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Originally published as JCO Early Release 10.1200/JCO.2005.01.5388 on December 19 2005 © 2006 American Society of Clinical Oncology. Impact of Vascular Endothelial Growth Factor-A Expression, Thrombospondin-2 Expression, and Microvessel Density on the Treatment Effect of Bevacizumab in Metastatic Colorectal CancerFrom the Departments of Pathology, Biostatistics, Bioinformatics, and BioOncology, Genentech Inc, South San Francisco; the University of California at Los Angeles, Los Angeles, CA; and the Department of Medical Oncology and Transplantation, Duke University Medical Center, Durham, NC Address reprint requests to Adrian Jubb, Academic Unit of Pathology, Algernon Firth Building, University of Leeds, Leeds, LS2 9JT, United Kingdom; e-mail: adrianjubb{at}gmail.com
PURPOSE: Bevacizumab is a monoclonal antibody to vascular endothelial growth factor-A (VEGF). In the pivotal trial in metastatic colorectal cancer (mCRC), addition of bevacizumab to first-line irinotecan, fluorouracil, and leucovorin (IFL) significantly prolonged median survival. The aim of these retrospective subset analyses was to evaluate VEGF, thrombospondin-2 (THBS-2), and microvessel density (MVD) as prognostic factors and/or predictors of benefit from bevacizumab. PATIENTS AND METHODS: In the pivotal trial, 813 patients with untreated mCRC were randomly assigned to receive IFL plus bevacizumab or placebo. Of 312 tissue samples collected (285 primaries, 27 metastases), outcome data were available for 278 (153 bevacizumab, 125 placebo). Epithelial and stromal VEGF expression were assessed by in situ hybridization (ISH) and immunohistochemistry on tissue microarrays and whole sections. Stromal THBS-2 expression was examined by ISH on tissue microarrays. MVD was quantified by Chalkley count. Overall survival was associated with these variables in retrospective subset analyses. RESULTS: In all subgroups, estimated hazard ratios (HRs) for risk of death were < 1 for bevacizumab-treated patients regardless of the level of VEGF or THBS-2 expression or MVD. Patients with a high THBS-2 score showed a nonsignificant improvement in survival following bevacizumab treatment (HR = 0.11; 95% CI, 0.02 to 0.51) compared to patients with a low score (HR = 0.65; 95% CI, 0.41 to 1.02); interaction analysis P = .22. VEGF or THBS-2 expression and MVD were not significant prognostic factors. CONCLUSION: These exploratory analyses suggest that in patients with mCRC addition of bevacizumab to IFL improves survival regardless of the level of VEGF or THBS-2 expression, or MVD.
Vascular endothelial growth factor-A (VEGF) is a secreted ligand with specific receptors that are expressed principally by angioblasts and endothelial cells.1 The interaction of VEGF with its receptors plays a major role in regulating both physiologic and pathologic angiogenesis, and a wide range of human malignancies upregulate their expression of VEGF to varying degrees during tumorigenesis.2 In preclinical studies a murine antihuman monoclonal antibody (mAb) directed against VEGF was shown to inhibit the growth of human tumor xenografts.3 This finding prompted a clinical trial program to assess the efficacy of a humanized variant of this antibody (bevacizumab) as a treatment for various cancers. Colorectal cancer (CRC) is the second most common cause of cancer deaths worldwide, with 945,000 new cases and 492,000 CRC-related deaths in 2000.4 In patients with metastatic CRC (mCRC), the addition of bevacizumab to an irinotecan, fluorouracil, and leucovorin (IFL) regimen was shown recently to significantly prolong the median duration of survival (from 15.6 to 20.3 months, corresponding to a hazard ratio (HR) for death of 0.66; P < .001).5 While this result demonstrates the potential of antiangiogenic therapy, the identification of biomarkers that may influence response to such treatment is of considerable interest. High levels of tumor VEGF expression, increased circulating VEGF, and increased microvessel density (MVD) have each been associated with poorer survival and an increased incidence of disease recurrence and distant metastasis in adenocarcinomas of the colon and rectum.6-21 As clinical responsiveness to some targeted therapies (for example, trastuzumab in breast cancer) is strongly influenced by the level of expression of the target,22 it would seem reasonable to evaluate tumor VEGF expression as a predictor of responsiveness to bevacizumab therapy. Endogenous negative regulators of angiogenesis may also play a prominent role in tumor behavior. For instance, members of the thrombospondin (THBS) gene family are reported to have antiangiogenic properties, (eg, inhibition of VEGF, matrix metalloproteinases, and basic fibroblast growth factor)23-26 and both preclinical and clinical studies suggest that they can inhibit tumor growth.27-36 Both THBS-1 and THBS-2 are expressed frequently by stromal cells in the tumor microenvironment and are reported to have prognostic significance in several malignancies.37-38 Expression of THBS-2 in CRC is associated with the inhibition of angiogenesis and a reduced frequency of distant metastasis,33 and has been implicated in the inhibition of angiogenesis even in the presence of high tumor-cell VEGF expression.31 Thus, there is a biologic rationale to suggest that host factors involved in the negative regulation of angiogenesis may serve as predictors of response to antiangiogenic therapies. In addressing these hypotheses, we conducted exploratory, retrospective subset analyses to evaluate VEGF expression, THBS-2 expression, and MVD as prognostic factors and/or predictors of benefit from bevacizumab in mCRC.
