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DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation
Human Pathology (2007) 38, 260 – 268
www.elsevier.com/locate/humpath
Original contribution
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation Lee-Ching Zhu MDa, Joon Yim MDa, Luis Chiriboga PhDa, Nicholas D. Cassai MSb, Gurdip S. Sidhu MDb, Andre L. Moreira MD, PhDa,* a
Department of Pathology, New York University School of Medicine, NY 10016, USA New York Harbor Health Care System–New York Campus and New York University Medical Center, NY 10016, USA
b
Received 25 May 2006; revised 26 July 2006; accepted 31 July 2006
Keywords: Clara cell; Type II pneumocyte; DC-LAMP; Surfactant protein; PE-10; Lung adenocarcinoma; Ultrastructure
Summary DC-LAMP is a molecule expressed in mature dendritic cells, but its mRNA is also found in the lung. This study compares the immunostaining spectrum of PE-10, an antisurfactant protein monoclonal antibody; thyroid transcription factor–1 (TTF-1); and DC-LAMP in normal and neoplastic lung in an attempt to characterize the cell type(s) that express DC-LAMP. Electron microscopy was used to define cell types. DC-LAMP marks pulmonary adenocarcinomas that show Clara cell characteristics by electron microscopy. In contrast, PE-10 labels tumors that have Clara cell and type II pneumocyte differentiation. DC-LAMP staining was lost in solid type adenocarcinomas but persisted in welldifferentiated areas. CC-10, an antibody that marks Clara cells, was also positive in tumors that labeled for DC-LAMP. There was no prognostic difference in tumors that reacted with DC-LAMP. DC-LAMP and CC-10 reactivity was also observed in endometrial adenocarcinomas but not in other tumor types. D 2007 Elsevier Inc. All rights reserved.
1. Introduction Pulmonary carcinomas are the leading cause of death in both men and women worldwide. Treatment and clinical management of patients with carcinomas of the lung are guided by the histologic classification of the tumor. Adenocarcinoma is the most common type of pulmonary carcinoma. Adenocarcinomas are a morphologically heterogeneous group, which includes a noninvasive tumor classified as bronchioloalveolar carcinoma (BAC) and invasive histologic subtypes showing acinar, papillary, or solid growth patterns. The heterogeneity of histologic types * Corresponding author. Department of Pathology, Memorial Sloan– Kettering Cancer Center, 1275 York Avenue, New York. NY 10021, USA. E-mail address: [email protected] (A. L. Moreira). 0046-8177/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.07.018
of pulmonary adenocarcinomas is recognized by the World Health Organization classification of lung tumors [1]. Adenocarcinomas more often occur in the periphery of the lung and are thought to originate from cells that line the alveoli, terminal, or respiratory bronchioles. Ultrastructurally, 2 cell types inhabit this region; Clara cells that are nonciliated columnar cells and have characteristic apical granules and type II pneumocytes, which are characterized by the presence of lamellar bodies. Clara cells are thought to represent reserve cells of the pulmonary bronchiole [2-4]. Adenocarcinomas with differentiation into either cell types have been described. Currently, the monoclonal antibody (mAb) PE-10, raised against surfactant proteins isolated from the lung lavage of patients with alveolar proteinosis, is being used as a marker for adenocarcinoma of the lung. PE-10 is, however, not specific, and reacts with
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation both Clara cells and type II alveolar pneumocytes [5,6]. Adenocarcinomas with Clara cell differentiation cannot be easily distinguished from adenocarcinomas with type II pneumocyte differentiation unless electron microscopy (EM), a costly technique, is used. Tumors with Clara cell differentiation can be identified by an antibody that reacts to CC-10, a 10-kd protein secreted by Clara cell [7]. A newly identified human lysosome-associated membrane, protein–3, was originally described as a molecule specifically expressed in mature dendritic cells and, thus, designated as DC-LAMP or CD208 [8]. We use the term DC-LAMP throughout the text because it is more frequently used in the medical literature. A definite function of DCLAMP has not been established. DC-LAMP is most homologous to CD68 [9], a lysosomal glycoprotein expressed by macrophages and platelets. Besides dendritic cells, DC-LAMP mRNA was found to be expressed in human and murine lungs [10-12], but the cellular source was not clearly established. In this study, the pattern of immunoreactivity to the antibodies DC-LAMP; CC-10; TTF-1, a nuclear transcription factor protein that is expressed in epithelial cells of the lung and thyroid gland [13]; and PE-10 in normal and neoplastic lung is compared with the objective of identifying in the lung parenchyma the cellular population that reacts with DC-LAMP. Electron microscopy (EM) is also used to better define this population.
2. Materials and methods 2.1. Patient population and tissue sample A total of 87 pulmonary adenocarcinomas resected at the New York University Medical Center (NYUMC) during the period 1992 to 2004 were included in this study. The mean age of the patients was 66 F 7.7 years (range, 49-82 years). The female-to-male ratio was 1.2:1. All tumors were classified according to the World Health Organization classification of lung tumors [1] and were staged using the TNM staging system [14]. Patients with pathologic stage 1A to 2B were included. For the purpose of this study, the tumors were further subclassified according to the amount of BAC component present in the histologic sections. There were 12 cases of pure BAC, 14 cases of mixed-type adenocarcinomas with more than 50% of the tumor mass composed of BAC, 25 cases of mixed type adenocarcinoma with less than 50% BAC component, and 36 cases of adenocarcinoma mixed subtype with no BAC component. Five cases of normal lung tissue resected for nonneoplastic lesions were also included as control. In addition, other types of pulmonary carcinomas such as small cell carcinoma (n = 7) and squamous cell carcinoma (n = 10) were included. Epithelial and mesenchymal tumors from other organs were also studied. These included endometrial adenocarcinoma (n = 28), ovarian papillary serous carcinoma (n = 2), colorectal adenocarcinoma (n = 2), gastric adenocarcinoma (n = 2), hepatocellular carcinoma (n = 2),
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renal cell carcinoma (n = 2), ductal carcinoma of the breast (n = 12), papillary adenocarcinoma of the thyroid gland (n = 5), melanoma (n = 5), granular cell tumor (n = 4), leiomyosarcoma (n = 4), rhabdomyosarcoma (n = 3), schwannoma (n = 3), phyllodes tumor of the breast (n = 4), fibroadenoma of the breast (n = 3), and alveolar soft part sarcoma (n = 2). Patient data were collected according to HIPPA (Health Insurance Portability and Accountability Act) regulations. The study was approved by the institutional review board.
