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In our study, we demonstrate that ccRCC cell lines with impaired function of pVHL to degrade HIFα express elevated levels of PD-L1. In vitro analysis provided evidence that both reconstitution of pVHL and silencing of HIF2α, but not of HIF1α, lead to reduced PD-L1 expression. The strong correlation of expression between the HIF2α-specific HIF target Glut1 and PD-L1 confirmed this finding in ccRCC cell lines and tissue. Soluble PD-L1 levels remained constant in the sera of ccRCC patients regardless of the PD-L1 expression status in their tumors. In conclusion, our data suggest PD-L1 as HIF2α target, which is upregulated in pVHL deficient ccRCC. The combination of PD-L1 targeting drugs with HIF inhibiting agents may be an additional option for the treatment of ccRCC.

To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvβ3, αvβ5, αvβ6 and αvβ8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvβ3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p

Although von Hippel-Lindau (VHL) tumor suppressor gene alterations dominate the genetic landscape of clear cell renal cell carcinoma (ccRCC), recent studies have identified new ccRCC genes, including SETD2, KDM6A, KDM5C, BAP1 and PBRM1. Strikingly, all these genes fall into a category of histone/chromatin regulators. Polybromo-1 (PBRM1) is the second most frequently mutated gene after VHL; however, the clinical relevance of its loss in ccRCC has not yet been reported. Here, we analyzed the expression of PBRM1, the product encoded by PBRM1, in ccRCC cell lines and in more than 300 RCC tumor samples. The data were correlated with clinicopathological parameters and VHL mutation status. We found that a significant number of ccRCC cancer cell lines lack detectable PBRM1 expression. Loss of PBRM1 was predominant in the clear cell subtype of RCC (∼ 70%) and correlated with advanced tumor stage (p < 0.0001), low differentiation grade (p = 0.0002) and worse patient outcome (p = 0.025), but not with the VHL mutation status. Our results indicate a critical role for PBRM1 in the suppression of ccRCC progression. Moreover, the results suggest that functional inactivation of PBRM1 in the context of pVHL loss-of-function may represent a key event in facilitating the development of key aspects of an aggressive tumor behavior. Given the role of PBRM1 in chromatin modification, the gene expression pathways disrupted by the inactivation of this protein may lead to new treatment strategies for ccRCC.

Epithelial cell adhesion molecule (EPCAM) has recently attained a renewed interest as a candidate protein in diagnosis, prognostication and therapy of various tumor entities. The molecular epidemiology and prognostic relevance of EPCAM in renal cell carcinoma (RCC) and amongst the histological subtypes of RCC is unclear. We analyzed the prevalence and prognostic significance of EPCAM in a tumor tissue micro-array (TMA) composed of 1088 independent RCCs samples by immunohistochemistry (IHC). We found significant variations of EPCAM IHC staining intensities in between the RCC subtypes: In papillary and chromophobe RCC, the majority of tumors (89-93%) showed an at least weak EPCAM protein expression. In the largest subgroup, the clear cell (cc)RCC (n=767), a negative EPCAM IHC was found in 1/3 of the patients and was associated with high-grade disease and nodal metastases. Kaplan-Meier analyses demonstrated a significant association between positive EPCAM IHC and prolonged overall survival, even in a subset of low risk ccRCC. In multivariable analyses, EPCAM represented an independent risk factor of survival throughout all subgroups. For localized, low-grade ccRCC, information of EPCAM IHC raised predictive accuracy of a multivariate model by ∼5%, compared to T-stage and grade alone. Our findings indicate, that EPCAM is an independent prognostic molecular marker in ccRCC and, especially in localized ccRCC, might be able to provide auxiliary information for a better prognostication.