Patients and Study Design Patient recruitment and trial design have been described elsewhere.5 In brief, previously untreated patients with mCRC were randomly assigned to receive IFL with placebo, IFL with bevacizumab, or fluorouracil and leucovorin with bevacizumab. At the time of enrollment, informed consent was obtained from trial participants to permit research on their archived tissue. The survival analyses described herein included tumor samples from only patients in the treatment arms that received irinotecan (placebo plus IFL [n = 411] or bevacizumab plus IFL [n = 402]). All experiments and analyses were performed blinded to clinical outcome.
Tissue Samples and Tissue Microarray Construction
In Situ Hybridization Hybridization of antisense ß-actin riboprobes was confirmed in all tissues before biomarker analysis. Whole sections of healthy human kidney and human tumor cell pellets were included in each experiment as controls for appropriate hybridization of antisense VEGF riboprobes (Figs 1 and 2). Sense VEGF and THBS-2 riboprobes were employed as negative controls for the specificity of hybridization. TMA cores were scored semiquantitatively on a scale of zero (no expression) to three (very strong signal), according to the overall intensity of the hybridization signal in the epithelium (VEGF) or stroma (THBS-2). VEGF expression in whole sections was scored on an identical scale to reflect the greatest signal intensity observed from at least 10% of neoplastic cells (VEGF maximum), the overall signal intensity observed from greater than 50% of neoplastic cells (VEGF overall), and the signal intensity observed from the stromal cells (VEGF stroma). TMA scoring was determined by a consensus of two pathologists (K.J.H. and H.K.) on a double-headed microscope; whole sections were scored by a single pathologist (H.K.). The hybridization signal intensities in the cell pellets were employed as reference points to assign VEGF scores in each experimental run; 786-0 cell pellets were scored three, and MCF7 cell pellets scored one by ISH (Figs 1 and 2). All ISH on TMAs, including all THBS-2 analyses, was performed in a single experiment. Representative VEGF and THBS-2 scores are illustrated in Figures 3 and 4.
Immunohistochemistry IHC was performed on freshly-cut tissue sections as described previously.2,41 Sections were incubated for 1 hour at room temperature with a primary antihuman VEGF mAb at 1 µg/mL (clone 26503, recognizing the 121 and 165 amino acid isoforms of VEGF; R&D Systems, Minneapolis, MN) or with a primary antihuman CD34 mAb at 2 µg/mL (clone QBEnd/10; NovoCastra, Newcastle on Tyne, United Kingdom). Negative control slides were incubated with an isotype-matched mouse immunoglobulin culture supernatant (Dako Cytomation, Copenhagen, Denmark) at 1 or 2 µg/mL, as appropriate, in place of the primary antibody. Whole sections of healthy human kidney and human tumor cell pellets were included in each experiment as controls for appropriate VEGF and CD34 immunostaining (Figs 1 and 2). To assess VEGF expression, TMA cores and whole sections were scored semiquantitatively on a scale of zero (no expression) to three (very strong signal), according to the intensity of chromogen deposition in the majority of neoplastic cells. TMAs and whole sections were scored by a single pathologist (H.K.). The hybridization signal intensities in the cell pellets were employed as reference points to assign VEGF scores in each experimental run; 786-0 cell pellets were scored three, and MCF7 cell pellets scored zero by IHC (Figs 1 and 2). All IHC on TMAs was performed in a single experiment. Representative scores are illustrated in Figure 5.
MVD Scoring CD34-stained whole sections were scanned into tiled, digital color images at 0.64-µ pixel size using a custom-automated image acquisition application for a Nikon E1000 microscope (Technical Instruments, Burlingame, CA). Images were processed by a custom software algorithm that automates the Chalkley counting procedure described previously.43,44 The algorithm exhaustively evaluated Chalkley scores over the image by varying the position and rotation of a virtual Chalkley field (equivalent to a 20x high-power objective with a 10x eyepiece magnification). The virtual Chalkley field was rotated through 360° in 1° increments at a rectangular array of positions spaced one Chalkley field radius apart and covering the entire image. The coincidence of a Chalkley point with an immunohistochemically-stained pixel was detected by colorimetric filtering. The three top-scoring locations, separated by at least one Chalkley field radius, were reported along with other statistics and diagnostic images. Images were reviewed for accuracy, and the mean vessel counts per Chalkley field were recorded. (Details of the algorithm are available from the authors by request.)
