The phase III CONFIRM clinical trials demonstrated that metastatic colorectal cancer patients with elevated serum lactate dehydrogenase (LDH) had improved outcome when the vascular endothelial growth factor receptor (VEGFR) inhibitor PTK/ZK (Vatalanib) was added to FOLFOX4 chemotherapy. We investigated the hypothesis that high intratumoral expression of genes regulated by hypoxia-inducible factor-1 alpha (HIF1α), namely LDHA, glucose transporter-1 (GLUT-1), VEGFA, VEGFR1, and VEGFR2, were predictive of outcome in CONFIRM-1. Tumor tissue was isolated by laser-capture microdissection from 85 CONFIRM-1 tumor specimens; FOLFOX4/placebo n=42, FOLFOX4/PTK/ZK n=43. Gene expression was analyzed using quantitative RT-PCR. In univariate analyses, elevated mRNA expression of LDHA, GLUT-1, and VEGFR1 were associated with response to FOLFOX4/PTK/ZK. In univariate and multivariate analyses, elevated LDHA and VEGFR1 mRNA levels were associated with improved progression-free survival in FOLFOX4/PTK/ZK patients. Furthermore, increased HIF1α and VEGFR2 mRNA levels were associated with decreased survival in FOLFOX/placebo patients but not in patients who received FOLFOX4/PTK/ZK. These are the first data suggesting intratumoral mRNA expression of genes involved in angiogenesis/HIF pathway may predict outcome to VEGFR-inhibitors. Biomarkers that assist in directing VEGFR-inhibitors toward patients with an increased likelihood of benefit will improve the cost-effectiveness of these promising agents.
Vascular endothelial growth factor (VEGF) and its receptors, VEGFR1 and VEFGR2, are intimately involved in the regulation of tumor-associated angiogenesis and have been correlated with poor prognosis in various human malignancies.1, 2 Their functions include increasing microvascular permeability, stimulating endothelial cell growth, and promoting angiogenesis and lymphangiogenesis.3, 4, 5 As the inhibition of VEGF-induced angiogenesis is highly selective for tumor-associated vessels, therapeutic approaches targeting angiogenesis mediated by VEGF and it's kinase receptor are expected to be safe and well tolerated in patients with cancer. The anti-VEGF monoclonal antibody bevacizumab has demonstrated improved clinical outcome and a favorable toxicity profile in patients with a variety of human carcinomas.6, 7, 8
Hypoxia is a common feature of many solid tumors and has been linked with treatment resistance.9 Hypoxia-inducible factor-1 alpha (HIF1α) is a key transcription factor that activates an aggressive network of genes involved in glycolytic energy metabolism, angiogenesis, cell survival, and erythropoiesis. Genes regulated by HIF1α include those encoding VEGF and it's type-1 receptor (VEGFR1), glucose transporter-1 (GLUT-1), and several glycolytic enzymes, such as lactate dehydrogenase 5 (LDHA).10, 11, 12 Recent data have suggested that high serum LDH is linked to poor prognosis in non-small cell lung and colorectal cancer, as well as tumor hypoxia, angiogenesis, and over-expression of both VEGF and LDHA.9, 10, 11, 12, 13, 14, 15 A direct association between LDH5 up-regulation and HIF1α and HIF2α accumulation in colorectal cancer cells has been reported13 and a subsequent study showed that patients with high serum LDH had significantly increased levels of intratumoral gene expression of VEGF and VEGFR1.16 The accumulating evidence supports the hypothesis that elevated serum LDH is directly associated with up-regulation of genes involved in the HIF1α and tumor-associated angiogenesis pathway.
