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Background: Liver metastases (LM) are ultimately present in ~50% of all patients with metastatic breast cancer (BC). Metastatic seeding to the liver relies on the interaction between cancer cells and the host microenvironment, resulting into two main histopathological growth patterns (HGPs): the replacement HGP (metastasis mimics the liver architecture and exploits liver vasculature) and the desmoplastic HGP (presence of a fibrotic rim separating the hepatocytes from the tumor cells). While the prognostic value of these HGPs is established for colorectal cancer, with the desmoplastic HGP being associated with better prognosis, investigation is needed for BC. It has also been reported that patients with LM have a poorer response to immune checkpoints inhibitors. A systematic evaluation of the HGP and LM-associated immune infiltrates (stromal tumor infiltrating lymphocytes, sTIL) is currently lacking. In this study, we aimed at: (i) investigating HGPs and sTIL in LM from patients with BC, and, (ii) evaluating the association of these HGPs and sTIL with standard variables and outcome. Patients and methods: The study currently includes clinical data and samples from: 1) a retrospective cohort of 122 patients from 7 hospitals with surgically resected LMs (represented by 548 hematoxylin and eosin (H&E)-sections), further referred to as ‘surgical cohort’ and, 2) LMs from 2 institutional post-mortem tissue donation studies for a total of 23 patients (97 H&E-sections). All available H&E-sections were used to assess HGP and sTIL. HGPs were scored according to a standardized method (PMIDs: 35650276) and categorized per patient as pure-replacement (rHGP, i.e. 100% of the tumor-liver interface is replacement) or any-desmoplastic (dHGP, i.e. at least 1% of the tumor-liver interface is desmoplastic). sTIL are expressed as the percentage of stromal area covered by mononuclear immune cells at the metastasis-liver interface. Associations were assessed using Fisher exact and Wilcoxon tests. Univariable and multivariable Cox regression analyses stratified by center were used to evaluate the role of HGP on progression-free (PFS) and overall survival (OS). Results: In the surgical cohort, 54 (44%) of patients displayed a rHGP and 68 (56%) a dHGP. Intra-patient, meaning inter-slide, heterogeneity of the HGP was observed in 24/122 (20%) of the patients suggesting that scoring multiple slides is needed for accurate assessment of the HGPs. We did not find any statistically significant association between HGP and clinico-pathological data. Higher sTIL were associated with dHGP (p=.003), as well as with a higher histological grade (p= .087), estrogen receptor-positivity (p=0.062) and ductal histology (p= .08) of the primary tumor. rHGP was associated with worse PFS and OS, both at the univariable and multivariable level (Table). In the post-mortem cohort, we observed a higher frequency of rHGP patients (19/23 patients, 83 %) and significantly lower levels of sTIL in the rHGP patients as compared to the rHGP patients from the surgical cohort (p= .009). Conclusion: This study represents the largest study evaluating HGPs and immune infiltrates of LMs from patients with BC. Approximately half of the surgically resected LMs have a dHGP, which is associated with higher sTIL and a better prognosis. The results from the LMs from the post-mortem cohort suggest that a more advanced stage of the disease is associated with an increase in rHGP and with a more immunosuppressed environment. Table 1: Univariable and multivariable analyses Citation Format: Sophia Leduc, Maxim De Schepper, Peter Vermeulen, Giuseppe Floris, Elia Biganzoli, Vincent Donckier, Ali Bohlok, Marco Gerling, François Richard, Marion Maetens, Joris Jaekers, Baki Topal, Emily Latacz, Karen Van Baelen, Tatjana Geukens, Ha Linh Nguyen, Luc Dirix, Denis Larsimont, Sophie Van Kerckhove, Rui Caetano Oliveira, Janina Kulka, Valerio Lucidi, Yannick Meyer, Cornelis Verhoef, Eva Santos, Ferenc Salamon, Lilla Madaras, A. Marcell Szasz, Székely Borbála, Kristòf Dede, Jennie Engstrand, Carlos Fernandez Moro, Christine Desmedt. Histopathological and immune characterization of liver metastases from patients with breast cancer [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 P6-14-06.

