Supplementary Table 1 Downregulation of HER3 membrane protein expression in skin biopsies following treatment with lumretuzumab and cetuximab or erlotinib Supplementary Table 2 Change of tumor biomarkers in tumor biopsies following treatment with lumretuzumab and cetuximab or erlotinib

Supplementary Figure 1 Quantile plots of baseline membrane HER3 IRS score (A) and HER3 mRNA log expression (B) in baseline FFPE tumor biopsy samples from patients with known adeno- and squamous cell carcinoma histology Supplementary Figure 2 Quantile plots of baseline membrane HER3 immuno-reactive scores in fresh FFPE tumor biopsies from NSCLC patients with non-squamous or squamous histology Supplementary Figure 3 Quantile plots of baseline HRG mRNA log expression determined by qRT-PCR in FFPE tumor biopsies of adeno- and squamous cell carcinoma histology (data obtained using the HRG research assay) Supplementary Figure 4 Baseline HRG mRNA log expression in FFPE tumor biopsies, determined by qRT-PCR, in non-sqNSCLC and sqNSCLC (data obtained using the HRG prototype diagnostic assay). Dotted line indicates prior determined median HRG expression in sqNSCLC. Supplementary Figure 5 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation and extension phase of the cetuximab part (n = 38). Red bars indicate PD patients, blue bars SD patients, light green bars PR patients and dark green bars CR patients. Eleven patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates a patient who received prior trastuzumab therapy. Supplementary Figure 6 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation phase and the two NRG1 fusion gene patients of the extension phase of the erlotinib part (n = 31). Red bars indicate PD patients, blue bars SD patients and green bars PR patients. Eight patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates two patients with NRG1 gene fusion.

Figure 1 Mean ({plus minus}SD) serum lumretuzumab profiles following multiple ascending doses from 100 mg up to 2000 mg Figure 2 Percentage change in standardized uptake value maximum from baseline as assessed by FDG-PET at (a) Cycle 1 Day 14 and (b) Cycle 4 Day 14 Table 1 Summary of baseline and change in HER3 expression measured by IHC in skin and tumor biopsy samples pre and post treatment with lumretuzumab Table 2 Summary of change in expression of HER3, HER2, EGFR and cMET in fresh tumor biopsies compared to primary archival samples Table 3 Peripheral blood immunophenotyping (T, B and NK cells and NK subsets) from patients dosed with 2000 mg lumretuzumab Table 4 Ex-vivo activation potential of peripheral NK lymphocytes for all patients at ba seline and following exposure to lumretuzumab in the 2000 mg dose cohort Table 5 Summary of tumor immune effector cell infiltration Table 6 Tumor response to treatment (RECIST)

Background This phase I, open-label, dose-escalation study evaluated the safety, pharmacokinetics and pharmacodynamics of combination therapy with the HDM2 inhibitor SAR405838 and the MEK1/2 inhibitor pimasertib administered orally once daily (QD) or twice daily (BID) in locally advanced or metastatic solid tumours (NCT01985191). Methods Patients with locally advanced or metastatic solid tumours with documented wild-type TP53 and RAS or RAF mutations were enroled. A 3 + 3 dose-escalation design was employed. The primary objective was to assess maximum tolerated dose (MTD). Results Twenty-six patients were treated with SAR405838 200 or 300 mg QD plus pimasertib 60 mg QD or 45 mg BID. The MTD was SAR405838 200 mg QD plus pimasertib 45 mg BID. The most common dose-limiting toxicity was thrombocytopenia. The most frequently occurring treatment-related adverse events were diarrhoea (81%), increased blood creatine phosphokinase (77%), nausea (62%) and vomiting (62%). No significant drug–drug interactions were observed. The biomarkers MIC-1 and pERK were, respectively, upregulated and downregulated in response to study treatment. In 24 efficacy-evaluable patients, one patient (4%) had a partial response and 63% had stable disease. Conclusions The safety profile of SAR405838 and pimasertib combined was consistent with the safety profiles of both drugs. Preliminary antitumour activity was observed.

