Anatomic versus non-anatomic liver resection for hepatocellular carcinoma-A European multicentre cohort study in cirrhotic and non-cirrhotic patients.

BACKGROUND: The incidence of hepatocellular carcinoma (HCC) is increasing in the western world over the past decades. As liver resection (LR) represents one of the most efficient treatment options, advantages of anatomic (ALR) versus non-anatomic liver resection (NALR) show a lack of consistent evidence. Therefore, the aim of this study was to investigate complications and survival rates after both resection types. METHODS: This is a multicentre cohort study using retrospectively and prospectively collected data. We included all patients undergoing LR for HCC between 2009 and 2020 from three specialised centres in Switzerland and Germany. Complication and survival rates after ALR versus NALR were analysed using uni- and multivariate Cox regression models. RESULTS: Two hundred and ninety-eight patients were included. Median follow-up time was 52.76 months. 164/298 patients (55%) underwent ALR. Significantly more patients with cirrhosis received NALR (n = 94/134; p < 0.001). Complications according to the Clavien Dindo classification were significantly more frequent in the NALR group (p < 0.001). Liver failure occurred in 13% after ALR versus 8% after NALR (p < 0.215). Uni- and multivariate cox regression models showed no significant differences between the groups for recurrence free survival (RFS) and overall survival (OS). Furthermore, cirrhosis had no significant impact on OS and RFS. CONCLUSION: No significant differences on RFS and OS rates could be observed. Post-operative complications were significantly less frequent in the ALR group while liver specific complications were comparable between both groups. Subgroup analysis showed no significant influence of cirrhosis on the post-operative outcome of these patients.

The short- and long-term outcome after the surgical management of common bile duct stones in a tertiary referral hospital.

BACKGROUND: The removal of common bile duct stones by endoscopic retrograde cholangiopancreatography (ERCP) shows excellent results with low complication rates and is therefore considered a gold standard. However, in case of stones non-removable by ERCP, surgical extraction is needed. The surgical approach is still controversial and clinical guidelines are missing. This study aims to analyze the outcomes of patients treated with choledochotomy or hepaticojejunostomy for common bile duct stones. METHODS: All patients who underwent choledochotomy or hepaticojejunostomy for common bile duct stones at a tertiary referral hospital over 11 years were included. The analyzed data contains basic demographics, diagnostics, surgical parameters, length of hospitalization, and morbidity and mortality. RESULTS: Over the study period, 4375 patients underwent cholecystectomy, and 655 received an ERCP with stone extraction, with 48 of these patients receiving subsequent surgical treatment. ERCP was attempted in 23/30 (77%) of the choledochotomy patients pre/intraoperatively and 11/18 (56%) in hepaticojejunostomy patients. The 30-day major complication rate (Clavien-Dindo > II) was 1/30 (3%) in the choledochotomy group and 2/18 (11%) in the hepaticojejunostomy group. Complications after 30 days occurred in 3/30 (10%) patients and 2/18 (11%), respectively, and no mortality occurred. CONCLUSION: ERCP should still be considered the gold standard, although due to low short- and long-term morbidity rates, choledochotomy and hepaticojejunostomy represent effective surgical solutions for common bile duct stones.

Association of Axillary Dissection With Systemic Therapy in Patients With Clinically Node-Positive Breast Cancer.

