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Japanese Journal of Clinical Oncology Pages 729-732

The Significance of Soluble Interleukin-2 Receptor in Monitoring Disease Relapse in Patients with Nasopharyngeal Cancer
Introduction
Materials And Methods
   Subjects
   Serum Collection
   Enzyme-linked Immunoabsorbant Assay for sIL-2R[alpha]
   Statistical Analysis
Results
   No Difference of sIL-2R[alpha] Seen Between Before and After Definitive Radiotherapy
   Increased sIL-2R[alpha] Concentration will be Found in Systemic Relapse but not in Locoregional Recurrence
Discussion
Acknowledgement
References
The Significance of Soluble Interleukin-2 Receptor in Monitoring Disease Relapse in Patients with Nasopharyngeal Cancer

The Significance of Soluble Interleukin-2 Receptor in Monitoring Disease Relapse in Patients with Nasopharyngeal Cancer

Le-Jung Wu1,2, Kuang Y. Chen1,2, Kwan-Hwa Chi1,2, Sen-Yu Chen1,2, Mien-Jung Liang1,2, Chen-Ying Shiau1,2, Ling-Wei Wang1,2, Yu-Ming Liu1,2, Kuan-Chih Chow1 and Sang-Hue Yen1,2

1Cancer Center, Veterans General Hospital-Taipei and 2National Yang-Ming University, Taipei, Taiwan

Background: Soluble interleukin-2 receptor [alpha] (sIL-2R[alpha]) is a well-known indicator of T-cell activation noted to be increasing in nasopharyngeal cancer. However, the significance of sIL-2R[alpha] in monitoring disease relapse is unclear. This study was initiated to address this issue.
Methods: Serum of 56 patients with NPC, which underwent either primary, salvage, or palliative treatments, from 1992 to 1993 at the Cancer Center, Veterans General Hospital-Taipei, were collected from our serum bank. According to their disease status at the time of study, at least two years after last treatments, the 56 patients were divided into four groups. The remission group represented those in remission at the time of study (n=24). The metastasis group represented those with distant metastasis present at the time of study (n=17). The recurrence group represented those with locoregional recurrence present at the time of study (n=11). The combined group represented those with locoregional recurrence as well as distant metastasis (n=4). The seral sIL-2R[alpha] concentrations of the 56 NPC patients were determined with enzyme-linked immunoabsorbent assay. The combined group was excluded in our statistical analysis. We performed statistical analysis on the differences of paired serum sIL-2R[alpha] concentrations between different periods of the diseases. The first analysis was on the differences of sIL-2R[alpha] concentrations between diagnosis and post-radiotherapy periods for 13 out of 24 patients in the remission group and 7 out of 11 patients in the recurrence group. The second analysis was on the differences of sIL-2R[alpha] concentration between follow-up before detection-of-relapse and after detection-of-relapse for 5 out of 17 patients in the metastasis group and six out of 11 patients in the recurrence group.
Results: The first statistical analysis revealed no significant differences of sIL-2R[alpha] concentrations for the remission group (P=0.946) and the recurrence group (P=0.156) between diagnosis and post-radiotherapy periods. The second statistical analysis revealed no significant differences of sIL-2R[alpha] concentrations between before and after detection-of-relapse for the recurrence group, neither (P=0.438). The results for the metastasis group were different. The sIL-2R[alpha] concentrations were shown to increase after the detection of metastasis for the 5 paired samples from the metastasis group, although the Wilcoxon signed ranks test on the differences only showed borderline significance (P=0.063).
Conclusions: Our findings show that sIL-2R[alpha] would be of no value in monitoring the development of locoregional recurrence but might be useful in monitoring distant metastasis. Although our current limited data did not provide strong support for the role of sIL-2R[alpha] in monitoring metastasis, it might be delineated in the future by collecting more data.

Key words: ELISA - locoregional recurrence - metastasis - nasopharyngeal cancer - sIL-2R[alpha]

INTRODUCTION

Interleukin-2 (IL-2), a glycoprotein secreted by activated T cells, is a growth factor stimulating the growth of T cells, NK cells, and B cells (1). The biological effects of IL-2 is mediated by binding to the IL-2 receptor (IL-2R), which has three component proteins [alpha], [beta] and [gamma]. On the membrane of resting T cells, only the low-affinity IL-2R[beta][gamma] is expressed. Activation of T cells stimulates the synthesis of cytosolic [alpha] chain and, subsequently, the formation of membrane-bound heterotrimeric receptor IL-2R[alpha][beta][gamma]. The membrane-bound IL-2R[alpha][beta][gamma], contrary to IL-2R[beta][gamma], has a high affinity for IL-2 and allows T cells to respond to very low concentrations of IL-2 (2). The expression of [alpha] molecule therefore is a useful marker for T cell activation (3). Soluble IL-2R[alpha] (sIL-2R[alpha]), a truncated IL-2R[alpha] protein without transmembrane domain being released into serum after T cell activation, could be detected in serum by enzyme-linked immunoabsorbant assay (ELISA) (2). The sIL-2R[alpha] levels measured by ELISA had been shown to increase in infection, autoimmune diseases (reviewed in (4)) and malignancy (reviewed in (4,5)). The increased levels noted in malignancy would indicate, in addition to T-cell activation, the disease activity and response to treatment as well [4].

