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Keywords:

  • renal cancer;
  • immunotherapy;
  • tumour stage;
  • grade;
  • T-lymphocytes;
  • C-reactive protein;
  • albumin;
  • interleukin;
  • cancer-specific survival

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

OBJECTIVE

To examine the longitudinal relationship between the systemic inflammatory response, circulating T-lymphocyte subpopulations, interleukin-6 and -10 in patients undergoing immunotherapy for metastatic renal cancer, as the inflammation-based Glasgow Prognostic Score (GPS) provides additional prognostic information in patients with advanced renal cancer, but the basis of the relationship between the systemic inflammatory response and poorer survival is not clear, and nor is the effect of immunotherapy on related variables.

PATIENTS AND METHODS

The study included 23 patients with metastatic renal cancer and starting immunotherapy. Samples of blood were drawn for routine laboratory analysis and to quantify cytokines using enzyme-linked immunosorbent assays before immunotherapy, and repeated after 2 weeks of treatment.

RESULTS

Most patients had a good performance status, favourable or intermediate Memorial Sloane-Kettering Cancer Center (MSKCC) risk scores, and with elevated C-reactive protein (>10 mg/L), GPS (1 or 2), interleukin-6 (>4 pg/mL) and interleukin-10 (>10 pg/mL). Patients who completed one cycle of immunotherapy were more likely to have a normal MSKCC (P < 0.05) or GPS (P < 0.05) scores, whilst the percentage of lymphocytes was lower (P < 0.05). The MSKCC and the GPS scores did not alter significantly during one cycle of immunotherapy. Similarly, leukocyte counts, CD4+ and CD8+ T-lymphocytes, interleukin-6 and -10 concentrations did not change significantly.

CONCLUSIONS

The pretreatment systemic inflammatory response and its related lymphopenia are important in determining the tolerance to immunotherapy in patients with metastatic renal cancer. Immunotherapy is not associated with changes in circulating T-lymphocytes, nor the systemic inflammatory response.


Abbreviations
SIR

systemic inflammatory response

CRP

C-reactive protein

GPS

Glasgow Prognostic Score

PS

performance status

ECOG

Eastern Cooperative Oncology Group

FITC

fluorescein isothiocyanate

PE

phycoerythrin

APC

antigen presenting cells

MSKCC

Memorial Sloan-Kettering Cancer Center

LDH

lactate dehydrogenase.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

RCC, although the 12th most common cause of cancer death, is one of the most lethal urological cancers. Each year in the UK, there are ≈3500 new cases and ≈30% of patients present with metastases, with a further 40% developing metastases after a potentially curative resection. The life-expectancy of patients with metastatic RCC is poor, with a median survival of ≈9 months [1].

Immunotherapy has been the traditional mainstay of treatment for patients with metastatic disease, with a survival advantage of ≈3 months reported from four randomized controlled trials [2–5]. However, the side-effects of immunotherapy are significant and include a ‘flu–like syndrome, fatigue, fever, anorexia, myalgia, arthralgia, dry skin and mucous membranes, mental state changes and depression.

It was previously shown that the systemic inflammatory response (SIR), as evidenced by an elevated level of C-reactive protein (CRP), predicts poor survival, independent of performance status, in patients receiving either interleukin-2- [6] or α-interferon-based immunotherapy [7]. More recently, we reported that an inflammation-based prognostic score (the Glasgow Prognostic Score, GPS), based on an elevated CRP level and hypoalbuminaemia, compares favourably with established scoring systems [8] in predicting cancer survival in patients undergoing immunotherapy [9].

However, the basis of the relationship between the SIR and poorer response rates and poorer survival in these patients is not clear. One explanation is that the SIR is associated with an impaired T-lymphocytic response to the tumour [10,11]. Indeed, mediators of the SIR, e.g. interleukin-6 and interleukin-10 [12], have been implicated in the reduced response rates and reduced survival in patients receiving interleukin-2 based immunotherapy [6,13].

To our knowledge the longitudinal relationships between the T-lymphocytic response and such inflammatory mediators have not been previously examined in patients undergoing immunotherapy for RCC. The aim of the present study was to examine the longitudinal relationship between the SIR, circulating T-lymphocyte subpopulations, and interleukin-6 and -10 in patients undergoing α-interferon-based immunotherapy for metastatic RCC.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Patients who, on the basis of clinical findings and CT of chest, abdomen and pelvis, were diagnosed with metastatic RCC and who were due to start out-patient immunotherapy, were included in the study between May 2005 and February 2006. The out-patient regimens were either s.c. α-interferon given at 10 MU three times weekly on a 12-weekly basis, or a modified regimen of Atzpodien et al.[14], comprising s.c. α-interferon, s.c. interleukin-2 for 5 weeks, with an additional 3 weeks of 5-fluorouracil given i.v., both defined as one cycle of treatment. No patients in the study had received systemic therapy or radiotherapy before starting immunotherapy.

