Comparative Analysis of miRNA and EMT Markers in Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignant condition in the world. Micro RNAs (miRNAs) as well as epithelial to mesenchymal transition (EMT) play an important role in the pathogenesis of CRC. We performed a comparative analysis of the expression of selected miRNA genes and EMT markers in bioptic samples from patients (n = 45) with primary CRC or metastatic (m)CRC to the regional lymph node using reverse transcription-quantitative PCR and IHC staining. Results: Out of all miRNA analyzed, the miR-17 expression was most significantly different and associated with lower risk of CRC spread to the lymph node. In addition, significant relationships were found between the tumor side localization and several miRNAs expressions (miR-9, miR-29b, miR-19a, miR-19b, miR-21, miR-106a, miR-20a and miR-17). In addition, of the examined EMT markers, only VEGFA expression correlated with tumor progression (tumor grade G2). In the examined set of patient samples and their matched healthy tissue, several specific molecular markers (miRNAs associated with EMT and tumor progression) were identified with a promising prognostic potential. Their further examination in larger patient cohorts is planned to validate the present data.

Pitx2 is a useful marker of midgut-derived neuroendocrine tumours - an immunohistochemical study of 224 cases.

Pitx2 is a transcription factor responsible for establishment of the right-left axis and development of the gut and pituitary. In mouse embryos, Pitx2 is expressed in the greater curvature of the stomach and midgut. Previously, Pitx2 was studied in pituitary neuroendocrine tumours but not in other NETs. Pitx2 expression was immunohistochemically assessed in whole sections and tissue microarrays in a cohort of 224 neuroendocrine neoplasms, and was analysed in 29 cases. The cohort included 18 cauda equina NETs, 38 paragangliomas, 98 cases of primary visceral NETs from different organs, 23 metastases of visceral NETs and 47 neuroendocrine carcinomas (NECs). Pitx2 expression was observed in 29.5% (29 of 98) NETs and 14.9% (7 of 47) NECs, but was not observed in any paraganglioma or cauda equina NET. Pitx2 was observed only in tumours of midgut-derived organs, including the small intestine (100%, 20 of 20), appendix (88.9%, eight of nine) and large intestine (9.1%, one of 11 - only caecal NET). The NETs of remaining locations were negative. Pitx2 was 96.7% sensitive and 100% specific for NETs of midgut origin. In NECs, Pitx2 positivity was observed in goblet cell adenocarcinoma (75%, three of four), medullary thyroid carcinoma (42.9, three of seven) and one Merkel cell carcinoma (25%, one of four). In metastatic NETs, Pitx2 was observed in all the tumours originating in the small intestine (n = 17) or caecum (n = 1). No positivity was observed in tumours from other locations (four pancreas, one lung). We observed no correlation between immunoreactivity and mRNA expression. Thus, Pitx2 immunohistochemistry can be helpful in assessing the midgut origin of NETs.

Is obesity a risk factor for coronary atherosclerosis?

A group of 279 adult autopsy patients (66 obese with BMI 30, versus 213 nonobese with BMI < 30) was retrospectively studied for the relation between body weight and coronary artery atherosclerosis. In the obese group, there was slightly higher grade of coronary narrowing than in the nonobese (2.31/2.5 versus 2.12/2). With increasing BMI in the obese, there was a statistically significant trend for milder coronary atherosclerosis, with least involvement in the extremely obese (BMI > 50). It seems that increased body weight by itself has little impact on coronary atherosclerosis, and extreme obesity may even by protective from it.

Oxaliplatin and irinotecan induce heterogenous changes in the EMT markers of metastasizing colorectal carcinoma cells.

In patients with advanced colorectal cancer (CRC), surgery is complemented with systemic therapy - chemotherapy and radiochemotherapy. Although the patients' overall survival has been significantly improved, tumor resistance is still a frequent cause of chemotherapy failure. Several factors contribute to chemoresistance of tumor cells including changes related with epithelial-mesenchymal transition (EMT). The present study was designed to verify the presence of EMT markers in paired CRC primary cell lines obtained from primary tumor sites and lymph node metastases of three patients and to investigate the effect of irinotecan and oxaliplatin treatment on these markers as well. The samples of the higher stage of CRC and positive for angioinvasion were selected and qPCR, western blot analysis, migration assay, cytotoxicity testing was performed. Results confirmed the increased expression of several markers characteristic of EMT and invasiveness in lymph node metastatic cells, with a significant variability between individual samples. Irinotecan and oxaliplatin decreased migration activity of the cells and to the varying degree affected the expression of EMT and invasiveness markers. In conclusion, in CRC EMT is present in metastatic cells over a phenotypic continuum whose expression is altered heterogeneously upon irinotecan and oxaliplatin treatment.

