Altered expression of 17­ß­hydroxysteroid dehydrogenase type 2 and its prognostic significance in non­small cell lung cancer.

Numerous studies have reported that oestrogens may contribute to the development of non­small cell lung cancer (NSCLC). Although different steroidogenic enzymes have been detected in the lung, the precise mechanism leading to an exaggerated accumulation of active oestrogens in NSCLC remains unexplained. 17­ß­Hydroxysteroid dehydrogenase type 2 (HSD17B2) is an enzyme involved in oestrogen and androgen inactivation by converting 17­ß­oestradiol into oestrone, and testosterone into 4­androstenedione. Therefore, the enzyme serves an important role in regulation of the intracellular availability of active sex steroids. This study aimed to determine the expression levels of HSD17B2 in lung cancer (LC) and adjacent histopathologically unchanged tissues obtained from 161 patients with NSCLC, and to analyse the association of HSD17B2 with clinicopathological features. For that purpose, reverse transcription­quantitative PCR, western blotting and immunohistochemistry were conducted. The results revealed that the mRNA and protein expression levels of HSD17B2 were significantly decreased in LC tissues compared with matched controls (P<10­6). Conversely, strong cytoplasmic staining of HSD17B2 was detected in the unchanged respiratory epithelium and in glandular cells. Notably, a strong association was detected between reduced HSD17B2 expression and advanced tumour stage, grade and size. Furthermore, it was revealed that HSD17B2 may have potential prognostic significance in NSCLC. A log­rank test revealed the benefit of high HSD17B2 protein expression for the overall survival (OS) of patients (P=0.0017), and multivariate analysis confirmed this finding (hazard ratio=0.21; 95% confidence interval=0.07­0.63; P=0.0043). Stratified analysis in the Kaplan­Meier Plotter database indicated that patients with higher HSD17B2 expression presented better OS and post­progression survival. This beneficial effect was particularly evident in patients with adenocarcinoma and during the early stages of NSCLC. Decreased expression of HSD17B2 appears to be a frequent feature in NSCLC. Retrospective analysis suggests that the HSD17B2 mRNA and protein status might be independent prognostic factors in NSCLC and should be further investigated.

Klotho expression and nodal involvement as predictive factors for large cell lung carcinoma.

INTRODUCTION: Klotho has been recently described as a carcinogenesis suppressor. Large cell neuroendocrine lung carcinoma (LCNEC) is a rare, highly malignant neoplasm. In the light of increasing incidence of neuroendocrine tumours, biomarkers predicting survival are needed. We consider that Klotho might be one. MATERIAL AND METHODS: We analysed records of all patients diagnosed with LCNEC, atypical carcinoid and typical carcinoid operated on in our institution between 2007 and 2015. Initially, we found 134 cases. Forty-six specimens were unattainable and thus excluded from research. All patients diagnosed with LCNEC according to the WHO classification were included in the study. Immunohistochemical staining for Klotho was performed. We retrospectively reviewed patient charts and analysed multiple variables. RESULTS: Positive staining for Klotho was present in 36 tissue specimens, while 12 patients were Klotho-negative. Survival length was significantly higher in Klotho-positive cases (p = 0.024), while advanced nodal status (N1 and N2) represented a marker of poor outcome (p = 0.011). In multivariate analysis, both Klotho presence (p = 0.015; HR = 0.37; 95% CI: 0.17-0.86) and nodal involvement (p = 0.007; HR = 3.04; 95% CI: 1.37-6.82) were independent prognostic factors. Tumour vessel invasion and visceral pleura infiltration were not associated with worse treatment results. Klotho presence predicted a favourable prognosis in these groups (p = 0.018; p = 0.007). CONCLUSIONS: Our results suggest that Klotho might be a positive factor for predicting survival in LCNEC and nodal involvement a negative one. Thus, these two markers may assist in the selection of subjects with unfavourable prognosis and to personalise therapy regimens.

The role of immunohistochemical examination in diagnosis of papillary thyroid cancer in struma ovarii.

