Interleukin-33 Expression Indicates a Favorable Prognosis in Malignant Salivary Gland Tumors.

The cytokine interleukin-33 (IL-33) is abundantly expressed in epithelial barrier tissues such as salivary glands. Here, we characterized nuclear IL-33 protein expression by immunohistochemistry in benign and malignant salivary gland tumors and associated it with disease outcome. Most benign salivary gland tumors expressed IL-33, and all Warthin's tumors showed strong and consistent IL-33 expression in the basally oriented cells of their bilayered epithelium. In the malignant group of neoplasms, nuclear IL-33 expression was limited to specific tumor entities-for example, to epithelial-myopepithelial carcinomas (n = 9/11), acinic cell carcinomas (n = 13/27), and oncocytic carcinomas (n = 2/2). IL-33 expression in the combined group of malignant salivary gland neoplasms was significantly associated with favorable histological parameters, lack of metastasis, and longer overall survival, compared with IL-33-negative tumors. We conclude that IL-33 expression is a novel prognostic marker for malignant salivary gland tumors with potential use in clinical diagnostics.

Clear cell papillary renal cell carcinoma and renal angiomyoadenomatous tumor: two variants of a morphologic, immunohistochemical, and genetic distinct entity of renal cell carcinoma.

Clear cell papillary renal cell carcinoma (ccpRCC) and renal angiomyoadenomatous tumor (RAT) share morphologic similarities with clear cell (ccRCC) and papillary RCC (pRCC). It is a matter of controversy whether their morphologic, immunophenotypic, and molecular features allow the definition of a separate renal carcinoma entity. The aim of our project was to investigate specific renal immunohistochemical biomarkers involved in the hypoxia-inducible factor pathway and mutations in the VHL gene to clarify the relationship between ccpRCC and RAT. We investigated 28 ccpRCC and 9 RAT samples by immunohistochemistry using 25 markers. VHL gene mutations and allele losses were investigated by Sanger sequencing and fluorescence in situ hybridization. Clinical follow-up data were obtained for a subset of the patients. No tumor recurrence or tumor-related death was observed in any of the patients. Immunohistochemistry and molecular analyses led to the reclassification of 3 tumors as ccRCC and TFE3 translocation carcinomas. The immunohistochemical profile of ccpRCC and RAT samples was very similar but not identical, differing from both ccRCC and pRCC. Especially, the parafibromin and hKIM-1 expression exhibited differences in ccpRCC/RAT compared with ccRCC and pRCC. Genetic analysis revealed VHL mutations in 2/27 (7%) and 1/7 (14%) ccpRCC and RAT samples, respectively. Fluorescence in situ hybridization analysis disclosed a 3p loss in 2/20 (10%) ccpRCC samples. ccpRCC and RAT have a specific morphologic and immunohistochemical profile, but they share similarities with the more aggressive renal tumors. On the basis of our results, we regard ccpRCC/RAT as a distinct entity of RCCs.

Characterization of the 19q12 amplification including CCNE1 and URI in different epithelial ovarian cancer subtypes.

BACKGROUND: CCNE1 is frequently amplified in high grade serous ovarian cancer and may serve as a target for ovarian cancer treatment. URI is closely related to CCNE1 at the 19q12 amplicon and may also contribute to the oncogenic effect. Our objective was to investigate the relevance of CCNE1 and URI gene amplification and protein expression in different histological subtypes of epithelial ovarian cancer (EOC). METHODS: A novel dual-color 19q12 in situ hybridization (ISH), covering CCNE1 and URI, and chromosome 19 as a surrogate using Ventana BenchMark XT platform was developed and applied to 148 EOCs. URI and CCNE1 amplifications were separately assessed by fluorescence in situ hybridization (FISH). Immunohistochemistry using a Cyclin E1 and a novel URI monoclonal antibody was performed. RESULTS: Amplification of 19q12 was found in 36.6%, CCNE1 in 21.7%, URI in 9.9%, and both genes simultaneously in 9% of EOC cases. High Cyclin E1 and URI protein expression were observed in 52.2% and 26.1%, respectively. Amplification of 19q12 occurred in all EOC subtypes and was associated with amplification and expression of CCNE1/Cyclin E1, URI, TP53 mutation, and advanced stage. CONCLUSION: The novel 19q12 ISH probe reliably detects both CCNE1 and URI amplifications as confirmed by FISH. The combination of 19q12 amplification with Cyclin E1 and URI protein expression may help to select patients more likely to benefit from CDK2 targeted therapies.

