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HGF is a hypoxia-induced secreted protein that binds to cMET and regulates IL8 expression. We evaluated the role of circulating HGF and IL8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin.
Patients with Stage III–IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or cisplatin plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL8 assay by ELISA and no major radiation deviations (N=498). Analyses included adjustment for major prognostic factors. p16INK4A staining (HPV surrogate) was performed on available tumors. 39 patients had hypoxia imaging with 18FAZA-PET.
Elevated IL8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (p=0.053): elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16INK4A negative patients. Four subgroups defined by high and low HGF/IL8 levels were examined for TPZ effect; the test for interaction with arm was p=0.099. TPZ/CIS appeared to be beneficial for patients with high HGF and IL8, but adverse for low HGF and high IL8. Only HGF correlated with 18FAZA tumor SUV.
IL8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL8/HGF levels appeared to do better with TPZ/CIS while others do worse; highlighting the complexity of hypoxia targeting in unselected patients.
Tumor hypoxia represents an imbalance between oxygen supply and consumption. Hypoxia has been shown to enhance radiation resistance and metastasis in solid tumors, including head and neck squamous cell carcinoma (HNSCC).(1, 2) Several approaches have been used to target tumor hypoxia with radiation therapy, including methods to increase tumor oxygen supply or reduce oxygen consumption, high linear energy transfer (LET) radiation, hypoxic cell radiosensitizers or hypoxic cell cytotoxins.(3, 4) Although several strategies were found promising in early phase II studies, many failed when tested in large randomized trials. Failure in hypoxia targeting could partially be attributed to poor patient selection for such therapies.(5) Therefore, it is important to identify markers that can be used to select for patients who would most benefit from a hypoxia-targeting approach.
Several molecular markers are induced by hypoxia, among which are the Hepatocyte Growth Factor (HGF), its receptor cMet and one of its downstream effectors, Interleukin-8 (IL8). Physiologically, the HGF/cMet pathway plays a major role in organ development and normal tissue homeostasis.(6–8) However, inappropriate activation of this pathway is linked to tumor transformation, growth, angiogenesis and metastasis.(9, 10) Both HGF and cMet are expressed at high levels in HNSCC(11, 12) and circulating HGF protein level is elevated in HNSCC patients compared to non-cancer controls.(13, 14) Importantly, the expressions of both HGF and cMet are induced by hypoxia, which enhances activation of this pathway.(15)
A downstream target of the HGF/cMet pathway is IL8, which is another hypoxia induced gene.(16, 17) Studies have shown that HGF engagement of cMet resulted in increased IL8 production and blockage of this pathway with either cMet shRNA or inhibitors resulted in decreased IL8 level.(11, 18, 19) Although the complete mechanism of hypoxia induction of IL8 has not been clearly delineated, the HGF/cMet pathway may mediate part of this effect. Therefore, both HGF and IL8 are attractive secreted molecules to evaluate as potential predictive markers for hypoxia-directed therapy.
Tirapazamine (TPZ) was developed as a hypoxic cell cytotoxin that can be administered concurrently with chemoradiation therapy (CRT). The TROG-02.02 (HeadSTART) trial was a large international phase III trial, which compared concurrent cisplatin-based CRT to a similar regimen with TPZ in patients with stage III-IV HNSCC. We have previously reported on the results of this trial, which showed that the addition of TPZ did not improve outcomes in unselected patients but major RT deviations resulted in worse locoregional control and survival.(20, 21) We hypothesized that high levels of plasma HGF and/or IL8 might identify patients with hypoxic tumors, who would have an adverse outcome with standard CRT. Furthermore, we hypothesized that plasma HGF and/or IL-8 levels may identify a population that benefits from TPZ, and conversely a population that does not benefit from it. To address these hypotheses, we measured the pre-treatment plasma levels of both cytokines in patients enrolled in this trial.
Recently, infection by the human papillomavirus (HPV) has been shown to be a significant prognostic marker for HNSCC.(22–28) We have reported that patients with HPV(+) oropharyngeal tumors on the TROG-02.02 trial had better survival than those with HPV(−) tumors.(29) To address the effect of HPV, we also stratified a subset of patients by p16INK4A status (a HPV surrogate marker), and evaluated the impact of HGF and IL8 in p16INK4A(+) and p16INK4A(−) patients.
