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Background: As the impact of unmanaged bias (i.e. systematic source of inaccuracy) in fecal immunochemical test (FIT) analytical performance on long-term colorectal cancer (CRC) outcomes is unknown, we assessed the impact bias in FIT performance in an ongoing FIT-based CRC screening program., Methods: This study consisted of two parts: cross-sectional observational data analysis to estimate change in short-term outcomes and microsimulation modelling to estimate change in long-term outcomes assuming different levels of bias by assuming 15 % lower up to 15 % higher Hemoglobin detected in the stool compared to observed. Two scenarios were considered: bias occurring 1) one-time only, due to the occasional bias associated with the FIT kits used in 2020 and 2) consistently due to a constant bias associated with the FIT kits used from 2020 onwards., Results: With a hypothetical bias of -15 % to +15 %, we observed a positivity rate ranging from 6.7 % to 7.8 %, and a detection rate for CRC between 0.65 % and 0.68 %. Single biases in FIT performance resulted in less than 0.1 % change in long-term CRC screening outcomes, while consistent biases resulted in a much larger change (up to 1.4 % in CRC cases and CRC-related deaths and up to 2.07 % in total costs). Detecting lower Hemoglobin concentrations resulted in a relatively larger change on long-term CRC outcomes in comparison to positive bias., Conclusions: Because of the substantial impact of consistent FIT bias, it is important to set evidence-based acceptance criteria of bias on long-term CRC screening outcomes and in particular, the introduction of an asymmetrical or upward shifted tolerance interval for FIT bias., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Dr. Iris Lansdorp-Vogelaar: Associate editor at Gastroenterology; expert at the Health Council; panel member of the European Commission Initiative on Colorectal Cancer; visiting scientist at IARC. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Male breast cancer (MBC) is extremely rare and accounts for less than 1% of all breast malignancies. Therefore, clinical management of MBC is currently guided by research on the disease in females. In this study, DNA obtained from 45 formalin-fixed paraffin-embedded (FFPE) MBCs with and 90 MBCs (52 FFPE and 38 fresh-frozen) without matched normal tissues was subjected to massively parallel sequencing targeting all exons of 1943 cancer-related genes. The landscape of mutations and copy number alterations was compared to that of publicly available estrogen receptor (ER)-positive female breast cancers (smFBCs) and correlated to prognosis. From the 135 MBCs, 90% showed ductal histology, 96% were ER-positive, 66% were progesterone receptor (PR)-positive, and 2% HER2-positive, resulting in 50, 46 and 4% luminal A-like, luminal B-like and basal-like cases, respectively. Five patients had Klinefelter syndrome (4%) and 11% of patients harbored pathogenic BRCA2 germline mutations. The genomic landscape of MBC to some extent recapitulated that of smFBC, with recurrent PIK3CA (36%) and GATA3 (15%) somatic mutations, and with 40% of the most frequently amplified genes overlapping between both sexes. TP53 (3%) somatic mutations were significantly less frequent in MBC compared to smFBC, whereas somatic mutations in genes regulating chromatin function and homologous recombination deficiency-related signatures were more prevalent. MDM2 amplifications were frequent (13%), correlated with protein overexpression (P = 0.001) and predicted poor outcome (P = 0.007). In conclusion, despite similarities in the genomic landscape between MBC and smFBC, MBC is a molecularly unique and heterogeneous disease requiring its own clinical trials and treatment guidelines.

Heredity, mostly due to BRCA germline mutations, is involved in 5% to 10% of all breast cancer cases. Potential BRCA germline mutation carriers may be missed following the current eligibility criteria for BRCA genetic testing. The purpose of this study was to, therefore, develop an immunohistochemistry-based model to predict likelihood of underlying BRCA1 and BRCA2 germline mutations in unselected female breast cancer patients. The study group consisted of 100 BRCA1-related, 46 BRCA2-related, and 94 sporadic breast carcinomas. Tumor expression of 44 proteins involved in (BRCA-related) breast carcinogenesis was assessed by immunohistochemistry. A prediction model for BRCA-related versus non-BRCA-related breast cancer was developed using Lasso logistic regression analysis with cross-validation. The model was assessed for its discriminative value and clinical usefulness. The optimal prediction model included 14 predictors (age, cyclinD1, ERα, ERβ, FGFR2, FGFR3, FGFR4, GLUT1, IGFR, Ki67, mitotic activity index, MLH1, p120, and TOP2A), showed excellent discriminative performance (area under the receiving operating characteristic curve=0.943; 95% confidence interval=0.909-0.978), and reasonable calibration. To enhance possible implementation, we developed an alternative model only considering more widely available immunostains. This model included 15 predictors (age, BCL2, CK5/6, CK8/18, cyclinD1, E-cadherin, ERα, HER2, Ki67, mitotic activity index , MLH1, p16, PMS2, PR, and vimentin), and still showed very good discriminative performance (area under the receiving operating characteristic curve=0.853; 95% confidence interval=0.795-0.911). We present a well-applicable and accurate tool to predict which breast cancer patients may have an underlying BRCA germline mutation, largely consisting of immunohistochemical markers independent of clinical characteristics. This may improve identification of potential BRCA germline mutation carriers and optimize referral for germline mutation testing.

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients.

Background: Medullary thyroid cancer (MTC) comprises only 4% of all thyroid cancers and originates from the parafollicular C-cells. HIF-1α expression has been implied as an indicator of worse prognosis in various solid tumors. However, whether expression of HIF-1α is a prognosticator in MTC remained unclear. Our aim was to evaluate the prognostic value of HIF-1α in patients with MTC., Methods: All patients with MTC who were operated on between 1988 and 2014 in five tertiary referral centers in The Netherlands were included. A tissue microarray was constructed in which 111 primary tumors could be analyzed for expression of HIF-1α, CAIX, Glut-1, VEGF and CD31 and correlated with clinicopathologic variables and survival., Results: The mean age of patients was 46.3 years (SD 15.6), 59 (53.2%) were male. Of the 111 primary tumors, 49 (44.1%) were HIF-1α negative and 62 (55.9%) were HIF-1α positive. Positive HIF-1α expression was an independent negative indicator for progression free survival (PFS) in multivariate cox regression analysis (HR 3.1; 95% CI 1.3 - 7.3). Five-years survival decreased from 94.0% to 65.9% for the HIF-1α positive group (p=0.007). Even within the group of patients with TNM-stage IV disease, HIF-1α positivity was associated with a worse prognosis, shown by a decrease in 5-years survival of 88.0% to 49.3% (p=0.020)., Conclusion: Expression of HIF-1α is strongly correlated with adverse prognosis of MTC. This could open up new ways for targeted systemic therapy of MTC.

