Molecular responses of rectal cancer to preoperative chemoradiation

doi:10.1016/j.radonc.2006.07.016

Radiotherapy and Oncology

Volume 80, Issue 2, August 2006, Pages 172-177

https://doi.org/10.1016/j.radonc.2006.07.016 Get rights and content

Abstract

Background and purpose

Some rectal cancers respond well to preoperative neoadjuvant therapy while others are inherently resistant or develop resistance during the treatment. To understand the mechanism underlying these differences, several markers that might be prognostic or predictive of downstaging in response to chemoradiotherapy in patients with rectal cancer were evaluated.

Material and methods

Thirty patients were enrolled in this study. All were treated with preoperative chemoradiation (45 Gy in 25 fractions + 5-FU). Paraffin-embedded sections obtained before and after therapy were stained by H&E, for COX-2, and Ki67. In addition, osteopontin and IL-6 concentrations were determined in blood samples obtained before, during, and after therapy.

Results

COX-2 expression increased in 67% (n = 8/12) of the patients from a median of 0% before to 74% after therapy (p = 0.009). Ki67 median positivity diminished from 90% to 45% in 83% (n = 10/12) of cases (p = 0.007). Osteopontin expression showed no significant changes during therapy, whereas IL-6 expression levels increased in 70% (n = 19/27) of all patients (p < 0.001). For osteopontin and IL-6, patients with a complete response tended to have lower pre-therapy levels. Moreover, osteopontin was much higher before (p = 0.02) and after therapy (p = 0.01) in patients who later developed metastases.

Conclusions

Chemoradiotherapy seems to affect expression of COX-2 and Ki67 which indicates that these proteins might be of importance in predicting long-term outcome. Moreover, osteopontin might be a marker of metastases.

Section snippets

Patients

Thirty patients were enrolled in a Phase II randomized, double-blind, placebo-controlled clinical trial with the COX-2 inhibitor Celecoxib. The patients were recruited from March 2003 until January 2006. This study was approved by the Ethical Committee of our hospital, the University Hospital of Leuven, Gasthuisberg, and all patients signed an informed consent.

The inclusion criteria were: histologically confirmed adenocarcinoma of the rectum, stage T_3–4 N₀M₀ or any T N_1–2M₀, age between 18 and

Clinical characteristics

The main clinical characteristics of the patients are given in Table 1. All biopsies were confirmed for malignancy by a pathologist and the response to therapy was determined following the RECIST criteria [21]. Twenty patients have been followed for more than 1 year. And, in this subgroup, four patients developed metastases and one patient died. No local recurrences occurred during this first year.

Immunohistochemical markers

COX-2 was expressed cytoplasmically in 43% (n = 9/21) of biopsies and in 93% (n = 13/14) of resection

Discussion

Although good results are obtained with the current multimodal treatment of rectal cancer, “patient-tailored” treatments are expected to give greater benefit. In this study, we analyzed the effect of preoperative chemoradiotherapy on the expression of several proteins (COX-2, Ki67, IL-6, osteopontin) and the potential value of these proteins in predicting the response of the tumours to preoperative chemoradiation.

Overexpression of COX-2 has been reported in several human malignancies including

Acknowledgement

Karin Haustermans is supported by a fundamental clinical mandate of the FWO.

