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Objective: To assess the expression pattern of X-linked inhibitor of apoptosis protein (XIAP), a cellular stress sensor, and delineate the associated changes in the tumor immune microenvironment (TiME) for prognostic value and new therapeutic targets in inflammatory breast cancer (IBC). Methods: Immunohistochemistry was conducted to assess the spatial localization of immune subsets, XIAP, and PDL1 expression in IBC and non-inflammatory breast cancer (nIBC) pretreatment tumors (n = 142). Validation and further exploration were performed by gene expression analysis of patient tumors along with signaling studies in a co-culture model. Results: High XIAP in 37/81 IBC patients correlated significantly with high PD-L1, increased infiltration of FOXP3+ Tregs, CD163+ tumor-associated macrophages (TAMs), low CD8/CD163 ratio in both tumor stroma (TS) and invasive margins (IM), and higher CD8+ T cells and CD79α+ B cells in the IM. Gene set enrichment analysis identified cellular stress response- and inflammation-related genes along with tumor necrosis factor receptor 1 (TNFR1) expression in high-XIAP IBC tumors. Induction of TNFR1 and XIAP was observed when patient-derived SUM149 IBC cells were co-cultured with human macrophage-conditioned media simulating TAMs, further demonstrating that the TNF-α signaling pathway is a likely candidate governing TAM-induced XIAP overexpression in IBC cells. Finally, addition of Birinapant, a pan IAP antagonist, induced cell death in the pro-survival cytokine-enriched conditions. Conclusion: Using immunophenotyping and gene expression analysis in patient biospecimens along with in silico modeling and a preclinical model with a pan-IAP antagonist, this study revealed an interplay between increased TAMs, TNF-α signaling, and XIAP activation during (immune) stress in IBC. These data demonstrate the potential of IAP antagonists as immunomodulators for improving IBC therapeutic regimens.

Abstract Liver is the third most common organ for breast cancer (BC) metastasis. Two main histopathological growth patterns (HGP) exist in liver metastases (LM): desmoplastic and replacement. Although a reduced immunotherapy efficacy is reported in patients with LM, tumor-infiltrating lymphocytes (TIL) have not yet been investigated in BCLM. Here, we evaluate the distribution of the HGP and TIL in BCLM, and their association with clinicopathological variables and survival. We collect samples from surgically resected BCLM (n = 133 patients, 568 H&E sections) and post-mortem derived BCLM (n = 23 patients, 97 H&E sections). HGP is assessed as the proportion of tumor liver interface and categorized as pure-replacement (‘pure r-HGP’) or any-desmoplastic (‘any d-HGP’). We score the TIL according to LM-specific guidelines. Associations with progression-free (PFS) and overall survival (OS) are assessed using Cox regressions. We observe a higher prevalence of ‘any d-HGP’ (56%) in the surgical samples and a higher prevalence of ‘pure r-HGP’ (83%) in the post-mortem samples. In the surgical cohort, no evidence of the association between HGP and clinicopathological characteristics is observed except with the laterality of the primary tumor (p value = 0.049) and the systemic preoperative treatment before liver surgery (p value = .039). TIL is less prevalent in ‘pure r-HGP’ as compared to ‘any d-HGP’ (p value = 0.001). ‘Pure r-HGP’ predicts worse PFS (HR: 2.65; CI: (1.45–4.82); p value = 0.001) and OS (HR: 3.10; CI: (1.29–7.46); p value = 0.011) in the multivariable analyses. To conclude, we demonstrate that BCLM with a ‘pure r-HGP’ is associated with less TIL and with the worse outcome when compared with BCLM with ‘any d-HGP’. These findings suggest that HGP could be considered to refine treatment approaches.

Abstract A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule’s extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.

Inflammatory breast cancer (IBC) is a rare but aggressive subtype of breast cancer, mainly characterized using primary tumor samples. Here, using public datasets, we compared the genomic alterations in primary and metastatic samples from patients with metastatic IBC versus patients with metastatic non-IBC. We observed a higher frequency of AURKA amplification in IBC. We further showed that AURKA amplification was associated with increased AURKA mRNA expression, which we demonstrated was higher in IBC. Finally, higher protein expression of AURKA was associated with worse prognosis in patients with IBC. These findings deserve further investigation given the existence of AURKA-inhibitors.

