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The in situ local immune response, tumour senescence and proliferation in colorectal cancer

Background: Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16ink4a, Ki-67 and immune infiltrates have similar prognostic value. Methods: Immunostaining of p16inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs). Results: Two hundred and thirty stage I–III cancers were studied. High nuclear p16ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16ink4a; P=0.002) were independently associated with poorer cancer survival. Conclusion: Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.

characterise these immune reactions, most work focusing on T lymphocytes and their subsets (CD3 þ , CD4 þ , CD8 þ , CD45RO þ and FOXP3 þ ) and macrophages (CD68 þ ; Naito et al, 1998;Pages et al, 2005;Galon et al, 2006;Roxburgh and McMillan, 2012). With the appreciation that density, type and location of intratumoural immune cells predict survival independent of stage (Galon et al, 2006), several validated scores of immune cell infiltrates have been developed including the immune score (based on C45RO þ and CD8 þ ) and the Klintrup-Makinen score (Klintrup et al, 2005;Roxburgh et al, 2009a,b;Mlecnik et al, 2011); consequently, there are increasing calls for immune scoring as part of the routine prognostication in CRC (Galon et al, 2012).
To date, it is unclear whether intratumoural immune responses represent tumour-or host-specific phenomena. High-grade infiltrates are commoner in early-stage disease (low T stage, absence of lymphovascular invasion or nodal/distant metastases) degrading as tumours enlarge and disseminate (Roxburgh et al, 2009a,b;Mlecknik et al, 2011). Associations with molecular tumour characteristics have also been reported, namely microsatellite instability, intratumoural HLA expression, and CpG island methylation (Mlecnik et al, 2011). Microsatellite unstable tumours have an established clinical phenotype including right-sided location, moderate-well differentiation as well as the presence of a pronounced lymphocytic infiltrate (Michael-Robinson et al, 2001a,b). However, immune cell infiltrates are consistently reported to offer additional prognostic information, independent of stage, pathological characteristics and mismatch repair status, potentially reflecting the complex interplay between host immune responses and the tumour (Michael-Robinson et al, 2001a;Baker et al, 2007;Laghi et al, 2009;Sinicrope et al, 2009;Deschoolmeester et al, 2010).
One potential tumour characteristic, that may in part account for immune cell infiltrates, is the presence of intratumoural cellular senescence and the 'senescence-associated secretory phenotype' (SASP). Cellular senescence, a state of stable cell cycle arrest, is thought to be an evolutionary mechanism to mitigate tumour development. The presence of senescent cells within a tumour may be a favourable prognostic feature in cancer, as such cells would not have bypassed cellular damage checkpoints (Narita and Lowe, 2005). Senescent cells are associated with a proinflammatory secretome including interleukins (ILs) and other cytokines (IL-6, IL-8, MMP 1 and 3, CXCR, IGF1 and 2; Campisi, 2005;Cairney et al, 2012). Work in animal models has identified increased infiltrates of innate immune cells (NK cells and macrophages) and CD4 þ lymphocytes drawn in by the proinflammatory secretome (Xue et al, 2007). This upregulation of intratumoural immune responses was thought to delay tumour progression. Therefore, one plausible explanation for the presence of high-grade lymphocytic infiltrates in colorectal tumours is that cellular senescence and the associated inflammatory secretome drive their presence.
To date, no single gold-standard biomarker for the detection and quantification of senescent cells in tissues has been developed. Biomarkers developed so far attempt to measure three main components related to senescence: cell cycle arrest (p16, p21, and Ki-67), chromatin (for example. HMGA1 and HMGA2) and the secretome (for example, IL-6, IL-8, MMP1 and so on; Cairney et al, 2012). Cell cycle markers of senescence include p16, p21 and Ki-67 expression. Of these markers, most work has utilised p16 ink4a expression, which is thought to represent an established and reliable marker of cellular senescence in tumours as well as normal tissues (Campisi, d 0 Adda di, 2007;Collado et al, 2007;Schmit et al, 2007;Raabe et al, 2011). P16 ink4a is a CDK inhibitor (Dimri et al, 1995;Campisi, 2005), the expression of which is upregulated in cellular senescence and ageing in normal tissues (Krishnamurthy et al, 2004;McGlynn et al, 2009). Importantly, p16 ink4a expression has previously ben associated with improved survival in colorectal tumours (Esteller et al, 2001;Lyall et al, 2006;Mitomi et al, 2010) but the mechanisms through which p16 ink4a expression influence cancer outcome are not yet established.
It is hypothesised that senescent tumour cells are associated with an immunogenic reaction in and around colorectal tumours. The aim of the present study was to establish whether markers of intratumoural senescence (evidence of nuclear p16 ink4a expression and low Ki-67 labelling) are associated with increased immune cell infiltration representative of an immunogenic phenotype in human CRCs and to determine whether senescence, proliferation and immune cell infiltration were associated with long-term survival.

