Publications by authors named "Steven Van Laere"

98 Publications

Association between the histopathological growth patterns of liver metastases and survival after hepatic surgery in breast cancer patients.

NPJ Breast Cancer 2020 Dec 18;6(1):64. Epub 2020 Dec 18.

Department of Surgery, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium.

Currently, there are no markers to identify patients with liver-only or liver-dominant metastases that would benefit from hepatic surgery. Here we characterized histopathological growth patterns (HGPs) of liver metastases in a consecutive series of 36 breast cancer patients who underwent hepatic surgery. Survival analyses showed that the presence of a desmoplastic HGP in the liver metastases (a rim of fibrous tissue separating cancer cells from the liver parenchyma, present in 20 (56%) patients) is independently associated with favorable progression-free and overall survival when compared with the replacement HGP (cancer cells growing into the liver parenchyma, present in 16 (44%) patients).
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http://dx.doi.org/10.1038/s41523-020-00209-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749172PMC
December 2020

Stereotactic Ablative Radiation Therapy to All Lesions in Patients With Oligometastatic Cancers: A Phase 1 Dose-Escalation Trial.

Int J Radiat Oncol Biol Phys 2020 Dec 8. Epub 2020 Dec 8.

Department of Radiation Oncology, Iridium Netwerk, Wilrijk (Antwerp), Belgium; Translational Cancer Research Unit (TCRU), Center for Oncological Research (CORE), University of Antwerp, Edegem (Antwerp), Belgium.

Purpose: Increasing evidence suggests that patients with a limited number of metastases benefit from SABR to all lesions. However, the optimal dose and fractionation remain unknown. This is particularly true for bone and lymph node metastases. Therefore, a prospective, single-center, dose-escalation trial was initiated.

Methods: Dose-Escalation trial of STereotactic ablative body RadiOtherapY for non-spine bone and lymph node metastases (DESTROY) was an open-label phase 1 trial evaluating SABR to nonspine bone and lymph node lesions in patients with up to 3 metastases. Patients with European Cooperative Oncology Group performance status ≤1, an estimated life expectancy of at least 6 months, and histologically confirmed nonhematological malignancy were eligible. Three SABR fractionation regimens, ie, 5 fractions of 7.0 Gy versus 3 fractions of 10.0 Gy versus a single fraction of 20.0 Gy, were applied in 3 consecutive patient cohorts. The rate of ≥grade 3 toxicity, scored according to the Common Toxicity Criteria for Adverse Events v. 4.03, up to 6 months after SABR, was the primary endpoint. The trial was registered on clinicaltrials.gov (NCT03486431).

Results: Between July 2017 and December 2018, 90 patients were enrolled. In total 101 metastases were treated. No ≥grade 3 toxicity was observed in any of the enrolled patients (95% CI 0.0%-12.3% for the first cohort with 28 analyzable patients; 95% CI 0.0%-11.6% for the second and third cohort with 30 analyzable patients each). Treatment-related grade 2 toxicities occurred in 4 out of 30 versus 2 out of 30 versus 2 out of 30 patients for the 5, 3 and 1 fraction schedule, respectively. Actuarial local control rate at 12 months was 94.5%.

Conclusion: All 3 treatment schedules were feasible and effective with remarkably low toxicity rates and high local control rates. From a patient and resource point of view, the single-fraction schedule is undoubtedly most convenient.
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http://dx.doi.org/10.1016/j.ijrobp.2020.11.066DOI Listing
December 2020

Automated enumeration and phenotypic characterization of CTCs and tdEVs in patients with metastatic castration resistant prostate cancer.

Prostate Cancer Prostatic Dis 2020 Nov 23. Epub 2020 Nov 23.

Translational Cancer Research Unit (TCRU), GZA Hospitals Sint-Augustinus, Antwerp, Belgium.

Background: Although most patients with metastatic castration-resistant prostate cancer (mCRPC) initially benefit from treatment with androgen receptor signaling inhibitors (ARSi), resistance inevitably occurs. Hence, we investigated the prognostic value of automated circulating tumor cell (CTC) and tumor-derived extracellular vesicle (tdEV) enumeration and their dynamics, in patients with mCRPC in the context of the initiation of treatment with ARSi. Furthermore, we hypothesize that CTC phenotypic heterogeneity might serve as a measurable biomarker under these circumstances.

Methods: Using an image analysis tool, we reanalyzed all CellSearch images previously acquired in the context of a prospective, multicenter clinical study for patients with mCRPC (n = 170) starting a new line of ARSi, for CTC and tdEV detection and enumeration. CTC (n = 19 129) phenotypic diversity was quantified by the Shannon index (SI). Progression-free survival (PFS) and overall survival (OS) were compared between groups of patients stratified according to CTC, tdEV, and SI levels.

