Knowledge Base

Authors: Paul Hofman 1,2, Cécile Badoual 3,4, Fiona Henderson 5 , Léa Berland 1 , Marame Hamila 1 , Elodie Long-Mira 1,2, Sandra Lassalle 1,2, Hélène Roussel 3,4, Véronique Hofman 1,2 , Eric Tartour 4,6 and Marius Ilié 1,2,*

1 Laboratory of Clinical and Experimental Pathology, Hospital-Integrated Biobank (BB-0033-00025), Nice Hospital University, FHU OncoAge, Université Côte d’Azur, Nice 06000, France; hofman.p@chu-nice.fr (P.H.); leaberland370@gmail.com (L.B.); hamila.m@chu-nice.fr (M.H.); long-mira.e@chu-nice.fr (E.L.-M.); lassalle.s@chu-nice.fr (S.L.); hofman.v@chu-nice.fr; ilie.m@chu-nice.fr (V.H.)

2 Team 4, Institute for Research on Cancer and Aging, Nice (IRCAN), INSERM U1081/UMR CNRS 7284, FHU OncoAge, Université Côte d’Azur, Nice 06107, France

3 Department of Pathology, Hôpital Européen Georges Pompidou, APHP, Paris 75015, France; cecile.badoual@aphp.fr (C.B.); helene.roussel@aphp.fr (H.R.)

4 INSERM U970, Université Paris Descartes Sorbonne Paris-Cité, Paris 75015, France; eric.tartour@aphp.fr

5 Department EMEA, Indica Labs, 2469 Corrales Rd Bldg. A-3 Corrales, NM 87048, USA; fhenderson@indicalab.com

As targeted molecular therapies and immuno-oncology have become pivotal in the management of patients with lung cancer, the essential requirement for high throughput analyses and clinical validation of biomarkers has become even more intense, with response rates maintained in the 20%–30% range. Moreover, the list of treatment alternatives, including combination therapies, is rapidly evolving. The molecular profiling and specific tumor-associated immune contexture may be predictive of response or resistance to these therapeutic strategies. Multiplexed immunohistochemistry is an effective and proficient approach to simultaneously identify specific proteins or molecular abnormalities, to determine the spatial distribution and activation state of immune cells, as well as the presence of immunoactive molecular expression. This method is highly advantageous for investigating immune evasion mechanisms and discovering potential biomarkers to assess mechanisms of action and to predict response to a given treatment. This review provides views on the current technological status and evidence for clinical applications of multiplexing and how it could be applied to optimize clinical management of patients with lung cancer.

Technical approaches of brightfield chromogenic mIHC include direct detection of antigens by primary antibodies from the same or different species that are directly labeled with different chromogens. Alternatively, an indirect mIHC detection method can be used with two or more layers of antibodies, allowing for increased amplification of signal [24]. The direct detection approach has several disadvantages, such as lower sensitivity for low abundance targets, the need for sizeable quantities of conjugated antibodies, which are usually more expensive, and the risk that antibody activity could be adversely affected by direct labeling [24]. The indirect approach can be limited by the number of available host species and the use of same species antibodies, which would thus require inactivation between successive cycles of immunolabeling [24]. The unwanted cross-reactivity between primary antibodies from different staining cycles is regarded as the main technical challenge in mIHC. The most frequent solution used to avoid such reactions is manual microwaving or heating of tissue slides to deactivate the preceding antibody [25,26]. Whereas microwaving is often used in research facilities when dealing with antibodies from the same host-species, it may not be an optimal method to be adopted in a routine clinical setting. Variable and heterogeneous results could be obtained by manual processing. Furthermore, microwaving can increase the damage of the tumor tissue and may remove small biopsies from the slides, especially if they have already been antigen retrieved by a previous heat-mediated procedure [27]. Another strategy for preventing cross-reactivity is the use of stripping buffers to elute the primary/secondary antibody complex [27,28]. A number of buffers with different pH, osmolality, detergent content and denaturing features were evaluated to strip the bound antibody complex from previous IHC staining cycles, however this produced variable results across studies. Certain buffers were found to be hazardous, to decolorize H&E stain and/or to reduce nuclear protein staining [27,28]. An alternative, more recent approach named “multiplexed immunohistochemical consecutive staining on single slide” (MICSSS), was developed for use on FFPE samples by applying repetitive cycles of immunoperoxidase labeling, image scanning, then chemical stripping of the chromogenic substrate [20,21]. However, this process can result in a labor-intensive protocol and a prolonged turnaround time to yield results that are not suitable for a routine clinical setting. Moreover, multiplexing may be limited due to tissue degradation after successive serial mIHC cycles [24,29]. More recently, a fully automated mIHC technology using a thermochemical process (heat deactivation; HD) to deactivate an antibody complex between staining cycles on an automated slide stainer was first developed for fluorescent detection, and further applied to brightfield chromogenic detection (Figure 1) [30,31].

