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Nuclear expression of p65 (RelA) in patients receiving post-operative radiotherapy for locally advanced squamous cell carcinoma of the head and neck.

Rades D, Huttenlocher S, Seibold ND, Gebhard MP, Thorns C, Hasselbacher K, Wollenberg B, Schild SE - BMC Cancer (2015)

Bottom Line: Univariate analyses were performed with Kaplan-Meier method and log-rank test, multivariate analyses with Cox proportional hazards model.On univariate analyses, p65-expression had a significant impact on OS (p < 0.001) and LRC (p < 0.001) but not on MFS (p = 0.29).P65-negativity was significantly associated with improved LRC and achieved borderline significance with respect to improved OS.

View Article: PubMed Central - PubMed

ABSTRACT

Background: This study investigated the prognostic role of nuclear expression of p65 in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) receiving post-operative radio(chemo)therapy.

Methods: Nuclear p65-expression (H-score ≤50 versus >50) plus twelve characteristics were analyzed in 151 patients for overall survival (OS), metastases-free survival (MFS) and loco-regional control (LRC). Additional characteristics included age, gender, Karnofsky performance score (KPS), pre-radiotherapy hemoglobin levels, tumor site, histological grading, human papilloma virus (HPV)-status, T-classification, N-classification, American Joint Committee on Cancer (AJCC)-stage, extent of resection and concurrent chemotherapy. Univariate analyses were performed with Kaplan-Meier method and log-rank test, multivariate analyses with Cox proportional hazards model.

Results: On univariate analyses, p65-expression had a significant impact on OS (p < 0.001) and LRC (p < 0.001) but not on MFS (p = 0.29). On multivariate analysis, KPS ≥80 (risk ratio [RR] 2.23; p = 0.012), HPV-positivity (RR 5.83; p = 0.020), T1-T2 (RR 1.38; p = 0.048), N0-N2a (RR 2.72; p = 0.005) and complete resection (RR 2.02; p = 0.049) were positively associated with OS; p65-negativity achieved borderline significance (RR 3.02; p = 0.052). Better MFS was associated with KPS ≥80 (RR 2.49; p = 0.015), T1-T2 (RR: 1.74; p = 0.005), N0-N2a (RR: 6.22; p < 0.001) and complete resection (RR 3.43; p = 0.003). Positive associations with LRC were found for p65-negativity (RR 5.06; p = 0.008), T1-T2 (RR: 1.49; p = 0.022), N0-N2a (RR: 2.97; p = 0.004) and favorable tumor site (RR 1.28; p = 0.025).

Conclusions: P65-negativity was significantly associated with improved LRC and achieved borderline significance with respect to improved OS. Thus, p65-expression may be an additional target for novel agents in the treatment of locally advanced SCCHN.

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Non-keratinizing carcinoma with immunostaining for p65: Brown staining of 30% nuclei in moderate intensity, original magnification 100x at microscope, bar = 100 μm.
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Fig1: Non-keratinizing carcinoma with immunostaining for p65: Brown staining of 30% nuclei in moderate intensity, original magnification 100x at microscope, bar = 100 μm.

Mentions: Nuclear expression of p65 (RelA) was analyzed in resected tumor specimens of 151 patients who had received obtained post-operative radiotherapy or radio-chemotherapy for locally advanced SCCHN. The study was approved by the local ethics committee (University of Lübeck). The patients have given written informed consent for the use of their data within retrospective studies in general. Children were not included in this study. Formalin-fixed and paraffin-embedded cancer tissues were sampled in a tissue micro array with cores of 2.0 mm (Manual Tissue Arrayer 1, Beecher Instruments, Silver Spring, MD, USA). Histological sections of 4 μm were de-paraffinized in xylene and rehydrated in graded alcohols. Antigene retrieval was processed according to standard operating procedure for our department of surgical pathology. Using 0.01 M citric acid pH6.0 for 5 min in a microwave at 850 W showed best results. Incubation with the primary antibody was done for 120 minutes in a dilution of 1:70. In a previous work by Balermpas et al., the polyclonal antibody ab31481 by Abcam plc, CB4 0FL Cambridge, UK) was used, but was not available anymore, when our study was performed [8]. After monoclonal antibody NF-kB p65 (clone L8F6) of Cell Signaling Technology Inc. (#6956) did not show any positive reaction, finally polyclonal rabbit Anti-NF-kB p65 antibody ab16502 (Abcam plc, CB4 0FL Cambridge, UK) was used. Visualization was performed with biotin-free poly-horseradish peroxidase-anti-mouse/rabbit/rat-IgG-kit (ImmunoLogic BV, 6921 VB Duiven, Netherlands). A diffuse large B-cell lymphoma served as positive control, and staining without primary antibody served as negative control. For differentiation between p65-negative and p65-positive tumors, the H-score was used [10]. The H-score was obtained as follows: % cells with weak staining × 1 plus % cells with moderate staining × 2 plus % cells with strong staining × 3. Tumors with an H-score of ≤50 were considered p65-negative and those tumors with an H-score of >50 were considered p65-positive. Microscopic evaluation was carried out by an experienced surgical pathologist, who was blinded to clinical and laboratory data. An example of a p65-positive tumor is given in Figure 1.Figure 1


Nuclear expression of p65 (RelA) in patients receiving post-operative radiotherapy for locally advanced squamous cell carcinoma of the head and neck.

