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Introduction: Even though randomized controlled trials could not show a significant survival benefit for the use of neoadjuvant systemic therapy (NST), it is increasingly recommended for patients with clinically node-positive breast cancer due to its implications on prognosis, locoregional downstaging and response-driven adjuvant systemic therapy. The aim of this study was to assess the need for international standardization of treatment recommendations by evaluating clinical practice heterogeneity in use of 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) after randomization of the first 500 patients with clinically node-positive breast cancer who underwent axillary lymph node dissection (ALND) or axillary radiation (ART) without ALND after tailored axillary surgery (TAS) in the context of extended regional nodal irradiation. Clinically node-positive breast cancer was defined by confirmed nodal disease at the time of initial diagnosis; in case of neoadjuvant therapy, residual nodal disease was mandatory. Investigators were encouraged to enroll all eligible patients consecutively. However, TAXIS is unique inasmuch as its pragmatic design allows both the neoadjuvant and adjuvant setting according to the preferences of the treating physicians and institutions and thus provides an excellent opportunity to study patterns and trends in use of NST in patients with clinically positive nodes in Europe. Results: A total of 500 patients with a median age of 57 years (IQR: 48-69 years) were included at 44 breast centers in 6 European countries from August 2018 to June 2022. Subtype was hormone receptor (HR) positive (+) and human epidermal growth factor receptor 2 (HER2) negative (-) in 393 (80.0%), HR+/HER2+ in 52 (10.6%), HR-/HER2+ in 5 (1.0%) and HR-/HER2- in 34 (6.9%) patients. The rate of patients undergoing NST was 31.4% with a significant upward trend over time during the study period (from 20.0% in 2018 to 38.1% in 2022; p=0.044). The use of NST varied significantly by country (p=< 0.001) and by site (p=0.015). For patients with clinical AJCC tumor stage II and III, the rates of patients undergoing NST in Switzerland were 26.5% (18 of 68) and 35.9% (92 of 256), in Germany 22.2% (2 of 9) and 30.4% (7 of 23), in Austria 50% (7 of 14) and 60% (9 of 15) and in Hungary 0% (0 of 15) and 20.7% (18 of 87), respectively (p=0.019 and 0.004). Large differences by country were found for ER+/HER2- breast cancer, ranging from 13.1% (11 of 84) in Hungary to 47.8% (11 of 23) in Austria (p=0.007). Within Switzerland, which was the country with most included patients (328 of 500) and participating sites (n=25), the rate of patients undergoing NST for ER+/HER2- breast cancer varied considerably by site, ranging from 10% (2 of 20) to 50% (11 of 22). Discussion: This study revealed substantial heterogeneity in clinical practice in Europe, indicating the need for development of and adherence to consistent guidelines to standardize the international use of NST. Citation Format: Walter P. Weber, Zoltan Matrai, Stefanie Hayoz, Guido Henke, Daniel R. Zwahlen, Günther Gruber, Frank Zimmermann, Thomas Ruhstaller, Simone Muenst, Markus Ackerknecht, Christian Kurzeder, Sherko Küemmel, Vesna Bjelic-Radisic, Viktor Smanykó, Conny Vrieling, Rok Satler, Inna Meyer, Charles Becciolini, Susanne Bucher, Colin Simonson, Peter M. Fehr, Natalie Gabriel, Robert Maráz, Dimitri Sarlos, Konstantin J. Dedes, Cornelia Leo, Gilles Berclaz, Hisham Fansa, Christopher Hager, Klaus Reisenberger, Ákos Sávolt, Christian F. Singer, Roland Reitsamer, Jelena Winkler, Giang Thanh Lam Lam, Mathias K. Fehr, Tatiana Naydina, Magdalena Kohlik, Karine Clerc, Valerijus Ostapenko, Florian Fitzal, Martin Heidinger, Nadia Maggi, Alexandra Schulz, Pagona Markellou, Loïc Lelièvre, Daniel Egle, Jörg Heil, Michael Knauer, Christoph Tausch. Trends in neoadjuvant systemic therapy rates in Europe: Pre-planned substudy of TAXIS (OPBC-03, SAKK 23/16, IBCSG 57-18, ABCSG-53, GBG 101) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-14-08.

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.

Supplementary Table from An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression

Supplementary Figure from An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression

Background Cylindroma of the breast is a rare benign neoplasm. Since its first description in 2001, 18 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.

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set Enrichment Analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top Differentially Expressed Genes (DEGs) between these cell states revealed a common set of partial EMT Transcriptions Factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

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., Highlights • Tailored axillary surgery is a novel concept for clinically node-positive breast cancer • Tailored axillary surgery selectively removes positive lymph nodes • Tailored axillary surgery is much less radical than axillary dissection • Tailored axillary surgery removes the clipped node in the vast majority of patients

