Autistic-like traits in laboratory rodents exposed to phthalic acid esters during early development - an animal model of autism?

Phthalates are chemical substances that are widely used to provide flexibility and durability to plastic materials. They leach from products in which they are mixed and reach living organisms. Results from experimental studies suggest that exposure to phthalates can have a negative impact on an individual's neuronal system and behavior. In this regard, exposure during early ontogenesis seems to be particularly dangerous due to the extensive growth and development of body structures and functions. Disruption during this critical time can result in alterations of behavior and the emergence of neurodevelopmental disorders, such as autism spectrum disorder (ASD). Various animal models have been used to elucidate the pathogenesis of this disease. They are fundamental for research, and although the translation of results to humans is difficult, new animal models are being developed. The aim of this review is to summarize laboratory rodent studies in which early developmental phthalate exposure resulted in brain alterations and autistic-like behavioral traits. We also discuss the possibility of using early developmental phthalate exposure in rodents to create a new animal model of autism.

Impact of prenatal hypoxia on the development and behavior of the rat offspring.

The healthy development of the fetus depends on the exact course of pregnancy and delivery. Therefore, prenatal hypoxia remains between the greatest threats to the developing fetus. Our study aimed to assess the impact of prenatal hypoxia on postnatal development and behavior of the rats, whose mothers were exposed to hypoxia (10.5 % O2) during a critical period of brain development on GD20 for 12 h. This prenatal insult resulted in a delay of sensorimotor development of hypoxic pups compared to the control group. Hypoxic pups also had lowered postnatal weight which in males persisted up to adulthood. In adulthood, hypoxic males showed anxiety-like behavior in the OF, higher sucrose preference, and lower levels of grimace scale (reflecting the degree of negative emotions) in the immobilization chamber compared to the control group. Moreover, hypoxic animals showed hyperactivity in EPM and LD tests, and hypoxic females had reduced sociability compared to the control group. In conclusion, our results indicate a possible relationship between prenatal hypoxia and changes in sociability, activity, and impaired emotion regulation in ADHD, ASD, or anxiety disorders. The fact that changes in observed parameters are manifested mostly in males confirms that male sex is more sensitive to prenatal insults.

A mixture of diethylhexyl, diisononyl and dibutyl phthalate decreased anogenital distance, postnatal testosterone levels, and changed social behavior in Wistar rats.

Phthalates are chemicals interfering with the function of testosterone and are suspected to play a role in the emergence of neurodevelopmental diseases. This could be due to interference with brain development for which optimal testosterone levels are essential. We investigated the effect of prenatal and early postnatal exposure to a phthalate mixture on the anogenital distance (AGD), plasma testosterone levels and social behavior in rats. Pregnant rats were exposed to a mixture of diethylhexyl, diisononyl and dibutyl phthalate, each at a dose of 4.5 mg/kg/day, from gestational day 15 to postnatal day 4. A social interaction test was performed to assess sociability in the three ontogenetic stages (weaning, puberty, adulthood). AGD was measured in adulthood to assess changes in prenatal testosterone levels. Plasma testosterone levels were measured in adults by a radioimmunoassay. The total frequency and time of socio-cohesive interactions were decreased in phthalate exposed females in weaning, puberty and adulthood. Phthalate exposed males showed a decrease in the frequency of social interactions in weaning only. Shorter anogenital distance was observed in adult males exposed to phthalates. Decreased testosterone levels were observed in the exposed group in both sexes. Our results suggest that early developmental phthalate exposure may play an important role in the hormonal and behavioral changes associated with several neurodevelopmental diseases.

An undifferentiated sarcoma with BCOR-CCNB3 fusion transcript - pathological and clinical retrospective study.

The BCOR-CCNB3 positive sarcoma is a recently identified sarcoma morphologically and clinically similar to Ewing sarcoma in adolescents and young adults. The BCOR-CCNB3 fusion transcript originates from a paracentric inversion on the X chromosome with an in-frame fusion between the last codon of BCOR and the exon 5 of CCNB3 gene. We report morphological and molecular genetic analysis of 8 undifferentiated sarcomas positive for the BCOR-CCNB3 fusion. Six of the eight BCOR-CCNB3 positive sarcoma patients were male. Five of the eight patients were in their second decade of life (median of all patients 14 years at diagnosis). The bone marrow involvement was demonstrated in 2 of 4 patients tested. Detection of the fusion transcripts BCOR-CCNB3 in the bone marrow suggests that patients with positive findings are at high risk of the tumor progression.