MATERIALS AND METHODS
Since 1997, all resectional CRC surgery performed at Glasgow Royal Infirmary (GRI) has been entered into a prospectively compiled and maintained database. For the present study, 230 patients undergoing surgery for stage I-III CRC between 1997-2005, and whose tumour biopsy was included in a CRC tissue microarray were studied. On the basis of the preoperative radiological staging and intraoperative findings, patients were considered to have undergone curative resection. Exclusion criteria were neoadjuvant treatment or death within 30 days of surgery. Local ethical committee approval was granted.
Routinely assessed pathological data were taken from reports issued by the GRI Department of Pathology at the time of surgery in accordance with the RCPath data set for colorectal cancer report (Williams et al, 2007). In addition, TNM stage (5th edition), other high-risk characteristics included tumour differentiation, serosal involvement, margin involvement, tumour perforation and venous invasion. The modified Glasgow Prognostic Score (mGPS), a measure of systemic inflammation based on serum C-reactive protein and albumin measured before surgery was also assessed (CRPo10 mg l À 1 , albumin 435 g dl À 1 ¼ mGPS 0; CRP410 mg l À 1 , allbumin 435 g dl À 1 ¼ mGPS 1; CRP410 mgl À 1 , albumin o35 g dl À 1 ¼ mGPS 2; Roxburgh and McMillan, 2012).
Tumour necrosis was graded in this cohort, scored semiquantitatively by two observers as 'absent' (none), 'focal' (o10% of tumour area), 'moderate' (10-30%) or 'extensive' (430%) according to published methodology (Pollheimer et al, 2010;Richards et al, 2012a,b). An assessment of inflammatory cell infiltrate at the IM using H&E slides (the Klintrup-Makinen score) has also been performed in all cancers within this cohort. The methods for inflammatory infiltrate scoring are described elsewhere (Klintrup et al, 2005;Roxburgh et al, 2009a,b). Briefly, the generalised inflammatory cell infiltrate at the margin is graded according to a 4-point score: 0 indicates there was no increase in inflammatory cells at the tumour's IM; 1 denotes a mild/patchy increase in inflammatory cells; 2 denotes a prominent inflammatory reaction forming a band at the IM: 3 denotes a florid cup-like inflammatory infiltrate at the invasive edge with destruction of cancer cell islands. These scores are subsequently given a binary classification as either low grade (scores 0 and 1) or high grade (scores 2 and 3).
Evaluation of T-cell density was undertaken blinded to outcome. Density was graded semiquantitatively as absent, weak, moderate or strong in three separate tumour compartments: (1) IM, (2) tumour stroma (ST) and (3)  In addition to assessing individual T-cell subtypes, a previously proposed 'immune score' was applied: the Galon Immune Score, a composite immunohistochemistry-based score, which grades CD45R0 þ and CD8 þ infiltration in both the margin and central tumour (Mlecnik et al, 2011). The scoring groups the tumours based on high or low density of each cell type in each area HiHiHiHi to LoLoLoLo for CD45RO þ and CD8 þ at the IM and tumour centre (Mlecnik et al, 2011).
TMA construction. In addition to full section analysis described above, CRC tissue microarrays (TMA) were used to assess p16 ink4a and Ki-67 expression. In brief, a trained pathologist with a specialist gastrointestinal interest identified tumour-rich area within each specimen and four 0.6-mm 2 tumour cores were used to construct the TMA (Tovey et al, 2005). TMA sections (2.5 mm) were cut and mounted on slides coated with aminopropyltriethoxysilane.