Introduction: Chemotherapy is recommended for patients with luminal breast cancer and more than three positive nodes. In addition, recent landmark trials raised the question if the exact number of positive nodes is required to indicate genomic testing. In the neoadjuvant setting, response-driven therapy is increasingly used and may be influenced by surgical staging of the axilla. The present study addressed the role of axillary lymph node dissection (ALND) as decision aid for systemic therapy in a contemporary cohort of patients with clinically node-positive breast cancer in the adjuvant and neoadjuvant setting. Methods: The study was preplanned in the international multicenter phase-III OPBC-03/TAXIS trial (ClinicalTrials.gov Identifier: NCT03513614). The first 500 patients with clinically node-positive breast cancer who were randomized after tailored axillary surgery (TAS) to undergo ALND or axillary radiotherapy (ART) without ALND in the context of extended regional irradiation were included from August 2018 to June 2022. Clinically node-positive breast cancer was defined by confirmed nodal disease at the time of initial diagnosis; in case of neoadjuvant therapy, the finding of residual nodal disease was mandatory for randomization. TAS consisted of removal of palpably suspicious findings and the sentinel nodes with the option of image guidance. In the ART arm, the total number of positive nodes was not known. We analyzed the impact of ALND on rate and type of systemic therapy. Results: A total of 500 patients with a median age of 57 years (IQR: 48-69 years) were included at 44 breast centers from six European countries. Subtype was hormone receptor (HR) positive (+) and human epidermal growth factor receptor 2 (HER2) negative (-) in 393 (80.0%), HR+/HER2+ in 52 (10.6%), HR-/HER2+ in 5 (1.0%) and HR-/HER2- in 34 (6.9%) patients. Of 343 patients (68.6%) who were treated in the adjuvant setting, 297 had HR+/HER2- disease. Of these 297 patients, 145 (48.8%) underwent ART without ALND and 152 (51.2%) underwent ALND after TAS. In the ART arm, the median number of lymph nodes removed was five (IQR 4-8), three (IQR 1-4) of which were positive and in the ALND arm, the number was 19 (IQR 14-26), four (IQR 2-9) of which were positive (p < 0.001). The use of ALND had no significant impact on the rate of patients with HR+/HER2- disease undergoing adjuvant chemotherapy (51.0% in the ART and 57.9% in the ALND arm, p=0.2), and there were no significant differences in type of systemic therapy with the exception of tamoxifen, which was 18.4% with ALND versus 9.0% without (p=0.018). A total of 143 patients (28.6%) underwent neoadjuvant chemotherapy, 13 had neoadjuvant antihormonal treatment and one had neoadjuvant double HER2-blockade without chemotherapy. Of the 143 patients who received neoadjuvant chemotherapy, 71 (49.7%) underwent ART without ALND and 72 (50.3%) underwent ALND. In the ART arm, the median number of lymph nodes removed was four (IQR 3-6), one (IQR 1-3) of which was positive and in the ALND arm, the number was 16 (IQR 12-19), two (IQR 1-5) of which were positive (p < 0.001). The use of ALND in patients after neoadjuvant treatment had no significant impact on the rate of adjuvant systemic therapy (71.8% in the ART and 65.3% in the ALND arm, p=0.4), with no significant differences in type of chemotherapy (e.g., capecitabine: 11.3% vs 12.5%, p=0.8; T-DM1: 11.3% vs. 11.1%, p>0.9) or antihormonal therapy (e.g., aromatase inhibitors: 49.3% vs. 41.7%, p=0.4; tamoxifen: 11.3% vs. 5.6%, p=0.2). Discussion: This study showed that although ALND significantly increased the number of positive nodes removed in the adjuvant and neoadjuvant setting, it had no relevant impact on rate and type of adjuvant systemic therapy. Citation Format: Walter P. Weber, Zoltan Matrai, Stefanie Hayoz, Christoph Tausch, Guido Henke, Daniel R. Zwahlen, Günther Gruber, Frank Zimmermann, Thomas Ruhstaller, Simone Muenst, Markus Ackerknecht, Sherko Küemmel, Vesna Bjelic-Radisic, Viktor Smanykó, Conny Vrieling, Rok Satler, Inna Meyer, Charles Becciolini, Susanne Bucher, Colin Simonson, Peter M. Fehr, Natalie Gabriel, Robert Maráz, Dimitri Sarlos, Konstantin J. Dedes, Cornelia Leo, Gilles Berclaz, Hisham Fansa, Christopher Hager, Klaus Reisenberger, Ákos Sávolt, Christian F. Singer, Roland Reitsamer, Jelena Winkler, Giang Thanh Lam Lam, Mathias K. Fehr, Tatiana Naydina, Magdalena Kohlik, Karine Clerc, Valerijus Ostapenko, Florian Fitzal, Martin Heidinger, Nadia Maggi, Alexandra Schulz, Pagona Markellou, Loïc Lelièvre, Daniel Egle, Jörg Heil, Michael Knauer, Christian Kurzeder. PD15-11 Axillary dissection to determine nodal burden to inform systemic therapy recommendations in patients with clinically node-positive breast cancer: Pre-planned substudy of TAXIS (OPBC-03, SAKK 23/16, IBCSG 57-18, ABCSG-53, GBG 101) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD15-11.

Background: Phyllodes tumor (PT), a rare fibroepithelial neoplasm of the breast, poses a significant diagnostic challenge in breast pathology, as there are no generally accepted histological criteria or cutoffs to unambiguously differentiate PT variants (benign, borderline, malignant) and fibroadenomas (FA) of the breast. In recent years, genome-wide DNA methylation analysis has gained wide interest as an objective tool to distinguish all sorts of neoplasms based on their individual methylation signatures. For brain tumors, methylation-based classification already outperforms classification by conventional histology and has led to changes in the WHO tumor classification. Materials and Methods: We have so far analyzed fresh frozen as well as formalin-fixed, paraffin-embedded tissue samples of PT (n=31) and FA (n=2) on Illumina Infinium Methylation EPIC bead chips. The resulting data were preprocessed, normalized, mapped to the genome and converted into beta values. Top differentially methylated probes were determined by the calculation of standard deviations across the dataset. Such filtered sets of methylation beta values were then compared by a dimension reduction algorithm alongside TCGA methylome datasets as well as collections from GEO and the entire diagnostic database of our institute (n=14342). In parallel, the genome-wide copy number alterations were calculated from array data for each case. Results: In analogy to other tumor entities included in our reference cohort (brain tumors, sarcomas, carcinomas, melanomas), PT and FA of the breast show distinct DNA methylation patterns that contain a breast-specific signature as well as tumor-derived patterns. Their methylomes are distinct from breast adenocarcinoma as well as from healthy breast tissue. Two distinct groups within the PT/FA spectrum were identified so far: One group contains a high fraction of histologically malignant PT and is enriched for complex copy number alterations while the other group, also containing the currently two included fibroadenomas and, features less prominent to no copy number variants and a higher similarity to the overall normal breast tissue methylation signature. Conclusion: The distinct methylation signatures of the histological PT/FA spectrum will not only allow the diagnostic discrimination of PTs from histological mimics such as FA or carcinomas, but will also enable the distinction of benign, borderline and malignant PTs, thus paving the way to an optimized prognostic patient stratification and clinical management. Citation Format: Simone Muenst, Tatjana Vlajnic, Savas Deniz Soysal, Stefan Frank, Jürgen Hench. Distinct methylation and copy number alteration patterns in phyllodes tumors of the breast and its mimics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-21-02.

