Association of the combined parameters including the frequency of primary cilia, PD-L1, Smoothened protein, membranous ß-catenin and cytoplasmic ß-catenin expression with the outcome of patients with clear cell renal cell carcinoma.

AIMS: The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and ß-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). METHODS: The PD-L1, Smoothened protein and ß-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human ß-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. RESULTS: Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic ß-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous ß-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous ß-catenin (P<0.001), cytoplasmic ß-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. CONCLUSION: The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and ß-catenin expression with the outcome in clear cell renal cell carcinoma.

Favorable prognostic significance of membranous ß-catenin expression and negative prognostic significance of cytoplasmic ß-catenin expression in pancreatic cancer.

The aim of this study was to investigate the prognostic significance of membranous ß-catenin and cytoplasmic ß-catenin expression in pancreatic cancer patients (pts). One hundred pts with histologically verified exocrine pancreatic ductal adenocarcinoma were retrospectively studied. The membranous ß-catenin, cytoplasmic ß-catenin, and cell nucleus ß-catenin expression were immunohistochemically evaluated. The expression of membranous ß-catenin was <5% in none of the pts, 5-25% in one patient, 26-50% in 2 pts, 51-75% in 14 pts, and >75% in 81 pts. The expression of cytoplasmic ß-catenin was <5% in 34 pts, 5-25% in 42 pts, 26-50% in 18 pts, 51-75% in 3 pts, and >75% in one patient. The expression of ß-catenin in the cell nucleus was negative in all pts. At the time of the last follow-up, 21 pts were alive and 79 pts had died. Median OS was 1.3 (0.4-2.3) years in pts with membranous ß-catenin expression ≤75% and 1.7 (1.3-2.1) years in pts with membranous ß-catenin expression >75% (p=0.045). Median OS was (1.3-2.0) 1.6 years in pts with cytoplasmic ß-catenin expression ≤25% and 0.9 (0.5-1.2) years in pts with cytoplasmic ß-catenin expression >25% (p=0.040). In the univariate Cox proportional hazard models HR (95% CI) was 0.556 (0.311-0.995) in pts with membranous ß-catenin expression >75% (p=0.048) and 2.200 (1.216-3.980) in pts with cytoplasmic ß-catenin expression >25% (p=0.009). The present results indicate a favorable prognostic significance of membranous ß-catenin expression in pancreatic cancer.

The clinical value of circulating free tumor DNA in testicular germ cell tumor patients.

BACKGROUND: Testicular germ cell tumors (TGCT) are unique malignancies of young adult men; their biology is, however, underexplored and there has not been much progress in their treatment for decades. Circulating free tumor DNA (cfDNA) analysis represents a promising way of discovering novel diagnostic and treatment options. OBJECTIVE: The study evaluates the clinical value of cfDNA detection in TGCT patients. DESIGN AND METHODS: Total cfDNA concentration and ratio of its 2 main fragments (180 and 360 bp) were evaluated by spectrophotometry, capillary electrophoresis and qPCR in peripheral blood plasma of 96 TGCT patients (173 samples) and 31 normal controls. Non-parametric tests were used for statistical analyses. RESULTS: The total cfDNA concentration was significantly higher in TGCT than in controls (P < 0.0001), with the highest levels at disease progression, but with no clear threshold between malignant and normal samples. Patients with positive tumor markers had higher cfDNA concentrations than those with negative markers (P = 0.01). Longer 360 bp cfDNA fragments were found in 58% of TGCT patients including almost all samples from relapse or disease progression but no normal controls (P < 0.0001). CONCLUSION: Total cfDNA levels are significantly increased in TGCT patients but without a clear threshold separating normal and tumor samples, thus total cfDNA amount itself is not a sensitive enough marker to identify or monitor TGCT. Longer cfDNA fragments have been found exclusively in a proportion of tumors and predominantly at disease progression, representing a novel potential marker for TGCT monitoring that would deserve further exploration.

Squamous cell carcinoma with sarcomatoid differentiation or carcinosarcoma of the uterine cervix associated with HPV33 infection: report of a rare case.

