Mykola Chekan

Application of Metabolomics in Thyroid Cancer Research

Thyroid cancer is the most common endocrine malignancy with four major types distinguished on the basis of histopathological features: papillary, follicular, medullary, and anaplastic. Classification of thyroid cancer is the primary step in the assessment of prognosis and selection of the treatment. However, in some cases, cytological and histological patterns are inconclusive; hence, classification based on histopathology could be supported by molecular biomarkers, including markers identified with the use of high-throughput “omics” techniques. Beside genomics, transcriptomics, and proteomics, metabolomic approach emerges as the most downstream attitude reflecting phenotypic changes and alterations in pathophysiological states of biological systems. Metabolomics using mass spectrometry and magnetic resonance spectroscopy techniques allows qualitative and quantitative profiling of small molecules present in biological systems. This approach can be applied to reveal metabolic differences between different types of thyroid cancer and to identify new potential candidates for molecular biomarkers. In this review, we consider current results concerning application of metabolomics in the field of thyroid cancer research. Recent studies show that metabolomics can provide significant information about the discrimination between different types of thyroid lesions. In the near future, one could expect a further progress in thyroid cancer metabolomics leading to development of molecular markers and improvement of the tumor types classification and diagnosis.

Detection of metabolites discriminating subtypes of thyroid cancer: Molecular profiling of FFPE samples using the GC/MS approach.

One of the critical issues in thyroid cancer diagnostic is differentiation between follicular adenoma, follicular carcinoma and the follicular variant of papillary carcinoma, which in some cases is not possible based on histopathological features only. In this paper we performed molecular profiling of thyroid tissue aiming to identify metabolites characteristic for different types of thyroid cancer. FFPE tissue specimens were analysed from 5 different types of thyroid malignancies (follicular, papillary/classical variant, papillary/follicular variant, medullary and anaplastic cancers), benign follicular adenoma and normal thyroid. Extracted metabolites were identified and semi-quantified using the GC/MS approach. There were 28 metabolites identified, whose abundances were significantly different among different types of thyroid tumours, including lipids, carboxylic acids, and saccharides. We concluded, that multi-component metabolome signature could be used for classification of different subtypes of follicular thyroid lesions. Moreover, potential applicability of the GC/MS-based analysis of FFPE tissue samples in diagnostics of thyroid cancer has been proved.

Detection of molecular signatures of oral squamous cell carcinoma and normal epithelium – application of a novel methodology for unsupervised segmentation of imaging mass spectrometry data

Intra‐tumor heterogeneity is a vivid problem of molecular oncology that could be addressed by imaging mass spectrometry. Here we aimed to assess molecular heterogeneity of oral squamous cell carcinoma and to detect signatures discriminating normal and cancerous epithelium. Tryptic peptides were analyzed by MALDI‐IMS in tissue specimens from five patients with oral cancer. Novel algorithm of IMS data analysis was developed and implemented, which included Gaussian mixture modeling for detection of spectral components and iterative k‐means algorithm for unsupervised spectra clustering performed in domain reduced to a subset of the most dispersed components. About 4% of the detected peptides showed significantly different abundances between normal epithelium and tumor, and could be considered as a molecular signature of oral cancer. Moreover, unsupervised clustering revealed two major sub‐regions within expert‐defined tumor areas. One of them showed molecular similarity with histologically normal epithelium. The other one showed similarity with connective tissue, yet was markedly different from normal epithelium. Pathologists re‐inspection of tissue specimens confirmed distinct features in both tumor sub‐regions: foci of actual cancer cells or cancer microenvironment‐related cells prevailed in corresponding areas. Hence, molecular differences detected during automated segmentation of IMS data had an apparent reflection in real structures present in tumor.

BRAFV600E-Associated Gene Expression Profile: Early Changes in the Transcriptome, Based on a Transgenic Mouse Model of Papillary Thyroid Carcinoma

