Gene-expression profiles of pretreatment biopsies predict complete response of rectal cancer patients to preoperative chemoradiotherapy.

PURPOSE: Preoperative (neoadjuvant) chemoradiotherapy (CRT) and total mesorectal excision is the standard treatment for rectal cancer patients (UICC stage II/III). Up to one-third of patients treated with CRT achieve a pathological complete response (pCR). These patients could be spared from surgery and its associated morbidity and mortality, and assigned to a "watch and wait" strategy. However, reliably identifying pCR based on clinical or imaging parameters remains challenging. EXPERIMENTAL DESIGN: We generated gene-expression profiles of 175 patients with locally advanced rectal cancer enrolled in the CAO/ARO/AIO-94 and -04 trials. One hundred and sixty-one samples were used for building, training and validating a predictor of pCR using a machine learning algorithm. The performance of the classifier was validated in three independent cohorts, comprising 76 patients from (i) the CAO/ARO/AIO-94 and -04 trials (n = 14), (ii) a publicly available dataset (n = 38) and (iii) in 24 prospectively collected samples from the TransValid A trial. RESULTS: A 21-transcript signature yielded the best classification of pCR in 161 patients (Sensitivity: 0.31; AUC: 0.81), when not allowing misclassification of non-complete-responders (False-positive rate = 0). The classifier remained robust when applied to three independent datasets (n = 76). CONCLUSION: The classifier can identify >1/3 of rectal cancer patients with a pCR while never classifying patients with an incomplete response as having pCR. Importantly, we could validate this finding in three independent datasets, including a prospectively collected cohort. Therefore, this classifier could help select rectal cancer patients for a "watch and wait" strategy. TRANSLATIONAL RELEVANCE: Forgoing surgery with its associated side effects could be an option for rectal cancer patients if the prediction of a pathological complete response (pCR) after preoperative chemoradiotherapy would be possible. Based on gene-expression profiles of 161 patients a classifier was developed and validated in three independent datasets (n = 76), identifying over 1/3 of patients with pCR, while never misclassifying a non-complete-responder. Therefore, the classifier can identify patients suited for "watch and wait".

PECAM-1 modulates liver damage induced by synergistic effects of TNF-α and irradiation.

The mechanisms of radiation-induced liver damage are poorly understood. We investigated if tumour necrosis factor (TNF)-α acts synergistically with irradiation, and how its activity is influenced by platelet endothelial cell adhesion molecule-1 (PECAM-1). We studied murine models of selective single-dose (25 Gy) liver irradiation with and without TNF-α application (2 µg/mouse; i.p.). In serum of wild-type (wt)-mice, irradiation induced a mild increase in hepatic damage marker aspartate aminotransferase (AST) in comparison to sham-irradiated controls. AST levels further increased in mice treated with both irradiation and TNF-α. Accordingly, elevated numbers of leucocytes and increased expression of the macrophage marker CD68 were observed in the liver of these mice. In parallel to hepatic damage, a consecutive decrease in expression of hepatic PECAM-1 was found in mice that received radiation or TNF-α treatment alone. The combination of radiation and TNF-α induced an additional significant decline of PECAM-1. Furthermore, increased expression of hepatic lipocalin-2 (LCN-2), a hepatoprotective protein, was detected at mRNA and protein levels after irradiation or TNF-α treatment alone and the combination of both. Signal transducer and activator of transcription-3 (STAT-3) seems to be involved in the signalling cascade. To study the involvement of PECAM-1 in hepatic damage more deeply, the liver of both wt- and PECAM-1-knock-out-mice were selectively irradiated (25 Gy). Thereby, ko-mice showed higher liver damage as revealed by elevated AST levels, but also increased hepatoprotective LCN-2 expression. Our studies show that TNF-α has a pivotal role in radiation-induced hepatic damage. It acts in concert with irradiation and its activity is modulated by PECAM-1, which mediates pro- and anti-inflammatory signalling.

Favorable radiation field decrease in gastric marginal zone lymphoma : Experience of the German Study Group on Gastrointestinal Lymphoma (DSGL).