The majority of prostate cancer harbors recurrent gene fusions involving ETS transcription factors, most commonly ERG. The second most common 5' fusion partner after TMPRSS2 is SLC45A3. The aim of our study was to quantify the protein expression of ERG, TMPRSS2 and SLC45A3 in prostate cancer to assess for diagnostic or prognostic utility. Six hundred and forty consecutive prostate cancer cases in tissue microarray format were immunohistochemically analyzed for ERG, TMPRSS2 and SLC45A3 protein. Resultant protein expression data was correlated to the respective gene rearrangement status and clinico-pathological parameters including PSA follow up times. ERG showed no expression in benign prostate glands. In cancer tissue, ERG protein expression showed a high rate of concordance with an underlying ERG rearrangement (91.5%). SLC45A3 showed a weaker expression in cancer as compared to benign tissue, which was pronounced in cases with SLC45A3-ERG fusion. Importantly, SLC45A3 down regulation was significantly associated with shorter PSA-free survival times. In contrast, TMPRSS2 was neither differentially expressed nor did it show a correlation between protein expression and rearrangement status. This study provides first evidence that the expression of SLC45A3 protein is down regulated through SLC45A3-ERG fusion in prostate cancer. Moreover, these cases may represent a distinct molecular subclass of ERG rearranged prostate cancer with distinct clinical features. This study also confirms that ERG protein expression is predominantly found in prostate carcinomas with ERG gene rearrangement and does not occur in benign glands.

In patients with early head and neck squamous cell carcinoma (HNSCC), occult lymph node metastasis is difficult to predict by clinical or pathological parameters. However, such parameters are necessary to select patients either for elective neck dissection or the sentinel lymph node (SLN) procedure. The membrane glycoprotein podoplanin is normally expressed in lymphatic endothelial cells. Recently, expression of podoplanin by cancer cells was demonstrated to promote tumor cell motility and tumor lymphangiogenesis in vitro. The value of cancer cell-expressed podoplanin was to be determined as a predictive marker for SLN metastasis in early HNSCC of the oral cavity and oropharynx. One hundred twenty patients with HNSCC of the oral cavity and oropharynx undergoing a SLN biopsy were enrolled in this prospective clinical trial of SLN biopsy. Cancer cell-expressed podoplanin was determined by immunohistochemistry using tissue microarrays. Podoplanin expression was quantified by the intensity reactivity score and categorized into expression and nonexpression. SLN examination revealed occult metastasis in 45 patients (37.5%). Twenty-nine of 120 (24.2%) primary HNSCC showed podoplanin expression. Podoplanin expression correlated significantly with SLN metastasis (p = 0.029) and remained a significant predictor for lymph node status even after controlling for tumor stage (p = 0.028). As a predictive marker for SLN metastasis, however, podoplanin expression reached a sensitivity of a mere 36% and a specificity of 83%. Podoplanin expression is associated with metastasis to lymph nodes in vivo. Podoplanin immunohistochemistry in early HNSCC of the oral cavity and oropharynx may help to select patients for the SLN procedure and to identify patients with increased risk for presence of occult lymph node metastasis in the neck.

The purpose of our study was to demonstrate that distinct cytogenetic alterations in the most common subtype of renal cell cancer, clear cell renal cell carcinoma (ccRCC), are reflected in protein expression profiles. We performed conventional cytogenetics and immunohistochemical analysis for cytokeratins (CKs) on 126 ccRCCs. Protein expression was evaluated in situ using a semiautomated quantitative system. The results were validated using an independent cohort of 209 ccRCCs with long-term follow-up. Cytogenetic alterations were identified in 96 of 126 ccRCCs, most of them involving chromosome 3 through loss, deletion or translocation. Expression of CKs and E-cadherin in ccRCC was associated with lack of cytogenetic alterations and low nuclear grade. In the validation set, CK7 and CK19 protein expression was associated with better clinical outcome. At the multivariate level, the best model included metastatic status and CK19 expression. Expression microarray analysis on 21 primary ccRCCs and 14 ccRCC metastases identified genes significantly associated with CK7 and CK19 expressing ccRCCs. Two novel ccRCC biomarkers associated with the CK7 positive ccRCC phenotype, PMS2 and MT1-MMP (MMP14), were further validated. We conclude that the variability observed for CK expression in ccRCC can be explained by genetic heterogeneity. Distinct molecular subtypes of ccRCC with prognostic relevance were identified, and the CK7/CK19 expressing subtype is associated with better outcome.