Patients were staged pathologically according to the 1997 TNM classification of renal tumours [15]. Tumours were graded according to criteria set out by Fuhrman et al.[16]. Clinical stage and performance status (PS, Eastern Cooperative Oncology Group, ECOG) were recorded before starting treatment. The Research Ethics Committee of North Glasgow NHS Trust approved the study.

Blood samples were collected before starting immunotherapy for routine laboratory analysis of haemoglobin, leukocyte and lymphocyte counts, lactate dehydrogenase, albumin and CRP. The limit of detection of the assay for CRP was <6 mg/L. The coefficients of variation of these methods, over the range of measurements, was <10%, as established by routine quality control procedures. Preoperative blood samples were also analysed for circulating lymphocyte subsets, analysed using flow cytometry (FACScantoTM, BD Bioscience, Oxford, UK) equipped with a 488-nm argon laser and a 635-nm red diode laser using FACScanto software. The monoclonal antibodies used were CD3 fluorescein isothiocyanate (FITC)/CD4 phycoerythrin (PE)/CD3 PenCPCy 5.5/CD8 antigen presenting cells (APC) and CD3 FITC/CD19 PE/CD16+ 56 PenCPCy 5.5/CD45 APC (BD Bioscience Oxford, UK). Absolute counts were from one platform using TruCOUNT tubes (BD Bioscience). The results were expressed as total number and percentage of positive cells counted.

A further blood sample was taken, centrifuged, and the serum stored at −80 °C before analysis of interleukin-6 and -10. Circulating concentrations of these cytokines were measured using an ELISA technique (Quantikine, R&D Systems Europe Ltd, Abingdon, UK). The minimum detectable concentrations were 2 pg/mL for interleukin-6 and 4 pg/mL for interleukin-10; inter- and intra-assay variability was <10% for both assays. Cytokine concentrations below the threshold of sensitivity of the respective assays were expressed as equal to this threshold.

The blood sampling and analyses were repeated 2–4 weeks after starting treatment, at the first outpatient review. The PS was recorded, with any dose reductions or treatment breaks. CT was used after 8–12 weeks of immunotherapy to assess the disease response or progression.

The Memorial Sloan-Kettering Cancer Center (MSKCC) scoring system [8] was used to stratify patients into favourable-, intermediate- and poor-risk groups. In this system an abnormal haemoglobin, calcium or lactate dehydrogenase (LDH) level three times greater than the upper limit of normal, Karnofsky PS <80% and the absence of previous nephrectomy are considered as risk factors. Patients with no risk factors are classified as favourable-, with one or two as intermediate-, and with three or more as poor-risk.

The GPS was constructed as previously described [17,18]. Briefly, patients with both an elevated CRP level (>10 mg/L) and hypoalbuminaemia (<35 g/L) were allocated a score of 2; those in whom only one of these biochemical abnormalities was present were allocated a score of 1, and those in whom neither of these abnormalities was present were allocated a score of 0.

The data are presented as the median (range) and the groups of patients compared using contingency table analysis (chi-square) as appropriate. The laboratory variables haemoglobin, leukocyte and lymphocyte counts and LDH were grouped using standard thresholds [19], as were the cytokines interleukin-6 and -10 [13]. Where appropriate, data were tested for statistical significance using the Mann–Whitney U-test and the Wilcoxon signed-rank test. Survival (cancer-specific) was analysed for the group variables using the Cox proportional hazard model. Deaths up to the end of June 2007 were included in the analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

The characteristics of 23 patients with advanced RCC and receiving immunotherapy are shown in Table 1; most were men, aged >60 years and had had a previous nephrectomy. Most were favourable or intermediate MSKCC risk, and had an ECOG PS of ≤1, had haemoglobin, adjusted calcium, LDH, albumin, leukocyte and lymphocyte counts in the normal range. By contrast, most patients had an elevated CRP level (>10 mg/L), GPS (1 or 2), interleukin-6 (>4 pg/mL) and -10 (>10 pg/mL). Of the seven patients with hypoalbuminaemia, six had an elevated CRP level.

Table 1.  The baseline characteristics and cancer-specific survival of 23 patients undergoing α-interferon-based immunotherapy for metastatic RCC; univariate analysis
Variablen patientsMean (95% CI) survival, monthsP
  • *

    One sample could not be analysed due to incorrect sampling technique.

