Expression of the cancer stem cell marker SSEA1 is associated with poor survival in metastatic high-grade serous carcinoma.

The objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (< 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 and p = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Metabolic reprogramming is associated with flavopiridol resistance in prostate cancer DU145 cells.

Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.

Cellular localization of CIP2A determines its prognostic impact in superficial spreading and nodular melanoma.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an important oncogene contributing to cancer progression partially by regulating cMYC and AKT. We examined CIP2A expression in cutaneous melanomas, its association with clinicopathological parameters and mapped molecular mechanisms regulated by CIP2A in vitro. CIP2A expression was analyzed by immunohistochemistry in 17 nevi, 132 primary melanomas and 49 metastases. Effects of siRNA-mediated down-regulation on proliferation, apoptosis and signaling pathways were assessed in melanoma cell lines. In superficial spreading melanomas (SSM), high nuclear CIP2A expression was associated with poor overall survival (OS) (P = 0.0018). Surprisingly, high cytoplasmic expression was related to improved relapse-free (P = 0.031) and OS (P = 0.014) in nodular melanomas (NM). In vitro experiments revealed that CIP2A can regulate proliferation and/or apoptosis partially through the PI3K/AKT pathway but also independently. In summary, CIP2A could represent a potential therapeutic target in SSM. However, in NM cytoplasmic CIP2A is associated with improved prognosis indicating that CIP2A has distinct, complex functions dependent on the molecular context and histological subtype. As seen in other cancer types, CIP2A can influence cMYC and AKT, but our data also suggest that in melanoma it has additional targets which need to be identified.

The diagnostic and research applications of flow cytometry in cytopathology.

Flow cytometry (FCM) is an established ancillary technique applied to the diagnosis of hematological malignancies and for measurement of DNA content. In recent years, the number of fluorochrome-conjugated antibodies available for immunofluorescence and FCM has greatly increased, making it possible to evaluate this technique in other diagnostic contexts, as well as to study a range of biological processes. Serous effusions are optimal for studies using FCM as they consist of viable cells in suspension. Molecules that have been studied for their expression and clinical role in effusions in recent years participate in central cellular functions, including adhesion, proliferation, cellular metabolism, and apoptosis, as well as intracellular signaling. FCM can further be applied to quantitative analysis of molecules related to chemotherapy response, which, together with apoptosis, may represent an important tool for evaluating treatment response and prognosis in advanced and/or recurrent cancer. As targeted therapy assumes an ever-growing role in the treatment of metastatic cancer, the ability to study living cells in effusions or fine-needle aspirates is becoming more important. This article reviews the currently available data in this area of cytopathology.

Synthetic retinoid CD437 induces apoptosis and acts synergistically with TRAIL receptor-2 agonist in malignant melanoma.

The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines.

Nucleoside transporters are widely expressed in ovarian carcinoma effusions.

OBJECTIVE: Equilibrative and concentrative nucleoside transporters (ENTs and CNTs) mediate the cellular uptake of anticancer nucleosides and sensitivity to such compounds. We studied the expression of ENTs and CNTs in ovarian carcinoma effusions. METHODS: ENT1, ENT2, ENT4 and CNT3 expression was analyzed in 66 ovarian carcinoma effusions (61 peritoneal, 5 pleural) from 64 ovarian carcinoma patients by flow cytometry. The majority of patients received platinum-based chemotherapy. Results were analyzed for association with clinicopathologic parameters and survival. RESULTS: With the exception of one ENT2-negative effusion, ENT1, ENT2, ENT4 and CNT3 protein was detected on carcinoma cells in all effusions, with expression observed in 1-95% of tumor cells. Nucleoside transporter expression was comparable between peritoneal and pleural effusions and was unrelated to age, tumor grade, International Federation of Gynecology and Obstetrics (FIGO) stage, residual tumor volume after surgery, previous exposure to chemotherapy and response to chemotherapy at diagnosis (P > 0.05). No correlation was found between ENT or CNT expression and overall survival or progression-free survival, although higher ENT2 expression was associated with a trend for longer overall (45 vs. 23 months; P = 0.055) and progression-free (17 vs. 5 months; P = 0.087) survival. CONCLUSION: Nucleoside transporters are frequently expressed in ovarian carcinoma effusions, but their expression generally appears to be unrelated to chemoresponse in this cancer in a cohort of patients treated by platinum-based chemotherapy. The role of ENT2 as a prognostic marker in this disease, as well as the role of these molecules in determining chemoresponse in patients treated by nucleoside analogs, merits further research.