Loss of E-cadherin expression is causal to the development of invasive lobular breast carcinoma (ILC). E-cadherin loss leads to dismantling of the adherens junction and subsequent translocation of p120-catenin (p120) to the cytosol and nucleus. Although p120 is critical for the metastatic potential of ILC through the regulation of Rock-dependent anoikis resistance, it remains unknown whether p120 also contributes to ILC development. Using genetically engineered mouse models with mammary gland-specific inactivation of E-cadherin, p120 and p53, we demonstrate that ILC formation induced by E-cadherin and p53 loss is severely impaired upon concomitant inactivation of p120. Tumors that developed in the triple-knockout mice were mostly basal sarcomatoid carcinomas that displayed overt nuclear atypia and multinucleation. In line with the strong reduction in ILC incidence in triple-knockout mice compared to E-cadherin and p53 double-knockout mice, no functional redundancy of p120 family members was observed in mouse ILC development, as expression and localization of ARVCF, p0071 or δ-catenin was unaltered in ILCs from triple-knockout mice. In conclusion, we show that loss of p120 in the context of the p53-deficient mouse models is dominant over E-cadherin inactivation and its inactivation promotes the development of basal, epithelial-to-mesenchymal-transition (EMT)-type invasive mammary tumors., Competing Interests: Compliance with Ethical Standards Grants Research was supported by grants from the Netherlands Organization for Scientific Research (NWO/ZonMW-VIDI 016.096.318), Foundation Vrienden UMC Utrecht (11.081) and the Dutch Cancer Society (KWF-UU-2011-5230). Conflict of Interest The authors declare that they have no conflict of interest.

Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ERα, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen's buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ERα-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen's buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ERα, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen's buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC., (© 2015 Wiley Periodicals, Inc.)

miRNA deregulation has been found to promote carcinogenesis. Little is known about miRNA deregulation in hereditary breast tumors as no miRNA expression profiling studies have been performed in normal breast tissue of BRCA1 and BRCA2 mutation carriers. miRNA profiles of 17 BRCA1- and 9 BRCA2-associated breast carcinomas were analyzed using microarrays. Normal breast tissues from BRCA1 and BRCA2 mutation carriers (both n = 5) and non-mutation carriers (n = 10) were also included. Candidate miRNAs were validated by qRT-PCR. Breast carcinomas showed extensive miRNA alteration compared to normal breast tissues in BRCA1 and BRCA2 mutation carriers. Moreover, normal breast tissue from BRCA1 mutation carriers already showed miRNA alterations compared to non-mutation carriers. Chromosomal distribution analysis showed several hotspots containing down- or up-regulated miRNAs. Pathway analysis yielded many similarities between the BRCA1 and BRCA2 axes with miRNAs involved in cell cycle regulation, proliferation and apoptosis. Lesser known pathways were also affected, including cellular movement and protein trafficking. This study provides a comprehensive insight into the potential role of miRNA deregulation in BRCA1/2-associated breast carcinogenesis. The observed extensive miRNA deregulation is likely the result of genome-wide effects of chromosomal instability caused by impaired BRCA1 or BRCA2 function. This study's results also suggest the existence of common pathways driving breast carcinogenesis in both BRCA1 and BRCA2 germ-line mutation carriers.

Background: Gene expression profiling revealed a strong signature predicting lymph node metastases in oral squamous cell carcinoma (OSCC). Four of the most predictive genes are secretory leukocyte protease inhibitor (SLPI), lipocalin-2 (LCN2), thrombospondin-2 (THBS2), and tumor-associated calcium signal transducer 2 (TACSTD2). This study correlates their protein expression with lymph node metastases, overall survival (OS), and disease-specific survival (DSS)., Methods: Two hundred twelve patients with OSCC were included for protein expression analysis by immunohistochemistry., Results: SLPI expression correlates with lymph node metastases in the whole cohort, not in a subgroup of cT1 to 2N0. SLPI expression correlates with OS (hazard ratio [HR] = 0.61) and DSS (HR = 0.47) in multivariate analysis. LCN2, THBS2, and TACSTD2 show no correlation with lymph node metastases, OS, or DSS., Conclusion: Although SLPI expression correlates with lymph node metastases, it has no additional value in determining lymph node metastases in early oral cancer. However, it is an independent predictor for both OS and DSS and therefore a relevant prognostic biomarker in OSCC., (© 2014 Wiley Periodicals, Inc.)

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Chronic gastrointestinal ischemia (CGI) is the result of decreased mucosal perfusion. Typical histological characteristics are lacking which hamper its early diagnosis. Hypoxia-inducible factor-1α (HIF-1α) is expressed under acute hypoxia. We investigated HIF-1α expression in chronic ischemic and inflammatory conditions of the human gastrointestinal (GI) tract. Immunohistochemical expression of HIF-1α was analyzed in 61 patients, including patients with CGI, Helicobacter pylori gastritis, ischemic colitis (IC), infectious colitis, and inflammatory bowel disease (IBD), and 22 controls. HIF-1α expression in >10 % of the cells was regarded as positive staining, and expression <10 % of the cells was considered as negative staining. In the upper GI tract, HIF-1α expression was found in 5/20 CGI patients, but not in controls (p = 0.08). The sensitivity and specificity of HIF-1α expression for diagnosing CGI were 25 and 84%, respectively. In the lower GI tract, HIF-1α was expressed in all patients with IC and infectious colitis and in a majority of IBD patients as well as in 7/12 controls. The sensitivity and specificity of HIF-1α for diagnosing IC were 100 and 51%, respectively. HIF-1α expression was more often (p = 0.02) observed in patients with histological signs of inflammation in the lower GI tract. HIF-1α is expressed in acute and chronic ischemic tissue, but also in normal colon tissue and inflammatory disorders.