Age, years
 ≤60 914.2 (7.5–20.8)0.566
 >601412.4 (7.5–17.4) 
Male1813.6 (8.8–18.3)0.993
Female 5 11.4 (3.7–19.0) 
Previous surgery
 Curative1317.4 (12.1–22.7)0.013
 Cytoreductive 313.0 (3.2–22.8) 
 None 7 5.4 (1.9–8.9) 
MSKCC score
 Favourable 920.4 (15.5–25.4)<0.001
 Intermediate12 9.6 (4.9–14.3) 
 Poor 2 1.5 (0–3.1) 
GPS
 0 818.4 (13.3–23.5)0.045
 1 913.2 (6.9–19.5) 
 2 6 5.1 (0.5–10.2) 
Leukocyte count, 109/L
 ≤11.02114.0 (9.7–18.2)0.095
 >11.0 2 5.4 (0–14.6) 
% total lymphocytes
 20–40 1115.5 (9.7–21.3)0.558
 12.0–19.9 8 11.8 (5.5–18.1) 
 0–11.9 4 9.6 (0.1–19.0) 
CD4+ T lymphocytes, 109/L*
 <0.54 1113.8 (7.8–19.8)0.840
 ≥0.54 1113.6 (7.8–19.4) 
CD8+ T-lymphocytes, 109/L*
 <0.27 815.9 (8.4–23.4)0.353
 ≥0.271412.3 (7.5–17.2) 
Interleukin-6, pg/mL
 ≤351616.2 (11.7–20.6)0.020
 >35 7 6.4 (0.4–12.3) 
Interleukin-10
 ≤351315.2 (10.1–20.30.289
 >3510 9.0 (4.3–13.8) 
Chemotherapy:
 α-interferon19 11.9 (7.7–16.1)0.151
 α-interferon plus 419.6 (9.8–29.3) 
Chemotherapy completed
 No12 7.2 (2.4–11.9)0.005
 Yes 1119.6 (15.7–23.6) 

The relationship between baseline clinicopathological criteria and cancer-specific survival of the 23 patients are also shown in Table 1. The minimum follow-up was 17 months and the median follow-up of survivors was 23 months. Fifteen (65%) patients died during the follow-up. On univariate survival analysis, absence of previous nephrectomy (P < 0.05), poor MSKCC score (P < 0.001), high GPS (P < 0.05), leukocyte count (P < 0.10) and interleukin-6 level (P < 0.05), and the failure to complete one cycle of chemotherapy (P < 0.01), were significantly associated with poorer survival. When the GPS and interleukin-6 were compared in a multivariate survival analysis, only the GPS (hazard ratio 2.23, 95% CI 1.09–4.57, P = 0.029) was independently associated with survival.

The patients were grouped according to whether or not they completed one cycle of treatment with no dose reduction of their immunotherapy regimen (Table 2). The patients who completed immunotherapy were more likely to have a favourable MSKCC score (P < 0.05) or GPS (P < 0.05) in the normal range. Also, the percentage of lymphocytes was lower (P < 0.05) and there was a trend to a higher interleukin-6 level (P < 0.10) in those patients who did not complete immunotherapy. There were significant inverse correlations between the percentage of lymphocytes and interleukin-6 level (rs = −0.43, P = 0.041) and CRP (rs = −0.52, P = 0.012). Of the patients who failed to complete one cycle of immunotherapy, three died during the first cycle of treatment, and the remaining nine required dose reduction due to toxicity.

Table 2.  The baseline characteristics in patients undergoing α-interferon based immunotherapy for metastatic RCC according to completion of immunotherapy
VariableImmunotherapyP
CompletedNot completed
No. of patients 1112 
Age group (≤60/>60 years) 4/7 5/70.791
Sex (M/F) 9/2 9/30.699
MSKCC score (favourable/intermediate/poor) 7/4/0 2/8/20.016
GPS (0, 1, 2) 6/4/1 2/5/50.033
Median (range):
 Leukocytes, 109/L 7.1 (5.7–10.9) 7.9 (3.4–38.5)0.566
 % total lymphocytes23.3 (13.1–33.5)15.5 (3.6–27.5)0.032
 CD4+ T-lymphocytes, 109/L 0.64 (0.18–1.16) 0.46 (0.33–1.04)0.622
 CD8+ T-lymphocytes, 109/L 0.47 (0.07–0.75) 0.38 (0.18–0.95)0.279
 Interleukin-6, pg/mL15 (12–41)39 (12–133)0.074
 Interleukin-10, pg/mL27 (12–238)28 (11–272)0.424
 Immunotherapy (α-interferon/α-interferon plus) 8/3 11/10.242

The laboratory values before and after therapy of those patients completing one cycle of immunotherapy are shown in Table 3. The MSKCC score and the GPS were not significantly changed over one cycle. Similarly, leukocyte counts, CD4+ and CD8+ T-lymphocytes, interleukin-6 and -10 levels did not change significantly. By contrast, there was a trend to an increase in the percentage of lymphocytes over one cycle of immunotherapy (P < 0.10).

