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. 2006 Dec;8(12):1055–1061. doi: 10.1593/neo.06577

Heparanase Localization and Expression by Head and Neck Cancer: Correlation with Tumor Progression and Patient Survival1

Ilana Doweck *, Victoria Kaplan-Cohen †, Inna Naroditsky ‡, Edmond Sabo ‡, Neta Ilan †, Israel Vlodavsky †
PMCID: PMC1783722  PMID: 17217623

Abstract

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate (HS) side chains of HS proteoglycans, the major proteoglycans in the extracellular matrix and cell surfaces. Traditionally, heparanase activity was implicated in cellular invasion associated with angiogenesis, inflammation, and cancer metastasis. More recently, heparanase upregulation was documented in an increasing number of primary human tumors, correlating with reduced postoperative survival rate and enhanced tumor angiogenesis. In the present study, we examined the expression of heparanase in squamous cell carcinoma of the head and neck by means of immunostaining, and we correlated expression levels with patient outcome. The intensity and extent of heparanase staining correlated with tumor stage (P = .049 and P = .027, respectively), and the extent of staining further correlated with tumor grade (P = .047). Moreover, heparanase expression inversely correlated with patient status at the end of the study (P = .012). Notably, heparanase localization was found to be an important parameter for patient status. Thus, 63% of patients with nuclear staining, compared to 19% of patients with cytoplasmic staining (P = .0043), were alive, indicating that nuclear localization of the enzyme predicts a favorable outcome.

Keywords: Head and neck, carcinoma, heparanase, nuclear localization, survival

Introduction

Squamous cell carcinoma of the head and neck (SCCHN) continues to be the sixth most common neoplasm in the world, where > 500,000 new cases are projected annually [1]. Approximately 200,000 deaths occur yearly as a result of cancer of the oral cavity and pharynx, and the outcome has not improved significantly in the past 25 years [2]. Tumor metastases are common among patients with head and neck cancer with uncontrolled local or regional disease, and autopsy studies revealed a 40% to a 47% overall incidence of distant metastases [3,4]. The incidence of distant metastases among patients who remain free of disease at local and regional sites is lower (18–20%) although still significant. With improvement in locoregional control of advanced head and neck cancer resulting from new treatment regimens, distant failure has emerged as the most common reason for disease recurrence [5,6]. Therapies for advanced SCCHN include combined chemoradiotherapy. Emerging evidence suggests the effectiveness of anti-epidermal growth factor receptor therapy, combined with chemotherapy and/or radiation [2]. Clearly, a better understanding of the molecular biology of head and neck tumors is required to define relevant targets and to develop novel therapeutic approaches.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate (HS) side chains of HS proteoglycans [7–9], the major proteoglycans in the extracellular matrix (ECM) and cell surfaces [10,11]. In addition to its structural role as a molecular link between different ECM components, contributing to ECM integrity and insolubility, HS side chains can bind a variety of biologic mediators, such as growth factors, cytokines, and chemokines, thus forming a readily available reservoir that can be liberated on local or systemic cues [12–15]. Traditionally, heparanase activity was implicated in cellular invasion associated with angiogenesis, inflammation, and cancer metastasis [16–20]. This notion gained further support by employing siRNA and ribozyme technologies, clearly depicting heparanase-mediated HS cleavage and ECM remodeling as critical requisites for metastatic spread [21]. Recently, heparanase upregulation was documented in an increasing number of primary human tumors. Heparanase upregulation correlated with reduced postoperative survival of pancreatic [22,23], bladder [24], gastric [25,26], cervical [27], and colorectal [28] cancer patients. Moreover, heparanase upregulation correlated with increased lymph node and distant metastases [23–26], providing strong clinical support for the prometastatic feature of heparanase. Similarly, heparanase upregulation was noted in head and neck tumors [29,30], yet studies examining a large number of tumor samples of this cancer have not been reported. Here, we examined heparanase expression in a cohort of head and neck patients and correlated clinical-pathological data with heparanase immunostaining and cellular localization. We report that heparanase expression correlates with head and neck tumor progression. Cytoplasmic staining of heparanase inversely correlates with patient survival and predicts poor prognosis, whereas nuclear heparanase predicts a favorable outcome.