NY-ESO-1 is a cancer testis antigen expressed in various malignancies and testicular germ cells. Because of its capacity to induce specific humoral and cellular immunity in patients with NY-ESO-1-positive carcinomas, it represents a promising target for cancer immunotherapy. In breast cancer, NY-ESO-1-mRNA was reported in up to 42%, but protein expression has not been determined to larger extent. In the present tissue microarray-based study, primary breast cancers (n = 1,444), in situ lesion (n = 148), recurrences (n = 88), lymph node (n = 525) and distant metastases (n = 91) were studied for NY-ESO-1 expression by immunohistochemistry. NY-ESO-1-protein expression was compared with mRNA expression by real-time PCR. NY-ESO-1-protein was detected in 3.1% (4/128) in situ lesions and in 2.1% (28/1355) invasive breast cancer. There were 1.8% (9/493) NY-ESO-1-positive lymph node and 5.1% (4/78) positive distant metastases. NY-ESO-1 was more frequently expressed in grade 3 (4.9%) than in grade 2 (0.8%) and grade 1 (0.5%) carcinomas (p < 0.0001). Presence of tumor-infiltrating CD8+ T-cells correlated with NY-ESO-1 (p < 0.0001) on the tissue microarray. On randomly selected large sections, 4 out of 9 NY-ESO-1-positive tumors displayed a brisk infiltrate of CD79a+ plasmocytes/B-cells, but none of 10 NY-ESO-1-negative tumors (p < 0.05). NY-ESO-1-mRNA expression was detected in frozen samples of NY-ESO-1-protein positive (n = 6) and negative breast cancers (n = 8) and in normal testis. Comparison between mRNA and protein expression revealed that only breast cancers with NY-ESO-1-mRNA levels comparable or higher than testis expressed NY-ESO-1-protein. These findings suggest that NY-ESO-1-positive breast cancers represent a small subset of poorly differentiated tumors with evidence of cellular and humoral immune response.

Microsatellite instability at mono- and dinucleotide repeats is the hallmark of the hereditary non-polyposis cancer syndrome (HNPCC) and is related to deficient DNA mismatch repair. In contrast, a distinct form of microsatellite instability at selective tetranucleotide repeats (EMAST or elevated microsatellite alterations at selected tetranucleotides) was described in several non-HNPCC cancer types. EMAST is probably unrelated to mismatch repair defects. We investigated the frequency of microsatellite instability at mononucleotide, dinucleotide and tetranucleotide repeats in a series of 75 ovarian carcinomas (53 serous and 22 non-serous). Microsatellite analysis was carried out using 5 mono- and dinucleotide markers from the National Cancer Institute Consensus Panel and 6 tetranucleotide markers, which have been reported as frequently unstable in the literature. High frequency of microsatellite instability (MSI-H) at mono- and dinucleotide repeats was observed in 9% and a low frequency (MSI-L) in 21% of serous carcinomas. MSI-H was detected in 4% and MSI-L in 18% of non-serous carcinomas. Nine percent of serous carcinomas showed instability at multiple and 9% at single tetranucleotide loci. All non-serous carcinomas were stable at tetranucleotide loci. In summary, EMAST (e.g., tumors with tetranucleotide instability without concomitant MSI-H) was observed in 13% of ovarian serous carcinomas. All EMAST positive tumors were of advanced stage. We conclude that EMAST occurs as a distinct form of microsatellite instability in ovarian cancer. EMAST seems to be particularly frequent in advanced serous carcinomas. Its clinical significance needs to be investigated.

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti-neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 M(r) cell surface protein. The protein was partially purified by immuno-affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH(2)-terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1-CAM), and both were characterized by the NH(2)-terminal deletion of 5 amino acids, comprising exon 2 of L1-CAM. Reverse trancription-polymerase chain reaction (RT-PCR) analysis of the regions spanning exon 2 and exon 27 of L1-CAM indicated that in neuroblastoma cells both transcripts for the full-length and exon-deleted forms are present, whereas in the renal carcinoma cell lines only the exon-deleted L1-CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1-CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1-CAM mRNA levels are correlated with protein expression.

