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Supplementary Figure from First-In-Human Phase I Study of the OX40 Agonist MOXR0916 in Patients with Advanced Solid Tumors

Supplementary Table from First-In-Human Phase I Study of the OX40 Agonist MOXR0916 in Patients with Advanced Solid Tumors

Purpose:OX40, a receptor transiently expressed by T cells upon antigen recognition, is associated with costimulation of effector T cells and impairment of regulatory T-cell function. This first-in-human study evaluated MOXR0916, a humanized effector-competent agonist IgG1 monoclonal anti-OX40 antibody.Patients and Methods:Eligible patients with locally advanced or metastatic refractory solid tumors were treated with MOXR0916 intravenously once every 3 weeks (Q3W). A 3+3 dose-escalation stage (0.2–1,200 mg; n = 34) was followed by expansion cohorts at 300 mg (n = 138) for patients with melanoma, renal cell carcinoma, non–small cell lung carcinoma, urothelial carcinoma, and triple-negative breast cancer.Results:MOXR0916 was well tolerated with no dose-limiting toxicities observed. An MTD was not reached. Most patients (95%) experienced at least one adverse event (AE); 56% of AEs, mostly grade 1–2, were related to MOXR0916. Most common treatment-related AEs included fatigue (17%), diarrhea (8%), myalgia (7%), nausea (6%), decreased appetite (6%), and infusion-related reaction (5%). Pharmacokinetic (PK) parameters were dose proportional between 80 and 1,200 mg and supported Q3W administration. The recommended expansion dose based on PK and OX40 receptor saturation was 300 mg Q3W. Immune activation and upregulation of PD-L1 was observed in a subset of paired tumor biopsies. One renal cell carcinoma patient experienced a confirmed partial response. Overall, 33% of patients achieved stable disease.Conclusions:Although objective responses were rarely observed with MOXR0916 monotherapy, the favorable safety profile and evidence of tumor immune activation in a subset of patients support further investigation in combination with complementary agents such as PD-1/PD-L1 antagonists.

Supplementary Data from First-In-Human Phase I Study of the OX40 Agonist MOXR0916 in Patients with Advanced Solid Tumors

Background T-DXd is a novel antibody-drug conjugate composed of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and membrane-permeable topoisomerase I inhibitor payload. In a phase 2 study in patients (pts) with HER2+ (IHC 3+ or IHC 2+/ISH+), unresectable or metastatic breast cancer (MBC) previously treated with trastuzumab emtansine (T-DM1), the confirmed ORR (cORR) with T-DXd was 60.9% and median PFS (mPFS) was 16.4 mo (Modi NEJM 2020). In pts with HER2-low (IHC 2+/ISH−, IHC 1+) advanced BC, cORR was 37.0% and mPFS 11.1 mo in a phase 1 study (Modi J Clin Oncol 2020). Recently, T-DXd was approved for the treatment of adult pts with HER2+, unresectable or MBC who have received ≥ 2 prior anti-HER2-based regimens (US) or had prior chemotherapy and are refractory to or intolerant of standard treatments (Japan). In preclinical models, T-DXd combined with an anti-PD-1 antibody had greater efficacy than either agent alone (Iwata Mol Cancer Ther 2018). We conducted a phase 1b, open-label, multicenter, 2-part study of T-DXd in combination with nivolumab (Nivo) in pts with HER2-expressing (by centrally testing) MBC or advanced urothelial cancer (DS8201-A-U105; NCT03523572); interim results for the BC cohorts are presented. Methods Pts were aged ≥18 y and immune checkpoint inhibitor naive. Pts were enrolled in the United States and Europe. In part 1 (dose escalation), pts received T-DXd 3.2 or 5.4 mg/kg intravenously (IV) every 3 weeks (q3w) and Nivo 360 mg IV q3w to determine the recommended dose for expansion (RDE). In part 2, the RDE was given to 2 MBC cohorts: HER2+ (IHC 3+ or IHC 2+/ISH+) disease that progressed on prior T-DM1 or HER2 low (IHC 1+ or IHC 2+/ISH−) disease that progressed on prior standard treatments. The primary efficacy endpoint is cORR by independent central review (ICR) per RECIST version 1.1 in part 2. Additional endpoints include duration of response (DOR), disease control rate (DCR), PFS, OS, safety, and pharmacokinetics. Results 52 pts with MBC were enrolled. In part 1, 4 pts received T-DXd 3.2 mg/kg (HER2+, n=3; HER2 low, n=1), 3 pts received 5.4 mg/kg (all HER2+). In part 2, 45 pts received the RDE of T-DXd 5.4 mg/kg and Nivo 360 mg (HER2+, n=29; HER2 low, n=16 [13 HR+]); median follow-up time was 7.0 and 6.9 mo, respectively. All pts (n=48) who received RDE were female; median duration of follow-up was 6.9 mo. HER2+ pts had a median age of 55 y and median of 5 prior lines of metastatic/locally advanced therapy. At data cutoff (June 8, 2020), 56.3% of pts remained on treatment (median treatment duration: T-DXd, 6.5 mo; Nivo, 5.2 mo). HER2 low pts had a median age of 47 y, median of 4 prior lines, and 50.0% remained on treatment (median: T-DXd, 6.3 mo; Nivo, 4.9 mo). In pts treated at RDE, the cORR by ICR in the HER2+ cohort was 59.4% (19/32, 18 PR) and for the HER2 low cohort was 37.5% (6/16, all PR). The DCR was 90.6% and 75.0% in the HER2+ and HER2-low cohorts, respectively. Median DOR was not reached in either cohort; median PFS was 8.6 mo (95% CI, 5.4-NE) for the HER2+ cohort and 6.3 mo (95% CI, 2.3-NE) for the HER2 low cohort. Adverse events (AEs) grade ≥3 occurred in 43.8% (18.8% related to T-DXd, 18.8% to Nivo) of pts treated at RDE (n=48); anemia (16.7%) and transaminase increase (6.3%) were most common. Nausea (54.2%), fatigue (45.8%), and alopecia (41.7%) were the most common any-grade AEs. 5 pts (10.4%, all HER2+) had treatment-related interstitial lung disease (ILD) as adjudicated by an independent committee (grade 5, n=1; grade 2, n=4). No other deaths associated with a drug-related AE occurred. Conclusions In pts with HER2-expressing MBC, T-DXd plus Nivo demonstrated antitumor activity consistent with prior studies of T-DXd and had an acceptable safety profile in this interim analysis; whether adding IO therapy to T-DXd benefits pts requires longer follow-up and additional studies. Citation Format: Erika Hamilton, Charles L Shapiro, Daniel Petrylak, Valentina Boni, Miguel Martin, Gianluca Del Conte, Javier Cortes, Laila Agarwal, Hendrik-Tobias Arkenau, Antoinette R. Tan, Philip Debruyne, Anna Minchom, Annemie Rutten, Frances Valdes-Albini, Evan Y Yu, Bincy Augustine, Anthony D'Amelio, Jr., Daniel Barrios, Sara A. Hurvitz. Trastuzumab deruxtecan (T-DXd; DS-8201) with nivolumab in patients with HER2-expressing, advanced breast cancer: A 2-part, phase 1b, multicenter, open-label study [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 PD3-07.