Background: Women with HR+ breast cancer experience a persistent risk of distant recurrence (DR) even after completion of 5 years of adjuvant endocrine therapy, with more than 50% of DR occurring after 5 years (late DR). The prognostic genomic signatures currently being used in the clinic were not developed or optimized specifically for late DR. We have previously shown that the Breast Cancer Index (BCI) and BCIN+ prognostic models were significantly prognostic for risk of overall (0-10y) and late (5-10y) distant recurrence (DR) in N0 and N1 HR+ patients in the Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial. Here, the prognostic performance of the BCI and BCIN+ models with alternative cut-points optimized for late DR were evaluated in patients from the TEAM trial, who were free from DR for at least 5 years. Methods: BCI testing was performed blinded to clinical outcome. The pre-specified alternative cut-points 4.4 and 1.8 for BCI and BCIN+ models were determined previously from Trans-aTTom and IDEAL studies, respectively (ESMO 2021). Kaplan-Meier analysis and log-rank test were used to evaluate the prognostic significance of BCI/BCIN+ risk groups based on DR. Univariate and multivariate Cox models were used to estimate hazard ratios (HRs) and the associated 95% confidence intervals (CIs). Results: 1285 HR+ N0 (median age 69.2, 54.2% T1, 92.5% G2-3, 21.3% chemotherapy) and 1762 N1 (median age 68.5, 49.7% T1, 80.8% G2-3, 42.6% chemotherapy) patients who remained free from DR at 5 years post randomization were included in the current analysis. For N0 patients, BCI identified 439 (34%) and 846 (66%) patients as low and high-risk with late 10-year DR rates of 3.8% (95% CI: 1.5-6.0%) and 9.1% (95% CI: 6.8-11.4%), respectively (HR: 2.6, 95% CI: 1.4-5.0; p=0.0025). For N1 patients, BCIN+ identified 287 (16%) and 1475 (84%) patients as low and high-risk with late 10-year DR rates of 3.4% (95% CI: 1.2-5.5%) and 12.3% (95% CI: 10.4-14.2%), respectively (HR: 3.5, 95% CI: 1.8-6.9; p< 0.0001). Similar results were observed in the HER2- patients. Notably, BCI/BCIN+ remained a statistically significant prognostic factor in the multivariate analysis after controlling for age, tumor size, grade, treatment. (Table). Conclusions: Compared to the original BCI/BCIN+ models, the optimized BCI and BCIN+ models showed improved prognostic performance for identifying low-risk patients with a very low risk of late DR (< 4%), for both N0 and N1 patients. These results provide further validation of BCI clinical utility as an aid in the decision-making for extended endocrine therapies for HR+ breast cancer, particularly in patients with N1 disease that may be spared extended endocrine treatment. Table Citation Format: John MS Bartlett, Keying Xu, Jenna Wong, Gregory R. Pond, Yi Zhang, Melanie Spears, Ranelle Salunga, Elizabeth Mallon, Karen J. Taylor, Annette Hasenburg, Christos Markopoulos, Luc Dirix, Caroline Seynaeve, Cornelis J.H. van de Velde, Daniel Rea, Catherine A. Schnabel, Kai Treuner, Jane Bayani. Validation of the Breast Cancer Index (BCI) prognostic models optimized for late distant recurrence in postmenopausal women with early-stage HR+ breast cancer in the TEAM trial [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 P2-11-10.

