Time patterns of changes in biomarkers, symptoms and histopathology during pelvic radiotherapy

Figures & data

Table I.  Grading of histologic changes

Structure	Characteristic
Surface epithelium	Loss of cellular height/flattening of cells
	Cellular inflammatory infiltrate
Glands (crypts)	Loss of goblet cells
	Loss of glands
	Cryptitis/crypt abcess
	Atrophy of epithel in crypts
	Distortion of glands
Lamina propria stroma	Inflammation
	Fibrosis

Normal: score = 0

Abnormal: mildly abnormal = 1; moderately abnormal = 2;

markedly abnormal = 3; severely abnormal = 4.

Table II.  Concentrations of vitamin E, LTB_4, TBARS, hemoglobin and leukocyte count

	Before radiotherapy (RT)		Week 2 of RT			Week 6 of RT			4 weeks after RT
Blood samples	Median	IR	Median	IR	p	Median	IR	p	Median	IR	p
Vitamin E (µg/ml)	13.7	(13.0–15.2)	12.8	(10.6–14.5)	0.057	12.0	(10.2–14.1)	0.002**	13.3	(11.7–15.5)	0.853
LTB4 (pg/ml)	33.9	(20.9–111.1)	33.7	(24.1–87.9)	0.604	36.8	(23.2–105.0)	0.586	36.1	(21.6–136.8)	0.723
TBARS (nmol/ml)	5	(3–7)	4	(3–5)	0.390	4	(3–5)	0.049*	4	(3–5)	0.162
Hgb (g/dl)	14.5	(13.7–14.9)	14.2	(13.6–14.8)	0.120	13.8	(12.9–14.3)	<0.001**	14.3	(13.3–14.7)	0.008**
Leukocyte count (10^9 /l)	6.6	(5.6–8.2)	5.5	(4.6–6.3)	<0.001**	5.8	(5.1–6.5)	0.029*	5.6	(4.7–6.4)	0.002**

p: comparisons with concentrations before RT by exact paired permutation tests

* p < 0.05 ** p < 0.01

IR: Interquartile range; 25 and 75 percentiles

LTB₄: leukotriene B4

TBARS: thiobarbituric acid reactive substances

n = 20; except leukocyte count: n = 18

Table III.  Concentrations of calprotectin, S100A12 and lactoferrin

	Before radiotherapy (RT)		Week 2 of RT			Week 6 of RT			4 weeks after RT
	Median	IR	Median	IR	p	Median	IR	p	Median	IR	p
Blood samples
Calprotectin (mg/L)	0.8	(0.6–1.0)	0.7	(0.5–0.9)	0.056	0.7	(0.6–1.1)	0.228	0.8	(0.6–1.1)	0.736
S100A12 (mg/L)	0.06	(0.04–0.10)	0.06	(0.03–0.08)	0.083	0.05	(0.03–0.08)	0.006**	0.06	(0.04–0.08)	0.689
Stool samples
Calprotectin (mg/kg)	13	(5–19)	15	(5–20)	0.794	14	(9–26)	0.842	11	(5–19)	0.646
S100A12 (mg/kg)	0.3	(0.1–0.5)	0.5	(0.2–1.0)	0.473	0.6	(0.2–1.6)	0.015*	0.2	(0.1–0.6)	0.631
Lactoferrin (mg/kg)	1.1	(0.5–4.4)	1.1	(0.4–3.6)	0.630	3.6	(0.5–19.6)	0.007**	1.1	(0.4–2.6)	0.297

p: comparisons with concentrations before RT by exact paired permutation tests

* p < 0.05 ** p < 0.01

IR: Interquartile range; 25 and 75 percentiles

Calprotectin measured in EDTA plasma, S100A12 measured in serum (n = 19)

n = 20 for stool samples

Figure 1.  Symptom scores (TSS) before, during and after radiotherapy. Median and interquartile range shown in black; individual patients’ TSS in grey. TSS: Total Symptom Scores: sum of scores from five questions grading nausea, abdominal pains, excessive gas, loose stools and painful defecations; “0” meaning no problem, “10” very much problem on each of the 5 questions. (n = 20)

Figure 2.  Biopsy scores before, during and after radiotherapy. Median and interquartile range shown in black; individual patients’ sums in grey. Individual patients’ sum from the biopsy evaluation (Table I). (n = 19)

Figure 3.  Histological sections from rectal mucosa biopsies. A and B: Hematoxylin & Eosin stained sections. A: Before RT. Note regularity of glands, goblet cells in epithelium and normal height of epithelium. B: Week 2. Reduced number of glands and goblet cells and atrophy of epithelium and fibrosis. C and D: Trichrom Blue staining of collagenous material. C: Before RT. Basal membrane is stained blue. D: Week 6. Increased collagen in lamina propria stroma and reduced number of glands. All photomicrographs at magnification×100.