Adverse events (AEs) of epidermal growth factor inhibitors (EGFRi) influence well-being with a risk to dose modifications (DMs). Hereby, clinical benefit of treatment might be affected. This retrospective cohort study was set up to gain insight into the suitability and added value of a patient-reported outcome measurement tool (PROM), together with a stepwise intervention management plan for EGFRi-related AEs in daily practice. The primary objective was to gain insight into total treatment duration and DMs, and the secondary objective to gain insight into patient-reported symptoms and well-being as well as healthcare professional-reported AEs. Sixty-eight patients on cetuximab and 19 on panitumumab treatment were included for analysis; 69% had squamous cell carcinoma of head and neck (SCCHN) and 26% metastatic colorectal carcinoma. DMs due to AEs occurred in 39% of the patients and dose discontinuations in 22%. Especially anorexia, dysphagia, oral pain and skin changes led to a decreased well-being. In patients on EGFRi, application of PROMs together with a stepwise symptom management plan enhances early recognition of symptom burden, pro-active symptom management and effect evaluation of interventions performed whereby well-being recovers. Since only SCCHN patients discontinued treatment due to AEs, patient-centred care focused on radiotherapy-related AEs, creates opportunities for amelioration.

Purpose: In tumours with wild-type TP53, the tumour-suppressive function of p53 is frequently inhibited by HDM2. This phase I, dose-escalating study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics and pharmacodynamics of SAR405838, an HDM2 inhibitor, in patients with advanced solid tumours (NCT01636479).Methods: In dose escalation, patients with any locally advanced/metastatic solid tumour with TP53 mutation prevalence below 40%, or documented as TP53 wild-type, were eligible. In the MTD expansion cohort, only patients with de-differentiated liposarcoma were included. Primary end-points were MTD and efficacy in the MTD expansion cohort. Secondary end-points included safety, pharmacokinetics and pharmacodynamics biomarkers.Results: Seventy-four patients were treated with SAR405838 (50-800 mg once daily [QD], 800-1800 mg weekly and 1800 mg twice weekly). Two patients treated with SAR405838 400 mg QD had thrombocytopaenia as a dose-limiting toxicity (DLT). The MTD for the QD schedule of SAR405838 was 300 mg QD. No DLTs were observed with the weekly schedule; one patient had a DLT of nausea with the 1800 mg twice-weekly dose. Treatment with SAR405838 was associated with increased plasma MIC-1, reflecting p53 pathway activation. In the de-differentiated liposarcoma MTD cohort, 89% of the patients had HDM2 amplification at baseline and no TP53 mutations were observed; best response was stable disease in 56% and progression free rate at 3 months was 32%.Conclusion: SAR405838 had an acceptable safety profile with limited activity in patients with advanced solid tumours. The MID of SAR405838 was 300 mg QD; MTD was not reached with the weekly schedule. (C) 2017 Elsevier Ltd. All rights reserved.

BACKGROUND:In this study, our aim was to identify molecular aberrations predictive for response to everolimus, an mTOR inhibitor, regardless of tumor type.METHODS:To generate hypotheses about potential markers for sensitivity to mTOR inhibition, drug sensitivity and genomic profiles of 835 cell lines were analyzed. Subsequently, a multicenter study was conducted. Patients with advanced solid tumors lacking standard of care treatment options were included and underwent a pre-treatment tumor biopsy to enable DNA sequencing of 1,977 genes, derive copy number profiles and determine activation status of pS6 and pERK. Treatment benefit was determined according to TTP ratio and RECIST. We tested for associations between treatment benefit and single molecular aberrations, clusters of aberrations and pathway perturbation.RESULTS:Cell line screens indicated several genes, such as PTEN (P = 0.016; Wald test), to be associated with sensitivity to mTOR inhibition. Subsequently 73 patients were included, of which 59 started treatment with everolimus. Response and molecular data were available from 43 patients. PTEN aberrations, i.e. copy number loss or mutation, were associated with treatment benefit (P = 0.046; Fisher's exact test).CONCLUSION:Loss-of-function aberrations in PTEN potentially represent a tumor type agnostic biomarker for benefit from everolimus and warrants further confirmation in subsequent studies.

