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

<p>Abstract</p> <p>Background</p> <p>Immunohistochemical 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).</p> <p>Materials and methods</p> <p>The 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.</p> <p>Results</p> <p>Strong 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 patient's age, sex, metastases presence and EABA was observed.</p> <p>Conclusion</p> <p>Among 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.</p

Expression of the selected adhesive molecules (cadherin E, CD44, LGAL3 and CA50) in papillary thyroid carcinoma

Wstęp: Celem pracy było określenie ekspresji wybranych molekuł adhezyjnych w tkance raka brodawkowatego tarczycy zarówno w guzie pierwotnym, jak i w przerzutach do węzłów chłonnych oraz ocena ich przydatności dla celów diagnostycznych i prognostycznych. Materiał i metody: Badaniem objęto grupę 47 chorych na raka brodawkowatego tarczycy i 11 chorych operowanych z powodu wola niezłośliwego. Badania przeprowadzono metodą immunohistochemiczną. Wyniki: Pod względem znaczenia diagnostycznego wśród badanych markerów galektyna-3 okazała się jednym z najbardziej czułych i swoistych, o czym świadczy różnica między guzem (91% przypadków dodatnich) a otoczeniem (5%). Molekuła adhezyjna CA50 występowała prawie tak samo często w guzie pierwotnym (86% przypadków), jak i przerzutowym (85%). Jej obecność w otoczeniu zaobserwowano tylko w jednym przypadku (3%), reakcja miała charakter ogniskowy. Kadheryna E występowała w 91% przypadków guza pierwotnego i 84% otoczenia. Dodatnią reakcję obserwowano również w 63% przypadków wola guzkowego. Molekuła CD44, klon DF1485, był obecny w 100% przerzutów do węzłów chłonnych, w 89% guzów pierwotnych i w 48% przypadków otoczenia guza. Molekuła CD44, klon BBA10, występował częściej w przerzutach. Wnioski: Spośród badanych markerów największą wartość diagnostyczną w raku brodawkowatym tarczycy wykazał antygen CA50 i galektyna-3. Ekspresja CD44 (DF1485) w guzie pierwotnym jest znacznie silniejsza niż w otoczeniu, jednak jego wartość diagnostyczna jest niepewna, gdyż występuje on często w zmianach łagodnych tarczycy. Zastosowanie przeciwciała BBA10 zwiększa czułość metody, ale zmniejsza jej swoistość. Częste występowanie kadheryny E w komórkach raka brodawkowatego tarczycy, otoczeniu guza i zmianach łagodnych tarczycy nie pozwala na jej zastosowanie diagnostyczne.Introduction: The aim of the study was to determine the expression of selected adhesive molecules in papillary thyroid carcinoma. Material and methods: 47 papillary thyroid carcinoma cases and 11 nonmalignant goiter cases were analyzed by immunohistochemistry. Results: Galectin-3 (LGAL3) was a sensitive and specific marker, present in 91% of analyzed tumors and only in 5% of tumor margin. The presence of CA50 was 86% and 3% respectively with only 3% positive non-malignant cases. Cadherin E expression was noted in 91% of primary tumors, in 84% of the surrounding tissue and in 63% of non-malignant goiter. CD44 (DF1485) was observed in 89% of primary tumors and 48% of surrounding tissue; the reaction with BBA10 was more characteristic for metastases. Conclusions: Our study confirms the high diagnostic value of galectin-3 in papillary thyroid carcinoma and reveals the similar efficiency of CA50. CD44 (DF1485) expression in primary tumor is more intensive than in surrounding tissue, but the diagnostical inportance is not high because it is often observed in benign lesions. Using of BBA10 is more sensitive, but less specific. High expression of cadherin E in benign lesions impairs its diagnostical application in papillary thyroid cancer

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues

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

Leczenie chirurgiczne i rekonstrukcyjne centralnego ziarniniaka olbrzymiokomórkowego żuchwy o agresywnym przebiegu – opis przypadku i przegląd piśmiennictwa

