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CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour’ approach to tumour host interaction in colorectal cancer

CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a... Molecular Diagnostics British Journal of Cancer (2009) 101, 1382 – 1392 & 2009 Cancer Research UK All rights reserved 0007 – 0920/09 $32.00 www.bjcancer.com CD8þ lymphocytes/ tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour’ approach to tumour host interaction in colorectal cancer 1 2 2 3 4 4 5 5 1 A Lugli , E Karamitopoulou , I Panayiotides , P Karakitsos , G Rallis , G Peros , G Iezzi , G Spagnoli , M Bihl , 1 ,1 L Terracciano and I Zlobec 1 2 Institute of Pathology, University of Basel, Scho¨nbeinstrasse 40, Basel 4031, Switzerland; Second Department of Pathology, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, Greece; Department of Diagnostic Cytopathology, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, 4 5 Greece; Fourth Department of Surgery, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, Greece; Institute for Surgical Research and Hospital Management, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland BACKGROUND: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial–mesenchymal transition and its hallmark ‘tumour budding’, is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a ‘pro-/anti-tumour’ approach defined by an established ‘pro-tumour’ (tumour budding) and host-related ‘anti-tumour’ factor of the adaptive immunological microenvironment (CD8þ lymphocytes). METHODS: Double immunostaining for CK22/CD8 on whole tissue sections (n¼ 279; Cohort 1) and immunohistochemistry for CD8þ using tissue microarrays (n¼ 191; Cohort 2) was carried out. Tumour buds, CD8þ and CD8þ T-lymphocytes : tumour buds indices were evaluated per high-power field. RESULTS: In Cohort 1, a low-CD8þ / buds index was associated with lymph node metastasis (Po0.001), vascular invasion (P¼ 0.009), worse survival in univariate (Po0.001) and multivariable (Po0.001) analysis, and furthermore in lymph node-negative patients (P¼ 0.002). In Cohort 2, the CD8þ / buds index was associated with T stage (Po0.001), N stage (P¼ 0.041), vascular invasion (P¼ 0.005) and survival in patients with TNM stage II (P¼ 0.019), stage III (P¼ 0.004), and adjuvantly untreated (P¼ 0.009) and treated patients (Po0.001). CONCLUSION: The CD8þ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease. British Journal of Cancer (2009) 101, 1382–1392. doi:10.1038/sj.bjc.6605318 www.bjcancer.com Published online 15 September 2009 & 2009 Cancer Research UK Keywords: tumour budding; CD8þ lymphocytes; colorectal cancer; prognosis During the Napoleonic Wars (1796–1815), one of the most factors, such as CD3þ,CD4þ,CD8þ,CD20þ lymphocytes, effective methods to attack in battle was the deployment of heavy Granzyme B, FoxP3þ regulatory T cells (Tregs), CD16þ cells, and cavalry, which the opponent tried to repel by organizing the mast and dendritic cells (Dadabayev et al,2004;Phillips et al, 2004; infantry into squares. Sato et al, 2005; Chaput et al,2009;Salama et al, 2009). A similar picture can be observed at the invasion front of colorectal Consequently, several groups focus on the invasive front of cancer. On one hand, the tumour mass invades the pericolic fat tissue, colorectal cancer using the term epithelial–mesenchymal transi- detaching clusters or single tumour cells (tumour buds) reflecting tion (EMT), which characterises tumour invasion by de-differ- tumour progression; whereas, on the other hand the host attempts to entiated colorectal carcinoma cells (Brabletz et al, 2005). EMT and confront this situation by building an ‘anti-tumour’ cytotoxic MET, the reverse transition from a mesenchymal to an epithelial inflammatory response. This ‘pro-/anti-tumour’ model is supported phenotype, are crucial steps not only in embryonic development by a range of studies proposing either independent tumour-related but also in tumour progression (Spaderna et al, 2007). prognostic factors (pro-tumour), such as tumour grade, tumour A histomorphological hallmark of EMT is the phenomenon of border configuration, medullary subtype, CEA level, microsatellite ‘tumour budding’ (Prall, 2007), which according to the third instability, loss of heterozygosity (LOH) 18q status, p53 levels, TGFB1 edition of ‘Prognostic Factors in Cancer’ published by the UICC in type II receptor levels, VEGF expression, proliferation rate and 2006, is considered an additional prognostic factor in colorectal metalloproteinase expression (Compton, 2006) or anti-tumour cancer (Compton, 2006). A tumour bud is typically defined as a single tumour cell or tumour cell cluster of up to five cells at the *Correspondence: Dr I Zlobec, Institute of Pathology, University of Basel, invasive tumour front (Prall, 2007). Indeed, tumour budding has Scho¨nbeinstrasse 40, Basel, 4031, Switzerland; E-mail: [email protected] been shown to be associated with lymph node positivity, poorly Received 7 May 2009; revised 3 August 2009; accepted 19 August 2009; differentiated tumours, presence of vascular and lymphatic published online 15 September 2009 invasion, local tumour recurrence and distant metastasis (Ueno Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al et al, 2004a,b; Nakamura et al, 2005; Kazama et al, 2006; Ishikawa Therefore, the aim of this study was to investigate on a potential et al, 2008; Wang et al, 2009). In particular, patients with stage III ‘pro-/anti-tumour’ model and to test the prognostic impact of disease have been reported to demonstrate a 5-year disease-free a ratio defined by an established pro-tumour (tumour budding) survival (DFS) of 62.1% in the absence of tumour budding and and anti-tumour (CD8þ lymphocytes) factor. To this end, two only a 35.1% DFS with this feature (Choi et al, 2007). Moreover, independent colorectal cancer patient cohorts from different the presence of tumour budding has repeatedly been linked to centres were investigated using two different approaches, namely poor clinical outcome, underlined by the adverse effect on overall whole tissue sections (n¼ 300) and the tissue microarray survival independent of TNM stage (Hase et al, 1993; Ueno et al, technique (n¼ 221). 2004b). Over the last 20 years, investigations on tumour immunity and host defence in colorectal cancer demonstrate promising results MATERIALS AND METHODS for immunotherapy. In most colorectal cancers, lymphocytic infiltration is composed predominantly of either CD4þ or Cohort 1-Whole tissue sections CD8þ T cells and both cell types appear to be significantly increased in tumour as compared with normal tissue (Ropponen Sample size determination To reach 85% power with an expected et al, 1997; Naito et al, 1998; Chiba et al, 2004; Koch et al, 2006). risk ratio of 1.8 between prognostic groupings and potential Several studies have shown that tumour infiltrating lymphocytes loss of patient samples in 10% of cases the appropriate sample (TILs) within the stroma and around the tumour along the size for this study was determined to be 255 cases. Owing to the invasive margin are significantly related to overall- and disease- availability of material this number was increased to 300 cases. specific survival in both univariate and multivariable analysis (Ali Specimens: Paraffin-embedded tissue blocks of 300 resection et al, 2004; Canna et al, 2005; Pages et al, 2005). Galon et al (2006) specimens of patients treated between 1987 and 1996 at the evaluated by gene-expression profiling and immunohistochemis- University Hospital of Basel were retrieved from the archives of try, the type, density and location (whether at the invasive margin the Institute of Pathology, University Hospital of Basel, as well or the tumour centre) of TILs in a large number of cases. They as at the Institute of Clinical Pathology, Basel, Switzerland. These evaluated CD3, CD8, granzyme B and memory CD45RO T cells, 300 cases were randomly selected from a larger previously demonstrating a significant independent and positive effect of described cohort of 938 colorectal cancer patients with full TILs on both recurrence and survival. clinico-pathological information (Zlobec et al, 2008). The use of In colorectal cancer, mismatch-repair status (microsatellite material for this study was approved by the local research ethics stable (MSS) and microsatellite instability-high (MSI-H)) seems committee. to relate highly to the number of CD8þ lymphocytes. Compared with MSS tumours, MSI-H cancers are characterised by prolonged Double immunostaining for CD8 and CK22 A double immuno- survival time, significantly more frequent peritumoural lympho- staining procedure using anti-CD8 (for detection of CD8þ cytic infiltration at the invasive front and by an inherent T-lymphocytes) and pan-cytokeratin (to facilitate visualization abundance of intra-epithelial TILs (Jass et al, 1998; Michael- of tumour buds at the invasive front) was carried out on one Robinson et al, 2001; Greenson et al, 2003; Jenkins et al, 2007). representative slide cut at 4 mm from paraffin-embedded tumour Nevertheless, many studies analyzing colorectal cancer samples blocks of all 300 colorectal cancer patients included in this study stratified by mismatch-repair status also report a positive effect of (Figure 1). Double staining was carried out using the BOND-MAX CD8þ lymphocytes in mismatch-repair proficient colorectal Automated Immunohistochemistry Vision Biosystem (Leica cancers (Baker et al, 2007). Microsystems GmbH, Wetzlar, Germany) according to the Although this wide range of possible additional prognostic factors following protocol. First, tissues were deparaffinised and pre- in colorectal cancer is currently being investigated, still missing is an treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH8.8) approach to include parameters reflecting the tumour dynamics. Such at 1001C for 20 min. After wash steps, peroxidase blocking was an approach has already been taken for the prognostication of breast carried out for 10 min using the Bond Polymer Refine Detection cancer, namely the Bloom–Richardson–Elston (BRE) score, which Kit DC9800 (Leica Microsystems GmbH). Tissues were again encompasses information on mitoses, tubule differentiation and washed, then incubated with primary antibody against CK22 nuclear pleomorphism, and is used as an important prognostic (Biomeda, Foster City, CA, USA, pan-CK22) for 30 min. Subse- feature additional to TNM staging. quently, tissues were incubated with polymer for 15 min and then Figure 1 Double immunostaining for CD8 (red) and CK22 (brown): (A) Overview ( 10) and (B) high power field ( 40) of the invasive front of colorectal cancer showing CK22 positive tumour buds surrounded by CD8þ T-lymphocytes. & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al with DAB-Chromogen for 10 min (Bond Polymer AP Red Table 1 Characteristics of patients in Cohort 1 (n¼ 279) Detection Kit DS9305, Leica Microsystems GmbH). CK22 positive Clinico-pathological features Frequency N (%) cells were therefore coloured in brown. After washing, incubation was carried out with anti-CD8 (DakoCytomation, Glostrup, Age (years) (n¼ 279) Denmark, clone CD8/144B) for 30 min followed by application of Mean, range 67.7, 37 – 96 AEC-substrate for 10 min and counterstaining with haematoxylin for 2 