Metastatic malignant melanoma mimicking benign breast cysts.

Benign cysts are one of the most common mass-occupying lesions of the breast and are often investigated with triple diagnostic trial (clinical examination, radiology, and cytology). Malignant melanoma is one of medicine's imitators, and metastatic disease can mimic cysts. Thorough investigation of any breast mass is essential to clarify its nature.

Mammalian target of rapamycin is a biomarker of poor survival in metastatic serous ovarian carcinoma.

The AKT signaling pathway is crucial for cancer cell survival. The objective of this study was to analyze the expression and clinical role of this pathway in serous ovarian carcinoma. Phospho-AKT and phospho-mammalian target of rapamycin protein expression was studied in 269 ovarian carcinomas (159 effusions, 38 primary carcinomas, 72 solid metastases) using immunohistochemistry. The association between AKT, mammalian target of rapamycin, and DJ-1 in effusions was quantitatively analyzed using flow cytometry. AKT phosphorylation status in effusions was further studied using Western blotting. Phospho-AKT and phospho-mammalian target of rapamycin were detected in the majority of tumors at all anatomical sites. Phospho-AKT expression in effusions was higher in grade 3 versus grades 1 and 2 tumors (P = .013). Flow cytometry analysis showed association between AKT, mammalian target of rapamycin, and DJ-1 expression (P < .001). Higher phospho-AKT Thr308/pan-AKT ratio by Western blotting was associated with more advanced International Federation of Gynecology and Obstetrics stage (P = .018) and a trend for poor response to chemotherapy at first disease recurrence (P = .051). Higher phospho-mammalian target of rapamycin protein expression in effusions by immunohistochemistry was associated with poor progression-free survival for patients with postchemotherapy effusions (P = .005). Phospho-mammalian target of rapamycin was an independent predictor of poor progression-free survival for patients with postchemotherapy effusions (P = .03). The association between activated AKT and mammalian target of rapamycin expression and clinicopathologic parameters of aggressive disease, including shorter patient survival, provides further evidence regarding the central role of this signaling pathway in ovarian carcinoma.

Evaluation of cell surface expression of phosphatidylserine in ovarian carcinoma effusions using the annexin-V/7-AAD assay: clinical relevance and comparison with other apoptosis parameters.

Phosphatidylserine cell surface exposure during apoptosis can be detected by its binding to the protein annexin-V. We investigated annexin-V expression in 76 ovarian carcinoma effusions using flow cytometry. Results were analyzed for association with clinicopathologic parameters and survival. Annexin-V expression was additionally compared with the previously studied apoptotic markers cleaved caspase-3, cleaved caspase-8, and deoxyuridine triphosphate (dUTP) incorporation into DNA fragments. Annexin-V was expressed in all specimens and was more frequently detected compared with cleaved caspases and dUTP incorporation (P < .001). Annexin-V expression was higher in grade 3 vs grades 1 and 2 tumors (P = .014). A higher percentage of annexin-V-expressing cells in postchemotherapy specimens was associated with poor overall (P = .005) and progression-free (P = .013) survival. We present the first evidence of annexin-V expression in ovarian carcinoma effusions. The higher annexin-V expression compared with other apoptosis parameters and its association with high-grade disease and poor survival in postchemotherapy patients suggest a role in cell survival rather than apoptosis in effusions.

Expression of the folate receptor genes FOLR1 and FOLR3 differentiates ovarian carcinoma from breast carcinoma and malignant mesothelioma in serous effusions.

