Nuclear survivin promoted by acetylation is associated with the aggressive phenotype of oral squamous cell carcinoma

Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.     

The distribution of IGF2 and IMP3 in osteosarcoma and its relationship with angiogenesis

The aim of this study was to investigate the expression patterns of IGF2 and IMP3 in osteosarcoma as well as its relationship with angiogenesis in the tumor. IGF2 and IMP3 expression was detected by immunohistochemical staining in the serial sections of the osteosarcoma. The impacts of IGF2 and IMP3 expression patterns on tumor angiogenesis were evaluated by statistics. The IGF2 and IMP3 staining had different expression patterns in different osteosarcoma. Twelve out of the sixty-four cases of conventional osteosarcoma showed nuclear staining patterns, and twenty-nine showed cytoplasmic staining of IGF2 and IMP3 simultaneously. On the other hand, fourteen cases showed nuclear IGF2 staining but cytoplasmic IMP3 expression, and nine cases showed nuclear IMP3 staining and cytoplasmic IGF2 expression. Twenty-eight out of forty-seven cases of parosteal osteosarcoma showed nuclear IGF2 and IMP3 expression, nine showed cytoplasmic IGF2 and IMP3 expression simultaneously. Seven out of forty-seven cases of parosteal osteosarcoma expressed IGF2 with nuclear staining but expressed IMP3 with cytoplasmic staining. Meanwhile, three cases expressed IGF2 with cytoplasmic staining but expressed IMP3 with nuclear staining. Similar to the parosteal osteosarcoma, the periosteal osteosarcoma expressed IGF2 and IMP3 mainly with nuclear staining simultaneously, forty out of fifty-five cases of periosteal osteosarcoma did that. Five out of fifty-five cases expressed IGF2 and IMP3 with cytoplasmic staining at the same time. Four cases showed nuclear IGF2 staining and cytoplasmic IMP3 staining. In the parosteal and periosteal osteosarcoma, there was no significant difference in IGF and IMP3 expression patterns (P = 0.216). However, compared with conventional osteosarcoma, the parosteal and periosteal osteosarcoma showed significant difference in IMP3 and IGF2 expression (P = 0.016, P = 0.023). IGF2 and IMP3 expression patterns were positive correlation in the different osteosarcoma (r = 0.1021, P = 0.032). The Microvessel density (MVD) in osteosarcoma with IGF2 and IMP3 cytoplasmic staining was more than that with nuclear expression of IGF2 and IMP3, and the difference was significant (P = 0.024). Moreover, the conventional osteosarcoma with cytoplasmic IGF and IMP3 showed more MVD than parosteal and periosteal osteosarcoma with cytoplasmic IGF and IMP3, and the difference was significant (P = 0.035). IGF2 and IMP3 had different expression patterns, which might be associated with angiogenesis. However, cytoplasmic and nuclear expression of IGF2 and IMP3 might play different roles in the angiogenesis of osteosarcoma.     

Differential subcellular localization of functionally divergent survivin splice variants

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.     

A multiple marker analysis of apoptosis-associated protein expression in non-small cell lung cancer in a Chinese population

