Myelofibrosis

Context.-Little is known about cellular and extracellular composition of fibrosis in bone marrows in the context of human immunodeficiency virus/acquired immunodeficiency syndrome.

Objective.-To evaluate the stromal composition of bone marrows affected by human immunodeficiency virus/ acquired immunodeficiency syndrome and to correlate this with laboratory parameters including CD4 lymphocyte counts.

Design.-We evaluated extracellular matrix and stromal cell composition in bone marrows and correlated these results with hematologic parameters. Extracellular matrix, stromal cells, and smooth muscle differentiation were evaluated by immunohistochemistry for collagen type IV expression and reticulin staining, an antibody directed against low-affinity nerve growth factor receptor (a marker of adventitial reticular cells), and actin staining, respectively. Concurrent laboratory information was collected, including white blood cell count, hemoglobin, platelet count, CD4 count, CD8 count, CD4/CD8 ratio, and absolute lymphocyte count.

Patients.-Bone marrows of 35 patients with human immunodeficiency virus/acquired immunodeficiency syndrome were evaluated.

Main Outcome Measures.-Correlation of reticulin, low-affinity nerve growth factor receptor, actin, and collagen IV staining with hematologic parameters.

Results.-More than half of the bone marrows showed moderate to severe reticulin fibrosis. The degree of reticulin fibrosis was correlated with the degree of low-affinity nerve growth factor receptor expression (P = .048). Actin expression was identified in only 3 of 35 cases and collagen IV in only 5 of 35 cases. No statistical relationship between degree of fibrosis and CD4 count was identified. Lower levels of low-affinity nerve growth factor receptor expression were associated with CD4 counts of >100 (P = .04). Marrow fibrosis was present in almost all cases studied (97%), and the staining of adventitial reticular cells correlated with the degree of reticulin fibrosis.

Conclusions.-There does not appear to be a correlation between CD4 count and degree of fibrosis, suggesting that the mechanism of fibrosis is independent of disease status.

(Arch Pathol Lab Med. 2005;129:1137-1140)

The bone marrow pathology of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) can be challenging. The constellation of changes, termed HiV myelopathy, includes a wide range of abnormal findings and can mimic a variety of primary bone marrow disorders, including myelodysplastic syndromes and myeloproliferative disorders.1 In addition, HIV/AIDS is associated with an increased incidence of opportunistic infections, hematologic and nonhematologic malignancies, and other hematologie complications, which typically worsen as the disease progresses.2

Bone marrow stroma is complex; it is a heterogeneous mixture of cellular and extracellular components that contribute to the unique microenvironment of the marrow cavity. Bone marrow stromal responses are a common pathway in both benign and malignant marrow disorders and account for some of the most striking changes seen in HIV/AIDS bone marrow. Defects of the bone marrow microenvironment contribute to the development of hematologie abnormalities in HIV/AIDS.2,3 It has been shown that stromal elements are directly infected and may act as a reservoir for the HIV virus.2-4 The HIV virus directly infects a variety of stromal elements, including monocytic/macrophage elements, microvascular endothelial cells, and fibroblastic and myoid cells.2,5 Infection of the stromal elements leads to a loss of the hematopoietic support function.6 In addition, megakaryocytes, which play an active role in the makeup of bone marrow stroma, are also infected by the HIV virus, as demonstrated by the presence of CD4 receptors on their surface.7 Human immunodeficiency virus infection of bone marrow elements may lead to loss of hematopoietic support function and may be responsible for some of the hematologic abnormalities.3 These changes may ultimately lead to cytopenias and susceptibility to infections, which are significant problems in the HIV-positive population.

We attempted to characterize the cellular and extracellular stromal reaction seen in the bone marrow in HIV/ AIDS, using an approach similar to that used to evaluate stromal changes in fibrotic myeloproliferative disorders and metastatic malignancy.8

The extracellular matrix was evaluated by reticulin staining and by immunohistochemistry for collagen type IV expression. Both reticulin fibers and collagen IV are known to be markedly increased in fibrotic marrow states, such as chronic idiopathic myelofibrosis.8-10 Low-affinity nerve growth factor receptor (LNGFR) has been recently described as a specific marker for a subset of bone marrow stromal cells known as adventitial reticular cells (ARC).11,12 In previous studies, the intensity of bone marrow LNGFR staining has been found to correlate with the degree of reticulin fibrosis.8-13 Actin staining was used to identify myofibroblastic differentiation in bone marrow stromal cells.14 Recent evidence has shown that bone marrow myoid cells may represent marrow reticular cells undergoing cytoskeletal remodeling in response to various stimuli, such as the presence of metastatic carcinoma.15

Because CD4^sup +^ T-lymphocyte counts are regularly used as a marker of disease progress and response to therapy in HIV/AIDS, we attempted to test correlation of the observed stromal responses with CD4^sup +^ lymphocyte counts and other hematologic parameters.