Background The molecular mechanisms driving the papillary thyroid carcinoma (PTC) are still poorly understood. The most frequent genetic alteration in PTC is the BRAFV600E mutation–its impact may extend even beyond PTC genomic profile and influence the tumor characteristics and even clinical behavior. Methods In order to identify BRAF-dependent signature of early carcinogenesis in PTC, a transgenic mouse model with BRAFV600E-induced PTC was developed. Mice thyroid samples were used in microarray analysis and the data were referred to a human thyroid dataset. Results Most of BRAF(+) mice developed malignant lesions. Nevertheless, 16% of BRAF(+) mice displayed only benign hyperplastic lesions or apparently asymptomatic thyroids. After comparison of non-malignant BRAF(+) thyroids to BRAF(−) ones, we selected 862 significantly deregulated genes. When the mouse BRAF-dependent signature was transposed to the human HG-U133A microarray, we identified 532 genes, potentially indicating the BRAF signature (representing early changes, not related to developed malignant tumor). Comparing BRAF(+) PTCs to healthy human thyroids, PTCs without BRAF and RET alterations and RET(+), RAS(+) PTCs, 18 of these 532 genes displayed significantly deregulated expression in all subgroups. All 18 genes, among them 7 novel and previously not reported, were validated as BRAFV600E-specific in the dataset of independent PTC samples, made available by The Cancer Genome Atlas Project. Conclusion The study identified 7 BRAF-induced genes that are specific for BRAF V600E-driven PTC and not previously reported as related to BRAF mutation or thyroid carcinoma: MMD, ITPR3, AACS, LAD1, PVRL3, ALDH3B1, and RASA1. The full signature of BRAF-related 532 genes may encompass other BRAF-related important transcripts and require further study.

Endogenous avidin biotin activity (EABA) in thyroid pathology: immunohistochemical study

BackgroundImmunohistochemical methods based on the high affinity of avidin and biotin (e.g. ABC, LSAB) are characterized by high sensitivity and are widely used for detection of immunologic reaction. However, a non-specific reaction, observed in frozen tissues and in paraffin-embedded material, increasing after heat induced epitope retrieval (HIER), and caused either by endogenous biotin or any another chemical compound with high affinity for avidin, may lead to diagnostic mistakes. The aim of our investigation is to study presence of endogenous avidin biotin activity (EABA) in thyrocytes originating from various thyroid pathological lesions (neoplastic and non-neoplastic).Materials and methodsThe immunohistochemical study was performed on paraffin-embedded specimens of surgically resected thyroid tissue from 97 patients with thyroid diseases: 65 patients with papillary carcinoma (PTC), 11 patients with nodular goiter in whom features of benign papillary hyperplasia were found, 9 with lymphocytic thyroiditis (LT), 8 with follicular adenoma, and 4 patients with follicular carcinoma. In PTC immunohistochemical study was performed both in primary tumors and in lymph node metastases. After HIER, incubation with streptavidin from LSAB+ (DakoCytomation) kit was done.ResultsStrong cytoplasmic EABA was observed in 56 of 65 (87.5%) PTC and in oxyphilic cells in 8 of 9 cases of LT. Significant correlation between EABA in primary PTC tumor and EABA in lymph node metastases was stated. Normal surrounding thyroid tissues showed absence or weak EABA. Aberrant intranuclear localization of biotin was noted in morules of cribriform-morular variant of PTC. No statistically significant correlation between patients age, sex, metastases presence and EABA was observed.ConclusionAmong thyroid lesions, false positive reactions are highly probable in papillary thyroid carcinoma and in lymphocytic thyroiditis if immunohistochemical detection is used on systems containing (strept)avidin. The most probable reason is the high endogenous biotin content.

Somatic mutation profiling of follicular thyroid cancer by next generation sequencing

The molecular etiology of follicular thyroid tumors is largely unknown, rendering the diagnostics of these tumors challenging. The somatic alterations present in these tumors apart from RAS gene mutations and PAX8/PPARG translocations are not well described. To evaluate the profile of somatic alteration in follicular thyroid tumors, a total of 82 thyroid tissue samples derived from 48 patients were subjected to targeted Illumina HiSeq next generation sequencing of 372 cancer-related genes. New somatic alterations were identified in oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK). Single nucleotide and large structural variants were most and least frequently identified, respectively. A novel translocation in DERL/COX6C was detected. Many somatic alterations in non-coding gene regions with high penetrance were observed. Thus, follicular thyroid tumor somatic alterations exhibit complex patterns. Most tumors contained distinct somatic alterations, suggesting previously unreported heterogeneity.

Molecular profiles of thyroid cancer subtypes: Classification based on features of tissue revealed by mass spectrometry imaging☆

Determination of the specific type of thyroid cancer is crucial for the prognosis and selection of treatment of this malignancy. However, in some cases appropriate classification is not possible based on histopathological features only, and it might be supported by molecular biomarkers. Here we aimed to characterize molecular profiles of different thyroid malignancies using mass spectrometry imaging (MSI) which enables the direct annotation of molecular features with morphological pictures of an analyzed tissue. Fifteen formalin-fixed paraffin-embedded tissue specimens corresponding to five major types of thyroid cancer were analyzed by MALDI-MSI after in-situ trypsin digestion, and the possibility of classification based on the results of unsupervised segmentation of MALDI images was tested. Novel method of semi-supervised detection of the cancer region of interest (ROI) was implemented. We found strong separation of medullary cancer from malignancies derived from thyroid epithelium, and separation of anaplastic cancer from differentiated cancers. Reliable classification of medullary and anaplastic cancers using an approach based on automated detection of cancer ROI was validated with independent samples. Moreover, extraction of spectra from tumor areas allowed the detection of molecular components that differentiated follicular cancer and two variants of papillary cancer (classical and follicular). We concluded that MALDI-MSI approach is a promising strategy in the search for biomarkers supporting classification of thyroid malignant tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Proteome profiles of different types of thyroid cancers