As seen in many malignant diseases including nasopharyngeal carcinoma (NPC), which is highly prevalent in the Chinese population, sIL-2R[alpha] concentrations were reported to increase in active disease and the degrees of increase were correlated to the clinical stages at the presentation (6,7). However, the significance of sIL-2R[alpha] as an indicator in monitoring the development of local recurrence and systemic relapse is still unknown. This study was initiated to address the question.

MATERIALS AND METHODS

Subjects

Serum was collected from our serum bank, where 56 NPC patients were identified to receive treatment from 1992 to 1993 at the Cancer Center, Veterans General Hospital-Taipei. The 56 patients were composed of two populations. One population consisted of 41 patients who were diagnosed initially and treated definitively from 1992 to 1993. The other consists of 15 patients diagnosed initially and treated definitively before 1992 and were treated subsequently salvagely or palliatively for relapse from 1992 to 1993. There were 45 males and 11 females. The distributions of initial stage, according to the staging manual published by the American Joint Committee on Cancer, were as follows; one in stage I, 8 in stage II, 30 in stage III, 17 in stage IV.

According to the disease status at the time of the study, all patients were divided into four groups. The remission group (n=24) represented those in remission. The metastasis group (n=17) indicated those with distant metastasis only. The recurrence group (n=11) specified those with locoregional recurrence only. The combined group (n=4) patients were those with both locoregional recurrence and metastasis. Disease relapse was diagnosed with pathological and/or imaging evidence. Locoregional recurrence was defined to be disease relapse at nasopharynx and/or neck. Distant metastasis was defined to be relapse at other sites.

Serum Collection

For patients treated definitively, the blood samples were collected intermittently from the time of initial diagnosis, i.e. 1992 to 1993, to the time of study initiation. For patients treated salvagely or palliatively, the blood samples were collected intermittently from the time of initial diagnosis i.e. before 1992, to the time of study initiated. Serum was collected from the clotted whole blood after the centrifugation 1500g for 10 min and was stored at -70°C before ELISA assay.

Enzyme-linked Immunoabsorbant Assay for sIL-2R[alpha]

Commercial ELISA kit was used to measure sIL-2R[alpha] concentrations (R&D Systems, Minneapolis, USA). All samples were assayed as recommended by the manufacturer. The concentrations were calculated according to the function of the best-fit curve derived from the plot of concentrations of standards versus selected optical densities. The function of the best fit curve was determined by computer program, SigmaPlot.

Statistical Analysis

The combined group was excluded to simplify our analysis. The blood samples spread over the whole period of disease. For the remission group, the periods were ranging from the time of diagnosis to during the follow-ups. For metastasis and recurrence groups, the periods ranged from the time of diagnosis, during follow-ups before detection-of-relapse, and during the follow-ups after detection-of-relapse.

The first statistical analysis was done to detect the differences of sIL-2R[alpha] concentrations between the diagnosis and during the post-radiotherapy (3 to 7 months after diagnosis) periods. The data of 13 patients from the remission group and 7 patients from the recurrence group with paired samples were available for analysis. The Wilcoxon signed ranks test comparing the differences of sIL-2R[alpha] concentrations between paired samples from the two periods was done for the two groups respectively. The second analysis was done to detect the differences of sIL-2R[alpha] concentrations between those paired samples from the follow-up before detection-of-relapse and from after detection-of-relapse for the metastasis group as well as the recurrence group patients. The data of five patients from the metastasis group and six patients from the recurrence group with paired samples were available for analysis. The Wilcoxon signed rank test comparing the differences of sIL-2R[alpha] concentrations between the paired samples from the two periods was done for the two groups respectively.

RESULTS

No Difference of sIL-2R[alpha] Seen Between Before and After Definitive Radiotherapy

The median (range) of sIL-2R[alpha] concentrations of the pre- and post-RT period from the paired samples of the 13 patients in the remission group were 1173.11 (609.39, 2345.54) pg/ml and 1274.07 (338.20, 1969.62) pg/ml, respectively. There is no statistical difference of sIL-2R[alpha] levels between these two periods as shown by the Wilcoxon signed ranks test (P=0.946, Table 1). Besides, the median of sIL-2R[alpha] concentrations of pre- and post-RT periods from the paired samples of the 7 patients in the recurrence group were 1502.20 (600.70, 1925.03) pg/ml and 952.95 (782.22, 1778.16) pg/ml, respectively. Similarly, there is no statistical difference of sIL-2R[alpha] between these two periods as shown by the Wilcoxon signed ranks test (P= 0.156, Table 1).