Surgical resection can lead to prolonged survival in patients with isolated liver metastases (LM) from various primary cancers. However, there are currently no validated predictive markers to discriminate between these oligo/argometastatic patients, who will benefit from surgery, and those with diffuse metastatic behavior in whom surgery will be futile. To evaluate whether the tumor microenvironment, or histopathological growth pattern (HGP), of LM reflects the type of metastatic progression independently of the origin of the primary cancer, we analyzed a combined series of patients who underwent surgery for colorectal LM (N=263) or non-colorectal LM (N=66). HGPs of LM were scored in each patient to distinguish between desmoplastic HGP (all LM showing a complete encapsulated pattern) and non-desmoplastic HGP (at least one LM with some infiltrating-replacement component). In the entire series, 5-year overall and progression-free survival were, 44.5% and 15.5%, respectively, with no significant differences between colorectal and non-colorectal LM. In patients with desmoplastic HGP, 5-year overall and progression-free survival were 57% and 32%, respectively, as compared to 41% and 12%, respectively, in patients with non-desmoplastic-HGP (p=0.03 and 0.005). Irrespective of cancer origin and compared to traditional risk factors, desmoplastic HGP was the most significant predictor for better post-operative overall survival (adjusted HR: 0.62; 95% CI: [0.49-0.97]; p=0.035) and progression-free survival (adjusted HR: 0.61; 95% CI: [0.42-0.87], p=0.006). This suggests that the HGP of LM may represent an accurate marker that reflects the mode of metastatic behavior, independently of primary cancer type.

Background In preclinical studies, combining M9241 (a novel immunocytokine containing interleukin (IL)-12 heterodimers) with avelumab (anti-programmed death ligand 1 antibody) resulted in additive or synergistic antitumor effects. We report dose-escalation and dose-expansion results from the phase Ib JAVELIN IL-12 trial investigating M9241 plus avelumab.Methods In the dose-escalation part of JAVELIN IL-12 (NCT02994953), eligible patients had locally advanced or metastatic solid tumors; in the dose-expansion part, eligible patients had locally advanced or metastatic urothelial carcinoma (UC) that had progressed with first-line therapy. Patients received M9241 at 4, 8, 12, or 16.8 µg/kg every 4 weeks (Q4W) plus avelumab 10 mg/kg every 2 weeks (Q2W, dose levels (DLs) 1–4) or M9241 16.8 µg/kg Q4W plus avelumab 800 mg once a week for 12 weeks followed by Q2W (DL5/dose expansion). Primary endpoints for the dose-escalation part were adverse events (AEs) and dose-limiting toxicities (DLTs), and those for the dose-expansion part were confirmed best overall response (BOR) per investigator (Response Evaluation Criteria in Solid Tumors V.1.1) and safety. The dose-expansion part followed a two-stage design; 16 patients were enrolled and treated in stage 1 (single-arm part). A futility analysis based on BOR was planned to determine whether stage 2 (randomized controlled part) would be initiated.Results At data cut-off, 36 patients had received M9241 plus avelumab in the dose-escalation part. All DLs were well tolerated; one DLT occurred at DL3 (grade 3 autoimmune hepatitis). The maximum-tolerated dose was not reached, and DL5 was declared the recommended phase II dose, considering an observed drug–drug interaction at DL4. Two patients with advanced bladder cancer (DL2 and DL4) had prolonged complete responses. In the dose-expansion part, no objective responses were recorded in the 16 patients with advanced UC; the study failed to meet the criterion (≥3 confirmed objective responses) to initiate stage 2. Any-grade treatment-related AEs occurred in 15 patients (93.8%), including grade ≥3 in 8 (50.0%); no treatment-related deaths occurred. Exposures for avelumab and M9241 concentrations were within expected ranges.Conclusions M9241 plus avelumab was well tolerated at all DLs, including the dose-expansion part, with no new safety signals. However, the dose-expansion part did not meet the predefined efficacy criterion to proceed to stage 2.