2.2. Immunohistochemistry Immunohistochemistry was performed on archival formalin-fixed, paraffin-embedded tissue. Sections were deparaffinized and hydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 minutes. The reaction was carried out using the streptavidin-biotin immunoperoxidase technique in an automated immunostainer (Benchmark, Ventana Medical System, Tucson, AZ). Antigen retrieval was performed in all sections by heating the slides in 0.01 mol/L of citrate buffer (pH 6.0) for 20 minutes, unless otherwise specified. The slides were then incubated with mouse antihuman surfactant protein mAb, clone PE-10 (Research Diagnostic, Inc, Minneapolis, MN; dilution, 1:50), CC-10 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100), or CD68 (Dako, Carpinteria, CA; dilution, 1:500) at 378C for 32 minutes. For DC-LAMP detection, sections were incubated with mAb DC-LAMP, clone 104G4 (Immunotech, Fullerton, CA; dilution 1:50), at room temperature overnight without antigen retrieval. In each analysis, a tumor section that had been previously confirmed to express targeted antigens was included as a positive control.
2.3. Electron microscopy For EM examination, samples of tumors were fixed in 3% phosphate-buffered glutaraldehyde in a phosphate buffer isomolar with plasma at a pH of 7.4, postfixed in 2% osmium tetroxide, dehydrated, and embedded in Epon 812. Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a JEOL 1010 electron microscope (Pleasonton, CA).
2.4. Statistical analysis The Fisher exact test was used as indicated. A P value of .05 was considered significant.
3. Results 3.1. Anti–DC-LAMP mAb stains Clara cells in normal lung Immunostains were performed on sections of normal lung to examine the expression pattern of PE-10,
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Fig. 1 Photomicrograph of normal and neoplastic lung tissue showing the localization of several markers studied. A, Normal lung tissue showing PE-10 staining in terminal bronchioles. Note staining on the surface of the cells of the terminal bronchioles and type II pneumocytes in the alveolar wall. B and C, Normal pulmonary tissue stained with antihuman DC-LAMP antibody. Note apical staining of cuboidal cells (B) and in granular staining pattern for this antibody (C). D, Normal pulmonary tissue stained with CC-10 antibody. Note apical staining of some of the cells in the terminal bronchiole. E, Pulmonary adenocarcinoma, mixed subtype with acinar and bronchioloalveolar patterns showing diffuse staining granular and cytoplasmic pattern in acinar component for DC-LAMP. F, Same tumor as in E, stained for CC-10.
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation DC-LAMP, and CC-10. PE-10 antibody showed a diffuse staining pattern in the cells lining the alveolar wall and bronchioles (Fig. 1A). DC-LAMP mAb showed an apical
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granular staining pattern in scattered cells in distal respiratory bronchiolar epithelium (Fig. 1B and C). The location and staining pattern resembles that of Clara cells. By light
Fig. 2 DC-LAMP antibody stains adenocarcinoma with Clara cell differentiation. A case of lung adenocarcinoma with EM-proven Clara cell differentiation (A-C) was reactive with PE-10 mAb (A) and DC-LAMP mAb (B) and showed typical Clara cell granules ultrastructurally (C). A case of lung adenocarcinoma with EM-proven type II pneumocyte differentiation (D-F) shows strong staining for PE-10 (D) but is negative for DC-LAMP (E). EM shows cytoplasmic lamellar bodies, which are characteristic of type II pneumocytes.
264 microscopy, Clara cells are barely discernible by histologic features, including nuclei located in the mid portion of the cell, and a dome-shaped apical protrusion of the cytoplasm above the apical tight junctions. A similar pattern of distribution of positive cells was seen with CC-10 antibody (Fig. 1D). Given that CD68 is the closest homologue of DC-LAMP, we also evaluated its staining pattern in normal lung and confirmed that the antibody stained alveolar macrophage,
L.-C. Zhu et al. with no staining in the epithelial cells of the respiratory bronchioles (not shown). There was nonspecific (cytoplasmic) staining of alveolar macrophages with DC-LAMP.
3.2. DC-LAMP mAb stains adenocarcinoma with Clara cell–type differentiation Bronchioloalveolar carcinomas of the lung are rare, well-differentiated carcinomas that show no stromal invasion. To define the expression pattern of DC-LAMP in neoplastic lung tissues, 12 cases of BAC were studied. These cases of pure BAC were actively collected over the period of 12 years from 3 independent hospitals that are part of the NYUMC, which reflects the rarity of this form of adenocarcinoma. Pure BAC represents approximately 5% of all lung adenocarcinomas resected with curative intent at NYUMC. All BAC studied stained strongly and diffusely for PE-10 and TTF-1. However, there was a heterogenous pattern of staining for DC-LAMP and CC-10. Among the 12 cases studied, only 4 cases showed a positive reaction for DC-LAMP. In these 4 cases, DC-LAMP mAb produced a diffuse, granular, and apical cytoplasmic staining pattern (Fig. 2). EM examination of these tumors demonstrated that the tumor cells had apical granules with a dense uniform content that are characteristic of Clara cell granules (Fig. 2). In contrast, in 3 of the 8 cases of BAC with negative staining for DC-LAMP, EM studies demonstrated that the tumor cells had cytoplasmic lamellar bodies, which are diagnostic of type II pneumocytes differentiation (Fig. 2). The data suggest that mAb DC-LAMP reacts with BAC with Clara cell differentiation.