Background Ramucirumab-an IgG1 vascular endothelial growth factor receptor 2 antagonistplus docetaxel was previously reported to improve progression-free survival in platinum-refractory, advanced urothelial carcinoma. Here, we report the secondary endpoint of overall survival results for the RANGE trial.Methods We did a randomised, double-blind, phase 3 trial in patients with advanced or metastatic urothelial carcinoma who progressed during or after platinum-based chemotherapy. Patients were enrolled from 124 investigative sites (hospitals, clinics, and academic centres) in 23 countries. Previous treatment with one immune checkpoint inhibitor was permitted. Patients were randomly assigned (1:1) using an interactive web response system to receive intravenous ramucirumab 10 mg/kg or placebo 10 mg/kg volume equivalent followed by intravenous docetaxel 75 mg/m 2 (60 mg/m 2 in Korea, Taiwan, and Japan) on day 1 of a 21-day cycle. Treatment continued until disease progression, unacceptable toxicity, or other discontinuation criteria were met. Randomisation was stratified by geographical region, Eastern Cooperative Oncology Group performance status at baseline, and visceral metastasis. Progression-free survival (the primary endpoint) and overall survival (a key secondary endpoint) were assessed in the intention-to-treat population. The study is registered with ClinicalTrials.gov, NCT02426125; patient enrolment is complete and the last patient on treatment is being followed up for safety issues.Findings Between July 20, 2015, and April 4, 2017, 530 patients were randomly allocated to ramucirumab plus docetaxel (n=263) or placebo plus docetaxel (n=267) and comprised the intention-to-treat population. At database lock (March 21, 2018) for the final overall survival analysis, median follow-up was 7.4 months (IQR 3.5-13.9). In our sensitivity analysis of investigator-assessed progression-free survival at the overall survival database lock, median progression-free survival remained significantly improved with ramucirumab compared with placebo (4.1 months [95% CI 3.3-4.8] vs 2.8 months [2.6-2.9]; HR 0.696 [95% CI 0.573-0.845]; p=0.0002). Median overall survival was 9.4 months (95% CI 7.9-11.4) in the ramucirumab group versus 7.9 months (7.0-9.3) in the placebo group (stratified HR 0.887 [95% CI 0.724-1.086]; p=0.25). Grade 3 or worse treatment-related treatment-emergent adverse events in 5% or more of patients and with an incidence more than 2% higher with ramucirumab than with placebo were febrile neutropenia (24 [9%] of 258 patients in the ramucirumab group vs 16 [6%] of 265 patients in the placebo group) and neutropenia (17 [7%] of 258 vs six [2%] of 265). Serious adverse events were similar between groups (112 [43%] of 258 patients in the ramucirumab group vs 107 [40%] of 265 patients in the placebo group). Adverse events related to study treatment and leading to death occurred in eight (3%) patients in the ramucirumab group versus five (2%) patients in the placebo group.Interpretation Additional follow-up supports that ramucirumab plus docetaxel significantly improves progression-free survival, without a significant improvement in overall survival, for patients with platinum-refractory advanced urothelial carcinoma. Clinically meaningful benefit might be restricted in an unselected population. Copyright (C) 2019 Elsevier Ltd. All rights reserved.

Purpose: OX40, a receptor transiently expressed by T cells upon antigen recognition, is associated with costimulation of effector T cells and impairment of regulatory T-cell function. This first-in-human study evaluated MOXR0916, a humanized effector-competent agonist IgG1 monoclonal anti-OX40 antibody. Patients and Methods: Eligible patients with locally advanced or metastatic refractory solid tumors were treated with MOXR0916 intravenously once every 3 weeks (Q3W). A 3+3 dose-escalation stage (0.2–1,200 mg; n = 34) was followed by expansion cohorts at 300 mg (n = 138) for patients with melanoma, renal cell carcinoma, non–small cell lung carcinoma, urothelial carcinoma, and triple-negative breast cancer. Results: MOXR0916 was well tolerated with no dose-limiting toxicities observed. An MTD was not reached. Most patients (95%) experienced at least one adverse event (AE); 56% of AEs, mostly grade 1–2, were related to MOXR0916. Most common treatment-related AEs included fatigue (17%), diarrhea (8%), myalgia (7%), nausea (6%), decreased appetite (6%), and infusion-related reaction (5%). Pharmacokinetic (PK) parameters were dose proportional between 80 and 1,200 mg and supported Q3W administration. The recommended expansion dose based on PK and OX40 receptor saturation was 300 mg Q3W. Immune activation and upregulation of PD-L1 was observed in a subset of paired tumor biopsies. One renal cell carcinoma patient experienced a confirmed partial response. Overall, 33% of patients achieved stable disease. Conclusions: Although objective responses were rarely observed with MOXR0916 monotherapy, the favorable safety profile and evidence of tumor immune activation in a subset of patients support further investigation in combination with complementary agents such as PD-1/PD-L1 antagonists.

BACKGROUND: Cancer seems to have an independent adverse prognostic effect on COVID-19-related mortality, but uncertainty exists regarding its effect across different patient subgroups. We report a population-based analysis of patients hospitalised with COVID-19 with prior or current solid cancer versus those without cancer. METHODS: We analysed data of adult patients registered until 24 May 2020 in the Belgian nationwide database of Sciensano. The primary objective was in-hospital mortality within 30 days of COVID-19 diagnosis among patients with solid cancer versus patients without cancer. Severe event occurrence, a composite of intensive care unit admission, invasive ventilation and/or death, was a secondary objective. These endpoints were analysed across different patient subgroups. Multivariable logistic regression models were used to analyse the association between cancer and clinical characteristics (baseline analysis) and the effect of cancer on in-hospital mortality and on severe event occurrence, adjusting for clinical characteristics (in-hospital analysis). RESULTS: A total of 13 594 patients (of whom 1187 with solid cancer (8.7%)) were evaluable for the baseline analysis and 10 486 (892 with solid cancer (8.5%)) for the in-hospital analysis. Patients with cancer were older and presented with less symptoms/signs and lung imaging alterations. The 30-day in-hospital mortality was higher in patients with solid cancer compared with patients without cancer (31.7% vs 20.0%, respectively; adjusted OR (aOR) 1.34; 95% CI 1.13 to 1.58). The aOR was 3.84 (95% CI 1.94 to 7.59) among younger patients (