Statistical Analysis
Study Group Characteristics and Frequency Data The cohort of patients in this study had demographic and pathologic characteristics that were representative of the pivotal trial population (Table 1). At the time of analysis, outcome data were available for 278 of the 312 patients examined (34% of pivotal trial participants). The remaining 34 of the 312 patients were treated with fluorouracil, leucovorin, and bevacizumab (arm 3 of the pivotal trial) and were not included in these analyses. The survival benefit from the addition of bevacizumab was not significantly different in the subset of 278 patients compared with the entire study population (HRs for bevacizumab plus IFL v placebo plus IFL = 0.57 [95% CI, 0.43 to 0.76] in the subset compared with 0.66 [95% CI, 0.54 to 0.81] in the entire study population; Table 1). Analyses of tumors from the 278 patients yielded informative data on 169 to 226 patients (Supplementary Fig 1, online only). Results were not available for the remaining cases because of limited amounts of tissue or technical assay failures.
Hybridization of the antisense VEGF riboprobe was more frequently observed over neoplastic cells than stromal cells, or healthy epithelium (Figs 1, 2, and 3). Considerable intratumor heterogeneity was noted in whole sections, although expression at the invasive edge of the cancer was not appreciably different from the remainder (data not shown). Less frequently, silver grains (indicative of mRNA expression) were seen over tumor-associated stromal cells (Fig 4). Signal intensity over neoplastic cells was often greatest in areas adjacent to tumor necrosis and declined with increasing distance from the necrotic focus (Fig 4). In certain cases, the inflammatory cell component of tumor-associated inflammatory infiltrates was found to express VEGF (Fig 4). Healthy crypts adjacent to the tumor were occasionally observed to express VEGF, in most cases at levels below the lowest-expressing tumor cells. Hybridization of the antisense THBS-2 riboprobe was most frequently observed covering tumor-associated stromal cells (Fig 3); THBS-2 message was absent from adjacent, healthy colon. Sense riboprobes did not demonstrate appreciable hybridization greater than background. Immunolabeling of VEGF demonstrated strong chromogen deposition over healthy kidney glomeruli in each run of experiments, similar to the ISH signal. IHC for VEGF in CRC specimens was most intense in the cytoplasm of neoplastic cells, where a granular pattern of staining could be observed (Fig 5). No appreciable staining was seen in adjacent healthy crypts. Weak immunoreactivity was observed frequently in the extracellular matrix and stromal cells, including ganglion cells of the myenteric plexus (Fig 5). Endothelium showed evidence of VEGF staining in both tumor-associated stroma and stroma found in healthy colonic mucosa adjacent cancer in the same tissue block (Fig 5). Tissue sections treated with isotype control antibodies did not exhibit appreciable staining.
Association of Biomarkers With Treatment Outcome
Association of Biomarkers With Prognosis VEGF and THBS-2 expression were not significant prognostic factors for duration of survival, irrespective of the treatment received (Fig 6B). Comparable results were found in analyses of progression-free survival and objective response rate (data not shown).
Association of MVD With Treatment Outcome and Prognosis
There is a biologic rationale to suggest that the level of VEGF expression by a tumor would determine its responsiveness to bevacizumab.6,7,10-13,45-47 Bevacizumab binds directly to VEGF, inhibiting its biologic activity3,48,49 and leaving the vasculature unable to support progressive tumor growth.3,50,51 Nevertheless, the examination of VEGF expression in this series did not reveal patient subgroups with differential responses to bevacizumab therapy (Fig 6A). Indeed, all CRCs in this series exhibited some level of VEGF expression in certain cell populations, and it may be that the distinction between high versus low VEGF expression is not clinically important. While these findings are of considerable interest, all analyses were performed retrospectively on relatively small subsets of predominantly primary tumors. Interpretation of the data must be performed with care, and these findings warrant confirmation in a larger series. In an initial survey, VEGF expression was examined in TMAs, permitting experimental standardization and the quantitation of ISH signal intensities. In this arrayed series, the level of VEGF expression was not a prognostic factor, nor a predictive factor of benefit from bevacizumab treatment (Fig 6). However, scoring VEGF expression by tumor region and cell type has also been reported previously to have prognostic significance.8,9 Therefore, a more detailed analysis of VEGF expression was conducted on whole sections. Nevertheless, in these analyses, both epithelial and stromal VEGF expression were independent of patient prognosis and response to bevacizumab in terms of overall survival (Fig 6). This is in keeping with the literature, which contains many conflicting reports on the prognostic significance of VEGF expression in CRC.6-13,47,52,53 Discrepancies within the literature most likely are a result of clinicopathologic differences between cohorts, and methodologic diversity. Specifically, our protocol stipulated collection of archived tissue specimens, the