Nicolson, Marianne C. MD, FRCP*; Fennell, Dean A. MD†; Ferry, David MD‡; O’Byrne, Kenneth MD§‖; Shah, Riyaz MD‖; Potter, Vanessa MD¶; Skailes, Geraldine MD#; Upadhyay, Sunil MD**; Taylor, Paul MD††; André, Valerie‡‡; Nguyen, Tuan S. PhD§§; Myrand, Scott P. MD§§; Visseren-Grul, Carla MD‖‖; Das, Mayukh MD¶¶; Kerr, Keith M. FRCPath*
Pemetrexed/cisplatin is the standard first-line treatment for wild-type epidermal growth factor receptor advanced nonsquamous non–small-cell lung cancer (NS-NSCLC), showing survival advantage over the gemcitabine/cisplatin doublet.1–4 For patients with a performance status of 0 to 1, and disease control after four cycles of first-line pemetrexed/cisplatin, maintenance treatment with pemetrexed increased median (m) overall survival (OS) from 11.1 to 13.9 months compared with placebo.5
Pemetrexed/cisplatin shows significant survival advantage in patients with NS-NSCLC over squamous histology.1 The biological hypothesis behind this finding is that pemetrexed, a multitargeted antifolate, is a potent direct inhibitor of thymidylate synthetase (TS); TS mRNA and protein expression is higher in squamous than in adenocarcinoma histopathological specimens.6,7 In two retrospective studies evaluating biopsy samples from NS-NSCLC patients treated with pemetrexed, low TS protein expression levels were associated with significantly better clinical outcomes.8,9 TS overexpression at protein and mRNA levels was associated with reduced sensitivity to pemetrexed.10,11
TS generates deoxythymidine monophosphate for DNA synthesis and is predominantly located in the cytoplasm,12 but it is also found in the nucleus of tumors with high TS-expression levels.13 Its activity takes place mainly in the nucleus, but the site of action is the same for pemetrexed.13
We conducted the first prospective phase II study to explore the association between TS protein and mRNA expression with clinical outcomes in NS-NSCLC patients treated with pemetrexed. To ascertain the biological activity of TS protein expression, evaluation of nuclear TS expression was a logical choice.
Evaluation of nuclear TS expression by immunohistochemistry (IHC) in routine clinical practice is feasible.14 In this phase II trial, eligible patients received up to four cycles of first-line pemetrexed/cisplatin treatment. Those who had not progressed and had a performance status 0 to 1 continued with pemetrexed maintenance until progression or maximum tolerability. The primary objective was to evaluate the association between TS nuclear IHC expression and progression-free survival (PFS). An exploratory objective was to identify the optimal cutpoint of TS expression (protein and mRNA) to separate patients into -high TS- and low TS-expression groups, resulting in maximal association between TS expression and PFS or secondary clinical outcomes—OS, overall response rate (ORR), and disease-control rate (DCR).
PATIENTS AND METHODS
H3E-BP-JMIK (NCT00887549) was an exploratory, single-arm, multicenter, phase II study of first-line pemetrexed plus cisplatin, followed by pemetrexed maintenance. The primary objective was to determine the association between nuclear TS expression and PFS.
Patients were enrolled from April to November 2009, at 14 centers in the United Kingdom and Ireland. Patients (≥18 years of age) with histologically confirmed, stage IIIB or IV NS-NSCLC were eligible and had one or more unidimensionally measurable lesion, meeting Response Evaluation Criteria in Solid Tumors criteria,15 with adequate tumor biopsy specimen available for TS assessment (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/JTO/A413) were considered eligible. Written informed consent was obtained from all patients. The study was approved by United Kingdom and Ireland ethics committees and performed in compliance with Good Clinical Practices.
Eligible patients received four cycles of pemetrexed/cisplatin treatment. On day (d)1 of each 21d-cycle, patients received 500 mg/m2 pemetrexed over 10 minutes, followed by 75 mg/m2 cisplatin over 2 hours as intravenous infusions. Patients achieving complete response (CR), partial response (PR), or stable disease (SD) continued on pemetrexed maintenance (500 mg/m2 every 21 d) until disease progression or discontinuation for intolerability. All patients received folic acid and vitamin B12 supplementation and prophylactic dexamethasone.
