Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy.

BACKGROUND:  Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. METHODS:  Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. RESULTS:  Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. CONCLUSION:  Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

The response to neoadjuvant chemoradiotherapy with 5-fluorouracil in locally advanced rectal cancer patients: a predictive proteomic signature.

BACKGROUND: Colorectal cancer is the third most common and the fourth most lethal cancer in the world. In the majority of cases, patients are diagnosed at an advanced stage or even metastatic, thus explaining the high mortality. The standard treatment for patients with locally advanced non-metastatic rectal cancer is neoadjuvant radio-chemotherapy (NRCT) with 5-fluorouracil (5-FU) followed by surgery, but the resistance rate to this treatment remains high with approximately 30% of non-responders. The lack of evidence available in clinical practice to predict NRCT resistance to 5-FU and to guide clinical practice therefore encourages the search for biomarkers of this resistance. METHODS: From twenty-three formalin-fixed paraffin-embedded (FFPE) biopsies performed before NRCT with 5-FU of locally advanced non-metastatic rectal cancer patients, we extracted and analysed the tumor proteome of these patients. From clinical data, we were able to classify the twenty-three patients in our cohort into three treatment response groups: non-responders (NR), partial responders (PR) and total responders (TR), and to compare the proteomes of these different groups. RESULTS: We have highlighted 384 differentially abundant proteins between NR and PR, 248 between NR and TR and 417 between PR and TR. Among these proteins, we have identified many differentially abundant proteins identified as having a role in cancer (IFIT1, FASTKD2, PIP4K2B, ARID1B, SLC25A33: overexpressed in TR; CALD1, CPA3, B3GALT5, CD177, RIPK1: overexpressed in NR). We have also identified that DPYD, the main degradation enzyme of 5-FU, was overexpressed in NR, as well as several ribosomal and mitochondrial proteins also overexpressed in NR. Data are available via ProteomeXchange with identifier PXD008440. CONCLUSIONS: From these retrospective study, we implemented a protein extraction protocol from FFPE biopsy to highlight protein differences between different response groups to RCTN with 5-FU in patients with locally advanced non-metastatic rectal cancer. These results will pave the way for a larger cohort for better sensitivity and specificity of the signature to guide decisions in the choice of treatment.

Radiomics strategies for risk assessment of tumour failure in head-and-neck cancer.

Quantitative extraction of high-dimensional mineable data from medical images is a process known as radiomics. Radiomics is foreseen as an essential prognostic tool for cancer risk assessment and the quantification of intratumoural heterogeneity. In this work, 1615 radiomic features (quantifying tumour image intensity, shape, texture) extracted from pre-treatment FDG-PET and CT images of 300 patients from four different cohorts were analyzed for the risk assessment of locoregional recurrences (LR) and distant metastases (DM) in head-and-neck cancer. Prediction models combining radiomic and clinical variables were constructed via random forests and imbalance-adjustment strategies using two of the four cohorts. Independent validation of the prediction and prognostic performance of the models was carried out on the other two cohorts (LR: AUC = 0.69 and CI = 0.67; DM: AUC = 0.86 and CI = 0.88). Furthermore, the results obtained via Kaplan-Meier analysis demonstrated the potential of radiomics for assessing the risk of specific tumour outcomes using multiple stratification groups. This could have important clinical impact, notably by allowing for a better personalization of chemo-radiation treatments for head-and-neck cancer patients from different risk groups.

Predictors of severe acute and late toxicities in patients with localized head-and-neck cancer treated with radiation therapy.

