Nurse-like cells impact on disease progression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is one of the most common B-cell malignancies in adults, characterized by an accumulation of monoclonal CD5 mature B cells in lymphoid tissues and peripheral blood. The complex interplay between malignant CLL cells and their surrounding bystander tumor microenvironment (TME) is critical for their survival and facilitates both their resistance to drug therapy and their orchestration of an immunosubversive milieu. CLL cells proliferate in distinct microanatomical tissue sites, termed proliferation centers, that allow intimate interactions with TME components notably nurse-like cells (NLC). These cells represent a leukemia-associated counterpart of tissue-associated macrophages, expressing high level of CD68 and CD163 and mediating chemoresistance particularly to ibrutinib, dexamethasone and chlorambucil. Even if these cells was well studied by in vitro approaches, the in vivo clinical impact of NLC outgrowth in the TME has not been definitively addressed to date in CLL, as described for the tumorassociated macrophages (TAMs) in solid tumors. If high TAM infiltration has been associated with a worse outcome in several solid cancers as well as in hematological malignancies including diffuse large B-cell lymphoma, in CLL studies were focused on indirect proof of evidence. Indeed, if CCL3 levels are associated with shorter treatment-free survival (TFS) and HMGB1 expression levels in the TME have been correlated with overall survival (OS), these prognostic factors do not clearly identify NLC infiltration as an important parameter associated with CLL outcome. In the present study, we demonstrated that TME, particularly NLC, could impact CLL progression. For this purpose, we reported for the first time that (i) CD163 NLC expression is correlated with CLL proliferation in lymph nodes (LNs), (ii) high soluble CD163 (sCD163) levels, the soluble counterpart of CD163, are linked with the worst prognostic factors in this disease, namely TP53 mutations, complex karyotype and unmutated immunoglobulin heavy-chain variable (IGHV) status and (iii) high levels of sCD163 are associated with shorter TFS and OS. To determine the clinical impact of NLC, we first compared the in vivo anatomical pattern of CD163 macrophages in both healthy donor (reactive tonsils, Figure 1a) and CLL LNs tissue sections (Figure 1b). In normal LNs and tonsils, CD163 macrophages were confined to the sub-capsular areas and along the lymphatic sinuses, whereas the B-cell zones were devoid of CD163 cells, as previously reported. Moreover, in CLL LNs, CD163 macrophages were present in the medulla, intertwined with leukemic cells and pseudofollicles. Confocal microscopy revealed that CD163 cells were consistently CD68 in CLL LNs, while some CD68 macrophages were devoid of CD163 expression (Supplementary Figure 1). Because CD68 can also be expressed by non-myeloid cells, we hypothesized that CD68 staining might over-interpret the exact quantity of NLC in LNs and we decided to use CD163 as the most relevant marker for NLC. We next questioned the reliability of CD163 NLC within TME with regards to disease progression. We then performed immunohistochemistry analyses on 27 previously untreated CLL patients for whom nodal tissue biopsies were available in our institution. These patients had either a suspicion of disease transformation or were biopsied before CLL was evidenced from the immunophenotyping of circulating lymphocytes. As shown in Figures 1c and d, some patients (for example, patients 1 and 2) had few Ki-67 proliferating CLL cells within pseudofollicles (and few CD163 cells), and had indolent CLL. Notably, these watch and wait patients still had more CD163 macrophages intertwined with tumoral B cells, as compared with normal LNs. In contrast, patients with active CLL (as defined by IWCLL2008 criteria) requiring therapy presented with (i) an increased frequency and size of pseudofollicles (for example, patients 3 and 4, Figures 1c and d), (ii) a higher fraction of Ki-67 CLL cells and (iii) a significantly higher number of CD163 cells. This is particularly relevant in the proliferating centers, as indolent CLL had very few CD163 cells in these areas. Altogether, these data from a large cohort of 27 patients suggested that CD163 cells load within the TME was significantly higher as CLL progressed towards first line of treatment. In previous studies, sCD163 may be used as a reliable marker of the total pool of CD163 cells in various conditions. Because of the limited availability of LN biopsies, and the absence of CD163 expression by circulating monocytes in CLL patients, we evaluated sCD163 levels (in serum) and other related soluble factor such as soluble CD68 (sCD68) and soluble HGMB1 (sHMGB1). Indeed, we found a higher amounts of sCD163, sCD68 and sHMGB1 in the sera of CLL patients than in age-matched normal donor sera (Figure 1e). Then, in this cohort of 94 patients naive to therapy (for thorough analysis of patient characteristics, see Supplementary Table 1), we correlated soluble factors levels with previously established prognostic markers. We found a statistically significant association between high sera levels of sCD163 and poor prognostic markers, such as unmutated IGHV status, complex karyotypes (defined by 43 anomalies), the presence of TP53 mutations and a trend towards an association with del(17p) and NOTCH1 mutational status (P= 0.055; Table 1). This was very relevant as these markers are considered as the worst prognostic markers in this disease. On the other hand, we found no such correlation with Binet stage, suggesting aggressiveness of disease reflected more faithfully than ‘crude accumulation’ of leukemic cells within tissues, and the amount of CD163 macrophages in these patients (Supplementary Figure 2). We did not find any evidence of association between sCD68 or sHMGB1 levels and CLL prognostic factors (Table 1). Lymphocytosis (that is, circulating tumor bulk) or monocytosis (that is, putative precursors of NLCs once recruited to tumor niches) also had no impact on the levels of the three soluble factors studied (Supplementary Figure 2). Altogether, as sCD163 levels did not correlate with sCD68/HMGB1 levels (Supplementary Figure 2), our data suggest for the first time that aggressive CLL modulate their TME to increase the education of monocytes into CD163 cells. Of the 94 patients included in our study, 78 required treatment, according to the IWCLL2008 criteria for active disease, and 24 died during follow-up. Median time to first treatment in the entire cohort was short (36 months; Supplementary Tables 2 and 3). By receiver operating characteristic analysis we found that 1000 ng/ml of sCD163 was the best threshold to segregate patients into two Citation: Blood Cancer Journal (2016) 6, e381; doi:10.1038/bcj.2015.108