Overexpression of hypoxia-inducible-factor 1α(HIF-1α) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage

1Oesophageal cancer is an aggressive tumour with a poor prognosis. Although diagnostic and surgical techniques have been advanced, survival rates have not been improved in the last decade, and the 5-year survival rate of patients with surgically treated oesophageal cancer remains less than 50% in spite of three-field lymph node dissection and combination chemotherapy and radiotherapy.

2The choice of therapeutic strategy is based primarily on whether lymph node metastasis has occurred. The presence of lymph node metastasis is the most important determinant of outcome. Patients with oesophageal cancer without nodal metastasis have a lower rate of recurrence after operation than those with nodal metastasis. However, it is difficult to determine whether lymph node metastases have occurred preoperatively in spite of new imaging techniques. Thus, the identification of a marker that predicts lymph node metastasis, and hence prognosis, is highly desirable.

3A hypoxic microenvironment is characteristic of many solid tumours. In the absence of neovascularisation, tumours cannot grow beyond several cubic millimeters, because the diffusion of oxygen, glucose, and other nutrients from blood vessels is limited. Cancer cell proliferation may outpace the rate of angiogenesis, resulting in tissue hypoxia. To surmount these limitations, the tumour needs to acquire abilities that allow it to adapt to a hypoxic microenvironment.

4Hypoxia-inducible-factor 1α(HIF-1α)is a 120 kDa nuclear protein. Hypoxia-inducible-factor 1 is a heterodimer, consisting of an α and a β subunit, both belonging to the basic–helix–loop–helix Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. Hypoxia-inducible-factor-1 is an important component of a widely operative transcriptional response, activated by hypoxia, cobaltous ions, and iron chelation. Hypoxia-inducible-factor 1 activates transcription of hypoxia-inducible genes, including those encoding erythropoietin, vascular endothelial growth factor (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase I. The C-terminal of HIF-1α binds to p300, and p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including VEGF.

5Induction of HIF-1α in response to hypoxia is instantaneous, and it can be expressed very early in carcinogenesis, before histologic evidence of angiogenesis or invasion exists.

6It has been shown that HIF-1α is a key player in the cancer cells response to low-oxygen tension in a variety of physiologic processes including embryogenesis, angiogenesis, tumorigenesis and metastases. Moreover, hypoxic regions have been shown to be both chemo- and radiation resistant (; Aebersold et al, 2001; ).

7In the current study, we examined archival material from 130 surgical specimens of oesophageal squamous cell carcinoma (OSCC) for HIF-1α immunoreactivity. The purpose was to determine the correlation between HIF-1α immunoreactivity and clinical and histopathologic factors.

8Surgical specimens were collected from 130 patients with primary OSCC, who underwent radical total oesophagectomy and three-field lymph node dissection from 1989 to 1999 at the Department of Surgical Oncology of Hokkaido University Hospital, Hokkaido Gastroenterology Hospital, or Teine Keijinkai Hospital. Cases of in-hospital death were excluded. The clinicopathologic stage was determined according to the TNM classification system of the International Union Against Cancer (UICC).

9The expression of HIF-1α was determined immunohistochemically in paraffin-embedded specimens fixed in 10% formalin. Histologic slides, 4 μm in thickness, were deparaffined in xylene and rehydrated through a series of graded ethanol. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 10 min. The sections were washed twice in phosphate-buffered saline (PBS) and incubated with 10% normal goat serum (Histofine SAB-PO kit, Nichirei Corporation, Tokyo, Japan) for 30 min. The slides were then exposed overnight to a monoclonal antibody against HIF-1α(HIF-1αAb-4, NEO MARKERS, Fremont, CA, USA) at a dilution of 1:400 at 4°C. After washing in PBS, a biotinylated goat antibody to mouse immunoglobulin (Histofine SAB-PO kit, Nichirei Corporation) was applied, followed by incubation at room temperature for 60 min. The immunohistochemical reactions were developed in freshly prepared 3,3′-diamino-benzidine tetrahydrochloride (Histofine SAB-PO kit, Nichirei Corporation). Slides were counterstained in haematoxylin and coverslipped in a systemic mounting medium. Tissue samples incubated with nonimmune serum served as negative controls. Immunostaining was evaluated in three visual fields at a power of × 200 under an Olympus microscope (Olympus Optical, Tokyo, Japan).

10Tumour cell immunoreactivity to HIF-1α protein was scored based on the number of cells exhibiting the nuclear or cytoplasmic staining using the following classification system: −, no staining; 1+, nuclear staining in less than 1% of cells; 2+, nuclear staining in 1%–10% of cells and/or with weak cytoplasmic staining; 3+, nuclear staining in 10% to 50% of cells and/or with distinct cytoplasmic staining; 4+, nuclear staining in more than 50% of cells and/or with strong cytoplasmic staining. Hypoxia-inducible-factor 1α 3+and 4+were considered high expression patterns while the remaining cases were considered to be low expression.

11All specimens were evaluated by three investigators who were blinded to the patients' clinical information.

