Strong Notch activation hinders bevacizumab efficacy ...

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Strong Notch activation hinders bevacizumab efficacy in advanced colorectal cancer Francesca V Negri*,1, Pellegrino Crafa2, Giuseppe Pedrazzi3, Cecilia Bozzetti1, Costanza Lagrasta2, Giorgio Gardini4, Ione Tamagnini4, Alessandra Bisagni4, Cinzia Azzoni2, Lorena Bottarelli2, Gallia Graiani2, Ida Romano5, Rosa Porzio6, Gian P Bacchini1, Carlo Paties7, Gianluca Tomasello8, Giovanni Marchetti9, Silvia Fanello10, Carmine Pinto1, Roberto Sala‡,11 & Andrea Ardizzoni‡,12

Aim: To assess the role of Notch activation in predicting bevacizumab efficacy in colorectal cancer (CRC). Materials & methods: Notch activation was evaluated by immunohistochemistry (IHC) on 65 CRC enrolled within randomized clinical trials assessing first-line bevacizumabbased chemotherapy and on 21 CRC treated with chemotherapy alone. Results: Strong Notch (IHC 3+) activation was negatively associated with response (18 vs 62% in low Notch cases [IHC 0, 1, 2+]; p = 0.016), progression-free survival (4.9 vs 12.1 months; p = 0.002) and overall survival (19.3 vs 30.4 months; p = 0.039). No correlation was found between Notch activation and clinical outcome in CRC treated with chemotherapy alone. Conclusion: A potential role of Notch activation in the antitumor activity of bevacizumab could be hypothesized. First draft submitted: 15 May 2015; Accepted for publication: 11 August 2015; Published online: 9 November 2015 VEGF-targeted agents improve the survival of patients with cancer [1–3] , but their success is limited by mechanisms of both intrinsic and adaptive resistance [4] , and in a number of models, by increased metastasis [5,6] . The vessel co-option [7] as well as the pre-existing expression of alternative proangiogenic pathways – that is, PDGFs, FGFs and PlGFs [8] , in the tumor have been previously reported as intrinsic properties of tumor cells that can lead to anti-VEGF resistance, by immediately compensating for the inhibition of VEGF signaling. In addition, hypoxia induced by VEGFsignaling blockade selects tumor cells which can cope with low oxygen conditions [9] . Endothelial Medical Oncology Unit, University Hospital, Via Gramsci 14, 43126 Parma, Italy Department of Pathology, University Hospital, Via Gramsci 14, 43126 Parma, Italy 3 Department of Neuroscience, University of Parma, Via Volturno 39, 43126 Parma, Italy 4 Pathology Unit, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS), Santa Maria Nuova Hospital, Viale Umberto I 50, 42123 Reggio Emilia, Italy 5 Department of Radiology, University Hospital, Via Gramsci 14, 43126 Parma, Italy 6 Medical Oncology Unit, Azienda Unità Sanitaria Locale, Via Taverna 49, 29121 Piacenza, Italy 7 Pathology Unit, Azienda Unità Sanitaria Locale, Via Taverna 49, 29121 Piacenza, Italy 8 Medical Oncology Unit, Azienda Istituti Ospitalieri, Viale Concordia 1, 26100 Cremona, Italy 9 Pathology Unit, Azienda Ospedaliera S.Maria Terni, Viale Tristano di Joannuccio 1, 05100 Terni, Italy 10 Medical Oncology Unit, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS), Santa Maria Nuova Hospital, Viale Umberto I 50, 42123 Reggio Emilia, Italy 11 Department of Biomedical Biotechnological & Translational Sciences (S.Bi.Bi.T), University of Parma, Via Volturno 39, 43126 Parma, Italy 12 Medical Oncology Unit, St Orsola-Malpighi Hospital, Via Albertoni 15, 40138 Bologna, Italy *Author for correspondence: Tel.: +39 052 170 2660; Fax: +39 052 199 5448; [email protected] ‡ Authors contributed equally