INTRODUCTION: Struma ovarii (SO) is a monodermal teratoma in which thyroid tissue comprises more than 50% of the tumour. Papillary thyroid cancer (PTC) in SO is a rare finding, as only 5% of SO cases undergo malignant transformation. Malignant SO is usually asymptomatic and infrequently diagnosed preoperatively. Because of its rarity, there is no consensus about diagnosis and management, while treatment and follow-up procedures are not clearly established. MATERIAL AND METHODS: Herewith, we report two cases of PTC in SO. The first patient was a 25-year-old woman diagnosed with bilateral ovarian tumours. The second patient, 19-year-old woman, presented with unilateral ovarian mass. Both patients were qualified for surgical excision of the tumours. Histopathological specimens underwent both conventional histopathological assessment and immunohistochemical staining. RESULTS: In the first patient histopathology revealed SO with two foci of PTC. Immunohistochemically a positive expression of CK7, CK19, p63 and thyroglobulin (Tg) confirmed the diagnosis. She underwent total thyroidectomy in 2016 in order to enable ablative radioiodine therapy and facilitate further thyroglobulin monitoring. Unfortunately, the patient was lost from follow-up. In the second patient, histopathological diagnosis was follicular variant of PTC in SO. Postoperatively, a pelvic CT revealed osteolytic lesion 6 cm in size, being a metastatic change. The patient underwent unilateral ovariectomy, total thyroidectomy and multiple cycles of radioiodine therapy. Currently, 9 years following the diagnosis, the patient achieved disease remission. CONCLUSIONS: PTC in SO still remains a diagnostic and therapeutic challenge. Immunostaining for CK7, CK19, p63 and Tg might be helpful in histopathological diagnosis. The decision on the need of total thyroidectomy and radioiodine therapy should be made individually. However, thyroid remnant ablation increases the sensitivity and specificity of follow-up testing using serum Tg level as a tumour marker.

18F-FDG PET/CT and nestin expression as prognostic tools in pulmonary neuroendocrine tumours.

OBJECTIVES: The aim of this study was to investigate the fluorine-18-fluorodeoxyglucose (F-FDG) uptake on integrated PET [PET/computed tomography (CT)] images and its correlation with nestin expression in a series of neuroendocrine lung tumours. As the incidence of neuroendocrine lung tumours is rising, tools predicting diagnosis, outcome and assisting in the selection of treatment regimens are needed. PATIENTS AND METHODS: We reviewed records of patients diagnosed with large cell neuroendocrine lung carcinoma, atypical carcinoid and typical carcinoid who were operated upon in our institution. Into the study, we included those who underwent F-FDG PET/CT before the operation. Immunohistochemical staining for nestin was performed. We retrospectively reviewed patient charts and analyzed multiple variables. RESULTS: Maximal standardized uptake value (SUVmax) was significantly higher in poorly differentiated than in well-differentiated tumours (P<0.001). The estimated SUVmax cut-off value, which distinguishes large cell neuroendocrine lung carcinoma from carcinoid with the highest sensitivity and specificity (88.6%; 85%), was 6.3. Positivity of the tumour on F-FDG PET/CT was associated with shorter survival of the patient (P=0.0057). Multivariate analysis showed that nodal involvement and SUVmax were predictors of adverse outcome. Nestin expression did not correlate with lymph node metastases (P=0.97), SUVmax (P=0.9), maximal size of the lesion (P=0.49) or Ki-67 (P=0.93). Nestin expression did not influence survival on multivariate analysis. CONCLUSION: The study revealed a comparable expression of nestin in tumours with different activity of glucose metabolism measured by F-FDG uptake at PET/CT. It did not show any significant influence of nestin expression on survival. The study confirmed that F-FDG PET/CT is useful in the preoperative evaluation of patients with pulmonary neuroendocrine tumours.

Clinical significance of nestin and its association with survival in neuroendocrine lung tumours.

Nestin is considered to be a cancer stem cell marker. Nestin expression in neuroendocrine tumours might be useful to predict prognosis and facilitate treatment planning. 88 patients with neuroendocrine lung tumours operated in the Department of Thoracic Surgery from 2007 to 2015 were included into the study. Immunohistochemical staining for nestin was performed. Clinicopathological and survival data were retrospectively analyzed. Nestin expression was detected in 15 (17%) specimens. Multivariate analysis showed that lymph node metastases (p = 0.0001; hazard ratio (HR) = 3.93; confidence interval (CI) 95%: 1.96-7.87), nestin expression (p = 0.034; HR = 2.30; CI 95%: 1.06-4.99) and patient's age (p = 0.024; HR = 1.04; CI 95%: 1.00-1.09) were independent negative prognostic factors. Nestin expression was significantly higher in large cell neuroendocrine carcinoma when compared with carcinoids (p = 0.001). Collected data support the thesis that nestin can be regarded as a biomarker in patients with neuroendocrine lung tumours.

Decreased expression of connective tissue growth factor in non-small cell lung cancer is associated with clinicopathological variables and can be restored by epigenetic modifiers.

PURPOSE: Recent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC. METHODS: We used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay. RESULTS: We found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation. CONCLUSIONS: Decreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.

Adverse histopathological findings in glottic cancer with anterior commissure involvement.