PURPOSE: Long-term impact of stage-adapted field reduction in a large cohort of gastric marginal zone lymphoma (gMZL) patients treated conservatively with curative radiation therapy (RT). PATIENTS AND METHODS: Prospective analysis of paper records of 290 patients with stage IE-IIE gMZL, treated in 78 radiotherapeutic institutions in Germany from 1992-2013. Stage-adapted radiation fields decreased from extended field (EF) to involved field (IF) over the course of three consecutive prospective trials of the German Study Group on Gastrointestinal Lymphoma (DSGL). Treatment results were compared between the three cohorts. RESULTS: Overall collective with median age of 60 years, slight male predominance (m:f = 1.1:1) and ratio of disease stage I:stage II = 2.1:1. Median follow-up 6.4 years in total: 13.0 years in the first gastrointestinal study (GIT 1992), 8.2 years in the second (GIT 1996) and 4.7 years in the third study (DSGL 01/2003). Stage-adapted radiation field decrease together with further technological development led to reduced relative frequencies of acute/chronic adverse effects and until now was accompanied by lower disease recurrence. The third study design with smallest field size (IF in stage I, locoregional EF in stage II) achieved the best survival outcome at the 5­year follow-up (overall survival 92.7%, event-free survival 89.5% and lymphoma-specific survival 100.0%). Disease relapse observed in 10 patients. Cumulative incidence of disease-specific death was 1.7% of the followed patients. Primary disease stage associated with lymphoma-specific survival. CONCLUSION: Stage-adapted reduction towards IF in gMZL resulted in favorable adverse effects, local control and survival rates. These results support further decreases in modern RT of gMZL.

Induction of Lipocalin2 in a Rat Model of Lung Irradiation.

Previously, we showed that lipocalin2 (LCN2) serum levels increased after liver irradiation and during acute-phase conditions. Here, we evaluate LCN2 expression and serum levels after single-dose lung irradiation with 25 Gy, percutaneously administered to the lung of randomly-paired male Wistar rats. Due to the concave anatomy of the lung recesses, the irradiation field included the upper part of the liver. No rat died due to irradiation. In control tissue, lung immunohistochemistry showed a high constitutive expression of LCN2+ granulocytes. LCN2 mRNA levels in lung tissue increased up to 24 h (9 ± 2.3-fold) after irradiation. However, serum LCN2 levels remained undetectable after lung irradiation. LCN2 expression in the upper part of the liver increased up to 4.2-fold after lung irradiation, but the lower liver showed an early decrease. Acute-phase cytokines (IL-1ß and TNF-α) showed a significant increase on transcript level in both lung and upper liver, whilst the lower liver did not show any considerable increase. In conclusion, constitutive expression of LCN2 in local immune cells demonstrates its local role during stress conditions in the lung. The absence of LCN2 in the serum strengthens our previous findings that the liver is the key player in secreting LCN2 during stress conditions with liver involvement.

Role of PECAM-1 in radiation-induced liver inflammation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is known to play an important role in hepatic inflammation. Therefore, we investigated the role of PECAM-1 in wild-type (WT) and knock-out (KO)-mice after single-dose liver irradiation (25 Gy). Both, at mRNA and protein level, a time-dependent decrease in hepatic PECAM-1, corresponding to an increase in intercellular cell adhesion molecule-1 (ICAM-1) (6 hrs) was detected in WT-mice after irradiation. Immunohistologically, an increased number of neutrophil granulocytes (NG) (but not of mononuclear phagocytes) was observed in the liver of WT and PECAM-1-KO mice at 6 hrs after irradiation. The number of recruited NG was higher and prolonged until 24 hrs in KO compared to WT-mice. Correspondingly, a significant induction of hepatic tumour necrosis factor (TNF)-α and CXC-chemokines (KC/CXCL1 interleukin-8/CXCL8) was detected together with an elevation of serum liver transaminases (6-24 hrs) in WT and KO-mice. Likewise, phosphorylation of signal transducer and activator of transcription-3 (STAT-3) was observed in both animal groups after irradiation. The level of all investigated proteins as well as of the liver transaminases was significantly higher in KO than WT-mice. In the cell-line U937, irradiation led to a reduction in PECAM-1 in parallel to an increased ICAM-1 expression. TNF-α-blockage by anti-TNF-α prevented this change in both proteins in cell culture. Radiation-induced stress conditions induce a transient accumulation of granulocytes within the liver by down-regulation/absence of PECAM-1. It suggests that reduction/lack in PECAM-1 may lead to greater and prolonged inflammation which can be prevented by anti-TNFα.