A failure to undergo apoptosis is widely thought to be an important event in cancer formation and progression. Although there have been many studies in vitro that provide evidence for this suggestion, the roles of apoptosis-associated proteins in cancer tissues in vivo are not as yet fully understood. Moreover, multiple marker analyses of apoptosis-associated protein expression in non-small cell lung cancer (NSCLC) tissues are scarce. In the present study, we investigate the expression of a group of apoptosis-associated proteins including bcl-2, caspase-3, fas, fas ligand (fasL) and survivin, and its clinical significance in NSCLC tissues using immunohistochemistry (IHC). Bcl-2 staining in cancer tissue cells was found in cytoplasm and the positive rate was 38.2% (29/76). Caspase-3 staining was mainly seen in cytoplasm of cancer tissue cells (53.9% [41/76]) with a few cases of nuclear staining (6.6% [5/76]). Fas staining was seen in cytomembrane (15.8% [12/76]) and cytoplasm (42.1% [32/76]) of cancer tissue cells. Likewise, fasL also showed staining in cytoplasm (55.3% [42/76]) and cytomembrane (44.7% [34/76]) of cancer tissue cells. Survivin staining was seen in cytoplasm but not nuclear of cancer tissue cells and the positive rate was 48.7% (37/76). Higher cytoplasm expression of bcl-2 was associated with large tumor size (≥ 3 cm) in NSCLC (p < 0.05). Decreased cytoplasm expression of fas was associated with poor grade in NSCLC (p < 0.05). A negative correlation was found between bcl-2 and cytoplasm caspase-3 expression in NSCLC (p < 0.001). No separate expression of the apoptosis-associated proteins in NSCLC was linked to overall survival of patients (p > 0.05). Multiple marker analyses revealed caspase-3+/cytomembrane fasL- to be linked to better survival of patients with NSCLC (p < 0.05). These results indicate that apoptosis- -associated proteins may impact a variety of clinicopathological features of NSCLC and may co-operatively influence the prognosis of patients with this malignant tumor.     

Prognostic significance of metallothionein expression in renal cell carcinoma

BACKGROUND: Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival. PATIENTS AND METHODS: The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival). RESULTS: Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25-50% of tumor cells in 7 cases, and in a percentage of 50-75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival. CONCLUSIONS: The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter.     

Nuclear shuttling of the peptidase nardilysin

The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.     

Nuclear and cytoplasmic expression of ErbB-4 in prostate cancer

PURPOSE: To evaluate cytoplasmic and nuclear ErbB-4 expression in prostate cancer specimens and its association with outcome. BASIC PROCEDURES: Specimens of 50 prostate cancer patients were investigated for ErbB-4 overexpression using Immunohistochemistry staining. Cytoplasmic and nuclear staining was graded as 0-3 according to its intensity. The prognostic parameters were tumor stage, PSA level, Gleason score, probability of positive lymph nodes (Partin's tables and Roach equation), and 5-year disease free survival (Kattan nomogram). MAIN FINDINGS: Overexpression of ErbB-4 (> or = 1) was detected in 30 (60%) patients and overexpression using cytoplasmic and nuclear staining was > or = 2 in 19 (38%) and 17 (34%) patients, respectively. In only one third of the specimens was there any similarity between the 2 types of staining. Advanced tumor stage, high pretreatment PSA levels and high Gleason scores were evenly distributed among the patients with low (< or = 1) and intermediate/high (> or = 2) ErbB-4 expression. The probability of lymph node involvement and 5-year disease free survival were similar in both types of staining. PRINCIPAL CONCLUSIONS: ErbB-4 was overexpressed (cytoplasmic and nuclear staining) in approximately one third of prostate cancer patients. The rate of similarity between the 2 staining types was only 33%: overexpression was evenly distributed among intermediate/high and low risk prostate cancer patients with both staining methods.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.     

CEA, ZGM and EMA localization in cells of pleural and peritoneal effusion: a preliminary study

Carcinoembryonic antigen (CEA), the zinc glycinate marker (ZGM) and epithelial membrane antigen (EMA) have been described as epithelial or tumour markers of varying specificity. These antigens were studied by immunoperoxidase localization in selected cell blocks of 62 pleural or peritoneal effusions and compared to cytological findings and review of the clinical records. By cytological criteria, 25 of the cell blocks were positive for malignancy, 30 negative, and 7 inconclusive. CEA, ZGM, and EMA by immunoperoxidase staining were localized on the cell surface and often in the cytoplasm of malignant cells, in 11/25 (44 per cent), 17/25 (68 per cent) and 22/25 (88 per cent) of the positive cell blocks respectively. Ten (40 per cent) of these cases were positive for all three antigens, 7 (28 per cent) for two, and 6 (24 per cent) for one. Of the 7 cases which were inconclusive on routine cytological reporting, 5 were positive for at least one marker. In 3 of the 5 a diagnosis of malignancy was confirmed, and in the other two was strongly suspected as malignant on clinical grounds. Macrophages were sometimes positive for one or more markers (but showed cytoplasmic staining only) and mesothelial cells in some cases stained positively for EMA but were always negative for CEA and ZGM. Localization of the 3 antigens in cells of malignant effusions was compared with their localization in the primary tumours in 9 cases. Localization corresponded for CEA in 7 of 9 cases, for EMA in 8 of 8 an for ZGM in only 2 of 9. Effusion fluid levels for CEA were compared with the cytological and immunocytochemical findings in 30 cases. Mucin stains performed on the cell blocks were also compared with the immunoperoxidase findings.     

