LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA.

BACKGROUND: The unmet challenge in prostate cancer (PCa) management is to discriminate it from benign prostate hyperplasia (BPH) due to the lack of specific diagnostic biomarkers. Contemporary research on potential PCa biomarkers is directed toward methylated cell-free DNA (cfDNA) from liquid biopsies since epigenetic mechanisms are strongly involved in PCa development. METHODS: In the present research, cfDNA methylation of the LGALS3 gene in blood and seminal plasma of PCa and BPH patients was assessed using pyrosequencing, as well as LGALS3 DNA methylation in tissue biopsies. Liquid biopsy samples were taken from patients with clinical suspicion of PCa, who were subsequently divided into two groups, that is, 42 with PCa and 55 with BPH, according to the histopathological analysis. RESULTS: Statistically significant higher cfDNA methylation of LGALS3 in seminal plasma of BPH than in PCa patients was detected by pyrosequencing. ROC curve analysis showed that it could distinguish PCa and BPH patients with 56.4% sensitivity and 70.4% specificity, while PSA did not differ between the two patient groups. In contrast, there was no statistically significant difference in LGALS3 cfDNA methylation in blood plasma between the two patient groups. In prostate tumor tissue, there was a statistically significant DNA hypermethylation of LGALS3 compared to surrounding nontumor tissue and BPH tissue. CONCLUSIONS: The DNA hypermethylation of the LGALS3 gene represents an event specific to PCa development. In conclusion, LGALS3 cfDNA methylation in seminal fluid discriminates early PCa and BPH presenting itself as a powerful novel PCa biomarker highly outperforming PSA.

CORRELATION OF EXPRESSION OF TGF- ß AND MMP2 BETWEEN PROSTATIC ADENOCARCINOMA AND ADJACENT UNAFFECTED PARENCHYMA.

In prostate adenocarcinoma, both tumorous stroma and epithelium have important role in tumor progression. Transforming growth factor beta (TGF- ß) is a promotor in advanced stages of prostate cancer. Matrix Metalloproteinase 2 (MMP2), the endopeptidase that degrades extracellular matrix is considered to be overexpressed in prostatic carcinoma related to its growth and aggressiveness. Therefore, the aim was to analyze the expression of proteins TGF- ß and MMP2 between both epithelium and stroma of prostatic adenocarcinoma and adjacent unaffected parenchyma. The intensity of TGF- ß and MMP2 expression in epithelium, tumorous stroma and adjacent unaffected parenchyma was analyzed in 62 specimens of prostatic adenocarcinoma by microarray-based immunohistochemistry. TGF- ß was more expressed in tumorous than in prostate stroma (p =0.000), while no statistical significance in case of MMP2 (p = 0.097) was found. MMP2 was more expressed in tumorous than in prostate epithelium (p =0.000), while no statistical significance in case of TGF- ß (p = 0.096) was observed. The study results indicate that both tumorous stroma and epithelium have a role in tumor progression and support potential role of TGF- ß and MMP2 in prostatic adenocarcinoma progression.

Different behaviour of DVL1, DVL2, DVL3 in astrocytoma malignancy grades and their association to TCF1 and LEF1 upregulation.