Figure 1. Chromogenic multiplexed immunohistochemistry assay scheme. The assay is using the sequential application of four unmodified primary antibodies with a specific heat deactivation (HD) step between staining cycles.

This technology allows the use of the first antibody from the same host species, detected by the anti-species secondary antibody conjugated to horseradish peroxidase (HRP). In the presence of its substrate, the active HRP, generates in-situ deposition of tyramide within the medium containing the chromogens. The bound primary antibody/secondary antibody complex is then eluted with a citrate/acetate buffer. Thus, the deposited chromogen-conjugated tyramide bounds covalently to the tissue near the first detected antigen. The same procedure is repeated to detect the following antigens [30,31]. Indeed, the sequential stripping may lead to wastage of the conjugated secondary antibodies, whereas the chromogen-conjugated tyramine remains stable by binding covalently to the electron-rich amino acids of detected proteins and by resisting to the elution with the stripping buffer [30,31]. Importantly, the automation allows for standardization of all critical mIHC steps, such as HD, reagent application, washing steps, control of temperatures, evaporation and humidity, while maintaining the integrity of the tissue architecture and the subsequent epitopes [32,33]. To setup a brightfield mIHC assay using sequential detection with unmodified primary antibodies and chromogenic detection, it is essential to optimize assay conditions on the tissue types of interest before testing clinical samples [34]. Thus, an optimal mIHC assay needs to assure several staining performances: (i) equivalent positive/negative signal to single “gold standard” IHC staining, (ii) robust dynamic proportion of low and high protein quantity, (iii) expected cellular staining topology (e.g., whole membrane, cytoplasmic, nuclear localization), and (iv) minimal overlap of chromogenic spectra for co-localized targets [34]. Recent developments have enabled optimal configurations suitable for testing on clinical samples. For instance, the order of chromogen deposition is determined by the effect of HD on each epitope, that is, the most HD-affected epitope is incubated first, with the least affected epitope incubated last. To offer the best detection sensitivity, other assay parameters must be taken into account such as the optimal epitope retrieval time to balance the signal/background ratio, and to protect the tissue architecture by optimizing the incubation time for each primary antibody [30]. Moreover, the number of antibodies for simultaneous immunolabeling on the same tissue slide has been extended up to six with the availability of additional chromogens [24,33]. In addition, a major technical challenge is the risk of insufficient deactivation of the primary antibody complexes, which could determine cross-reactions and may give false-positive signals. Besides efforts to optimize HD steps during assay validation, the imaging tools can help to anticipate or to detect potential cross-reactions [35]. 2.2. Immunofluorescent Multiplexing Many newly identified or discovered biomarkers, especially for cancer immunotherapy, are linked to the tumor microenvironment and need to be analyzed with new methodological tools. For years, it has become increasingly essential to develop staining and interpretation techniques for the different cell populations infiltrating or composing a tissue. This is particularly true in oncology. To date, as previously described, the use of immunohistochemistry can help the visualization of an antibody-antigen conjugation. It has been showed in the last subsection, that an antibody is conjugated to an enzyme, like a peroxidase, can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged with a fluorophore. Nowadays the use of immunofluorescence is far easier due to technical improvement, like the use of stable fluorophores or the possibility to perform staining in paraffin embedded slides. Since years, research teams proposed immunoscoring, using single staining per slide, to identify prognostic factors [36]. However, the tumor microenvironment is too complex to be summarized by the exploration of a single marker. Chromogenic mIHC is one of the alternatives, and even if this technique is much easier to be used routinely, it is limited by the use of 4 antibodies on the same slide. In addition, fluorescence reveals membrane co-localizations (in the membrane or the nucleus), which is more difficult to obtain with the latter technique. Nevertheless, the use of multiple antibodies (mixed or used step by step) was restricted to the specificity of the primary and the risk of false positivity due to cross reactivity between them. Until recently, the single-parametric Cancers 2019, 11, 283 5 of 22 or even multiparametric (double or triple) staining, revealed by chromomeric or fluorescent staining, were most often read and interpreted directly by the researchers [37], with a lot of technical constraints. The microenvironment can now be studied using the multiplex fluorescence technique based on tyramide coupled to a fluorophore (e.g., Opal®, PerkinElmer, Waltham, MA, USA). This allows the simultaneous detection of several markers of interest on FFPE tissues. The concept of the technique is very close the one described above, the chromogenic mIHC assay using sequential application of four unmodified primary antibodies with a specific HD step between staining cycles. The main advantage of this technique is the multiplicity of the staining. The technique is based on a conventional fixation on the epitope of interest. The secondary antibody then binds to the primary antibody followed by Opal® HRP polymer and one of the Opal®fluorophore adjunction. After deposition of Opal® reagents, antibodies are stripped after use of a specific microwave to allow subsequent staining of other antigens. These cycles can be repeated at least seven to nine times. This seven to nine color multiplex staining technique makes it possible to more precisely characterize different cells and their interactions with their environment, on the same paraffin slide [38,39]. However, the use of these new techniques requires the acquisition of specific expertise for in situ multiple staining. Automation of this different process is now efficient and several autostainers are able to execute most of the steps previously described. For the validation of the different panels of multiparametric IHC markers, in particular for the exploration of the immune system, staining can be performed on tonsil tissue sections as this contains lympho-epithelial structures (Figure 2). Before any application on a cohort, especially when it concerns lung sections, the validation of staining on pulmonary tissue sections as a positive control is highly recommended. In addition, the same positive tissue control could be run on the same slide tested with mIHC, such as is currently performed for clinical diagnostic IHC.

