Integrative Analyses of Colorectal Cancer Show Immunoscore Is a Stronger Predictor of Patient Survival Than Microsatellite Instability.

Microsatellite instability in colorectal cancer predicts favorable outcomes. However, the mechanistic relationship between microsatellite instability, tumor-infiltrating immune cells, Immunoscore, and their impact on patient survival remains to be elucidated. We found significant differences in mutational patterns, chromosomal instability, and gene expression that correlated with patient microsatellite instability status. A prominent immune gene expression was observed in microsatellite-instable (MSI) tumors, as well as in a subgroup of microsatellite-stable (MSS) tumors. MSI tumors had increased frameshift mutations, showed genetic evidence of immunoediting, had higher densities of Th1, effector-memory T cells, in situ proliferating T cells, and inhibitory PD1-PDL1 cells, had high Immunoscores, and were infiltrated with mutation-specific cytotoxic T cells. Multivariate analysis revealed that Immunoscore was superior to microsatellite instability in predicting patients' disease-specific recurrence and survival. These findings indicate that assessment of the immune status via Immunoscore provides a potent indicator of tumor recurrence beyond microsatellite-instability staging that could be an important guide for immunotherapy strategies.

Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.

The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence.

Colibactin-positive Escherichia coli induce a procarcinogenic immune environment leading to immunotherapy resistance in colorectal cancer.

Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APCMin/+ mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APCmin/+ mice were chronically infected with a CoPEC strain (11G5). Immune cells (neutrophils and T-cell populations) were then quantified by immunofluorescent staining of the colon. The quantification of lymphoid populations was also performed in the mesenteric lymph nodes (MLNs). Here, we show that the colonization of CRC patients by CoPEC is associated with a decrease of tumor-infiltrating T lymphocytes (CD3+ T-cells). Similarly, we demonstrated, in mice, that CoPEC chronic infection decreases CD3+ and CD8+ T-cells and increases colonic inflammation. In addition, we noticed a significant decrease in antitumor T-cells in the MLNs of CoPEC-infected mice compared to that of controls. Moreover, we show that CoPEC infection decreases the antimouse PD-1 immunotherapy efficacy in MC38 tumor model. Our findings suggest that CoPEC could promote a procarcinogenic immune environment through impairment of antitumor T-cell response, leading to tumoral resistance to immunotherapy. CoPEC could thus be a new biomarker predicting the anti-PD-1 response in CRC.

International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study.

BACKGROUND: The estimation of risk of recurrence for patients with colon carcinoma must be improved. A robust immune score quantification is needed to introduce immune parameters into cancer classification. The aim of the study was to assess the prognostic value of total tumour-infiltrating T-cell counts and cytotoxic tumour-infiltrating T-cells counts with the consensus Immunoscore assay in patients with stage I-III colon cancer. METHODS: An international consortium of 14 centres in 13 countries, led by the Society for Immunotherapy of Cancer, assessed the Immunoscore assay in patients with TNM stage I-III colon cancer. Patients were randomly assigned to a training set, an internal validation set, or an external validation set. Paraffin sections of the colon tumour and invasive margin from each patient were processed by immunohistochemistry, and the densities of CD3+ and cytotoxic CD8+ T cells in the tumour and in the invasive margin were quantified by digital pathology. An Immunoscore for each patient was derived from the mean of four density percentiles. The primary endpoint was to evaluate the prognostic value of the Immunoscore for time to recurrence, defined as time from surgery to disease recurrence. Stratified multivariable Cox models were used to assess the associations between Immunoscore and outcomes, adjusting for potential confounders. Harrell's C-statistics was used to assess model performance. FINDINGS: Tissue samples from 3539 patients were processed, and samples from 2681 patients were included in the analyses after quality controls (700 patients in the training set, 636 patients in the internal validation set, and 1345 patients in the external validation set). The Immunoscore assay showed a high level of reproducibility between observers and centres (r=0·97 for colon tumour; r=0·97 for invasive margin; p<0·0001). In the training set, patients with a high Immunoscore had the lowest risk of recurrence at 5 years (14 [8%] patients with a high Immunoscore vs 65 (19%) patients with an intermediate Immunoscore vs 51 (32%) patients with a low Immunoscore; hazard ratio [HR] for high vs low Immunoscore 0·20, 95% CI 0·10-0·38; p<0·0001). The findings were confirmed in the two validation sets (n=1981). In the stratified Cox multivariable analysis, the Immunoscore association with time to recurrence was independent of patient age, sex, T stage, N stage, microsatellite instability, and existing prognostic factors (p<0·0001). Of 1434 patients with stage II cancer, the difference in risk of recurrence at 5 years was significant (HR for high vs low Immunoscore 0·33, 95% CI 0·21-0·52; p<0·0001), including in Cox multivariable analysis (p<0·0001). Immunoscore had the highest relative contribution to the risk of all clinical parameters, including the American Joint Committee on Cancer and Union for International Cancer Control TNM classification system. INTERPRETATION: The Immunoscore provides a reliable estimate of the risk of recurrence in patients with colon cancer. These results support the implementation of the consensus Immunoscore as a new component of a TNM-Immune classification of cancer. FUNDING: French National Institute of Health and Medical Research, the LabEx Immuno-oncology, the Transcan ERAnet Immunoscore European project, Association pour la Recherche contre le Cancer, CARPEM, AP-HP, Institut National du Cancer, Italian Association for Cancer Research, national grants and the Society for Immunotherapy of Cancer.

