Gene Positive Cases Research Articles

Abstract P5-12-07: Polygenic Risk Score in a cohort of 105 Breast Cancer patients previously tested with a multi gene panel for hereditary cancer

Abstract AIM: Most patients tested by a 44-gene panel for hereditary cancer (HerediGene), even though they had a strong family history, have a negative result or a finding in a low-risk gene. This creates a question about the risk for these individuals to develop breast cancer. The Polygenic Risk Score is a tool used to identify and calculate the lifetime risk of developing breast cancer and thus we are investigating whether it can be used in our cohort of patients to identify this risk. PATIENTS AND METHODS: Among 111 patients analyzed with a 44-gene hereditary cancer panel, we were able to produce the Polygenic Risk Score for 105 of them. All the patients were diagnosed with breast cancer, and they all had a strong family history. We analyzed the ability of the PRS to identify the risk of the patients in combination with the findings of the 44-gene panel. RESULTS: Overall, 74% of patients with a family history had a negative PRS (with a cut-off of 20%). Among the BRCA positive patients all of them had a positive PRS and among all the high-risk gene positive cases, 70% had a positive PRS. On the other hand, among the low-risk genes and the negative cases 18.5% had a positive PRS. There is a positive correlation between the findings from the NGS panel analysis and the PRS. CONCLUSION: Our analysis shows that PRS is correlated to the findings of the 44-gene NGS panel having the majority of the PRS positive patients carry high risk mutations. In addition to this, in 18.5% of the patients with low-risk findings or negative result from the 44-gene panel, the PRS was positive which can explain the outcome of the patient since breast cancer had developed. This is an indication that the combination of PRS with the findings from the 44-gene panel can identify individuals with a higher risk of developing breast cancer. Citation Format: Nikolaos Tsoulos, Konstantinos Agiannitopoulos, Helen Goga, Theodoros Troupis, Christos Markopoulos. Polygenic Risk Score in a cohort of 105 Breast Cancer patients previously tested with a multi gene panel for hereditary cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-12-07.

A Pilot Study on Presence of Parkinson’s Disease Risk Gene PARK7 in Population of West Bengal, India: A Preliminary Observation

Introduction: The neurodegenerative Parkinson’s Disease (PD) is associated with different risk genes along with some environmental factors. Till date, there are few identified genes which play a pivotal role in the early onset of PD among the general population. Aim: To screen the presence of different risk genes of Parkinson’s disease along with associated environmental factors among randomly selected 62 urban participants. Materials and Methods: The present randomised control trial single blind pilot study was conducted in the Department of Biotechnology, Heritage Institute of Technology, Kolkata, West Bengal, India, from March 2021 to June 2021. The study participants were informed about the study and blood was collected from them. The genomic Deoxyribonucleic Acid (DNA) extraction was carried out following phenol chloroform extraction protocol and Polymerase Chain Reaction (PCR) was done with specific primers of the risk genes namely, Parkinson Disease 1 (PARK1) Alpha-synuclein (α-synuclein), Parkinson Disease 2 (PARK2) (Parkin), Parkinson Disease Protein 7 (PARK7) or DJ -1 and PINK 1. Protein Deglycase-1 (DJ-1) and PTEN Induced Kinase 1 (PINK1). The statistical analysis of the findings was done using Statistical package for the Social Sciences (SPSS) software version 21.0. Results: Out of 62 particpants, 47 were males and 15 were females and the age distribution of the participants was in between 21 years to >60 years. After obtaining specific band in agarose gel electrophoresis, 35 positive cases of PARK7 gene and 1 positive case of PARK2 gene were identified. Conclusion: There was a statistically significant association of different bacterial and viral infections with the PARK7 gene positive cases. PARK7 gene responsible for early onset of PD is significantly prevalent in the general population of West Bengal.

394P Research of the algorithm for rare driver genes in non-small cell lung cancer using pathological images and artificial intelligence

Target therapy for driver gene-positive cases is one of the standard treatments in advanced or recurrent non-small cell lung cancer (NSCLC). However, there is a need for a simple method to find rare driver genes due to their rarity. We developed and evaluated the algorithm to detect rare driver gene in NSCLC comprehensively using pathological images and AI.

