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The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer

The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and... British Journal of Cancer (2005) 92, 651 – 654 & 2005 Cancer Research UK All rights reserved 0007 – 0920/05 $30.00 www.bjcancer.com The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer 1 1 ,1 2 2 1 1 K Canna , PA McArdle , DC McMillan , A-M McNicol , GW Smith , RF McKee and CS McArdle 1 2 University Department of Surgery, Royal Infirmary, Glasgow G31 2ER, UK; Department of Pathology, Royal Infirmary, Glasgow G31 2ER, UK There is increasing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of common solid tumours. The aim of the present study was to examine the relationship between tumour T-lymphocyte subset infiltration, the systemic inflammatory response and cancer-specific survival in patients with colorectal cancer. In all, 147 patients undergoing potentially curative resection for colorectal cancer were studied. Circulating concentrations of C-reactive protein were measured prior to surgery. CD4þ and CD8þ T-lymphocyte infiltration of the tumour was assessed using immunohistochemistry and a point counting technique. When patients were grouped according to the percentage tumour volume of CD4þ T-lymphocytes, there was no difference in terms of age, sex, tumour site, stage and tumour characteristics. However, there was an inverse relationship between percentage tumour CD4þ T-lymphocytes and C-reactive protein (Po0.01). On univariate analysis, both C-reactive protein concentrations (Po0.001) and percentage tumour volume of CD4þ (Po0.05) T-lymphocytes were associated with cancer-specific survival. The results of the present study show that low tumour CD4þ T-lymphocyte infiltration is associated with elevated C-reactive protein concentrations and both predict poor cancer-specific survival. British Journal of Cancer (2005) 92, 651–654. doi:10.1038/sj.bjc.6602419 www.bjcancer.com Published online 8 February 2005 & 2005 Cancer Research UK Keywords: colorectal cancer; CD4þ and CD8þ T-lymphocytes; C-reactive protein; survival It has long been recognised that disease progression in cancer recurrence and poorer survival in patients undergoing potentially patients is not solely determined by the characteristics of the curative resection for colorectal cancer (McMillan et al, 1995, 2003; tumour but also by the host response. Indeed, there is increasing Nielsen et al, 2000). evidence that both local and systemic inflammatory responses To date, the inter-relationships between the local and systemic play an important role in the progression of a variety of common inflammatory responses and outcome does not appear to have solid tumours (O’Byrne and Dalgleish, 2001; Vakkila and Lotze, been examined in patients with colorectal cancer. The aim of the 2004). present study was therefore to examine the relationship between In patients with colorectal cancer, there is good evidence that, tumour T-lymphocyte subset infiltration, circulating concentra- on simple staining of tumour sections, the presence of a tions of C-reactive protein and cancer-specific survival in patients pronounced lymphocytic infiltration within the tumour is who had undergone potentially curative resection for colorectal associated with improved survival (Jass et al, 1987; Ropponen cancer. et al, 1997; Nielsen et al, 1999). More recently, the ability to identify lymphocyte subsets by immunohistochemistry has led to renewed interest in the relationship between the tumour inflam- PATIENTS AND METHODS matory infiltrate and outcome. Indeed, increased infiltration of the tumour by CD8þ (Naito et al, 1998) and CD4þ T-lymphocytes Patients (Ali et al, 2004) has been shown to be associated with increased survival in patients with colorectal cancer. Patients with histologically proven colorectal cancer who, on the There is also increasing evidence that the presence of a systemic basis of preoperative imaging and the surgeons’ assessment at inflammatory response, as evidenced by elevated circulating operation, were considered to have undergone potentially curative concentrations of C-reactive protein, is associated with early resection for Dukes’ B and C colorectal cancer between January 1997 and August 2001 in a single surgical unit at Glasgow Royal Infirmary were included in the study. A blood sample was taken *Correspondence: Dr DC McMillan; prior to surgery for the measurement of C-reactive protein. The E-mail: [email protected] tumours were staged using conventional Dukes’ classification Received 25 October 2004; revised 23 December 2004; accepted 4 (Dukes and Bussey, 1958). All patients were followed up at a January 2005; published