Figure 4.  Vitamin E in plasma before, during and after radiotherapy. Median and interquartile range shown in black; individual patients’ concentrations shown in grey. (n = 20)

Table IV.  Fatty acids in rectal mucosa biopsies

	Before radiotherapy (RT)		Week 2 of RT			Week 6 of RT			4 weeks after RT
Fatty acids (mg/g)	Median	IR	Median	IR	p	Median	IR	p	Median	IR	p
∑ saturated	4.1	(3.8–4.6)	4.1	(3.3–4.3)	0.115	3.8	(3.3–4.3)	0.977	4.0	(3.1–4.2)	0.026*
16:0	1.8	(1.6–2.2)	1.9	(1.4–2.1)	0.402	1.6	(1.6–2.2)	0.932	1.8	(1.3–2.0)	0.096
18:0	2.1	(2.0–2.2)	1.9	(1.7–2.1)	0.038*	1.8	(1.6–2.2)	0.162	1.9	(1.6–2.1)	0.014*
∑ monounsaturated	3.4	(3.2–3.9)	2.6	(2.3–3.1)	0.016*	2.5	(2.1–2.8)	0.568	2.8	(2.6–3.3)	0.015*
18:1n − 7	0.3	(0.3–0.4)	0.3	(0.2–0.3)	0.003**	0.2	(0.2–0.3)	0.049*	0.3	(0.2–0.3)	0.094
18:1n − 9	3.0	(2.8–3.1)	2.0	(1.7–2.7)	0.012*	2.0	(1.7–2.3)	0.471	2.3	(2.1–2.8)	0.015*
20:1n − 9	0.1	(0.1–0.1)	0.1	(0.0–0.1)	0.523	0.1	(0.0–0.1)	0.569	0.0	(0.0–0.1)	0.018*
∑ n − 6 PUFAs	3.6	(3.2–4.1)	3.4	(2.9–4.5)	0.818	3.2	(2.7–4.4)	0.420	3.2	(2.9–3.8)	0.325
18:2n − 6	2.1	(1.8–2.4)	1.8	(1.5–2.6)	0.966	1.6	(1.4–2.6)	0.482	2.0	(1.7–2.0)	0.452
20:3n − 6	0.2	(0.2–0.2)	0.2	(0.2–0.3)	0.829	0.2	(0.1–0.2)	0.158	0.2	(0.1–0.2)	0.526
20:4n − 6	1.3	(1.2–1.5)	1.4	(1.1–1.6)	0.356	1.2	(1.1–1.5)	0.617	1.0	(1.0–1.5)	0.220
∑ n − 3 PUFAs	1.0	(0.8–1.2)	1.0	(0.9–1.3)	0.181	1.0	(0.8–1.3)	0.936	0.9	(0.8–1.0)	0.291
20:5n − 3	0.3	(0.2–0.4)	0.3	(0.2–0.3)	0.321	0.2	(0.2–0.4)	0.560	0.2	(0.2–0.3)	0.272
22:5n − 3	0.1	(0.1–0.2)	0.2	(0.1–0.2)	0.006**	0.2	(0.2–0.2)	0.123	0.1	(0.1–0.1)	0.537
22:6n − 3	0.5	(0.4–0.6)	0.6	(0.4–0.7)	0.258	0.5	(0.5–0.7)	0.937	0.5	(0.4–0.5)	0.304
AA/EPA	4.2	(3.8–6.5)	5.3	(4.1–7.3)	0.243	4.5	(3.9–7.2)	0.732	4.1	(3.6–5.8)	0.840
n − 6/n − 3 PUFAs	3.7	(3.4–4.3)	3.7	(2.9–4.0)	0.161	3.5	(3.1–4.0)	1.000	3.6	(3.3–4.2)	0.879

p: comparisons with concentrations before RT by exact paired permutation tests

* p < 0.05 ** p < 0.01

IR: Interquartile range; 25 and 75 percentiles

∑: sum of groups of fatty acids

PUFAs: polyunsaturated fatty acids

Omega nomenclature: first digits indicate number of carbon in chain; next number indicate number of double bonds; n − 3 indicates at which carbon the first double bond is positioned (e.g. omega-3 in this example);

AA/EPA: ratio arachidonic acid ( 20:4n − 6 ) and eicosapentaenoic acid (20:5n − 3)

(n = 11)

