Advancing drug discovery using the power of the human genome.

Human genetics plays an increasingly important role in drug development and population health. Here we review the history of human genetics in the context of accelerating the discovery of therapies, present examples of how human genetics evidence supports successful drug targets, and discuss how polygenic risk scores could be beneficial in various clinical settings. We highlight the value of direct-to-consumer platforms in the era of fast-paced big data biotechnology, and how diverse genetic and health data can benefit society. © 2021 23andMe, Inc. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

A death receptor 6-amyloid precursor protein pathway regulates synapse density in the mature CNS but does not contribute to Alzheimer's disease-related pathophysiology in murine models.

Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aß peptides derived from APP. Given the link between APP, Aß, and Alzheimer's disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

LRF-mediated Dll4 repression in erythroblasts is necessary for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.

Pathology in drug discovery and development.

The rapid pace of drug discovery and drug development in oncology, immunology and ophthalmology brings new challenges; the efficient and effective development of new targeted drugs will require more detailed molecular classifications of histologically homogeneous diseases that show heterogeneous clinical outcomes. To this end, single companion diagnostics for specific drugs will be replaced by multiplex diagnostics for entire therapeutic areas, preserving tissue and enabling rapid molecular taxonomy. The field will move away from the development of new molecular entities as single agents, to which resistance is common. Instead, a detailed understanding of the pathological mechanisms of resistance, in patients and in preclinical models, will be key to the validation of scientifically rational and clinically effective drug combinations. To remain at the heart of disease diagnosis and appropriate management, pathologists must evolve into translational biologists and biomarker scientists. Herein, we provide examples of where this metamorphosis has already taken place, in lung cancer and melanoma, where the transformation has yet to begin, in the use of immunotherapies for ophthalmology and oncology, and where there is fertile soil for a revolution in treatment, in efforts to classify glioblastoma and personalize treatment. The challenges of disease heterogeneity, the regulatory environment and adequate tissue are ever present, but these too are being overcome in dedicated academic centres. In summary, the tools necessary to overcome the 'whens' and 'ifs' of the molecular revolution are in the hands of pathologists today; it is a matter of standardization, training and leadership to bring these into routine practice and translate science into patient benefit. This Annual Review Issue of the Journal of Pathology highlights the central role for pathology in modern drug discovery and development.

PAK1 mediates pancreatic cancer cell migration and resistance to MET inhibition.

Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signalling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signalling to cytoskeletal effectors and tumour cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or by direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumour growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDACs (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signalling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.

Predicting benefit from anti-angiogenic agents in malignancy.

A high probability of benefit is desirable to justify the choice of anti-angiogenic therapy from an ever-expanding list of expensive new anticancer agents. However, biomarkers of response to cytotoxic agents are not optimal for predicting benefit from anti-angiogenic drugs. This discussion will focus on both preclinical and clinical research to identify biomarkers for anti-angiogenic therapies that can inform dosing, early clinical benefit, initial drug choice, emerging resistance and second-line treatments.

Targeting p21-activated kinase 1 (PAK1) to induce apoptosis of tumor cells.

p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.

In situ validation of an intestinal stem cell signature in colorectal cancer.

OBJECTIVE: Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer. DESIGN: 19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas. RESULTS: Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance. CONCLUSION: These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.

Impact of MET expression on outcome in BRAF(V600E/K) advanced melanoma.

AIMS: Preclinical data suggest that signalling through the HGF-MET pathway may confer resistance to BRAF inhibition in BRAF(V600E/K) melanoma. Therefore, blockade of HGF-MET signalling might be a valid therapeutic strategy, in combination with BRAF inhibition, in BRAF(V600E/K) melanoma. The aim of this study was to investigate the clinical relevance of these observations by evaluating the survival impact of MET expression in patients with BRAF(V600E/K) advanced melanoma treated with vemurafenib. METHODS AND RESULTS: Formalin-fixed tissue blocks were obtained of tumours from patients enrolled in the BRIM2 (n = 59) and BRIM3 (n = 150) trials of vemurafenib in advanced BRAF(V600E/K) melanoma. Immunohistochemistry for MET (SP44 rabbit monoclonal antibody) was performed with a highly validated assay and clinically validated scoring system. Pretreatment MET expression was frequent at the ≥1 + cutoff (BRIM3, 31%; BRIM2, 49%), but relatively infrequent at the ≥2 + cutoff (BRIM3, 9%; BRIM2, 19%). Retrospective subset analyses showed that, irrespective of the cutoff used or the treatment arm, MET expression did not show prognostic significance, in terms of objective response rate, progression-free survival, or overall survival. CONCLUSIONS: MET is expressed in a proportion of BRAF(V600E/K) advanced melanomas. Further analyses on appropriately powered subsets are needed to determine the prognostic and predictive significance of MET in vemurafenib-treated melanoma.