18F-fluoroazomycin arabinoside (18FAZA) is a novel hypoxia tracer used in positron emission tomography (PET) imaging and has been shown to predict the success of TPZ in combination with CRT in animal models.(30–32). It has been used to image hypoxia in human HNSCC.(31) In a small subset of patients enrolled in the TROG-02.02 trial, we performed pretreatment 18FAZA imaging and correlated tumor 18FAZA standard up take values (SUV) with pre-treatment HGF and IL8 levels.
We have previously reported on the study design, eligibility criteria, treatment details, follow up and outcomes of the TROG-02.02 trial.(20) For this sub-study additional eligibility criteria were available plasma samples for cytokine assay and no major radiation deviations predicted to have an impact on tumor control. Using plasma samples from the same patient population, we have also measured the levels of circulating Osteopontin and Vascular Endothelial Growth Factors (VEGF), two other hypoxia induced secreted markers, and have reported the results for Osteopontin in a separate manuscript.(33) Here we focus on HGF and IL8 as independent hypoxia markers. All studies were approved by the local institutional review board.
The primary end point was overall survival (OS) adjusted for the following prognostic factors: site (oropharynx/larynx vs. oral cavity/hypopharynx), T category (T1–2 vs. 3–4), N category (N0–1 vs. 2–3), hemoglobin (high vs. low), and ECOG performance status (0 vs. 1–2). This same endpoint was defined for the main analysis.(20) Another evaluated endpoint was failure-free survival (FFS).
Standardized plasma collection, isolation and storage procedures were used by all sites. Peripheral blood samples were collected in 5–10-ml EDTA tubes pre-treatment. Plasma was isolated, aliquoted and stored at −80°C until assayed by a central site (Amgen). A fibrinogen assay was used ensure that all tested samples were plasma and not serum. HGF measurement was performed using a modified ELISA system (R&D Systems), where Assay Diluent GF2 (Meso Scale Discovery) was utilized to ensure assay linearity.(34)
We initially prospectively validated the prognostic value of HGF using a test-set and validating-set approach. We measured HGF level in an initial 168 patients, who were balanced for clinical characteristics. Upon determining that a higher HGF level was associated with worse survival in the test-group, we validated its prognostic significance in the remaining patients (data not shown).
IL8 was quantified by ELISA (R&D Systems) per the manufacturer’s instruction at the Peter MacCallum Cancer Center.
p16INK4A immunohistochemistry was performed and quantified as previously described.(29) p16INK4A staining intensity was scored as 0 (none), 1 (weak), 2 (moderate) or 3 (strong); with 0–1 defined as negative, and 2–3 defined as positive.(29)
18FDG and 18FAZA synthesis and imaging were performed as described.(31) Briefly, at one hour after FDG injection or two hours after FAZA injection (5.2 MBq/kg), a static PET acquisition was performed and reconstructed.(31) 18FAZA images were co-registered with 18FDG PET images using a mutual information algorithm and the 18FDG PET was then used to identify tumor and involved nodal areas as regions of interest (ROI) for 18FAZA analysis. The maximum standard uptake values (SUV) for 18FAZA and 18FDG were calculated separately for the tumor and the involved nodes using the RT_Image software.(35)
The distributions of marker variables according to baseline factors, and correlations between markers and SUVs were assessed using exact nonparametric tests (Wilcoxon, Fisher and Spearman). The Kaplan-Meier method was used to estimate OS curves. OS was measured from the end of radiotherapy, because of the radiation deviation eligibility criterion. The groups were compared with respect to OS, using the log rank test and Cox proportional hazards model, adjusting for previously identified prognostic factors described above. We decided a priori to analyse HGF and IL8 as dichotomous variables (median cut points). However, we also evaluated the markers as continuous variables. Median levels of HGF and IL8 were calculated on all patients assayed with the particular marker. Hazard ratios (HR) for two-group comparisons refer to high (≥ median):low (<median) for marker comparisons and TPZ:CIS for treatment arm comparisons. In order to identify patient subgroups based on the two markers, such identification was guided by tests for statistical interaction between treatment the two arms and the two markers with three degree of freedom. All P-values are two-sided. Statistical analyses were carried out using the R statistical package.(36)
Of 853 eligible patients, 596 had plasma available for HGF and IL8 assays. Ninety-eight were excluded because of major radiotherapy deviations, leaving 498 for marker analysis (Supplementary Table A). Of these 165 patients had died, 199 had failed or died and 111 had experienced locoregional failure as a first failure. Table 1 shows the characteristics of the radiation compliant patients who did and did not have marker data; those with marker data available had lower haemoglobin, more laryngeal and fewer hypopharyngeal cancers.
