Chapter 4 Immunologic Interpretation of Cancer Biology ... · Chapter 4 Immunologic Interpretation...

Chapter 4Immunologic Interpretation of CancerBiology: Impact on Clinical Outcome

Maria Libera Ascierto, Francesco M. Marincola and Ena Wang

Abstract The biology of tumors cannot be understood by simply studying thetraits of the neoplastic cells in isolation. Instead, the contributions of the ‘‘tumormicroenvironment’’ to tumor genesis must be considered. The complexity of theinteraction between not transformed host cells with cancer cells and the molecularpathways involved in such interaction could be recently appreciated by high-throughput tools capable of providing a global view of biological processes. Basedon this approach numerous studies have defined a new portrait of cancer,describing a linkage between the immune infiltrate, prognosis and response totherapy. Here, we provide an overview of the current status of the field describingthe immune cells involved in this phenomenon. Molecular events and biomarkersassociated with the favorable clinic outcome will be described, analyzing alsocommonalities and discrepancies among studies.

Keywords Tumor microenvironment � Tumor immune infiltrate � Prognosticvalue of immune infiltrate � Genetic alterations of host � Genetic alterations oftumor

M. L. Ascierto (&) � E. Wang (&)National Institutes of Health, Building 10, Room 1N224, 10 Center Drive,Bethesda, MD 20892, USAe-mail: [email protected]; [email protected]

E. Wange-mail: [email protected]

M. L. Ascierto � F. M. Marincola � E. WangInfectious Disease and Immunogenetics Section (IDIS), Department of TransfusionMedicine, Clinical Center and Trans-NIH Center for Human Immunology (CHI),National Institutes of Health, Bethesda, MD 20892, USAe-mail: [email protected]

M. L. AsciertoDepartment of Internal Medicine, University of Genoa, Genoa, Italy

M. R. Shurin et al. (eds.), The Tumor Immunoenvironment,DOI: 10.1007/978-94-007-6217-6_4,� Springer Science+Business Media Dordrecht 2013

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4.1 Introduction

Traditionally, anti-cancer therapies have targeted exclusively transformed cancercells. However, more recently tumors are not only considered as masses of pro-liferating cells, but it is widely accepted that non-transformed host cells, whichinclude both innate and acquired immunity, such as endothelial, fibroblasts,mesenchymal cells, MDSCs, macrophages, DCs, mast cells, eosinophils, neutro-phils, NK cells, and lymphocytes, interact with malignant tumor cells to form adynamic environment in which the non-transformed cells exert immune surveil-lance or promote spread of cancer cells. In turn, also cancer cells affect the phe-notype of the non transformed host cells by express suppressive molecules orprovide negative feedback to compromise their function.

Viewed from this prospective, the biology of tumors can no longer be restrictedto the study of the cancer cells. Instead, the contributions of the ‘‘tumor micro-environment’’ to tumor genesis must be considered. Due to the high complexity ofthe tumor microenvironment, understanding the networking between not trans-formed host’s and cancer cells and the molecular pathways involved in suchinteraction is a ‘‘must’’. Such complexity could be recently appreciated in itsextent by high-throughput tools, such as gene profiling or DNA sequencingcapable of providing a global view of biological processes defining possiblebiomarkers of tumor regression or therapy’s response. Based on this approachoncologists and immunologists are combining their efforts to reshape the biologyof cancer to provide better diagnostic, prognostic and predictive power.

4.2 The Role of Immunity in Cancer

The first studies able to re-classify cancer biology according to global transcrip-tional analysis were published about a decade ago (Alizadeh et al. 2000; Lossoset al. 2000; Perou et al. 2000). Since then, numerous studies described a cancerphenotype where the infiltration of immune cells of both the innate and adaptivearms of the immune system as well as the combined with the expression ofinterferon–stimulated genes (ISGs) and immune effectors functions (IEFs), sustaina self-perpetuating inflammatory process which influences tumor growth and/orresponsiveness to anti-cancer immunotherapy (Ascierto et al. 2011; Disis 2010).

These observations emphasized the host immune cells both within and sur-rounding tumors as critical determinants of cancer biology and key factors for thesuccess or failure of human cancer therapy. Those statements changed the field oftumor immunology as pointed out by a recent review of (Hanahan and Weinberg2011) in which ‘‘avoiding immune destruction’’ is being considered an emerginghallmark of cancer. However, it is also quite confusing when we contemplate thatjust a decade ago the same authors claimed that the presence of immune cells andtumor-associated inflammatory responses promote tumor growth (Hanahan andWeinberg 2000).

84 M. L. Ascierto et al.

To better understand this apparent paradox the intensity of inflammatory pro-cesses must be considered. Usually the inflammation associated with cancer issimilar to a chronic inflammatory process, where the production of growth andangiogenic factors stimulates tissue repair and growth. Occasionally, however, it isobserved a cancer inflammatory process similar to acute inflammation character-ized by the presence of innate and adaptive T cells responses which favor theactivation of immune effector mechanisms able to generate spontaneous or treat-ment induced cancer regression.

This phenomenon strongly indicates that even established cancer hold thecharacteristic of plasticity of cancer-related inflammation and should be consid-ered as a dynamic event (Mantovani et al. 2008). Before tumor establishment,immune surveillance can control or eliminate some premalignant lesions. How-ever, tumor cells can become resistant to the first line of defense and develop aphenotype able of manipulating immune cells, inducing a process referred to as‘‘immune editing’’. Trough the immune editing mechanism, the tumor cells areable to escape immune recognition, by down regulate the expression of majorhistocompatibility complex, by decrease expression of co-stimulatory moleculesimportant for T cell activation, and by enhance surface expression of molecules,which suppress the activation of T cells, (PD-L1/B7-H1 and B7-H4). Cancer cellscan also limit the function of the immune system through the secretion of solublefactors able to inhibit the activation, proliferation, and differentiation of the var-ious components of the immune response (Mantovani et al. 2008). However, at onepoint a threshold is reached: the immune cells and the secreted chemokines andcytokines turn an indolent process that favors tumor growth into an acute processthat promotes tumor destruction.

In theory lesions can regress and all cancer cells are eliminated, disappear.Immunotherapy aimed at manipulate and optimized immune cells by blockadeimmune checking points, induce production of pro inflammatory cytokines andchemokines which in turn helps this process through the recruiting and activationof T cells and natural killer cells and gear toward reaching the invisible threshed totip the balance in favor of tumor elimination. In some cases tumor cells are stillable to escape these ‘‘acute’’ immune—related destructive phenomena, thusleading to tumor progression or recurrence.

The observation of a similar behavior in other tissues undergoing different typesof immune-mediated tissue-specific destruction (TSD) such as flares of autoim-munity, clearance of pathogen infected cells during acute infection, allograftrejection and graft versus host disease, leaded us to propose that distinct immuneprocesses ultimately converge into a ‘‘immunological constant of rejection’’mechanism (Wang et al. 2008) (Fig. 4.1). Thus, although distinct immune phe-nomena are prompted by different mechanisms, the requirements for endogenousor exogenous inflammatory stimuli reach at one point a threshold which turns achronic inflammatory process into an acute one, converging into in a conservedtissue destruction mechanism.

In the following discussion, we provide a description of the immune cells whichare mostly involved in these phenomena; our attention will be particularly focused

4 Immunologic Interpretation of Cancer Biology 85

on the infiltration of T cells, NK cells and B cell in tumor side. Molecular eventsand biomarkers associated with the favorable clinic outcome will be described,analyzing also commonalities and discrepancies among studies.