Diagnostic utility of GLUT-1 expression in the cytologic evaluation of serous fluids

OBJECTIVE: To evaluate the extent to which adenocarcinomas in body cavity fluids express GLUT-1 in comparison to currently available markers for adenocarcinomas. STUDY DESIGN: Archival paraffin-embedded cell blocks of serous fluids from 25 cases of benign effusions containing reactive mesothelial cells and 39 cases of malignant effusions with metastatic adenocarcinoma (11 ovarian, 11 pulmonary, 9 gastrointestinal and 8 breast) were retrieved from the surgical pathology files. All cases were stained with antibodies for GLUT-1, Ber-Ep4, B72.3 and CEA. Positive staining was defined as distinct linear membrane staining for GLUT-1 and Ber-EP4, cytoplasmic staining for CEA, and cytoplasmic or membrane staining for B72.3. Strong staining in at least 10% of the tumor cells was required in order to consider the case positive for the particular marker. RESULTS: GLUT-1 was expressed in 72% (28 of 39) of cases of malignant effusions: 100% (11 of 11) from the ovary, 91% (10 of 11) from the lung, 67% (6 of 9) from the gastrointestinal tract and 12% (1 of 8) from the breast. None (0 of 25) of the benign effusions expressed GLUT-1. Malignant effusions expressed CEA in 74% (29 of 39), Ber-Ep4 in 85% (33 of 39), and B72.3 in 62% (24 of 39). Benign effusions expressed CEA in 3 cases and B72.3 in 2 cases. CONCLUSION: GLUT-1 is a useful marker that can be applied to cytologic specimens. It can be used as a reliable component of an antibody panel to distinguish reactive mesothelial cells from metastatic adenocarcinoma in particular adenocarcinomas of body cavity effusions, in particular adenocarcinomas of ovarian and pulmonary origin.     

Nuclear Expression of the Deubiquitinase CYLD Is Associated with Improved Survival in Human Hepatocellular Carcinoma

Background & Aims

The deubiquitinase CYLD removes (K-63)-linked polyubiquitin chains from proteins involved in NF-κB, Wnt/ß-catenin and Bcl-3 signaling. Reduced CYLD expression has been reported in different tumor entities, including hepatocellular carcinoma (HCC). Furthermore, loss of CYLD has been shown to contribute to HCC development in knockout animal models. This study aimed to assess subcellular CYLD expression in tumor tissues and its prognostic significance in HCC patients undergoing liver resection or liver transplantation.

Methods

Subcellular localization of CYLD was assessed by immunohistochemistry in tumor tissues of 95 HCC patients undergoing liver resection or transplantation. Positive nuclear CYLD staining was defined as an immunhistochemical (IHC) score ≥3. Positive cytoplasmic CYLD staining was defined as an IHC score ≥6. The relationship with clinicopathological parameters was investigated. Cell culture experiments were performed to analyze subcellular CYLD expression in vitro.

Results

Cytoplasmic CYLD expression was observed in 57 out of 95 (60%) HCC specimens (^cyt°CYLD⁺). Nuclear CYLD staining was positive in 52 out of 95 specimens (55%, ^nucCYLD⁺). 13 out of 52 ^nucCYLD⁺ patients (25%) showed a lack of cytoplasmic CYLD expression. ^nucCYLD⁺ was associated with prolonged overall survival in patients after resection or liver transplantation (P = 0.007). 5-year overall survival rates were 63% in ^nucCYLD⁺ vs. 26% in ^nucCYLD^- patients. Nuclear CYLD staining strongly correlated with tumor grading (P<0.001) and Ki67 positivity (P = 0.005). ^nucCYLD⁺ did not prove to be an independent prognostic parameter. In vitro, Huh7, Hep3B and HepG2 showed reduced CYLD levels compared to the non-malignant liver cell line THLE-2. Induction of CYLD expression by doxorubicin treatment led to increased cytoplasmic and nuclear expression of CYLD.

