Prevalence and prognosis of PIK3CA mutations in Bulgarian patients with metastatic breast cancer receiving endocrine therapy in first-line setting.

BACKGROUND AND AIMS: In approximately 40% of patients with HER2-negative/HR-positive breast cancer tumors, the PIK3CA gene is mutated. Despite this, clinical outcomes vary between studies in this cohort. We aimed to ascertain the prevalence of PIK3CA mutations in patients with metastatic HR+/HER2- breast in Bulgaria, as well the evaluation and comparison of progression free survival (PFS) between wild-type (WT) and mutation-positive groups in the real-world setting. METHODS: Three oncology centers in Bulgaria collected 250 tissue samples between 2016 and 2022 for this multicentric retrospective study. PIK3CA mutations were identified using Real-Time qPCR. The median follow-up period was 35 months. RESULTS: The mean age of the mutant cohort was 57.6 ± 11.6 years, compared to 56.5 ± 12.2 years for the WT cohort (p = .52). The percentage of patients with visceral metastasis was 58.8% (n = 147). Approximately 84.3% (n = 210) of the patients had reached postmenopause. 29.2% (n = 73) of the patients had PIK3CA mutations. The predominant mutation was present in exon 20, H1047R (46.5%). We found a significant correlation only between the presence of a mutation and the metastatic diseases at diagnosis (p = .002). As first-line therapy, 67.1% of patients received endocrine therapy (ET) plus cyclin dependent kinase (CDK4/6) inhibitor, while the remainder received ET alone. The median PFS of patients in the group with the mutation was 32 months (95%, CI: 22-40) compared to 24 months in the WT cohort ((95%, CI: 21-36) (p = .45)); HR = 0.86 (95%, CI: 0.5-1.3) (p = .46). We corroborated our conclusion using propensity matching score analysis, (36 months [95% CI: 20-40] vs. 26 months [95% CI: 21-38], [p = .69]). CONCLUSIONS: We found that the prevalence of PIK3CA mutations in our patients was comparable to what has been reported in other nations. Our results suggest that PIK3CA mutational status has no bearing to ET efficacy in first-line setting.

Comprehensive Genetic Evaluation of Bulgarian Children with Syndromic Craniosynostosis.

Syndromic craniosynostosis (SC) is a genetically determined premature closure of one or more of the cranial sutures, which may result in severe dysmorphism, increased intracranial pressure along with many other clinical manifestations. The considerable risk of complications along with their significant incidence makes these cranial deformations an important medical problem. Aiming to elucidate the complex genetic etiology of syndromic craniosynostosis, we investigated 39 children, screened systematically with a combination of conventional cytogenetic analysis, multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridisation (aCGH). Pathological findings were established in 15.3% (6/39) of the cases using aCGH, in 7.7% (3/39) using MLPA and 2.5% (1/39) using conventional karyotyping. About 12.8% (5/39) of the patients with normal karyotype carried submicroscopic chromosomal rearrangements. Duplications were found to be more common than deletions. Conclusion: The systematic genetic evaluation of children with SC revealed a high prevalence of submicrosopic chromosomal rearrangements (most commonly duplications). This suggests the leading role of those defects in the pathogenesis of syndromic craniosynostosis. The genetic complexity of SC was reaffirmed by the dis Bulgaria covery of pathological findings in various chromosomal regions. Certain genes were discussed in conjunction with craniosynostosis.

Detecting EGFR Mutations in Patients with Non-small Cell Lung Cancer.

Mutations in the receptor of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) are used as biomarkers for predicting the response of treatment with EGFR tyrosine kinase inhibitors (EGFR TKIs). Non-small cell lung cancer patients usually have activating EGFR mutations that leads to a very good response when they are treated with EGFR TKIs. Our tumor samples were examined for the presence of sensitive mutations in the EGFR gene, resistant mutations or the absence of mutations. To identify the types of the mutation, we used a real-time polymerase chain reaction (RT-PCR) method. Additionally, we evaluated the frequency of EGFR mutations and their association with smoking status, gender and histology. The tumor samples (n = 551) were tested for 29 somatic mutations in the EGFR gene. Sensitive mutations in the EGFR genes were found in 55 NSCLC samples (10.0%). The prevalence of EGFR mutations was much higher for females than for males (27.1 vs. 3.9%, p <0.001). The prevalence of EGFR mutations was greater in subjects who had never smoked than in smokers (15.0 vs. 6.08%, p <0.003). Additionally, the frequency of EGFR mutations was higher in adenocarcinomas than in other histological types (14.9 vs. 5.1%; p <0.001). Our results show that activating mutations on the EGFR gene are more frequent in females than in males, in adenocarcinoma than other histological types and in non smokers than smokers.

Benign, Pathogenic and Copy Number Variations of Unknown Clinical Significance in Patients with Congenital Malformations and Developmental Delay.

