Abstract
The therapeutic effect of intratumoural application of Interleukin-2 (IL-2) was studied in patients with stage III–IV nasopharyngeal carcinoma (NPC) that received radiotherapy. Patients with stage III–IV NPC receiving a standard treatment of 7,000 cGy external beam irradiation have a mean disease-free survival of about 1.5 years. In this paper, we describe ten of these patients who were treated with additional peritumoural and intratumoural injections with 3×104 U IL-2 on 5 days in weeks 2, 4, and 6 of the 7-weeks’ irradiation period. This combined treatment group was compared with a historical group of patients treated with standard irradiation alone. Local IL-2 therapy showed a marked clinical and statistical significant improvement of disease-free survival. After 5 years, 63% of the IL-2 treated patients were disease-free versus 8% of the control patients. These results suggest that the therapeutic results of radiotherapy can be significantly improved by combining it with local IL-2 treatment. To our knowledge, this is the first clinical report showing that local IL-2 therapy is effective against an infiltrative and locally metastasizing tumour in human patients.
Keywords: Nasopharyngeal carcinoma, Interleukin-2, Human cancer, Local IL-2, Radiotherapy
Introduction
Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, with the exception of South China, the Inuits in Labrador and some areas in East Africa. In low incidence areas, most tumours of the nasopharynx are squamous cell carcinomas (75–100%) [29]. NPCs can be keratinizing, non-keratinizing and undifferentiated carcinomas, which correspond with the World Health Organization (WHO) type 1, 2 and 3, respectively. WHO type 1 is a squamous cell carcinoma and WHO types 2 and 3 can be considered together as undifferentiated carcinomas of the nasopharyngeal type [3]. About 10% of the patients with the keratinizing type and about 30% of the patients with the other types are under 40 years of age. WHO type 1 is predominantly found in Western countries, whereas WHO types 2 and 3 are predominantly found in endemic areas. Epstein-Barr virus (EBV) is associated with the aetiology of NPC [9]. More specifically, EBV appears to be involved in all cases of WHO type 2 and 3, and in the majority of cases of WHO type 1.
In Western countries, the disease is usually far advanced at the time of initial presentation due to the rarity of this disease, the aspecific symptoms, and diagnostic problems related to the inaccessibility of the tumour. Prognosis of the patients is directly related to the size of the primary lesion and the presence of nodal metastases. The principal determinant of survival is local recurrence after treatment.
Treatment of cancer with interleukin-2 (IL-2) directly applied at the site of the tumour is very effective in mice with transplanted lymphoma [22], in bovine ocular squamous cell carcinoma [9], and in human recurrent superficial bladder carcinoma [8]. In addition we found that combined therapy with irradiation and local IL-2 application is therapeutically more effective than each of these modalities alone [16].
Intratumoural IL-2 exerts its anti-tumour action through two mechanisms: it causes vascular leakage and tissue necrosis inside the tumour [1, 6, 15], and it causes systemic immunity exerted by CD4 and CD8 T lymphocytes [22].
Materials and methods
Inclusion criteria
Ten patients who presented themselves in August 1995–February 1997, who met the inclusion criteria and gave informed consent, were treated with standard radiotherapy and additional local IL-2 injections. Criteria for admission were (a) all stages of NPC that are treated with megavoltage external beam irradiation; (b) histologically proven malignancy; and (c) WHO-performance status 0–2. Criteria for exclusion were (a) tumour type other than squamous cell or undifferentiated carcinoma; (b) WHO-performance status 3 and 4; (c) white blood count below 2,000/mm3 and/or platelet count below 50,000/mm3; (d) previous or concurrent cancer at other sites (other than adequately treated basal or squamous cell carcinoma of the skin); (e) expected follow-up less than 6 months; (f) previous treatment with IL-2; and (g) younger than 18 years of age.
