Background: Cetuximab has shown significant clinical activity in metastatic colon cancer. However, cetuximab-containing neoadjuvant chemoradiation has not been shown to improve tumor response in locally advanced rectal cancer patients in recent phase I/II trials. We evaluated functional germline polymorphisms of genes involved in epidermal growth factor receptor pathway, angiogenesis, antibody-dependent cell-mediated cytotoxicity, DNA repair, and drug metabolism, for their potential role as molecular predictors for clinical outcome in locally advanced rectal cancer patients treated with preoperative cetuximab-based chemoradiation.

Methods: 130 patients (74 men and 56 women) with locally advanced rectal cancer (4 with stage II, 109 with stage III, and 15 with stage IV, 2 unknown) who were enrolled in phase I/II clinical trials treated with cetuximab-based chemoradiation in European cancer centers were included. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor samples and genotyping was done by using PCR-RFLP assays. Fisher's exact test was used to examine associations between polymorphisms and complete pathologic response (pCR) that was determined by a modified Dworak classification system (grade III vs. grade IV: complete response).

Results: Patients with the epidermal growth factor (EGF) 61 G/G genotype had pCR of 45% (5/11), compared with 21% (11/53) in patients heterozygous, and 2% (1/54) in patients homozygous for the A/A allele (P < 0.001). In addition, this association between EGF 61 G allele and pCR remained significant (P = 0.019) in the 59 patients with wild-type KRAS.

Conclusion: This study suggested EGF A+61G polymorphism to be a predictive marker for pCR, independent of KRAS mutation status, to cetuximab-based neoadjuvant chemoradiation of patients with locally advanced rectal cancer. Clin Cancer Res; 17(15); 5161–9. ©2011 AACR.

Translational Relevance

Our study suggests epidermal growth factor A+61G polymorphism is significantly associated with pathologic response to cetuximab-based neoadjuvant chemoradiation in locally advanced rectal cancer patients, independent of KRAS mutation status. This is the first study suggesting this polymorphism might be predictive marker for response to cetuximab-based therapy.

It is estimated that 142,570 Americans will be diagnosed with colorectal cancer (CRC) in 2010, of which 39,670 are rectal cancers. The incidence rate of rectal cancer has decreased from 18.42 to 12.77 per 100,000 over the past 20 years, largely attributed to the improved screening programs. From 1999 to 2006, 77% of diagnosed rectal cancer patients had localized or regional advanced disease and these patients had significant 5-year survival advantage (88.6% and 67.1%) compared with 12.3% for distantly metastasized patients (SEER's data). Similar trend has been observed in Western Europe. The widely used surgical technique, total mesorectal excision alone, has decreased the local recurrence rate from 39.4% to 9.8% and improved the observed 5-year survival rate from 50% to 71% (1). In addition, preoperative therapy with concurrent radiation and infusion of 5FU has shown to further decrease the local recurrence rate in stages II and III rectal cancer (2–4). Therefore neoadjuvant chemoradiation is now a standard of care for locally advanced rectal cancer patients. Interestingly, neither pelvic radiation nor chemotherapy before surgery has shown significant survival benefits. Ongoing clinical trials are examining newer agents such as capecitabine, oxaliplatin, irinotecan, cetuximab, or bevacizumab, to further optimize local control, reduce distant metastases and ultimately improve survival.

Epidermal growth factor receptor (EGFR) signals through 2 major pathways, the RAS-mitogen-activated protein kinase and the PI3K-AKT pathway. Hyperactivity of the EGFR pathways is associated with more aggressive phenotype and poor prognosis of cancers (5) and can render cancer cells resistant to radiation (6). Cetuximab is an EGFR monoclonal antibody and has shown significant clinical activity in metastatic colon cancer (7, 8). In addition, concomitant high-dose radiotherapy plus cetuximab has been shown to improve locoregional control and reduce mortality in squamous carcinoma of the head and neck (9). Recent phase I/II clinical trials have evaluated EGFR inhibitors as a radiosensitizer in CRC and showed that cetuximab can be safely given without unexpected toxicity (10, 11). However, cetuximab-containing neoadjuvant chemoradiation has not been shown to improve tumor response and complete pathologic response (pCR) rates in locally advanced rectal cancer patients (12–14). To identify the subgroup of patients who may benefit from the cetuximab protocol, we designed a retrospective study within a cohort of 4 prospectively conducted phase I/II clinical trials. We evaluated functional germline polymorphisms of genes involved in the EGFR signaling pathway (EGF, EGFR, COX-2, KRAS, CyclinD1), angiogenesis (VEGF, IL-8), antibody-dependent cell-mediated cytotoxicity (FCGR2A/3A), DNA repair (XRCC3, Rad51), and drug metabolism (TS, MTHFR), for their potential role as molecular predictors for clinical outcome in locally advanced rectal cancer patients treated with preoperative cetuximab-based chemoradiation followed by surgery.

Patients

130 patients (74 men and 56 women, median age 61) with locally advanced rectal cancer (4 with stage II, 109 with stage III, and 15 with stage IV, 2 unknown) who were enrolled in phase I/II clinical trials treated with cetuximab-based chemoradiation in 4 European cancer centers (Brussels/Leuven, Belgium; Ljubljana, Slovenia; Halle/Erlangen/Göttingen/Regensburg, Germany, and Cologne, Germany) were included (Table 1; refs. 15, 16). Informed consent was obtained from each patient and the studies were approved by the Institutional Review Boards of the participating centers. Clinical staging was based on results from endoscopy, endoscopic ultrasound, and computed tomography/MRI of the chest and abdomen. Patients underwent radical resection within 4 to 8 weeks after completion of chemoradiation by either a low anterior resection or an abdomino-perineal rectal amputation with total mesorectal excision.

Table 1.

Patient characteristics and treatments

N = 130%
Age, y 
 Median 61  
 Range 33–83  
Sex 
 Male 74 57 
 Female 56 43 
Stage 
 II 
 III 109 85 
 IV 15 12 
Type of neoadjuvant therapy 
 5-FU/Cetuximab/Radiation (Cologne, Germany) 16 12 
 Capecitabine/Cetuximab/Radiation (Belgium/Slovenia) 72 56 
 Capecitabine/Oxaliplatin/Cetuximab/Radiation 42 32 
 (Halle/Erlangen/Göttingen/Regensburg, Germany)   
N = 130%
Age, y 
 Median 61  
 Range 33–83  
Sex 
 Male 74 57 
 Female 56 43 
Stage 
 II 
 III 109 85 
 IV 15 12 
Type of neoadjuvant therapy 
 5-FU/Cetuximab/Radiation (Cologne, Germany) 16 12 
 Capecitabine/Cetuximab/Radiation (Belgium/Slovenia) 72 56 
 Capecitabine/Oxaliplatin/Cetuximab/Radiation 42 32 
 (Halle/Erlangen/Göttingen/Regensburg, Germany)   

Treatment protocols

Brussels/Leuven, Belgium.