Utility of Multi-target Nested PCR and ELISPOT Assays for the Detection of Paucibacillary Leprosy: A Possible Conclusion of Clinical Laboratory Misdiagnosis.

The diagnosis of paucibacillary (PB) leprosy often possesses a diagnostic challenge, especially for pure neuritic and lesser skin lesions with the zero bacillary load, requiring a sensitive and accurate diagnostic tool. We have included 300 clinically diagnosed new leprosy cases (comprising 98 PB cases) and analyzed the sensitivity and specificity of PB leprosy cases by nested PCR with folP, gyrA, rpoB, RLEP, and 16SrRNA and Enzyme-linked Immunospot Assay test (ELISPOT) with MMPII, NDO-BSA, and LID-1 antigens by detecting interferon gamma (IFN-γ) release. The overall positivity rates of genes tested in 300 clinical specimens were identified as 55% of 16SrRNA, 59% of RLEP, 59.3% of folP, 57.3% of rpoB, 61% of gyrA while 90% of nested folP, 92.6% of nested rpoB, and 95% of nested gyrA, and 285 (95%) of at least one gene positive cases. For PB specimens, 95% PCR positivity was achieved by three tested genes in nested PCR. The data obtained from ELISPOT for three antigens were analyzed for IFN-γ expression with 600 subjects. Among 98 PB leprosy cases, the sensitivity of MMP II, LID-1, and NDO-BSA was 90%, 87%, and 83%, respectively, and the specificity was 90%, 91%, and 86%, respectively. The total number of cases positive for at least one antigen was 90 (91.8%) in PB, which is significantly higher than that in multibacillary (MB) leprosy (56.7%). The combination of multi-targets nested PCR and ELISPOT assay provides a specific tool to early clinical laboratory diagnosis of PB leprosy cases. The two assays are complementary to each other and beneficial for screening PB patients.

MPC-06 Cutting-edge of Cancer Genomic Medicine for brain tumors

Kyushu University Hospital was designated a Cancer Genome Core Hospital in April 2018, and the multi-gene panel test has been introduced since August 2019. The expert panel has been held for 21 cases of the central nervous system (11 adult glioma, 5 pediatric brain tumors, 5 extramedullary tumors). Actionable gene abnormalities were newly detected in two cases. First case is epithelioid glioblastoma with BRAF V600E mutation, and second is embryonal tumor with VCL-ALK fusion. For the first case, BRAF/MEK inhibitor can be used by the prospective trial of patient-proposed healthcare services with multiple targeted agent based on the result of gene profiling by multigene panel test (NCCH1901). For the second case, we are planning to introduce ALK inhibitor by indicator-initiated clinical trial while continuing ICE therapy. The current approved agents for tumor-agnostic treatment are immune checkpoint inhibitors for mismatch repair deficient (dMMR) cases and TRK inhibitors for NTRK fusion gene-positive cases. We selected microsatellite instability (MSI) test and immunostaining of MMR gene for the indication of immune checkpoint inhibitor for recurrent glioma and Lynch syndrome that require dMMR evaluation, but FoundationOne CDx (F1CDx) allows simultaneous evaluation of MSI and MMR gene abnormalities. Regarding the indication of TRK inhibitors, F1CDx assay is selected as a companion diagnosis for ALK, NTRK1/2/3 fusion gene analysis for pediatric cases. At present, the actionable gene abnormalities are detected by multi-gene panel tests in about 10% of brain tumors. Development of tumor-agnostic treatment will expand the molecular target therapy for brain tumor in the future. Based on the experience of different schemes for molecular targeted therapy, it became clear that it is necessary to establish a cancer genome medical system for prompt introduction of precision medicine for highly malignant brain tumors.