Key regulators of the Wnt signalling, DVL1, DVL2 and DVL3, in astrocytomas of different malignancy grades were investigated. Markers for DVL1, DVL2 and DVL3 were used to detect microsatellite instability (MSI) and gross deletions (LOH), while immunohistochemistry and immunoreactivity score were used to determine the signal strengths of the three DVL proteins and transcription factors of the pathway, TCF1 and LEF1. Our findings demonstrated that MSI at all three DVL loci was constantly found across tumour grades with the highest number in grade II (P = 0.008). Collectively, LOHs were more frequent in high-grade tumours than in low grade ones. LOHs of DVL3 gene were significantly associated with grade IV tumours (P = 0.007). The results on protein expressions indicated that high-grade tumours expressed less DVL1 protein as compared with low grade ones. A significant negative correlation was established between DVL1 expression and malignancy grades (P < 0.001). The expression of DVL2 protein was found similar across grades, while DVL3 expression significantly increased with malignancy grades (P < 0.001). The signal strengths of expressed DVL1 and DVL3 were negatively correlated (P = 0.002). However, TCF1 and LEF1 were both significantly upregulated and increasing with astrocytoma grades (P = 0.001). A positive correlation was established between DVL3 and both TCF1 (P = 0.020) and LEF1 (P = 0.006) suggesting their joint involvement in malignant progression. Our findings suggest that DVL1 and DVL2 may be involved during early stages of the disease, while DVL3 may have a role in later phases and together with TCF1 and LEF1 promotes the activation of Wnt signalling.

Somatostatin receptor SSTR2A and SSTR5 expression in neuroendocrine breast cancer.

Neuroendocrine breast cancer (NEBC) is a group of rare tumors, which could benefit from therapy targeting the somatostatin receptors (SSTRs). In particular, SSTR2A and SSTR5 are potential targets given their consistent expression in gastrointestinal and pancreatic primary and metastatic neuroendocrine cancers. Currently, there are no studies describing the expression of SSTRs in NEBC. The purpose of our study was to characterize the immunohistochemical expression of SSTR2A and SSTR5 in a cohort of NEBC. Thirty-one primary NEBC cases were analyzed, and SSTR2A and SSTR5 immunohistochemistry performed and scored using the modified immunoreactive score proposed by Remmele and Stanger. All patients were females with a mean age of 66.6 years (SD = 14). 77% of cases were histological grade 2. SSTR2A showed a weak positivity in 11 cases (35.5%), moderate positivity in 6 cases (19.4%) and strong positivity in 5 cases (16.1%). Nine cases were negative for SSTR2A (29%). SSTR5 showed a weak positivity in 16 cases (51.6%), moderate positivity in 6 cases (19.4%), while no cases showed strong positivity. Nine cases were negative for SSTR5 (29%). Five cases were negative for both SSTR2A and SSTR5. A weak to moderate SSTR2A and SSTR5 expression was observed in 50-70% of the cases. A subset of NEBCs with strong SSR2A expression may benefit from SSTRs targeted therapy. These results need further validation in a larger series including metastatic NEBC, to provide potential therapeutic targets for patients with advanced disease.

Genetic changes of MLH1 and MSH2 genes could explain constant findings on microsatellite instability in intracranial meningioma.

Postreplicative mismatch repair safeguards the stability of our genome. The defects in its functioning will give rise to microsatellite instability. In this study, 50 meningiomas were investigated for microsatellite instability. Two major mismatch repair genes, MLH1 and MSH2, were analyzed using microsatellite markers D1S1611 and BAT26 amplified by polymerase chain reaction and visualized by gel electrophoresis on high-resolution gels. Furthermore, genes DVL3 (D3S1262), AXIN1 (D16S3399), and CDH1 (D16S752) were also investigated for microsatellite instability. Our study revealed constant presence of microsatellite instability in meningioma patients when compared to their autologous blood DNA. Altogether 38% of meningiomas showed microsatellite instability at one microsatellite locus, 16% on two, and 13.3% on three loci. The percent of detected microsatellite instability for MSH2 gene was 14%, and for MLH1, it was 26%, for DVL3 22.9%, for AXIN1 17.8%, and for CDH1 8.3%. Since markers also allowed for the detection of loss of heterozygosity, gross deletions of MLH1 gene were found in 24% of meningiomas. Genetic changes between MLH1 and MSH2 were significantly positively correlated (p = 0.032). We also noted a positive correlation between genetic changes of MSH2 and DVL3 genes (p = 0.034). No significant associations were observed when MLH1 or MSH2 was tested against specific histopathological meningioma subtype or World Health Organization grade. However, genetic changes in DVL3 were strongly associated with anaplastic histology of meningioma (χ2 = 9.14; p = 0.01). Our study contributes to better understanding of the genetic profile of human intracranial meningiomas and suggests that meningiomas harbor defective cellular DNA mismatch repair mechanisms.