Downstage migration after neoadjuvant chemoradiotherapy for rectal cancer: the reverse of the Will Rogers phenomenon?

Downstaging after neoadjuvant treatment is increasingly used as a prognostic factor and surrogate endpoint in clinical trials. However, in recent trials of neoadjuvant 5-fluorouracil-based chemoradiotherapy for rectal cancer, downstaging did not translate into a benefit with regard to either disease-free survival (DFS) or overall survival. By analyzing the 10-year outcome data of the German CAO/ARO/AIO-94 phase 3 trial, the authors demonstrated that significantly fewer patients had poor prognostic features (eg, ypT3-4, ypN1-2) after preoperative 5-fluorouracil-based chemoradiotherapy. Nevertheless, these patients with International Union for Cancer Control stage II disease were found to be at a higher risk of developing distant metastases and had poorer DFS compared with patients with corresponding TNM tumor (sub)groups in the postoperative treatment arm, whereas patients with International Union for Cancer Control stage III disease demonstrated a nonsignificant trend toward a worse outcome after preoperative treatment. Overall, DFS remained identical in both treatment arms. Thus, "downstage migration" after neoadjuvant treatment resembles the reverse of the Will Rogers phenomenon and therefore may not be a reliable endpoint for long-term outcomes.

High-grade acute organ toxicity and p16(INK4A) expression as positive prognostic factors in primary radio(chemo)therapy for patients with head and neck squamous cell carcinoma.

BACKGROUND: Superior treatment response and survival for patients with human papilloma virus (HPV)-positive head and neck cancer (HNSCC) are documented in clinical studies. However, the relevance of high-grade acute organ toxicity (HGAOT), which has also been correlated with improved prognosis, has attracted scant attention in HPV-positive HNSCC patients. Hence we tested the hypothesis that both parameters, HPV and HGAOT, are positive prognostic factors in patients with HNSCC treated with definite radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS: Pretreatment tumor tissue and clinical records were available from 233 patients receiving definite RT (62 patients) or RCT (171 patients). HPV infection was analysed by means of HPV DNA detection or p16(INK4A) expression; HGAOT was defined as the occurrence of acute organ toxicity >grade 2 according to the Common Toxicity Criteria. Both variables were correlated with overall survival (OS) using Cox proportional hazards regression. RESULTS: Positivity for HPV DNA (44 samples, 18.9 %) and p16(INK4A) expression (102 samples, 43.8 %) were significantly correlated (p < 0.01), and HGAOT occurred in 77 (33 %) patients. Overall, the 5-year OS was 23 %; stratified for p16(INK4A) expression and HGAOT, OS rates were 47 %, 42 %, 20 % and 10 % for patients with p16(INK4A) expression and HGAOT, patients with HGAOT only, patients with p16(INK4A) expression only, and patients without p16(INK4A) expression or HGAOT, respectively. After multivariate testing p16(INK4A) expression (p = 0.003) and HGAOT (p < 0.001) were significantly associated with OS. CONCLUSION: P16(INK4A) expression and HGAOT are independent prognostic factors for OS of patients with HNSCC, whereas p16(INK4A) expression is particularly important for patients without HGAOT.

The anti-TNF-α antibody infliximab inhibits the expression of fat-transporter-protein FAT/CD36 in a selective hepatic-radiation mouse model.