The Immunoscore in the Clinical Practice of Patients with Colon and Rectal Cancers.

In the fine balance between tumor invasion and our defensive systems, the role played by the adaptive immune response at the tumor site is critical. Beyond the fact that all the immune components of the innate and adaptive response can be observed to varying degrees in the tumor microenvironment, it appears that a high density of T cytotoxic and memory lymphocytes, in a context of Th1 immune orientation in the tumor and its invasion front, provides a prognostic marker of paramount importance for colorectal cancer and more generally all solid tumors. The understanding of the role of immunity in cancer, tailored during one century of intensive research, has led to a complete paradigm shift.based on a sharp dissection In order to show the major impact of this conceptual revolution, we herein retrace through the example of colorectal cancer, how an effective immune test, namely the "Immunoscore", has been developed. We also provide up to date data demonstrating the capacity of the Immunoscore to prognosticate with a better accuracy than the TME classification clinical outcomes and to guide therapeutic strategies.

Immunotherapy: a new standard of care in thoracic malignancies? A summary of the European Respiratory Society research seminar of the Thoracic Oncology Assembly.

In May 2017, the second European Respiratory Society research seminar of the Thoracic Oncology Assembly entitled "Immunotherapy, a new standard of care in thoracic malignancies?" was held in Paris, France. This seminar provided an opportunity to review the basis of antitumour immunity and to explain how immune checkpoint inhibitors (ICIs) work. The main therapeutic trials that have resulted in marketing authorisations for use of ICIs in lung cancer were reported. A particular focus was on the toxicity of these new molecules in relation to their immune-related adverse events. The need for biological selection, currently based on immunohistochemistry testing to identify the tumour expression of programmed death ligand (PD-L)1, was stressed, as well as the need to harmonise PD-L1 testing and techniques. Finally, sessions were dedicated to the combination of ICIs and radiotherapy and the place of ICIs in nonsmall cell lung cancer with oncogenic addictions. Finally, an important presentation was dedicated to the future of antitumour vaccination and of all ongoing trials in thoracic oncology.

World-Wide Immunoscore Task Force: meeting report from the "Melanoma Bridge", Napoli, November 30th-December 3rd, 2016.

The predictive accuracy of the traditional staging system is based on disease progression as a tumour cell-autonomous process, but it fails to incorporate the effects of the host immune response. A precise analysis of the immune component of the tumour microenvironment by computer-based analysis may be essential to managing patients better, opening the road to an expertise in this new emerging field. The Immunoscore as a new possible approach in the classification of cancer, designated TNM-Immune, studied in colon cancer patients with predictive and prognostic value. This new scoring system is derived from the immune contexture, and is based on the numeration of lymphocyte populations, both in the core of the tumour and in the invasive margin of tumours. The Immunoscore demonstrated to be quantitative, reproducible and robust. The usefulness of Immunoscore in advanced melanoma cancer patients has been as well demonstrated; the correlation of marker expression profile with clinical outcome is ongoing. More recently, the Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. A multivariable Cumulative "Suppression Index" scoring system has been also studied in Oral Squamous Cell Carcinoma patients: it evaluates both the tumor and stromal microcompartments at the invasive margin and summarizes them into the score, providing an accurate stratification, independent of stage, tumour classification. The introduction of Immunoscore requires a redefinition of the Laboratory system according to the LEAN Management process, which has been already implemented in referral research labs. The definition and test of hundreds of biomarkers, in the tumour contexture represents a definitive scientific progression. However, there is still a need of substantial body of work to reach the end of the tunnel to assure a personalize treatment.

