PD-L1 expression is regulated by microphthalmia-associated transcription factor (MITF) in nodular melanoma.

Malignant melanoma (MM) is known to avoid the host's immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10-6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma.

Matrix metalloproteinases MMP-1, MMP-2, and MMP-13 are overexpressed in primary nodular melanoma.

BACKGROUND: The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This study analyzed the expression of MMP-1, MMP-2, and MMP-13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival. METHODS: Immunohistochemistry for MMP-1, MMP-2, and MMP-13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method. RESULTS: A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP-1 and MMP-13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05). CONCLUSION: Cox analysis revealed that Clark categories, Breslow thickness, and MMP-1 high protein expression are predictive factors for shorter overall survival (P < 0.05).

EGFR and cyclin D1 in nodular melanoma: correlation with pathohistological parameters and overall survival.

Considering that nodular melanoma (NM) has the potential to show an early distant metastasis, there is an urgent need for the discovery and evaluation of new diagnostic and prognostic biomarkers. We aimed to investigate the protein expression of membrane and nuclear epidermal growth factor receptor (EGFR), cyclin D1, and the corresponding gene status in NM samples and correlate the results obtained with clinicopathological parameters and overall survival of patients. Immunohistochemical and fluorescence in-situ hybridization analyses were carried out on tissue microarrays constructed from 110 NM samples, 30 compound nevi, and 38 dysplastic nevi. NM samples showed 24% strong cyclin D1 and 37% strong Ki67 protein expression compared with 3 and 0% strong cyclin D1 and Ki67 expression in the control group. Membrane EGFR expression was detected in 50% of NM cases, whereas EGFR gene amplification was detected in only 4% of NM cases. Multiple NM samples presented simultaneous membrane and nuclear EGFR expression. We found a negative correlation between tumor thickness and membrane EGFR expression. It was also observed that membrane EGFR 3+ NM samples presented ulceration significantly more often than membrane EGFR-negative (0) NM samples. In univariate analysis, carried out on 44 patients with follow-up data, both nuclear and membrane EGFR overexpression showed a correlation with a shorter overall survival. Nuclear EGFR (++, +++) showed 3.06 and membrane EGFR (2+, 3+) showed 2.76 higher risk of mortality compared with patients with low and negative nuclear and membrane EGFR expression (P<0.05).

A controlled trial of photodynamic therapy of actinic keratosis comparing different red light sources.

BACKGROUND: Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) with red light is well established for actinic keratosis (AK). Differences have been observed concerning pain and efficacy rates with different red light sources. OBJECTIVES: To compare pain scores, short- and long-term efficacy rates of PDT of multiple AKs when employing different red light sources. MATERIAL AND METHODS: In a controlled trial, 88 patients (310 AK lesions) received ALA-PDT in combination with either visible light (VIS) + water-filtered infrared A (wIRA) light (PhotoDyn(®) 750 (PD750), 580-1400 nm) for 30 min or incoherent light (Waldmann(®) 1200L (Wa1200L), 600-720 nm) for 10-11 min. Follow-up visits were performed after 1, 3, 6, and 12 months. If there was no complete cure after 1, 3 or 6 months, a second cycle of PDT was performed. RESULTS: Pain scores were significantly lower in patients illuminated with PD750 rather than Wa1200L. Patient complete clearance rates were 85% and 91% after 1 month, 79% and 92% after 3 months, 97% and 92% after 6 months, and 69% and 85% after 12 months in the PD750 and Wa1200L groups, respectively. Lesion complete clearance rates were 94% and 92% after 1 month, 88% and 97% after 3 months, 96% and 95% after 6 months, and 81% and 89% after 12 months in the PD750 and Wa1200L group, respectively. The efficacy rates were not significantly different. CONCLUSION: A VIS + wIRA light source produced considerably less pain, while efficacy was not much affected in contrast to previously published studies, probably because the illumination time was longer in this study.

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma.

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells. Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples. Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p=0.015) and protein (p<0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p=0.006 and p=0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.

Fulminant primary manifestations of Wegener's granulomatosis might not be pauci-immune.

Wegener's granulomatosis is an ANCA-associated small vessel vasculitis. Because histologically immune complex deposits are frequently lacking, the term pauci-immune has been introduced for this subgroup. We report a patient with fulminant, severe PR3-ANCA-positive Wegener's granulomatosis and multi-organ involvement (upper respiratory tract, lung, kidneys, skin and general symptoms), who showed pronounced immunoglobulin and complement deposits within the skin biopsy. Our observation supports the hypothesis that immune complex deposits may be under-recognized in early lesions of ANCA-associated Wegener's granulomatosis.