MATERIALS AND METHODS

Bone marrow biopsies of patients with HIV/AIDS were obtained from the files of Wishard Memorial Hospital and Clarian Health Partners (Indianapolis, Ind). In all, formalin-fixed, paraffin-embedded bone marrow biopsies of 35 patients were studied. In all cases, either significant hematologie abnormalities or possible involvement by infectious disease prompted bone marrow examination. Concurrent clinical information was collected including age, white blood cell count, hemoglobin, platelet count, CD4 count, CDS count, CD4/CD8 ratio, and absolute lymphocyte count. There was no evidence on chart review of transfusions within 7 days preceding the bone marrow biopsies.

Standard hematoxylin-eosin sections were evaluated in all cases. Bone marrow stroma was evaluated using immunohistochemical staining for collagen type IV (CIV; DakoCorporation, Carpinteria, Calif), actin (HHF-35; Dako), and LNGFR (ME20-4; Dako) expression (Table 1) and histochemical staining for reticulin. Density of stromal cell staining was graded semiquantitatively, as follows: 0+ indicates absent or rare; 1+, focal weak staining; 2+ , focally intense or diffuse staining; and 3+ , diffuse intense staining.

Immunoperoxidase stains were performed using an automated immunostainer (Dako), which employs a standard streptavidinbiotin-peroxidase complex technique. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol, and endogenous biotin activity was blocked using avidin and biotin. The peroxidase activity was developed with 3,3-diaminobenzidine and counterstained with hematoxylin. Controls were evaluated and stained appropriately in all cases.

Extracellular Matrix

The extracellular matrix was evaluated by reticulin staining, using the Gomori technique, and by immunohistochemistry for collagen type IV expression.

Stromal Cell Composition

Low-affinity nerve growth factor receptor staining was used to identify bone marrow stromal ARCs. Actin staining was used to evaluate myofibroblastic differentiation in bone marrow stromal cells.14

Statistical Analysis

Statistical analyses were performed using LogXact (version 2.1) (Cytel Software Corporation, Cambridge, Mass) and SAS (version 8.2) (SAS Institute Inc, Gary, NC) software packages using a P of .05 for all tests performed. Comparison of CD4 (

RESULTS

Demographics and Hematologie Parameters

The patients included in the study consisted of 9 women and 26 men. The patients' ages ranged from 18 to 57 years, with a mean age of 38 years. The mean hematologie parameters were as follows: white blood cell count, 4.3 × 10^sup 3^/µL; hemoglobin, 10.6 g/dL; and platelet count, 140 × 10^sup 3^/µL. The mean absolute lymphocyte count was 1014/ µL (normal range, 1400-3800/µL), with all but 1 patient having a decreased CD4/CD8 ratio (

Histologie Parameters

Histology.-Hematoxylin-eosin-stained sections were reviewed in all cases (Figure, A).

Reticulin Fibrosis and LNGFR.-With one exception, all cases showed at least 1+ reticulin fibrosis (0+, 1 case; 1+, 15 cases; 2+, 15 cases; and 3+, 4 cases). A major proportion of cases (19/35, or 54%) showed moderate or severe reticulin fibrosis (Figure, B). All cases but one showed an increase in LNGFR staining (0+, 1 case; 1+ , 15 cases; 2+ , 15 cases; and 3+, 4 cases) (Figure, C). In cases with increased reticulin fibrosis, the expression of LNGFR in ARC was typically upregulated. The degree of reticulin fibrosis paralleled the degree of LNGFR expression (P = .048).

Actin and Collagen IV-Actin (HHF-35) expression was identified focally in only 9% (3/35) of cases (Figure, D). Collagen IV demonstrated weak staining of vessel walls in only 5 of 35 cases of HIV/AIDS marrow biopsies (Figure, E). Linear staining was noted only in large-caliber vessel walls. Focal staining of small-caliber vessels was seen only rarely.

Correlation of Hematologie and Histologie Parameters

Statistical analysis did not reveal any correlation between CD4 count and the degree of reticulin fibrosis. However, lower levels of LNGFR expression were associated with CD4 counts of 100 or more (P = .04 in univariate and multiple regression). In analyses of continuous CD4 count, decreased LNGFR was associated with increased CD4 count (P = .03 and .02 in univariate and multiple regression, respectively).

COMMENT

A wide variety of hematologic abnormalities occur in HIV/AIDS patients, and many of these arise as a result of direct and indirect effects of the disease on the bone marrow. Stromal changes in the bone marrow and their relationship to hematologic parameters have not been closely examined in previous studies of HIV/AIDS-affected bone marrows.