The objective of this study was to analyze the diagnostic and clinical role of the folate receptor-alpha (FOLR1) and folate receptor-gamma (FOLR3) genes in effusion cytology. Expression of the FOLR1 protein product, FR-alpha, was additionally studied. Ninety-one effusions (71 ovarian carcinomas, 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for FOLR1 and FOLR3 gene expression using quantitative polymerase chain reaction. FR-alpha expression was analyzed using flow cytometry. Ovarian carcinoma expression levels were analyzed for association with clinicopathologic parameters and survival. Quantitative polymerase chain reaction analysis showed significantly higher FOLR1and FOLR3 mRNA levels in ovarian carcinomas compared with both breast carcinomas and mesotheliomas (P < .001). FOLR1 and FOLR3 mRNA levels were directly interrelated in ovarian carcinoma (P < .001). FR-alpha protein levels were similarly higher in ovarian carcinoma compared with the 2 other cancer types (P < .001). FOLR1and FOLR3 mRNA and FR-alpha protein expression in ovarian carcinoma effusions showed no association with clinical parameters or survival. Our data suggest that folate receptor levels effectively differentiate ovarian carcinoma from other cancers affecting the serosal cavities and that folate receptor genes are coexpressed in this tumor. The high expression of folate receptors in ovarian carcinoma supports their validity as molecular therapeutic targets in this disease.

Cleaved caspase-3 and nuclear factor-kappaB p65 are prognostic factors in metastatic serous ovarian carcinoma.

Tumor progression and treatment failure in ovarian carcinoma are frequently associated with metastasis to effusions. The present study analyzed the expression and clinical role of nuclear factor-kappaB p65, nuclear factor-kappaB inhibitor alpha, and parameters of apoptosis in serous carcinoma. Cleaved caspase-3 and caspase-8 levels and deoxyuridine triphosphate incorporation were measured in 65 effusions using flow cytometry. Effusions (n = 209) and corresponding primary carcinomas and solid metastases (n = 114) were immunohistochemically analyzed for nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expression. Effusions (n = 75) were further analyzed for nuclear factor-kappaB phospho-p65 (Ser536) levels using immunoblotting. Results were analyzed for association with anatomic site, clinicopathologic parameters, and survival. Caspase cleavage and deoxyuridine triphosphate incorporation were limited to less than 10% of cells in most effusions. Nuclear factor-kappaB p65 expression was frequently detected at all anatomic sites, with less frequent cytoplasmic nuclear factor-kappaB p65 and nuclear factor-kappaB inhibitor alpha expressions. Immunoblotting showed nuclear factor-kappaB p65 phosphorylation in 72 (96%) of 75 effusions. Higher than median cleaved caspase-3 levels correlated with improved overall and progression-free survival in univariate analysis of all patients (P = .024 and P = .046, respectively) and of those with postchemotherapy effusions (P = .042 and P = .036, respectively). Cleaved caspase-3 expression was an independent predictor of longer progression-free survival for patients with postchemotherapy effusions (P = .029). Nuclear factor-kappaB p65 expression correlated with poor progression-free survival for all patients (P = .048) and for those with postchemotherapy effusions (P = .025). Ovarian carcinoma cells in effusions undergo little apoptosis, but high levels of cleaved caspase-3 are associated with improved survival. Nuclear factor-kappaB p65 is frequently expressed in advanced-stage serous ovarian carcinoma, and its nuclear localization is associated with poor progression-free survival.

The chemokine receptor CXCR4 is more frequently expressed in breast compared to other metastatic adenocarcinomas in effusions.