Rational bases for the use of the Immunoscore in routine clinical settings as a prognostic and predictive biomarker in cancer patients.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) tumor, nodes, metastasis (TNM) classification system based on tumor features is used for prognosis estimation and treatment recommendations in most cancers. However, the clinical outcome can vary significantly among patients within the same tumor stage and TNM classification does not predict response to therapy. Therefore, many efforts have been focused on the identification of new markers. Multiple tumor cell-based approaches have been proposed but very few have been translated into the clinic. The recent demonstration of the essential role of the immune system in tumor progression has allowed great advances in the understanding of this complex disease and in the design of novel therapies. The analysis of the immune infiltrate by imaging techniques in large patient cohorts highlighted the prognostic impact of the in situ immune cell infiltrate in tumors. Moreover, the characterization of the immune infiltrates (e.g. type, density, distribution within the tumor, phenotype, activation status) in patients treated with checkpoint-blockade strategies could provide information to predict the disease outcome. In colorectal cancer, we have developed a prognostic score ('Immunoscore') that takes into account the distribution of the density of both CD3(+) lymphocytes and CD8(+) cytotoxic T cells in the tumor core and the invasive margin that could outperform TNM staging. Currently, an international retrospective study is under way to validate the Immunoscore prognostic performance in patients with colon cancer. The use of Immunoscore in clinical practice could improve the patients' prognostic assessment and therapeutic management.

[Focus on the Immunoscore and its potential clinical implications]. / Mise au point sur l'Immunoscore et ses potentielles implications cliniques.

The role of the immune response at the tumor site is now recognized as crucial in the clinical course of patients with cancer. The importance of the immune cell type, their functional orientation, their density and location within the tumor's regions (tumor/invasion margin) has recently been shown and were grouped together under the term "immune contexture". A strong infiltration by cytotoxic and memory T cells in a Th1-polarized tumor microenvironment appears to have a major prognosis impact. A test called Immunoscore taking into account these various parameters has been suggested to measure in a simple, reproducible and robust manner the intra- and peritumoral immune response. The prognostic value of Immunoscore has recently been validated in colon cancers by a large international retrospective study under the aegis of the Society for Immunotherapy of Cancer (SITC). The Immunoscore could have several potential clinical applications such as prognostic as well as theranostic.

Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

[Intratumoral immune microenvironment and survival: the immunoscore]. / Microenvironnement immunitaire et cancer - intérêt de l'immunoscore pour prédire l'évolution clinique.

The natural history of cancer involves interactions between the tumor and the host immune system. In humans, clinical and experimental data support the existence of a natural immune response against cancer. We provided evidence that the type, the density and the location of immune cells within the tumor strongly influence the prognosis, independently of the TNM classification. We established a methodology named "immunoscore" to assess in clinical practice the immune infiltrate. An international consortium of expert laboratories is currently testing the immunoscore in routine clinical settings for cancer classification. The availability of the mmunoscore could significantly improve the prognostic assessment of patients and better guide the therapeutic decision. This could result in the implementation of the immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-immune).

Prognostic and Predictive Value of Immunoscore in Stage III Colorectal Cancer: Pooled Analysis of Cases From the SCOT and IDEA-HORG Studies.

PURPOSE: Immunoscore (IS) is prognostic in stage III colorectal cancer (CRC) and may predict benefit of duration (6 v 3 months) of adjuvant infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy. We sought to determine IS prognostic and predictive value in stage-III CRC treated with adjuvant FOLFOX or oral capecitabine and infusional oxaliplatin (CAPOX) in the SCOT and IDEA-HORG trials. METHODS: Three thousand sixty-one cases had tumor samples, of which 2,643 (1,792 CAPOX) were eligible for IS testing. Predefined cutoffs (IS-Low and IS-High) were used to classify cases into two groups for analysis of disease-free survival (3-year DFS) and multivariable-adjusted hazard ratios (mvHRs) by Cox regression. RESULTS: IS was determined in 2,608 (99.5%) eligible cases, with 877 (33.7%) samples classified as IS-Low. IS-Low tumors were more commonly high-risk (T4 and/or N2; 52.9% IS-Low v 42.2% IS-High; P < .001) and in younger patients (P = .024). Patients with IS-Low tumors had significantly shorter DFS in the CAPOX, FOLFOX, and combined cohorts (mvHR, 1.52 [95% CI, 1.28 to 1.82]; mvHR, 1.58 [95% CI, 1.22 to 2.04]; and mvHR, 1.55 [95% CI, 1.34 to 1.79], respectively; P < .001 all comparisons), regardless of sex, BMI, clinical risk group, tumor location, treatment duration, or chemotherapy regimen. IS prognostic value was greater in younger (≤65 years) than older (>65 years) patients in the CAPOX cohort (mvHR, 1.92 [95% CI, 1.50 to 2.46] v 1.28 [95% CI, 1.01 to 1.63], PINTERACTION = .026), and in DNA mismatch repair proficient than deficient mismatch repair disease (mvHR, 1.68 [95% CI, 1.41 to 2.00] v 0.67 [95% CI, 0.30 to 1.49], PINTERACTION = .03), although these exploratory analyses were uncorrected for multiple testing. Adding IS to a model containing all clinical variables significantly improved prediction of DFS (likelihood ratio test, P < .001) regardless of MMR status. CONCLUSION: IS is prognostic in stage III CRC treated with FOLFOX or CAPOX, including within clinically relevant tumor subgroups. Possible variation in IS prognostic value by age and MMR status, and prediction of benefit from extended adjuvant therapy merit validation.