Previously, we reported a radiation-induced inflammation triggering fat-accumulation through fatty-acid-translocase/cluster of differentiation protein 36 (FAT/CD36) in rat liver. Furthermore, inhibition of radiation-induced FAT/CD36-expression by anti-tumor necrosis factor-α (anti-TNF-α) (infliximab) was shown in vitro. The current study investigates fat-accumulation in a mouse-model of single-dose liver-irradiation (25-Gray) and the effect of anti-TNF-α-therapy on FAT/CD36 gene-expression. Mice livers were selectively irradiated in vivo in presence or absence of infliximab. Serum- and hepatic-triglycerides, mRNA, and protein were analyzed by colorimetric assays, RT-PCR, Immunofluorescence and Western-Blot, respectively. Sudan-staining was used demonstrating fat-accumulation in tissue. In mice livers, early (1-3 h) induction of TNF-α-expression, a pro-inflammatory cytokine, was observed. It was followed by elevated hepatic-triglyceride level (6-12 h), compared to sham-irradiated controls. In contrast, serum-triglyceride level was decreased at these time points. Similar to triglyceride level in mice livers, Sudan staining of liver cryosections showed a quick (6-12 h) increase of fat-droplets after irradiation. Furthermore, expression of fat-transporter-protein FAT/CD36 was increased at protein level caused by radiation or TNF-α. TNF-α-blockage by anti-TNF-α showed an early inhibition of radiation-induced FAT/CD36 expression in mice livers. Immunohistochemistry showed basolateral and cytoplasmic expression of FAT/CD36 in hepatocytes. Moreover, co-localization of FAT/CD36 was detected with α-smooth muscle actin (α-SMA+) cells and F4/80+ macrophages. In summary, hepatic-radiation triggers fat-accumulation in mice livers, involving acute-phase-processes. Accordingly, anti-TNF-α-therapy prevented early radiation-induced expression of FAT/CD36 in vivo.

A disease-specific enteral nutrition formula improves nutritional status and functional performance in patients with head and neck and esophageal cancer undergoing chemoradiotherapy: results of a randomized, controlled, multicenter trial.

BACKGROUND: In patients with head and neck and esophageal tumors, nutritional status may deteriorate during concurrent chemoradiotherapy (CRT). The aim of this study was to investigate the influence of enteral nutrition enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on body composition and nutritional and functional status. METHODS: In a controlled, randomized, prospective, double-blind, multicenter study, 111 patients with head and neck and esophageal cancer undergoing concurrent CRT received either an enteral standard nutrition (control group) or disease-specific enteral nutrition Supportan®-containing EPA+DHA (experimental group) via percutaneous endoscopic gastrostomy. The primary endpoint was the change of body cell mass (BCM) following CRT at weeks 7 and 14 compared with the baseline value. Secondary endpoints were additional parameters of body composition, anthropometric parameters, and nutritional and functional status. RESULTS: The primary endpoint of the study, improvement in BCM, reached borderline statistical significance. Following CRT, patients with experimental nutrition lost only 0.82 ± 0.64 kg of BCM compared with 2.82 ± 0.77 kg in the control group (P = .055). The objectively measured nutritional parameters, such as body weight and fat-free mass, showed a tendency toward improvement, but the differences were not significant. The subjective parameters, in particular the Kondrup score (P = .0165) and the subjective global assessment score (P = .0065) after follow-up improved significantly in the experimental group, compared with the control group. Both enteral regimens were safe and well tolerated. CONCLUSION: Enteral nutrition with EPA and DHA may be advantageous in patients with head and neck or esophageal cancer by improving parameters of nutritional and functional status during CRT.

Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII.

Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes' Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes' systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction.

Serum Lipocalin2 is a potential biomarker of liver irradiation damage.