Importance: The role of axillary lymph node dissection (ALND) to determine nodal burden to inform systemic therapy recommendations in patients with clinically node (cN)-positive breast cancer (BC) is currently unknown. Objective: To address the association of ALND with systemic therapy in cN-positive BC in the upfront surgery setting and after neoadjuvant chemotherapy (NACT). Design, Setting, and Participants: This was a prospective, observational, cohort study conducted from August 2018 to June 2022. This was a preplanned study within the phase 3 randomized clinical OPBC-03/TAXIS trial. Included were patients with confirmed cN-positive BC from 44 private, public, and academic breast centers in 6 European countries. After NACT, residual nodal disease was mandatory, and a minimum follow-up of 2 months was required. Exposures: All patients underwent tailored axillary surgery (TAS) followed by ALND or axillary radiotherapy (ART) according to TAXIS randomization. TAS removed suspicious palpable and sentinel nodes, whereas imaging-guidance was optional. Systemic therapy recommendations were at the discretion of the local investigators. Results: A total of 500 patients (median [IQR] age, 57 [48-69] years; 487 female [97.4%]) were included in the study. In the upfront surgery setting, 296 of 335 patients (88.4%) had hormone receptor (HR)-positive and Erb-B2 receptor tyrosine kinase 2 (ERBB2; formerly HER2 or HER2/neu)-negative disease: 145 (49.0%) underwent ART, and 151 (51.0%) underwent ALND. The median (IQR) number of removed positive lymph nodes without ALND was 3 (1-4) nodes compared with 4 (2-9) nodes with ALND. There was no association of ALND with the proportion of patients undergoing adjuvant chemotherapy (81 of 145 [55.9%] vs 91 of 151 [60.3%]; adjusted odds ratio [aOR], 0.72; 95% CI, 0.19-2.67) and type of systemic therapy. Of 151 patients with NACT, 74 (51.0%) underwent ART, and 77 (49.0%) underwent ALND. The ratio of removed to positive nodes was a median (IQR) of 4 (3-7) nodes to 2 (1-3) nodes and 15 (12-19) nodes to 2 (1-5) nodes in the ART and ALND groups, respectively. There was no observed association of ALND with the proportion of patients undergoing postneoadjuvant systemic therapy (57 of 74 [77.0%] vs 55 of 77 [71.4%]; aOR, 0.86; 95% CI, 0.43-1.70), type of postneoadjuvant chemotherapy (eg, capecitabine: 10 of 74 [13.5%] vs 10 of 77 [13.0%]; trastuzumab emtansine-DM1: 9 of 74 [12.2%] vs 11 of 77 [14.3%]), or endocrine therapy (eg, aromatase inhibitors: 41 of 74 [55.4%] vs 36 of 77 [46.8%]; tamoxifen: 8 of 74 [10.8%] vs 6 of 77 [7.8%]). Conclusion: Results of this cohort study suggest that patients without ALND were significantly understaged. However, ALND did not inform systemic therapy recommendations.

Third International Consensus Conference on lesions of uncertain malignant potential in the breast (B3 lesions).

The heterogeneous group of B3 lesions in the breast harbors lesions with different malignant potential and progression risk. As several studies about B3 lesions have been published since the last Consensus in 2018, the 3rd International Consensus Conference discussed the six most relevant B3 lesions (atypical ductal hyperplasia (ADH), flat epithelial atypia (FEA), classical lobular neoplasia (LN), radial scar (RS), papillary lesions (PL) without atypia, and phyllodes tumors (PT)) and made recommendations for diagnostic and therapeutic approaches. Following a presentation of current data of each B3 lesion, the international and interdisciplinary panel of 33 specialists and key opinion leaders voted on the recommendations for further management after core-needle biopsy (CNB) and vacuum-assisted biopsy (VAB). In case of B3 lesion diagnosis on CNB, OE was recommended in ADH and PT, whereas in the other B3 lesions, vacuum-assisted excision was considered an equivalent alternative to OE. In ADH, most panelists (76%) recommended an open excision (OE) after diagnosis on VAB, whereas observation after a complete VAB-removal on imaging was accepted by 34%. In LN, the majority of the panel (90%) preferred observation following complete VAB-removal. Results were similar in RS (82%), PL (100%), and FEA (100%). In benign PT, a slim majority (55%) also recommended an observation after a complete VAB-removal. VAB with subsequent active surveillance can replace an open surgical intervention for most B3 lesions (RS, FEA, PL, PT, and LN). Compared to previous recommendations, there is an increasing trend to a de-escalating strategy in classical LN. Due to the higher risk of upgrade into malignancy, OE remains the preferred approach after the diagnosis of ADH.

Cylindroma of the breast with CYLD gene mutation: a case report and review of the literature.

BACKGROUND: Cylindroma of the breast is a rare benign neoplasm. Since its first description in 2001, 20 cases have been reported in the literature. METHODS AND RESULTS: We report another case of this rare tumor in a 60-year-old woman with demonstration of the underlying molecular alteration. Histologically, the tumor showed the typical "jigsaw" pattern of a dual population of cells with a triple-negative phenotype. The pathognomonic mutation of the CYLD gene mutation was detected by whole exome sequencing. Cylindromas show morphological overlap with the solid-basaloid variant of adenoid cystic carcinoma, which renders this differential diagnosis difficult. However, distinction of these two lesions is of outmost importance, since cylindromas, in contrast to solid-basaloid variant of adenoid cystic carcinoma, behave in an entirely benign fashion. CONCLUSIONS: Careful evaluation of morphological features such as mitotic figures and cellular atypia is crucial in the diagnostic work-up of triple-negative breast lesions. It is important to keep cylindroma in mind as a pitfall and possible differential diagnosis for the solid-basaloid variant of adenoid cystic carcinoma. Molecular detection of CYLD gene mutation is helpful in cases with ambiguous histology. With this case report, we aim to contribute to a better understanding of mammary cylindroma and facilitate the diagnosis of this rare entity.

