Charles Pottier

MALDI mass spectrometry imaging: A cutting-edge tool for fundamental and clinical histopathology.

Histopathological diagnoses have been done in the last century based on hematoxylin and eosin staining. These methods were complemented by histochemistry, electron microscopy, immunohistochemistry (IHC), and molecular techniques. Mass spectrometry (MS) methods allow the thorough examination of various biocompounds in extracts and tissue sections. Today, mass spectrometry imaging (MSI), and especially matrix‐assisted laser desorption ionization (MALDI) imaging links classical histology and molecular analyses. Direct mapping is a major advantage of the combination of molecular profiling and imaging. MSI can be considered as a cutting edge approach for molecular detection of proteins, peptides, carbohydrates, lipids, and small molecules in tissues. This review covers the detection of various biomolecules in histopathological sections by MSI. Proteomic methods will be introduced into clinical histopathology within the next few years.

A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

Langerhans cell number is a strong and independent prognostic factor for head and neck squamous cell carcinomas

OBJECTIVES Head and neck squamous cell carcinomas (HNSCCs) exhibit great biological heterogeneity and relatively poor prognosis. Tobacco and alcohol consumption is involved in the cause of the majority of these cancers, but over the last several years, Human Papilloma Virus (HPV) infection has increased specifically in oropharyngeal cancers and become an additional risk factor. Here, we evaluated the number of Langerhans cells (LCs) in HNSCC and reporting its prognostic power in comparison to other risk factors. MATERIALS AND METHODS Our clinical series was composed of 25 tumor-free peritumoral epithelium, 64 low-grade dysplasia, 54 high-grade dysplasia and 125 carcinoma samples. HPV was detected by E6/E7 qPCR and p16 immunohistochemistry. CD1a-positive LCs were counted in intra-tumoral and stromal compartments as well as lymph nodes. MIP-3α was assessed in carcinomas using immunohistochemistry. RESULTS Univariate Cox regression analyses demonstrated that high LC number is associated with longer recurrence-free survival in both intra-tumoral and stromal compartments and longer overall survival in stromal compartment. Tobacco and alcohol habits, but not HPV status, are also correlated with poor prognoses in terms of recurrence. Multivariate analyses reported stromal LC number as a strong prognostic factor independent of tobacco, alcohol and HPV status. Moreover, LC number is higher in tumors and invaded lymph nodes than dysplastic lesions but it decreases in HPV-positive cancer patients. Further, LC number correlates with MIP-3α expression. CONCLUSION These findings suggest that LC number is a significant and independent prognostic factor for HNSCC. LC infiltration is increased in cancer lesions but decrease with HPV infection.

The importance of the tumor microenvironment in the therapeutic management of cancer.

Tumor prognosis is generally defined by various tumor parameters. However, it is well known that paracrine, endocrine and cell–cell interactions between the tumor and its microenvironment contribute to its growth. The tumor microenvironment (TME) can also influence disease prognosis and is likely to be considered as an important prognostic factor. In addition, conventional therapies can influence the microenvironment and antitumor immunity. Similarly, the TME will influence the effectiveness of therapy. The purpose of this review is to demonstrate how TME is important in therapeutic management. Key interactions between TME and different cancer therapies as well as their current clinical consequences have been described. More research is needed to establish the important network between tumor cells and their environment to highlight their relationships with conventional therapies and develop global therapeutic strategies.

MALDI Imaging-Guided Microproteomic Analyses of Heterogeneous Breast Tumors – A Pilot Study

Matrix‐assisted laser desorption/ionization (MALDI) imaging is an ideal tool to study intratumor heterogeneity (ITH) and its implication in prognostic stratification of patients. However, there are some drawbacks concerning protein identification. On the other hand, laser microdissection (LMD)‐based microproteomics allows retrieving thousands of protein identifications from small tissue pieces. As a proof of concept, the authors combine these two complementary approaches to analyze heterogeneous regions in breast tumors. Invasive ductal breast cancer FFPE tissue sections from five patients are analyzed by MALDI imaging and the dataset is processed by segmentation. Heterogeneous regions within tumors are processed by LMD‐based microproteomics, in duplicates. Liquid chromatography‐tandem mass spectrometry data are classified by hierarchical clustering. Heterogeneous tissue regions are discriminated on the basis of their actual molecular heterogeneity. The dataset is correlated with MALDI imaging to identify m/z values discriminating heterogeneous regions. The molecular characterization of cell clones in tumors related to bad patient outcome could have great impact for pathology. A combined application of LMD‐based microproteomics and MALDI imaging for ITH studies is presented.