YIELD OF GENETIC TESTING FOR HYPERTROPHIC CARDIOMYOPATHY ACCORDING TO CONTEMPORARY VARIANT INTERPRETATION GUIDELINES: RESULTS FROM A LARGE CANADIAN REFERRAL CENTRE

The yield of genetic testing for hypertrophic cardiomyopathy (HCM) has been reported to be between 30% and 60%. Differences in populations studied are likely to be one explanation for this wide variation. Changes in the way we interpret genetic variants is likely to be a contributing factor. We aimed to analyze the genetic results from a large Canadian referral centre interpreted according to contemporary guidelines in order to examine the yield of genetic testing in the Canadian population. HCM index cases who underwent genetic testing between 2005 and January 2018 were included. All variants were re-classified according to the guidelines of the American College of Medical Genetics (ACMG) and subsequent gene-specific adaptations. Of 1,730 cases tested 393 (23%) were tested for 5 sarcomeric genes and 1,337 (77%) were tested for 8. In 325 patients (19%) a pathogenic (P) or likely pathogenic (LP) variant was identified and variants of unknown significance in 247 (14%). The majority (57%) of P/LP variants were found in the MYBPC3 gene, followed by the MYH7 gene (31%). The yield of TNNI3, TPM1 and TNNT2 genes was 4%, 3% and 2%, respectively. The 3 remaining genes combined (MYL2, MYL3 and ACTC1) were accountable for 3% of P/LP variants. Of the MYBPC3 variants 89% were loss-of-function (nonsense, frameshift or splice-site), one was an in-frame deletion and the rest (11%) missense. The yield of genetic testing in our centre was lower than previously reported. Possible explanations include lack of a founder mutation in the heterogenous Canadian population and higher referral rates of non-familial cases which are less likely to be gene-positive. Finally, using current and stricter guidelines for variant interpretation may have led to less variants being classified as P/LP, and may also explain the higher percentage of MYBPC3 variants among gene-positive cases. Further studies are required in order to determine whether this reflects the true genetic landscape of HCM or the limitations of current variant interpretation methods.

Abstract P2-05-03: Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas

Abstract Introduction: Breast adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer associated with an indolent clinical behavior. AdCCs provide a clear example of genotypic-phenotypic correlation with the majority harboring the MYB-NFIB fusion gene. In this study, we sought to identify alternative driver genetic alterations in breast AdCCs lacking the MYB-NFIB fusion gene. Methods: Nucleic acids obtained from four breast AdCCs lacking the MYB-NFIB fusion gene as defined by reverse transcription (RT)-PCR and/or fluorescence in situ hybridization (FISH) were subjected to RNA-sequencing (n=3), whole-genome (n=2) and/or targeted (n=1) massively parallel sequencing. Sequencing data were analyzed using state-of-the-art bioinformatics algorithms, and potential alternative driver genetic alterations were validated using orthogonal sequencing and molecular pathology methods. Results: RNA-sequencing revealed the presence of MYBL1-ACTN1 or MYBL1-NFIB fusion genes in two breast AdCCs, which were validated by whole-genome sequencing and/or MYBL1 FISH analysis. Both MYBL1 fusion gene-positive cases were found to overexpress MYBL1 as defined by quantitative RT-PCR analysis. In the third MYB-NFIB-negative breast AdCC studied, a high-level MYB gene amplification coupled with overexpression of MYB at the mRNA and protein levels was identified. In the fourth breast AdCC, which expressed high levels of MYB, whole-genome and RNA-sequencing revealed no definite alternative driver alteration, however, a MYBL2 intronic mutation was found in this case, which was associated with high levels of MYBL2 mRNA expression. In this case, single sample gene set enrichment analysis revealed activation of pathways similar to those activated in AdCCs harboring the MYB-NFIB or MYBL1 fusions genes. Conclusion: We demonstrate that in breast AdCCs lacking the MYB-NFIB fusion gene MYBL1 rearrangements and MYB amplification are likely alternative driver genetic events. Given that activation of MYB/MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification or other yet to be validated mechanisms (e.g. MYBL2 non-coding mutations), our findings further suggest that breast AdCCs constitute a convergent phenotype. Citation Format: Kim J, Geyer FC, Martelotto LG, Ng CKY, Lim RS, Selenica P, Li A, Pareja F, Fusco N, Edelweiss M, Mariani O, Badve S, Vincent-Salomon A, Norton L, Reis-Filho JS, Weigelt B. Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-03.

