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Immune mediators as potential diagnostic tools for colorectal cancer: from experimental rationale to early clinical evidence Expert Rev. Mol. Diagn. 14(3), 387–399 (2014)

Giuseppe Di Caro*1, Federica Marchesi1,2, Maria Rosaria Galdiero1,3 and Fabio Grizzi*1 1 Humanitas Clinical and Research Center, Via Manzoni 56, 20089, Rozzano, Milan, Italy 2 Department of Biotechnologies and Translational Medicine, University of Milan, 20089, Milan, Italy 3 Division of Clinical Immunology and Allergy, University of Naples Federico II, Via Pansini 5, 80131, Naples, Italy *Authors for correspondence: Tel.: +39 028 224 5113; +39 028 224 5252 Fax: +39 028 224 4590 [email protected]; [email protected]

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At the tumor site, solid tumors recruit native and adaptive infiltrating cell subtypes with a unique pattern, varying according to the organ of origin and the stage of the disease, which contributes to the complexity of the cancer microenvironment. The recruitment and activation of immune cells depend on a plethora of soluble immune mediators, including cytokines and chemokines that have a critical role in the process of cancer onset and progression. In colorectal cancer, measurement of soluble immune mediators in the serum seems to reflect the specific inflammatory reaction at the tumor site, and thus they might serve in clinical practice to improve available colorectal cancer detection and screening strategies. Clinical translation of data from experimental models could lead to the earlier detection of colorectal cancer resulting in a decreased burden of metastatic disease. These models and the most promising candidates for immune-based serum screening tests in colorectal cancer are discussed here. KEYWORDS: chemokines • colorectal cancer • cytokines • diagnostic test • immune-based serum tests • immunity • inflammation • proinflammatory mediators • screening

Colorectal cancer (CRC) ranks second as a cause of death related to cancer in the USA [1]. Despite advancements in diagnostic tools aimed to provide an earlier detection of CRC, about 20–25% of patients with CRC have metastases at diagnosis, resulting in relatively high overall mortality rates [2]. The use of endoscopic screening has increased lately, even though it is an invasive and expensive procedure with inadequate compliance [3], which is also related to the patient’s medical privilege. Thus, there is the need for novel non-invasive and economical tests aimed to provide an earlier cancer detection, which might in turn reduce the burden of metastatic disease at diagnosis. To date, the only available non-invasive methodology that is employed to this aim in clinical practice is fecal occult blood test (FOBT) [4]. In large randomized clinical trials, FOBT has been shown to be the only non-invasive test associated with a better

10.1586/14737159.2014.900443

survival [5], although the sensitivity and specificity of the assay was hindered by intermittent tumor bleeding and frequent comorbidities other than CRC [6]. In a survey, 88.8% of patients reported that they would perform FOBT if asked by a doctor, while 84.9% of patients would undergo colonoscopy if FOBT was positive [4]. Thus, it is conceivable to hypothesize that cost-effective serum tests would reach high compliance rates, at least equal to those of FOBT. The identification of serum markers with improved CRC detection rates would provide clinicians with a costeffective and highly compliant tool to select high-risk individuals to undergo colonoscopy, while excluding those with low risk to avoid unnecessary interventions and improving economic performances. Therefore, the development of alternative tools for early diagnosis is gaining relevance in CRC research since serum tests are not implemented to date in clinical


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programs of disease prevention, despite reports revealing these tools as promising markers for this aim. In this regard, soluble immune mediators appear as valid candidates and laboratory markers of systemic inflammation have been tested as biomarkers in several neoplastic diseases. Thus, to improve the development of new diagnostic tools, research in cancer immunology is moving from understanding the role of immune cells during tumor development toward the translation of these data into the clinical practice. The use of laboratory assays for evaluating immune mediators in the serum, which we define as immune serum-based screening tests (IST), might represent a promising innovation by increasing the yield of CRC detection provided by FOBT. In this scenario, IST might become a substitute to colonoscopy in screening routine and dramatically enhancing patient compliance. The critical role of the tumor microenvironment in the process of cancer initiation and progression is now widely accepted [7–10], with its ability to contribute to the evasion of cancer immune destruction being included as one of the hallmarks of cancer [9]. Accordingly, cancer development is associated with the accumulation of immune cells at the tumor site, as evidenced by histopathological analyses in a variety of human malignancies [11–13]. Tumor immune infiltration includes cells of the innate and adaptive immune system. Despite the fact that the role of tumor-infiltrating T cells during cancer development has been the most investigated issue, the recognition of the diversity of the immune microenvironment in solid tumors has led to evaluate the immune infiltrate with a more comprehensive approach. The result has been the generation of specific ‘immune signatures’, that is, immune profiles describing the type, density and activation of immune cells infiltrating the tumor tissue that uniquely characterize each solid tumor [14–16]. These immune scores have great clinical perspectives, since they have been shown to be helpful in identifying post-surgical recurrences [17], although this phenomenon might be more efficient among early-stage CRCs [18–20]. Important components of these immune profiles are soluble immune mediators, including cytokines and chemokines, which are responsible for the recruitment of immune cells at the tumor site and for the interaction between immune cells, tumor cells and other cellular components of the microenvironment [21,22]. For these important functions, they contribute to define the behavioral orientation of the local immune response in tumor tissues [23]. The considerable presence of soluble immune mediators in the tumor microenvironment is not surprising, considering the association of inflammatory conditions and tumor development. Cytokines and chemokines are pivotal players of the network of inflammatory mediators associated with neoplasia [8]. A great amount of experimental data obtained on tumor models functionally relate cytokine networks to tumor progression [8]. Mechanistic and functional models that summarize the role of the immune system in tumor progression might translate into prognostically relevant phenomena and novel diagnostic potential platforms. Here, we will discuss the role of soluble immune mediators of the 388