Gastrointestinal stromal tumors - quantitative detection of the Ki-67, TPX2, TOP2A, and hTERT telomerase subunit mRNA levels to determine proliferation activity and a potential for aggressive biological behavior.

Gastrointestinal stromal tumors (GISTs) have an unpredictable biological potential ranging from benign to malignant. Molecular markers involved in the mechanisms of proliferation and cellular senescence may provide additional information about biological behavior of the tumor. The aim of the present study was to investigate Ki-67, TPX2, TOP2A and hTERT mRNA expression levels in specimens from patients with GISTs to define relationships between proliferation activity and biological potential and progression of the disease. We measured Ki-67, TPX2, TOP2A and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ RT PCR). The highest Ki-67, TPX2, TOP2A and hTERT mRNA expression levels were found in the highly proliferative BLs (18 specimens), in comparison with GISTs (137 specimens) and LMSs (9 specimens). Patients with GISTs and adequate information about mitotic activity, tumor size and anatomical site (84 specimens) were divided into two groups - GISTs with benign (29 patients) and with malignant (55 patients) potential. We observed association between higher Ki-67, TPX2 and hTERT mRNA levels and the GISTs with malignant potential. Univariate analysis (57 patients with available follow-up information) of survival (Kaplan Meier curves method) revealed a correlation between higher levels of TPX2, Ki-67 and hTERT markers and shorter event-free survival (EFS) or poorer overall survival (OS). The results demonstrate the importance of quantitative assessment of the proliferation activity in GISTs. Proliferation markers of Ki-67, TPX2, TOP2A and hTERT are suitable markers for detection the proliferation activity and telomerase activity of these tumors. Furthermore, the assessment of TPX2, Ki-67 and hTERT expression levels is appropriate for determination of malignant potential of GISTs.

Rhabdomyosarcoma: molecular analysis of Igf2, MyoD1 and Myogenin expression.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. There are two major histopathological types of RMS ­ embryonal (eRMS) and alveolar (aRMS). A molecular study of Igf2, MyoD1 and Myogenin was performed to determine the expression profiles and to assess the possible utility of these genes as potential treatment targets. Patients with RMS showed up to 100-fold increase of Igf2 transcription in comparison with normal skeletal muscle. Our data suggest that overexpression of Igf2 occurs in RMS of both histological subtypes. No correlation between the results of Igf2 mRNA expression and LOH at the 11p15 region (p= 0.12) was observed, but there was a trend of a higher expression of Igf2 mRNA in RMS samples with LOH. We observed a high level of MyoD1 mRNA in both aRMS and eRMS, and we detected a similar level of MyoD1 mRNA in RMS and normal skeletal muscles. There was a correlation between the results of MyoD1 mRNA expression and LOH at the 11p15 region.We did not observe any statistical difference in the level of Myogenin mRNA in the subgroups of RMS. Analogous to MyoD1, we observed a similar level of Myogenin mRNA in RMS and normal skeletal muscles.

[Clinical relevance of chromosomal aberrations in bone and soft tissue tumors in children and young adults]. / Klinický význam chromozomálních zmen u nádoru kostí a mekkých tkání detí a mladistvých.

BACKGROUND: We present the results of a cytogenetic and molecular cytogenetic analysis of a series of patients with bone and soft tissue tumors. PATIENTS ANDMETHODS: We analyzed a cohort of 26 patients with Ewing sarcoma/PNET, 15 patients with rhabdomyosarcoma, 5 with synovial sarcoma and one patient with an undifferentiated sarcoma using the cytogenetic and molecular cytogenetic techniques M-FISH and arrayCGH. RESULTS: We found nonrandom chromosomal structural and numerical changes with diagnostic and prognostic relevance in most patients. Eight patients with ES/PNET had only a t(11;22)(q24;q12), eight patients had secondary aberrations as well and six had only secondary aberrations. In the RMS patients we detected the t(1;13)(p36;q14) once and the t(2;13)(q35;q14) four times, both of them characteristic for the alveolar subtype with poor prognosis and numerical aberrations, characteristic for the embryonal subtype, in five patients. Four patients with synovial sarcoma had the diagnostic t(X;18)(p11.2;q11.2), one of them had a complex karyotype with a complex t(X;18;21) (p11.2;q11.2;q11.2) together with t(2;5)(q24-32;p13-14) and t(12;20)(p11;q13). We correlated the karyotype of cancer cells with histopathologic morphologic analysis, clinical outcome and foreign published results. CONCLUSION: Cytogenetic and molecular cytogenetic analysis is a valuable diagnostic tool in bone and soft tissue tumors, especially in less differentiated subtypes, and as such it should be an integral part of curative care.

Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinomas.

The frequency and prognostic relevance of RET proto-oncogene somatic mutations in sporadic medullary thyroid carcinoma (MTC) remain controversial. In order to study somatic mutations in the RET proto-oncogene in sporadic MTCs found in the Czech population and to correlate these mutations with clinical and pathological characteristics, we investigated 48 truly sporadic MTCs by sequencing classical risk exons 10, 11, 13, 14, 15 and 16. From the 48 tumors studied, 23 (48%) had somatic mutation in the RET proto-oncogene in exons 10, 11, 15 or 16. The classical somatic mutation Met918Thr in exon 16 was only found in 13 tumors (27%). In five cases, multiple somatic mutations and deletions were detected. A statistically significant correlation between the presence of somatic mutation with more advanced pathological TNM stages was observed. Other clinical and pathological characteristics did not show any statistical significant association with the presence or absence of somatic mutation.

Rhabdomyosarcoma: molecular diagnostics of patients classified by morphology and immunohistochemistry with emphasis on bone marrow and purged peripheral blood progenitor cells involvement.

Two histologically distinct subtypes of rhabdomyosarcomas (RMS), embryonal and alveolar, are different in many aspects, such as age distribution, primary site, and clinical outcome. We analyzed a group of 30 patients with RMS. The aim was to broaden the spectrum of diagnostic tools in evaluating the primary tumors, their recurrences and/or metastases, and to extend the diagnostic boundary to bone marrow and purged peripheral progenitor blood cell samples. We have performed the RT-PCR assay to analyze RMS for the presence of expression of MyoD1 gene and for the presence of chimeric transcripts PAX3/FKHR or PAX7/FKHR. MyoD1 gene expression was found in all 30 patients in samples from primary tumors. The chimeric transcripts PAX/FKHR were identified in 13 of 15 patients with alveolar RMS. Furthermore, the fusion transcript PAX7/FKHR was identified in 2 of 15 patients with RMS classified as embryonal by histology. Bone marrow samples (12) and peripheral blood progenitor cell specimens (13) in ten patients were examined by RT-PCR. We were able to identify 7 patients with bone marrow involvement and/or with contamination of peripheral blood progenitor cells by the tumor cells. We demonstrate that employing molecular diagnostics has an impact on staging, therapy monitoring and recognition of malignant cells at the tumor resection margins.

[Molecular diagnosis of malignant B-cell lymphoma: detection of clonality and a brief review of problems with our results]. / Molekulární diagnostika maligních lymfomu rady B: detekce klonality a strucný prehled problematiky s vlastními výsledky.

BACKGROUND: Diagnostic approach to non-Hodgkin's lymphomas requires a combination of laboratory methods. Methods used in morphology constitute the basis of the diagnostics; in many instances it is necessary to combine them with methods of molecular genetics. The latter method plays a key role in the detection of B cell clonality using identification of the rearrangement of IGH and/or IGK genes and in detection of the chromosomal translocations specific for some lymphomas. METHODS AND RESULTS: Using PCR we investigated 113 patients with malignant B cell lymphomas of different types (follicular--FL, mantle cell--MCL, small cell--CLL/SLL, diffuse large cell--DLBCL). We established the IGH gene clonal rearrangement in 85% of the cases (96/113), and the clonal rearrangement of the IGK gene in 58.3% patients (42/72). Combination of both approaches (IGH and IGK) revealed a positive result in 90.3% (102/113). The highest yield was rendered in patients with CLL/SLL and with MCL (100%), and it was 86 and 87% in cases with FL and DLBCL. CONCLUSIONS: The detection of clonality in lymphomas helps to distinguish a malignant disease from polyclonal hyperplastic and lymphoproliferative disorders of B cells. The recognition of clonal rearrangements of the IGH and IGK genes serves for a long term monitoring of the disease activity in cases in which there are no other molecular markers available. The demonstration of lymphoma characteristic translocations is relatively specific and useful but at present its usefulness is reduced in cases with variable breakpoint regions.