Figure 2. Immunofluorescent multiplexing, image scanned with a spectral scanner (Polaris®; PerkinElmer, Waltham, MA, USA) using 20× magnification. The tissue is a paraffin embedded tonsil. The stains are as follows: pan-Cytokeratin (CK, teal), CD4 (green), CD68 (purple), PD-1 (red), PD-L1 (yellow) and dapi (blue). The central picture compiles the entire staining (merge).

The principle of a multiplex analysis of the tumor microenvironment is the automatic acquisition of a large surface, or the entire slide, quickly and sustainably. Having a fast acquisition time (milliseconds for each illuminated spot) is fundamental for fluorescence techniques because it prevents the "bleaching" which is the progressive extinction of the fluorescent signal after excitation.

Figure 3. Interpretation of the programmed death-ligand 1 (PD-L1) staining in serial whole-tissue formalin-fixed paraffin embedded samples from a lung adenocarcinoma case. (a) PD-L1 expression revealed by conventional immunoperoxidase staining; (b) PD-L1 expression revealed by chromogenic multiplexed immunohistochemistry, with the anti-TTF1 antibody colored in purple, anti-AE1/AE3 in yellow and anti-PD-L1 SP263 in teal. Blue dotted line: tumor area; red dotted line, immune cells.

As the restricted tissue size is a major issue for the management of the vast majority of solid tumors, and individual antibodies rarely demonstrate 100% specificity in the determination of malignancy or cell lineage, a chromogenic mIHC approach with specific multiple protein markers can provide valuable diagnostic information and has the potential to enhance the clinical significance of histological subtyping by delivering substantial prognostic information with therapeutic consequences [54,55].

3.2.1. Localization of Immune Cells and Their Relationships with Immunosuppressive Markers in the Tumor Microenvironment

The multiplex immunofluorescence techniques better distinguish the stromal and the tumor compartment and thus have allowed for a more detailed description of the topography of immune Cancers 2019, 11, 283 8 of 22 cells in cancer. Cruz et al. found that T lymphocytes were predominantly concentered in stromal compartment instead of the epithelial compartment in NSCLC [56]. Based on a quantitative immunofluorescence study, a comparative analysis of the expression of immunosuppressive molecules (e.g., PD-L1, IDO-1, B7H4) with the infiltration of intratumoral cells in lung cancer showed that PD-L1 and IDO-1 were consistently associated with prominent B- and T-cell infiltrates, but B7-H4 was not [57]. This could be explained by the role of IFNγ produced by immune cells in regulating PD-L1 and IDO-1 in the tumor microenvironment [58]. 3.2.2. Novel Prognostic Composite Biomarker based on Fluorescence in Situ Multiplexing One of the first clinical studies based on fluorescent digital pathology was the work of Schalper et al., who reported that the infiltration of intratumoral CD3+ and CD8+T cells was associated with a better overall survival in lung cancer patients [59]. For the CD8+T cell infiltration, this prognostic impact was independent from age, tumor size, histology and stage in multivariate analyses [59]. This technology also allows us to better define the prognostic value of immune cells depending on their localization in the tumor microenvironment. For example, after neoadjuvant chemotherapy, high levels of epithelial but not stromal CD4+CD3+T lymphocytes correlated with better survival in patients with NSCLC [60]. A more complex cell phenotype could also be better characterized with this multiparametric analysis. A novel subpopulation of CD8+T cells called resident memory T cells appear to play a major role in immunosurveillance, as they localize in close contact with epithelial tumor cells [61]. They are defined by a composite phenotype including various biomarkers such as CD103, CD49a, CD69 (Figure 4).