This objective of this study was to investigate the expression of chemokine receptors in tumor cells and leukocytes in breast carcinoma effusions. The expression of leukocyte markers (CD3/4/8/14/16/19) and chemokine receptors (CXCR1/4, CCR2/5/7) was studied in 21 breast carcinoma effusions using flow cytometry. Breast carcinoma cells expressed CXCR4 in 7/21 (33%) effusions, with less frequent expression of CXCR1, CCR5, and CCR7. CXCR2 and CCR2 were absent. Lymphocytes showed frequent CXCR4, CCR5, and CCR7 expression, while CXCR1, CXCR2, CCR2 were rarely or never detected. Macrophages expressed all six receptors except for CXCR2. Comparative analysis of breast carcinoma effusions with previously studied ovarian and cervical/endometrial adenocarcinomas (ACs) showed significantly higher CXCR4 expression in breast carcinoma cells compared to the other gynecological ACs (p = 0.001). Breast and cervical/endometrial carcinoma effusions showed different expression of chemokine receptors in lymphocytes (lower CXCR1, higher CXCR4 and CCR7 levels; p = 0.012, p = 0.005, p < 0.001, respectively) and macrophages (higher CCR7 levels; p < 0.001), as well as lower CD8 counts (p < 0.001) and higher CD19 counts (p = 0.001) compared to ovarian carcinoma effusions. Higher numbers of CD8-positive lymphocytes (p = 0.080) and higher CCR7 monocyte expression (p = 0.087) were associated with a trend for shorter disease-free survival. In conclusion, breast carcinoma cells express CXCR4, a unique feature among metastatic ACs in effusions, with rare expression of other chemokine receptors. Chemokine receptor expression in leukocytes and lymphocyte counts significantly differ from those of ovarian carcinoma effusions. The prognostic role of CCR7 expression in monocytes and CD8 counts in breast carcinoma effusions merits further research.

Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry.

We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.

Death receptor expression is associated with poor response to chemotherapy and shorter survival in metastatic ovarian carcinoma.

BACKGROUND: Death receptors mediate both apoptosis and survival in cancer cells. The authors analyzed death receptor expression in metastatic ovarian carcinoma. METHODS: Viable tumor cells in ovarian carcinoma effusions (n = 95) were analyzed for DR4, DR5, Fas, TNFR1, and TNFR2 expression using flow cytometry. Results were analyzed for association with clinicopathologic parameters, chemotherapy response, and survival. RESULTS: DR4, DR5, and Fas were expressed by the majority of specimens, with less frequent expression of TNFR1 and TNFR2. DR4 (P = .005) and TNFR2 (P = .041) expression was higher in FIGO stage IV compared with stage III tumors. Effusions from patients who responded poorly to chemotherapy administered at disease recurrence had significantly higher DR4 (P = .006), DR5 (P = .01), and Fas (P = .001) expression. In univariate survival analysis, higher DR4 expression in viable cells correlated with poor overall (P = .0352) and progression-free (P = .0411) survival. DR4 expression was found to be an independent predictor of overall (P = .008) and progression-free (P = .003) survival. CONCLUSIONS: The authors have presented the first evidence of death receptor coexpression in ovarian carcinoma effusions. The association of death receptor expression in effusions with advanced stage, poor response to chemotherapy, and shorter survival suggests that these molecules are linked to an aggressive clinical course in metastatic ovarian carcinoma.

Flow cytometric immunophenotyping of cancer cells in effusion specimens: diagnostic and research applications.

Flow cytometry (FCM) immunophenotyping is frequently used as an ancillary technique for the diagnosis of hematological malignancies or for measurement of DNA content. In recent years, we applied FCM to the diagnosis of metastatic adenocarcinoma and malignant mesothelioma in effusions. We established a panel of antibodies that allows for rapid and effective differentiation between epithelial cells, mesothelial cells, and leukocytes. FCM was subsequently used for quantitative analysis of integrin subunits. Recently, we studied different parameters of the immune response, including HLA molecules and chemokine receptors, using this method. Our data suggest that FCM is an effective method for the characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and that this method is comparable to immunohistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. This review discusses previous work in this area and the future potential of this method in the characterization of tumor cells in serous effusions.

Chemokine receptors are infrequently expressed in malignant and benign mesothelial cells.