Patients
The selected patients had histologically confirmed NPC (International Classification of Diseases (ICD) code 147). Histological classification of the tumours was done according to the standard WHO classification [28]. Most Dutch patients had histological type 1. All patients had a TNM-stage corresponding to disease stage III or IV [2]. Twenty control patients were selected from the hospital’s database until the end of 1997. Three of these control patients, YL, YR, and YT were treated at the time of this study, but refused to receive IL-2. The control patients were matched to the IL-2 treated patients with respect to their T, N, and M status. Two control patients were selected and matched for every IL-2 treated patient, e.g. patient ZB was matched with controls YA and YB. If more historical controls for a certain disease status were available, the most recently treated were selected. All IL-2 treated and control patients were males. The average age was 54.6 and 54.5 years for IL-2-treated and control patients, respectively. Details of IL-2 treated and control patients are given in Table 1.
Table 1.
Patient characteristics and therapeutic effects
| Patient | Site | Age (years) | Date | Stage | T | N | T surv. | Td-f | Recurrence |
|---|---|---|---|---|---|---|---|---|---|
| Radiotherapy only | |||||||||
| YA | 147.X | 64 | 1984 | IV | 1 | 3 | 2.9 | 2.0 | Distant |
| YB | 147.1 | 57 | 1985 | IV | 1 | 3 | 2.5 | 1.6 | Distant |
| YC | 147.0 | 58 | 1991 | III | 2 | 1 | 2.8 | 1.9 | Distant |
| YD | 147.0 | 30 | 1982 | IV | 2 | 2 | 3.3 | 1.5 | Local |
| YE | 147.1 | 31 | 1988 | IV | 2 | 2 | >5.0 | 1.4 | Local |
| YF | 147.1 | 40 | 1985 | IV | 2 | 2 | >5.0 | >5.0 | No |
| YH | 147.1 | 66 | 1989 | III ? | 3 | X | >5.0 | 1.7 | Local |
| YI | 147.0 | 52 | 1993 | III | 3 | 0 | 1.6 | – | Local* |
| YJ | 147.0 | 83 | 1983 | III | 3 | 0 | 1.1 | 1.1 | Distant |
| YK | 147.1 | 72 | 1993 | III | 3 | 1 | 2.7 | 2.7 | No |
| YG | 147.0 | 71 | 1991 | IV | 3 | 2a | 3.3 | 2.5 | Local |
| YL | 147.1 | 44 | 1995 | IV | 3 | 2c | 2.8 | 1.1 | Local |
| YM | 147.1 | 53 | 1997 | IV | 3 | 2c | >0.4# | 0.4 | Distant |
| YN | 147.2 | 51 | 1992 | IV | 3 | 2c | >5.0 | 3.3 | Distant |
| YO | 147.0 | 61 | 1987 | IV | 4 | 0 | 1.8 | 1.8 | Local |
| YQ | 147.1 | 29 | 1991 | IV | 4 | 0 | 0.5 | 0.5 | Local |
| YP | 147.1 | 70 | 1988 | IV | 4 | 0 | 1.2 | 1.2 | Local |
| YR | 147.1 | 57 | 1996 | IV | 4 | 2c | 1.3 | 1.3 | No |
| YS | 147.1 | 57 | 1985 | IV | 4 | 2b | 0.7 | 0.7 | Local |
| YT | 147.2 | 44 | 1996 | IV | 4 | X | 0.7 | – | Local* |
| IL-2 plus radiotherapy | |||||||||
| ZB | 147.1 | 70 | 1997 | IV | 1 | 3 | 1.5 | 1.5 | No |
| ZD | 147.1 | 72 | 1997 | III | 2 | 1 | 2.0 | 1.0 | Local |
| ZF | 147.0/1 | 45 | 1996 | IV | 2 | 2 | >5.0 | >5.0 | No |
| ZH | 147.0 | 42 | 1996 | III | 3 | 0 | >5.0 | >5.0 | No |
| ZJ | 147.9 | 25 | 1996 | III | 3 | 0 | 1.9 | 1.8 | Distant |
| ZL | 147.0 | 56 | 1996 | IV | 3 | 2a | >5.0 | >5.0 | No |
| ZN | 147.X | 76 | 1995 | IV | 3 | 3 | 4.0 | 4.0 | No |
| ZP | 147.2 | 56 | 1995 | IV | 4 | 0 | >5.0 | >5.0 | No |
| ZR | 147.0 | 53 | 1996 | IV | 4 | 1 | 1.2 | 0.9 | Local** |
| ZT | 147.0 | 51 | 1996 | IV | 4 | X | 1.6 | 1.3 | Local |
All patients were males
Site site localization; Date start of treatment; T, N classification given (all patients had M zero, i.e. no distant metastases); T surv. survival time (in years); Td-f time disease-free survival (in years); recurrence recurrence of tumour after therapy; Distant distant metastases; Local locoregional recurrence (primary tumour or lymph node); >, greater than, No no tumour recurrence (remained tumour-free); ?, classification unsure due to lack of data of N-stage (could be stage IV); X unknown; *, partial remission (calculated as locoregional); #, lost for follow-up; **, bronchosarcoma (calculated as locoregional)
Treatments and follow-up