tumor microenvironment in regulating the anti-tumor immune response and the progression of CRC. In addition, we will move to the possible diagnostic translation of these mediators as innovative serum-based biomarkers, aimed to design novel promising screening tests for earlier detection of CRC. Innate & adaptive immune cells in the colorectal microenvironment Tumor-associated macrophages

Macrophages are the most abundant immune cell population in the tumor microenvironment [24,25]. Tissue macrophages are classically considered as terminally differentiated cells derived from circulating monocytes recruited at the tumor site through chemokines such as CCL2 and CCL5, or growth factors including VEGF, TGF-b, M-CSF or GM-CSF [26]. Plasticity is a widely known feature of macrophages [27–29] and consists of the ability of these cells to modify their phenotypic and functional aspects based on the signals derived from the microenvironment. It is now widely accepted that under the influence of soluble mediators, namely IFN-g or IL-4/ IL-13, macrophages may give rise to a ‘classical’ M1 or ‘alternative’ M2 phenotype, respectively. Other stimuli can also influence macrophage polarization, for example, CCL2 and CXCL4 promote an M2-like phenotype [30,31] and immunoglobulin complexes, glucocorticoids, TGF-b and IL-10 give rise to M2-like functional phenotypes similar to IL-4- or IL-13-activated macrophages [32]. Of note, macrophage plasticity comprises a wide variety of distinct functional states, with M1 and M2 as the extreme limits [33–35]. M1 macrophages express typical Th1 cell-attracting chemokines such as CXCL9 and CXCL10, produce high levels of IL-12 and IL-23, as well as effector molecules (i.e., reactive nitrogen intermediates) and inflammatory cytokines (IL-1b, TNF, IL-6), but low levels of the immunoregulatory cytokine IL-10. M1 cells have been reported to participate in polarized Th1 responses and to exert anti-microbial and anti-tumor activity. On the contrary, M2-polarized macrophages mainly express CCL17, CCL22 and CCL24, chemokines involved in Treg lymphocytes, Th2, eosinophil and basophil recruitment [8,36,37]; they produce high levels of IL-10, low levels of IL-12 and IL-23 and present a variable capacity to produce inflammatory cytokines. M2 macrophages exert immunoregulatory functions, promote tissue remodeling, angiogenesis and are involved in tumor progression [35,38]. It has been ascertained that tumor-associated macrophages (TAMs) recapitulate many aspects of M2 macrophages. TAMs have been described as promoters of the metastatic potential of several human tumors [39,40] and experimental models [41–43]. TAMs may express a wide range of mediators involved in new vessel formation, such as TGF-b, VEGF-A, VEGF-C, PDGF, thymidine phosphorylase and chemokines including CXCL8 [44–48]. TAMs display a poor antigenpresenting activity and favor the development of an immunosuppressive environment, given the release of molecules such as TGF-b, indoleamine 2,3-dioxygenase and IL-10, which may Expert Rev. Mol. Diagn. 14(3), (2014)

Immune mediators as potential diagnostic tools for CRC

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contribute to immunosuppression [49,50]. In CRC, the role of TAMs is still controversial, and evidence suggests that the proversus anti-tumor functions of TAMs may depend on their location within the tumor (i.e., invasive front vs intratumoral location) [51], even though, among the reported data, a general trend toward the association between macrophage infiltration and improved patient prognosis may be described [52,53]. Tumor-associated neutrophils