Background: Due to rapid technical advances, steeply declining sequencing costs, and the ever-increasing number of targeted therapies, it can be expected that extensive tumor sequencing such as whole-exome and whole-genome sequencing will soon be applied in standard care. Clinicians will thus be confronted with increasingly complex genetic information and multiple test-platforms to choose from. General medical training, meanwhile, can hardly keep up with the pace of innovation. Consequently, there is a rapidly growing gap between clinical knowledge and genetic potential in cancer care. Multidisciplinary Molecular Tumor Boards (MTBs) have been suggested as a means to address this disparity, but shared experiences are scarce in literature and no quality requirements or guidelines have been published to date. Methods: Based on literature review, a survey among hospitals in The Netherlands, and our own experience with the establishment of a nationally operating MTB, this article evaluates current knowledge and unmet needs and lays out a strategy for successful MTB implementation. Results: Having access to an MTB can improve and increase the application of genetics-guided cancer care. In our survey, however, <50% of hospitals and only 5% of nonacademic hospitals had access to an MTB. In addition, current MTBs vary widely in terms of composition, tasks, tools, and workflow. This may not only lead to variation in quality of care but also hinders data sharing and thus creation of an effective learning community. Conclusions: This article acknowledges a leading role for MTBs to govern (extensive) tumor sequencing into daily practice and proposes three basic necessities for successful MTB implementation: (i) global harmonization in cancer sequencing practices and procedures, (ii) minimal member and operational requirements, and (iii) an appropriate unsolicited findings policy. Meeting these prerequisites would not only optimize MTB functioning but also improve general interpretation and application of genomics-guided cancer care.

Abstract Purpose: This study investigated the safety, clinical activity, and target-associated biomarkers of lumretuzumab, a humanized, glycoengineered, anti-HER3 monoclonal antibody (mAb), in combination with the EGFR-blocking agents erlotinib or cetuximab in patients with advanced HER3-positive carcinomas. Experimental Design: The study included two parts: dose escalation and dose extension phases with lumretuzumab in combination with either cetuximab or erlotinib, respectively. In both parts, patients received lumretuzumab doses from 400 to 2,000 mg plus cetuximab or erlotinib according to standard posology, respectively. The effect of HRG mRNA and HER3 mRNA and protein expression were investigated in a dedicated extension cohort of squamous non–small cell lung cancer (sqNSCLC) patients treated with lumretuzumab and erlotinib. Results: Altogether, 120 patients were treated. One dose-limiting toxicity (DLT) in the cetuximab part and two DLTs in the erlotinib part were reported. The most frequent adverse events were gastrointestinal and skin toxicities, which were manageable. The objective response rate (ORR) was 6.1% in the cetuximab part and 4.2% in the erlotinib part. In the sqNSCLC extension cohort of the erlotinib part, higher tumor HRG and HER3 mRNA levels were associated with a numerically higher disease control rate but not ORR. Conclusions: The toxicity profile of lumretuzumab in combination with cetuximab and erlotinib was manageable, but only modest clinical activity was observed across tumor types. In the sqNSCLC cohort, there was no evidence of meaningful clinical benefit despite enriching for tumors with higher HRG mRNA expression levels. Clin Cancer Res; 23(18); 5406–15. ©2017 AACR.

Objective: To investigate whether next-generation sequencing (NGS) in ovarian and endometrial tumours can discover mutations with a relevant prognostic or predictive value. Methods: After a literature search, selected studies were critically appraised using the Quality in Prognostic Studies tool. Data on mutation incidence and correlations with prognostic and predictive items were extracted from relevant studies and compared to our own cohort consisting of 28 patients analysed using NGS. Results: Eight out of 739 articles were found eligible, including different tumour types. Prevalence of mutations in the KRAS gene ranged between 5.34 and 58.8% in ovarian cancer. Two studies showed a significant correlation between KRAS mutations and an improved disease free- and overall survival. Clinical data were available for 17 of our patients, mostly cases of endometrial carcinomas. KRAS, PIK3CA, CTNNB1, and TP53were the most frequently mutated genes in endometrial carcinomas, and PTEN and CTNNB1 correlated with a higher FIGO stage. Conclusion: In the ovary KRAS mutation is associated with type I ovarian tumours (low-grade serous, mucinous, endometrioid, and clear-cell) and may seem to have a more favourable prognosis. The prognostic value of TP53 is still controversial. In endometrial tumours, PTEN shows a positive correlation with better prognosis. PIK3CA may have a correlation with poorer prognosis. CTNNB1 mutations in endometrial carcinomas could predict a worse prognosis.