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. A single-cell, spatially resolved analysis of breast cancer demonstrates the heterogeneity of tumour and stroma tissue and provides a more-detailed method of patient classification than the current histology-based system.

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.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.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.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.

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., Cancer Research, 82 (4), ISSN:0008-5472, ISSN:1538-7445

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.

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. Discussion 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.

Objective Numerous publications describe percutaneous cholecystostomy (PC) as a possible treatment option for acute cholecystitis (AC) in selected cases where laparoscopic or open cholecystectomy (CHE) is not feasible due to limited health conditions. Whereas certain experts propose PC as a definitive therapy option for AC, a number of studies question the use of PC, due to high complication rates, no additional benefit of PC compared to CHE, and an increased mortality. The aim of our study was to retrospectively analyze the outcome of patients treated with PC over an extended period of time. Methods We conducted a retrospective study of patients who underwent PC for AC at a tertiary referral hospital during the last 10 years. The collected data included basic demographics, details about PC procedure, outcome, surgical-rate and final histologic diagnosis. Results Out of 158 patients (median age 75 years) treated with PC for AC, 47 (30%) died without undergoing subsequent CHE. Half of the PC patients (79) underwent subsequent CHE (8% in the hot phase), with 97% of these patients undergoing subsequent CHE within one year after PC. Seven (5%) of them died within the first year. The overall Charlson Comorbidity Index (CCI) was 6.4 (CHE vs. no CHE 5.3 vs. 7.4). Histologically, 22 (29%) of the 75 analyzed specimens showed chronic cholecystitis (CC), and 57 patients (68%) had signs of an AC. In 48 patients (30%), a complication after PC occurred. Conclusion In our collective, the 1-year survival after PC was 72%. The majority of these patients were in limited health conditions with a mean pre-PC CCI > 5, which implies a potential one-year mortality rate of over 85%. Histologic examination of almost all cholecystectomy specimens showed persistent inflammation. To our knowledge, this is the first extensive report of histologic findings in gallbladder specimens after PC. Based on our findings, especially in view of the high mortality rate of PC patients, we propose CHE as the treatment of choice in AC, even in chronically ill and elderly patients after stabilization, e.g. with a PC. PC represents no definitive treatment for AC and should remain a short-term solution because of the persistent inflammatory focus. Because CHE in a critically ill patient can be challenging, it should be performed by the most experienced surgeons.

Myeloperoxidase (MPO) is an enzyme secreted by neutrophil granulocytes as a result of phagocytosis during inflammation. In colorectal cancer, tumour infiltration by MPO expressing cells has been shown to be independently associated with a favourable prognosis. In this study, we explored the role of MPO-positive cell infiltration and its prognostic significance in invasive breast cancer. We performed immunohistochemical staining for MPO on multiple tissue microarrays comprising a total of 928 human breast cancer samples with detailed clinical-pathological annotation and outcome data. MPO-positive cell infiltration (≥ 5 cells/tissue punch) was found in 150 (16%) of the 928 evaluable breast cancer cases. In univariate survival analyses, infiltration by MPO-positive cells was associated with a significantly better overall survival (p

Background: Hepatocellular carcinoma (HCC) accounts for 75-85% of all primary liver malignancies. HCC represents an attractive target for immune checkpoint inhibitors (ICI) and tumor microenvironment (TME) agents. The development and success of ICI therapies requires a comprehensive model to study interactions of immune networks in the TME. Patient-derived organoids (PDO) have emerged as promising individualized models for ex vivo drug testing. While PDO are superior to previous models, due to their inherent heterogeneity and their enhanced capacity to capture tumor characteristics, the lack of mesenchymal and immune components limit their use for testing drug classes such as ICI or study tumor-TME interaction. Here, we developed and characterized a perfusion-based cultivated ex vivo HCC tissue model that preserves the tumor tissue architecture and maintains the pathophysiological microenvironment of human tissue for testing of TME-related treatments. Materials and methods: Fresh tumor tissue was obtained from surgical specimens of six patients undergoing liver resection for HCC. Tissue fragments of 2x2x2 mm were installed between two discs of collagen type 1 porous scaffold and placed in a perfusion-based bioreactor (U-shaped Culture Under Perfusion; U-CUP) for tissue engineering or cultured in static condition. At different time points, cultured tissue fragments were formalin-fixed and paraffin-embedded for downstream analysis. Cell viability and morphology was monitored on day 0, 5 and 7 by using hematoxylin and eosin staining. The changes in TME composition were monitored by immunohistochemistry using cell-type specific antibodies like CD34 for endothelial cells, CD3 for T-cells, alpha-smooth muscle actin (a-SMA) for fibroblasts and arginase-L for urea cycle. Results: Microscopic analysis revealed that, compare to static culture, the morphology and the viability of the cells were preserved during the culture period in the U-CUP. Tissue histology as well as the immunophenotypic pattern observed in the original tissue was maintained. Similarly, the presence of endothelial cells (CD34+) and fibroblasts (a-SMA+) were detected in the U-CUP cultured samples in a similar proportion to the original tumor samples. The presence of T-cells (CD3+) was stable at all time points. Compared to the samples cultured under static conditions, the U-CUP tended to show less necrosis during the seven-day course. Conclusion: Compared to PDO, U-CUP bioreactor tumor culture seems to preserve mesenchymal and immune components. This enables further investigation and assessment of TME-related treatments, such as ICI. Citation Format: Gabriel Fridolin Hess, Manuele Giuseppe Muraro, Mairene Coto-Llerena, Silvio Däster, Caner Ercan, Simone Muenst, Otto Kollmar, Salvatore Piscuoglio, Savas Deniz Soysal. Perfusion-based bioreactor preserves microenvironment of hepatocellular carcinoma ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6041.