An Aggressive Hypoxia Related Subpopulation of Melanoma Cells is TRP-2 Negative.

Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells. Analysing over 200 melanomas including primaries, partly matched metastases and patients' cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival. Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation.

Identification of molecular tumor markers in renal cell carcinomas with TFE3 protein expression by RNA sequencing.

TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study, we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNA read-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P < .001) and 22 papillary RCCs (P < .05-.07). Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) and Contactin 3 (CNTN3) expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both).

Whole genome and transcriptome amplification: practicable tools for sustainable tissue biobanking?

The use of whole genome amplification (WGA) and whole transcriptome amplification (WTA) techniques enables the enrichment of DNA and RNA from very small amounts of tissue. Here, we tested the suitability of WGA and WTA for tumor tissue biobanking. DNA and RNA from 13 standardized and seven non-standardized frozen and 12 formalin-fixed, paraffin-embedded (FFPE) clear cell renal cell carcinoma specimens (>9 years old) served to test the robustness of the WGA and WTA products by reidentifying von Hippel-Lindau (VHL) gene mutations known to exist in these samples. The enrichment of DNA and RNA from frozen tissue was up to 1,291-fold and 423-fold, respectively. The sizes and yields (10- to 73-fold) of the amplified DNA obtained from the 12 FFPE samples were generally lower. The quality of the RNA from the FFPE samples was too low to reliably perform WTA. Our results demonstrate that frozen tumor tissue is very suitable for WGA and WTA. All 20 VHL mutations were verified with WGA. Notably, we were able to show that 18 of the 20 (90 %) VHL mutations are also transcribed. In FFPE tumor tissue, 8 of 12 cases (67 %) showed the expected mutations after the first WGA. Accurate WTA with FFPE material is sophisticated and strongly depends on the modification and degradation status of the fixed tissue. We conclude that for sustainable tissue biobanking, the use of WGA and WTA is a unique opportunity to provide researchers with sufficient amounts of nucleic acids, preferably from limited frozen tissue material.

Automated ERCC1 immunochemistry on hybrid cytology/tissue microarray of malignant effusions: evaluation of antibodies 8F1 and D-10.

BACKGROUND: The excision repair cross-complementation group 1 (ERCC1) protein is the key enzyme of the nucleotide excision repair (NER) pathway. Loss of protein expression on immunohistochemistry is predictive for platinum-based chemotherapy response. Frequently, the diagnosis of malignancy is made on cytologic effusion samples. Therefore, we evaluated the staining quality of monoclonal anti-ERCC1 antibodies 8F1 and D-10 on microarrays of malignant pleural and peritoneal effusions by automated immunochemistry. METHODS: Cores from effusion cell blocks of 117 patients with > 40 malignant cell clusters per whole section (pleural n = 75, peritoneal n = 42) were assembled together with 30 histologic control cores from large tissue blocks (lung, breast and ovarian carcinoma, each n = 10) on hybrid cytology-tissue microarrays (C/TMA). Four immunochemistry protocols (Mab 8F1 and D-10, CC1-mono Ventana and H2-60 Bond automat) were performed. Immunoreactivity was semi-quantitatively scored for intensity and intensity multiplied by percentage staining (H-score). RESULTS: Tumors were classified into female genital tract carcinoma (n = 39), lung adenocarcinoma (n = 23), mesothelioma (n = 15), unknown primary (n = 14), breast carcinoma (n = 10), gastro-intestinal carcinoma (n = 12) and other (n = 4). On both platforms, reproducible nuclear ERCC1 immunoreactivity was achieved with both antibodies, although D-10 was slightly weaker and presented more background staining as well as more variation in the low expression range. No significant differences were found between cytologic and histologic cores. Using the 8F1 CC1-mono protocol, lung and breast carcinomas had lower ERCC1 expression in comparison to the other entities (p-value < 0.05). CONCLUSIONS: Cytology microarrays (CMA) are suitable for investigation of clinical biomarkers and can be combined with conventional TMA's. Dichotomization of ERCC1 immunoreactivity scores is most suitable for patient stratification since definition of negativity is antibody-dependent.

VHL mutations and dysregulation of pVHL- and PTEN-controlled pathways in multilocular cystic renal cell carcinoma.