Loss of p53 expression is accompanied by upregulation of beta-catenin in meningiomas: a concomitant reciprocal expression.

Crosstalk between Wnt and p53 signalling pathways in cancer has long been suggested. Therefore in this study we have investigated the involvement of these pathways in meningiomas by analysing their main effector molecules, beta-catenin and p53. Cellular expression of p53 and beta-catenin proteins and genetic changes in TP53 were analysed by immunohistochemistry, PCR/RFLP and direct sequencing of TP53 exon 4. All the findings were analysed statistically. Our analysis showed that 47.5% of the 59 meningiomas demonstrated loss of expression of p53 protein. Moderate and strong p53 expression in the nuclei was observed in 8.5% and 6.8% of meningiomas respectively. Gross deletion of TP53 gene was observed in one meningioma, but nucleotide alterations were observed in 35.7% of meningiomas. In contrast, beta-catenin, the main Wnt signalling molecule, was upregulated in 71.2%, while strong expression was observed in 28.8% of meningiomas. The concomitant expressions of p53 and beta-catenin were investigated in the same patients. In the analysed meningiomas, the levels of the two proteins were significantly negatively correlated (P = 0.002). This indicates that meningiomas with lost p53 upregulate beta-catenin and activate Wnt signalling. Besides showing the reciprocal relationship between proteins, we also showed that the expression of p53 was significantly (P = 0.021) associated with higher meningioma grades (II and III), while beta-catenin upregulation was not associated with malignancy grades. Additionally, women exhibited significantly higher values of p53 loss when compared to males (P = 0.005). Our findings provide novel information about p53 involvement in meningeal brain tumours and reveal the complex relationship between Wnt and p53 signalling, they suggest an important role for beta-catenin in these tumours.

Brain metastases from lung cancer show increased expression of DVL1, DVL3 and beta-catenin and down-regulation of E-cadherin.

The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p=0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p=0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.

Leiomyosarcoma of the spermatic cord with scalp metastasis: case report and literature review.

Herein we present 82-year-old man with leiomyosarcoma arising from the spermatic cord with scalp metastasis, five years after primary surgical treatment. Complete surgical excision is required in such cases, as well as precise evaluation of further therapy. Paratesticular leiomyosarcoma is a rare entity, malignant mesenchimal tumor of smooth muscle differentiation. Although leiomyosarcomas of different localizations have well-known metastatic potential, cutaneous metastases are extremely rare with only 16 cases described in the literature. To our knowledge there are no reported cases of the paratesticular leiomyosarcoma metastatic to the skin. This article reviews the literature regarding paratesticular leiomyosarcoma presentation, diagnosis and treatment.

The Relationship between Tumor Budding and Tumor Deposits in Patients with Stage III Colorectal Carcinoma.

Background/Objectives: Recently, some new morphological features of colorectal cancer have been discovered as important prognostic factors; in this paper, we study the relationship between tumor budding (TB) and tumor deposits (TDs). Methods: The retrospective cohort study included 90 patients with pathohistologically confirmed stage III CRC who were treated with radical surgical resection. All hematoxylin and eosin (H and E)-stained slides from each patient were reviewed, and histological parameters were recorded. The samples were divided into two groups with similar sizes: a group without TDs (N = 51) and a control group with TDs (N = 39). The presence and TB grade were further analyzed in these groups and compared with other clinical and histological features. Results: The prevalence of TB in the investigated cohort was unexpectedly high (94.4%). Overall, there were 23 (25.6%) Bd1, 20 (22.2%) Bd2, and 47 (52.2%) Bd3 cases. The presence of TDs was significantly associated with a higher number of TB (p < 0.001, OR 16.3) and, consequently, with a higher TB grade (p = 0.004, OR 11.04). A higher TB grade (p = 0.001, HR 2.28; 95% CI 1.93-4.76) and a growing number of TDs (p = 0.014, HR 1.52; 95% CI 1.09-2.1) were statistically significantly associated with shorter survival. Conclusions: TDs appear more often in patients with higher TB grades in stage III CRC. A higher TB grade and a growing number of TDs were statistically significantly associated with shorter overall survival. These results could give additional emphasis to the importance of TB as an adverse prognostic factor since a strong relationship with TDs has been demonstrated.