Aims: Members of the claudin family are involved in cancer progression and are differentially expressed in subtypes of breast cancer. Breast cancers in BRCA1 germ line mutation carriers have distinct clinicopathological characteristics. Biomarkers that discriminate between BRCA1-related and sporadic breast cancer cases are needed to improve early identification of mutation carriers. In this study we evaluated protein expression of five major claudins in BRCA1-related breast cancers in comparison with sporadic controls., Methods and Results: Forty breast cancers in BRCA1 mutation carriers and 40 age-matched sporadic breast cancers were immunohistochemically stained for claudins 1, 3, 4, 6 and 7. Total intratumoural expression levels were compared to those in the surrounding normal tissue. In addition, subcellular claudin expression was scored. Higher overexpression rates were observed for all five claudins in BRCA1-related breast cancers when compared to sporadic controls. In multivariate analysis, overexpression of claudin 3, 4, and 7 was mainly dependent on ER-status, whereas overexpression of claudin 6 and high membranous expression of claudin 1 were independent of other characteristics., Conclusions: BRCA1-related breast cancers are characterized by frequent overexpression of claudins. Especially claudin 1 and 6 expression may help to discriminate mutation carriers from sporadic breast cancer cases., (© 2014 John Wiley & Sons Ltd.)

Cancer initiation and progression is characterized by (epi)genetic aberrations. However, little is known about the changes that occur during breast cancer metastasis. In the present study, multiplex ligation-dependent probe amplification was used to compare copy numbers of 21 established oncogenes and tumor suppressor genes between 55 primary breast cancer samples and corresponding distant metastases. Distant breast cancer metastases generally showed similar gene copy number aberrations compared to their corresponding primary tumors. The few genes that showed differences between primary tumor and metastasis (PRDM14, MED1, CCNE1, TRAF4, MTDH, CDH1) have been implicated in the development of therapy resistance., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: We studied discordance in estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status between multiple distant metastases from the same breast cancer patient., Material and Methods: Multiple distant metastases from 55 female patients were stained for ERα, PR and HER2 by immunohistochemistry and in situ hybridization for confirmation of the HER2 status., Results: Different metastatic sites within the same patient showed discordance in ERα receptor status in 7.3% or 10.9% of patients (using a 10% or 1% threshold for positivity, respectively). For PR, 29.1% or 30.9% of patients showed discordance. Taking ERα and PR together, 36.4% of cases (both thresholds) showed discrepancy between metastases. In 10.9% (10% threshold) or 14.5% of patients (1% threshold), such discordance could have clinical consequences with regard to hormonal treatment. For HER2, there was 3.6% discordance on the immunohistochemical level but 0% on the gene level., Conclusion: In a significant proportion of metastatic breast cancer patients, discordance in ERα and PR receptor status between different metastatic sites was observed. This implies that multiple metastases may need to be biopsied to optimally reassess receptors.

Background: Focal boosting of prostate tumours to improve outcome, requires accurate tumour delineation. For this, the apparent diffusion coefficient (ADC) derived from diffusion-weighted MR imaging (DWI) seems a useful tool. On voxel level, the relationship between ADC and histological presence of tumour is, however, ambiguous. Therefore, in this study the relationship between ADC and histological variables was investigated on voxel level to understand the strengths and limitations of DWI for prostate tumour delineation., Material and Methods: Twelve radical prostatectomy patients underwent a pre-operative 3.0T DWI exam and the ADC was calculated. From whole-mount histological sections cell density and glandular area were retrieved for every voxel. The distribution of all variables was described for tumour, peripheral zone (PZ) and central gland (CG) on regional and voxel level. Correlations between variables and differences between regions were calculated., Results: Large heterogeneity of ADC on voxel level was observed within tumours, between tumours and between patients. This heterogeneity was reflected by the distribution of cell density and glandular area. On regional level, tumour was different from PZ having higher cell density (p = 0.007), less glandular area (p = 0.017) and lower ADCs (p = 0.017). ADC was correlated with glandular area (r = 0.402) and tumour volume (r = -0.608), but not with Gleason score. ADC tended to decrease with increasing cell density (r = -0.327, p = 0.073). On voxel level, correlations between ADC and histological variables varied among patients, for cell density ranging from r = -0.439 to r = 0.261 and for glandular area from r = 0.593 to r = 0.207., Conclusions: The variation in ADC can to a certain extent be explained by the variation in cell density and glandular area. The ADC is highly heterogeneous, which reflects the heterogeneity of malignant and benign prostate tissue. This heterogeneity might however obscure small tumours or parts of tumours. Therefore, DWI has to be used in the context of multiparametric MRI.

The prediction of progression of individual tumours, prognosis, and survival in squamous cell carcinoma (SCC) of the head and neck is difficult. Cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptor expression is related to survival in several types of cancer, and the aim of this study was to find out whether the expression of CB1 and CB2 receptors is associated with survival in primary SCC of the head and neck. We made immunohistochemical analyses of the cannabinoid receptors on tissue arrays from 240 patients with the disease. Receptor immunoreactivity was classified as none, weak, moderate, or strong staining. Overall survival and disease-specific survival were plotted using Kaplan-Meier survival curves. A multivariate Cox proportional hazard model was created with all the relevant clinical and pathological features. Strong immunoreactivity of the CB2 receptor was significantly associated with reduced disease-specific survival (p=0.007). Cox-proportional hazard ratio (HR) showed that CB2 receptor immunoreactivity contributed to the prediction of survival (HR 3.6, 95% CI 1.5-8.7, p=0.004). Depth of invasion (HR 2.2, 95% CI 1.2-4.2, p=0.01) and vascular invasion (HR 2.5, 95% CI 1.4-4.5, p=0.001) were also associated with survival., (Copyright © 2013 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal's website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.

Metastatic breast cancer remains the chief cause of cancer-related death among women in the Western world. Although loss of cell-cell adhesion is key to breast cancer progression, little is known about the underlying mechanisms that drive tumor invasion and metastasis. Here, we show that somatic loss of p120-catenin (p120) in a conditional mouse model of noninvasive mammary carcinoma results in formation of stromal-dense tumors that resemble human metaplastic breast cancer and metastasize to lungs and lymph nodes. Loss of p120 in anchorage-dependent breast cancer cell lines strongly promoted anoikis resistance through hypersensitization of growth factor receptor (GFR) signaling. Interestingly, p120 deletion also induced secretion of inflammatory cytokines, a feature that likely underlies the formation of the prometastatic microenvironment in p120-negative mammary carcinomas. Our results establish a preclinical platform to develop tailored intervention regimens that target GFR signals to treat p120-negative metastatic breast cancers., (©2013 AACR.)