3.3. Loss of DC-LAMP expression in solid areas of lung adenocarcinoma In an effort to determine the expression level of DCLAMP in lung adenocarcinoma, 75 cases of primary lung adenocarcinomas were studied. All tumors were mixed subtype adenocarcinomas. The carcinomas were further divided into 3 groups according to the content of the BAC component. Group I (14 cases) was composed of mixedtype adenocarcinomas with a predominance of BAC component (more than 50% of the tumor mass); group 2 (25 cases) was composed of mixed-type carcinomas with both BAC and invasive components in which the BAC Fig. 3 Loss of DC-LAMP expression in solid areas of lung adenocarcinoma. A, Section of lung adenocarcinoma, mixed subtype with solid and acinar components showed DC-LAMP– positive staining in the more differentiated areas of the tumor (acinar) and loss of DC-LAMP expression in the adjacent solid tumor areas. B and C, Positive DC-LAMP staining was consistently detected at the periphery of the tumor nodules and in the lung immediately abutting the tumor nodules. Note positive reaction in cells lining the alveolar wall that have type II pneumocyte appearance (C). Higher magnification of the same area shown in (B).
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation component was less than 50% of the tumor mass; and Group III (36 cases) was composed of invasive adenocarcinoma with no BAC component. DC-LAMP showed positive staining in 21.4% (3/14) of the tumors in group 1 (2 women and 1 man), 36% (9/25) of the tumors in Group II (5 women and 4 men), and 25% (9/36) of the tumors of group 3 (4 women and 5 men). Therefore, DC-LAMP appears to mark a subset of pulmonary adenocarcinomas independent of the amount of BAC component. There was no sex predilection for DC-LAMP–positive tumors. Of all adenocarcinomas studied, 86% were also positive for TTF-1, whereas 74% were positive for PE-10. All DC-LAMP– positive adenocarcinomas were positive for TTF-1 and PE10. All tumors that were negative for TTF-1 were also negative for DC-LAMP. Therefore, DC-LAMP does not seem to be a better marker than TTF-1 and PE-10 in the diagnosis of pulmonary adenocarcinoma. Furthermore, EM examination of 6 of the 14 cases from group 1 demonstrated Clara cell type phenotype in the 3 tumors with strong DC-LAMP expression, and type II pneumocyte phenotype in the 3 tumors with negative DCLAMP staining. It was consistently noted that DC-LAMP was mainly expressed in the relatively well-differentiated glandular (acinar) component, even in the group with no BAC component. Solid tumor areas usually lost expression of the antigen (Fig. 3A). Similar results were obtained with CC-10. All tumors that were positive for DC-LAMP were also reactive to CC-10 (Fig. 1E and F), with the exception of 1 tumor that only showed reactivity to CC-10 and no DC-LAMP stain. Strong DC-LAMP and CC-10 positivity was usually detected in reactive cells at the junction of tumor nodules and areas of organizing pneumonia close to the tumor mass (not shown), thus indicating that the antigens (DC-LAMP and CC-10) were expressed in reactive cells in response to injury (Fig. 3B-C). These reactive cells lining the alveolar space had morphologic characteristics of type II pneumocytes.
3.4. Mortality rate of patients with DC-LAMP–positive tumors To investigate whether there was a difference in biologic behavior from tumors that showed Clara cell differentiation, as compared with the rest of the pulmonary adenocarcinomas, survival data from the time of diagnosis were obtained from patients’ medical charts. The average time of follow-up was 5 years, with a range from 1 to 12 years. Most patients who succumbed to the disease died within 1 year of diagnosis. There were no recorded deaths in the group of 12 patients whose tumors were diagnosed as pure BAC. There was no death in group 1 (patients with tumors with a predominant BAC component). In group 2 (mixed adenocarcinomas with small amount of BAC component), there were 2 deaths of 25 patients, representing a mortality rate of 8% in this
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group. When stratified for DC-LAMP reactivity, 1 death was seen in the DC-LAMP–positive tumor (1 [11%] of 9 patients), and 1 death in a DC-LAMP–negative tumor group (1 [6%] of 16 patients). In group 3, which is composed of tumors with no BAC component, there were 8 deaths in 36 patients, representing a mortality rate of 22%. Two deaths occurred in patients that had DC-LAMP–positive tumors (2 of 9, ie, 22%), and 6 deaths in DC-LAMP– negative tumors (6 of 27, ie, 22%). Although the number of cases studied was small, there was no statistical difference in death rate when stratified per DC-LAMP reactivity ( P N .05). Therefore, the histologic classification of the tumor and the amount of BAC component of the adenocarcinoma seem to correlate with the prognosis as previously reported [15,16] but not the cell type differentiation.
3.5. DC-LAMP expression in other epithelial and mesenchymal tumors There was no reactivity with DC-LAMP antibody in pulmonary squamous cell carcinoma and small cell carcinoma. No stain was seen in other nonpulmonary carcinomas and mesenchymal tumors such as renal cell carcinoma, gastric, prostatic, mammary, colorectal adenocarcinomas, hepatocellular carcinoma, papillary adenocarcinoma of the thyroid, ovarian papillary serous carcinoma, malignant melanoma, granular cell tumors, leiomyosarcoma, rhabdomyosarcoma, schwannoma, phyllodes tumors of the breast, fibroadenomas of the breast, and alveolar soft part sarcoma. The lone exception was endometrial adenocarcinoma. In the cases of endometrial adenocarcinoma studied, DCLAMP showed a similar apical and granular staining pattern, as seen in BAC of the lung (Fig. 4) in 22% of the endometrial adenocarcinomas evaluated (6 of 28 cases), thus demonstrating a similar heterogeneous staining pattern for endometrial adenocarcinoma, as observed in the lung. Reactivity for DC-LAMP was also noted in complex hyperplasia of the endometrium but not in simple hyperplasia and normal endometrial cells (data not shown). Similar to DC-LAMP, anti–CC-10 antibody also marked endometrial carcinomas and not other tumor types. PE-10 reactivity was seen only in pulmonary adenocarcinomas. TTF-1 reactivity was seen in pulmonary adenocarcinoma, papillary adenocarcinoma of the thyroid gland, and pulmonary small cell carcinoma.
4. Discussion This study demonstrates that DC-LAMP stains well differentiated pulmonary adenocarcinoma with Clara cell differentiation without cross-reactivity, with adenocarcinomas showing type II pneumocyte differentiation. In normal human lung, DC-LAMP stains cells in the most distal part of the respiratory bronchiole where Clara cells are the predominant cell type [17].
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Fig. 4 DC-LAMP expression in endometrial adenocarcinoma. A, Microphotograph of a well-differentiated endometrial adenocarcinoma (hematoxylin and eosin stain). B, Endometrial adenocarcinoma stained with anti-human DC-LAMP. Insert shows similar diffuse and granular pattern of staining, as seen in lung adenocarcinomas with Clara cell differentiation.