12Either the χ2 test or Fisher's exact test was used to analyse the correlation between HIF-1α expression and clinicopathologic features. The cumulative survival rate was calculated by the Kaplan–Meier method, and the significance of differences in survival was analysed by the log-rank test. The univariate and multivariate analyses were performed using the Cox proportional hazard regression model; P< 0.05 was considered significant in all analyses. Computations were performed using the Statview J version 4.5 (SAS Institute, Inc., Cary, NC, USA) software package.

13Specimens from 130 patients were included in the current study (113 male and 17 female patients). The median patient age was 63 years (range, 38–82 years). A relatively large number of patients had early-stage disease (81 patients, 62%). Sixty-six patients (51%) had lymph node metastases and 22 patients (17%) had distant nodal metastases. No patient had distant organ metastasis at the time of operation. The study population had the following performance status (PS): PS0, 114 patients; PS1, 15 patients; and PS2, one patient. Following radical operation, adjuvant therapy was administered to 52 patients (Stage I; seven cases, Stage II; 23 cases; Stage III; 11 cases, and Stage IV; 11 cases). Chemotherapy, radiotherapy, and chemoradiotherapy were treated in 12, 18, and 22 patients, respectively (Table 1). The median follow-up period was 29 months (range, 2–114 months).

14A total of 130 OSCCs were grouped as 42 HIF-1α negative tumours; 15 HIF-1α 1+tumours; 33 HIF-1α 2+tumours; 30 HIF-1α 3+tumours; and 10 HIF-1α 4+tumours (Figure 1). Thus, 40 tumours (30.8%) were classified as showing high HIF-1α expression.

15The frequency of high HIF-1α expression increased with tumour stage according to pTNM system: 15.0% of stage I (six of 40 cases), 26.8% of stage II (11 of 41 cases), 44.8% of stage III (13 of 29 cases), and 50.0% of stage IV (10 of 20 cases; Table 2).

16High HIF-1α expression correlated with the depth of tumour invasion (P=0.0186), lymph node metastasis (P=0.0035), distant metastasis (P=0.0320), pTNM stage (P=0.0019), lymphatic invasion (P=0.0492), and positive surgical margin (P=0.0156) (Table 3).

17HIF-1α immunoreactivity had already been identified in carcinoma in situ of oesophagus (Figure 1F).

18The overall 5-year survival rate was 50.4%. The survival curve of patients with a high HIF-1α expression tumours was worse than that of patients with low-expression tumours (log-rank test, P=0.0007; Figure 2).

19Univariate analysis performed by Cox regression identified depth of tumour invasion (P<0.0001), distant metastasis (P=0.0002), lymph node metastasis (P<0.0001), lymphatic invasion (P=0.0021), positive surgical margin (P<0.0001), and High HIF-1α expression (P=0.0011) as correlating with survival (Table 4).

20Cox regression multivariate analysis identified depth of tumour invasion (P=0.0091), lymph node metastasis (P=0.0003), and positive surgical margin (P=0.0375), as independent unfavorable factors. High HIF-1α expression was not an independent prognostic factor (Table 4).

21Survival in patients with a high HIF-1α expression pattern was significantly worse than in those with a low-expression pattern in the patient treated with adjuvant therapy (P=0.0464; Figure 3).

22The current results show that

23high HIF-1α expression correlates with depth of tumour invasion, lymph node metastasis, distant metastasis, pTNM stage, lymphatic invasion and a positive surgical margin, andsurvival in patients with a high HIF-1α pattern was worse than in those with low-expression pattern.

24high HIF-1α expression correlates with depth of tumour invasion, lymph node metastasis, distant metastasis, pTNM stage, lymphatic invasion and a positive surgical margin, and

25survival in patients with a high HIF-1α pattern was worse than in those with low-expression pattern.

26Although HIF-1α was not an independent unfavourable prognostic factor, its expression may strongly influence both tumour proliferation and lymph node metastasis in OSCC. However, it has been reported that HIF-1α overexpression was not significantly correlated with pathological parameter in other cancers, including head and neck cancer, and oropharyngeal cancer. Thus, HIF-1α expression seems to behave in a tissue-dependent manner.

27Hypoxia has been shown to compromise the beneficial effects of chemotherapeutic drugs and interfere with the response of tumours to radiation. Pretreatment oxygenation levels have been found to be predictive of the radiation response and survival of patients with cancer of the uterine cervix, head and neck, oropharyngeal, and early oesophageal cancer. In the current study, of all pathological stages, overexpression of HIF-1α in OSCC significantly correlates with an unfavourable prognosis in the patients treated with adjuvant therapies.

28Preoperative studies on biopsy specimens obtained by endoscopy might allow clinicians to make better-informed therapeutic decisions in conjunction with this marker.

29In conclusion, we have suggested that (1) high HIF-1α expression may be a marker for lymph node metastasis; and (2) high HIF-1α expression may predict an unfavourable prognosis in the patient treated with OSCC.