Materials and Methods

Experimental Design

The study included 74 patients with head and neck cancer who were diagnosed at the Department of Otolaryngology, Head and Neck Surgery, Carmel Medical Center (Haifa, Israel) whose archival paraffin-embedded pathological specimens were available for immunohistochmical analysis. The study protocol was approved by the Institutional Review Board. The clinical data of all patients were reviewed, and patients were restaged according to the American Joint Committee of Cancer 2003 staging system. Clinical data included demographics, tumor site, tumor-node-metastasis staging, treatment modality, status at the end of the study (dead or alive), failure (local, regional, or distant), timeto failure, follow-up, and survival. Pathological data included histology, tumor grade, local and regional spread of the disease (pT and pN), and extracapsular extension (ECE).

Heparanase Immunostaining

Staining of formalin-fixed, paraffin-embedded, 5-µm sections for heparanase was performed essentially as described [31,32]. Briefly, slides were deparaffinized and rehydrated, and endogenous peroxidase activity was quenched (for 30 minutes) by 3% hydrogen peroxide in methanol. Slides were then subjected to antigen retrieval by boiling (for 20 minutes) in 10 mM citrate buffer, pH 6. Slides were incubated with 10% normal goat serum in phosphate-buffered saline (PBS) for 60 minutes to block nonspecific binding and were incubated (20 h, 4°C) with anti-heparanase 733 antibody diluted 1:100 in blocking solution. Antibody 733 was raised in rabbits against a 15-amino-acid peptide (KKFKNSTYSRSSVDC) that maps at the N-terminus of a 50-kDa heparanase subunit and preferentially recognizes the 50-kDa heparanase subunit versus a 65-kDa latent proenzyme [31]. Slides were extensively washed with PBS containing 0.01% Triton X-100 and incubated with a secondary reagent (Envision kit; Dako, Glostrup, Denmark) according to the manufacturer's instructions. Following additional washes, color was developed with AEC reagent (Dako), and sections were counterstained with hematoxylin and mounted, as described [31]. Immunostained specimens were examined by senior pathologists (E.S. and I.N.) blinded to the clinical data of patients and were scored according to intensity of staining (0 = none; 1 = weak-moderate; 2 = strong) and the percentage (extent staining) of tumor cells that were stained (0, < 10%; 1, 10–50%; 2, > 50%). Specimens that were similarly stained with preimmune serum or application of the above procedure without the primary antibody yielded no detectable staining. For statistical analysis, we combined cases diagnosed as negative heparanase staining with cases diagnosed as weak heparanase staining compared to cases with strong heparanase intensity and/or extent (subgroups 0 and 1 vs subgroup 2).

Statistical Analysis

Univariate associations between heparanase parameters (intensity and extent of staining) and clinical and pathological findings, as well as patient outcome, were analyzed using chisquare test (Pearson and Fisher's exact test). Multivariable logistic regression was performed to detect independent parameters that affect patient status and to estimate relevant odds ratio (OR) with 95% confidence interval (95% CI). Univariate association with survival and cause-specific survival was evaluated by Kaplan-Meier curves and tested using log-rank test. A multivariable Cox's proportional hazard model was performed with stepwise selection to identify independent predictors of survival and cause-specific survival (P for enter and P to stay were set at .1). The model included all parameters with P < .2 by univariate analysis.

Results

We have developed a polyclonal antibody (pAb no. 733) that preferentially recognizes the 50-kDa heparanase subunit over the latent 65-kDa proenzyme [31]. Thus, immunostaining of archival material can be correlated with heparanase enzymatic activity. We employed antibody 733 to examine heparanase expression in specimens collected from head and neck cancer patients. Seventy-four patients (68 males and 6 females) were included in this study (the median age at diagnosis was 66.7 ± 12 years). The mean follow-up was 43.9 ± 6.04 months for the entire group and 64.8 ± 11.05 months for patients who are still alive. A clinical description of patients is presented in Table 1. Larynx was the predominant site (77%; 57 of 74), followed by the pharynx, oral cavity, carcinoma with unknown primary origin, and others. Most patients had advanced disease, as indicated by T (primary tumor) and N (nodal metastasis) staging criteria (Table 1), because patients with early disease were treated with radiotherapy and tumor specimens were not available. Neck dissection was carried out in 35 patients. Metastatic nodes were found in 30 specimens: 13 with one node and 17 with two or more metastatic nodes. ECE was observed in 14 metastatic specimens.