Introduction: Central giant cell granuloma (CGCG) is a benign tumor-like lesion of a bone, mainly localized in the mandible. It usually occurs in children and young adults under 30 y/o., predominantly in females. The etiology of the disease remains unknown. Clinically, two types of CGCG have been distinguished – a non-aggressive one, in which the granuloma grows slowly, often asymptomatically, and aggressive type for which the following features are characteristic: increased bone destruction, severe pain, large size, rapid growth, high recurrence rate and complications such as root resorption, tooth displacement or cortical bone perforation. The treatment of CGCG depends on its type. In cases of granulomas of the aggressive type, the following therapeutic procedures have been proposed: intralesional corticosteroid injections, interferon and calcitonin therapy as well as immunotherapy with anti-bone resorptive human monoclonal antibody like denosumab. However, in most cases nonsurgical treatment is insufficient. Local curettage of the lesion also entails a high risk of relapse. Therefore, radical surgical resection, often combined with bone reconstruction, is the recommended way of treatment for aggressive CGCG. Case report: The authors present a case of a 31-year-old female patient treated for central giant cell granuloma of the mandible at the Department of Oncological and Reconstructive Surgery, Maria Sklodowska Curie Memorial Cancer Centre and Institute of Oncology in Gliwice. The resection of CGCG localized in the mandible on the right side together with fibular free flap reconstruction has been performed, with satisfactory aesthetic effect. The immunohistochemical examination indicated a positive stain reaction for CD68 and CD31 and expression of Ki67 marker was 13%. No complications were reported in the postoperative period. The six-month follow up revealed no relapse. Conclusions: The authors claim that radical surgical management should be performed in all patients with CGCG of the aggressive type. Fibular free flap is recommended for reconstruction in large bone defects. This allows tumor-free margins at the resection and satisfactory cosmetic outcome. Quality of life and facial appearance can be improved with dental implantation after a certain period of remission. A regular follow-up is essential as an element of holistic oncological process.Wstęp: CGCG jest łagodną, guzopodobną zmianą kości, najczęściej lokalizującą się w obrębie żuchwy. Występuje ona głównie u dzieci i młodych dorosłych poniżej 30 r.ż., z przewagą płci żeńskiej. Etiologia schorzenia nie została do końca poznana. Klinicznie wyróżnia się dwie postacie CGCG: (1) nieagresywną, kiedy ziarniniak rośnie powoli, często bezobjawowo, oraz (2) agresywną, charakteryzującą się: nasilonym niszczeniem kości, dolegliwościami bólowymi, większymi rozmiarami guza, jego gwałtownym wzrostem i powikłaniami, takimi jak: resorpcja korzeni czy perforacja blaszki kostnej, oraz wysoką tendencją do nawrotów. Leczenie ziarniniaka zależy od jego postaci. W przypadkach CGCG o agresywnym przebiegu zastosowanie znajduje leczenie zachowawcze pod postacią sterydoterapii w infekcjach miejscowych, aplikacji interferonu, kalcytoniny czy denosumabu. Jednak w większości przypadków ta forma terapii bywa nieskuteczna. Również wyłyżeczkowanie zmiany niesie wysokie ryzyko nawrotu choroby. Stąd najlepszym sposobem postępowania jest radykalne chirurgiczne usunięcie zmiany, często wymagające operacji rekonstrukcyjnej ubytków kostnych. Opis przypadku: W niniejszej pracy autorzy przedstawiają przypadek 31-letniej chorej, leczonej w Klinice Chirurgii Onkologicznej i Rekonstrukcyjnej Centrum Onkologii – Instytutu im. Marii Curie-Skłodowskiej Oddziału w Gliwicach z powodu centralnego ziarniniaka olbrzymiokomórkowego żuchwy. U pacjentki wykonano operację resekcji fragmentu żuchwy po stronie prawej wraz z guzem z następową rekonstrukcją wolnym płatem strzałkowym, uzyskując zadowalający efekt kosmetyczny. Przebieg pooperacyjny był niepowikłany. W badaniu immunohistochemicznym wykazano dodatnią reakcję z przeciwciałami CD68 i CD31, a współczynnik Ki67 wyniósł 13%. W półrocznym okresie obserwacji nie odnotowano wznowy ziarniniaka. Wnioski: Autorzy uważają, że radykalne leczenie chirurgiczne powinno być stosowane u wszystkich pacjentów z agresywną postacią centralnego ziarniniaka olbrzymiokomórkowego. Z uwagi na częste naciekanie kości w tych przypadkach, zalecają rekonstrukcję wolnymi płatami kostnymi z mikrozespoleniem naczyniowym. Pozwala to na uzyskanie radykalności onkologicznej w połączeniu z dobrym efektem kosmetycznym. Jakość życia i estetyka twarzy może być udoskonalona dzięki implantom zębowym, zastosowanym po odpowiednim czasie remisji choroby. W całościowym leczeniu onkologicznym ogromną rolę odgrywa ścisła, regularna kontrola w ośrodku prowadzącym

Classification of Thyroid Tumors Based on Mass Spectrometry Imaging of Tissue Microarrays; a Single-Pixel Approach

The primary diagnosis of thyroid tumors based on histopathological patterns can be ambiguous in some cases, so proper classification of thyroid diseases might be improved if molecular biomarkers support cytological and histological assessment. In this work, tissue microarrays representative for major types of thyroid malignancies&mdash;papillary thyroid cancer (classical and follicular variant), follicular thyroid cancer, anaplastic thyroid cancer, and medullary thyroid cancer&mdash;and benign thyroid follicular adenoma and normal thyroid were analyzed by mass spectrometry imaging (MSI), and then different computation approaches were implemented to test the suitability of the registered profiles of tryptic peptides for tumor classification. Molecular similarity among all seven types of thyroid specimens was estimated, and multicomponent classifiers were built for sample classification using individual MSI spectra that corresponded to small clusters of cells. Moreover, MSI components showing the most significant differences in abundance between the compared types of tissues detected and their putative identity were established by annotation with fragments of proteins identified by liquid chromatography-tandem mass spectrometry in corresponding tissue lysates. In general, high accuracy of sample classification was associated with low inter-tissue similarity index and a high number of components with significant differences in abundance between the tissues. Particularly, high molecular similarity was noted between three types of tumors with follicular morphology (adenoma, follicular cancer, and follicular variant of papillary cancer), whose differentiation represented the major classification problem in our dataset. However, low level of the intra-tissue heterogeneity increased the accuracy of classification despite high inter-tissue similarity (which was exemplified by normal thyroid and benign adenoma). We compared classifiers based on all detected MSI components (n = 1536) and the subset of the most abundant components (n = 147). Despite relatively higher contribution of components with significantly different abundance and lower overall inter-tissue similarity in the latter case, the precision of classification was generally higher using all MSI components. Moreover, the classification model based on individual spectra (a single-pixel approach) outperformed the model based on mean spectra of tissue cores. Our result confirmed the high feasibility of MSI-based approaches to multi-class detection of cancer types and proved the good performance of sample classification based on individual spectra (molecular image pixels) that overcame problems related to small amounts of heterogeneous material, which limit the applicability of classical proteomics