BACKGROUND/AIM: IL-6 - IL-1- lipocalin2 (LCN2) - liver irradiation - oxidative stress - TNF-a Lipocalin2 (LCN2) is an acute phase protein. The source of its increased serum level in oxidative stress conditions (ROS) remains still unknown. We prospectively evaluate the serum LCN2 increase after single dose liver irradiation along with hepatic LCN2 gene and protein expression. METHODS: A single dose of 25 Gray was administered percutaneously to the liver of randomly paired rats after a planning CT scan. Male Wistar rats were sacrificed 1, 3, 6, 12, 24 and 48 h after irradiation along with sham-irradiated controls. ELISA, RT-PCR, Western blot and immunofluorescence staining was performed. Furthermore, hepatocytes, myofibroblasts and Kupffer cells were isolated from the liver of healthy rats and irradiated ex-vivo. RESULTS: After liver irradiation, LCN2 serum levels increased significantly up to 2.7 µg/ml within 6 h and stayed elevated over 24 h. LCN2 specific transcripts increased significantly up to 552 ± 109-fold at 24 h after liver irradiation, which was further confirmed at protein level. α2-macroglobulin and hemoxygenase-1 also showed an increase, but the magnitude was less as compared to LCN2. LCN2+ granulocytes were detected within 1 h after irradiation around central and portal fields and remained high during the course of study. Ex-vivo irradiated hepatocytes (2.4 ± 0.6-fold) showed a higher LCN2 gene expression as compared to myofibroblasts and Kupffer cells. IL-1ß treatment further increased LCN2 gene expression in cultured hepatocytes. CONCLUSIONS: Single dose liver irradiation induces a significant increase in LCN2 serum levels, comparable to the induction of acute phase proteins. We suggest LCN2 as marker for the early phase of radiation-induced tissue damage.

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects.

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small "scars" on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of "provisional clot"-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.

Preoperative chemoradiotherapy and postoperative chemotherapy with fluorouracil and oxaliplatin versus fluorouracil alone in locally advanced rectal cancer: initial results of the German CAO/ARO/AIO-04 randomised phase 3 trial.

BACKGROUND: Preoperative chemoradiotherapy, total mesorectal excision surgery, and adjuvant chemotherapy with fluorouracil is the standard combined modality treatment for rectal cancer. With the aim of improving disease-free survival (DFS), this phase 3 study (CAO/ARO/AIO-04) integrated oxaliplatin into standard treatment. METHODS: This was a multicentre, open-label, randomised, phase 3 study in patients with histologically proven carcinoma of the rectum with clinically staged T3-4 or any node-positive disease. Between July 25, 2006, and Feb 26, 2010, patients were randomly assigned to two groups: a control group receiving standard fluorouracil-based combined modality treatment, consisting of preoperative radiotherapy of 50·4 Gy plus infusional fluorouracil (1000 mg/m(2) days 1-5 and 29-33), followed by surgery and four cycles of bolus fluorouracil (500 mg/m(2) days 1-5 and 29; fluorouracil group); and an experimental group receiving preoperative radiotherapy of 50·4 Gy plus infusional fluorouracil (250 mg/m(2) days 1-14 and 22-35) and oxaliplatin (50 mg/m(2) days 1, 8, 22, and 29), followed by surgery and eight cycles of adjuvant chemotherapy with oxaliplatin (100 mg/m(2) days 1 and 15), leucovorin (400 mg/m(2) days 1 and 15), and infusional fluorouracil (2400 mg/m(2) days 1-2 and 15-16; fluorouracil plus oxaliplatin group). Randomisation was done with computer-generated block-randomisation codes stratified by centre, clinical T category (cT1-4 vs cT4), and clinical N category (cN0 vs cN1-2) without masking. DFS is the primary endpoint. Secondary endpoints, including toxicity, compliance, and histopathological response are reported here. Safety and compliance analyses included patients as treated, efficacy endpoints were analysed according to the intention-to-treat principle. This study is registered with ClinicalTrials.gov, number NCT00349076. FINDINGS: Of the 1265 patients initially enrolled, 1236 were evaluable (613 in the fluorouracil plus oxaliplatin group and 623 in the fluorouracil group). Preoperative grade 3-4 toxic effects occurred in 140 (23%) of 606 patients who actually received fluorouracil and oxaliplatin during chemoradiotherapy and in 127 (20%) of 624 patients who actually received fluorouracil chemoradiotherapy. Grade 3-4 diarrhoea was more common in those who received fluorouracil and oxaliplatin during chemoradiotherapy than in those who received fluorouracil during chemoradiotherapy (73 patients [12%] vs 52 patients [8%]), as was grade 3-4 nausea or vomiting (23 [4%] vs nine [1%]). 516 (85%) of the 606 patients who received fluorouracil and oxaliplatin-based chemoradiotherapy had the full dose of chemotherapy, and 571 (94%) had the full dose of radiotherapy; as did 495 (79%) and 601 (96%) of 624 patients who received fluorouracil-based chemoradiotherapy, respectively. A pathological complete response was achieved in 103 (17%) of 591 patients who underwent surgery in the fluorouracil and oxaliplatin group and in 81 (13%) of 606 patients who underwent surgery in the fluorouracil group (odds ratio 1·40, 95% CI 1·02-1·92; p=0·038). In the fluorouracil and oxaliplatin group, 352 (81%) of 435 patients who began adjuvant chemotherapy completed all cycles (with or without dose reduction), as did 386 (83%) of 463 patients in the fluorouracil group. INTERPRETATION: Inclusion of oxaliplatin into modified fluorouracil-based combined modality treatment was feasible and led to more patients achieving a pathological complete response than did standard treatment. Longer follow-up is needed to assess DFS. FUNDING: German Cancer Aid (Deutsche Krebshilfe).