vast majority of which are primary tumor tissue and may not accurately reflect the biomarker status of metastatic disease. VEGF expression is reported to be relatively higher in colorectal metastases2 compared with the primary disease, and may also vary with the location of the metastasis.54,55 However, Kuramochi et al56 recently reported no significant difference in the level of VEGF mRNA expression in a series of matched primary CRCs and liver metastases. Until this issue is resolved, in future clinical trials it will be important to obtain metastatic biopsies prospectively to allow the assessment of biomarker status in the treated disease. In addition to their synthesis of proangiogenic molecules, tumor-associated stromal cells also express antiangiogenic factors, including members of the THBS gene family. Both THBS-1 and THBS-2 are expressed in the stroma of CRC,33 although analyses have conferred a greater clinical significance to THBS-2 expression.33,37,38,57 One may conjecture that tumors with a high level of THBS expression are more dependent on the expression of proangiogenic molecules to tip the balance in favor of angiogenesis. In this environment, the impact of bevacizumab might be bolstered by the antiangiogenic effects of THBS-2.23-26 These observations, in addition to the availability of a robust THBS-2 assay, led us to investigate the prognostic and predictive impact of THBS-2 in our substudy population. Although THBS-2 was not a significant prognostic factor (Fig 6B), the overall survival benefit from the addition of bevacizumab was greater in the subgroup that scored high for THBS-2 expression (Fig 6A). However, this observation did not reach statistical significance in an interaction analysis (Table 2). Therefore, these data do not have implications for clinical practice, as all patient subgroups benefited from the addition of bevacizumab to first-line IFL. In addition, the limitations of this retrospective, subset analysis must be addressed prospectively in a larger population before any definitive conclusions can be drawn. MVD is believed to summarize the effects of all angiogenic regulators, and therefore may better predict patient outcome than the analysis of a single growth factor or signal-transduction pathway. Assessments of MVD are frequently employed as surrogate measures of angiogenesis in CRC7,8,45-47,53,58 and there are numerous reports describing associations of MVD with both VEGF expression and prognosis.7,45,47 In this series, MVD was not a significant prognostic factor or a predictor of benefit from bevacizumab treatment. This discrepancy may reflect differences in the clinical behavior of primary malignancies and metastatic disease. Angiogenesis is a reactive phenomenon, and not necessarily a stable phenotype that is comparable in primary cancers and their metastases. Moreover, the prognostic impact of MVD may be reduced in more advanced malignancies. Scoring the fraction of immature vessels, which are most susceptible to anti-VEGF therapy,51,59 might provide additional information. At present, though, the significance of MVD in assessing response to antiangiogenic therapy remains unclear. Human mCRCs treated with bevacizumab eventually progress.5,50 Although we cannot exclude the possibility that a different dosing regimen may be more efficacious, response may be best predicted by screening for the acquisition of a phenotype that permits escape from VEGF inhibition.50 This could be achieved by expression profiling60-62 or analysis of tumor physiology51,63 before and after treatment with bevacizumab. Specific tumor types demonstrate coexpression of VEGF and other angiogenic factors,64,65 raising the possibility that complete inhibition of angiogenesis might require the blockade of more than one signaling pathway.66,67 For instance, it has been suggested that predominantly immature endothelium, devoid of pericytes, regresses on treatment with bevacizumab.51,67,68 Endothelial survival signals produced by neoplastic cells,65 tumor stroma,69 and/or pericytes68 are believed to confer resistance to anti-VEGF treatment. Examples include angiopoietin-1/tie-2 and platelet-derived growth factor signaling.67,68,70 Therefore, examining the expression of genes involved in pericyte recruitment and pericyte-endothelial paracrine signaling may yield biomarkers that predict escape from bevacizumab and new strategies for combination antiangiogenic therapy. These exploratory analyses suggest that, in patients with mCRC, addition of bevacizumab to first-line IFL improves survival regardless of the level of VEGF or THBS-2 expression, or MVD. More fundamental research on the appropriate clinical material is required to fully understand the complexity of angiogenesis and patient response to antiangiogenic therapy.
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed discription of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000–99,000 (C)
We thank all patients and investigators who participated in the phase III clinical trial of bevacizumab in metastatic colorectal cancer. Members of Genentech’s anatomical pathology and oligonucleotide synthesis groups were instrumental in the implementation of this study.
Presented in part at the American Society of Clinical Oncology Gastrointestinal Cancers Symposium, Miami, FL, January 27-29, 2005. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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