Table 1.

Clinical Description of Patients (n = 74).

Parameter Patients [n (%)]
Site of tumor
Larynx 57 (77)
Pharynx 6 (8)
Oropharynx 4
Hypopharynx 2
Oral cavity 3 (4)
Other 8 (11)
T stage
T0–2 21 (28)
T3 29 (39)
T4 24 (33)
N stage
N0 39 (53)
N1 8 (11)
N2–3 27 (36)
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Expression and Cellular Localization of Heparanase

Positive heparanase staining was found in 86% (64 of 74) of tumor specimens (Figure 1B), whereas 14% (10 of 74) of the specimens were negative (Figure 1A). The heparanase-positive group was further categorized according to the intensity and extent of staining (Table 2). Thus, weak staining (+1) was found in 58% (37 of 64) of positive specimens, whereas 42% (27 of 64) stained strongly (+2) for heparanase (Table 2, Figure 1). According to the extent criteria (see Materials and Methods section), 81% (52 of 64) of the specimens that positively stained for heparanase were scored as high extent (+2), whereas the rest 19% were scored as low extent. Heparanase expression in terms of intensity and, even more so, extent positively correlated with head and neck tumor progression (P = .049 and .027, respectively; Table 2). For example, although only 50% of the tumors that expressed no or low levels of heparanase were diagnosed as T3–4, 81% of the tumors with strong heparanase staining were diagnosed as such (Table 2, extent). A similar trend was noted with respect to the intensity criterion (Table 2, intensity). Moreover, the extent of heparanase staining also correlated with tumor grade (P = .047; Table 2), further supporting a role for heparanase in head and neck tumor progression. A close examination of the heparanase-positive group revealed a distinct cellular localization pattern (Figure 1). Thus, in 50% of the specimens (32 of 64), heparanase staining appeared cytoplasmic (Figure 1B); in 12.5% (8 of 64), heparanase was found in the cell nucleus (Figure 1C); and, in the rest of the specimens, 37.5% (24 of 64) of heparanase-positive specimens assumed both nuclear and cytoplasmic localization (Figure 1D).

Figure 1.

Figure 1

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Immunohistochemical staining of heparanase in SCCHN tumor specimens. Formalin-fixed, paraffin-embedded, 5-µm sections of head and neck tumors were subjected to immunostaining of heparanase by applying anti-heparanase 733 antibody, as described under Materials and Methods section. Shown are representative photomicrographs of heparanase-negative (A) and heparanase-positive (B–D) specimens: (B) cytoplasmic; (C) nuclear; and (D) cytoplasmic and nuclear. Specimens that were similarly stained with preimmune serum (E) or application of the above procedure but without the primary antibody (F) yielded no detectable staining. (A–D) Original magnification, x400; (E and F) original magnification, x200.

Table 2.

Association of Heparanase Intensity and Extent of Staining with T Stage and Tumor Grade (Univariate Analysis).

T Stage T0–2 [n (%)] T3–4 [n (%)] Total P
Heparanase intensity
0 5 (50) 5 (50) 10 .049
1 12 (32) 25 (68) 37
2 4 (15) 23 (85) 27
Total 21 53 74
Heparanase extent
0 5 (50) 5 (50) 10 .027
1 6 (50) 6 (50) 12
2 10 (19) 42 (81) 52
Total 21 53 74
Grade Carcinoma In Situ and Well-Differentiated Carcinoma [n (%)] Moderately to Poorly Differentiated Carcinoma [n (%)]
Heparanase extent
0–1 8 (40) 12 (60) 20 .047
2 9 (18) 42 (82) 51
Total 17 52 71
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Heparanase intensity: 0 = no staining; 1 = weak to moderate staining; 2 = strong staining.