International Validation of the Immunoscore Biopsy in Patients With Rectal Cancer Managed by a Watch-and-Wait Strategy.

PURPOSE: No biomarker capable of improving selection and monitoring of patients with rectal cancer managed by watch-and-wait (W&W) strategy is currently available. Prognostic performance of the Immunoscore biopsy (ISB) was recently suggested in a preliminary study. METHODS: This international validation study included 249 patients with clinical complete response (cCR) managed by W&W strategy. Intratumoral CD3+ and CD8+ T cells were quantified on pretreatment rectal biopsies by digital pathology and converted to ISB. The primary end point was time to recurrence (TTR; the time from the end of neoadjuvant treatment to the date of local regrowth or distant metastasis). Associations between ISB and outcomes were analyzed by stratified Cox regression adjusted for confounders. Immune status of tumor-draining lymph nodes (n = 161) of 17 additional patients treated by neoadjuvant chemoradiotherapy and surgery was investigated by 3'RNA-Seq and immunofluorescence. RESULTS: Recurrence-free rates at 5 years were 91.3% (82.4%-100.0%), 62.5% (53.2%-73.3%), and 53.1% (42.4%-66.5%) with ISB High, ISB Intermediate, and ISB Low, respectively (hazard ratio [HR; Low v High], 6.51; 95% CI, 1.99 to 21.28; log-rank P = .0004). ISB was also significantly associated with disease-free survival (log-rank P = .0002), and predicted both local regrowth and distant metastasis. In multivariate analysis, ISB was independent of patient age, sex, tumor location, cT stage (T, primary tumor; c, clinical), cN stage (N, regional lymph node; c, clinical), and was the strongest predictor for TTR (HR [ISB High v Low], 6.93; 95% CI, 2.08 to 23.15; P = .0017). The addition of ISB to a clinical-based model significantly improved the prediction of recurrence. Finally, B-cell proliferation and memory in draining lymph nodes was evidenced in the draining lymph nodes of patients with cCR. CONCLUSION: The ISB is validated as a biomarker to predict both local regrowth and distant metastasis, with a gradual scaling of the risk of pejorative outcome.

Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND: The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS: An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS: Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS: The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes.

Tumor immunosurveillance in human cancers.

Until now, the anatomic extent of tumor (TNM classification) has been by far the most important factor to predict the prognosis of colorectal cancer patients. However, in recent years, data collected from large cohorts of human cancers demonstrated that the immune contexture of the primary tumors is an essential prognostic factor for patients' disease-free and overall survival. Tumoral and immunological markers predicted by systems biology methods are involved in the shaping of an efficient immune reaction and can serve as targets for novel therapeutic approaches. Global analysis of tumor microenvironment showed that the nature, the functional orientation, the density, and the location of adaptive immune cells within distinct tumor regions influence the risk of relapse events. The density and the immune cell location within the tumor have a prognostic value that is superior to the TNM classification, and tumor invasion is statistically dependent on the host-immune reaction. Thus, the strength of the immune reaction could advance our understanding of cancer evolution and have important consequences in clinical practice.

The immune score as a new possible approach for the classification of cancer.