min. Tumour diameter (mm) (n¼ 279) Mean, range 54.0, 13 – 170 Evaluation of tumour buds and CD8þ T-lymphocytes Tumour Gender (n¼ 279) budding was defined as an isolated single cancer cell or a cluster Male 122 (43.7) of up to five cells at the invasive front of colorectal cancer Female 157 (56.3) (Prall, 2007). The tumour border was scanned at a  100 magnification and the area of most intense budding was identified Tumour location (n¼ 279) (Ueno et al, 2002). After selecting this field, the number of buds Left-sided 71 (25.4) was counted using a 40 objective lens to focus specifically Right-sided 111 (39.8) on the presence of CD8þ T-cells most highly related to the Rectum 97 (34.7) microenvironment surrounding the tumour buds. Within this Histological subtype (n¼ 277) same field, all CD8þ lymphocytes were individually counted. In Adenocarcinoma 252 (91.0) 10 cases, the abundance of CD8þ infiltrate led to cell counts Mucinous 21 (7.6) exceeding 200 cells per field, and thus single cell counting was Other 4 (1.4) not feasible. These cases were assigned a CD8þ score of 200 cells. The ratio of CD8þ T-lymphocytes to the number of tumour pT stage (n¼ 274) buds (CD8þ / buds index) was obtained. In 13 cases when zero pT1 6 (2.2) pT2 35 (12.8) buds were identified, the count of CD8þ lymphocytes was not pT3 187 (68.3) carried out. pT4 46 (16.8) Clinico-pathological characteristics Of these 300 cases, 279 were pN stage (n¼ 273) evaluable for CD8 and CK22 protein expression, simultaneously. pN0 140 (51.3) Hematoxylin and eosin (H&E) stained slides were reviewed pN1 76 (27.8) and histomorphological data included histological subtype, pT pN2 57 (20.9) stage, pN stage and tumour grade. The tumour border configura- Tumour grade (n¼ 274) tion and the presence of conspicuous peritumoural lymphocytic G1 5 (1.8) infiltration were defined according to Jass et al (1986). Clinical G2 247 (90.2) data were retrieved from patient records and included age at G3 22 (8.0) diagnosis, gender, tumour location and follow up. Clinical outcome of interest was disease-specific survival time, which was Vascular invasion (n¼ 274) available for all 279 patients. Median follow-up time was 60 Absent 206 (75.2) Present 68 (24.8) months. In all, 128 patients died of disease. Patient characteristics are listed in Table 1. Tumour border configuration (n¼ 271) Infiltrating 186 (68.6) Microsatellite instability (MSI) status, KRAS and BRAF gene Pushing 85 (31.4) status Genomic DNA was obtained from primary tumours using NucleoMag 96 Tissue Kit (Macherey Nagel, Oensingen, Peritumoural lymphocytic infiltration (n¼ 274) Switzerland) protocol and processed in the Xiril X-100 robot (Xiril, Absent 204 (74.5) Hombrechtikon, Switzerland). Briefly, punched tissue was lysed in Present 70 (25.6) proteinase K. B-beads and MB2 buffer were added to the cleared Microsatellite instability status (n¼ 125) lysate and shaken for 5 min at room temperature. The supernatant Stable/low 95 (76.0) was removed and MB3 was added followed by shaking and Instable/high 30 (24.0) supernatant removal. The genomic DNA was eluted with MB6 buffer. Genomic DNA was amplified by PCR using AmpliTaq Gold KRAS gene status (n¼ 117) polymerase (Applied Biosystem, Foster City, CA, USA). KRAS Wild-type 82 (70.1) (exon 2, codon 12 and 13) and BRAF (exon 15, codon 600) were Mutation 35 (29.9) amplified by a first and a nested PCR. Residual primers BRAF gene status (n¼ 106) were removed using the EXOSAPit (Amersham, Otelfingen, Wild-type 85 (80.2) Switzerland). Samples were then subjected to direct sequencing Mutation 21 (19.8) of single-stranded PCR products using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) and the ABI Prism 3130 5-year survival (%) (n¼ 279) genetic analyser (Applied Biosystems). All products were se- Rate (95%CI) 60.3 (54 – 66) quenced bi-directionally. Analysis of MSI status was based on the Continuous variables age and tumour diameter are described as the mean and range multiplex amplification of the five microsatellites (BAT25, BAT26, of values, whereas categorical variables are represented by the number of cases (% of D2S123, D5S346 and D17S250). An initial denaturation step at cases). Survival time was obtained using the Kaplan – Meier method. 951C for 10 min was followed by 42 cycles at 951C for 40 s, 541C for 40 s and 721C for 60 s. For the analysis, 1 ml of the DNA weight marker ROX 500 (Applied Biosystem) was added and 10 ml of deionised formamide in 3 ml of the PCR amplified solution. (Applied Biosystems). MSS and MSI-low (MSI-L) status were DNA was denaturated by incubation for 2 min at 951C. The defined as instability at zero and one markers, respectively. MSI-H POP-7 polymer solution (Applied Biosystem) was used for was characterised by the presence of instability in two or more the electrophoresis on the ABI Prism 3130 genetic analyser markers (Umar et al, 2004). British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Cohort 2-Tissue microarray Table 2 Characteristics of patients in Cohort 2 (n¼ 191) Colorectal cancer tissue microarray construction Atissuemicro- Frequency N (%) array of 221 unselected, non-consecutive colorectal cancer patients Age (years) (n¼ 184) treated at the Second Department of Pathology, University of Athens Mean, range 67.9 (35 – 93) between the years 2004 and 2006 was constructed at the Institute for Pathology, University Hospital of Basel. The use of this material was Tumour diameter (mm) (n¼ 189) approved by the local ethics committee of the University of Athens. Mean, range 4.6 (1.0 – 12.0) Each patient had multiple tissue punches taken from formalin-fixed, paraffin-embedded blocks using a tissue cylinder with a diameter of Gender (n¼ 189) 0.6 mm, which were subsequently transferred into one recipient Male 88 (46.6) paraffin block (3 2.5 cm )using ahomemadesemi-automatedtissue Female 101 (53.4) arrayer. Tissues were obtained from the tumour centre, the invasive Tumour location (n¼ 190) tumour front within the representative area of most intense tumour Left-sided 113 (59.5) budding in all sections of the tumour, as determined from Right-sided 27 (14.2) corresponding H&E slides, the normal adjacent mucosa (if available) Rectum 50 (26.3) and the transitional zone where tumour and normal adjacent mucosa first interact (if available). Each patient on average had 5.1 tissue Histological subtype (n¼ 189) punches included on this array. The final tissue microarray contained Mucinous 24 (12.7) 1079 tissues, namely 437 tissues from the tumour centre, 430 from the Other 165 (87.3) invasive front, 90 from normal adjacent mucosa and 122 from the transitional zone. For the purposesofthisstudy,onlytissuepunches pT stage (n¼ 189) from the invasive tumour front per patient were analysed. pT1 – 2 52 (27.5) pT3 – 4 137 (72.5) Clinico-pathological features H&E slides were reviewed and pN stage (n¼ 189) histomorphological data included histological subtype, pT stage, pN0 102 (54.0) pN stage, pM stage, tumour grade, and vascular and lymphatic pN1 – 2 87 (46.0) invasion. Clinical data were retrieved from patient records and included age at diagnosis, gender, tumour location and follow up. TNM stage (n¼ 189) Information on adjuvant therapy was available for all patients. Stage I 45 (23.8) Patients with TNM stage IV disease were removed from this study, Stage II 57 (30.2) thus 191 patients with stage I–III disease constituted the final Stage III 87 (46.0) patient cohort. Clinical outcome of interest was disease-specific Tumour grade (n¼ 189) survival time, which was available for all 191 patients. Median G1 – 2 122 (64.6) follow-up time is 35 months and 44 patients died of the disease. G3 67 (35.5) Patient characteristics are listed in Table 2. Vascular or lymphatic invasion (n¼ 191) Immunohistochemistry Immunohistochemistry was carried out Absent 147 (91.1) usinganti-CD8antibodyatthe Second Department of Pathology, Present 17 (8.9) University of Athens. Briefly, 5-mm TMA sections were deparaffinised, and pre-treated with the Envision FlexTarget Retrieval Solution pH8.8 Adjuvant therapy (n¼ 191) (DM812, Dako) at 8001C for 6 min followed by incubation at room None 69 (36.1) temperature for 10 min. After wash steps, peroxidase blocking was Treated 122 (63.9) carried out for 10 min. Sections were again washed and then incubated with primary antibody against CD8 (DakoCytomation; clone CD8/ 5-year survival (%) (n¼ 191) Rate (95%CI) 67.5 (57 – 76) 144B) for 60 min. Subsequently, sections were washed with TBS and incubated with Real Envision Solution (Dako) for 30 min, then washed Continuous variables age and tumour diameter are described as the mean and range again in TBS and incubated with DAB-Chromogen for 10 min. After of values, whereas categorical variables are represented by the number of cases (% of washing, sections were counterstained with haematoxylin for 2 min. cases). Survival time was obtained using the Kaplan – Meier method. Only peritumoural CD8þ T-lymphocytes in punches taken from the invasive tumour front were counted. w and Fisher’s exact tests, where appropriate. Univariate survival Cut-off score for low vs high budding, CD8þ and CD8þ / buds analysis was carried out using the Kaplan–Meier and log-rank indices All cut-off scores to classify patients as having a ‘low’ or tests. Multivariable analysis was carried out using Cox’s propor- ‘high’ index were obtained by receiver operating characteristic tional hazards regression analysis after the verification of curve (ROC) analysis (Zlobec et al, 2007b). 50% of the data was proportional hazards assumption. Hazard ratio (HR) and 95%CI randomly selected for this purpose. Using this method, a plot of were obtained to determine the prognostic effect of each index the sensitivity and false positive rate (1-specificity) for discrimi- after adjustment. With logistic regression analysis, the odds ratios nating between survivors and non-survivors was assessed. The (OR) and 95%CI were obtained to determine the odds of death at most discriminating cut-off score was determined as the point on 5 years with the use of different indices. All statistical analyses the ROC curve with the shortest distance to the coordinate (0, 1), were carried out using SAS (V9, The SAS Institute, NC, Cary, USA). namely with the maximum sensitivity and specificity for survival. The reliability of all cut-off scores was tested by re-sampling of the data (500 bootstrapped replications). The discriminatory ability of RESULTS each feature for survival was also evaluated by analyzing the area under the ROC curve (AUC) and 95% confidence interval (CI). Cohort 1-Whole tissue sections Additional statistical analyses The association of indices with Tumour-budding index A high tumour-budding index was categorical clinico-pathological features was obtained using the defined, using ROC curve analysis, as at least 16 buds per  40 & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al A B 0.9 0.8 0.8 0.7 Low budding index 0.6 0.6 Number of buds Cut-off score = 16 buds 0.5 High budding index 0.4 0.4 0.3 0.2 0.2 0.1 AUC (95% Cl) = 0.59 (0.52−0.67) P = 0.022 0 12 24 36 48 60 72 84 96 108 120 0 0.2 0.4 0.6 0.8 1 1–specificity Time (months) C D 0.9 0.8 0.8 CD8+ T-cell High CD8+ index 0.7 Cut-off score = 40 cells 0.6 0.6 0.5 0.4 Low CD8+ index 0.4 0.3 0.2 0.2 0.1 AUC (95% Cl) = 0.64 (0.59−0.7) P < 0.001 0 0.2 0.4 0.6 0.8 1 0 