In contrast, PE 10, an antibody to surfactant proteins, will react with any cell type in the lungs that secretes or stores surfactant proteins or surfactant protein accumulated in the alveolar space. TTF-1 stains pulmonary epithelium with exception of ciliated bronchial epithelial cells. Thus, PE-10 and TTF-1 can be used clinically as a tool for the identification of adenocarcinomas of the lung. DC-LAMP is, however, not a useful antibody for the clinical characterization of metastatic pulmonary adenocarcinoma because it only stains a subset of lung tumors that shows Clara cell differentiation. In addition, all tumors that were reactive with DC-LAMP are also reactive for TTF-1 and PE-10. Our results are in contrast with prior investigations that report that DC-LAMP is a marker for type II pneumocytes. In elegant experiments, Salaun et al [12] showed that an mAb against mouse DC-LAMP stains cells in the alveolar wall that have type II pneumocyte morphology. In addition, the antibody seems to colocalize with lamellar bodies, an
L.-C. Zhu et al. ultrastructural marker of type II pneumocytes, in which surfactant protein is stored before its release. This mouse antibody is also reported to cross-react with human DCLAMP. The authors used samples of human adenocarcinomas and showed that in these tumors, only a portion of adenocarcinomas stains with the mouse DC-LAMP antibody, a finding similar to our observations. The differences in cell type that expressed DC-LAMP may be related to a difference in animal species (mouse versus human) or in the antibody used. The authors also show that mouse DCLAMP antibody stains Jaagsiekte sheep retrovirus–associated ovine pulmonary adenocarcinoma. Interestingly, these tumors may also have type II pneumocytes and Clara cell differentiation [18]. We observed that there is an up-regulation of the expression of DC-LAMP in reactive cells around the tumor or around areas of tissue damage. In these areas, the expression of DC-LAMP extended beyond the terminal bronchiole into the alveolar wall. The presence of DCLAMP in these cells could be a result of proliferation of Clara cells in response to injury [19] or proliferation of a progenitor cell population of the lung that can differentiate toward type II pneumocytes and Clara cells [19,20]. In fact, it has been shown that reactive alveolar cell seen in areas of diffuse alveolar damage and idiopathic pulmonary fibrosis can show duo differentiation of Clara cells and type II pneumonocytes, as seen ultrastructurally by the presence of lamellar bodies and dense granules in the same cell [21]. In addition, duo differentiation of Clara cell and type II pneumocytes have been seen in atypical adenomatous hyperplasia, a preneoplastic process that occurs around the terminal bronchioles [22,23]. Pulmonary papillomas with duo differentiation of Clara cells and type II pneumocytes have also been described [24,25]. Alternatively, DC-LAMP staining of these reactive cells could be a result of upregulation of the molecule in type II pneumocytes. It has been shown that hormone-induced differentiation of type II pneumocytes in vitro leads to transient expression of DCLAMP in these cells [26]. Therefore, it is possible to suggest that DC-LAMP may be a marker of Clara cells or an epithelial progenitor cell in the lung that could differentiate toward Clara cell and type II pneumocytes. Our observations that antihuman DC-LAMP stains Clara cells is further corroborated by the fact that the antibody CC10, a specific marker for Clara cells, marks the same tumors that are positive for DC-LAMP. In similarity to DC-LAMP, CC-10 also labels endometrial adenocarcinoma. There was no detectable labeling in any other adenocarcinomas, mesenchymal tumors, and melanomas. The protein CC-10 is homologous to human uteroglobin, a protein that is expressed in endometrial cells [27]. The reasons for the colocalization of CC-10 and DC-LAMP in endometrial adenocarcinomas is not clear. The two protein however, do not share structural homology. In addition, DC-LAMP is located on chromosome 3q27 [8,10,11], whereas CC-10 is localized on chromosome 11q13 [28], which suggests that
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation these two molecules are not regulated under the same promoter. DC-LAMP seems to colocalize with lysosomes, and CC-10 is a protein secreted by Clara cells and thought to have anti-inflammatory properties [7,27]. CC-10 expression by endometrial cells seems to be hormonal, regulated with higher expressions during the progesterone phase of the menstrual cycle [29]. It has been reported that DC-LAMP expression in the lung can be induced by steroid hormones [26]. Our studies also suggest that DC-LAMP is lost during tumorigenesis because poorly differentiated adenocarcinomas of the lung shows very little expression of DC-LAMP, but the antigen is present in the well-differentiated areas of the adenocarcinoma. Similar observations have been made with the antibody against CC-10, the Clara cell marker. This antigen is also lost during tumor differentiation and tumorigenesis [30]. However, there was no down-regulation of DC-LAMP labeling in the normal lung adjacent to adenocarcinomas, as has been reported for CC-10 [31]. The role of DC-LAMP in Clara cells and tumorigenesis is unknown. In a recent report, cell lines transfected with human DC-LAMP expressed increased mobility in in vitro and in vivo assays [32]. In addition, high expression of DC-LAMP by uterine adenocarcinoma and cervical squamous cell carcinoma is associated with increased metastatic potential and poor outcome [32]. In the lung however, the expression of DC-LAMP shows no correlation with prognosis. The function of DC-LAMP is still unknown, but DCLAMP may play a role in cell migration through release of lysosomal enzymes because the molecule is up-regulated in dendritic cells during and upon maturation and migration from skin to regional lymph nodes [33]. It is possible that a similar role is playing in Clara cells, or in a pulmonary precursor cell, because mobility is one of the intrinsic characteristics of stem cells [34]. DC-LAMP is not likely to be a suitable antibody for clinical diagnostic purposes because it does not stain all pulmonary adenocarcinomas, but it might be a useful tool to understanding the biology of Clara cells in lung development and pathology because the marker appears to be expressed in bronchiolar Clara cells, a reserve cell of the bronchiole, and also it appears to be up-regulated in peritumoral benign reactive cells with type II pneumocytes morphology. DC-LAMP could be a candidate as a marker for a pulmonary stem cell.
Acknowledgments The authors thank Ms Olena Ardacheva for her expert technical support on immunochemistry.