In addition to the well-characterized TAMs, neutrophils have recently appeared as tumor-infiltrating myeloid cells playing important roles in tumor initiation and progression [54]. As for macrophages, a dual role in tumorigenesis has been proposed. More in details, at least in mice, neutrophils are driven to acquire a pro-tumoral ‘N2’ phenotype by TGF-b, whereas TGF-b inhibition promotes an anti-tumoral ‘N1’ phenotype, which displays cytotoxic activity on cancer cells and an immunostimulatory profile (i.e., TNF-ahigh, CCL3high, ICAM-1high, arginaselow) [55]. The dual aspect of tumor-associated neutrophils (TANs) is first of all due to their ability to induce genotoxic stress and damage, which is considered as a hallmark of cancer [9]. Activated neutrophils represent an important source of nitric oxide derivatives and reactive oxygen species (ROS), able to induce DNA damage and genomic instability [56,57]. Moreover, activated neutrophils may stimulate ROS and nitric oxide accumulation in neighboring epithelial cells through the release of cytokines such as TNF-a, which is implicated in the process of carcinogenesis [58,59]. ROS can also directly inactivate mismatch repair enzymes [60]. On the other hand, ROS may also exert an anti-tumoral effect. In a murine model of orthotopic breast cancer, neutrophils accumulated in the pre-metastatic lung and prevented tumor cell seeding through an H2O2-dependent mechanism [61]. Neutrophils also express a wide armamentarium of pro-angiogenic factors able to modulate tumor neovascularization. For instance, CXCL1 exerts its angiogenic potential through the recruitment of neutrophils that in turn release biologically active VEGF-A, resulting in angiogenesis in vivo [62]. On the other hand, neutrophils may also release anti-angiogenic factor, such as elastase, which is able to activate angiostatin and degrade VEGF-A, and a-defensins, which are able to inhibit endothelial cell proliferation [63–65]. Neutrophilderived proteins and growth factors may also play several roles in cancer development and progression. It has been shown that breast cancer cells induce the release of oncostatin M by neutrophils through GM-CSF and neutrophil-derived oncostatin M stimulates breast cancer cells to produce VEGF, promotes cancer cell detachment and enhances their invasiveness [66]. Neutrophils are also a source of HGF, which enhances tumor cell migration in bronchoalveolar carcinoma, in human cholangiocarcinoma and hepatocellular carcinoma [67,68]. On the other hand, in BCG immunotherapy for superficial bladder cancer it has been demonstrated that neutrophils may express TNF-related apoptosis-inducing ligand, which possesses antitumoral activity [69,70]. In chronic myeloid leukemia patients, informahealthcare.com

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neutrophils may exert cytotoxic activity toward CD34+ chronic myeloid leukemia cells, through the IFNs-induced release of TNF-related apoptosis-inducing ligand [71]. In regard to digestive carcinogenesis, in particular, neutrophils play different roles, through the release of ROS, proteases, angiogenic or angiostatic mediators and other soluble factors [72]. Human neutrophil peptides 1–3 have been found to be elevated in tissues and plasma of patients with digestive carcinomas [73]. Other molecules, such as neutrophil gelatinase-associated lipocalin or neutrophil elastase are able to either inhibit or promote the invasive behavior of carcinoma cells [74,75]. The relationship between TAN infiltration and cancer patient prognosis has been recently discussed [76]. For what concerns CRC, previous studies indicated an association between neutrophil infiltration and good patient prognosis [77–80]. Conversely, some investigators have associated TAN with poor patient prognosis [81,82]. Recently, a new research questions once more the negative prognostic value of neutrophil infiltration [83]. These controversial results may depend on the type of tumor and on the method used to assess neutrophil density within the tumors (i.e., H&E stain vs immunohistochemistry, tissue macroarray vs whole histological sections). T cells

Tumor-infiltrating T cells (TILs) include CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ T-helper cells. CTLs are the effector cellular mediators of the anti-tumor immune response, as they are equipped to recognize and eliminate tumor cells. As to CD4+ T cells, their main function is to sustain activation of other cells, including macrophages, B cells and CTLs, by the release of several cytokines, such as IL-2, TNF-a and IFN-g, which coordinate a Th1 immune response or IL-4, IL-5, IL-6, driving a Th2 immune response. T-cell traffic through lymphoid organs and homing at peripheral tumor sites are tightly regulated by chemokine receptor surface expression and local production of specific chemokines. The expression of selected chemokine receptors by TILs not only designates their homing capability, but also reflects their differentiation status and polarization, thus indicating the extent of the local immune response. CD8+ CTLs and Th1 cells are mainly responsive to CXC chemokines through CXCR3, and to CX3CL1 acting through CX3CR1; Th2 cells are attracted through the action of CCR3 and CCR4 agonists [84]. The prevalence of specific T-cell populations at the tumor site is associated with local chemokine production. Both tumor and stromal cells produce a variety of chemokines, which contribute to recruitment of effector T cells and dictate the type of immune infiltrate. The CXC chemokines CXCL9 and CXCL10 are the main attracting stimuli for TILs, which express high levels of the cognate receptor CXCR3. Increased expression of these chemokines can elicit anti-tumoral responses correlated with increased infiltration of CD4+ and CD8+ lymphocytes, as reported in renal [85], colorectal [86,87] and gastric cancers [88]. The presence of CD8+ T lymphocytes in the melanoma microenvironment is associated with local production of CCL2, CCL3, CCL4, CCL5, 389