PURPOSE: Radiation therapy (RT) causes acute and late toxicities that affect various organs and functions. In a large cohort of patients treated with RT for localized head and neck cancer (HNC), we prospectively assessed the occurrence of RT-induced acute and late toxicities and identified characteristics that predicted these toxicities. METHODS AND MATERIALS: We conducted a randomized trial among 540 patients treated with RT for localized HNC to assess whether vitamin E supplementation could improve disease outcomes. Adverse effects of RT were assessed using the Radiation Therapy Oncology Group Acute Radiation Morbidity Criteria during RT and one month after RT, and the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer Late Radiation Morbidity Scoring Scheme at six and 12 months after RT. The most severe adverse effect among the organs/tissues was selected as an overall measure of either acute or late toxicity. Grade 3 and 4 toxicities were considered as severe. Stepwise multivariate logistic regression models were used to identify all independent predictors (p < 0.05) of acute or late toxicity and to estimate odds ratios (OR) for severe toxicity with their 95% confidence intervals (CI). RESULTS: Grade 3 or 4 toxicity was observed in 23% and 4% of patients, respectively, for acute and late toxicity. Four independent predictors of severe acute toxicity were identified: sex (female vs. male: OR = 1.72, 95% confidence interval [CI]: 1.06-2.80), Karnofsky Performance Status (OR = 0.67 for a 10-point increment, 95% CI: 0.52-0.88), body mass index (above 25 vs. below: OR = 1.88, 95% CI: 1.22-2.90), TNM stage (Stage II vs. I: OR = 1.91, 95% CI: 1.25-2.92). Two independent predictors were found for severe late toxicity: female sex (OR = 3.96, 95% CI: 1.41-11.08) and weight loss during RT (OR = 1.26 for a 1 kg increment, 95% CI: 1.12-1.41). CONCLUSIONS: Knowledge of these predictors easily collected in a clinical setting could help tailoring therapies to reduce toxicities among patients treated with RT for HNC.

Influence of smoking and alcohol drinking behaviors on treatment outcomes of patients with squamous cell carcinomas of the head and neck.

PURPOSE: To retrospectively evaluate the prognostic value of smoking and drinking status in patients with head-and-neck squamous cell carcinomas. METHODS AND MATERIALS: All patients with all stages and sites were included if complete information was available on baseline smoking and alcohol behavior (never, former, active), disease stage, primary site, radiation dose, sex, and age. Treatment was radiotherapy in 973 patients, postoperative radiotherapy in 469, and chemoradiotherapy in 429. Statistical analysis was performed with Kaplan-Meier and Cox methods. RESULTS: Data from 1,871 patients were available. At baseline, 9% of patients never smoked, 40% were former smokers, and 51% were active smokers; 20% never drank, 25% were former drinkers, and 55% were active drinkers. Smoking was associated with inferior local control and survival. For local control, the hazard ratio (HR) of active smokers vs. former smokers was 1.5 (p = 0.0001). For survival, the HRs of former smokers and active smokers vs. those who never smoked were also statistically significant (1.3 and 1.7, respectively, p = 0.000001). Alcohol drinking was associated with local control (p = 0.03), and was associated with survival. For survival, HRs of former and active drinkers compared with those who never drank were, respectively, 1.1 (p = 0.01) and 1.28 (p = 0.001). Adjusted 5-year local control and survival rates for those who never smoked and never drank were 87% and 77%, respectively, and for those who were both active smokers and active drinkers were 72% (p = 0.007) and 52% (p = 0.0009), respectively. CONCLUSION: Smoking and drinking at baseline were associated with poor outcomes in these patients.

A comparison of treatment outcomes by radiochemotherapy and postoperative radiotherapy in locally advanced squamous cell carcinomas of head and neck.

PURPOSE: To investigate the treatment outcomes by concomitant radiochemotherapy (CHEMORAD), and by surgery and postoperative radiotherapy (PORT) in patients with locally advanced head and neck cancers. METHOD: A retrospective study included all patients with Stage III and IV (except T1-2 N1) diseases between 1989 and 2002 treated with CHEMORAD (163 patients) or PORT (424 patients) at the L'Hôtel-Dieu de Québec. Chemotherapy was realized with Cisplatin (DDP) 100 mg/m(2), d1, d23, and d45 during radiotherapy or weekly DDP 40 mg/m(2) for 6 weeks in case of patients with poor general condition. Neck dissection was performed if residual disease was diagnosed on CHEMORAD. For the PORT group, 410 patients had neck dissection before adjuvant treatment. RESULTS: The 2-year loco-regional control was 82% for CHEMORAD, and 72% for PORT (P = 0.01). The 2-year disease-free survival was 72% for CHEMORAD, and 64% for PORT (P = 0.02). The results were further confirmed by a Cox proportional hazard model. CONCLUSION: The results indicated that CHEMORAD is a better approach than PORT in the management of locally advanced head and neck cancers. Further randomized study will be needed to compare radical CHEMORAD with surgery plus adjuvant CHEMORAD to determine an appropriate treatment in the management of locally advanced head and neck squamous cell carcinomas.