The aim of this study was to investigate the association between the immunohistochemical expression of hypoxia-inducible factor (HIF)-1α and HIF-2α and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters Ktrans and kep in prostate cancer. Therefore, 15 patients with biopsy-confirmed prostate cancer underwent a pre-operative 3T DCE-MRI scan. Immunohistochemical analysis of HIF-1α and HIF-2α, and of CD31 for microvessel density (MVD) was performed. Tumor areas were delineated on whole-mount histopathological sections. Nuclear HIF expression was correlated with the quantitative DCE-MRI parameters Ktrans and kep, MVD and Gleason score. HIF expression was highly heterogeneous within tumors and between patients. Pronounced expression of HIF-2α was present, while HIF-1α expression was more limited. Larger tumors showed higher HIF-2α expression (p=0.041). A correlation between HIF-2α and Ktrans p5th was found (r=0.30, p=0.02), but no differences in Ktrans, kep and MVD were observed for different levels of HIF expression. HIF expression was not associated with Gleason score. In conclusion, in this whole-mount prostate cancer study, larger prostate tumors showed frequently high HIF-2α expression, suggesting that larger tumors are clinically most relevant. However, HIF-1α and HIF-2α were not correlated with DCE-MRI parameters. Given the pronounced expression of HIF-2α and independence of Gleason score, HIF expression may function as a biomarker to guide boost dose prescription.

Aims: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer., Methods and Results: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α., Conclusions: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors.

Recent studies have revealed that BRCA1 and BRCA2 germline mutation-related breast cancers show frequent overexpression of hypoxia inducible factor-1α (HIF-1α), the key regulator of the hypoxia response. However, the question remained whether hypoxia is a late stage bystander or a true carcinogenetic event in patients with hereditary predisposition. We therefore studied HIF-1α overexpression in ductal carcinoma in situ (DCIS), an established precursor of invasive breast cancer.We used immunohistochemistry to examine the expression of the hypoxia markers HIF-1α, CAIX and Glut-1 in DCIS and available invasive carcinoma lesions of 32 BRCA1, 16 BRCA2 and 77 non-BRCA mutation-related cases. HIF-1α expression was detected in 63% of BRCA1 and 62% of BRCA2 as compared to 34% of non-BRCA mutation-related DCIS cases (p = 0.005). CAIX overexpression was present in 56% of BRCA1 and 44% of BRCA2 as compared to 6% of non-BRCA mutation-related DCIS cases (p = 0.000). Glut-1 overexpression was observed in 59% of BRCA1, 75% of BRCA2 and 67% of non-BRCA mutation-related DCIS cases (p = 0.527). Overall, HIF-1α, CAIX and Glut-1 expression in BRCA mutation-related DCIS matched the expression in the accompanying invasive cancers in 60% or more of cases. In non-BRCA mutation-related cases the expression of the hypoxia markers in DCIS matched the expression in the invasive part in 46% or more of the cases.Although BRCA1 and BRCA2 germline mutation-related invasive breast cancers are different in many ways, the hypoxia-related proteins HIF-1α, CAIX and Glut-1 are expressed in both DCIS and invasive lesions of BRCA1 and BRCA2 mutation carriers. This suggests that hypoxia may already play a role in the DCIS stage of BRCA1 and BRCA2 germline mutation related breast carcinogenesis, and may also drive cancer progression. Hypoxia-related proteins are therefore putative targets for therapy and molecular imaging for early detection and monitoring therapy response in BRCA mutation patients.

Introduction: Male breast cancer accounts for 0.5-1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer., Methods: Tissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers., Results: Growth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers., Conclusions: Expression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for male breast cancer.

Background: NOTCH signaling is involved in every step of metazoan development and maintenance of adult tissue homeostasis. It is frequently deregulated by mutations and overexpression in different cancer types including solid tumors such as breast cancer. Another common feature of solid tumors is hypoxia, which occurs due to defective or insufficient vascularization. Hypoxia-inducible factors (HIFs) are key regulators of the homeostatic response to low oxygen levels. HIF-1α is overexpressed in many solid tumors, including breast cancer. Hypoxia-induced stabilization of HIF transcription factors has been shown to lead to NOTCH activation in vitro in different contexts and tissues, causing differentiation arrest and induction of proliferation and migration., Methods: Since the link between HIF-1α and NOTCH signalling has hardly been studied, we set out to closely investigate associations between the expression of HIF-1α and NOTCH pathway members in primary and metastatic human breast cancer specimens and their prognostic value., Results: Co-expression of NOTCH1 intracellular domain (N1ICD) and HIF-1α was associated with a high grade and a high proliferation rate in invasive breast cancer. HIF-1α expression was low in classic, but high in pleomorphic lobular cancers, which also frequently showed stromal HIF-1α expression. NOTCH1 pathway activation was prognostically unfavorable., Conclusion: In breast cancer, NOTCH pathway activation appears to be associated with a poor prognosis, but NOTCH and HIF signaling do not seem to be functionally associated.

Recent molecular data pointed towards the possibility of a stepwise dedifferentiation in a subgroup of invasive breast cancer (BC) cases. It was hypothesized that oestrogen receptor positive (ER+) grade 3 (G3) ductal invasive BCs are the end stage of a dedifferentiation process of luminal BC. A progression of luminal A towards luminal B BCs associated with a 'progression through grade' and an increased cell proliferation seemed the obvious explanation. In order to verify this hypothesis on a morphological and immunohistochemical level, we investigated 865 invasive BC cases. All cases were reviewed for the presence of intratumoural heterogeneity in grade of the invasive cancer and the presence of associated ductal carcinoma in situ (DCIS). With the use of tissue microarrays, the molecular subtype was determined and correlated with clinico-pathological features. In addition, all cases were stained for p21, p27, Ki-67, Cyclin D1, bcl-2, p53, and p16 and the results subjected to a biomathematical dependency analysis. The frequency of ER-positivity decreased with tumour size. The frequency of luminal A BC decreased as well, whereas the number of luminal B BCs remained constant. A gradual increase of the frequency of basal-like, HER2-driven and non-expressor BCs with tumour size was seen. In only 1 out of 865 BC cases, both a G1 and a G3 invasive cancer component was seen within the same BC. In two cases, a ductal invasive G1 carcinoma was associated with a poorly-differentiated DCIS. The frequency of columnar cell lesions was evenly distributed over ER+ and ER- ductal invasive G3 carcinomas. The biomathematical analysis gave striking hints against an obligate progression of BC trough grade. In conclusion, our results show that a morphological recognizable striking 'progression through grade' at least in its extreme form from G1 towards G3 is a very rare event in the natural course of invasive BC, including luminal BC.