Background: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Curative treatments in early-stage HCC are hampered by high recurrence rates, and targeted therapies against HCC are rare. Little is known about the impact of tumor microenvironment (TME) in this tumor. Manipulation of the immune system by tumors leads to compromised antigen recognition and an immunosuppressive microenvironment. One immunomodulatory strategy employed by malignant tumors is the expression of the programmed cell death receptor 1 ligand (PD-L1), whose receptor is expressed on activated T cells (PD-1). The use of immune checkpoint inhibitors (ICI) has shown antitumor activity and unprecedented rates of durable clinical responses in various cancer types. In HCC, nivolumab and pembrolizumab are described as possible second line therapies. Expression of PD-1 and PD-L1 in the TME has prognostic importance and can predict response to ICI. The aim of our study was to investigate the impact of PD-1 and PD-L1 expression on HCC recurrence and the relation to their cancer-immune phenotypes. Materials and methods: Immunohistochemical staining was performed on archival tissue from 91 HCC, primary and recurrence, using the following antibodies: NAT105 (PD-1) and Ventana SP263 (PD-L1). On the basis of protein expression, tumors were classified as immunologically active, excluded or deserted. PD-1 and PD-L1 immunoreactivity was evaluated as the proportion of positive staining of the total number of immune cells (0%, 1%). Results: Basic tumor characteristics were not related to recurrence of HCC. However, the immune-excluded subclass showed significant association with recurrence in HCC. The majority of non-recurrent HCC showed PD-1 expression >1% (78%; 38/49), also statistically significant. Conversely, recurrent HCC showed low PD-1 expression (0% or 1%) (77%; 49/64). An association between HCC recurrence and PD-L1 expression on tumor cells was not found. Conclusion: In concordance with previous reports, we found an association between low frequency of PD-1 expression and increased HCC recurrence, as well as the lack of impact of PD-L1 expression on HCC recurrence. Furthermore, our results suggest that HCC recurrence may be more frequent in immune-exclusive tumors. Pembrolizumab as adjuvant instead of 2nd line therapy should be further investigated. Citation Format: Gabriel Fridolin Hess, Mairene Coto-Llerena, Jasmin Zeindler, Silvio Däster, Simone Muenst, Jürg Vosbeck, Caner Ercan, Otto Kollmar, Salavtore Piscuoglio, Savas Deniz Soysal. PD1 expression and immune-active lymphocytes can predict recurrence of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6146.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

CD8+ tumor-infiltrating T cells can be regarded as one of the most relevant predictive biomarkers in immune-oncology. Highly infiltrated tumors, referred to as inflamed (clinically “hot”), show the most favorable response to immune checkpoint inhibitors in contrast to tumors with a scarce immune infiltrate called immune desert or excluded (clinically “cold”). Nevertheless, quantitative and reproducible methods examining their prevalence within tumors are lacking. We therefore established a computational diagnostic algorithm to quantitatively measure spatial densities of tumor-infiltrating CD8+ T cells by digital pathology within the three known tumor compartments as recommended by the International Immuno-Oncology Biomarker Working Group in 116 prospective metastatic melanomas of the Swiss Tumor Profiler cohort. Workflow robustness was confirmed in 33 samples of an independent retrospective validation cohort. The introduction of the intratumoral tumor center compartment proved to be most relevant for establishing an immune diagnosis in metastatic disease, independent of metastatic site. Cut-off values for reproducible classification were defined and successfully assigned densities into the respective immune diagnostic category in the validation cohort with high sensitivity, specificity, and precision. We provide a robust diagnostic algorithm based on intratumoral and stromal CD8+ T-cell densities in the tumor center compartment that translates spatial densities of tumor-infiltrating CD8+ T cells into the clinically relevant immune diagnostic categories “inflamed”, “excluded”, and “desert”. The consideration of the intratumoral tumor center compartment allows immune phenotyping in the clinically highly relevant setting of metastatic lesions, even if the invasive margin compartment is not captured in biopsy material., The authors present a robust diagnostic algorithm based on digital pathology and image analysis that quantifies intratumoral and stromal CD8+ T-cell densities in the tumor center and invasive margin compartment in metastatic melanoma. Spatial CD8+ T-cell densities are translated into the clinically relevant immune diagnostic categories “inflamed”, “excluded”, and “desert”. Their approach also allows efficient immune phenotyping of metastatic lesions, on biopsy material or even in the absence of material from the invasive margin.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

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.

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.

International audience; 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.

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.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.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.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.

Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors. Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays. Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition. Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells. Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-13.