BackgroundTo determine the safety and efficacy of the anti-colony-stimulating factor 1 receptor (anti-CSF1R) monoclonal antibody AMG 820 in combination with pembrolizumab in patients with select solid tumors.Patients and methodsPatients had advanced, refractory mismatch repair-proficient colorectal cancer, pancreatic cancer, or non-small cell lung cancer (NSCLC) with low (ResultsOverall, 116 patients received ≥1 dose of AMG 820 plus pembrolizumab (18 at 1400 mg AMG 820; 98 at 1100 mg AMG 820). Most patients (64%) were male; the median age was 64 (range 30–86) years. Seven patients had DLTs (1 at 1400 mg AMG 820; 6 at 1100 mg AMG 820). Almost all patients (99.1%) had AEs, 87.9% with grade ≥3 AEs. The most common AEs were increased aspartate aminotransferase (59.5%), fatigue (48.3%), periorbital/face edema (48.3%), and rash/maculopapular rash (37.1%). The best response was immune-related partial response in 3 patients (3%; duration of response 9.2, 10.0, 12.5 months) and immune-related stable disease in 39 patients (34%). None of the completed phase II cohorts met the predefined threshold for efficacy. Post-treatment there was accumulation of serum colony-stimulating factor 1 (CSF1) and interleukin-34, reduction in CSF1-dependent CD16-expressing monocytes, and increased PD-L1 expression and CD4 and CD8 cell numbers in tumor biopsies.ConclusionsThe recommended combination dose of 1100 mg AMG 820 plus 200 mg pembrolizumab had an acceptable safety profile. Although pharmacodynamic effects were observed, antitumor activity was insufficient for further evaluation of this combination in selected patient populations.Trial registration numberNCT02713529

BACKGROUND The outcome to treatment administered to patients with metastatic castration-resistant prostate cancer (mCRPC) greatly differs between individuals, underlining the need for biomarkers guiding treatment decision making. OBJECTIVE To investigate the prognostic value of circulating tumor cell (CTC) enumeration and dynamics, in the context of second-line endocrine therapies (ie, abiraterone acetate or enzalutamide), irrespective of prior systemic therapies. DESIGN, SETTINGS, AND PARTICIPANTS In a prospective, multicentre study blood samples for CTC enumeration were collected from patients with mCRPC at baseline (n = 174). In patients who responded for minimally 10-12 weeks a follow-up sample was collected. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS For baseline analysis, patients were stratified in

Background: Toll-like receptors (TLRs), including TLR7, are involved in innate immune signaling. LHC165 is a TLR7 agonist which activates signaling pathways, increases cytokine release and promotes effector T-cell antitumor activity. In mouse models LHC165 treatment led to inhibition of tumor growth, and combination with an anti-PD-1 agent had more effective inhibition than either treatment alone. Methods: LHC165X2101 is a Phase I/Ib open-label dose-escalation/-expansion trial of LHC165 single agent (SA) ± spartalizumab, an anti-PD-1 mAb (NCT03301896). Eligible pts (≥18 yrs, ECOG 0-2) with advanced/metastatic solid tumors were treated with LHC165 SA (100-600 µg, Q2W intratumoral injection) ± spartalizumab (400 mg, Q4W IV). Primary objective was to assess safety and tolerability. Dose-escalation data are presented. Results: As of the data cutoff Sept 18, 2020, 39 pts enrolled; 20 pts received LHC165 SA and 19 pts combination. Median age 56 y, ECOG ≤1, median prior IO therapies: 2 (1-8) in SA pts, 3 (1-7) in combination pts. 37 (95%) pts discontinued: 27 (69%) progressive disease; 4 (10%) pt decision; 2 (5%) AEs; 2 (5%) completed treatment; and 2 (5%) pts died, treatment unrelated. 1 DLT of Gr 3 reversible pancreatitis was observed (LHC165 400 µg + spartalizumab 400 mg). AEs suspected to be treatment related observed in 22 (56%) pts; Gr ≥3 in 2 (5%) pts, 1 pt with lymphopenia and neutropenia, and 1 pt with pancreatitis. BOR in all pts was 3 PRs (all IO pretreated) and 4 SDs (1 IO pretreated). DCR (95%) was 18% (7.5%-33.5%). Median DOE (wks) was 4.5 (1.1-25.0) for SA pts, 11.9 (4.0-48.3) for combination pts. RDE was declared LHC165 600 µg ± spartalizumab 400 mg, with promising results in pts with melanoma and HNSCC. Systemic cytokine increases (CXCL10, IFNγ, IL6) were notable in a subset of pts following LHC165 ± spartalizumab. Immunohistochemistry tended to show higher baseline tumoral levels of CD8 and CD68 in pts with PR and on-treatment increases in pts with PR/SD. Tumoral RNA-sequencing showed higher T-cell inflammation at baseline in pts with PR and a consistent increase in T-cell inflammation in pts with PR/SD after treatment. Dose response relationships were not observed with available data. In most pts maximum LHC165 concentration was reached at around 1 hr, the first PK sampling time. Exposure of LHC165 ± spartalizumab increased with increasing dose of LHC165. Terminal half-life of LHC165 600 µg ranged from 9.4-58.9 hrs. No evidence of LHC165 accumulation over time or interaction between LHC165 and spartalizumab was noted. Conclusions: LHC165 ± spartalizumab demonstrated safety, tolerability, and evidence of preliminary antitumor activity. Exposure to LHC165 increased with dose, no interaction was observed with spartalizumab. Increased systemic cytokines and T-cell inflammation after LHC165 suggests active TLR7 agonism. Biomarker data suggest an active immune microenvironment at baseline is required for a response. Citation Format: Giuseppe Curigliano, Miguel Martin Jimenez, Toshio Shimizu, Bhumsuk Keam, Funda Meric-Bernstam, Annemie Rutten, John Glaspy, Nehal S. Parikh, Mary Ising, Nadia Hassounah, Jincheng Wu, Kun Xu, Somesh Choudhry, Elena Garralda. Phase I study of LHC165 ± spartalizumab (PDR001) in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT103.

PJP is a major cause of morbidity and mortality in immunocompromised individuals. Diagnosing PJP is often difficult because respiratory signs might be minimal or absent and the sensitivity of a chest X-ray (CXR) is low. We studied the clinical risk factors in order to increase awareness and facilitate the diagnosis.We studied a prospective case series over a one year period (from 1 January 2015 to 1 January 2016) and did a retrospective analysis (from 2013 to 2016) of all PJP positive bronchoalveolar lavage (BAL) analysis.Seven patients with metastatic solid tumors were diagnosed with symptomatic PJP (based on positive polymerase chain reaction) over a one year period. The median age was 61 years. Three patients had brain metastasis. Four of them were on steroids, the median dose was 16 mg methylprednisolone with three of them at a tapered dose. Respiratory failure developed in four cases and prompted intensive care monitoring. Two patients needed non-invasive ventilation and the third patient was intubated and mechanically ventilated. No patient died of PJP. A retrospective analysis on microbiological results obtained from BAL in our hospital from 2013 to 2016 shows a striking high percentage PJP positivity in cancer patients (including hematologic cancers) of nearly 22% (15/69) as opposed to the overall number of PJP-positives 7.3% (59/803).The incidence of clinical PJP in patients treated for metastatic cancer is substantial. A high index of suspicion, especially in cases with unexplained respiratory symptoms, concurrent or recent use of steroids, a normal CXR and otherwise unexplained increased lactate dehydrogenase levels, is critical. The threshold for performing a CT-scan must be low and the diagnosis needs to be confirmed microbiologically.