Background: Invasive lobular carcinoma (ILC) represents up to 15% of all breast carcinomas. The majority of ILC express the estrogen receptor (ER) and have no amplification/overexpression of the human epidermal growth factor receptor 2 (HER2). A high body mass index (BMI) has been associated with an increased risk of developing ILC in postmenopausal women, similar to what is seen for breast cancer of no special type (NST). It is however unknown if BMI impacts the clinicopathological features and the prognosis of ILC. Methods: We performed a multicentric retrospective study in 5 European centers of patients diagnosed between January 2000 and December 2020 with ER+/HER2- non-metastatic pure (i.e., not mixed) ILC. Patient and tumor characteristics and event-related data were collected. BMI was categorized into underweight (≤18.5kg/m2), lean (>18.5kg/m2 and < 25kg/m2), overweight (≥25kg/m2 and < 30kg/m2) and obese (≥30kg/m2). The association of BMI as either a continuous or a categorical variable with clinicopathological variables was assessed using linear regression or ordinal logistic regression, respectively. Median follow-up was calculated using the reverse Kaplan-Meier estimator. Survival analyses using univariable (stratified by center) and multivariable (adjusted for all included variables and stratified by center) Cox regression were performed to evaluate the association of BMI with disease free survival (DFS), distant recurrence free survival (DRFS) and overall survival (OS). DFS and DRFS were analyzed in the presence of death without event as the competing risk. Results: The data of 2476 patients were collected and BMI was available for 2346 patients. In total, 1299 (55%) patients were lean, 638 (27%) overweight and 339 (14%) obese. Underweight patients only represented 3% of all patients and were thus excluded from further analyses. A higher age at diagnosis, higher grade, larger tumor size, nodal involvement and multifocality were significantly associated with higher BMI (Table 1). The median follow-up was 8,5 years (interquartile range 59.24 – 142.13 months). In univariable analysis, higher BMI was associated with worse survival outcomes (Table 2). However, this association was not seen in multivariable analysis while grade, tumor size and nodal involvement were still prognostic for all endpoints. Similar results were seen with BMI as a continuous variable. Conclusion: Larger tumors and nodal involvement were more likely to be found in patients with ER+/HER2- ILC with higher BMI which might be explained by a delayed diagnosis in these patients. Higher grade also seemed to be associated with higher BMI. In multivariable analyses, BMI was not found to be an independent prognostic factor. Tumor grade, tumor size, and nodal status remained strongly prognostic for survival outcomes in multivariable survival analyses which is consistent with their known prognostic importance in luminal tumors. We hypothesize that the prognostic effect of BMI is mediated through these variables for patients with ER+/HER2- ILC. Table 1. Association of clinicopathological features of ER+/HER2- ILC with categorical BMI. Table 2. Association of categorical BMI and other clinicopathological features of ER+/HER2- ILC with survival. Citation Format: Karen Van Baelen, Ha-Linh Nguyen, François Richard, Anne-Sophie Hamy, Aullène Toussaint, Fabien Reyal, Anne Salomon, Luc Dirix, Peter Vermeulen, Hilde Wuyts, Maria Karsten, Adam D. Dordevic, Guilherme Nader Marta, Evandro de Azambuja, Christos Sotiriou, Denis Larsimont, Ottavia Amato, Marion Maetens, Maxim De Schepper, Tatjana Geukens, Sileny Han, Thaïs Baert, Kevin Punie, Hans Wildiers, Chantal Remmerie, Ann Smeets, Ines Nevelsteen, Giuseppe Floris, Elia Biganzoli, Patrick Neven, Christine Desmedt. Association of body mass index with clinicopathological features and survival in patients with primary ER+/HER2- invasive lobular breast cancer [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 P3-05-40.

Background. Liquid biopsies have been studied as an accessible method to capture spatial tumour heterogeneity and repeatedly evaluate targetable aberrations in cancer. Here, we analysed pure single and pools of circulating and disseminated tumour cells (CTC and DTC) to study genetic heterogeneity. Methods. From, three patients with metastatic breast cancer, CTC and DTC (bone marrow, pleural fluid, and cerebrospinal fluid) were CellSearch® enriched and DEPArray® sorted into 136 samples of singles and pools up to 150 cells. Sorted cells, primary tumour, circulating free (cf)DNA, and enriched CTC/DTC fractions were analysed for mutations and copy number alterations (CNA). Results. Two patients harboured a driver PIK3CA mutation in all samples. Variant allele frequencies matched the ploidy status calculated from copy number data. The majority of CNA were homogeneously present in most samples of each patient, including breast cancer subtype specific CNA. One patient with rapidly progressive disease harboured additional CNA in the CTC/DTC compartment, reflecting this aggressive course and a possible subtype switch. These clones emerged at distinct timepoints in the different compartments. Furthermore, various mutations were present in unique samples. Conclusion. Repeated tumour analysis with liquid biopsies unveils changing molecular characteristics over time, and even a difference between simultaneous primary and metastatic disease. We show that all major subclones can be captured by combined sequencing of enriched CTC and cfDNA at disease progression.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) & gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P< 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P< 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