Immunohistochemistry for p16 ink4a and Ki-67. Before p16 ink4a antibody staining of TMAs, the antibody was validated using a p16 ink4a blocking peptide competitor assay on tissue sections, in the presence of p16 ink4a antibody. Specificity of the p16 ink4a antibody was evident by an absence of staining in the presence of the blocking peptide. Paraffin-embedded TMAs were baked, dewaxed, and rehydrated followed by antigen retrieval in Tris/EDTA (pH 8.0 for p16 ink4a and pH 6.0 for Ki-67) with boiling for 5 min in a microwave under pressure. After cooling, endogenous peroxidase was quenched by incubating sections in 3% hydrogen peroxide for 10 min. Slides were blocked for 1 h at 25 1C in 1 Â casein (Vector Laboratories, SP-5020) diluted in TBS buffer. Slides were then incubated at 4 1C overnight with the primary antibodiy: 1 in 300 dilution of p16 ink4a (Santa Cruz sc-468; Santa Cruz Biotechnology, Dallas, TX, USA) antibody in Dako diluent (Dako, Glostrup, Denmark, S0809), and 1 in 50 dilution of Ki-67 antibody (Monoclonal mouse antihuman, Ki-67 antigen, Clone MIB-1, CodeM7240) with a negative (no primary antibody) slide control. Slides were then washed and incubated with Dako REAL Envision HRP Rabbit/Mouse (Dako, K5007) for 1 h at 25 1C before developing with DAB (Vector Laboratories, SK-4100). Slides were washed and counterstained with haematoxylin before being dehydrated and mounted. Scoring of the nuclear p16 ink4a was performed using the weighted histoscore method. Histoscoring is a widely applied and validated method of obtaining reliable and reproducible assessments of scoring immunohistochemical stains in heterogeneous tissues (Kirkegaard et al, 2006). The percentage of positive nuclei within tumour tissue was multiplied by the strength of staining: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Only tumour tissue was scored. On the basis of the distribution of histoscores in the whole cohort, the median p16 histoscore was 19.5. Therefore, nuclear p16 expression was considered high if 420. For Ki-67 scoring, 108 specimens were co-scored by two observers blinded to outcome (AP and LM). The ICCC was 0.94. For p16 ink4a scoring, 65 specimens were co-scored by two observers blinded to outcome. The ICCC was 0.72. In addition to ICCC to ensure correlation between observers, bland Altman plots were performed to ensure there was no bias. Figures 1E and F show both strong and weak nuclear staining for p16 ink4a .
In order to calculate the Ki-67 proliferation index, all tumour cells within each core was scored providing the total number of positive and negative cells and allowing calculation of the percentage positive cells. The mean representative score was taken across all cores. The threshold of 15% was taken based on previously published work (Mohammed et al, 2012).
Statistical analysis. Information on date and cause of death was derived from data linkage with cancer registry data performed by the West of Scotland Cancer Surveillance Unit. Date and cause of death was cross-checked with information from clinical notes where possible.
All variables were grouped according to standard or previously published thresholds. Associations were examined using w 2 -tests for linear trend as appropriate. Death records were complete until 1 December 2011, which served as the censor date. Univariate survival analyses were performed using the Kaplan-Meier survival curves with log-rank tests. Multivariate analyses were performed using Cox proportional hazards regression with a backwards method. The proportionality assumptions for age, mGPS, TNM and necrosis were explored using log-log plots and were found to be satisfactory. A P-value of o0.05 was required to enter the multivariate regression model. Statistical analyses were performed using SPSS version 19.0 (SPSS, Chicago, IL, USA).
The relationships between intratumoural p16 ink4a expression, Ki-67 labelling and clinicopathological characteristics in CRC are shown in Supplementary Table 1. p16 ink4a expression was not related to any patient or tumour characteristics, including age, sex, systemic inflammatory response (mGPS), TNM stage or venous invasion. Similarly, a high Ki-67 index did not relate to any patient or tumour characteristic. A weak association was observed between low tumour proliferation and surgical margin involvement (P ¼ 0.042; Supplementary Table 1).

DISCUSSION
The present study reports for the first time that in a large cohort of curative CRC patients with mature follow-up, immune cell infiltrates and intratumoural senescence (measured with p16 ink4a and Ki-67 labelling) are stage-independent prognostic features. It was hypothesised that based on tumour p16 ink4a expression and Ki-67 proliferative index, strong relationships would be observed between markers of senescence and increased density of immune cell infiltrates, potentially drawn in by a proinflammatory SASP. Further, we hypothesised that p16 ink4a expression would at least partly explain the associations between immune cell infiltrates and survival in CRC. Although p16 ink4a expression was related to improved cancer-specific survival, the present study's results suggest that this is unlikely to be mediated via immune interactions involving tumour-infiltrating T lymphocytes (TILs; CD3 þ , CD8 þ , CD45RO þ and FOXP3 þ ). If anything, higher p16 ink4a expression alone and in combination with low Ki-67 labelling was associated with reduced density of several T-cell markers at the margin, within the stroma and within the CCN (Table 2). Therefore, we would conclude that the method by which the presence of intratumoural senescence slows cancer progression in CRC does not appear to relate to overt upregulation of antitumour effector T-cell immune responses. These results provide further evidence that the in situ immune response has a key role in determining CRC outcome independent of tumour stage and makers of senescence.
The fact that no significant relationship between p16ink4a and immune cell infiltrates invalidates the present study's hypothesis. Further, tumours with high levels of senescence should demonstrate a low proliferative index. The fact that immune cell infiltrates demonstrated a strong relationship with proliferative tumours further strengthens the argument that the presence of tumour lymphocytic infiltrates exist independently of senescent cells. Data from murine models previously reported that intratumoural senescence was associated with generation of an antitumour immune response represented by the presence of CD4 þ cells, macrophages and natural killer cells (Xue et al, 2007). The majority of published evidence on the prognostic value of immune cell infiltrates in human CRC has focused on T cells, particularly effector T cells (for example, CD8 þ cytotoxic T cells). We chose a panel of T-cell markers representative of the most validated and studied adaptive immune cells, CD3 þ (generic T-cell marker) memory T cells (CD45RO), T-regulatory cells (FOXP3 þ ) and cytotoxic T cells (CD8 þ ). Although we cannot say p16 ink4a expression did not relate to innate (macrophages or NK cellpredominant infiltrates) or Th2-type adaptive (CD4 þ infiltrates) intratumoural immune responses, we can conclude that p16 ink4a expression does not appear to relate to a Th1-type response represented by higher densities of intratumoural effector CD8 þ infiltrates.