1224364860728496 108 120 1–specificity Time (months) EF 0.9 0.8 CD8+/buds ratio 0.8 High CD8+/buds index cut-off score = 3/1 0.7 0.6 0.6 0.5 0.4 0.4 Low CD8+/buds index 0.3 0.2 0.2 0.1 AUc (95% Cl) = 0.68 (0.61−0.75) P < 0.001 0 0 0 0.2 0.4 0.6 0.8 1 0 1224364860728496 108 120 Time (months) 1–specificity Figure 2 Cohort 1. (A, C and E): Receiver operating characteristic (ROC) curves highlighting the ability of tumour budding (A), CD8þ lymphocytes (C) and CD8þ / buds index (E) to discriminate survivors and non-survivors. AUC¼ area under the ROC curve. Larger the AUC, more discriminating the feature. Arrows are drawn from the point on the curve used to classify patients into ‘low’ or ‘high’ indices based on their proximity to the coordinate (0,1). (B, D and F) corresponding Kaplan –Meier survival curves for ‘low’ and ‘high’ indices. field (Figure 2A). However, the ROC curve also highlights a (P¼ 0.028) and with an infiltrating tumour border configuration relatively poor discrimination of survivors and non-survivors at (P¼ 0.005) (Table 3). Patients with MSI-H cancers were signifi- 5-year follow up with an AUC value of 0.59 (95%CI: 0.52–0.67). cantly less prone to a high tumour-budding index (P¼ 0.022), A high-budding index was significantly associated with a more whereas no association was observed with KRAS or BRAF advanced pT stage (P¼ 0.017), with the presence of lymph node mutation. The odds of death from disease at 5 years in the group metastasis (P¼ 0.002), with the presence of vascular invasion with a high-budding index was OR (95%CI)¼ 1.89 (1.1–3.3) British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Sensitivity Sensitivity Sensitivity Survival probability Survival probability Survival probability Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Table 3 Association of budding index, CD8+ index and CD8þ / buds index with clinico-pathological features – Cohort 1 (n¼ 279) Budding index CD8+ index CD8+/buds index Clinico-pathological feature Low High P-value Low High P-value Low High P-value Gender Female 107 (56.9) 50 (55.0) 0.797 61 (54.0) 87 (57.2) 0.598 72 (54.1) 85 (58.2) 0.492 Male 81 (43.1) 41 (45.1) 52 (46.0) 65 (42.8) 61 (45.9) 61 (41.8) Tumour location Left-sided 48 (25.5) 23 (25.3) 0.06 30 (26.5) 37 (40.7) 0.551 34 (25.6) 37 (25.3) 0.053 Right-sided 82 (43.6) 29 (31.9) 42 (37.2) 62 (40.8) 45 (33.8) 66 (45.2) Rectum 58 (30.9) 39 (42.9) 41 (36.3) 53 (34.9) 54 (40.6) 43 (29.5) pT stage pT1 – 2 34 (18.6) 7 (7.7) 0.017 15 (13.5) 20 (13.4) 0.983 15 (11.4) 26 (18.3) 0.107 pT3 – 4 149 (81.4) 84 (92.3) 96 (86.5) 129 (86.6) 117 (88.6) 116 (81.7) pN stage pN0 106 (57.9) 34 (37.8) 0.002 50 (45.5) 81 (54.4) 0.156 53 (40.5) 87 (61.3) o 0.001 pN1 – 2 77 (42.1) 56 (62.2) 60 (54.6) 68 (45.6) 78 (59.5) 55 (38.7) Tumour grade G1 – 2 166 (90.7) 86 (94.5) 0.276 104 (63.7) 135 (90.6) 0.366 125 (94.7) 127 (89.4) 0.109 G3 17 (9.3) 5 (5.5) 7 (6.3) 14 (9.4) 7 (5.3) 15 (10.6) Vascular invasion Absent 145 (79.2) 61 (67.0) 0.028 79 (71.2) 115 (77.2) 0.271 90 (68.2) 116 (81.7) 0.009 Present 38 (20.8) 30 (33.0) 32 28.8) 34 (22.8) 42 (31.8) 26 (18.3) Tumour border configuration Infiltrating 114 (63.0) 72 (80.0) 0.005 80 (73.4) 102 (68.9) 0.435 100 (76.9) 86 (61.0) 0.005 Pushing 67 (37.0) 18 (20.0) 29 (26.6) 46 (31.1) 30 (23.1) 55 (39.0) Peritumoural lymphocytic inflammation Absent 137 (74.9) 67 (73.6) 0.825 93 (83.8) 100 (67.1) 0.002 108 (81.8) 96 (67.6) 0.007 Present 46 (25.1) 24 (26.4) 18 (16.2) 49 (32.9) 24 (18.2) 46 (32.4) KRAS gene status Wild-type 55 (75.3) 27 (61.4) 0.11 31 (64.6) 47 (74.6) 0.253 38 (64.4) 44 (75.9) 0.176 Mutation 18 (24.7) 17 (38.6) 17 (65.4) 16 (25.4) 21 (35.6) 14 (24.1) BRAF gene status Wild-type 54 (77.1) 31 (86.1) 0.273 36 (90.0) 45 (75.0) 0.063 41 (85.4) 44 (75.9) 0.219 Mutation 16 (22.9) 5 (13.9) 4 (10.0) 15 (25.0) 7 (14.6) 14 (24.1) Microsatellite instability status (MSS) MSS/MSI-L 54 (69.2) 41 (87.2) 0.022 43 (86.0) 47 (69.1) 0.033 51 (83.6) 44 (68.8) 0.052 MSI-H 24 (30.8) 6 (12.8) 7 (14.0) 21 (30.9) 10 (16.4) 20 (31.2) Survival rate (%) (95% CI) 5-year 63.9 (57 – 70) 53.0 (42 – 63) 0.033 47.2 (38 – 56) 67.9 (60 – 75) o 0.001 46.6 (38 – 55) 72.8 (65 – 79) o 0.001 Table 4 Comparison of the discriminatory ability of each feature for identifying survivors and non-survivors at 5-year follow up Feature Sensitivity Specificity Overall accuracy (95% CI) OR (95% CI) P-value Budding count (high vs low) 0.413 0.737 0.59 (0.52 – 0.67) 1.89 (1.1 – 3.3) 0.022 CD8+ count (low vs high) 0.597 0.683 0.64 (0.59 – 0.7) 3.18 (1.8 – 5.5) o0.001 CD8 : buds index (low vs high) 0.721 0.61 0.68 (0.61 – 0.75) 4.12 (2.4 – 7.1) o0.001 Odds ratio (OR) indicate that the odds of disease-specific death at 5-years is 1.89 times higher in the high-budding count group compared with the low group; for CD8+ count, 3.18 times higher in the low compared with the high CD8+ count group and for the CD8 : buds index, is 4.12 times higher in those with a low compared with a high index. compared with those with a low index (P¼ 0.022) (Table 4). In all, more advanced pT stage (P¼ 0.019), pN stage (P¼ 0.002), vascular 5-year disease-specific survival rate of patients with high and low invasion (P¼ 0.004) and an infiltrating tumour border configu- tumour-budding index was 53.0 (95%CI 42–63) and 63.9 (95%CI ration (P¼ 0.021) and worse survival in univariate analysis 57–70), suggesting a significantly worse prognosis in patients with (P¼ 0.02). In MSI-H patients, the high tumour-budding index high tumour-budding index (P¼ 0.033) (Figure 2B). In patients was only related to an infiltrating tumour border configuration with MSS/MSI-L tumours, a high-budding index was related to (P¼ 0.015). & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al CD8þ index A high CD8þ index was characterised by the inflammation at the invasive front (P¼ 0.007) and a marginal presence of at least 40 CD8þ cells per  40 field (Figure 2C). The association with MSI-H status (P¼ 0.052). Most notably, a discriminatory ability of CD8þ as indicated by the AUC was 0.64 considerable difference in survival time between patients with (95%CI 0.59–0.7), suggesting an improvement compared with the low and high CD8þ / buds index was observed with patients with a budding index alone. The odds of death from disease at 5 years low index demonstrating a highly adverse prognosis (Po0.001) was OR (95%CI)¼ 3.18 (1.8–5.5) in patients with a low compared (Figure 2F). This prognostic difference was also maintained in the with a high CD8þ index. A high CD8þ index was associated group of MSS/MSI-L patients (P¼ 0.001). with significantly more frequent peritumoural lymphocytic inflam- mation at the tumour front (P¼ 0.002), with MSI-H status Independent prognostic effects of budding, CD8þ and CD8þ / (P¼ 0.033) and marginally with BRAF mutation. Patients with buds indices A comparison of the performance of each of these a high CD8þ index demonstrated a significantly prolonged indices in multivariable survival analysis is outlined in Table 5. survival time compared with those with a low CD8þ index The effect of each of these factors was re-evaluated along with age, (Po0.001) (Figure 2D). In MSS/MSI-L and MSI-H patients, the gender, pT stage, pN stage, tumour grade, vascular invasion and CD8þ index was not linked to any features of tumour progression the tumour border configuration. Analysis was thus carried out although in MSS/MSI-L those with a high CD8þ index only for patients with complete clinico-pathological information experienced a significant prolonged survival time (P¼ 0.011). for all these features (n¼ 226, 101 deaths). The budding index was not found to add independent prognostic information when CD8þ / buds index A ratio of at least three times more analysed along with these additional features (P¼ 0.85), whereas CD8þ T-cells vs the number of tumour buds led to a strong the CD8þ index (Po0.001) and the CD8þ / buds index discrimination of survivors and non-survivors of colorectal cancer, (Po0.001) were highly relevant as prognostic factors in multi- as indicated by an AUC value of 0.68 (95%CI 0.61–0. 75) variable analysis, with similar relative risks of death suggesting a (Figure 2E). The odds of death at 5 years in patients with a low comparable performance of these two features as prognostic CD8þ / buds index compared with those with a high index was indices. OR (95%CI)¼ 4.12 (2.4–7.1) indicating that on average the odds In order to prevent over-fitting of multivariable models, only the of death was 4.12 times greater in patients with a low compared most relevant prognostic factors were included into sub-group with a high CD8þ / buds index (Table 4). Patients with a high analyses of lymph node-negative patients (n¼ 140, 42 deaths), as CD8þ / buds index demonstrated more favourable features, such well as of those with colon (n¼ 176; 79 deaths) or rectal cancers as early T stage (Po0.001), absence of vascular invasion (n¼ 94; 44 deaths). In lymph node-negative patients, multivariable (P¼ 0.009), presence of a pushing/expanding tumour border analysis with pT stage, vascular invasion and tumour border configuration (P¼ 0.005), presence of peritumoural lymphocytic configuration revealed independent prognostic effects for both the Table 5 Comparison of relative risks of buds, CD8+ and CD8+/ buds index in multivariable analysis with age, gender, pT stage, pN stage, tumour grade, vascular invasion and tumour border configuration in Cohort 1 Buds index only CD8+ index only CD8+/ buds index HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Index Low 1.0 0.85 1.0 o0.001 1.0 o0.001 High 1.04 (0.7 – 1.6) 0.45 (0.3 – 0.7) 0.44 (0.3 – 0.7) Age (years) Baseline 1.0 0.041 1.0 0.026 1.0 0.021 Increasing by year 1.02 (1.0 – 1.1) 1.02 (1.0 – 1.1) 1.02 (1.0 – 1.1) Gender Male 1.0 0.468 1.0 0.507 1.0 0.443 Female 0.86 (0.6 – 1.3) 0.87 (0.6 – 1.3) 0.86 (0.6 – 1.3) pT stage pT1 – 2 1.0 0.042 1.0 0.048 1.0 0.022 pT3 – 4 2.41 (1.0 – 5.6) 2.35 (1.0 – 5.5) 2.69 (1.2 – 6.3) pN stage pN0 1.0 o0.001 1.0 o0.001 1.0 0.001 pN1 – 2 2.27 (1.5 – 3.5) 2.12 (1.4 – 3.3) 2.06 (1.4 – 3.2) Tumour grade G1 – 2 1.0 0.205 1.0 0.738 1.0 0.873 G3 0.63 (0.3 – 1.3) 0.88 (0.4 – 1.9) 0.94 (0.4 – 2.0) Vascular invasion Absent 1.0 o0.001 1.0 0.001 1.0 0.014 Present 2.2 (1.4 – 3.5) 2.1 (1.3 – 3.3) 1.79 (1.1 – 2.8) Tumour border configuration Pushing 1.0 0.758 1.0 0.945 1.0 0.915 Infiltrating 1.09 (0.6 – 1.8) 1.02 (0.6 – 1.8) 1.03 (0.6 – 1.8) British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Table 6 Association of budding index, CD8+ index and CD8þ / buds index with clinico-pathological features – Cohort 2 (n¼ 191) Budding index CD8+ index CD8+/ buds index Low High Low High Low High Clinico-pathological feature (n¼ 131) (n¼ 60) P-value (n¼ 69) (n¼ 121) P-value (n¼ 68) (n¼ 122) P-value Gender Male 65 (50.4) 23 (38.3) 0.122 34 (49.3) 54 (54.4) 0.606 33 (48.5) 55 (45.8) 0.722 Female 64 (49.6) 37 (61.7) 35 (50.7) 65 (54.6) 35 (51.5) 65 (54.2) Tumour location Left-sided 80 (61.5) 33 (55.0) 0.311 38 (55.1) 74 (61.7) 0.673 35 (51.5) 77 (63.6) 0.054 Right-sided 20 (15.4) 7 (11.7) 11 (15.9) 16 (13.3) 8 (11.8) 19 (15.7) Rectum 30 (23.1) 20 (33.3) 20 (16.0) 30 (25.0) 25 (36.8) 25 (20.7) pT stage pT1 – 2 42 (32.3) 10 (17.0) 0.029 15 (21.7) 37 (31.1) 0.167 9 (13.4) 43 (35.5) 0.001 pT3 – 4 88 (67.7) 49 (83.0) 54 (78.3) 82 (68.9) 58 (86.6) 78 (64.5) pN stage pN0 79 (71.2) 23 (67.7) 0.694 30 (60.0) 71 (75.5) 0.053 