Multilocular cystic renal cell carcinoma is a rare renal cell carcinoma with an excellent prognosis. To clarify the relationship with typical clear cell renal cell carcinoma, we evaluated 15 cases of multilocular cystic renal cell carcinomas diagnosed according to the 2004 WHO classification. Von Hippel Lindau (VHL) gene mutations were determined by whole genome amplification and direct sequencing. Carbonic anhydrase 9 (CAIX), a hypoxia-inducible factor (HIF) target, paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor p27 and glycogen synthase kinase 3-ß (GSK3ß) were immunohistochemically evaluated as members of the VHL protein (pVHL)- and phosphatase and tensin homolog (PTEN)-controlled pathways. VHL mutations were identified in 3 of 12 (25%) tumors. Inactivated GSK3ß, decreased PTEN expression and PAX2 positivity were observed in the vast majority of the multilocular cystic renal cell carcinomas. Strong nuclear staining of p27 was seen in 14 of 15 cases. Compared with multilocular cystic renal cell carcinomas, expression frequencies of PAX2, p-GSK3ß, PTEN and CAIX were similar in a set of low-grade, early-stage clear cell renal cell carcinomas, whereas only 30% had strong p27 positivity. These results are consistent with the hypothesis that multilocular cystic renal cell carcinomas are related at the molecular level with clear cell renal cell carcinomas. Maintenance of a strong subcellular p27 expression in all multilocular cystic renal cell carcinomas analyzed may in part explain the excellent prognosis of these tumor patients.

Podoplanin expression correlates with sentinel lymph node metastasis in early squamous cell carcinomas of the oral cavity and oropharynx.

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.

IGFBP2 and IGFBP3 protein expressions in human breast cancer: association with hormonal factors and obesity.

PURPOSE: The insulin-like growth factor (IGF) signaling system is involved in breast cancer initiation and progression. The prognostic relevance of tumor expression patterns of IGFI-related proteins remains poorly understood. This study associates the expression of selected IGF proteins with breast tumor and patient characteristics. EXPERIMENTAL DESIGN: IGFI, IGFI receptor, IGF-binding protein (IGFBP)2, and IGFBP3 expression was measured in 855 primary breast carcinomas by immunohistochemistry using tissue microarrays. We investigated the association of tumor and nodal stage, grade, hormone receptor status, HER2 gene amplification, menopausal status, body mass index, and survival with IGF protein expression. RESULTS: In contrast to IGFI, the expression of IGFI receptor, IGFBP2, and IGFBP3 was associated with estrogen receptor status. In addition, IGFBP3 was positively correlated with body mass index and premenopausal status. Importantly, IGFBP2 was an independent and positive predictor of overall survival (hazard ratio, 0.48; 95% confidence interval, 0.24-0.95; P = 0.04). There was a weak suggestion for IGFBP2 and overweight to modify each other's effect on survival. CONCLUSIONS: According to these results, which need confirmation in larger patient series, the prognostic relevance of IGFBP2 and IGFBP3 protein expressions in breast cancer may depend on the hormonal context and body weight.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC).

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.

Association of cytokeratin 7 and 19 expression with genomic stability and favorable prognosis in clear cell renal cell cancer.

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.

A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology.

Different methods for snap freezing surgical human tissue specimens exist. At pathology institutes with higher work loads, solid carbon dioxide, freezing sprays, and cryostat freezing are commonly used as coolants for diagnosing frozen tissue sections, whereas for tissue banking, liquid nitrogen or isopentane cooled with liquid nitrogen is preferred. Freezing tissues for diagnostic and research purposes are therefore often time consuming, laborious, even hazardous, and not user friendly. In tissue banks, frozen tissue samples are stored in cryovials, capsules, cryomolds, or cryocassettes. Tissues are additionally embedded using freezing media or wrapped in plastic bags or aluminum foils to prevent desiccation. The latter method aggravates enormously further tissue handling and processing. Here, we describe an isopentane-based workflow which concurrently facilitates tissue freezing and processing for both routine intra-operative frozen section and tissue banking and satisfies the qualitative demands of pathologists, cancer researchers, laboratory technicians, and tissue bankers.

Distinct expression patterns of the immunogenic differentiation antigen NY-BR-1 in normal breast, testis and their malignant counterparts.

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).

Automated immunofluorescence analysis defines microvessel area as a prognostic parameter in clear cell renal cell cancer.