28 EORTC – NCI – AACR Symposium on Molecular Targets and Cancer Therapeutics 29 November 2016 - 02 December 2016

Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. Conclusion Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

BACKGROUND Early signs of efficacy are critical in drug development. Response Evaluation Criteria in Solid Tumors (RECIST) are commonly used to determine the efficacy of anti-cancer therapy in clinical trials. RECIST, however, emphasizes the value of tumor shrinkage, while many targeted agents induce prolonged tumor growth arrest. This limits its use for the detection of treatment efficacy for these more cytostatic regimens. Therefore, we designed an individualized variant of a time to progression (TTP) end point based on prospective volumetric measurements and an intra-patient control, the TTP ratio. PATIENTS AND METHODS Patients with any metastatic malignancy, without regular treatment options, were treated with the mTOR inhibitor everolimus. Treatment response was determined using both RECIST and the TTP ratio. The TTP ratio was defined as the volumetric pretreatment TTP divided by the volumetric on-treatment TTP. A patient was classified as a responder if the TTP ratio was <0.7. Consistency and reproducibility of volumetric measurements were determined. RESULTS Seventy-three patients were included of whom 59 started treatment. A TTP ratio could be established in 73% (n = 43) of the treated patients. The inter-observer agreement for volumetric progression was 0.78 (95% confidence interval 0.70-0.87) (Krippendorff's α-coefficient). According to RECIST, 35 patients (59%) had stable disease (SD) and 1 patient demonstrated a partial response (PR), whereas only 21 patients (36%) met the prespecified criteria for treatment efficacy according to the TTP ratio. Treatment response according to both the TTP ratio and RECIST (SD + PR) correlated with overall survival (OS) [P(log-rank) < 0.001]. The TTP ratio, however, was also able to differentiate which patients had a better OS within the RECIST SD group [P(log-rank) = 0.0496]. CONCLUSION The TTP ratio had a high inter-observer agreement, correlated with OS and identified which patients within the RECIST SD group had a longer OS. CLINICALTRIALSGOV IDENTIFIER NCT01566279.

PURPOSE:Pazopanib is a tyrosine kinase inhibitor approved for the treatment of renal cell carcinoma and soft tissue sarcoma. Retrospective analyses have shown that an increased median progression-free survival and tumor shrinkage appear in patients with higher plasma trough levels (Cmin). Therefore, patients with low Cmin might benefit from pharmacokinetically guided individualized dosing. EXPERIMENTAL DESIGN:We conducted a prospective multicenter trial in 30 patients with advanced solid tumors. Pazopanib Cmin was measured weekly by LC-MS/MS. At weeks 3, 5, and 7, the pazopanib dose was increased if the measured Cmin was <20 mg/L and toxicity was <grade 3. RESULTS:In total, 17 patients had at least one Cmin <20 mg/L at weeks 3, 5, and 7. Of these, 10 were successfully treated with a pharmacokinetically guided dose escalation, leading to daily dosages ranging from 1,000 to 1,800 mg. Cmin in these patients increased significantly from 13.2 (38.0%) mg/L [mean (CV%)] to 22.9 mg/L (44.9%). Thirteen patients had all Cmin levels ≥20.0 mg/L. Of these, 9 patients with a high Cmin of 51.3 mg/L (45.1%) experienced ≥grade 3 toxicity and subsequently required a dose reduction to 600 or 400 mg daily, yet in these patients, Cmin remained above the threshold at 28.2 mg/L (25.3%). CONCLUSIONS:A pharmacokinetically guided individualized dosing algorithm was successfully applied and evaluated. The dosing algorithm led to patients being treated at dosages ranging from 400 to 1,800 mg daily. Further studies are needed to show a benefit of individualized dosing on clinical outcomes, such as progression-free survival. Clin Cancer Res; 22(23); 5738-46. ©2016 AACRSee related commentary by Ornstein and Rini, p. 5626.