Patients (n = 41) received capecitabine (4 patients at first dose level of 650 mg/m2 twice a day; 37 patients at second dose level of 825 mg/m2 twice a day) and intravenous cetuximab (400 mg/m2 as initial dose 1 week before CRT followed by 250 mg/m2/week for 5 weeks) and 45 Gy of radiation (25 × 1.8 Gy).

Ljubljana, Slovenia.

Patients (n = 31) received capecitabine 1,250 mg/m2 twice daily for 2 weeks followed by intravenous cetuximab 400 mg/m2 at week 3, then weekly intravenous 250 mg/m2 cetuximab plus CRT including capecitabine 825 mg/m2 twice daily (including weekends during radiotherapy) with radiotherapy of 45 Gy (25 × 1.8 Gy), 5 days a week for 5 weeks.

Halle/Erlangen/Göttingen/Regensburg,Germany.

Patients (n = 42) received cetuximab given as an initial dose of 400 mg/m2 7 days before the start of RT, and then at 250 mg/m2 once weekly during RT (50.4 Gy, 28 × 1.8 Gy). Capecitabine and oxaliplatin were administered according to an established schedule of oxaliplatin (50 mg/m2 on Days 1, 8, 22, and 29) and capecitabine (Days 1–14 and 22–35) at 3 dose levels: 1,000, 1,300, and 1,650 mg/m2/d.

Cologne, Germany.

Patients (n = 16) where treated with 45 Gy of radiation (25 × 1.8 Gy) and cetuximab (400 mg/m2 as initial dose 1 week before CRT followed by 250 mg/m2/wk for 5 weeks) in combination with 5-FU.

Tissue samples and pCR evaluation

Tumor biopsies from the study patients were obtained at the time of diagnosis (pretherapeutic biopsy), and at the time of surgery (posttherapeutic biopsy). Pretreatment biopsies from 109 (84%) patients and posttreatment biopsies from 117 (90%) patients were collected and analyzed in this study. For diagnosis, 3 sections representative of the beginning, middle, and end of the tissue were collected. The resected specimens were fixed in 10% formalin, embedded in paraffin, and cut into 5-μm thick slices. The sections were then stained with hematoxylin and eosin stain and all samples were reviewed by a staff pathologist in each study center. To determine pCR, Dworak's regression grading system (grade 0: no regression; grade 1: minimal regression; grade 2: moderate regression; grade 3: good regression; and grade 4: total regression) was used in all of the participating centers (17). Grade 4 was defined as complete pCR.

DNA extraction

Formalin-fixed paraffin-embedded tissues from the patients were dissected as described previously. Ten micrometer thick slides were obtained from the identified areas with the highest tumor concentration and were mounted on uncoated glass slides. Sections were deparafinized in xylene for 10 minutes, hydrated with 100%, 95%, and 70% ethanol and then washed in H2O for 30 seconds. The sections were then stained with nuclear fast red (American Master Tech Scientific, Inc.) for 20 seconds and rinsed in H2O for 30 seconds, dehydrated with 70% ethanol, 95% ethanol, and 100% ethanol for 30 seconds each, followed by xylene for 10 minutes and then completely air-dried. Laser capture microdissection (P.A.L.M. Microlaser Technologies AG) was used in all tumor samples. Genomic DNA was then extracted from the dissected tumor samples by using the QIAamp kit (Qiagen) according to manufacture's instructions.

KRAS analysis

KRAS analysis of 7 KRAS mutations (codon 12 and codon 13) was done according to a proprietary procedure defined by Response Genetics Inc. (United States patent number 6, 248, 535).

PCR-RFLP

Genotyping was done by using PCR-RFLP assays. Briefly, forward primers and reverse primers (Table 2) were used for PCR amplification, PCR products were digested by restriction enzymes (New England Biolab), and alleles were separated on 4% NuSieve ethidium bromide-stained agarose gel.

Table 2.