Table 1. Wilcoxon signed ranks test for analysis of the sIL-2R[alpha] difference between the time at diagnosis date and post-radiotherapy period
  (A) Remission group
(n=13)		 (B) Recurrence group
(n=7)		 (A)+(B)
(n=20)
Diagnosis Post-RT Diagnosis Post-RT Diagnosis Post-RT
Media 1173.11 1274.07 1502.20 952.95 1320.10 1133.56
(sIL-2R[alpha] concentration)
P-value 0.946 0.156 0.388

Table 2. Wilcoxon signed ranks test for analysis of the sIL-2R[alpha] difference between disease-free period and disease-relapsed period
  Metastasis group
(n=5)		 Recurrence group
(n=6)
Disease-free Relapse Disease-free Relapse
Median 909.87 2403.7 931.41 1028.61
(sIL-2R[alpha] concentration)
P-value 0.063 (borderline) 0.438

The collective data of these 20 patients in the two groups also indicated no significant differences of the sIL-2R[alpha] between the two periods as shown by the Wilcoxon signed ranks test (P= 0.388, Table 1).

Increased sIL-2R[alpha] Concentration will be Found in Systemic Relapse but not in Locoregional Recurrence

To find out the significance of sIL-2R[alpha] for monitoring disease relapse, we embarked on statistical analysis of the differences of sIL-2R[alpha] concentrations between before and after detection-of-relapse periods. The results were illustrated in Table 2. The median of sIL-2R[alpha] concentrations of before and after detection-of-metastasis periods from the paired samples of the five patients in metastasis group were 909.87 (679.84, 1597.77) pg/ml and 2403.7 (1471.29, 3646.41) pg/ml, respectively. The Wilcoxon signed ranks test showed borderline significances (P=0.063, Table 2). Besides, the median of sIL-2R[alpha] concentrations of before and after detection-of-recurrence periods from the paired samples of the six patients in the recurrence group were 931.41 (782.22, 1073.96) pg/ml and 1028.61 (697.70, 1407.91) pg/ml (Table 2), respectively. The Wilcoxon signed rank test showed no statistical difference either (P=0.438, Table 2).

DISCUSSION

Immune reaction is a well-known feature seen in NPC. The tumor is markedly infiltrated by lymphocytes (8). In vitro studies on these tumor-infiltrating lymphocytes (TIL) showed that these were predominantly activated T cells expressing IL-2R (9) with decreased cytotoxic and mitogenic responses (10,11). Besides, the systemic immune responses to NPC included elevated seral antibodies against Epstein-Barr Virus (EBV) antigens, e.g. viral capsid antigen, early antigen and nuclear antigen (12-14); and the selective expansion of the CD8+ subset of T cells (15). In contrast to TILs, the in vitro investigation on peripheral blood lymphocytes (PBL) from NPC patients displayed normal mitogenic response and impaired cytotoxicity (16). These results imply that the existence of local disease would induce tumor immunogenicity. Since sIL-2R[alpha] is an indicator of T cell activation (4), sIL-2R[alpha] levels had also been found to be increased in patients with NPC and the degree of increase to correlate with disease activity at presentation (6,7).

However, in our study, we could not detect significant changes of the sIL-2R[alpha] concentrations whether local disease was eradicated or not with radiotherapy (remission group in Table 1 and recurrence group in Table 2). It might suggest that T cell activation, once initiated by the presence of a tumor, will not be desensitized by the shrinkage of local tumor. On the contrary, the metastatic tumor might display a different expression of sIL-2R[alpha] from locoregional disease, i.e. an increase of sIL-2R[alpha] after the detection of metastasis. Therefore, we wondered if the nature of metastatic tumor has changed in some way compared to primary local tumor.

Our preliminary observation on biopsied and resected metastatic tumors showed less extensive degree of TIL infiltration in metastatic tumor (data not shown). Since TILs in NPC are predominated by activated T cell expressing IL-2R (9), it is highly probable that the T cell activation in metastatic tumor is reduced compared to that in primary tumor. The contradictory finding of reduced local T cell activation of and increased sIL-2R[alpha] levels in metastatic tumor could be explained that the metastatic tumor cells have evolved to be able to secrets sIL-2R[alpha]. In other words, NPC metastatic tumor is a major, if not predominant, source of sIL-2R[alpha] in addition to activated T cells. This hypothesis could be supported by the studies of McMillan et al. and Yasumura et al. (17,18). In their reports, they had shown that, in vitro, sIL-2R[alpha] expression was found in solid tumor cell and carcinoma cell lines. To validate this presence of tumor secretion of sIL-2R[alpha], an in vivo investigation employing in situ hybridization to identify the mRNA of sIL-2R[alpha] is now in progress in our laboratory.