[Anaplastic large-cell lymphoma: review]. / Anaplastický velkobunecný lymfom: prehled problematiky.

Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of malignant lymphoproliferative diseases. Most of the cases are of T-cell line with a loss of cell surface receptors but with a production of cytotoxic cytoplasmatic granules--immunohistochemically (IHC) positive perforin, granzyme B, and TIA-1. The diagnostics of ALCL is based on morphological findings and results of IHC, which further stratify ALCLs to basic immunophenotypes according to ALK (anaplastic lymphoma kinase) protein expression--ALCL CD30+ ALK+ and ALCL CD30+ ALK+. The morphological investigations are supplemented by karyotyping and/or by a demonstration of breakpoint at 2p23 harboring ALK gene (FISH), and by molecular detection of chimeric genes characteristic of ALK+ lymphomas (NPM-ALK, ATIC-ALK, TPM3-ALK, TFG-ALK, and some even rarer rearrangements). Molecular diagnostics is important in monitoring minimal residual disease. As some of the characteristic molecular changes were demonstrated in healthy individuals and in Hodgkin's disease by quantitative PCR, the validation of these findings demands further studies. ALK protein positive ALCLs affect patients in age categories up to the third decade, whereas ALK protein negative cases occur in older patients with an average age of 60 years. Both subgroups of lymphomas are aggressive but ALK+ lymphomas react well to systemic treatment, and have a more favorable prognosis. Primary skin ALCLs belong to a group of T-cell lymphoproliferative diseases of the skin and have, in the majority of cases, a favorable course without generalization.

[New aspects of MALT lymphomas of the stomach]. / Nové pohledy na problematiku MALT lymfomu zaludku.

The knowledge about MALT lymphomas has dramatically changed in the last few years. The genesis of MALT lymphomas has become clearer, and new tools in modern diagnostics are available. This article, as a concise review, summarises the latest data and the clinical and morphological characteristics with the aspect of routine diagnostics. The characteristic translocation t(11;18) and its chimeric transcript API2-MALT1 with antiapoptotic function is emphasised.

[Follicular lymphomas: molecular diagnosis using t(14;18)(q32;q21)--fluorescence in situ hybridization, qualitative and quantitative PCR]. / Folikulární lymfomy: molekulární diagnostika t(14;18)(q32;q21)--fluorescencní in situ hybridizace, kvalitativní a kvantitativní PCR.

Diagnosis of follicular lymphoma (FL) is based on histology and immunohistochemical profile (CD20+, CD79alfa+, CD10+, BCL-2+, CD5-). A chromosomal marker--translocation t(14;18)(q32;q21) supporting the tumor diagnosis and useful for monitoring bone marrow or peripheral blood infiltration by the tumor cells is also used. The BCL2 gene (18q21) is controlled by an enhancer of the IGH gene (14q32) resulting in BCL-2 protein overexpression. The translocation is present in the majority of patients with FL. The aim of the study was to introduce the quantitative PCR (RQ-PCR, real-time quantification) method for the assessment of the quantity of cells bearing the translocation t(14;18) in patients with FL. The fluorescence in situ hybridization on interphasic nuclei (I-FISH) in histologic sections was used for screening of patients with the t(14;18). A search for the break of the BCL2 gene at the major breakpoint region (mbr) was performed by means of qualitative PCR. We determined the relative number of the tumor cells bearing t(14;18) translocation (mbr) in patients with FL by the RQ-PCR. The relative quantity of these cells was significantly higher in the lymph nodes than in the bone marrow or peripheral blood. The RQ-PCR is a tool of choice to monitor the activity of the disease in individual patients, and to detect an early disease relapse before its manifestation at the level diagnosed by morphology.

