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Purpose: Hereditary breast and ovarian cancer has long been established to affect a considerable number of patients and their families. By identifying those at risk ideally before they have been diagnosed with breast and/or ovarian cancer, access to preventive measures, intensified screening and special therapeutic options can be obtained, and thus, prognosis can be altered beneficially. Therefore, a standardized screening and counseling process has been established in Germany under the aegis of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC). As one of these specialized clinics, the HBOC-Center at Charité offers genetic counseling as well as genetic analysis based on the GC-HBOC standards. This analysis aims first at depicting this process from screening through counseling to genetic analysis as well as the patient collective and second at correlating the results of genetic analysis performed. Thus, real-world data from an HBOC-Center with a substantial patient collective and a high frequency of pathogenic variants in various genes shall be presented., Methods: The data of 2531 people having been counseled at the HBOC-Center at Charité in 2016 and 2017 were analyzed in terms of patient and family history as well as pathogenic variants detected during genetic analysis with the TruRisk ® gene panel when genetic analysis was conducted. This standardized analysis is compiled and regularly adjusted by the GC-HBOC. The following genes were included at time of research: BRCA1, BRCA2, ATM, CDH1, CHEK2, PALB2, RAD51C, RAD51D, NBN, and TP53., Results: Genetic analysis was conducted in 59.8% of all cases meeting the criteria for genetic analysis and 286 pathogenic variants were detected among 278 (30.3%) counselees tested using the TruRisk ® gene panel. These were primarily found in the genes BRCA1 (44.8%) and BRCA2 (28.3%) but also in CHEK2 (12.2%), ATM (5.6%) and PALB2 (3.5%). The highest prevalence of pathogenic variants was seen among the families with both ovarian and breast cancer (50.5%), followed by families with ovarian cancer only (43.2%) and families with breast cancer only (35.6%)-these differences are statistically significant (p < 0.001). Considering breast cancer subtypes, the highest rate of pathogenic variants was detected among patients with triple-negative breast cancer (40.7%) and among patients who had had been diagnosed with triple-negative breast cancer before the age of 40 (53.4%)-both observations proved to be statistically significant (p = 0.003 and p = 0.001)., Conclusion: Genetic counseling and analysis provide the foundation in the prevention and therapy of hereditary breast and ovarian cancer. The rate of pathogenic variants detected is associated with family history as well as breast cancer subtype and age at diagnosis, and can reach considerable dimensions. Therefore, a standardized process of identification, genetic counseling and genetic analysis deems mandatory., (© 2022. The Author(s).)

As a member of the 11-gene "death-from-cancer" gene expression signature, overexpression of the Ubiquitin-Specific Protease 22 (USP22) was associated with poor prognosis in various human malignancies. To investigate the function of USP22 in cancer development and progression, we sought to detect common USP22-dependent molecular mechanisms in human colorectal and breast cancer cell lines. We performed mRNA-seq to compare gene expression profiles of various colorectal (SW837, SW480, HCT116) and mammary (HCC1954 and MCF10A) cell lines upon siRNA-mediated knockdown of USP22. Intriguingly, while USP22 depletion had highly heterogeneous effects across the cell lines, all cell lines displayed a common reduction in the expression of Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1). The downregulation of HSP90AB1 was confirmed at the protein level in these cell lines as well as in colorectal and mammary tumors in mice with tissue-specific Usp22 deletions. Mechanistically, we detected a significant reduction of H3K9ac on the HSP90AB1 gene in USP22-deficient cells. Interestingly, USP22-deficient cells displayed a high dependence on HSP90AB1 expression and diminishing HSP90 activity further using the HSP90 inhibitor Ganetespib resulted in increased therapeutic vulnerability in both colorectal and breast cancer cells in vitro. Accordingly, subcutaneously transplanted CRC cells deficient in USP22 expression displayed increased sensitivity towards Ganetespib treatment in vivo. Together, we discovered that HSP90AB1 is USP22-dependent and that cooperative targeting of USP22 and HSP90 may provide an effective approach to the treatment of colorectal and breast cancer.

Gains of chromosomes 7p and 8q are associated with poor prognosis among oestrogen receptor-positive (ER+) stage I/II breast cancer. To identify transcriptional changes associated with this breast cancer subtype, we applied suppression subtractive hybridisation method to analyse differentially expressed genes among six breast tumours with and without chromosomal 7p and 8q gains. Identified mRNAs were validated by real-time RT-PCR in tissue samples obtained from 186 patients with stage I/II breast cancer. Advanced statistical methods were applied to identify associations of mRNA expression with distant metastasis-free survival (DMFS). mRNA expression of the key enzyme of cholesterol biosynthesis, squalene epoxidase (SQLE, chromosomal location 8q24.1), was associated with ER+ 7p+/8q+ breast cancer. Distant metastasis-free survival in stage I/II breast cancer cases was significantly inversely related to SQLE mRNA in multivariate Cox analysis (P<0.001) in two independent patient cohorts of 160 patients each. The clinically favourable group associated with a low SQLE mRNA expression could be further divided by mRNA expression levels of the oestrogen-regulated zinc transporter LIV-1. The data strongly support that SQLE mRNA expression might indicate high-risk ER+ stage I/II breast cancers. Further studies on tumour tissue from standardised treated patients, for example with tamoxifen, may validate the role of SQLE as a novel diagnostic parameter for ER+ early stage breast cancers.