Effect of pretreatment anemia on treatment outcome of concurrent radiochemotherapy in patients with head and neck cancer.

PURPOSE: To investigate the effect of anemia on outcome of treatment with radiochemotherapy in patients with head-and-neck cancer. METHODS AND MATERIALS: The data of 196 patients with Stage II-IV head-and-neck cancer treated with concomitant cisplatin-based radiochemotherapy were retrospectively reviewed. Anemia was defined according to World Health Organization criteria as hemoglobin <130 g/L in men and <120 g/L in women. RESULTS: Fifty-three patients were classified as anemic, 143 as nonanemic. The 3-year local control rate of anemic and nonanemic patients was 72% and 85%, respectively (p = 0.01). The 3-year overall survival rate of anemic and nonanemic patients was 52% and 77%, respectively (p = 0.004). In multivariate analysis, anemia was the most significant predictor of local control (hazard ratio, 0.37, p = 0.009) and survival (hazard ratio, 0.47, p = 0.007). A dose-effect relationship was also found for local control (p = .04) and survival (0.04) when grouping by hemoglobin concentration: <120, 120-140, and >140 g/L. CONCLUSIONS: Anemia was strongly associated with local control and survival in this cohort of patients with head-and-neck cancer receiving radiochemotherapy.

[Proteases by reactive stromal cells in cancer: an attractive therapeutic target]. / Protéases des cellules stromales réactionnelles du cancer: une cible thérapeutique attrayante.

Cancer-associated or reactive stromal cells are composed of endothelial and inflammatory cells as well as of spindle cells such as fibroblasts and myofibroblasts. In addition to participating to the tumor tissue frame, these cells contribute actively to tumor nutrition and progression through neo-angiogenesis and production of a variety of molecules including numerous proteases, of which a number (MMP14, MMP11, FAP and uPA) are almost exclusively produced by reactive stromal cells. Cancer cells interact with reactive stromal cells which involves a large number of proteases. Several molecules (TGFbeta, PDGF, EMMPRIN) produced by cancer cells induce the production of stromal proteases which in turn stimulate cancer cells through binding to a receptor (for example, MMP-2 and integrin alpha v beta 3). Our experience shows that protease overexpression by reactive stromal cells (cathepsin D, MMP-11, MMP-14) leads to an adverse clinical course in breast cancer. Phenotypic and genotypic differences were found between reactive stromal cells and fibroblasts of normal tissue and our research team found that reactive stromal cells also respond differently to similar stimulations in different individuals. These results support the hypothesis that the biologic behaviour of cancer is not only dependent on tumour characteristics but also on those of patients'stromal cells and that comparable tumours in two individuals may follow different clinical courses. These studies and our experience underscores the importance of characterising cancer-associated reactive stromal cells because of the therapeutic potential of this approach. Furthermore, reactive stromal cells should be genetically more stable that cancer cells and, in theory, should less likely develop mutations and treatment resistance.

The influence of MMP-14, TIMP-2 and MMP-2 expression on breast cancer prognosis.

INTRODUCTION: Matrix metalloproteinase (MMP)-2 is very active at degrading extracellular matrix. It is under the influence of an activator, membrane type 1 MMP (MMP-14), and the tissue inhibitor of metalloproteases (TIMP)-2. We hypothesized that the individual expression of these three markers or their balance may help to predict breast cancer prognosis. METHODS: MMP-2, MMP-14 and TIMP-2 expression has been evaluated by 35S mRNA in situ hybridization on paraffin material of 539 breast cancers without distant metastasis at diagnosis and with a median follow-up of 9.2 years. RESULTS: MMP-2 and MMP-14 mRNA was detected primarily in reactive stromal cells whereas TIMP-2 mRNA was expressed by both stromal and cancer cells. Of the three molecules, an adjusted Cox model revealed that high MMP-14 mRNA (> or = 10% cells) alone predicted a significantly shorter overall survival (p = 0.031) when adjusted for clinical factors (tumor size and number of involved lymph nodes). Prognostic significance was lost when further adjusted for Her-2/neu and urokinase-type plasminogen activator (p = 0.284). Furthermore, when all three components were analyzed together, the survival was worst for patients with high MMP-2/high MMP-14/low TIMP-2 (5 year survival = 60%) and best with low MMP-2/low MMP-14/high TIMP-2 (5 year survival = 74%), but the difference did not reach statistical significance (p = 0.3285). CONCLUSION: Of the MMP-14/TIMP-2/MMP-2 complex, MMP-14 was the factor most significantly associated with the outcome of breast cancer and was an independent factor of poor overall survival when adjusted for clinical prognostic factors, but not for certain ancillary markers.