Neuropilin-1 expression in cancer and development.

Neuropilin (NRP)-1 is a co-receptor for vascular endothelial growth factor (VEGF). Preclinical data suggest that blockade of NRP1 suppresses tumour growth by inhibiting angiogenesis, in addition to directly inhibiting tumour cell proliferation in certain models. A humanized monoclonal antibody to NRP1 is currently being evaluated as a potential anti-cancer therapy in clinical trials. However, the expression of NRP1 in cancer and physiological angiogenesis has yet to be systematically described. Here we characterize the in situ expression of NRP1 in human cancer and during mammalian development. A monoclonal antibody to human NRP1 was generated and validated for immunohistochemistry by western blotting, use of formalin-fixed cell pellets transfected with NRP1, immunofluorescence, and comparison with in situ hybridization. NRP1 expression was assessed in whole sections of 65 primary breast carcinomas, 95 primary colorectal adenocarcinomas, and 90 primary lung carcinomas. An additional 59 human metastases, 16 xenografts, and three genetically engineered mouse tumour models were also evaluated. Immunoreactivity for NRP1 was seen in vessels from normal tissues adjacent to cancer and in 98-100% of carcinomas. Tumour cell expression of NRP1 was also observed in 36% of primary lung carcinomas and 6% of primary breast carcinomas, but no colorectal adenocarcinomas. NRP1 was evaluated in mouse embryos, where expression was limited to the nervous system, endocardium, vascular smooth muscle, and, focally, endothelium on subsets of vessels. Moreover, in a model of VEGF-dependent angiogenesis in the postnatal mouse trachea, blockade of NRP1 signalling resulted in defective angiogenesis and recapitulated the effects of anti-VEGF treatment. These observations confirm NRP1 as a valid anti-angiogenic target in malignancy, and as a potential direct anti-tumour target in a subset of cancers. The data also confirm a role for NRP1 in physiological, VEGF-mediated angiogenesis.

Anti-VEGF antibody therapy does not promote metastasis in genetically engineered mouse tumour models.

Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another.

Neuropilin-2 expression in cancer.

AIMS: Neuropilin-2 is a coreceptor for vascular endothelial growth factor family members. Blockade of neuropilin-2 is able to suppress lymphogenous metastasis in preclinical models. The aim of this study was to validate a protocol for the evaluation of neuropilin-2 protein expression in situ, by comparison with in-situ hybridization, western blotting, and mRNA expression levels. METHODS AND RESULTS: Immunohistochemistry was performed on normal human tissues, and whole sections for 79 primary non-small-cell lung carcinomas, 65 primary breast carcinomas, 79 primary colorectal cancers, and 52 metastases. Neuropilin-2 expression was observed in lymphatic and blood vessels from all normal and malignant tissues examined. In addition, 32% of primary non-small-cell lung carcinomas, 15% of primary breast carcinomas and 22% of primary colorectal cancers showed tumour cell expression. Fifty-five primary and nine secondary malignant melanomas were also examined for neuropilin-2 expression by in-situ hybridization. All showed vascular expression, and 85% of primary malignant melanomas showed tumour cell expression. CONCLUSIONS: In the majority of lung, breast and colorectal cancers, the effects of anti-neuropilin-2 are likely to be restricted to the vasculature. These results will assist in pharmacokinetic evaluations, tolerability assessments and the choice of setting to evaluate the activity of anti-neuropilin-2 therapies.

Expression of vascular Notch ligands Delta-like 4 and Jagged-1 in glioblastoma.

AIMS: The coordinated expression of the Notch ligands Delta-like 4 (Dll4) and Jagged (Jag)1 is believed to define appropriate endothelial sensitivity to vascular endothelial growth factor (VEGF). Preclinical data suggest that Dll4-Notch signalling may confer resistance to anti-VEGF therapy with bevacizumab, and Jag1 may antagonize Dll4-Notch. The aims of this study were to characterize the expression of Dll4 and Jag1 in primary glioblastomas. METHODS AND RESULTS: Immunohistochemistry was performed on 40 glioblastomas and normal brain using validated antibodies against Dll4 and Jag1. In-situ hybridization for Dll4 was performed on serial sections and compared with protein expression. Dll4 expression was localized to the cytoplasm and membrane of endothelial cells in all glioblastomas; it was weak or absent in normal brain. Jag1 expression was observed in the cytoplasm and membrane of glomeruloid and non-glomeruloid endothelial cells from 76% and 67% of glioblastomas, respectively. However, endothelial Jag1 expression was less intense and less prevalent than Dll4. There was no association between Dll4 and Jag1 expression. CONCLUSIONS: In summary, Dll4 and Jag1 are expressed in glioblastoma vasculature. These data may define subsets of glioblastoma that might be sensitive (Dll4(+) /Jag1(+) ) or resistant (Dll4(+) /Jag1(-) ) to bevacizumab. Our data also suggest that anti-Dll4 therapy should be evaluated experimentally in glioblastoma.