34 Hour QT analysis in the prediction of genotype positivity in suspected LQTS: a study in both proband and family screening populations

Introduction LQTS is an inheritable condition with a suspected prevalence on 1/2000 and has a risk of sudden cardiac death due to ventricular arrhythmia if untreated. 24 hour QT analysis is a method of averaging QT measurements obtained via holter monitoring over a 24 hour period. The utility of 24 hour QT analysis to predict genotype positivity has not been widely studied. Methods 28 probands with genetic testing results and 107 first degree relatives of genotyped LQTS patients were included in the analysis. Retrospective review of the medical charts of all patients was performed. Parameters analysed included corrected QT (QTc) interval on 12 lead ECG, 24 hour ambulatory ECG, the QTc after 4 minutes of recovery post exercise stress testing (EST), and the Schwartz score based on clinical and ECG criteria. All these parameters were then compared to the results of subsequent genetic testing. Results 28 probands were analysed with 7 returning a positive gene test and 21 returning a negative gene test. Gene positive individuals had significantly greater QTc by resting ECG (p = 0.047), 24 hour ECG (p = 0.005) and after 4 minutes recovery post EST (p = 0.001) (independent t-test). Using standard cut offs (450 ms for males and 460 ms for females) the association between clinically positive resting ECG and positive genotype was not statistically significant (p = 0.17). Schwartz score >3.5 showed no significant difference between gene positive and gene negative groups (p = 0.33). 24 hour QTc (p = 0.029) was significantly associated with positive genotype and QTc at 4 minutes recovery post EST showed a trend toward significance (p = 0.069). Of 107 first degree relatives of LQTS cases there were 50% gene positive cases. There were statistically significant differences between gene positive and gene negative family members in QTc (p 3.5 was 98% specific for genotype positive LQTS but this test had a very low sensitivity of 29%. The most sensitive test was 24 hour QTc with a cut off of 450 ms for males and 460 ms for females with a sensitivity of 69% and specificity of 83%. Conclusions Family screening and prevention of sudden cardiac death is central to the management of LQTS. Current individual clinical testing methods are not sufficiently sensitive to diagnose LQTS alone and must be used in combination to assess the risk of LQTS. 24 hour QT analysis is the most sensitive method of predicting positive genotype in both probands and family members of affected individuals.

Application of Combined Detection of Fusion Gene and BIOMED-2 Standardized Ig Gene Rearrangement System in Childhood B-cell Acute Lymphoblastic Leukemia

To explore the application of combined detection of fusion gene and BIOMED-2 standardized immunoglobulin (Ig) gene rearrangement system in diagnosis and treatment of children with acute lymphoblastic leukemia (ALL). Multiplex-PCR amplifications and RQ-PCR of RNA/DNA were performed using ALL fusion gene detection kit and BIOMED-2 primer. The Ig gene rearrangements were analyzed by using PCR fragment analysis system. Out of 251 children with B-ALL, 77 cases were TEL-AML1(+) , 28 cases were E2A-PBX1(+) , 10 cases were MLL-AF4(+) , 11 cases were BCR-ABL(+) , the total positive rate was 50.2%, 82.5% showed IgH VH-JH rearrangement, 53.4% showed IgK rearrangement. The positive rate of combined detection of fusion gene and gene rearrangement was 99%. E2A-PBX1(+) and MLL-AF4(+) with IgK(+) gene rearrangement group was compared with negative control group, the difference was statistically significant (P < 0.001 or P = 0.005); 105 ALL fusion gene positive cases had been detected by fluorescence in situ hybridization (FISH) simultaneously, the accordance rate of fusion gene and FISH was more than 94%. The combined detection of ALL fusion gene and BIOMED-2 standardized clonality analysis system can improve the positive detected rate of B-ALL dramatically, and make the grouping of disease prognosis more accurately; this combined detection is a more faster and sensitive method than FISH.