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CXCL9 and CXCL10 [89]. In CRC, CX3CL1 and CXCL16 can also contribute to recruitment of TILs and the expression of these chemokines is correlated with the number of TILs and with a better prognosis [87,90,91]. However, in the majority of solid tumors, CTLs are rendered dysfunctional by tumor-induced immunosuppressive mechanisms, which can selectively interfere with T-cell proliferation, activation, cytokine production, cytotoxicity or cell migration. Both soluble and cellular immunosuppressive mediators are responsible for the disarmament of the adaptive immune response and the inhibition of tumor immune destruction. The main cytokines involved in the immunosuppressive function are IL-10 and TGF-b. As a result, in solid tumors, including cervical, colorectal and prostate cancer, the prevalence of an effective antitumor or immunosuppressive immune response is reflected by the gene expression profile of the tumor. An ‘adaptive immune’ Th1 profile (i.e., T-bet+, IRF1+, IFN-g +, CD8a+, granzyme B+, granulysin+) has been associated with better prognosis in CRC, while an ‘inflammatory’ (i.e., IL-8+, VEGF+, CEACAM-1+, MMP-7+) or ‘immunosuppressive’ profile (i.e., TGF-b+, IL-10+, B7-H3+, CD32b+) has been associated with worse outcome [22,92]. As regards to chemokines, CCL17 and CCL22 are able to recruit CCR4+ Treg and polarized Th2 cells, therefore, their expression is often correlated with poor prognosis. These chemokines are produced by cancer and infiltrating cells in a variety of neoplasia, including breast [93], ovarian [94], prostate [95] and gastric carcinoma [96]. T regulatory cells

During tumor progression, CD4+/FOXP3+/CD25 high Treg lymphocytes, a small subset of CD4+ T cells with regulatory function, accumulate in tumor tissues and peripheral blood of cancer patients [97]. Both the frequency and immunosuppressive activity of Treg lymphocytes increase in tumor conditions, contributing to the generation of the local and systemic immunosuppression involved in tumor outgrowth [98,99]. Suppression of CD8+ T-cell proliferation and IFN-g production is the strategy adopted by Treg to inhibit the anti-tumor immune response locally and involves IL-10 and TGF-b. These two cytokines are key players for the immunosuppressive function of Treg, as they are both involved in the induction of adaptive Treg in the periphery [100] and mediate Treg immunosuppression [101]. However, the immunoregulatory function of Treg in physiological conditions may turn into a favorable anti-tumor activity in those tumors strongly associated with chronic inflammation, due to the property of Treg to dampen pro-tumor inflammation [102]. Thus, clinical association of Treg density with better prognosis has been reported in CRC [103]. The earlier classification of Treg in natural and inducible subsets, according to their thymic or peripheral origin has been recently integrated with a functional classification [104], which has underlined the complexity of the Treg population in terms of immunosuppressive activity and phenotype. The majority of functional human Treg express the transcription factor FOXP3. Hence, enumeration of FOXP3 cells at tumor site might not be a reliable assay 390

to evaluate the presence of regulatory immunosuppressive T cells, as the tumor microenvironment apparently impacts on the function of Treg [97]. Thus, there is an urgent need for new markers to identify Treg. Among others, chemokine receptors might be good candidates, since CCR4 [93,94,105], CCR6 [106] and CCR7 [107] are expressed by human Treg, although they are also found on other T cells. B cells

The effect of intratumoral B lymphocytes in cancer is far from clear. B and T cells are often localized in tight association, possibly reflecting how T cells, mainly Th2, modulate B-cell activation and antibody production. At the same time, B cells modulate T-lymphocyte responses by presenting antigens, providing co-stimulation and secreting cytokines [15,108]. B cells can be functionally classified according to their cytokine profile. When primed by T cells in the presence of Th1-type cytokines, B cells secrete IFN-g and IL-12. These B cells do not secrete substantial amounts of IL-4, IL-13 or IL-2, but can secrete IL-10, TNF and IL-6 [109,110]. By contrast, B cells primed by T lymphocytes in the presence of Th2-type cytokines secrete IL-2, IL-4 and IL-13 and can also secrete IL-10, TNF and IL-6 [109,110]. Finally, regulatory B cells produce IL-10 and can suppress CD4+ T-cell responses [111,112]. Overall, it is clear that B cells can influence CD4+ T-cell responses by several antibody-independent mechanisms, playing opposite roles according to the cytokine profile, ranging from suppression to enhancement of T-cell responses. Therefore, in the tumor setting, distinct B-cell subpopulations can accumulate at the tumor site and play either a pro-tumoral or an anti-tumoral action [110,113]. A recent comprehensive analysis of the immune landscape of CRC has revealed that B cells are associated with increased survival [15]. B cells express the chemokine receptor CXCR5, endowing them with the capability to migrate to the chemokine CXCL13. Genomic instability of the chemokine CXCL13 has been proposed as the mechanism involved in Bcell recruitment in CRC [15]. Patients with CXCL13 deletion had significantly decreased CXCL13 expression levels and a significantly higher risk of relapse than patients without aberrations. In a recent translational study, the cellular composition and structure of tertiary lymphoid structures (TLT) was defined in the CRC microenvironment as a clinically relevant histological feature rich in CD20+ B cells and CD3+ T cells. Notably, the anti-tumor algorithm represented by interspersed CD3+ TILs coordinated with the presence of TLT at the tumor margin, thus revealing TLT as a relevant immune player in the progression of early-stage CRC [114]. The presence of such structures is a novel potential prognostic platform in CRC and the development of novel immunotherapies aimed to enhance TLT presence in the tumor microenvironment should be designed by balancing their efficacy according to TNM staging. Immune serum biomarkers of CRC