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Osteopontin (OPN) is a highly phosphorylated glycophosphoprotein having acidic characteristics and rich in aspartic acid. OPN, a multifunctional protein, has important functions on cardiovascular diseases, cancer, diabetes and kidney stone diseases and in the process of inflammation, biomineralization, cell viability and wound healing. Osteopontin acts on organisms by playing a key role in secretion levels of interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-3 (IL–3), interferon-γ (IFN-γ), integrin αvB3, nuclear factor kappa B (NF-kB), macrophage and T cells, regulating the osteoclast function and affecting CD44 receptors. The aim of the present review is to address majority of different functions of OPN protein which are known, suspected or suggested through the data obtained about this protein yet.
Quantitative imaging outperforms molecular markers when predicting response to chemoradiotherapy for rectal cancer
2017, Radiotherapy and Oncology
To explore the integration of imaging and molecular data for response prediction to chemoradiotherapy (CRT) for rectal cancer.
Eighty-five rectal cancer patients underwent preoperative CRT. ¹⁸F-FDG PET/CT and diffusion-weighted imaging (DWI) were acquired before (TP1) and during CRT (TP2) and prior to surgery (TP3). Inflammatory cytokines and gene expression were analysed. Tumour response was defined as ypT0-1N0. Multivariate models were built combining the obtained parameters. Final models were calculated on the data combination with the highest AUC.
Twenty-two patients (26%) achieved ypT0-1N0 response. ¹⁸F-FDG PET/CT had worse predictive performance than DWI and T2-volumetry (AUC 0.61 ± 0.04, 0.72 ± 0.03, and 0.72 ± 0.02, respectively). Combining all imaging parameters increased the AUC to 0.81 ± 0.03. Adding cytokines or gene expression did not improve the AUC (AUC of 0.72 ± 0.06 and 0.79 ± 0.04 respectively). Final models combining ¹⁸F-FDG PET/CT, DWI, and T2-weighted volumetry at all TPs and using only TP1 and TP3, allowed ypT0-1N0 prediction with a 75% sensitivity, 94% specificity and PPV of 80%.
Combining ¹⁸F-FDG PET/CT, DWI, and T2-weighted MRI volumetry obtained before CRT and prior to surgery may help physicians in selecting rectal cancer patients for organ-preservation.
Longitudinal analysis of cytokine expression during neoadjuvant chemoradiotherapy and subsequent surgery in esophageal cancer patients
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Surgical stress and necessity of OLV are responsible for a massive release of pro-inflammatory cytokines and a depressed host immune response by T helper type 1 (Th1) and 2 (Th2) cells.7,8 Systemic effects of both chemotherapy and radiotherapy induce pro-inflammatory responses through IL-1β, IL-6, IL-8, and TNF-α within few hours after exposure.4,13–15 With present data, we were not able to confirm all these statements; we observed significant elevated concentrations of I-FABP after NCRT.
The purpose of this study was to provide more insight in the course of cytokine concentrations related to pathologic response (pR) and complications after neoadjuvant chemoradiotherapy (NCRT) and esophagectomy in esophageal cancer patients.
Patients treated with NCRT followed by transthoracic esophagectomy (n = 35) or transthoracic esophagectomy alone (n = 8) were included. Eight different cytokine concentrations were determined during NCRT, esophagectomy, and the first postoperative week.
Platelet-activating factor before NCRT was associated with pR (P = .011) and remained elevated in patients with a better response. Concentrations of intestinal fatty acid–binding protein and angiopoietin 1 (Ang-1) were different between patients with and without NCRT. Decreased concentrations of Ang-1 on the third postoperative day were associated with postoperative complications (P = .046).
In this observational study, elevated platelet-activating factor concentrations before NCRT were associated with pR. NCRT is associated with decreased Ang-1 concentrations, whereas reduced Ang-1 concentrations were associated with postoperative complications.
Predictive and prognostic biomarkers for neoadjuvant chemoradiotherapy in locally advanced rectal cancer
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The authors attributed the lack of p53 induction to loss of p53 function, which correlated with mutations in the p53 genes. Other studies demonstrated a decrease in Ki67 and p21, in conjunction with an increase in apoptosis [34–37]. A panel of molecular markers, including p53 and p21, DNA mismatch repair proteins (MLH-1 and MSH2), MIB-1, thymidylate synthase, EGFR and VEGF in 91 patients at biopsy and at time of surgery showed baseline over-expression for p53, p21, MLH-1, MLSH2, MIB-1, thymidylate synthase, EGFR and VEGF.
Locally advanced rectal cancer is regularly treated with trimodality therapy consisting of neoadjuvant chemoradiation, surgery and adjuvant chemotherapy. There is a need for biomarkers to assess treatment response, and aid in stratification of patient risk to adapt and personalise components of the therapy. Currently, pathological stage and tumour regression grade are used to assess response. Experimental markers include proteins involved in cell proliferation, apoptosis, angiogenesis, the epithelial to mesenchymal transition and microsatellite instability. As yet, no single marker is sufficiently robust to have clinical utility. Microarrays that screen a tumour for multiple promising candidate markers, gene expression and microRNA profiling will likely have higher yield and it is expected that a combination or panel of markers would prove most useful. Moving forward, utilising serial samples of circulating tumour cells or circulating nucleic acids can potentially allow us to demonstrate tumour heterogeneity, document mutational changes and subsequently measure treatment response.