The newly detected mutations in the RET proto-oncogene in exon 16 as a cause of sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) occurs as a sporadic form or, less frequently, as an autosomal dominant inherited familial disorder. Germline mutations in the RET proto-oncogene in exons 10, 11, 13, 14, 15, and 16 are found in most of the familial cases (nearly 95%). Somatic mutations in sporadic MTC are detected in 23-69% of patients. The most frequent somatic mutation is located in exon 16 at codon 918, and only a small percentage of mutations are found in exons 10, 11, 13, and 15. We have searched for somatic mutations in Czech MTC patients using direct sequencing. We report here two new somatic missense mutations in exon 16 of the RET proto-oncogene associated with the sporadic MTC detected in two Czech men. A homozygous mutation at codon 922 TCC(Ser)-->CCC(Pro) as a result of loss of heterozygosity was revealed in the first patient. In the second one a heterozygous mutation at codon 930 ACG(Thr)-->ATG(Met) was found.

Long-term follow-up of chimerical state of the patients transplanted for different haematologic diseases.

Long-term follow-up of peripheral cellular chimerism in patients treated with BMT or PBSCT revealed the usefulness of their continuous monitoring at molecular level. Our results are based on monitoring of 120 patients, who were followed for at least 24 months. Comparison of the patients treated for chronic myelogenous leukemia (CML), acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS) and aplastic anaemia (AA) revealed that mixed chimerism was practically absent in MDS and relatively long-lasting in ALL and AA (regardless to substantially different post-transplantation treatment). The first disease relapses signalized by molecular checking of mixed peripheral chimerism were observed also after a period of remission lasting for several years. Molecular watching enables us to detect relapses at their very beginning that would remain hidden to less sensitive methods. We believe that all of the transplanted patients ought to be monitored for residual disease i.e. cellular chimerism using molecular methods without time limits. On the other hand low level of mixed cellular chimerism is not necessarily a sign of disease progression and can remain unchanged as "status quo" for a very long period.

[Large-cell diffuse B-cell lymphoma: heterogenous origin and prognosis from the aspect of modern diagnosis]. / Velkobunecné difuzní B lymfomy: heterogenní puvod a prognóza z hlediska soucasné diagnostiky.

BACKGROUND: Diffuse large B-cell lymphomas represent a heterogeneous group of tumors with a different origin, morphological findings and a variable clinical prognosis. These tumors have been recently classified into two prognostically relevant subgroups differing in the gene expression. The key genes suitable for routine diagnostics of DLBCL have not been yet identified. The aim of this work was to study changes and expression of several genes and proteins participating in the genesis of DLBCL. METHODS AND RESULTS: We analysed a group of 31 patients with diffuse large B-cell lymphomas. Basic clinical data including follow-up of the patients were available. Tumors were examined by a panel of immunohistochemical reactions with antibodies against CD20, CD79a, BCL-2, BCL-6, CD10, Ki-67 and TP53. FISH was used to detect a translocation t(14;18)(q32;q21) and/or a break in BCL6 region (3q27) suggestive of a translocation with a variable translocation partner t(3;?). PCR was utilized to detect the translocation t(14;18) and a clonal rearrangement of heavy and/or kappa chain of the immunoglobin genes. CONCLUSIONS: The expression of BCL-2 protein appeared to correlate with a higher mortality rate. The expression of other proteins examined in the study did not correspond significantly with the clinical development of the disease. Tumors with follicular lymphoma as a component had significantly higher mortality rate than the tumors developing de novo. Moreover, higher mortality was evident in cases with higher values of the International Prognostic Index (IPI).

Ethanol fixation of lymphoma samples as an alternative approach for preservation of the nucleic acids.

Molecular methods play an important role in diagnostic pathology of lymphomas. PCR based demonstration of clonality or detection of a specific chromosomal translocation may determine the exact classification of the lymphoma. Hence the final diagnosis may depend on the quality of preserved nucleic acids in the bioptic specimen. The integrity of DNA and RNA may be damaged by formalin fixation, which destroys the nucleic acids by fragmentation. Therefore, a portion of each lymphoma sample should be frozen. To substitute freezing techniques we utilized ethanol as a fixative, which preserves nucleic acids. We compared PCR and RT-PCR products from lymphoma samples, which were differently pre-treated by ethanol fixation, formalin fixation and freezing. The ethanol fixed samples retained a high quality of both DNA and RNA and provided reproducible PCR products similar to frozen samples and significantly better then those extracted from formalin fixed samples. We may recommend ethanol as a complementary fixative for all pathology laboratories where deep freezing in not routinely available.