Immunohistochemical detection of EGFRvIII in glioblastoma - Anti-EGFRvIII antibody validation for diagnostic and CAR-T purposes.

The emergence of therapies such as CAR-T has created a need for reliable, validated methods for detecting EGFRvIII in patient tumor cells. Particularly so since previous studies have already suggested that some anti-EGFRvIII antibodies may be non-specific. The present paper evaluates the use of the L8A4 antibody in the immunohistochemical (IHC) and immunocytochemical (ICC) detection of EGFRvIII in 30 glioblastoma specimens, and compares it with other methods such as RT-PCR, MLPA, and FISH. The results indicate that Real-time PCR appears to be a very specific and sensitive method of EGFRvIII detection. ICC analysis with L8A4 also appears specific but requires cell culture. IHC analyses of EGFRvIII returned a number of false positives when using L8A4. Due to the growing need for an effective diagnostic tool before starting immunotherapy methods, such as the CAR-T anti-EGFRvIII or SynNotch CAR-T recognizing EGFRvIII, it is necessary to identify a more reliable and simple method of EGFRvIII detection or improve the specificity of the anti-EGFRvIII antibody, until then, immunocytochemistry may temporarily replace immunohistochemistry.

WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

BACKGROUND: The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). OBJECTIVE: The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. METHODS: WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). RESULTS: WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. CONCLUSIONS: There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

Expression Profiles of AQP3 and AQP4 in Lung Adenocarcinoma Samples Generated via Bronchoscopic Biopsies.

Aquaporins (AQPs) are highly conserved channel proteins which are mainly responsible for the exchange of water and small molecules and have shown to play a pivotal role in the development and progression of cancer. Lung adenocarcinoma is the most common primary lung cancer seen in patients in Europe and the United States. However, in patients it is often not diagnosed until the advanced tumor stage is present. Previous studies provided strong evidence that some members of the AQP family could serve as clinical biomarkers for different diseases. Therefore, we aimed to investigate how AQP3 and AQP4 protein expression in lung adenocarcinoma (ADC) biopsy samples correlate with clinical and pathological parameters. The protein expression of AQP3 and AQP4 was analyzed based on immunohistochemical staining. AQP3 protein was observed in the cytoplasmic membrane of cancer tissue in 82% of lung samples. Significant differences in relative protein expression of AQP3 were noted between advanced age patients compared to younger counterparts (p = 0.017). A high expression of AQP3 was significant in cancer tissue when compared to the control group (p < 0.001), whereas a low AQP4 membrane expression was noted as significantly common in cancer tissue compared to non-neoplastic lung tissue (p < 0.001). Moreover, a low AQP4 membrane expression was positively correlated with a more advanced disease status, e.g., lymph node metastases (p = 0.046). Based on our findings, AQP3 and AQP4 could be used as biomarkers in ADC patients.

Colonic Adenocarcinomas Harboring NTRK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 16 Cases and Review of the Literature.

This study was undertaken to determine the frequency, and the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age: 63 y). The tumors involved the right (n=12) and left colon unequally and were either stage T3 (n=12) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component (n=10), including 1 case with sheets of signet ring cells. DNA mismatch repair-deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. Programmed death-ligand 1 positive tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS, and PIK3CA were identified. However, other genes of the PI3K-AKT/MTOR pathway were mutated. In several cases, components of Wnt/ß-catenin (APC, AMER1, CTNNB1), p53, and TGFß (ACVR2A, TGFBR2) pathways were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.

WNT5A gene and protein expression in endometrial cancer.