online 8 February 2005 specialist colorectal cancer clinic. Clinical Studies Clinical Studies T-lymphocyte infiltration and colorectal cancer K Canna et al C-reactive protein concentrations were measured by a fluores- Statistics cence polarisation immunoassay and using an Abbott TDXTM Data are presented as median and range. Where appropriate, analyser and Abbott reagents (Abbott Laboratories, Abbott Park, comparison of patient groups of patients was carried out using IL, USA). The limit of detection of the assay is a CRP concentra- 1 contingency table analysis (w ) and the Kruskal–Wallis test for tion lower than 5 mg l . The coefficient of variation, over the analysis of variance. For the purpose of analysis, T-lymphocyte range of measurement, was less than 5%, as established by routine subsets were grouped by tertiles as described previously (Nielsen quality control procedures. Based on previous work, a C-reactive 1 et al, 1999). Survival analysis was performed using the Cox protein concentration of greater than 10 mg l was considered to proportional-hazard model. Deaths up to 31st March 2004 have indicate the presence of a systemic inflammatory response been included in the analysis. Analysis was performed using SPSS (O’Gorman et al, 2000). software (SPSS Inc., Chicago, IL, USA). The study was approved by the local ethics committee. RESULTS The baseline clinicopathological characteristics of the patients Immunohistochemistry (n¼ 147) who underwent potentially curative resection for colo- Blocks from the primary tumour were fixed in 10% buffered rectal cancer are shown in Table 1. The majority were over the age formalin and embedded in paraffin wax. One representative of 65years and had Dukes’ stage B tumours. In all, 53 (36%) block of tumour was selected for each patient. Sections (4mm) patients had an elevated C-reactive protein concentration prior to were cut and mounted on slides coated with aminopropyl- surgery. triethoxysilane. Sections were then immunostained using the Patients grouped according to tertiles of the percentage tumour peroxidase-based Envision (Dako, Cambridgeshire, UK) techn- volume of CD4þ T-lymphocytes are shown in Table 2. The tertiles ique as described previously (Bromwich et al, 2003). The primary antibody for CD4 was mouse monoclonal (Vector, Table 1 Clinicopathological characteristics in patients undergoing Peterborough, UK) and that for CD8 was mouse monoclonal potentially curative resection for colorectal cancer (Dako, Cambridgeshire, UK). Patients (n¼ 147) Age group (years) (o65/65 – 74/X75) 46/44/57 Sex (male/female) 78/69 Morphometry Site (colon/rectum) 105/42 Quantitative analysis of the lymphoid infiltrate was performed Dukes’ stage (B/C) 91/56 using point counting (Anderson and Dunnill, 1965) with a random C-reactive protein (p10/410 mg l ) 94/53 sampling technique. With this method, the volume occupied by Tumour characteristics any given component (volume density) is expressed as a Diameter (mm) 40 (10 – 130) percentage of the total volume of the tissue. A 100-point ocular Ulceration (no/yes) 72/75 grid was used at 400 magnification and 30 fields were counted Differentiation (well/moderate/poor) 18/116/13 per case for CD4þ and CD8þ immunopositive cells. Only fields Lymphatic invasion (negative/positive) 124/22 within the tumour (including cancer cell nests and surrounding Venous invasion (negative/positive) 118/28 tissue stroma) were counted. Any normal tissue on the slide was excluded from the analysis. % Tumour volume CD4+ T-lymphocytes 0.90 (0.03 – 3.57) This final method was designed on the basis of a pilot study, CD8+ T-lymphocytes 1.13 (0.23 – 6.30) which demonstrated that the volume density of CD4þ and CD8þ CD4+ plus CD8+ T-lymphocytes 2.17 (0.50 – 8.27) of two observers reached a plateau after 25–30 fields. This pilot study also demonstrated that CD4þ and CD8þ counts were Adjuvant therapy (no/yes) 116/31 equivalent to the CD3þ counts (unpublished data). The observers Alive/dead 92/55 (Canna and McArdle) were blinded to the clinical outcome of the Cancer-specific/intercurrent disease 39/16 patient. Table 2 Relationship between increasing percentage volume of CD4+ T-lymphocytes and tumour characteristics in patients undergoing potentially curative resection for colorectal cancer Percentage volume of CD4+ T-lymphocytes Tertile (n¼ 49) Tertile (n¼ 49) Tertile (n¼ 49) P-value Age group (years) (o65/65 – 74/X75) 12/13/24 19/11/19 15/20/14 0.122 Sex (male/female) 22/27 24/25 32/17 0.101 Site (colon/rectum) 35/14 36/13 34/15 0.905 Dukes’ stage (B/C) 30/19 33/16 28/21 0.578 C-reactive protein (p10/410 mg l ) 22/27 32/17 39/10 0.002 Tumour characteristics Diameter (tertiles) 15/17/17 16/12/21 18/20/11 0.250 Ulceration (no/yes) 21/28 22/27 29/20 0.212 Differentiation (well/moderate/poor) 6/38/5 7/37/5 5/41/3 0.883 Lymphatic invasion (negative/positive) 40/8 43/6 41/8 0.794 Venous invasion (negative/positive) 38/10 42/7 38/11 0.554 British Journal of Cancer (2005) 92(4), 651 – 654 & 2005 Cancer Research UK T-lymphocyte infiltration and colorectal cancer K Canna et al 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 6 12 18 24 30 36 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Survival (months) CD4+ T-lymphocytes (%) Figure 3 Relationship between preoperative C-reactive protein (p10/ Figure 1 Relationship between percentage tumour CD4þ T-lympho- 410 mg l from top to bottom) and cancer-specific survival in patients cyte infiltration and preoperative C-reactive protein in patients undergoing undergoing potentially curative resection for colorectal cancer. potentially curative resection for colorectal cancer. CD8þ T-lymphocytes and cancer-specific survival failed to reach 1.0 statistical significance (P¼ 0.074). On multivariate analysis, only age (HR 2.05, 95% CI 1.30–3.23, 0.9 P¼ 0.002), stage (HR 4.39, 95% CI 2.14–9.00, Po0.001) and C- 0.8 reactive protein (HR 4.66, 95% CI 2.20–9.89, Po0.001) retained independent significance. 0.7 0.6 DISCUSSION 0.5 In the present study, a poor tumour CD4þ T-lymphocyte 0.4 infiltrate was associated with an elevated circulating C-reactive protein concentration in patients undergoing potentially curative 0.3 resection for colorectal cancer. Furthermore, both CD4þ T- 0.2 lymphocytes and C-reactive protein were associated with a poor outcome. Therefore, the results of the present study indicate that 0.1 both local and systemic inflammatory responses are linked and 0.0 predict outcome independent of tumour stage. 0 6 12 18 24 30 36 These results appear to be consistent with previous work by Survival (months) Naito et al (1998), who showed that tumour CD8þ T-lymphocyte infiltrate had prognostic value in patients with colorectal cancer. Figure 2 Relationship between tumour CD4þ T-lymphocyte infiltra- However, they did not assess the tumour CD4þ T-lymphocyte tion (tertiles decreasing from top to bottom) and cancer-specific survival in infiltrate and used a less extensive sampling method. Furthermore, patients undergoing potentially curative resection for colorectal cancer. they included patients with Dukes’ A tumours who were unlikely to progress and patients with Dukes’ D tumours who had already progressed (Naito et al, 1998). were similar in terms of age, sex, tumour site, stage and tumour In the present study, tumour T-lymphocyte subset density was characteristics. There was an inverse relationship between assessed using extensive sampling and a point counting technique. percentage tumour CD4þ T-lymphocytes and C-reactive protein This approach provided an objective assessment of lymphocytic (r ¼0.245, P¼ 0.003, Figure 1). However, there was no relation- infiltration and circumvents the problem of variation in distribu- ship between percentage tumour CD8þ T-lymphocytes and C- tion of lymphocytes within an individual tumour. The present reactive protein (r ¼0.091, P¼ 0.273). There was a positive study was also confined to patients with Dukes’ B and C tumours. relationship between percentage tumour CD4þ and CD8þ T- It was of interest that C-reactive protein (a systemic inflamma- lymphocytes (r ¼ 0.440, Po0.001). tory response) was superior to tumour T-lymphocytic infiltration The minimum follow-up was 30 months; the median follow-up (a local inflammatory response) in predicting cancer specific of the survivors was 62 months. During the course of the study, 55 survival. One possible explanation is that C-reactive protein can patients died, 39 patients of their cancer and 16 of intercurrent be measured with greater accuracy and precision than tumour disease. On univariate analysis, increased age (Po0.001), sex T-lymphocytic infiltration. Alternatively, the systemic inflamma- (P¼ 0.052), Dukes’ stage (Po0.001) and venous invasion tory response may be more important in determining survival in (P¼ 0.002) were associated with poorer cancer-specific survival. these patients. A decreased percentage tumour volume of CD4þ T-lymphocytes The relationship between tumour CD4þ T-lymphocytic infil- (P¼ 0.025, Figure 2) and an elevated C-reactive protein (Po0.001, tration and cancer-specific survival is the opposite of that Figure 3) were also associated with poorer cancer-specific survival. previously reported for both renal and prostate cancer (Bromwich However, the relationship between percentage tumour volume of et al, 2003; McArdle et al, 2004a). The reasons for this are as yet & 2005 Cancer Research UK British Journal of Cancer (2005) 92(4), 