BACKGROUND:The discovery of novel biomarkers that predict treatment response in advanced cancer patients requires acquisition of high-quality tumor samples. As cancer evolves over time, tissue is ideally obtained before the start of each treatment. Preferably, samples are freshly frozen to allow analysis by next-generation DNA/RNA sequencing (NGS) but also for making other emerging systematic techniques such as proteomics and metabolomics possible. Here, we describe the first 469 image-guided biopsies collected in a large collaboration in The Netherlands (Center for Personalized Cancer Treatment) and show the utility of these specimens for NGS analysis.PATIENTS AND METHODS:Image-guided tumor biopsies were performed in advanced cancer patients. Samples were fresh frozen, vital tumor cellularity was estimated, and DNA was isolated after macrodissection of tumor-rich areas. Safety of the image-guided biopsy procedures was assessed by reporting of serious adverse events within 14 days after the biopsy procedure.RESULTS:Biopsy procedures were generally well tolerated. Major complications occurred in 2.1%, most frequently consisting of pain. In 7.3% of the percutaneous lung biopsies, pneumothorax requiring drainage occurred. The majority of samples (81%) contained a vital tumor percentage of at least 30%, from which at least 500 ng DNA could be isolated in 91%. Given our preset criteria, 74% of samples were of sufficient quality for biomarker discovery. The NGS results in this cohort were in line with those in other groups.CONCLUSION:Image-guided biopsy procedures for biomarker discovery to enable personalized cancer treatment are safe and feasible and yield a highly valuable biobank. The Oncologist 2017;22:33-40Implications for Practice: This study shows that it is safe to perform image-guided biopsy procedures to obtain fresh frozen tumor samples and that it is feasible to use these biopsies for biomarker discovery purposes in a Dutch multicenter collaboration. From the majority of the samples, sufficient DNA could be yielded to perform next-generation sequencing. These results indicate that the way is paved for consortia to prospectively collect fresh frozen tumor tissue.

AbstractPurpose: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer.Experimental Design: Twenty-five patients with histologically confirmed HER3-expressing tumors received lumretuzumab (100, 200, 400, 800, 1,600, and 2,000 mg) every two weeks (q2w) in 3+3 dose-escalation phase. In addition, 22 patients were enrolled into an extension cohort at 2,000 mg q2w.Results: There were no dose-limiting toxicities. Common adverse events (any grade) included diarrhea (22 patients, 46.8%), fatigue (21 patients, 44.7%), decreased appetite (15 patients, 31.9%), infusion-related reactions (13 patients, 27.7%), and constipation (10 patients, 21.3%). The peak concentration (Cmax) and area under the concentration–time curve up to the last measurable concentration (AUClast) of lumretuzumab increased more than dose proportionally from 100 mg up to 400 mg. Linear PK was observed with doses ≥400 mg q2w indicating target-mediated drug disposition saturation. Downregulation of HER3 membranous protein was observed in on-treatment tumor biopsies from 200 mg, and was maximal at and above 400 mg. An ex vivo assay demonstrated increased activation potential of peripheral NK lymphocytes with lumretuzumab compared with a non-glycoengineered anti-HER3 antibody. Ten patients (21.3%) had stable disease and remained on study at a median of 111 days (range, 80–225 days).Conclusions: Lumretuzumab was well tolerated and showed evidence of clinical activity. Linear serum PK properties and plateauing of PD effects in serial tumor biopsies indicate optimal biologically active doses of lumretuzumab from 400 mg onwards. Clin Cancer Res; 22(4); 877–85. ©2015 AACR.