Primers and enzymes for PCR-RFLP

GeneForward-Primer (5′-3′)Reverse-Primer (5′-3′)EnzymeAnnealing
FCGR2A H131R GGAAAATCCCAGAAATTCTCGC CAACAGCCTGACTACCTATTACGCGGG BstU I 55° 
FCGR3A V158F CTGAAGACACATTTTTACTCCCAAA/C TCCAAAAGCCACACTCAAAGAC n.a. 64° 
VEGF C+936T AAGGAAGAGGAGACTCTGCGCAGAG C TAAATGTATGTATGTGGGTGGGTGTGT CTACAG G Nla III 60° 
EGF A+61G CATTTGCAAACAGAGGCTCA TGTGACAGAGCAAGGCAAAG Alu I 60° 
EGFR (CA)n Repeat ACCCCAGGGCTCTATGGGAA TGAGGGCACAAGAAGCCCCT n.a. 55° 
EGFR G+497A TGCTGTGACCCACTCTGTCT CCAGAAGGTTGCACTTGTCC Bst-N I 59° 
MTHFR C677T GCACTTGAAGGAGAAGGTGTCT TGTGTCAGCCTCGAAAGAAAAGCT Hin fI 68° 
IL8 T-251A TTGTTCTAACACCTGCCACTCT GGCAAACCTGAGTCTCACA Mfe I 60° 
MTHFR A-1298C GGAGGAGCTGCTGAAGATGTG CCCGAGAGGTAAAGAACAAAGACTT Mbo II 68° 
COX-2 G-765C ATTCTGGCCATCGCCGCTTC CTCCTTGTTTCTTGGAAA GAGACG Aci I 55° 
Cyclin D1A+870G GTGAAGTTCATTTCCAATCCGC GGGACATCACCCTCACTTAC ScrF I 61° 
COX-2 G-765C ATTCTGGCCATCGCCGCTTC CTCCTTGTTTCTTGGAAA GAGACG Aci I 55° 
RCC3Thr241 Met GCCTGGTGGTCATCGACTC ACAGGGCTCTGGAAGGCACTGCTCA GCTCACGC ACC Nco I 60° 
S 3′UTR 6bp+/− CAAATCTGAGGGAGCTGAGT CAGATAAGTGGCAGTACAGA Dra I 58° 
S 5′UTR 2R/3R GAAAAGGCGCGCGGAAGG GCTCCGAGCCGGCCACA n.a 62° 
RAD G51C TGGGAACTGCAACTCATCTGG GCGCTCCTCTCTCCAGCAG Mva I 60° 
KRAS let-7 lcs6 TGACTGAATATAAACTTGTGGTAGTT G TCGTCCACAAAATGATTCTGAA Tfi I 60° 
GeneForward-Primer (5′-3′)Reverse-Primer (5′-3′)EnzymeAnnealing
FCGR2A H131R GGAAAATCCCAGAAATTCTCGC CAACAGCCTGACTACCTATTACGCGGG BstU I 55° 
FCGR3A V158F CTGAAGACACATTTTTACTCCCAAA/C TCCAAAAGCCACACTCAAAGAC n.a. 64° 
VEGF C+936T AAGGAAGAGGAGACTCTGCGCAGAG C TAAATGTATGTATGTGGGTGGGTGTGT CTACAG G Nla III 60° 
EGF A+61G CATTTGCAAACAGAGGCTCA TGTGACAGAGCAAGGCAAAG Alu I 60° 
EGFR (CA)n Repeat ACCCCAGGGCTCTATGGGAA TGAGGGCACAAGAAGCCCCT n.a. 55° 
EGFR G+497A TGCTGTGACCCACTCTGTCT CCAGAAGGTTGCACTTGTCC Bst-N I 59° 
MTHFR C677T GCACTTGAAGGAGAAGGTGTCT TGTGTCAGCCTCGAAAGAAAAGCT Hin fI 68° 
IL8 T-251A TTGTTCTAACACCTGCCACTCT GGCAAACCTGAGTCTCACA Mfe I 60° 
MTHFR A-1298C GGAGGAGCTGCTGAAGATGTG CCCGAGAGGTAAAGAACAAAGACTT Mbo II 68° 
COX-2 G-765C ATTCTGGCCATCGCCGCTTC CTCCTTGTTTCTTGGAAA GAGACG Aci I 55° 
Cyclin D1A+870G GTGAAGTTCATTTCCAATCCGC GGGACATCACCCTCACTTAC ScrF I 61° 
COX-2 G-765C ATTCTGGCCATCGCCGCTTC CTCCTTGTTTCTTGGAAA GAGACG Aci I 55° 
RCC3Thr241 Met GCCTGGTGGTCATCGACTC ACAGGGCTCTGGAAGGCACTGCTCA GCTCACGC ACC Nco I 60° 
S 3′UTR 6bp+/− CAAATCTGAGGGAGCTGAGT CAGATAAGTGGCAGTACAGA Dra I 58° 
S 5′UTR 2R/3R GAAAAGGCGCGCGGAAGG GCTCCGAGCCGGCCACA n.a 62° 
RAD G51C TGGGAACTGCAACTCATCTGG GCGCTCCTCTCTCCAGCAG Mva I 60° 
KRAS let-7 lcs6 TGACTGAATATAAACTTGTGGTAGTT G TCGTCCACAAAATGATTCTGAA Tfi I 60° 

Statistical analysis

Tumor response to neoadjuvant chemoradiotherapy evaluated by Dworak criteria was the primary endpoint. Patients with grade 4 Dworak response were classified as having pCR. Patients having grade 0 to 3 Dworak response were categorized as not having pCR. Fisher's exact test was used to examine associations between polymorphisms and pCR or KRAS mutation status and pCR. The exact conditional test was used to evaluate the associations between polymorphisms and ordinal Dworak response. The independent effects of polymorphisms on pCR were examined by using the Logistic regression model when stratifying the study center and patient baseline characteristics.

Although the genetic model of inheritance for the polymorphisms had not established, a codominant model was considered first. A dominant model was used whenever the patients carrying the genotype of variant/variant were too few (<5%).

The false discovery rate (FDR) of multiple testing was controlled by using the Benjamini and Hochberg method (18). The FDR adjusted P values less than 15% were considered as significant.

An internal validation method, leave-one-out cross validation was used to evaluate the process of polymorphism selection to predict pCR.

All statistical tests were 2-sided and done by using the SAS statistical package version 9.2 (SAS Institute Inc.).

pCR

Data from 125 of 130 patients were available to evaluate pCR with cetuximab-based chemoradiation after surgery. Among them, 21 (17%) patients had grade 0 to 1, 55 (44%) patients had grade 2, 30 (24%) patients had grade 3, and 19 (15%) patients had grade 4 response.

EGF 61 polymorphism and complete pCR

Genotyping for EGF 61 was successful in 118 (91%) of 130 cases. In 12 patients (9%), genotyping was not successful because of limited quantity and quality of extracted genomic DNA or biopsy samples. Forty-six percent (54/118) of patients were homozygous for A/A allele, 45% (53/118) were heterozygous A/G, and 9% (11/118) were homozygous for the G/G allele. Patients with the EGF G/G genotype had a pCR of 45% (5/11), compared with 21% (11/53) in patients heterozygous, and 2% (1/54) in patients homozygous for the A/A allele, respectively (Fig. 1). There was statistically significant association between EGF A61G polymorphism and pCR after treatment of these patients (P < 0.001, Fisher's exact test, Table 3). EGF A61G polymorphism remained significantly associated with pCR in the multivariable logistic regression model stratified by the study center and age (Table 4).

Figure 1.

EGF 61 polymorphism status and pCR.

Figure 1.

EGF 61 polymorphism status and pCR.

Close modal
Table 3.