Notch3 signalling promotes tumour growth in colorectal cancer.

Increased Notch1 activity has been observed in intestinal tumours, partially accomplished by ß-catenin-mediated up-regulation of the Notch ligand Jagged-1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved is unclear. Microarray data indicated that Notch3 transcript levels are significantly up-regulated in primary and metastatic CRC samples compared to normal mucosa. Moreover, Notch3 protein was expressed at strong/moderate levels by 19.7% of 158 CRC samples analysed, and at weak levels by 51.2% of the samples. Intrigued by these findings, we sought to investigate whether Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumourigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including Notch3, Delta-like 4 (DLL4), and Jagged-1 ligands. Stimulation with immobilized recombinant DLL4 or transduction with DLL4-expressing vectors dramatically increased Notch3 expression in CRC cells, associated with accelerated tumour growth. Forced expression of an active form of Notch3 mirrored the effects of DLL4 stimulation and increased tumour formation. Conversely, attenuation of Notch3 levels by shRNA resulted in perturbation of the cell cycle followed by reduction in cell proliferation, clonogenic capacity, and inhibition of tumour growth. Altogether, these findings indicate that Notch3 can modulate the tumourigenic properties of CRC cells and contributes to sustained Notch activity in DLL4-expressing tumours.

Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer.

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Biomarkers to predict the clinical efficacy of bevacizumab in cancer.

Bevacizumab is a monoclonal antibody against vascular endothelial growth factor that has antiangiogenic activity and improves progression-free survival in many solid malignancies when combined with cytotoxic chemotherapy, but has little effect on overall survival. Despite the effects of this drug in unselected patients, the Response Evaluation Criteria in Solid Tumours are suboptimum to predict benefit. Additionally, tumours show inherent and emerging resistance to regimens that include bevacizumab. The ability to target therapy towards well selected subgroups of patients would increase the likelihood of benefits and would improve cost-effectiveness and therapeutic outcomes. In this review we discuss putative clinical, radiological, and molecular markers of bevacizumab efficacy, derived from data obtained in clinical trials. Current evidence indicates some predictive value for hypertension, vascular imaging, and polymorphisms affecting components of the vascular endothelial growth factor pathway in patients receiving bevacizumab. Many questions relating to these and other surrogate biomarkers, however, remain unanswered and their clinical usefulness has yet to be proven.

Assessment of tumour hypoxia for prediction of response to therapy and cancer prognosis.

Tumour cells exploit both genetic and adaptive means to survive and proliferate in hypoxic microenvironments, resulting in the outgrowth of more aggressive tumour cell clones. Direct measurements of tumour oxygenation, and surrogate markers of the hypoxic response in tumours (for instance, hypoxia inducible factor-1alpha, carbonic anhydrase 9 and glucose transporter-1) are well-established prognostic markers in solid cancers. However, individual markers do not fully capture the complex, dynamic and heterogeneous hypoxic response in cancer. To overcome this, expression profiling has been employed to identify hypoxia signatures in cohorts or models of human cancer. Several of these hypoxia signatures have demonstrated prognostic significance in independent cancer datasets. Nevertheless, individual hypoxia markers have been shown to predict the benefit from hypoxia-modifying or anti-angiogenic therapies. This review aims to discuss the clinical impact of translational work on hypoxia markers and to explore future directions for research in this area.

Targeting DLL4 in tumors shows preclinical activity but potentially significant toxicity.

Evaluation of: Yan M, Callahan CA, Beyer JC et al.: Chronic DLL4 blockade induces vascular neoplasms. Nature 463, E6-E7 (2010). Delta-like ligand 4 (DLL4) is a Notch ligand that is critical in the formation of a functional vascular network in tumors. Blockade of DLL4-mediated Notch signaling strikingly increases nonproductive angiogenesis, but significantly inhibits tumor growth in preclinical mouse models. Thus, DLL4 has emerged as an attractive target for cancer therapy. Anti-DLL4 antibodies have recently entered clinical trials. However, the potential toxic effects of anti-DLL4 are poorly understood. In this article, Yan et al. reported that chronic DLL4 blockade abnormally activates endothelial cells, causes pathological changes of multiple organs and induces vascular neoplasms. The findings need confirmation in further studies using different tumor-bearing animals but, nevertheless, raise important safety concerns regarding the use of anti-DLL4 agents and warrant monitoring for these effects in clinical trials for targeting DLL4.