Results: Automated CTC enumeration provided similar clinical prognostication compared with operator-based counts. Patients demonstrating high CTC phenotypic heterogeneity before therapy had a shorter median PFS (4.82 vs. 8.49 months, HR 1.79; P = 0.03) and OS (12.6 months vs. not reached, HR 2.32; P = 0.03), compared to patients with low diversity, irrespective of CTC level. Multivariable analysis showed how the prognostic value of the baseline SI was lost by pretreatment chemotherapy status, CTC counts, and PSA levels.

Conclusions: Automated CTC counts are a reliable substitute for reviewer-based enumeration, as they are equally informative for prognosis assessment in patients with mCRPC. Beyond enumeration, we demonstrated the added value of studying CTC phenotypic diversity for patient prognostication, warranting future investigation.
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http://dx.doi.org/10.1038/s41391-020-00304-1DOI Listing
November 2020

Genomic landscape of inflammatory breast cancer identifies potential actionable genetic alterations.

Oncoscience 2020 Jul 30;7(7-8):57-59. Epub 2020 Jun 30.

Laboratoire d'Oncologie Prédictive, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, CNRS UMR7258, Institut Paoli-Calmettes, Aix-Marseille Université, Marseille F-13009, France.

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http://dx.doi.org/10.18632/oncoscience.515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458333PMC
July 2020

Oncological outcome, postoperative complications, and mammographic changes after intraoperative radiotherapy with electrons (IOERT) as a boost in a large single-institution cohort of breast cancer patients.

Breast J 2020 10 11;26(10):1937-1945. Epub 2020 Aug 11.

Department of Radiation Oncology, Iridium Kankernetwerk, Antwerp, Belgium.

Advantages of using intraoperative radiotherapy with electrons (IOERT) as a boosting modality in breast-conserving therapy include the direct visualization of the tumor bed, a reduced skin dose, and patient convenience. We report oncological outcome, postoperative complication rate, and mammographic changes on follow-up imaging in women treated at our institution with IOERT as a boost modality in breast-conserving therapy for early-stage breast carcinoma. Between January 2007 and June 2018, 763 consecutive patients were enrolled. During breast-conserving surgery, an IOERT boost of 9 Gy was applied, followed by whole breast irradiation (WBI). At a median follow-up of 62.2 months (range: 0.5-135), 13 in-breast recurrences were observed, yielding a local tumor control rate of 98.4% at 5 years. In multivariable analysis, high tumor grading was predictive for local recurrence (HR = 5.6; 95%CI: 1.19-26.2). A total of 27 (3.5%) patients developed any kind of postoperative complication. None of the tumor characteristics nor any of the IOERT technical parameters were predictive for development of a postoperative complication. On follow-up imaging, 145 patients with mammographic changes BIRADS score ≥3 were found of which 50.3% required a biopsy. Only 17 patients had positive biopsies; none of the IOERT parameters were predictive for false-positive imaging. A 9 Gy IOERT boost combined with postoperative WBI provided outstanding local control rates, was well-tolerated, with limited postoperative complications. However, radiologists must be aware of a presumable higher prevalence of mammographic changes after IORT as a boost.
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http://dx.doi.org/10.1111/tbj.13986DOI Listing
October 2020

Can medical imaging identify the histopathological growth patterns of liver metastases?

Semin Cancer Biol 2020 Jul 28. Epub 2020 Jul 28.

Translational Cancer Research Unit, GZA Hospitals, Sint-Augustinus, Antwerp, Belgium; Iridium Kankernetwerk, Antwerp, Belgium; Center for Oncological Research (CORE), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Antwerp, Belgium. Electronic address:

The histopathological growth patterns (HGPs) of liver metastases of colorectal cancer and of several other tumor types predict outcome of patients in multiple studies. The HGPs of liver metastases have a prognostic but also a predictive value with one of the growth patterns, the replacement growth pattern, related to resistance to systemic treatment. Given that the HGP can only be assessed in a reliable manner when a surgical resection of the metastasis has been performed, this biomarker cannot be exploited to the full. For example, HGPs can at this moment, not be used to decide whether patients with liver metastatic breast or colorectal cancer will benefit or not from locoregional treatment, such as surgery or radiotherapy, and from peri-operative systemic treatment. In this review we highlight studies that suggest that the HGPs of liver metastases can be identified by medical imaging. Although still to be confirmed by a prospective multicenter approach, some studies indeed achieve a high accuracy in predicting the HGPs by applying radiomic algorithms on CT- or MR-images of liver metastases. This is an important step towards a treatment planning of patients with liver metastatic cancer that takes into account the biology and the progression kinetics of the metastases.
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http://dx.doi.org/10.1016/j.semcancer.2020.07.002DOI Listing
July 2020

Outcome and toxicity of hypofractionated image-guided SABR for spinal oligometastases.

Clin Transl Radiat Oncol 2020 Sep 25;24:65-70. Epub 2020 Jun 25.

Department of Radiation Oncology, Iridium Kankernetwerk, Wilrijk (Antwerp), Belgium.

