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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: 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.

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.

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.

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.

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.