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".

The impact of rectal/bladder filling and applicator positioning on in vivo rectal dosimetry in vaginal cuff brachytherapy using an enhanced therapy setting.

PURPOSE: The impact of rectal filling and bladder volume on in vivo rectal dosimetry (IVD) in vaginal cuff brachytherapy (VCBT) is unknown. The purpose of this study was to compare rectal doses from IVD with those calculated from treatment planning and to identify influencing factors. MATERIALS AND METHODS: We collected data of 80 VCBT sessions, four for each of 20 patients. Each was retrospectively compared with doses determined by the treatment planning system. Factors potentially predicting the IVD rectum dose were analyzed. RESULTS: For a series of 80 brachytherapy applications, the calculated mean dose to the rectum was 2.52 Gy. The mean difference between all calculated and measured doses for the 80 applications with five probe positions each was 0.09 Gy (p = 0.952) proving high overall accordance between IVD and calculated doses at the rectum. The mean volume of the rectum was 119 ± 57 cm³. The rectal volume was not statistically significantly associated with the IVD or the calculated rectum doses. At the third and fourth rectal probe position in craniocaudal ordering, increased filling of the urinary bladder resulted in decreased measured and calculated doses (p < 0.05 for both). A rectum pointing position of the applicator significantly increased the maximum rectum dose compared with a bladder-oriented position (p < 0.05). CONCLUSIONS: IVD provided valuable data for rectal exposure in VCBT. Increased bladder filling and vaginal applicator positioning off the rectum elicited related with less rectal radiation exposure, whereas rectal filling did not. Further confirmation including assessment of IVD in bladder is pending to define optimal dosimetric conditions in VCBT.

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.

Rituximab With Involved Field Irradiation for Early-stage Nodal Follicular Lymphoma: Results of the MIR Study.

The MabThera and Involved field Radiotherapy study investigated efficacy and safety of involved field (IF) radiotherapy in combination with the anti-CD20 antibody Rituximab for early-stage follicular lymphoma (FL) in a prospective, single-arm multicenter phase 2 design. Eighty-five stage I-II FL patients received 8 cycles of Rituximab (375 mg/m2) and IF irradiation (30/40 Gy). The primary endpoint was progression-free survival (PFS) 2 years from treatment start. Secondary endpoints were overall survival (OS), complete response rates, toxicity, quality of life, and minimal residual disease (MRD) response with protocol defined visits up to month 30. For the primary endpoint, PFS at 2 years was 85% for the intention-to-treat set. Long-term data were captured in selected sites and evaluated as post hoc analysis in the per protocol (PP) set: PFS and OS were 78% and 96% at 5 years with a median follow-up of 66 or 78 months, respectively. There were 17/76 recurrences in the PP set, of which 14 were outside the radiation volume only. MRD analyses revealed a clonal marker in 36% of patients at diagnosis. All but 1 marker positive patients experienced a molecular treatment response. There were 13 serious adverse events (4 related to the therapy) during the first 30 months. IF radiotherapy combined with Rituximab is well tolerated and highly efficient with low rates of recurrence in the first years in early-stage FL. The efficacy is comparable with more aggressive therapy approaches without compromising the quality of life and maintains for an extended follow-up of more than 5 years.

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.

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.

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.

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.

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.

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.

Gold marker displacement due to needle insertion during HDR-brachytherapy for treatment of prostate cancer: a prospective cone beam computed tomography and kilovoltage on-board imaging (kV-OBI) study.

PURPOSE: To evaluate gold marker displacement due to needle insertion during HDR-brachytherapy for therapy of prostate cancer. PATIENTS AND METHODS: 18 patients entered into this prospective evaluation. Three gold markers were implanted into the prostate during the first HDR-brachytherapy procedure after the irradiation was administered. Three days after marker implantation all patients had a CT-scan for planning purpose of the percutaneous irradiation. Marker localization was defined on the digitally-reconstructed-radiographs (DRR) for daily (VMAT technique) or weekly (IMRT) set-up error correction. Percutaneous therapy started one week after first HDR-brachytherapy. After the second HDR-brachytherapy, two weeks after first HDR-brachtherapy, a cone-beam CT-scan was done to evaluate marker displacement due to needle insertion. In case of marker displacement, the actual positions of the gold markers were adjusted on the DRR. RESULTS: The value of the gold marker displacement due to the second HDR-brachytherapy was analyzed in all patients and for each gold marker by comparison of the marker positions in the prostate after soft tissue registration of the prostate of the CT-scans prior the first and second HDR-brachytherapy. The maximum deviation was 5 mm, 7 mm and 12 mm for the anterior-posterior, lateral and superior-inferior direction. At least one marker in each patient showed a significant displacement and therefore new marker positions were adjusted on the DRRs for the ongoing percutaneous therapy. CONCLUSIONS: Needle insertion in the prostate due to HDR-brachytherapy can lead to gold marker displacements. Therefore, it is necessary to verify the actual position of markers after the second HDR-brachytherapy. In case of significant deviations, a new DRR with the adjusted marker positions should be generated for precise positioning during the ongoing percutaneous irradiation.

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.

Guidelines for delineation of lymphatic clinical target volumes for high conformal radiotherapy: head and neck region.

The success of radiotherapy depends on the accurate delineation of the clinical target volume. The delineation of the lymph node regions has most impact, especially for tumors in the head and neck region. The purpose of this article was the development an atlas for the delineation of the clinical target volume for patients, who should receive radiotherapy for a tumor of the head and neck region. Literature was reviewed for localisations of the adjacent lymph node regions and their lymph drain in dependence of the tumor entity. On this basis the lymph node regions were contoured on transversal CT slices. The probability for involvement was reviewed and a recommendation for the delineation of the CTV was generated.

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.

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.