Moreover, studies supporting interplay between immune contexture of tumormicroenvironment, genetic background of the host as well as genetic instabilityof neoplastic cells, and their association with cancer patients’ outcome will bediscussed. It is important to point out that while many of the results reported

Allograft rejection

Viral clearance

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Autoimmunity

Tissue-specific disruption

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Th17Th17

CTL

CTL

CTLTh2

Th2Th2

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Crosstalk between innate and adaptive immune system

Crosstalk between adaptive and humoralimmune system

Fig. 4.1 Immunological constant of rejection. Immune molecular pathways observed duringtissue rejection and inflammatory responses to cancer are also observed in curse of theinflammatory response against viral infection, allograft rejection as well as autoimmunity disease.This suggests that although the tissue-specific destruction varies in distinct pathologic states, theeffector immune response converges into a single mechanism that includes the activation ofadaptive and innate cytotoxic mechanisms and the crosstalk between them. The keys genesinvolved in those processes we can observed that they are mostly centered around IFN-csignaling, TNF signaling, Interferon Regulatory Factors (IRFs) as well as Immune EffectorFunction (IEF) genes such as granzyme B, perforin. Those inflammatory key genes, in severalcondition are associate with better prognosis and in other conditions are instead associated withnegative prognosis. To better understand this apparent paradox the intensity of inflammatoryprocesses must be considered


below are quite impressive in terms of similar conclusions, there are also someconflicting observations. These can potentially be explained by differences inmethodologies used. Older studies used light microscopy, and in some cases nodistinction was made between different cell types. The advancement of tech-nologies such as Polymerase Chain Reaction (PCR), and gene expression pro-filing has addressed the study of tumor-infiltrating immune cell populations inmore details reducing the number of conflicting observations (Bedognetti et al.2010).

However, we need to consider that sometimes it is possible to observe dis-crepancies between these similar techniques mostly due to the usage of differentstandards for expression normalization. Moreover, if we consider that sometimesimmune cell types found in tumors can vary in degree of maturation and/oractivation, and many diverse cell types can share similar markers, the descriptionof an objective and real picture becomes even more challenging.

4.3 Prognostic Significance of T Cells

Increasing evidence indicates that some patients with cancer can generate anadaptive immune response specifically directed against antigenic proteinsexpressed by tumors (Marincola 2005). In particular, an adaptive T cell response,which is composed of both cytotoxic CD8+ T cells (CTLs) and CD4+ T cells, canpromote the secretion of cytokines such as interferon gamma (IFN-c) and tumornecrosis factor alpha (TNF-a) generating an acute inflammation which results inexpansion of cytotoxic CD8+ T cells, tissue destruction and control of cancergrowth. Although in animal models the in vivo eradication of tumors is mostlydependent by CTLs, there has been increased appreciation over the last years forthe importance of the CD4+ T cells, which through the secretion of cytokinesinvolved in the regulation of acute inflammatory responses, are able to enhance orlimit CTLs function or proliferation (Disis 2010). Thus, CD4+ cells are referred toas T-helper (Th) cells which manifest with different phenotypes.

Th1 cells are stimulated by type I dendritic cells (DCs). Together these cells,generate a tumor environment rich of cytokines such as IFN-c, TNF-a, Inter-leukin (IL)-12 (IL-12), IL-2 and support CTL and tissue destruction; the resultsis a potential control or elimination of tumor growth. Th2 cells are stimulated bytumor associated macrophages (TMAs) and myeloid–derived suppressor cells(MDSCs). Together these cells types generate a tumor environment rich ofcytokines such as IL-10, IL-4, IL-5, IL-6, which enhance B cell responses andlimit CTL proliferation. Th17 cells secrete IL-17 and are operative in pathologicautoimmune disease. Regulatory T cells (Tregs) secrete IL-10 and TumorGrowth Factor (TGF-b), which limit CTL response resulting in potential pro-liferation of tumor cells. We could assume that the overall immune responseelicited within each individual tumor results from the balance of the type of cellsand cytokines secreted.


4.3.1 CTLs, Th1 Cells and T Memory Cells

Gene expression studies performed in distinct solid tumors types indicated that animmune response characterized by the expression of gene related to adaptive andinnate effector immunity such as antigen presentation, IFN-c production, activa-tion of T cell receptor signaling, is associated with improved prognosis in mela-noma, head and neck, breast, bladder, urothelial, ovarian, colorectal, renal,prostatic, and lung cancer (Ascierto et al. 2011). One important investigationdemonstrating this association was performed in colon cancer patients through theidentification of a cluster of genes inversely correlated with tumor recurrence. Theidentified predictive signatures, encoding Interferon (IFN) regulatory factor 1(IRF1), IFN-c, CD3, CD8, granulysin, and grazyme B is mostly associated withTh1 immunity and CTLs generation (Galon et al. 2006).

The same observation was also previously described by Guidoboni et al. (2001)in colorectal cancer where high numbers of activated CTLs correlated withimproved overall and disease-free survival, particularly in patients with stage IIItumors (n = 109, P \ 0.001). Moreover, a high frequency of microsatelliteinstability was correlated to survival. Multivariate analysis revealed that patientswith both features had a risk of death and relapse markedly lower than thatassociated with microsatellite status or intra-tumoral cytotoxic lymphocytesseparately. Similar observations were then reported in other cancer types (Asciertoet al. 2011).

In melanoma, a pilot study of 19 patients vaccinated with a combination of fourtumor antigens plus IL-12, (Gajewski 2011) observed by global transcriptionalanalysis that tumors of patients likely to respond to therapy have a pre-existingover expression of interferon stimulated genes (ISGs) and Th1 cell attractingchemokines.

These findings were associated with the histopathological demonstration of thepresence of CTLs in the same tumors. Interestingly, together with the effectorcomponent of the immune response, the tumors derived from the respondingpatients also displayed the presence of immune inhibitory mechanisms such asexpression of Programmed Death Ligand 1 (PD-L1) which upon binding with PD-1 mediate the de-phosphorylation of signaling molecules downstream of the T cellreceptor, thus dampening T cell sensitivity to antigenic stimulation (Ahmadzadehet al. 2009). The co-expression of indoleamine-2,3-dioexygenase (IDO), whichcauses immune suppression through breakdown of tryptophan in the tumormicroenvironment producing toxic tryptophan catabolites which consequentlycause growth arrest and functional suppression of effector T cells, was alsoobserved in the same group of patients (Masferrer et al. 2000; Sinha et al. 2007).These findings can be interpreted as demonstration that tumors phenotype do notdiscriminate among various components of the immune response as they cansustain immune effector and immune regulatory functions simultaneously; it isprobably the overall balance between the immunosuppressive and immune-activefactors that determines the ultimate fate of individual cancers.


In addition to the Th1 signature, the second key feature of a potentially effectiveimmune response is the capacity of T cells to travel into the site of the tumor andinfiltrate deeply into the tumor parenchyma. Although a connotation of lympho-cytic infiltrates as a favorable prognostic biomarker in cancer was originallyreported by Cochran (1969), much progress has been made in the last decade.Clemente et al. (1996), reported that the presence of tumor infiltrating lympho-cytes in the vertical growth phase of primary cutaneous melanoma was an inde-pendently favorable prognostic factor.

Similar observations were subsequently showed in a study conducted on 186patients with ovarian cancer were T cells infiltrates turned out to be a significantprognostic factor (Zhang et al. 2003). The authors observed significant differencesin the distributions of progression-free survival and overall survival according tothe presence or absence of intratumoral T cells (P \ 0.001). The presence ofintratumoral T cells was independently correlated with delayed recurrence ordelayed death in multivariate analysis and was associated with increased expres-sion of IFN-c, IL2, and lymphocyte-attracting chemokines within the tumor. Onthe other end the absence of intratumoral T cells was associated with increasedlevels of vascular endothelial growth factor.