// Simone Muenst 1,* , Robert Mechera 2,3,* , Silvio Daster 2 , Salvatore Piscuoglio 1 , Charlotte K.Y. Ng 1 , Fabienne Meier-Abt 4 , Walter P. Weber 2,* and Savas D. Soysal 2,* 1 Institute of Pathology, University Hospital Basel, Basel, Switzerland 2 Department of Surgery, University Hospital Basel, Basel, Switzerland 3 Department of Biomedicine, University Hospital Basel, Basel, Switzerland 4 Institute of Hematology, University Hospital Zurich, Zurich, Switzerland * These authors have contributed equally to this work Correspondence to: Savas D. Soysal, email: // Keywords : early pregnancy, breast cancer, Wnt signaling pathway, breast cancer prevention, Pathology Section Received : November 18, 2016 Accepted : February 22, 2017 Published : March 08, 2017 Abstract Background: Pregnancy at early age is the most significant modifiable factor which consistently decreases lifetime breast cancer risk. However, the underlying mechanisms haven’t been conclusively identified. Studies in mice suggest a reduction in progesterone-receptor (PR) sensitive epithelial cells as well as a downregulation of the Wnt signaling pathway as being one of the main mechanisms for the protective effect of early pregnancy. The aim of our study was to validate these findings in humans. Methods: We collected benign breast tissue of 125 women who had been stratified according to age at first pregnancy and the occurrence of subsequent breast cancer, and performed immunohistochemistry for PR, Wnt4 and the Wnt-target Versican. Results: The number of PR positive epithelial cells was significantly lower in the group of women with early pregnancy and no subsequent breast cancer compared to the group of nulliparous women with subsequent invasive breast cancer ( p = 0.0135). In women with early pregnancy, expression of Versican and Wnt4 was significantly lower compared to nulliparous women ( p = 0.0036 and p = 0.0241 respectively), and Versican expression was also significant lower compared to women with late pregnancy ( p < 0.0001). Discussion: Our results confirm prior observations in mice and suggest a role of downregulation of epithelial Wnt signaling in the protective effect of early pregnancy in humans. This results in a decreased proliferation of stem/progenitor cells; therefore, the Wnt signaling pathway may represent a potential target for breast cancer prevention in humans.

Summary Breast cancer is a heterogeneous disease. Tumor cells and associated healthy cells form ecosystems that determine disease progression and response to therapy. To characterize features of breast cancer ecosystems and their associations with clinical data, we analyzed 144 human breast tumor and 50 non-tumor tissue samples using mass cytometry. The expression of 73 proteins in 26 million cells was evaluated using tumor and immune cell-centric antibody panels. Tumors displayed individuality in tumor cell composition, including phenotypic abnormalities and phenotype dominance. Relationship analyses between tumor and immune cells revealed characteristics of ecosystems related to immunosuppression and poor prognosis. High frequencies of PD-L1+ tumor-associated macrophages and exhausted T cells were found in high-grade ER+ and ER− tumors. This large-scale, single-cell atlas deepens our understanding of breast tumor ecosystems and suggests that ecosystem-based patient classification will facilitate identification of individuals for precision medicine approaches targeting the tumor and its immunoenvironment., Graphical Abstract, Highlights • Single-cell proteomics reveals tumor and immune cell diversity in tumor ecosystems • Breast cancer exhibits tumor cell phenotypic abnormalities and tumor individuality • PD-L1+ TAMs and exhausted T cells are abundant in high-grade ER− and ER+ tumors • Tumor-immune relationships in the tumor ecosystem are patient-stratifying, A single-cell atlas of cancer and immune cells reveals distinct tumor ecosystems across breast cancer patients, informing prognosis and, potentially, therapy selection.

Background The Rearranged during Transfection (RET) protein is overexpressed in a subset of Estrogen Receptor (ER) positive breast cancer, with both signalling pathways functionally interacting. This cross-talk plays a pivotal role in the resistance of breast cancer cells to anti-endocrine therapies, and RET expression is assumed to correlate with poor prognosis based on findings in small patient cohorts. The aim of our study was to investigate the impact of RET expression on patient outcome in human breast cancer. Methods We performed an immunohistochemical analysis of RET protein expression on a tissue microarray encompassing 990 breast cancer patients and correlated its expression with clinicopathological parameters and survival data. Results Expression of RET was detected in 409 out of 990 cases (41.3%). RET and ER expression significantly correlated (p