Imaging study currently is the standard method monitoring the development of relapse from NPC. Its high cost and/or radiation hazard preclude frequent whole-body imaging work-up. Consequently, due to infrequent image monitoring, relapsed tumor usually would become so advanced at the time of detection that it could hardly be eradicated. Therefore, finding a safe and economical method capable of early detection of relapse is imperative to improve the treatment result with early intervention. Measuring the concentration differences of serum tumor marker would be one good method to achieve this goal.

It is interesting to find, in our study, that different profiles of sIL-2R[alpha] changes were seen in the locoregional versus systemic relapse groups. First, there was insignificant difference of sIL-2R[alpha] concentrations noted before and after detection of locoregional recurrence. It implies that sIL-2R[alpha] would be of no value for monitoring local recurrence. Secondly, in contrast to local recurrence, an obvious increase of sIL-2R[alpha] concentrations was noted after the detection of systemic metastasis (Figure 1). Unfortunately, due to our limited data, the obvious differences of sIL-2R[alpha] concentrations had been only shown borderline significance with Wilcoxon signed ranks test. However, we think that it is probable that sIL-2R[alpha] might be effective as a tumor marker monitoring the development of metastasis in NPC. Whether there is significant differences of sIL-2R[alpha] concentrations in NPC metastasis could be validated with more data in the future.


Figure 1. The difference of sIL-2R[alpha] levels in patients with relapses between the disease-free and relapse periods.

Acknowledgement

The authors would like to thank Ms Jing Yi Chao for help in statistical analysis, Ms Shu-cheng Yang for her invaluable statistical analysis and manuscript preparation, and to Ms Shuan-lan Chou for her excellent work on ELISA. This study was supported by grant DOH87-HR-524 from The Department of Health of the R.O.C.

References

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4. Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology, function, and clinical application. Ann Int Med 1990;113:619-27.

5. Lissoni P, Barni S, Rovelli F, Viviani S, Maestroni GJ, Conti A, et al. The biological significance of soluble interleukin-2 receptors in solid tumors. Eur J Cancer 1990;26:33-6. MEDLINE Abstract

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9. Herait P, Ganem G, Lipinski M, Carlu C, Micheau C, Schwaab G, et al. Lymphcyte subsets in tumour of patients with undifferentiated nasopharyngeal carcinoma: presence of lymphocytes with the phenotype of activated T cells. Br. J. Cancer 1987;55:135-9. MEDLINE Abstract

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11. Ferradini L, Miescher S, Stoeck M, Busson P, Barras C, Cerf-Bensussan N, et al. Cytotoxic potential despite impaired activation pathways in T lymphocytes infiltrating nasopharyngeal carcinoma. Int J Cancer 1991;47:362-70. MEDLINE Abstract

12. Liebowitz D, Kieff E, Epstein-Barr virus. In: Roizman B, Whitley RJ, Lopez C, editors. The Human Herpes Viruses. New York: Raven Press, 1993:107-72.

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16. Lakhdar M, Ellouz R, Kammoun H, Ben H, Tira S, Khedhiri N, et al. Presence of in vivo-activated T-cells expressing HLA-DR molecules and IL-2 receptors in peripheral blood of patients with nasopharyngeal carcinoma. Int J Cancer 1987;39:663-9. MEDLINE Abstract

17. McMillan DN, Kernohan NM, Flett ME, Heys SD, Deehan DJ, Sewell HF, et al. Interleukin-2 receptor expression and interleukin 2 localization in human solid tumor cells in situ and in vitro: evidence for a direct role in the regulation of tumor cell proliferation. Int J Cancer 1995;60:766-72. MEDLINE Abstract

18. Yasumura S, Lin WC, Weidmann E, Hebda P, Whiteside TL. Expression of interleukin 2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin 2. Int J Cancer 1994;59:225-34. MEDLINE Abstract

Received August 28, 1998; accepted October 6, 1998
For reprints and all correspondence: Sang-Hue Yen, Cancer Center, Veterans General Hospital-Taipei No. 201 Sec. 2 Shih-Pai Rd. Taipei, 11217 Taiwan, Republic of China. E-mail: shyen{at}vghtpe.gov.tw
Abbreviations: sIL-2R[alpha], soluble interleukin-2 receptor [alpha]; IL-2, interleukin-2; IL-2R, IL-2 receptor; ELISA, enzyme-linked immunoabsorbant assay; NPC, nasopharyngeal carcinoma; TIL, tumor-infiltrating lymphocytes; EBV, Epstein-Barr Virus; PBL, peripheral blood lymphocytes

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Copyright©Japanese Journal of Clinical Oncology, 1998.
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