Abstract Single cell technologies allow the interrogation of tumor heterogeneity, providing insights into tumor evolution and treatment resistance. To better understand whether circulating tumor cells (CTCs) could complement metastatic biopsies for tumor genomic profiling, we characterized 11 single CTCs and 10 pooled CTC samples at the mutational and copy number aberration (CNA) levels, and compared these results with matched synchronous tumor biopsies from 3 metastatic breast cancer patients with triple-negative (TNBC), HER2-positive and estrogen receptor-positive (ER+) tumors. Similar CNA profiles and the same patient-specific driver mutations were found in bulk tissue and CTCs for the HER2-positive and TNBC tumors, whereas different CNA profiles and driver mutations were identified for the ER+ tumor, which presented two distinct clones in CTCs defined by mutations in ESR1 Y537N and TP53, respectively. Furthermore, de novo mutational signatures derived from CTCs described patient-specific biological processes. These data suggest that tumor tissue and CTCs provide complementary clinically relevant information to map tumor heterogeneity and tumor evolution.

Abstract Inflammatory breast cancer (IBC) is an aggressive disease for which the spectrum of preclinical models was rather limited in the past. More recently, novel cell lines and xenografts have been developed. This study evaluates the transcriptome of an extended series of IBC preclinical models and performed a comparative analysis with patient samples to determine the extent to which the current models recapitulate the molecular characteristics of IBC observed clinically. We demonstrate that the IBC preclinical models are exclusively estrogen receptor (ER)-negative and of the basal-like subtype, which reflects to some extent the predominance of these subtypes in patient samples. The IBC-specific 79-signature we previously reported was retrained and discriminated between IBC and non-IBC preclinical models, but with a relatively high rate of false positive predictions. Further analyses of gene expression profiles revealed important roles for cell proliferation, MYC transcriptional activity, and TNFɑ/NFκB in the biology of IBC. Patterns of MYC expression and transcriptional activity were further explored in patient samples, which revealed interactions with ESR1 expression that are contrasting in IBC and nIBC and notable given the comparatively poor outcomes of ER+ IBC. Our analyses also suggest important roles for NMYC, MXD3, MAX, and MLX in shaping MYC signaling in IBC. Overall, we demonstrate that the IBC preclinical models can be used to unravel cancer cell intrinsic molecular features, and thus constitute valuable research tools. Nevertheless, the current lack of ER-positive IBC models remains a major hurdle, particularly since interactions with the ER pathway appear to be relevant for IBC.

Abstract Multiparametric assays for risk stratification are widely used in the management of both node negative and node positive hormone receptor positive invasive breast cancer. Recent data from multiple sources suggests that different tests may provide different risk estimates at the individual patient level. The TEAM pathology study consists of 3284 postmenopausal ER+ve breast cancers treated with endocrine therapy Using genes comprising the following multi-parametric tests OncotypeDx®, Prosigna™ and MammaPrint® signatures were trained to recapitulate true assay results. Patients were then classified into risk groups and survival assessed. Whilst likelihood χ 2 ratios suggested limited value for combining tests, Kaplan–Meier and LogRank tests within risk groups suggested combinations of tests provided statistically significant stratification of potential clinical value. Paradoxically whilst Prosigna-trained results stratified Oncotype-trained subgroups across low and intermediate risk categories, only intermediate risk Prosigna-trained cases were further stratified by Oncotype-trained results. Both Oncotype-trained and Prosigna-trained results further stratified MammaPrint-trained low risk cases, and MammaPrint-trained results also stratified Oncotype-trained low and intermediate risk groups but not Prosigna-trained results. Comparisons between existing multiparametric tests are challenging, and evidence on discordance between tests in risk stratification presents further dilemmas. Detailed analysis of the TEAM pathology study suggests a complex inter-relationship between test results in the same patient cohorts which requires careful evaluation regarding test utility. Further prognostic improvement appears both desirable and achievable.

Abstract Currently, there are no markers to identify patients with liver-only or liver-dominant metastases that would benefit from hepatic surgery. Here we characterized histopathological growth patterns (HGPs) of liver metastases in a consecutive series of 36 breast cancer patients who underwent hepatic surgery. Survival analyses showed that the presence of a desmoplastic HGP in the liver metastases (a rim of fibrous tissue separating cancer cells from the liver parenchyma, present in 20 (56%) patients) is independently associated with favorable progression-free and overall survival when compared with the replacement HGP (cancer cells growing into the liver parenchyma, present in 16 (44%) patients).