PTK787/ZK 222584 (PTK/ZK, vatalanib) is a novel oral angiogenesis tyrosine kinase inhibitor (TKI) that is active against all known VEGFR tyrosine kinases and platelet-derived growth factor receptor (PDGFR) tyrosine kinases and therefore offers a novel approach to inhibiting tumor growth facilitated by angiogenesis.17, 18, 19, 20 Two randomized, double-blind, placebo-controlled, phase III studies were carried out in metastatic colorectal cancer (mCRC) patients who were receiving first-line (CONFIRM-1) or second-line (CONFIRM-2) chemotherapy with folinic acid (leucovorin), 5-fluorouracil, oxaliplatin (FOLFOX4), and either PTK/ZK or placebo.21, 22 Although the primary efficacy objectives of both trials were not met, subgroup analysis on the final data from CONFIRM-1 and CONFIRM-2 demonstrated with statistical significance and striking similarity, that patients with elevated serum LDH levels (more than 1.5 times the upper limit of normal) derived a greater clinical benefit when PTK/ZK was added to a standard FOLFOX4 regimen, compared with FOLFOX4 plus placebo.21, 22 The hypothesis proposed herein is that serum LDH may serve as a surrogate predictive marker of an activated tumoral HIF1α and angiogenic gene expression profile with the potential to predict response to VEGFR-inhibitor in combination with chemotherapy. This hypothesis is further supported by a recent study that analyzed LDH5 (the most active LDH isozyme encoded by the LDHA gene) by immunohistochemistry in a balanced subset of patients from the CONFIRM trials and found that tumors with high LDH5 expression had improved response to the FOLFOX4 plus PTK/ZK combination compared with tumors with low LDH5 expression.23
Understanding the molecular mechanisms that govern resistance to VEGFR TKIs will provide critical insight that will assist in the future development of this class of agents and will be paramount in improving cost-effectiveness through the successful selection of patients who may show optimum benefit from VEGFR TKI therapies. Therefore, to investigate the role of genes involved in angiogenesis and HIF-signaling in predicting the efficacy of PTK/ZK in mCRC, we tested the hypothesis that patients with high serum LDH would have increased intratumoral gene expression of VEGFR1, VEGFR2, and additional genes involved in the HIF1α pathway and that such patients would benefit from the addition of PTK/ZK to chemotherapy.
Patients and methods
The phase III multinational CONFIRM-1 trial was designed to investigate the first-line therapeutic potential of the VEGFR TKI PTK/ZK in combination with oxaliplatin-based chemotherapy in patients with mCRC. In the CONFIRM-1 trial, 1168 patients were randomized to receive FOLFOX4 with either PTK/ZK or placebo as first line therapy. Progression-free survival (PFS) was the primary endpoint with secondary endpoints of overall safety and overall response rate.21 Of note, in both the CONFIRM trials, patients were stratified according to performance status (PS) and serum LDH levels. PTK/ZK improved the PFS of patients with high baseline serum LDH levels in CONFIRM 1: Hazard ratio for PFS=0.67; 95% confidence interval (CI): 0.49–0.91; P=0.009.21 For comparative purposes, similar results were observed for patients with elevated serum LDH in CONFIRM-2, which evaluated the same therapy as second line: Hazard ratio for PFS=0.63; 95% CI: 0.48–0.83; P<0.001.22
Where applicable, this study was conducted and is reported according to Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria.24
All patients on the CONFIRM 1 study provided informed consent for the analysis of molecular correlates. All analyses herein were approved by the University of Southern California Institutional Review Board.
A total of 85 formalin-fixed paraffin-embedded (FFPE) primary colorectal adenocarcinoma samples from patients enrolled in the CONFIRM-1 trial were available for analysis. A pathologist reviewed FFPE tumor blocks for quality and tumor content. Ten sections, each of 1-μm thickness, were obtained from the areas identified with the highest tumor concentration and mounted on uncoated glass slides. Three sections representative of the beginning, middle, and end of the tumor were taken for histological diagnosis and stained with hematoxylin and eosin using the standard method. Before micro-dissection, the sections were deparaffinized in xylene for 10 min, hydrated with 100, 95, and 70% ethanol, and then washed in water for 30 s. Following this, the sections were stained with nuclear fast red (American Master Tech Scientific, Lodi, CA, USA) for 20 s and rinsed in water for 30 s. Samples were dehydrated with 70% ethanol, 95% ethanol, and 100% ethanol for 30 s each, followed by xylene for 10 min, and then the slides were air dried. Laser capture micro-dissection (PALM Microlaser Technologies AG, Munich, Germany) was performed on all tumor samples to ensure that only tumor cells were dissected.25 The dissected areas of tissue were then transferred to a reaction tube containing 400 μl of RNA lysis buffer.
