Clinical outcome
In the selected subset of patients from the IVICA trial, all patients reported compliance with OI therapy. Patients were similar at recruitment for age, sex, Dukes stage, haemoglobin, ferritin and transferrin saturations. Patients had a significantly higher increase in ferritin in the IV group (median ferritin 588 ng/mL IV versus 22 ng/mL oral, p=0.001). Haemoglobin and transferrin saturations were also higher in the IV group by day of surgery (Table 1).
Table 1
Clinical outcome
|
Oral iron (n=15)
|
IV iron (n=15)
|
p
|
Age (years)1
|
74 (46-82)
|
74 (53-85)
|
0.662
|
Sex ratio (M:F)
|
10:5
|
8:7
|
0.758
|
Haemoglobin (g/dL)2
Recruitment
Day of surgery
|
10.3 (1.0)
11.4 (1.1)
|
10.0 (1.7)
12.3 (2.1)
|
0.616
0.131
|
Ferritin (ng/mL)3
Recruitment
Day of surgery
|
21 (14-45)
22 (16-51)
|
39 (12-204)
588 (318-1415)
|
0.384
0.001*
|
Transferrin saturations (%)3
Recruitment
Day of surgery
|
7 (4-16)
7.5 (4-13)
|
5.5 (2-14)
20 (17-24)
|
0.301
0.290
|
Dukes n (%)
A
B
C
|
0
9 (60)
6 (40)
|
2 (13.3)
10 (66.7)
3 (20)
|
0.845
|
Site n (%)
Caecum
Ascending colon
Hepatic flexure
Transverse colon
Splenic flexure
Descending colon
Sigmoid
Rectum
|
6 (40)
0
1 (6.7)
3 (20)
2 (13.3)
0
2 (13.3)
1 (6.7)
|
9 (60)
0
1 (6.7)
0
1 (6.7)
1 (6.7)
0
3 (20)
|
0.938
|
Iron therapy
|
Ferrous sulphate
200mg BD PO
|
Ferric carboxymaltose
1000mg Single dose IV
|
-
|
Days before surgery1
|
25 (16-36)
|
26 (15-34)
|
0.798
|
Transfusions
|
0
|
0
|
-
|
1 Median (range) 2 Mean (Standard deviation) 3 Median (Interquartile range) * p<0.05.
|
Patients in the OI group remained iron deficient, ferritin 22 ng/ml. No patients had a pre-operative transfusion (Fig.1).
Microsatellite instability
MSI was present in 23% of tumours, with five cancers demonstrating loss of MLH1 and two cancers demonstrating loss of both MSH2 and MLH1. Of these seven tumours, four MSI tumours were in the OI group and the remaining three in the IV group. MSS and MSI tumours were compared for all RT-PCR and immunohistochemistry outcomes and across treatment groups (Additional file 4 and 5). Sub-analyses are discussed below.
Cellular proliferation and Wnt signalling
No significant differences between tumours in oral and IV groups were seen. Exclusion of MSI phenotype did not alter results between treatment groups, (Additional file 4 and 5).
The proliferation marker Ki67 immune staining was mainly nuclear with low immuno-reactivity in normal tissue but significantly higher in tumours (p=0.002) (Fig. 2a and b). No difference was seen between treatment groups.
Membranous and cytoplasmic β-Catenin, the main intracellular signal transducer in the Wnt signalling pathway, had immunoreactivity in both normal and tumour tissues. However, nuclear staining was only seen in some tumour tissues and no normal tissues (Fig. 2c).
There were no significant differences in β-Catenin membranous expression between normal and tumour tissues. In contrast, both cytoplasmic and nuclear immunoreactivity for β-Catenin showed significant differences between normal and tumour tissue (p<0.001) (Fig. 2d). No differences were seen between treatment groups. The p21-activated kinase 1 (PAK1), a downstream effector of GTPases overexpressed in many tumours, showed cytoplasmic immunoreactivity in all tissues, both normal and tumour (Fig. 2e). There were no significant differences between normal and tumour or between treatment groups.
Wnt signalling target gene c-MYC mRNA fold-changes were significantly higher in tumour cells compared with normals (p<0.0001) and increased in both treatment groups (Fig. 2f). IRP2 mRNA levels positively correlated with MYC mRNA levels (R2=39%, ANOVA p=0.010) and SLC11A2 (R2=67%, p<0.001) (Fig. 2g).
DNA damage and apoptosis
The tumour suppressor genes p53 revealed no positive immunoreactivity in the nuclei in 89% of normal tissue. In comparison, only 20% of tumours had no positive nuclei (Fig. 3a) and there was a statistically significant difference in immunoreactivity between normal and tumour tissue (p<0.002) (Fig. 3b). There were no significant differences in p53 expression between the treatment groups. MSI tumours were associated with higher p53 expression compared to the MSS group (p=0.01) (Additional file 5). DNA double strand breaks, detected through γH2AX protein staining showed no or low nuclear immuno-reactivity in all normal tissue. Tumour, as expected, had higher immuno-reactivity (Fig. 3c). There was a statistically significant difference between normal and tumour tissues (p<0.0001) (Fig. 3d). No difference was seen between treatment groups in normal or tumour tissue. There was no positive staining for the apoptosis marker cleaved caspase 3 (CC3) protein in normal tissue. However, all tumours except one had some CC3 immunoreactivity (Fig. 3e) which was statistically significant compared to that of normal tissues (Fig. 3f). There were no differences between treatment groups.
Tissue iron loading and storage
Iron loading was significantly increased in tumour tissues compared to normal tissues from the OI group (p=0.005) (Fig. 4a and b). similar staining was observed between the normal and tumour tissues from the IV group (data not shown). In tumour tissues, Perls Prussian blue expression was significantly higher in the epithelial cells from the oral group compared with the IV group (p=0.01) whereas in the IV group it was significantly distributed to the stroma (p=0.0001) (Fig. 4c). FTH1 mRNA that encodes the heavy subunit of ferritin, was reduced in tumours compared to normal tissues with a greater reduction in the IV group, p<0.001 (Fig. 4d), (Additional file 6).
Cellular iron transport and iron regulation
The iron transport gene SLC11A2 expression in OI versus IV and the immunoreactivity of its encoded protein DMT1 were higher in tumours compared to normal tissues but this increase did not reach statistical significance (p<0.07) (Fig. 5a - c respectively). In OI compared to IV, RT-PCR showed IRP2 (IREB2) was reduced (Fig. 5d) and MSS versus MSI groups were significantly different with an increased IRP2 (p=0.001) and SLC11A2 (p<0.009) in the MSI group, (Additional file 4). IRP2 mRNA levels positively correlated with SLC11A2 (R2=67%, p<0.001, Fig. 5e).
TFRC gene expression in OI treatment was not significantly different to that in IV treatment group (Fig. 6a). However, the immunoreactivity of its protein TfR1 was significantly higher in tumour tissues compared with normal tissues (p<0.0001) (Fig. 6b and c respectively). Furthermore, immune-reactivity of ferroportin was significantly higher in tumour compared to normal tissue (p<0.001) (Fig. 6d and e respectively). Staining for DMT1, TfR1 and ferroprotin was altered in tumours and localised to the cytoplasm whereas in normal tissues their expression was membranous (Fig. 5b, 6b and 6d respectively). No significant differences in the iron transporters were seen between treatment groups and sub-analysis of microsatellite instability did not alter these results.