Proteomics profiling of tissue specimens representative for major types of thyroid cancers: papillary (classical and follicular variant), follicular, anaplastic and medullary, as well as benign follicular adenoma, was performed using shotgun LC-MS/MS approaches. A combination of Orbitrap and MALDI-TOF approach allowed to identify protein products of 3700 unique genes and revealed large differences between medullary, anaplastic and epithelium-derived differentiated cancers (papillary and follicular). Proteins characteristic for medullary and anaplastic cancers included factors associated with neuroendocrine functions and factors typically associated with advanced malignancies, respectively. Proteomes of different types of epithelium-derived differentiated cancers and follicular adenoma were compared using multi-enzyme LC-MS/MS approach, which revealed products of 4800 unique genes. A comparable overall similarity of follicular cancers to both variants of papillary cancers was found. Moreover, follicular adenoma showed higher overall similarity to follicular cancer than to either variant of papillary cancer. Proteins discriminating differentiated thyroid neoplasms included factors associated with lipid and hormone metabolism, regulation of gene expression and maintenance of DNA structure. Importantly, proteome data matched several features of transcriptome and metabolome profiles of thyroid cancers contributing to systems biology of this malignancy.

Discrimination of papillary thyroid cancer from non-cancerous thyroid tissue based on lipid profiling by mass spectrometry imaging

INTRODUCTION The distinction of papillary thyroid carcinomas from benign thyroid lesions has important implication for clinical man-agement. Classification based on histopathological features can be supported by molecular biomarkers, including lipidomic signatures, identified with the use of high-throughput mass spectrometry techniques. Formalin fixation is a standard procedure for stabilization and preservation of tissue samples, therefore this type of samples constitute highly valuable source of clinical material for retrospective molecular studies. In this study we used mass spectrometry imaging to detect lipids discriminating papillary cancer from not cancerous thyroid directly in formalin-fixed tissue sections. MATERIAL AND METHODS For this purpose imaging and profiling of lipids present in non-malignant and cancerous thyroid tissue specimens were conducted. High resolution MALDI-Q-Ion Mobility-TOF-MS technique was used for lipidomic analysis of formalin fixed thyroid tissue samples. Lipids were identified by the comparison of the exact molecular masses and fragmentation pathways of the protonated molecule ions, recorded during the MS/MS experiments, with LIPID MAPS database. RESULTS Several phosphatidylcholines (32:0, 32:1, 34:1 and 36:3), sphingomyelins (34:1 and 36:1) and phosphatidic acids (36:2 and 36:3) were detected and their abundances were significantly higher in cancerous tissue compared to non-cancerous tissue. The same lipid species were detected in formalin-fixed as in fresh-frozen tissue, but [M + Na]+ ions were the most abundant in formalin fixed whereas [M + K]+ ions were predominant in fresh tissue. CONCLUSIONS Our results prove the viability of MALDI-MSI for analysis of lipid distribution directly in formalin-fixed tissue, and the potential for their use in the classification of thyroid diseases.

Coexistence of TERT Promoter Mutations and the BRAF V600E Alteration and Its Impact on Histopathological Features of Papillary Thyroid Carcinoma in a Selected Series of Polish Patients

TERT promoter (TERTp) mutations are important factors in papillary thyroid carcinomas (PTCs). They are associated with tumor aggressiveness, recurrence, and disease-specific mortality and their use in risk stratification of PTC patients has been proposed. In this study we investigated the prevalence of TERTp mutations in a cohort of Polish patients with PTCs and the association of these mutations with histopathological factors, particularly in coexistence with the BRAF V600E mutation. A total of 189 consecutive PTC specimens with known BRAF mutational status were evaluated. TERTp mutations were detected in 8.5% of cases (16/189) with the C228T mutation being the most frequent. In six of the PTC specimens (3.2%), four additional TERTp alterations were found, which included one known polymorphism (rs2735943) and three previously unreported alterations. The association analysis revealed that the TERTp hotspot mutations were highly correlated with the presence of the BRAF V600E mutation and their coexistence was significantly associated with gender, advanced patient age, advanced disease stage, presence of lymph node metastases, larger tumor size, and tumor-capsule infiltration. While correlations were identified, the possibility of TERTp mutations being key molecular modulators responsible for PTC aggressiveness requires further studies.