24 (58.5) 77 (74.8) 0.055 pN1 – 2 32 (28.8) 11 (32.4) 20 (40.0) 23 (24.5) 17 (41.5) 26 (25.2) Tumour grade G1 – 2 93 (71.5) 29 (49.2) 0.003 37 (53.6) 85 (71.4) 0.014 29 (43.3) 93 (76.9) o0.001 G3 37 (28.5) 30 (50.8) 32 (46.4) 34 (28.6) 38 (56.7) 28 (23.1) Vascular invasion Present 8 (6.1) 9 (15.0) 0.045 12 (17.4) 5 (4.1) 0.002 12 (17.7) 5 (4.1) 0.002 Absent 123 (93.9) 51 (85.0) 57 (82.6) 116 (95.9) 56 (82.4) 117 (95.9) Survival rate (%) (95%CI) 5 years 75.0 (62 – 84) 52.9 (36 – 67) 0.003 49.4 (30 – 66) 77.1 (66 – 85) 0.031 43.8 (26 – 60) 81.5 (69 – 89) o0.001 CD8þ index (P¼ 0.001) and the CD8þ / buds index (P¼ 0.002), the independent adverse prognostic effect of a low CD8þ / buds but not for the budding index. Moreover, in patients with colon or index after adjusting for these additional factors. rectal cancer, the CD8þ index (P¼ 0.015 and P¼ 0.006, respec- tively) as well as the CD8þ / buds index (P¼ 0.002 and P¼ 0.016, DISCUSSION respectively) maintained a significant independent prognostic effect after adjusting for pT stage, pN stage and vascular invasion. The aim of this study was to investigate an alternative approach to reflect the dynamic at the tumour front of colorectal cancer using an index including tumour budding and CD8þ lymphocytes. Cohort 2-Tissue microarray Briefly summarised, our results confirm the prognostic impact In Cohort 2, a high tumour-budding index in stages I–III patients of the selected tumour and host-related factors. In both patient was defined as at least six buds per punch and was related to a more cohorts, a high tumour budding and a low CD8þ lymphocytes advanced pT stage (P¼ 0.029), more poorly differentiated tumours index were associated with tumour progression and worse survival. (P¼ 0.003), presence of vascular invasion (P¼ 0.045) and was highly Indeed, tumour budding has previously been found to be related to a worse prognosis (P¼ 0.003) (Table 6). The threshold associated with higher N stage, higher tumour grade and presence value for a high CD8þ index was 13 cells per punch and was linked of vascular invasion, local tumour recurrence, distant metastasis as to lymph node negativity (P¼ 0.053), early tumour grade (P¼ 0.014), well as worse survival (Ueno et al, 2004a,b; Nakamura et al, 2005; absence of vascular invasion (P¼ 0.002) and a prolonged survival Kazama et al, 2006; Prall, 2007; Ishikawa et al, 2008; Wang et al, time compared with patients with a low CD8þ index (P¼ 0.031). 2009). There is also strong evidence for an association between a A ratio of three times more CD8þ lymphocytes to the number high number of CD8þ lymphocytes and absence of lymph node of tumour buds was identified as the most discriminating cut-off metastases, lower tumour grade, presence of peritumoural value to classify survivors and non-survivors. Patients with a lymphocytes, mismatch repair-deficient status and better survival high CD8þ / buds index had tumours that were more early pT (Ropponen et al, 1997; Naito et al, 1998; Chiba et al, 2004; Galon stage (P¼ 0.001), lymph node-negative (P¼ 0.055), early tumour et al, 2006; Koch et al, 2006; Baker et al, 2007). grade (Po0.001) and vascular invasion negative (P¼ 0.002). Low The question, therefore, arises as to whether a CD8þ CD8þ / buds index, led to a highly unfavourable prognosis lymphocytes : budding index is more advantageous over the use compared with patients with a high CD8þ / buds index (Po0.001). of one out of the two parameters alone. With the exception of This difference was also maintained in sub-group analysis of TNM 5-year survival, a certain intra- and inter-group variability stage II (P¼ 0.019) and stage III (P¼ 0.004) patients (Figure 3A is observed when analysing the relationship of tumour budding and B). In particular, in both untreated and treated patients, a low or CD8þ lymphocytes with clinico-pathological parameters. CD8þ / buds index was linked to a shorter survival time (P¼ 0.009 In contrast, the use of a CD8þ lymphocytes : budding index and Po0.001, respectively) (Figure 3B and C). Multivariable analysis better summarises these associations with key endpoints of for the budding index, CD8þ index and CD8þ / buds index was tumour progression, namely presence of lymph node metastases, carried out accounting for pN stage, vascular and lymphatic invasion vascular invasion and worse survival, and moreover, in both and treatment effect. Results outlined in Table 7 highlight once more cohorts. In addition, the CD8þ / buds index shows an increased & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al High CD8+/buds index (n =37; 2 deaths) 1.00 1.00 High CD8+/buds index (n =44; 10 deaths) 0.75 0.75 Low CD8+/buds index 0.50 (n =19; 5 deaths) 0.50 Low CD8+/buds index (n =43; 23 deaths) 0.25 0.25 TNM stage II; P = 0.019 TNM stage III; P =0.004 0.00 0.00 0 10203040506070 0 10203040506070 Time (months) Time (months) High CD8+/buds index High CD8+/buds index (n =69; 9 deaths) 1.00 1.00 (n=53; 7 deaths) 0.75 0.75 Low CD8+/buds index 0.50 0.50 (n =15; 7 deaths) Low CD8+/buds index (n =53; 21 deaths) 0.25 0.25 No adjuvant therapy; P =0.009 Adjuvant therapy; P< 0.001 0.00 0.00 0 10203040506070 0 10203040506070 Time (months) Time (months) Figure 3 Cohort 2. Kaplan –Meier survival curves illustrating the poorer outcome in patients with a low CD8þ / buds index in (A) TNM stage II patients, (B) TNM stage III patients, (C) patients not receiving adjuvant therapy and (D) patients treated with adjuvant therapy. Table 7 Comparison of relative risks of buds, CD8+ and CD8þ / buds index in multivariable analysis with pN stage, vascular and lymphatic invasion and adjuvant therapy in Cohort 2 Buds index only CD8+ index only CD8+/ buds index Cohort 2 HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Index Low 1.0 0.029 1.0 0.473 1.0 0.005 High 1.96 (1.1 – 3.6) 0.79 (0.4 – 1.5) 0.39 (0.2 – 0.8) pN stage pN0 1.0 o0.001 1.0 o0.001 1.0 0.002 pN1 – 2 4.11 (1.8 – 9.2) 4.41 (2.0 – 9.7) 3.58 (1.6 – 7.9) Vascular/lymphatic invasion Absent 1.0 o0.001 1.0 0.006 1.0 0.007 Present 3.41 (1.7 – 7.1) 2.97 (1.4 – 6.4) 2.76 (1.3 – 5.7) Adjuvant therapy Untreated 1.0 0.057 1.0 0.068 1.0 0.076 Treated 0.5 (0.2 – 1.0) 0.52 (0.3 – 1.1) 0.53 (0.3 – 1.1) discriminatory ability for identifying survivors and non-survivors of death at 5 years compared with a three-fold or under two-fold at 5-year follow up compared with tumour budding or CD8þ risk for patients with low CD8þ counts or high number of tumour lymphocytes count themselves. More specifically, patients with a buds, respectively. In addition, the CD8þ / buds index maintained low CD8þ lymphocyte : buds index have more than four-fold odds its significant impact on outcome in patients with stage II disease, British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Survival probability Survival probability Survival probability Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al in both colon and rectal cancer patients and also, independently of completely independent cohorts of colorectal cancer patients treated adjuvant therapy. Our previous work describes the absence of in Switzerland and Greece, respectively. In addition to whole tissue tumour budding in cases with marked peritumoural inflammatory sections, we used the tissue microarray technique, a powerful tool reaction at the invasive margin of colorectal cancers and a strong for the investigation of novel or potential biomarkers, which has correlation between this inflammation and the presence of allowed us to evaluate hundreds of tissue cores for CD8þ . TILs(Zlobec et al, 2007a). We hypothesised that an immune Moreover, the evaluation of multiple tissue punches per tumour in response to colorectal cancer could account for an improved this study exceeds the number of cores required for adequate prognosis in patients with abundant TIL count by specifically representativity of tumour heterogeneity (Goethals et al,2006). targeting budding cells, a so-called ‘nipping in the bud’ effect Although double staining was carried out for Cohort 1, only leading to improved prognosis. Although we cannot directly allude immunohistochemistry for CD8 was carried out on Cohort 2. These to the functional interaction of CD8þ lymphocytes and tumour differences in methodology were chosen based on the ease of budding in this study, our data nonetheless point to a strong link identification of buds. In whole tissue sections, budding can often be between favourable prognosis and a high CD8þ lymphocyte : missed because of peritumoural inflammation, whereas tissue tumour-budding index. microarrays may aid the observer to focus on one specific area Several points in this study should still be considered. Although containing tumour buds. It is important to note that the independent the impact of tumour budding on clinical outcome is validated by prognostic effects of the CD8þ / buds index were found in both sub- several groups, a standardised scoring system for this feature has groups despite the different laboratory circumstances and meth- not yet been established, although several similar methods have odologies, thereby underlining the biological consistency of the been proposed (Ueno et al, 2002; Shinto et al, 2005; Prall and CD8þ / buds index in colorectal cancer. Although we recognise that Ostwald, 2007). This study highlights a new approach, which may this particular index is unlikely to be a final solution for daily be helpful in furthering the understanding of colorectal pathogen- diagnostic practice, it may however serve as a basis that can be esis and prognosis, but requires validation by other research further extended to include other strong and validated ‘pro-tumour’ groups both in retrospective and prospective settings. Finally, our or ‘anti-tumour’. This approach could potentially lead to a new findings, particularly with regard to the test group may be colorectal cancer prognostic score, which can be applied in addition somewhat influenced by the lack of clinical information regarding to TNM staging, as is the case for the BRE score for breast cancers. recurrence, metastasis and treatment although the majority of In conclusion, the CD8þ lymphocyte to tumour-budding index clinico-pathological features are adequately covered. is an independent prognostic factor in colorectal cancer and On the other hand, several factors strongly support the validity of represents biologically a ‘pro-/anti-tumour’ model that could be a our findings. The selection of parameters included in the proposed promising approach for a future prognostic score in colorectal index is evidence based, as both tumour budding and the CD8þ cancer. lymphocytes have been tested and validated in colorectal cancer by different research groups. 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JClinOncol 27: 186– 192 1112– 1116 British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour’ approach to tumour host interaction in colorectal cancer

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Springer Journals
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Copyright © 2009 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.6605318