Recent results suggest that p53 inactivation is required for cervical-carcinoma development. The mdm-2 oncogene, which forms an auto-regulatory feedback loop with the normal p53 protein, has been found amplified in human carcinomas, thus abolishing the anti-proliferative function of p53. To investigate whether the mdm-2/p53 interaction plays a role in cervical neoplasms, we performed an immunohistochemical study in archival fixed, embedded specimens that included 178 pre-cancerous lesions (CIN) and invasive squamous-cell carcinomas of clinical stage IB. In addition to p53, we assessed the p53-associated protein, mdm-2, and the Ki-67 labelling index (LI). The presence of HPV was assessed by in situ DNA hybridization. Tumor expression of all nuclear proteins was scored as fraction of positive CIN or cancer nuclei. The analysis demonstrated a significant association of the Ki-67 LI with grade of atypia in cervical neoplasms. p53 accumulation and mdm-2 expression are higher in invasive carcinomas than in pre-cancerous lesions. No correlation was observed with HPV status. An inverse correlation was found between increased tumor-cell proliferation and mdm-2 expression in invasive carcinomas (p < 0.0001). mdm-2 expression was significantly associated with p53 accumulation (p < 0.02). However, the investigated nuclear proteins were not associated with overall survival in patients with invasive carcinomas. Cox stepwise-regression analysis revealed regional lymph node status and depth of invasion to be independent parameters.

To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvβ3, αvβ5, αvβ6 and αvβ8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvβ3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p0.01). Similarly, integrin αvβ8 expression was increased in brain metastases compared to primary tumors of breast cancer (p0.0001), lung cancer (p0.01) and renal cancer (p0.0001), with a similar trend in metastatic melanoma. Integrin αvβ5 was expressed in most primary tumors (98% breast cancer; 67% lung cancer; 90% renal cancer; 89% melanoma) and showed a stronger expression in brain metastases compared to primary tumors from lung cancer and melanoma (p0.05). Also integrin αvβ6 expression was increased in brain metastases compared to primary breast cancer (p0.001).The stronger αv-integrin expression in brain metastases, especially of αvβ3 and αvβ8 integrins, suggests that certain αv integrin are involved in the process of brain metastasis. αv Integrins may be therapeutic targets for patients with metastatic cancer in brain.

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.

Cancer testis (CT) antigens are attractive targets for immunotherapy in cancer patients. Immunohistochemistry was used to study the expression of the CT antigens MAGE-C2/CT-10, MAGE-C1/CT-7, GAGE, MAGE-A4 and NY-ESO-1 in 146 hepatocellular carcinomas, 13 intrahepatic cholangiocarcinomas, 37 extrahepatic cholangiocarcinomas and 32 gallbladder carcinomas. Immunopositivity was correlated with clinicopathological parameters, MHC Class 1 expression, intratumoral CD4+, CD8+ and FOXP3+ T cells and CD163+ antigen-presenting cells. Of the 146 hepatocellular carcinomas, 34% were positive for MAGE-C2/CT-10, 12% for MAGE-C1/CT-7, 11% for GAGE and 2% for NY-ESO-1, respectively. MHC Class 1 coexpression was identified in almost all CT antigen-positive tumors. The number of intratumoral FOXP3+ regulatory T cells was increased in CT antigen-positive hepatocellular carcinomas (p < 0.004), suggesting inhibition of immune response in such tumors. Furthermore, MAGE-C1/CT-7 and GAGE positivity was correlated with reduced overall survival in patients with hepatocellular carcinoma (p = 0.03 and 0.01, respectively). Four (13%) gallbladder carcinomas stained positive for MAGE-C2/CT-10, of which 1 tumor (3%) was also positive for NY-ESO-1 and GAGE. CT antigens were not expressed in intra- and extrahepatic cholangiocarcinomas. Our results suggest that MAGE-C2/CT-10 may be a good candidate for peptide vaccination in patients with hepatocellular carcinoma. © 2008 Wiley-Liss, Inc.

The purpose of our study was to demonstrate that distinct cytogenetic alterations in the most common subtype of renal cell cancer, clear cell renal cell carcinoma (ccRCC), are reflected in protein expression profiles. We performed conventional cytogenetics and immunohistochemical analysis for cytokeratins (CKs) on 126 ccRCCs. Protein expression was evaluated in situ using a semiautomated quantitative system. The results were validated using an independent cohort of 209 ccRCCs with long-term follow-up. Cytogenetic alterations were identified in 96 of 126 ccRCCs, most of them involving chromosome 3 through loss, deletion or translocation. Expression of CKs and E-cadherin in ccRCC was associated with lack of cytogenetic alterations and low nuclear grade. In the validation set, CK7 and CK19 protein expression was associated with better clinical outcome. At the multivariate level, the best model included metastatic status and CK19 expression. Expression microarray analysis on 21 primary ccRCCs and 14 ccRCC metastases identified genes significantly associated with CK7 and CK19 expressing ccRCCs. Two novel ccRCC biomarkers associated with the CK7 positive ccRCC phenotype, PMS2 and MT1-MMP (MMP14), were further validated. We conclude that the variability observed for CK expression in ccRCC can be explained by genetic heterogeneity. Distinct molecular subtypes of ccRCC with prognostic relevance were identified, and the CK7/CK19 expressing subtype is associated with better outcome.