Table 3.  The clinicopathological characteristics before and during chemotherapy in patients completing one cycle of treatment
VariableBeforeDuringP
No. of patients 11 11 
MSKCC score (favourable/intermediate/poor) 7/4/0 6/5/00.564
GPS (0, 1, 2) 6/4/1 4/6/10.317
Median (range):
 Leukocyte count, 109/L 7.1 (5.7–10.9) 5.2 (2.5–17.7)0.374
 % total lymphocytes23.3 (13.1–33.4)28.9 (14.9–61.5)0.075
 CD4+ T-lymphocytes, 109/L 0.66 (0.21–0.83) 0.55 (0.32–1.11)0.674
 CD8+ T-lymphocytes, 109/L 0.42 (0.07–0.64) 0.34 (0.15–0.68)0.327
 Interleukin-6, pg/mL17 (13–41)19 (12–82)0.859
 Interleukin-10, pg/mL24 (12–238)65 (17–240)0.214

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Despite the change in the treatment of metastatic RCC brought about by the new small-molecule inhibitors, immunotherapy might still have a role, especially in fit patients with a good PS. As such, ongoing efforts to examine the mechanism of action, and prognostic markers for patients treated with immunotherapy, remain relevant. In the present prospective study of patients undergoing immunotherapy for metastatic RCC the failure to complete immunotherapy and poor cancer-specific survival was significantly associated with the established MSKCC risk score, the SIR, as shown by the GPS and pro-inflammatory cytokine release assessed by interleukin-6. Moreover, the percentage of total lymphocytes was associated inversely with CRP and interleukin-6 levels, and the likelihood of completing one cycle of immunotherapy. On receiving immunotherapy, there was no change in the SIR, but there was an increase in the percentage of total lymphocytes. Taken together, these results suggest that the SIR before treatment [7,9], interleukin-6 [6,13] and its related lymphopenia [20], are important in determining tolerance and outcome after immunotherapy in patients with metastatic RCC. Therefore, a measurement of the SIR is likely to be useful in assessing patient suitability for immunotherapy, in addition to recognized scoring systems and PS.

In the present study the proportions of CD4+ and CD8+ T-lymphocytes were not significantly associated with cancer- specific survival and, the failure to complete immunotherapy, and were not significantly altered after immunotherapy. Several studies examined the impact of circulating T-lymphocytes subpopulations on the efficacy of immunotherapy or the effect of immunotherapy on T-lymphocyte populations [21]. A few small studies suggested that elevated CD4+ and CD8+ T-lymphocytes, before or during immunotherapy, are associated with an improved clinical response [22,23]. For example, Gohring et al.[22] reported that, in 22 patients who received interleukin-2/α-interferon and 5-fluorouracil in one to three treatment cycles over 8 weeks, CD4+, CD8+ and CD25+ T-lymphocytes correlated significantly with the clinical response. By contrast, other small studies suggested no such relationship [24,25]. For example, Donskov et al.[25] reported that, in 26 patients receiving interleukin-2/α-interferon and histamine treatment, CD4+ and CD8+ T-lymphocytes, before or during immunotherapy, were not associated with an improved clinical response to immunotherapy and survival. These results would suggest that further work is required to establish whether an impaired T-lymphocytic response is associated with a poor outcome in patients with RCC. Irrespective, in the present study CD4+ and CD8+ T-lymphocytes appear to offer little value compared with established prognostic variables such as the MSKCC risk score and the GPS.

In the present study there was a trend for a higher interleukin-6 level to be associated with the failure to complete immunotherapy and with poorer cancer-specific survival, and interleukin-6 levels were not significantly altered after immunotherapy. However, the prognostic value of interleukin-6 was poorer than another marker of the SIR, the GPS. Therefore, these results do not confirm a major role for interleukin-6, as previously proposed by others [6,13], in determining the outcome of immunotherapy in patients with metastatic RCC. Furthermore, the present results do not confirm a predictive role for interleukin-10, as proposed by Wittke et al.[26].

In summary, the present results show that SIR before treatment, and its related lymphopenia, are important in determining the tolerability of toxic immunotherapy in patients with metastatic RCC. They also show that interferon-based immunotherapy is not associated with major changes in circulating T-lymphocytes, nor with the SIR.

CONFLICT OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

None declared. Source of funding: self-funded.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES
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