651 – 654 −1 Preoperative C-reactive protein (mg l ) Cumulative survival Cumulative survival Clinical Studies Clinical Studies T-lymphocyte infiltration and colorectal cancer K Canna et al unclear. However, given that tumour lymphocytic infiltration have important implications for the treatment of the systemic parallels that of other inflammatory cells (Nielsen et al, 1999; Lin inflammatory response in patients with different tumours. and Pollard, 2004) and that an elevated C-reactive protein is In the present study, we attempted to assess interleukin-6 within associated with poor outcome in all three tumours (Lewenhaupt the tumour using different methods of antigen retrieval and et al, 1990; Blay et al, 1992; McMillan et al, 2003), it appears likely staining and the use of negative and positive controls. However, we that the source of interleukin-6, the primary stimulus for the were unable to reliably identify regions of IL-6 expression in the production of C-reactive protein, (Gabay and Kushner, 1999), colorectal tumours due to deep background staining, which differs in different tumours. precluded accurate scoring of IL-6-positive cells in the tumour tissue. It is of interest therefore that McArdle et al (2004b) have In summary, the results of the present study show that, in recently reported that the relationship between interleukin-6 and patients undergoing curative resection for colorectal cancer, low C-reactive protein was similar in benign prostatic hyperplasia and tumour CD4þ T-lymphocyte infiltration is associated with prostate cancer, and that there was no relationship between elevated C-reactive protein concentrations. Furthermore, both a interleukin-6 and PSA concentrations. This would suggest that, in low tumour CD4þ T-lymphocyte infiltration and an elevated prostate cancer at least, interleukin-6 is produced by the C-reactive protein predict poor cancer-specific survival. inflammatory cells. In colorectal cancer, it has been reported that interleukin-6 concentrations increase with tumour stage and correlate with CEA ACKNOWLEDGEMENTS concentrations. This might therefore suggest that interleukin-6 is produced by the tumour cells (Kinoshita et al, 1999; Belluco et al, We gratefully acknowledge the assistance of Mr JH Anderson, and 2000; Miki et al, 2004). If this were to prove to be the case, it would Mr P Horgan and the technical expertise of Mr D Murray. REFERENCES Ali AA, McMillan DC, Matalka II, McNicol AM, McArdle CS (2004) Tumour McArdle PA, McMillan DC, Sattar N, Wallace AM, Underwood MA (2004b) T-lymphocyte subset infiltration and tumour recurrence following The relationship between interleukin-6 and C-reactive protein in patients curative resection for colorectal cancer. Eur J Surg Oncol 30: 292–295 with benign and malignant prostate disease. Br J Cancer 91: 1755–1757 Anderson JA, Dunnill MS (1965) Observations on the estimation of the McMillan DC, Canna K, McArdle CS (2003) Systemic inflammatory quantity of emphysema in the lungs by the point-sampling method. response predicts survival following curative resection of colorectal Thorax 20: 462–466 cancer. Br J Surg 90: 215–219 Belluco C, Nitti D, Frantz M, Toppan P, Basso D, Plebani M, Lise M, Jessup McMillan DC, Wotherspoon HA, Fearon KC, Sturgeon C, Cooke TG, JM (2000) Interleukin-6 blood level is associated with circulating McArdle CS (1995) A prospective study of tumor recurrence and the carcinoembryonic antigen and prognosis in patients with colorectal acute-phase response after apparently curative colorectal cancer surgery. cancer. Ann Surg Oncol 7: 133–138 Am J Surg 170: 319–322 Blay JY, Negrier S, Combaret V, Attali S, Goillot E, Merrouche Y, Mercatello Miki C, Konishi N, Ojima E, Hatada T, Inoue Y, Kusunoki M (2004) A, Ravault A, Tourani JM, Moskovtchenko JF (1992) Serum level of C-reactive protein as a prognostic variable that reflects uncontrolled interleukin 6 as a prognosis factor in metastatic renal cell carcinoma. up-regulation of the IL-1-IL-6 network system in colorectal carcinoma. Cancer Res 52: 3317–3322 Dig Dis Sci 49: 970–976 Bromwich EJ, McArdle PA, Canna K, McMillan DC, McNicol A-M, Brown Naito Y, Saito K, Shiiba K, Ohuchi A, Saiqenji K, Naqura H, Ohtani H M, Aitchison M (2003) The relationship between T-lymphocyte (1998) CD8+ T cells infiltrated within cancer cell nests as prognostic infiltration, stage, tumour grade and survival in patients undergoing factor in human colorectal cancer. Cancer Res 58: 3491–3494 curative surgery for renal cell cancer. Br J Cancer 89: 1906–1908 Nielsen HJ, Christensen IJ, Sorensen S, Moesgaard F, Brunner N (2000) Dukes CE, Bussey HJR (1958) The spread of rectal cancer and its effect on Preoperative plasma plasminogen activator