CD146+ bone marrow osteoprogenitors increase in the advanced stages of primary myelofibrosis

CD146⁺ bone marrow stromal cells have been recently recognizedas clonogenic osteoprogenitors able to organize a complete hematopoieticmicroenvironment. In this study we used immunohistochemicalanalysis to investigate the contribution of CD146⁺ bone marrowosteoprogenitors to the stromal remodeling occurring in thedifferent stages of primary myelofibrosis. We found that CD146⁺cells sited at the abluminal side of the bone marrow vesselsand branching among hematopoietic cells significantly increasedin the advanced stages of primary myelofibrosis (p<0.001),paralleling the extent of fibrosis ( ${rho}$ =0.916, p<0.0001) andthe microvascular density (r=0.883, p<0.0001). Coherentlywith a mural cell function, such cells also displayed smooth-muscleactin expression. Our data providing evidence of CD146⁺ cellinvolvement in bone marrow stromal changes occurring in primarymyelofibrosis are consistent with the capability of these cellsto participate in fiber deposition, angiogenesis, and bone formation.They could also represent rationale for new therapies targetingthe bone marrow stroma in primary myelofibrosis.

Key words: bone marrow osteoprogenitors, primary myelofibrosis.

Introduction

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Introduction

Primary myelofibrosis is Ph- hematopoietic stem cell (HSC) disordercharacterized by the clonal proliferation of the myeloid andmegakaryocytic populations and by the progressive developmentof reticulin and collagen fibrosis in the bone marrow (BM) parenchyma.¹ Due to its fibrogenic nature, leading to BM failure, primarymyelofibrosis (PMF) has a poor prognosis and still lacks a validtherapeutic approach.

Besides the proliferation of the hematopoietic cell lineages,also the BM stromal reaction seems to paly a role in the pathogenesisof this disease. This is suggested by the increase in vesselneoformation and microvascular sprouting which can be observedthroughout the different phases of the disease, and by the boneremodeling and formation (osteosclerosis) occurring in the advancedstages.² The active role of the BM stroma is also supportedby the increase in the microvessel density that can be observedin PMF paralleling the degree of BM fibrosis and retaining,along with fibrosis, a prognostic significance.^3–5

Recently, the growing attention on the BM stromal niches andtheir cellular constituents has led to the identification ofCD146 (MCAM)-expressing BM stromal cells as clonogenic BM osteoprogenitors,deriving from hematopoietic stem cells, able to induce boneformation and to organize a hematopoietic microenvironment.^6,7In the human BM in situ, such a BM stromal stem-cell (BM-SSC)phenotype has been shown to be shared by subendothelial (adventitial)reticular cells displaying mural-cell properties (e.g. smoothmuscle actin expression, reticulin and collagen fiber deposition).^6–8Furthermore, CD146⁺ BM subendothelial cells have been shownto be able to sustain microvessel assembly and remodeling throughthe production of Angiopoietin-1 and its interaction with Tie-2receptor on endothelial cells.⁶ On the basis of these observations,we hypothesized that such cells could play a role in the BMstromal changes occurring in PMF and wanted to assess the presenceand distribution of CD146⁺ osteoprogenitors in the bone marrowof patients with different stages of PMF, namely early- (grade0/1 fibrosis) and advanced-stages (grade 2/3 fibrosis), by themeans of immunohistochemical analysis.

Design and Methods

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Design and Methods

This study was performed with the approval and permission ofthe Institutional Review Board of the University Hospital "PoliclinicoPaolo Giaccone" of Palermo. All the procedures followed werein accordance with the Helsinki Declaration.

Sample selection
The study was performed on bone marrow trephine biopsies obtainedfrom 32 patients who were diagnosed with PMF according to theWHO criteria⁹ between January 2005 and December 2007 at theDepartment of Human Pathology of the University of Palermo.Only representative bone marrow trephine biopsies (i.e. non-tangentialbiopsies of more than 1.5 cm in length) collected at the timeof diagnosis, before any treatment was started, were includedin the study. Ten bone marrow biopsies from age-matched patientswith Hodgkin’s lymphoma without BM involvement were usedas controls. BM biopsies were fixed in formalin and paraffinembedded and 4 mm-thick sections were cut from paraffin blocksfor histochemical and immunophenotypical evaluation. The followingclinical data were retrospectively gathered from the patients’medical records: age, gender, white blood cell count, plateletcount, hemoglobin value, presence of one or more risk factorsincluded in the Mayo prognostic score¹⁰ (Hb<10 g/dL, WBC<4or >30x10⁹/L, PLT<100x10⁹/L, AMC >1x10⁹/L), and JAK2(V617F) mutational status.