Image_1_Proteomic and metabolomic signatures of rectal tumor discriminate patients with different responses to preoperative radiotherapy.pdf

BackgroundNeoadjuvant radiotherapy (neo-RT) is widely used in locally advanced rectal cancer (LARC) as a component of radical treatment. Despite the advantages of neo-RT, which typically improves outcomes in LARC patients, the lack of reliable biomarkers that predict response and monitor the efficacy of therapy, can result in the application of unnecessary aggressive therapy affecting patients’ quality of life. Hence, the search for molecular biomarkers for assessing the radio responsiveness of this cancer represents a relevant issue.MethodsHere, we combined proteomic and metabolomic approaches to identify molecular signatures, which could discriminate LARC tumors with good and poor responses to neo-RT.ResultsThe integration of data on differentially accumulated proteins and metabolites made it possible to identify disrupted metabolic pathways and signaling processes connected with response to irradiation, including ketone bodies synthesis and degradation, purine metabolism, energy metabolism, degradation of fatty acid, amino acid metabolism, and focal adhesion. Moreover, we proposed multi-component panels of proteins and metabolites which could serve as a solid base to develop biomarkers for monitoring and predicting the efficacy of preoperative RT in rectal cancer patients.ConclusionWe proved that an integrated multi-omic approach presents a valid look at the analysis of the global response to cancer treatment from the perspective of metabolomic reprogramming.</p

Image_6_Proteomic and metabolomic signatures of rectal tumor discriminate patients with different responses to preoperative radiotherapy.pdf

BackgroundNeoadjuvant radiotherapy (neo-RT) is widely used in locally advanced rectal cancer (LARC) as a component of radical treatment. Despite the advantages of neo-RT, which typically improves outcomes in LARC patients, the lack of reliable biomarkers that predict response and monitor the efficacy of therapy, can result in the application of unnecessary aggressive therapy affecting patients’ quality of life. Hence, the search for molecular biomarkers for assessing the radio responsiveness of this cancer represents a relevant issue.MethodsHere, we combined proteomic and metabolomic approaches to identify molecular signatures, which could discriminate LARC tumors with good and poor responses to neo-RT.ResultsThe integration of data on differentially accumulated proteins and metabolites made it possible to identify disrupted metabolic pathways and signaling processes connected with response to irradiation, including ketone bodies synthesis and degradation, purine metabolism, energy metabolism, degradation of fatty acid, amino acid metabolism, and focal adhesion. Moreover, we proposed multi-component panels of proteins and metabolites which could serve as a solid base to develop biomarkers for monitoring and predicting the efficacy of preoperative RT in rectal cancer patients.ConclusionWe proved that an integrated multi-omic approach presents a valid look at the analysis of the global response to cancer treatment from the perspective of metabolomic reprogramming.</p

Table_5_Proteomic and metabolomic signatures of rectal tumor discriminate patients with different responses to preoperative radiotherapy.xlsx

BackgroundNeoadjuvant radiotherapy (neo-RT) is widely used in locally advanced rectal cancer (LARC) as a component of radical treatment. Despite the advantages of neo-RT, which typically improves outcomes in LARC patients, the lack of reliable biomarkers that predict response and monitor the efficacy of therapy, can result in the application of unnecessary aggressive therapy affecting patients’ quality of life. Hence, the search for molecular biomarkers for assessing the radio responsiveness of this cancer represents a relevant issue.MethodsHere, we combined proteomic and metabolomic approaches to identify molecular signatures, which could discriminate LARC tumors with good and poor responses to neo-RT.ResultsThe integration of data on differentially accumulated proteins and metabolites made it possible to identify disrupted metabolic pathways and signaling processes connected with response to irradiation, including ketone bodies synthesis and degradation, purine metabolism, energy metabolism, degradation of fatty acid, amino acid metabolism, and focal adhesion. Moreover, we proposed multi-component panels of proteins and metabolites which could serve as a solid base to develop biomarkers for monitoring and predicting the efficacy of preoperative RT in rectal cancer patients.ConclusionWe proved that an integrated multi-omic approach presents a valid look at the analysis of the global response to cancer treatment from the perspective of metabolomic reprogramming.</p