Introduction: We developed tailored axillary surgery (TAS) to selectively remove positive nodes and omit axillary lymph node dissection (ALND) in patients with clinically node-positive breast cancer irrespective of the use of neoadjuvant chemotherapy. In this study, we evaluate the performance of this novel surgical concept that tailors the extent of axillary surgery to the extent of axillary disease. Methods: A prospective study was pre-specified to assess the performance of TAS in the international multicenter phase-III TAXIS trial randomizing patients with clinically node-positive breast cancer to undergo ALND or axillary radiation after TAS. TAS consists of selective removal of all palpably suspicious findings and the SLNs followed by specimen radiography to document removal of the clip placed in the sampled node. Imaging-guided localization is encouraged to increase the chances of clip removal. Only patients with confirmed nodal disease at the time of surgery can be randomized in TAXIS; the first 200 randomized patients were analyzed together with the ones achieving nodal pCR in this study. ClinicalTrials.gov Identifier: NCT03513614. Results: A total of 296 patients with a median age of 56.5 years (range: 25-88 years) were included at 28 breast centers from four European countries, 125 (42.3%) of whom underwent NACT and 75 (25.3%) of whom had nodal pCR. Subtype was hormone receptor (HR) positive (+) and human epidermal growth factor receptor 2 (HER2) negative (-) in 194 (65.5%), HR+/HER2+ in 40 (13.5%), HR-/HER2+ in 17 (5.7%) and HR-/HER2- in 39 (13.2%) patients. Breast-conserving surgery was performed in 178 patients (60%) and mastectomy in 117 (40%). Imaging-guided localization was attempted in 258 patients (87.2%) and was successful in 243 (82.1%). TAS removed a median of two (interquartile range [IQR] 0-3) palpably suspicious lesions and two (IQR 1-3) SLNs, thereby successfully removing the clip in 279 (94.3%) patients. There were no significant differences by use of imaging-guided localization (94.6% with vs 92.1% without, p=0.47) or type of clip (p=0.19), but a trend toward lower rate of clip removal after NACT (91.2% with vs 96.5% without NACT, p=0.075). Palpable disease was left behind after TAS in two (2.1%) patients and no SLN was detected in three (3.1%). In the 200 randomized patients with confirmed nodal disease at the time of surgery, lymph node metastases were palpable at the time of initial diagnosis in 102 (51%) patients and detectable only by imaging in 98 (49%). The median number of lymph nodes removed by TAS was four (IQR 2-8), two (IQR 1-4) of which were positive. Completion ALND following TAS removed additional positive nodes in 71 of 100 (71%) patients in the control group (20% with one additional node, 9% with 2, 8% with 3, 6% with 4, and 28% with >4). The median number of additional lymph nodes removed by ALND was 14 (IQR 10-18), two (IQR 0-6) of which were positive. Of the 200 randomized patients, one in the TAS group received a radiotherapy boost and one in the ALND group returned to the operating room for residual suspicious findings on imaging. Discussion: The present results suggest that TAS has the potential to become the new axillary surgery standard in patients with clinically node-positive breast cancer. TAS was successfully performed in the vast majority of patients, with no further improvement by imaging-guided localization, which makes the procedure feasible at most breast centers. TAS selectively removed positive lymph nodes and was much less radical than ALND, but ALND removed additional positive nodes in more than two thirds of patients. Disease-free survival and quality of life will be assessed in the randomized trial. Citation Format: Walter Paul Weber, Guido Henke, Stefanie Hayoz, Karin Ribi, Stefanie Seiler, Charlotte Maddox, Thomas Ruhstaller, Daniel Rudolf Zwahlen, Simone Muenst, Markus Ackerknecht, Florian Fitzal, Mihály Újhelyi, Christian Kurzeder, Loïc Lelièvre, Christoph Tausch, Daniel Egle, Jörg Heil, Zoltan Matrai, Michael Knauer. Tailored axillary surgery to omit axillary lymph node dissection independently from the use of neoadjuvant chemotherapy in patients with clinically node-positive breast cancer: Pre-specified subproject within TAXIS (SAKK 23/16 / IBCSG 57-18 / ABCSG-53 / GBG 101) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD4-04.

Background Main weaknesses of neoadjuvant chemotherapy (NACT) to avoid axillary dissection (ALND) in patients with clinically node-positive breast cancer are frequent failure of achieving nodal pathologic complete response (pCR) and administration of chemotherapy even though not indicated otherwise in many cases. Tailored axillary surgery (TAS) was designed to selectively remove positive nodes and omit ALND in patients with clinically node-positive breast cancer either in the upfront surgery setting or in case of residual nodal disease after neoadjuvant therapy, which distinguishes this trial from all others ongoing and published. Trial design In this international, multi-center, phase-III, non-inferiority randomized controlled trial, including 61 study sites from six countries, we plan to randomize 1500 patients to either receive TAS followed by ALND and regional nodal irradiation excluding the dissected axilla, or receive TAS only followed by regional nodal irradiation including the full axilla. TAS consists of selective removal of the sentinel lymph nodes (SLNs) and all palpably suspicious findings, thereby tailoring the extent of axillary surgery to the extent of axillary disease, followed by specimen radiography to document removal of the clip placed in the sampled node. Imaging-guided localization is encouraged to increase the chances of clip removal. All patients undergo adjuvant whole-breast irradiation after breast conserving surgery and chest wall irradiation after mastectomy. Inclusion of internal mammary nodes is recommended irrespective of treatment arm. ClinicalTrials.gov Identifier: NCT03513614. Inclusion criteria - Clinically node-positive breast cancer (all molecular subtypes allowed) - Node-positivity palpable or detectable only by imaging at time of initial diagnosis - Newly diagnosed or isolated in-breast recurrence or second ipsilateral breast cancer after previous breast conserving surgery and sentinel procedure and at least 3 years disease free and no prior axillary dissection or axillary RT. - In case of prior neoadjuvant treatment: residual disease (including residual ITCs) confirmed by pathology at the time of surgery - Clipping of sampled axillary lymph node Exclusion criteria - Absence of clip in the specimen radiography - Palpable disease left behind in the axilla after TAS - No SLN identified in the axilla Specific aims To test the hypothesis that treatment with TAS and axillary radiotherapy is non-inferior to ALND in terms of disease-free survival (DFS) of clinically node-positive breast cancer patients. Secondary objective is to test if quality of life is significantly better with TAS and axillary radiotherapy compared to ALND. Statistical methods With type I error 5% and power 80%, 385 events will be needed to show non-inferiority of TAS and axillary RT in comparison to ALND with a non-inferiority hazard ratio (HR) of 1.289 (corresponding to a DFS at 5 years of 80% in the ALND arm and 75% in the TAS and axillary RT arm), including one interim analysis for efficacy/futility after 20% of the required events have occurred. The sample size needed is 1500 patients (750 per arm). The HR and one-sided 95% confidence interval will be calculated using a Cox regression model based on the per-protocol set. Present accrual and target accrual The trial was activated on 31 July 2018 and the first patient was randomized on 07 August 2018. As of 03 July 2020, 291 patients have been randomized. Accrual is currently running according to protocol and is planned until end of 2023 with the primary endpoint analysis expected in 2029. Contact information Prof. Dr. Walter Paul Weber, University Hospital Basel; Tel: +41 61 328 61 49; Walter.Weber@usb.ch Citation Format: Walter Paul Weber, Guido Henke, Stefanie Hayoz, Karin Ribi, Stefanie Seiler, Charlotte Maddox, Thomas Ruhstaller, Daniel Rudolf Zwahlen, Simone Muenst, Markus Ackerknecht, Florian Fitzal, Mihály Újhelyi, Christian Kurzeder, Loïc Lelièvre, Christoph Tausch, Daniel Egle, Jörg Heil, Zoltan Matrai, Michael Knauer. Tailored axillary surgery with or without axillary lymph node dissection followed by radiotherapy in patients with clinically node-positive breast cancer (SAKK 23/16 / IBCSG 57-18 / ABCSG-53 / GBG 101 - TAXIS): A multicenter randomized phase III trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-04-03.