Trends in use of neoadjuvant systemic therapy in patients with clinically node-positive breast cancer in Europe: prospective TAXIS study (OPBC-03, SAKK 23/16, IBCSG 57-18, ABCSG-53, GBG 101).

PURPOSE: The aim of this study was to evaluate clinical practice heterogeneity in use of neoadjuvant systemic therapy (NST) for patients with clinically node-positive breast cancer in Europe. METHODS: The study was preplanned in the international multicenter phase-III OPBC-03/TAXIS trial (ClinicalTrials.gov Identifier: NCT03513614) to include the first 500 randomized patients with confirmed nodal disease at the time of surgery. The TAXIS study's pragmatic design allowed both the neoadjuvant and adjuvant setting according to the preferences of the local investigators who were encouraged to register eligible patients consecutively. RESULTS: A total of 500 patients were included at 44 breast centers in six European countries from August 2018 to June 2022, 165 (33%) of whom underwent NST. Median age was 57 years (interquartile range [IQR], 48-69). Most patients were postmenopausal (68.4%) with grade 2 and 3 hormonal receptor-positive and human epidermal growth factor receptor 2-negative breast cancer with a median tumor size of 28 mm (IQR 20-40). The use of NST varied significantly across the countries (p < 0.001). Austria (55.2%) and Switzerland (35.8%) had the highest percentage of patients undergoing NST and Hungary (18.2%) the lowest. The administration of NST increased significantly over the years (OR 1.42; p < 0.001) and more than doubled from 20 to 46.7% between 2018 and 2022. CONCLUSION: Substantial heterogeneity in the use of NST with HR+/HER2-breast cancer exists in Europe. While stringent guidelines are available for its use in triple-negative and HER2+ breast cancer, there is a need for the development of and adherence to well-defined recommendations for HR+/HER2-breast cancer.

Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model.

Interplay between non-cancerous cells (immune, fibroblasts, mesenchymal stromal cells (MSC), and endothelial cells (EC)) has been identified as vital in driving tumor progression. As studying such interactions in vivo is challenging, ex vivo systems that can recapitulate in vivo scenarios can aid in unraveling the factors impacting tumorigenesis and metastasis. Using the synthetic tumor microenvironment mimics (STEMs)-a spheroid system composed of breast cancer cells (BCC) with defined human MSC and EC fractions, here we show that EC organization into vascular structures is BC phenotype dependent, and independent of ERα expression in epithelial cancer cells, and involves MSC-mediated Notch1 signaling. In a 3D-bioprinted model system to mimic local invasion, MDA STEMs collectively respond to serum gradient and form invading cell clusters. STEMs grown on chick chorioallantoic membrane undergo local invasion to form CAM tumors that can anastomose with host vasculature and bear the typical hallmarks of human BC and this process requires both EC and MSC. This study provides a framework for developing well-defined in vitro systems, including patient-derived xenografts that recapitulate in vivo events, to investigate heterotypic cell interactions in tumors, to identify factors promoting tumor metastasis-related events, and possibly drug screening in the context of personalized medicine.

PD-L1 Expression in Triple-negative Breast Cancer-a Comparative Study of 3 Different Antibodies.