Open partial horizontal laryngectomy (OPHL) specimens include cartilage and lymph nodes. Pathological adverse findings (PAF): perichondrium, cartilage, perineural invasion, microvessel spread and prelaryngeal metastases can be detected histologically. We aimed at examining PAF in OPHL specimens and examining the interdependence with oncological outcomes. Prospective analysis of 254 glottis cancers: 87-T2a, 77-T2b and 90-T3 with anterior commissure (AC) involvement treated by OPHL at tertiary referral centre between 2001 and 2008. In 38/254 patients (15 %) PAF were found (16 prelaryngeal metastases, 22 other); more often in stage T2b/T3 versus T2a (p = 0.008). PAF other than prelaryngeal metastases were found more often in T2b than T2a tumours (p = 0.005). Outcomes revealed that out of 36 patients with local recurrence, 19 had PAF. Comparison of 216 patients with no PAF and 16 patients with prelaryngeal metastases revealed, respectively: 7.9 versus 81.3 % local recurrences (p = 0.034), 3.2 versus 68.8 % nodal recurrences (p = 0.011), 90.7 versus 43.8 % of 5-year organ preservation (p = 0.021) and 92.6 versus 75 % 5-year overall survival (p = 0.022). Out of 10 patients with close margins, 1 developed the local recurrence; impact for organ preservation and overall survival was not significant. In 22/254 cases including the cartilage into the operating specimen were therapeutically meaningful; in 16/254 with prelaryngeal metastases, the impact for larynx preservation and overall survival was significant. T2b patients had higher probability of PAF than T2a. PAF significantly influenced higher local recurrence rate. This finding ensures the authors, that OPHL type II is not the "overtreatment" compared to the TLM.

Increased expression of 17-beta-hydroxysteroid dehydrogenase type 1 in non-small cell lung cancer.

OBJECTIVES: Recent studies indicated that estrogens may influence the development of non-small cell lung cancer (NSCLC). The 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) catalyzes the reduction of estrone (E1) to the highly potent E2. Although the significance of aromatase in an intratumoral E2 production in NSCLC is well established, the role of HSD17B1 remains largely unknown. Therefore, we investigated the expression of HSD17B1 in lung cancerous and corresponding histopathologically unchanged tissues from NSCLC patients and the association between HSD17B1 expression and clinicopathological features. Than, we examined the biological significance of HSD17B1 in NSCLC cells in vitro. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) on HSD17B1 expression and activity. MATERIALS AND METHODS: We used Real Time quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry to evaluate HSD17B1 expression in tissues obtained from 48 patients with NSCLC. The methylation status of the promoter region of HSD17B1 in A549 and Calu-1 cells was evaluated by bisulfite sequencing. We investigated the effect of 5-dAzaC on HSD17B1 transcript levels (by RT-qPCR) and on HSD17B1 enzyme activity by measuring the conversion of E1 to E2. The xCELLigence System was used for monitoring of cell proliferation. RESULTS: We found a substantial increase of HSD17B1 mRNA and protein amount in NSCLC tissues compared with histopathologically unchanged tissues in the group of male patients. An overexpression of HSD17B1 was associated with squamous cell carcinoma and with lung cancer stage 3A. We showed that 5-dAzaC induces DNA demethylation of HSD17B1 promoter, leading to increased HSD17B1 mRNA levels and protein activity in NSCLC cells. It resulted in enhanced E2 production in both cell lines and supported the proliferation of Calu-1 cells but not A549 cells. CONCLUSION: Increased expression of HSD17B1 in NSCLC may contribute to an elevated intratissue level of E2 and consequently may support the development and spread of cancer.

Changes of B and T lymphocytes and selected apopotosis markers in Hashimoto's thyroiditis.

The aim was to assess changes of B and T lymphocytes and selected apoptotic markers in Hashimoto thyroiditis (HT) cases on the basis of quantitative immunohistochemical studies (CD20, CD43, CD8, Bcl-2, caspase-3). The control group comprised colloid goitres without inflammatory infiltrate taken from 10 female patients. Thyroid specimens were obtained retrospectively from 40 patients. The immunohistochemical reactions were subject to quantitative evaluation performed using image-processing methods, including a spatial visualisation of the markers' expression. The percentage of Bcl-2 reactions in HT (mean 3.65%, SD 2.94%) was significantly lower than in the control group (mean 13.99%, SD 5.04%), while the thyroid follicles in HT samples exhibited a higher degree of staining for caspase-3 (mean 1.10%, SD 1.03%) in contrast to normal control tissues (mean 0.48%, SD 1.02%). The results from this study indicate that apoptosis plays a major role in the patogenesis of autoimmune thyroid diseases containing the main pathogenic events in the lesion of thyroid follicular cells in HT. Moreover, the reactivity of CD43 and CD20 was significantly higher in Hashimoto disease, while CD8 was not significantly different from the control group.