Clinical Outcomes and Safety
PFS and OS were defined as time from cycle 1 d1 to first date of progressive disease (PD) (PFS) or death from any cause (OS). Patients were followed until 18 months after the last patient had started chemotherapy. Objective tumor response was assessed on alternate cycles by computed tomography scan (Response Evaluation Criteria in Solid Tumors 1.0).15 Patients were assessed for adverse events before each cycle using Common Terminology Criteria for Adverse Events, Version 3.0, NCI 2006.
Diagnostic histological material was centrally reviewed by an expert pathologist, blinded to the diagnostic information to homogenize NS-NSCLC diagnosis. A formalin-fixed, paraffin-embedded diagnostic tumor tissue (FFPE) sample was obtained to evaluate TS expression at the protein IHC and mRNA level (quantitative reverse-transcription polymerase chain reaction, qRT-PCR). Almac Diagnostics, Craigavon, United Kingdom, coordinated all laboratory work under specifically developed standard operating procedures.
TS protein expression in the nucleus and cytoplasm
Standard protocols were used for IHC staining and assessment, using antibody clone 4H4B1 (Zymed, South San Francisco, CA; Catalog No. 18–0399).14 A pathologist and medical scientist, blinded to clinical information, evaluated TS nuclear and cytoplasmic staining, using an intensity scale (0–3) and calculated the percentage of cells in each category, resulting in semiquantitative H-scores ranging from 0 to 300. Total TS H-scores were calculated posthoc as the sum of nuclear and cytoplasm TS H-scores (range, 0–600).
TS mRNA expression
mRNA was isolated from each FFPE sample and amplified by qRT-PCR.16,17 TS mRNA was quantified based on the cycle threshold (Cq), which corresponds to the number of cycles required to observe a predefined signal intensity. qRT-PCR data are presented as Δ-Cq, calculated as TS gene Cq minus reference Cq (mean Cq of 4 normalized reference control genes: topoisomerase I, succinate dehydrogenase complex subunit A, hydroxymethylbilane synthase, and glucuronidase β). High Δ-Cq values correspond to low TS mRNA expression.
All treated patients with valid TS scores were included in the analyses. All patients who received at least one dose of chemotherapy were evaluated for safety. Data were analyzed using SAS 9.2 software (SAS Institute, Cary, NC). Statistical analyses were conducted at a significance level of α = 0.05.
Monte Carlo simulations18 were used to estimate a sample size that achieved a power of approximately 90% for detecting a significant association between TS expression and PFS at a two-sided significance level of 0.05. Simulations resulted in a planned sample size of approximately 60 patients, to obtain 54 patients with valid TS scores (assuming failure of IHC assessment in 10% of FFPE samples). For the purpose of analysis, TS expression was assumed to be categorical (staining intensity), although the underlying variable (H-score) was semiquantitative.
Association between TS expression (H-score and qRT-PCR, continuous variable) and clinical outcomes
A Cox proportional hazard regression model was applied, including nuclear or cytoplasm TS H-score, or as posthoc analysis minus nuclear plus cytoplasm (total) TS H-score as continuous variable (hazard ratio [HR] per 1-unit increase of H-score), or Δ-Cq (HR per 1-unit decrease of Δ-Cq) as continuous variable, and PFS as dependent variable; no additional covariates were included.
Corresponding Cox regression models were used to explore the association of TS expression with OS. Similar logistic regression analyses were applied to explore the association between TS expression (IHC and qRT-PCR) and ORR or DCR (odds ratio per 1-unit increase in H-score or Δ-Cq value).
Exploratory analysis of low versus high TS- expression groups
Maximum χ2 analysis19 was used to identify the optimal cutpoint for each TS expression class to divide patients into TS high- and low-expression groups, scanning the inner 80% of marker values. The optimal cutpoint was the H-score or Δ-Cq, which resulted in maximal association between TS expression class and the clinical outcome of interest (PFS, OS, ORR, DCR). Regression models, including PFS/OS (Cox) or ORR/DCR (logistic) as dependent variables and high or low TS expression (based on the identified optimal cutpoint) as the independent variable, were then used to explore the association between TS expression class and clinical outcomes. p Values were adjusted based on the asymptotic probability of the observed maximal χ2 statistic, limiting the search to the inner 80% of marker values.19
The Kaplan–Meier technique20 was used to estimate mPFS and mOS together with 95% confidence intervals (CIs) computed, using the complementary log-log transformation.21 ORR was calculated as the proportion of evaluable patients who achieved a best response of CR or PR, including the exact 95%CI.22 The DCR was calculated correspondingly (CR+PR+SD). PFS was censored at the date of the last objective progression-free disease assessment; OS was censored at the last contact date.