Background: To investigate progression free survival (PFS), local control (LC) and overall survival (OS) outcomes for patients treated with spine hypofractionated stereotactic ablative radiotherapy (SABR) and to evaluate possible predictors of rapid progression in view of a correct patient selection for this potentially curative SABR.

Materials And Methods: A cohort of 59 patients with spinal metastases were treated with SABR. Patient selection criteria were the following: histologically proven diagnosis of a solid tumor, a World Health Organization (WHO) score ≤ 2, life expectancy > 6 months, Spinal Instability Neoplastic Score (SINS) ≤ 12 points and presenting with radically treated oligometastatic disease (≤5 lesions) or stable polymetastatic disease with an oligoprogressive lesion.

Results: From March 2015 to June 2019, 59 patients were treated with Linac-based SABR to 64 spinal metastases with a median follow-up of 55 months. SABR was standard delivered every other day in 3 to 10 fractions with median prescription dose of 27 Gy (range 21-49 Gy).The 1-,2- and 5-year PFS was 98%, 85% and 75% for all patients. OS at 5 years for all patients was 92%. Metachronous lesions (p < 0.01; HR = 7.1) and oligometastatic (vs. oligoprogressive) lesions (p = 0.02; HR = 0.3) were associated with higher PFS in uni- and multivariate Cox regression analysis. No significant predictors in multivariate analysis were demonstrated for rapid progressors.Vertebral compression fractures developed de novo in 6.3% (4/64) of cases. The median time to fracture was 11 months (range 7-15) after treatment. No other adverse events ≥ 3 grade were observed.

Conclusions: Tumor control and toxicity after high-dose hypofractionated SABR was evaluated in patients with spinal oligometastases. High rates of efficacy and minimal toxicity were demonstrated. Oligometastatic patients with metachronous spinal metastases seem to benefit the most from SABR.
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http://dx.doi.org/10.1016/j.ctro.2020.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334439PMC
September 2020

Quantitative hormone receptor (HR) expression and gene expression analysis in HR+ inflammatory breast cancer (IBC) vs non-IBC.

BMC Cancer 2020 May 18;20(1):430. Epub 2020 May 18.

Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA.

Background: The purpose of this study was to determine the prognostic role of hormone receptor (HR) on inflammatory breast cancer (IBC) to elucidate its aggressive biological behavior.

Methods: We evaluated the expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemical staining and determined the predictive and prognostic role of HR expression on 189 patients with HR+/HER2- IBC and 677 patients with HR+/HER2- stage III non-IBC. Furthermore, we performed gene expression (GE) analyses on 137 patients with HR+/HER2- IBC and 252 patients with HR+/HER2- non-IBC to detect genes that are specifically overexpressed in IBC.

Results: The expression of ER% was significantly associated with longer distant disease-free survival and overall survival. However, there was no significant relationship between ER% and neoadjuvant chemotherapy outcome. In the GE study, 84 genes were identified as significantly distinguishing HR+ IBC from non-IBC. Among the top 15 canonical pathways expressed in IBC, the ERK/MAPK, PDGF, insulin receptor, and IL-7 signaling pathways were associated with the ER signaling pathway. Upregulation of the MYC gene was observed in three of these four pathways. Furthermore, HR+/HER2- IBC had significantly higher MYC amplification, and the genetic alteration was associated with poor survival outcome.

Conclusions: Higher ER expression was significantly associated with improved survival in both HR+/HER2- IBC and HR+/HER2- stage III non-IBC patients. HR+/HER2- IBC had several activated pathways with MYC upregulation, and the genetic alteration was associated with poor survival outcome. The results indicate that MYC may be a key gene for understanding the biology of HR+/HER2- IBC.
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http://dx.doi.org/10.1186/s12885-020-06940-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236459PMC
May 2020

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

J Pathol 2020 05 24;251(1):63-73. Epub 2020 Mar 24.

Institute of Cancer and Genomic Sciences, The University of Birmingham, Birmingham, UK.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6β1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5415DOI Listing
May 2020

Inflammatory breast cancer cells are characterized by abrogated TGFβ1-dependent cell motility and SMAD3 activity.

Breast Cancer Res Treat 2020 Apr 10;180(2):385-395. Epub 2020 Feb 10.

Center for Oncological Research (CORE), Faculty of Medicine and Health Sciences - University of Antwerp, Campus Drie Eiken - Universiteitsplein 1, 2610, Wilrijk - Antwerp, Belgium.

Purpose: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with elevated metastatic potential, characterized by tumor emboli in dermal and parenchymal lymph vessels. This study has investigated the hypothesis that TGFβ signaling is implicated in the molecular biology of IBC.

Methods: TGFβ1-induced cell motility and gene expression patterns were investigated in three IBC and three non-IBC (nIBC) cell lines. Tissue samples from IBC and nIBC patients were investigated for the expression of nuclear SMAD2, SMAD3, and SMAD4. SMAD protein levels were related to gene expression data.