Microvessel density (MVD) has been reported to have prognostic relevance for clear cell renal cell carcinoma (ccRCC). However, this finding is controversial because of the difficulty of MVD evaluation in this complex vascularized tumor type. The present study evaluates the use of an automated quantitative analysis (AQUA) system for objective and reproducible determination of tumor vascularization in clear cell renal cell carcinoma (ccRCC). The AQUA system was applied to tissue microarrays with 284 primary ccRCC tumors. To determine angiogenesis in ccRCC, we created an epithelial/stromal mask consisting of CD10, epithelial membrane antigen, and vimentin to distinguish epithelial tumor cells from CD34-positive endothelial cells. Using immunofluorescence and computer-aided quantification of CD34 expression, we measured the relative microvessel area (MVA) and compared the MVA to the manually counted MVD. The MVA determined by AQUA in a test set with 209 ccRCCs ranged from 0% to 30.3% (mean +/- SD, 10.1% +/- 6.3%). The manually determined MVD ranged from 6 to 987 vessels/mm(2) (416.8 +/- 252.8 vessels/mm(2)). MVA and MVD were significantly correlated (P < .001). A larger MVA was associated with histologic grade (P < .001), tumor stage (P =.008), presence of metastasis (P = .005), presence of sarcomatoid areas (P < .001), and tumor-specific survival (P < .001). Using MVA as defined in the test set, all associations with clinical and pathologic parameters were confirmed in a second independent validation set. MVA determination by AQUA is an objective and reliable method to quantify tumor vascularization in ccRCC. A large MVA correlates with a high MVD and is associated with better patient prognosis.

Consistent expression of the stem cell renewal factor BMI-1 in primary and metastatic melanoma.

Stem cell-like cells have recently been identified in melanoma cell lines, but their relevance for melanoma pathogenesis is controversial. To characterize the stem cell signature of melanoma, expression of stem cell markers BMI-1 and nestin was studied in 64 cutaneous melanomas, 165 melanoma metastases as well as 53 melanoma cell lines. Stem cell renewal factor BMI-1 is a transcriptional repressor of the Ink4a/Arf locus encoding p16(ink4a) and p14(Arf). Increased nuclear BMI-1 expression was detectable in 41 of 64 (64%) primary melanomas, 117 of 165 melanoma metastases (71%) and 15 of 53 (28%) melanoma cell lines. High nestin expression was observed in 14 of 56 primary melanomas (25%), 84 of 165 melanoma metastases (50%) and 21 of 53 melanoma cell lines (40%). There was a significant correlation between BMI-1 and nestin expression in cell lines (p = 0.001) and metastases (p = 0.02). These data indicate that cells in primary melanomas and their metastases may have stem cell properties. Cell lines obtained from melanoma metastases showed a significant higher BMI-1 expression compared to cell lines from primary melanoma (p = 0.001). Further, primary melanoma lacking lymphatic metastases at presentation (pN0, n = 40) was less frequently BMI-1 positive than melanomas presenting with lymphatic metastases (pN1; n = 24; 52% versus 83%; p = 0.01). Therefore, BMI-1 expression appears to induce a metastatic tendency. Because BMI-1 functions as a transcriptional repressor of the Ink4a/Arf locus, p16(ink4a) and p14(Arf) expression was also analyzed. A high BMI-1/low p16(ink4a) expression pattern was a significant predictor of metastasis by means of logistic regression analysis (p = 0.005). This suggests that BMI-1 mediated repression of p16(ink4a) may contribute to an increased aggressive behavior of stem cell-like melanoma cells.

NY-BR-1 protein expression in breast carcinoma: a mammary gland differentiation antigen as target for cancer immunotherapy.

NY-BR-1 is a recently identified differentiation antigen of the mammary gland. To use NY-BR-1 for T-cell-based immunotherapy, analysis of its co-expression with HLA class I antigens is required. In the present tissue microarray study, primary breast cancers (n = 1,444), recurrences (n = 88), lymph node (n = 525) and distant metastases (n = 91) were studied for NY-BR-1 expression using a novel monoclonal antibody. NY-BR-1 expression was compared with prognosis, estrogen receptor, HER2-status, EGFR and HLA class I antigen expression. NY-BR-1 was more frequently expressed in grade 1 (82%) than in grade 2 (69%) and grade 3 (46%) carcinomas (P < 0.0001). Moreover, NY-BR-1 expression correlated directly with estrogen receptor expression (P < 0.0001) and inversely correlated with HER2-status and EGFR expression (P < 0.0001 for both). Considering high expression level of co-expression, 198/1,321 (15%) primary breast carcinomas and 4/65 (6%) distant metastases expressed NY-BR-1 and HLA class I, suggesting that active immunotherapy can be applied to about 10% of breast cancer patients. Survival analysis showed an association of NY-BR-1 expression with better patient outcome (P = 0.015). No difference between NY-BR-1 expression of primary tumors and metastases could be found, indicating that the presence of NY-BR-1 in metastases can be deduced from their corresponding primary. Forty-three paired biopsies taken from patients before and after chemotherapy suggest that NY-BR-1 expression is not influenced by preceding chemotherapy (kappa = 0.89, P < 0.0001). In summary, the co-expression of NY-BR-1 with HLA class I antigens and its expression in metastases without modification by chemotherapy suggest that NY-BR-1 targeted immunotherapy represents a viable strategy in addition to other targeted cancer drug therapies of breast cancer.