The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.

Cancer treatments are increasingly focusing on the molecular mechanisms underlying the oncogenic processes present in tumors of individual patients. Fibroblast growth factor receptors (FGFRs) are among the many molecules that are involved in oncogenesis and are currently under investigation for their potential as drug targets in breast cancer patients. These receptor tyrosine kinases play a role in several processes including proliferation, angiogenesis, and migration. Alterations in these basal processes can contribute to the development and progression of tumors. Among breast cancer patients, several subgroups have been shown to harbor genetic aberrations in FGFRs, including amplifications of FGFR1, FGFR2, and FGFR4 and mutations in FGFR2 and FGFR4. Here, we review in vitro and in vivo models that have partly elucidated the molecular implications of these different genetic aberrations, the resulting tumor characteristics, and the potential of FGFRs as therapeutic targets for breast cancer treatment.

Background: Mammographic population screening in The Netherlands has increased the number of breast cancer patients with small and non-palpable breast tumors. Nevertheless, mammography is not ultimately sensitive and specific for distinct subtypes. Molecular imaging with targeted tracers might increase specificity and sensitivity of detection. Because development of new tracers is labor-intensive and costly, we searched for the smallest panel of tumor membrane markers that would allow detection of the wide spectrum of invasive breast cancers., Methods: Tissue microarrays containing 483 invasive breast cancers were stained by immunohistochemistry for a selected set of membrane proteins known to be expressed in breast cancer., Results: The combination of highly tumor-specific markers glucose transporter 1 (GLUT1), epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), hepatocyte growth factor receptor (MET), and carbonic anhydrase 9 (CAIX) 'detected' 45.5% of tumors, especially basal/triple negative and HER2-driven ductal cancers. Addition of markers with a 2-fold tumor-to-normal ratio increased the detection rate to 98%. Including only markers with >3 fold tumor-to-normal ratio (CD44v6) resulted in an 80% detection rate. The detection rate of the panel containing both tumor-specific and less tumor-specific markers was not dependent on age, tumor grade, tumor size, or lymph node status., Conclusions: In search of the minimal panel of targeted probes needed for the highest possible detection rate, we showed that 80% of all breast cancers express at least one of a panel of membrane markers (CD44v6, GLUT1, EGFR, HER2, and IGF1-R) that may therefore be suitable for molecular imaging strategies. This study thereby serves as a starting point for further development of a set of antibody-based optical tracers with a high breast cancer detection rate.

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4)., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Molecular subtyping of breast cancer by gene expression has proven its significance in females. Immunohistochemical surrogates have been used for this classification, because gene expression profiling is not yet routinely feasible. Male breast cancer is rare and large series are lacking. In this study, we used immunohistochemistry for molecular subtyping of male breast cancer. A total of 134 cases of male breast cancer were immunohistochemically stained on tissue microarrays for estrogen receptor (ER), progesterone receptor (PR), HER2 and epidermal growth factor receptor (EGFR), as well as for CK5/6, CK14, and Ki67. HER2 was also assessed by chromogen in situ hybridization. Cases were classified as luminal A (ER+ and/or PR+, and HER2- and Ki67 low), luminal B (ER+ and/or PR+, and HER2+ or Ki67 high), HER2 driven (ER-, PR-, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+ and/or CK14+ and/or EGFR+), or unclassifiable triple-negative (negative for all six markers). Luminal type A was by far the most encountered type of male breast cancers, representing 75% of the cases. Luminal type B was seen in 21% and the remaining 4% of cases were classified as basal-like (n=4) and unclassifiable triple-negative (n=1). No HER2 driven cases were identified. Patients with basal-like cancer were significantly younger (P=0.034). Luminal B type cancers showed significantly higher histological grade (P<0.001), mitotic index (P<0.001), and PR negativity (P=0.005) compared with luminal type A cancers. In conclusion, most male breast cancers are luminal A and luminal B types, whereas basal-like, unclassifiable triple-negative, and HER2 driven male breast cancers are rare. Luminal type B seem to represent a subtype with an aggressive phenotype. This distribution of molecular subtypes in male breast cancer is clearly different compared with female breast cancers, pointing to possible important differences in carcinogenesis.

BRCA1/2 germ line mutation carriers have a high risk of developing fallopian tube carcinoma (FTC), thought to occur through different early (p53 signatures) and later (dysplasia, intra-epithelial carcinoma) premalignant stages. Promoter hypermethylation of tumour suppressor genes is known to play a key role in (early) carcinogenesis. However, little is known about methylation in normal and (pre)malignant fallopian tube tissue. We identified 14 areas of p53 accumulation in the fallopian tubes of BRCA mutation carriers. Cells from these areas were harvested together with cells from adjacent benign appearing areas. An age-matched non-BRCA sporadic control group (n=13) and eight sporadic FTCs were included as negative and positive controls respectively. Methylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 70 tumour suppressor genes in all samples. We observed a gradual increase in methylation from sporadic control tissue (median cumulative methylation index (CMI) 568.19) through normal tissue and from areas of p53 accumulation in BRCA carriers (median CMI 687.54 and 676.72) to FTC (median CMI 780.97). Furthermore, the methylation percentage of many individual tumour suppressor genes differed significantly between these groups, gradually increasing as for CMI. Between areas with and without p53 accumulation in BRCA mutation carriers no significant differences were found. In this paper, we have shown that BRCA mutation carriers display increased methylation of tumour suppressor genes in their non-malignant fallopian tube epithelium, closer to methylation levels in FTC than to normal sporadic tissue. Methylation could, therefore, play an important role in the increased risk of gynaecological malignancies in BRCA mutation carriers.

Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC) (p = 0.007), while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC) (p = 0.006). Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023), ERα negativity (p = 0.001), and the HER2-driven and basal/triple-negative breast cancers (p = 0.018). Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001) and invasive breast cancer overexpressing EGFR (p = 0.019). We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120) (p<0.01). In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005). We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.

Background: Hypoxia Inducible Factor-1α (HIF-1α) expression in breast cancer is associated with a poor clinical outcome. HIF-1α shows two expression patterns: the canonical poor prognosis hypoxia-related perinecrotic pattern and a diffuse expression pattern that seems to have less downstream effects and is clearly associated with poor survival. Factor-inhibiting hypoxia-inducible factor 1 (FIH-1) inhibits HIF-1 activity by hydroxylating the C-terminal trans-activation domain of the HIF-1α subunit, thus preventing HIF-1 from recruiting co-activators CPB/p300, which are important for inducing the transcription of target genes. The aim of this study was to investigate the expression patterns of FIH-1 in breast cancer and evaluate the relationship between FIH-1 and HIF-1α expression in breast cancer as a possible explanation for apparently less downstream effects of diffuse HIF-1α expression., Methods: Tissue sections from 92 consecutive invasive breast carcinomas were stained by immunohistochemistry for FIH-1, HIF-1α, glucose transporter 1 (GLUT-1) and carbonic anhydrase IX (CAIX)., Results: 45 cases overexpressed HIF-1α, 5 of which in a perinecrotic fashion while FIH-1 was positive in 73 of the 92 cases studied. Contrary to our expectations, three out of five cases with perinecrotic HIF-1α expression were also positive for FIH1. Cytoplasmic FIH-1 correlated with HIF-1α expression (P = 0.03) and tumor grade (P = 0.01). HIF-1α overexpression predicted poorer prognosis as usual (P = 0.02). FIH expression had no additional prognostic value to HIF-1α., Conclusions: FIH1 is expressed in the majority of invasive breast carcinomas and shows distinct subcellular localization patterns. FIH-1 expression does not seem to explain the proposed functional differences between diffuse and perinecrotic HIF-1α expression in breast cancer.

Metastatic breast cancer is the major cause of cancer-related death among women in the Western world. Invasive carcinoma cells are able to counteract apoptotic signals in the absence of anchorage, enabling cell survival during invasion and dissemination. Although loss of E-cadherin is a cardinal event in the development and progression of invasive lobular carcinoma (ILC), little is known about the underlying mechanisms that govern these processes. Using a mouse model of human ILC, we show here that cytosolic p120-catenin (p120) regulates tumor growth upon loss of E-cadherin through the induction of anoikis resistance. p120 conferred anchorage independence by indirect activation of Rho/Rock signaling through interaction and inhibition of myosin phosphatase Rho-interacting protein (Mrip), an antagonist of Rho/Rock function. Consistent with these data, primary human ILC samples expressed hallmarks of active Rock signaling, and Rock controlled the anoikis resistance of human ILC cells. Thus, we have linked loss of E-cadherin - an initiating event in ILC development - to Rho/Rock-mediated control of anchorage-independent survival. Because activation of Rho and Rock are strongly linked to cancer progression and are susceptible to pharmacological inhibition, these insights may have clinical implications for the development of tailor-made intervention strategies to better treat invasive and metastatic lobular breast cancer.

Background: Hereditary breast cancer runs in families where several members in different generations are affected. Most of these breast cancers are caused by mutations in the high penetrance genes BRCA1 and BRCA2 accounting for about 5% of all breast cancers. Other genes that include CHEK2, PTEN, TP53, ATM, STK11/LKB1, CDH1, NBS1, RAD50, BRIP1 and PALB2 have been described to be high or moderate penetrance breast cancer susceptibility genes, all contributing to the hereditary breast cancer spectrum. However, in still a part of familial hereditary breast cancers no relationship to any of these breast cancer susceptibility genes can be found. Research on new susceptibility genes is therefore ongoing., Design: In this review we will describe the function of the today known high or moderate penetrance breast cancer susceptibility genes and the consequences of their mutated status. Furthermore, we will focus on the histology, the immunophenotype and genotype of breast cancers caused by mutations in BRCA1 and BRCA2 genes and the other high or moderate penetrance breast cancer susceptibility genes. Finally, an overview of the clinical implications of hereditary breast cancer patients will be provided., Conclusion: This information leads to a better understanding of the morphological, immunohistochemical and molecular characteristics of different types of hereditary breast cancers. Further, these characteristics offer clues for diagnosis and new therapeutic approaches.

Introduction: The BRCA1 protein makes mammary stem cells differentiate into mature luminal and myoepithelial cells. If a BRCA1 mutation results in a differentiation block, an enlarged stem cell component might be present in the benign tissue of BRCA1 mutation carriers, and these mammary stem cells could be the origin of BRCA1 related breast cancer. Since ALDH1 is a marker of both mammary stem cells and breast cancer stem cells, we compared ALDH1 expression in malignant tissue of BRCA1 mutation carriers to non-carriers., Methods: Forty-one BRCA1 related breast cancers and 41 age-matched sporadic breast cancers were immunohistochemically stained for ALDH1. Expression in epithelium and stroma was scored and compared., Results: Epithelial (P = 0.001) and peritumoral (P = 0.001) ALDH1 expression was significantly higher in invasive BRCA1 related carcinomas compared to sporadic carcinomas. Intratumoral stromal ALDH1 expression was similarly high in both groups. ALDH1 tumor cell expression was an independent predictor of BRCA1 mutation status., Conclusion: BRCA1 related breast cancers showed significantly more frequent epithelial ALDH1 expression, indicating that these hereditary tumors have an enlarged cancer stem cell component. Besides, (peritumoral) stromal ALDH1 expression was also more frequent in BRCA1 mutation carriers. ALDH1 may therefore be a diagnostic marker and a therapeutic target of BRCA1 related breast cancer.