Genomic polymorphisms and tumor response in patients with rectal cancer treated with neoadjuvant chemoradiation

pCRDworak response
PolymorphismNcYesNo1234
IL-8 T-251A 
 TT 38 8 (22%) 28 12 
 TA 62 7 (12%) 53 29 15 
 AA 26 4 (16%) 21 12 
Pa  0.37 (0.74)   0.54 (0.70)   
MTHFR C677T 
 CC 59 12 (20%) 47 23 15 12 
 CT 56 4 (8%) 48 27 12 
 TT 11 3 (30%) 
Pa  0.070 (0.37)   0.50 (0.70)   
MTHFR A-1298C 
 AA 54 8 (16%) 43 26 10 
 CA 62 8 (13%) 52 12 25 15 
 CC 3 (38%) 
Pa  0.18 (0.72)   0.30 (0.70)   
FCGR2A H131R 
 HH 35 4 (12%) 30 13 11 
 HR 69 11 (17%) 55 11 31 13 11 
 RR 19 4 (22%) 14 
Pa  0.61 (0.85)   0.77 (0.82)   
FCGR3A V158F 
 VV 49 6 (12%) 43 23 13 
 VF 37 3 (9%) 32 15 
 FF 15 3 (20%) 12 
Pa  0.49 (0.85)   0.55 (0.70)   
CYCLIND1 A870G 
 AA 32 5 (17%) 25 15 
 AG 58 7 (12%) 49 23 17 
 GG 34 6 (18%) 27 15 
Pa  0.76 (0.85)   0.55 (0.70)   
XRCC3 Thr241Met 
 TT 51 6 (12%) 42 11 21 10 
 CT 55 9 (17%) 45 26 12 
 CC 12 2 (18%) 
Pa  0.74 (0.85)   0.059 (0.24)   
VEGF C936T 
 CC 93 13 (15%) 75 16 40 19 13 
 CTb 30 6 (19%) 26 13 
 TTb       
Pa  0.58 (0.85)   0.33 (0.70)   
EGFR G497A 
 GG 71 10 (15%) 57 13 24 20 10 
 AG 42 8 (19%) 34 23 
 AA 12 1 (9%) 10 
Pa  0.75 (0.85)   0.61 (0.70)   
EGFR (CA)n repeat 
 Both <20 58 10 (18%) 47 23 15 10 
 Any ≥20 47 9 (21%) 34 20 
Pa  0.80 (0.85)   0.83 (0.83)   
EGF A61G 
 AA 54 1 (2%) 49 14 24 11 
 AG 53 11 (21%) 41 25 12 11 
 GG 11 5 (45%) 
Pa  <0.001 (0.002)   <0.001 (<0.001)   
TS 3′ UTR 
 +/+ 55 8 (15%) 44 21 16 
 +/− 56 9 (16%) 46 10 25 11 
Pa−/− 14 2 (15%) 11 
  1.00 (1.00)   0.24 (0.70)   
TS 5′ UTR 
 2R/2R 15 5 (36%) 
 2R/3R 56 9 (16%) 46 11 23 12 
 3R/3R 24 0 (0%) 23 14 
Pa  0.006 (0.045)   0.015 (0.12)   
COX-2 G-765C 
 GG 84 15 (19%) 66 12 37 17 15 
 GCb 38 4 (10%) 35 16 11 
 CCb       
Pa  0.30 (0.72)   0.47 (0.70)   
RAD G51C 
 GG 102 14 (14%) 84 16 43 25 14 
 GC 21 5 (25%) 15 
Pa  0.31 (0.72)   0.61 (0.70) 
KRAS let-7 lcs6 poly 
 TT 109 15 (14%) 92 20 48 24 15 
 TGb 14 4 (29%) 10 
 GGb       
Pa  0.23 (0.72)   0.024 (0.13)   
pCRDworak response
PolymorphismNcYesNo1234
IL-8 T-251A 
 TT 38 8 (22%) 28 12 
 TA 62 7 (12%) 53 29 15 
 AA 26 4 (16%) 21 12 
Pa  0.37 (0.74)   0.54 (0.70)   
MTHFR C677T 
 CC 59 12 (20%) 47 23 15 12 
 CT 56 4 (8%) 48 27 12 
 TT 11 3 (30%) 
Pa  0.070 (0.37)   0.50 (0.70)   
MTHFR A-1298C 
 AA 54 8 (16%) 43 26 10 
 CA 62 8 (13%) 52 12 25 15 
 CC 3 (38%) 
Pa  0.18 (0.72)   0.30 (0.70)   
FCGR2A H131R 
 HH 35 4 (12%) 30 13 11 
 HR 69 11 (17%) 55 11 31 13 11 
 RR 19 4 (22%) 14 
Pa  0.61 (0.85)   0.77 (0.82)   
FCGR3A V158F 
 VV 49 6 (12%) 43 23 13 
 VF 37 3 (9%) 32 15 
 FF 15 3 (20%) 12 
Pa  0.49 (0.85)   0.55 (0.70)   
CYCLIND1 A870G 
 AA 32 5 (17%) 25 15 
 AG 58 7 (12%) 49 23 17 
 GG 34 6 (18%) 27 15 
Pa  0.76 (0.85)   0.55 (0.70)   
XRCC3 Thr241Met 
 TT 51 6 (12%) 42 11 21 10 
 CT 55 9 (17%) 45 26 12 
 CC 12 2 (18%) 
Pa  0.74 (0.85)   0.059 (0.24)   
VEGF C936T 
 CC 93 13 (15%) 75 16 40 19 13 
 CTb 30 6 (19%) 26 13 
 TTb       
Pa  0.58 (0.85)   0.33 (0.70)   
EGFR G497A 
 GG 71 10 (15%) 57 13 24 20 10 
 AG 42 8 (19%) 34 23 
 AA 12 1 (9%) 10 
Pa  0.75 (0.85)   0.61 (0.70)   
EGFR (CA)n repeat 
 Both <20 58 10 (18%) 47 23 15 10 
 Any ≥20 47 9 (21%) 34 20 
Pa  0.80 (0.85)   0.83 (0.83)   
EGF A61G 
 AA 54 1 (2%) 49 14 24 11 
 AG 53 11 (21%) 41 25 12 11 
 GG 11 5 (45%) 
Pa  <0.001 (0.002)   <0.001 (<0.001)   
TS 3′ UTR 
 +/+ 55 8 (15%) 44 21 16 
 +/− 56 9 (16%) 46 10 25 11 
Pa−/− 14 2 (15%) 11 
  1.00 (1.00)   0.24 (0.70)   
TS 5′ UTR 
 2R/2R 15 5 (36%) 
 2R/3R 56 9 (16%) 46 11 23 12 
 3R/3R 24 0 (0%) 23 14 
Pa  0.006 (0.045)   0.015 (0.12)   
COX-2 G-765C 
 GG 84 15 (19%) 66 12 37 17 15 
 GCb 38 4 (10%) 35 16 11 
 CCb       
Pa  0.30 (0.72)   0.47 (0.70)   
RAD G51C 
 GG 102 14 (14%) 84 16 43 25 14 
 GC 21 5 (25%) 15 
Pa  0.31 (0.72)   0.61 (0.70) 
KRAS let-7 lcs6 poly 
 TT 109 15 (14%) 92 20 48 24 15 
 TGb 14 4 (29%) 10 
 GGb       
Pa  0.23 (0.72)   0.024 (0.13)   

aNominal P values were based on Fisher's exact test for pCR and exact conditional test for Dworak response. FDR-adjusted P values were shown in parentheses.

bCombined in the analysis.

cNumber of patients by each genotype. Some of the patients had response not evaluable.