All patients were treated locoregionally with external beam irradiation given for 7 weeks, five times 200 cGy per week. Dose prescription was performed according to the International Commission on Radiation Units and Measurements (ICRU) criteria for external beam irradiation (ICRU-report 50–62). IL-2 was injected intratumourally on five consecutive days in week 2, 4, and 6 of irradiation. Within 60 min after the daily irradiation, a dose of 3×104 IU IL-2 (Aldesleukin; Chiron, Amsterdam) diluted in 0.5 ml containing 5% dextrose and 0.1% serum albumin was applied transnasally using long needles. After decongestion of the nose (1% xylometazoline) the IL-2 was injected under direct vision (Storz endoscope) in the tumour, preferably in more than one site. Some patients needed local anaesthesia of the nasal mucosa as well. Patients were consulted weekly and possible side effects of therapy were discussed. Since usually only minor locoregional and no systemic side effects were noticed, no scoring system was used.
Statistical analysis
Survival curves were plotted with the Kaplan–Meier method and subjected to the Mantel–Haenszel log-rank tests. The average survival was analysed with Student’s t test. The ethical committee of the Utrecht Medical Centre accepted the protocol.
Results
Comparing treatments
Table 1 shows the individual characteristics of all IL-2 and radiotherapy treated and matched radiotherapy-only treated patients. Table 2 summarizes these data for the IL-2 and radiotherapy group, and for the radiotherapy-only group. These comparisons show that the groups were similar with respect to age of the patients, T and N stage. Interesting significant differences were found in the percentage of people having tumour recurrences and the 5-year disease-free survival (both average time and censored percentage surviving all 5 years). The 5-year overall survival and the first appearance of local or distant recurrences differed also, albeit not significantly.
Table 2.
Comparison of IL-2 treated group with the control group
| Radiotherapy only | IL-2 plus radiotherapy | p | |||
|---|---|---|---|---|---|
| Average | SD | Average | SD | ||
| Number of patients | 20 | 10 | |||
| Age (years) | 55 | 15 | 55 | 15 | NS |
| Year of treatment | 1990 | 5 | 1996 | 1 | 0.000 |
| Stagea | 3.8 | 0.4 | 3.7 | 0.5 | NS |
| T | 2.9 | 1.0 | 2.9 | 1.0 | NS |
| N | 1.2 | 1.0 | 1.3 | 1.2 | NS |
| Censored overall survival (years) | |||||
| Mean | 2.6 | 1.6 | 3.2 | 1.7 | NS |
| Median | 2.8 | >5.0 | |||
| Censored disease-free survival (years) | |||||
| Mean | 1.6 | 1.2 | 3.0 | 1.9 | 0.04 |
| Median | 1.5 | >5.0 | |||
| 5-years censored data in percentages | |||||
| Overall survival | 25% | 63% | NS | ||
| Disease-free survival | 8% | 63% | 0.01 | ||
| Tumour recurrenceb | 92% | 42% | 0.03 | ||
| Local recurrenceb, c | 48% | 27% | NS | ||
| Distant recurrenceb, d | 45% | 17% | NS | ||
SD standard deviation, NS not significant
aAverages of stage, T, and N are shown solely to indicate the similar distribution of disease progression in the historical controls and IL-2 treated patients
bPatients that died without tumours are censored
cPatients with distant recurrences are censored
dPatients with local recurrences are censored
Overall survival
Overall survival was defined as the percentage of patients being alive and patients who died without a detectable tumour were censored. The median censored overall survival is 2.8 years for control-treated patients, and extends beyond 5 years for IL-2 treated patients. The mean values are 2.6 and 3.2 years, respectively. Figure 1 shows that censored 5-years overall survival is 25% in the controls and 63% in the IL-2 treated patients. The hazard ratio for overall survival of receiving additional IL-2=0.37 (95% confidence interval 0.16–1.18; p=0.10). This censored overall survival of control patients is in line with published data of 30% and 25% (stage III and IV, respectively) [13].