inhibitor type-1 and serum prognosis. Br J Cancer 12: 309–320 C-reactive protein levels in patients with colorectal cancer. The RANX05 Gabay C, Kushner I (1999) Acute-phase proteins and other systemic Colorectal Cancer Study Group. Ann Surg Oncol 7: 617–623 responses to inflammation. N Engl J Med 340: 448–454 Nielsen HJ, Hansen U, Christensen IJ, Reimert CM, Brunner N, Moesgaard Jass JR, Love SB, Northover JM (1987) A new prognostic classification of F, The RANX05 Study Group (1999) Independent prognostic value of rectal cancer. Lancet 1(8545): 1303– 1306 eosinophil and mast cell infiltration in colorectal cancer tissue. J Pathol Kinoshita T, Ito H, Miki C (1999) Serum interleukin-6 level reflects the 189: 487–495 tumor proliferative activity in patients with colorectal carcinoma. Cancer O’Byrne KJ, Dalgleish AG (2001) Chronic immune activation and 85: 2526–2531 inflammation as the cause of malignancy. Br J Cancer 85: 473–483 Lewenhaupt A, Ekman P, Eneroth P, Nilsson B (1990) Tumour markers as O’Gorman P, McMillan DC, McArdle CS (2000) Prognostic factors in prognostic aids in prostatic carcinoma. Br J Urol 66: 182 –187 advanced gastrointestinal cancer patients with weight loss. Nutr Cancer Lin EY, Pollard JW (2004) Role of infiltrated leucocytes in tumour growth 37: 36–40 and spread. Br J Cancer 90: 2053 –2058 Ropponen KM, Eskelinen MJ, Lipponen PK, Alhava PK, Kosma VM (1997) McArdle PA, Canna K, McMillan DC, McNicol AM, Campbell R, Under- Prognostic value of tumour-infiltrating lymphocytes (TILs) in colorectal wood MA (2004a) The relationship between T-lymphocyte subset cancer. J Pathol 182: 318–324 infiltration and survival in patients with prostate cancer. Br J Cancer Vakkila J, Lotze MT (2004) Inflammation and necrosis promote tumour 91: 541–543 growth. Nat Rev Immunol 4: 641–648 British Journal of Cancer (2005) 92(4), 651 – 654 & 2005 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer

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Springer Journals
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Copyright © 2005 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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10.1038/sj.bjc.6602419
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Abstract

British Journal of Cancer (2005) 92, 651 – 654 & 2005 Cancer Research UK All rights reserved 0007 – 0920/05 $30.00 www.bjcancer.com The relationship between tumour T-lymphocyte infiltration, the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer 1 1 ,1 2 2 1 1 K Canna , PA McArdle , DC McMillan , A-M McNicol , GW Smith , RF McKee and CS McArdle 1 2 University Department of Surgery, Royal Infirmary, Glasgow G31 2ER, UK; Department of Pathology, Royal Infirmary, Glasgow G31 2ER, UK There is increasing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of common solid tumours. The aim of the present study was to examine the relationship between tumour T-lymphocyte subset infiltration, the systemic inflammatory response and cancer-specific survival in patients with colorectal cancer. In all, 147 patients undergoing potentially curative resection for colorectal cancer were studied. Circulating concentrations of C-reactive protein were measured prior to surgery. CD4þ and CD8þ T-lymphocyte infiltration of the tumour was assessed using immunohistochemistry and a point counting technique. When patients were grouped according to the percentage tumour volume of CD4þ T-lymphocytes, there was no difference in terms of age, sex, tumour site, stage and tumour characteristics. However, there was an inverse relationship between percentage tumour CD4þ T-lymphocytes and C-reactive protein (Po0.01). On univariate analysis, both C-reactive protein concentrations (Po0.001) and percentage tumour volume of CD4þ (Po0.05) T-lymphocytes were associated with cancer-specific survival. The results of the present study show that low tumour CD4þ T-lymphocyte infiltration is associated with elevated C-reactive protein concentrations and both predict poor cancer-specific survival. British Journal of Cancer (2005) 92, 651–654. doi:10.1038/sj.bjc.6602419 www.bjcancer.com Published online 8 February 2005 & 2005 Cancer Research UK Keywords: colorectal cancer; CD4þ and CD8þ T-lymphocytes; C-reactive protein; survival It has long been recognised that disease progression in cancer recurrence and poorer survival in patients undergoing potentially patients is not solely determined by the characteristics of the curative resection for colorectal cancer (McMillan et al, 1995, 2003; tumour but also by the host response. Indeed, there is increasing Nielsen et al, 2000). evidence that both local and systemic inflammatory responses To date, the inter-relationships between the local