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Original contribution
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation Lee-Ching Zhu MDa, Joon Yim MDa, Luis Chiriboga PhDa, Nicholas D. Cassai MSb, Gurdip S. Sidhu MDb, Andre L. Moreira MD, PhDa,* a
Department of Pathology, New York University School of Medicine, NY 10016, USA New York Harbor Health Care System–New York Campus and New York University Medical Center, NY 10016, USA
b
Received 25 May 2006; revised 26 July 2006; accepted 31 July 2006
Keywords: Clara cell; Type II pneumocyte; DC-LAMP; Surfactant protein; PE-10; Lung adenocarcinoma; Ultrastructure
Summary DC-LAMP is a molecule expressed in mature dendritic cells, but its mRNA is also found in the lung. This study compares the immunostaining spectrum of PE-10, an antisurfactant protein monoclonal antibody; thyroid transcription factor–1 (TTF-1); and DC-LAMP in normal and neoplastic lung in an attempt to characterize the cell type(s) that express DC-LAMP. Electron microscopy was used to define cell types. DC-LAMP marks pulmonary adenocarcinomas that show Clara cell characteristics by electron microscopy. In contrast, PE-10 labels tumors that have Clara cell and type II pneumocyte differentiation. DC-LAMP staining was lost in solid type adenocarcinomas but persisted in welldifferentiated areas. CC-10, an antibody that marks Clara cells, was also positive in tumors that labeled for DC-LAMP. There was no prognostic difference in tumors that reacted with DC-LAMP. DC-LAMP and CC-10 reactivity was also observed in endometrial adenocarcinomas but not in other tumor types. D 2007 Elsevier Inc. All rights reserved.
1. Introduction Pulmonary carcinomas are the leading cause of death in both men and women worldwide. Treatment and clinical management of patients with carcinomas of the lung are guided by the histologic classification of the tumor. Adenocarcinoma is the most common type of pulmonary carcinoma. Adenocarcinomas are a morphologically heterogeneous group, which includes a noninvasive tumor classified as bronchioloalveolar carcinoma (BAC) and invasive histologic subtypes showing acinar, papillary, or solid growth patterns. The heterogeneity of histologic types * Corresponding author. Department of Pathology, Memorial Sloan– Kettering Cancer Center, 1275 York Avenue, New York. NY 10021, USA. E-mail address: [email protected] (A. L. Moreira). 0046-8177/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.07.018
of pulmonary adenocarcinomas is recognized by the World Health Organization classification of lung tumors [1]. Adenocarcinomas more often occur in the periphery of the lung and are thought to originate from cells that line the alveoli, terminal, or respiratory bronchioles. Ultrastructurally, 2 cell types inhabit this region; Clara cells that are nonciliated columnar cells and have characteristic apical granules and type II pneumocytes, which are characterized by the presence of lamellar bodies. Clara cells are thought to represent reserve cells of the pulmonary bronchiole [2-4]. Adenocarcinomas with differentiation into either cell types have been described. Currently, the monoclonal antibody (mAb) PE-10, raised against surfactant proteins isolated from the lung lavage of patients with alveolar proteinosis, is being used as a marker for adenocarcinoma of the lung. PE-10 is, however, not specific, and reacts with
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation both Clara cells and type II alveolar pneumocytes [5,6]. Adenocarcinomas with Clara cell differentiation cannot be easily distinguished from adenocarcinomas with type II pneumocyte differentiation unless electron microscopy (EM), a costly technique, is used. Tumors with Clara cell differentiation can be identified by an antibody that reacts to CC-10, a 10-kd protein secreted by Clara cell [7]. A newly identified human lysosome-associated membrane, protein–3, was originally described as a molecule specifically expressed in mature dendritic cells and, thus, designated as DC-LAMP or CD208 [8]. We use the term DC-LAMP throughout the text because it is more frequently used in the medical literature. A definite function of DCLAMP has not been established. DC-LAMP is most homologous to CD68 [9], a lysosomal glycoprotein expressed by macrophages and platelets. Besides dendritic cells, DC-LAMP mRNA was found to be expressed in human and murine lungs [10-12], but the cellular source was not clearly established. In this study, the pattern of immunoreactivity to the antibodies DC-LAMP; CC-10; TTF-1, a nuclear transcription factor protein that is expressed in epithelial cells of the lung and thyroid gland [13]; and PE-10 in normal and neoplastic lung is compared with the objective of identifying in the lung parenchyma the cellular population that reacts with DC-LAMP. Electron microscopy (EM) is also used to better define this population.
2. Materials and methods 2.1. Patient population and tissue sample A total of 87 pulmonary adenocarcinomas resected at the New York University Medical Center (NYUMC) during the period 1992 to 2004 were included in this study. The mean age of the patients was 66 F 7.7 years (range, 49-82 years). The female-to-male ratio was 1.2:1. All tumors were classified according to the World Health Organization classification of lung tumors [1] and were staged using the TNM staging system [14]. Patients with pathologic stage 1A to 2B were included. For the purpose of this study, the tumors were further subclassified according to the amount of BAC component present in the histologic sections. There were 12 cases of pure BAC, 14 cases of mixed-type adenocarcinomas with more than 50% of the tumor mass composed of BAC, 25 cases of mixed type adenocarcinoma with less than 50% BAC component, and 36 cases of adenocarcinoma mixed subtype with no BAC component. Five cases of normal lung tissue resected for nonneoplastic lesions were also included as control. In addition, other types of pulmonary carcinomas such as small cell carcinoma (n = 7) and squamous cell carcinoma (n = 10) were included. Epithelial and mesenchymal tumors from other organs were also studied. These included endometrial adenocarcinoma (n = 28), ovarian papillary serous carcinoma (n = 2), colorectal adenocarcinoma (n = 2), gastric adenocarcinoma (n = 2), hepatocellular carcinoma (n = 2),
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renal cell carcinoma (n = 2), ductal carcinoma of the breast (n = 12), papillary adenocarcinoma of the thyroid gland (n = 5), melanoma (n = 5), granular cell tumor (n = 4), leiomyosarcoma (n = 4), rhabdomyosarcoma (n = 3), schwannoma (n = 3), phyllodes tumor of the breast (n = 4), fibroadenoma of the breast (n = 3), and alveolar soft part sarcoma (n = 2). Patient data were collected according to HIPPA (Health Insurance Portability and Accountability Act) regulations. The study was approved by the institutional review board.