Hypoxia inducible factor-1alpha correlates with vascular endothelial growth factor A and C indicating worse prognosis in clear cell renal cell carcinoma.

BACKGROUND: The role of angiogenesis in the pathogenesis of renal cell carcinoma is well recognized, however, the influence of tumor cells in this activity has not yet been fully clarified. The aim of this study was to analyze the expression of hypoxia inducible factor-1alpha (HIF-1alpha), a regulatory factor of angiogenic switch, in comparison to vascular endothelial growth factor A and C (VEGF-A and VEGF-C), recognized to be involved in blood and lymph vessel neoangiogenesis, with potential association in the prognosis of patients with renal cell carcinoma. METHODS: Ninety-four patients with diagnosis of clear cell renal cell carcinomas (CCRCC), all clinicopathological characteristics and overall survival were unrolled in this study. Immunohistochemicaly VEGF-A, VEGF-C, HIF-1alpha and Ki67 were detected on tumor cells and the staining was performed on tissue microarrays (TMA). The staining was evaluated as a percentage of cytoplasmic or nuclear positive tumor cells. RESULTS: Variable expression of all three proteins was confirmed. Both angiogenic factors demonstrated perimembranous or diffuse cytoplasmic staining, with diffuse pattern positively associated (p < 0.001). Nuclear HIF-1alpha expression (nHIF-1alpha) showed inverse correlation with diffuse cytoplasmic VEGF-A (p = 0.002) and VEGF-C (p = 0.053), while cytoplasmic HIF-1alpha expression (cHIF-1alpha) showed positive correlation with diffuse staining of both angiogenic factors (p < 0.001; p < 0.001, respectively). In comparison to clinicopathological characteristics, a higher nuclear grade (p = 0.006; p < 0.001, respectively), larger tumor size (p = 0.009; p = 0.015, respectively), higher stage (p = 0.023; p = 0.027, respectively) and shorter survival (p = 0.018; p = 0.024, respectively) were associated with overexpression of cHIF-1alpha and diffuse cytoplasmic VEGF-A expression. In contrary, overexpression of nHIF-1alpha was associated with better diagnostic parameters i.e. lower nuclear grade (p = 0.006), smaller tumor size (p = 0.057), and longer survival (p = 0.005). CONCLUSION: Overexpression of VEGF-A and cHIF-1alpha in tumor cells highlights a more aggressive subtype of CCRCC that might have some clinical implications. The significance of nHIF-1alpha expression associated with better differentiated tumors should be further elucidated.

Comparison of molecular assays for detection and typing of human papillomavirus.

OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

Prevalence of human papillomavirus among Croatian women attending regular gynecological visit.

Human papillomavirus (HPV) infection has been identified as major risk factor for cervical intraepithelial neoplasia (CIN) and invasive cervical cancer. About 40 HPV viral types are commonly found in the genital tract. Most HPV infections resolve spontaneously, while persistent infection with oncogenic types, namely HPV 16 and 18 is necessary for CIN to occur and progress to cancer. Cervical screening is presently based on the Pap smear that is designed to diagnose precancerous lesions and cervical cancer The aim of this study was to investigate the prevalence of HPV DNA and to determine HPV types distribution among 361 women attending regular gynecological visit. There were 205 women (29+/-8 years old) without determined abnormal cervical lesions and 156 women (34+/-15 years old) with abnormal Pap smear; low grade squamous intraepitehelial lesions (LSIL, n=69), high grade squamous intraepithelial lesions (HSIL, n=72) and atypical squamous cells of undetermined significance (ASCUS, n=15). HPV DNA detection and genotyping was performed by Hybrid Capture 2 assay and additionally by consensus and type-specific primers directed PCR. The overall prevalence of high-risk HPV (hrHPV) in women with abnormal Pap smears was 67.9% (106/156), of which in ASCUS 33.4% (5/15), LSIL 62.3% (43/69) and HSIL 80.6% (58/72). In HPV positive specimens, HPV 16 was found as predominant type in 60.4% cases, followed by HPV 31 (8.5%), HPV 33 (6.6%) and HPV 18 (3.7%). In the group of women without obvious cervical changes the overall hrHPV prevalence was 35.6% with HPV 16 found in 43.8% cases, followed by HPV 31 (17.8%), HPV33 (9.5%) and HPV18 (6.8%). In both study groups, women with and without cervical lesions, the prevalence of HPVof indeterminate type was 14.2% and 13.7%, respectively. Our results indicate that cervical intraepithelial lesions are largely associated with HPV type 16, followed by HPV types 31, 33, 18 and HPV of indeterminate type. Although there is a significant difference in hrHPV DNA prevalence among two groups, no significant differences between particular hrHPV types distribution were observed.