Background A growing understanding of the molecular biology of cancer and the identification of specific aberrations driving cancer evolution have led to the development of various targeted agents. Tumors can exhibit significant heterogeneity and this may change over time, also as the result of selective pressure. Circulating tumor cells (CTCs), shed from multiple tumor sites, have demonstrated to represent of the overall tumor burden. We report the results of an ongoing comparative study on mutation and copy number profiles of primary and metastatic tissue, CTCs, and synchronously isolated DTCs from metastatic effusions of patients with clinically progressive MBC. Materials & Methods CTCs and DTCs were enriched from 7.5 ml blood or effusion using the CellSearch system and were further purified and sorted with the DEPArray system. For this study we isolated both 70 single and 70 pools of 10-200 CTCs or DTCs, in order have enough power to detect 5% subclones and analyse heterogeneity. Single and pooled WBCs were isolated as technical controls. DNA was isolated and amplified using the Ampli1-kit and subjected to Illumina WGS and Ion Torrent AmpliSeq panel sequencing. Fresh frozen tissue from solid metastases and the primary tumor, and bulk CTC (CellSearch Profile) were sequenced as comparators for mutation and copy number profiles. DNA of buffy coat was sequenced to enable germline variant detection. For mutational analysis, only somatic variants with good quality metrics, >20x coverage, variant allele frequencies >10%, and being non-synonymous or splice site variants, were taken into account. Results AmpliSeq panel sequencing was performed on 153 unique samples of three patients with a mean coverage depth of 1000x. In patient 1, a PIK3CA hotspot mutation was found clonally in all tumor samples at heterozygous level. Furthermore, various private mutations were found in both CTCs and DTCs, however not in WBC, including several TP53 hotspot mutations. In patient 2, another PIK3CA hotspot was present in all CTCs at heterozygous frequencies. In patient 3, an enormous heterogeneity was observed between all CTC and DTC samples. For patient 3, disease evolution was detected during multiple events of progressive disease over 2 years. At the moment, low pass WGS for CN detection for all samples is being performed and results will be present prior to the SABCS. Conclusion Based on the mutational status we conclude that both clonal mutations as well as various private variants are present in single and pools of CTCs and DTCs. In addition to the detection of targetable aberrations, the evaluation of heterogeneity is of clinical importance, as the effect of targeting subclones is currently being explored in clinical trials. Citation Format: Brouwer A, van Dam P-J, Rutten A, Prové A, Peeters M, Van Laere S, Dirix L. Evaluation of subclonality in the CTC and DTC compartment of patients with metastatic breast cancer using low pass whole genome and AmpliSeq panel sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-01.

2641 Background: ECI-006 is a combination of TriMix (mRNAs encoding for dendritic cell [DC] activating molecules [CD40L, CD70 and caTLR4]), and mRNAs encoding for melanoma-specific tumor-associated antigens (TAAs): tyrosinase, gp100, MAGE-A3, MAGE-C2, and PRAME. DCs transfected ex vivo with TriMix and TAAs mRNAs showed significant clinical activity in combination with ipilimumab in metastatic melanoma without increasing toxicity. This study aims to assess the safety and immunogenicity of ECI-006 vaccine administered intranodally (i.n.) in an adjuvant setting for patients with resected melanoma. Methods: Twenty patients who underwent resection of stage IIc/III/IV cutaneous melanoma received 5 administrations of ECI-006 (either 600 µg or 1800 µg [n = 10, each]) injected i.n. on Day 1 and after 2, 4, 6 and 14 weeks. Treatment-emergent adverse events (TEAEs) were graded using CTCAE version 4.0.3. Blood samples for immune monitoring (ELISPOT and intracellular cytokine staining [ICS]) were collected pre-dose and at weeks 4, 7, 14 and 15. Results: Nineteen patients completed the treatment. One patient in the low dose group discontinued the study after 4 doses due to disease relapse. Administration of ECI-006 was well tolerated. No serious adverse events or TEAEs Grade 3 or higher were reported. Of all TEAEs, myalgia and fatigue were the most reported in 3 (15%) and 5 (25%) patients, respectively. ELISPOT and ICS were performed on T cells pre-stimulated in vitro for 10-12 days, using a previously in-house validated protocol. Vaccine-induced immune responses according to predefined criteria were detected in 4/10 and 3/9 patients treated with the low and high dose, respectively. Samples from these patients are currently being subjected to T-cell receptor repertoire analysis. Conclusions: Among patients undergoing resection of stage IIc/III/IV melanoma, i.n. administration of ECI-006 at 600 or 1800 µg was generally well tolerated. ECI-006 demonstrated to be immunogenic in a proportion of patients. These results warrant further development of ECI-006 in combination with anti-PD-1 therapy in melanoma patients. Clinical trial information: NCT03394937.

Background: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. Methods: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with >= 5 CTCs were classified as Stage IVaggresive, those with < 5 CTCs as Stage IVindolent. Survival was analyzed using Kaplan-Meier curves and the log rank test. Results: For all patients, Stage IVindolent patients had longer median overall survival than those with Stage IVaggresive (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IVindolent vs. 18.7 months Stage IVaggresive p < 0.0001). Moreover, patients with Stage IVindolent disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P < 0.0001), HER2-positive (36.7 months vs. 20.4 months, P < 0.0001), and triple negative (23.8 months vs. 9.0 months, P < 0.0001). Similar results were obtained regardless of prior treatment or disease location. Conclusions: We confirm the identification of two subgroups of MBC, Stage IVindolent and Stage IVaggresive, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.

Purpose: To infer the prognostic value of simultaneous androgen receptor (AR) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi). Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC (n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan–Meier analysis, and independent associations were determined using multivariable Cox regression models. Results: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18–3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort (n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001). Conclusions: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis. See related commentary by Rebello et al., p. 1699