D2-40 and calretinin - a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation.

Anti-calretinin antibodies are useful to differentiate adenocarcinomas from malignant mesotheliomas of the lung. Therefore, calretinin expression is rarely reported for sarcomatoid mesotheliomas. Anti-podoplanin antibodies (eg D2-40) react with lymphatic endothelia, Kaposi's sarcoma, lymphangioma and mesotheliomas. For the interpretation of spindle cell lesions of the pleura, knowledge of calretinin and D2-40 expression frequencies in sarcomatoid mesothelioma is desirable. To systematically investigate the sensitivity of calretinin and D2-40 antibodies in epithelioid and sarcomatoid areas of malignant mesotheliomas, a tissue microarray with 341 malignant mesotheliomas, including 112 epithelioid, 46 sarcomatoid and 183 biphasic tumors was constructed. Epithelioid and sarcomatoid differentiated tumor areas were clearly separated within the tissue microarray. Expression of calretinin and D2-40 was separately studied in epithelioid and sarcomatoid areas by immunohistochemistry. Calretinin expression was found in 91% of epithelioid and 57% of sarcomatoid tumor areas. D2-40 immunostaining was present in 66% of the epithelioid and 30% of the sarcomatoid tumor areas. A combination of calretinin and D2-40 increased the sensitivity in epithelioid tumor areas to 0.96 and in sarcomatoid tumor areas to 0.66. These data indicate that a combination of calretinin and D2-40 will improve diagnostic accuracy for spindle cell lesions of the pleura, whereas almost all epithelioid mesotheliomas are identified by calretinin alone.

Influence of slide aging on results of translational research studies using immunohistochemistry.

Several reports have shown that a long delay between cutting sections and immunohistochemical (IHC) staining can decrease the IHC reaction intensity. However, systematic large-scale studies to investigate to what extent this problem may influence the outcome of translational research studies are lacking. In this study, we used a tissue microarray (TMA) approach to investigate the influence of slide age on comparisons between the results of IHC analyses for estrogen receptor (ER), progesterone receptor (PR), cyclin D1, HER2 (HercepTest), and E-cadherin and clinical outcome in a series of 522 breast cancer patients. Old TMA sections stored for 6 months at 4 degrees C and freshly cut sections were analyzed under exactly identical experimental conditions. As compared to results obtained on freshly cut sections, the frequency of positivity on old sections decreased from 65 to 46% for ER (P<0.0001), from 33 to 18.5% for PR (P<0.0001), from 16.3 to 9.6% for HER2 (P=0.0047), from 45.1 to 37.7% for cyclin D1 (P=0.10), and from 58.9 to 32.9% for E-cadherin (P<0.0001). Despite the lower fraction of positive cases, most associations between IHC data and tumor phenotype that were observed in fresh section analysis were also found when old section data were analyzed. The results confirm that slide aging has a great influence on the intensity of IHC staining in individual cases, but they also suggest that many clinicopathological associations can be detected if suboptimally processed sections are used for IHC.

Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples.

Calretinin is a calcium-binding protein expressed in different normal and neoplastic tissues. Early studies suggested that calretinin is a useful marker to differentiate adenocarcinomas from malignant mesotheliomas of the lung, but subsequent work has shown that calretinin can be expressed in several other tumor types. To systematically investigate the epidemiology of calretinin expression in normal and neoplastic tissues, we used tissue microarrays (TMAs) to analyze the immunohistochemically detectable expression of calretinin in 5233 tissue samples from 128 different tumor categories and 76 different normal tissue types. At least 1 case with weak expression could be found in 74 of 128 (58%) different tumor types and 46 entities (36%) had at least 1 tumor with strong positivity. In normal tissues, a particularly strong expression was found in Leydig cells of the testis, neurons of the brain, theca-lutein and theca interna cells of the ovary, and mesothelium. In tumors, strong calretinin expression was most frequently found in malignant mesotheliomas (6 of 7), Leydig cell tumors of the testis (5 of 5), adenomas of adrenal gland (5 of 9), and adenomatoid tumors (4 of 9). In summary, calretinin is frequently expressed in many different tumor types. Metastases of various different origins must be included in the differential diagnosis of calretinin-positive pleura tumors.