Figure 4. Infiltration of resident memory T cells (CD103+CD8+T cells) in human lung cancer. Frozen tissue sections derived from lung adenocarcinoma patients were stained by immunofluorescence with antibodies directed against human (a) CD8 (green), and (b) CD103 (red). (c) The co-localization of CD8 and CD103 markers can be detected by merging the mono-stained pictures. The arrows indicate double positive cells. Staining with isotype controls was included for each experiment (20× magnification).

We previously demonstrated that high levels of intratumoral infiltration with a resident memory CD8+T cells are associated with a better clinical outcome of NSCLC patients, both in univariate and multivariate analyses [62]. These were a more powerful prognostic marker than the infiltration of total CD8+T cells. These data were then confirmed by various clinical studies [63,64]. This technique also allows us to focus beyond just one cell type, and to integrate the relationships that exist between immune cells in the various compartments of tumors and the relative impact of these cellular relationships on the future of patients. For example, a high effector CD8+T cell/regulatory T cell ratio in the tumor nest is correlated with a better overall survival than when each cell measured independently [65].

3.2.3. Fluorescence Multiplexing Technique to Predict Clinical Response to Immunotherapy

Various parameters such as PD-L1, the expression of PD-1 and the intratumoral infiltration of CD8+T cells are considered, especially when combined together, as potential predictive biomarkers of clinical response to immunotherapy [66]. Parra et al., observed higher levels of PD-L1 expression on Cancers 2019, 11, 283 9 of 22 tumor cells and an increase in the infiltration of T cells and PD-1+T cells in the tumor microenvironment of NSCLC after neoadjuvant chemotherapy [60]. These findings confirm studies in other cancers reporting that neoadjuvant chemotherapy, whatever the regimen, makes the tumor microenvironment more permissive to immunotherapy [67,68]. These results suggest that it would be worthwhile to combine chemotherapy and immunotherapy before surgical resection of locally advanced lung cancer. Using a quantitative multiplex immunofluorescence technique, we reported that EGFR-mutated NSCLC weakly expressed PD-L1 and was not infiltrated by CD8+T cells suggesting that it would not be prone to respond to immunotherapy [69]. This hypothesis was then clinically confirmed in various clinical trials [70]. Interestingly, we found that a subpopulation of NSCLC displaying chromosomal rearrangement of the ALK gene expressed significant levels of PD-L1 on their tumor cells and were infiltrated by PD-1+CD8+T cells [69]. However, other studies showed that concurrent CD8+T cells and high PD-L1 expression on tumor cells tend to be rare in ALK positive NSCLCs [71,72]. Clinical trials did not confirm the sensitivity of this cancer subtype to the blockade of PD-1/PD-L1 axis [71]. This may suggest that other resistance mechanisms occur in this population such as the possible co-expression of inhibitory receptors on T cells or the infiltration of immunosuppressive cells [73,74]. Finally, an increase of T cells with a quiescent phenotype defined by a low proliferation and activation status (Ki67 and Granzyme negative) correlated with better overall survival in NSCLC patients treated by anti-PD-1/PD-L1 [75]. Interestingly, in NSCLC patients not treated by immunotherapy, this population of “dormant” T cells did not correlate with a better clinical outcome, supporting the fact that these cells could represent a true predictive biomarker of response to immunotherapy and not a prognostic marker [75].

Figure 5. Tissue classification using the random forest classifier in non-small cell lung cancer tissue (20× magnification). (a) The multiplexed immunohistochemistry (mIHC) image was scanned with a Nanozoomer 2.0-HT Scanner (Hamamatsu photonics, Hamamatsu, Japan). The stains are as follows: Pan-cytokeratin (yellow), CD8 (brown), Ki67 (purple), PD-L1 (teal) and hematoxylin (dark purple). (b) The random forest classifier in HALO was used to separate the image into three classes: tumor, stroma and microscope glass slide. The classifier mask is shown overlaying the mIHC image where classified tumor regions are shown in yellow, stroma regions in purple, and the microscope glass slide in pale pink. (c) The probability threshold used by the random forest to detect tumor regions was increased to 70%. A heatmap is displayed where the red regions represent areas most likely to be tumor regions, and the green regions that are less likely. No mask will appear in areas where pixels have below 70% probability of being in the tumor class. (d) The classifier to annotations option was used whereby regions can automatically be annotated from the classification mask; only the tumor has been annotated (shown in yellow).