Of 70 patients starting pemetrexed/cisplatin, 60 had valid TS IHC scores and were included in the primary analysis (Fig. 1); 61 had valid qRT-PCR data. The results below relate to the 60 patients with valid TS IHC assays (Table 1), constituting the evaluable patient population. Baseline characteristics were similar for the 70 patients treated and the 60 patients with valid TS IHC assays (Table 1).
For the 60 patients with IHC assessments, mPFS was 5.5 months (95%CI, 3.9–6.9 months; 31.7% of patients censored) at a minimum of 18 months’ follow-up, with mOS of 9.6 months (95%CI, 7.3–15.7 months; 10.0% of patients censored). The ORR was 30.0% (18 PRs; no CR; n = 60); 20 of 60 patients achieved SD (DCR 63.3%).
Association between TS Expression (Continuous) and Clinical Outcomes
In the primary Cox regression analysis, a statistically significant association was observed between nuclear TS expression and PFS (p < 0.001; HR per 1-unit increase in H-score of 1.015 [95%CI, 1.008–1.021]), indicating that lower nuclear TS expression levels were associated with longer PFS (Table 2). Supplementary Figure 1A (Supplemental Digital Content 2, http://links.lww.com/JTO/A414) illustrates this trend by plotting the individual TS nuclear H-scores versus the respective PFS.
The associations between nuclear TS expression and OS or DCR were also significant (p < 0.0001 and p = 0.0112, respectively; Table 2), whereas the association with ORR was not significant (p = 0.0919). The associations between TS cytoplasm expression (H-score), or TS mRNA expression and PFS or OS, were less pronounced than those of nuclear TS expression, but also statistically significant, and all analyses were directionally consistent; no significant association was found between TS-expression biomarkers and tumor response (ORR, DCR) in this analysis (Table 2).
Identification of Optimal Cutpoints for Dividing Patients into Low–TS- and High–TS-Expression Groups
Using the maximal χ2 method, the optimal H-score cutpoint for dividing patients into low- and high-nuclear TS IHC expression groups was consistently identified as 70 for PFS, OS, and ORR (Table 3). The optimal cutpoint for DCR was 50. Figure 2A and Supplementary Figure 2A (Supplemental Digital Content 3, http://links.lww.com/JTO/A415) present the effect of moving the cutpoint along the H-score continuum on the association with PFS and OS. The association between nuclear H-scores and PFS or OS was statistically significant (p < 0.05) across a wide range of cutpoints (Wald χ2 statistic above the horizontal line of 3.84), including the identified optimal cutpoint of 70. Regardless of which cutpoint was chosen, mPFS and mOS of the low–TS-expression group were consistently higher than the mPFS and mOS of the corresponding high-expression group (Fig. 2B; Supplementary Figure 2B, Supplemental Digital Content 3, http://links.lww.com/JTO/A415). The association with ORR and DCR was weaker (Supplementary Figs. 3 and 4, Supplemental Digital Content 4 and 5, http://links.lww.com/JTO/A416 and http://links.lww.com/JTO/A417).
For TS expression in the cytoplasm, no consistent optimal H-score cutpoint was identified across the different outcomes (Table 3); associations were less pronounced, but otherwise, showed the same direction as those for nuclear TS expression (Table 3; and Supplementary Figs. 5–8, Supplemental Digital Content 6, 7, 8, and 9 http://links.lww.com/JTO/A418, http://links.lww.com/JTO/A419, http://links.lww.com/JTO/A420, and http://links.lww.com/JTO/A421).
For TS mRNA expression, the optimal cutpoint dividing patients into TS mRNA low- and high-expression groups was identified as a Δ-Cq value of –1.30 for both PFS and OS, and a value of –1.28 for both ORR and DCR (Table 3). Supplementary Figures 9A, 10A, 11A, and 12A (Supplemental Digital Content 10, 11, 12, and 13, http://links.lww.com/JTO/A422, http://links.lww.com/JTO/A423, http://links.lww.com/JTO/A424, and http://links.lww.com/JTO/A425) present the effect of moving the cutpoint along the Δ-Cq score continuum on the association with the different clinical outcomes. The separations of median values between low and high mRNA-expression groups showed the same trend, but were much less pronounced, than those for the nuclear TS protein expression (Supplementary Figs 9B, 10B, 11B, and 12B, Supplemental Digital Content 10, 11, 12, and 13, http://links.lww.com/JTO/A422, http://links.lww.com/JTO/A423, http://links.lww.com/JTO/A424, and http://links.lww.com/JTO/A425).