However, the study that best characterized the phenotype of the T cells able topenetrate into tumor parenchyma came from gene expression analysis performedon colon cancer patients. Investigators showed that high levels of intra-tumoralCD45RO+ (memory) T cells correlated with better survival. Also the localizationwas important for the prognostic significance which was very favorable when theT cells were accumulating in the center and in the invasive margins of the tumors.Gene expression analysis performed on the same cohort of patients demonstratedthat the Th1 signature, described above, facilitate the infiltration of intra-tumoralCD45+RO T cells (Pages et al. 2005).

All together, these investigations suggested that prognosis in patients withcancer is positively affected by the presence of type I adaptive immune response intumor environment and by the ability of T cells to penetrate through tumor stromaand infiltrate deeply into parenchyma.

4.3.2 T Regulatory Cells

In contrast to CTLs, Th1 CD4+ cells and memory T cells, the relevance of other CD4+T cell populations on clinical outcome has been more controversial (Fridman et al.2012). The case of regulatory T (Treg) cells serves as a good example of conflictingdata that lead to difficult interpretation. Although, there are different subpopulations ofTreg, most studies define them as a population of CD4+ T cells expressing high levelsof CD25 and the transcription factor forkhead box protein P3 (FOXP3). However, noneof these markers is fully restricted to Treg cells. Indeed, CD25 and FOXP3 are alsoexpressed by activated effector T cells, and there are also FOXP3- suppressor cells.Thus, the initial report by Curiel et al. (2004) which demonstrated a correlation of


intra-tumoural Treg cells and poor survival in ovarian cancer and which have beenfollowed by similar observations in other cancer type such as hepatocellular carcinoma(Gao et al. 2007) has to be carefully reconsidered. In fact, several analyses of othercancer types have found no impact of Treg infiltration on survival (Ascierto et al. 2011;Heimberger et al. 2008; Hillen et al. 2008; Mahmoud et al. 2011). By contrast, otherreports have demonstrated a positive clinical correlation between the density of intra-tumoral Treg cells and the local immune control of tumors (Badoual et al. 2006;Carreras et al. 2006; Frey et al. 2010; Salama et al. 2009). In colorectal cancer, Tosoliniet al. (2011) recently described two clusters of genes associated with regulatoryfunctions. Although the first cluster (IL-10/TGF-b) was not associated with a favorableoutcome, FOXP3 (second cluster) mRNA expression and the presence of high densityFOXP3 positive cells were associated with better survival. We recently describedsimilar observation in patients with metastatic melanoma receiving high-dose inter-leukin-2 plus the gp100:209–217(210 M) peptide vaccine melanoma patients. Theresults showed that the vaccine–interleukin-2 group, as compared with the interleukin-2–only group, experienced a significant improvement in overall clinical response andlonger progression-free survival. In study, an increase in T regulatory cells(CD4+FOXP3+) was observed in patients responding to treatment (Schwartzentruberet al. 2011).

The reasons for Treg discrepancies are not evident; they may be due theimperfect markers used to phenotype suppressive cells or due to technical dif-ferences. Regulatory T cells can lose FOXP3 (Hoffmann et al. 2009), effectorT cells can transiently express FOXP3 without acquisition of suppressive functionsand FOXP3 acts as a tumor suppressor gene when expressed by tumor cells. Theseobservations complicate the interpretation of the aforementioned studies inabsence of functional and cell-specific analyses (Walker et al. 2003; Zuo et al.2007; Wang et al. 2009).

4.3.3 Th17 Cells

The analysis of other CD4+ T cell populations has also yielded apparently con-tradictory results (Wilke et al. 2011). Th17 cells have been reported associatedwith poor prognosis in colorectal, lung and hepatocellular carcinoma (HCC)(Jochems and Schlom 2011). Zhang et al. (2009) have examined IL-17+ cells inpatients with HCC and suggested a potential pro-tumor role for IL-17. In this caseincreased IL-17- producing cell density within the tumors of HCC patients wascorrelated with both micro vessel density and poor prognosis. Notably, HCC isfrequently associated with chronic viral hepatitis which can strongly affect thegeneration and function of Th17 cells in cancer patients.

In non-small-cell lung cancer patients, higher levels of IL-17 within the tumorcorrelated with higher blood vessel density and shorter survival (Chen et al. 2010).On the contrary in other studies Th17 cells have been reported to predict bettersurvival (Tassi et al. 2008). In ovarian cancer, tumor-associated Th17 levels


correlate positively with microenvironment Th1 cells, cytotoxic CD8+ T cells andnatural killer cells (Kryczek et al. 2006). The same group also observed a com-bined infiltration of IL-17 expressing CD4+ T cells and CD8+ effector T cells;through synergistic action between IL-17 and IFN-c expression Th17 cells wereobserved to stimulate the expression of CXCL-9 and -10 by ovarian cancer cells aswell as tumor infiltrating macrophages in order to recruit more effector T and NKcells to the tumor microenvironment. This combination was observed to positivelypredict patient outcome in the context of ovarian, colon, melanoma and pancreaticcarcinoma. Altogether these studies demonstrated that Th17 cell infiltration inseveral tumor types was quantitatively and positively correlated with NK cell-mediated innate and adaptive immune responses (Kryczek et al. 2009). Still veryfew studies have focused on primary Th17 cells in the human tumor microenvi-ronment, so it is difficult to deduce the exact role(s) they may have in cancerpatients. Moreover, Th17 cell biology has been partially examined in patients withwell-established cancer. It may be important to investigate the roles of Th17 cellsand IL-17 in the very early phases of human tumor growth to better understandhow these roles may change during disease progression (Wilke et al. 2011).

4.3.4 Th2 Cells

Th2 cells, through the activation of B cells or through the production ofthe immunosuppressive cytokine IL-10, seem to be associated with aggressivetumours. Recently (De Monte et al. 2011) showed that in pancreatic cancer theTh2 immune deviation has an active role in tumor progression. Moreover,the quantity of Th2 with respect to Th1 cells present in the tumor stroma has adirect correlation with prognosis in surgically resected patients. However, this isnot a general phenomenon, as Th2 cells are also associated with favorable outcomein Hodgkin’s lymphoma and breast cancer, which suggests a protective effect ofantibodies in these diseases (Schreck et al. 2009; Yoon et al. 2010).

4.4 Prognostic Significance of Innate and HumoralImmune Response: Focus on NK Cells and B Cells

Numerous lines of evidence suggest that presence of T cells identifies cancerpatients with an improved prognosis while the role of B cells and that of innateimmune effector mechanisms and their cross talk with adaptive immune responsesas played a Cinderella role (Shanker and Marincola 2011). This may also be due tothe fact that cells of the innate immune system are involved in tissue repair andremodeling and the factors secreted by these cells are usually believed to enhancerather than inhibit tumor growth. Indeed, several types of innate immune cells


have been shown to be independent predictors of poor prognosis and cancerprogression (Martuza et al. 1991; Kelley et al. 2007; Zhu et al. 2008). Theseinclude mast cells, eosinophils, neutrophils, type 2 macrophages, and MDSCs(Ostrand-Rosenberg and Sinha 2009; Allavena et al. 2008). Even DCs, as pro-fessional antigen-presenting cells (APCs), may participate in tumor immuneevasion by failing to stimulate effective adaptive responses (Jensen et al. 2011;Chaput et al. 2008).