Immune cells infiltrating the tumor microenvironment are critical players in tumor onset and progression [18]. The recruitment Expert Rev. Mol. Diagn. 14(3), (2014)

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Immune mediators as potential diagnostic tools for CRC

of innate and adaptive cells contributes to establish an immune milieu that can either impair immune-surveillance activities or favor tumor progression or mediate an effective anti-tumor immune response. To date, a consistent amount of literature has been focused on the abilities of immune cells infiltrating tumors to predict post-surgical tumor recurrence [115,116]. Here, we will report several studies, which addressed the hypothesis of whether a circulating cytokine pattern or a specific immunological repertoire of circulating immune cells might reflect the in situ immune response. Overall, these studies highlight, with a translational approach, the potential IST available with regard to their relevance in CRC detection and early diagnosis. Unspecific circulating inflammatory response

Unspecific inflammatory response in the circulatory system of patients, represented by levels of C-reactive protein (CRP), has been lately employed as a prognostic marker of CRC recurrence [117]. CRP is an acute phase protein produced by the liver whose blood levels increase in response to acute and chronic inflammatory states, trauma and infections [118]. For this reason, CRP is commonly regarded as an unspecific inflammatory marker. Despite this, the Glasgow prognostic score, based on the combination of acute phase proteins (such as CRP and albumin) in the serum, has been suggested to be included in the staging system as a prognostic factor in CRC [119]. Several prospective studies challenged CRP blood levels to test its diagnostic performance in identifying the occurrence of CRC in subjects (TABLE 1) and in this regard data were equivocal. A metaanalysis revealed that the association between higher levels of CRP and the occurrence of CRC is unconvincing [120] and while two large studies validated this trend [121,122], six prospective studies opposed this idea [123–129]. Interestingly, in the study from Joshu et al., adults undergoing colonoscopy had blood levels of CRP that were not associated with signs of histological inflammation in the colonic mucosa, although they associated with obesity [130]. These data reflect the idea that the prognostic behavior of CRP blood levels is critically confounded by other comorbidities. Thus, CRP blood levels is not a specific marker of the colonic inflammatory status and, while it seems to reflect other sources of inflammation, it is not likely to increase the detection rate of early CRC provided by FOBT. The identification of novel diagnostic tools in CRC screening should adhere to common guidelines available for biomarker discovery. Statistical significance in associating subgroups of patients with differing quantity of serum IST with their risk to develop CRC is not sufficient to properly test and compare their clinical relevance with regard to FOBT. The evaluation of continuous distributions of quantitative data retrieved by clinical tests is a critical issue and ROC curve analysis of such ‘big data’ should be employed to efficiently define optimal cut-off values as a function of the optimal proportion of sensitivity and specificity that can be achieved. ROC curve provides a common methodological platform, wherein the diagnostic accuracy and efficacy of different IST with FOBT can be compared. However, as shown in TABLE 1, most of the studies did informahealthcare.com

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not provide the sensitivity and specificity of IST in detecting CRC occurrence. For these reasons, prospective studies should be properly designed in order to avoid statistical inconsistencies that might explain discrepant results found in the literature. In the attempt to provide a more specific characterization of the inflammatory status, the prognostic value of different hematologic components have been tested in the serum of CRC patients [131]. The evaluation of neutrophil-to-lymphocyte ratio (NLR) is commonly used as a measure of systemic inflammation, which is known to be associated with neutrophilia together with concomitant lymphocytopenia and it has been shown that elevated circulating neutrophils were associated with Glasgow prognostic score [118,131]. Moreover, NLR has been tested for its association with tumor development and response to therapy. However, the prognostic abilities of this score remain controversial, since only 4 out of 11 studies reported its independent association with operable CRC survival [131–134]. Overall, a high NLR count is associated with worst cancer survival and seems to retain a more consistent prognostic value among cancers with an advanced stage of disease, which are also more likely to receive chemotherapy treatments or being not operable [131]. Three studies on CRC reported NLR to be prognostic of chemotherapy-treated CRC progression [135,136]. Kishi et al. reported that NLR count independently predicted outcome in patients with CRC [136] and decreased to low levels as a consequence of preoperative chemotherapy treatment in 68% of patients. Patients with reverted levels of NLR after chemotherapy had better prognosis compared with those with higher NLR [136], thus despite this effect might be related to the general immunosuppression induced by chemotherapy, NLR levels might be relevant to predict responsiveness to chemotherapy. A better understanding of the molecular bases of the interactions between NLR and chemotherapy promises to identify novel strategies to improve patient’s responsiveness to this treatment. However, NLR is not a specific biomarker since its level might be critically confounded by other comorbidities not related to cancer occurrence and progression, thus making NLR not suitable as a specific and sensitive diagnostic test for CRC screening. Alternative studies that tested soluble immune mediators in the serum of CRC patients more likely to reflect the local tumor microenvironment identified more specific diagnostic biomarkers of tumor detection. Circulating cytokine & chemokine levels

