Tumor clock protein PER2 as a determinant of survival in patients (pts) receiving oxaliplatin-5-FU- leucovorin as 1st line chemotherapy for metastatic colorectal cancer (mCRC) S. IACOBELLI, P. Innominato, M. Piantelli, G. Bjarnason, B. Coudert, C. Focan, S. Giacchetti, A. Poncet, C. Garufi, F. Levi, and the ARTBC Chronotherapy Group

Authors’ disclosure The Authors indicate no potential conflicts of interest

Learning Objectives: Role of the Circadian Timing System in malignant processes Evaluate the correlations between the expression of clock proteins in healthy colonic mucosa and primary colon cancer Assess the prognostic value of the molecular clock in human cancer

The Molecular Clock Levi F, Schibler U. Annu Rev Pharmacol Toxicol 2007;47: Harmer SL, Panda S, Kay SA. Annu Rev Cell Dev Biol 2001;17:215-53

Circadian control of drug metabolism; Tight link between molecular clock components and cell cycle, DNA repair and apoptosis Mutant clock genes and cancer risk PER1 & PER2 functioning as oncosuppressors Role of the molecular clock in malignant processes Levi F, Schibler U. Annu Rev Pharmacol Toxicol 2007;47: Hunt T, Sassone-Corsi P. Cell 2007 May 4;129(3):461-4 Zhu Y, Leaderer D, Guss C, et al. Int J Cancer 2007 Jan 15;120(2):432-5 Zhu Y, Brown HN, Zhang Y, et al. Cancer Epidemiol Biomarkers Prev 2005 Jan;14(1): Fu L, Pelicano H, Liu J, Huang P, et al. Cell 2002 Oct 4;111(1): Gery S, Komatsu N, Baldjyan L, et al. Mol Cell 2006 May 5;22(3):375-82

OBJECTIVE To assess whether the expression patterns of PER1, PER2 and PER3 proteins in primary tumor or healthy mucosa predicts for clinical outcome in chemo-naive patients with mCRC receiving a combination of oxaliplatin, 5-fluorouracil and folinic acid in a randomized phase III trial (EORTC trial 05963) of flat vs. chronomodulated infusion

METHODS PER1, PER2 and PER3 expression evaluated by immunohistochemistry; Healthy mucosa samples: categorized according to the % of positive cells –Negative: ≤ 5% –Weakly positive: >5%- ≤ 20% –Strongly positive: > 20% Primary tumors: % of labelled cells categorized according to terciles.

The correlations between the expression patterns of each PER between healthy mucosa and primary tumor were computed Univariate and multivariate analyses were performed from time of inclusion in the study till the event using the aforementioned categories. STATISTICAL ANALYSIS

Paraffin wax embedded samples of primary tumor were collected for 198 patients. In 169 of them, healthy mucosa was also present. RESULTS – sample size

RESULTS –patients’ characteristics CharacteristicNo% Age (yrs) median range Sex Male Female Treatment Conv Chrono WHO PS CharacteristicNo% No Mets sites CEA normal abnormal

RESULTS – PERs expression in mucosa Negative Weak Strong PER1 PER2 PER3

RESULTS – PER2 in Mucosa Negative Weak Strong

RESULTS – PER2 in Tumor 0% 100%

The expression pattern of any PER protein is not influenced by gender, age, site of primary tumor and no. of metastatic sites. RESULTS – clinical features

The expression pattern of any PER protein in healthy mucosa was not associated with statistically significant differences in OS. No association was found between the % of labelled tumor cells for PER1 or PER3 and OS. RESULTS – overall survival

RESULTS – PER2 & overall survival The greater the % of stained tumor cells for PER2, the longer the survival at univariate analysis (Log-rank, p=0.04; Log-rank for trend, p= 0.013) Median (months) 2 yrs (%)5 yrs (%) 1 st tercile nd tercile rd tercile

No. At risk 1° tercile ° tercile ° tercile Log-rank, p = Log-rank for trend p= RESULTS – PER2 & overall survival

A multivariate Cox model was computed using PER2%+Tum terciles, WHO-PS, # of metastatic sites, presence of metastases at diagnosis, age, gender and treatment arm as covariates. RESULTS – multivariate analysis

The independent prognostic value of PER2%+Tum terciles was confirmed, together with PS and # of metastatic sites involved. HR (95% CI)p value PER2 terciles 1 st vs 2 nd 1 st vs 3 rd 0.74 ( ) 0.58 ( ) WHO PS 0 vs 1 0 vs ( ) 3.7 ( ) No. Mets sites 1 vs 2 1 vs ≥3 1.5 ( ) 2.2 ( )

Summary PER3 clock protein expression in healthy mucosa and primary tumor were correlated. Lower expression of PER2 in tumor cells was associated with a shorter survival.

Conclusions Down-regulation of core clock gene Per2 in tumors predicted for poor survival in chemo-naive pts with metastatic colorectal cancer. This is the first report of an association between clock gene down regulation and outcome in metastatic colorectal cancer.

RESULTS – PERs expression in tumor PER1 PER2 PER3 1 st tercile 2 nd tercile 3 rd tercile

RESULTS - correlations A positive correlation between the expression pattern of PER3 in healthy mucosa and primary tumor was found (p=0.002, Pearson’s  2 ). Mucosa 1 st tercile2 nd tercile3 rd tercile Negative Weak Strong PER3