Background: The European Pooled Analysis of CTC (EPAC) in metastatic breast cancer, based on 1,944 individual data from patients with various tumor types and clinical settings (Bidard et al, Lancet Oncol 2014), has established CTC count (CellSearch) at baseline and during therapy as a level of evidence 1 independent prognostic biomarker and demonstrated its superiority over serum blood markers. As part of the study pre-planned objectives, we sought to establish nomograms allowing accurate individual survival predictions. Methods: Using individual data from 17 centers, we built simplified multivariate prognostic models taking into account the independent prognostic clinico-pathological (CP) characteristics including CTC count, dichotomized using the 5CTC/7.5ml threshold, at baseline and at 3-5 weeks after the start of a new treatment regimen, and derived nomograms for progression-free survival (PFS) and overall survival (OS) prediction at baseline and after 3-5 weeks of treatment. We report here the internal validation of these nomograms. Discrimination of the models was assessed using the c-index estimated by a jackknife procedure and the calibration was visually assessed through 10-fold crossvalidated calibration plots at 1,2,3 years for OS and 1,2 years for PFS. Results: Multivariate models at baseline for PFS and OS were fitted on 1501 and 568 individual patient data with CTC count at baseline and CTC count at baseline and after 3-5 weeks, respectively. Models include tumor subtype, the number of previous chemotherapy lines (0/1/≥2), PS, age (50-65/>65 years), metastasis-free intervals (0/>0-3/>3 years), metastatic sites (liver and CNS) and CTC count at baseline and eventually at 3-5 weeks of treatment. The C-index increased from 0.722 to 0.755 (increase in C-index:0.033, 95% CI [0.019;0.045]) when adding baseline CTC to the CP only model for OS (n=1501). For those patients with CTC values at 3-5 weeks (n=568), there was an additional increase in the C-index when adding CTC at 3-5 weeks to a model with already CP and baseline CTC from 0.731 to 0.743 (increase in C-index 0.013, 95% CI [-0.004;0.025]). The model with CP and baseline CTC counts showed a good calibration for OS at 1,2,3 years and the model with CP, baseline CTC and CTC count at 3-5 weeks a moderately good calibration. Similar results were obtained for PFS. Conclusion: From the largest database with individual CTC data, we were able to build PFS and OS survival nomograms, with satisfactory discrimination and calibration. Our planned next step is to validate the nomogram in an additional cohort. Citation Format: Bidard F-C, Peeters D, Fehm T, Nole F, Gisbert-Criado R, Mavroudis D, Grisanti S, Generali D, Garcia-Saenz JA, Stebbing J, Caldas C, Gazzaniga P, Manso L, Zamarchi R, Fernandez de Lascoiti A, de Mattos-Arruda L, Ignatiadis M, van Laere SJ, Meier-Stiegen F, Sandri M-T, Vidal-Martinez J, Politaki E, Consoli F, Bottini A, Diaz-Rubio E, Krell J, Dawson S-J, Raimondi C, Rutten A, Janni W, Munzone E, Carañana V, Agelaki S, Almici C, Dirix L, Solomayer E, Zorzino L, Reis-Filho JS, Squifflet P, Pantel K, Beije N, Sleijfers S, Pierga J-Y, Michiels S. Circulating tumor cells count-based nomograms to predict survival of metastatic breast cancer patients: Results from the European pooled analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-08-08.

Introduction In metastatic breast cancer (MBC), discordant expression levels of the human epidermal growth factor receptor 2 (HER2) have been noted between primary tumors (PT) and matched metastatic lesions. Reassessment of HER2 status during treatment decisions in patients with advanced disease might help to optimize outcome. Circulating tumor cells (CTCs) offer the potential to provide a repeatedly accessible source of tumor cells for the real-time assessment of actual tumor characteristics. However, little is known on the concordance of HER2 expression on CTCs measured by immunofluorescence and the amplification status. Here we report on a preclinical study, using five spiked breast cancer cell lines, comparing semi-quantitative HER2 scoring on CellSearch (Riethdorf 2010) with objective DEPArray analysis, and subsequent FISH analysis on DEPArray-sorted tumor cells. At the moment these data are also being generated for 10 patient CTC samples. Expression and amplification status of CTCs will be compared with primary tumor tissue. Materials and methods MDA-MB-436, MCF-7, BT-20, KPL-4, and SKBR3 cells (increasing HER2 status) were spiked into donor blood and subjected to CellSearch enrichment. HER2/FITC intensity was scored manually on the CellSearch analyzer. All cell lines were injected into the DEPArray and exposure settings were optimized (FITC: exposure time 800 ms, gain 5%). These settings are further used for all preclinical and clinical samples. HER2 scoring was based on relative fluorescent units (rfu) of the HER2/FITC signal with background subtraction. Cells were sorted into pure batches of HER2 positive (DAPI+/CK+/HER2+/CD45-) and negative (DAPI+/CK+/HER2-/CD45-) tumor cells. Cytospins were formalin fixed and subjected to DAKO IQFISH. Results HER2 expression on CellSearch turned out to be very heterogeneous within the same cell line. DEPArray data was highly reproducible for all cell lines (p4). MDA-MB-436 and MCF-7 cells showed no gene amplification on FISH, while in KPL-4 there was a HER2/CEN17 ratio of >2. Four patient samples with HER2 positive status on CellSearch have been run on the DEPArray. For patient 1, 1005 CTC were analyzed, 32.4% were HER2+. This was 53 (69.8% HER2+), 352 (5.7% HER2+), and 622 (6.7% HER2+) for patient 2-4 respectively. These numbers are comparable with CellSearch analysis. Discussion HER2 expression analysis by immunofluorescence is comparable between CellSearch and DEPArray, however DEPArray has the advantage that it is user-independent and highly reproducible. Furthermore, CTCs can be sorted into pure batches for downstream analysis. The FISH technique on DEPArray sorted cells is now optimized and will be used to determine the correlation between the immunofluorescent HER2 scoring and the actual amplification status of the CTCs. These data will be incorporated prior to upcoming SABCS. Citation Format: Brouwer A, Verhoest F, Vermeulen P, Rutten A, Prové A, Van Laere S, Peeters M, Dirix L. Evaluation of HER2 expression and amplification on CTCs using DEPArray analysis and sorting followed by FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-10.

The complexity of delivering precision medicine to oncology patients has led to the creation of molecular tumourboards (MTBs) for patient selection and assessment of treatment options. New technologies like the liquid biopsy are augmenting available therapeutic opportunities. This report aims to analyse the experience of our MTB in the implementation of personalised medicine in a cancer network., info:eu-repo/semantics/published

BackgroundThere 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.MethodsA 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.ResultsctDNA 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-ARstructural variation, from 15.4% during first line mCRPC 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 ofPTEN, RB1andTP53detected 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 inPTENand RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays.ConclusionsctDNA 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 challenge 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.

Background Many observational studies investigated the prognostic significance of angiogenesis and lymphangiogenesis in patients with melanoma. However, the obtained results are rather contradictory, probably due to the lack of the consensus methodology. Methods To investigate the prognostic significance of angiogenesis and lymphangiogenesis-related parameters in patients with melanoma, we performed a retrospective investigation following the consensus recommendations for angiogenesis and lymphangiogenesis quantification in solid tumors and reporting recommendations for tumor marker (REMARK) criteria for reporting the results. Blood and lymphatic vessel Chalkley scores, endothelial cell proliferation fractions and microvessel densities were quantified using a double immunostaining for endothelial marker CD34 or lymphendothelial marker D240 and the proliferation marker Ki-67 in 196 patients with melanoma. These parameters were evaluated separately for peritumoral (PT) and intratumoral areas and were correlated with outcome. Results In multivariate analysis PT D240 Chalkley score was identified as a strongest predictor for sentinel lymph node metastases, non-sentinel lymph node metastases, distant metastases, disease free survival and overall survival in patients with melanoma. Conclusions If additional studies corroborate our findings, we believe that the inclusion of PT D240 Chalkley counts to the routine pathology examination of melanoma samples would provide additional information for identifying high-risk patients.

Ado- trastuzumab emtansine (T-DM1) is a human epidermal growth factor receptor 2 (HER2)-targeted antibody-drug conjugate composed of trastuzumab, a stable linker (MCC), and the cytotoxic agent DM1 (derivative of maytansine; mertansine). T-DM1 retains the mechanisms of action of trastuzumab, but also acts as a, selectively delivered, tubulin inhibitor. Following antigen-mediated binding to the tumor cell, T-DM1 is endocytosed and intracellularly catabolized resulting in the release of its cytotoxic moiety.T-DM1 has completed Phase III development and compared favorably with the lapatinib/capecitabine combination with a superior response rate (objective response rate [ORR]) and duration of response, longer duration of disease control (progression-free survival [PFS]), prolonged overall survival and improved tolerability and quality of life in patients with prior treatment with trastuzumab and a taxane. In a separate Phase III, T-DM1 was compared with any other chosen regimen in patients who had at least received two prior HER2-directed therapies. T-DM1 nearly doubled PFS.T-DM1 (Kadcyla) has become the treatment of choice in second-line and beyond for patients with advanced HER2-expressing breast cancer.