Heparanase extent: 0, < 10% staining; 1, 10–50% staining; 2, > 50% staining.

Patients with no heparanase staining (0) were combined with the low-expressing group (1) for statistical analysis.

Prognostic Value of Heparanase for Head and Neck Carcinomas

To determine the prognostic value of heparanase for SCCHN, we analyzed patient status, cumulative survival, and cause-specific survival according to heparanase expression levels and localization. The extent of heparanase expression inversely correlated with patient status (P = .011; Table 3, Figure 2), and a similar trend was noted for the intensity of staining, although borderline significant (P = .085; Table 3). Even more impressive was the correlation between the cellular localization of heparanase and patient outcome. Clearly, patients with nuclear staining had an outcome better than that of patients with cytoplasmic staining. Although 63% of patients assuming nuclear staining survived, only 19% of patients exhibiting cytoplasmic staining and 54% of cases with both nuclear and cytoplasmic staining were alive at the end of the study (P = .0043; Table 3, Figure 3). For further analysis, we combined the patients with nuclear heparanase staining with those with both nuclear and cytoplasmic stainings and compared them to patients with cytoplasmic staining only. We further employed multivariate logistic regression analysis to examine the contribution of different parameters to patient status (including T and N stages), heparanase staining intensity, extent, and cellular localization (Table 4). The most significant and independent parameter that influenced patient status was heparanase localization (cytoplasmic versus nuclear, P = .002), followed by T stage (P = .006) (Table 4). Univariate analysis of overall survival and cause-specific survival was similarly performed for clinical, pathological (T and N stages, ECE), and heparanase staining parameters. Patients exhibiting nuclear localization of heparanase had significantly higher overall survival at 5 years (69%) than patients with only cytoplasmic staining (26%) (P = .03; Table 5, Figure 3). For cause-specific survival, the N stage (P = .0007), ECE (P = .0006), and nuclear localization of heparanase (P = .04) were the most significant parameters. Cox's proportional hazard model was subsequently performed for overall survival and included all heparanase parameters and clinical parameters (Tstage, N stage, heparanase extent staining, and localization; Table 6). The analysis was adjusted to the age of the patients at diagnosis. The most significant independent parameters that influenced the overall survival of SCCHN patients included heparanase localization (P = .007, for cytoplasmic versus nuclear staining), age at the time of diagnosis (P = .008), T stage (P = .07), and N stage (P = .08) (Table 6). Cox's proportional hazard model for cause-specific survival of the group revealed that the most significant parameters are N stage (P = .002) and heparanase staining extent (P = .05) (Table 7). Age was not a significant independent parameter for cause-specific survival. Thus, both the extent of staining and the localization of heparanase are critical parameters for the survival of SCCHN patients. These results indicate that heparanase cellular localization stands as an independent parameter for the overall survival of head and neck cancer patients and supports a role for heparanase in the progression of SCCHN, suggesting that the enzyme is a valid target for the development of anticancer drugs and, possibly, is a diagnostic marker for this malignancy.

Table 3.

Heparanase Parameters in Correlation with Patient Status.

Alive [n (%)] Died [n (%)] Total P
Heparanase intensity
0 7 (70) 3 (30) 10 .085
1 16 (43) 21 (57) 37
2 8 (30) 19 (70) 27
Heparanase extent
0 7 (70) 3 (30) 10 .0116
1 8 (67) 4 (33) 12
2 16 (31) 36 (69) 54
Heparanase localization
Nuclear/cytoplasmic
0 7 (70) 3 (30) 10 .0043
Nuclear 5 (63) 3 (37) 8
Nuclear + cytoplasmic 13 (54) 11 (46) 24
Cytoplasmic 6 (19) 26 (81) 32
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Figure 2.

Figure 2

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Cause-specific survival (Kaplan-Meier survival plot) stratified by extent of heparanase staining. Note that patients with low levels of heparanase staining (< 10%) had a 5-year cause-specific survival of 100%.