and systemic play an important role in the progression of a variety of common inflammatory responses and outcome does not appear to have solid tumours (O’Byrne and Dalgleish, 2001; Vakkila and Lotze, been examined in patients with colorectal cancer. The aim of the 2004). present study was therefore to examine the relationship between In patients with colorectal cancer, there is good evidence that, tumour T-lymphocyte subset infiltration, circulating concentra- on simple staining of tumour sections, the presence of a tions of C-reactive protein and cancer-specific survival in patients pronounced lymphocytic infiltration within the tumour is who had undergone potentially curative resection for colorectal associated with improved survival (Jass et al, 1987; Ropponen cancer. et al, 1997; Nielsen et al, 1999). More recently, the ability to identify lymphocyte subsets by immunohistochemistry has led to renewed interest in the relationship between the tumour inflam- PATIENTS AND METHODS matory infiltrate and outcome. Indeed, increased infiltration of the tumour by CD8þ (Naito et al, 1998) and CD4þ T-lymphocytes Patients (Ali et al, 2004) has been shown to be associated with increased survival in patients with colorectal cancer. Patients with histologically proven colorectal cancer who, on the There is also increasing evidence that the presence of a systemic basis of preoperative imaging and the surgeons’ assessment at inflammatory response, as evidenced by elevated circulating operation, were considered to have undergone potentially curative concentrations of C-reactive protein, is associated with early resection for Dukes’ B and C colorectal cancer between January 1997 and August 2001 in a single surgical unit at Glasgow Royal Infirmary were included in the study. A blood sample was taken *Correspondence: Dr DC McMillan; prior to surgery for the measurement of C-reactive protein. The E-mail: [email protected] tumours were staged using conventional Dukes’ classification Received 25 October 2004; revised 23 December 2004; accepted 4 (Dukes and Bussey, 1958). All patients were followed up at a January 2005; published online 8 February 2005 specialist colorectal cancer clinic. Clinical Studies Clinical Studies T-lymphocyte infiltration and colorectal cancer K Canna et al C-reactive protein concentrations were measured by a fluores- Statistics cence polarisation immunoassay and using an Abbott TDXTM Data are presented as median and range. Where appropriate, analyser and Abbott reagents (Abbott Laboratories, Abbott Park, comparison of patient groups of patients was carried out using IL, USA). The limit of detection of the assay is a CRP concentra- 1 contingency table analysis (w ) and the Kruskal–Wallis test for tion lower than 5 mg l . The coefficient of variation, over the analysis of variance. For the purpose of analysis, T-lymphocyte range of measurement, was less than 5%, as established by routine subsets were grouped by tertiles as described previously (Nielsen quality control procedures. Based on previous work, a C-reactive 1 et al, 1999). Survival analysis was performed using the Cox protein concentration of greater than 10 mg l was considered to proportional-hazard model. Deaths up to 31st March 2004 have indicate the presence of a systemic inflammatory response been included in the analysis. Analysis was performed using SPSS (O’Gorman et al, 2000). software (SPSS Inc., Chicago, IL, USA). The study was approved by the local ethics committee. RESULTS The baseline clinicopathological characteristics of the patients Immunohistochemistry (n¼ 147) who underwent potentially curative resection for colo- Blocks from the primary tumour were fixed in 10% buffered rectal cancer are shown in Table 1. The majority were over the age formalin and embedded in paraffin wax. One representative of 65years and had Dukes’ stage B tumours. In all, 53 (36%) block of tumour was selected for each patient. Sections (4mm) patients had an elevated C-reactive protein concentration prior to were cut and mounted on slides coated with aminopropyl- surgery. triethoxysilane. Sections were then immunostained using the Patients grouped according to tertiles of the percentage tumour peroxidase-based Envision (Dako, Cambridgeshire, UK) techn- volume of CD4þ T-lymphocytes are shown in Table 2. The tertiles ique as described previously (Bromwich et al, 2003). The primary antibody for CD4 was mouse monoclonal (Vector, Table 1 Clinicopathological characteristics in patients undergoing Peterborough, UK) and that for CD8 was mouse monoclonal potentially curative resection for colorectal cancer (Dako, Cambridgeshire, UK). Patients (n¼ 147) Age group (years) (o65/65 – 74/X75) 46/44/57 Sex (male/female) 