1

Introduction

Postoperative radiotherapy (PORT) improves locoregional control and survival rates for selected patients with advanced-stage laryngeal squamous cell carcinoma (LSCC) treated with primary surgery . In LSCC, the indications for PORT to reduce the risk of local recurrence currently include: close/positive surgical margins (irradical resection); perineural, lymphatic, or vascular spread; extralaryngeal carcinoma extension; carcinoma growth through the laryngeal cartilage; and subglottic extension . PORT is also given to reduce the regional recurrence risk in cases of extracapsular spread, multiple metastatic nodes, or metastatic nodes ≥ 3 cm . The available literature has reported very different (and sometimes disappointing) locoregional recurrence rates in LSCC patients treated with PORT, however . The main limitations of the research on outcomes after PORT include the paucity of prospective trials and the variety of modalities of RT and patients’ characteristics . We need novel, objective parameters to enable head and neck surgeons and oncologists, and radiotherapists to detect patients at higher risk of locoregional recurrence after PORT, who should be given combined postoperative chemo-radiotherapy to improve locoregional disease control and survival – as demonstrated for head and neck carcinoma by the EORTC (#22931) and RTOG (# 9501) clinical trials .

Predictive markers are needed for LSCC (particularly for advanced cases) to orient the choice of the most appropriate therapy for individual patients, tumor molecular biomarkers have been attracting increasing interest. Cancer cells display a broad spectrum of genetic alterations that lead to disruptions in the molecular pathways, triggering uncontrolled proliferation, cell death down-regulation, increased vascularization, and an altered metabolism . The epidermal growth factor receptor (EGFR) is a trans-membrane tyrosine kinase of the ErbB family involved in many physiological and pathological cell pathways that acts as a proto-oncogene in cell transformation . An increase in EGFR expression is associated with an aggressive tumor behavior. It has been demonstrated that this receptor regulates the angiogenesis, development, and progression of head and neck cancer, including LSCC . EGFR is probably the most amply studied biomarker for laryngeal cancer. Monoclonal antibodies targeting EGFR (i.e. cetuximab) are the only FDA-approved molecular treatment for head and neck cancer. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase located downstream from the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. It is a proto-oncogene often activated in several neoplastic diseases , with a key role in regulating cancer cell proliferation, apoptosis, cell migration, and angiogenesis. Marioni et al. tested for mTOR expression in 103 consecutive primarily-operated LSCCs and found that, among the mTOR-positive cases, the locoregional recurrence rate was higher and the disease-free survival (DFS) was shorter in patients whose mTOR expression was > 50.7%. In a multivariate statistical setting, mTOR status was also an independent indicator of a poor prognosis . Survivin is the smallest mammalian member of the family of inhibitor of apoptosis proteins (IAP). Being active simultaneously in tumor cell division and apoptosis inhibition, survivin has a pivotal role in cell survival . Survivin protein expression has been correlated strongly with more aggressive forms of malignancies. In 86 patients consecutively operated for LSCC, Marioni et al. investigated survivin expression, and survivin splice variant expression by reverse transcription and quantitative real-time PCR. Wild-type survivin was the most frequently detected splice variant in LSCC tissues. Nuclear survivin expression appeared to influence the aggressiveness of LSCC, higher nuclear survivin levels correlating with a higher recurrence rate and a shorter DFS . Anti-apoptotic members of the B-cell lymphoma-2 family (Bcl-2, Bcl- _XL , Bcl _W , Bfl-1, and Mcl-2) are pivotal regulators of apoptotic cell death. Bcl-2 is implicated in cancer development and progression and in protecting cells against a variety of cytotoxic insults, including irradiation and chemotherapeutic drugs. Bcl-2-related radio-resistance has been hypothesized in LSCC, and investigated in series of patients given RT alone . Angiogenin (ANG) is a member of the ribonuclease super-family involved in regulating angiogenesis in both physiological and pathological conditions. Some studies have even suggested that ANG might be released by tumor cells to drive this process. ANG expression is up-regulated in various types of human malignancy . Endoglin (CD105) is a proliferation-associated, hypoxia-inducible cell membrane glycoprotein, a component of the receptor complex of transforming growth factor beta (TGF-b), a cytokine that modulates angiogenesis by regulating cell proliferation, differentiation, and migration. CD105 has recently been studied in several upper airway cancers and it is considered an important independent predictor of clinical outcome in head and neck carcinoma . In 2010, our research group analyzed 108 consecutively-operated cases of LSCC: ANG expression in carcinoma cells was higher in patients who experienced locoregional recurrences of the disease, and DFS was shorter in cases with carcinoma cells showing ANG expression > 21% . A higher locoregional carcinoma recurrence rate and shorter DFS were also found in patients with high CD105 expression levels . The nm23 gene family encodes for proteins involved in regulating a number of cell processes, including proliferation, differentiation, molecular transport, and apoptosis . Ten nm23 genes (from nm23-H1 to nm23-H10) have been identified in humans to date . The best-characterized member of this family is nm23-H1, which is believed to have oncosuppressive functions because a lower expression of this gene has been significantly associated with a more aggressive behavior in several human malignancies . Studying a series of 104 LSCCs treated with primary surgery, Marioni et al. found lower mean nuclear nm23-H1 levels in patients with recurrent disease, and a longer DFS in patients with nuclear levels of nm23-H1 > 2%. On multivariate analysis, nuclear nm23-H1 expression proved prognostically significant in relation to DFS.

Using a multivariate logistic model, the aim of the present study was to identify a panel of LSCC tissue markers (chosen also in the light of our previous experience in this field) for pinpointing patients at higher risk of recurrence after PORT. Given that cancerogenesis involves several molecular pathways, it is reasonable to assume that a panel of appropriate biomarkers would be more reliable than a single marker in recognizing patients at high risk of recurrence. Such patients should be considered for postoperative chemo-radiotherapy, which is generally believed to be superior to PORT for high-risk patients .