Summary Background Few treatments with a distinct mechanism of action are available for patients with platinum-refractory advanced or metastatic urothelial carcinoma. We assessed the efficacy and safety of treatment with docetaxel plus either ramucirumab—a human IgG1 VEGFR-2 antagonist—or placebo in this patient population. Methods We did a randomised, double-blind, phase 3 trial in patients with advanced or metastatic urothelial carcinoma who progressed during or after platinum-based chemotherapy. Patients were enrolled from 124 sites in 23 countries. Previous treatment with one immune-checkpoint inhibitor was permitted. Patients were randomised (1:1) using an interactive web response system to receive intravenous docetaxel 75 mg/m 2 plus either intravenous ramucirumab 10 mg/kg or matching placebo on day 1 of repeating 21-day cycles, until disease progression or other discontinuation criteria were met. The primary endpoint was investigator-assessed progression-free survival, analysed by intention-to-treat in the first 437 randomised patients. This study is registered with ClinicalTrials.gov, number NCT02426125. Findings Between July, 2015, and April, 2017, 530 patients were randomly allocated either ramucirumab plus docetaxel (n=263) or placebo plus docetaxel (n=267). Progression-free survival was prolonged significantly in patients allocated ramucirumab plus docetaxel versus placebo plus docetaxel (median 4·07 months [95% CI 2·96–4·47] vs 2·76 months [2·60–2·96]; hazard ratio [HR] 0·757, 95% CI 0·607–0·943; p=0·0118). A blinded independent central analysis was consistent with these results. An objective response was achieved by 53 (24·5%, 95% CI 18·8–30·3) of 216 patients allocated ramucirumab and 31 (14·0%, 9·4–18·6) of 221 assigned placebo. The most frequently reported treatment-emergent adverse events, regardless of causality, in either treatment group (any grade) were fatigue, alopecia, diarrhoea, decreased appetite, and nausea. These events occurred predominantly at grade 1–2 severity. The frequency of grade 3 or worse adverse events was similar for patients allocated ramucirumab and placebo (156 [60%] of 258 vs 163 [62%] of 265 had an adverse event), with no unexpected toxic effects. 63 (24%) of 258 patients allocated ramucirumab and 54 (20%) of 265 assigned placebo had a serious adverse event that was judged by the investigator to be related to treatment. 38 (15%) of 258 patients allocated ramucirumab and 43 (16%) of 265 assigned placebo died on treatment or within 30 days of discontinuation, of which eight (3%) and five (2%) deaths were deemed related to treatment by the investigator. Sepsis was the most common adverse event leading to death on treatment (four [2%] vs none [0%]). One fatal event of neutropenic sepsis was reported in a patient allocated ramucirumab. Interpretation To the best of our knowledge, ramucirumab plus docetaxel is the first regimen in a phase 3 study to show superior progression-free survival over chemotherapy in patients with platinum-refractory advanced urothelial carcinoma. These data validate inhibition of VEGFR-2 signalling as a potential new therapeutic treatment option for patients with urothelial carcinoma. Funding Eli Lilly and Company.

Summary Malignant melanoma represents < 10% of all skin cancers but is responsible for the majority of skin-cancer-related deaths. Metastatic melanoma has historically been considered as one of the most therapeutically challenging malignancies. Fortunately, for the first time after decades of basic research and clinical investigation, new drugs have produced major clinical responses. Angiogenesis has been considered an important target for cancer treatment. Initial efforts have focused primarily on targeting endothelial and tumour-related vascular endothelial growth factor signalling. Here, we review different mechanisms of tumour vascularization described in melanoma and discuss the potential clinical implications.

Improvements in the treatment of patients with MBC and the inefficiency of many drugs in crossing the BBB have led to an increased incidence of symptomatic CNS metastasis. CNS metastases are clinically characterized by two distinct phenotypes, although their combined occurrence is possible. Most patients suffer from solid CNS metastases, but metastatic deposits limited to the leptomeningeal surfaces occur (LMM). In addition to its poor prognosis, neurological impairment in LMM is devastating. Methods: 27 patients with breast cancer (MBC) were diagnosed with LMM and treated with intrathecal (IT) therapy from 2008 to 2018 in a single centre (GZA Hospitals Antwerp). Clinicopathologic and treatment information were collected. Conventional diagnosis was based on positive cytology on CSF and/or on the combination of clinical signs and neuro-imaging findings. For a subset of patients (n=12), we have enumerated circulating tumor cells (CTCs) and CSF DTCs at time of LMM diagnosis. Whole blood and CSF was processed on the CellSearch platform according to the standard method for CTC enumeration in blood. Results: The 27 MBC pts diagnosed with LMM had a median time to LMM of 70.5 months (6.5-241) from the diagnosis of BC. Median age at LMM diagnosis was 56 y (36-75). At time of last follow-up, 23 patients (85%) had died. The median OS from LMM diagnosis was 27 months (4.5-128). From the 27 patients, 52% (14/27) were hormone receptor-positive (HR+), 33% (9/27) were HER2 overexpressing and 15% (4/27) were triple-negative. Coexisting brain metastases are present in 37% of patients. In 11 cases (41%), LMM was not accompanied by extra cranial disease at time of diagnosis. The biochemical data matched the classic CSF findings of LMM including a high protein concentration (75%, 9/12) and a low glucose concentration (42%, 5/12) (Table 1.). CSF lactate was elevated (>2.2 mmol/l) in 100% (10/10). In 70% (7/10) CSF lactate was >3.5 mmol/l. Cytologic evaluation of the CSF demonstrated unequivocal malignant cells in 10/12 (83%). DTC enumeration in the CSF of these patients, however, revealed the presence of tumor cells 12/12 (100%). Furthermore, the cell count ranged between 10 to 3523 Epcam-positive cells/mL CSF. Additional evaluation proved these to be cancer cells. Results off the CSF analysis in 12 patients with LMMPatientWBCGlucoseProteinCTCCytologyDTCVolume mL125 10005413339608neg> 10007.554042280pos114427.56772064410pos63667.5756261211151pos264237.581675161NApos>100039894840pos39311062313200pos191657.511296802.40pos591441225605,113neg7994 Conclusion: Symptomatic LMM has a variable prognosis, with some patients experiencing prolonged survival. LMM occurs in all breast cancer subtypes and can be the sole location (40%). CSF lactate is a sensitive but aspecific marker. DTC enumeration in the CSF is a robust tool to diagnose LMM with apparently a superior sensitivity over conventional cytology. It furthermore has a broad dynamic range rendering it more suitable for treatment effect evaluation. This methodology should be considered in routine workup in MBC patients with suspected LMM. Citation Format: Van Goethem A, Rypens C, Rutten A, Prove A, Van den Mooter T, Erven K, Van Havenbergh T, Van Laere S, Vermeulen P, Dirix L. Sensitivity and dynamic range of CellSearch based DTC enumeration in the CSF in patients with leptomeningeal metastases (LMM) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-02-02.

