Christos Perisanidis

Evaluation of immunohistochemical expression of p53, p21, p27, cyclin D1, and Ki67 in oral and oropharyngeal squamous cell carcinoma

BACKGROUND The purpose of this study was to evaluate whether the immunohistochemical expression of p53, p21, p27, cyclin D1, and Ki67 can predict therapy response and survival in patients with oral and oropharyngeal squamous cell carcinoma treated with preoperative chemoradiation. METHODS Biomarker expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded pretreatment biopsies of 111 homogenously treated patients. We assessed the association between clinicopathological variables including response to neoadjuvant chemoradiotherapy as well as the survival of the patients and the expression of the biomarkers as both dichotomized (positive vs. negative) and continuous variables. RESULTS Biomarker overexpression on the basis of pre-selected cutoff points was seen in 66 of 111 (59%) cases for p53, in 77 (69%) for p21, in 48 (43%) for p27, in 81 (73%) for cyclin D1, and in 54 (49%) cases for Ki67, respectively. None of the examined biomarkers was able to predict response to neoadjuvant chemoradiotherapy or was associated with survival outcome. Post-treatment pathologic TNM stage (P < 0.001), pathologic response (P < 0.001), and perineural invasion (P < 0.001) were the only factors having a significant effect on recurrence-free survival. Post-treatment pathologic N stage (P = 0.005), post-treatment pathologic TNM stage (P < 0.001), pathologic response (P < 0.001), and perineural invasion (P = 0.001) had a significant impact on overall survival. CONCLUSIONS Our results suggest that the biomarkers p53, p21, p27, cyclin D1, and Ki67 have no impact on treatment response and survival in patients with oral and oropharyngeal cancer treated with preoperative chemoradiation.

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Purpose: Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). Experimental Design: A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. Results: PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Conclusions: Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors. Clin Cancer Res; 22(3); 704–13. ©2015 AACR.

Complications after free flap surgery: do we need a standardized classification of surgical complications?

Our main objective was to apply a standard classification to surgical complications after free flap surgery for reconstructions of the head and neck. We used the modified Clavien-Dindo classification in a cohort of 79 patients who were having reconstructions with jejunal free flaps simultaneously with resections of oral and oropharyngeal cancer. The most common minor complication was the need for a blood transfusion, and the most common major complication of a respiratory nature. The medical complications, and those at the recipient site and the donor site were 53/79 (67%), 44/79 (56%), and 9/79 (11%), respectively. The Clavien-Dindo classification is suitable and can easily be used to evaluate postoperative complications after free tissue transfer.

Markers of epithelial to mesenchymal transition in association with survival in head and neck squamous cell carcinoma (HNSCC).

Background Elucidating the molecular phenotype of cancers with high metastatic potential will facilitate the development of novel therapeutic approaches to the disease. Gene expression profiles link epithelial to mesenchymal transition (EMT) phenotype with high-risk HNSCC. We sought to determine the role of protein biomarkers of EMT in head and neck squamous carcinoma (HNSC) prognosis. Methods Protein expression analysis of EGFR, β-catenin and E-cadherin was performed on a cohort of 102 patients with HNSCC recruited between 1992 and 2005 using automated quantitative protein analysis (AQUA). We evaluated associations with clinicopathological parameters and prognosis. Results There were 67 patients with primary squamous cell carcinoma of the head and neck in this cohort who met inclusion criteria and for whom we had complete E-cadherin, beta-catenin and EGFR expression data. High E-cadherin expressers had longer 5-year progression-free survival (PFS) compared to those with low E-cadherin (59.7% versus 40.6%, p = 0.04) and overall survival (OS) (69.6% versus 44.3%, p  = 0.05). Kaplan-Meier analysis showed that patients with low beta-catenin-expressing tumors trended toward worse 5-year PFS (p = 0.057). High EGFR expressers had inferior OS compared to low EGFR expressers (27.7% vs. 54%, p = 0.029). In the multivariable analysis context, E-cadherin remained an independent predictor of improved OS (HR = 0.204, 95% CI 0.043 to 0.972, p = 0.046) while EGFR trended towards significance for OS. Conclusions The putative markers of EMT defined within a panel of HNSCC using AQUA are associated with tumors of poor prognosis.

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma

Background Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. Patients and methods We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). Results PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). Conclusions We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

Molecular profile of head and neck squamous cell carcinomas bearing p16 high phenotype

BACKGROUND We sought to determine biomarker expression differences in head and neck squamous cell cancers (HNSCCs) based on p16/human papillomavirus (HPV) classification. In addition, our aim was to explore how expression of biomarkers is modulated after E6/E7 repression in HPV16⁺ oropharyngeal cancer cells. METHODS HPV16⁺ and HPV⁻ HNSCC cells were infected with retroviruses expressing short hairpin RNA targeting HPV16 E6/E7. Components of the epidermal growth factor receptor (EGFR) pathway before and after E6/E7 gene silencing were analyzed by immunoblotting and qRT-PCR. Protein expression of 13 biomarkers was analyzed using AQUA on a tissue microarray (TMA). The HPV16 status was determined using HPV16 in situ hybridization (ISH). RESULTS In HPV16⁺ cells, E6/E7 silencing was associated with PTEN upregulation and reduction of phosphorylated EGFR. Tumors were classified into four categories based on the HPV and p16 status. HPV⁺/p16⁺ tumors expressed significantly higher levels of E-cadherin (P = 0.003), PTEN (P = 0.004), lower levels of PI3Kp110 and β-catenin (P = 0.07). There was a significant difference in overall survival (OS, P = 0.016) among the four subsets. The median OS was 24.83 months for p16⁻/HPV⁻ patients, 11.63 for p16⁻/HPV⁺ patients and was not reached for p16⁺/HPV⁻ and p16⁺/HPV⁺ groups. CONCLUSIONS Aberrant EGFR signaling contributes to malignant conversion of HPV16⁺ HNSCC cells. These results validate β-catenin as a distinct biomarker in HPV⁺/p16⁺ HNSCC. Wnt signaling inhibitors merit exploration in HPV⁺/p16⁺ HNSCC.

