for VCAM-1, VLA-4, CXCL12 and CXCR4 protein expression. The IRS was calculated as follows: no color reaction (0 point), weak (1 point), moderate (2 points) and strong (3 points); staining intensity was multiplied by the percentage score of positive cells (1–25%, 1 point; 26–50%, 2 points; 51–75%, 3 points, 76–100%, 4 points), thus leading to scores of 1 to 4, 6, 8, 9 and 12. w/o, without; osteol., osteolytic; typ., typical; HPF, high power field.

Because of the mild thrombocytopenia and anemia, a trephine BM biopsy and an open bone biopsy of the trochanter major were performed. Immunohistochemical assessment revealed an 80–90% BM infiltration by blasts positive for CD79a, CD10 (both not shown), CD34 and nuclear terminal deoxynucleotidyl transferase (TdT) [Figure 1(C)]. On the other hand, these blasts were negative for CD3, CD20, pan cytokeratin, CK 8/18, S-100, myeloperoxidase, CD56, Vs38c, CD138, CD117 and CD45RO as well as kappa and lambda light chain (not shown), suggesting a common-ALL (c-ALL) with aleukemic presentation. Additional staining of CD68 indicated a higher incidence of macrophages/monocytes and osteoclast-like cells in lytic regions of atypical BM as compared to biopsies of patients with c-ALL with a classical clinical presentation [Figure 1(C)].

The patient was treated with a standard polychemotherapy regimen (doxorubicin, vincristine, asparaginase, cyclophosphamide, mercaptopurin and methotrexate) but achieved only a partial remission. In consequence, an allogeneic transplant with a matched unrelated donor was performed, resulting in a continuous complete remission (16 + months).

To determine whether the atypical presentation of B-ALL was associated with deregulation of the VLA-4/VCAM-1 and CXCR4/CXCL12 signaling axes, a second immunohistochemical analysis was conducted. The expression of these proteins in osteolytic and non-osteolytic BM areas of the discussed patient was analyzed and compared to the BM of four cases of typical c-ALL and two non-leukemic BM specimens [Figure 1(D)].

In both osteolytic and non-osteolytic bone regions of the patient with atypical c-ALL, VLA-4 expression on leukemic cells was highly increased and accompanied by an abundant and strong up-regulation of the respective ligand VCAM-1 on stromal and reticular cells [Figure 1(D), second and first column, respectively]. These findings were further quantified as an immune reactive score (IRS, see figure legend) [Figure 1(E)] with a VLA-4 IRS = 6 in osteolytic BM regions and IRS = 12 in non-osteolytic regions. VCAM-1 expression in osteolytic areas was scored with IRS = 4 due to the high percentage of VCAM-1-positive stromal cells. In contrast, leukemic cells of patients with typical c-ALL displayed lower VLA-4 (IRS = 1–2) and a scarce expression of VCAM-1 (IRS = 1–2) on remaining stromal cells. CXCL12 was presented on an extremely high number of endothelial cells within all BM areas of the osteolytic patient (60 CXCL12 + cells per high power field [HPF]). Within infiltrated regions of typical c-ALL BM only a few vascular cells were positive for CXCL12 (0–12 CXCL12 + cells per HPF). CXCR4 expression of both leukemic cells and residual hematopoietic cells was elevated solely in osteolytic regions (IRS = 8). CXCR4 was exclusively expressed by progenitor cells such as megakaryocytes (IRS = 1–3).

In conclusion, we found elevated numbers of CD68 expressing cells in lytic BM sites of the patient with atypical c-ALL that are presumably involved in osteoclast recruitment, maturation and function [8,9]. The unusual up-regulation of VCAM-1 and CXCL12 expression on BM endothelium and other stromal cells may further influence the attraction of VLA-4 + osteoclast progenitors, similar to observations of an involvement of VCAM-1 expression in osteolytic bone metastases in breast cancer [2]. Others demonstrated that high levels of multiple myeloma cell-derived CXCL12 stimulated the recruitment of osteoclast precursors to local sites within the BM and enhanced the motility and bone-resorbing activity of mature osteoclasts, thereby contributing to the formation of osteolytic lesions [7,10]. Our findings of elevated VCAM-1 and CXCL12 levels point to similar mechanisms in establishing osteolytic niches, providing a reasonable basis for further functional investigation of VLA-4/VCAM-1 and CXCR4/CXCL12 in an atypical presentation of ALL.

The interaction of the integrin–ligand pair VLA-4/VCAM-1 assists in retaining hematopoietic stem cells in the BM and contributes to B cell development therein [4,11]. Therefore, the observed increase in VCAM-1 expression on a subset of stromal cells and sinusoid endothelial cells within BM from the patient with atypical c-ALL and its respective ligand VLA-4 on c-ALL cells could be responsible for retention of the leukemic cells and, in turn, for deceptive normal blood counts. Elevated CXCL12/CXCR4 signaling may further add to this phenomenon as CXCL12 activates VLA-4 [12] and plays a crucial role in retention of developing B cells within fetal liver and BM [6]. Additionally, Mansour et al. [13] showed that osteoclast activity modulates B-cell development in the BM. Based on this finding, we hypothesize that an enhanced presence of macrophages/monocytes and osteoclast-like CD68 + cells can provide an explanation not only for the lytic processes, but also for the deregulated retention of c-ALL cells in their niche.

In summary, our data suggest potential molecules and mechanisms involved in an atypical and osteolytic presentation of precursor B-ALL, raising the basis for future exploration of VCAM-1/VLA-4 and CXCL12/CXCR4 axes in tumor-associated osteolyses. Targeting these signaling pathways may provide a therapeutic approach for the treatment of rare but severe cases of B-ALL, accompanied by skeletal osteolyses.

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