We studied chemokine receptor expression in malignant mesothelioma (MM), reactive mesothelium (RM), and leukocytes in effusions. The expression of leukocyte markers (CD3, CD4, CD8, CD14, CD16, and CD19) and chemokine receptors (CXCR1, CXCR4, CCR2, CCR5, and CCR7) was studied in 11 MM and 16 RM specimens using flow cytometry. RM specimens showed higher lymphocyte counts (mean rank, 17.6 vs 8.8; P = .004), whereas monocyte numbers were higher in MM (mean rank, 19.5 vs 10.2; P = .002). CXCR1 (P =.006) and CXCR4 (P = .036) expression was higher in MM monocytes. Chemokine receptors were infrequently expressed in MM (0-2/11 effusions per receptor), whereas RM specimens were uniformly negative. Chemokine receptors are widely expressed on leukocytes in MM and RM effusions but are infrequently found on cells of mesothelial origin. This finding suggests a major role for an autocrine chemokine pathway in leukocytes but not in MM cells. The increased monocyte infiltration and their higher chemokine receptor expression in MM effusions may have a tumor-promoting rather than tumor-inhibiting effect.

NK- and B-cell infiltration correlates with worse outcome in metastatic ovarian carcinoma.

We studied the clinical role of leukocyte infiltration and chemokine receptor expression in ovarian carcinoma effusions. Expression of leukocyte markers (CD3, CD4, CD8, CD4/CD8 ratio, CD16, CD19, and CD14) and chemokine receptors (CXCR1, CXCR4, CCR2, CCR5, and CCR7) was studied in 73 effusions by using flow cytometry. CXCR4, CCR5, and CCR7 were expressed abundantly on leukocytes, but all receptors were expressed rarely on cancer cells. The presence of natural killer cells (P = .042) and International Federation of Gynecology and Obstetrics (FIGO) stage IV disease (P = .024) predicted worse overall survival (OS). A higher percentage of CD19+ cells (P = .015) and stage IV disease (P = .008) predicted poor survival for patients with postchemotherapy effusions. Only FIGO stage retained significance as a predictor of OS (P = .035) in multivariate analysis. Chemokine receptors are expressed widely on leukocytes but rarely on carcinoma cells in ovarian carcinoma effusions, arguing against an autocrine chemokine pathway in this malignancy. Immune response parameters in ovarian cancer effusions are weak predictors of outcome.

Expression of HLA-G in malignant mesothelioma and clinically aggressive breast carcinoma.

The aim of the present study was to evaluate HLA-G expression in breast carcinoma and malignant mesothelioma (MM). Malignant breast carcinoma effusions (46) and corresponding solid tumors (39) and 104 MM (26 effusions, 78 solid tumors) were analyzed using immunohistochemistry (IHC). HLA-G protein and mRNA expression were further studied using immunoblotting (IB) and RT-PCR. HLA-ABC expression was analyzed using flow cytometry (FCM). IHC showed predominantly focal HLA-G expression in 12 of 46 (26%) breast carcinoma effusions and 16 of 39 (41%) solid lesions. In MM, 20 of 78 (26%) solid lesions and 14 of 26 (54%) effusions were focally HLA-G positive. Expression in MM was higher in effusions (p=0.008). IB showed more frequent HLA-G expression in MM compared with breast carcinoma effusions, while RT-PCR showed HLA-G mRNA expression in both tumors. FCM showed conserved HLA-ABC expression in 15 of 15 effusions. Breast cancer patients with HLA-G-positive tumor cells had shorter disease-free survival (mean 37 vs 85, median 25 vs 31 months), though not significantly (p=0.14). In conclusion, HLA-G is focally expressed in MM and breast carcinoma, while HLA-ABC expression is conserved. However, the up-regulated expression of HLA-G in MM effusions and its possible association with shorter disease-free survival in advanced stage of breast carcinoma suggest a possible role in immune response evasion in some tumors.

Quantitative analysis of integrin expression in effusions using flow cytometric immunophenotyping.