Early placenta insulin-like growth factor (EPIL) is expressed by a subpopulation of the Her2-positive SKBR3 breast cancer cell line displaying high motility and transendothelial invasiveness in vitro, as recently shown by our group. As a consequence of this, we established cellular models by generating an EPIL-overexpressing SKBR3 cell line, knocked down EPIL by adding specific small interfering RNA (siRNA) to those cells and produced EPIL-enriched and depleted serum-free culture media. EPIL-expressing cells as well as EPIL-induced SKBR3 cells acquired a high capacity for transendothelial invasiveness. We observed a thin and outspread morphology caused by enhanced formation of lamellipodia, i.e. protrusions in the initial phase of motility. In parallel, Her2-positive MDAHer2 breast cancer cells also showed increased invasiveness when induced by EPIL-conditioned medium. A downstream signaling impact of EPIL could be observed in the form of reduced phosphorylation of Her2, erk1/2 and akt, while phospholipase Cgamma1 phophorylation remained unaffected. As an in vivo model for highly motile tumor cells, Paget's disease of the nipple showed simultaneous EPIL and Her2 expression upon immunohistochemical examination using specific antibodies. Such experimental data have been translated to a clinical setting by using a prognostic tissue microarray established from 603 breast cancer cases. Survival data analysis found a significant association between expression levels of EPIL and 5-year overall survival that was dose dependent: EPIL (negative) 84%, EPIL (moderately positive) 77%, EPIL (strongly positive) 48% (P < 0.005). One particular subgroup (7.6% of the cases with full clinical records) that comprised tumors simultaneously expressing EPIL and Her2 represented patients with the poorest 5-year overall survival. The results suggested that EPIL might be a cancer cell-produced growth factor that influences lateral Her2 signaling. Moreover, EPIL may be induced by factors apart from Her2 and may independently provide signaling for cancer invasion and motility.

Aims: Recently, we were able to show that the expression of early placenta insulin like growth factor (EPIL) is expressed by highly motile HER-2-positive breast cancer cells in vitro (Brandt et al., Cancer Res. 2002) in Paget cells in vivo and indicates a poor clinical prognosis, irrespectively of other prognostic factors., Methods: In order to demonstrate the interplay between HER-2 and Epil we established a cellular model for high simultaneous Epil and HER-2 expression. The HER-2-positive breast cancer cell line SKBR3 was modified with an EPIL expression vector. In addition, an assay for the knockdown of EPIL-expression via siRNA was established. Erk1/2 expression was measured via Western Blot. The phenotype of the viable cells was determined by laser scan microscopy., Results: Epil overexpression in SKBR3 cells resulted in fast and frequent protrusion formation of the cells shown by laser scan microscopy. The cells were further characterized by a significantly increased invasiveness, which could be reversed by Epil specific siRNA treatment. Increased invasiveness and morphological changes were associated with a decreased erk1/2 phosphorylation., Conclusions: These data further supports the assumption that EPIL might provide an autocrine loop in HER-2-positive breast cancer cells that enforce metastasis, conceivably escape from adjuvant therapy and in consequence poor clinical outcome. A tight interaction between HER-2 and EPIL in invasive breast cancer cells is therefore likely. The exact mechanims remain to be elucidated.

Distinct parallel cytogenetic pathways in breast carcinogenesis could be identified in recent years. Nevertheless, it remained unclear as to which tumours may have progressed in grade or which patterns of cytogenetic alteration may define the switch from an in situ towards an invasive lesion. In order to gain more detailed insights into cytogenetic mechanisms of the pathogenesis of breast cancer, the chromosomal imbalances of 206 invasive breast cancer cases were characterised by means of comparative genomic hybridisation (CGH). CGH data were subjected to hierarchical cluster analysis and the results were further compared with immunohistochemical findings on tissue arrays from the same breast cancer cases. The combined analysis of immunohistochemical and cytogenetic data provided evidence that carcinomas with gains of 7p, and to a lesser extent losses of 9q and gains of 5p, are a distinct subgroup within the spectrum of ductal invasive grade 3 breast carcinomas. These aberrations were associated with a high degree of cytogenetic instability (16.6 alterations per case on average), 16q-losses in over 70% of these cases, strong oestrogen receptor expression and absence of strong expression of p53, c-erbB2 and Ck 5. These characteristics provide strong support for the hypothesis that these tumours may develop through stages of well- and perhaps intermediately differentiated breast cancers. Our results therefore underline the existence of several parallel and also stepwise progression pathways towards breast cancer.