Efficacy and prognostic risk factors of childhood relapsed acute lymphoblastic leukemia: analysis from a single center

Objective To investigate the efficacy and prognostic risk factors of ALL-R-2003 protocol in the treatment of relapsed childhood relapsed acute lymphoblastic leukemia (ALL) in single center. Methods A retrospective study of clinical data of 51 children with relapsed ALL from January 2004 to December 2014 was performed by using SPSS version 19.0 statistical software for statistical analysis. Results The median age at initial diagnosis of 51 patients was 5.5 years (range, 0.8-13.4 years). The median time from initial diagnosis to relapse was 25 months (range, 3-68 months) and follow-up time was 39 months (range, 3-116 months). The relapse rate in the standard-risk, intermediate-risk and the high-risk groups were 27.5% (14/51), 29.4% (15/51) and 43.1% (22/51), respectively. The probability of 3-year overall survival (pOS) after relapse was (18.8±5.9)% and the probability of event free survival (pEFS) was (16.2±5.8)%. The 3-year pOS in very early relapse, early relapse and late relapse were 0, (11.7±7.7)% and (51.7±14.8)%, respectively (P= 0.000). There was no statistical difference in survival rate of different immunophenotype groups and sites of relapse (P> 0.05). The 3-year pOS of group S1, S2, S3, S4 were (50.0±35.4)%, (39.9±1.3)%, (10.0±9.5)% and 0, respectively (P= 0.000). The 3-year pOS of bcr-abl and MLL gene positive groups were (25.0±21.7)% and 0, respectively, with no statistically significance compared with the negtive group [(24.1±12.0)%] (P> 0.05). The 3-year pOS rates of children with bone marrow transplantation and without transplantation were (40.0±15.5)% and (13.0±5.9)% respectively (P= 0.038). Conclusions The children who in high risk group at initial diagnose are easily to meet earlier relapse and poorer prognosis. The survival period after relapse of bcr-abl or MLL gene positive cases is very short. Bone marrow transplantation can improve survival rate. Risk group at initial diagnose, relapse time and transplantation are the main factors influencing prognosis, and the relapse time and transplantation are the independent prognostic factors for relapsed childhood ALL. Key words: Leukemia, lymphocytic, acute; Relapse; Risk factors; Prognosis; Child

MECHANISMS OF DISEASE IN THE HYPEREKPLEXIAS

AimsTo identify mutations associated with hyperekplexia and to investigate the underlying pathophysiological mechanism of novel mutations identified, whilst providing a genetic diagnosis of hyperekplexia in the cases referred.MethodAs part of...

Onset of Huntington's disease: can it be purely cognitive?

Knowledge of the cognitive manifestation of Huntington's disease has burgeoned over the past two decades. Many studies from independent datasets have shown that cognitive impairment is evident before motor diagnosis, and annual cognitive decline is a robust marker of disease progression. Additionally, cognition is a critical concern to patients and families and is associated with meaningful outcomes, including functional capacity, driving, loss of accustomed work, and quality of life. In the past few years, Huntington's disease animal models of cognition have increased, preparing for preclinical experimental therapeutics with cognitive endpoints. A longitudinal analysis of cognitive variables was conducted with 559 gene-positive cases and 233 controls showing no signs of motor abnormalities over approximately a 3-year period. Results show statistically significant differences in rate of annual change for some cognitive variables, such that the cases group had worsening performance over time. These findings show that cognitive deterioration can be seen in persons with the Huntington's disease gene expansion with no overt motor signs or symptoms, suggesting that cognitive onset of Huntington's disease may precede motor.