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Molecular Diagnostics British Journal of Cancer (2009) 101, 1382 – 1392 & 2009 Cancer Research UK All rights reserved 0007 – 0920/09 $32.00 www.bjcancer.com CD8þ lymphocytes/ tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour’ approach to tumour host interaction in colorectal cancer 1 2 2 3 4 4 5 5 1 A Lugli , E Karamitopoulou , I Panayiotides , P Karakitsos , G Rallis , G Peros , G Iezzi , G Spagnoli , M Bihl , 1 ,1 L Terracciano and I Zlobec 1 2 Institute of Pathology, University of Basel, Scho¨nbeinstrasse 40, Basel 4031, Switzerland; Second Department of Pathology, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, Greece; Department of Diagnostic Cytopathology, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, 4 5 Greece; Fourth Department of Surgery, Attikon University Hospital, Rimini 1, Haidari, Athens 12464, Greece; Institute for Surgical Research and Hospital Management, University of Basel, Hebelstrasse 20, Basel 4031, Switzerland BACKGROUND: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial–mesenchymal transition and its hallmark ‘tumour budding’, is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a ‘pro-/anti-tumour’ approach defined by an established ‘pro-tumour’ (tumour budding) and host-related ‘anti-tumour’ factor of the adaptive immunological microenvironment (CD8þ lymphocytes). METHODS: Double immunostaining for CK22/CD8 on whole tissue sections (n¼ 279; Cohort 1) and immunohistochemistry for CD8þ using tissue microarrays (n¼ 191; Cohort 2) was carried out. Tumour buds, CD8þ and CD8þ T-lymphocytes : tumour buds indices were evaluated per high-power field. RESULTS: In Cohort 1, a low-CD8þ / buds index was associated with lymph node metastasis (Po0.001), vascular invasion (P¼ 0.009), worse survival in univariate (Po0.001) and multivariable (Po0.001) analysis, and furthermore in lymph node-negative patients (P¼ 0.002). In Cohort 2, the CD8þ / buds index was associated with T stage (Po0.001), N stage (P¼ 0.041), vascular invasion (P¼ 0.005) and survival in patients with TNM stage II (P¼ 0.019), stage III (P¼ 0.004), and adjuvantly untreated (P¼ 0.009) and treated patients (Po0.001). CONCLUSION: The CD8þ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease. British Journal of Cancer (2009) 101, 1382–1392. doi:10.1038/sj.bjc.6605318 www.bjcancer.com Published online 15 September 2009 & 2009 Cancer Research UK Keywords: tumour budding; CD8þ lymphocytes; colorectal cancer; prognosis During the Napoleonic Wars (1796–1815), one of the most factors, such as CD3þ,CD4þ,CD8þ,CD20þ lymphocytes, effective methods to attack in battle was the deployment of heavy Granzyme B, FoxP3þ regulatory T cells (Tregs), CD16þ cells, and cavalry, which the opponent tried to repel by organizing the mast and dendritic cells (Dadabayev et al,2004;Phillips et al, 2004; infantry into squares. Sato et al, 2005; Chaput et al,2009;Salama et al, 2009). A similar picture can be observed at the invasion front of colorectal Consequently, several groups focus on the invasive front of cancer. On one hand, the tumour mass invades the pericolic fat tissue, colorectal cancer using the term epithelial–mesenchymal transi- detaching clusters or single tumour cells (tumour buds) reflecting tion (EMT), which characterises tumour invasion by de-differ- tumour progression; whereas, on the other hand the host attempts to entiated colorectal carcinoma cells (Brabletz et al, 2005). EMT and confront this situation by building an ‘anti-tumour’ cytotoxic MET, the reverse transition from a mesenchymal to an epithelial inflammatory response. This ‘pro-/anti-tumour’ model is supported phenotype, are crucial steps not only in embryonic development by a range of studies proposing either independent tumour-related but also in tumour progression (Spaderna et al, 2007). prognostic factors (pro-tumour), such as tumour grade, tumour A histomorphological hallmark of EMT is the phenomenon of border configuration, medullary subtype, CEA level, microsatellite ‘tumour budding’ (Prall, 2007), which according to the third instability, loss of heterozygosity (LOH) 18q status, p53 levels, TGFB1 edition of ‘Prognostic Factors in Cancer’ published by the UICC in type II receptor levels, VEGF expression, proliferation rate and 2006, is considered an additional prognostic factor in colorectal metalloproteinase expression (Compton, 2006) or anti-tumour cancer (Compton, 2006). A tumour bud is typically defined as a single tumour cell or tumour cell cluster of up to five cells at the *Correspondence: Dr I Zlobec, Institute of Pathology, University of Basel, invasive tumour front (Prall, 2007). Indeed, tumour budding has Scho¨nbeinstrasse 40, Basel, 4031, Switzerland; E-mail: [email protected] been shown to be associated with lymph node positivity, poorly Received 7 May 2009; revised 3 August 2009; accepted 19 August 2009; differentiated tumours, presence of vascular and lymphatic published online 15 September 2009 invasion, local tumour recurrence and distant metastasis (Ueno Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al et al, 2004a,b; Nakamura et al, 2005; Kazama et al, 2006; Ishikawa Therefore, the aim of this study was to investigate on a potential et al, 2008; Wang et al, 2009). In particular, patients with stage III ‘pro-/anti-tumour’ model and to test the prognostic impact of disease have been reported to demonstrate a 5-year disease-free a ratio defined by an established pro-tumour (tumour budding) survival (DFS) of 62.1% in the absence of tumour budding and and anti-tumour (CD8þ lymphocytes) factor. To this end, two only a 35.1% DFS with this feature (Choi et al, 2007). Moreover, independent colorectal cancer patient cohorts from different the presence of tumour budding has repeatedly been linked to centres were investigated using two different approaches, namely poor clinical outcome, underlined by the adverse effect on overall whole tissue sections (n¼ 300) and the tissue microarray survival independent of TNM stage (Hase et al, 1993; Ueno et al, technique (n¼ 221). 2004b). Over the last 20 years, investigations on tumour immunity and host defence in colorectal cancer demonstrate promising results MATERIALS AND METHODS for immunotherapy. In most colorectal cancers, lymphocytic infiltration is composed predominantly of either CD4þ or Cohort 1-Whole tissue sections CD8þ T cells and both cell types appear to be significantly increased in tumour as compared with normal tissue (Ropponen Sample size determination To reach 85% power with an expected et al, 1997; Naito et al, 1998; Chiba et al, 2004; Koch et al, 2006). risk ratio of 1.8 between prognostic groupings and potential Several studies have shown that tumour infiltrating lymphocytes loss of patient samples in 10% of cases the appropriate sample (TILs) within the stroma and around the tumour along the size for this study was determined to be 255 cases. Owing to the invasive margin are significantly related to overall- and disease- availability of material this number was increased to 300 cases. specific survival in both univariate and multivariable analysis (Ali Specimens: Paraffin-embedded tissue blocks of 300 resection et al, 2004; Canna et al, 2005; Pages et al, 2005). Galon et al (2006) specimens of patients treated between 1987 and 1996 at the evaluated by gene-expression profiling and immunohistochemis- University Hospital of Basel were retrieved from the archives of try, the type, density and location (whether at the invasive margin the Institute of Pathology, University Hospital of Basel, as well or the tumour centre) of TILs in a large number of cases. They as at the Institute of Clinical Pathology, Basel, Switzerland. These evaluated CD3, CD8, granzyme B and memory CD45RO T cells, 300 cases were randomly selected from a larger previously demonstrating a significant independent and positive effect of described cohort of 938 colorectal cancer patients with full TILs on both recurrence and survival. clinico-pathological information (Zlobec et al, 2008). The use of In colorectal cancer, mismatch-repair status (microsatellite material for this study was approved by the local research ethics stable (MSS) and microsatellite instability-high (MSI-H)) seems committee. to relate highly to the number of CD8þ lymphocytes. Compared with MSS tumours, MSI-H cancers are characterised by prolonged Double immunostaining for CD8 and CK22 A double immuno- survival time, significantly more frequent peritumoural lympho- staining procedure using anti-CD8 (for detection of CD8þ cytic infiltration at the invasive front and by an inherent T-lymphocytes) and pan-cytokeratin (to facilitate visualization abundance of intra-epithelial TILs (Jass et al, 1998; Michael- of tumour buds at the invasive front) was carried out on one Robinson et al, 2001; Greenson et al, 2003; Jenkins et al, 2007). representative slide cut at 4 mm from paraffin-embedded tumour Nevertheless, many studies analyzing colorectal cancer samples blocks of all 300 colorectal cancer patients included in this study stratified by mismatch-repair status also report a positive effect of (Figure 1). Double staining was carried out using the BOND-MAX CD8þ lymphocytes in mismatch-repair proficient colorectal Automated Immunohistochemistry Vision Biosystem (Leica cancers (Baker et al, 2007). Microsystems GmbH, Wetzlar, Germany) according to the Although this wide range of possible additional prognostic factors following protocol. First, tissues were deparaffinised and pre- in colorectal cancer is currently being investigated, still missing is an treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH8.8) approach to include parameters reflecting the tumour dynamics. Such at 1001C for 20 min. After wash steps, peroxidase blocking was an approach has already been taken for the prognostication of breast carried out for 10 min using the Bond Polymer Refine Detection cancer, namely the Bloom–Richardson–Elston (BRE) score, which Kit DC9800 (Leica Microsystems GmbH). Tissues were again encompasses information on mitoses, tubule differentiation and washed, then incubated with primary antibody against CK22 nuclear pleomorphism, and is used as an important prognostic (Biomeda, Foster City, CA, USA, pan-CK22) for 30 min. Subse- feature additional to TNM staging. quently, tissues were incubated with polymer for 15 min and then Figure 1 Double immunostaining for CD8 (red) and CK22 (brown): (A) Overview ( 10) and (B) high power field ( 40) of the invasive front of colorectal cancer showing CK22 positive tumour buds surrounded by CD8þ T-lymphocytes. & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al with DAB-Chromogen for 10 min (Bond Polymer AP Red Table 1 Characteristics of patients in Cohort 1 (n¼ 279) Detection Kit DS9305, Leica Microsystems GmbH). CK22 positive Clinico-pathological features Frequency N (%) cells were therefore coloured in brown. After washing, incubation was carried out with anti-CD8 (DakoCytomation, Glostrup, Age (years) (n¼ 279) Denmark, clone CD8/144B) for 30 min followed by application of Mean, range 67.7, 37 – 96 AEC-substrate for 10 min and counterstaining with haematoxylin for 2 min. Tumour diameter (mm) (n¼ 279) Mean, range 54.0, 13 – 170 Evaluation of tumour buds and CD8þ T-lymphocytes Tumour Gender (n¼ 279) budding was defined as an isolated single cancer cell or a cluster Male 122 (43.7) of up to five cells at the invasive front of colorectal cancer Female 157 (56.3) (Prall, 2007). The tumour border was scanned at a  100 magnification and the area of most intense budding