Keywords

• bevacizumab • colorectal cancer • DLL4 • Notch

activation

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Research Article Negri, Crafa, Pedrazzi et al. cells can also induce pericytes recruitment to retain slim and functional vessels and protect themselves from death [10] . Besides, a variety of bone marrow-derived cells within the tumor microenvironment mediate resistance to VEGF pathway inhibition leading to upregulation of the above-mentioned proangiogenic factors [4] . With multiple potential mechanisms of resistance to anti-VEGF inhibitors within the tumor or the microenvironment, discovery of clinical markers of drug resistance may provide a rationale for clinical testing of alternative treatment regimens. Notch signaling pathway plays an important role in both biological and cancer processes [11] . In mammals, this pathway consists of four Notch receptors (Notch 1–4) and five membrane-anchored ligands (Jagged1, Jagged2 and DLL1, 3, 4) [12] . Ligand receptor binding leads to intramembrane cleavage catalyzed by γ-secretase and subsequent translocation of Notch intracellular cleaved domain (NICD) from the cell membrane to the nucleus where it binds to transcription factors. Despite multiple ligands, recent findings demonstrate that the DLL4–Notch interaction is the dominant functioning component in vascular development. DLL4 is predominantly expressed in arterial endothelial cell during embryonic development, and haploinsufficiency of DLL4 results in embryonic lethality from severe vascular defects in mice [13] . DLL4 expression has been shown to be upregulated in tumor vasculature [14,15] . The Notch pathway interacts with the VEGF signaling by crosstalks with VEGFR [11] . VEGF-A through VEGFR-2 upregulates DLL4 expression in sprouting endothelial tip cells which, by activating Notch signaling on adjacent cells, reduces VEGFR-2 expression and suppresses sprouting [16,17] . Notch pathway mediated by ligand Jagged1 supports instead sprouting. On the basis of DLL4/Jagged1 ratio, Notch signaling has opposing effects on angiogenesis [18] . Moreover, a considerable amount of endothelial Notch is maintained without VEGFR-2 activation. By using inducible loss-offunction genetics in combination with inhibitors in vivo, it has been established that DLL4 expression in retinal vasculature is weakly regulated by VEGFR-2 signaling [19] . DLL4–Notch signaling has been involved in tumor resistance to antihuman VEGF antibody. When transduced by retroviral vector encoding Notch DLL4, human glioblastoma cells promote the formation of large vessels in vivo and

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are insensitive to the humanized antihuman VEGF antibody, bevacizumab. The large vessels triggered by Notch signaling activation by increasing blood perfusion reduce necrosis and hypoxia in DLL4-expressing tumors, leading to a decreased VEGF production and a diminished tumor dependency on VEGF. DLL4–Notch signaling mediates tumor resistance also to VEGFR multikinase inhibitor sorafenib [20] . Based on these preclinical reports, the objective of this study was to assess the role of Notch and DLL4 expression in predicting bevacizumab efficacy in metastatic colorectal cancer (CRC). Materials & methods This study included 65 consecutive CRC enrolled into six prospective clinical trials assessing first-line bevacizumab-based regimens at the Medical Oncology Units of University Hospital, Parma; Santa Maria Nuova Hospital, Reggio Emilia; Azienda USL, Piacenza and Istituti Ospitalieri, Cremona, Italy, between July 2005 and May 2011 [21–24] . Patients were selected because of the availability in archives of their tumor samples. Patients received bevacizumab at the dose of 5 mg/kg on day 1 given every 2 weeks or 7.5 mg/kg on day 1 given every 3 weeks until disease progression, unacceptable toxic effects or withdrawal of consent. As controls, we evaluated 21 CRC among the randomly enrolled patients who received first-line chemotherapy without bevacizumab within the aforementioned clinical trials [23,24] . The Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) were used for disease response assessment. This study was conducted according to a protocol approved by the institutional review board/independent ethics committee and informed consent was obtained from all patients for the use of tissue samples and the analysis of clinical information. Formalin-fixed, paraffin-embedded tissue blocks from pretreatment primary lesions, selected on the basis of quality and representativeness of the sample, were cut into 5 μm thick sections and incubated overnight at 4°C with a polyclonal antibody to DLL4 (1:50, SIGMA Life Science, MO, USA) and NICD (1:50, Cell Signaling Technology, MA, USA). Sections were incubated with ADVANCE HRP Link and ADVANCE HRP Enzyme (Dako Corp., CA, USA) and counterstained with hematoxylin eosin solution. NICD expression was quantified using a semiquantitative scoring system based on intensity