Osteopontin-integrin α<inf>v</inf>β<inf>3</inf> axis is crucial for 5-fluorouracil resistance in oral squamous cell carcinoma
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Plasma OPN is an independent prognostic marker for OSCC [29–32]. In particular, OPN was recently proposed as a surrogate marker for hypoxia, the most important predictor for a response to radiation, and thus for radiotherapy outcome in head and neck cancer [30,33–38]. Our present study using microarray and qRT-PCR analyses showed that OPN gene expression was significantly higher in OSCC tissues in patients with pretreatment resistance to 5-FU-based chemoradiotherapy compared with patients who had sensitivity to this chemoradiotherapy.
Clinical applications of a chemotherapeutic agent, 5-fluorouracil (5-FU) in oral squamous cell carcinoma (OSCC) have been limited because of drug resistance. This study aimed to identify novel mechanisms of 5-FU resistance. Here we found increased osteopontin (OPN) gene expression in OSCC tissues with resistance to 5-FU-based chemoradiotherapy. OPN overexpression in OSCC cells led to 5-FU resistance and abrogated the prosurvival effect of the drug in a mouse xenograft model. OPN-induced 5-FU resistance required integrin α_vβ₃. Targeting integrin α_vβ₃ reversed the resistance in a 5-FU-resistant clone highly expressing OPN. Our data suggest that the OPN-integrin α_vβ₃ axis is crucial for 5-FU resistance in OSCC.
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Although the role of osteopontin as a prognostic factor has been studied extensively in a broad range of solid tumors, only one study looked at the predictive value of osteopontin levels and response to CRT in rectal cancer. Debucquoy et al. found an association between lower osteopontin levels and better response in 30 rectal cancer patients, which is in line with our findings, but this did not reach statistical significance [13]. In our patient group it was a significant predictor in univariate analysis, but it lost significance in multivariate analysis.
Chemoradiation (CRT) has been shown to lead to downsizing of an important portion of rectal cancers. In order to tailor treatment at an earlier stage during treatment, predictive models are being developed. Adding blood biomarkers may be attractive for prediction, as they can be collected very easily and determined with excellent reproducibility in clinical practice. The hypothesis of this study was that blood biomarkers related to tumor load, hypoxia and inflammation can help to predict response to CRT in rectal cancer.
295 patients with locally advanced rectal cancer who were planned to undergo CRT were prospectively entered into a biobank protocol (NCT01067872). Blood samples were drawn before start of CRT. Nine biomarkers were selected, based on a previously defined hypothesis, and measured in a standardized way by a certified lab: CEA, CA19-9, LDH, CRP, IL-6, IL-8, CA IX, osteopontin and 25-OH-vitamin D. Outcome was analyzed in two ways: pCR vs. non-pCR and responders (defined as ypT0-2N0) vs. non-responders (all other ypTN stages).
276 patients could be analyzed. 20.7% developed a pCR and 47.1% were classified as responders. In univariate analysis CEA (p = 0.001) and osteopontin (p = 0.012) were significant predictors for pCR. Taking response as outcome CEA (p < 0.001), IL-8 (p < 0.001) and osteopontin (p = 0.004) were significant predictors. In multivariate analysis CEA was the strongest predictor for pCR (OR 0.92, p = 0.019) and CEA and IL-8 predicted for response (OR 0.97, p = 0.029 and OR 0.94, p = 0.036). The model based on biomarkers only had an AUC of 0.65 for pCR and 0.68 for response; the strongest model included clinical data, PET-data and biomarkers and had an AUC of 0.81 for pCR and 0.78 for response.
CEA and IL-8 were identified as predictive biomarkers for tumor response and PCR after CRT in rectal cancer. Incorporation of these blood biomarkers leads to an additional accuracy of earlier developed prediction models using clinical variables and PET-information. The new model could help to an early adaptation of treatment in rectal cancer patients.

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View full text

Molecular radiobiologyMolecular responses of rectal cancer to preoperative chemoradiation

Abstract

Background and purpose

Material and methods

Results

Conclusions

Section snippets

Patients

Clinical characteristics

Immunohistochemical markers

Discussion

Acknowledgement

Exp Cell Res

Gastroenterology

Trends Cell Biol

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Biochem Biophys Res Commun

Am J Gastroenterol

Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling

J Natl Cancer Inst

GSH level and IL-6 production increased in Sertoli cells and astrocytes after gamma irradiation

Anticancer Res

Ki67 protein: the immaculate deception?

Histopathology

Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies

Clin Cancer Res

Differential expression of cyclooxygenase 2 in human colorectal cancer

Gut

Molecular radiobiology
Molecular responses of rectal cancer to preoperative chemoradiation