Fig. 1.
Censored overall survival of control patients (n=20; open squares) and IL-2 treated patients (n=10; closed squares). Censored data are indicated by a plus
Disease-free survival
Disease-free survival was defined as the percentage of patients being alive without having any form of cancer; and patients who died without a detectable tumour were censored. The median censored disease-free survival is 1.5 years for control-treated patients, and extends beyond the 5 years of this study for IL-2 treated patients. The mean values are 1.6 and 3.0 years, respectively. Figure 2 shows that after 5 years the censored disease-free survival is 8% in the controls and 63% in the IL-2 treated patients. The hazard ratio for disease-free survival is 0.25 (95% confidence interval 0.13–0.79; p=0.014). To our knowledge, data concerning 5-year disease-free survival of NPC have not been published. After 5 years of follow-up the average disease-free survival of control patients was 1.57 years and of the IL-2 treated patients 3.04 years (p<0.05).
Fig. 2.
Censored disease-free survival of control patients (n=20; open squares) and IL-2 treated patients (n=10; closed squares). Censored data are indicated by a plus
Validity of historical controls
Table 2 shows that our controls were treated at a significantly different time-point than our test patients. A putative problem is the evolution of radiotherapeutic equipment and treatment schedules at these different time-points. Therefore, the difference in year of treatment (Table 2), might contribute to the differences found in (disease-free) survival. Therefore, we selected from our database all stage III–IV patients in the period 1982–2001 treated with radiotherapy alone and established the disease-free survival. Figure 3 shows that during this period the disease-free survival was always about 1.5 years and did not increase.
Fig. 3.
Censored disease-free survival of NPC patients receiving standard radiotherapy only. The used historical controls (1982–1995) and all stage III–IV patients in our database from 1995–2001 are included. The data show a small decrease in time of disease-free survival (y = −0.0453x + 91.794) with a low correlation (R2=0.0548) that lacks clinical significance
Tumour recurrences
Tumour recurrences decreased significantly in patients treated with radiotherapy and IL-2, versus radiotherapy only. Both local tumour recurrences and the development of distant metastases were decreased, but neither was significant due to the low numbers of patients (Table 2). A study using a larger number of patients will be required to investigate the relative effects of local IL-2 on either type of recurrence. If IL-2 stimulates anti-tumour immunity, increased anti-tumour immunity would inhibit both local recurrence and the development of distant tumour metastases.
Side effects
Side effects due to IL-2 injections were minimal and only regional. Sometimes pain was felt at the injection site. Occasionally scarring was formed between the concha inferior and the lateral nasal mucosa without causing complaints. The scarring was not at the site of injection, and probably due to mechanical damage. One patient had an ulcer with a granulocyte infiltrate at the roof and the back of the nasopharynx. One patient became blind 2 years after treatment due to a bilateral neuritis of the optical nerve. No systemic side effects were observed.
Discussion
Side effects
We did not observe adverse toxic side effects in the IL-2-treated patients. Only local side effects were observed, which were most likely due to irradiation and/or mechanical damage with the needle. The local ulcer could be caused by irradiation, IL-2 and/or injection. One patient went blind 2 years after treatment due to a bilateral neuritis of the optical nerve. This very rare complication was reported after the use of irradiation doses higher than 60 Gy to the optical nerve [26]. Our patients received 70 Gy, and therefore, we think that this blindness was due to irradiation and not due to IL-2.
The very limited local side effects and the absence of systemic side effects are probably due to the local application of the extremely low total dose of IL-2 (15×3×104=4.5×105 IU IL-2). Higher doses of local IL-2 have been given in a single injection in mice (16×106 IU), dogs (1×106 IU) and cows (2×106 IU) without observed side effects [7].