Purpose: LY2334737 is an orally available prodrug of gemcitabine. The objective of this study was to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of daily administration of LY2334737 with or without erlotinib. Experimental Design: Patients with advanced or metastatic cancer were treated with escalating doses of LY2334737 monotherapy or in combination with continuous daily administration of 100 mg erlotinib. LY2334737 was given once daily for 14 days of a 21-day cycle. The study was extended with a bioequivalence trial to investigate a novel LY2334737 drug formulation. Results: A total of 65 patients were treated in this study. The MTD was 40 mg LY2334737. Fatigue was the most frequent DLT for LY2334737 monotherapy (4 patients) followed by elevated transaminase levels (2 patients), both observed at the 40- to 50-mg dose levels. Among the 10 patients in the combination arm, 2 had DLTs at the 40-mg dose level. These were fatigue and elevated liver enzyme levels. The most common adverse events were fatigue (n = 38), nausea (n = 27), vomiting (n = 24), diarrhea (n = 23), anorexia (n = 20), pyrexia (n = 18), and elevated transaminase levels (n = 14). The pharmacokinetics showed dose proportional increase in LY2334737 and gemcitabine exposure. The metabolite 2′,2′-difluorodeoxyuridine accumulated with an accumulation index of 4.3 (coefficient of variation: 20%). In one patient, complete response in prostate-specific antigen was observed for 4 cycles, and stable disease was achieved in 22 patients overall. Pharmacokinetic analysis showed that the 2 investigated LY2334737 drug formulations were bioequivalent. Conclusions: LY2334737 displays linear pharmacokinetics and the MTD is 40 mg with or without daily administration of 100 mg erlotinib. Signs of antitumor activity warrant further development. Clin Cancer Res; 17(18); 6071–82. ©2011 AACR.

Conventional chemotherapy has shown its effectivity in the treatment of many cancers types. Nevertheless, because of the non-specificity of the agents, this tumor destroying capacity goes hand in hand with damage to the healthy tissues, expressed as unwanted toxicity. The past decades this treatment emerging toxicity seemed to be well managed by standard care strategies. Still, it is desirable to only target the tumor cells, resulting in a more effective therapeutical potential of anti cancer agents and less unwanted side effects. From that point of view, in the treatment of cancer the focus has shifted from general cytotoxic agents to a more cell specific, targeted therapy. A multitude of ‘targeting’ agents has been explored and some of them have already proven their therapeutical activity and are nowadays implemented as standard care for certain cancer types. In this dissertation I focussed on endothelial cells and their role and implications in the treatment of cancer. These cells reconstitute a specific target for therapy as they are the building blocks for the vasculature, and in specific tumor vasculature. Endothelial cells can be inhibited by interference in the vascular endothelial growth factor (VEGF) signalling pathway, a growth factor involved in endothelial cell proliferation, migration and function. VEGF inhibiting agents reconstitute a powerful target for therapy, as single agent therapy, but also as part of combinatorial regimens. Within this spectrum, as having an important function in normal physiology, endothelial cells also play an important role in VEGF inhibition induced toxicity. Contrary to expectations, despite the specificity of this kind of treatment, these endothelial cells play also an important role in normal physiology and this is reflected in a specific toxicity profile. Endothelial cell/ angiogenesis inhibition by blocking VEGF-mediated signaling can result in serious side effects in a substantial proportion of patients. Most of these side effects seem to be manageable, but likely will become more problematic when survival increases. Therefore it is desirable to elucidate the underlying pathophysiological mechanisms, to provide new insights into the pathogenesis of this toxicity in order to manage these side effects effectively. Finally this could result in the development of more specific angiogenesis inhibitors. Although being claimed as promising agents, major encumbrances still exist consisting of the considerable side effects especially when treatment is continued for a longer period, the apparently inevitable resistance to this kind of agents on the long run and the substantial costs of the treatment. For that reason, it is desirable to be able to select only those patients who will benefit from treatment and to recognize resistance. By means of a predictieve biomarker it might be possible to make this selection, and consequently to prevent overtreatment and unneeded toxicity and to early recognize resistance to the drug. The final implication of endothelial cells discussed in this thesis is that they would be candidates for the role as a surrogate biomarker in angiogenesis inhibition. Before this last application can be implemented, general agreement of the true identity and the most proper way to enumerate these cells should be achieved.