Due to its crucial involvement in the regulation of the acute phase response [118], IL-6 is now accepted as a critical factor in regulating the systemic inflammatory status. In tumor settings, IL-6 serum levels have been found to be positively associated with tumor stage, local invasion, size and metastasis at diagnosis and poor patients prognosis [131,137–139]. Notably, patients with polymorphisms in IL-6 genes had a lower level of CRP [140] and accordingly other studies showed a linear correlation between these two markers in cancers other than CRC [141–143]. For these reasons, it is still unknown whether the increased amount of circulating IL-6 reflects the local 391

392

CXCL16

CXCL10

Matsushita et al. (2012)

Toiyama et al. (2012)

n.a.

n.a.

n.a.

199 pg/ml

3.15 ng/ml

1.53 ng/ml

n.a.

n.a.

n.a.

n.a.

n.a.

Overall

Overall

Overall

Overall

Overall

Overall

Men

Overall

Overall

CS

#

218/17

314/20

255/33

99/107

50/50

148/86

274/532

70/34

164/20

555/280

R

R

R

R

R

P

P

P

R

P

CS

118/218# 242/631

P

P

P

P

P

Series design

988/953

6684/32

10’520/93

9836/166

1096/1096

CRC§

[160]

[161]

[159]

[157]

[163]

[151]

[129]

[146]

[137]

[128]

[127]

[150]

[126]

[124]

[123]

[122]

[121]

Ref.

Hazard ratio (95% CI) in multivariate models. ‡ By ROC curve analysis. § Number of CRC cases/controls. { OR for colon cancers only. # Screening for adenoma. †† For lean men only. ‡‡ Biochip analysis of: IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-8 and IL-10, TNF-a, IFN-g, MCP-1. AUC: Area under the curve; CR: Cross-sectional; CRC: Colorectal cancer; CRP: C-reactive protein; IST: Immune serum-based screening tests; n.a.: Not available; P: Prospective; R: Retrospective; SB: Sensibility; SF: Specificity.

†

CXCL5

Kawamura et al. (2012)

n.a.

SB: 0.28, SF: 0.94, AUC: 0.73

IL-2, IL-4, IL-6, IFN-g

n.a.

Berghella et al. (1998)

Kantola et al. (2012)

n.a.

n.a.

n.a.

IL-6, CRP, TNFR2

Song et al. (2013)

12 ng/ml

IL-8‡‡

IL-6

Kinoshita et al. (1999)

n.a.

Bunger et al. (2011)

IL-6

Chung and Chang (2003)

Women

n.a.

2.29 mg/l 2.05 pg/ml 3083 pg/ml

No risk No risk 1.67 (1.05–2.68)

SB: 0.68, SF: 0.95, AUC: 0.89

CRP, IL-6, TNFR2

Chan et al. (2011)

Overall

n.a.

0.36 pg/ml 2.24 pg/ml 12 ng/ml

1.85 (1.24–2.75); 1.66 (1.10–2.52); No risk

ROC model

IL-6, TNF-a, CRP

Kim et al. (2008)

Overall

n.a.

Women

1.62 pg/ml

AUC: 0.62

Overall

Overall

Overall

Overall

Participants selected

n.a.

2.5 mg/ml 5.3 mg/ml

n.a.

0.02 (0.01–0.22) 52.1 (4.3–632) 8665.4 (201.1–37343) 0.041 (0.0052–0.31) 0.029 (0.0057–0.14)

IL-6

Sasaki et al. (2012)

1.50 (1.12–2.00){

n.a.

n.a.

IL-1ra, IL-6, IL-8, IP-10, MCP-1

CRP SAA score

Toriola et al. (2013)

No risk No risk

3 mg/l

n.a.

n.a.

CRP IL-6

Heikkila et al. (2009)

No risk

5.64 mg/l

n.a.

1.39 pg/ml 1.11 mg/l 2725 pg/ml

CRP

Allin et al. (2009)

1.97 (1.13–3.43)

3 mg/l

ROC curve‡

1.99 (1.02–3.90)†† No risk No risk

CRP

Prizment et al. (2011)

1.36 (1.00–1.85){

Score threshold

8.3 pg/ml

CRP

Aleksandrova et al. (2010)

Risk of CRC†

n.a.