Figure 6. Automated digital analysis of multiplexed immunohistochemistry (mIHC) using the HALO software in non-small cell lung cancer tissue. (a) The mIHC image was scanned with a Nanozoomer 2.0-HT Scanner (Hamamatsu photonics, Hamamatsu, Japan) using 20x magnification. The stains are as follows: Pan-cytokeratin (yellow), CD8 (brown), Ki67 (purple), PD-L1 (teal) and hematoxylin (dark purple). (b) The HALO mark-up image shows colors similar to the original stain color and in the same cell compartment (nucleus/cytoplasm/membrane as the stain is found. The user can select different colors to be used in the mark-up image if they wish.

When this is run in conjunction with the pre-made classifier, information about the number of cells with a specific phenotype in both the tumor and the stroma can be obtained. Additionally, HALO’s interactive cell-by-cell data table allows easy localization of the phenotyped cells on the image. In the example analysis in Figure 7, outputs will include those for the entire image, those specific to the tumor and those specific to the stroma. Other outputs include the number of cells positive for each stain in each compartment, number of cells with different stain co-localizations, the average optical density values for each stain in each compartment, cell/nucleus/cytoplasm/membrane area, and tissue areas in square microns.

Figure 7. Results of a multiplexed immunohistochemistry (mIHC) analysis in HALO. The top left table provides the summary results from the analysis; important outputs in this analysis are the density of cells co-expressing both CD8 and Ki67 in the tumor and stroma, and so the data relating to this has been highlighted. The bottom left table is HALO’s interactive cell-by-cell data table, which can be mined to find specific cell types. Here, only cells that are positive for CD8 and Ki67 and are in the stroma have been selected. HALO will find the cells selected in the image viewer (right, 20× magnification) by putting a black box around each cell.

After running a mIHC analysis in HALO, the pathologist then has the option to generate spatial information using the spatial analysis module. As outlined above, spatial information is becoming increasingly important in cancer research, prominently in the immune-oncology field [36,85]. Three different types of spatial analysis can be performed in HALO: nearest neighbor, proximity analysis and invasive margin analysis. Nearest neighbor outputs will calculate the average distance of two cell populations based on their nearest neighbors. Proximity analysis allows you to calculate the number of cells of one phenotype (e.g., CD8+ cytotoxic T lymphocytes) within a defined distance of another cell type. Lastly, the invasive margin analysis allows you to count the number of cells within a user defined distance of the invasive margin. Similarly, the HALO image analysis software was recently used to demonstrate the divergent state of exhaustion of the PD-1 receptor in T cells with impaired effector cytokine production, while producing CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures [80]. Cancers 2019, 11, 283 13 of 22 Importantly, the presence of PD-1high cells was strongly predictive for both response and survival in a cohort of NSCLC patients treated with a PD-1 blocking agent [80]. In the immunofluorescence multiplexing field, the use of scanners (fluorescent or spectral) represents a major technological advance by enabling the utilization of multiple and sometimes unstable fluorochromes (e.g., phycoerythrins) and thus more than 7 different antibodies on the same slide. For example, Vectra® systems or Polaris®(PerkinElmer) allow the capture of information by spectral resolution in the visible and in the near infrared band (bandwidth between 420 and 900 nm). Vectra® or Polaris® allows extremely precise quantitative (cell-by-cell) management of the markings of different tissue samples, in brightfield or fluorescence detection. Detection and phenotypic characterization of cells in tissues, combined with bioinformatic image analysis is possible thanks to the InForm® software (PerkinElmer). This software allows automatic analysis of parameters that cannot be accurately discerned by the human eye (cell forms, multiple molecule networks, vascular network). Franchising of autofluorescence by the "Autofluorescence Reduction Technology” (ART™, PerkinElmer) technique is possible with the Inform® software (PerkinElmer). Of course, the technologies developed for a specific type of cancer are subsequently transposable to the majority of other tumor proliferations or inflammatory diseases. Finally, virtual slides can be analyzed automatically (cell counting, surface measurements, etc.) using dedicated image analysis software (Figure 8). In particular, some software enables the quantification of weakly expressing and overlapping biomarkers within cells and cellular compartments.

