Introduction 1 2 3 4 3 5 6 7 8 4 9 10 11 11 12 13 12 14 15 12 The aim of this study is to evaluate BrdUrd LI and S-phase fraction (SPF) as the possible indicators of tumor proliferation rate and predictors of the tumor response to neoadjuvant RT in patients with rectal cancer, and to suggest an optimal interval between short RT course and surgery. Methods and Materials Patients 16 The criteria for exclusion were: locally nonresectable tumor; plan to perform only local tumor excision; known metastatic disease; previous radiotherapy of pelvis region; other malignant disease; and patient’s refusal. Preoperative Radiotherapy The patients assigned to preoperative radiotherapy received a total tumor dose of 25 Gy. The treatment was given in five fractions over 5 days, one posterior and two lateral wedged fields were irradiated with photons of maximum 6 MV energy. According to the random selection surgery was performed the following week (schedule I) or after longer interval of 4–5 weeks (schedule II). Surgery Anterior resection of rectum or abdominoperineal excision was performed within a week or a month after the completion of RT. Type of surgery was resection of the rectum and lower sigmoid with involved adjacent tissue and regional lymph nodes up to or above the origin of inferior mesenteric artery. A minimal touch technique was used with high tight ligation of the inferior mesenteric artery. The decision whether the patient should have an abdominoperineal resection or a sphincter-preserving surgery was made by the surgeon during the operation. An abdominoperineal resection of rectum was performed in 41 (44.6%) of the patients, and sphincter preserving surgery was performed in 51 (55.4%). Biological Assessment of Tumor Response Tumor samples were taken twice: before radiotherapy (through a rectoscope) and during surgery from the same place, i.e., at the lowest edge of the tumor mass. Each biopsy was divided into two parts: one was used for BrdUrd LI assessment, and the second was used for immunohistochemical analysis (these results will be the subject of a separate study). Bromodeoxyuridine Labeling Index 3 in vitro 17 3 1/0 Clinical Assessment of Tumor Response 18 Complete response (CR): 100% disappearance; partial response (PR): 30–99% decrease; stable disease (SD): neither CR, PR or PD criteria met; progression of disease (PD): 20% increase in sums of tumor longest diameters. Pathological Assessment of Tumor Response 19 D0—no regression; D1—dominant tumor mass with obvious fibrosis and/or vasculopathy; D2—dominantly fibrotic changes with few tumor cells or groups; D3—very few (difficult to find microscopically) tumor cells in fibrotic tissue with or without mucous substance; D4—no tumor cells, only fibrotic mass (total regression or response). Statistical Methods t P Results Patients 1 Table 1 Selected Characteristics of Patients and Treatment Parameters Characteristics Schedule I Schedule II Total Age mean (±SD) years a (54) 61.9± 9.5 (92) 61.6 ± 10.6 Sex  Male 30 38 68  Female 8 16 24 Histological grade  G1 6 20 26  G2 29 34 63  G3 3 0 3 Tumor stage  T1 8 19 27  T2 25 30 55  T3 5 5 10 PTNM  1 16 25 41  2 8 6 14  3 13 17 30  4 1 2 3 Interval between RT and surgery  Mean (range) days a (54) 32.9 (17–45) (92) 22.9 (2–45)  OTT mean (range) days (38) 13.8 (7–19) (54) 37.9 (22–50) (92) 27.9 (7–50) Surgery  Sphincter-preserving 20 (52.6 %) 31(57.4 %) 51  Abdominoperineal resection 18 23 41 a 1 1 1 Biologic, Pathologic, and Clinical Assessment of Tumor Response 2 P 3 2 1 P P Table 2 Status of Biological Parameters Before and After RT Group BrdUrd LI (%) Mean (range) S-phase fraction (%) Mean (range) Apoptosis (%) Mean (range) All patients Before RT 8.5 (1.0–24.2) 