Abstract Background The purpose of this study was to determine the prognostic role of hormone receptor (HR) on inflammatory breast cancer (IBC) to elucidate its aggressive biological behavior. Methods We evaluated the expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemical staining and determined the predictive and prognostic role of HR expression on 189 patients with HR+/HER2– IBC and 677 patients with HR+/HER2– stage III non-IBC. Furthermore, we performed gene expression (GE) analyses on 137 patients with HR+/HER2– IBC and 252 patients with HR+/HER2– non-IBC to detect genes that are specifically overexpressed in IBC. Results The expression of ER% was significantly associated with longer distant disease-free survival and overall survival. However, there was no significant relationship between ER% and neoadjuvant chemotherapy outcome. In the GE study, 84 genes were identified as significantly distinguishing HR+ IBC from non-IBC. Among the top 15 canonical pathways expressed in IBC, the ERK/MAPK, PDGF, insulin receptor, and IL-7 signaling pathways were associated with the ER signaling pathway. Upregulation of the MYC gene was observed in three of these four pathways. Furthermore, HR+/HER2– IBC had significantly higher MYC amplification, and the genetic alteration was associated with poor survival outcome. Conclusions Higher ER expression was significantly associated with improved survival in both HR+/HER2– IBC and HR+/HER2– stage III non-IBC patients. HR+/HER2– IBC had several activated pathways with MYC upregulation, and the genetic alteration was associated with poor survival outcome. The results indicate that MYC may be a key gene for understanding the biology of HR+/HER2– IBC.

Inflammatory breast cancer (IBC) is the most pro‐metastatic form of breast cancer. Better understanding of its pathophysiology and identification of actionable genetic alterations (AGAs) are crucial to improve systemic treatment. We aimed to define the DNA profiles of IBC vs noninflammatory breast cancer (non‐IBC) clinical samples in terms of copy number alterations (CNAs), mutations, and AGAs. We applied targeted next‐generation sequencing (tNGS) and array‐comparative genomic hybridization (aCGH) to 57 IBC and 50 non‐IBC samples and pooled these data with four public datasets profiled using NGS and aCGH, leading to a total of 101 IBC and 2351 non‐IBC untreated primary tumors. The respective percentages of each molecular subtype [hormone receptor‐positive (HR+)/HER2−, HER2+, and triple‐negative] were 68%, 15%, and 17% in non‐IBC vs 25%, 35%, and 40% in IBC. The comparisons were adjusted for both the molecular subtypes and the American Joint Committee on Cancer (AJCC) stage. The 10 most frequently altered genes in IBCs were TP53 (63%), HER2/ERBB2 (30%), MYC (27%), PIK3CA (21%), BRCA2 (14%), CCND1 (13%), GATA3 (13%), NOTCH1 (12%), FGFR1 (11%), and ARID1A (10%). The tumor mutational burden was higher in IBC than in non‐IBC. We identified 96 genes with an alteration frequency (p

Summary: Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.

Background. Anti-PD1/PDL1 immune checkpoint inhibitors (ICIs) showed promising results in breast cancer, and exploration of additional actionable immune checkpoints is ongoing. Inflammatory breast cancer (IBC) is an aggressive form of disease, the immune tumor microenvironment (TME) of which is poorly known. We aimed at providing the first comprehensive immune portrait of IBCs. Methods. From the gene expression profiles of 137 IBC and 252 non-IBC clinical samples, we measured the fractions of 22 immune cell types, expression of signatures associated with tertiary lymphoid structures (TLS) and with the response to ICIs (T cell-inflamed signature: TIS) and of 18 genes coding for major actionable immune checkpoints. The IBC/non-IBC comparison was adjusted upon the clinicopathological variables. Results. The immune profiles of IBCs were heterogeneous. CIBERSORT analysis showed profiles rich in macrophages, CD8+ and CD4 + T-cells, with remarkable similarity with melanoma TME. The comparison with non-IBCs showed significant enrichment in M1 macrophages, γδ T-cells, and memory B-cells. IBCs showed higher expression of TLS and TIS signatures. The TIS signature displayed values in IBCs close to those observed in other cancers sensitive to ICIs. Two-thirds of actionable immune genes (HAVCR2/TIM3, CD27, CD70, CTLA4, ICOS, IDO1, LAG3, PDCD1, TNFRSF9, PVRIG, CD274/PDL1, and TIGIT) were overexpressed in IBCs as compared to normal breast and two-thirds were overexpressed in IBCs versus non-IBCs, with very frequent co-overexpression. For most of them, the overexpression was associated with better pathological response to chemotherapy. Conclusion. Our results suggest the potential higher vulnerability of IBC to ICIs. Clinical trials.