BACKGROUND: Squamous cell carcinoma is the most common malignant tumor of the uterine cervix with a well-documented link to infection with human papillomaviruses (HPV). According to a recent classification, there are several morphological variants of cervical squamous carcinoma, without reference to sarcomatoid squamous cell carcinoma, which is well described in other organs. CASE PRESENTATION: In this paper, we describe an extremely rare case of a 77-year-old woman with primary malignant cervical tumor displaying biphasic histomorphology with an epithelioid and sarcomatoid part; the latter was immunohistochemistry positive for cytokeratin and vimentin. The association with a high-grade squamous intraepithelial lesion and molecular proof of HPV33 infection in the tumor tissue supported our diagnosis of carcinoma with partial sarcomatoid differentiation. CONCLUSION: We report a rare case of a primary cervical epithelial tumor with a partial sarcomatoid phenotype, an unequivocal HPV infection, and an associated precancerous lesion in the cervical mucosa. This is the first description of an HPV33 infection underlying a biphasic epithelioid-sarcomatous tumor of the uterine cervix. The terminology overlap between sarcomatoid carcinoma and carcinosarcoma is also discussed.

Negative prognostic significance of primary cilia, CD8+ tumor infiltrating lymphocytes and PD1+ cells expression in clear cell renal cancer.

PURPOSE: The aim of this study was to investigate the potential association and combined prognostic significance of the frequency of primary cilia (PC), programmed cell death protein-1 receptor (PD1) and CD8+ tumor infiltrating lymphocytes (TIL) in patients with clear cell renal cancer (ccRCC). METHODS: The frequency of PC, PD1 expression and the frequency of intratumoral CD8+ TIL were evaluated in 104 ccRCC patients. RESULTS: The median frequency of PC was 0.003. The expression of PD1+ cells were <5% in 52 patients, 5-25% in 34 patients and 26-50% in 13 patients and >50% in 5 patients. Intratumoral CD8+ TIL were evaluable in all patients: negative in 1 patient, <25% in 63, 26-50% in 29 and >50% in 11 patients. Overall survival (OS) according to the frequency of PC was significantly shorter in patients with higher frequency (≥0.002) than in patients with lower frequency (<0.002) (p<0.001). Median OS was significantly shorter in patients with higher (25%) CD8+ TIL and higher (>25%) PD1+ expression than in patients with lower (<25%) expression (4.6 vs. 97. years, p=0.006 and 2.9 vs. 8.9 years, p=0.006, respectively). CONCLUSIONS: The present study provides the first data on the potential association and combined prognostic significance of frequency of PC, PD1+ cells and CD8+ TIL in patients with clear cell renal cancer.

Molecular Basis of Cisplatin Resistance in Testicular Germ Cell Tumors.

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients' samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients' samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.

Frozen section of lung, pleura and mediastinum specimen: Retrospective analysis of 5-years practical experiences and review of the literature.

Thoracic frozen section biopsies including lungs, pleura and mediastinum have crucial role in differential diagnosis of solitary lesions of the lungs and mediastinum. There is a significant difference in patient´s therapeutical management, if the patients suffer from lung primary malignant tumour, metastasis or non-malignant lesion. In this review, we summarize the usefulness of frozen section biopsies in thoracic surgery and provide 5-years of experiences with thoracic frozen section biopsies performed at the Department of Pathology and Molecular Medicine of Thomayer Hospital in Prague. Keywords: frozen section - lung - mediastinum.

Nontuberculous mycobacterial infection after therapy with temsirolimus for metastatic renal cell carcinoma.

We describe the case of a patient with metastatic renal cell carcinoma (mRCC) who developed a nontuberculous mycobacteria (NTM)-related pulmonary nodule during therapy with the mammalian target of rapamycin (mTOR) inhibitor temsirolimus. After discontinuation of temsirolimus treatment, a small pulmonary nodule with increased glucose uptake was detected on a positron emission tomography (PET) scan. A lung resection carried out to confirm and treat the suspected solitary metastasis of RCC yielded the surprising finding of a caseating granuloma containing NTM. A single PET-positive nodule presents a significant differential diagnostic dilemma in the setting of mRCC treated with mTOR inhibitors. Although the treatment of mRCC with temsirolimus can lead to immunosuppression and opportunistic infections, there is no report to our knowledge on the occurrence of NTM infections in mRCC patients treated with mTOR inhibitors. These infections should be included in the differential diagnosis of lung nodules. Interestingly, there is strong preclinical evidence pointing to direct and indirect antimycobacterial activity of mTOR inhibitors. We therefore hypothesize that while the seeding of NTM can occur during temsirolimus therapy due to T-lymphocyte suppression, the infection may only become active after the discontinuation of mTOR inhibitor treatment.