IST

Study (year)

Table 1. List of studies addressing the ability of immune serum-based screening tests to detect colorectal cancers.

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Immune mediators as potential diagnostic tools for CRC

production of the cytokine in the tumor milieu or an unspecific systemic inflammatory reaction. In a recent study, a model was proposed, wherein IL-6 was produced in an autocrine fashion by progenitor cancer cells, and was required for tumor progression from a very early stage of cancer occurrence [144]. Accordingly, several studies documented that the levels of IL-6 in the serum of CRC patients increased compared with normal subjects [137,145–149]. Importantly, serum levels of IL-6 linearly correlated with its immunohistochemical expression in tumor gland matched from the same patients [146]. In two retrospective cross-sectional studies based on colonoscopy with a case–control approach, an increase of IL-6 serum levels [150] and TNF-a [127] was associated with the presence of colorectal polyps, thus predicting their presence among healthy subjects better than CRP. [127]. A prospective study from Chan et al. showed that women with high plasma levels of TNF receptor 2 had higher risk to develop CRC independently of IL-6 or CRP levels [128]. In a more recent retrospective study from the same group, but conducted in a population of men, suggested that the ability of IL-6 blood levels to predict the risk of developing CRC might vary according to the body mass index, while no association between CRP and TNF receptor 2 and the risk of developing CRC was detected [129]. Thus, although the employment of IL-6 assessment as an IST for the diagnosis and detection of CRC precursor lesions seems very promising, it deserves wider prospective population-based validation studies for its translation into clinical practice. Kantola et al. analyzed a wide array of serum cytokines in a series of CRC patients and a control group [151]. They showed that IL-1ra, IL-6 and IL-8 were higher in CRC patients compared with healthy donors, while MCP-1 and IFN-g-inducible protein-10 decreased. The authors showed by ROC analysis that a model built on the levels of these cytokines had an area under the curve of 0.890 with a sensitivity of 0.687 and a specificity of 0.952 in detecting CRC patients. This study demonstrated the idea that the detection of serum ‘cytokine footprints’ seems to be a promising tool retaining a great accuracy to be applied in diagnostic screening of CRC. In the same study, the increase in IL-6 and IL-8 was consistent along with the stage of CRC, thus reflecting their likely involvement in the severity of the disease [151]. The decrease in MCP-1 expression in CRC is counterintuitive in light of the pro-tumoral role of macrophages [152,153] and might reflect the paradoxical association between the number of CD68+ cells at the CRC invasive margin and better patient survival, which has been shown in other retrospective studies [52,154,155]. A case–control study from Saltzman et al. revealed that a single-nucleotide polymorphism at the TGF-b1 locus was associated with levels of circulating serum TGF-b1 and a lower risk to develop CRC [156]. These data are clinical evidence of the important role of this cytokine in the tumor molecular strategies to evade immune surveillance and increase tumor progression. Berghella et al. have shown that sIL-2R, IL-4, IL-6, IL-10 serum levels increased in CRC patients compared with healthy donors, while IL-2 and IFN-g decreased [157]. In a informahealthcare.com

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study by Shibata and Takekawa, sIL-2R inversely correlated with the extent of disease in cachectic patients [158]. In different studies, CXCL5, CXCL10 and CXCL16 serum levels were higher in CRC patients compared with controls and were both shown to be independent prognostic markers of poor prognosis [159,160]. The employment of larger cohort studies [159], matched with cytokine array or a chip-based assay [158], is crucial to the generation of promising data [162,163]. In a large case–control screening study, serum levels of IL-8 were benchmarked and when matched with carcinoembryonic antigen, they had 86% specificity and 47% sensitivity in detecting CRC and were better than CRP combined with carcinoembryonic antigen at this task. The authors compared the ability of IL-8 and CRP to detect CRC in terms of sensitivity and specificity of FOBT available in literature and underlined [163] that a score based on IL-8 detection might be combined with FOBT to improve their performance in population screenings. Expert commentary

The idea that tumor development and progression is the result of the interactions between inflammatory networks and adaptive anti-tumor immune response is well established. Most of these complex interactions occur in the tumor microenvironment among TAM and TAN and adaptive immune cell defenses, with a wide armamentarium of cytokines and chemokines being the mediators of this intricate network. While the prognostic value of the infiltration of lymphocytes in primary CRC has been today widely assessed, its clinical exploitation for the identification of post-surgical recurrences still has to be confirmed by properly designed prospective clinical trials. In a different perspective, an earlier diagnosis would decrease the burden of patients with metastatic CRCs and mortality rate, and while endoscopic screening is employed to this aim, it is a screening tool procedure with low compliance. While the possibility to increase the identification of earlier CRCs is still underestimated, unspecific circulating inflammatory markers such as levels of CRP, NLR or lymphocytic count are not reliably associated with the number of cells in the tumor milieu and are unlikely to reflect the inflammatory status of the CRC microenvironment. Prospective studies available to date concerning the diagnostic value of CRP in CRC were conflicting and revealed inconsistencies. Data concerning specificity and sensitivity of diagnostic biomarkers by ROC analysis would provide a shared reproducible paradigm to benchmark their yield of diagnostic ability. However, most of the prospective studies on this issue did not share such information and as a result it is difficult to build diagnostic comparative models, for this reason, more efforts in standardizing procedures in diagnostic biomarkers validation are much needed. In a new perspective, the quantification of the levels of soluble immune mediators in the serum mirrors the ongoing inflammatory reaction at the tumor site. Cohort studies revealed IL-6 as a promising tool, which seems to have the potential to become a paradigm in non-invasive diagnostic screenings for the detection of colonic early precursors. 393