Angiotensin-converting enzyme insertion/deletion polymorphism in hypertrophic cardiomyopathy: An Egyptian case control study

Hypertrophic Cardiomyopathy (HCM) is a disease characterized by genetic and phenotypic heterogeneity. Renin–angiotensin–aldosteron be system (RAAS) is a potential disease modifier. The aim of the present case control study is evaluation of the controversial role of ACE I/D polymorphism in HCM among Egyptians. The study comprised 211 unrelated HCM patients (138 sporadic, 73 familial) and 203 age and sex matched ECG screened healthy volunteers. ACE I/D polymorphism was determined using previously described PCR and gel electrophoresis based method. Distribution of ACE genotype among the Egyptian controls was in Hardy-Weinberg equilibrium ( P = 0.778) but not in HCM patients ( P = 0.0010). The ACE DD genotype was significantly higher among HCM patients ( P = 0.049), particularly in sporadic HCM group compared with familial cases ( P = 0.0001). In addition, the distribution of D allele was significantly higher in HCM patients carrying sarcomeric mutations in TNNT2 and MYH7 , ( P = 0.0476). There was no observed significant effect of the ACE genotypes on the phenotypic expression of the disease. The finding of higher frequency of DD genotype among HCM patients compared to healthy volunteers, particularly so, in sporadic cases suggests that HCM expression is possibly influenced by a genetically predisposed milieu partially determined by the ACE I/D variants. Despite the lack of significant correlation between I/D variants and clinicopathologic characteristics of the HCM patients, however, the higher prevalence of D allele among TNNT2 and MYH7 mutation carriers may contribute to the variable disease outcome among sarcomeric gene positive cases, such a correlation can only be proven through long term follow up studies.

RET-targeting molecular stratified non-small-cell lung cancers.

Recent advances in lung cancer genomics have successfully characterized therapeutic targets of lung cancer. RET fusion gene products are among the newest target molecules for lung adenocarcinoma. Preclinical findings and preliminary reports regarding potential tumor control by RET-targeting multi-kinase inhibitors encourage further clinical trials. The infrequent prevalence of RET fusion gene-positive cases may be a major obstacle hindering the development of RET-targeted therapy. Thus, it is necessary to recruit appropriate participants for trials to develop an efficient RET fusion gene detection system to achieve targeted therapy for lung adenocarcinomas stratified by this molecular target.

Genotype-phenotype correlations in hyperekplexia: apnoeas, learning difficulties and speech delay

Congenital hyperekplexia is a rare, potentially treatable neuromotor disorder. Three major genes of effect are known, and all three affect glycinergic neurotransmission. Two genes encode for subunits of the postsynaptic inhibitory glycine receptor, GLRA1 encoding the α1 subunit and GLRB encoding the β subunit. The third, SLC6A5, encodes the cognate presynaptic glycine transporter 2. Ninety-seven individuals had a clinical diagnosis of hyperekplexia confirmed by genetic testing: 61 cases had mutations in GLRA1, 24 cases in SLC6A5 and 12 in GLRB. Detailed retrospective clinical analysis ascertained that all gene-positive cases present in the neonatal period (occasionally prenatally) and that clonazepam is the treatment of choice (95% found it to be efficacious). We confirm that hyperekplexia is predominantly a recessive condition but dominant cases are seen (16%). We found no genetic evidence for 'major' or 'minor' forms of hyperekplexia on a population basis. Thirty-five gene-negative cases were studied for comparison, their cardinal feature was presentation after the first month of life (P < 0.001). In addition to the characteristic 'stiffness, startles and stumbles' of hyperekplexia, apnoea attacks (50 of 89) and delayed development (47 of 92) were frequently reported. Patients with SLC6A5 mutations were significantly more likely to have had recurrent infantile apnoeas (RR1.9; P < 0.005) than those with GLRA1 mutations. Patients with GLRB and SLC6A5 mutations were more likely to have developmental delay (RR1.5 P < 0.01; RR1.9 P < 0.03) than those with GLRA1 mutations; 92% of GLRB cases reported a mild to severe delay in speech acquisition. Molecular modelling of pathogenic mutations demonstrates specific patterns of protein disruption that can be used to predict phenotype severity. The developmental delay in hyperekplexia, and speech acquisition in particular, may represent failure of developmental neural networks or subtle neurogenic migration defects in the absence of presynaptic glycine release. We recommend early genetic testing for symptomatic neonates and possibly preconception counselling for those at risk for GLRB and SLC6A5 mutations, because of the more challenging phenotype.