Results: TGFβ1-induced cell motility was strongly abrogated in IBC cells (P = 0.003). Genes differentially expressed between IBC and nIBC cells post TGFβ1 exposure revealed attenuated expression of SMAD3 transcriptional regulators, but overexpression of MYC target genes in IBC. IBC patient samples demonstrated a near absence of SMAD3 and -4 expression in the primary tumor compared to nIBC patient samples (P < 0.001) and a further reduction of staining intensity in tumor emboli. Integration of gene and protein expression data revealed that a substantial fraction of the IBC signature genes correlated with SMAD3 and these genes are indicative of attenuated SMAD3 signaling in IBC.

Conclusion: We demonstrate attenuated SMAD3 transcriptional activity and SMAD protein expression in IBC, together with obliterated TGFβ1-induced IBC cell motility. The further reduction of nuclear SMAD expression levels in tumor emboli suggests that the activity of these transcription factors is involved in the metastatic dissemination of IBC cells, possibly by enabling collective invasion after partial EMT.
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http://dx.doi.org/10.1007/s10549-020-05571-zDOI Listing
April 2020

Comparative Profiling of Metastatic 4T1- vs. Non-metastatic Py230-Based Mammary Tumors in an Intraductal Model for Triple-Negative Breast Cancer.

Front Immunol 2019 17;10:2928. Epub 2019 Dec 17.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

The transition of ductal carcinoma (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models.
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http://dx.doi.org/10.3389/fimmu.2019.02928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927949PMC
November 2020

NOTCH and DNA repair pathways are more frequently targeted by genomic alterations in inflammatory than in non-inflammatory breast cancers.

Mol Oncol 2020 03 5;14(3):504-519. Epub 2020 Feb 5.

Translational Cancer Research Unit and Center for Oncological Research (CORE), Faculty of Medicine and Health Sciences, GZA Hospitals Sint-Augustinus and University of Antwerp Wilrijk, Antwerp, Belgium.

Inflammatory breast cancer (IBC) is the most pro-metastatic form of breast cancer. Better understanding of its pathophysiology and identification of actionable genetic alterations (AGAs) are crucial to improve systemic treatment. We aimed to define the DNA profiles of IBC vs noninflammatory breast cancer (non-IBC) clinical samples in terms of copy number alterations (CNAs), mutations, and AGAs. We applied targeted next-generation sequencing (tNGS) and array-comparative genomic hybridization (aCGH) to 57 IBC and 50 non-IBC samples and pooled these data with four public datasets profiled using NGS and aCGH, leading to a total of 101 IBC and 2351 non-IBC untreated primary tumors. The respective percentages of each molecular subtype [hormone receptor-positive (HR+)/HER2-, HER2+, and triple-negative] were 68%, 15%, and 17% in non-IBC vs 25%, 35%, and 40% in IBC. The comparisons were adjusted for both the molecular subtypes and the American Joint Committee on Cancer (AJCC) stage. The 10 most frequently altered genes in IBCs were TP53 (63%), HER2/ERBB2 (30%), MYC (27%), PIK3CA (21%), BRCA2 (14%), CCND1 (13%), GATA3 (13%), NOTCH1 (12%), FGFR1 (11%), and ARID1A (10%). The tumor mutational burden was higher in IBC than in non-IBC. We identified 96 genes with an alteration frequency (p < 5% and q < 20%) different between IBC and non-IBC, independently from the molecular subtypes and AJCC stage; 95 were more frequently altered in IBC, including TP53, genes involved in the DNA repair (BRCA2) and NOTCH pathways, and one (PIK3CA) was more frequently altered in non-IBC. Ninety-seven percent of IBCs displayed at least one AGA. This percentage was higher than in non-IBC (87%), notably for drugs targeting DNA repair, NOTCH signaling, and CDK4/6, whose pathways were more frequently altered (DNA repair) or activated (NOTCH and CDK4/6) in IBC than in non-IBC. The genomic landscape of IBC is different from that of non-IBC. Enriched AGAs in IBC may explain its aggressiveness and provide clinically relevant targets.
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http://dx.doi.org/10.1002/1878-0261.12621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053236PMC
March 2020

Pathological features of vessel co-option versus sprouting angiogenesis.

Angiogenesis 2020 02 26;23(1):43-54. Epub 2019 Oct 26.

Translational Cancer Research Unit, GZA Hospitals, Sint-Augustinus, Antwerp, Belgium.