Rades D, Huttenlocher S, Seibold ND, Gebhard MP, Thorns C, Hasselbacher K, Wollenberg B, Schild SE - BMC Cancer (2015)

Non-keratinizing carcinoma with immunostaining for p65: Brown staining of 30% nuclei in moderate intensity, original magnification 100x at microscope, bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352566&req=5

Fig1: Non-keratinizing carcinoma with immunostaining for p65: Brown staining of 30% nuclei in moderate intensity, original magnification 100x at microscope, bar = 100 μm.
Mentions: Nuclear expression of p65 (RelA) was analyzed in resected tumor specimens of 151 patients who had received obtained post-operative radiotherapy or radio-chemotherapy for locally advanced SCCHN. The study was approved by the local ethics committee (University of Lübeck). The patients have given written informed consent for the use of their data within retrospective studies in general. Children were not included in this study. Formalin-fixed and paraffin-embedded cancer tissues were sampled in a tissue micro array with cores of 2.0 mm (Manual Tissue Arrayer 1, Beecher Instruments, Silver Spring, MD, USA). Histological sections of 4 μm were de-paraffinized in xylene and rehydrated in graded alcohols. Antigene retrieval was processed according to standard operating procedure for our department of surgical pathology. Using 0.01 M citric acid pH6.0 for 5 min in a microwave at 850 W showed best results. Incubation with the primary antibody was done for 120 minutes in a dilution of 1:70. In a previous work by Balermpas et al., the polyclonal antibody ab31481 by Abcam plc, CB4 0FL Cambridge, UK) was used, but was not available anymore, when our study was performed [8]. After monoclonal antibody NF-kB p65 (clone L8F6) of Cell Signaling Technology Inc. (#6956) did not show any positive reaction, finally polyclonal rabbit Anti-NF-kB p65 antibody ab16502 (Abcam plc, CB4 0FL Cambridge, UK) was used. Visualization was performed with biotin-free poly-horseradish peroxidase-anti-mouse/rabbit/rat-IgG-kit (ImmunoLogic BV, 6921 VB Duiven, Netherlands). A diffuse large B-cell lymphoma served as positive control, and staining without primary antibody served as negative control. For differentiation between p65-negative and p65-positive tumors, the H-score was used [10]. The H-score was obtained as follows: % cells with weak staining × 1 plus % cells with moderate staining × 2 plus % cells with strong staining × 3. Tumors with an H-score of ≤50 were considered p65-negative and those tumors with an H-score of >50 were considered p65-positive. Microscopic evaluation was carried out by an experienced surgical pathologist, who was blinded to clinical and laboratory data. An example of a p65-positive tumor is given in Figure 1.Figure 1

Bottom Line: Univariate analyses were performed with Kaplan-Meier method and log-rank test, multivariate analyses with Cox proportional hazards model.On univariate analyses, p65-expression had a significant impact on OS (p < 0.001) and LRC (p < 0.001) but not on MFS (p = 0.29).P65-negativity was significantly associated with improved LRC and achieved borderline significance with respect to improved OS.

View Article: PubMed Central - PubMed

ABSTRACT

Background: This study investigated the prognostic role of nuclear expression of p65 in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN) receiving post-operative radio(chemo)therapy.

Methods: Nuclear p65-expression (H-score ≤50 versus >50) plus twelve characteristics were analyzed in 151 patients for overall survival (OS), metastases-free survival (MFS) and loco-regional control (LRC). Additional characteristics included age, gender, Karnofsky performance score (KPS), pre-radiotherapy hemoglobin levels, tumor site, histological grading, human papilloma virus (HPV)-status, T-classification, N-classification, American Joint Committee on Cancer (AJCC)-stage, extent of resection and concurrent chemotherapy. Univariate analyses were performed with Kaplan-Meier method and log-rank test, multivariate analyses with Cox proportional hazards model.

Results: On univariate analyses, p65-expression had a significant impact on OS (p < 0.001) and LRC (p < 0.001) but not on MFS (p = 0.29). On multivariate analysis, KPS ≥80 (risk ratio [RR] 2.23; p = 0.012), HPV-positivity (RR 5.83; p = 0.020), T1-T2 (RR 1.38; p = 0.048), N0-N2a (RR 2.72; p = 0.005) and complete resection (RR 2.02; p = 0.049) were positively associated with OS; p65-negativity achieved borderline significance (RR 3.02; p = 0.052). Better MFS was associated with KPS ≥80 (RR 2.49; p = 0.015), T1-T2 (RR: 1.74; p = 0.005), N0-N2a (RR: 6.22; p < 0.001) and complete resection (RR 3.43; p = 0.003). Positive associations with LRC were found for p65-negativity (RR 5.06; p = 0.008), T1-T2 (RR: 1.49; p = 0.022), N0-N2a (RR: 2.97; p = 0.004) and favorable tumor site (RR 1.28; p = 0.025).

Conclusions: P65-negativity was significantly associated with improved LRC and achieved borderline significance with respect to improved OS. Thus, p65-expression may be an additional target for novel agents in the treatment of locally advanced SCCHN.

Show MeSH
Related in: MedlinePlus