[Identification of an optimal algorithm for effective diagnostics of non-small cell lung cancer with ALK gene rearrangement - implementation of the method and practical experiences with routine diagnostics]. / Stanovení optimálního vysetrovacího algoritmu pro efektivní vyhledávání nemalobunecných karcinomu plic s prestavbou genu ALK - zavedení metodiky a praktické zkusenosti z rutinního vysetrování.

The aim of the retrospective part of the study was a) to select an optimal clone of immunohistochemical (IHC) antibody against the ALK protein with specificity and sensitivity high enough to use this antibody as a screening method for selecting non-small cell lung cancer (NSCLC) cases for fluorescence in situ hybridization (FISH) testing of ALK gene rearrangement and b) to determine the diagnostic yield of "small" biopsies i.e. endobronchial, transbronchial and transthoracic biopsies and cytoblocks for ALK gene rearrangement testing. The best IHC method of ALK protein detection (clone D5F3, dilution 1:100, Cell Signaling Technology, Danvers, MA, USA) was then verified in prospective routine testing of patients with NSCLC. ALK status was correlated with tumor morphology and clinical data. In the retrospective part of the study, 170 EGFR-nonmutated cases of NSCLC were IHC and FISH tested. In the prospective part, 557 cases of NSCLC were tested by IHC and 76 by FISH. There were 8/154 (5.2%) cases with ALK gene rearrangement detected in the retrospective part and 24/557(4.3 %) in the prospective part. Sensitivity and specificity of the best IHC method were 100 % and 99 % in the retrospective part and 100 % and 80 % in the prospective part. The diagnostic yield of "small" biopsies was between 74 - 80 % retrospectively, depending on IHC variant, and 88 % prospectively. No case with ALK gene rearrangement detected prospectively had EGFR mutation. A high diagnostic yield confirms that ALK status testing can be used in this type of specimen. A prevalence of 5.2 % in the retrospective part (EGFR-nonmutated cases) and 4.3 % in the prospective part (without known EGFR mutation status), tumor morphology (solid and acinar type, mucinous type or at least partial mucin production (extra- and/or intracellular) as well as lower average age and male/female ratio of patients with ALK positive tumors in the prospective part (57.5 y vs. 65.2 y, 8 men and 16 women vs. 336 men and 197 women) are consistent with global data.

Contribution of in vitro comparison of colorectal carcinoma cells from primary and metastatic lesions to elucidation of mechanisms of tumor progression and response to anticancer therapy.

Colorectal cancer has been a leading cause of cancer-related morbidity and mortality. For the research and individualization of therapy, primary cell lines of the colorectal cancer appear to be still an invaluable tool. We evaluated the differences in metastatic potential between four isolated primary colon cancer cells and cells derived from their lymph node metastasis. These results were compared with correspond immortalized cells-SW480 and SW620, respectively. The ability to migrate was tested using real-time measurement in xCELLigence system. Expressions of molecules involved in adhesion and invasion processes were examined using RT-PCR and western blot analysis. Furthermore, impact of cytotoxic effect of selected chemotherapeutics (irinotecan, oxaliplatin) and biological therapy (bevacizumab, cetuximab, panitumumab) was assessed by the WST assay. As expected, cell lines derived from lymph node migrated more aggressively and higher expression of adhesion molecules ICAM-1, EpCAM, and N-cadherin was detected. The expression of MMP-2 and -9 was elevated, on the other hand, in cell lines derived from primary tumor cancer cells as well as the expression of miR-21, miR-29a, and miR-200a. The most pronounced cytotoxic effect has been recorded with oxaliplatin and irinotecan (IC50 = 48.23 resp. 0.11 µg/ml), especially in cells originating from lymph node metastases. In total, comparison of isolated cell lines and immortalized cell lines has shown many similarities, as well as several differences. Adhesion/invasion molecules and several miRNAs, which play an important role in tumor development and the invasive and migratory behavior, could be a useful therapeutic target in malignant colorectal cancer.

Isolated thoracic aortitis.

Isolated thoracic aortitis is a new pathological entity. We review the histopathological features of this disease, the role of imaging, and diagnostic modalities necessary to make the diagnosis of aortitis and discuss the management of patients with an established diagnosis of isolated thoracic aortitis.

Imaging the elemental distribution within human malignant melanomas using Laser-Induced Breakdown Spectroscopy.