Table 4.

Logistic regression model of EGF A61G and TS 5′ UTR polymorphisms and pCR

Univariate analysisMultivariate analysisb
PolymorphismOR (95% CI)OR (95% CI)
EGF A61G 
 AA 1 (reference) 1 (reference) 
 AG or GG 16.68 (95% CI: 2.13–130.82) 13.08 (95% CI: 1.51–113.54) 
Pa 0.007 0.020 
TS 5′ UTR 
 2R/2R 1 (reference) 1 (reference) 
 2R/3R or 3R/3R 0.24 (95% CI: 0.06–0.86) 0.30 (95% CI: 0.06–1.52) 
Pa 0.028 0.14 
Univariate analysisMultivariate analysisb
PolymorphismOR (95% CI)OR (95% CI)
EGF A61G 
 AA 1 (reference) 1 (reference) 
 AG or GG 16.68 (95% CI: 2.13–130.82) 13.08 (95% CI: 1.51–113.54) 
Pa 0.007 0.020 
TS 5′ UTR 
 2R/2R 1 (reference) 1 (reference) 
 2R/3R or 3R/3R 0.24 (95% CI: 0.06–0.86) 0.30 (95% CI: 0.06–1.52) 
Pa 0.028 0.14 

aBased on Wald test.

bStratified by study center and age (<60 vs. 60+ years old).

TS 5′UTR polymorphism and complete pCR

Genotyping for TS 5′UTR was successful in 95 (73%) of 130 cases. In 35 patients (27%), genotyping was not successful because of limited quantity and quality of extracted genomic DNA or biopsy samples. Sixteen percent (15/95) of patients were homozygous for 2R/2R allele, 59% (56/95) were heterozygous 2R/3R, and 25% (24/95) were homozygous for the 3R/3R allele. Patients with the TS 5′UTR 2R/2R genotype had a pCR of 33% (5/15), compared with 16% (9/56) in patients heterozygous, and 0% (0/24) in patients homozygous for the 3R/3R genotype, respectively. There was statistically significant association between TS 5′UTR 2R allele and pCR after treatment of these patients (P = 0.006, Fisher's exact test, Table 3). The association was no longer statistically significant in the multivariate logistic regression model (Table 4).

KRAS mutation status and pCR

KRAS mutation status was available from pretherapeutic tumor biopsies of 101 patients, 42% of which (42/101) had KRAS mutation, 58% (59/101) had wild-type KRAS. The mutation status (wild type vs. mutant) was not significantly correlated with pCR (P = 0.214, Fischer exact test). KRAS mutant patients showed 15% (5/34) pCR to cetuximab-based neoadjuvant CRT compared with KRAS wild-type patients with 10% (5/52) pCR.

Gene polymorphisms in patients with wild-type KRAS

When gene polymorphism and tumor response were analyzed in the 59 patients with wild-type KRAS, EGF 61 remains to be significantly associated with the pCR, with the EGF G/G genotype had a pCR of 40% (2/5), compared with 12% (3/25) in patients heterozygous, and 0% (0/24) in patients homozygous for the A/A allele (P = 0.019, Fisher's exact test). On the other hand, TS 5′UTR polymorphism was no longer significantly associated with pCR (P = 0.67, data not shown).

Analysis of other tested germline polymorphisms

We did not observe statistically significant associations between other tested gene polymorphisms and pCR (Table 3).

EGF A61G SNP was the only polymorphism predicted pCR, which was validated by the leave-one-out cross validation method (118 of 121 leave-one-out datasets) through all the steps of analyses including the univariate, multivariate analysis, and subgroup analysis among KRAS wild-type patients.

With the development of the total mesorectal excision technique and the current neoadjuvant chemoradiation regimen, the local recurrence rate in locally advanced rectal cancer patients has decreased significantly from 20% to 30% to less than 10%, and is no longer the reason for poor survival in these patients (19). Interestingly, this drop in local recurrence has not been correlated with better survival, and the high rate of distant metastasis appeared to be the problem for this failure (20). Therefore, targeted therapies have been added to the standard care of neoadjuvant chemoradiation for better local control and survival. Complete pCR has been shown to be strongly associated with the effectiveness of treatments and has been used to predict survival following neoadjuvant therapy (21). However, recent phase I/II clinical trials with cetuximab-based chemoradiation therapy have failed to show improved pCR or survival in locally advanced rectal cancer patients. Question has been raised whether a subset of patients might benefit from this treatment. Our study suggests EGF A+61G polymorphism is significantly associated with pCR to cetuximab-based neoadjuvant chemoradiation in locally advanced rectal cancer patients, independent of KRAS mutation status. This is the first study suggesting this polymorphism might be a predictive marker for response to cetuximab-based therapy.

The EGF/EGFR pathway plays important roles in the pathogenesis and progression of human cancers (22) and both EGF and EGFR are commonly overexpressed in CRC. Epidermal growth factor (EGF) is encoded by a single gene on chromosome 4q25–q27. In 2002, Shahbazi and colleagues identified a G to A polymorphism at position 61 in the 5′ untranslated region of EGF that is significantly correlated with production of EGF (23). Individuals with the G/G or A/G genotypes had higher EGF production in vitro than individuals with the A/A genotype, and homozygosity of the G allele was shown to be associated with the risk of developing malignant melanoma and several other malignancies, including gastric cancer (24), glioma (25), hepatocellular carcinoma in cirrhotic patients (26), CRC, and recurrence of liver metastasis from CRC (27), although subsequent studies showed conflicting results (28–31). On the other hand, several recent studies have shown that the G/G genotype is associated with improved OS and progression-free survival (PFS) or less tumor recurrence in patients with metastatic colorectal and esophageal cancers (32–34). Graziano and colleagues investigated 110 metastatic CRC patients who were treated with cetuximab-irinotecan salvage therapy and found significant association between EGF 61 G/G genotype and a favorable OS in these patients. In another group of 130 mCRC patients who were treated with single agent cetuximab, EGF + 61 G/G genotype was associated with better PFS (35). These apparently paradoxical results are possibly due to the different effect of increased EGF activity in different stages of cancer development. Overproduction of the EGF can impose an increased risk to develop cancer on normal cells, however, it might also increase cancer cells' susceptibility to treatment, especially targeted therapy such as cetuximab. Indeed, in our study, EGF61 G allele is strongly associated with pCR in this group of locally advanced rectal patients treated with cetuximab-based neoadjuvant chemoradiation. Our data are in line with the result of Khambata-Ford and colleagues that mCRC patients whose tumors with increased expression of EGF ligands epiregulin and amphiregulin are more likely to have disease control and PFS when treated with cetuximab (36). Whether this association of EGF 61 polymorphism and pCR is due to enhanced inhibition of the EGFR pathway or increased sensitivity to radiation therapy requires further investigation. KRAS mutation status has been shown to predict response to cetuximab-based therapy in mCRC patients (37) and mCRC patients with mutant-type KRAS are more likely to have a worse response, PFS, and OS when treated with cetuximab (38). On the other hand, studies of cetuximab-based neoadjuvant therapy in rectal cancer patients are few and have generated inconclusive results about the significance of KRAS mutation status and response to cetuximab therapy (39), likely due to small patient numbers of these studies. Our data showed that the KRAS mutation status is not statically significantly associated with the pCR. However, only 10 of 101 patients with known KRAS mutation status had pCR to cetuximab-based neoadjuvant chemoradiation therapy. This small sample size limited our ability to interpret the result and larger trials are required to make the conclusion. However, our data showed that the EGF61 A>G polymorphism has significant association with pCR to the cetuximab-based therapy in patients with both KRAS wild-type and mutant tumors.