However, experimental as well as clinical observations suggest that immune-mediated tumor destruction is dependent upon coordinate activation of immuneeffector genes expressed by cells of the innate and also humoral immune systems(Wang et al. 2008; Zuo et al. 2007; Shanker et al. 2007). Thus, a description of theeffect of these parallel arms of the immune system is necessary and an associationwith patient’s clinical outcome needs to be evaluated.

4.4.1 NK Cells

Among cells of the innate immune system, Natural Killer (NK) cells play a majorrole against tumors and they participate in the shaping of the adaptive immuneresponse thought the secretion of cytokines such as IFN-c (Vivier et al. 2011,2012; Moretta et al. 1994). An important feature of NK cells is their capacity todistinguish stressed (such as tumor cells, infected cells and cells have undergonephysical or chemical injuries) from healthy cells. NK cells were initially identifiedthrough their ability to kill tumor cell, hence their name (Oldham and Herberman1973; Herberman et al. 1975; Kiessling et al. 1975). Recognition of tumor cells byNK cells is mediated by the interaction of activating receptors with ligandsexpressed on tumor target cells which at the same time do not express ligandsspecific for the NK inhibitory receptors. NK cells also express adhesion molecules,thereby interacting with tumor cells mediating their disruption (Moretta andMoretta 2004).

However, the role of NK cells in controlling the growth of human tumors hasbeen less extensively explored. In mice, tumor rejection is positively correlatedwith precursor frequency of both tumor-specific CD8+ T cells and NK effectorcells are able to interact with tumor cells via the activating receptor killer celllectin-like receptor subfamily K, member 1 (KLRK1 also known as NKG2D)(Shanker et al. 2010). Additionally, our group observed that nude mice treatedwith oncolytic viruses can reject tumor xenografts (Worschech et al. 2009). Thisrejection was associated with the activation of ISGs (both IFN-c and IFN-astimulated genes), upregulation of CXCR3 and CCR5 ligands, and activation ofIEF genes (granzyme B, caspase 8). Since these mice lack T cells and secondarilylack B cell responses, this immune-mediated tissue destruction is thought to beinduced by innate immune effectors such as NK cells and activated macrophages.This study suggested that, at least in this model, innate immunity can be an


independent effector of tissue-specific destruction not requiring adaptiveimmunity.

In human, it was a decade ago when it has been showed for the first time acorrelation between a partial regression of primary tumor growth in melanomapatients and NK cell activity, suggesting that NK cells may represent a potentialprognostic marker (Jochems and Schlom 2011). Subsequently, Coca et al. (1997)showed that intratumoral natural killer cells analyzed in a cohort of 157 patientswith colorectal cancer was positively correlated with favorable prognosis; patientswith little and moderate NK infiltration showed significantly shorter survival rates(overall and disease free survival) than those with extensive infiltration (P \ 0.01).Three significant factors affecting survival were selected in a stepwise fashion inincreasing order as follows: TNM stage, NK infiltration, and lymphocytic infil-tration. Patients with TNM Stage III disease and extensive NK infiltration showedsignificantly longer survival rates than those with little or moderate infiltration(P \ 0.001). The same observations were described in other cancer types such asin renal, lung cancer and hepatocellular carcinoma (Ishigami et al. 2000; Villegaset al. 2002; Donskov and von der Maase 2006). However, in the majority of thesestudies NK cells have been detected according to the expression of CD57 or CD56markers which has been shown to inaccurately identify NK cells since they arealso expressed by other tumor-infiltrating lymphoid cells. The advent of highthroughput technology, mostly based on gene expression profiling has shown therelevance of the prognostic infiltration of NK cell markers at the tumor site.

In breast cancer, a meta analysis conducted on a large publicly available set ofmicroarray data from primary tumors suggested that all the major histologicalsubtypes of breast cancer displayed variable expression of ligands for NK cellreceptors. In particular, NKG2D-ligands and DNAX Accessory Molecule-1(DNAM1) ligands, known for their NK cell activating function, were found to bewidely expressed across all breast cancer subtypes (Mamessier et al. 2011). Thesame group reported that a decrease in the expression of the activating receptorNKG2D, DNAM1 and NKp46 (considered a gene uniquely expressed by NK cells)was associated with tumor progression.

Recently, our group also observed that NK cell molecular signatures are pre-dictive of relapse free survival of favorable prognosis of breast cancer patients(manuscript in preparation). Tumors were obtained from patients experiencingeither 5–9 years relapse-free survival or tumor relapse within 1–6 years followinginitial treatment. Based on differential expression of CD56 and CD16, NK cells didnot vary between relapse free and progressing groups. However, tumors frompatients with no recurrence were characterized by up-regulation of activatingreceptors NKp46, NKp30, NKG2D and DNAM1 as well as molecules involved inthe interaction of NK cells with tumor cells. On the contrary, expression of NKcells inhibitory receptors transcripts was not significantly different in patients withwidely diverging.

An involvement of activating receptors in cancer prognosis has also beenrecently observed in gastrointestinal stromal tumor, where an increased expressionof the NK activating receptor NKp30 (also known as NCR3) was associated with


prolonged survival of patients as well as in renal cancer, where high expression ofthe activating receptor NKp46 was associated with higher survival probability(Delahaye et al. 2011; Eckl et al. 2012).

Taken together, the following observations suggest that although NK cellsrepresent only a minor component of tumor microenvironments and of lympho-cytes infiltrates, they play an important role in cancer immune surveillance as theyare able to interact with tumor cells mediating their description or with other targetcells, such as DCs, favoring their activation which positively affect the adaptiveimmune response.

4.4.2 B Cells

The prognostic significance of intra-tumoral B cells remains unclear. Mousemodels support a negative role of B cells in cancer immune surveillance. Initially,this activity was mostly associated with an increased production of IL-10 by Bcells, which is considered an immunosuppressive cytokine (Wong et al. 2010).However, intravenous administration of recombinant IL-10 to humans producespro-inflammatory effects by enhancing release of IFN-c, IFN-inducible protein 10,TNF-a, and IL-1 and appears to induce activation of CTL and NK cells, asreflected by increased plasma levels of granzyme-B (Mocellin et al. 2004). Theseobservations lead to the hypothesis that IL-10 might contribute to the immune-mediated rejection of cancer, at least under some circumstances. Thus, a delete-rious role of B cells based on the production of IL-10 is not fully supported byexperimental observations (Mocellin et al. 2003; Rossi et al. 2004).

Another negative effect of intra-tumoral B cells could be due to the productionof IgG, forming antigen-IgG antibody complexes which may activate an M2 pro-toumor phenotype in macrophages promoting the early stage of carcinogenesis(Andreu et al. 2010). However, recent evidence, suggest a favorable associationbetween B cell infiltration and prognosis in epithelial ovarian cancer. In particular,tissue microarray (TMA) performed in a cohort of 198 patients showed thatintraepithelial CD20+ cells occurred in 41.9 % (83/198) of evaluable tumors.Moreover, presence of CD20+ infiltrates was strongly associated with that ofT cell subsets (CD3, CD4, and CD8), with the activation markers CD45RO andCD25 and with Granzyme B and FoxP3. Finally, CD20+ infiltrates were associ-ated with increased disease specific survival (DSS). This effect was mostlyascribed to the promotion of the opsonization of tumor antigens, complement-mediated destruction of tumor cells, or antibody-dependent cellular cytotoxicityand to the fact that B cells can also present antigen to both CD4+ and CD8+ T cells(Milne et al. 2009).