Renal adenocarcinoma presenting as a spontaneous perirenal hematoma in a patient on warfarin therapy--case report and literature review.

We are presenting a rare case of a spontaneous extensive perirenal hematoma caused by ruptured renal adenocarcinoma in a patient who was on warfarin therapy because she had atrial fibrillation and three myocardial infarctions. A 77-year-old woman was admitted to our department with acute right flank pain and hemorrhagic shock. The anamnestic data revealed no trauma and hematuria. Abdominal ultrasonography and computed tomography scan showed large retroperitoneal hematoma. The patient underwent urgent surgery and radical nephrectomy was performed. A large retroperitoneal hematoma was found originating from a ruptured renal neoplasm in the upper pole of the right kidney. The pathohistological diagnosis was chromophobe renal cell carcinoma. The clinical, diagnostic and therapeutic peculiarities of this rare condition are presented, along with the literature review on the topic.

Flat Warts (Verrucae Planae) Confined to the Pigment of a Tattoo: A Rare Tattoo-associated Complication.

Tattoos are a form of decorative body art in which pigment dyes of different colors are inoculated into the skin. It is estimated that 15-25% of general population has one or more tattoos (1), and the reasons for the popularity of this procedure may include greater social acceptance, aesthetic appeal, or perhaps the option of using laser removal techniques to eliminate unwanted tattoos. Even though modern professional tattoos are usually performed in sterile conditions, complications still occur, and with increasing numbers of people getting tattoos, the incidence of tattoo-associated side-effects presenting to dermatologists, which may be as high as 2%, is likely to increase (2). Herein we present a case of a 43-year-old male patient with multiple HPV-associated flat warts (verrucae planae) confined to the black pigment of a tattoo done 15 years ago. A 43-year-old patient presented to our clinic due to eczema on the trunk. However, during skin examination, we observed asymptomatic verrucous papules confined to the black ink of a tattoo done 15 years ago on the lateral side of his right lower leg (Figure 1a). Clinical examination showed multiple, discrete, skin-colored, verrucous papules disseminated exclusively within the lines of the black-colored tattoo. Full skin examination did not reveal any similar lesions anywhere else on the body. Dermoscopically, papules showed a discretely papillomatous surface and sharp borders (Figure 1b). The patient had another black tattoo on his trunk, in which no similar lesions were found. All his tattoos had been done more than 15 years ago in a professional tattoo salon and with no previous history of cutaneous lesions within tattoos. The patient had no other medical conditions and was not taking any medications. Additionally, no history of warts or other HPV-related lesions of the skin or mucosal membranes could be established. A biopsy of an individual papule was taken and sent for a histopathological analysis, which subsequently showed hyperkeratotic, orthokeratotic, and parakeratotic acanthotic epidermis with hypergranulosis and rare cells with perinuclear halo indicative of koilocytes (Figure 3b). Immunohistochemical analysis showed negative reaction for p16 and p53, while Ki67 was positive only in rare basal and suprabasal cells. These findings were indicative of low-risk HPV, and the diagnosis of HPV-induced verruca plana was ultimately established. The patient was then successfully treated with cautious curettage of the lesions, leaving no scars. Due to the growing popularity of tattoos, especially among younger populations, it is necessary to emphasize the possibility of various tattoo-related side-effects that can still occur due to improper preparation of the tattoo location, contamination of ink products, improperly sterilized instruments, or due to insufficient personal hygiene following