Figure 8. Automated digital analysis of fluorescent multiplexing using Inform software in tonsil tissue (20× magnification). (a) Multiparametric fluorescent staining Pan-Cytokeratin (turquoise), CD4 (green), CD68 (purple), PD-1 (red), PD-L1 (yellow) and dapi (blue). (b) Tissue segmentation: identification and recognition of tumor areas (red) or stroma (green). (c) Individual cells identification and segmentation, with nuclear, membranous and cytoplasmic segmentation. (d) Phenotyping: identification of the cells on the slide, with their phenotypes, among all the cells present in the image, or among the cells stained.

These new approaches allow us to explore cellular interactions to find biomarkers in a non-supervised manner. The education of the software remains long and tedious, with a phase of learning or "teaching". However, an approach without a priori knowledge can also be developed in parallel. Several companies developed such software (e.g., Definiens AG, Munich, Germany; TRIBVN Healthcare, Châtillon, France; Owkin, Paris, France; Imstar, Paris, France; Indica Labs; PerkinElmer). These software systems are becoming more and more efficient, and they can differentiate anatomical structures, such as glands [86], but the recognition of cell units is more delicate. The results obtained in the context of cross-sectional research studies are, however, very impressive and we must expect a change in diagnostic habits with the implementation of deep learning [87]. Finally, an important issue for mIHC digital analysis and relevant data extraction is the calibration of the signal acquisition technology and the control of variations caused by the different staining techniques when several batches are required to analyze large clinical series (e.g., for biomarker validation). These controls are also necessary for the valid comparison of different series or studies and ultimately for clinical application [88].

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PDF Version:  cancers-11-00283.pdf

Author Contributions:

Conceptualization, P.H., C.B. and M.I.; methodology, P.H., E.T., F.H., C.B., and M.I.; software, E.T., F.H., C.B., and M.I.; validation, P.H., E.T., F.H., C.B., and M.I.; formal analysis, P.H., E.T., F.H., C.B., and M.I.; investigation, P.H., E.T., F.H., L.B., E.L-M., S.L., H.R., V.H., C.B., and M.I.; resources, P.H., E.T., F.H., L.B., E.L-M., S.L., H.R., V.H., C.B., and M.I.; data curation, P.H., E.T., F.H., L.B., E.L-M., S.L., H.R., V.H., C.B., and M.I.; writing—original draft preparation, P.H., E.T., F.H., C.B., and M.I.; writing—review and editing, P.H., E.T., F.H., L.B., E.L-M., S.L., H.R., V.H., C.B., and M.I.; visualization, P.H., E.T., F.H., C.B., and M.I.; supervision, P.H., E.T., F.H., C.B., and M.I.; project administration, P.H., E.T., F.H., C.B., and M.I.; funding acquisition, P.H., E.T., F.H., C.B., and M.I. Cancers 2019, 11, 283 16 of 22

Funding: This research was funded in part by the Initiative of excellence IDEX - UCAJEDI, Université Côte d’Azur; the Cancéropole PACA; the French Government (Agence Nationale de Recherche, ANR) through the ‘Investments for the Future’ LABEX SIGNALIFE [ANR-11-LABX-0028-01]; CARPEM (SIRIC); Labex Immuno-Oncology, INCA (2016-PL-Bio); the “Fondation ARC pour la Recherche sur le Cancer” (ARC SL220110603478); the CANC’AIR Genexposomic project; the “Ligue Départementale 06 de Lutte contre le Cancer”, the “Conseil Départemental des Alpes-Maritimes”; the “Région Sud-Provence Alpes-Côte d’Azur”, France. The funders had no role in the design of the study; in the collection, analyses, and interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Acknowledgments: To Julien Fayada and Marion Mandavit for their technical support.

Conflicts of Interest: P.H. has received travel grants and honoraria from AstraZeneca, Roche, Bristol-Myers Squibb, Qiagen, ThermoFisher, Pfizer, and Merck & Co. F.H. is an employee of Indica Labs. C.B. has received grant travel and/or honoraria from AstraZeneca, Roche, Bristol-Myers Squibb, and Merck & Co. E.T. has received travel grants and honoraria from AstraZeneca, Bristol-Myers Squibb. M.I. has received travel grants and honoraria from AstraZeneca, Merck & Co., Roche, Boehringer-Ingelheim and Bristol-Myers Squibb. There are no other competing financial interests or other conflicts.

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