BACKGROUND: Assessment of programmed death protein-ligand 1 (PD-L1) in triple-negative breast cancer (TNBC) has entered daily practice to identify patients eligible for treatment with immune checkpoint inhibitors. However, different antibodies and different cut-offs for PD-L1 positivity are used, and the interchangeability of these methods is not clear. The aim of our study was to analyze whether different PD-L1 antibodies can be used interchangeably to identify TNBC patients as PD-L1 positive. METHODS: A tissue microarray encompassing 147 TNBC cases was immunohistochemically analyzed using 3 different antibodies against PD-L1: SP142, SP263, and E1L3N. PD-L1 positivity was determined as ≥1% of positive tumor-associated immune cells. The staining patterns of the 3 antibodies were compared and correlated with clinicopathological data. RESULTS: A total of 84 cases were evaluable for PD-L1 analysis with all 3 antibodies. PD-L1 was positive in 50/84 patients (59.5%) with SP263, in 44/84 (52.4%) with E1L3N, and in 29/84 (34.5%) with SP142. There was no statistical difference between the performance of SP263 and E1L3N, but both antibodies stained significantly more cases than the SP142 antibody. CONCLUSIONS: Our results show that the 3 PD-L1 antibodies identify different TNBC patient subgroups as PD-L1 positive and, therefore cannot be used interchangeably. Additional studies are needed to further investigate the use and impact of different PD-L1 antibody clones for predictive selection of TNBC patients for treatment with immune checkpoint inhibitors.

High ratio of pCXCR4/CXCR4 tumor infiltrating immune cells in primary high grade ovarian cancer is indicative for response to chemotherapy.

BACKGROUND: Ovarian cancer (OC) is the fifth most common malignant female cancer with a high mortality, mainly because of aggressive high-grade serous carcinomas (HGSOC), but also due to absence of specific early symptoms and effective detection strategies. The CXCL12-CXCR4 axis is considered to have a prognostic impact and to serve as potential therapeutic target. Therefore we investigated the role of pCXCR4 and CXCR4 expression of the tumor cells and of tumor infiltrating immune cells (TIC) in high-grade serous OC and their association with the recurrence-free (RFS) and overall survival (OS). METHODS: A tissue microarray of 47 primary high grade ovarian serous carcinomas and their recurrences was stained with primary antibodies directed against CXCR4 and pCXCR4. Beside the evaluation of the absolute tumor as well as TIC expression in primary and recurrent cancer biopsies the corresponding ratios for pCXCR4 and CXCR4 were generated and analyzed. The clinical endpoints were response to chemotherapy, OS as well as RFS. RESULTS: Patients with a high pCXCR4/CXCR4 TIC ratio in primary cancer biopsies showed a significant longer RFS during the first two years (p = 0.025). However, this effect was lost in the long-term analysis including a follow-up period of 5 years (p = 0.128). Interestingly, the Multivariate Cox regression analysis showed that a high pCXCR4/CXCR4 TIC ratio in primary cancer independently predicts longer RFS (HR 0.33; 95CI 0.13 - 0.81; p = 0.015). Furthermore a high dichotomized distribution of CXCR4 positive tumor expression in recurrent cancer biopsies showed a significantly longer 6-month RFS rate (p = 0.018) in comparison to patients with low CXCR4 positive tumor expression. However, this effect was not independent of known risk factors in a Multivariate Cox regression (HR 0.57; 95CI 0.24 - 1.33; p = 0.193). CONCLUSIONS: To the best of our knowledge we show for the first time that a high pCXCR4/CXCR4 TIC ratio in primary HGSOC biopsies is indicative for better RFS and response to chemotherapy. HIGHLIGHTS: • We observed a significant association between high pCXCR4/CXCR4 TIC ratio and better RFS in primary cancer biopsies, especially during the early postoperative follow-up and independent of known risk factors for recurrence. • High CXCR4 tumor expression in recurrent HGSOC biopsies might be indicative for sensitivity to chemotherapy. We found evidence that at the beginning of the disease (early follow-up) the role of the immune response seems to be the most crucial factor for progression. On the other hand in recurrent/progressive disease the biology of the tumor itself becomes more important for prognosis. • We explored for the first time the predictive and prognostic role of pCXCR4/CXCR4 TIC ratio in high-grade serous ovarian cancer.

An Integrated Epigenomic and Genomic View on Phyllodes and Phyllodes-like Breast Tumors.

