Excision repair cross-complementing group-1 (ERCC1) induction kinetics and polymorphism are markers of inferior outcome in patients with colorectal cancer treated with oxaliplatin

Devika Rao, Atrayee Basu Mallick, Titto Augustine, Cecilia Daroqui, Jeeshan Jiffry, Amartej Merla, Imran Chaudhary, Raviraja Seetharam, Arjun Sood, Srikanth Gajavelli, Santiago Aparo, Lakshmi Rajdev, Andreas Kaubisch, Jennifer Chuy, Abdissa Negassa, John M Mariadason, Radhashree Maitra, Sanjay Goel, Devika Rao, Atrayee Basu Mallick, Titto Augustine, Cecilia Daroqui, Jeeshan Jiffry, Amartej Merla, Imran Chaudhary, Raviraja Seetharam, Arjun Sood, Srikanth Gajavelli, Santiago Aparo, Lakshmi Rajdev, Andreas Kaubisch, Jennifer Chuy, Abdissa Negassa, John M Mariadason, Radhashree Maitra, Sanjay Goel

Abstract

Background: ERCC1, a component of nucleotide excision repair pathway, is known to repair DNA breaks induced by platinum drugs. We sought to ascertain if ERCC1 expression dynamics and a single nucleotide polymorphism (SNP) rs11615 are biomarkers of sensitivity to oxaliplatin therapy in patients with colorectal cancer (CRC).

Methods: Western blot and qPCR for ERCC1 expression was performed from PBMCs isolated from patients receiving oxaliplatin-based therapy at specified timepoints. DNA was also isolated from 59 biorepository specimens for SNP analysis. Clinical benefit was determined using progression free survival (PFS) for metastatic CRC.

Results: ERCC1 was induced in PBMC in response to oxaliplatin in 13/25 patients with mCRC (52%). Median PFS with ERCC1 induction was 190d compared to 237d in non-induced patients (HR 2.35, CI 1.005-5.479; p=0.0182). ERCC1 rs11615 SNP analysis revealed that 43.3% harbored C/C, 41.2%-T/C and 15.5%-T/T genotype. Median PFS was significantly lower with C/C or T/C (211 and 196d) compared to T/T (590d; p=0.0310).

Conclusions: ERCC1 was induced in a sub-population of patients undergoing oxaliplatin treatment, which was associated with poorer outcome, suggesting this could serve as a marker of oxaliplatin response. C/C or C/T genotype in ERCC1 rs11615 locus decreased benefit from oxaliplatin.

Keywords: ERCC1; FOLFOX; colorectal cancer; oxaliplatin; resistance.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare that they have no conflict of interest. DISCLOSURES The authors have no conflicts of interest to disclose.

Copyright: © 2019 Rao et al.

Figures

Figure 1. A schematic representation of the…
Figure 1. A schematic representation of the study workflow.
54 patients were consented for blood draw to assess ERCC1 kinetics. Expression levels in all patients were assessed by either Western blot or polymerase chain reaction. 43/54 patients had analysis by both modalities. 38/54 samples were additionally assessed for presence of ERCC1 rs11615 SNP. This was based on availability of residual sample. For validation of ERCC1 rs11615 SNP analysis 59 individual samples were additionally obtained from an institutional biorepository which stores excised tumors as Formalin Fixed Paraffin Embedded (FFPE) tissue blocks.
Figure 2. A scatter plot which depicts…
Figure 2. A scatter plot which depicts the PFS among patients with mCRC.
Correlation is made with baseline, 0 hour, ERCC1 expression identified by qPCR after normalization with the housekeeping gene, GAPDH. R2 value indicates no significant correlation exists.
Figure 3. A line graph depicting the…
Figure 3. A line graph depicting the pattern of change in ERCC1 expression over time in each individual patient (n=50) where mRNA expression was quantified by qPCR.
The graph is censored at 20 to allow for graphical representation. The lowest expression was seen in patient 21 at 2 hours where ERCC1 level was suppressed to 2% of baseline levels. Contrarily, the highest expression was in patient 1 at 48 hours, where ERCC1 level was 400% of baseline.
Figure 4. Western blot (WB) images from…
Figure 4. Western blot (WB) images from three representative patients.
The superior band indicates expression of a housekeeping gene, β -actin, which served as internal control. The inferior band represents the gene of interest, ERCC1. Each patient had expression analyzed at four time points: 0, 2, 48 Hour and 15 Day. The first patient did not show change in ERCC1 expression over time, assessed by band intensity remaining equal over time. Patient 2 had an increase in ERCC1 band intensity at the 15 Day time point compared to 0 Hour, while Patient 3 showed the maximum ERCC1 expression at 0 Hour. In this case, there was a decrease in expression over the other three time points.
Figure 5. A graphical representation of the…
Figure 5. A graphical representation of the ERCC1 kinetics in patients with mCRC over time.
13 patients had an increase in ERCC1 expression, with early increase, at the 2 hour mark, being seen in 7/13 patients. 5 patients had a decrease in ERCC1 expression, with all showing the change as early as the 2-hour time point. Eighteen patients with mCRC had a change in ERCC expression, of whom 12 (67%) demonstrated this at 2 hours. Six patients had no change in expression at any time point.
Figure 6. The progression free survival (PFS)…
Figure 6. The progression free survival (PFS) analysis of metastatic colorectal cancer patients who underwent evaluation of ERCC1 kinetics (n=24).
13/24 had an increase in ERCC1 expression, with a corresponding median PFS of 190 days. This was significantly lower than the PFS of patients who had no change or decrease in ERCC1 expression kinetics (237 days, p= 0.018).
Figure 7. A representation of the baseline…
Figure 7. A representation of the baseline ERCC1 mRNA expression levels level in relation to ERCC1 rs11615 SNP in the study cohort.
This graph is censored at 20, while the highest expression in the C/C group is 126 (n=1) and in the heterozygote group is 77 (n=1).
Figure 8. The progression free survival (PFS)…
Figure 8. The progression free survival (PFS) analysis in the study (n=19) and validation (n=59) cohorts of patients with metastatic colorectal cancer wherein tumor tissues were obtained from an institutional biorepository.
Samples were analyzed for ERCC1 rs11615 SNP. Patients were categorized into homozygous (C/C, T/T) and heterozygous (T/C) genotypes.

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