Background:. Inflammatory breast cancer (IBC) represents a rare (~1-5% of all breast cancers, BC) and particularly aggressive type of BC, accounting for roughly 10% of BC-related mortality annually. So far IBC has been mainly characterized at the genomic level using samples from the primary tumor. Here, using publicly available data from two large cohorts, we compared the genomic alterations in primary and metastatic samples from patients with metastatic IBC to alterations in samples of patients with metastatic non-IBC. Patients and methods:. We retrieved publicly available clinical and genomic data from primary (P) and metastatic (M) samples from MSK-IMPACT (MSK) and the Metastatic Breast Cancer project (MBC) from cbioportal. IBC was defined as cT4d. We considered female metastatic patients with invasive BC of no special type (commonly referred to as invasive ductal BC): 34 patients with IBC and 602 with non-IBC. To focus on alterations with potential clinical relevance, we only retained the genomic alterations annotated as oncogenic by OncoKB. We applied Firth logistic regression adjusted for hormonal receptor status (positive vs negative), HER2 status (positive vs negative) stage (II vs I, III vs I, IV vs I) and cohort (MSK vs MBC) with genomic alterations or metastatic sites as outcome variables and IBC status as independent variable (yes vs no). A minimum of 5 patients harboring the alterations were required to perform the analysis. Chromosomal instability (CIN) score and Tumor Mutational Burden (TMB) were investigated by linear regression and quantile regression respectively, adjusted as described above and considering the median for the latest. P and M samples were analyzed separately, a patient was considered as ‘having’ the alteration if at least one of her samples was altered. All analyses were performed in R 4.0.2. Results:. Metastatic samples from chest wall and skin metastases were significantly more represented in patients with IBC as compared to patients with non-IBC (Adjusted Odds Ratio (ORadj): 6.16, 95% confidence interval (95CI): [1.94-18.38], p-value: .003 and ORadj: 7.60, 95CI: [1.66-30.05], p-value: .012 respectively), which is in line with the course of the IBC disease. Several studies have suggested IBC to have a higher TMB than non-IBC. Here we confirmed an increase in TMB in P and M IBC (Adjusted coefficient (coefadj): 3.00, 95CI: [1.60-4.20], p-value: .003 and coefadj: 4.00, 95CI: [0.85-4.15], p-value Table 1.Patients with primary samples. (279 patients with clinical information available - 279 samples)Patients with metastatic samples. (311 patients with clinical information available - 339 samples)AlterationAlteration countOR [95CI]p-valueAlteration countOR [95CI]p-valueTP53 mutation4120.18 [4.95-96.38] Citation Format: François Richard, Maxim De Schepper, Marion Maetens, Sophia Leduc, Edoardo Isnaldi, Tatjana Geukens, Karen Van Baelen, Ha-Linh Nguyen, Peter Vermeulen, Steven Van Laere, Luc Dirix, Giuseppe Floris, Elia Biganzoli, Christine Desmedt. Comparison of the genomic alterations in metastatic inflammatory and non-inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-08-07.

Supplementary Figures PDF file - 529K, Supplementary Figure 1: Application of the Gaussian mixture model to ESR1- and ERBB2-expression data Supplementary Figure 2: Normalization Supplementary Figure 3: Silhouette Analysis Supplementary Figure 4: Silhouette Analysis (P-Values) Supplementary Figure 5: Validation of Limma-corrected IBC/nIBC signature Supplementary Figure 6: Networks overrepresented in IBC Supplementary Figure 7: Networks overrepresented in nIBC Supplementary Figure 8: Prognostic value of the IBC/nIBC signature in nIBC

PDF file - 45K, Summary of adverse events suspected to be study-drug related by grade (safety set).

Purpose:Report results of the phase Ib dose-escalation/expansion study of triplet therapy with cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor (ribociclib), mTOR inhibitor (everolimus), and endocrine therapy (exemestane).Patients and Methods:Postmenopausal women with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−), pretreated, advanced breast cancer (ABC) were enrolled. The primary objective of the dose-escalation phase was to estimate the MTD and recommended phase II dose (RP2D) of triplet therapy through evaluation of the incidence of dose-limiting toxicities. Safety, tolerability, and efficacy of the RP2D were evaluated in the dose-expansion phase in patients naïve or refractory to CDK4/6 inhibitor therapy.Results:Patients (N = 116) received triplet therapy (n = 83 in the dose-escalation phase; n = 33 in the dose-expansion phase). A dose-dependent drug–drug interaction was observed for everolimus, with exposure increasing two- to fourfold in the presence of ribociclib. The RP2D was determined to be ribociclib 300 mg once daily, 3 weeks on/1 week off in a 4-week cycle, plus everolimus 2.5 mg once daily, plus exemestane 25 mg once daily taken with food. The safety profile was consistent with the known profiles of the combination partners, and preliminary evidence of antitumor activity was observed. Higher ESR1 gene expression trended with better treatment response to triplet therapy; higher gene expression of MAPK pathway genes trended with worse treatment response.Conclusions:Triplet therapy with endocrine therapy and mTOR and CDK4/6 inhibition provides clinical benefit and an acceptable safety profile in previously treated postmenopausal women with HR+, HER2− ABC.