A similar portrait was recently described by our group for breast cancer patientswhere the expression of the B cells marker immunoglobulin K C (IGKC), predictedrelapse-free survival with higher than 85 % accuracy (Ascierto et al. 2012). Inter-estingly, genes involved in primary immunodeficiency signaling, T cell apoptosis,


CTLA-4 signaling and production of NO and reactive oxygen species were also up-regulated in the tumor specimens of relapse-free patients. Such paradoxical findingsas to simultaneous up-regulation of immune effector genes and immune suppressorgenes may suggest that tumor-derived factors were responsible for the expression ofimmune suppressor genes thereby facilitating cancer progression even in the pres-ence of the increased immune effector genes.

A subsequent comprehensive analysis of human gene expression profiles con-firmed the stromal IGKC as a prognostic marker in breast cancer (Schmidt et al.2012) and other solid tumor types including lung adenocarcinoma and colorectalcarcinoma. Interestingly, any significant association instead was found for ovariancarcinoma (Schmidt et al. 2012).

The observations that IGKC in tumor is dependent upon plasma cells con-tradict the assumption that tumor cells are capable of producing immunoglob-ulins to promote tumor growth and survival (Qiu et al. 2003). Rather it supportsa previous report that cancer specimens typically have tumor infiltration of IgG-positive plasma cells (Ito et al. 1986). Although the biological role of the IGKhave to be further addressed, the prognostic impact shared by breast, lung andcolorectal carcinoma represents a comprehensive biomarker predicting of theimmune system in a variety of cancer types. Moreover, the evidences that thepresence of both CD8+ and CD20+ Tumor-Infiltrating Lymphocytes (TILs) wasassociated with increased survival compared with CD8+ TILs alone providesevidence that CD8+ and CD20+ TIL act cooperatively to promote immunity(Nielsen et al. 2012). The authors proposed three roles for CD20+ TILs inpromoting antitumor immunity. First, B cell can bind tumor antigens via surfaceIg molecules, process them and then present peptides to CD8+ and CD4+ T cellsvia MHC Class I and Class II, respectively. Second, B cells trough the secretionof lymphotoxin, can induce stromal cells to express adhesion molecules, cyto-kines and chemokines which in turn can recruit and retain other lymphocytes.Third, Type -1 and Type 2 B effector cells (Be1 and Be2) can secrete cytokinessuch as IFN-c, and IL-4, which can activate T-cell responses toward Th1, Th2 orother functional states.

In summary cells of the innate, adaptive and humoral immunity and the cyto-kines that they produce are associated with good clinical outcome for all cancertypes. In Fig. 4.2 it is possible to note that although this concept is strongly evidentand supported for Th1 cells, CTLs and CD8 memory cells, is less appreciated forNK cells and remain still controversial for other T helper cell populations and forB cells. This may be due to the different status of their maturation, on the balancebetween immune cells in the tumor microenvironment and on the tumor type inconsideration.


4.5 The Immune Score Approach: A Novel Approachfor Cancer Classification

Usually, outcome prediction in cancer is based on evaluation of tissue samplesobtained during surgical removal of the primary tumor focusing on their histo-pathological characteristics. In addition, histological as well as radiologicalanalysis of tumor draining, regional lymph nodes and distant organs are performedto identity evidence of metastases. Until now tumor staging (AJCC/UICC-TNMclassification) summarizes data on tumor burden (T), presence of cancer cells indraining and regional lymph nodes (N), and evidence for metastases (M). How-ever, this classification provides inaccurate information for prognosis since canceroutcomes can vary significantly among patients within the same stage and does notpredict response to therapy.

Based on numerous reports suggesting that cancer development is controlled bythe host’s immune system the importance of including immunological biomarkersfor the prediction of prognosis and response to therapy appeared to be necessary.By analyzing 400 patients with colorectal cancer, Mlecnik et al. (2011) suggestthat immune cell infiltration by cytotoxic CD8-positive and memory CD45RO-positive T cells has prognostic discriminatory power that is superior to standardstaging. The results demonstrate two key findings: patients with high immunescores have increased disease-free and overall survival as compared with patientswhose tumors demonstrate low immune scores, and the immune score wassuperior in predicting disease outcome as compared with a host of importantprognostic clinical parameters, including TNM staging. Thus, although there arestill few issues that need to be addressed (Broussard and Disis 2011), it seemsimportant to consider immune scoring as a prognostic factor and to introduce thisparameter as a marker to classify cancers, as part of the routine diagnostic and

Fig. 4.2 Association of immune cell infiltrates with prognosis in cancer


prognostic assessment of tumors. At the same time, the inherent complexity ofquantitative immunohistochemistry, in conjunction with variable assay protocolsacross laboratories, the different immune cell types analyzed, different regionselection criteria, and variable ways to quantify immune infiltration underscore theurgent need to reach assay harmonization (Emens et al. 2012).

In an effort to promote the immunoscore in routine clinical settings worldwide,the Society for Immunotherapy of Cancer (SITC), the European Academy ofTumor Immunology, the Cancer and Inflammation Program, the National CancerInstitute, National Institutes of Health, USA and ‘‘La Fondazione Melanoma’’initiated on February 2012 in Naples a task force on Immunoscoring as a NewPossible Approach for the Classification of Cancer. An immune-classification oftumors has been proposed based on an immune score, performed by the quanti-fication of two lymphocyte populations (CD3/CD8, or CD3/CD45RO, or CD8/CD45RO), both in the core of the tumor and the invasive margin of the tumor, toestablish prognosis of clinical outcome in patients. Initially, the Immune scorescreening, which involves the participation of 21 international centers, will beperformed on colon cancer patients where high densities of T cells (CD3+), ofcytotoxic T cells (CD8+), and of memory T cells (CD45RO+) are clearly asso-ciated with a longer disease-free (after surgical resection of the primary tumor)and/or overall survival (Pages et al. 2009). However, the validation of Immunescore on other cancer types is under discussion.

A ‘‘Workshop on Immune Scoring’’ organized in Naples in December of 2012will lead to the preparation of a summary document providing recommendationsfor the harmonization and implementation of the Immune Score as a new com-ponent for the classification of cancer (Galon 2012).

4.6 The Immune Phenotype Associated with ImmuneResponsiveness in Partly Dependent Upon GeneticBackground of the Host and Somatic Alterationof the Tumor

All together the results derived from the studies mentioned above stronglyunderline the relative role played by the innate, adaptive a humoral immunesystems in the tumor milieu and their coordinated action in tumor rejection whichin our idea is limited not only by immunosuppressant induced by tumor growth butalso by a natural immune response of our system that try to prevent a perceivedautoimmune reaction.

There have been several demonstrations of a linkage between the induction ofautoimmunity with immune based cancer therapies and antitumor response (Disis2010; Wang et al. 2008). Autoimmunity and tumor regression have occurred afterthe use of a variety of immune based treatments. The development of vitiligo anduveitis has been reported after tumor regression was induced with the adoptive


transfer of T cells expanded ex vivo from tumor-infiltrating lymphocytes inpatients with metastatic melanoma (Dudley et al. 2002).

In a study performed on 200 patients with melanoma undergoing high doseIFN-a-2b adjuvant treatment, it was observed that 26 % developed autoantibodiesand clinical symptoms consist in autoimmunity. Moreover, autoimmunity in thisstudy was showed to be predictive of response free survival (RFS) and overallsurvival (OR) (P \ 0.001) (Gogas et al. 2006). If we assume that if autoimmunityis associated with cancer immune responsiveness, polymorphisms of genes relatedto autoimmunity itself might be associated with cancer regression (Wang et al.2012). A recent observation supporting this hypothesis came from a group ofpatients with melanoma treated with the adoptive transfer of ex vivo activatedtumor infiltrating lymphocytes (Uccellini et al. 2012).