NY-BR-1 is a differentiation antigen and a potential target for cancer immunotherapy. Its mRNA expression is restricted to breast, testis, prostate and breast cancer by RT-PCR. In this study, we correlated NY-BR-1 protein and mRNA expression on tissue microarrays of mammary, prostatic and testicular malignancies using immunohistochemistry and in situ hybridization with probes for exon 4-7 and 30-33. NY-BR-1 mRNA was confined to primary spermatocytes, suggesting a role in spermatogenesis. Exon 4-7 and 30-33 were equally expressed this cell type. However, NY-BR-1 was absent in all germ cell tumours analyzed (n = 475) and present in one of 56 (2%) prostate carcinomas. In breast, NY-BR-1 mRNA expression was detected in 307 of 442 (70%) primary carcinomas, with strong correlation to its protein expression (p < 0.0001). mRNA expression was significantly stronger and more frequently detected by the exon 30-33 probe than by the exon 4-7 probe (70% vs. 35%, p < 0.0001), indicating the presence of alternative splice variants that lack 5-prime sequences. A similar restricted mRNA pattern was also observed in the normal breast epithelium. NY-BR-1 protein and mRNA correlated significantly with estrogen receptor alpha (ER alpha) protein expression (p < 0.0001), with stronger association to NY-BR-1 mRNA than protein (odds ratio 7.7 compared to 4.6). We identified 4 estrogen response elements (ERE)-like sequences nearby the promoter region, suggesting that NY-BR-1 transcription might be controlled by ER alpha. Accordingly, analysis of matching pairs of primary tumors with their recurrences showed a marked decrease of NY-BR-1 expression in recurrences after tamoxifen treatment (p < 0.0001).

A loss of chromosome-9 material is one of the most frequent genomic aberrations known in bladder cancer. In order to better understand the role of chromosome-9 losses in bladder cancer, 125 formalin-fixed and 37 unfixed bladder tumors were examined using fluorescence in situ hybridization (FISH). A repetitive probe for a pericentromeric region on 9q12 (pHUR98) was applied for chromosome-9 copy-number enumeration. Under-representation of chromosome 9 was found in 74 of 162 cases. There was no association between loss of chromosome 9 and increased grade or stage, papillary growth pattern, p53 protein expression, or tumor-cell proliferation (Ki-67). These data show that chromosome-9 loss is an early event in bladder-cancer development, occurring independently of p53 alterations. In order to determine the prevalence of large sub-regional chromosome-9 deletions, dual hybridizations with pHUR98 and cosmid probes for 9q34, 9q22, and 9p21 were performed. Partial deletion was detected in only 1 of 36 cases for 9q34 and in 1 of 24 cases for 9p21. Surprisingly, amplification of the interferon alpha locus on 9p21 was seen in 1 of 24 tumors. The finding of 9p amplification may indicate the site of an oncogene relevant for bladder cancer.

Epidermal-growth-factor-receptor (EGF-r) expression has been proposed as a prognostic marker in bladder cancer and is associated with rapid proliferation in cell lines. Ninety-three fresh and 74 formal in-fixed bladder tumors were examined by fluorescence in situ hybridization (FISH) and immunohistochemistry to assess the relationship between EGF-r expression and proliferation as well at the prevalence of epidermal-growth-factor-receptor (EGF-r) gene amplification. EGF-r expression was strongly associated with BUdr labeling index, grade and stage. EGF-r expression emerged as a stronger predictor of tumor proliferation than grade or stage in analysis of variance. Rapid tumor proliferation might be responsible for bad prognosis reported in EGF-r positive bladder tumors. Also chromosome 7 copy number was associated with grade and stage. EGF-r gene amplification was uncommon (5 of 107 tumors). However, FISH analysis allowed characterization of the pattern of amplification, with clustering of signals suggestive of intrachromosomal amplification more common than diffuse distribution consistent with extrachromosomal amplification. © 1994 Wiley-Liss, Inc.