The efficacy and sequencing of a short course of androgen suppression on freedom from biochemical failure when administered with radiation therapy for T2-T3 prostate cancer.

PURPOSE: We evaluated the benefits and sequencing of androgen suppression (AS) administered with external beam radiation therapy (EBRT) in T2-T3 prostate cancers. MATERIALS AND METHODS: Between 1990 and 1999, 481 patients were entered in 2 successive, prospective, randomized studies, including 161 in the study 1 and 325 in study 2. Eligible patients had clinical stages T2-T3 prostate cancer. In the first study (L-101) subjects were randomly allocated among EBRT alone (group 1), EBRT preceded by 3 months of AS (group 2), and neoadjuvant, concomitant and adjuvant AS for a total of 10 months (group 3). In the second study (L-200) we analyzed neoadjuvant and concomitant AS (total 5 months) vs neoadjuvant, concomitant and short course adjuvant (total 10 months) AS with EBRT. In each study we used a total AS (a luteinizing hormone-releasing hormone agonist plus an antiandrogen) and a standard dose of radiation therapy at that time. Patient characteristics were well balanced in regard to age, stage, prostate specific antigen and Gleason score. No biochemical evidence of disease (BNED) was defined as an end point according to the Vancouver rule. RESULTS: In the study 1 at a median followup of 5 years 7-year biochemical-free survival rates were 42%, 66% and 69% in groups 1 to 3, respectively. BNED was significantly different between groups 1 and 2 (p = 0.009) and between groups 1 and 3 (p = 0.003) but not between groups 2 and 3 (p = 0.6). Multivariate analysis using a Cox proportional hazards model showed an HR of 6.1 for Gleason score (p = 0.001), 1.4 for PSA (p = 0.002), 0.5 for group 1 vs group 2 (p = 0.01) and 0.35 for group 1 vs group 3 (p = 0.008). In study 2 BNED at 4 years was 65%. There was no significant difference between arms 1 and 2 (p = 0.55). CONCLUSIONS: The analysis of study 1 shows a benefit of using a short course of neoadjuvant AS with EBRT vs EBRT alone for localized T2-T3 prostate cancers. Moreover, in each study adding a short course of adjuvant AS after neoadjuvant 1 provided no more advantage in these patients.

Stromelysin-3 expression by mammary tumor-associated fibroblasts under in vitro breast cancer cell induction.

To understand better the influence of the host stromal phenotype on stromal expression of stromelysin-3 (ST3) in breast cancer, we have investigated ST3 expression by host stromal cells isolated from 9 different primary breast carcinomas. These tumor-associated fibroblasts were cocultivated with 3 epithelial cancer cell lines of mammary origin (MDA-MB-231, SK-BR-3 and MCF-7), as well as with normal human mammary epithelial cells (NME and 184A1) and keratinocytes, using both direct and indirect coculture systems. ST3 expression was demonstrated by both in situ hybridization and immunocytochemistry. The results showed that ST3 expression by stromal cells was cancer-specific. Indeed, ST3 expression by tumor-associated stromal cells was induced by 3 malignant cancer cell lines (MDA-MB-231, SK-BR-3 and MCF-7), whereas no ST3 was expressed under normal mammary epithelial cell stimulation. ST3 expression was weak or absent in unstimulated tumor-associated fibroblasts. However, after direct coculture with cancer cells, expression of ST3 transcripts reappeared in 8 of the 9 cases and was observed only in fibroblasts located in close contact with tumor cells. Under similar coculture conditions and using the same cancer cell line stimulation, ST3 expression was, however, quite variable among these 9 cases, reflecting the difference of protease expression observed on the sections of the original tumors. Tumor induction of ST3 expression was much more important by direct cell-cell contact than by indirect stimulation and was not influenced by the addition of basic fibroblast growth factor (bFGF) and anti-bFGF to the culture medium. Our results suggest that the host stromal cell phenotype may significantly influence host stromal cell protease expression under cancer cell stimulation.