Cancer cells can use existing blood vessels to acquire a vasculature. This process is termed 'vessel co-option'. Vessel co-option is an alternative to the growth of new blood vessels, or angiogenesis, and is adopted by a wide range of human tumour types growing within numerous tissues. A complementary aspect of this process is extravascular migratory tumour spread using the co-opted blood vessels as a trail. Vessel co-opting tumours can be discriminated from angiogenic tumours by specific morphological features. These features give rise to distinct histopathological growth patterns that reflect the interaction of cancer cells with the microenvironment of the organ in which they thrive. We will discuss the histopathological growth patterns of vessel co-option in the brain, the liver and the lungs. The review will also highlight evidence for the potential clinical value of the histopathological growth patterns of cancer. Vessel co-option can affect patient outcomes and resistance to cancer treatment. Insight into the biological drivers of this process of tumour vascularization will yield novel therapeutic strategies.
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http://dx.doi.org/10.1007/s10456-019-09690-0DOI Listing
February 2020

HER-2 status of circulating tumor cells in a metastatic breast cancer cohort: A comparative study on characterization techniques.

PLoS One 2019 4;14(9):e0220906. Epub 2019 Sep 4.

Centre for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium.

Background: Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categories have been described. Major questions in CTC research are how these IF categories reflect gene expression and amplification, and if we should consider 'medium' HER-2 expressing CTCs for patient selection.

Methods: Tumor cells from spiked cell lines (n = 8) and CTCs (n = 116 samples) of 85 metastatic breast cancer patients were enriched using CellSearch. Comparative analysis of HER-2 expression by IF imaging (ACCEPT, DEPArray, and visual scoring) with qRT-PCR and HER-2/neu FISH was performed.

Results: Automated IF HER-2-profiling by DEPArray and ACCEPT delivered comparable results. There was a 98% agreement between 17 trained observers (visual scoring) and ACCEPT considering HER-2neg and HER-2high expressing CTCs. However, 89% of HER-2med expressing CTCs by ACCEPT were scored negative by observers. HER-2high expressing tumor cells demonstrated HER-2/neu gene amplification, whereas HER-2neg and HER-2med expressing tumor cells and CTCs by ACCEPT were copy-number neutral. All patients with HER-2-positive archival tumors had ≥1 HER-2high expressing CTCs, while 80% of HER-2-negative patients did not. High relative gene expression of HER-2 measured on enriched CTC lysates correlated with having ≥1 HER-2high expressing CTCs.

Conclusion: Automated images analysis has enormous potential for clinical implementation. HER-2 characterization and clinical trial design should be focused on HER-2high expressing CTCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220906PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726188PMC
March 2020

Identification of Macrophage Genotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome.

Stem Cells Int 2019 2;2019:9545261. Epub 2019 May 2.

Laboratory of Cellular and Molecular Cardiology, Antwerp University Hospital, Edegem, Belgium.

Background: Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC.

Methods And Results: Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CAC). Genes and pathways of interest were further evaluated using qPCR comparing CAC versus CD14 monocytic cells. The CAC transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CAC was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CAC, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation.

Conclusions: CAC are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation.
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http://dx.doi.org/10.1155/2019/9545261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525806PMC
May 2019

The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.

Crit Rev Oncol Hematol 2019 Feb 19;134:39-45. Epub 2018 Dec 19.

Department of Tumor Biology, Center of Experimental Medicine, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

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 IV, those with < 5 CTCs as Stage IV Survival was analyzed using Kaplan-Meier curves and the log rank test.

Results: For all patients, Stage IV patients had longer median overall survival than those with Stage IV (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IV vs. 18.7 months Stage IV, p < 0.0001). Moreover, patients with Stage IV 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 IV and Stage IV, 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.
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http://dx.doi.org/10.1016/j.critrevonc.2018.12.004DOI Listing
February 2019

The circular RNome of primary breast cancer.

Genome Res 2019 03 28;29(3):356-366. Epub 2019 Jan 28.

Erasmus MC Cancer Institute and Cancer Genomics Netherlands, University Medical Center Rotterdam, Department of Medical Oncology, 3015GD Rotterdam, the Netherlands.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative ( < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of , , and in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.
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http://dx.doi.org/10.1101/gr.238121.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396421PMC
March 2019

Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer.

Breast Cancer Res 2019 01 23;21(1):11. Epub 2019 Jan 23.

Metastasis Research Laboratory, GIGA-Cancer, University of Liège (ULiège), Pathology Tour, +4 level, Building 23, Avenue Hippocrate 13, 4000, Liège, Belgium.

Background: Elevated aerobic glycolysis rate is a biochemical alteration associated with malignant transformation and cancer progression. This metabolic shift unavoidably generates methylglyoxal (MG), a potent inducer of dicarbonyl stress through the formation of advanced glycation end products (AGEs). We have previously shown that the silencing of glyoxalase 1 (GLO1), the main MG detoxifying enzyme, generates endogenous dicarbonyl stress resulting in enhanced growth and metastasis in vivo. However, the molecular mechanisms through which MG stress promotes metastasis development remain to be unveiled.

Methods: In this study, we used RNA sequencing analysis to investigate gene-expression profiling of GLO1-depleted breast cancer cells and we validated the regulated expression of selected genes of interest by RT-qPCR. Using in vitro and in vivo assays, we demonstrated the acquisition of a pro-metastatic phenotype related to dicarbonyl stress in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cellular models. Hyperactivation of MEK/ERK/SMAD1 pathway was evidenced using western blotting upon endogenous MG stress and exogenous MG treatment conditions. MEK and SMAD1 regulation of MG pro-metastatic signature genes in breast cancer cells was demonstrated by RT-qPCR.