The median HGF level was 823 pg/ml (range: 190–42,300) and the median IL8 level was 6.48 pg/ml (range: 0–365). Table 1 also shows the distribution for the low (<median) versus high (≥ median) HGF and IL8 groups, respectively. High HGF level was significantly associated with higher T-stage (T3–4), worse performance status (ECOG PS > 0), more oral cavity and hypopharyngeal primaries and being a current smoker; whereas high IL8 was only associated with higher T-stage. Consistent with the known biological association, there was a correlation between HGF and IL8; the patient distribution for the marker groups was: 35% for both markers being low, 28% for both being high, 16% for IL8-low/HGF-high and 21% for IL8-high/HGF-low group (p < 0.001).
In the cohort with known p16INK4A status (n=223), there was a correlation between p16INK4A and HGF levels (p=0.001) and between p16INK4A and IL8 levels (p=0.003), with high levels of each marker being more common in p16INK4A(−) patients (Supplementary Table B).
Both pre-treatment plasma HGF and IL8 levels were prognostic for OS and FFS on univariate analysis in the whole population. For HGF, the hazard ratio was 1.50 (p=0.008) for OS and 1.43 (p=0.011) for FFS when analyzed as a dichotomous variable (by the median) and 1.42 (per doubling; p=0.001) for OS and 1.39 (p=0.001) for FFS, respectively, when evaluated as a continuous variable (log-transformed). For IL8, the hazard ratio was 1.86 (p<0.001) for OS and 1.59 (p=0.001) for FFS when analyzed as a dichotomous variable (by the median) whereas it was 1.12 (per doubling; p=0.002) for OS and 1.08 (p=0.013) for FFS, respectively, when assessed as a continuous variable (log-transformed).
However, when these analyses were repeated adjusting for known prognostic factors, in order to address the main aims of the study, only IL8 remained significant: the HR for HGF was 1.20 (p=0.27) and for IL8 was 1.55 (p=0.007) (Supplementary Figures A and B). However, there was an interaction between HGF and treatment arm (the p-value for the test of interaction = 0.053). High HGF levels predicted for worse OS in the control arm, but not in the TPZ/CIS arm (Figure 1). The 2 year OS on the control arm was 63% for the high HGF versus 76% for the low HGF group (HR: 1.62, p=0.028) (Table 2). On the TPZ/CIS arm, the 2 year OS was 72% versus 69% for high and low HGF, respectively (HR: 0.84, p=0.46). In contrast, there was no interaction between IL8 (analysed by median) and treatment (p=0.66). High IL8 level was associated with worse OS, regardless of the treatment received (Figure 2).
As there was an interaction between HGF and arm we examined the effect of treatment in the high and low HGF groups, adjusting for prognostic factors using a three-degree of freedom test. There was a suggestion TPZ/CIS may be associated with an adverse outcome in low HGF patients and a better outcome in high HGF patients, but these differences were not statistically significant. Within the low HGF group the 2 year OS was 76% for CIS versus 69% for the TPZ/CIS (HR: 1.43, p=0.12), and within the high HGF group the two-year OS was 63% for CIS versus 72% for TPZ/CIS (HR: 0.76, p=0.21) (Table 2).
We next sought to determine whether there was an interaction between the four possible high/low IL8/HGF combinations and treatment arm. The p-value for this interaction adjusting for prognostic factors was 0.099. Although not statistically significant, the p-value was small enough to suggest that there may be differences in the relative efficacy of TPZ among the four subgroups. Based on this we proceeded to see if there were any significant differences by arm within the IL8/HGF subgroups (Figure 3A – interaction analysis, Table 2). While none of the tests was significant, the HRs for 2 of the groups were large and in opposite directions. This suggests that TPZ/CIS may be advantageous in the IL8-high/HGF-high group (HR=0.64, p=0.12) and adverse in the IL8-high/HGF-low group (HR=1.86, p=0.07) (Figure 3B).