Clinical treatment of breast cancer is successful. What remains a challenge for individualized therapy is the prediction of which cancer responds to which treatment. Emerging evidence indicates that mechanical alterations on a (sub-) cellular level are markers of cancer aggressiveness. Detecting these could serve as biomarker and help optimize cancer diagnosis, therapy choice, and patient follow-up. We developed a diagnostic platform called ARTIDIS (Automated and Reliable Tissue Diagnostics) based on atomic force microscopy (AFM)-type nanomechanical testing. ARTIDIS uses a 20-nanometer-sharp tip measuring 10'000 miniscule indentation across the biopsy's surface and quantifies stiffness on a molecular level. The relative size and distribution of stiffness values is integrated into a data analytics tool that systematically analyses tissue biomechanics in correlation to clinical parameters, histological prognostic markers, and treatment yielding prognostic and predictive value. To demonstrate ARTIDIS' clinical utility for diagnosis and development of prognostic and predictive biomarkers we conducted a prospective blinded clinical study on 545 patients. All patients undergoing a core needle or vacuum biopsy of the breast qualified. Within the routine clinical workflow, ARTIDIS analyzed fresh biopsies in a physiological solution preserving the biopsy's viability for histological and genetic analysis. Per patient, one representative biopsy was measured, marked and reunited with the patient's other biopsies for standard histological procedure. In total, 583 biopsies from N=545 patients were analyzed: 62.1% were B1-B4 (normal, benign, of uncertain malignant potential, or suspicious); a third (36.9%) malignant (B5a-B5d), and six specimens (1%) not assignable. Half of all B5b lesions were Luminal B, followed by Luminal A, Luminal B-like, HER2-positive or triple negative. The analysis of the primary endpoint (N=520 patients qualified, N=3 were excluded due to missing information) demonstrated ARTIDIS' ability to detect cancer in all samples including lesions with We could separate more aggressive cases within the Luminal B subtype (CI 95%; sensitivity 83%, specificity 82%, AUC = 0.86). Secondary analysis also indicated a similarity between the majority of B3 (uncertain) and B2 (benign) lesions and distinct differences from all B5a and B5b, which could help reduce overtreatment of B3 lesions. This large prospective single center study demonstrated ARTIDIS' ability to differentiate benign from malignant lesions and to further subclassify specific breast cancer subtypes. In addition, Luminal B cancers exhibited nanomechanical profiles associated with better or worse prognosis and treatment outcome. The final analysis results provide cutoffs that will be assessed in the upcoming large multicenter prospective trial. Finally, this study helped to determine clinical utility of nanomechanical characterization of breast and other solid cancers for prediction and treatment optimization. Citation Format: Rosemarie Burian, Ahmed Jizawi, Flora Scott, Sabine Schädelin, Christian Raez, Simone Muenst-Soysal, Tatjana Vlaijnic, Ellen Obermann, Sophie Dellas, Serafino Forte, Zlatko Marušić, Tobias Appenzeller, Philipp Oertle, Nam Seung-Zln, Gregory Zaugg, Milan Liepelt, Gabriel Zihlman, Roderick Lim, Marko Loparic, Marija Plodinec. First clinical validation of the physical biomarker based on the nanomechanical tissue profiling for rapid breast cancer diagnosis, prognosis and predication of treatment outcome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-273.

Background Complete lymph node removal through conventional axillary dissection (ALND) has been standard treatment for breast cancer patients for almost a century. In the 1990s, however, and in parallel with the advent of the sentinel lymph node (SLN) procedure, ALND came under increasing scrutiny due to its association with significant patient morbidity. Several studies have since provided evidence to suggest omission of ALND, often in favor of axillary radiation, in selected clinically node-negative, SLN-positive patients, thus supporting the current trend in clinical practice. Clinically node-positive patients, by contrast, continue to undergo ALND in many cases, if only for the lack of studies re-assessing the indication for ALND in these patients. Hence, there is a need for a clinical trial to evaluate the optimal treatment for clinically node-positive breast cancer patients in terms of surgery and radiotherapy. The TAXIS trial is designed to fill this gap by examining in particular the value of tailored axillary surgery (TAS), a new technique for selectively removing positive lymph nodes. Methods In this international, multicenter, phase-III, non-inferiority, randomized controlled trial (RCT), including 34 study sites from four different countries, we plan to randomize 1500 patients to either receive TAS followed by ALND and regional nodal irradiation excluding the dissected axilla, or receive TAS followed by regional nodal irradiation including the full axilla. All patients undergo adjuvant whole-breast irradiation after breast-conserving surgery and chest-wall irradiation after mastectomy. The main objective of the trial is to test the hypothesis that treatment with TAS and axillary radiotherapy is non-inferior to ALND in terms of disease-free survival of clinically node-positive breast cancer patients in the era of effective systemic therapy and extended regional nodal irradiation. The trial was activated on 31 July 2018 and the first patient was randomized on 7 August 2018. Discussion Designed to test the hypothesis that TAS is non-inferior to ALND in terms of curing patients and preventing recurrences, yet is significantly superior in reducing patient morbidity, this trial may establish a new worldwide treatment standard in breast cancer surgery. If found to be non-inferior to standard treatment, TAS may significantly contribute to reduce morbidity in breast cancer patients by avoiding surgical overtreatment. Trial registration ClinicalTrials.gov, ID: NCT03513614. Registered on 1 May 2018. www.kofam.ch, ID: NCT03513614. Registered on 17 June 2018. EudraCT No.: 2018–000372-14. Electronic supplementary material The online version of this article (10.1186/s13063-018-3021-9) contains supplementary material, which is available to authorized users.

