Recruitment of the Mammalian Histone-modifying EMSY Complex to Target Genes Is Regulated by ZNF131.

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.

p120-Catenin Is Critical for the Development of Invasive Lobular Carcinoma in Mice.

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.

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer.

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.

Copy number profiling by array comparative genomic hybridization identifies frequently occurring BRCA2-like male breast cancer.

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.

Distinct claudin expression characterizes BRCA1-related breast cancer.

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.

Regulation of E2F1 by the von Hippel-Lindau tumour suppressor protein predicts survival in renal cell cancer patients.

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.

Modelling the impact of bias in fecal immunochemical testing on long-term outcomes of colorectal cancer screening.

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.

Why prostate tumour delineation based on apparent diffusion coefficient is challenging: an exploration of the tissue microanatomy.

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.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers.

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).

Is there 'progression through grade' in ductal invasive breast cancer?

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.

Molecular subtyping of male breast cancer by immunohistochemistry.

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.

Immunophenotyping invasive breast cancer: paving the road for molecular imaging.

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.

Receptor conversion in distant breast cancer metastases.

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.

Lympho-vascular invasion in BRCA related breast cancer compared to sporadic controls.

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.

Necrosis related HIF-1alpha expression predicts prognosis in patients with endometrioid endometrial carcinoma.

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.

The molecular genetic make-up of male breast cancer.

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.

Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer.

Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A-M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10-20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.

Comprehensive Proteomic Profiling-derived Immunohistochemistry-based Prediction Models for BRCA1 and BRCA2 Germline Mutation-related Breast Carcinomas.

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, ERß, 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.

Characterizing steroid hormone receptor chromatin binding landscapes in male and female breast cancer.

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.

Hypoxia and angiogenesis in endometrioid endometrial carcinogenesis.

BACKGROUND: Hypoxia-inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia, triggering biologic events associated with aggressive tumor behavior. METHODS: Expression of HIF-1alpha and proteins in the HIF-1alpha pathway (Glut-1, CAIX, VEGF) in paraffin-embedded specimens of normal (n=17), premalignant (n=17) and endometrioid endometrial carcinoma (n=39) was explored by immunohistochemistry, in relation to microvessel density (MVD). RESULTS: HIF-1alpha overexpression was absent in inactive endometrium but present in hyperplasia (61%) and carcinoma (87%), with increasing expression in a perinecrotic fashion pointing to underlying hypoxia. No membranous expression of Glut-1 and CAIX was noticed in inactive endometrium, in contrast with expression in hyperplasia (Glut-1 0%, CAIX 61%, only focal and diffuse) and carcinoma (Glut-1 94.6%, CAIX 92%, both mostly perinecrotically). Diffuse HIF-1alpha was accompanied by activation of downstream targets. VEGF was significantly higher expressed in hyperplasias and carcinomas compared to inactive endometrium. MVD was higher in hyperplasias and carcinomas than in normal endometrium (p<0.001). CONCLUSION: HIF-1alpha and its downstream genes are increasingly expressed from normal through premalignant to endometrioid adenocarcinoma of the endometrium, paralleled by activation of its downstream genes and increased angiogenesis. This underlines the potential importance of hypoxia and its key regulator HIF-1alpha in endometrial carcinogenesis.