MSACL 2018 US Abstract

Topic: Tissue Imaging & Analysis

Combining MALDI MS Imaging with 2D Gel-based Proteomics to Reveal Potential Penile Carcinoma Biomarkers

Elisangela Silva (Presenter)
A.C.Camargo Cancer center

Bio: Bachelor's degree in Biomedicine from the Federal University of Pernambuco (2009), a Master's Degree in Human Health and Environment from the Federal University of Pernambuco(2012), and a PhD in Oncology from A.C.Camargo Cancer Center (2016). Currently is a post-Doc fellow at the Investigative Pathology research group at A.C. Camargo Cancer Center. Has experience in the application of analytical chemistry techniques in cancer field, including 2D Electrophoresis, LC-MS/MS and Imaging Mass Spectrometry.

Authorship: Elisβngela J Silva (1), Diego Magno Assis (2), Isabela W Cunha (1 ), Stênio C Zequi (1), Gustavo C Guimarães (1), Fernando A Soares (1), Nilson A Assunção (3), Jose Vassallo (3,4)
(1) A.C.Camargo Cancer Center, Sao Paulo, Brazil, (2) Bruker Daltonics, Atibaia, Sao Paulo, Brazil, (3) Federal University of Sao Paulo, Sao Paulo, Brazil, (4) University of Campinas Medical School (Unicamp), Sao Paulo, Brazil

Short Abstract

Penile cancer (PeCa) is a mutilating men’s malignant tumor, especially in underprivileged socioeconomic regions of developing countries. New markers are still needed for early diagnosis, prognosis and prediction of therapy. Supervised and unsupervised analysis, using MALDI Mass Spectrometry Imaging (MSI) integrated with 2D gel data, provided evidence that the protein calreticulin might be a potential biomarker of PeCa. Immunohistochemistry validation was performed in PeCa tumors and controls. Calreticulin expression was associated with poorly prognosis in PeCa patients. Our approach proved to be useful for the in situ analysis of target protein and for the development of biomarkers with clinical value.

Long Abstract

Introduction

Penile cancer (PeCa) is a mutilating men’s malignant tumor, especially in underprivileged socioeconomic regions of developing countries. New markers are still needed for early diagnosis, prognosis and prediction of therapy. Our previous study evidenced high levels of Calreticulin (CRT) in PeCa samples when compared to normal specimens by 2D gel-based proteomic. This protein is a resident of endoplasmic reticulum, and is implicated in multiple cellular functions, including calcium homeostasis and regulation of transcriptional activity. It seems to be related to tumor progression; however, the molecular mechanisms involved in this process are still unclear [1]. We investigated if this protein could be a potential biomarker for PeCa through a MALDI MSI supervised and unsupervised analysis. Immunohistochemistry and clinicopathological correlations were also performed in an independent group of PeCa tumors.

Methods

Tissue specimens: Twenty fresh-frozen specimens of Penile Squamous Cell Carcinoma (PeSCC), usual type, and 10 normal control specimens [postectomy (n=4) and adjacent normal skin tissue (n=6)], were obtained from patients and used in MALDI MSI analysis. For immunohistochemistry, 158 paraffin-embedded PeCa samples, which were included in a tissue microarray, were used.

MALDI MS Imaging: All fresh-frozen tissues sections were digested and submitted to peptide MALDI MSI analysis. Then, we integrated the imaging result with previous 2D gel-based proteomics data to evaluate the spatial distribution of CRT. An unsupervised clustering was also performed in four of the fresh-frozen PeSCC samples to confirm if CRT had the potential to distinguish tumor and non-neoplastic adjacent regions. To assist in this effort, we obtained a segmentation map that coincide with histological annotation for tumor and non-neoplastic adjacent regions and applied correlation analysis to detect m/z-values co-localized with clusters. After, we searched for CRT peaks at the differential m/z-values list and used ROC curve to evaluate the potential of CRT to discriminate the two regions of interest. All experiments were carried out on an Autoflex MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany), using a lateral resolution of 200 µm, covering the range of m/z 700-3500. Data analysis were conducted using Flex Control, Flex Imaging 4.0, ImageID software package (Bruker Daltonics, Bremen, Germany) and SCiLS Lab package software (SCiLS, Bremen, Germany).

Immunohistochemistry: The slides were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer pH 6.0. Immunoreactivity of CRT (Clone FMC, diluted at 1:1000, Abcam, Cambridge, UK) were visualized using the Dako advance detection system (Agilent Technologies). The association between the clinicopathologic characteristics of the patients and CRT immunoreactivity was analyzed by Mann-Whitney U test, qui-square test, Log-Rank statistic and Cox regression. The significance level was 5% for all statistical tests. All analyses were performed using SPSS (23 version) and R (3.3.0 version) software.

Results

MALDI-MSI and 2D based-proteomic integration visually resulted in a high signal intensity of CRT peptide peak (m/z-value 1410) in tumor regions when compared to adjacent non-neoplastic tissues. Unsupervised analysis confirmed that m/z-value 1410 was a significant differential peak (P<0.001; AUC=0.962) and its signal intensity coincides to tumor regions of the segmentation maps. Immunohistochemistry validation demonstrated that higher levels of CRT were associated with poor overall (Hazard ratio [HR] 2.3, Confidence Interval [CI]-1.46-3.96; P<0,001) and cancer-specific survival in PeCa patients (HR 4.37; CI-1.66-11.51; P=0,002). CRT also played a role in predicting lymph node metastasis in this study (HR 14.18; CI-3.29-61.12; P <0,001).

Conclusions & Discussion

Immunohistochemistry validation performed in 158 PeCa samples and 10 controls demonstrated that higher levels of CRT were associated with poor overall and cancer-specific survival in PeCa patients. CRT also played a role in predicting lymph node metastasis in this study. This acknowledgment is relevant for PeCa clinical management, as CRT level was associated with a significantly more unfavorable outcome. Therefore, MALDI-MSI proved to be useful to search for potential cancer biomarkers through in situ protein analysis. Our results might represent the basis for further studies on the characterization of PeCa and for the development of biomarkers with significant clinical value.


References & Acknowledgements:

[1] Lu Y-C, Weng W-C, Lee H. Functional roles of calreticulin in cancer biology. Biomed Res Int 2015; 2015:526524.

We thank the Brazilian Biosciences National Laboratory (LnBio) for the mass spectrometry analysis. Financial Support was received from São Paulo Research Foundation (Fapesp 2009/52088-3), from National Counsel of Technological Scientific Development (CNPq 446368/2014) and Brazilian Innovation Agency (FINEP).


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