Fibroepithelial lesions (FL) of the breast, in particular, phyllodes tumors (PT) and fibroadenomas, pose a significant diagnostic challenge. There are no generally accepted criteria that distinguish benign, borderline, malignant PT and fibroadenomas. Combined genome-wide DNA methylation and copy number variant (CNV) profiling is an emerging strategy to classify tumors. We compiled a series of patient-derived archival biopsy specimens reflecting the FL spectrum and histological mimickers including clinical follow-up data. DNA methylation and CNVs were determined by well-established microarrays. Comparison of the patterns with a pan-cancer dataset assembled from public resources including "The Cancer Genome Atlas" (TCGA) and "Gene Expression Omnibus" (GEO) suggests that FLs form a methylation class distinct from both control breast tissue as well as common breast cancers. Complex CNVs were enriched in clinically aggressive FLs. Subsequent fluorescence in situ hybridization (FISH) analysis detected respective aberrations in the neoplastic mesenchymal component of FLs only, confirming that the epithelial component is non-neoplastic. Of note, our approach could lead to the elimination of the diagnostically problematic category of borderline PT and allow for optimized prognostic patient stratification. Furthermore, the identified recurrent genomic aberrations such as 1q gains (including MDM4), CDKN2a/b deletions, and EGFR amplifications may inform therapeutic decision-making.

An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.

Persistent acute cholecystitis after cholecystostomy - increased mortality due to treatment approach?

BACKGROUND: Percutaneous cholecystostomy (PC) is a treatment option for acute cholecystitis (AC) in cases where cholecystectomy (CCY) is not feasible due to limited health conditions. The use of PC remains questionable. The aim was to retrospectively analyse the outcome of patients after PC. METHODS: All patients who underwent PC for AC at a tertiary referral hospital over 10 years were included. Descriptive statistics, analysed mortality with and without CCY after PC, and a multivariable logistic regression for potential confounder and a landmark sensitivity analysis for immortal time bias were used. RESULTS: Of 158 patients, 79 were treated with PC alone and 79 had PC with subsequent CCY. Without CCY, 48% (38 patients) died compared to 9% with CCY. In the multivariable analysis CCY was associated with 85% lower risk of mortality. The landmark analysis was compatible with the main analyses. Direct PC-complications occurred in 17% patients. Histologically, 22/75 (29%) specimens showed chronic cholecystitis, and 76% AC. CONCLUSION: Due to the high mortality rate of PC alone, performing up-front CCY is proposed. PC represents no definitive treatment for AC and should remain a short-term solution because of the persistent inflammatory focus. According to these findings, almost all specimens showed persistent inflammation.

Tailored axillary surgery in patients with clinically node-positive breast cancer: Pre-planned feasibility substudy of TAXIS (OPBC-03, SAKK 23/16, IBCSG 57-18, ABCSG-53, GBG 101).

AIM: We developed tailored axillary surgery (TAS) to reduce the axillary tumor volume in patients with clinically node-positive breast cancer to the point where radiotherapy can control it. The aim of this study was to quantify the extent of tumor load reduction achieved by TAS. METHODS: International multicenter prospective study embedded in a randomized trial. TAS is a novel pragmatic concept for axillary surgery de-escalation that combines palpation-guided removal of suspicious nodes with the sentinel procedure and, optionally, imaging-guided localization. Pre-specified study endpoints quantified surgical extent and reduction of tumor load. RESULTS: A total of 296 patients were included at 28 sites in four European countries, 125 (42.2%) of whom underwent neoadjuvant chemotherapy (NACT) and 71 (24.0%) achieved nodal pathologic complete response. Axillary metastases were detectable only by imaging in 145 (49.0%) patients. They were palpable in 151 (51.0%) patients, of whom 63 underwent NACT and 21 had residual palpable disease after NACT. TAS removed the biopsied and clipped node in 279 (94.3%) patients. In 225 patients with nodal disease at the time of surgery, TAS removed a median of five (IQR 3-7) nodes, two (IQR 1-4) of which were positive. Of these 225 patients, 100 underwent ALND after TAS, which removed a median of 14 (IQR 10-17) additional nodes and revealed additional positive nodes in 70/100 (70%) of patients. False-negative rate of TAS in patients who underwent subsequent ALND was 2.6%. CONCLUSIONS: TAS selectively reduced the tumor load in the axilla and remained much less radical than ALND.

Identification of Somatic Mutations in Thirty-year-old Serum Cell-free DNA From Patients With Breast Cancer: A Feasibility Study.