Selected markers of proliferation and apoptosis in the parathyroid lesions: a spatial visualization and quantification.

The aim of the paper was to apply a method for quantitative assessment of proliferation and apoptosis markers, based on their 3D visualization, in cases of parathyroid adenoma and hyperplasia. Material was obtained from 49 patients (32 females and 17 males) with primary hyperparahyroidism. Quantitative immunohistochemistry studies of Ki-67, proliferating cell nuclear antigen (PCNA) and bcl-2 were performed on digital microscopy images with the use of 3D visualization. The use of spatial visualization method allowed us to perform objective quantitative assessment of the studied immunohistochemical markers. The average cell nuclear fraction of Ki67+ was 1.8% in hyperplasia and 1.9% in adenoma cases while 3.5% in the controls. The highest expression of PCNA was found in parathyroid hyperplasia (22.9%) and significantly decreased in adenoma (12.5%) and in the control group (16.8%). The lower expression of bcl-2 in hyperplasia cases (mean area fraction of 0.172 per 1 mum(2), in contrast to 0.643 in adenomas and 0.648 in control) suggested that principal cells can be ready for apoptosis and may confirm the important role of bcl-2 protein in etiopathogenesis of hyperplasia of the parathyroid gland while PCNA might be a useful marker for differentiating adenoma from early hyperplasia in primary hyperparahyroidism cases.

[The role of immunohistochemical staining (protein p53, cyclin D1) in the prognosis of adenoid cystic carcinoma salivary gland tumors]. / Rola badan immunohistochemicznych (bialka p53, cykliny D1) w prognozowaniu raka gruczolowato-torbielowatego (carcinoma adenoides cysticum) slinianek.

INTRODUCTION: Adenoid cystic carcinoma of the salivary glands (carcinoma adenoides cysticum) is malignant epithelial tumor of rare occurrence. Tumor of this kind has among salivary glands tumors uncertain prognosis and unpredictable course. The aim of the study was to characterize the patient population and the immunohistochemical analyses (p53 protein, cyclin D1). MATERIAL AND METHODS: The examined group consisted of 30 patients with adenoid cystic carcinoma of the salivary glands. The expression of p53 protein and D1 cyclin in the tumor was evaluated and the correlation between these proteins and the organ and clinical grading was defined. RESULTS: The immunohistochemical studies showed in 70% the positive staining for p53 protein and 90% for cyclin D1. There was not statistically significant difference between the advanced grading of the organic and clinical adenoid cystic carcinoma.

[Histological charakteristics of tongue and floor of the mouth neoplasms and their clinical prognostic value]. / Cechy histologiczne raków jezyka i dna jamy ustnej a prognozowanie kliniczne.

INTRODUCTION: The number of histological classifications has been introduced to asses the squamous cell carcinoma in head and neck. The Jakobsson histological system employs 8 different morphological characteristics. Four of them concerning tumor cell population and another four describe relation to the adjacent tissues. MATERIAL AND METHODS: The material comprised tissue specimens from 80 patients with squamous cell carcinoma of the tongue and floor of the mouth who underwent the primary surgery. All the data concerning Jakobsson classification, histological differentiation G, TNM classification, and recurrence and survival rates were recorded and analyzed. RESULTS: Any significant correlation has been found between Jakobsson classification and histological differentiation. Nevertheless statistically significant correlation between Jakobsson classification and T of the tumor, nodal metastases and local and nodal relapse has been recorded. CONCLUSIONS: The Jakobsson classification constitute the important supplement of the routine histological assessment and may significantly contribute to the choice of therapy.

[Correlation between ultrastructural changes and the presence of metastases in adenoid cystic carcinoma of salivary glands]. / Badania ultrastrukturalne a wystepowanie przerzutów w wezlach chlonnych w raku gruczolowato-torbielowatym slinianek.

Adenoid-cystic carcinoma is a malignant epithelial neoplasm which has different histological types. Still, an open question is, whether adenoid-cystic carcinoma of the salivary glands with metastases to lympho nodes and specific micro- and macroscopic features, allows us to define their progression. A group of 46 patients with adenoid-cystic cancer of the salivary glands was studied and was subjected to histological and ultrastructural assessment. In analysed group in 12 patient metastases to lympho nodes were confirmed. In 10 cases it was solid type of tumour and in 2 canaliculars type. In tumours with metastases to lympho nodes solid types prevailed and there was no a typical cribriform type. In all cases they were found fields of mixted texture of the tumours structure.