Background: The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer. Methods: CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of >= 5 CTCs per 7.5 ml blood. Results: No significant differences were observed in the absolute CTC counts (P = 0.120) or in CTC positivity rates according to >= 1 and >= 5 CTCs per 7.5 ml blood detection thresholds (P = 0.165 and P = 0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as >= 80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P = 0.024). In the total study population, the presence of X5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P < 0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease. Conclusion: The detection of EpCAM + CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.

We present a single-cell application to determine PIK3CA mutations in CTCs, which uncovered the degree of intra-patient heterogeneity in patients with metastatic hormone receptor-positive breast cancer (HR+ MBC) and high CTC count (>10 CTCs/7.5mL). Using CellSearch and DEPArray we isolated circulating tumor cells (CTCs) and white blood cells (WBCs) from peripheral blood and sequenced PIK3CA exons 9 and 20 by targeted amplicon sequencing. Comparative analysis between the primary tumor (PT, n=27 patients), circulating cell-free DNA (cfDNA, n=31 patients), single (n=146 CTCs) and pools (n=70 CTC suspensions, ranging 5-120 cells/suspension) of CTCs from 26 patients and metastases/DTCs (n=11 patients) was performed. Mutations were frequent in PT (15/27 (55.5%)) and showed slight and substantial agreement with cfDNA (n=21; kappa=0.14) and CTCs (n=22; kappa=0.6733), respectively. A wild-type genotype in WBCs indicates a high specificity. Inter-compartmental concordance was observed in 13/18 (72.2%) patients and temporal heterogeneity in 4/18 patients (22.2%). CTC analysis reveals both mutational homo- and heterogeneity with cases showing the presence of different mutant and wild-type CTC subpopulations. Additionally, unique double-mutated CTCs were detected in 8/26 patients (30.7%). The developed liquid biopsy provides an insight into inter and intra-patient PIK3CA mutational heterogeneity in patients with HR+ MBC, paving the way towards the application of a more personalized medicine.

e18511Background: Molecular Tumor Boards (MTBs) for patient selection and assessment of treatment options were created for adequate precision medicine delivering. We evaluated the implementation of...

We have shown that in up to half of the patients with metastatic breast cancer (MBC), higher numbers of circulating tumour cells (CTCs) are present in the central venous blood (CVB) compared to the peripheral venous blood (PVB), suggesting that the lungs might retain a substantial number of CTCs. Here we report the presence of tumour cell emboli (TCE) in the microvasculature of the lungs in three out of eight patients with MBC and one patient with metastatic cervical carcinoma who had markedly elevated numbers of CTCs in the blood. All these patients suffered from symptomatic dyspnoea not easily attributable to other causes. No TCE were observed in five patients with MBC and elevated CTC counts and three patients with MBC who had low CTC counts (

Many observational studies investigated the prognostic significance of angiogenesis and lymphangiogenesis in patients with melanoma. However, the obtained results are rather contradictory, probably due to the lack of the consensus methodology.To investigate the prognostic significance of angiogenesis and lymphangiogenesis-related parameters in patients with melanoma, we performed a retrospective investigation following the consensus recommendations for angiogenesis and lymphangiogenesis quantification in solid tumors and reporting recommendations for tumor marker (REMARK) criteria for reporting the results. Blood and lymphatic vessel Chalkley scores, endothelial cell proliferation fractions and microvessel densities were quantified using a double immunostaining for endothelial marker CD34 or lymphendothelial marker D240 and the proliferation marker Ki-67 in 196 patients with melanoma. These parameters were evaluated separately for peritumoral (PT) and intratumoral areas and were correlated with outcome.In multivariate analysis PT D240 Chalkley score was identified as a strongest predictor for sentinel lymph node metastases, non-sentinel lymph node metastases, distant metastases, disease free survival and overall survival in patients with melanoma.If additional studies corroborate our findings, we believe that the inclusion of PT D240 Chalkley counts to the routine pathology examination of melanoma samples would provide additional information for identifying high-risk patients.

We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data.We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics.17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model.These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not.Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

Summary In malignant melanoma (MM) there is an urgent need to identify new markers with predictive value superior to the traditional clinical and histological parameters. Angiogenesis and lymphangiogenesis have been recognized as critical processes in tumour growth and metastasis development, and numerous studies have evaluated the significance of these parameters in predicting the prognosis in solid tumours, including MM. We set out to determine whether angiogenesis, lymphangiogenesis and lymphatic invasion (LI) are valuable prognostic markers in MM. We systematically reviewed the available literature and subsequently performed a meta-analysis on the compiled data. To be eligible for the systematic review, a study had to provide the microvessel density (MVD), the lymphatic vessel density (LVD) or information about LI, assessed by immunohistochemistry on the primary site in patients with MM. To be evaluable for the meta-analysis, a study also had to provide information on clinical outcome. We approached selected studies with the Reporting recommendations for tumour marker (REMARK) criteria, verifying whether they had followed the recommendations. In total, nine angiogenesis, seven lymphangiogenesis and 10 LI studies were included in our meta-analysis, representing 419, 474 and 802 patients, respectively. Using meta-analysis, we showed that peritumoral LVD and the presence of LI have prognostic value for patients with MM. In contrast, MVD and intratumoral LVD did not have prognostic value in these patients. LVD and LI seem to have prognostic value for patients with MM.

Trastuzumab emtansine (T-DM1) is a human epidermal growth factor receptor 2 (HER2)-targeted antibody-drug conjugate (ADC) composed of trastuzumab, a stable linker (MCC), and the cytotoxic agent DM1 (derivative of maytansine). Administration of T-DM1 leads to limited systemic exposure of free DM1, with no evidence of DM1 accumulation after repeated dosing.Phase I and Phase II clinical trials with T-DM1 as a single agent and in combination with paclitaxel, docetaxel, and pertuzumab have shown substantial clinical activity and a favorable safety profile. A randomized, open-label, first-line trial comparing trastuzumab and docetaxel with single agent T-DM1 showed a significant improved progression-free survival for T-DM1.T-DM1 has successfully completed second-line Phase III development for advanced HER2-positive breast cancer. The Phase III EMILIA study demonstrated an overall survival benefit for T-DM1 compared to the combination of lapatinib and capecitabine in taxane-trastuzumab pretreated patients. T-DM1 may offer delivery on a personalized basis of very potent cytotoxic agents in a cellular selective manner.

Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation., info:eu-repo/semantics/published

Hedgehog pathway signaling is important for embryonic development; however, inappropriate reactivation of this pathway in adults has been linked to several forms of cancer. Vismodegib (Erivedge™), a first-in-class hedgehog pathway inhibitor, blocks the pathway by inhibiting the activity of the signaling protein SMO. Preclinical studies have provided promising indications of potential tumor-reducing activity in several cancers. Thus far, clinical pharmacology and Phase I studies have demonstrated the unique pharmacokinetic profile of vismodegib, its efficacy in certain types of tumors and a generally tolerable adverse-event profile. A pivotal Phase II clinical trial confirmed the favorable benefit:risk profile of vismodegib in advanced basal cell carcinoma.