Radiation-induced damage in different segments of the rat intestine after external beam irradiation of the liver.

INTRODUCTION: The out-of-field effects on the intestine, caused by radiation treatment of a parenchymatous organ, have not previously been studied. METHODS: A single dose of 25Gy was administered percutaneously to the liver of male Wistar rats after a planning CT-scan. Sham-irradiated animals served as controls. At 1, 6, 24, 96h, 1.5 and 3months the duodenum, jejunum, ileum and distal colon were removed, washed and deep-frozen or prepared for paraffin staining. RESULTS: All animals survived the treatment. Epithelial cell damage occurred in all small-intestinal segments. However, prolonged denudation of the villi together with destruction of the crypt lining was only observed in the ileum, resulting in deficient regeneration. In the colon, changes were minor. Radiation mucositis with granulocyte (MP0+) infiltration was seen from 1 to 24h in the duodenum and jejunum, when ED1+ macrophages, CD3+ T-lymphocytes, and CD34+ hematopoietic precursor cells were recruited, accompanied by an increase in the chemokines MCP-1, MIP-1α, MIP3α and Il-8. In the ileum, early granulocyte infiltration was delayed but continuous. Recruitment of macrophages and lymphocytes was deficient and induction of chemokines as of the adhesion molecules PECAM-1, ICAM-1 was lacking. CONCLUSION: Post-irradiation damage to the ileum was delayed and followed by an altered repair process with structural changes of the villi. The observed changes might result from a higher sensitivity to oxidative stress mechanisms with subsequent damage of the regenerative capacity of the crypt-villus axis, accompanied by a sustained "inflammatory response" and vascular damage with a lack of regeneratory cell recruitment.

Gender-specific acute organ toxicity during intensified preoperative radiochemotherapy for rectal cancer.