We found that marrow fibrosis, as measured by reticulin staining, was present in all cases studied. As in other studies, we found a statistically significant association between the staining of ARCs and the degree of reticulin fibrosis.11-13 No statistical correlation between CD4^sup +^ lymphocyte count and degree of reticulin fibrosis was identified. This appears to suggest that the mechanism of marrow fibrosis is independent of disease status, as measured by CD4^sup +^ lymphocyte counts.

No significant expression of collagen IV was seen in the HIV/AIDS-affected bone marrows, except staining normally seen in the walls of large vessels. Also, no significant actin staining was seen in the bone marrows studied. This suggests that the development of a "myofibroblastic" phenotype is not a common pathway of stromal response in HIV/AIDS-affected bone marrow.

Megakaryocytes, ARCs, and monocyte/macrophages may all play a role in the development of stromal responses in bone marrow. With such diverse components contributing to the development of stromal changes, it is possible that different marrow disorders develop different "stromal phenotypes." Our previous studies of bone marrow stroma in myeloproliferative disorders and metastatic malignancies suggest significant differences in the stromal response to these disorders.8

Our findings in HIV/AIDS-affected bone marrows suggest that the stromal responses are similar to those seen in other hematologic disorders in which the stromal response is characterized by an increase in the number of ARCs (as assessed by LNGFR expression), with a concomitant increase in reticulin fibrosis (Table 3). In contrast to metastatic malignancies, there was no significant increase of actin expression, suggesting a lack of myofibroblastic differentiation. These are most likely "common pathway" effects of cytokine production by hematopoietic and stromal cells in response to changes in the marrow microenvironment.

We did not attempt to extend our findings to include type or duration of therapy in this study. However, several of the patients were on combination antiretroviral therapies (eg, highly active antiretroviral therapy). In spite of these therapies, there were still clinically significant stromal alterations in these marrows. We speculate that the stromal alterations in the bone marrow may persist in spite of highly active antiretroviral therapy, perhaps because of the effects of the nucleoside analogues.2 It is possible that bone marrow changes and hematologic abnormalities may persist in spite of treatments that improve other symptomatology.

Our evaluation of stroma in bone marrows of patients with HIV/AIDS suggests that fibrosis with increase of marrow ARCs is common in HIV/AIDS patients. Further, these appear to be independent of CD4^sup +^ lymphocyte counts. Further research into stromal responses to different marrow disorders may provide insight into the pathophysiology of these diseases and may improve our understanding of these disorders.

The authors thank Lee Ann Baldridge, HT(ASCP) QIHC, and Cecelia Dodson, HT(ASCP) QIHC, for their excellent technical assistance.

References

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12. Orazi A, Cattoretti G, Schiro R, et al. Recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor administered in vivo after high-dose cyclophosphamide cancer chemotherapy: effect on hematopoiesis and microenvironment in human bone marrow. Blood. 1992;79:2610-2619.

13. Cattoretti G, Schiro R, Orazi A, Soligo D, Colombo MP. Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro. Blood. 1993:81:1726-1738.

14. Bonanno E, Ercoli L, Missori P, Rocchi G, Spagnoli LG. Homogeneous stromal cell population from normal human adult bone marrow expressing alphasmooth muscle actin filaments. Lab Invest. 1994;71:308-315.

15. Papadopoulos N, Simopoulos C, Kotini A, Lambropoulou M, Tolparidou I, Tamiolakis D. Differential expression of alpha-smooth muscle actin molecule in a subset of bone marrow stromal cells, in B-cell chronic lymphocytic leukemia, autoimmune disorders and normal fetuses. EurJ Cynaecol Oncol. 2001:22:447-450.

Dennis P. O'Malley, MD; Joy Sen, MD; Beth E. Juliar, MA, MS; Attilio Orazi, MD, FRCPath

Accepted for publication May 12, 2005.

From the Department of Pathology and Laboratory Medicine, Division of Hematopathology (Drs O'Malley and Orazi), the Department of Pathology and Laboratory Medicine (Dr Sen), and the Department of Medicine, Division of Biostatistics (Ms Juliar), Indiana University School of Medicine, Indianapolis.

The authors have no relevant financial interest in the products or companies described in this article.

Presented in abstract form at the annual meeting of the United States and Canadian International Academy of Pathology, Vancouver, British Columbia, March 2004.

Reprints: Dennis P. O'Malley, MD, Indiana University School of Medicine, Department of Pathology and Laboratory Medicine, Division of Hematopathology, 702 Barnhill Dr, Riley 0969, Indianapolis, IN 46202 (e-mail: dpomalle@iupui.edu).

Copyright College of American Pathologists Sep 2005
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