Quantitative reverse transcription polymerase chain reaction
Isolation of RNA from FFPE tumor samples was performed according to a proprietary procedure defined by Response Genetics (Los Angeles, CA, USA; United States patent number 6 248 535). Briefly, FFPE tumor samples were deparaffinized followed by homogenization using mechanical and sonic homogenization in RNA lysis buffer. The homogenized samples were heated, RNA recovered by phenol–chloroform extraction and integrity confirmed by spectrophotometry. cDNA was synthesized and gene expression was analyzed using a quantitative real-time reverse transcription PCR method as described previously.16 Relative mRNA levels were expressed as ratios between the target gene and an internal reference gene (β-actin). Quantification of LDHA, GLUT-1, HIF1α, VEGF, VEGFR1, VEGFR2, and β-actin was performed using a TaqMan-based real-time detection method on board an ABI PRISM 7900 Sequence detection System (Applied Biosystems, Foster City, CA, USA). The PCR reaction mixture consisted of the following: 1200 nmol l−1 of each primer, 200 nmol l−1 of probe, 0.4 U of AmpliTaq Gold Polymerase, 200 nmol l−1 each of dATP, dCTP, dGTP, dTTP, 3.5 mmol l−1 MgCl2, and 1 × TaqMan Buffer A containing a reference dye, added to a final volume of 20 μl (all reagents from PE Applied Biosystems, Foster City, CA, USA). Cycling conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 46 cycles of 95 °C for 15 s, then 60 °C for 1 min. The primer sequences and details of PCR conditions have been published previously.16
Tumor response in CONFIRM-1 was assessed according to modified RECIST criteria.21 Non-responders were defined as patients with stable or progressive disease. PFS was calculated as the period from the first day of randomization until the first observation of disease progression or death from any cause. If a patient had not progressed or died, PFS was censored at the time of the last follow-up. Overall survival (OS) was calculated as the time from the first day of randomization until death from any cause or until the date of the last follow-up.
Gene expression values are expressed as ratios between two absolute measurements: the gene of interest versus the internal reference gene (β-actin). To assess the associations between the expression level of each gene and tumor response, PFS or OS, the expression level was categorized into a low and a high value at optimal cutoffs. The maximal χ2 method of Miller and Siegmund26 and Halpern27 was used to determine which gene expression value (optimal cut-offs) best segregated patients into poor-prognosis and good-prognosis subgroups, in terms of likelihood of response. The cutoff values chosen in analyzing response were applied for analysis of PFS and OS. The analysis was conducted separately by therapy. The Cox proportional hazards regression model was used to evaluate the independent effects of gene expression levels on PFS and OS, when adjusting baseline PS and serum LDH level. Interactions between treatment and expression values were tested by comparing corresponding likelihood ratio statistics between the baseline and nested models that included the multiplicative product terms. No adjustment for multiple comparisons was performed because this study was exploratory in nature and conducted to generate hypotheses for future studies. All reported P-values were two-sided. All analyses were performed using the SAS statistical package version 9.0 (SAS Institute, Cary, NC, USA).