Supplementary Data from Relapse-Free Survival in Breast Cancer Patients Is Associated with a Gene Expression Signature Characteristic for Inflammatory Breast Cancer

Supplementary Data File and Legend PDF file - 113K, Supplementary Data File and legend

Supplementary Information

Purpose: This phase I/II study evaluated safety, efficacy, and pharmacokinetics of escalating, multiple doses of siltuximab, a chimeric anti-interleukin (IL)-6 monoclonal antibody derived from a new Chinese hamster ovary (CHO) cell line in patients with advanced/refractory solid tumors.Experimental Design: In the phase I dose-escalation cohorts, 20 patients with advanced/refractory solid tumors received siltuximab 2.8 or 5.5 mg/kg every 2 weeks or 11 or 15 mg/kg every 3 weeks intravenously (i.v.). In the phase I expansion (n = 24) and phase II cohorts (n = 40), patients with Kirsten rat sarcoma-2 (KRAS)-mutant tumors, ovarian, pancreatic, or anti-EGF receptor (EGFR) refractory/resistant non–small cell lung cancer (NSCLC), colorectal, or H&N cancer received 15 mg/kg every 3 weeks. The phase II primary efficacy endpoint was complete response, partial response, or stable disease >6 weeks.Results: Eighty-four patients (35 colorectal, 29 ovarian, 9 pancreatic, and 11 other) received a median of three (range, 1–45) cycles. One dose-limiting toxicity occurred at 5.5 mg/kg. Common grade ≥3 adverse events were hepatic function abnormalities (15%), physical health deterioration (12%), and fatigue (11%). Ten percent of patients had siltuximab-related grade ≥3 adverse events. Neutropenia (4%) was the only possibly related adverse event grade ≥3 reported in >1 patient. Serious adverse events were reported in 42%; most were related to underlying disease. The pharmacokinetic profile of CHO-derived siltuximab appears similar to the previous cell line. No objective responses occurred; 5 of 84 patients had stable disease >6 weeks. Hemoglobin increased ≥1.5 g/dL in 33 of 47 patients. At 11 and 15 mg/kg, completely sustained C-reactive protein suppression was observed.Conclusions: Siltuximab monotherapy appears to be well tolerated but without clinical activity in solid tumors, including ovarian and KRAS-mutant cancers. The recommended phase II doses were 11 and 15 mg/kg every 3 weeks. Clin Cancer Res; 20(8); 2192–204. ©2014 AACR.

PDF file - 99KB, Supplementary Methods: The supplemental material contains additional detail on the pharmacokinetic and pharmacodynamic methods used during the study as well as a summary table of safety events and a figure detailing patient disposition. Supplemental Table 1: Summary of safety events in all study cohorts.

Supplementary Figure from Assessing CSF ctDNA to Improve Diagnostic Accuracy and Therapeutic Monitoring in Breast Cancer Leptomeningeal Metastasis