Association between TS Expression (Low and High) and Clinical Outcomes
Using the identified optimal H-score cutpoint of 70 for nuclear TS expression, low nuclear TS expression was significantly associated with longer PFS and OS (low- versus high-expression group: mPFS 7.1 versus 2.6 months; HR [95%CI] 0.283 [0.155–0.516]; [adjusted p = 0.0015; mOS 17.5 versus 4.6 months; HR [95%CI] 0.306 [0.161–0.585]; adjusted p = 0.0003; Table 3, Fig. 3). For ORR and DCR, the differences between high–TS- and low–TS-expression groups were not significant, but data showed the same direction as for PFS and OS (Table 3). If the prespecified H-score cutpoint of 70 was used, the association between nuclear TS expression and outcome was significant (unadjusted p value) for all four clinical outcomes (Fig. 2A; Supplementary Figs. 2A, 3A, and 4A, Supplemental Digital Content 3, 4, and 5, http://links.lww.com/JTO/A415, http://links.lww.com/JTO/A416, and http://links.lww.com/JTO/A417). If TS-negative and TS-positive expression groups were compared (H-score cutpoint prespecified as 0), a significant association was observed only for OS.
For the association between cytoplasmic TS or TS mRNA expression and clinical outcomes, the differences between high–TS- and low–TS-expression groups were less pronounced, but consistently showed the same direction as for nuclear TS expression (Table 3; Supplementary Figs. 13–15 Supplemental Digital Content, 14, 15, and 16, http://links.lww.com/JTO/A426, http://links.lww.com/JTO/A427, and http://links.lww.com/JTO/A428).
In summary, low nuclear TS expression was significantly associated with longer PFS and OS (continuous and optimal cutpoint analyses) (Table 4). H-scores for nuclear or total TS expression and Δ-Cq for TS mRNA expression were highly correlated (Spearman correlation coefficient r = −0.616 and –0.535, respectively; p < 0.001 for both); the correlation between cytoplasm H-scores and Δ-Cq was less pronounced (r = −0.286; p = 0.03). The correlation between H-scores for nuclear and cytoplasmic TS expression was also highly significant (r = 0.406; p = 0.001).
Treatment Exposure and Safety
Of 70 patients treated (60 with valid IHC), 51 received all four cycles of pemetrexed/cisplatin, and 43 (61.4%; 37 with valid TS IHC) started pemetrexed maintenance. Patients received median seven treatment cycles (range, 1–24); median relative dosage intensity for pemetrexed was 93% (cisplatin 97.6%).
Five patients died during or within 30 days after chemotherapy: one from neutropenic sepsis during pemetrexed/cisplatin (cycle 3); one from pneumonia not related to study drugs; three from underlying NSCLC. Forty-one patients (58.6%) experienced at least one serious adverse event (related to study drug 24 patients; 34.4%). Supplementary Table 2 (Supplemental Digital Content 17, http://links.lww.com/JTO/A429) summarizes the most frequent study–drug–related grade 3/4 toxicities.
This is the first prospective study to show a statistically significant association between low TS expression and better clinical outcomes in NS-NSCLC patients, treated with first-line pemetrexed/cisplatin, followed by maintenance pemetrexed. The study is unique for its prospective design measurement of TS expression at the protein and mRNA levels, with comprehensive statistical analyses. The association between low TS expression and longer PFS and OS was statistically significant for all four TS assays (continuous TS analyses).
A separate analysis applied the maximal χ2 method to identify optimal cutpoints for dividing patients into low and high TS-expression groups. The optimal cutpoint was the H-score or Δ-Cq value, which resulted in maximal association between TS expression class and the clinical outcome of interest; for nuclear TS expression, an H-score of 70 was consistently identified as the optimal cutpoint for three different outcomes (PFS, OS, ORR). In the maximal χ2 analysis using this optimal cutpoint, the nuclear low TS-expression group was significantly associated with longer PFS and OS. The maximal χ2analysis for DCR identified an H-score of 50 as the optimal cutpoint, but was not significant after adjusting for testing multiple points. If a cutpoint of 70 was analyzed for DCR, a similar general relationship was observed, as with the other efficacy endpoints. Many pathologists define TS expression as positive or negative; looking at TS-negative versus TS-positive expression groups (nuclear H-score cutpoint specified as 0), a significant association was observed only with OS. However, the direction of the results was consistent with the optimal cutpoint approach for all four outcomes. Optimal cutpoint analyses for TS cytoplasm and mRNA expression showed the same pattern, but the effect was less pronounced, indicating that IHC assessment of nuclear TS expression may be the more relevant biomarker. Most previous studies assessed TS mRNA expression by qRT-PCR, but an IHC-based assay would be better for routine use because the assay is simpler, more robust, does not require fresh tumor tissue, and directly reflects only tumor-cell TS.