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Notch activation hinders bevacizumab efficacy in colorectal cancer and on percentage of immunostained cells (0, no staining; 1+, weakly positive; 2+, moderately positive; 3+, strongly positive staining when more than 50% of cells were positive) [25] . Thymus and fetal heart were used as positive controls. For the purpose of the present analysis, intensity levels 0–2 were grouped together and defined low expression whereas level 3 was considered high expression. Membranous and/or cytoplasmatic DLL4 expression was observed in large endothelium vessels of the submucosa and muscularis propria under the neoplasm, as well as in tumor-associated vessels. The whole histological section was scored semi-quantitatively on a scale of 0 (no expression) to 3 (almost all vessels). Pairs of pretreatment primary and matched metastatic lesions were analyzed for comparison of biomarkers expression levels. The immunostains were evaluated by two observers blinded to the clinical and pathological data. Data were analyzed with the IBM-SPSS Statistical package, version 20.0 and the widespread open source statistical system ‘R,’ version ‘3.0.1.’ The correlation between ordinal variables has been evaluated by the Spearman coefficient, while association between nominal or dichotomized variables and clinical outcome parameters was estimated by chi-square test and Fisher exact test for 2 × 2 tables. Overall survival (OS) and progression-free survival (PFS) was calculated using the Kaplan–Meier method. Univariate and multivariate Cox proportional hazard models were fitted to investigate the associations between OS and PFS, and covariates. Variables included were: CEA, KRAS status, age, gender, chemotherapy protocol, second-line chemotherapy, DLL4, NICD expression. Only variables with p < 0.05 were included in multivariate models. Results were expressed as hazard-ratios (HRs) with 95% CIs. All tests were two-sided. Results There was no significant difference in pretreatment features and in percentages of NICD and DLL4-positive staining between patients treated with bevacizumab and controls (Table 1) . NICD expression was evaluable in 61/65 CRC analyzed. Eleven patients (18%) showed high NICD expression (IHC 3+; Figure 1A). In total, 5% (3/61) were NICD negative (IHC 0), 26% (16/61) had a score of 1 and 51% (31/61) had a score of 2. NICD immunostaining demonstrated


Research Article

a prevalent cytoplasmic pattern, except five mucinous CRC showing a prevalent nuclear localization. NICD level was not correlated with any of the common clinicopathological characteristics (Table 2) . High NICD expressing tumors had a higher percentage of KRAS mutations compared with low expressing tumors, although this difference did not reach statistical significance (82 vs 47%; p = 0.08). The comparative analysis between seven primary and matched metastases did not reveal discrepancy in NICD expression levels. In six of these pairs, the primary lesion and the paired metastatic lesion showed low NICD expression. In one additional patient, both the primary and the paired liver metastasis showed strong NICD expression. The majority of patients achieving a partial or complete response had low NICD expression (IHC 0, 1, 2+; 31/50, 62%), whereas patients with high NICD levels (IHC 3+) reported a greater rate of stable or progressive disease (9/11, 82%; p = 0.016) (Table 3) . Table 1. Baseline characteristics. Characteristic

Patients (n = 65), n (%)

Controls (n = 21), n (%)

Age (range); years Sex: – Male – Female KRAS: – Mutant – Wild-type Number metastatic sites: –1 – ≥2 Metastatic sites: – Liver – Lung – Other NICD: – High – Low DLL4: – High – Low Subsequent chemotherapy: – Yes – Received cetuximab Response rate: – PR + CR – SD – PD

67 (36–81) 38 (58) 27 (42) 26 (51) 25 (49) 41 (63) 24 (37) 47 (72) 20 (30) 23 (34) 11 (18) 50 (82) 21 (46) 25 (54) 47 (72) 19 (29) 34 (52) 19 (29) 12 (19)

59 (41–81) 12 (57) 9 (43) 7 (54) 6 (46) 12 (57) 9 (43) 19 (90) 8 (38) 4 (19) 5 (28) 13 (72) 4 (31) 9 (69) 12 (57) 4 (19) 10 (50) 4 (20) 6 (30)

CR: Complete response; PD: Progressive disease; PR: Partial response; SD: Stable disease.

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Research Article Negri, Crafa, Pedrazzi et al.

Figure 1. Example of colorectal cancer tissues immunostained for NICD and DLL4. Immunostaining for (A) NICD and (B) DLL4. Original magnification 20×, immunostaining developed in DAB (3,3’-diaminobenzidine). Strong cytoplasmic reactivity is seen for (A) NICD. Strong membranous and/or cytoplasmatic expression is seen for (B) DLL4. Bar length: 0.1 mm.