Assessment of the Multifactorial Effect on Antimicrobial Sensitivity in Positive Staphylococcus Aureus Clinical Isolates from Assir Region, Saudi Arabia

General objectives: This study aimed at assessment of factors affecting antimicrobial sensitivity in Staphylococcus aureus clinical isolates from Assir region, Saudi Arabia. Materials and Methods: In this study, eighty one patients presented with Staph. aureus infections either nosocomial or community acquired infections were involved by collecting nasal swabs from them at Aseer Central Hospital General Lab. These patients were from all age groups and from males and females during the period of Jan 2011- Jun 2011. These samples were undergone variable laboratory procedures mainly; bactech, culture media, antibiotics sensitivity test using diffusion disc test (MIC) and molecular (PCR) for detection of mec A gene. Clinical and laboratory data were recorded in special formats and analyzed by statistical computer program (SPSS). Results: Showed that; Descriptive and analytical statistical analysis were performed and final results were plotted in tables. In Staph aureus MecA gene positive cases (50) showed: Oxacillin/ Mithicillin, Ciprofloxacin and Fusidin resistant in diabetic patients were 13, 26.0%, 9, 18% and 7, 14% respectively and in non diabetic patients were 37, 74.0%, 22, 44% and 20, 40% respectively. While no sensitivity in diabetic and non diabetic patients using Oxacillin/ Mithicillin. In Staph aureus MecA gene negative cases (31) showed: Oxacillin/ Mithicillin, sensitivity in diabetic patients (5, 16.1%) and in non diabetic were (26, 83.9%). While no resistant in diabetic and non diabetic patients. In Ciprofloxacin and Fusidin resistant in diabetic patients were 1, 3.2% and 1, 3.2% respectively and in non diabetic patients were 12, 38.7% and 7, 22.6%respectively. Erythromycin in Staph aureus ( MecA gene) positive cases (50) showed: resistant in age (0-15) years were (5, 10%), (16-50) years were (16, 32%) and ( ›50 years) were (12, 24%). Erythromycin in Staph aureus (MecA gene) negative cases (31) showed: resistant in age (0-15) years were (6, 19.3%), (16-50) years were (5, 16.1%) and ( ›50 years) were (3, 9.7%). Conclusion: Drugs resistance is a major progressive multifactorial problem facing the treatment of Staph aureus infections. DOI: http://dx.doi.org/10.3329/jom.v13i2.12750 J Medicine 2012; 13 : 152-159

Broad and unexpected phenotypic expression in Greek children with steroid-resistant nephrotic syndrome due to mutations in the Wilms’ tumor 1 (WT1) gene

Mutations in the Wilms' tumor suppressor gene 1 (WT1), most commonly within exons 8 or 9 or intron 9, are found in cases with the overlapping conditions of Denys-Drash and Frasier syndromes, as well as in patients with steroid-resistant nephrotic syndrome (SRNS). This study investigated the presence of WT1 gene mutations in cases with childhood SRNS, along with an evaluation of their clinical outcome. Twenty-seven Greek children with sporadic (19 cases) and familial (8 cases) SRNS were tested. Four phenotypically female patients with sporadic SRNS were found to carry de novo WT1 mutations, including two cases with p.R394W, and one case each with p.R366H, or n.1228+5G>A. Karyotype analysis found 46XX in three cases, but 46XY in one. No phenotype-genotype correlations were apparent in the WT1 gene positive cases since their clinical presentation varied broadly. Interestingly, one patient with a pathological WT1 nucleotide variation responded fully to combined therapy with cyclosporine A and corticosteroids. This study further illustrates that investigation of WT1 gene mutations is clinically useful to support definitive diagnosis in children presenting with SRNS in order to direct the most appropriate clinical management.