Background: Hypoxia inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia and is associated with aggressive tumour behaviour. We have shown p27kip1, which is generally reduced in endometrial cancer, to be re-expressed in hypoxic regions. This possibly contributes to survival of cancer cells. The aim of this study was to evaluate the prognostic value of HIF-1alpha and p27kip expression in patients with endometrioid endometrial cancer., Methods: Expression levels of HIF-1alpha, CAIX, Glut-1, and p27kip1 were analyzed by immunohistochemistry. Percentage of positive cells, staining pattern (perinecrotic, diffuse, or mixed) and presence of necrosis were noted., Results: Necrosis was correlated with shortened disease free survival (DFS) (p = 0.008) and overall survival (OS) (p = 0.045). For DFS, perinecrotic HIF-1alpha expression was also prognostic (p = 0.044). Moreover, high p27kip1 expression was an additional prognostic factor for these patients with perinecrotic HIF-1alpha expression. In multivariate Cox regression, perinecrotic HIF-expression emerged as an independent prognostic factor. Perinecrotic HIF-1alpha expression was significantly associated with CAIX and Glut-1 expression, pointing towards functional HIF-1., Conclusions: In patients with endometrioid endometrial cancer, necrosis and necrosis-related expression of HIF-1alpha are important prognostic factors. More aggressive adjuvant treatment might be necessary to improve the outcome of patients with these characteristics.

Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.

Background: Germline mutations in the BRCA1 gene predispose to the development of breast cancer, exhibiting a specific histological phenotype. Identification of possible hallmarks of these tumors is important for selecting patients for genetic screening and provides inside in carcinogenetic pathways.Since BRCA1-associated breast cancers have pushing borders that prevent them from easily reaching vessels and are often of the medullary (like) type that is known to have a low rate of lympho-vascular invasion (LVI), we hypothesized that absence of LVI could characterize BRCA1 related breast cancer., Methods: A population of 68 BRCA1 related invasive breast cancers was evaluated for LVI by an experienced breast pathologist blinded to mutation status, and compared to a control group matched for age, grade and tumor type., Results: LVI was present in 25.0% of BRCA1 related cases, compared to 20.6% of controls (P = 0.54, OR = 1.29, CI 0.58-2.78)., Conclusion: LVI is frequent in BRCA1 germline mutation related breast cancers, but seems to occur as often in sporadic controls matched for age, grade and tumor type. Apparently, these hereditary cancers find their way to the blood and lymph vessels despite their well demarcation and often medullary differentiation.

Objectives: To uncover novel biomarkers that will help predict which patients are at risk and may guide future treatment decisions. A significant proportion of prostate cancer patients treated with radical prostatectomy will experience a recurrence of the disease. Given the substantial role of hypoxia in prostate cancer development and treatment, this investigation focuses on the Hypoxia Inducible Factor (HIF-1alpha) pathway., Methods: A tissue microarray was constructed of prostate cancer tissue collected from 71 patients undergoing radical prostatectomy. The expression of proteins involved in the HIF-1alpha pathway was investigated by an immunohistochemical approach and correlated to clinical features including the time to biochemical recurrence., Results: Expression of GLUT1 correlated significantly (P <.05) with a shorter time to biochemical recurrence after radical prostatectomy and was independent from the Gleason grade and stage of cancer. Furthermore, our studies revealed for the first time that accumulation of prolyl-4-hydroxylases 1 especially in the nucleus, is a significant indicator for a worse prognosis (P <.001)., Conclusions: This study confirms the upregulation of proteins involved in the HIF-1alpha hypoxia pathway in prostate cancer cells, indicative of a hypoxic tumor state. Importantly, we report the identification of 2 novel markers, GLUT1 and prolyl-4-hydroxylases 1, with prognostic significance for patients undergoing radical prostatectomy., (Copyright 2010 Elsevier Inc. All rights reserved.)

A unique feature of the retina is the presence of photoreceptors, which require an enormous amount of oxygen for the conversion of light to an electrical signal. Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a transcription factor that is the master regulator of cellular adaptation to low oxygen tension. Only in hypoxic conditions is HIF-1alpha protein stabilized and translocated to the nucleus, where it induces transcription of target genes involved in oxygen delivery and energy metabolism. We hypothesized that HIF-1alpha is constitutively stabilized and active in the normal human retina. We investigated the cellular distribution of HIF-1alpha and the expression of its downstream targets, vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), and carbonic anhydrase IX (CAIX), by immunohistochemistry and immunoblotting in the retina of normal rats and human donor eyes. Both human and rat retinas displayed prominent staining of HIF-1alpha in nuclei of most cell types in inner and outer nuclear layers and the ganglion cell layer, a cellular distribution pattern which was confirmed in human retina by immunoblotting of nuclear extracts. A negative correlation was found between HIF-1alpha protein levels and postmortem times. In human retina, staining of VEGF, GLUT-1, and CAIX was found. Our observations indicate that active HIF-1 signaling occurs constitutively in the normal human and rat retina, suggesting that HIF-1 has a physiological role in the retina.

Objectives: To investigate the relationship between angiogenesis and coagulation markers in tumor tissues of primary renal cell carcinoma (RCC). Tumors stimulate angiogenesis and activate the coagulation cascade. The importance of the interplay between these pathways for RCC is unknown., Methods: In all, 69 clear cell RCC specimens were analyzed by immunohistochemical staining applied to tissue microarrays. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1alpha, fibrinogen and fibrin, and microvessel density were visually analyzed. Finally, staining patterns were related to clinical variables and survival., Results: The VEGF expression was detected in 100% of tumors, with 68% showing a high expression, whereas hypoxia-inducible factor-1alpha staining was low (only 26% had a moderate to high staining). Fibrinogen was expressed adjacent to the tumor cells in 26% of cases, whereas in 84% it was expressed around the blood vessels. In 30% of tumors, expression of fibrin was detected. High tumor VEGF expression correlated with high fibrin staining (P = .05). From a multivariate analysis, microvessel density (P = .033) and fibrinogen adjacent to tumor cells (P = .046) were independent factors related to VEGF expression., Conclusions: In this study, we found clinical evidence for the permeability activity of VEGF as reflected by extravascular fibrinogen expression adjacent to tumor cells in the extracellular matrix. In addition, VEGF and fibrin expression were associated, indicative for concomitant activation of the coagulation cascade and angiogenesis in RCC. Taken together, these data indicate that activation of angiogenesis and coagulation are related in RCC., (2010 Elsevier Inc. All rights reserved.)