Therapeutic results
Both control and IL-2 treated patients with end-stage III/IV NPC were treated with external beam irradiation. The results show that the addition of IL-2 significantly improved censored disease-free survival time of the patients. Local IL-2 therapy has been reported to be very effective in experimental end-stage cancer in mice [22] and in human patients [18, 19].
Systemic versus local IL-2
Systemic IL-2 has been used in combination with radiotherapy for treatment of NPC patients [4]. Chi et al. [4] injected approximately 15 times 5 million IU IL-2 intravenously per week. The total dose given was about 150 million IU IL-2 in 2 weeks, and this was repeated again after 5 or 6 weeks. Thus, the total dose of IL-2 by Chi et al. [4] was more than 600 times higher than our dose of IL-2. Our methods and results contrast sharply with this study of Chi et al., who found no therapeutic effect, but observed major side effects, including one treatment-related death.
We think that local IL-2 therapy is more effective than systemic IL-2 therapy [10, 22], because intratumoural IL-2 induces extensive vascular leakage at the site of injection [1, 6]. Locoregional IL-2 injected around the draining lymph nodes was not effective in head and neck cancer [5]. Repeated local injections of IL-2, as has also been done in our NPC patients, causes repeated vascular leakage inside the tumour, which improves the therapeutic efficiency [20]. The intratumoural vascular leakage is directly associated with the increase in therapeutic efficiency of intratumoural versus peritumoural IL-2 [15].
Remarkably, the major side effect of systemic IL-2 is the systemic vascular leakage syndrome, which can be fatal [11, 14, 27]. Vascular leakage diminished the circulation in tumour tissue [1, 6, 20], which causes tumour cell death [15]. After local IL-2 therapy, this side effect is no problem for patients as only the treated tumour will be affected by decreased blood flow. In case of systemic IL-2, vascular leakage causes liver necrosis [14]. Moreover, systemic IL-2 diminished the microcirculation [12] and causes oedema in the lungs [27], both contributing to hypotension and hypoxemia. [11].
Chemotherapy
The treatment of advanced NPC has undergone several developments since the start of this study in 1995. A major improvement is the addition of chemotherapy to radiotherapy. In a Chinese clinical phase III study, the 5-year progression-free survival increased from 53% to 72% in patients treated with radiotherapy alone versus concurrent chemo-radiotherapy [21].
A direct comparison of the results of Lin et al. with our findings is difficult, since the histological grading of the Chinese tumour cases differs considerably with ours. Chinese patients most commonly have WHO type 3, whereas Dutch people often have WHO type 1 NPC [23]. The prominence of WHO type 1 in our patients implies a significantly worse 5-year survival, i.e. survival in type 1 is 6–37% versus 51–63% survival in type 2 and 3 [23, 25]. Clearly, a direct comparison between therapies applied in patients with different histological tumour types is impossible.
Chemotherapy is especially effective in cases with distant failure after radiotherapy. Distant failure occurs more often in WHO type 2 and 3 [17], but they have a better local control rate after treatment with radiotherapy [3]. Thus chemotherapy seems most appropriate (and probably most effective) in these undifferentiated histological types of NPC [24]. The side effects we described after local IL-2 are minimal compared to the side effects of chemotherapy [24].
Conclusions
This paper describes successful treatment of a highly malignant tumour. This treatment is cheap and—after obtaining the necessary technical skills—does not further require expensive and complex interventions. Local IL-2 treatment does not entail additional hospitalization or a complex laboratory work-up.
Local IL-2 treatment (in addition to radiotherapy) of stage III/IV, NPC significantly improved the disease-free survival when compared to historical controls.
To our knowledge, this is the first report showing that local IL-2 therapy has large (from 8% to 63%) and statistically significant effects on the censored 5-year disease-free survival in advanced human cancer. These data call for a randomized prospective clinical phase III study to confirm these findings in a more controlled setting.
Acknowledgements
We thank Nanny Scheepmaker for data management. IL-2 was a kind gift from Chiron (Amsterdam).
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