2.2. Immunohistochemistry Immunohistochemistry was performed on archival formalin-fixed, paraffin-embedded tissue. Sections were deparaffinized and hydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 minutes. The reaction was carried out using the streptavidin-biotin immunoperoxidase technique in an automated immunostainer (Benchmark, Ventana Medical System, Tucson, AZ). Antigen retrieval was performed in all sections by heating the slides in 0.01 mol/L of citrate buffer (pH 6.0) for 20 minutes, unless otherwise specified. The slides were then incubated with mouse antihuman surfactant protein mAb, clone PE-10 (Research Diagnostic, Inc, Minneapolis, MN; dilution, 1:50), CC-10 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:100), or CD68 (Dako, Carpinteria, CA; dilution, 1:500) at 378C for 32 minutes. For DC-LAMP detection, sections were incubated with mAb DC-LAMP, clone 104G4 (Immunotech, Fullerton, CA; dilution 1:50), at room temperature overnight without antigen retrieval. In each analysis, a tumor section that had been previously confirmed to express targeted antigens was included as a positive control.
2.3. Electron microscopy For EM examination, samples of tumors were fixed in 3% phosphate-buffered glutaraldehyde in a phosphate buffer isomolar with plasma at a pH of 7.4, postfixed in 2% osmium tetroxide, dehydrated, and embedded in Epon 812. Ultrathin sections were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a JEOL 1010 electron microscope (Pleasonton, CA).
2.4. Statistical analysis The Fisher exact test was used as indicated. A P value of .05 was considered significant.
3. Results 3.1. Anti–DC-LAMP mAb stains Clara cells in normal lung Immunostains were performed on sections of normal lung to examine the expression pattern of PE-10,
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Fig. 1 Photomicrograph of normal and neoplastic lung tissue showing the localization of several markers studied. A, Normal lung tissue showing PE-10 staining in terminal bronchioles. Note staining on the surface of the cells of the terminal bronchioles and type II pneumocytes in the alveolar wall. B and C, Normal pulmonary tissue stained with antihuman DC-LAMP antibody. Note apical staining of cuboidal cells (B) and in granular staining pattern for this antibody (C). D, Normal pulmonary tissue stained with CC-10 antibody. Note apical staining of some of the cells in the terminal bronchiole. E, Pulmonary adenocarcinoma, mixed subtype with acinar and bronchioloalveolar patterns showing diffuse staining granular and cytoplasmic pattern in acinar component for DC-LAMP. F, Same tumor as in E, stained for CC-10.
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation DC-LAMP, and CC-10. PE-10 antibody showed a diffuse staining pattern in the cells lining the alveolar wall and bronchioles (Fig. 1A). DC-LAMP mAb showed an apical
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granular staining pattern in scattered cells in distal respiratory bronchiolar epithelium (Fig. 1B and C). The location and staining pattern resembles that of Clara cells. By light
Fig. 2 DC-LAMP antibody stains adenocarcinoma with Clara cell differentiation. A case of lung adenocarcinoma with EM-proven Clara cell differentiation (A-C) was reactive with PE-10 mAb (A) and DC-LAMP mAb (B) and showed typical Clara cell granules ultrastructurally (C). A case of lung adenocarcinoma with EM-proven type II pneumocyte differentiation (D-F) shows strong staining for PE-10 (D) but is negative for DC-LAMP (E). EM shows cytoplasmic lamellar bodies, which are characteristic of type II pneumocytes.
264 microscopy, Clara cells are barely discernible by histologic features, including nuclei located in the mid portion of the cell, and a dome-shaped apical protrusion of the cytoplasm above the apical tight junctions. A similar pattern of distribution of positive cells was seen with CC-10 antibody (Fig. 1D). Given that CD68 is the closest homologue of DC-LAMP, we also evaluated its staining pattern in normal lung and confirmed that the antibody stained alveolar macrophage,
L.-C. Zhu et al. with no staining in the epithelial cells of the respiratory bronchioles (not shown). There was nonspecific (cytoplasmic) staining of alveolar macrophages with DC-LAMP.
3.2. DC-LAMP mAb stains adenocarcinoma with Clara cell–type differentiation Bronchioloalveolar carcinomas of the lung are rare, well-differentiated carcinomas that show no stromal invasion. To define the expression pattern of DC-LAMP in neoplastic lung tissues, 12 cases of BAC were studied. These cases of pure BAC were actively collected over the period of 12 years from 3 independent hospitals that are part of the NYUMC, which reflects the rarity of this form of adenocarcinoma. Pure BAC represents approximately 5% of all lung adenocarcinomas resected with curative intent at NYUMC. All BAC studied stained strongly and diffusely for PE-10 and TTF-1. However, there was a heterogenous pattern of staining for DC-LAMP and CC-10. Among the 12 cases studied, only 4 cases showed a positive reaction for DC-LAMP. In these 4 cases, DC-LAMP mAb produced a diffuse, granular, and apical cytoplasmic staining pattern (Fig. 2). EM examination of these tumors demonstrated that the tumor cells had apical granules with a dense uniform content that are characteristic of Clara cell granules (Fig. 2). In contrast, in 3 of the 8 cases of BAC with negative staining for DC-LAMP, EM studies demonstrated that the tumor cells had cytoplasmic lamellar bodies, which are diagnostic of type II pneumocytes differentiation (Fig. 2). The data suggest that mAb DC-LAMP reacts with BAC with Clara cell differentiation.
3.3. Loss of DC-LAMP expression in solid areas of lung adenocarcinoma In an effort to determine the expression level of DCLAMP in lung adenocarcinoma, 75 cases of primary lung adenocarcinomas were studied. All tumors were mixed subtype adenocarcinomas. The carcinomas were further divided into 3 groups according to the content of the BAC component. Group I (14 cases) was composed of mixedtype adenocarcinomas with a predominance of BAC component (more than 50% of the tumor mass); group 2 (25 cases) was composed of mixed-type carcinomas with both BAC and invasive components in which the BAC Fig. 3 Loss of DC-LAMP expression in solid areas of lung adenocarcinoma. A, Section of lung adenocarcinoma, mixed subtype with solid and acinar components showed DC-LAMP– positive staining in the more differentiated areas of the tumor (acinar) and loss of DC-LAMP expression in the adjacent solid tumor areas. B and C, Positive DC-LAMP staining was consistently detected at the periphery of the tumor nodules and in the lung immediately abutting the tumor nodules. Note positive reaction in cells lining the alveolar wall that have type II pneumocyte appearance (C). Higher magnification of the same area shown in (B).