Thymidylate synthase catalyze the reaction from dUMP to dTMP, the only de novo source of dTMP in cells, and is the therapeutic target for 5FU and its derivatives. High TS expression levels and activity have been associated with poor prognosis and response rates in CRC patients (40). The variable number of tandem repeats (VNTR) of a 28-bp sequence (2R and 3R) located within the 5′ untranslated region (5′UTR) is one of the 3 polymorphisms identified to affect the expression of TS gene, located at 18p11.32. Studies have shown that the wild-type 3R enhance TS protein translation and elevates enzyme activity (41). However, data on the prognostic and predictive value of the VNTR has been mixed and conflicting. TS 5′ UTR 3R genotype was linked to lower response rate (42), better survival outcome (43), better response to chemotherapy, or no significant difference in outcome (44) in advanced CRC patients treated with 5FU. On the other hand, heterozygous 2R/3R genotype was associated with worse overall survival in one study (45) and was a favorable marker in others (46). Possible explanations for these inconsistent results include the multiple polymorphisms existing in the TS gene including the G/C SNP in the 3G allele (47), the 6bp deletion within the 3′UTR (48), and variations of TS gene copy number due to loss of heterozygosis (49) or gene amplification. In our study, TS 5′UTR 3R/3R genotype was significantly associated with the worst response rate (0% pCR) to cetuximab-based neoadjuvant chemoradiation therapy, compared with 33% pCR in patients with 2R/2R genotype, and 16% pCR in patients with heterozygous 2R/3R genotype (P = 0.006). Interestingly, this association is not significant in patients with wild-type KRAS. This result might be confounded by the treatment of all patients with 5FU or capecitabine.

In conclusion, our study suggests intratumoral EGF61 A/G polymorphism to be a potential predictive marker for pCR to cetuximab-based neoadjuvant chemoradiation in patients with locally advanced rectal cancer. The retrospective nature and relatively small sample size limited the significance of this study. Furthermore, the patients all received cetuximab-based therapy and there is no untreated control group. Therefore the results of this study should be interpreted carefully and validated in larger prospective trials.

D. Arnold, honoraria (Speakers'Bureau), Roche, Merck Serono, sanofiaventis and research support, Roche, Sanofi-aventis. C. Rödel, research funds, Merck Pharma GmbH, Darmstadt, Germany and speacker honoraria, Hoffmann-La Roche AG, Grenzach-Whylen, Germany and Sanofi-aventis Pharma GmbH, Berlin, Germany. H.-J. Lenz, consult, BMS, Imclone and Merck KG.

This study was done in the Sharon A. Carpenter Laboratory at USC/Norris Comprehensive Cancer Center.