tattooing (3). In the past, tattoo-associated infections were significantly more frequent, with the highest prevalence of Staphylococcus and Streptococcus infections causing impetigo, folliculitis, cellulitis, erysipelas, or sepsis (2), but recent improvement and efforts in using sterile techniques in tattooing has led to a significant drop in the number of tattoo-related infections. In this short report, we present a case of a different and a relatively rare type of tattoo-associated infection - flat warts i.e., verrucae planae. Flat warts are usually caused by HPV-3, -6B, -10, -28, and -49. Typical predilection sites are the face, dorsal sides of the hands or feet, arms, and legs, and they usually appear as skin-colored, pink, or brown, flat-topped discrete papules. It is believed that HPV can be inoculated through contaminated ink, instruments, the artist's saliva, or that it may be a pre-existing unnoticed wart in the tattooed area (4-6). The latency period between tattooing and HPV infection can range from several months to 10 years, with a mean period of 5 years (3). This may suggest that the immune system can control the infection for some time, and that some form of immune suppression may result in the development of a clinical disease. In our case, the latency period could not be established due to the patient's unawareness of the lesions, and no potential trigger could be identified. The occurrence of lesions on only one of the patient's tattoos as well as their confinement to the black pigmented ink may indicate a correlation to this specific pigment. Ramey et al. (6) conducted a study in which they assessed the localization of warts in differently colored tattoos. The results showed that black ink tattoos had a seven times higher risk of developing warts when compared with colored ink. At first it was thought that this was due to HPV inoculation via contaminated instruments, ink, or autoinoculation of the patient's own warts, but some evidence indicates that it is unlikely for HPV to survive in ink and that if the warts were inoculated they would occur equally in all ink colors. A different theory by Ruocco et al. (7) explains this phenomenon through an "immunocompromised district" mechanism, in which polycyclic aromatic hydrocarbons found in black ink produce reactive oxygen species (ROS) that can damage cellular structures and consequently increase the risk of a variety of infections, including HPV. Moreover, black ink contains almost pure nanoparticles, which are associated with greater ROS production than the larger particles found in colored ink (7,8). There are several treatment options for verrucae planae, such as liquid nitrogen cryotherapy, topical 5% fluorouracil, topical 5% imiquimod, 0.025-0.050% tretinoin, 10% salicylic acid, or 10-30% glycolic acid. These treatments have differing success rates. Destructive modalities, such as surgical excision, curetting, or laser ablation may significantly damage the tattoo and cause scarring, and are thus not regularly performed. It's necessary to emphasize that despite today's sterile methods of tattooing, complications may still occur and medical professionals, namely dermatologists, should be aware of them. A person who wishes to get a tattoo should be advised to visit a licensed tattoo artist at a licensed tattoo parlour only. Patients with pre-existing dermatoses characterized by an isomorphic phenomenon, such as psoriasis or lichen planus, are particularly prone to developing a tattoo-associated adverse reaction, and those with severe dermatoses should be advised to avoid tattooing. Additionally, people with a previous history of warts should be aware of the possibility of warts occurrence, even years or decades after tattooing. Even though most of tattoo-related side effects are merely inconveniences, there is a potential for serious complications and patients should be advised as such.

Expression and Prognostic Significance of PD-L1 and NY-ESO1 in Gallbladder Carcinoma.