Intratumoral gene expression levels of LDHA, GLUT-1, HIF1α, VEGF, VEGFR1, and VEGFR2
A total of 85 FFPE tumor samples from patients enrolled in CONFIRM-1 were available for analysis; 43 who received FOLFOX4 plus PTK/ZK and 42 who received FOLFOX4 plus placebo. There were no statistically significant differences in clinical and demographic characteristics, including serum LDH, sex, PS, age, and gene expression levels and treatment received (Table 1). Baseline characteristics and clinical outcome in patients from whom FFPE tissue was recovered for this study (response rate, 62%; PFS, 9 months; OS, 25 months) were similar to the entire CONFIRM-1 patient population21 and historical outcomes for first-line therapy in similar patient populations.28
Gene expression levels by treatment and response
In CONFIRM-1, elevated mRNA levels of LDHA (P=0.033), GLUT-1 (P=0.045), and VEGFR1 (P=0.012) were predictive of a response to treatment in patients who received FOLFOX4 plus PTK/ZK but not in patients treated with FOLFOX4 plus placebo. Elevated expression of VEGFR2 also demonstrated a statistical trend in favor of improved response to FOLFOX4 plus PTK/ZK (P=0.071). Furthermore, there was a significant interaction between treatments to predict PTK/ZK activity for GLUT-1 (P=0.046) and VEGFR1 (P=0.05) with VEGFR2 again demonstrating a trend toward predicting PTK/ZK treatment (P=0.074) (Table 2).
Gene expression levels by treatment and PFS
Increased mRNA levels of LDHA (P=0.004) and VEGFR1 (P=0.023) were significantly associated with improved PFS in patients treated with FOLFOX4 plus PTK/ZK in CONFIRM-1. (Table 3, Figure 1a and b). All associations remained significant in the multivariate Cox model when adjusting for baseline PS and LDH level (P=0.013 for LDHA and P=0.036 for VEGFR1; Table 4).
Gene expression levels and OS by treatment
No significant association between gene expression levels and OS was demonstrated in patients treated with FOLFOX4 plus PTK/ZK (Table 5). However, elevated HIF1α and VEGFR2 expression were significantly associated with decreased OS in patients receiving FOLFOX4 plus placebo in CONFIRM-1 (P=0.026 and P=0.003, respectively; Table 5). Patients who received FOLFOX4 plus PTK/ZK showed no difference in OS between high and low VEGFR2 and HIF1α expression. The associations between HIF1α and VEGFR2 expression and OS remained significant after adjusting for baseline PS and LDH level (P=0.008 for HIF1α and P=0.007 for VEGFR2; Table 4).
The CONFIRM trials ended with the conclusion that elevated serum LDH was a negative prognostic factor but that these patients appeared to derive significant benefit from inclusion of VEGFR-targeted agent alongside chemotherapy.21, 22 Hypoxia-triggered or oncogene-activated HIF pathways are common events in human malignancies and are linked to aggressive tumor biology, resistance to chemotherapy, and poor prognosis.9, 14, 15 Activation of the VEGF/VEGFR axis triggers multiple signaling pathways that result in the promotion of endothelial cell survival, mitogenesis, migration, differentiation, changes to vascular permeability, and mobilization of endothelial progenitor cells.3, 4, 5 Over-expression of VEGF mRNA and protein is associated with tumor size, progression, and poor prognosis in a variety of malignancies. Despite the overall poor prognosis of these tumors, there is a significant rationale suggesting that these tumors may in fact show greater benefit from therapies that counter tumor angiogenesis driven by hypoxia and the VEGF pathway.
The data presented herein supports this hypothesis and suggests that intratumoral expression profiling of genes involved in the HIF1α pathway may be predictive and prognostic in patients with mCRC treated with FOLFOX4 in combination with PTK/ZK. These data are consistent with our previous results16 and with the hypothesis that patients with increased serum LDH have significant up-regulation of VEGF and VEGFR1 gene expression in the tumor and may benefit from VEGFR TKI therapy. A recent study analyzed serum LDH and tumoral expression of LDH5, the LDH isozyme encoded by the LDHA gene, in a balanced subset of patients from the same CONFIRM trial populations. The authors reported that LDH5 was a significant negative prognostic marker only in patients who did not receive PTK/ZK and the addition of this angiogenesis inhibitor countered the adverse prognostic effect observed in patients with elevated LDH5.23 It is plausible that overexpression of LDH5 is a surrogate marker for the presence of aggressive HIF-activated tumor biology that includes increased angiogenesis mediated by VEGF and it's receptors that is attenuated by PTK/ZK. We previously confirmed a direct link between elevated expression of HIF1α pathway genes, such as LDHA, VEGF, VEGFR1, VEGFR2, and GLUT-1 in patients with high serum LDH.16 In the present study, we expanded upon these initial observations to confirm a direct link between increased expression of these genes and response to PTK/ZK.