This work was funded by the NIH grant 5 P30CA14089-27I and the Dhont Family Foundation. This work was also funded by the Deutsche Forschungsgemeinschfat (DFG), grant No. VA 506/1-1; 535112, and Cologne Fortune.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Kockerling
F
,
Reymond
MA
,
Altendorf-Hofmann
A
,
Dworak
O
,
Hohenberger
W
. 
Influence of surgery on metachronous distant metastases and survival in rectal cancer
.
J Clin Oncol
1998
;
16
:
324
9
.
2.
Kapiteijn
E
,
Marijnen
CA
,
Nagtegaal
ID
,
Putter
H
,
Steup
WH
,
Wiggers
T
, et al
Preoperative radiotherapy combined with total mesorectal excision for resectable rectal cancer
.
N Engl J Med
2001
;
345
:
638
46
.
3.
Sauer
R
,
Becker
H
,
Hohenberger
W
,
Rödel
C
,
Wittekind
C
,
Fietkau
R
, et al
Preoperative versus postoperative chemoradiotherapy for rectal cancer
.
N Engl J Med
2004
;
351
:
1731
40
.
4.
Bosset
JF
,
Collette
L
,
Calais
G
,
Mineur
L
,
Maingon
P
,
Radosevic-Jelic
L
, et al
Chemotherapy with preoperative radiotherapy in rectal cancer
.
N Engl J Med
2006
;
355
:
1114
23
.
5.
Kopp
R
,
Rothbauer
E
,
Mueller
E
,
Schildberg
FW
,
Jauch
KW
,
Pfeiffer
A
. 
Reduced survival of rectal cancer patients with increased tumor epidermal growth factor receptor levels
.
Dis Colon Rectum
2003
;
46
:
1391
9
.
6.
Wollman
R
,
Yahalom
J
,
Maxy
R
,
Pinto
J
,
Fuks
Z
. 
Effect of epidermal growth factor on the growth and radiation sensitivity of human breast cancer cells in vitro
.
Int J Radiat Oncol Biol Phys
1994
;
30
:
91
8
.
7.
Van Cutsem
E
,
Köhne
CH
,
Hitre
E
,
Zaluski
J
,
Chang
Chien CR
,
Makhson
A
, et al
Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer
.
N Engl J Med
2009
;
360
:
1408
17
.
8.
Bokemeyer
C
,
Bondarenko
I
,
Makhson
A
,
Hartmann
JT
,
Aparicio
J
,
de Braud
F
, et al
Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer
.
J Clin Oncol
2009
;
27
:
663
71
.
9.
Bonner
JA
,
Harari
PM
,
Giralt
J
,
Azarnia
N
,
Shin
DM
,
Cohen
RB
, et al
Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck
.
N Engl J Med
2006
;
354
:
567
78
.
10.
Hofheinz
RD
,
Horisberger
K
,
Woernle
C
,
Wenz
F
,
Kraus-Tiefenbacher
U
,
Kähler
G
, et al
Phase I trial of cetuximab in combination with capecitabine, weekly irinotecan, and radiotherapy as neoadjuvant therapy for rectal cancer
.
Int J Radiat Oncol Biol Phys
2006
;
66
:
1384
90
.
11.
Machiels
JP
,
Sempoux
C
,
Scalliet
P
,
Coche
JC
,
Humblet
Y
,
Van Cutsem
E
, et al
Phase I/II study of preoperative cetuximab, capecitabine, and external beam radiotherapy in patients with rectal cancer
.
Ann Oncol
2007
;
18
:
738
44
.
12.
Bertolini
F
,
Chiara
S
,
Bengala
C
,
Antognoni
P
,
Dealis
C
,
Zironi
S
, et al
Neoadjuvant treatment with single-agent cetuximab followed by 5-FU, cetuximab, and pelvic radiotherapy: a phase II study in locally advanced rectal cancer
.
Int J Radiat Oncol Biol Phys
2009
;
73
:
466
72
.
13.
Bengala
C
,
Bettelli
S
,
Bertolini
F
,
Salvi
S
,
Chiara
S
,
Sonaglio
C
, et al
Epidermal growth factor receptor gene copy number, K-ras mutation and pathological response to preoperative cetuximab, 5-FU and radiation therapy in locally advanced rectal cancer
.
Ann Oncol
2009
;
20
:
469
74
.
14.
Rödel
C
,
Arnold
D
,
Hipp
M
,
Liersch
T
,
Dellas
K
,
Iesalnieks
I
, et al
Phase I-II trial of cetuximab, capecitabine, oxaliplatin, and radiotherapy as preoperative treatment in rectal cancer
.
Int J Radiat Oncol Biol Phys
2008
;
70
:
1081
6
.
15.
Velenik
V
,
Ocvirk
J
,
Oblak
I
,
Anderluh
F
. 
A phase II study of cetuximab, capecitabine and radiotherapy in neoadjuvant treatment of patients with locally advanced resectable rectal cancer
.
Eur J Surg Oncol
2010
;
36
:
244
50
.
16.
Semrau
R VD
,
Kocher
M
, et al
Pre-operative radio-chemo-immunotherapy with cetuximab and 5-FU in patients with locally advanced rectal cancer. First results of a phase I/II trial
.
Annual meeting of the German and Austrian Society of Radiooncology
,
Vienna;
2008
;
abstract 169
.
17.
Dworak
O
,
Keilholz
L
,
Hoffmann
A
. 
Pathological features of rectal cancer after preoperative radiochemotherapy
.
Int J Colorectal Dis
1997
;
12
:
19
23
.
18.
Benjamini
Y
,
Hochberg
Y
. 
Controlling the false discovery rate: a practical and powerful approach to multiple testing
.
J Royal Stat Soc
1995
;
57
:
289
300
.
19.
Peeters
KC
,
Marijnen
CA
,
Nagtegaal
ID
,
Kranenbarg
EK
,
Putter
H
,
Wiggers
T
, et al
The TME trial after a median follow-up of 6 years: increased local control but no survival benefit in irradiated patients with resectable rectal carcinoma
.
Ann Surg
2007
;
246
:
693
701
.
20.
Collette
L
,
Bosset
JF
,
den Dulk
M
,
Nguyen
F
,
Mineur
L
,
Maingon
P
, et al
Patients with curative resection of cT3-4 rectal cancer after preoperative radiotherapy or radiochemotherapy: does anybody benefit from adjuvant fluorouracil-based chemotherapy? A trial of the European Organisation for Research and Treatment of Cancer Radiation Oncology Group
.
J Clin Oncol
2007
;
25
:
4379
86
.
21.
Rödel
C
,
Martus
P
,
Papadoupolos
T
,
Füzesi
L
,
Klimpfinger
M
,
Fietkau
R
, et al
Prognostic significance of tumor regression after preoperative chemoradiotherapy for rectal cancer
.
J Clin Oncol
2005
;
23
:
8688
96
.
22.
Fisher
DA
,
Lakshmanan
J
. 
Metabolism and effects of epidermal growth factor and related growth factors in mammals
.
Endocr Rev
1990
;
11
:
418
42
.
23.
Shahbazi
M
,
Pravica
V
,
Nasreen
N
,
Fakhoury
H
,
Fryer
AA
,
Strange
RC
, et al
Association between functional polymorphism in EGF gene and malignant melanoma
.
Lancet
2002
;
359
:
397
401
.
24.
Hamai
Y
,
Matsumura
S
,
Matsusaki
K
,
Kitadai
Y
,
Yoshida
K
,
Yamaguchi
Y
, et al
A single nucleotide polymorphism in the 5′ untranslated region of the EGF gene is associated with occurrence and malignant progression of gastric cancer