Bone marrow histochemical and immunophenotypical analysis
On histopathological evaluation, all the previous diagnosesof PMF were confirmed. Assessment of the BM fibrosis was performedon Gomori’s silver stained sections on the basis of theEuropean Myelofibrosis Network (EUMNET) consensus grading andscored accordingly from grade 0 to 3.¹¹ All the cases were independentlyassessed by four of the authors (UG, EB, CT, AMF) and interobserveragreement on BM fibrosis was reached by re-examining discordantcases on a multi-headed microscope. Immunohistochemistry wasperformed on BM sections by means of the streptavidinbiotin-peroxidasecomplex with rhodamine-red amino-ethyl carbazole as chromogen.The following primary antibodies were used: mouse monoclonalanti-CD34 (clone QBEnd/10, Novocastra, UK), mouse monoclonalanti-CD146 (clone N1238, Novocastra, UK), mouse monoclonal anti-SMA(clone 1A4, Dako, Denmark). The microvascular density was assessedby counting the absolute number of CD34⁺ vessels out of 10 high-power(400x) microscopical fields (HPF) and was expressed as the meanvalue. CD146 was similarly evaluated by counting the numberof positive cells out of 10 HPF and was expressed as the meanvalue.

Statistical analysis
The Mann Whitney U-test was performed to compare two independentgroups of data. Bivariate correlation was tested by the meansof Spearman’s rho test. Results of the statistical analysiswere considered significant for p values <0.05. All calculationsand plots were performed using the SPSS 13.0 statistical softwarepackage.

Results and Discussion

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Results and Discussion

Clinical, histological, and immunophenotypical features of early-and advanced-PMF cases are summarized in Table 1. The assessmentof CD34 immunostaining revealed a noticeable increase in theMVD in PMF cases as compared to control cases (p=0.003) and,consistently with previous observations, in PMF cases the MVDparalleled the increase in fiber content (Spearman’s ${rho}$ =0.880,p<0.001) (Figure 1A).^3,4 CD146 proved to be expressed byreticular cells sited at the abluminal side of the BM vasculatureextending dendritic processes among hematopoietic cells (Figure 2A).These cells with adventitial position and reticular morphologyalso expressed SMA, which confirmed their mural cell phenotype(Figure 2B). In control samples very few CD146⁺ cells were detected.No labeling for CD146 was detected in hematopoietic cells orin other BM stromal cell types (adipocytes, macrophages, osteoblastsand osteocytes, endosteal cells) in controls and PMF cases.In the early stages of PMF (grade 0/1 cases), CD146 expressionwas similar to that observed in control samples. A marked increasein CD146 expression was found in the advanced phases of PMF,with CD146⁺ subendothelial meshwork being more extended anddenser than that of early phases (p<0.001) (Figure 1B, Figure 2C–H).Furthermore, CD146 proved to be significantly associated withthe degree of fibrosis (Spearman’s rho=0.916, p<0.0001)(Figure 1C) as well as with the MVD (Spearman’s ${rho}$ =0.883,p<0.0001) (Figure 1D). However, the slight increase in themicrovascular density which was observed in the early phasesof the disease (grade 0/1) was not paralleled by a correspondingincrease in CD146⁺ cells (Figure 2C–D). This finding furthersupports the evidence that the increase in microvascular densityoccurs as an early, though variably intense, phenomenon in thePMF pathogenesis,¹² and suggests a prevalent contribution ofCD146⁺ cells to the advanced stages of the disease.

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Table 1. Clinical and pathological characteristics of the 32 primary myelofibrosis patients.

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Figure 1. (A) Comparison between microvascular density in controls, early and advanced stage of primary myelofibrosis. Microvascular density shows a remarkable increase in primary myelofibrosis cases. (B) Comparison between CD146 expression in controls, early and advanced primary myelofibrosis cases. Advanced primary myelofibrosis cases display a noticeable increase in CD146 positive cells compared to early stage and controls, while the differences between early stage cases and controls are less remarkable. (C) The graph shows the significant association between the number of CD146 positive cells and the degree of fibrosis, graded according to the EUMNET consensus criteria. Larger dots represent a higher number of cases. (D) The graph shows a significant association between number of CD146 positive cells and the microvascular density in different phases of primary myelofibrosis. Larger dots represent a higher number of cases.

