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Even though the BCR-ABL tyrosine kinase inhibitor imatinib significantly improves the prognosis of chronic myelogenous leukaemia (CML) patients, drug resistance is a major obstacle to better management. We examined the interaction of recently defined bone marrow microenvironment factors CXCL12 and ATP-binding cassette (ABC) transporters in the bone marrow of CML patients in the chronic phase and blast crisis.Expression levels of mRNA extracted from frozen specimens of CML patients were measured by real-time polymerase chain reaction. The expression of the ABC transporters MDR1, ABCC1, ABCG2, and CXCL12 was significantly higher in the bone marrow samples of CML blast crisis than in those of CML chronic phase. Immunohistochemical staining for CXCL12 revealed that the proportion of CXCL12 positive reticular cell areas correlated well with the mRNA levels of CXCL12 in CML bone marrow. Finally, co-culture experiments of K562 CML cells with CXCL12 expressing mesenchymal cells (OP9 cells or human CXCL12 transfected 3T3 cells) revealed enhanced mRNA levels for MDR1 in a CXCL12 rich environment.These results suggest that imatinib treatment restores the bone marrow microenvironment in CML with the presence of CXCL12 expressing reticular cells but in turn induces the overexpression of MDR1 in haematopoietic cells due to up-regulated expression of CXCL12.

The bone marrow microenvironment, known as 'hematopoietic stem cell niche,' is essential for the survival and maintenance of hematopoietic stem cells. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell diseases, which eventually result in leukemic transformation (acute myelogenous leukemia with myelodysplasia-related changes, AML-MRC). However, the precise components and functions of the MDS niche remain unclear. Recently, CXCL12-abundant reticular cells were shown to act as a hematopoietic stem cell niche in the murine bone marrow. Using immunohistochemistry, we show here that CXCL12(+) cells were located in the cellular marrow or perivascular area, and were in contact with CD34(+) hematopoietic cells in control and MDS/AML-MRC bone marrow. MDS bone marrow exhibited higher CXCL12(+) cell density than control or AML, not otherwise specified (AML-NOS) bone marrow. Moreover, AML-MRC bone marrow also exhibited higher CXCL12(+) cell density than control bone marrow. CXCL12(+) cell density correlated positively with bone marrow blast ratio in MDS cases. CXCL12 mRNA level was also higher in MDS bone marrow than in control or AML-NOS bone marrow. In vitro coculture analysis revealed that overexpression of CXCL12 in stromal cells upregulated BCL-2 expression of leukemia cell lines. Triple immunostaining revealed that the CD34(+) hematopoietic cells of MDS bone marrow in contact with CXCL12(+) cells were BCL-2-positive and TUNEL-negative. In the bone marrow of MDS cases, CXCL12-high group showed significantly higher Bcl-2(+)/CD34(+) cell ratio and lower apoptotic cell ratio than CXCL12-low group. Moreover, CXCL12-high refractory cytopenia with multilineage dysplasia (RCMD) cases had a greater tendency to progress to refractory anemia with excess blasts (RAEBs) or AML-MRC than CXCL12-low RCMD cases. These results suggest that CXCL12(+) cells constitute the niche for CD34(+) hematopoietic cells, and may be associated with the survival/antiapoptosis of CD34(+) hematopoietic cells and disease progression in MDS. Thus, CXCL12(+) cells may represent a novel MDS therapeutic target.

Background: Mycosis fungoides (MF) classically presents from patch stage to plaque stage over a number of years and finally progresses to tumour stage with nodal or visceral involvement. The mechanism of progression remains incompletely elucidated. Chemokines and their receptors are known to be involved in disease mechanisms, with CXCL12 and CXCR4 playing a critical role in carcinogenesis, invasion and cancer cell migration in various carcinomas., Objectives: To investigate the expression of CXCL12 and CXCR4 in different cutaneous stages of MF., Methods: Formalin-fixed, paraffin-embedded skin samples from 40 patients with MF (21 patch stage, 10 plaque stage, nine tumour stage) and 30 non-neoplastic control skin samples were analysed. CXCL12 and CXCR4 were assessed by quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining., Results: The expression level of mRNA for CXCL12 in plaque-stage MF was significantly higher than in control skin (P = 0.0035), or patch-stage (P = 0.0108) or tumour-stage disease (P = 0.0089). The CXCR4 mRNA expression level in plaque-stage disease was significantly higher than in control skin (P = 0.0090) or patch-stage disease (P = 0.0387). CXCL12- and CXCR4-positive cell rates in patch-stage and plaque-stage MF were significantly higher than those in control skin (P < 0.0001). CXCL12- and CXCR4-positive cell rates in tumour-stage MF were significantly lower than those in patch- and plaque-stage disease (P = 0.0274 and P = 0.0492, respectively)., Conclusions: Our data suggest that neoplastic T cells in MF are exposed to the microenvironment, given the abundance of CXCL12 during its progression, and also that neoplastic T cells express CXCR4, especially in the pretumour stage. We reveal that the CXCL12-CXCR4 axis plays a critical role in MF progression., (© 2014 British Association of Dermatologists.)

Aims: To determine the expression of CXCL12 in human spleens with extramedullary haematopoiesis (EMH) for clarifying the association of splenic haematopoietic stem cells (HSCs) with CXCL12, which has been demonstrated to be a marker of bone marrow niches., Methods: We examined the expression of mRNA for CXCL12 by quantitative reverse transcription polymerase chain reaction (RT-PCR) and localised the CXCL12 protein by immunohistochemical staining in EMH negative and positive spleen samples from autopsy cases., Results: Expression of CXCL12 was significantly higher in samples from EMH positive cases than those from EMH negative cases. CXCL12 was localised to the endothelial cells of the sinuses of the red pulp in EMH positive spleens while vascular endothelial cells of the white pulp expressed CXCL12 throughout the spleen. c-kit positive/CD34 negative cells were identified in contact with CXCL12 positive endothelial cells of sinuses in EMH positive cases, although the number was few. In contrast, erythroblastic islands were frequently observed in EMH positive cases and dominantly localised to the intrasinusoidal spaces in association with CD68 positive macrophages., Conclusions: Our results suggest that endothelial cells of splenic sinuses expressing CXCL12 may contribute to attracting circulating haematopoietic precursor cells and constitute bone marrow niche-like regions of EMH in humans. Differentiating haematopoietic cells may move into intrasinusoidal spaces to form EMH foci such as erythroblastic accumulation.