Proteoglycan-based diversification of disease outcome in head and neck cancer patients identifies NG2/CSPG4 and syndecan-2 as unique relapse and overall survival predicting factors

Abstract

Background: Tumour relapse is recognized to be the prime fatal burden in patients affected by head and neck squamous cell carcinoma (HNSCC), but no discrete molecular trait has yet been identified to make reliable early predictions of tumour recurrence. Expression of cell surface proteoglycans (PGs) is frequently altered in carcinomas and several of them are gradually emerging as key prognostic factors.

Methods: A PG expression analysis at both mRNA and protein level, was pursued on primary lesions derived from 173 HNSCC patients from whom full clinical history and 2 years post-surgical follow-up was accessible. Gene and protein expression data were correlated with clinical traits and previously proposed tumour relapse markers to stratify high-risk patient subgroups.

Results: HNSCC lesions were indeed found to exhibit a widely aberrant PG expression pattern characterized by a variable expression of all PGs and a characteristic de novo transcription/translation of GPC2, GPC5 and NG2/CSPG4 respectively in 36%, 72% and 71% on 119 cases. Importantly, expression of NG2/CSPG4, on neoplastic cells and in the intralesional stroma (Hazard Ratio [HR], 6.76, p = 0.017) was strongly associated with loco-regional relapse, whereas stromal enrichment of SDC2 (HR, 7.652, p = 0.007) was independently tied to lymphnodal infiltration and disease-related death. Conversely, down-regulated SDC1 transcript (HR, 0.232, p = 0.013) uniquely correlated with formation of distant metastases. Altered expression of PGs significantly correlated with the above disease outcomes when either considered alone or in association with well-established predictors of poor prognosis (i.e. T classification, previous occurrence of precancerous lesions and lymphnodal metastasis). Combined alteration of all three PGs was found to be a reliable predictor of shorter survival.

Conclusions: An unprecedented PG-based prognostic portrait is unveiled that incisively diversifies disease course in HNSCC patients beyond the currently known clinical and molecular biomarkers.

Figure 1

Cell surface-associated PGs are differentially expressed in primary lesions of oral cavity HNSCC patients. (a) Heat map and hierarchical clustering of the relative expression levels (columns) of the 8 most modulated cell surface-associated PGs in primary lesions of 119 oral cavity HNSCC patients (rows): red, up-regulation; green, down-regulation, black: no change in comparison to the healthy epithelial tissue. Distance between clusters was calculated as reported in Additional file 3. (b) PG expression profiles in oral cavity HNSCC specimens derived from T1-T2-N-, T1-T2-N+, T3-T4-N- and T3-T4-N+ graded tumours. SDC1-4, syndecans 1–4; GPC1-6, glypicans 1–6.

Figure 2

In situ immunolocalization of GPCs and NG2/CSPG4 in oral cavity HNSCC primary lesions. Representative patterns of GPC and NG2/CSPG4 distribution in oral cavity HNSCC lesions. (a, b) representative views of GPC1 expression in lesions with different degrees of keratinizing neoplastic cells. (c, d) representative images of GPC1 expression in stromal cells of pre-malignant lesions (c) and lack of expression in the stromal cells of HNSCC tissue (d). GPC3 was detected in neoplastic cells (e), but not stromal fibroblasts (f), whereas GPC4 (g) and GPC6 (h) were primarily found to be associated with the neoplastic cells. (i) Shows the lack of expression of GPC6 in the intralesional stroma. NG2/CSPG4 was found to be abundantly expressed in both well- (k) and moderately-differentiated (l) oral cavity HNSCC lesions, whereas it was similarly absent from potentially pre-malignant lesions (j).

Figure 3

Immunodetection of SDCs in oral cavity HNSCC primary lesions. Representative view of the SDC1 expression pattern, inversely correlating with the overall differentiation status of the tumour (a, displatyc tissue; b, c, well-differentiated; d, poorly differentiated), while being particularly abundant in the center of neoplastic nests (p) and in the stromal compartment (e-f). SDC2 was seen strongly associated with tumour vessels (g, n, o) and was the only PG to be widely expressed in the different degree of dysplastic tissue (h-j). SDC3 (k) and SDC4 (m) immunolocalized in the epithelial tumour cells, but not in the stromal compartment (l, SDC3; m, SDC4).

Figure 4

Differential PG expression correlates with clinical outcome. Survival and probability curves for the following correlations: (a) loco-regional relapse vs de novo expression of NG2/CSPG4, (b) loco-regional relapse vs T group classification; (c) loco-regional relapse vs coincident NG2/CSPG4 expression and advanced T classification; (d) lymphnodal metastases vs enhanced SDC2 expression in stromal cells; (e) lymphnodal metastases vs manifestation of precancerous lesions; (f) lymphnodal metastases vs the combination of both previous prognostic indicators; (g) distant metastases vs up-regulated SDC1 expression; (h) distant metastases vs infiltration of cervical lymph nodes; (i) distant metastases vs the combination of both previous prognostic indicators; (j) overall survival vs enhanced SDC2 expression in the stromal compartment; (k) overall survival vs N classification; (l) overall survival vs the coincidence of both previously indicated events; (m) the occurrence of any of the clinical outcomes vs up-regulated SDC1 expression; (n) the occurrence of any of the clinical outcomes vs N classification; (o) the occurrence of any of the clinical outcomes vs the combination of both above listed events; (p) overall survival, (q) distant metastases and (r) the occurrence of any of the clinical outcomes vs the combination of SDC1 up-regulation, de novo expression of NG2/CSPG4 and stromal enhancement of SDC2 positivity. Abbreviations: LRFS, loco-regional relapse-free survival; LMFS, lymphnodal metastasis-free survival; DMFS, distant metastasis-free survival; OS, overall survival, COsFS, clinical outcomes-free survival.

Figure 5

Cox proportional hazard analysis. Plot overview of Cox proportional hazard estimated regression coefficients of the resulting independent prognostic factors for loco-regional recurrence, lymphnodal metastases, distant metastases, disease-related death and the occurrence of any of the clinical outcomes.