The high frequency (3.0-5.0%) of congenital anomalies (CA) and intellectual disabilities (IDs), make them a serious problem, responsible for a high percentage (33.0%) of neonatal mortality. The genetic cause remains unclear in 40.0% of cases. Recently, molecular karyotyping has become the most powerful method for detection of pathogenic imbalances in patients with multiple CAs and IDs. This method is with high resolution and gives us the opportunity to investigate and identify candidate genes that could explain the genotype-phenotype correlations. This article describes the results from analysis of 81 patients with congenital malformations (CMs), developmental delay (DD) and ID, in which we utilized the CytoChip ISCA oligo microarray, 4 × 44 k, covering the whole genome with a resolution of 70 kb. In the selected group of patients with CAs, 280 copy number variations (CNVs) have been proven, 41 were pathogenic, 118 benign and 121 of unknown clinical significance (average number of variations 3.5). In six patients with established pathogenic variations, our data revealed eight pathogenic aberrations associated with the corresponding phenotype. The interpretation of the other CNVs was made on the basis of their frequency in the investigated group, the size of the variation, content of genes in the region and the type of the CNVs (deletion or duplication).

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Genome-wide methylation profiling of schizophrenia.

Schizophrenia is one of the major psychiatric disorders. It is a disorder of complex inheritance, involving both heritable and environmental factors. DNA methylation is an inheritable epigenetic modification that stably alters gene expression. We reasoned that genetic modifications that are a result of environmental stimuli could also make a contribution. We have performed 26 high-resolution genome-wide methylation array analyses to determine the methylation status of 27,627 CpG islands and compared the data between patients and healthy controls. Methylation profiles of DNAs were analyzed in six pools: 220 schizophrenia patients; 220 age-matched healthy controls; 110 female schizophrenia patients; 110 age-matched healthy females; 110 male schizophrenia patients; 110 age-matched healthy males. We also investigated the methylation status of 20 individual patient DNA samples (eight females and 12 males. We found significant differences in the methylation profile between schizophrenia and control DNA pools. We found new candidate genes that principally participate in apoptosis, synaptic transmission and nervous system development (GABRA2, LIN7B, CASP3). Methylation profiles differed between the genders. In females, the most important genes participate in apoptosis and synaptic transmission (XIAP, GABRD, OXT, KRT7), whereas in the males, the implicated genes in the molecular pathology of the disease were DHX37, MAP2K2, FNDC4 and GIPC1. Data from the individual methylation analyses confirmed, the gender-specific pools results. Our data revealed major differences in methylation profiles between schizophrenia patients and controls and between male and female patients. The dysregulated activity of the candidate genes could play a role in schizophrenia pathogenesis.

[Genomic study in patients with idiopathic azoospermia and oligoasthenoteratozoospermia].

Genome disbalances are related to the different types of infertility and they play a role in the treatment of human infertility. Comparative genome hybridization (CGH) combined with microchips is a modern high resolution technique for all human chromosomes investigations. We analysed the genome disbalances in 16 blood samples of men with an idiopathic oligoastenozoospermia or azoospermia using CGH and microchips for the whole human genome. Our data showed a few affected loci, including: 3q26.32 deletion accompanied by 14q11.1 deletion, 9q12 deletion, 5q35.1 amplification, 7p22.3 amplification and 17q12-17q21.2 amplification. In this study we have match the deletions: in two patients in the same area in the 8 chromosome, as well as in 5 patients in 14 chromosome. The deleted region contains 25 genes. Two of them (SPAG11B and SPAG11A) are associated with stages of spermatogenesis and in particular formation and maturation of the spermatozoa. These genes play a role during spermatogenesis and fertilization. Loss. chr.14q11.2 (EDDM3A and EDDM3B) affected the proteins that are synthesized and secreted by epididymal epithelial cells that has been found up-regulated in epididimis of nonobstructive azoospermic men. Our results displayed the significance of CGH and microchip analysis as a promising area of research with serious clinical application for resolving the problems of the male infertility and still have an important annex for selecting the most appropriate methods for the treatment in these patients as a perspective scientific field of investigations with a clinical appliance.

Analysis of the genes encoding neuroligins NLGN3 and NLGN4 in Bulgarian patients with autism.

Many studies have supported a genetic aetiology for autism. Neuroligins are postsynaptically located cell-adhesion molecules. Mutations in two X-linked neuroligin genes, NLGN3 and NLGN4, have been implicated in pathogenesis of autism. In order to confirm these causative mutations in our autistic population and to determine their frequency we screened 20 individuals affected with autism. We identified one patient with a point mutation in NLGN4 gene that substituted a Met for Thr 787 - c.2360C > T, p.(Thr787Met) and three patients with identical polymorphisms in the same gene: c.933C > T, p.(Thr311Thr) in combination with c.[1777C > T+1779C > G, p.(Leu593Leu)]. All patients tested for NLGN3 mutations were negative. These results indicate that mutations in these genes are responsible for at most a small fraction of autism cases.