Results: High-throughput transcriptome profiling of GLO1-depleted breast cancer cells highlighted a pro-metastatic signature that establishes novel connections between MG dicarbonyl stress, extracellular matrix (ECM) remodeling by neoplastic cells and enhanced cell migration. Mechanistically, we showed that these metastasis-related processes are functionally linked to MEK/ERK/SMAD1 cascade activation in breast cancer cells. We showed that sustained MEK/ERK activation in GLO1-depleted cells notably occurred through the down-regulation of the expression of dual specificity phosphatases in MG-stressed breast cancer cells. The use of carnosine and aminoguanidine, two potent MG scavengers, reversed MG stress effects in in vitro and in vivo experimental settings.

Conclusions: These results uncover for the first time the key role of MG dicarbonyl stress in the induction of ECM remodeling and the activation of migratory signaling pathways, both in favor of enhanced metastatic dissemination of breast cancer cells. Importantly, the efficient inhibition of mitogen-activated protein kinase (MAPK) signaling using MG scavengers further emphasizes the need to investigate their therapeutic potential across different malignancies.
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http://dx.doi.org/10.1186/s13058-018-1095-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343302PMC
January 2019

Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

Nature 2019 02;566(7742):E1

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected.
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http://dx.doi.org/10.1038/s41586-019-0883-2DOI Listing
February 2019

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis.

Genome Med 2018 11 21;10(1):85. Epub 2018 Nov 21.

Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.

Background: There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited.

Methods: A combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients.

Results: ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays.

Conclusions: ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.
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http://dx.doi.org/10.1186/s13073-018-0595-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247769PMC
November 2018

Rates of immune cell infiltration in patients with triple-negative breast cancer by molecular subtype.

PLoS One 2018 12;13(10):e0204513. Epub 2018 Oct 12.

Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) are associated with improved survival. Lehmann et al. identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)], and an immunomodulatory (IM) gene expression signature indicates the presence of TILs and modifies these subtypes. The association between TNBC subtype and TILs is not known. Also, the association between inflammatory breast cancer (IBC) and the presence of TILs is not known. Therefore, we studied the IM subtype distribution among different TNBC subtypes. We retrospectively analyzed patients with TNBC from the World IBC Consortium dataset. The molecular subtype and the IM signature [positive (IM+) or negative (IM-)] were analyzed. Fisher's exact test was used to analyze the distribution of positivity for the IM signature according to the TNBC molecular subtype and IBC status. There were 88 patients with TNBC in the dataset, and among them 39 patients (44%) had IBC and 49 (56%) had non-IBC. The frequency of IM+ cases differed by TNBC subtype (p = 0.001). The frequency of IM+ cases by subtype was as follows: BL1, 48% (14/29); BL2, 30% (3/10); LAR, 18% (3/17); and M, 0% (0/21) (in 11 patients, the subtype could not be determined). The frequency of IM+ cases did not differ between patients with IBC and non-IBC (23% and 33%, respectively; p = 0.35). In conclusion, the IM signature representing the underlying molecular correlate of TILs in the tumor may differ by TNBC subtype but not by IBC status.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204513PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193579PMC
March 2019

Outperforms Other Androgen Receptor Biomarkers to Predict Abiraterone or Enzalutamide Outcome in Metastatic Castration-Resistant Prostate Cancer.

Clin Cancer Res 2019 03 12;25(6):1766-1773. Epub 2018 Sep 12.

Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium.

Purpose: To infer the prognostic value of simultaneous androgen receptor () and profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of signaling inhibitors (ARSi). Between March 2014 and April 2017, we recruited patients with mCRPC ( = 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 splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of and 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 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. inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; = 0.008], and outperformed ARV expression and detection of genomic alterations. Using Cox coefficient analysis of clinical parameters and status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; < 0.0001), which was validated in an independent mCRPC cohort ( = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; < 0.0001).

Conclusions: In an all-comer cohort, tumor burden estimates and outperform any perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-1943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330086PMC
March 2019

A phase I dose-escalation trial of stereotactic ablative body radiotherapy for non-spine bone and lymph node metastases (DESTROY-trial).

Radiat Oncol 2018 Aug 20;13(1):152. Epub 2018 Aug 20.

Department of Radiation Oncology, Iridium Cancer Network, Wilrijk, Antwerp, Belgium.

Background: In an oligometastatic setting, metastasis-directed treatment could render patients disease free, possibly for a protracted interval. Stereotactic ablative radiotherapy (SABR) is one of the treatment modalities that can be offered to these patients. In addition, the radiobiological qualities of SABR are promising for the use in perceived radioresistant tumours. There is also emerging evidence that SABR can stimulate the immune response, and a specific therapeutic window may exist for the optimal use of radiotherapy as an immune adjuvant. However, when SABR is considered for non-spine bone or lymph node metastases, the optimal fractionation schedule is not yet known.