Figure 3.

Figure 3

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Survival analysis. Overall survival stratified by nuclear versus cytoplasmic staining of heparanase (Kaplan-Meier survival plot). Log-rank test, P = .03.

Table 4.

Multivariate Logistic Regression for Patient Status at the End of the Study (Dead or Alive).

Term OR 95% CI P
Heparanase localization: cytoplasmic/nuclear 2.4 1.36–4.35 .0023
T stage (3/4-0/1/2) 5.3 1.65–18.36 .0068
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OR, odds ratio; CI, confidence interval.

Table 5.

Univariate 5-Year Overall Survival and Cause-Specific Survival Analyses Including Clinical and Heparanase Parameters.

Parameter 5-Year Cause-Specific Survival (%) P 5-Year Overall Survival (%) P Patients at Risk
T stage
T0–2 68 .1 59 .08 21
T3 46 37 29
T4 66 28 24
N stage
N0 76 .0007 47 .096 39
N1 44 31 8
N2–3 28 26 27
ECE
Without ECE 73 .0006 55 .0005 21
With ECE 30* 24* 14
Heparanase intensity
0 100 .13 44 .4 10
1 58 41 37
2 47 34 27
Heparanase extent
0 100 .11 44 .19 10
1 69 69 12
2 51 33 52
Heparanase localization
0 100 .04 44 .03 10
Nuclear and nuclear + cytoplasmic 60 69 32
Cytoplasmic 47 26 32
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Statistical significance was tested using the log-rank test.

*

Survival estimated at 24 months.

ECE, extracapsular extension.

Table 6.

Cox's Proportional Hazard Model for Overall Survival: Hazard Ratio and 95% CI of All Independent Parameters.

Parameter Hazard Ratio P 95% CI
Heparanase localization: cytoplasmic/nuclear 2.42 .007 1.27–4.62
Age 1.04 .008 1.01–1.07
T stage .075
T3vs T0–2 2.24 .12 0.81–6.02
T4vs T0–2 3.16 .024 1.16–8.58
N stage .08
N1vs N0 1.24 .68 0.45–3.39
N2vs N0 2.27 .025 1.1–4.66
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Table 7.

Cox's Proportional Hazard Model for Cause-Specific Survival: Hazard Ratio and 95% CI of Independent Parameters.

Parameter Hazard Ratio P 95% CI
N stage .002
N1vs N0 3.42 .078 0.87–13.4
N2vs N0 5.23 .001 2.05–13.3
Heparanase extent 2.76 .053 0.99–7.75
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Discussion

