Tumour expression of leptin is associated with chemotherapy resistance and therapy-independent prognosis in gastro-oesophageal adenocarcinomas

Abstract

Background: Cytotoxic chemotherapy remains the main systemic therapy for gastro-oesophageal adenocarcinoma, but resistance to chemotherapy is common, resulting in ineffective and often toxic treatment for patients. Predictive biomarkers for chemotherapy response would increase the probability of successful therapy, but none are currently recommended for clinical use. We used global gene expression profiling of tumour biopsies to identify novel predictive biomarkers for cytotoxic chemotherapy.

Methods: Tumour biopsies from patients (n=14) with TNM stage IB-IV gastro-oesophageal adenocarcinomas receiving platinum-based combination chemotherapy were used as a discovery cohort and profiled with Affymetrix ST1.0 Exon Genechips. An independent cohort of patients (n=154) treated with surgery with or without neoadjuvant platinum combination chemotherapy and gastric adenocarcinoma cell lines (n=22) were used for qualification of gene expression profiling results by immunohistochemistry. A cisplatin-resistant gastric cancer cell line, AGS Cis5, and the oesophageal adenocarcinoma cell line, OE33, were used for in vitro validation investigations.

Results: We identified 520 genes with differential expression (Mann-Whitney U, P<0.020) between radiological responding and nonresponding patients. Gene enrichment analysis (DAVID v6.7) was used on this list of 520 genes to identify pathways associated with response and identified the adipocytokine signalling pathway, with higher leptin mRNA associated with lack of radiological response (P=0.011). Similarly, in the independent cohort (n=154), higher leptin protein expression by immunohistochemistry in the tumour cells was associated with lack of histopathological response (P=0.007). Higher leptin protein expression by immunohistochemistry was also associated with improved survival in the absence of neoadjuvant chemotherapy, and patients with low leptin protein-expressing tumours had improved survival when treated by neoadjuvant chemotherapy (P for interaction=0.038). In the gastric adenocarcinoma cell lines, higher leptin protein expression was associated with resistance to cisplatin (P=0.008), but not to oxaliplatin (P=0.988) or 5fluorouracil (P=0.636). The leptin receptor antagonist SHLA increased the sensitivity of AGS Cis5 and OE33 cell lines to cisplatin.

Conclusions: In gastro-oesophageal adenocarcinomas, tumour leptin expression is associated with chemoresistance but a better therapy-independent prognosis. Tumour leptin expression determined by immunohistochemistry has potential utility as a predictive marker of resistance to cytotoxic chemotherapy, and a prognostic marker independent of therapy in gastro-oesophageal adenocarcinoma. Leptin antagonists have been developed for clinical use and leptin and its associated pathways may also provide much needed novel therapeutic targets for gastro-oesophageal adenocarcinoma.

Figure 1

Mean leptin mRNA from Affymetrix Exon 1.0 ST Array in discovery set (n=14) in radiological responders and nonresponders (Student's t-test, P=0.011). Error bars±s.e.m. There is no significant correlation between tumour leptin mRNA expression and the tumour cell content of biopsies (P=0.779).

Figure 2

Leptin IHC in gastro-oesophageal adenocarcinomas. (A) Representative IHC (weak and medium × 200; strong × 400). (B) Leptin IHC and histopathological response to neoadjuvant platinum-based combination chemotherapy (χ², P=0.007). Mandard TRG 4–5 have a medium survival of 23.8 months and Mandard TRG 1–3 median survival not reached but >72 months (Kaplan–Meier analysis, log rank P=0.009, data not shown) in this series and survival of Mandard TRG 4–5 is not different to those who had surgery only and no neoadjuvant chemotherapy in this cohort (median survival=34.4 months, Kaplan–Meier log rank P=0.757, data not shown). (C) Leptin IHC and overall survival (Kaplan–Meier analysis, log rank P=0.021). (D) Leptin IHC and survival stratified by neoadjuvant chemotherapy (P for interaction=0.038). Leptin positive=leptin IHC strong; leptin negative=leptin IHC negative, weak or moderate.

Figure 3

Leptin IHC and in vitro chemosensitivity to (A) cisplatin, (B) oxaliplatin and (C) 5FU in gastric adenocarcinoma cell panel (n=22 cell lines). Figures show mean IC₅₀ (drug concentration for 50% growth inhibition) performed in triplicate and a minimum of three replicates, P-values, Student's t-test and error bars±s.e.m. Representative examples of (C) weak, (D) moderate and (F) strong leptin IHC staining in cell lines. Magnification × 100 in all cases. Effects of leptin receptor antagonist on cisplatin sensitivity of (G) a highly cisplatin-resistant gastric adenocarcinoma cell line AGS Cis5, IC₅₀=16 μM, cisplatin=8 μM, SHLA=0.32 ng ml⁻¹, and (H) oesophageal adenocarcinoma cell line OE33, IC₅₀=1.5 μM, cisplatin=0.5 μM, SHLA=0.64 ng ml⁻¹. (G and H) Mean growth from four independent experiments repeated in triplicate, relative cell survival (%)=(MTT OD value for cells only/MTT OD value for cells+treatment as indicated) × 100, with P-values for ANOVA with Tukey's post hoc test shown where significant (P<0.05), and error bars±s.e.m.