INTRODUCTION: The aim of this study was to assess the feasibility of cell-free DNA (cfDNA) extraction and circulating tumor DNA sequencing in 30-year-old serum samples. MATERIALS AND METHODS: We evaluated serum samples from 52 patients with breast cancer, which were collected between 1983 and 1991, with correlating clinicopathologic data. cfDNA was extracted by using the QIAamp Circulating Nucleic Acid Extraction Kit (Qiagen). Of these 52 cfDNA samples, 10 were randomly selected and sequenced with the Oncomine Breast cfDNA Assay (A31183). In a second step, high-depth targeted sequencing of 15 additional cfDNA samples was performed using a custom Ampliseq Ion Torrent panel targeting breast cancer-related genes. RESULTS: cfDNA extraction was successful in 52 (100%) of 52 patients with a total concentration of 0.2 to 54 ng/uL. A total of 24 cancer-specific mutations were found in 22 (88%) of the 25 samples undergoing sequencing. Of the 52 patients, 32 (62%) had died from breast cancer after a median follow-up of 7.9 years (interquartile range, 3.7-15.5 years). CONCLUSION: The present study shows that current next generation sequencing technology is sufficiently robust and specific to analyze 30-year-old serum. Therefore, longitudinal studies can be designed with storage of serum samples over many years, thereby obviating the need for timely and continuous cfDNA extraction and sequencing. The samples can be pooled and processed at once with the most modern technology available at the end of the study, when accumulation of events allows correlation of clinical outcomes with adequate power.

Tall cell carcinoma of the breast with reversed polarity (TCCRP) with mutations in the IDH2 and PIK3CA genes: a case report.

Tall cell carcinoma with reversed polarity (TCCRP) is a rare breast carcinoma with low malignant potential, initially named "breast tumor resembling the tall cell variant of papillary thyroid carcinoma", which has recently been recognized as a separate entity in the 5th edition of the WHO (World Health Organization) classification of breast tumors. Since the first report of this entity in 2003, more than 40 cases have been reported in the literature. Here, we report another case of this rare tumor in a 60-year-old woman. We performed immunohistochemical analyses and next-generation-sequencing (NGS) using the Oncomine™ Comprehensive DNA Panel (Thermo Fisher Scientific). The tumor showed the typical morphological features of TCCRP and a "triple-negative" phenotype. Moreover, we identified pathogenic mutations in the IDH2 (p.R172G) and PIK3CA (p.H1047R) genes. We report a case of TCCRP of the breast showing the characteristic morphologic, immunohistochemical and molecular features of this entity. There is still a limited number of cases with comprehensive molecular analyses reported in the literature. Therefore, we herewith contribute to a better understanding of the morphological and molecular characteristics as well as the clinical behavior of this rare entity.

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance.

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

The single-cell pathology landscape of breast cancer.

Single-cell analyses have revealed extensive heterogeneity between and within human tumours1-4, but complex single-cell phenotypes and their spatial context are not at present reflected in the histological stratification that is the foundation of many clinical decisions. Here we use imaging mass cytometry5 to simultaneously quantify 35 biomarkers, resulting in 720 high-dimensional pathology images of tumour tissue from 352 patients with breast cancer, with long-term survival data available for 281 patients. Spatially resolved, single-cell analysis identified the phenotypes of tumour and stromal single cells, their organization and their heterogeneity, and enabled the cellular architecture of breast cancer tissue to be characterized on the basis of cellular composition and tissue organization. Our analysis reveals multicellular features of the tumour microenvironment and novel subgroups of breast cancer that are associated with distinct clinical outcomes. Thus, spatially resolved, single-cell analysis can characterize intratumour phenotypic heterogeneity in a disease-relevant manner, with the potential to inform patient-specific diagnosis.

High density of CD66b in primary high-grade ovarian cancer independently predicts response to chemotherapy.