BACKGROUND/AIM: Gallbladder cancer is a rare malignancy with a very high mortality, usually due to diagnosis in an advanced stage of the disease. Therefore, the aim of this study was to evaluate the clinical significance of cancer/testis antigen 1A (CTAG1A, NY-ESO1) and CD274 molecule (PD-L1, the ligand for programmed cell death protein 1) and their impact on the overall survival of patients with gallbladder cancer. PATIENTS AND METHODS: Using immunohistochemical staining, we determined the expression of NY-ESO1 in tumor cells (positivity: cytoplasmic/nuclear staining of any intensity in ≥50%) and PD-L1 in tumor cells and intratumoral immune cells (positivity: cytoplasmic/membranous staining of any intensity in ≥1%). RESULTS: The median overall survival (OS) of 58 patients with gallbladder cancer in our cohort was 7 months, and depended on the clinical stage of the disease; the 5-year OS rate was 10%. NY-ESO1 was expressed in 69.1% of cases. Immune cells were PD-L1-positive in 36.4% of cases, while tumor cells expressed PD-L1 in only 10.9% of cases. In six cases (10.9%), neither of the studied proteins were expressed. NY-ESO1 expression was negatively correlated with PD-L1 expression in immune cells (p=0.021). NY-ESO1 showed no correlation with any clinicopathological parameters or OS. PD-L1 expression in immune cells was significantly higher in tumors with perineural invasion (rs=0.318; p=0.018) and higher clinical disease stage (rs=0.339; p=0.013) but showed no correlation with OS. CONCLUSION: Patients whose gallbladder cancer expresses NY-ESO1 or PD-L1 might be candidates for immunotherapy.

Methylation pattern of caveolin-1 in prostate cancer as potential cfDNA biomarker.

High prevalence and mortality of prostate cancer (PCa) are well known global health issues. Novel biomarkers for better identifying patients with PCa are the subject of extensive research. Prostate specific antigen (PSA) shows low specificity in screening and diagnostics, leading to unnecessary biopsies and health costs. Eighty patients with PCa and benign prostate hyperplasia (BPH) were included in the study. We analyzed CAV1 gene expression and methylation in tissue. CAV1 cfDNA methylation from blood and seminal plasma was accessed as a potential PCa biomarker. Although methylation in blood plasma did not differ between PCa and BPH patients, methylation in seminal plasma showed better PCa biomarker performances than tPSA (AUC 0.63 vs. AUC 0.52). Discrimination of BPH and Gleason grade group 1 PCa patients from patients with higher Gleason grade groups revealed very good performance as well (AUC 0.72). CAV1 methylation is useful biomarker with potential for further seminal plasma cfDNA research, but its diagnostic accuracy should be improved, as well as general knowledge about cfDNA in seminal plasma.

Frequency of loss of heterozygosity of the NF2 gene in schwannomas from Croatian patients.

AIM: To identify gross deletions in the NF2 gene in a panel of schwannomas from Croatian patients in order to establish their frequencies in Croatian population. METHODS: Changes of the NF2 gene were tested by polymerase chain reaction/loss of heterozygosity (LOH) using two microsatellite markers, D22S444 and D22S929. RESULTS: The analysis with both markers demonstrated that 43.75% of schwannomas exhibited LOH of the NF2 gene. The D22S444 region exhibited 45.5% of LOHs and the D22S929 region exhibited 14.3% of LOHs. Four LOHs were found in Antoni B, 2 in Antoni A, and 1 in Antoni A and B type tumors. CONCLUSION: The frequency of changes observed in Croatian patients is broadly similar to that reported in other populations and thus confirms the existing hypothesis regarding the tumorigenesis of schwannomas and contributes to schwannoma genetic profile helping us to better understand its etiology and treatment.

Atypical Mixed Tumor of the Skin in the Gluteal Region with Strong and Diffuse Nuclear Expression of the p16 Stain: A Case Report.

Benign, atypical, or malignant chondroid syringoma (mixed tumor of the skin) have almost identical clinical presentation with similar histological features, except for infiltrative growth, and perineural and vascular invasion in the malignant type. Tumors with borderline features are described as atypical chondroid syringoma. Immunohistochemical profiles in all three types are similar, with the the main difference in the expression of the p16 stain. We present a case of an atypical chondroid syringoma in an 88-year-old female patient with a subcutaneous, painless nodule in the gluteal region and with diffuse, strong nuclear immunohistochemical staining for p16. To our knowledge, this is the first such reported case.