With a median follow-up of 13.9 months, the median OS of CRC patients treated with bevacizumab-based therapy was 28.0 months. The median PFS to bevacizumab-based therapy was 10.8 months. Patients with high NICD expressing tumors (IHC 3+) had a significantly shorter PFS (4.9 vs 12.1 months; HR: 2.51; 95% CI: 0.96–6.58; p = 0.002; Figure 2A) and OS (19.3 vs 30.4 months; HR: 2.21; 95% CI: 0.79–6.15; p = 0.039; Figure 2B ) when compared with patients with low NICD-expressing tumors (IHC 0, 1, 2+). Multivariate analysis confirmed that high NICD expression correlated with shorter PFS (HR: 1.37; 95% CI: 1.08–1.74; p= 0.009) and OS (HR: 1.33; 95% CI: 1.01–1.74; p = 0.045). Among the cohort of patients treated with chemotherapy alone, 19/21 could be analyzed for NICD expression and no correlation was found with OS and PFS. Patients with low NICD-expressing tumors had a median OS significantly shorter if treated only with chemotherapy (n = 13) compared with patients treated with bevacizumab (n = 50; 16.5 vs 30.4 months; p = 0.002; Figure 2C). High cytoplasmic DLL4 protein levels (Figure 1B) of large and small tumor vessels were observed in 21/46 (46%) and in 10/58 (17%) evaluable CRC, respectively. No significant difference in clinical response, PFS and OS to bevacizumab treatment by DLL4 expression levels was found (data not shown). With the purpose to evidence a link between Notch activation score and DLL4 expression score, NICD and DLL4 expression score

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was investigated in subsequent sections of the same histological sample (number of samples analyzed = 59) and no correlation was found (Spearman R = -0.07; p = 0.61) within the same tumor sample. Discussion The regulatory approval of bevacizumab for the treatment of several tumor types, including CRC, provides clinical validation of tumor dependence on VEGF signaling. Nevertheless, the therapeutic benefit of this agent is often shortlasting and drug-resistant disease may develop within few months [5,6] . The data provided here suggest for the first time a link between Notch signaling and the efficacy of bevacizumab-containing therapy in patients with metastatic CRC. In this study, high NICD expression in CRC is inversely correlated with objective response, PFS and OS to bevacizumab-containing firstline treatment. Despite the small-sized cohort and the retrospective nature, this conclusion is based on analyses of patients from randomized clinical trials. Our results on the frequency of NICD staining on primary CRC and the concordance of the stain between primary and secondary lesions confirms that Notch signaling is an early event in CRC progression [26] . Although the exact mechanisms between the Notch and VEGF pathways remain incompletely understood, a high endothelial DLL4 expression seems to sustain the resistance against VEGF inhibitors in endothelial cell lines, and in vivo [27] . However, our clinical data on CRC, do not seem to support


Notch activation hinders bevacizumab efficacy in colorectal cancer

Research Article

Table 2. Patients characteristics according to Notch intracellular cleaved domain. Characteristic

Strong NICD: IHC 3+ (n = 11), n (%)

Low NICD: IHC 0–1–2+ p-value (n = 50), n (%)

Age (years) Sex: – Male – Female CEA: – 30 KRAS: – Mutant – Wild-type Number metastatic sites: –1 –≥2 Subsequent chemotherapy: – Yes – Received cetuximab

62.7 5 (46) 6 (54) 5 (45) 6 (55) 9 (82) 2 (18) 6 (55) 5 (45) 9 (82) 3 (27)

63.5 32 (63) 18 (37) 27 (54) 21 (46) 17 (47) 19 (53) 33 (66) 17 (34) 34 (68) 12 (24)

0.79 0.31 0.73 0.08 0.50 0.48 1.00

IHC: Immunohistochemistry.