In this study, univariate analysis of response rates to chemotherapy showed that high expression of LDHA, GLUT-1, and VEGFR1 were predictive of response following treatment with FOLFOX4 plus PTK/ZK but not in patients treated with FOLFOX4 plus placebo. In multivariate analysis, the association for LDHA and VEGFR1 and PFS remained significant, suggesting that tumors with an HIF1α-induced pathway benefit the most from VEGFR-TKI when given in combination with chemotherapy. In terms of OS, there was no significant difference in survival benefit for patients with high vs low expression of VEGF, VEGFR1, or VEGFR2 when treated with FOLFOX4 plus PTK/ZK, whereas patients with high expression of VEGFR2 and HIF1α treated with FOLFOX4 plus placebo had a significantly shorter OS in multivariate analysis. This was a highly statistically significant interaction, suggesting a strong negative prognostic influence for these genes that was countered by the addition of PTK/ZK. Response (or disease stabilization) to an antiangiogenic inhibitor and/or chemotherapy is transient and is typically followed by a distinct change in tumor biology leading to the development of resistance and subsequent disease progression in the majority of patients. The importance of the genes that regulate treatment outcome are likely to change during this process and may switch from those involved in a predictive role (i.e., those genes that determine response to therapy) to those whose prognostic influence is likely to dictate the subsequent course of the disease and OS as resistance develops. It is possible that therapeutic intervention with PTK/ZK in patients with elevated HIF1α and VEGFR2 resulted in a shift in tumor biology to alternate angiogenic pathways that facilitated continued disease progression and ultimately impacted OS. This is supported by the results of CONFIRM-1 where a statistically significant improvement in PFS but no OS benefit was observed in patients with elevated LDH.21
The current study is a retrospective subset analysis but represents a focused and concerted research effort based on the current biological understanding to generate hypotheses for future testing in clinical trials. As with all retrospective pilot studies, this analysis has potential limitations. First, our findings are based on a relatively small number of patients who were recruited from a large international study over a period of 4 years; second, owing to the retrospective nature, additional analyses of tumor angiogenesis such as microvessel density or venous/lymphatic vascular invasion were not possible; and third, only six genes within the angiogenesis pathway were examined. Nonetheless, this type of retrospective study is an ideal platform for testing a novel hypothesis and for generating data that can be confirmed prospectively in future clinical trials evaluating this class of agents. Care was taken to select candidate genes with a documented role in the angiogenesis pathway, which have been found to be associated with prognosis in previous studies at our institution or in published manuscripts. In addition, internal validation analysis was performed to reduce the likelihood of overanalyzing this dataset.
Limitations aside, we have identified independent molecular markers associated with clinical outcome in patients treated with FOLFOX4 plus PTK/ZK. Importantly, the significant observations we report are entirely consistent with the underlying tumor biology and the subsequent hypothesis tested. Elevated expression levels of genes involved in angiogenesis and HIF-tumor biology, and in particular VEGFR1, were independently associated with clinical outcome in patients enrolled in CONFIRM-1 treated with PTK/ZK, indicative of a potential role in predicting the efficacy of VEGFR TKI therapy. Recently, additional VEGFR TKI agents have been developed, but they have not been shown to increase the efficacy of FOLFOX4.29 It is plausible that a subgroup of patients who are likely to benefit from a VEGFR-TKI could be identified by molecular predictive markers. However, as with all retrospective analyses, larger independent, prospective, biomarker-embedded clinical trials are needed to confirm and validate these preliminary findings.
This study was sponsored by Novartis and Bayer Schering Pharma as part of a co-development program.
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Biochemical Pharmacology (2014)