Introduction: When breast cancer patients develop distant metastases, the choice of systemic treatment is usually based on tissue characteristics of the primary tumor as determined by immunohistochemistry (IHC) and/or molecular analysis. Several previous studies have shown that the immunophenotype of distant breast cancer metastases may be different from that of the primary tumor ("receptor conversion"), leading to inappropriate choice of systemic treatment. The studies published so far are however small and/or methodologically suboptimal. Therefore, definite conclusions that may change clinical practice could not yet be drawn. We therefore aimed to study receptor conversion for estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in a large group of distant (non-bone) breast cancer metastases by re-staining all primary tumors and metastases with current optimal immunohistochemical and in situ hybridization methods on full sections., Methods: 233 distant breast cancer metastases from different sites (76 skin, 63 liver, 43 lung, 44 brain and 7 gastro-intestinal) were IHC stained for ERα, PR and HER2, and expression was compared to that of the primary tumor. HER2 in situ hybridization (ISH) was done in cases of IHC conversion or when primary tumors or metastases showed an IHC 2+ result., Results: Using a 10% threshold, receptor conversion by IHC for ERα, PR occurred in 10.3%, 30.0% of patients, respectively. In 10.7% of patients, conversion from "ER+ or PR+" to ER-/PR- and in 3.4% from ER-/PR- to "ER+ or PR+" was found. Using a 1% threshold, ERα and PR conversion rates were 15.1% and 32.6%. In 12.4% of patients conversion from "ER+ or PR+" to ER-/PR-, and 8.2% from ER-/PR- to "ER+ or PR+" occurred. HER2 conversion occurred in 5.2%. Of the 12 cases that showed HER2 conversion by IHC, 5 showed also conversion by ISH. One further case showed conversion by ISH, but not by IHC. Conversion was mainly from positive in the primary tumor to negative in the metastases for ERα and PR, while HER2 conversion occurred equally both ways. PR conversion occurred significantly more often in liver, brain and gastro-intestinal metastases., Conclusions: Receptor conversion by immunohistochemistry in (non-bone) distant breast cancer metastases does occur, is relatively uncommon for ERα and HER2, and more frequent for PR, especially in brain, liver and gastro-intestinal metastases.

Aims: Ductal carcinoma in situ (DCIS) is an established late precursor of sporadic invasive breast cancer and to a large extent parallels its invasive counterpart with respect to molecular changes and immunophenotype. Invasive breast cancers in germline BRCA1 and BRCA2 mutation carriers have a distinct "basal" and "luminal" immunophenotype, respectively, but the immunophenotype of their precursor lesions has hardly been studied, and this was the aim of this study., Methods: DCIS lesions of 25 proven BRCA1 and 9 proven BRCA2 germline mutation carriers and their 22 and 6, respectively, accompanying invasive lesions were stained by immunohistochemistry for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER)2/neu, cytokeratin (CK)5/6, CK14, epidermal growth factor receptor (EGFR) and Ki67., Results: DCIS lesions in BRCA1 mutation carriers were mostly of the basal molecular type with low ER/PR/HER2 expression, while they frequently expressed CK5/6, CK14 and EGFR, and were mostly grade 3 and highly proliferative. DCIS lesions in BRCA2 mutation carriers were mostly of luminal molecular type with frequent expression of ER/PR, and infrequent expression of CK5/6, CK14 and EGFR, and they were mostly grade 3 and showed low proliferation. In BRCA1 and BRCA2 mutation carriers there was a high concordance between DCIS lesions and their concomitant invasive counterpart with regard to expression of individual markers as well as "molecular" subtype., Conclusions: Although the number of cases studied was low, DCIS lesions in BRCA1 and BRCA2 mutations carriers are usually of the basal and luminal molecular type, respectively, similar to their accompanying invasive cancers, thereby providing evidence that DCIS is a direct precursor lesion in these hereditary predisposed patients. This also suggests that crucial carcinogenetic events leading to these phenotypes in hereditary predisposed patients occur before the stage of invasion.

Therapeutic approaches aimed at targeting tumor surface markers using monoclonal antibodies provide a powerful strategy in cancer treatment. Here we report selection of single variable domains (VHH) of llama heavy chain antibodies, using a VHH-phage-display library. A reverse proteomic approach was used to identify the cognate proteins recognized by enriched VHH on HeLa cells. One of these VHH bound the integrin alpha 3 beta 1 (VLA-3) and was further characterized. Most interestingly, this VHH could inhibit VLA-3 mediated cell-matrix adhesion. Our approach provides a fast and efficient method to screen for novel cell surface markers on normal and tumor cells that may find diagnostic or therapeutic application in disease management or treatment.

Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1alpha (HIF-1alpha) is the key regulator of the hypoxia response. HIF-1alpha is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1alpha in hereditary breast carcinogenesis. A recent study suggests a role for BRCA1 in HIF-1alpha regulation. We therefore examined the expression of HIF-1alpha in BRCA1 related breast cancers. By immunohistochemistry we studied expression of HIF-1alpha and some of its downstream targets in 30 hereditary invasive breast cancers in comparison with 200 sporadic controls. HIF-1alpha overexpression was significantly more frequent in BRCA1 related breast cancers (27/30, 90%) than in sporadic controls (88/200, 44%) (P < 0.0001). 19/30 (63%) of BRCA1 tumors showed perinecrotic (hypoxia induced) and 8/30 (27%) a diffuse HIF-1alpha overexpression pattern, the latter more likely related to genetic alterations in oncogenes and tumor suppressor genes. In contrast, sporadic breast cancer HIF-1 expressing tumors showed an inverse ratio of perinecrotic/diffuse expression (31 vs. 69%, P = 0.0002). Glut-1 and CAIX, downstream HIF1 targets, were expressed in 27/30 (90%) and 15/21 (71%) of hereditary cases versus 54/183 (29%) and 24/183 (13%) in sporadic cases. p300 levels, necessary for HIF-1 downstream activation, were significantly higher in hereditary cases (20/21, 95%) compared to sporadic cases (91/183, 50%, P = 0.0001). In conclusion, in BRCA1 germline mutation related breast cancer, functional HIF-1alpha overexpression is seen at a much higher frequency than in sporadic breast cancer, mostly hypoxia induced. This points to an important role of hypoxia and its key regulator HIF-1alpha in hereditary breast carcinogenesis.