A specific immune and lymphatic profile characterizes the pre-metastatic state of the sentinel lymph node in patients with early cervical cancer

ABSTRACT The lymph node (LN) pre-metastatic niche is faintly characterized in lymphophilic human neoplasia, although LN metastasis is considered as the strongest prognostic marker of patient survival. Due to its specific dissemination through a complex bilateral pelvic lymphatic system, early cervical cancer is a relevant candidate for investigating the early nodal metastatic process. In the present study, we analyzed in-depth both the lymphatic vasculature and the immune climate of pre-metastatic sentinel LN (SLN), in 48 cases of FIGO stage IB1 cervical neoplasms. An original digital image analysis methodology was used to objectively determine whole slide densities and spatial distributions of immunostained structures. We observed a marked increase in lymphatic vessel density (LVD) and a specific capsular and subcapsular distribution in pre-metastatic SLN when compared with non-sentinel counterparts. Such features persisted in the presence of nodal metastatic colonization. The inflammatory profile attested by CD8+, Foxp3, CD20 and PD-1expression was also significantly increased in pre-metastatic SLN. Remarkably, the densities of CD20+ B cells and PD-1 expressing germinal centers were positively correlated with LVD. All together, these data strongly support the existence of a pre-metastatic dialog between the primary tumor and the first nodal relay. Both lymphatic and immune responses contribute to the elaboration of a specific pre-metastatic microenvironment in human SLN. Moreover, this work provides evidence that, in the context of early cervical cancer, a pre-metastatic lymphangiogenesis occurs within the SLN (pre-metastatic niche) and is associated with a specific humoral immune response.

High stromal Foxp3-positive T cell number combined to tumor stage improved prognosis in head and neck squamous cell carcinoma

OBJECTIVES Head and neck squamous cell carcinomas (HNSCC), one of the most frequent cancers in the world, are largely infiltrated by inflammatory immune cells. Our aim was to evaluate the number of Foxp3+ T cells in HNSCC, reporting its prognostic power in comparison to other risk factors. MATERIAL AND METHODS Our clinical series was composed of 21 tumor-free peri-tumoral epithelia, 49 low grade dysplasia, 43 high grade dysplasia and 110 carcinoma samples including some cases with HPV infection. In vivo experiments were conducted on 80 C3H/HeN mice which were orthotopically injected with SCCVII CT, E7, E6 and E6/E7 cell lines. RESULTS Foxp3+ T cell infiltration increased with tumor progression from normal epithelia, dysplasia to carcinoma and the increase is more important in HPV+ patients than in negative ones. Animal experiments revealed that E7 oncoprotein expression was significantly associated with an increase in Foxp3+ T cell recruitment in tumor, a delay in tumor onset and improved animal survival. Univariate Cox regression analyses demonstrated that high Foxp3+ T cell number in stromal compartment is associated with longer patient recurrence-free and overall survivals. Foxp3+ T cell number improved the prognostic value of tumor stage. Multivariate analyses reported that stromal Foxp3+ T cell number is a strong prognostic factor independent of classical risk factors such as tobacco, alcohol, and HPV status. CONCLUSION Foxp3+ T cell number is a significant prognostic factor for HNSCC, improving the tumor stage, and that viral E7 may play a role in the Foxp3+ T cell infiltration to the tumor.

Microproteomic Profiling of High‐Grade Squamous Intraepithelial Lesion of the Cervix: Insight into Biological Mechanisms of Dysplasia and New Potential Diagnostic Markers

High‐grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC).