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Figure 2. A–B. Sections from a case of advanced (grade-2) primary myelofibrosis showing that subendothelial cells, branching into the surrounding hematopoietic parenchyma (violet arrows), stain positive either for CD146 (A) or smooth muscle actin (B), while endothelial cells are negative (black arrows) for both antibodies. Inset: the distinction between CD34 positive endothelial cells (blue) and CD146 positive adventitial cells (red) is highlighted by double immunostaining. (Strept-ABC, original magnification 400x).
C–H. Differences in microvascular density and in the number of CD146 positive cells betwen an early (C, D, E; grade-1 fibrosis) and an advanced primary myelofibrosis (F, G, H; grade-2 fibrosis). CD146 positive subendothelial cells form a wider and denser meshwork in the advanced stage (G) of primary myelofibrosis than in the early subendothelial stage (D). The slight increase in microvascular density observed in the early stage (C) is not associated with a remarkable increase in CD146 expression (D) (C, F: CD34 immunostaining; D, G: CD146 immunostaining; E, H: Gomori’s staining; original magnification 200x).

The strong association we found between the CD146 cell countand the degree of fibrosis prompted us to test for possibleassociations occurring between these two variables and the clinicaldata of the 32 PMF patients (listed in Table 1). Both the CD146cell count and the degree of fibrosis proved to be significantlyassociated with a lower Hb value ( ${rho}$ =– 0.745, p=0.001 and ${rho}$ = –0.798 p<0.0001, respectively) and PLT count ( ${rho}$ = –0.630,p=0.009 and ${rho}$ = –0.657, p=0.006 respectively) on diagnosis.These data are in keeping with those from previous observationsindicating that the progression of the BM stromal reaction isoften matched by the decline of hematopoietic function.^5,13

Interestingly, the CD146 cell count also correlated with thepresence of risk factors on diagnosis ( ${rho}$ =0.634, p=0.008), whilethe association of this latter with the extent of fibrosis didnot reach statistical significance. No associations were foundwith the remaining presenting features, including the JAK2 mutationalstatus. However, the analysis of the JAK2V617F allele burden,which was not performed in this study, could possibly lead todifferent results. Moreover, due to the descriptive design ofthis study and the small number of cases included, the resultscoming from such a clinicopathological analysis should be consideredwith caution.

Overall, our results suggest the involvement of CD146⁺ subendothelialreticular cells the stromal remodeling that occurs in the advancedstages of PMF. In this setting, CD146⁺ cells might play a rolemainly through their recently reported function of clonogenicosteoprogenitor cells responsible for new bone formation⁶ andthrough the direct production and deposition of reticulin andcollagen fibers. CD146⁺ cells might also support BM neoangiogenesisthrough the angiopoietin-1/Tie-2 pathway, complementary to thatof VEGF which has been recently described in Ph- CMPDs.¹⁴

In conclusion, our data on the participation of CD146⁺ subendothelialosteoprogenitors in PMF stromal changes could represent a rationalefor new studies focusing on these cells as targets for new therapiesaffecting the BM stroma. To this purpose, along with CD146,other recently identified BM-SSC markers like CD200, and CD73could possibly be taken into consideration.¹⁵

Further studies are also needed to investigate the clonal natureof CD146⁺ BM cells in PMF and their possible prognostic relevance.

Acknowledgments

The authors would like to acknowledge Menarini Diagnostics,Italy for kindly providing the anti-CD146 monoclonal antibody.

Footnotes

Authorship and Disclosures

CT and ADB designed the study; EI and GF selected the casesand contributed to drawing up the manuscript; UG, EB, and AMFperformed the histopathological evaluation of the cases andscored the degree of fibrosis; CG, RP, and SI performed theimmunohistochemical reactions; ADB and MPT evaluated the immunoistochemicaldata and performed the statistical analysis; A.M. Florena revisedthe manuscript. All the authors approved the final version ofthe manuscript.

The authors reported no potential conflicts of interest.

Received for publication July 1, 2008. Revision received August 25, 2008. Accepted for publication September 5, 2008.

References

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