Extensive retraction artefact predicts biochemical recurrence-free survival in prostatic carcinoma.

AIMS: To determine whether the presence and extent of peritumoral retraction artefact could be used to predict biochemical recurrence-free survival in prostatic carcinoma. METHODS AND RESULTS: The study included 162 consecutive patients treated by radical retropubic prostatectomy and bilateral lymphadenectomy for clinically localized prostatic carcinoma. A variable degree of retraction artefact was present in all 162 analysed tumours. The extent of retraction artefact in prostatic carcinomas ranged from 5% to 55% with a median value of 15% (interquartile range 10-25%). We found no correlation between the extent of retraction artefact in the tumours and patient's age (P=0.608), preoperative (P=0.362) and postoperative (P=0.279) Gleason score or lymph node metastases (P=0.084). In contrast, the extent of retraction artefact correlated with high preoperative prostate-specific antigen (P<0.001), short follow-up time (P<0.001), seminal vesicle invasion and/or extracapsular extension of the tumour (T3 stage tumours) (P<0.001) and positive surgical margins (P<0.001). Furthermore, extensive retraction artefact was associated with poor biochemical recurrence-free survival in both univariate (P<0.001) and multivariate analyses (P=0.013). CONCLUSION: The presence of extensive retraction artefact in prostatic carcinoma correlates with tumour characteristics signifying aggressive behaviour and indicates poor biochemical recurrence-free survival.

C-myc expression in the microvessels of medulloblastoma.

The increased expression of c-myc is related to neoplastic transformation and angiogenesis. Therefore, the assessment of expression of c-myc in endothelial cells and neovascularization could help to determine the biological behavior of the tumor. We analyzed neovascularization and c-myc expression in 36 medulloblastoma specimens. The results were shown by determining immunohistochemical staining index (ISI), the sum of staining intensity (SI) and the percentage of positive cells (PPC) in the blood vessels endothelium of the tumor. We also performed the microvessel count (MVC) in 10 high-power fields (400X) with the most prominent vascularization and expressed it as microvessel density per mm2 (MVD). C-myc immunostaining intensity index in blood vessel endothelium is grouped into four groups, 0--no reaction, I-weak reaction (ISI = 1 or 2), II--moderate reaction (ISI = 3 or 4), III--strong reaction (ISI = 5 or 6). Statistically significant differences (p = 0.0214) have been found between groups 0 and 1 compared to groups 2 and 3. A higher percentage of positive cells has been found in male patients than in female ones (p = 0.0483). C-myc PPC 0 or 1 has on the average smaller density of blood vessels per mm2 than c-myc PPC 2 or 3, but the difference is not statistically significant. C-myc ISI 0 or 1 has, on the average, smaller density of blood vessels per mm2 than c-myc ISI 2 or 3, but the difference is not statistically significant. We concluded that c-myc staining intensity was associated with higher microvessels density.

AXIN-1 protein expression and localization in glioblastoma.

The etiology and pathogenesis of tumors of the central nervous system are still inadequately explained. In the present study the expression patterns of a critical molecular component of wnt signaling pathway - axin I was investigated in 42 patients with glioblastoma, the most aggressive form of glial tumors. Immunostaining and image analysis revealed the quantity and localization of the protein. Downregulation of this tumor suppressor expression was observed in 31% of tumors when compared to the levels of axin in healthy brain tissues. Axin was observed in the cytoplasm in 69% of glioblastoma samples, in 21.4% in both the cytoplasm and nucleus and 9.5% had expression solely in the nucleus. Mean values of relative axin's expression obtained by image analysis showed that the highest relative quantity of axin was measured when the protein was in the nucleus and the lowest relative quantity of axin when the protein was localized in the cytoplasm. Investigation on axin's existence at the subcellular level in glioblastomas suggests that axin's expression and spatial regulation is a dynamic process. Despite increasing knowledge on glioma biology and genetics, the prognostic tools for glioblastoma still need improvement. Our findings on expression of axin 1 may contribute to better understanding of glioblastoma molecular profile.