Purpose:Patients with advanced urothelial carcinoma who fail platinum-containing chemotherapy (treatment fails) have a poor prognosis and limited treatment options. Recent approvals of immune-checkpoint inhibitors confirmed the value of immunomodulatory therapy in urothelial carcinoma. Tremelimumab is a selective human immunoglobulin G2 (IgG2) monoclonal antibody against cytotoxic T-lymphocyte–associated antigen 4 with demonstrated durable response rate in metastatic melanoma. This is the first study to report the efficacy and safety of tremelimumab in urothelial carcinoma.Patients and Methods:We report the results of the urothelial carcinoma cohort from a phase II, open-label, multicenter study of patients with advanced solid tumors (NCT02527434). Patients with locally advanced/metastatic urothelial carcinoma were treated with tremelimumab monotherapy (750 mg via intravenous infusion every 4 weeks for seven cycles, then every 12 weeks for two additional cycles) for up to 12 months or until disease progression, initiation of other anticancer therapy, unacceptable toxicity, or consent withdrawal.Results:In 32 evaluable patients with metastatic urothelial carcinoma, objective response rate was 18.8% (95% confidence interval, 7.2–36.4), including complete response (CR) in 2 (6.3%), and partial response in 4 patients (12.5%). Median duration of response has not been reached. Stable disease of ≥12 months was reported in 1 patient (3.1%), yielding a disease control rate at 12 months of 21.9%. Overall, tremelimumab was generally well tolerated; safety results were consistent with the known safety profile.Conclusions:Tremelimumab monotherapy demonstrated clinical activity and durable responses in patients with metastatic urothelial carcinoma. This study is the first in which CR has been observed with tremelimumab as a single agent in urothelial carcinoma.

Supplementary Tables XLS file - 236K, Supplementary Table 1: Comparison of ER- and ErbB2-status determined using gene expression profiling and immunohistochemistry Supplementary Table 2: Validation of the algorithm to identify transcription factor activation Supplementary Table 3: Description of the public nIBC data sets Supplementary Table 4: Molecular data of IBC and nIBC samples Supplementary Table 5: List of 491 probe sets differentially expressed between IBC and nIBC samples in a molecular subtype-independent manner Supplementary Table 6: IPA analysis of the list of 443 unique genes differentially expressed between IBC and nIBC Supplementary Table 7: Networks identified by IPA analysis of the list of 443 unique genes differentially expressed between IBC and nIBC Supplementary Table 8: Uni- and multivariate analyses for DMFS in the public pooled nIBC data set

Supplementary Data from Assessing CSF ctDNA to Improve Diagnostic Accuracy and Therapeutic Monitoring in Breast Cancer Leptomeningeal Metastasis

Supplementary Fig 2

PDF file - 67K, Bayesian logistic regression model: dose-response curve and model inference results at the time of determination of the recommended Phase II dose.

PDF file - 65K, Criteria for defining dose-limiting toxicities.

Purpose:Cerebrospinal fluid (CSF) cytology is the gold standard diagnostic test for breast cancer leptomeningeal metastasis (BCLM), but has impaired sensitivity, often necessitating repeated lumbar puncture to confirm or refute diagnosis. Further, there is no quantitative response tool to assess response or progression during BCLM treatment.Experimental Design:Facing the challenge of working with small-volume samples and the lack of common recurrent mutations in breast cancers, cell-free DNA was extracted from the CSF and plasma of patients undergoing investigation for BCLM (n = 30). ctDNA fraction was assessed by ultra-low-pass whole genome sequencing (ulpWGS), which does not require prior tumor sequencing.Results:In this proof-of-concept study, ctDNA was detected (fraction ≥0.10) in the CSF of all 24 patients with BCLM+ (median ctDNA fraction, 0.57), regardless of negative cytology or borderline MRI imaging, whereas CSF ctDNA was not detected in the six patients with BCLM− (median ctDNA fraction 0.03, P < 0.0001). Plasma ctDNA was only detected in patients with extracranial disease progression or who had previously received whole brain radiotherapy. ctDNA fraction was highly concordant with mutant allele fraction measured by tumor mutation-specific ddPCR assays (r = 0.852; P < 0.0001). During intrathecal treatment, serial monitoring (n = 12 patients) showed that suppression of CSF ctDNA fraction was associated with longer BCLM survival (P = 0.034), and rising ctDNA fraction was detectable up to 12 weeks before clinical progression.Conclusions:Measuring ctDNA fraction by ulpWGS is a quantitative marker demonstrating potential for timely and accurate BCLM diagnosis and therapy response monitoring, with the ultimate aim to improve management of this poor-prognosis patient group.