Patients with locally advanced rectal cancer (cUICC stages II/III) are typically treated with preoperative 5-fluorouracil-based (5-FU-based) radiochemotherapy (RCT). However, trials are currently being conducted to improve the complete remission rates and the systemic control by combining 5-FU with oxaliplatin. The primary objective was to identify the subgroups of rectal cancer patients who were at risk for high-grade toxicity. All 196 patients who were included in the present study were treated with 50.4 Gy and chemotherapy that included either 5-FU (n = 115) or 5-FU+oxaliplatin (n = 81). The preoperative RCT was followed by a total mesorectal excision and adjuvant chemotherapy. Acute toxicity was monitored weekly and a toxicity grade ≥3 (Common Toxicity Criteria) for a skin reaction, cystitis, proctitis, or enteritis was defined as high-grade acute organ toxicity. After RCT with 5-FU+oxaliplatin, complete tumor remission was achieved in 13.6% of the patients and in 11.3% after RCT with 5-FU alone. Complete irradiation dosages of 50.4 Gy were given to 99% (5-FU) and 95% (5-FU+oxaliplatin) of the patients. Concomitant chemotherapy was fully administered in 95% of the patients treated with 5-FU compared with the 84% of patients treated with 5-FU+oxaliplatin. A significantly higher proportion of acute organ toxicity was found in the patients who were treated with 5-FU+oxaliplatin compared with those who were treated with 5-FU. Additionally, women with a low body mass index were at the highest risk for acute organ toxicity. These results suggest that there are basic clinical parameters, such as gender and body mass index, that may be potential markers for generating individual risk profiles of RCT-induced toxicity.

High-grade acute organ toxicity as positive prognostic factor in adjuvant radiation and chemotherapy for locally advanced head and neck cancer.

PURPOSE: To test for an association between high-grade acute organ toxicity during adjuvant radiation and chemotherapy and treatment outcome in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Institutional review board approval was obtained for this retrospective study. From September 1994 to October 2008, 294 HNSCC patients were treated with adjuvant radiation and chemotherapy at the authors' department. They received normofractionated (2 Gy per fraction) irradiation to include associated nodal drainage sites, for a cumulative dose of 60-64 Gy. From January 2002 to December 2009, 91 patients received additional concomitant cisplatin-based chemotherapy. Toxicity during treatment was monitored weekly according to the common toxicity criteria (CTC); any CTC toxicity grade 3 or higher, including mucositis, dysphagia, or skin reaction, was considered high-grade acute organ toxicity. The influence of possible prognostic factors on overall survival and locoregional control was studied by means of uni- and multivariate Cox regression. RESULTS: A statistically significant association was found between high-grade acute organ toxicity and both overall survival and locoregional control. Patients with CTC grade 3 or greater acute organ toxicity had a 5-year overall survival and locoregional control rate of 90% and 97%, respectively, as compared with 24% and 74%, respectively, in patients without such toxicity (P < .01). Multivariate analyses revealed that this association was independent from other factors that may influence treatment toxicity, especially concomitant chemotherapy and/or radiation therapy. CONCLUSION: The data suggest that normal tissue and tumor tissue may behave similarly with respect to treatment response, as high-grade acute organ toxicity during radiation and chemotherapy was associated with better outcomes in the patient population; therefore, the hypothesis should be further analyzed on the biomolecular and clinical level and with other tumor entities in prospective clinical trials.

Single-dose gamma-irradiation induces up-regulation of chemokine gene expression and recruitment of granulocytes into the portal area but not into other regions of rat hepatic tissue.

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Comparison of the micronucleus and chromosome aberration techniques for the documentation of cytogenetic damage in radiochemotherapy-treated patients with rectal cancer.

PURPOSE: The goal of the interdisciplinary Clinical Research Unit KFO179 (Biological Basis of Individual Tumor Response in Patients with Rectal Cancer) is to develop an individual Response and Toxicity Score for patients with locally advanced rectal cancer treated with neoadjuvant radiochemotherapy. The aim of the present study was to find a reliable and sensitive method with easy scoring criteria and high numbers of cell counts in a short period of time in order to analyze DNA damage in peripheral blood lymphocytes. Thus, the cytokinesis-block micronucleus (CBMN) assay and the chromosome aberration technique (CAT) were tested. MATERIALS AND METHODS: Peripheral blood lymphocytes obtained from 22 patients with rectal cancer before (0 Gy), during (21.6 Gy), and after (50.4 Gy) radiochemotherapy were stimulated in vitro by phytohemagglutinin (PHA); the cultures were then processed for the CBMN assay and the CAT to compare the two methods. RESULTS: A significant increase of chromosomal damage was observed in the course of radiochemotherapy parallel to increasing radiation doses, but independent of the chemotherapy applied. The equivalence of both methods was shown by Westlake's equivalence test. CONCLUSION: The results show that the CBMN assay and the CAT are equivalent. For further investigations, we prefer the CBMN assay, because it is simpler through easy scoring criteria, allows high numbers of cell counts in less time, is reliable, sensitive, and has higher statistical power. In the future, we plan to integrate cytogenetic damage during radiochemotherapy into the planned Response and Toxicity Score within our interdisciplinary Clinical Research Unit.