INTRODUCTION: WNT5A (Wnt family member 5A) belongs to the WNT family of secreted signaling glycoproteins that play essential role in developmental, physiological and pathological processes. WNT5A was shown to take part in carcinogenesis process playing both oncogenic and suppressor functions in various types of human malignancies. This study aimed to assess the expression of the WNT5A gene at the mRNA and protein levels in the specimens derived from endometrial cancer (EC) or unchanged control endometrium. The associations between the WNT5A expression levels and clinicopathological characteristics and survival of EC patients were evaluated. MATERIALS AND METHODS: Total RNA was isolated in order to assess the relative amounts of WNT5A mRNA by quantitative polymerase chain reaction (QPCR) in samples of unchanged endometrial control (n = 8) and tumor samples of EC patients (n = 28). Immunohistochemistry (IHC) was used to determine the presence of WNT5A protein in the sections of formalin-fixed, paraffin-embedded tissue specimens derived from unchanged endome-trial controls (n = 6) and EC tumors (n = 19). Significance of differences in WNT5A expression levels between the studied groups of EC patients and correlations between the WNT5A and demographic data, pathological features, hematological parameters and overall survival of the patients were evaluated by statistical analysis. RESULTS: The level of WNT5A mRNA was decreased in EC in comparison to unchanged endometrium. WNT5A expression was associated with primary tumor invasion status exhibiting reduced level of transcripts in EC that involved organs beyond the uterus when compared to the uterus-confined cancers. WNT5A immunoreactivity was visualized in the cytoplasm and nuclei of EC cells as well as in the luminal and glandular epithelial cells of unchanged endometrium. WNT5A mRNA expression levels correlated negatively with cytoplasmic, and positively with nuclear immunoreactivity of the WNT5A protein in the EC cells. In addition, the relationships between blood leucocyte count (in particular granulocytes and lymphocytes) of patients with EC and their WNT5A mRNA and protein expression levels were established. A positive correlation between the nuclear immunoexpression of WNT5A protein in the cancer cells in cell nuclei and mean platelet volume in blood was also found. CONCLUSIONS: The results of the first study of WNT5A expression at the transcript and protein levels indicate that it could be considered as a potential marker of molecular changes that take place during EC development.

[Glioblastoma with BRAFV600E mutation and numerous metastatic foci: a case report].

Glioblastoma, the most malignant astrocytic tumour, is associated with limited survival and thus rare metastases. We analysed a particularly interesting case - a 51-year-old male diagnosed within 2 years with primary and recurrent glioblastoma, isocitrate dehydrogenase (IDH)-wild type, as well as with numerous extra-central nervous system (CNS) metastatic foci. Genetic material obtained from primary and recurrent tumours, as well as from pulmonary metastasis was analysed and compared at a molecular level. Next generation sequencing (NGS) analysis revealed BRAFV600E mutation, detected only in 2-5% of glioblastomas, in both the primary tumour and pulmonary metastases. Importantly, this mutation provides a possible therapeutic option as it constitutes a target for clinically approved inhibitors. This case study not only demonstrates a molecular comparison of primary, recurrent and metastatic glioblastoma, but also emphasizes the need for precise molecular diagnostics, which may facilitate treatment choice, especially in tumours currently lacking efficient treatment.

The molecular pattern of histopathological progression to anaplastic meningioma - A case report.

Meningiomas (MGs) are the most frequent primary tumours of the central nervous system (CNS) and exhibit a large spectrum of histological types and clinical phenotypes. The WHO classification of CNS tumours established strict diagnostic criteria of the benign (Grade 1), atypical (Grade 2) and anaplastic (Grade 3) subtypes. Combined with the resection rate, WHO grading has the most crucial role as the prognostic factor. Additionally, such biomarkers as Ki-67/MIB-1, progesterone receptors and phosphor-histone H3 were correlated with MG progression. Recently, it was suggested that the aggressive behaviour of some MGs is attributed to molecular alterations, regardless of their histopathology. The analysis of loss of heterozygosity (LOH) at chromosomes 1, 9, 10, 14 and 22 was performed. The presented case of WHO Grade 2 MG initially exhibited LOH at chromosomes 10, 14 and 22. In the first recurrence, the tumour genetic profiling revealed additional LOH at chromosome 1p and atypical histopathology. During the second recurrence, an aggressive phenotype was observed and tumour progressed to an anaplastic form. Considering the appearance of the tumour relapses, the set of molecular changes overtook the histopathological progression. The genetic and histopathological imbalance in the tumour progression in secondary anaplastic MGs has not been previously described. The evolution of genetic and histopathological changes was presented in the same patient. In the future, the individualised therapy of potentially more aggressive forms of MGs could be based on certain chromosome aberrations.