this scenario as no relation was found between DLL4 expression and Notch activation within the same tumor. Mutational activation of KRAS is found in almost 50% of CRC. It has been suggested that the link between KRAS and Notch relies on the appropriate cellular context in which KRAS is most effective as oncogene. Without reaching a statistical significance, a high percentage of KRAS mutations gathered among the cell population with strong NICD. If both events are independent variables in CRC outcome, it must be taken into account that KRAS expression in colonic epithelium induces hyperproliferation and the loss of stem cell differentiation in Apc-mutant CRC [28] . So it could also be hypothesized that Notch activation might cope with KRAS activation to maintain a stem cell population within the cancer lesions which affects the response to therapy. Given that activation of Notch signaling may contribute to chemoresistance by inducing prosurvival signals, such as PI3K/Akt [29] , we also evaluated NICD on baseline biopsy specimens from CRC patients undergoing first-line chemotherapy without bevacizumab within the same randomized clinical trials. The lack of prognostic relevance of Notch activation in patients who received chemotherapy alone is consistent with the hypothesis that this molecular marker might be a predictor of bevacizumab activity rather than a simple unspecific prognostic factor. Notch activation maintains tissue stem cells and stem cells are able to promote angiogenesis


throughout several factors such as MDK, EGF, IL-8, besides VEGF [30] . Thus, the anti-VEGF therapy in such scenario could be of reduced relevance. This study shows also a high level of concordance for NICD levels between primary and paired metastatic site, suggesting that analysis of secondary tumor biopsies may be useful to personalized treatment based on the expression of markers of sensitivity or resistance to therapy by each individual tumor. Conclusion In conclusion, this is the first study that suggests strong NICD expression as a potential marker of primary resistance to bevacizumab and identifies a patient population where Notch signal is constitutively activated and that could benefit from alternative treatment strategies targeting the Notch pathway. Future perspective Selecting patients based on Notch expression can avoid about 20% (proportion of high Notch-expressing tumors among all CRC) of Table 3. Response according to Notch intracellular cleaved domain. NICD

CR + PR

SD + PD

Low Strong

31 (62) 2 (18)

19 (38) 9 (82)

p = 0.016. CR: Complete response; PD: Progressive disease; PR: Partial response; SD: Stable disease.


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Research Article Negri, Crafa, Pedrazzi et al.

1.0

+

12.1 months (8.8–15.3) 4.9 months (1.0–8.3)

0.8

p = 0.002 0.6

+

0.4 + +++

0.2

+

+

++

Low NICD Strong NICD

+ ++

0.8 Cumulative OS

Cumulative PFS

1.0

Median PFS (95% CI)

Low NICD Strong NICD

++

+++ ++ + ++++++

0.6

+

0.0

p = 0.039

++ + ++

0.4 +

0.2

+

Median OS (95% CI) 30.4 months (25.2–35.7) 19.3 months (13.2–25.5)

+ +

0.0 0

10

20

30

40

0

10

20

Time (months)

1.0

40

50

60

70

Median OS (95% CI)

+ ++

0.8 Cumulative OS

30

Time (months)

0.6

+++ ++ +++++

++ +

16.5 months (9.8 – 23.0) 30.4 months (25.2 – 35.7) p = 0.002 ++ + ++

0.4

Low NICD – chemotherapy Low NICD – bevacizumab

+ + +

0.2 0.0 0

10

20

30 40 Time (months)

50

60

70

Figure 2. Clinical outcome according to NICD status. (A) PFS and (B) OS in strong and low NICD stained tumors. (C) OS in low NICD-stained tumors treated with bevacizumab and with chemotherapy alone. OS: Overall survival; PFS: Progression-free survival.

inappropriate therapies. This finding, if confirmed in further studies, provides novel insights on using bevacizumab in CRC and suggest Notch pathway as a key target in clinical practice. Acknowledgements The authors are grateful to R Nizzoli, University Hospital of Parma, Italy and to G Carlinfante, Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS), Santa Maria Nuova Hospital, Reggio Emilia, Italy for assistance with immunohistochemical analysis.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a

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financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.


Notch activation hinders bevacizumab efficacy in colorectal cancer

Research Article

Executive summary ●●

evacizumb is a recognized powerful tool in the first- or second-line therapy of metastatic colorectal cancer, but so far B without any indication on the specific phenotype.

●●

definition of cancer phenotype contributes to the transition from a disease-focused medicine to a patient-focused A medicine.

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Upregulation of the Notch signaling pathway in cancer has been extensively investigated.

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I n our study, cancers with intracellular domain of activated Notch had a poor prognosis when treated with bevacizumab and, in contrast, they were more responsive to traditional chemotherapy, thus defining a population which may require a different therapeutic approach.

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