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However, it is conceivable to hypothesize that profiles of chemokines and cytokines in serum by multiplex assay detection might be a more efficient technology to detect CRCs in population screening compared with single markers. Properly designed prospective studies with wide arrays of IST are greatly required to test this hypothesis and provide to clinicians an efficient, cost-effective and accessible tool to identify individuals who will get advantage from colonoscopy.

translate these experimental evidence have to be designed to generate reproducible clinical data. The development of a shared benchmarking platform to compare the diagnostic power of IST profiles will have the ultimate aim to provide a widespread consensus for their clinical implementation. Thus, in the long journey from experimental rationale to clinical validation great efforts will be required in the future to identify and test the most promising mediators of inflammation to be used as IST in the diagnostic screening of CRC.

Five-year view

Preclinical models greatly contributed to the discovery of a wide array of soluble mediators of inflammation, which have been shown to play a pivotal role in tumor progression since its very early steps of disease occurrence. Inflammatory models of CRC are representative of colitis-associated cancer, which accounts for about 1% of the overall population of CRCs, while genetic models of adenomatous polyposis coli and syngeneic and xenograft methodologies have inherent and unpredictable limitations to reproduce human CRC. It is important to underline that cutting-edge non-inflammatory, site-specific and spontaneous genetically engineered preclinical models of CRC properly recapitulating the disease progression along its different stages are greatly required. Appropriate experimental standards applied to the identification of clinical relevant mediators of inflammation will provide a reliable rationale to implement expensive prospective validating screening studies. In the next years, large population-based clinical studies required to

Acknowledgements

The authors are grateful to N Cortese for her precious comments to improve the manuscript. Financial & competing interests disclosure

This work was supported by the Italian Association for Cancer Research (AIRC) Italy (grant number MFAG-11677 to Federica Marchesi) and the Italian Ministry of University and Research, FIRB grant (RBAP11H2R9). G Di Caro is supported by a fellowship from Fondazione Umberto Veronesi. MR Galdiero was supported by a fellowship from P.O.R. Campania FSE 2007–2013, Project cre`me. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • Innate immune cells are main components of the tumor microenvironment and critically contribute to the onset and progression of colorectal cancer (CRC). • Adaptive immune cells are endowed with anti-tumor activity, which is importantly regulated by the presence of tumor-associated macrophage and tumor-associated neutrophil in the tumor milieu. • Tumor-associated macrophage and tumor-associated neutrophil modify their phenotypic and functional aspects based on the signals derived from the microenvironment. • A plethora of soluble mediators of inflammation (cytokines and chemokines) are involved in the recruitment of innate and adaptive immune cells to the tumor microenvironment and have been shown to play a fundamental role in tumor progression from its earlier steps. • An earlier detection would decrease the rate of metastatic CRC at diagnosis. Colonoscopy is used for this purpose, but it is an invasive and painful procedure, costly, time consuming and thus with a low compliance. Fecal occult blood test is the only non-invasive screening strategy available in CRC, it is a methodology with a high compliance but not sufficiently specific and sensitive in detecting CRC occurrence. Thus, novel immune serum tests are greatly required in CRC population screening. • Prospective trials available suggest C-reactive protein (CRP) as an unreliable diagnostic biomarker for CRC screening. Unspecific systemic inflammatory markers such as levels of CRP, NLR and lymphocytic count are not reliably associated with the number of cells in the tumor milieu and are unlikely to reflect the inflammatory status of the CRC microenvironment. Serum levels of immune mediators are likely to efficiently reflect the specific local immune reaction in the tumor milieu compared with CRP. • Several translational studies suggest that arrays of chemokines and cytokines in the serum might be implemented to clinical practice as novel screening markers to provide an earlier detection of CRC. • Arrays of circulating levels of chemokines and cytokines need to be tested in properly designed prospective clinical trials with the aim to identify immune serum-based screening tests with potential diagnostic value, which might be implemented in clinical practice to improve the compliance of colonoscopy and reduce the burden of metastatic CRC at diagnosis.

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clinical impact. Cancer Microenviron 2013; 6(2):117-22

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