Genome-wide association study on bipolar disorder in the Bulgarian population.

Bipolar disorder is a severe psychiatric disorder influenced by environmental and genetic factors. Genetic studies have implicated many variants in the disease's etiology but only few have been successfully replicated. We conducted a genome-wide association study (GWAS) on bipolar disorder in the Bulgarian population followed by a replication study of the top 100 single nucleotide polymorphisms (SNPs) showing the smallest P values. The GWAS was performed on 188 bipolar disorder patients and 376 control subjects genotyped on the Illumina 550 platform. The replication study was conducted on 122 patients and 328 controls. Although our study did not show any association P value that achieved genome-wide significance, and none of the top 100 SNPs reached the Bonferroni-corrected P value in the replication study, the plausible involvement of some variants cannot be entirely discarded. Three polymorphisms, rs8099939 [P = 2.12 × 10(-6), odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.43-2.67] in GRIK5, rs6122972 (P = 3.11 × 10(-6), OR = 2.02, 95% CI = 1.46-2.80) in PARD6B and rs2289700 (P = 9.14 × 10(-6), OR = 2.13, 95% CI = 1.53-2.95) in CTSH remained associated at a similar level after Mantel-Haenszel test for combining the results from the genome-wide and replication studies. A modest association was also detected for SNP rs1012053 (GWAS P = 4.50 × 10(-2)) in DGKH, which has already been reported as the most significant variant in a previous genome-wide scan on bipolar disorder. However, further studies using larger datasets are needed to identify variants with smaller effects that contribute to the risk of bipolar disorder.

[Clinical, histopathological and cytogenetical findings in six cases with primary glioblastoma multiforme].

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. There is an increasing amount of data demonstrating that the routine histological examination has limited value to predict the tumor biological behaviour. AIM: To compare the clinical, histological and cytogenetical findings in a group of six patients with primary GBM surgically treated in the Department of Neurosurgery at University Hospital "St. Ivan Rilski"- Sofia, Bulgaria. MATERIAL AND METHODS: The studied group consisted of three women and three men with average age of 51 years and 6 months. In all patients the diagnosis was histologically confirmed. A microarray comparative genomic hybridization (CGH) analysis of fresh-frozen tumor tissue samples was also made. RESULTS: In two of the patients the tumor was localized in frontotemporal region, in another two- in frontoparietal, and in the other two- in parietal and occipital respectively. The onset was with headache in three of the cases. The median time between the onset and admission in the clinic was 70 days. Gross-total tumor removal was performed in one patient. In the other five subtotal excision was made. Three of the patients improved after the intervention. One patient deteriorated after the surgery. All patients demonstrated typical histological findings except one who had giant cell subtype of GBM. The microarray CGH analysis determined chromosome 10 monosomy in five patients, trisomy 7 - in four, trisomy 20 - in three, 3q23 deletion - in three, and Yp11.2 deletion - in three. CONCLUSION: GBMs are genetically heterogeneous tumors with different clinical response to standard multimodal treatment regimens. The microarray CGH analysis is a powerful method which can demonstrate the presence of a number of molecular markers with possible predictive value.

Long-Term Survival of a Patient with Giant Cell Glioblastoma: Case Report and Review of the Literature.

Glioblastoma multiforme (GBM) is the most common glial tumor of the central nervous system. Overall survival is less than a year in most of the cases in spite of multimodal treatment approaches. A 45-year-old female with histologically confirmed giant cell GBM was treated at our institution. Subtotal excision of the lesion situated in the right precentral area was performed during the initial stay in August 2005. The patient improved after the procedure with no hypertension and additional neurological deficit. Radiotherapy plus concomitant and adjuvant temozolomide was performed. The patient was symptom-free for 35 months after initial surgery. From July 2008 the patient developed partial motor seizures in the left side of the body and progressive hemiparesis. Local tumor progression was demonstrated on the neuroimaging studies. In December 2008, a second operative intervention was performed with subtotal excision of the tumor. Forty-five months after the initial diagnosis the patient is still alive with moderate neurological deficit. Microarray analysis of the tumor found the following numeric chromosomal aberrations: monosomy 8, 10, 13, 22, and trisomy 21, as well as amplifications in 4q34.1, 4q28.2, 6q16.3, 7q36.1, 7p21.3, and deletions in 1q42.12, 1q32.2, 1q25.2, 1p33, 2q37.2, 18q22.3, 19p13.2, Xq28, and Xq27.3. GBMs seem to be a heterogeneous group of glial tumors with different clinical course and therapeutic response. Microarray analysis is a useful method to establish a number of possible molecular predictors.