The diagnosis of malignant melanoma, often an inconspicuous but highly aggressive tumor, is most commonly done by histological examination, while additional diagnostic methods on the level of elements and molecules are constantly being developed. Several studies confirmed differences in the chemical composition of healthy and tumor tissue. Our study presents the potential of the LIBS (Laser-Induced-Breakdown Spectroscopy) technique as a diagnostic tool in malignant melanoma (MM) based on the quantitative changes in elemental composition in cancerous tissue. Our patient group included 17 samples of various types of malignant melanoma and one sample of healthy skin tissue as a control. To achieve a clear perception of results, we have selected two biogenic elements (calcium and magnesium), which showed a dissimilar distribution in cancerous tissue from its healthy surroundings. Moreover, we observed indications of different concentrations of these elements in different subtypes of malignant melanoma, a hypothesis that requires confirmation in a more extensive sample set. The information provided by the LIBS Imaging method could potentially be helpful not only in the diagnostics of tumor tissue but also be beneficial in broadening the knowledge about the tumor itself.

Assessing spatial distribution of bioindicator elements in various cutaneous tumors using correlative imaging with laser-ablation-based analytical methods.

Correlative imaging of cutaneous tumors provides additional information to the standard histopathologic examination. However, the joint progress in the establishment of analytical techniques, such as Laser-Induced Breakdown Spectroscopy (LIBS) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) in clinical practice is still limited. Their combination provides complementary information as it is also shown in our study in terms of major biotic (Ca, Mg, and P) and trace (Cu and Zn) elements. To elucidate changes in the elemental composition in tumors, we have compiled a set of malignant tumors (Squamous Cell Carcinoma, Basal Cell Carcinoma, Malignant Melanoma, and Epithelioid Angiosarcoma), one benign tumor (Pigmented Nevus) and one healthy-skin sample. The data processing was based on a methodological pipeline involving binary image registration and affine transformation. Thus, our paper brings a feasibility study of a practical methodological concept that enables us to compare LIBS and LA-ICP-MS results despite the mutual spatial distortion of original elemental images. Moreover, we also show that LIBS could be a sufficient pre-screening method even for a larger number of samples according to the speed and reproducibility of the analyses. Whereas LA-ICP-MS could serve as a ground truth and reference technique for preselected samples.

Cardiac sarcoidosis.

Cardiac sarcoidosis is a rare entity and may be difficult to diagnose prior to cardiac surgery. We review the imaging and diagnostic studies necessary to make the diagnosis and discuss therapeutic algorithms to manage this disease.

Histopathological findings of diseased ascending aortae with clinicopathological correlation - A single-centre study of 160 cases.

The most common reason for ascending aorta resection is an aneurysm or dissection. Aortic dissection is a life-threatening condition in which an aneurysm is a crucial risk factor. The essential criteria for aneurysm resection include the diameter, genetic predisposition, and aortic valve disease. This study aimed to compare the histological findings in aneurysms and dissections and correlate them with clinical parameters to determine whether histopathological findings correspond with the current clinical approach. A total of 160 ascending aorta surgical specimens, separate or with an aortic valve, were collected and divided into four groups: aneurysm-tricuspid (n = 40; median 67 y), aneurysm-malformed (n = 68; median 50 y), dissection-tricuspid (n = 48; median 65.5 y), and dissection-malformed (n = 4; median 52.5 y). Male preponderance was observed in all groups; the youngest patients were in the aneurysm-malformed group. None of the specimens showed normal aortic histology. The most common finding in the aortic samples was medial degeneration, which was the most severe and most common in dissection. The mildest findings were found in the aneurysm-malformed group. Atherosclerosis was predominant and most severe in the aneurysm-tricuspid group, while only mild in both dissection groups, suggesting its protective effect against this complication. Chronic aortitis was the least common pathology, found only in the aneurysm-tricuspid group. The aortic valve was resected and examined simultaneously with the ascending aorta in 76 cases, most commonly in the aneurysm-malformed group (n = 53). Myxoid degeneration was the major finding in the tricuspid aortic valves, with calcifications in the malformed. Comparing the histopathological results with the clinical aspects, aneurysms with a malformed aortic valve seem to be managed appropriately, with the findings not reaching the severity as in patients with a tricuspid valve. In contrast, in patients with a tricuspid valve, there were more dissections than aneurysms, with a significant subset of aneurysms showing histological findings almost identical to those of dissections. Supported by histological findings, patients with a diseased ascending aorta and tricuspid aortic valve represent an underdiagnosed risk group that would benefit from earlier diagnosis and intervention to prevent dissection. There is a need to find a marker for dissection risk other than the aortic diameter.

Leiomyoma with Bizarre Nuclei: a Study of 108 Cases Focusing on Clinicopathological Features, Morphology, and Fumarate Hydratase Alterations.