PURPOSE: Ovarian carcinoma (OC) is the most lethal female genital cancer. After a primary curative surgical approach followed by chemotherapy, a fraction of the patients recur with chemoresistant disease. Data indicate a favorable therapeutic effect of tumor-infiltrating neutrophils (TIN) in OC. Our aim was to investigate the prognostic role of CD66b expression, corresponding to neutrophilic infiltration for recurrence-free survival (RFS) and overall survival (OS) in patients with OC. METHODS: A collective of 47 primary serous ovarian carcinoma and their matching recurrences were processed and stained with CD66b using immunohistochemistry. Tumors from patients with RFS of more than 6 months were defined as chemosensitive. Statistical analysis of CD66b expression was performed to assess the clinical endpoints. RESULTS: High density of CD66b expressing neutrophils in primary carcinoma was associated with chemosensitivity (p = 0.014) and longer RFS (p = 0.001). Univariate analysis identified high density of CD66b expressing neutrophils as a predictor for favorable RFS (HR 0.41, 95% CI 0.22-0.76, p < 0.005). Residual disease > 2 cm (HR 3.67, 95% CI 1.62-8.31, p < 0.002) and higher number of chemotherapy cycles (HR 1.28, 95% CI 1.05-1.55, p < 0.013) were associated with worse RFS. Multivariate analysis showed that high density of CD66b expressing neutrophils (HR 0.22, 95% CI 0.10-0.48, p < 0.001) and residual disease > 2 cm (HR 3.69, 95% CI 1.43-9.53, p < 0.007) were independent predictors of RFS but had no impact on OS. CONCLUSION: High CD66b neutrophil density in primary high-grade OC predicts good response to initial chemotherapy and longer recurrence-free survival independent of known risk factors.

Nectin-4 Expression Is an Independent Prognostic Biomarker and Associated With Better Survival in Triple-Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) represents about 10-20% of all invasive breast cancers and is associated with a poor prognosis. The nectin cell adhesion protein 4 (Nectin-4) is a junction protein involved in the formation and maintenance of cell junctions. Nectin-4 has previously shown to be expressed in about 60% of TNBC as well as in TNBC metastases, but to be absent in normal breast tissue, which makes it a potential specific target for TNBC therapy. Previous studies have shown an association of Nectin-4 protein expression with worse prognosis in TNBC in a small patient cohort. The aim of our study was to explore the role of Nectin-4 in TNBC and confirm its impact on survival in a larger TNBC patient cohort. Material and Methods: We performed immunohistochemical staining for Nectin-4 on a tissue microarray encompassing 148 TNBC cases with detailed clinical annotation and outcomes data. Results: A high expression of Nectin-4 was present in 86 (58%) of the 148 TNBC cases. In multivariate survival analysis, high expression of Nectin-4 was associated with a significantly better overall survival when compared with low expression of Nectin-4 (p < 0.001). Nectin-4-high expression was also significantly associated with a lower tumor stage (p = 0.025) and pN0 lymph node stage (p = 0.034). Conclusion: Our results confirm that expression of Nectin-4 serves as a potential prognostic marker in TNBC and is associated with a significantly better overall survival. In addition, Nectin-4 represents a potential target in TNBC, and its role in molecular defined breast cancer subtype should be investigated in larger patient cohorts.

Genetic Alterations in Benign Breast Biopsies of Subsequent Breast Cancer Patients.

Background: Fibrocystic changes are associated with an increased risk of breast cancer. Genetic alterations have been found in fibrocystic changes with or without epithelial changes, suggesting that critical oncogenic events are occurring at an early stage. Methods: We investigated a unique collective of 17 breast cancer patients who, prior to the diagnosis of invasive breast cancer, underwent open surgical biopsy showing fibrocystic changes of the breast. The time span between biopsy for fibrocystic changes and invasive carcinoma ranged from 1 to 11 years (average 5.3 years). Ten (58.8%) of the patients had an ipsilateral invasive carcinoma, and 7 (41.2%) of the patients developed an invasive carcinoma of the contralateral breast. Massive parallel sequencing targeting genes frequently mutated in breast cancer was performed on the fibrocystic breast tissue as well as the ensuing cancer tissue. Results: In 9 cases, somatic mutations were found in the tumor tissue, the most prevalent being PIK3CA mutations (n = 4), followed by TP53 mutations (n = 2). None of these mutations were present in the previously removed mastopathy tissue. In one of the cases, an ERBB3 E928G mutation was present in the mastopathy as well as in the tumor tissue, with the variant allele frequency in the mastopathy being <0.1%. In two patients, we found two mutations (MAP3K1 L380fs and PIK3CA I391M, respectively) present in the mastopathy as well as in the subsequent breast cancer. These two mutations, however, could also be due to fixation artifacts. Conclusion: Since no significant somatic mutations in the fibrocystic breast tissue, and only doubtful shared mutations between benign and associated cancer tissue were detected, it remains unclear why women with fibrocystic breast disease have a statistically significant increased risk of breast cancer. Further analyses, maybe on the level of gene expression, could help to clarify the role of these benign alterations in the development of breast cancer and help to identify women at greater risk of developing subsequent invasive cancer.