The emerging role of immunotherapy in head and neck squamous cell carcinoma (HNSCC): anti-tumor immunity and clinical applications

Head and neck squamous cell carcinoma (HNSCC) carries a poor prognosis, with low survival rates for advanced stage tumors and minimal improvement in survival trends through the past decades. It is becoming increasingly clear that HNSCC oncogenesis and evolution is characterized by profound immune defects, as cancer cells evade immunosurveillance due to accumulation of genetic mutations and tumor heterogeneity. Improved understanding of the role of the immune system in cancer has led to the identification of novel therapeutic targets, which are being investigated for their potential to provide durable responses. In this review, we will summarize the role of the immune system in HNSCC, the rationale behind immunotherapy strategies and their clinical applications.

Transfusion of allogenic leukocyte-depleted packed red blood cells is associated with postoperative morbidity in patients undergoing oral and oropharyngeal cancer surgery

Evidence indicates that allogenic packed red blood cell transfusion results in the hosts immunomodulation, and is associated with adverse clinical outcomes after surgery. The aim of this study was to test whether allogenic leukocyte-depleted blood transfusion represents a significant risk factor for postoperative morbidity after oral and oropharyngeal cancer surgery. A total of 142 patients, diagnosed for the first time with oral and oropharyngeal squamous cell carcinoma, and receiving neoadjuvant chemoradiotherapy followed by surgery between 2000 and 2008 were retrospectively included in this study. Univariate and multivariate logistic regression models were calculated to identify predictors of postoperative complications. We found a significantly higher complication rate in the group of transfused patients compared to patients not exposed to transfusion (complication rate of 84% and 39%, respectively, p<0.001). On multivariate analysis, the amount of packed red blood cells transfused (for 1-4 units transfused: adjusted OR, 2.59; 95% CI, 1.24-5.39; p=0.011; for more than >4 units transfused: adjusted OR, 5.29; 95% CI, 2.01-13.88; p=0.001) and Charlsons comorbidity score ≥1 (adjusted OR, 2.81; 95% CI, 1.38-5.70; p<0.004) were independently associated with the development of postoperative complications. Allogenic leukocyte-depleted blood transfusion is independently associated with increased postoperative complications in patients undergoing surgery for oral and oropharyngeal cancer. This association follows a dose-response relationship, as patients who received larger amounts of packed red blood cells showed a significant trend toward higher postoperative morbidity.

The promise of immunotherapy in head and neck squamous cell carcinoma

Squamous cell cancers of the head and neck (HNSCC) comprise a diverse group of malignancies that includes tobacco-related tumors in addition to an increasing number of human papillomavirus-associated cancers. Independently of cause, there is a growing body of evidence supporting that the immune system plays a pivotal role in HNSCC development, as tumor cells evade immunosurveillance by exploiting inhibitory checkpoint pathways that suppress anti-tumor T-cell responses. HNSCC cells have the ability to manipulate the immune system through a variety of different mechanisms, forcing it to promote tumor growth and spread. Over the last decade, discoveries in immunologic research resulted in increased understanding of complex interactions between HNSCC and the host immune system as well as T-cell regulatory mechanisms, promoting the development of a variety of novel immunotherapies. Following the availability of novel immunotherapeutic strategies, the challenge for clinicians is to understand how and in which clinical setting to use these agents in order to provide greater clinical benefit for patients. Combination of immunotherapies with standard treatment approaches also represents an evolving field of research. Herein, we provide a comprehensive review of immune escape mechanisms in HNSCC, as well as current immunotherapy approaches under investigation.

HCRP1 expression status is a significant prognostic marker in oral and oropharyngeal cancer

OBJECTIVE The hepatocellular carcinoma-related protein 1 (HCRP1) is a key factor in the degradation of the epidermal growth factor receptor. In this study, we assessed the prognostic significance of HCRP1 expression in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). METHODS HCRP1 expression was determined by immunohistochemistry on tissue biopsy sections of 111 patients with locally advanced OOSCC undergoing neoadjuvant chemoradiotherapy followed by surgery. The Kaplan-Meier method and Cox regression models were used for survival analyses. RESULTS Low HCRP1 expression was associated with poor recurrence-free survival (P = 0.046) and overall survival (P = 0.03). Multivariate analysis revealed that low HCRP1 expression remained an independent risk factor for relapse (HR 2.98, 95% CI 1.19-7.49, P = 0.02) and death (HR 3.04, 95% CI 1.19-7.79, P = 0.02). CONCLUSION Low HCRP1 expression was found to be of adverse prognostic significance in patients with OOSCC who received preoperative chemoradiotherapy.