SCCHN is characterized by poor prognosis due to aggressive tumor growth and high rate of tumor cell dissemination [1]. In spite of improved treatment, the outcome of SCCHN patients did not improve significantly in the past 25 years [1,2], clearly implying a need for a better understanding of cellular and molecular aspects of this malignancy to establish better therapeutic modalities. Here, we examined the expression of heparanase in biopsy specimens derived from 74 SCCHN patients, using immunohistochemistry. The anti-heparanase 733 antibody employed has been previously shown to preferentially recognize the 50-kDa heparanase subunit versus the 65-kDa latent protein [31]. The 50-kDa subunit comprises, together with an 8-kDa subunit, the active enzyme heterodimer [33–35], so that positive staining with the 733 antibody most likely correlates with heparanase enzymatic activity. This antibody was successfully employed to study heparanase expression and regulation by nasopharyngeal and colon carcinomas [32,36], supporting its diagnostic potential. Heparanase upregulation was noted in the majority (82%) of the SCCHN specimens examined, in agreement with heparanase overexpression found in many other human cancers [37,38]. Importantly, intensity and, even more so, the extent (i.e., percentage of positively stained cells) of heparanase staining correlated with the progression of these tumors (T stage and grade criteria; Table 2), suggesting an important role of the enzyme in the etiology of this type of malignancy. In agreement with this notion was the inverse correlation found between heparanase expression levels and the survival of SCCHN patients (Figure 2, Table 3). This observation is in agreement with previous findings analyzing 17 cases of primary SCCHN and the localization of heparanase to the invasion front of these tumors [30]. Even more impressive was the correlation between the cellular localization of heparanase and patient survival. Clearly, nuclear localization of heparanase predicts a favorable outcome, whereas cytoplasmic localization of the enzyme correlates with poor prognosis (Figure 3, Tables 3–6). We further employed the Cox's proportional hazard model statistical analysis and demonstrated that cytoplasmic versus nuclear heparanase localization, age, T stage, and N stage are the most significant parameters that affect the survival of head and neck cancer patients (Table 6). Moreover, heparanase localization stands as an independent parameter for patient status (Table 4). Further analysis indicated that heparanase staining extent is a critical parameter that affects cause-specific survival (Table 7), suggesting that both heparanase expression levels (heparanase extent) and localization are crucial for SCCHN tumor progression. These results support and further extend previous findings correlating heparanase expression with the poor prognosis of patients with head and neck [30], as well as colorectal [28], bladder [39], and cervical [27] carcinomas. The mechanism by which heparanase contributes to tumor progression is not entirely clear but likely involves tumor angiogenesis. Heparanase upregulation in several primary human tumors has been shown to correlate with increased microvessel density [24,27,28,40–42], an angiogenic feature that has been recapitulated in several in vitro [43] and in vivo experimental settings [44–49]. Moreover, heparanase downregulation by specific anti-heparanase ribozyme or siRNA constructs resulted in xenografts that appeared less vascularized [21], further supporting heparanase as a proangiogenic mediator. More recently, heparanase was found to induce the expression of vascular endothelial growth factor in an Src-dependent manner [49], clearly depicting its angiogenic function. The role that heparanase may play in the nucleus is less understood. Nuclear localization of heparanase was demonstrated by cell fractionation [50] and by immunostaining of cultured cells [50] and tumor biopsies [26,51]. Notably, nuclear localization was correlated with sustained cellular differentiation and a favorable outcome in patients with gastric and esophageal carcinomas [26,51], in agreement with our study (Tables 3–6, Figure 3). Gene transcription may be due to a direct interaction of nuclear heparanase with DNA or may be a consequence of heparanase-mediated remodeling of nuclear HS. The latter possibility is supported by a recent publication by Kobayashi et al. [52] studying the differentiation of esophageal keratinocytes. In this system, heparanase was noted to be translocated from the cytoplasm to the nucleus during esophageal cell differentiation, correlating with induction of keratinocyte differentiation markers such as p27 and involucrin. Notably, p27 and involucrin induction was significantly reduced on inhibition of heparanase activity by a novel pharmacological inhibitor SF4 [52], suggesting that HS remodeling by heparanase directly affects gene expression, in agreement with previous studies demonstrating nuclear localization of HS [53,54]. Moreover, transfection of breast carcinoma MCF-7 cells with a plasmid vector that targets heparanase exclusively to the nucleus induced the appearance of lipid droplets typical of differentiated mammary epithelial cells [55], thus further supporting a role for heparanase in epithelial cell differentiation. Taken together, our results indicate that heparanase expression is enhanced in head and neck tumors and that the outcome of these patients correlates with heparanase levels and with its cellular localization. These findings are novel and contribute to our understanding of head and neck tumor biology, suggesting that heparanase is a promising target for the development of anticancer therapeutics for head and neck malignancies.

Acknowledgement

We would like to acknowledge Ofra Barnett-Griness (Department of Community Medicine and Epidemiology, Carmel Medical Center) for assistance with statistical analyses.

Abbreviations

ECM

extracellular matrix

ECE

extracapsular extension

SCCHN

squamous cell carcinoma of the head and neck

HS

heparan sulfate

Footnotes

1

This work was supported by grants from the Israel Science Foundation (grant 532/02), the National Cancer Institute, the National Institutes of Health (grant RO1-CA106456), the Israel Cancer Research Fund, the Rappaport Family Institute Fund, the Israel Cancer Association (grant 20040085), and the Ronni Udassin Memorial Fund (to I.D.).

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