Background Anti-programmed cell death ligand 1 (PD-L1)/programmed cell death 1 antibodies have shown clinical activity in platinum-treated metastatic urothelial carcinoma, resulting in regulatory approval of several agents, including avelumab (anti-PD-L1). We report ≥2-year follow-up data for avelumab treatment and exploratory subgroup analyses in patients with urothelial carcinoma.Methods Patients with previously treated advanced/metastatic urothelial carcinoma, pooled from two cohorts of the phase Ib JAVELIN Solid Tumor trial, received avelumab 10 mg/kg every 2 weeks until disease progression, unacceptable toxicity or withdrawal. End points included best overall response and progression-free survival (PFS) per RECIST V.1.1, overall survival (OS) and safety. Post hoc analyses included objective response rates (ORRs) in subgroups defined by established high-risk/poor-prognosis characteristics and association between time to response and outcome.Results 249 patients received avelumab; efficacy was assessed in 242 postplatinum patients. Median follow-up was 31.9 months (range 24–43), and median treatment duration was 2.8 months (range 0.5–42.8). The confirmed ORR was 16.5% (95% CI 12.1% to 21.8%; complete response in 4.1% and partial response in 12.4%). Median duration of response was 20.5 months (95% CI 9.7 months to not estimable). Median PFS was 1.6 months (95% CI 1.4 to 2.7 months) and the 12-month PFS rate was 16.8% (95% CI 11.9% to 22.4%). Median OS was 7.0 months (95% CI 5.9 to 8.5 months) and the 24-month OS rate was 20.1% (95% CI 15.2% to 25.4%). In post hoc exploratory analyses, avelumab showed antitumor activity in high-risk subgroups, including elderly patients and those with renal insufficiency or upper tract disease; ORRs were numerically lower in patients with liver metastases or low albumin levels. Objective response achieved by 3 months versus later was associated with longer OS (median not reached (95% CI 18.9 months to not estimable) vs 7.1 months (95% CI 5.2 to 9.0 months)). Safety findings were consistent with previously reported 6-month analyses.Conclusions After ≥2 years of follow-up, avelumab showed prolonged efficacy and acceptable safety in patients with platinum-treated advanced/metastatic urothelial carcinoma, including high-risk subgroups. Survival appeared longer in patients who responded within 3 months. Long-term safety findings were consistent with earlier reports with avelumab treatment in this patient population.

Background Avelumab, a human anti–programmed death-ligand 1 immunoglobulin G1 monoclonal antibody, showed favorable efficacy and safety in patients with metastatic Merkel cell carcinoma (mMCC) in the phase II JAVELIN Merkel 200 trial, leading to approval in multiple countries. We describe real-world experience with avelumab in patients with mMCC from an expanded access program.Methods Eligible patients had mMCC and progressive disease during or after chemotherapy or were ineligible for chemotherapy or clinical trial participation. Patients received an initial 3-month supply of avelumab (administered as 10 mg/kg intravenously every 2 weeks until progressive disease or unacceptable toxicity); resupply was allowed following complete response, partial response, stable disease, or clinical benefit per physician assessment.Results Between December 15, 2015, and March 4, 2019, 558 of 620 requests from 38 countries were medically approved, and 494 patients received avelumab. Among 240 evaluable patients, the objective response rate was 46.7% (complete response in 22.9%, including 3 of 16 potentially immunocompromised patients), and the disease control rate was 71.2%. The median duration of treatment in evaluable patients with response was 7.9 months (range, 1.0–41.7) overall and 5.2 months (range, 3.0–13.9) in immunocompromised patients. No new safety signals were identified. The expanded access program closed for new requests on December 31, 2018, as required after regulatory approval; benefitting patients continued to receive avelumab.Conclusions The avelumab expanded access program for patients with mMCC demonstrated efficacy and safety in a real-world setting, consistent with the results from JAVELIN Merkel 200, and provided a treatment for patients with limited options.