Methods: The DESTROY-trial is a non-randomized prospective phase I trial determining a regimen of choice for patients with non-spine bone and lymph node metastases. A total of 90 patients will be included in three different treatment regimens. They will be offered stereotactic ablative radiotherapy in 5, 3 or 1 fractions. Dose-limiting toxicity will be recorded as primary endpoint. Acute and late toxicity, local response and local recurrence, and progression-free survival are secondary endpoints. Liquid biopsies will be collected throughout the course of this study from the second fractionation schedule on.

Discussion: Despite its almost universal use in (oligo-)metastatic patients, the level of evidence supporting radical local treatment in general, and stereotactic radiotherapy in particular, is low. This prospective phase I trial will evaluate different SABR regimens for metastases and the differences in immune-stimulatory effects.

Trial Registration: The Ethics committee of the GZA Hospitals (B099201732915) approved this study on 05/07/2017. Amendment for translational research was approved on 06/02/2018. Trial registered on Clinicaltrials.gov ( NCT03486431 ) on 03/04/2018 - Retrospectively registered.
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http://dx.doi.org/10.1186/s13014-018-1096-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102883PMC
August 2018

Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer.

J Exp Clin Cancer Res 2018 Aug 15;37(1):191. Epub 2018 Aug 15.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

Background: Murine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.

Methods: Intraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.

Results: Mice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.

Conclusions: The current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.
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http://dx.doi.org/10.1186/s13046-018-0860-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094904PMC
August 2018

Multidisciplinary molecular tumour board: a tool to improve clinical practice and selection accrual for clinical trials in patients with cancer.

ESMO Open 2018 23;3(5):e000398. Epub 2018 Jul 23.

Phase I, Early Clinical Trials Unit, Oncology, Universitair Ziekenhuis Antwerpen, Edegem, Belgium.

Background: 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.

Materials And Methods: Patients diagnosed with solid tumours progressing to standard treatments were referred to our Phase I unit. They underwent comprehensive next generation sequencing (NGS) of either tumour tissue or cell-free circulating tumour DNA (ctDNA) or both. The MTB expressed either a positive or negative opinion for the treatment of the patients with discovered druggable alterations inside a clinical trial, in an expanded access programme, with a compassionate use. Afterwards, discovered alterations were matched with OncoKB levels of evidence for the choice of alteration-specific treatments in order to compare MTB outcomes with a standardised set of recommendations.

Results: NGS was performed either on ctDNA or tumour tissue or in both of them in 204 patients. The MTB evaluated 173 of these cases. Overall, the MTB proposed alteration-specific targeted therapy to 72 patients (41.6%). 49 patients (28.3% of the total evaluated) were indicated to enter a clinical trial. In 29 patients with matched liquid biopsy NGS (lbNGS), tumour tissue NGS (ttNGS) and MTB evaluation, the MTB changed the treatment strategy coming from standardised recommendations based on lbNGS and ttNGS alone in 10 patients (34.5%), thanks to the evaluation of other clinical parameters. In our cohort, lbNGS was more likely, compared with ttNGS, to detect point mutations (OR 11, 95% CI 2.9 to 24.1, p<0.001) and all-type alterations (OR 13.6, 95% CI 5.5 to 43.2, p<0.001) from the same genes of matched patients.

Conclusions: Our MTB allows patients with refractory cancer to be included in clinical trials and improves the precision of clinical decisions compared with a standardised set of mutation-driven recommendations.
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http://dx.doi.org/10.1136/esmoopen-2018-000398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069914PMC
July 2018

Potential new biomarkers for squamous carcinoma of the uterine cervix.

ESMO Open 2018 28;3(4):e000352. Epub 2018 Jun 28.

Centre of Oncologic Research (CORE) Antwerp University, Edegem, Belgium.

Aim: An in silico pathway analysis was performed in an attempt to identify new biomarkers for cervical carcinoma.

Methods: Three publicly available Affymetrix gene expression data sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total 9 cervical cancer cell lines, 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples. An Agilent data set (GSE7410; 5 normal cervical samples, 35 samples from invasive cervical cancer) was selected as a validation set. Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. We compared the lists of differentially expressed genes between normal and CIN3 samples on the one hand (n=1923) and between CIN3 and invasive cancer samples on the other hand (n=628).

Results: Seven probe sets were identified that were significantly overexpressed (at least 2 fold increase expression level, and false discovery rate <5%) in both CIN3 samples respective to normal samples and in cancer samples respective to CIN3 samples. From these, five probes sets could be validated in the Agilent data set (P<0.001) comparing the normal with the invasive cancer samples, corresponding to the genes DTL, HMGB3, KIF2C, NEK2 and RFC4. These genes were additionally overexpressed in cervical cancer cell lines respective to the cancer samples. The literature on these markers was reviewed.

