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Loss of Membrane Expression of E-Cadherin in Leukemic Erythroblasts.

Normal hematopoiesis in adult humans occurs within the bone marrow and is critically dependent on interactions of the hematopoietic progenitor cells with the surrounding microenvironment,[1,2] including various stromal cells, cytokines, and a complex extracellular matrix. These influences all contribute to an environment that allows hematopoietic progenitor cells to proliferate and differentiate normally. There are several indications that regulatory interactions between the microenvironment and hematopoietic cells are determined, at least partially, by mutual recognition and adhesion,[3] and there may be a link between the adhesive characteristics of hematopoietic cells and their maturation. Egress of developing hematopoietic cells is also a highly regulated process governed by interactions with the microenvironment.[1]

Various adhesion molecules, including the integrins and members of the immunoglobulin superfamily, but also adhesive and antiadhesive molecules of the extracellular matrix, have been implicated in interactions of developing hematopoietic progenitors and their microenvironment.[2-5] A recent report[6] suggested that E-cadherin, which is mainly present on epithelial cells, is expressed on erythroid precursors in early stages of maturation.

In leukemia, there is a breakdown in the mechanisms determining the maturation-linked distribution of hematopoietic cells, leading to dysregulation of hematopoiesis.[1,3] In such cases, immature blasts are not retained within the marrow, suggesting a breakdown of specific adhesive mechanisms.[3] However, few studies have been done on the adhesive properties of leukemic cells, and the molecular basis of the above abnormality is unknown.

In this study, we characterize for the first time, to our knowledge, the expression pattern of E-cadherin by bone marrow erythroid precursors using immunohistochemistry, and we demonstrate the loss of membrane expression of E-cadherin in erythroleukemia.


Fourteen trephine bone marrow biopsy specimens from iliac crest biopsies of 12 patients diagnosed with erythroleukemia were retrieved from the surgical pathology files of the University of Pennsylvania Medical Center (Philadelphia, Penn). The diagnosis of erythroleukemia was based on established morphologic, histochemical, immunohistochemical, and flow cytometric criteria (French-American-British [FAB] M6).[7,8] According to the recently revised classification of erythroleukemia,[9,10] all cases can be classified as M6A (myeloblastic/erythroleukemia). Five normal bone marrow samples from rib resections and 15 bone marrow biopsy specimens from the iliac crest of patients with breast carcinoma (5 cases), metastatic anaplastic thyroid carcinoma (1 case), multiple myeloma (2 cases), non-Hodgkin lymphoma (4 cases), Hodgkin disease (2 cases), and reactive erythroid hyperplasia (1 case) were also studied. The biopsy and the rib resection specimens were fixed in B5 and 10% buffered formalin fixative, respectively.

Air-dried bone marrow aspirate smears from 6 patients with breast carcinoma (2 cases), non-Hodgkin lymphoma (2 cases), multiple myeloma (1 case), and reactive erythroid hyperplasia (1 case), and smears of bone marrow obtained from 2 normal ribs resected during nephrectomy were fixed in 95% ethanol for 10 minutes and postfixed in 10% buffered formalin for 30 minutes. No bone marrow aspirate smears were available from patients with erythroleukemia.

Immunoperoxidase staining for E-cadherin (HECD-1 clone, 1: 200 dilution, Zymed Laboratories, South San Francisco, Calif) was performed following heat-induced antigen retrieval, using the avidin-biotin peroxidase technique. Sections of in situ and infiltrating ductal breast carcinoma, known to show strong E-cadherin immunostaining, were used as positive controls.


In 2 bone marrow smears from normal ribs and 6 bone marrow aspirate smears, strong membrane expression of E-cadherin was seen in the erythroid precursors (Figure 1, A). Similar staining reaction was seen in paraffin-embedded bone marrow biopsy and rib specimens, in which strong membrane expression of E-cadherin was seen in the erythroid precursors in all 20 cases of the control group. E-cadherin expression was stronger in erythroblasts of earlier maturational stages than in more mature erythroblasts. Sixteen of 20 cases showed weak cytoplasmic staining in the erythroid cells. Perinuclear, large, blocklike granular staining was present in 12 cases (Figure 1, B). Weak to moderate cytoplasmic reactivity was seen in megakaryocytes in 17 cases (Figure 2). However, megakaryocytes did not show membrane expression of E-cadherin. Cells of other lineages did not show either membrane or cytoplasmic staining for E-cadherin.


No membrane expression of E-cadherin was present in any of the 14 bone marrow biopsy specimens from patients with erythroleukemia (Figure 3). However, perinuclear dotlike positivity and weak cytoplasmic reactivity was present in 8 cases. Cytoplasmic staining of megakaryocytes was seen in 5 cases.



