HER-3 molecular classification, expression of PD-L1 and clinical importance in breast cancer.

Receptors of the large HER family play an important role in breast cancer, which is undergoing a gradual development in connection with biological development, both in the field of diagnostics and therapy. Dimerization of HER-2 with other HER members, such as HER-3, is the biggest driver of tumor cell growth and survival. Numerous studies show that HER-3 gene overexpression correlates with poor prognosis. However, other studies have shown HER-3 overexpression to be a positive prognostic factor. HER-3 may confer resistance to certain EGFR or HER-2 receptor therapeutics. An interesting fact, however, is that HER-3 expression can serve as a marker in immunotherapy for triple-negative breast cancer (TNBC). It is thought to be involved not only in cell survival and proliferation, but also in the regulation of PD-L1 expression. In breast cancer, PD-L1 expression is heterogeneous and is generally associated with the presence of tumor­infiltrating lymphocytes and a number of factors with poor prognosis such as young age, hormone receptor negativity, and high HER-2 expression and proliferation index. Our results showed amplification of HER-3 (CERB3) in 2 out of a sample of 20 patients with TNBC, and 13 of 20 HER-2­positive patients. PD-L1 expression was demonstrated in 3 out of 13 HER-3­positive patients and 2 out of 2 HER-3­positive TNBC patients. There was a strong correlation between positive HER-3 and PD-L1 TNBC expression (p = Keywords: breast cancer, HER family, overexpression, HER-3, HER-2, PD-L1, TNBC.

Bacterial DNA detection in very preterm infants assessed for risk of early onset sepsis.

OBJECTIVES: The aim of this study is to evaluate the diagnostic ability of multiplex real-time polymerase chain reaction (PCR) in very preterm infants assessed for risk of early onset neonatal sepsis (EOS). METHODS: Prospective observational cohort study. Blood samples of preterm neonates ≤32 weeks of gestation were evaluated by commercial multiplex real-time PCR within 2 h after delivery. The definition of EOS was based on positive blood culture and clinical signs of infection or negative blood culture, clinical signs of infection and abnormal neonatal blood count and serum biomarkers. RESULTS: Among 82 subjects analyzed in the study, 15 had clinical or confirmed EOS. PCR was positive in four of these infants (including the only one with a positive blood culture), as well as in 15 of the 67 infants without sepsis (sensitivity 27%, specificity 78%). Out of 19 PCR positive subjects, Escherichia coli was detected in 12 infants (63%). Statistically significant association was found between vaginal E. coli colonization of the mother and E. coli PCR positivity of the neonate (p=0.001). No relationship was found between neonatal E. coli swab results and assessment findings of bacterial DNA in neonatal blood stream. CONCLUSIONS: Multiplex real-time PCR had insufficient diagnostic capability for EOS in high risk very preterm infants. The study revealed no significant association between PCR results and the diagnosis of clinical EOS. Correlation between maternal vaginal swab results and positive PCR in the newborn needs further investigation to fully understand the role of bacterial DNA analysis in preterm infants.

Molecular pathology of cholangiocellular carcinomas.

Cholangiocellular carcinoma is a relatively rare malignant tumor, originating from cholangiocytes, with poor prognosis and late diagnosis. It is a malignancy with a variable biological etiology, numerous genetic and epigenetic changes. Its incidence in the Czech Republic is about 1.4 per 100,000 people per year. For good prognosis and long-term survival, early diagnosis with surgical treatment is important. In these cases, a 5-year survival rate is about 20-40 %. In the early diagnosis imaging methods and histopathological verification play an essential role, whereas laboratory oncomarkers are not yet sufficiently accurate. The same applies for genetic markers. This leads to the search of new molecular targets and the high effort in the introduction of cytological and molecular-biological methods with high specificity and sensitivity into routine practice. Current early diagnosis is based on the use of efficient imaging methods. The use of genetic testing, and especially knowledge of the molecular basis of this disease, will be of a great benefit. The observation of the association between the genetic pathways, IDH1, RAS-MAPK etc., and genetic mutations of genes, such as TP53, KRAS, SMAD4, BRAF, IDH1/2, may be significant. From the molecular point of view, it is also interesting to monitor oncogenic potential in HBV/HCV infection.

Molecular pathological predictive diagnostics in a patient with non-small cell lung cancer treated with crizotinib therapy: A case report.

Lung cancer is one of the most common malignant cancers in the Czech Republic in men, with the highest mortality rate of all the malignant diseases. The development of biological treatment enables study into novel personalized treatment options. This type of treatment is usually of high quality, and is often demanding of predictive and biopsy diagnostics, which is dependent on the quality of the collected material and close cooperation among particular departments. The present study describes the complete biopsy and predictive examinations performed in a male patient with lung adenocarcinoma, with an emphasis on the logistics of the whole process and the application of the tyrosine kinase inhibitors, crizotinib and LDK378. The patient experienced a long overall survival time of 28 months from diagnosis.