Consistent with the concept that autoimmunity and cancer rejection mightrepresent different facets of the same phenomenon, we observed that polymor-phisms protecting against the susceptibility to develop systemic lupus erythema-tosus (SLE) such as the IRF5 rs10954213 GG genotype, were significantly moreprevalent among patients who did not respond to adoptive TIL therapy. It isinteresting to observe that the IRF5 genotype appeared to segregate two differentpatterns. When genes differentiating melanoma cell lines in vitro according togenotype were applied for class prediction, a segregation of responding and nonresponding cases was observed and it was only partially predictive of the IRF5genotype in vivo. The resulting segregation of cases according to genotype wasassociated with likelihood of responsiveness suggesting that germline variants canaffect directly the intrinsic biology of cancer cells besides affecting the behavior ofhost’s cells (Uccellini et al. 2012).

Thus, we can conclude that the immune phenotype associated with immuneresponsiveness in partly dependent genetic background of the host (Wang et al.2012).

Another demonstration, of the impact of genetic alteration of the host on tumorregression and response to cancer therapy was also showed by our group in theevaluation of polymorphisms of CXCR3 and CCR3,-ligand chemokines, (whichare considered critical in tumor microenvironment for the recruitment of T lym-phocytes and subsequent immune mediated rejection), as predictive biomarkers ofclinical response to adoptive therapy in melanoma patients (manuscript in prep-aration). A common single nucleotide polymorphism of CXCR3 (rs2280964) hasbeen associated with the variation in chemotactic activity. CCR5, polymorphismD32, (a deletion of 32 bases encoding a protein not expressed on cell surface), hasbeen correlated with poor prognosis in metastatic melanoma patients. In our studyit was postulated that polymorphisms of CXCR3 and CCR5 genes may influencethe migration of TILs on tumor side and eventually tumor regression. The resultsshowed that no significant correlation was detected between CXCR3 polymor-phisms and response. Surprisingly CCR5-D32 carriers had a better overall survivalcompared to wild type patients. These results allowed us to generate newhypotheses on the role of this molecule in the modulation of stimulatoryconditions.


Together with genetic alteration of the host, we need to consider that the immuneresponsiveness and in general tumor regression is also related to acquired alterationsof cancer cells genetics. This explains the phenomenon of mixed responses. In fact, aproportion of cancer patients experience a mix response to therapy characterized bysimultaneous regression of the some metastases, while other progress. These casesare very rare and unique because they only consider the tumor’s aspects of immuneresponsiveness excluding the genetic background of the patients.

We recently performed a comparative gene expression analysis of 15 metas-tases (10 regressing and 5 progressing) obtained from 2 melanoma patientsexperiencing a mixed response following immunotherapy. The obtained resultsindicated that the regression of melanoma metastases is associated with acuteinflammation mediated by up regulation of genes involved in antigen presentation.This unique study provided a clear demonstration that within the same geneticbackground tumor can behave differently supporting the hypothesis that tumorrejection is in part dependent upon tumor biology.

Based on these last mentioned observations, it looks like one side the geneticbackground of the host’s have an important impact on immune responsiveness, onthe other side it looks like tumor can behave differently within the same geneticbackground.

This apparent paradox can be explained by a multifactor model of cancerimmune responsiveness.

It should be emphasized that host and cancer genetics are mostly overlappingsince cancer cells carry the majority of the host genetics. Thus, inherited geneticfactors may affect the biology of the cancer besides that of normal cells. Then, itcould be hypothesized that some patients carry a genetic background that makethem resistant to immunotherapy by affecting either the biology of immuneresponse, the biology of cancer cells, or both.

On the other hand, an ‘‘immune –responsive genotype’’ may still be limited bythe genetics of the tumors: although the patients may be predisposed to cancerrejection the tumor lacks additional properties necessary for its recognition by theimmune system. In the model we propose a favorable genetic background of thehost is necessary but not sufficient for tumor rejection. A good example is providedby the analysis of patients with IRF5 polymorphisms where the immune resistantphenotypes appears to exclusively preclude cancer rejection during adoptivetherapy with tumor infiltrating lymphocytes; however the ‘‘immune responsivephenotype’’ can be segregated into two categories. One enriched in patientsresponding to therapy and the other of non-responding.

4.7 Conclusions

It is becoming clear that tumors can be segregated into at least two categoriesindependently of their histology. Of them, one is characterized by a molecularsignature consistent with a Th1 type of immune activation which is also associated


with, lymphocytic infiltrate, better prognosis and enhanced likelihood to respondto therapy. This dichotomy may be determined by a continuum interaction of amultitude of factors including the host’s genetic background, somatic mutationsand external factors such as intensity and effectiveness of treatment or generalcondition of the patient. This multistep inference may also explain why it isgenerally easier to predict accurately lack of responsiveness than responsiveness.

Although it is still unclear whether future progresses will cause drastic changesin understanding the biology of cancer or will just add new details in order tobetter elaborate regulatory circuits that have been already mapped out, lookingahead we prospect significant advances during the coming decade in our under-standing of biology of cancer.

Acknowledgments Researcher in the Infectious Disease and Immunogenetics Section (FMMarincola’s laboratory) are supported by NIH Intramural Research Program. ML Ascierto thanksDr. Ermelinda Ascierto and Dr. Domenico Ascierto for insightful suggestions.

References

Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE et al (2009)Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and arefunctionally impaired. Blood 114:44–1537

Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A et al (2000) Distinct types ofdiffuse large B-cell lymphoma identified by gene expression profiling. Nature 403:11–503

Allavena P, Sica A, Solinas G, Porta C, Mantovani A (2008) The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages. Crit Rev OncolHematol 66:1–9

Andreu P, Johansson M, Affara NI, Pucci F, Tan T, Junankar S et al (2010) FcRgamma activationregulates inflammation-associated squamous carcinogenesis. Cancer Cell 17:121–134

Ascierto ML, De Giorgi V, Liu Q, Bedognetti D, Spivey TL, Murtas D et al (2011) Animmunologic portrait of cancer. J Transl Med 9:146

Ascierto ML, Kmieciak M, Idowu MO, Manjili R, Zhao YD, Grimes M et al (2012) A signatureof immune function genes associated with recurrence-free survival in breast cancer patients.Breast Cancer Res Tr 131:871–880

Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay Nel H et al (2006) Prognosticvalue of tumor-infiltrating CD4 ? T-cell subpopulations in head and neck cancers. ClinCancer Res 12:72–465

Bedognetti D, Wang E, Sertoli MR, Marincola FM (2010) Gene-expression profiling in vaccinetherapy and immunotherapy for cancer. Expert Rev Vaccines 9:65–555

Broussard EK, Disis ML (2011) TNM staging in colorectal cancer: T is for T cell and M is formemory. J Clin Oncol 29:601–603

Carreras J, Lopez-Guillermo A, Fox BC, Colomo L, Martinez A, Roncador G et al (2006) Highnumbers of tumor-infiltrating FOXP3-positive regulatory T cells are associated with improvedoverall survival in follicular lymphoma. Blood 108:64–2957

Chaput N, Conforti R, Viaud S, Spatz A, Zitvogel L (2008) The Janus face of dendritic cells incancer. Oncogene 27:5920–5931

Chen X, Wan J, Liu J, Xie W, Diao X, Xu J et al (2010) Increased IL-17-producing cells correlatewith poor survival and lymphangiogenesis in NSCLC patients. Lung Cancer 69:348–354


Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, Cascinelli N (1996) Prognosticvalue of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneousmelanoma. Cancer 77:10–1303

Coca S, Perez-Piqueras J, Martinez D, Colmenarejo A, Saez MA, Vallejo C et al (1997) Theprognostic significance of intratumoral natural killer cells in patients with colorectalcarcinoma. Cancer 79:2320–2328

Cochran AJ (1969) Histology and prognosis in malignant melanoma. J Pathol 97:68–459Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P et al (2004) Specific recruitment of

regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reducedsurvival. Nat Med 10:9–942

De Monte L, Reni M, Tassi E, Clavenna D, Papa I, Recalde H et al (2011) Intratumor T helpertype 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietinproduction and reduced survival in pancreatic cancer. J Exp Med 208:469–478

Delahaye NF, Rusakiewicz S, Martins I, Menard C, Roux S, Lyonnet L et al (2011) Alternativelyspliced NKp30 isoforms affect the prognosis of gastrointestinal stromal tumors. Nat Med17:700–707

Disis ML (2010) Immune regulation of cancer. J Clin Oncol 28:8–4531Donskov F, von der Maase H (2006) Impact of immune parameters on long-term survival in

metastatic renal cell carcinoma. J Clin Oncol 24:1997–2005Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ et al (2002)

Cancer regression and autoimmunity in patients after clonal repopulation with antitumorlymphocytes. Science 298:850–854

Eckl J, Buchner A, Prinz PU, Riesenberg R, Siegert SI, Kammerer R et al (2012) Transcriptsignature predicts tissue NK cell content and defines renal cell carcinoma subgroupsindependent of TNM staging. J Mol Med 90:55–66 (Berl)

Emens LA, Silverstein SC, Khleif S, Marincola FM, Galon J (2012) Toward integrative cancerimmunotherapy: targeting the tumor microenvironment. J Transl Med 10:70

Frey DM, Droeser RA, Viehl CT, Zlobec I, Lugli A, Zingg U et al (2010) High frequency oftumor-infiltrating FOXP3(+) regulatory T cells predicts improved survival in mismatchrepair-proficient colorectal cancer patients. Int J Cancer 126:2635–2643

Fridman WH, Pages F, Sautes-Fridman C, Galon J (2012) The immune contexture in humantumours: impact on clinical outcome. Nat Rev Cancer 12:298–306

Gajewski TF (2011) Molecular profiling of melanoma and the evolution of patient-specifictherapy. Semin Oncol 38:42–236

Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C et al (2006)Type, density, and location of immune cells within human colorectal tumors predict clinicaloutcome. Science 313:4–1960

Galon J, Pages F, Marincola FM, Thurin M, Trinchieri G, Fox BA et al (2012) The immune scoreas a new possible approach for the classification of cancer. J Transl Med 3:10–11

Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS et al (2007) Intratumoral balance of regulatoryand cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection.J Clin Oncol 25:93–2586

Gogas H, Ioannovich J, Dafni U, Stavropoulou-Giokas C, Frangia K, Tsoutsos D et al (2006)Prognostic significance of autoimmunity during treatment of melanoma with interferon.N Engl J Med 354:709–718

Guidoboni M, Gafa R, Viel A, Doglioni C, Russo A, Santini A et al (2001) Microsatelliteinstability and high content of activated cytotoxic lymphocytes identify colon cancer patientswith a favorable prognosis. Am J Pathol 159:297–304

Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:74–646Heimberger AB, Abou-Ghazal M, Reina-Ortiz C, Yang DS, Sun W, Qiao W et al (2008)

Incidence and prognostic impact of FoxP3 ? regulatory T cells in human gliomas. ClinCancer Res 14:72–5166


Herberman RB, Nunn ME, Holden HT, Lavrin DH (1975) Natural cytotoxic reactivity of mouselymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells.Int J Cancer 16:230–239

Hillen F, Baeten CI, van de Winkel A, Creytens D, van der Schaft DW, Winnepenninckx V et al(2008) Leukocyte infiltration and tumor cell plasticity are parameters of aggressiveness inprimary cutaneous melanoma. Cancer Immunol Immunother 57:97–106

Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G et al (2009) Loss of FOXP3expression in natural human CD4 ? CD25 ? regulatory T cells upon repetitive in vitrostimulation. Eur J Immunol 39:1088–1097

Ishigami S, Natsugoe S, Tokuda K, Nakajo A, Che X, Iwashige H et al (2000) Prognostic value ofintratumoral natural killer cells in gastric carcinoma. Cancer 88:577–583

Ito T, Saga S, Nagayoshi S, Imai M, Aoyama A, Yokoi T et al (1986) Class distribution ofimmunoglobulin-containing plasma cells in the stroma of medullary carcinoma of breast.Breast Cancer Res Treat 7:97–103

Jensen TO, Schmidt H, Moller HJ, Donskov F, Hoyer M, Sjoegren P et al (2012) Intratumoralneutrophils and plasmacytoid dendritic cells indicate poor prognosis and are associated withpSTAT3 expression in AJCC stage I/II melanoma. Cancer 118(9):2476–2485

Jochems C, Schlom J (2011) Tumor-infiltrating immune cells and prognosis: the potential linkbetween conventional cancer therapy and immunity. Exp Biol Med 236:567–579 (Maywood)

Kelley T, Beck R, Absi A, Jin T, Pohlman B, Hsi E (2007) Biologic predictors in follicularlymphoma: importance of markers of immune response. Leuk Lymphoma 48:2403–2411

Kiessling R, Klein E, Pross H, Wigzell H (1975) ‘‘Natural’’ killer cells in the mouse. II. Cytotoxiccells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell. EurJ Immunol 5:117–121

Kryczek I, Wei S, Zou L, Zhu G, Mottram P, Xu H et al (2006) Cutting edge: induction of B7–H4on APCs through IL-10: novel suppressive mode for regulatory T cells. J Immunol 177:40–44

Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S et al (2009) Phenotype, distribution,generation, and functional and clinical relevance of Th17 cells in the human tumorenvironments. Blood 114:1141–1149

Lossos IS, Alizadeh AA, Eisen MB, Chan WC, Brown PO, Botstein D et al (2000) Ongoingimmunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas. Proc Natl Acad Sci U S A 97:13–10209

Mahmoud SM, Paish EC, Powe DG, Macmillan RD, Lee AH, Ellis IO et al (2011) An evaluationof the clinical significance of FOXP3 ? infiltrating cells in human breast cancer. BreastCancer Res Treat 127:99–108

Mamessier E, Sylvain A, Thibult ML, Houvenaeghel G, Jacquemier J, Castellano R et al (2011)Human breast cancer cells enhance self tolerance by promoting evasion from NK cellantitumor immunity. J Clin Invest 121:3609–3622

Mantovani A, Romero P, Palucka AK, Marincola FM (2008) Tumour immunity: effectorresponse to tumour and role of the microenvironment. Lancet 371:83–771

Marincola FM (2005) A balanced review of the status T cell-based therapy against cancer.J Transl Med 3:16

Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM (1991) Experimental therapy ofhuman glioma by means of a genetically engineered virus mutant. Science 252:854–856

Masferrer JL, Leahy KM, Koki AT, Zweifel BS, Settle SL, Woerner BM et al (2000)Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res60:11–1306

Milne K, Kobel M, Kalloger SE, Barnes RO, Gao D, Gilks CB et al (2009) Systematic analysis ofimmune infiltrates in high-grade serous ovarian cancer reveals CD20, FoxP3 and TIA-1 aspositive prognostic factors. PLoS ONE 4:e6412