Immature hematopoietic cells express a variety of cell adhesion receptors,[1] which are thought to be involved in cell-cell and cell-matrix interactions.[11,12] Functionally, these receptors may serve for localization of hematopoietic progenitors within the marrow, but they may also mediate various intracellular signal-transduction pathways, leading to regulated cell growth and differentiation.[2,3,5,13] The special societal relationships existing between various cell types in the marrow suggest functional consequences for these cell-cell interactions.

E-cadherin, an adhesion molecule mainly found on the surface of epithelial cells,[11,14] has been demonstrated to play major roles during morphogenesis,[15] in the maintenance of epithelial tissues, and in malignant tumors.[16] It was recently demonstrated by flow cytometric analysis that E-cadherin is transiently expressed on bone marrow erythropoietic cells.[6,17] Double-labeling experiments with anti-E-cadherin antibody revealed coexpression with the molecules glycophorin A and the transferrin receptor, while no coexpression was found with lymphoid (CD2, CD3, CD10, CD19, CD56, and CD62L) and myeloid (CD13, CD33, and CD41) markers.[6,17] E-cadherin appeared to be strongly expressed on erythroblasts and to decrease during maturation of erythroid precursors. Mature erythrocytes did not express E-cadherin. It was suggested that E-cadherin represents a selective marker of immature erythropoietic cells.[17] Our results also indicate that E-cadherin is selectively expressed on normal bone marrow erythroid elements. Membrane expression of E-cadherin was previously reported in megakaryocytes[18]; however, we observed only cytoplasmic staining in these cells. Thus, the significance of the latter findings is unclear.

Several lines of evidence suggest that cadherin-mediated interactions can induce cell differentiation.[11] Experimental data suggest that E-cadherin may be involved in the regulation of erythroid differentiation, because antibodies against E-cadherin inhibited erythropoietic differentiation in vitro.[6] However, it is unclear -whether differentiation of the erythroblasts is a molecular event triggered by cell-cell adhesion. Through their interactions with the cytoplasmic catenins,[19] cadherins may be involved in signal-transduction events. Thus, E-cadherin might not function as an adhesion factor; instead, it might have a role as a signal-transduction molecule in this differentiation process.[6] Our demonstration of strong membrane expression suggests that erythroid precursors express a functional E-cadherin molecule, which may also play an essential role in the formation of erythroid islands and in the retention of maturing erythroid elements in the marrow.

There is compelling evidence suggesting that cell adhesion plays a central role in regulating cell growth and that adhesive mechanisms are major targets affected by transformation.[11] Abnormal interaction between the developing hematopoietic cells and their microenvironment may, at least partially, cause the premature egress of hematopoietic cells in leukemia,[3] but it is not yet clear whether a "leukemic" bone marrow microenvironment exists.[20] Several studies have focused on the expression of cell adhesion receptors on leukemic cells[1,4]; an altered expression pattern of integrins was observed on leukemic CD34-positive cells compared to normal progenitor cells.[21] Progenitor cells of chronic myeloid leukemia have been demonstrated to have diminished adhesion capacity for binding to normal bone marrow stroma due to impaired [Beta]1 integrin function.[22]

We showed that erythroleukemia (FAB M6) cells lack membrane expression of E-cadherin, suggesting that leukemic erythroid precursors do not express functional E-cadherin molecules, in contrast to their normal counterparts. Using flow cytometric analysis, Buhring et al[17] found that the E-cadherin-specific monoclonal antibody 67A4 did not react with erythroleukemia cell lines. Interestingly, our results also showed that the leukemic cells retained the perinuclear, large, blocklike granular staining in 8 of 14 cases. We have observed a similar staining pattern in rare cases of lobular carcinoma in situ of the breast.[23] It was suggested that perinuclear staining might result from mutant E-cadherin that is incorrectly processed within the Golgi apparatus or from accelerated protein turnover.[24]

Decreased expression of E-cadherin is thought to be associated with invasiveness in various tumors.[16,24,25] Somatic mutations of the E-cadherin gene (located at chromosomal band 16q22.1[15]) have been detected in diffuse-type gastric carcinomas[24] and in lobular breast carcinomas[26] lacking Ecadherin immunoreactivity. Recently, E-cadherin germline mutations in gastric and lobular breast carcinomas were reported,[27,28] emphasizing the importance of E-cadherin mutations in tumorigenesis. Cytogenetic aberrations involving the long arm of chromosome 16 have been reported in erythroleukemia,[9] and development of acute leukemia was also reported in families with E-cadherin germline mutations (the type of leukemia was not specified)[27] Further studies are needed to establish whether E-cadherin gene mutations play a role in the development of erythroleukemia.

In summary, we showed that immunohistochemical detection of membrane expression of E-cadherin may be a useful tool for identification of normal erythroid precursors. In addition, we demonstrated that leukemic erythroblasts do not show membrane expression of E-cadherin. While currently the biological significance of this finding is not clear, further studies are needed to define the potential role of E-cadherin in the maturation and malignant transformation of erythroid precursors.