was identified Tumour location (n¼ 279) (Ueno et al, 2002). After selecting this field, the number of buds Left-sided 71 (25.4) was counted using a 40 objective lens to focus specifically Right-sided 111 (39.8) on the presence of CD8þ T-cells most highly related to the Rectum 97 (34.7) microenvironment surrounding the tumour buds. Within this Histological subtype (n¼ 277) same field, all CD8þ lymphocytes were individually counted. In Adenocarcinoma 252 (91.0) 10 cases, the abundance of CD8þ infiltrate led to cell counts Mucinous 21 (7.6) exceeding 200 cells per field, and thus single cell counting was Other 4 (1.4) not feasible. These cases were assigned a CD8þ score of 200 cells. The ratio of CD8þ T-lymphocytes to the number of tumour pT stage (n¼ 274) buds (CD8þ / buds index) was obtained. In 13 cases when zero pT1 6 (2.2) pT2 35 (12.8) buds were identified, the count of CD8þ lymphocytes was not pT3 187 (68.3) carried out. pT4 46 (16.8) Clinico-pathological characteristics Of these 300 cases, 279 were pN stage (n¼ 273) evaluable for CD8 and CK22 protein expression, simultaneously. pN0 140 (51.3) Hematoxylin and eosin (H&E) stained slides were reviewed pN1 76 (27.8) and histomorphological data included histological subtype, pT pN2 57 (20.9) stage, pN stage and tumour grade. The tumour border configura- Tumour grade (n¼ 274) tion and the presence of conspicuous peritumoural lymphocytic G1 5 (1.8) infiltration were defined according to Jass et al (1986). Clinical G2 247 (90.2) data were retrieved from patient records and included age at G3 22 (8.0) diagnosis, gender, tumour location and follow up. Clinical outcome of interest was disease-specific survival time, which was Vascular invasion (n¼ 274) available for all 279 patients. Median follow-up time was 60 Absent 206 (75.2) Present 68 (24.8) months. In all, 128 patients died of disease. Patient characteristics are listed in Table 1. Tumour border configuration (n¼ 271) Infiltrating 186 (68.6) Microsatellite instability (MSI) status, KRAS and BRAF gene Pushing 85 (31.4) status Genomic DNA was obtained from primary tumours using NucleoMag 96 Tissue Kit (Macherey Nagel, Oensingen, Peritumoural lymphocytic infiltration (n¼ 274) Switzerland) protocol and processed in the Xiril X-100 robot (Xiril, Absent 204 (74.5) Hombrechtikon, Switzerland). Briefly, punched tissue was lysed in Present 70 (25.6) proteinase K. B-beads and MB2 buffer were added to the cleared Microsatellite instability status (n¼ 125) lysate and shaken for 5 min at room temperature. The supernatant Stable/low 95 (76.0) was removed and MB3 was added followed by shaking and Instable/high 30 (24.0) supernatant removal. The genomic DNA was eluted with MB6 buffer. Genomic DNA was amplified by PCR using AmpliTaq Gold KRAS gene status (n¼ 117) polymerase (Applied Biosystem, Foster City, CA, USA). KRAS Wild-type 82 (70.1) (exon 2, codon 12 and 13) and BRAF (exon 15, codon 600) were Mutation 35 (29.9) amplified by a first and a nested PCR. Residual primers BRAF gene status (n¼ 106) were removed using the EXOSAPit (Amersham, Otelfingen, Wild-type 85 (80.2) Switzerland). Samples were then subjected to direct sequencing Mutation 21 (19.8) of single-stranded PCR products using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) and the ABI Prism 3130 5-year survival (%) (n¼ 279) genetic analyser (Applied Biosystems). All products were se- Rate (95%CI) 60.3 (54 – 66) quenced bi-directionally. Analysis of MSI status was based on the Continuous variables age and tumour diameter are described as the mean and range multiplex amplification of the five microsatellites (BAT25, BAT26, of values, whereas categorical variables are represented by the number of cases (% of D2S123, D5S346 and D17S250). An initial denaturation step at cases). Survival time was obtained using the Kaplan – Meier method. 951C for 10 min was followed by 42 cycles at 951C for 40 s, 541C for 40 s and 721C for 60 s. For the analysis, 1 ml of the DNA weight marker ROX 500 (Applied Biosystem) was added and 10 ml of deionised formamide in 3 ml of the PCR amplified solution. (Applied Biosystems). MSS and MSI-low (MSI-L) status were DNA was denaturated by incubation for 2 min at 951C. The defined as instability at zero and one markers, respectively. MSI-H POP-7 polymer solution (Applied Biosystem) was used for was characterised by the presence of instability in two or more the electrophoresis on the ABI Prism 3130 genetic analyser markers (Umar et al, 2004). British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Cohort 2-Tissue microarray Table 2 Characteristics of patients in Cohort 2 (n¼ 191) Colorectal cancer tissue microarray construction Atissuemicro- Frequency N (%) array of 221 unselected, non-consecutive colorectal cancer patients Age (years) (n¼ 184) treated at the Second Department of Pathology, University of Athens Mean, range 67.9 (35 – 93) between the years 2004 and 2006 was constructed at the Institute for Pathology, University Hospital of Basel. The use of this material was Tumour diameter (mm) (n¼ 189) approved by the local ethics committee of the University of Athens. Mean, range 4.6 (1.0 – 12.0) Each patient had multiple tissue punches taken from formalin-fixed, paraffin-embedded blocks using a tissue cylinder with a diameter of Gender (n¼ 189) 0.6 mm, which were subsequently transferred into one recipient Male 88 (46.6) paraffin block (3 2.5 cm )using ahomemadesemi-automatedtissue Female 101 (53.4) arrayer. Tissues were obtained from the tumour centre, the invasive Tumour location (n¼ 190) tumour front within the representative area of most intense tumour Left-sided 113 (59.5) budding in all sections of the tumour, as determined from Right-sided 27 (14.2) corresponding H&E slides, the normal adjacent mucosa (if available) Rectum 50 (26.3) and the transitional zone where tumour and normal adjacent mucosa first interact (if available). Each patient on average had 5.1 tissue Histological subtype (n¼ 189) punches included on this array. The final tissue microarray contained Mucinous 24 (12.7) 1079 tissues, namely 437 tissues from the tumour centre, 430 from the Other 165 (87.3) invasive front, 90 from normal adjacent mucosa and 122 from the transitional zone. For the purposesofthisstudy,onlytissuepunches pT stage (n¼ 189) from the invasive tumour front per patient were analysed. pT1 – 2 52 (27.5) pT3 – 4 137 (72.5) Clinico-pathological features H&E slides were reviewed and pN stage (n¼ 189) histomorphological data included histological subtype, pT stage, pN0 102 (54.0) pN stage, pM stage, tumour grade, and vascular and lymphatic pN1 – 2 87 (46.0) invasion. Clinical data were retrieved from patient records and included age at diagnosis, gender, tumour location and follow up. TNM stage (n¼ 189) Information on adjuvant therapy was available for all patients. Stage I 45 (23.8) Patients with TNM stage IV disease were removed from this study, Stage II 57 (30.2) thus 191 patients with stage I–III disease constituted the final Stage III 87 (46.0) patient cohort. Clinical outcome of interest was disease-specific Tumour grade (n¼ 189) survival time, which was available for all 191 patients. Median G1 – 2 122 (64.6) follow-up time is 35 months and 44 patients died of the disease. G3 67 (35.5) Patient characteristics are listed in Table 2. Vascular or lymphatic invasion (n¼ 191) Immunohistochemistry Immunohistochemistry was carried out Absent 147 (91.1) usinganti-CD8antibodyatthe Second Department of Pathology, Present 17 (8.9) University of Athens. Briefly, 5-mm TMA sections were deparaffinised, and pre-treated with the Envision FlexTarget Retrieval Solution pH8.8 Adjuvant therapy (n¼ 191) (DM812, Dako) at 8001C for 6 min followed by incubation at room None 69 (36.1) temperature for 10 min. After wash steps, peroxidase blocking was Treated 122 (63.9) carried out for 10 min. Sections were again washed and then incubated with primary antibody against CD8 (DakoCytomation; clone CD8/ 5-year survival (%) (n¼ 191) Rate (95%CI) 67.5 (57 – 76) 144B) for 60 min. Subsequently, sections were washed with TBS and incubated with Real Envision Solution (Dako) for 30 min, then washed Continuous variables age and tumour diameter are described as the mean and range again in TBS and incubated with DAB-Chromogen for 10 min. After of values, whereas categorical variables are represented by the number of cases (% of washing, sections were counterstained with haematoxylin for 2 min. cases). Survival time was obtained using the Kaplan – Meier method. Only peritumoural CD8þ T-lymphocytes in punches taken from the invasive tumour front were counted. w and Fisher’s exact tests, where appropriate. Univariate survival Cut-off score for low vs high budding, CD8þ and CD8þ / buds analysis was carried out using the Kaplan–Meier and log-rank indices All cut-off scores to classify patients as having a ‘low’ or tests. Multivariable analysis was carried out using Cox’s propor- ‘high’ index were obtained by receiver operating characteristic tional hazards regression analysis after the verification of curve (ROC) analysis (Zlobec et al, 2007b). 50% of the data was proportional hazards assumption. Hazard ratio (HR) and 95%CI randomly selected for this purpose. Using this method, a plot of were obtained to determine the prognostic effect of each index the sensitivity and false positive rate (1-specificity) for discrimi- after adjustment. With logistic regression analysis, the odds ratios nating between survivors and non-survivors was assessed. The (OR) and 95%CI were obtained to determine the odds of death at most discriminating cut-off score was determined as the point on 5 years with the use of different indices. All statistical analyses the ROC curve with the shortest distance to the coordinate (0, 1), were carried out using SAS (V9, The SAS Institute, NC, Cary, USA). namely with the maximum sensitivity and specificity for survival. The reliability of all cut-off scores was tested by re-sampling of the data (500 bootstrapped replications). The discriminatory ability of RESULTS each feature for survival was also evaluated by analyzing the area under the ROC curve (AUC) and 95% confidence interval (CI). Cohort 1-Whole tissue sections Additional statistical analyses The association of indices with Tumour-budding index A high tumour-budding index was categorical clinico-pathological features was obtained using the defined, using ROC curve analysis, as at least 16 buds per  40 & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al A B 0.9 0.8 0.8 0.7 Low budding index 0.6 0.6 Number of buds Cut-off score = 16 buds 0.5 High budding index 0.4 0.4 0.3 0.2 0.2 0.1 AUC (95% Cl) = 0.59 (0.52−0.67) P = 0.022 0 12 24 36 48 60 72 84 96 108 120 0 0.2 0.4 0.6 0.8 1 1–specificity Time (months) C D 0.9 0.8 0.8 CD8+ T-cell High CD8+ index 0.7 Cut-off score = 40 cells 0.6 0.6 0.5 0.4 Low CD8+ index 0.4 0.3 0.2 0.2 0.1 AUC (95% Cl) = 0.64 (0.59−0.7) P < 0.001 0 0.2 0.4 0.6 0.8 1 0 1224364860728496 108 120 1–specificity Time (months) EF 0.9 0.8 CD8+/buds ratio 0.8 High CD8+/buds index cut-off score = 3/1 0.7 0.6 0.6 0.5 0.4 0.4 Low CD8+/buds index 0.3 0.2 0.2 0.1 AUc (95% Cl) = 0.68 (0.61−0.75) P < 0.001 0 0 0 0.2 0.4 0.6 0.8 1 0 1224364860728496 108 120 Time (months) 1–specificity Figure 2 Cohort 1. (A, C and E): Receiver operating characteristic (ROC) curves highlighting the