Purpose: We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC).Experimental Design: RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples. Samples were classified as IBC-like or nIBC-like. The IBC signature classification was compared with the classifications according to other prognostically relevant gene signatures and clinicopathologic variables. In addition, relapse-free survival (RFS) was compared by the Kaplan-Meyer method.Results: Classification according to the IBC signature is significantly (P < 0.05) associated with the cell-of-origin subtypes, the wound healing response, the invasive gene signature, the genomic grade index, the fibroblastic neoplasm signature, and the 70-gene prognostic signature. Significant associations (P < 0.01) were found between the IBC signature and tumor grade, estrogen receptor status, ErbB2 status, and patient age at diagnosis. Patients with an IBC-like phenotype show a significantly shorter RFS interval (P < 0.05). Oncomine analysis identified cell motility as an important concept linked with the IBC signature.Conclusions: We show that nIBC carcinomas having an IBC-like phenotype have a reduced RFS interval. This suggests that IBC and nIBC show comparable phenotypic traits, for example augmented cell motility, with respect to aggressive tumor cell behavior. This observation lends credit to the use of IBC to study aggressive tumor cell behavior.

Purpose: Phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway activation in patients with HER2-positive (HER2+) breast cancer has been implicated in de novo and acquired trastuzumab resistance. The purpose of this study was to determine the clinical activity of the PI3K inhibitor buparlisib (BKM120) in patients with HER2+ advanced/metastatic breast cancer resistant to trastuzumab-based therapy.Experimental Design: In the dose-escalation portion of this phase I/II study, patients with trastuzumab-resistant locally advanced or metastatic HER2+ breast cancer were treated with daily oral doses of buparlisib and weekly intravenous trastuzumab (2 mg/kg). Dose escalation was guided by a Bayesian logistic regression model with overdose control.Results: Of 18 enrolled patients, 17 received buparlisib. One dose-limiting toxicity of grade 3 general weakness was reported at the 100-mg/day dose level (the single-agent maximum tolerated dose) and this dose level was declared the recommended phase II dose (RP2D) of buparlisib in combination with trastuzumab. Common (>25%) adverse events included rash (39%), hyperglycemia (33%), and diarrhea (28%). The pharmacokinetic profile of buparlisib was not affected by its combination with trastuzumab. At the RP2D, there were two (17%) partial responses, 7 (58%) patients had stable disease (≥6 weeks), and the disease control rate was 75%. Pharmacodynamic studies showed inhibition of the PI3K/AKT/mTOR and RAS/MEK/ERK pathways.Conclusions: In this patient population, the combination of buparlisib and trastuzumab was well tolerated, and preliminary signs of clinical activity were observed. The phase II portion of this study will further explore the safety and efficacy of this combination at the RP2D. Clin Cancer Res; 20(7); 1935–45. ©2014 AACR.

Background: Inflammatory breast cancer (IBC) is a poorly characterized form of breast cancer. So far, the results of expression profiling in IBC are inconclusive due to various reasons including limited sample size. Here, we present the integration of three Affymetrix expression datasets collected through the World IBC Consortium allowing us to interrogate the molecular profile of IBC using the largest series of IBC samples ever reported.Experimental Design: Affymetrix profiles (HGU133-series) from 137 patients with IBC and 252 patients with non-IBC (nIBC) were analyzed using unsupervised and supervised techniques. Samples were classified according to the molecular subtypes using the PAM50-algorithm. Regression models were used to delineate IBC-specific and molecular subtype-independent changes in gene expression, pathway, and transcription factor activation.Results: Four robust IBC-sample clusters were identified, associated with the different molecular subtypes (P < 0.001), all of which were identified in IBC with a similar prevalence as in nIBC, except for the luminal A subtype (19% vs. 42%; P < 0.001) and the HER2-enriched subtype (22% vs. 9%; P < 0.001). Supervised analysis identified and validated an IBC-specific, molecular subtype-independent 79-gene signature, which held independent prognostic value in a series of 871 nIBCs. Functional analysis revealed attenuated TGF-β signaling in IBC.Conclusion: We show that IBC is transcriptionally heterogeneous and that all molecular subtypes described in nIBC are detectable in IBC, albeit with a different frequency. The molecular profile of IBC, bearing molecular traits of aggressive breast tumor biology, shows attenuation of TGF-β signaling, potentially explaining the metastatic potential of IBC tumor cells in an unexpected manner. Clin Cancer Res; 19(17); 4685–96. ©2013 AACR.

Supplementary Appendix

PDF file - 25KB, Supplementary Figure S1. Patient disposition: AE, adverse event. KRAS, Kirsten rat sarcoma-2. PD, progressive disease. q2w, every 2 weeks. q3w, every 3 weeks.