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation component was less than 50% of the tumor mass; and Group III (36 cases) was composed of invasive adenocarcinoma with no BAC component. DC-LAMP showed positive staining in 21.4% (3/14) of the tumors in group 1 (2 women and 1 man), 36% (9/25) of the tumors in Group II (5 women and 4 men), and 25% (9/36) of the tumors of group 3 (4 women and 5 men). Therefore, DC-LAMP appears to mark a subset of pulmonary adenocarcinomas independent of the amount of BAC component. There was no sex predilection for DC-LAMP–positive tumors. Of all adenocarcinomas studied, 86% were also positive for TTF-1, whereas 74% were positive for PE-10. All DC-LAMP– positive adenocarcinomas were positive for TTF-1 and PE10. All tumors that were negative for TTF-1 were also negative for DC-LAMP. Therefore, DC-LAMP does not seem to be a better marker than TTF-1 and PE-10 in the diagnosis of pulmonary adenocarcinoma. Furthermore, EM examination of 6 of the 14 cases from group 1 demonstrated Clara cell type phenotype in the 3 tumors with strong DC-LAMP expression, and type II pneumocyte phenotype in the 3 tumors with negative DCLAMP staining. It was consistently noted that DC-LAMP was mainly expressed in the relatively well-differentiated glandular (acinar) component, even in the group with no BAC component. Solid tumor areas usually lost expression of the antigen (Fig. 3A). Similar results were obtained with CC-10. All tumors that were positive for DC-LAMP were also reactive to CC-10 (Fig. 1E and F), with the exception of 1 tumor that only showed reactivity to CC-10 and no DC-LAMP stain. Strong DC-LAMP and CC-10 positivity was usually detected in reactive cells at the junction of tumor nodules and areas of organizing pneumonia close to the tumor mass (not shown), thus indicating that the antigens (DC-LAMP and CC-10) were expressed in reactive cells in response to injury (Fig. 3B-C). These reactive cells lining the alveolar space had morphologic characteristics of type II pneumocytes.
3.4. Mortality rate of patients with DC-LAMP–positive tumors To investigate whether there was a difference in biologic behavior from tumors that showed Clara cell differentiation, as compared with the rest of the pulmonary adenocarcinomas, survival data from the time of diagnosis were obtained from patients’ medical charts. The average time of follow-up was 5 years, with a range from 1 to 12 years. Most patients who succumbed to the disease died within 1 year of diagnosis. There were no recorded deaths in the group of 12 patients whose tumors were diagnosed as pure BAC. There was no death in group 1 (patients with tumors with a predominant BAC component). In group 2 (mixed adenocarcinomas with small amount of BAC component), there were 2 deaths of 25 patients, representing a mortality rate of 8% in this
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group. When stratified for DC-LAMP reactivity, 1 death was seen in the DC-LAMP–positive tumor (1 [11%] of 9 patients), and 1 death in a DC-LAMP–negative tumor group (1 [6%] of 16 patients). In group 3, which is composed of tumors with no BAC component, there were 8 deaths in 36 patients, representing a mortality rate of 22%. Two deaths occurred in patients that had DC-LAMP–positive tumors (2 of 9, ie, 22%), and 6 deaths in DC-LAMP– negative tumors (6 of 27, ie, 22%). Although the number of cases studied was small, there was no statistical difference in death rate when stratified per DC-LAMP reactivity ( P N .05). Therefore, the histologic classification of the tumor and the amount of BAC component of the adenocarcinoma seem to correlate with the prognosis as previously reported [15,16] but not the cell type differentiation.
3.5. DC-LAMP expression in other epithelial and mesenchymal tumors There was no reactivity with DC-LAMP antibody in pulmonary squamous cell carcinoma and small cell carcinoma. No stain was seen in other nonpulmonary carcinomas and mesenchymal tumors such as renal cell carcinoma, gastric, prostatic, mammary, colorectal adenocarcinomas, hepatocellular carcinoma, papillary adenocarcinoma of the thyroid, ovarian papillary serous carcinoma, malignant melanoma, granular cell tumors, leiomyosarcoma, rhabdomyosarcoma, schwannoma, phyllodes tumors of the breast, fibroadenomas of the breast, and alveolar soft part sarcoma. The lone exception was endometrial adenocarcinoma. In the cases of endometrial adenocarcinoma studied, DCLAMP showed a similar apical and granular staining pattern, as seen in BAC of the lung (Fig. 4) in 22% of the endometrial adenocarcinomas evaluated (6 of 28 cases), thus demonstrating a similar heterogeneous staining pattern for endometrial adenocarcinoma, as observed in the lung. Reactivity for DC-LAMP was also noted in complex hyperplasia of the endometrium but not in simple hyperplasia and normal endometrial cells (data not shown). Similar to DC-LAMP, anti–CC-10 antibody also marked endometrial carcinomas and not other tumor types. PE-10 reactivity was seen only in pulmonary adenocarcinomas. TTF-1 reactivity was seen in pulmonary adenocarcinoma, papillary adenocarcinoma of the thyroid gland, and pulmonary small cell carcinoma.
4. Discussion This study demonstrates that DC-LAMP stains well differentiated pulmonary adenocarcinoma with Clara cell differentiation without cross-reactivity, with adenocarcinomas showing type II pneumocyte differentiation. In normal human lung, DC-LAMP stains cells in the most distal part of the respiratory bronchiole where Clara cells are the predominant cell type [17].
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Fig. 4 DC-LAMP expression in endometrial adenocarcinoma. A, Microphotograph of a well-differentiated endometrial adenocarcinoma (hematoxylin and eosin stain). B, Endometrial adenocarcinoma stained with anti-human DC-LAMP. Insert shows similar diffuse and granular pattern of staining, as seen in lung adenocarcinomas with Clara cell differentiation.