Complete diagnostics and clinical approach for a female patient with unusual glioblastoma: A case study.

The present study reports a case of a 44-year-old female patient with a large frontal lobe tumor who underwent surgery using a modern navigation system SonoWand that combines the advantages of a non-frame navigation system with intraoperative real-time ultrasound imaging. The right frontal lobe tumor consisted of two morphologically different sections. A diffuse astrocytoma grade II and a glioblastoma grade IV were identified. These tumors were relatively substantially separated. A 17 p deletion, including TP53, was detected in a diffuse astrocytoma but not in a glioblastoma. EGFR and MDM2 amplifications were detected only in a glioblastoma. Detection of these amplifications is typical for primary glioblastomas. These findings support our assumption of two independent tumors. The KRAS, BRAF and EGFR gene mutations were also detected in a glioblastoma. Such an accumulation of molecular mutations is rare in one tumor. Following oncological treatment the patient was cared for in the oncological center and survived for 15 months after the surgery without any signs of a disease. This is an unusual case, and to the best of our knowledge, is not frequently published in literature.

Atypical Polypoid Adenomyoma of the Uterus: An Immunohistochemical and Molecular Study of 21 Cases.

Atypical polypoid adenomyoma (APA) is an uncommon uterine lesion that commonly recurs after local excision and is occasionally associated with or precedes the development of atypical hyperplasia or endometrioid adenocarcinoma. Despite the fact that about 230 cases have been reported in the literature, only 2 studies of 6 and of 7 cases have investigated the molecular aspects; as such, molecular alterations that occur in APA remain largely unknown. We undertook a comprehensive immunohistochemical and molecular analysis of 21 cases of APA in 17 patients (including 4 recurrent/persistent lesions). The analyzed genes were PTEN and TP53 (by fluorescence in situ hybridization) and KRAS, BRAF, EGFR, and NRAS (all by polymerase chain reaction). Immunohistochemical staining was performed for PTEN, p53, mTOR, ß-catenin, HNF-1ß, and GLUT1 and for the mismatch-repair proteins MLH-1, MSH-2, MSH-6, and PMS-2. In most cases, there was nuclear expression of ß-catenin in squamous morules and expression of HNF-1ß, mTOR, and GLUT1 in the glandular component. All cases exhibited "wild-type" expression of p53. A common finding was loss of PTEN expression (6/19 cases). In 1 of these cases, loss of PTEN expression was accompanied by PTEN deletion. Mutation of the KRAS gene was found in 5/19 cases. Intact mismatch-repair protein expression was present in all cases, and TP53 abnormalities or mutations of EGFR, NRAS, or BRAF genes were not found. Given the association with atypical hyperplasia and endometrioid adenocarcinoma and the shared immunohistochemical and molecular features, we feel that, conceptually, APA is best regarded as analogous to a localized form of atypical hyperplasia.

Pathomorphology of inflammatory response following traumatic brain injury, serum values of interleukins, and gene polymorphisms.

UNLABELLED: Our present study was aimed to investigate time-profile kinetics of interleukins, vascular endothelial growth factor (VEGF) in acute inflammatory response following traumatic brain injury, and the influence of activated microglial cells in patients who developed severe space occupying lesion (SOL) of secondary traumatic brain injury. Interleukins IL-6, monocyte chemo attractant protein (MCP-1), and VEGF had a significant different time-profile kinetics (p<0.05) in patient with, and without expansive traumatic brain contusions (SOL). The serum VEGF was significantly higher in trauma patients with uncomplicated brain contusions, and lower in patients with SOL. The patients with septic complications developed the sudden increase of TNF alpha and IL-8 within the first 72 hours. Our data suggested PSGL and CD68 immunopositivity of microglial cells in both focal and diffuse TBI, predominantly in perivascular space correlated with telolysosome formation in cytoplasma. Polymorphism of PAI-1, MTHFR, eNOS, VEGF, and Apo E genes in TBI were in patients with SOL were bound to show up leucocyte plugging in capillaries. KEYWORDS: traumatic brain injury - acute inflammatory response - microglial cells - interleukins - vascular endothelial growth factor - monocyte chemoattractant protein - gene polymorphisms.

Comparison of immunohistochemistry, four in situ hybridization methods and quantitative polymerase chain reaction for the molecular diagnosis of HER2 status in gastric cancer: a study of 55 cases.