We also evaluated the prognostic importance of HGF by p16INK4A status. Of 498 patients, 223 patients had tissue slides available and analysable for p16INK4A; 95 tumors were p16INK4A(+) (81 oropharyngeal, 14 non-oropharyngeal) and 128 were p16INK4A(−). With the caveat that these are small cohorts, there was no apparent difference in outcomes by HGF level for either arm in the p16INK4A(+) patients (Supplementary Figure C), while in the p16INK4A(−) patients, there was a trend for worse OS with high HGF level in the control but not in the TPZ/CIS arm (Supplementary Figure D). The 2 year OS on the control arm was 52% (high HGF) versus 68% (low HGF); HR = 1.90, p = 0.099. The p-value of the test of interaction between HGF and treatment in the p16INK4A(−) patients was 0.15.
39 patients from the Peter MacCallum Cancer Centre also had pre-treatment 18FAZA hypoxia PET imaging together with plasma HGF and IL8. HGF levels significantly correlated with the maximum 18FAZA SUV and 18FDG SUV (SUVmax) in the primary tumor (Table 3). No correlation was noted for IL8 with any 18FAZA or 18FDG parameters.
Despite extensive knowledge of tumor hypoxia, attempts to target it, including TPZ, have not been successful in large phase III trials.(3, 20, 37–41) This may be due to inappropriate patient selection, hence diluting the patient pool with individuals who would not benefit and may even be harmed by the drug.(42) One HNSCC patient group who may not benefit is those with HPV(+) oropharyngeal carcinoma. These patients are known to have an excellent prognosis with conventional therapy and considerations are being made to de-intensify their treatment. A prior study has shown that they did not benefit from nimorazole, a hypoxic cell radiosensitizer.(43) These patients made up a large percentage of the patients in the TROG-02.02 trial and may partially explain the negative results here. Subset analysis showed a trend for improved locoregional control with TPZ in the HPV(−) patients.(29) Therefore, it’s crucial to identify molecular predictors for hypoxia-targeted therapy.
Since both HGF and IL8 can be induced by hypoxia, we investigated the prognostic and predictive significance of these markers on TPZ efficacy in this randomized study. We found that IL8 was a prognostic factor for survival in both arms, whereas there was an interaction between HGF and treatment with elevated HGF level being associated with worse OS in the control but not in the TPZ/CIS arm. Within the high HGF group, there was a trend for better OS favoring TPZ/CIS; however the p-value did not reach statistical significance. When HGF and IL8 were combined, the patients who had the largest hazard reduction of death with the addition of TPZ were those with elevated pretreatment level of both markers. In contrast, patients with high IL8 but low HGF level had worse survival on TPZ/CIS. The reason for the differential impact of TPZ on these 2 patient groups is unclear though may be related to IL8 regulation. Hypoxia can induce both markers with HGF induction occurring prior to IL8.(44) HGF can directly promote IL8 expression in HNSCC through both MEK- and PI3K- dependent pathways.(18) While HGF is secreted primarily from HNSCC tumor-derived fibroblasts, IL8 is often produced by tumor cells.(11, 18) In addition to being regulated by hypoxia and HGF, tumor IL8 expression can be affected by other factors, including inflammation, oxidative stress, the Src kinases, and the NFB/AP1 pathway. (45, 46) We hypothesize that patients with high IL8 level belong to 2 different groups: those with IL8 being induced by hypoxia via stromal-derived HGF (IL8-high/HGF-high) and those with IL8 being promoted by factors unrelated to hypoxia (IL8-high/HGF-low). Since hypoxia is a major driver of tumor progression in the first group, targeting hypoxia with TPZ would be beneficial here. In contrast, since hypoxia is unlikely to be the cause of tumor aggression in the second group, the use of TPZ would not benefit these patients. Moreover, these patients, while deriving no benefit from TPZ, may be adversely affected by the lower dose of cisplatin in the TPZ/CIS arm (75mg/m2 in TPZ/CIS versus 100mg/m2 in the control arm). These data suggest that biomarker-guided studies, using multiple markers, may be useful to define patient groups for future hypoxia targeted therapy.