CD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitro and in vivo upon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially “re-educated” M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.

Zusammenfassung: Interventionelle kardiovaskuläre Eingriffe nehmen stetig zu. Die verwendeten Führungskatheter sind mit hydrophilem Material beschichtet, das Einführen wie Kontrolle der Katheter erleichtert und gleichzeitig vaskuläre Spasmen verhindert. Wir berichten von 2Fällen, bei denen die Verwendung eines ChoICE®-PT-Führungskatheters bei Koronarangiographie zu einer Embolisation dieser hydrophilen Beschichtung mit nachfolgendem Verschluss kleiner intramyokardialer Arterien führte

Adenomyoepithelioma of the breast is a rare tumor characterized by epithelial−myoepithelial differentiation, whose genetic underpinning is largely unknown. Here we show through whole-exome and targeted massively parallel sequencing analysis that whilst estrogen receptor (ER)-positive adenomyoepitheliomas display PIK3CA or AKT1 activating mutations, ER-negative adenomyoepitheliomas harbor highly recurrent codon Q61 HRAS hotspot mutations, which co-occur with PIK3CA or PIK3R1 mutations. In two- and three-dimensional cell culture models, forced expression of HRASQ61R in non-malignant ER-negative breast epithelial cells with or without a PIK3CAH1047R somatic knock-in results in transformation and the acquisition of the cardinal features of adenomyoepitheliomas, including the expression of myoepithelial markers, a reduction in E-cadherin expression, and an increase in AKT signaling. Our results demonstrate that adenomyoepitheliomas are genetically heterogeneous, and qualify mutations in HRAS, a gene whose mutations are vanishingly rare in common-type breast cancers, as likely drivers of ER-negative adenomyoepitheliomas., Adenomyoepithelioma is a rare tumor of the breast with an unknown genetic basis. Here the authors perform a genomic analysis of adenomyoepitheliomas revealing that their repertoire of somatic mutations vary according to the estrogen receptor (ER) status, and that ER-negative tumors harbor recurrent mutations in HRAS and PI3K pathway genes.

The co-inhibitory receptor programmed death 1 (PD-1) and its ligands are key regulators in a wide spectrum of immune responses and play a critical role in autoimmunity and self-tolerance as well as in cancer immunology. Emerging evidence suggests that cancer cells might use the PD-1/PD-ligand (PD-L) pathway to escape anti-tumor immunity. Based on this evidence, early phase human clinical trials targeting the PD-1/PD-L pathway are currently underway for multiple human cancers.The role of the PD-1/PD-L pathway in autoimmune disease, viral infections as well as in malignant neoplasms is discussed and an overview of the existing therapeutics as well as the results of clinical trials targeting this pathway in cancer is given.The PD-1/PD-L pathway represents an important mechanism of immune evasion for malignant neoplasms. Early clinical trials indicate effectiveness of PD-1/PD-L pathway blockade in several solid cancers. However, greater insight into the exact mechanisms by which tumors are able to evade anti-tumor immunity is needed to increase clinical effectiveness, for example by combination blockade of diverse co-inhibitory receptors.

Interaction between cancerous, non-transformed cells, and non-cellular components within the tumor microenvironment plays a key role in response to treatment. However, short-term culture or xenotransplantation of cancer specimens in immunodeficient animals results in dramatic modifications of the tumor microenvironment, thus preventing reliable assessment of compounds or biologicals of potential therapeutic relevance. We used a perfusion-based bioreactor developed for tissue engineering purposes to successfully maintain the tumor microenvironment of freshly excised breast cancer tissue obtained from 27 breast cancer patients and used this platform to test the therapeutic effect of antiestrogens as well as checkpoint-inhibitors on the cancer cells. Viability and functions of tumor and immune cells could be maintained for over 2 weeks in perfused bioreactors. Next generation sequencing authenticated cultured tissue specimens as closely matching the original clinical samples. Anti-estrogen treatment of cultured estrogen receptor positive breast cancer tissue as well as administration of pertuzumab to a Her2 positive breast cancer both had an anti-proliferative effect. Treatment with anti-programmed-death-Ligand (PD-L)-1 and anti-cytotoxic T lymphocyte-associated protein (CTLA)-4 antibodies lead to immune activation, evidenced by increased lymphocyte proliferation, increased expression of IFNγ, and decreased expression of IL10, accompanied by a massive cancer cell death in ex vivo triple negative breast cancer specimens. In the era of personalized medicine, the ex vivo culture of breast cancer tissue represents a promising approach for the pre-clinical evaluation of conventional and immune-mediated treatments and provides a platform for testing of innovative treatments.