Our findings are in line with published preclinical and retrospective clinical data.8–11,23 This is consistent with the hypothesis that the reduced efficacy of pemetrexed in patients with squamous NSCLC is related to higher mean TS expression.1,7,24 Tumors may acquire resistance to pemetrexed by up-regulating TS gene expression.10,25
The few previous studies that assessed TS expression by IHC in NS-NSCLC used different scoring and cutpoint systems, and did not differentiate between the nucleus and cytoplasm. Sun et al.8 applied H-scores ranging from 0 to 200 and defined tumors with H-scores at or below the median as TS negative. TS negativity was associated with a higher RR (33.7% versus 14.1%; p = 0.002) and prolonged PFS (mPFS: 4.1 versus 2.0 months; p = 0.001).8 Chen et al. 9 defined low- and high-expression groups as H-scores lesser than or equal to 120 and more than 120, respectively. For 49 patients with tested specimens, mPFS was 4.8 months in the low–TS- and 3.4 months in the high–TS-expression groups (p = 0.01).9 In the subgroup of patients with adenocarcinoma, the low–TS-expression group also had prolonged PFS and OS (mPFS 4.8 versus 3. 8 months; mOS 21.4 versus 10.0 months; p = 0.03 for both).9 On the basis of the optimal cutpoint for TS nuclear expression identified in this study, differences between low–TS- and high–TS-expression groups were more pronounced (mPFS 7.1 versus 2.6 months; p = 0.0015; mOS 17.5 versus 4.6 months; p = 0.0100). Further studies in broader patient populations are required to clarify whether clinically relevant cutpoints for nuclear TS expression can be validated for use in routine clinical practice.
Because of the small sample size and the lack of a comparator arm, the evaluation of TS expression in this study does not allow robust assessment of its predictive role, because of the possibility that TS may also be prognostic. On the individual patient level, PFS varied widely among tumors with low nuclear TS expression, indicating that TS expression is not an exclusive predictive factor and additional determinants of sensitivity to this agent remain to be discovered. Further evidence is needed to determine whether IHC assessment of nuclear TS expression may be developed as a biomarker for the clinical outcome of pemetrexed treatment in NSCLC and other tumors.
Regarding the clinical efficacy of pemetrexed maintenance, mPFS and mOS were 5.5 and 9.6 months (18 months of follow-up), respectively. A single-arm Japanese study evaluated pemetrexed maintenance after four cycles of pemetrexed/carboplatin in 109 NS-NSCLC patients; mPFS was similar (5.7 months) to that of the present study but OS data were not reported.26 These values were lower than those reported for maintenance trials of pemetrexed because those studies excluded patients with PD on first-line treatment from the reported OS values.5,27,28
In conclusion, this prospective study showed consistent results across different assays and clinical outcomes, and provides the best evidence to date that low TS expression in NS-NSCLC patients treated with pemetrexed/cisplatin is associated with better clinical outcome. The association was most pronounced for low nuclear TS expression assessed by IHC. Our data suggest that an optimal cutpoint can be identified to define low versus high nuclear TS expression.
The authors thank all investigators, patients, and their carers for their participation in the study. The authors thank Perry Maxwell, Belfast Hospitals’ Trust, Belfast, United Kingdom, for performing TS IHC assays, and Stephen Moore, Almac Diagnostics, for coordinating all laboratory tests. The authors also thank Rebecca Hozak and her oncology statistics group, Eli Lilly, Indianapolis for conducting the pharmacogenomic analyses, and Nicola Murray, Eli Lilly, United Kingdom, for managing the study. Annemarie Hütz and Karin Helsberg, Trilogy Writing and Consulting, Frankfurt, Germany, provided medical writing support on behalf of Eli Lilly and Company.
This study concept was developed as part of an American Society of Clinical Oncology/American Association for Cancer Research workshop on methods in clinical research, Vail, CO, September 2008. The authors sincerely thank the mentoring group comprising Mary L. (Nora) Disis, MD, Quynh-Thu Le, MD, and Danille Normolle, PhD.
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