Table V.  Fatty acids in plasma samples

	Before radiotherapy (RT)			Week 2 of RT			Week 6 of RT			4 weeks after RT
Fatty acids (mg/ml)	Median	IR	Median	IR	p	Median	IR	p	Median	IR	p
∑ saturated	1.7	(1.2–2.0)	1.5	(1.2–2.0)	0.719	1.5	(1.2–2.1)	0.903	1.8	(1.2–2.1)	0.367
14:0	0.1	(0.1–0.2)	0.1	(0.1–0.2)	0.517	0.1	(0.1–0.2)	0.379	0.1	(0.1–0.2)	0.904
16:0	1.2	(0.9–1.5)	1.1	(0.8–1.5)	0.617	1.1	(0.8–1.5)	0.962	1.3	(0.9–1.5)	0.385
18:0	0.3	(0.3–0.4)	0.3	(0.2–0.3)	0.677	0.3	(0.2–0.3)	0.225	0.3	(0.3–0.4)	0.185
∑ monounsaturated	1.4	(1.0–1.6)	1.2	(0.9–1.7)	0.789	1.2	(0.9–1.6)	0.948	1.3	(0.9–1.6)	0.407
16:1n − 7	0.1	(0.1–0.2)	0.1	(0.1–0.2)	0.812	0.1	(0.1–0.2)	0.419	0.1	(0.1–0.2)	0.219
18:1n − 7	0.1	(0.1–0.1)	0.1	(0.1–0.1)	0.380	0.1	(0.1–0.1)	0.694	0.1	(0.1–0.1)	0.208
18:1n − 9	1.0	(0.7–1.2)	0.9	(0.7–1.1)	0.695	0.9	(0.7–1.2)	0.679	1.0	(0.7–1.2)	0.578
∑ n-6 PUFAs	2.1	(1.6–2.3)	2.1	(1.5–2.3)	0.257	2.1	(1.5–2.2)	0.001**	2.1	(1.7–2.5)	0.315
18:2n − 6	1.7	(1.2–1.9)	1.6	(1.1–1.8)	0.317	1.6	(1.1–1.7)	0.002**	1.7	(1.3–2.0)	0.310
20:3n − 6	0.1	(0.0–0.1)	0.1	(0.0–0.1)a	0.025*	0.1	(0.0–0.1)a	0.003**	0.1	(0.0–0.1)	0.933
20:4n − 6	0.3	(0.2–0.3)	0.3	(0.2–0.3)	0.479	0.3	(0.2–0.4)	0.439	0.3	(0.2–0.4)	0.642
∑ n-3 PUFAs	0.3	(0.3–0.5)	0.4	(0.3–0.6)	0.100	0.4	(0.4–0.6)	0.034*	0.4	(0.3–0.7)	0.142
18:3n − 3	0.1	(0.0–0.1)	0.1	(0.0–0.1)	0.562	0.1	(0.0–0.1)	0.477	0.1	(0.0–0.1)	0.234
20:5n − 3	0.1	(0.1–0.2)	0.1	(0.1–0.2)	0.215	0.1	(0.1–0.2)a	0.028*	0.1	(0.1–0.2)	0.707
22:6n − 3	0.2	(0.1–0.2)	0.2	(0.2–0.2)	0.104	0.2	(0.2–0.3)	0.033*	0.2	(0.2–0.3)	0.308
AA/EPA	2.7	(1.8–3.5)	2.1	(1.5–2.9)	0.028*	2.0	(1.5–2.7)	0.004**	2.4	(1.7–3.9)	0.818
n − 6/n − 3 PUFAs	5.0	(3.9–6.2)	4.0	(3.2–5.7)	0.026*	4.1	(3.2–5.3)	0.002**	4.8	(3.4–6.9)	0.825

p: comparisons with concentrations before RT by exact paired permutation tests

* p < 0.05 ** p < 0.01

IR: Interquartile range; 25 and 75 percentiles

∑: sum of groups of fatty acids

PUFAs: polyunsaturated fatty acids

Omega nomenclature: first digits indicate number of carbon in chain;next number indicate number of double bonds; n − 3 indicates at which carbon the first double bond is positioned (e.g. omega-3 in this example);

AA/EPA: ratio arachidonic acid (20:4n − 6) and eicosapentaenoic acid (20:5n − 3)

a: 20:3n − 6 increased from baseline to week 2 and 6; 20:5n − 3 increased in week 6

(n = 20)

Figure 5.  Lactoferrin in stool samples before, during and after radiotherapy. Median and interquartile range shown in black; individual patients’ concentrations in grey. (n = 20)