78/69 Morphometry Site (colon/rectum) 105/42 Quantitative analysis of the lymphoid infiltrate was performed Dukes’ stage (B/C) 91/56 using point counting (Anderson and Dunnill, 1965) with a random C-reactive protein (p10/410 mg l ) 94/53 sampling technique. With this method, the volume occupied by Tumour characteristics any given component (volume density) is expressed as a Diameter (mm) 40 (10 – 130) percentage of the total volume of the tissue. A 100-point ocular Ulceration (no/yes) 72/75 grid was used at 400 magnification and 30 fields were counted Differentiation (well/moderate/poor) 18/116/13 per case for CD4þ and CD8þ immunopositive cells. Only fields Lymphatic invasion (negative/positive) 124/22 within the tumour (including cancer cell nests and surrounding Venous invasion (negative/positive) 118/28 tissue stroma) were counted. Any normal tissue on the slide was excluded from the analysis. % Tumour volume CD4+ T-lymphocytes 0.90 (0.03 – 3.57) This final method was designed on the basis of a pilot study, CD8+ T-lymphocytes 1.13 (0.23 – 6.30) which demonstrated that the volume density of CD4þ and CD8þ CD4+ plus CD8+ T-lymphocytes 2.17 (0.50 – 8.27) of two observers reached a plateau after 25–30 fields. This pilot study also demonstrated that CD4þ and CD8þ counts were Adjuvant therapy (no/yes) 116/31 equivalent to the CD3þ counts (unpublished data). The observers Alive/dead 92/55 (Canna and McArdle) were blinded to the clinical outcome of the Cancer-specific/intercurrent disease 39/16 patient. Table 2 Relationship between increasing percentage volume of CD4+ T-lymphocytes and tumour characteristics in patients undergoing potentially curative resection for colorectal cancer Percentage volume of CD4+ T-lymphocytes Tertile (n¼ 49) Tertile (n¼ 49) Tertile (n¼ 49) P-value Age group (years) (o65/65 – 74/X75) 12/13/24 19/11/19 15/20/14 0.122 Sex (male/female) 22/27 24/25 32/17 0.101 Site (colon/rectum) 35/14 36/13 34/15 0.905 Dukes’ stage (B/C) 30/19 33/16 28/21 0.578 C-reactive protein (p10/410 mg l ) 22/27 32/17 39/10 0.002 Tumour characteristics Diameter (tertiles) 15/17/17 16/12/21 18/20/11 0.250 Ulceration (no/yes) 21/28 22/27 29/20 0.212 Differentiation (well/moderate/poor) 6/38/5 7/37/5 5/41/3 0.883 Lymphatic invasion (negative/positive) 40/8 43/6 41/8 0.794 Venous invasion (negative/positive) 38/10 42/7 38/11 0.554 British Journal of Cancer (2005) 92(4), 651 – 654 & 2005 Cancer Research UK T-lymphocyte infiltration and colorectal cancer K Canna et al 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 6 12 18 24 30 36 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Survival (months) CD4+ T-lymphocytes (%) Figure 3 Relationship between preoperative C-reactive protein (p10/ Figure 1 Relationship between percentage tumour CD4þ T-lympho- 410 mg l from top to bottom) and cancer-specific survival in patients cyte infiltration and preoperative C-reactive protein in patients undergoing undergoing potentially curative resection for colorectal cancer. potentially curative resection for colorectal cancer. CD8þ T-lymphocytes and cancer-specific survival failed to reach 1.0 statistical significance (P¼ 0.074). On multivariate analysis, only age (HR 2.05, 95% CI 1.30–3.23, 0.9 P¼ 0.002), stage (HR 4.39, 95% CI 2.14–9.00, Po0.001) and C- 0.8 reactive protein (HR 4.66, 95% CI 2.20–9.89, Po0.001) retained independent significance. 0.7 0.6 DISCUSSION 0.5 In the present study, a poor tumour CD4þ T-lymphocyte 0.4 infiltrate was associated with an elevated circulating C-reactive protein concentration in patients undergoing potentially curative 0.3 resection for colorectal cancer. Furthermore, both CD4þ T- 0.2 lymphocytes and C-reactive protein were associated with a poor outcome. Therefore, the results of the present study indicate that 0.1 both local and systemic inflammatory responses are linked and 0.0 predict outcome independent of tumour stage. 0 6 12 18 24 30 36 These results appear to be consistent with previous work by Survival (months) Naito et al (1998), who showed that tumour CD8þ T-lymphocyte infiltrate had prognostic value in patients with colorectal cancer. Figure 2 Relationship between tumour CD4þ T-lymphocyte infiltra- However, they did not assess the tumour CD4þ T-lymphocyte tion (tertiles decreasing from top to bottom) and cancer-specific survival in infiltrate and used a less extensive sampling method. Furthermore, patients undergoing potentially curative resection for colorectal cancer. they included patients with Dukes’ A tumours who were unlikely to progress and patients with Dukes’ D tumours who had already progressed (Naito et al, 1998). were similar in terms of age, sex, tumour site, stage and tumour In the present study, tumour T-lymphocyte