.
Pathobiology
2005
;
72
:
133
8
.
25.
Costa
BM
,
Ferreira
P
,
Costa
S
,
Canedo
P
,
Oliveira
P
,
Silva
A
, et al
Association between functional EGF+61 polymorphism and glioma risk
.
Clin Cancer Res
2007
;
13
:
2621
6
.
26.
Tanabe
KK
,
Lemoine
A
,
Finkelstein
DM
,
Kawasaki
H
,
Fujii
T
,
Chung
RT
, et al
Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis
.
JAMA
2008
;
299
:
53
60
.
27.
Kovar
FM
,
Thallinger
C
,
Marsik
CL
,
Perkmann
T
,
Puhalla
H
,
Haslacher
H
, et al
The EGF 61A/G polymorphism - a predictive marker for recurrence of liver metastases from colorectal cancer
.
Wien Klin Wochenschr
2009
;
121
:
638
43
.
28.
Randerson-Moor
JA
,
Gaut
R
,
Turner
F
,
Whitaker
L
,
Barrett
JH
,
Silva
Idos S
, et al
The relationship between the epidermal growth factor (EGF) 5′UTR variant A61G and melanoma/nevus susceptibility
.
J Invest Dermatol
2004
;
123
:
755
9
.
29.
Goto
Y
,
Ando
T
,
Goto
H
,
Hamajima
N
. 
No association between EGF gene polymorphism and gastric cancer
.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
2454
6
.
30.
Vauleon
E
,
Auger
N
,
Benouaich-Amiel
A
,
Laigle-Donadey
F
,
Kaloshi
G
,
Lejeune
J
, et al
The 61 A/G EGF polymorphism is functional but is neither a prognostic marker nor a risk factor for glioblastoma
.
Cancer Genet Cytogenet
2007
;
172
:
33
7
.
31.
Qi
P
,
Wang
H
,
Chen
YM
,
Sun
XJ
,
Liu
Y
,
Gao
CF
. 
No association of EGF 5′UTR variant A61G and hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection
.
Pathology
2009
;
41
:
555
60
.
32.
Lanuti
M
,
Liu
G
,
Goodwin
JM
,
Zhai
R
,
Fuchs
BC
,
Asomaning
K
, et al
A functional epidermal growth factor (EGF) polymorphism, EGF serum levels, and esophageal adenocarcinoma risk and outcome
.
Clin Cancer Res
2008
;
14
:
3216
22
.
33.
Graziano
F
,
Ruzzo
A
,
Loupakis
F
,
Canestrari
E
,
Santini
D
,
Catalano
V
, et al
Pharmacogenetic profiling for cetuximab plus irinotecan therapy in patients with refractory advanced colorectal cancer
.
J Clin Oncol
2008
;
26
:
1427
34
.
34.
Lurje
G
,
Leers
JM
,
Pohl
A
,
Oezcelik
A
,
Zhang
W
,
Ayazi
S
, et al
Genetic variations in angiogenesis pathway genes predict tumor recurrence in localized adenocarcinoma of the esophagus
.
Ann Surg
2010
;
251
:
857
64
.
35.
Lurje
G
,
Nagashima
F
,
Zhang
W
,
Yang
D
,
Chang
HM
,
Gordon
MA
, et al
Polymorphisms in cyclooxygenase-2 and epidermal growth factor receptor are associated with progression-free survival independent of K-ras in metastatic colorectal cancer patients treated with single-agent cetuximab
.
Clin Cancer Res
2008
;
14
:
7884
95
.
36.
Khambata-Ford
S
,
Garrett
CR
,
Meropol
NJ
,
Basik
M
,
Harbison
CT
,
Wu
S
, et al
Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab
.
J Clin Oncol
2007
;
25
:
3230
7
.
37.
Lièvre
A
,
Bachet
JB
,
Boige
V
,
Cayre
A
,
Le Corre
D
,
Buc
E
, et al
KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab
.
J Clin Oncol
2008
;
26
:
374
9
.
38.
Qiu
LX
,
Mao
C
,
Zhang
J
,
Zhu
XD
,
Liao
RY
,
Xue
K
, et al
Predictive and prognostic value of KRAS mutations in metastatic colorectal cancer patients treated with cetuximab: A meta-analysis of 22 studies
.
Eur J Cancer
2010
;
46
:
2781
7
.
39.
Debucquoy
A
,
Haustermans
K
,
Daemen
A
,
Aydin
S
,
Libbrecht
L
,
Gevaert
O
, et al
Molecular response to cetuximab and efficacy of preoperative cetuximab-based chemoradiation in rectal cancer
.
J Clin Oncol
2009
;
27
:
2751
7
.
40.
Popat
S
,
Matakidou
A
,
Houlston
RS
. 
Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and meta-analysis
.
J Clin Oncol
2004
;
22
:
529
36
.
41.
Morganti
M
,
Ciantelli
M
,
Giglioni
B
,
Putignano
AL
,
Nobili
S
,
Papi
L
, et al
Relationships between promoter polymorphisms in the thymidylate synthase gene and mRNA levels in colorectal cancers
.
Eur J Cancer
2005
;
41
:
2176
83
.
42.
Pullarkat
ST
,
Stoehlmacher
J
,
Ghaderi
V
,
Xiong
YP
,
Ingles
SA
,
Sherrod
A
, et al
Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy
.
Pharmacogenomics J
2001
;
1
:
65
70
.
43.
Dotor
E
,
Cuatrecases
M
,
Martínez-Iniesta
M
,
Navarro
M
,
Vilardell
F
,
Guinó
E
, et al
Tumor thymidylate synthase 1494del6 genotype as a prognostic factor in colorectal cancer patients receiving fluorouracil-based adjuvant treatment
.
J Clin Oncol
2006
;
24
:
1603
11
.
44.
Stoehlmacher
J
,
Park
DJ
,
Zhang
W
,
Yang
D
,
Groshen
S
,
Zahedy
S
, et al
A multivariate analysis of genomic polymorphisms: prediction of clinical outcome to 5-FU/oxaliplatin combination chemotherapy in refractory colorectal cancer
.
Br J Cancer
2004
;
91
:
344
54
.
45.
Fernandez-Contreras
ME
,
Sanchez-Hernandez
JJ
,
Gonzalez
E
,
Herráez
B
,
Domínguez
I
,
Lozano
M
, et al
Combination of polymorphisms within 5′ and 3′ untranslated regions of thymidylate synthase gene modulates survival in 5 fluorouracil-treated colorectal cancer patients
.
Int J Oncol
2009
;
34
:
219
29
.
46.
Suh
KW
,
Kim
JH
,
Kim
YB
,
Kim
J
,
Jeong
S
. 
Thymidylate synthase gene polymorphism as a prognostic factor for colon cancer
.
J Gastrointest Surg
2005
;
9
:
336
42
.
47.
Mandola
MV
,
Stoehlmacher
J
,
Muller-Weeks
S
,
Cesarone
G
,
Yu
MC
,
Lenz
HJ
, et al
A novel single nucleotide polymorphism within the 5′ tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity
.
Cancer Res
2003
;
63
:
2898
904
.
48.
Ulrich
CM
,
Bigler
J
,
Velicer
CM
,
Greene
EA
,
Farin
FM
,
Potter
JD
. 
Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene
.
Cancer Epidemiol Biomarkers Prev
2000
;
9
:
1381
5
.
49.
Uchida
K
,
Hayashi
K
,
Kawakami
K
,
Schneider
S
,
Yochim
JM
,
Kuramochi
H
, et al
Loss of heterozygosity at the thymidylate synthase (TS) locus on chromosome 18 affects tumor response and survival in individuals heterozygous for a 28-bp polymorphism in the TS gene
.
Clin Cancer Res
2004
;
10
:
433
9
.