We have previously shown that flow cytometric (FCM) immunophenotyping is a useful adjunct to morphology, in the diagnosis of serous effusions. The objective of the present study was to evaluate the possible application of FCM to quantitative analysis of adhesion molecule expression in this clinical setting. Fresh frozen cells from 67 effusions underwent quantitative analysis of alphaV, alpha6, beta1, and beta3 integrin subunit expression, using FCM. Specimens were diagnosed as carcinoma (n = 48), reactive (n = 12), or malignant mesothelioma (MM; n = 7) using morphology and, in selected cases, immunocytochemistry prior to FCM analysis. Antibodies against established epithelial, lymphoid, and mesothelial cell epitopes (Ber-EP4, anti-epithelial membrane antigen; (EMA), anti-CD45, anti-CD14, and anti-CD15) completed the panel. Results (percentage of cells expressing the antigen) were analyzed for relationship with the morphologic diagnosis. Frequent expression of the alphaV, alpha6, and beta1 subunits was seen in all diagnostic groups, with significantly higher expression of the alpha6 subunit in MM (P = 0.029, Kruskal-Wallis H test). The beta3 integrin subunit was not detected in any of the specimens. Ber-EP4 and CD15 expression was significantly higher in carcinomas compared with reactive effusions and MM (P < 0.001 and P = 0.001, Kruskal-Wallis H test), and EMA expression was higher in carcinomas and MM, compared with reactive specimens (P < 0.001, Kruskal-Wallis H test). In conclusion, FCM is an efficient tool for quantitative analysis of adhesion molecules in effusions. The high alpha6 integrin subunit expression in MM suggests involvement of this receptor in tumor attachment to laminin. The frequent expression of the alphaV and beta1 subunits support attachment to fibronectin and vitronectin as the major ECM ligands in body cavities.

Altered expression of metastasis-associated and regulatory molecules in effusions from breast cancer patients: a novel model for tumor progression.

PURPOSE: The aim of this study was to characterize phenotypic alterations along the progression of breast carcinoma from primary tumor to pleural effusion through analysis of the expression of proteases, laminin receptors (LRs), and transcription factors involved in invasion and metastasis. EXPERIMENTAL DESIGN: The material studied consisted of 60 malignant pleural effusions from breast cancer patients and 68 corresponding solid tumors (37 primary and 31 metastatic tumors). Expression of matrix metalloproteinases [MMPs (MMP-1, MMP-2, MMP-9, and MMP-14)], the MMP inhibitor tissue inhibitor of metalloproteinase-2, the MMP inducer EMMPRIN, the 67-kDa LR, the alpha6 integrin subunit, and the transcription factors AP-2, Ets-1, and PEA3 was studied using immunohistochemistry, mRNA in situ hybridization, reverse transcription-polymerase chain reaction, zymography, and flow cytometry. Hormone receptor (estrogen receptor and progesterone receptor) status and c-erbB-2 status were also studied. RESULTS: Significantly reduced estrogen receptor (P < 0.001) and progesterone receptor (P = 0.001) expression was seen in effusions compared with primary tumors, with opposite findings for c-erbB-2 (P = 0.003). Tumor cell MMP-2 protein expression in effusions was higher than that in primary tumors (P < 0.001) and lymph node metastases (P = 0.01). In situ hybridization demonstrated higher MMP-2 (P = 0.007), PEA3 (P = 0.038), and EMMPRIN (P = 0.026) mRNA expression in effusions. The time to progression from primary tumor to effusion was significantly shorter for patients whose primary tumors expressed MMP-1 (P = 0.016) and who expressed the 67-kDa LR protein in primary tumor (P = 0.007) and effusion (P = 0.015). CONCLUSIONS: Our data provide documented evidence of molecular events that occur during the progression of breast carcinoma from primary tumor to effusion. The coordinated up-regulation of MMP-2 and Ets transcription factors in carcinoma cells in effusions is in full agreement with our previous reports linking these factors to poor prognosis in ovarian cancer. The rapid progression to effusion in cases showing MMP-1 and 67-kDa LR expression in primary tumor cells links aggressive clinical behavior with expression of metastasis-associated molecules in this setting.