(Reactive) Eccrine Syringofibroadenoma with Foci of Squamous Cell Carcinoma in situ: A Case Report.

We describe a rare case of an eccrine syringofibroadenoma with a foci of squamous cell carcinoma in situ, which has to best of our knowledge been reported only twice in the English literature. An excisional biopsy of an elevated, lobular tumor of the lower leg in an 86-year-old male patient was performed. Histologic examination revealed a tumor consisting of anastomosing strands of epithelial cells originating from the epidermis, occasionally showing ductal eccrine differentiation. Foci of squamous cell carcinoma in situ were observed within the described lesion. The diagnosis of eccrine syringofibroadenoma with squamous cell carcinoma in situ was established. Eccrine syringofibroadenoma is a rare lesion, mostly considered to be a reactive process arising secondarily in association with other cutaneous diseases such as dermatoses or neoplasms, although some researchers do not exclude the possibility that it is a primary neoplasm with a potential for malignant transformation.

Expression of adipokine ghrelin and ghrelin receptor in human colorectal adenoma and correlation with the grade of dysplasia.

BACKGROUND: Ghrelin is an adipokine that plays an important role in energy balance. Expression of ghrelin and ghrelin receptor has been investigated in different tissues and tumors. Studies regarding expression of ghrelin and ghrelin receptor in colorectal tumors are scarce and no data on expression of ghrelin and its receptor in colorectal adenomas has been published. Ghrelin and ghrelin receptor were highly expressed in colon carcinoma cells while expression was decreased in less differentiated tumors, presuming that ghrelin might be important in early phases of tumorigenesis. AIM: To investigate the expression of ghrelin and ghrelin receptor in human colorectal adenomas and adjacent colorectal tissue. METHODS: In this prospective study (conducted from June 2015 until May 2019) we included 92 patients (64 male and 28 female) who underwent polypectomy for colorectal adenomas in the Department of Gastroenterology and Hepatology, "Sestre milosrdnice" Clinical Hospital Center in Zagreb, Croatia. After endoscopic removal of colorectal adenoma, an additional sample of colon mucosa in the proximity of the adenoma was collected for pathohistological analysis. Adenomas were graded according to the stage of dysplasia, and ghrelin and ghrelin receptor expression were determined immunohistochemically in both adenoma and adjacent colon tissue using the polyclonal antibody for ghrelin (ab150514, ABCAM Inc, Cambridge, United States) and ghrelin receptor (ab48285, ABCAM Inc, Cambridge, United States). Categorical and nominal variables were described through frequencies and proportions and the difference between specific groups were analyzed with Fisher's and Fisher-Freeman-Halton's method respectively. Spearman's rank correlation coefficient was determined for correlation of expression of ghrelin and ghrelin receptor in adenoma and adjacent colon tissue with the grade of adenoma dysplasia. RESULTS: Among 92 patients with colorectal adenoma 43 had adenomas with high-grade dysplasia (46.7%). High expression of ghrelin was 7 times more common in high-grade adenoma compared to low-grade adenomas (13.95% to 2.04%, P = 0.048), while the expression of ghrelin in adjacent colon tissue was low. We found no correlation between ghrelin receptor expression in adenoma and adjacent colon tissue and the grade of colorectal adenoma dysplasia. The most significant correlation was found between ghrelin and ghrelin receptor expression in adenomas with high-grade dysplasia (rho = 0.519, P < 0.001). CONCLUSION: Ghrelin and ghrelin receptor are expressed in colorectal adenoma and adjacent tissue with ghrelin expression being more pronounced in high grade dysplasia as a possible consequence of increased local synthesis.