In contrast, PE 10, an antibody to surfactant proteins, will react with any cell type in the lungs that secretes or stores surfactant proteins or surfactant protein accumulated in the alveolar space. TTF-1 stains pulmonary epithelium with exception of ciliated bronchial epithelial cells. Thus, PE-10 and TTF-1 can be used clinically as a tool for the identification of adenocarcinomas of the lung. DC-LAMP is, however, not a useful antibody for the clinical characterization of metastatic pulmonary adenocarcinoma because it only stains a subset of lung tumors that shows Clara cell differentiation. In addition, all tumors that were reactive with DC-LAMP are also reactive for TTF-1 and PE-10. Our results are in contrast with prior investigations that report that DC-LAMP is a marker for type II pneumocytes. In elegant experiments, Salaun et al [12] showed that an mAb against mouse DC-LAMP stains cells in the alveolar wall that have type II pneumocyte morphology. In addition, the antibody seems to colocalize with lamellar bodies, an
L.-C. Zhu et al. ultrastructural marker of type II pneumocytes, in which surfactant protein is stored before its release. This mouse antibody is also reported to cross-react with human DCLAMP. The authors used samples of human adenocarcinomas and showed that in these tumors, only a portion of adenocarcinomas stains with the mouse DC-LAMP antibody, a finding similar to our observations. The differences in cell type that expressed DC-LAMP may be related to a difference in animal species (mouse versus human) or in the antibody used. The authors also show that mouse DCLAMP antibody stains Jaagsiekte sheep retrovirus–associated ovine pulmonary adenocarcinoma. Interestingly, these tumors may also have type II pneumocytes and Clara cell differentiation [18]. We observed that there is an up-regulation of the expression of DC-LAMP in reactive cells around the tumor or around areas of tissue damage. In these areas, the expression of DC-LAMP extended beyond the terminal bronchiole into the alveolar wall. The presence of DCLAMP in these cells could be a result of proliferation of Clara cells in response to injury [19] or proliferation of a progenitor cell population of the lung that can differentiate toward type II pneumocytes and Clara cells [19,20]. In fact, it has been shown that reactive alveolar cell seen in areas of diffuse alveolar damage and idiopathic pulmonary fibrosis can show duo differentiation of Clara cells and type II pneumonocytes, as seen ultrastructurally by the presence of lamellar bodies and dense granules in the same cell [21]. In addition, duo differentiation of Clara cell and type II pneumocytes have been seen in atypical adenomatous hyperplasia, a preneoplastic process that occurs around the terminal bronchioles [22,23]. Pulmonary papillomas with duo differentiation of Clara cells and type II pneumocytes have also been described [24,25]. Alternatively, DC-LAMP staining of these reactive cells could be a result of upregulation of the molecule in type II pneumocytes. It has been shown that hormone-induced differentiation of type II pneumocytes in vitro leads to transient expression of DCLAMP in these cells [26]. Therefore, it is possible to suggest that DC-LAMP may be a marker of Clara cells or an epithelial progenitor cell in the lung that could differentiate toward Clara cell and type II pneumocytes. Our observations that antihuman DC-LAMP stains Clara cells is further corroborated by the fact that the antibody CC10, a specific marker for Clara cells, marks the same tumors that are positive for DC-LAMP. In similarity to DC-LAMP, CC-10 also labels endometrial adenocarcinoma. There was no detectable labeling in any other adenocarcinomas, mesenchymal tumors, and melanomas. The protein CC-10 is homologous to human uteroglobin, a protein that is expressed in endometrial cells [27]. The reasons for the colocalization of CC-10 and DC-LAMP in endometrial adenocarcinomas is not clear. The two protein however, do not share structural homology. In addition, DC-LAMP is located on chromosome 3q27 [8,10,11], whereas CC-10 is localized on chromosome 11q13 [28], which suggests that
DC-LAMP stains pulmonary adenocarcinoma with bronchiolar Clara cell differentiation these two molecules are not regulated under the same promoter. DC-LAMP seems to colocalize with lysosomes, and CC-10 is a protein secreted by Clara cells and thought to have anti-inflammatory properties [7,27]. CC-10 expression by endometrial cells seems to be hormonal, regulated with higher expressions during the progesterone phase of the menstrual cycle [29]. It has been reported that DC-LAMP expression in the lung can be induced by steroid hormones [26]. Our studies also suggest that DC-LAMP is lost during tumorigenesis because poorly differentiated adenocarcinomas of the lung shows very little expression of DC-LAMP, but the antigen is present in the well-differentiated areas of the adenocarcinoma. Similar observations have been made with the antibody against CC-10, the Clara cell marker. This antigen is also lost during tumor differentiation and tumorigenesis [30]. However, there was no down-regulation of DC-LAMP labeling in the normal lung adjacent to adenocarcinomas, as has been reported for CC-10 [31]. The role of DC-LAMP in Clara cells and tumorigenesis is unknown. In a recent report, cell lines transfected with human DC-LAMP expressed increased mobility in in vitro and in vivo assays [32]. In addition, high expression of DC-LAMP by uterine adenocarcinoma and cervical squamous cell carcinoma is associated with increased metastatic potential and poor outcome [32]. In the lung however, the expression of DC-LAMP shows no correlation with prognosis. The function of DC-LAMP is still unknown, but DCLAMP may play a role in cell migration through release of lysosomal enzymes because the molecule is up-regulated in dendritic cells during and upon maturation and migration from skin to regional lymph nodes [33]. It is possible that a similar role is playing in Clara cells, or in a pulmonary precursor cell, because mobility is one of the intrinsic characteristics of stem cells [34]. DC-LAMP is not likely to be a suitable antibody for clinical diagnostic purposes because it does not stain all pulmonary adenocarcinomas, but it might be a useful tool to understanding the biology of Clara cells in lung development and pathology because the marker appears to be expressed in bronchiolar Clara cells, a reserve cell of the bronchiole, and also it appears to be up-regulated in peritumoral benign reactive cells with type II pneumocytes morphology. DC-LAMP could be a candidate as a marker for a pulmonary stem cell.
Acknowledgments The authors thank Ms Olena Ardacheva for her expert technical support on immunochemistry.
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