In the current study, the sensitivity and specificity of methods of HER2 status detection were studied in 55 patients presenting with gastric/gastroesophageal junction carcinoma (30 intestinal and 25 diffuse), in small biopsy (endoscopy; n=33) and resection specimens (n=22). The primary objective of the present study was to compare various methods for the assessment of HER2 status, with regards to the sensitivity and specificity of each method, as well as their concordance. In all cases, the status of HER2 was determined using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH), and quantitative polymerase chain reaction (qPCR). The concordance rate between IHC and ISH was 100% for IHC 0 and 3+. The concordance rate for IHC 1+ was 100% between IHC and SISH, and 92.9% between IHC and FISH. The concordance rate among different FISH methods was 100%, between FISH and SISH it was 96.2%, and between qPCR and ISH methods it was 88.5%. Thus, the results demonstrate that different in situ hybridization methods are comparable and that none were superior. Furthermore, the IHC and FISH methods were found to be comparable and the concordance rate was particularly good. qPCR analysis correlated well with the other methods and appears to be a possible alternative tool for detection of the HER2 status. However, the concordance rate of qPCR with other methods was identified to be lower in the diffuse carcinoma group of endoscopy biopsy specimens; therefore investigation of further cases is required.

[Molecular testing in malignant melanoma]. / Molekulární testování melanocytárních lézí

The use of molecular biology and cytogenetic methods and the development of knowledge regarding the molecular basis of melanocytic tumors in recent years has been on the rise. Topics of interest include not only diagnostic and predictive pathology, but also genetic counseling and molecular classification of melanocytic lesions. The following text discusses the current knowledge on the molecular basis of malignant melanoma, the use of molecular biology and cytogenetic methods, especially in the differential diagnosis of melanocytic lesions, and the difficulties of predictive testing.

Administration of a plasmid that expresses SDF-1α affects the oncogenic potential of mouse bcr-abl-transformed cells.

Stromal-derived factor 1α (SDF­1α, also known as CXCL12) is a chemokine that exerts its effects through the G-protein coupled receptors, C-X-C chemokine receptor type 4 (CXCR4) and 7 (CXCR7). There is marked evidence that the SDF-1/CXCR4 axis is involved in the pathogenesis of leukemia and therapies that target this axis are under development. The present study aimed to increase the efficacy of a DNA-based bcr-abl vaccine by simultaneously immunizing mice with a plasmid carrying the whole SDF-1α gene. Bcr-abl­transformed 12B1 cells were used to challenge the mice. These cells have the oncogenic potential to induce both leukemia following intravenous inoculation and lymphoma-type solid tumors after subcutaneous inoculation. Administering an SDF­1 carrying plasmid together with the bcr-abl vaccine resulted in increased survival following a challenge with subcutaneously administered 12B1 cells, although the difference was not statistically significant. However, there was a difference when the animals that developed subcutaneous tumors were only taken into consideration. In doubly-treated mice, significantly more mice failed to develop solid tumors than mice that had only received the bcr-abl vaccine. By contrast, the occurrence of fatal leukemia was significantly higher in the mice that were treated with the SDF-1 plasmid, regardless of whether they were immunized with the bcr-abl-vaccine. No humoral or cellular immune responses against SDF­1 were detected in the treated mice, which suggested that the changes in oncogenic potential of 12B1 cells were due to the activity of SDF-1 itself.

[Ultrastruktural diagnosis of hypertrophic kardiomyopathy with ß-aktin mutation in sudden death - case report]. / Ultrastrukturální diagnostika hypertrofické kardiomyopatie s mutací ß-aktinu u náhlého úmrtí - kazuistika.

INTRODUCTION: Light microscopy and electron microscopy rank among methods to diagnose of cardiomyopathy in forensic medicine, and, recently, the methods of molecular biology. METHODS: Investigation of 27 year old man who collapsed on his way to work. The Rescuers did not succeed resuscitation of vital function. Samples were H-E stained and processed for the electron microscopy. RNA was isolated from the tissue for the alpha, beta, gama actine primer investigation. RESULTS: By H-E staining we proved irregular hypertrophic cardiomyocytes (disarray) with the links and loci patches of thin fibrosis. Ultrastructurally we diagnosed a disarray of Z-bands, accumulation of mitochondria, rectangular nuclei of cardiomyocytes. We have detected rare plasmocytes and leucocytes with specific granules in cytoplasma. In the electronogrames we can see myofibriles oriented longitudinally and transversally. A genetic examination demonstrated beta actin mutation. CONCLUSION: Cardiomyopathy can be a cause of sudden and unexpected death in young individuals and its diagnostics requires an interdisciplinary collaboration. KEYWORDS: Sudden and unexpected death - hypertrophic cardiomyopathy - ultrastructure of cardiomyocyte - gene mutation.