Conclusions

Expression of nuclear CYLD is a novel prognostic factor for improved survival in patients with HCC undergoing liver resection or transplantation.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A “nuclear” cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

p53突变蛋白在胃癌组织中的表达及免疫电镜观察

作者应用抗ｐ５３单克隆抗体Ｐａｂ１８０１（Ａｂ２美国癌基因公司产品）对３８例手术切除的胃癌组织及癌旁胃粘膜的冰冻切片标本进行ｐ５３突变蛋白表达的检测,并进一步用胶体全免疫电镜技术对ｐ５３突变蛋白的分布特征进行观察。结果：３８例胃癌组织中,２４例有ｐ５３突变蛋白高表达,阳性率６３．２％。在对应的癌旁胃粘膜中１０例为ｐ５３的弱表达,正常组织无表达。伴有淋巴结转移的２３例胃癌标本中,１８例ｐ５３高表达（７８．３％）。免疫电镜结果表现,ｐ５３蛋白主要分布于核内染色质中,胞浆中有散在的阳性区,但以核膜周边为主,紧靠核膜。本研究结果提承胃癌的发生及其肿瘤的生物学行为与抑癌基因ｐ５３的突变密切相关,ｐ５３突变蛋白可能是通过对ＤＮＡ复制的影响而参与肿瘤的形成。     

Histone deacetylase 6 (HDAC6) deacetylates survivin for its nuclear export in breast cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.     

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions

Objective: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. Methods: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. Results: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. Conclusions: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.     

The G Protein-Coupled Estrogen Receptor (GPER) Is Expressed in Two Different Subcellular Localizations Reflecting Distinct Tumor Properties in Breast Cancer

Introduction

The G protein-coupled estrogen receptor (GPER) is a novel estrogen receptor that mediates proliferative effects induced by estrogen but also by tamoxifen. The aim of our study was to analyze the frequency of GPER in a large collective of primary invasive breast carcinomas, with special emphasis on the subcellular expression and to evaluate the association with clinicopathological parameters and patient overall survival.

Methods

The tissue microarrays from formalin-fixed, paraffin embedded samples of primary invasive breast carcinomas (n = 981) were analyzed for GPER expression using immunohistochemistry. Expression data were compared to the clinicopathological parameters and overall survival. GPER localization was also analyzed in two immortalized breast cancer cell lines T47D and MCF7 by confocal immunofluorescence microscopy.

Results

A predominantly cytoplasmic GPER expression was found in 189 carcinomas (19.3%), whereas a predominantly nuclear expression was observed in 529 cases (53.9%). A simultaneous comparable positive expression of both patterns was found in 32 of 981 cases (3.2%), and negative staining was detected in 295 cases (30%). Confocal microscopy confirmed the occurrence of cytoplasmic and nuclear GPER expression in T47D and MCF7. Cytoplasmic GPER expression was significantly associated with non-ductal histologic subtypes, low tumor stage, better histologic differentiation, as well as Luminal A and B subtypes. In contrast, nuclear GPER expression was significantly associated with poorly differentiated carcinomas and the triple-negative subtype. In univariate analysis, cytoplasmic GPER expression was associated with better overall survival (p = 0.012).

Conclusion

Our data suggest that predominantly cytoplasmic and/or nuclear GPER expression are two distinct immunohistochemical patterns in breast carcinomas and may reflect different biological features, reason why these patterns should be clearly distinguished in histological evaluations. Prospective studies will be needed to assess whether the expression status of GPER in breast carcinomas should be routinely observed by clinicians, for instance, before implementing endocrine breast cancer treatment.