Abstract Background Enadenotucirev is a chimeric adenovirus with demonstrated preclinical tumor-selective cytotoxicity and a short half-life. Further clinical mechanism of action data showed that enadenotucirev can gain access to and replicate within different types of epithelial tumors. This phase 1 dose escalation study assessed intravenous (IV) dose escalation with enadenotucirev to establish the maximum tolerated dose (MTD) and subsequently identify a suitable schedule for repeated cycles. Methods Sixty-one patients with advanced epithelial tumors unresponsive to conventional therapy were enrolled and received enadenotucirev monotherapy as part of this study. During the phase 1a dose escalation (n = 22) and expansion (n = 9), delivery of enadenotucirev between 1 × 1010 and 1 × 1013 viral particles (vp) on days 1, 3, and 5 (single cycle) was used to determine an appropriate MTD. Subsequent treatment cohorts (phase 1a, n = 6 and phase 1b, n = 24) examined the feasibility of repeated dosing cycles in either 3-weekly or weekly dosing regimens. Results Enadenotucirev displayed a predictable and manageable safety profile at doses up to the MTD of 3 × 1012 vp, irrespective of infusion time or dosing schedule. The most commonly reported treatment-emergent adverse events (TEAEs) of grade 3 or higher were hypoxia, lymphopenia, and neutropenia. The frequency of all TEAEs (notably pyrexia and chills) was highest within 24 h of the first enadenotucirev infusion and decreased upon subsequent dosing. Additionally, delivery of three doses of enadenotucirev over 5 days optimized pharmacokinetic and chemokine profiles in the circulation over time. Conclusions This study provides key clinical data in patients with solid epithelial tumors following treatment with IV enadenotucirev monotherapy and supports further investigation of enadenotucirev in combination with other therapeutic agents at doses up to the MTD of 3 × 1012 vp. Trial registration (ClinicalTrials.gov Identifier: NCT02028442). Trial registration date: 07 January 2014 – Retrospectively registered.

Abstract Background There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. Methods A combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. Results ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. Conclusions ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.

Genetics: Expression signature identifies high-risk patients A gene expression signature identifies breast cancer patients who do poorly after endocrine therapy and might benefit from extra treatment. A team led by John Bartlett and Paul Boutros from the Ontario Institute for Cancer Research in Toronto, Canada, measured the activity levels of 165 genes known to be involved in breast cancer development in tumor samples from 3825 patients with early estrogen receptor-positive disease. The patients received either endocrine therapies (tamoxifen or an aromatase inhibitor) alone or additional chemotherapy as well. The researchers identified a 95-gene expression signature that, when combined with a determination of whether the cancer has spread into the lymph nodes, can help predict which patients are at high risk of disease progression, regardless of whether they received chemotherapy or not. These patients could be prioritized for additional drug therapies.

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models.