Conclusion: Novel biomarkers in combination with primary human papilloma virus (HPV) testing may allow complete cervical screening by objective, non-morphological molecular methods, which may be particularly important in developing countries.
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http://dx.doi.org/10.1136/esmoopen-2018-000352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045706PMC
June 2018

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

Breast Cancer Res Treat 2018 Sep 1;171(2):283-293. Epub 2018 Jun 1.

Departments of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Introduction: We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype.

Materials And Methods: Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44CD49fCD133/2 mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples.

Results: 8 of 8 IBC samples expressed isolated CD44CD49fCD133/2 stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44CD49fCD133/2 cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001).

Conclusions: Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.
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http://dx.doi.org/10.1007/s10549-018-4835-6DOI Listing
September 2018

Scientific Summary from the Morgan Welch MD Anderson Cancer Center Inflammatory Breast Cancer (IBC) Program 10 Anniversary Conference.

J Cancer 2017 9;8(17):3607-3614. Epub 2017 Oct 9.

MD Anderson Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX.

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10 Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.
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http://dx.doi.org/10.7150/jca.21200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687177PMC
October 2017

MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation.

PLoS One 2018 23;13(2):e0193173. Epub 2018 Feb 23.

Laboratory of Cellular and Molecular Cardiology, Research Group Cardiovascular Diseases, Department of Translational Pathophysiological Research, University of Antwerp, Antwerp, Belgium.

MicroRNA (miRNA) regulate gene expression through posttranscriptional mRNA degradation or suppression of translation. Many (pre)analytical issues remain to be resolved for miRNA screening with TaqMan Low Density Arrays (TLDA) in plasma samples, such as optimal RNA isolation, preamplification and data normalization. We optimized the TLDA protocol using three RNA isolation protocols and preamplification dilutions. By using 100μL elution volume during RNA isolation and adding a preamplification step without dilution, 49% of wells were amplified. Informative target miRNA were defined as having quantification cycle values ≤35 in at least 20% of samples and low technical variability (CV across 2 duplicates of 1 sample <4%). A total of 218 miRNA was considered informative (= 59% of all target miRNA). Different normalization strategies were compared: exogenous Ath-miR-159a, endogenous RNA U6, and three mathematical normalization techniques: geNorm (Qbase, QB) and NormFinder (NF) normalization algorithms, and global mean calculation. To select the best normalization method, technical variability, biological variability, stability, and the extent to which the normalization method reduces data dispersion were calculated. The geNorm normalization algorithm reduced data dispersion to the greatest extent, while endogenous RNA U6 performed worst. In conclusion, for miRNA profiling in plasma samples using TLDA cards we recommend: 1. Implementing a preamplification step in the TLDA protocol without diluting the final preamplification product 2. A stepwise approach to exclude non-informative miRNA based on quality control parameters 3. Against using snoRNA U6 as normalization method for relative quantification 4. Using the geNorm algorithm as normalization method for relative quantification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193173PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825041PMC
May 2018

Circulating tumor cells and survival in abiraterone- and enzalutamide-treated patients with castration-resistant prostate cancer.

Prostate 2018 05 12;78(6):435-445. Epub 2018 Feb 12.

Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium.

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 <5 or ≥5 CTCs/7.5 mL, whereas for the analysis of CTC dynamics at 10-12 weeks, in patients with stable, increasing or decreasing CTC counts. Progression-free survival (PFS), overall survival (OS), and PSA changes at 10-12 weeks were compared between groups.

Results: Patients demonstrating increasing CTCs on therapy had a shorter median PFS (4.03 vs 12.98 vs 13.67 months, HR 3.6, 95%CI 1.9-6.8; P < 0.0001) and OS (11.2 months vs not reached, HR 9.5, 95%CI 3.7-24; P < 0.0001), compared to patients with decreasing or stable CTCs. Multivariable Cox regression showed that prior chemotherapy (HR 4.1, 95%CI 1.9-8.9; P = 0.0003), a high baseline CTC count (HR 1.5, 95%CI 1.2-1.9; P = 0.002) and increasing CTCs at follow-up (HR 3.3, 95%CI 1.4-7.6; P = 0.005) were independent predictors of worse PFS. Previous chemotherapy (HR 7, 95%CI 1.9-25; P = 0.003), high baseline CTC counts (HR 2.2, 95%CI 1.4-3.7; P = 0.002) and increasing CTCs during therapy (HR 4.6, 95%CI 1.4-15; P = 0.01) were independently associated with shorter OS. ≥30% and ≥50% PSA responses less frequently occurred in patients with CTC inclines at 10-12 weeks on therapy (χ test: P < 0.01).

Conclusions: CTC dynamics during therapy are associated with PSA response and provide independent clinical prognostication over PSA declines. Hence the study demonstrates the pharmacodynamic properties of CTCs.
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http://dx.doi.org/10.1002/pros.23488DOI Listing
May 2018