[1.] Klein G. The extracellular matrix of the hematopoietic microenvironment. Experientia. 1995;51:914-926.

[2.] Long MW. Blood cell cytoadhesion molecules. Exp Hematol. 1992;20:288-301.

[3.] Gordon MY. Adhesive properties of haemopoietic stem cells. Br J Haematol. 1988;68:149-151.

[4.] Liesveld JL, Winslow JM, Frediani KE, Ryan DH, Abboud CN. Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells. Blood. 1993;81:112-121.

[5.] Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol. 1990;8:111-137.

[6.] Armeanu S, Buhring H, Reuss-Borst M, Muller CA, Klein G. E-cadherin is functionally involved in the maturation of the erythroid lineage. J Cell Biol. 1995; 131:243-249.

[7.] Head DR. Revised classification of acute myeloid leukemia. Leukemia. 19,96;10:1826-1831.

[8.] Society for Hematopathology Program. Proposed World Health Organization classification of neoplastic diseases of hematopoietic and lymphoid tissues. Am J Surg Pathol. 1997;21:114-121.

[9.] Mazzella FM, Kowal-Vern A, Shrit A, et al. Acute erythroleukemia: evaluation of 48 cases with reference to classification, cell proliferation, cytogenetics, and prognosis. Am J Clin Pathol. 1998;110:590-598.

[10.] Mazzella FM, Cotelingam JD, Kowal-Vern A, et al. Correspondence re: Brunning R: proposed WHO classification of acute myeloid leukemia and myelodysptastic syndromes: Mod Pathol 1999:1:102-104. Mod Pathol. 2000;13:101-102.

[11.] Geiger 13, Ayalon O. Cadherins. Annu Rev Cell Biol. 1992;8:307-332.

[12.] Springer TA. Adhesion receptors of the immune system. Nature. 1990;346: 425-434.

[13.] Mohandas N. Cell-cell interactions and erythropoiesis. Blood Cells. 1991; 17:59-64.

[14.] Shimoyama Y, Hirohashi S, Hirano S, et al. Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res. 1989;49:2128-2133.

[15.] Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251:1451-1455.

[16.] Takeichi M. Cadherins in cancer: implications in invasion and metastasis. Curr Opin Cell Biol. 1993;5:806-811.

[17.] Buhring H, Muller T, Herbst R, et al. The adhesion molecule E-cadherin and a surface antigen recognized by the antibody 9C4 are selectively expressed on erythroid cells of defined maturational stages. Leukemia. 1996;10:106-116.

[18.] Mbalaviele G, Chen H, Boyce BF, Mundy GR, Yoneda T. The role of cadherins in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow. J Clin Invest. 1995;95:2757-2765.

[19.] Kemler R. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet. 1993;9:317-321.

[20.] Greenberger JS. Is the marrow stroma of AML patients a "leukemic" stroma? Exp Hematol. 1992;20:1041-1042.

[21.] Reuss-Borst MA, Klein G, Waller HD, Muller CA. Differential expression of adhesion molecules in acute leukemia. Leukemia. 1995;9:869-874.

[22.] Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves ME Altered adhesive interactions with marrow stroma of hematopoietic progenitor cells in chronic myelogenous leukemia. Nature. 1987;328:342-344.

[23.] Acs G, Lawton TJ, Rebbeck TR, et al. Differential expression of E-cadherin in ductal and lobular neoplasms of the breast and its biologic and diagnostic implications. Am J Clin Pathol. In press.

[24.] Handschuh G, Candidus S, Luber B, et al. Tumour-associated E-cadherin mutations alter cellular morphology, decrease cellular adhesion and increase cellular motility. Oncogene. 1999;18:4301-4312.

[25.] Tang A, Amagai M, Granger LG, Stanley JR, Udey MC. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature. 1993; 361:82-85.

[26.] Berx G, Cleton-Jansen AM, Nollet F, et al. E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J. 1995;14: 6107-6115.

[27.] Guilford PJ, Hopkins JBW, Grady WM, et al. E-cadherin germline mutations define an inherited cancer syndrome dominated by diffuse gastric cancer. Hum Mutat. 1999;14:249-255.

[28.] Keller G, Vogelsang H, Becker I, et al. Diffuse type gastric and lobular breast carcinoma in a familial gastric cancer patient with an E-cadherin germline mutation. Am J Pathol. 1999;155:337-342.

Accepted for publication September 19, 2000.

From the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pa.

Reprints: Geza Acs, MD, PhD, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 6 Founders Pavilion, 3400 Spruce St, Philadelphia, PA 19104.
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Author:Acs, Geza; LiVolsi, Virginia A.
Publication:Archives of Pathology & Laboratory Medicine
Date:Feb 1, 2001
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