[Polymerase chain reaction: basic principles and applications in molecular pathology]. / Polymerázová retezová reakce: princip metody a vyuzití v molekulární patologii.

Polymerase chain reaction (PCR) is a technology used for quick and easy amplifying DNA sequences, which is based on the principle of enzymatic replication of the nucleic acids. This method has in the field of molecular biology an irreplaceable role and constitutes one of the basic methods for DNA analysis. In the following article we describe the basic principles of PCR, and its importance especially in the field of pathology.

Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony stimulating factor (GM-CSF): II. Adverse effects of GM-CSF.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is considered to be the most effective immunostimulating factor for the construction of gene-engineered anti-cancer vaccines. In some tumour cells, this type of genetic modification has resulted in the loss of the oncogenic potential. This was not the case with bcr-abl-transformed mouse 12B1 cells. A cell line, designated 12B1/GM-CSF/cl-5 producing more than 100 ng/106 cells/24 h, displayed higher pathogenicity than the parental, non-transduced cells. Although the tumours induced by the parental 12B1 cells and 12B1/GM-CSF/cl-5 cells appeared nearly at the same time and then grew at an approximately equal rate, the latter mice were in a much poorer clinical condition. In these animals the growth of the tumours was associated with gradually increasing blood levels of GM-CSF. In both groups of animals splenomegaly was observed; it was much more pronounced in the case of 12B1/GM-CSF/cl-5-inoculated animals. While in the case of animals inoculated with the parental cells the splenomegaly was probably mainly due to infiltration with tumour cells, in the animals inoculated with the GM-CSF-secreting cells splenomegaly and derangement of parenchymal organs, such as lungs, liver and kidneys, were more complex, including congestion and infiltration with hemopoietic cells, predominantly immature cells of myeloid lineage. The most conspicuous of these changes was the hyperaemia of the lungs. No such alterations were seen in animals inoculated with the parental cells. On the other hand, the contents of T regulatory cells were comparable in both groups and they increased in parallel at the end of the observation period. When GM-CSF neutralizing antibody was administered to animals inoculated with the 12B1/GM-CSF/cl-5 cells, the pathological changes observed within the organs were suppressed, this proving that the overproduced GM-CSF and not any other substance, played the key role in their induction.

Apoptosis - associated genes and their role in predicting responses to neoadjuvant breast cancer treatment.

BACKGROUND: Neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. The present study investigated biomarkers that can predict a pathologic response to the therapy. MATERIAL/METHODS: The role of apoptosis in regression of the tumors after neoadjuvant chemotherapy was determined by TUNEL and anti-active caspase 3 assay. The transcriptional profile of 84 key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific real-time PCR assay. Obtained data were analyzed by hierarchical cluster analysis and correlation analysis. The immunohistochemical profile of each tumor was determined using the standard ABC method. RESULTS: On the basis of a hierarchical cluster analysis of 13 significantly changed genes, we divided patients into good and poor prognosis groups, which correlate well with progression-free survival. In the good prognosis group, we found a statistically significant down-regulation of the expression of MCL1 and IGF1R genes after neoadjuvant treatment. We also found a statistically significant overexpression of BCL2L10, BCL2AF1, CASP8, CASP10, CASP14, CIDEB, FADD, HRK, TNFRSF25, TNFSF8 and CD70 genes. In contrast, we found up-regulation of IGF1R after the treatment in the group with poor prognosis. CONCLUSIONS: Gene expression profiling using real-time PCR assay is a valuable research tool for the investigation of molecular markers, which reflect tumor biology and treatment response.

Unexpected properties of endostatin-producing mouse BCR-ABL-transformed cells.

We investigated whether a genetic modification of BCR-ABL-transformed mouse cells that resulted in endostatin (ES) production altered their oncogenic potential. Mouse B210 cells, which express p210bcr-abl fusion protein and induce leukemia-like disease and extremely rarely solid tumors after intravenous (i.v.) administration, were used. The cells were transfected with a plasmid carrying genes for mouse ES and resistance to blasticidine. Transduced cells were isolated in media supplemented with blasticidine. Production of ES was determined by Western blotting. For further tests, two clones were selected, and their pathogenicity after i.v. inoculation was tested. Compared with the parental B210 cells, the capability of both gene-modified cell clones to induce lethal leukemia was reduced. However, mice that did not succumb to leukemia subsequently developed solid tumors. They were composed of poorly differentiated cells with irregular nuclei and roughly granular chromatin and were well vascularized. FISH revealed the presence of the BCR-ABL fusion gene both in tumors and spleens. Immunohistological investigation of the tumors demonstrated the production of ES in vivo and the cell lines derived from the tumors produced detectable amounts of ES, this demonstrating that the formation of solid tumors was not associated with the loss or silencing of the ES gene.