High infiltration of CD68+ macrophages is associated with poor prognoses of head and neck squamous cell carcinoma patients and is influenced by human papillomavirus

Incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HNSCCs) has increased over the last few decades. The reaction of the host immune system to these tumors remains biologically complex. Here, we investigated CD68+ macrophage numbers, reporting the prognostic value in comparison to other risk factors. We also examined CD68+ macrophage infiltration during disease progression regarding the impact of HPV infection, and we studied the role of HPV16-E6/E7 oncoproteins in CD68+ macrophage recruitment. CD68+ macrophage numbers were evaluated in 10 cases of tumor-free peri-tumoral epithelia, 43 cases of low-grade dysplasia, 45 cases of high-grade dysplasia and 110 cases of carcinoma. Our in vivo model was developed in 80 C3H/HeN mice orthotopically injected with HPV16-E6, -E7 or -E6/E7-transfected SCC-VII cell lines. High CD68+ macrophage numbers in the intra-tumoral compartment were associated with shorter patient survival (recurrence-free survival: p = 0.001; overall survival: p = 0.01). Multivariate analyses reported that CD68+ macrophage infiltration and tumor stage were strong and independent prognostic factors of HNSCC. CD68+ macrophage numbers increased during HNSCC progression both in intra-epithelial (p < 0.001) and stromal compartments (p < 0.001). A higher density of CD68+ macrophages was observed in advanced stages (p = 0.004). Patients with transcriptionally active HPV infections had higher CD68+ macrophage density than did HPV-negative patients (p = 0.003). CD68+ macrophage infiltration was higher in HPV-E7+ and −E6/E7+ mouse tumors than in -E6+ tumors (p = 0.029 and p < 0.001). In conclusion, the extent of CD68+ macrophage infiltration is a significant prognostic factor for HNSCC patients. The recruitment of macrophages increases during disease progression and is influenced by the HPV virus.

Abstract LB-187: MALDI imaging-guided microproteomics workflow for intratumor heterogeneity studies

A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding and therapy resistance [1]. MALDI imaging has proven its efficiency for revealing hidden molecular features offering an insight into distinct cellular regions based on their molecular content. Further, proteomics applied to these regions could allow depicting the molecular context associated to particular cells groups and enable the collection of qualitative, quantitative and spatial information for each protein. Breast cancer Formalin Fixed and Paraffin Embedded (FFPE) tissues, from patients whose outcome had been recorded over a period of 10 years, were used for this study. After Citric Acid Antigen Retrieval and trypsin digestion, images were obtained by MALDI-TOF/TOF-MS (Bruker, Germany). Analytical data analysis were applied to the measured large normalized datasets using the cloud software Multimaging (ImaBioTech, France) in order to find potential biomarkers that could be correlated to different patient survivals. Small tissue areas were obtained by laser microdissection (LEICA LMD 700, Germany), upon which a combination of chemical processes was applied to ensure optimal protein antigen retrieval, extraction and digestion. Finally, the tissue pieces obtained were analyzed by LC-MS/MS using UPLC Waters Nanoacquity and Thermo Q-Exactive instruments. Based on mathematical calculations carried out on the MALDI imaging datasets, ROIs could be detected in a single tumor. We aimed to compare the proteomic profiles of these intra-tumoral ROIs for several MALDI datasets. Recently, Longuespee [2] published a method to retrieve the identification of more than 1400 proteins from microdissected tissue pieces containing only 2700 cells. This whole procedure allowed us to identify a panel of proteins that characterizes tissue heterogeneity within a single tumor and to associate their presence with the information of each patient, such as their prognosis. This proves the applicability of the combination of MALDI imaging for the discovery of tumoral heterogeneity without a priori, on a mathematical basis, and classical proteomics applied on laser-microdissected tissue samples of very restricted areas. This way, we will be able to retrieve the extensive molecular context associated to a bad patient prognosis and/or therapy resistance. The described workflow combines the unique advantages of MALDI imaging for de novo molecular feature characterization and LMD-based microproteomics. It offers the possibility to identify protein/peptide markers that will have the power to predict the outcome of the breast cancer patient at the beginning of their treatment, and thus, improve the clinical care for the benefit of the patients. [1] Zardavas et al., Nature Rev. Clin. Onc. (2015) [2] Longuespee et al., Methods (2015) Citation Format: Deborah Alberts, Remi Longuespee, Charles Pottier, Nicolas Smargiassio, Gabriel Mazzucchelli, Dominique Baiwir, Philippe Delvenne, Gregory Hamm, Stefan Linehan, Fabien Pamelard, Gael Picard de Muller, Edwin De Pauw. MALDI imaging-guided microproteomics workflow for intratumor heterogeneity studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-187.