Background PIK3CA is the most frequent somatic mutated oncogene in estrogen receptor (ER) positive breast cancer. We previously observed an association between PIK3CA genotype and aromatase inhibitors (AI) treatment outcome. This study now evaluates whether expression of mRNAs and miRs are linked to PIK3CA genotype and are independently related to AI therapy response in order to define potential expressed biomarkers for treatment outcome. Materials and methods The miR and mRNA expression levels were evaluated for their relationship with the PIK3CA genotype in two breast tumor datasets, i.e. 286 luminal cancers from the TCGA consortium and our set of 84 ER positive primary tumors of metastatic breast cancer patients who received first line AI. BRB Array tools class comparison was performed to define miRs and mRNAs whose expression associate with PIK3CA exon 9 and 20 status. Spearman correlations established miR–mRNA pairs and mRNAs with related expression. Next, a third dataset of 25 breast cancer patients receiving neo‐adjuvant letrozole was evaluated, to compare expression levels of identified miRs and mRNAs in biopsies before and after treatment. Finally, to identify potential biomarkers miR and mRNA levels were related with overall survival (OS) and progression free survival (PFS) after first‐line AI therapy. Results Expression of 3 miRs (miR‐449a, miR‐205‐5p, miR‐301a‐3p) and 9 mRNAs (CCNO, FAM81B, LRG1, NEK10, PLCL1, PGR, SERPINA3, SORBS2, VTCN1) was related to the PIK3CA status in both datasets. All except miR‐301a‐3p had an increased expression in tumors with PIK3CA mutations. Validation in a publicly available dataset showed that LRG1, PGR, and SERPINA3 levels were decreased after neo‐adjuvant AI‐treatment. Six miR–mRNA pairs correlated significantly and stepdown analysis of all 12 factors revealed 3 mRNAs (PLCL1, LRG1, FAM81B) related to PFS. Further analyses showed LRG1 and PLCL1 expression to be unrelated with luminal subtype and to associate with OS and with PFS, the latter independent from traditional predictive factors. Conclusion We showed in two datasets of ER positive and luminal breast tumors that the expression of 3 miRs and 9 mRNAs associate with the PIK3CA status. Expression of LRG1 is independent of luminal (A or B) subtype, decreased after neo‐adjuvant AI‐treatment, and is proposed as potential biomarker for AI therapy outcome.

BACKGROUND:Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categories have been described. Major questions in CTC research are how these IF categories reflect gene expression and amplification, and if we should consider 'medium' HER-2 expressing CTCs for patient selection. METHODS:Tumor cells from spiked cell lines (n = 8) and CTCs (n = 116 samples) of 85 metastatic breast cancer patients were enriched using CellSearch. Comparative analysis of HER-2 expression by IF imaging (ACCEPT, DEPArray, and visual scoring) with qRT-PCR and HER-2/neu FISH was performed. RESULTS:Automated IF HER-2-profiling by DEPArray and ACCEPT delivered comparable results. There was a 98% agreement between 17 trained observers (visual scoring) and ACCEPT considering HER-2neg and HER-2high expressing CTCs. However, 89% of HER-2med expressing CTCs by ACCEPT were scored negative by observers. HER-2high expressing tumor cells demonstrated HER-2/neu gene amplification, whereas HER-2neg and HER-2med expressing tumor cells and CTCs by ACCEPT were copy-number neutral. All patients with HER-2-positive archival tumors had ≥1 HER-2high expressing CTCs, while 80% of HER-2-negative patients did not. High relative gene expression of HER-2 measured on enriched CTC lysates correlated with having ≥1 HER-2high expressing CTCs. CONCLUSION:Automated images analysis has enormous potential for clinical implementation. HER-2 characterization and clinical trial design should be focused on HER-2high expressing CTCs.

Abstract Interpreting molecular profiles in a biological context requires specialized analysis strategies. Initially, lists of relevant genes were screened to identify enriched concepts associated with pathways or specific molecular processes. However, the shortcoming of interpreting gene lists by using predefined sets of genes has resulted in the development of novel methods that heavily rely on network-based concepts. These algorithms have the advantage that they allow a more holistic view of the signaling properties of the condition under study as well as that they are suitable for integrating different data types like gene expression, gene mutation, and even histological parameters.

The exponential use of robotic surgery is not the result of evidence-based benefits but mainly driven by the manufacturers, patients and enthusiastic surgeons. The present review of the literature shows that robot-assisted surgery is consistently more expensive than video-laparoscopy and in many cases open surgery. The average additional variable cost for gynecological procedures was about 1600 USD, rising to more than 3000 USD when the amortized cost of the robot itself was included. Generally most robotic and laparoscopic procedures have less short-term morbidity, blood loss, intensive care unit, and hospital stay than open surgery. Up to now no major consistent differences have been found between robot-assisted and classic video-assisted procedures for these factors. No comparative data are available on long-term morbidity and oncologic outcome after open, robotic, and laparoscopic gynecologic surgery. It seems that currently only for very complex surgical procedures, such as cardiac surgery, the costs of robotics can be competitive to open surgical procedures. In order to stay viable, robotic programs will need to pay for themselves on a per case basis and the costs of robotic surgery will have to be reduced.