P16 ink4a and Ki-67 are considered part of a panel of potential biomarkers of senescence. Other biomarkers include markers of  the senescence-associated secretome including the proinflammatory cytokine IL-6. Although no measure of intratumoural cytokines were available here, we included serum measures of systemic inflammation (mGPS based on C-reactive protein and albumin). Interleukin-6 is responsible for 90% of the hepatic production of C-reactive protein (Gabay and Kushner, 1999). No significant relationships were seen between p16 ink4a expression, C-reactive protein and albumin. It is possible that the IL-6 produced in the presence of the SASP does not represent a systemic phenomenon and may have more subtle intratumoural effects. Further, the presence of a systemic inflammatory response is known to persist independent of local intratumoural inflammation (Roxburgh et al, 2009a,b). The present study also demonstrated significant relationships between T-cell infiltrates and highly proliferative tumours. The reasons for such observations have not been fully elucidated, but previous studies have demonstrated associations between microsatellite instability and higher proliferative index (Michael-Robinson et al, 2001). Highly proliferative tumours were associated with improved survival in this cohort; this relationship may be partly explained by increased immunogenicity of these tumours drawing in immune reactions. Unfortunately, at present no measure of microsatellite instability is available in this cohort, but the high Ki-67 labelling may be a surrogate measure for genomic instability in part explaining these relationships with immune cell infiltrates and improved survival. Alternatively, one may construct the hypothesis that an altered tumour secretome overrides any effect the SASP has. Highly proliferative tumour cells may then produce factors modulating immune activity, based on altered tumour cell metabolism and consequently the tumour cell secretome (Sebastián et al, 2012).
The role of p16 ink4a expression has previously been examined in CRC using immunohistochemistry (Zhao et al, 2003;Cui et al, 2004;Gonzalez-Quevedo et al, 2004;Lyall et al, 2006;Wassermann et al, 2009;Shima et al, 2011). To date, published data on loss of p16 ink4a expression is variable, reported between 8-91% in stage I-IV disease. Many studies had a low number of cancers studied (n ¼ 32-117). The largest study (n ¼ 802) reported that p16 ink4a was expressed in 75%, more comparable with the current study (63%) (Shima et al, 2011   that p16 ink4a loss was associated with poorer overall survival (HR: 1.30, 95% CI: 1.03-1.63, P ¼ 0.026), also comparable with the present study's findings for poorer cancer-specific survival (univariate analysis, HR: 1.85, 95% CI: 1.18-2.90, P ¼ 0.007). However, the study by Shima et al (2011) reported that the prognostic value of p16 ink4a was lost on multivariate analysis; age and tumour grade were the principal confounders. In the present study of 230 patients, p16 ink4a expression was an independent prognostic factor when age and grade (differentiation) were considered.
There is an increasing literature on the role of immune scoring in CRC prognostication with calls for the introduction of these stage-independent prognostic biomarkers into routine clinical practice in addition to utilisation within clinical trials of cancer therapeutics for stratification purposes (Galon et al, 2012). The basis or driving force for the immune cell infiltrates is largely unclear. Some have suggested that this response is representative of the local in situ host immune reaction against the tumour and once lost, or evaded, the tumour can grow and metastasise or 'escape' (Mlecnik et al, 2011;Roxburgh and McMillan, 2012). This is in keeping with previous reports that suggest the response is lost with increasing tumour size or T and N stage (Roxburgh et al, 2009a,b;Mlecnik et al, 2011). It is probable that intratumoural factors have a partial role in determining the specific densities of immune cell infiltrates including microsatellite instability, CpG island methylation and intratumoural HLA expression (Mlecnik et al, 2011), but at this stage these features do not yet seem to explain fully their presence.
In summary, the present study highlights the prognostic value of intratumoural senescence. Importantly, the presence of senescence determined by p16 ink4a expression does not appear to mediate its effect on oncological outcome through TILs. On the other hand, high tumour proliferation is strongly associated with the presence of a local immune response. However, TILs and the in situ local inflammatory response remain strong prognostic factors independent of these intratumoural characteristics. p16 ink4a expression and low tumour proliferation as markers of intratumoural senescence do not explain the presence of TILs in CRCs.