subset density was characteristics. There was an inverse relationship between assessed using extensive sampling and a point counting technique. percentage tumour CD4þ T-lymphocytes and C-reactive protein This approach provided an objective assessment of lymphocytic (r ¼0.245, P¼ 0.003, Figure 1). However, there was no relation- infiltration and circumvents the problem of variation in distribu- ship between percentage tumour CD8þ T-lymphocytes and C- tion of lymphocytes within an individual tumour. The present reactive protein (r ¼0.091, P¼ 0.273). There was a positive study was also confined to patients with Dukes’ B and C tumours. relationship between percentage tumour CD4þ and CD8þ T- It was of interest that C-reactive protein (a systemic inflamma- lymphocytes (r ¼ 0.440, Po0.001). tory response) was superior to tumour T-lymphocytic infiltration The minimum follow-up was 30 months; the median follow-up (a local inflammatory response) in predicting cancer specific of the survivors was 62 months. During the course of the study, 55 survival. One possible explanation is that C-reactive protein can patients died, 39 patients of their cancer and 16 of intercurrent be measured with greater accuracy and precision than tumour disease. On univariate analysis, increased age (Po0.001), sex T-lymphocytic infiltration. Alternatively, the systemic inflamma- (P¼ 0.052), Dukes’ stage (Po0.001) and venous invasion tory response may be more important in determining survival in (P¼ 0.002) were associated with poorer cancer-specific survival. these patients. A decreased percentage tumour volume of CD4þ T-lymphocytes The relationship between tumour CD4þ T-lymphocytic infil- (P¼ 0.025, Figure 2) and an elevated C-reactive protein (Po0.001, tration and cancer-specific survival is the opposite of that Figure 3) were also associated with poorer cancer-specific survival. previously reported for both renal and prostate cancer (Bromwich However, the relationship between percentage tumour volume of et al, 2003; McArdle et al, 2004a). The reasons for this are as yet & 2005 Cancer Research UK British Journal of Cancer (2005) 92(4), 651 – 654 −1 Preoperative C-reactive protein (mg l ) Cumulative survival Cumulative survival Clinical Studies Clinical Studies T-lymphocyte infiltration and colorectal cancer K Canna et al unclear. However, given that tumour lymphocytic infiltration have important implications for the treatment of the systemic parallels that of other inflammatory cells (Nielsen et al, 1999; Lin inflammatory response in patients with different tumours. and Pollard, 2004) and that an elevated C-reactive protein is In the present study, we attempted to assess interleukin-6 within associated with poor outcome in all three tumours (Lewenhaupt the tumour using different methods of antigen retrieval and et al, 1990; Blay et al, 1992; McMillan et al, 2003), it appears likely staining and the use of negative and positive controls. However, we that the source of interleukin-6, the primary stimulus for the were unable to reliably identify regions of IL-6 expression in the production of C-reactive protein, (Gabay and Kushner, 1999), colorectal tumours due to deep background staining, which differs in different tumours. precluded accurate scoring of IL-6-positive cells in the tumour tissue. It is of interest therefore that McArdle et al (2004b) have In summary, the results of the present study show that, in recently reported that the relationship between interleukin-6 and patients undergoing curative resection for colorectal cancer, low C-reactive protein was similar in benign prostatic hyperplasia and tumour CD4þ T-lymphocyte infiltration is associated with prostate cancer, and that there was no relationship between elevated C-reactive protein concentrations. Furthermore, both a interleukin-6 and PSA concentrations. This would suggest that, in low tumour CD4þ T-lymphocyte infiltration and an elevated prostate cancer at least, interleukin-6 is produced by the C-reactive protein predict poor cancer-specific survival. inflammatory cells. In colorectal cancer, it has been reported that interleukin-6 concentrations increase with tumour stage and correlate with CEA ACKNOWLEDGEMENTS concentrations. This might therefore suggest that interleukin-6 is produced by the tumour cells (Kinoshita et al, 1999; Belluco et al, We gratefully acknowledge the assistance of Mr JH Anderson, and 2000; Miki et al, 2004). If this were to prove to be the case, it would Mr P Horgan and the technical expertise of Mr D Murray. 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Journal

British Journal of Cancer – Springer Journals

Published: Feb 8, 2005

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