Our group has shown that hypoxia imaging with 18F-Misonidazole (18FMISO), a closely related compound to 18FAZA, could be used to predict for TPZ benefit in a randomized phase II study.(47) We used 18FAZA imaging here because it has a better signal to noise ratio than 18FMISO.(30, 48) In a 13 patient study of intra-patient comparison of the two tracers, we noted a higher tumor to background ratio with FAZA.(49) Here, we found that HGF, but not IL8 levels significantly correlated with both maximum 18FDG and 18FAZA tumor SUV. These findings indicate for the first time that circulating HGF levels may partially reflect tumor hypoxia in HNSCC. The correlation between HGF and FDG may reflect the hypoxia inducibility of both the glucose transporter GLUT1 and the glycolytic enzyme hexokinase. Correlations between hypoxia and FDG have been noted for HNSCC, although variability due to other influences on tumor FDG uptake is significant.(50)
The conduct of this study followed the guidelines suggested by an expert panel from the NCI and FDA.(51) Samples were collected prospectively from most participating patients using standardized collection, handling and storage procedures and connected to a well-annotated clinical database. The HGF ELISA system has been rigorously tested for assay performance and reproducibility. Although HGF is stable for up to 5 freeze-thaw cycles, the tested samples were freeze-thawed only once for alliquoting prior to assayed. The coefficient of variation (CV) of intra-assay precision is within 10% and inter-assay precision is <15%, which are consistent of with most commercial ELISA systems. Stringent statistical analyses were used with a priori defined cut points for each biomarker, adjustments for known prognostic markers and employment test of statistical interactions between markers and treatment. These safeguards ensure that the results are not likely due to chance alone.
As indicated in the method section, we made an a priori decision to exclude patients with major radiation deviations from all analyses of prognostic markers in this trial because we have found that these patients had a markedly inferior outcome compared to those without a major deviation, presumably due to inadequate treatment of their cancer.(21) Consequently, survival had to be measured from the end of radiation and not at the time of enrolment. This exclusion of a small patient subgroup in theory can introduce biases when comparing arms but in reality is unlikely to be significant since the proportion of patients receiving poor RT was similarly present in the two arms (18% CIS, 20% TPZ, p=0.40). Moreover, such potential biases are much less of a concern than the potential bias introduced by inclusion of the patients with known poor survival from radiation deviations. However, because of this exclusion, any future inferences regarding the results of this study should be restricted to patients who have received radiation per plan.
In summary, we found that IL8 is an independent prognostic factor irrespective of treatment and that there is an interaction between treatment arm and HGF level. Such an interaction means that the arms must differ for patients with low HGF, or for patients with high HGF or for both. The examination of the differences between arms within the HGF subgroups provided no definitive answer as to where these differences may lie but indicated that either TPZ/CIS is superior for high HGF or TPZ/CIS is inferior for low HGF or both are true. Furthermore, using a combination of IL8 and HGF, our data suggested that the patients with IL8-high/HGF-high may benefit from TPZ/CIS while those with IL8-high/HGF-low may do better with standard treatment. These biomarker-defined groups highlight the complexity of conducting hypoxia targeted trials in unselected patients and suggest that prospective collection of blood and tumor samples for biomarker validation is critical for success of future hypoxia targeted strategies.
The presence of tumor hypoxia is consistently associated with an adverse prognosis in head and neck cancers (HNC). Despite the importance of this microenvironment factor, the results of trials targeting hypoxia in unselected patient populations have been disappointing. Here we measured the pretreatment plasma levels of HGF and IL8 in 498 patients enrolled in a large randomized trial evaluating the efficacy of Tirapazamine, a hypoxic cell cytotoxin, in HNC. We showed that certain patient subgroups, based on the combination HGF and IL8, may fare better with hypoxia targeted therapy while others may do worse. The results of our study highlight the importance of identifying appropriate markers to select patients for hypoxia targeted therapy.
Supported by: 1 R01 CA118582 (QTL, RF, RJY, HC, CK, EG, RJH, GAM, AG, DR) NHMRC project grant (RF, RJY, GAM, LP, DR) and P01- CA67166 (QTL, HC, AG)
Conflict of Interest: None
Presented at ASCO Annual Meeting, Chicago June 2010