Analysis of chemokine and chemokine receptor expression in squamous cell carcinoma of the head and neck (SCCHN) cell lines.

The purpose of this work was to analyze chemokine and chemokine receptor expression in untreated and in irradiated squamous cell carcinoma of the head and neck (SCCHN) tumor cell lines, aiming at the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy. Five low passage and 10 established SCCHN lines, as well as two normal cell lines, were irradiated at 2 Gy or sham-irradiated, and harvested between 1 and 48 h after treatment. For chemokines with CC and CXC structural motifs and their receptors, transcript levels of target and reference genes were quantified relatively by real-time PCR. In addition, CXCL1 and CXCL12 protein expression was analyzed by ELISA. A substantial variation in chemokine and chemokine receptor expression between SCCHN was detected. Practically, all cell lines expressed CCL5 and CCL20, while CCL2 was expressed in normal cells and in some of the tumor cell lines. CXCL1, CXCL2, CXCL3, CXCL10, and CXCL11 were expressed in the vast majority of the cell lines, while the expression of CXCL9 and CXCL12 was restricted to fibroblasts and few tumor cell lines. None of the analyzed cell lines expressed the chemokines CCL3, CCL4, or CCL19. Of the receptors, transcript expression of CCR1, CCR2, CCR3, CCR5, CCR7, CCXR2, and CCXR3 was not detected, and CCR6, CXCR1, and CXCR4 expression was restricted to few tumor cells. Radiation caused up- and down-regulation with respect to chemokine expressions, while for chemokine receptor expressions down-regulations were prevailing. CXCL1 and CXCL12 protein expression corresponded well with the mRNA expression. We conclude that the substantial variation in chemokine and chemokine receptor expression between SCCHN offer opportunities for the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy.

High-grade acute organ toxicity as positive prognostic factor in primary radio(chemo)therapy for locally advanced, inoperable head and neck cancer.

PURPOSE: To test for a possible correlation between high-grade acute organ toxicity during primary radio(chemo)therapy and treatment outcome in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: From 05/1994 to 01/2009, 216 HNSCC patients were treated with radio(chemo)therapy in primary approach. They received normofractionated (2 Gy/fraction) irradiation including associated nodal drainage sites to a cumulative dose of 70 Gy. 151 patients received additional concomitant chemotherapy (111 patients 5-fluorouracil/mitomycin C, 40 patients cisplatin-based). Toxicity during treatment was monitored weekly according to the Common Toxicity Criteria (CTC), and any toxicity grade CTC >or= 3 of mucositis, dysphagia or skin reaction was assessed as high-grade acute organ toxicity for later analysis. RESULTS: A statistically significant coherency between high-grade acute organ toxicity and overall survival as well as locoregional control was found: patients with CTC >or= 3 acute organ toxicity had a 5-year overall survival rate of 44% compared to 8% in patients without (p < 0.01). Thereby, multivariate analyses revealed that the correlation was independent of other possible prognostic factors or factors that may influence treatment toxicity, especially concomitant chemotherapy and radiotherapy technique or treatment-planning procedure. CONCLUSION: These data indicate that normal tissue and tumor tissue may behave similarly with respect to treatment response, as high-grade acute organ toxicity during radio(chemo)therapy showed to be an independent prognostic marker in the own patient population. However, the authors are aware of the fact that a multivariate analysis in a retrospective study generally has statistical limitations. Therefore, their hypothesis should be further analyzed on biomolecular and clinical levels and other tumor entities in prospective trials.