The outcome prediction in cancer is usually achieved by evaluating tissue samples obtained during surgical removal of the primary tumor focusing on their histopathological characteristics. Tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N), and evidence for metastases (M). However, this classification provides limited prognostic information in estimating the outcome in cancer and does not predict response to therapy. It is recognized that cancer outcomes can vary significantly among patients within the same stage. Recently, many reports suggest that cancer development is controlled by the host's immune system underlying the importance of including immunological biomarkers for the prediction of prognosis and response to therapy. Data collected from large cohorts of human cancers demonstrated that the immune-classification has a prognostic value that may be superior to the AJCC/UICC TNM-classification. Thus, it is imperative to begin incorporating immune scoring as a prognostic factor and to introduce this parameter as a marker to classify cancers, as part of the routine diagnostic and prognostic assessment of tumors. At the same time, the inherent complexity of quantitative immunohistochemistry, in conjunction with variable assay protocols across laboratories, the different immune cell types analyzed, different region selection criteria, and variable ways to quantify immune infiltration underscore the urgent need to reach assay harmonization. In an effort to promote the immunoscore in routine clinical settings worldwide, the Society for Immunotherapy of Cancer (SITC), the European Academy of Tumor Immunology, the Cancer and Inflammation Program, the National Cancer Institute, National Institutes of Health, USA and "La Fondazione Melanoma" will jointly initiate a task force on Immunoscoring as a New Possible Approach for the Classification of Cancer that will take place in Naples, Italy, February 13th, 2012. The expected outcome will include a concept manuscript that will be distributed to all interested participants for their contribution before publication outlining the goal and strategy to achieve this effort; a preliminary summary to be presented during the "Workshop on Tumor Microenvironment" prior to the SITC annual meeting on October 24th - 25th 2012 in Bethesda, Maryland, USA and finally a "Workshop on Immune Scoring" to be held in Naples in December of 2012 leading to the preparation of a summary document providing recommendations for the harmonization and implementation of the Immune Score as a new component for the classification of cancer.

Cancer classification using the Immunoscore: a worldwide task force.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Immune infiltration in human cancer: prognostic significance and disease control.

The interplay between tumors and their immunologic microenvironment is complex and difficult to decipher, but its understanding is of seminal importance for the development of novel prognostic markers and therapeutic strategies. This chapter discusses tumor-immune interactions in several human cancers that illustrate various aspects of this complexity and proposes an integrated scheme of the impact of local immune reactions on clinical outcome. Thus, the fact that a strong infiltration of memory T cells with a Th1 and cytotoxic pattern is the strongest predictor for recurrence and metastasis is exemplified in colorectal cancer in which intratumoral chemokines shape an efficient immune reaction. Based on these data, we propose an immune score that predicts recurrence in early stage (UICC-TNM stage I-II) cancers. Studies on non-small lung cancers have confirmed findings of colorectal cancers and have addressed the question of the sites where antitumor immune reactions may take place. Tertiary lymphoid structures (TLS) adjacent to the tumor nest are sites of intense activity with mature dendritic cells in contact with T cells and germinal-like centers with proliferating B cells. The large number of these TLS being correlated with disease specific and overall survival tempts to postulate that they are privileged sites to mount an efficient antitumor reaction. Inflammation is a major component of human tumors and chronic inflammation is generally of bad prognosis. Head and neck cancers are highly inflammatory and two ways to modulate inflammation in these diseases are presented here: soluble IL-15 receptor α (IL-15 Rα) increases the pro-inflammatory effect of IL-15 and aggravates inflammation resulting in poor prognosis when found at high levels in the plasma of patients. By contrast, infiltration of regulatory T cells is paradoxically beneficial for local control of head and neck tumors, probably by "cooling down" the inflammatory process. The modulation of other aspects of innate immunity may also result in paradoxical effects such as the signaling through Toll like receptors 7 and 8 expressed on lung tumor cells which induce an aggressive tumoral phenotype. Finally, the analysis of primary intraocular lymphoma, which develops in the eye, exemplifies the induction of an antitumor immune reaction in an "immune sanctuary," presenting all the complexities of the tumor-immune interplay in "open" tissues such as the colon or the lung.

Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation.

BACKGROUND: Cases of the spontaneous regression of multiple pulmonary metastases, after radiofrequency ablation (RFA), of a single lung metastasis, have been documented to be mediated by the immune system. The interaction of immune checkpoints, e.g., PD-1/PD-L1 and CTLA-4/CD80, may explain this phenomenon. The purpose of this study is to identify and quantify immune mechanisms triggered by RFA of pulmonary metastases originating from colorectal cancer. METHODS: We used two-site time-resolved Förster resonance energy transfer as determined by frequency-domain FLIM (iFRET) for the quantification of receptor-ligand interactions. iFRET provides a method by which immune checkpoint interaction states can be quantified in a spatiotemporal manner. The same patient sections were used for assessment of ligand-receptor interaction and intratumoral T-cell labeling. CONCLUSION: The checkpoint interaction states quantified by iFRET did not correlate with ligand expression. We show that immune checkpoint ligand expression as a predictive biomarker may be unsuitable as it does not confirm checkpoint interactions. In pre-RFA-treated metastases, there was a significant and negative correlation between PD-1/PD-L1 interaction state and intratumoral CD3+ and CD8+ density. The negative correlation of CD8+ and interactive states of PD-1/PD-L1 can be used to assess the state of immune suppression in RFA-treated patients.