Mlecnik B, Tosolini M, Kirilovsky A, Berger A, Bindea G, Meatchi T et al (2011)Histopathologic-based prognostic factors of colorectal cancers are associated with the stateof the local immune reaction. J Clin Oncol 29:610–618


Mocellin S, Panelli MC, Wang E, Nagorsen D, Marincola FM (2003) The dual role of IL-10.Trends Immunol 24:36–43

Mocellin S, Panelli M, Wang E, Rossi CR, Pilati P, Nitti D et al (2004) IL-10 stimulatory effectson human NK cells explored by gene profile analysis. Genes Immun 5:621–630

Moretta L, Moretta A (2004) Unravelling natural killer cell function: triggering and inhibitoryhuman NK receptors. EMBO J 23:255–259

Moretta L, Ciccone E, Mingari MC, Biassoni R, Moretta A (1994) Human natural killer cells:origin, clonality, specificity, and receptors. Adv Immunol 55:341–380

Nielsen JS, Sahota RA, Milne K, Kost SE, Nesslinger NJ, Watson PH et al (2012)CD20 ? tumor-infiltrating lymphocytes have an atypical CD27- memory phenotype andtogether with CD8 ? T cells promote favorable prognosis in ovarian cancer. Clin Cancer Res18:3281–3292

Oldham RK, Herberman RB (1973) Evaluation of cell-mediated cytotoxic reactivity againsttumor associated antigens with 125I-iododeoxyuridine labeled target cells. J Immunol111:862–871

Ostrand-Rosenberg S, Sinha P (2009) Myeloid-derived suppressor cells: linking inflammationand cancer. J Immunol 182:4499–4506

Pages F, Berger A, Camus M, Sanchez-Cabo F, Costes A, Molidor R et al (2005) Effectormemory T cells, early metastasis, and survival in colorectal cancer. N Engl J Med353:66–2654

Pages F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G et al (2009) In situcytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer.J Clin Oncol 27:5944–5951

Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA et al (2000) Molecularportraits of human breast tumours. Nature 406:52–747

Qiu X, Zhu X, Zhang L, Mao Y, Zhang J, Hao P et al (2003) Human epithelial cancers secreteimmunoglobulin g with unidentified specificity to promote growth and survival of tumor cells.Cancer Res 63:6488–6495

Rossi CR, Testori A, Mocellin S, Campana L, Lejeune F (2004) Melanoma—what is new insentinel node biopsy and locoregional treatments in 2003? Report of a workshop at the thirdresearch meeting on Melanoma, Milan, Italy, May 2003. Melanoma Res 14:329–332

Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D et al (2009) Tumor-infiltratingFOXP3 ? T regulatory cells show strong prognostic significance in colorectal cancer. J ClinOncol 27:186–192

Schmidt M, Hellwig B, Hammad S, Othman A, Lohr M, Chen Z et al (2012) A comprehensiveanalysis of human gene expression profiles identifies stromal immunoglobulin kappa C as acompatible prognostic marker in human solid tumors. Clin Cancer Res 18:2695–2703

Schreck S, Friebel D, Buettner M, Distel L, Grabenbauer G, Young LS et al (2009) Prognosticimpact of tumour-infiltrating Th2 and regulatory T cells in classical Hodgkin lymphoma.Hematol Oncol 27:31–39

Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, Treisman J et al (2011)Gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med364:2119–2127

Shanker A, Marincola FM (2011) Cooperativity of adaptive and innate immunity: implicationsfor cancer therapy. Cancer Immunol Immunother 60:1061–1074

Shanker A, Verdeil G, Buferne M, Inderberg-Suso EM, Puthier D, Joly F et al (2007) CD8 T cellhelp for innate antitumor immunity. J Immunol 179:6651–6662

Shanker A, Buferne M, Schmitt-Verhulst AM (2010) Cooperative action of CD8 T lymphocytesand natural killer cells controls tumour growth under conditions of restricted T-cell receptordiversity. Immunology 129:41–54

Sinha P, Clements VK, Fulton AM, Ostrand-Rosenberg S (2007) Prostaglandin E2 promotestumor progression by inducing myeloid-derived suppressor cells. Cancer Res 67:13–4507


Tassi E, Gavazzi F, Albarello L, Senyukov V, Longhi R, Dellabona P et al (2008)Carcinoembryonic antigen-specific but not antiviral CD4 ? T cell immunity is impaired inpancreatic carcinoma patients. J Immunol 181:6595–6603

Tosolini M, Kirilovsky A, Mlecnik B, Fredriksen T, Mauger S, Bindea G et al (2011) Clinicalimpact of different classes of infiltrating T cytotoxic and helper cells (Th1, th2, treg, th17) inpatients with colorectal cancer. Cancer Res 71:1263–1271

Uccellini L, De Giorgi V, Zhao Y, Tumaini B, Erdenebileg N, Dudley ME et al (2012) IRF5 genepolymorphisms in melanoma. J Transl Med 10:170

Villegas FR, Coca S, Villarrubia VG, Jimenez R, Chillon MJ, Jareno J et al (2002) Prognosticsignificance of tumor infiltrating natural killer cells subset CD57 in patients with squamouscell lung cancer. Lung Cancer 35:23–28

Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL et al (2011) Innate oradaptive immunity? The example of natural killer cells. Science 331:44–49

Vivier E, Ugolini S, Blaise D, Chabannon C, Brossay L (2012) Targeting natural killer cells andnatural killer T cells in cancer. Nat Rev Immunol 12:239–252

Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH et al (2003)Induction of FoxP3 and acquisition of T regulatory activity by stimulated humanCD4 ? CD25- T cells. J Clin Invest 112:1437–1443

Wang E, Worschech A, Marincola FM (2008) The immunologic constant of rejection. TrendsImmunol 29:62–256

Wang XY, Han M, Xiao ZP, Chen L, Kong LH, Fu K et al (2009) The comparison ofCD4(+);CD25(high); Foxp3(+); regulatory T cells between neonatal cord blood and adultperipheral blood. Bao X, Yu Fen Zi Mian Yi Xue Za Zhi. 25:1152–1154

Wang E, Uccellini L, Marincola FM (2012) A genetic inference on cancer immuneresponsiveness. Oncoimmunology 1:520–525

Wilke CM, Kryczek I, Wei S, Zhao E, Wu K, Wang G et al (2011) Th17 cells in cancer: help orhindrance? Carcinogenesis 32:643–649

Wong SC, Puaux AL, Chittezhath M, Shalova I, Kajiji TS, Wang X et al (2010) Macrophagepolarization to a unique phenotype driven by B cells. Eur J Immunol 40:2296–2307

Worschech A, Chen N, Yu YA, Zhang Q, Pos Z, Weibel S et al (2009) Systemic treatment ofxenografts with vaccinia virus GLV-1h68 reveals the immunologic facet of oncolytic therapy.BMC Genomics 10:301

Yoon NK, Maresh EL, Shen D, Elshimali Y, Apple S, Horvath S et al (2010) Higher levels ofGATA3 predict better survival in women with breast cancer. Hum Pathol 41:1794–1801

Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G et al (2003)Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med348:13–203

Zhang JP, Yan J, Xu J, Pang XH, Chen MS, Li L et al (2009) Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients. J Hepatol50:980–989

Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ et al (2008) High expression ofmacrophage colony-stimulating factor in peritumoral liver tissue is associated with poorsurvival after curative resection of hepatocellular carcinoma. J Clin Oncol 26:2707–2716

Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W et al (2007) FOXP3 is an X-linked breastcancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell129:1275–1286


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