22.0 (3.8–49.9) 5.9 (0–52.8) After RT 4.1* (0.4–18.3) 16.8** (1.5–101.0) 9.8*** (0–45.9) RT schedule I Before RT 8.4 (1.1–24.2) 21.5 (6.1–49.2) 6.6 (0–32.4) After RT 3.8*(0.8–12.6) 14.1**** (1.5–47.9) 10.5 (0–43.3) RT schedule II Before RT 8.6 (1.0–20.0) 22.3 (3.8–49.9) 5.4 (0–52.8) After RT 4.5* (0.4–18.3) 17.2 (2.6–101.0) 9.5***** (0–45.9) P P P P P Table 3 The Relationship Between Tumor Biological Parameters and Histological Grade Histological grade N BrdUrd LI (%) Mean (range) S-phase fraction (%) Mean (range) Apoptosis (%) Mean (range) G1 26 8.5 (1.1–17.1) 23.7 (5.8–49.9) 3.4 (0–32.4) G2 61 8.2 (1.0–20.0) 21.0 (3.8–45.6) 7.2 (0–52.8) G3 3 16.2*, ** (9.5–24.2) 27.7 (18.9–34.7) 2.1 (0.4–4.8) P P Figure 1 P  P 1 1 P 2 P 16 20 19 2 2 Figure 2 Association between biological and pathological assessment (Dworak classification) of early tumor regression for (A) total group of patients and (B) for slowly (BrdUrdLI ≤ 8.5%) and fast proliferating (BrdUrdLI > 8.5%) tumors. Mean values ± SE are shown. For stages D0–D1, statistically significant lower inhibition of tumor cell proliferation after RT was observed for slowly than fast proliferating tumors.  3 3 Figure 3 Association between biological and clinical assessment of early tumor regression after RT for all tumors (A) and separately for slowly and fast proliferating tumors (B). Mean value ± SE are shown.  Discussion This study provides evidence of a clinically significant biological effect of a short preoperative course of RT on tumor proliferation rate. The impact of irradiation on biological tumor response was assessed by BrdUrd LI, SPF, and the degree of subsequent pathologic and clinical down-staging of the tumors after surgery. The study showed differences in the pretreatment proliferation rate of the tumor. Mean BrdUrd LI before RT was equal to 8.5% and ranged from 1 to 24.2%. Mean SPF was 22.0% and ranged from 3.8 to 49.9%. The proportion of cells in S-phase as estimated by the DNA content overestimates the labeling index determined by the uptake of BrdUrd. This may be so because the exposure time is quite short and there may be subpopulations in the tumors that are synthesizing DNA at a very slow rate, or there may indeed be cells with an S-phase DNA content that are not synthesizing DNA (as a result of nutrient or oxygen supply, lack of growth factors, inadequate vascularity). 21 22 23 21 In our study, pretreatment BrdUrd LI or SPF was not predictive for early clinical and pathologicl tumor response, probably because of different tumor microenvironment. However, BrdUrd LI after/before RT ratio gave information on the different significant biological processes that take place after irradiation, and have impact on cell death like redistribution, repopulation, and reoxygenation. 24 25 P 26 27 28 29 30 31 32 25 14 25 14 14 33 10 4 34 11 15 15 33 35 35 In conclusion, our study shows that pretreatment BrdUrd LI or SPF were not predictive for early clinical and pathologic tumor response. After/before BrdUrd LI ratios showed inhibition of proliferation in responsive tumors, but this was not reflected in the number of sphincter preserving procedures performed. As 1 month after RT, accelerated proliferation of tumor cells is observed only in slowly proliferating tumors, we think that longer interval between RT and surgery is inadvisable. If late tumor response confirms that patients having tumors with increased proliferative activity have statistically significantly less recurrences and improved survival rates compared with patients with less proliferative tumors, then we will be able to suggest a prognostic factor for individual rectal cancer patient, and a basis for selection to postoperative adjuvant chemotherapy.