2599 Background: The oral PARP inhibitor olaparib is well tolerated at doses ≤400 mg bid (capsule formulation) and has shown efficacy in patients (pts) with BRCA-mutated ovarian cancer (Ledermann e...

Introduction: The enumeration of circulating tumour cells (CTCs) with the EPCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). However, breast cancer has been shown to be a molecularly heterogeneous disease. The aim of this study was to assess potential differences in the detection and prognostic significance of CTCs according to the immunohistochemically defined molecular subtypes of breast cancer. Methods: CellSearch CTC counts were obtained from 110 patients with MBC prior to first line systemic treatment, treated at GZA Hospitals Sint-Augustinus between november 2007 and december 2011. Clinicopathological variables were prospectively entered in a database. Based on the St-Gallen surrogate definitions of intrinsic breast cancer subtypes (Goldhirsch et al. Ann Oncol 2011), patients were divided in 5 groups: luminal A (ER/PR+, HER2−, Bloom-Richardson histological grade I-II), luminal B – HER2 negative (ER/PR+, Her2−, grade III), luminal B – HER2 positive (ER/PR+, HER2+, any grade), HER2 positive – non luminal (ER/PR−, HER2+), and triple negative (TN) (ER/PR−, HER2−). Differences in progression free survival (PFS) and overall survival (OS) according to the FDA approved prognostic cut-off of ≥5 CTC/7.5 ml blood were estimated using Kaplan Meier and Cox proportional hazard statistics. Results: CTC were detected in 78 of 110 (71%) patients. Higher detection rates and numbers of CTC were observed in patients with luminal A and TN breast cancer as compared to patients with luminal B and HER2 positive disease. However, no differences in positivity rates were observed between molecular subtypes according to the 5 CTC prognostic cut-off point (table 1). After a median FU time of 3.1 years, 39 patients had died. In the total study population, the presence of ≥5 CTC was an independent predictor of PFS and OS in multivariate analysis (PFS: HRCTC≥5=2.236 (1.366–3.658), p = 0.001; OS: HRCTC≥5=3.180 (1.553–6.509), p = 0.002). When analyzing subgroups separately, a lower prognostic power was observed in the HER2 positive and luminal B subgroups. Conclusion: Significant differences were observed in the detection and prognostic significance of EPCAM positive CTC according to the immunohistochemically defined breast cancer subtypes. Interestingly, CTC were detected more frequently in patients with luminal A and TN tumors. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2 positive patients with MBC. Our data independently confirm those reported by Giordano et al. (Ann Oncol 2010) in a large clinically uniform population of patients with MBC before the start of first-line treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-08.

Background: We have shown that in up to 50% of patients with metastatic breast cancer (MBC) significantly higher numbers of circulating tumor cells (CTCs) can be detected in central venous blood (CVB) as compared to peripheral venous blood (PVB), suggesting that the lungs might retain a substantial number of CTCs from the blood stream (Peeters et al. Br J Cancer 2011). The aim of this study was 1) to investigate the relation between elevated numbers of CTCs and the presence of (intravascular) tumor cell emboli (TCE) in the lung in patients with advanced carcinomas, and 2) to investigate whether CTCs derived from CVB and PVB exhibit differential transcriptional characteristics. Methods: Seven patients with MBC and 1 patient with metastatic cervical carcinoma, all suffering from end-stage disease, were included in the first part of this study. CTCs were isolated and enumerated with the CellSearch system (Veridex, Raritan, NJ, USA) in 7.5 ml blood obtained from the central venous access catheter (CVB) and/or a peripheral vein (PVB). All blood samples were obtained within 5 days prior to death. The presence of TCE was studied in lung tissue samples obtained at autopsy. For the second study aim, paired CVB and PVB CTC samples were collected from an additional 10 MBC and 2 LABC patients. Transcriptional profiles were obtained for 91 breast cancer related genes as described by Sieuwerts et al. (Clin Cancer Res 2011). Results: Multiple TCE were observed in 4 out of 6 patients with highly elevated numbers of CTCs (>100 CTC/7.5 ml blood). These TCE were located exclusively intravascularly in 2 patients, while the other 2 patients had a more diffuse infiltration pattern with perivascular and lymphovascular TCE. All 4 patients had a history of rapidly evolving respiratory distress in the last week of life although radiological examination of the lungs did not show significant interval changes. In another 2 MBC patients with >100 CTCs and 2 MBC patients with Conclusions: TCE were observed in 4 out of 6 patients with highly elevated numbers of CTCs. In these patients, cumulative entrapment of CTCs in the lung might have contributed to respiratory dysfunction. High numbers of CTC might therefore represent an oncological emergency. Transcriptional profiling of 91 breast cancer related genes revealed no substantial difference in gene expression of CTCs derived from CVB and PVB, suggesting that CTC entrapment by the lung is a rather passive process in advanced cancer patients. These findings will be further challenged by comparing the obtained profiles with gene expression profiles of 13 additionally selected homing markers in these samples. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-09.

e14647 Background: Pleural and peritoneal effusions are common in advanced malignant disease and relative resistant to traditional treatments. VEGF-A is considered to be a crucial factor in the biology of accumulation of malignant effusions (ME). Sunitinib is a multi-targeted TKI with a high potency of inhibiting VEGFR2. A treatment that could symptomatically reduce ME would be useful, regardless of its anti-tumoural or anti-angiogenic effect. Methods: Refractory patients with symptomatic ME were enrolled to receive 2x6 weeks of daily 37.5mg sunitinib regardless of their primary histology or site. Outcome was measured by time to re-puncture or by measurement of volume ME by CT-scan every 6 weeks or at the end of treatment. Puncture fluids and blood samples were collected for extensive biomarker analysis (VEGFA&C, VEGFR1–3, b-FGF, PDGFA-B, PDGFRα-β, PlGF1–2) on consecutive samples. Results: At present 6 patients have been enrolled. Patients had ovarian cancer (N=3), breast cancer (N=2) and squamous cervical cancer (N=1) as primary tumours. All patients had been extensively pre-treated with 4 to 8 lines of systemic therapy. Efficacy: Three (3/6) patients showed regression of effusion fluid by consecutive CT-scans. Two (2/6) patients had unchanged volume and one (1/6) patient had an increase of fluid volume. Follow-up: Two (2/6) patients stopped the trial early due to toxicity. Two patients (2/6) died during treatment due to disease progression elsewhere. Two patients (2/6) completed the 2 cycles of 6 weeks treatment. Of which, one had sustained reduction in volume fluid accumulation, while the other one had initial stabilisation after one cycle but eventually had increasing volume of ascites. Conclusions: These findings support the hypothesis that sunitinib could be useful in the management of ME. Although the numbers are low, in a majority of patients (5/6) there was reduction or stabilisation of fluid accumulation. Toxicity led to study withdrawal in a third of our patients. An update of patient numbers as well as results of translational/biomarker analysis will be presented at the meeting. No significant financial relationships to disclose.