Leiomyoma with bizarre nuclei (LBN) is an uncommon variant of uterine smooth muscle neoplasm. Involvement of fumarate hydratase (FH) has been suggested in the pathogenesis of a subset of LBN. The goal of our study is to assess the clinicopathological, morphological, immunohistochemical and molecular findings focusing on FH in LBNs (n = 108) and compare it with the findings in usual leiomyomas (UL; n = 50) and leiomyosarcomas (LMS; n = 42). Immunohistochemically, loss of FH expression was found in 67/108 of LBN, 1/50 of UL and in no LMS. Class 4/5 FH mutations were detected in 15/53 LBN with sufficient DNA quality for molecular analysis. Pathogenic variants of the FH gene were detected in neither UL nor LMS. Local recurrence after surgery was present in 18/92 of LBN patients, 7 of which were histologically verified and 2 of which were found to be LBN. Our results confirmed that LBN behave in a benign fashion, although they may relapse. FH gene mutations were a common finding only in LBN, but not in UL and LMS. Immunohistochemistry with an antibody against FH seems to have a good sensitivity (87%) and moderate specificity (58%) with regard to predicting FH gene mutations and could be used as a screening method in tumors with features suggestive of FH alterations to identify patients who are at risk for the FH aberrations.

Derivation and basic characterization of colorectal carcinoma primary cell lines.

BACKGROUND: Colorectal carcinoma is one of the most common malignancies in western countries. Among different approaches to its research, primary cancer cell lines can play an important role. AIM: The main purposes of this study were: 1) to establish an effective and reproducible method of colorectal cancer cell isolation and cultivation from primary tumours and lymph node metastases and 2) to elucidate the biological features of the tumours favouring successful cell cultivation. MATERIALS AND METHODS: The tumour cells were obtained from colectomy specimens. Primary tumour and lymph node metastasis tissue was used for establishing the tissue cultures. Colectomy samples were further processed for routine histopathological assessment: tumour grade, stage, angioinvasion and perineural spread were evaluated. Features of tissue culture cells were assessed using phase contrast microscopy and immune-histochemical techniques. WST-1 assay and X-CELLigence real time analysis were carried out for viability and proliferation testing before and after treatment with irinotecan and oxaliplatin. Molecular features of the tumour including K-RAS/B-RAF/N-RAS mutations were tested using allele-specific PCR. Results of the cultivation process were compared to the histopathological and molecular features of the tumours. RESULTS: In total, we obtained 33 samples from the primary site of tumours and 20 samples from lymph node metastases; in total, 27 cell lines were successfully isolated. Morphologic features characteristic of tumour cells in primary cell lines and epithelial differentiation (positive for AE1/AE3 cytokeratin) were evaluated. Higher tumour stage, angioinvasion and presence of perineural spread in primary tumour correlated positively with successful cell isolation from lymph node metastasis. All samples tested were NRAS wild-type. No correlation was found between molecular phenotype and the cell culture features. A higher proliferation potential was observed in the primary tumour cells, whereas higher sensitivity to irinotecan was found in the lymph node metastatic cells. CONCLUSIONS: Using mechanical dissociation, we successfully derived and cultivated CRC cells from primary tumours and lymph node metastases with success rate 3 % and 70% respectively. Primary tumour features favouring successful establishment of cell cultures were identified.

Calcific aortic valve stenosis: Immunohistochemical analysis of inflammatory infiltrate.

Calcific aortic valve disease is considered a form of atherosclerosis and, like the latter, possibly of inflammatory origin. The aim of our work was to study the pattern of cellular infiltrate in calcific aortic valve stenosis (CAS). Fifteen operatively excised calcified aortic valves were examined by histology and immunohistochemistry (CD20, CD79α, CD3, CD4, CD8, CD68, CD138, CD117, BJK, BJL, IgA, IgD, IgG, IgG4 and IgM). The findings revealed that in CAS, there were chronic inflammatory features with infiltrates comprising lymphocytes, polyclonal plasma cells, histiocytes and mast cells. In T-lymphocytes, CD4 prevailed over CD8. In B-lymphocytes, there was a slight preponderance of CD20 over CD79α. The BJL (lambda)-positive plasma cells prevailed over the BJK (kappa) ones. The CD138-positive plasma cells comprised 24% IgA-, 20% IgD-, 41% IgG- (including 11% of IgG4-) and 15% IgM-positive cells. CAS did not fulfill the criteria of the recently described clinicopathological entity IgG4-related sclerosing systemic disease. The inflammatory process was the same in both subsets of CAS - those with trileaflet (normally formed) valves and those with congenitally bicuspid valves.