ability of tumour budding (A), CD8þ lymphocytes (C) and CD8þ / buds index (E) to discriminate survivors and non-survivors. AUC¼ area under the ROC curve. Larger the AUC, more discriminating the feature. Arrows are drawn from the point on the curve used to classify patients into ‘low’ or ‘high’ indices based on their proximity to the coordinate (0,1). (B, D and F) corresponding Kaplan –Meier survival curves for ‘low’ and ‘high’ indices. field (Figure 2A). However, the ROC curve also highlights a (P¼ 0.028) and with an infiltrating tumour border configuration relatively poor discrimination of survivors and non-survivors at (P¼ 0.005) (Table 3). Patients with MSI-H cancers were signifi- 5-year follow up with an AUC value of 0.59 (95%CI: 0.52–0.67). cantly less prone to a high tumour-budding index (P¼ 0.022), A high-budding index was significantly associated with a more whereas no association was observed with KRAS or BRAF advanced pT stage (P¼ 0.017), with the presence of lymph node mutation. The odds of death from disease at 5 years in the group metastasis (P¼ 0.002), with the presence of vascular invasion with a high-budding index was OR (95%CI)¼ 1.89 (1.1–3.3) British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Sensitivity Sensitivity Sensitivity Survival probability Survival probability Survival probability Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Table 3 Association of budding index, CD8+ index and CD8þ / buds index with clinico-pathological features – Cohort 1 (n¼ 279) Budding index CD8+ index CD8+/buds index Clinico-pathological feature Low High P-value Low High P-value Low High P-value Gender Female 107 (56.9) 50 (55.0) 0.797 61 (54.0) 87 (57.2) 0.598 72 (54.1) 85 (58.2) 0.492 Male 81 (43.1) 41 (45.1) 52 (46.0) 65 (42.8) 61 (45.9) 61 (41.8) Tumour location Left-sided 48 (25.5) 23 (25.3) 0.06 30 (26.5) 37 (40.7) 0.551 34 (25.6) 37 (25.3) 0.053 Right-sided 82 (43.6) 29 (31.9) 42 (37.2) 62 (40.8) 45 (33.8) 66 (45.2) Rectum 58 (30.9) 39 (42.9) 41 (36.3) 53 (34.9) 54 (40.6) 43 (29.5) pT stage pT1 – 2 34 (18.6) 7 (7.7) 0.017 15 (13.5) 20 (13.4) 0.983 15 (11.4) 26 (18.3) 0.107 pT3 – 4 149 (81.4) 84 (92.3) 96 (86.5) 129 (86.6) 117 (88.6) 116 (81.7) pN stage pN0 106 (57.9) 34 (37.8) 0.002 50 (45.5) 81 (54.4) 0.156 53 (40.5) 87 (61.3) o 0.001 pN1 – 2 77 (42.1) 56 (62.2) 60 (54.6) 68 (45.6) 78 (59.5) 55 (38.7) Tumour grade G1 – 2 166 (90.7) 86 (94.5) 0.276 104 (63.7) 135 (90.6) 0.366 125 (94.7) 127 (89.4) 0.109 G3 17 (9.3) 5 (5.5) 7 (6.3) 14 (9.4) 7 (5.3) 15 (10.6) Vascular invasion Absent 145 (79.2) 61 (67.0) 0.028 79 (71.2) 115 (77.2) 0.271 90 (68.2) 116 (81.7) 0.009 Present 38 (20.8) 30 (33.0) 32 28.8) 34 (22.8) 42 (31.8) 26 (18.3) Tumour border configuration Infiltrating 114 (63.0) 72 (80.0) 0.005 80 (73.4) 102 (68.9) 0.435 100 (76.9) 86 (61.0) 0.005 Pushing 67 (37.0) 18 (20.0) 29 (26.6) 46 (31.1) 30 (23.1) 55 (39.0) Peritumoural lymphocytic inflammation Absent 137 (74.9) 67 (73.6) 0.825 93 (83.8) 100 (67.1) 0.002 108 (81.8) 96 (67.6) 0.007 Present 46 (25.1) 24 (26.4) 18 (16.2) 49 (32.9) 24 (18.2) 46 (32.4) KRAS gene status Wild-type 55 (75.3) 27 (61.4) 0.11 31 (64.6) 47 (74.6) 0.253 38 (64.4) 44 (75.9) 0.176 Mutation 18 (24.7) 17 (38.6) 17 (65.4) 16 (25.4) 21 (35.6) 14 (24.1) BRAF gene status Wild-type 54 (77.1) 31 (86.1) 0.273 36 (90.0) 45 (75.0) 0.063 41 (85.4) 44 (75.9) 0.219 Mutation 16 (22.9) 5 (13.9) 4 (10.0) 15 (25.0) 7 (14.6) 14 (24.1) Microsatellite instability status (MSS) MSS/MSI-L 54 (69.2) 41 (87.2) 0.022 43 (86.0) 47 (69.1) 0.033 51 (83.6) 44 (68.8) 0.052 MSI-H 24 (30.8) 6 (12.8) 7 (14.0) 21 (30.9) 10 (16.4) 20 (31.2) Survival rate (%) (95% CI) 5-year 63.9 (57 – 70) 53.0 (42 – 63) 0.033 47.2 (38 – 56) 67.9 (60 – 75) o 0.001 46.6 (38 – 55) 72.8 (65 – 79) o 0.001 Table 4 Comparison of the discriminatory ability of each feature for identifying survivors and non-survivors at 5-year follow up Feature Sensitivity Specificity Overall accuracy (95% CI) OR (95% CI) P-value Budding count (high vs low) 0.413 0.737 0.59 (0.52 – 0.67) 1.89 (1.1 – 3.3) 0.022 CD8+ count (low vs high) 0.597 0.683 0.64 (0.59 – 0.7) 3.18 (1.8 – 5.5) o0.001 CD8 : buds index (low vs high) 0.721 0.61 0.68 (0.61 – 0.75) 4.12 (2.4 – 7.1) o0.001 Odds ratio (OR) indicate that the odds of disease-specific death at 5-years is 1.89 times higher in the high-budding count group compared with the low group; for CD8+ count, 3.18 times higher in the low compared with the high CD8+ count group and for the CD8 : buds index, is 4.12 times higher in those with a low compared with a high index. compared with those with a low index (P¼ 0.022) (Table 4). In all, more advanced pT stage (P¼ 0.019), pN stage (P¼ 0.002), vascular 5-year disease-specific survival rate of patients with high and low invasion (P¼ 0.004) and an infiltrating tumour border configu- tumour-budding index was 53.0 (95%CI 42–63) and 63.9 (95%CI ration (P¼ 0.021) and worse survival in univariate analysis 57–70), suggesting a significantly worse prognosis in patients with (P¼ 0.02). In MSI-H patients, the high tumour-budding index high tumour-budding index (P¼ 0.033) (Figure 2B). In patients was only related to an infiltrating tumour border configuration with MSS/MSI-L tumours, a high-budding index was related to (P¼ 0.015). & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al CD8þ index A high CD8þ index was characterised by the inflammation at the invasive front (P¼ 0.007) and a marginal presence of at least 40 CD8þ cells per  40 field (Figure 2C). The association with MSI-H status (P¼ 0.052). Most notably, a discriminatory ability of CD8þ as indicated by the AUC was 0.64 considerable difference in survival time between patients with (95%CI 0.59–0.7), suggesting an improvement compared with the low and high CD8þ / buds index was observed with patients with a budding index alone. The odds of death from disease at 5 years low index demonstrating a highly adverse prognosis (Po0.001) was OR (95%CI)¼ 3.18 (1.8–5.5) in patients with a low compared (Figure 2F). This prognostic difference was also maintained in the with a high CD8þ index. A high CD8þ index was associated group of MSS/MSI-L patients (P¼ 0.001). with significantly more frequent peritumoural lymphocytic inflam- mation at the tumour front (P¼ 0.002), with MSI-H status Independent prognostic effects of budding, CD8þ and CD8þ / (P¼ 0.033) and marginally with BRAF mutation. Patients with buds indices A comparison of the performance of each of these a high CD8þ index demonstrated a significantly prolonged indices in multivariable survival analysis is outlined in Table 5. survival time compared with those with a low CD8þ index The effect of each of these factors was re-evaluated along with age, (Po0.001) (Figure 2D). In MSS/MSI-L and MSI-H patients, the gender, pT stage, pN stage, tumour grade, vascular invasion and CD8þ index was not linked to any features of tumour progression the tumour border configuration. Analysis was thus carried out although in MSS/MSI-L those with a high CD8þ index only for patients with complete clinico-pathological information experienced a significant prolonged survival time (P¼ 0.011). for all these features (n¼ 226, 101 deaths). The budding index was not found to add independent prognostic information when CD8þ / buds index A ratio of at least three times more analysed along with these additional features (P¼ 0.85), whereas CD8þ T-cells vs the number of tumour buds led to a strong the CD8þ index (Po0.001) and the CD8þ / buds index discrimination of survivors and non-survivors of colorectal cancer, (Po0.001) were highly relevant as prognostic factors in multi- as indicated by an AUC value of 0.68 (95%CI 0.61–0. 75) variable analysis, with similar relative risks of death suggesting a (Figure 2E). The odds of death at 5 years in patients with a low comparable performance of these two features as prognostic CD8þ / buds index compared with those with a high index was indices. OR (95%CI)¼ 4.12 (2.4–7.1) indicating that on average the odds In order to prevent over-fitting of multivariable models, only the of death was 4.12 times greater in patients with a low compared most relevant prognostic factors were included into sub-group with a high CD8þ / buds index (Table 4). Patients with a high analyses of lymph node-negative patients (n¼ 140, 42 deaths), as CD8þ / buds index demonstrated more favourable features, such well as of those with colon (n¼ 176; 79 deaths) or rectal cancers as early T stage (Po0.001), absence of vascular invasion (n¼ 94; 44 deaths). In lymph node-negative patients, multivariable (P¼ 0.009), presence of a pushing/expanding tumour border analysis with pT stage, vascular invasion and tumour border configuration (P¼ 0.005), presence of peritumoural lymphocytic configuration revealed independent prognostic effects for both the Table 5 Comparison of relative risks of buds, CD8+ and CD8+/ buds index in multivariable analysis with age, gender, pT stage, pN stage, tumour grade, vascular invasion and tumour border configuration in Cohort 1 Buds index only CD8+ index only CD8+/ buds index HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Index Low 1.0 0.85 1.0 o0.001 1.0 o0.001 High 1.04 (0.7 – 1.6) 0.45 (0.3 – 0.7) 0.44 (0.3 – 0.7) Age (years) Baseline 1.0 0.041 1.0 0.026 1.0 0.021 Increasing by year 1.02 (1.0 – 1.1) 1.02 (1.0 – 1.1) 1.02 (1.0 – 1.1) Gender Male 1.0 0.468 1.0 0.507 1.0 0.443 Female 0.86 (0.6 – 1.3) 0.87 (0.6 – 1.3) 0.86 (0.6 – 1.3) pT stage pT1 – 2 1.0 0.042 1.0 0.048 1.0 0.022 pT3 – 4 2.41 (1.0 – 5.6) 2.35 (1.0 – 5.5) 2.69 (1.2 – 6.3) pN stage pN0 1.0 o0.001 1.0 o0.001 1.0 0.001 pN1 – 2 2.27 (1.5 – 3.5) 2.12 (1.4 – 3.3) 2.06 (1.4 – 3.2) Tumour grade G1 – 2 1.0 0.205 1.0 0.738 1.0 0.873 G3 0.63 (0.3 – 1.3) 0.88 (0.4 – 1.9) 0.94 (0.4 – 2.0) Vascular invasion Absent 1.0 o0.001 1.0 0.001 1.0 0.014 Present 2.2 (1.4 – 3.5) 2.1 (1.3 – 3.3) 1.79 (1.1 – 2.8) Tumour border configuration Pushing 1.0 0.758 1.0 0.945 1.0 0.915 Infiltrating 1.09 (0.6 – 1.8) 1.02 (0.6 – 1.8) 1.03 (0.6 – 1.8) British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al Table 6 Association of budding index, CD8+ index and CD8þ / buds index with clinico-pathological features – Cohort 2 (n¼ 191) Budding index CD8+ index CD8+/ buds index Low High Low High Low High Clinico-pathological feature (n¼ 131) (n¼ 60) P-value (n¼ 69) (n¼ 121) P-value (n¼ 68) (n¼ 122) P-value Gender Male 65 (50.4) 23 (38.3) 0.122 34 (49.3) 54 (54.4) 0.606 33 (48.5) 55 (45.8) 0.722 Female 64 (49.6) 37 (61.7) 35 (50.7) 65 (54.6) 35 (51.5) 65 (54.2) Tumour location Left-sided 80 (61.5) 33 (55.0) 0.311 38 (55.1) 74 (61.7) 0.673 35 (51.5) 77 (63.6) 0.054 Right-sided 20 (15.4) 7 (11.7) 11 (15.9) 16 (13.3) 8 (11.8) 19 (15.7) Rectum 30 (23.1) 20 (33.3) 20 (16.0) 30 (25.0) 25 (36.8) 25 (20.7) pT stage pT1 – 2 42 (32.3) 10 (17.0) 0.029 15 (21.7) 37 (31.1) 0.167 9 (13.4) 43 (35.5) 0.001 pT3 – 4 88 (67.7) 49 (83.0) 54 (78.3) 82 (68.9) 58 (86.6) 78 (64.5) pN stage pN0 79 (71.2) 23 (67.7) 0.694 30 (60.0) 71 (75.5) 0.053 24 (58.5) 77 (74.8) 0.055 pN1 – 2 32 (28.8) 11 (32.4) 20 (40.0) 23 (24.5) 17 (41.5) 26 (25.2) Tumour grade G1 – 2 93 (71.5) 29 (49.2) 0.003 37 (53.6) 85 (71.4) 0.014 29 (43.3) 93 (76.9) o0.001 G3 37 (28.5) 30 (50.8) 32 (46.4) 34 (28.6) 38 (56.7) 28 (23.1) Vascular invasion Present 8 (6.1) 9 (15.0) 0.045 12 (17.4) 5 (4.1) 0.002 12 (17.7) 5 (4.1) 0.002 Absent 123 (93.9) 51 (85.0) 57 (82.6) 116 (95.9) 56 (82.4) 117 (95.9) Survival rate (%) (95%CI) 5 years 75.0 (62 – 84) 52.9 (36 – 67) 0.003 49.4 (30 – 66) 77.1 (66 – 85) 0.031 43.8 (26 – 60) 81.5 (69 – 89) o0.001 CD8þ index (P¼ 0.001) and the CD8þ / buds index (P¼ 0.002), the independent adverse prognostic effect of a low CD8þ / buds but not for the budding index. Moreover, in patients with colon or index after adjusting for these additional factors. rectal cancer, the CD8þ index (P¼ 0.015 and P¼ 0.006, respec- tively) as well as the CD8þ / buds index (P¼ 0.002 and P¼ 0.016, DISCUSSION respectively) maintained a significant independent prognostic effect after adjusting for pT stage, pN stage and vascular invasion. The aim of this study was to investigate an alternative approach to reflect the dynamic at the tumour front of colorectal cancer using an index including tumour budding and CD8þ lymphocytes. Cohort 2-Tissue microarray Briefly summarised, our results confirm the prognostic impact In Cohort 2, a high tumour-budding index in stages I–III patients of the selected tumour and host-related factors. In both patient was defined as at least six buds per punch and was related to a more cohorts, a high tumour budding and a low CD8þ lymphocytes advanced pT stage (P¼ 0.029), more poorly differentiated tumours index were associated with tumour progression and worse survival. (P¼ 0.003), presence of vascular invasion (P¼ 0.045) and was highly Indeed, tumour budding has previously been found to be related to a worse prognosis (P¼ 0.003) (Table 6). The threshold associated with higher N stage, higher tumour grade and presence value for a high CD8þ index was 13 cells per punch and was linked of vascular invasion, local tumour recurrence, distant metastasis as to lymph node negativity (P¼ 0.053), early tumour grade (P¼ 0.014), well as worse survival (Ueno et al, 2004a,b; Nakamura et al, 2005; absence of vascular invasion (P¼ 0.002) and a prolonged survival Kazama et al, 2006; Prall, 2007; Ishikawa et al, 2008; Wang et al, time compared with patients with a low CD8þ index (P¼ 0.031). 2009). There is also strong evidence for an association between a A ratio of three times more CD8þ lymphocytes to the number high number of CD8þ lymphocytes and absence of lymph node of tumour buds was identified as the most discriminating cut-off metastases, lower tumour grade, presence of peritumoural value to classify survivors and non-survivors. Patients with a lymphocytes, mismatch repair-deficient status and better survival high CD8þ / buds index had tumours that were more early pT (Ropponen et al, 1997; Naito et al, 1998; Chiba et al, 2004; Galon stage (P¼ 0.001), lymph node-negative (P¼ 0.055), early tumour et al, 2006; Koch et al, 2006; Baker et al, 2007). grade (Po0.001) and vascular invasion negative (P¼ 0.002). Low The question, therefore, arises as to whether a CD8þ CD8þ / buds index, led to a highly unfavourable prognosis lymphocytes : budding index is more advantageous over the use compared with patients with a high CD8þ / buds index (Po0.001). of one out of the two parameters alone. With the exception of This difference was also maintained in sub-group analysis of TNM 5-year survival, a certain intra- and inter-group variability stage II (P¼ 0.019) and stage III (P¼ 0.004) patients (Figure 3A is observed when analysing the relationship of tumour budding and B). In particular, in both untreated and treated patients, a low or CD8þ lymphocytes with clinico-pathological parameters. CD8þ / buds index was linked to a shorter survival time (P¼ 0.009 In contrast, the use of a CD8þ lymphocytes : budding index and Po0.001, respectively) (Figure 3B and C). Multivariable analysis better summarises these associations with key endpoints of for the budding index, CD8þ index and CD8þ / buds index was tumour progression, namely presence of lymph node metastases, carried out accounting for pN stage, vascular and lymphatic invasion vascular invasion and worse survival, and moreover, in both and treatment effect. Results outlined in Table 7 highlight once more cohorts. In addition, the CD8þ / buds index shows an increased & 2009 Cancer Research UK British Journal of Cancer (2009) 101(8), 1382 – 1392 Molecular Diagnostics Molecular Diagnostics Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al High CD8+/buds index (n =37; 2 deaths) 1.00 1.00 High CD8+/buds index (n =44; 10 deaths) 0.75 0.75 Low CD8+/buds index 0.50 (n =19; 5 deaths) 0.50 Low CD8+/buds index (n =43; 23 deaths) 0.25 0.25 TNM stage II; P = 0.019 TNM stage III; P =0.004 0.00 0.00 0 10203040506070 0 10203040506070 Time (months) Time (months) High CD8+/buds index High CD8+/buds index (n =69; 9 deaths) 1.00 1.00 (n=53; 7 deaths) 0.75 0.75 Low CD8+/buds index 0.50 0.50 (n =15; 7 deaths) Low CD8+/buds index (n =53; 21 deaths) 0.25 0.25 No adjuvant therapy; P =0.009 Adjuvant therapy; P< 0.001 0.00 0.00 0 10203040506070 0 10203040506070 Time (months) Time (months) Figure 3 Cohort 2. Kaplan –Meier survival curves illustrating the poorer outcome in patients with a low CD8þ / buds index in (A) TNM stage II patients, (B) TNM stage III patients, (C) patients not receiving adjuvant therapy and (D) patients treated with adjuvant therapy. Table 7 Comparison of relative risks of buds, CD8+ and CD8þ / buds index in multivariable analysis with pN stage, vascular and lymphatic invasion and adjuvant therapy in Cohort 2 Buds index only CD8+ index only CD8+/ buds index Cohort 2 HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Index Low 1.0 0.029 1.0 0.473 1.0 0.005 High 1.96 (1.1 – 3.6) 0.79 (0.4 – 1.5) 0.39 (0.2 – 0.8) pN stage pN0 1.0 o0.001 1.0 o0.001 1.0 0.002 pN1 – 2 4.11 (1.8 – 9.2) 4.41 (2.0 – 9.7) 3.58 (1.6 – 7.9) Vascular/lymphatic invasion Absent 1.0 o0.001 1.0 0.006 1.0 0.007 Present 3.41 (1.7 – 7.1) 2.97 (1.4 – 6.4) 2.76 (1.3 – 5.7) Adjuvant therapy Untreated 1.0 0.057 1.0 0.068 1.0 0.076 Treated 0.5 (0.2 – 1.0) 0.52 (0.3 – 1.1) 0.53 (0.3 – 1.1) discriminatory ability for identifying survivors and non-survivors of death at 5 years compared with a three-fold or under two-fold at 5-year follow up compared with tumour budding or CD8þ risk for patients with low CD8þ counts or high number of tumour lymphocytes count themselves. More specifically, patients with a buds, respectively. In addition, the CD8þ / buds index maintained low CD8þ lymphocyte : buds index have more than four-fold odds its significant impact on outcome in patients with stage II disease, British Journal of Cancer (2009) 101(8), 1382 – 1392 & 2009 Cancer Research UK Survival probability Survival probability Survival probability Prognostic impact of CD8þ / tumour buds on colorectal cancer A Lugli et al in both colon and rectal cancer patients and also, independently of completely independent cohorts of colorectal cancer patients treated adjuvant therapy. Our previous work describes the absence of in Switzerland and Greece, respectively. In addition to whole tissue tumour budding in cases with marked peritumoural inflammatory sections, we used the tissue microarray technique, a powerful tool reaction at the invasive margin of colorectal cancers and a strong for the investigation of novel or potential biomarkers, which has correlation between this inflammation and the presence of allowed us to evaluate hundreds of tissue cores for CD8þ . TILs(Zlobec et al, 2007a). We hypothesised that an immune Moreover, the evaluation of multiple tissue punches per tumour in response to colorectal cancer could account for an improved this study exceeds the number of cores required for adequate prognosis in patients with abundant TIL count by specifically representativity of tumour heterogeneity (Goethals et al,2006). targeting budding cells, a so-called ‘nipping in the bud’ effect Although double staining was carried out for Cohort 1, only leading to improved prognosis. Although we cannot directly allude immunohistochemistry for CD8 was carried out on Cohort 2. These to the functional interaction of CD8þ lymphocytes and tumour differences in methodology were chosen based on the ease of budding in this study, our data nonetheless point to a strong link identification of buds. In whole tissue sections, budding can often be between favourable prognosis and a high CD8þ lymphocyte : missed because of peritumoural inflammation, whereas tissue tumour-budding index. microarrays may aid the observer to focus on one specific area Several points in this study should still be considered. Although containing tumour buds. It is important to note that the independent the impact of tumour budding on clinical outcome is validated by prognostic effects of the CD8þ / buds index were found in both sub- several groups, a standardised scoring system for this feature has groups despite the different laboratory circumstances and meth- not yet been established, although several similar methods have odologies, thereby underlining the biological consistency of the been proposed (Ueno et al, 2002; Shinto et al, 2005; Prall and CD8þ / buds index in colorectal cancer. Although we recognise that Ostwald, 2007). This study highlights a new approach, which may this particular index is unlikely to be a final solution for daily be helpful in furthering the understanding of colorectal pathogen- diagnostic practice, it may however serve as a basis that can be esis and prognosis, but requires validation by other research further extended to include other strong and validated ‘pro-tumour’ groups both in retrospective and prospective settings. Finally, our or ‘anti-tumour’. This approach could potentially lead to a new findings, particularly with regard to the test group may be colorectal cancer prognostic score, which can be applied in addition somewhat influenced by the lack of clinical information regarding to TNM staging, as is the case for the BRE score for breast cancers. recurrence, metastasis and treatment although the majority of In conclusion, the CD8þ lymphocyte to tumour-budding index clinico-pathological features are adequately covered. is an independent prognostic factor in colorectal cancer and On the other hand, several factors strongly support the validity of represents biologically a ‘pro-/anti-tumour’ model that could be a our findings. The selection of parameters included in the proposed promising approach for a future prognostic score in colorectal index is evidence based, as both tumour budding and the CD8þ cancer. lymphocytes have been tested and validated in colorectal cancer by different research groups. The index, which encompasses a tumour- and host-related feature, and therefore represents the more advantageous ‘multi-marker’ approach to prognosis, better reflects ACKNOWLEDGEMENTS the tumour dynamic at the invasive front, compared with either This study is funded by the Krebsliga Beider Basel (IZ, LT and AL). feature alone. This study also benefits from the inclusion of two REFERENCES Ali AA, McMillan DC, Matalka II, McNicol AM, McArdle CS (2004) Tumour Velde CJ, Kuppen PJ (2004) Dendritic cells in colorectal cancer correlate T-lymphocyte subset infiltration and tumour recurrence following with other tumor-infiltrating immune cells. Cancer Immunol Immun- curative resection for colorectal cancer. 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British Journal of Cancer – Springer Journals

Published: Sep 15, 2009

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