Fusion Gene–Negative Alveolar Rhabdomyosarcoma Is Clinically and Molecularly Indistinguishable From Embryonal Rhabdomyosarcoma

To determine whether the clinical and molecular biologic characteristics of the alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) subtypes have relevance independent of the presence or absence of the PAX/FOXO1 fusion gene. The fusion gene status of 210 histopathologically reviewed, clinically annotated rhabdomyosarcoma samples was determined by reverse transcriptase polymerase chain reaction. Kaplan-Meier analysis was used to assess event-free survival and overall survival in fusion gene-negative ARMS (ARMSn; n = 39), fusion gene-positive ARMS (ARMSp; n = 94), and ERMS (n = 77). A total of 101 RMS samples were also profiled for whole-genome expression, and 128 were profiled for genomic copy number imbalances. Profiling data were analyzed by supervised and unsupervised methods to compare features related to histopathology and fusion gene status. Results were also projected by meta-analysis techniques across three separate publically available data sets. Overall and event-free survival, frequency of metastases, and distribution of site at initial presentation were not significantly different between ARMSn and ERMS. Consistent with this, analysis of gene expression signatures could not reproducibly distinguish ARMSn from ERMS whereas fusion gene-positive cases were distinct. ARMSn and ERMS frequently show whole-chromosome copy number changes, notably gain of chromosome 8 with associated high levels of expression of genes from this chromosome. The clinical behavior and molecular characteristics of alveolar cases without a fusion gene are indistinguishable from embryonal cases and significantly different from fusion-positive alveolar cases. This implies that fusion gene status irrespective of histology is a critical factor in risk stratification of RMS.

Clonal Origins of 'late' Relapses in ETV6-RUNX1 Acute Lymphoblastic Leukemia..

Abstract 1583Poster Board I-609Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits' in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver' CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (<2 to 9.9 years), the relapse clone appeared to be derived from either a major or minor clone at diagnosis with none (0/6) of the very late relapses (>5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL.Relapse typeRemission duration (years)< 22 - 5> 51••2••••••••••3•••••••4••Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2194: Predictors of RyR2 Genotype Among Patients Referred for Long QT Syndrome Genetic Testing

Introduction : Type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1) is pursuant to mutations in the RyR2- encoded cardiac ryanodine receptor. Recently, we discovered nearly as many CPVT1-causing mutations as LQT3-causing mutations in a large cohort of unrelated LQTS referral patients. Here, we examine potential predictors of RyR2 genotype (CPVT1) among LQTS referral patients. Methods : Previously, comprehensive mutational analysis of the LQT1–10 susceptibility genes was completed for 541 unrelated patients (358 females, average age at diagnosis = 24 years, average QTc = 482 ms) referred for LQTS genetic testing. Using PCR, DHPLC, and DNA sequencing, we performed targeted analysis of RyR2 involving 44 exons (8–15, 37–49, 83–105) for the 260 unrelated referrals (172 females, average age at diagnosis = 25 years, average QTc = 470 ms) that remained “genotype-negative” following LQTS genetic testing. Results : Overall, 22 distinct RyR2 mutations (19 novel) were seen in 23 (8.8%, 12 females, average age at diagnosis = 22 ±14 years) unrelated “genotype-negative” LQTS individuals. Compared to patients with either genetically proven LQTS or the remaining genotype negative cohort, the RyR2 -positive cohort had significantly lower average QTc (419 ms vs 494 ms, p =2.4 ×10 −6 ; vs 472 ms, p = 0.005) and was more likely to experience either syncope (88% vs 66%, p = 0.06 vs 53%, p = 0.008) or cardiac arrest (56% vs 23%, p = 0.006 vs 17%, p = 0.001). Overall, 123 of the 541 LQTS referrals (23%) exhibited exertion-induced cardiac events (68 LQTS gene positive; 42 gene negative, and 13 RyR2-positive). Every RyR2 -positive case with an exertionally-triggered event had a QTc ≤ 460 ms, compared to only 17% of LQTS gene positive cases (p = 0.1 × 10 −8 ) and 24% of LQT1 cases. None of the exertion-induced LQTS gene positive cases had a QTc ≤ 420 ms. Conclusions: Failure to distinguish CPVT from LQTS can be a fatal mistake, as individuals with CPVT1 have a more severe phenotype and are much less protected by beta blocker therapy than those with LQT1. CPVT1, rather than concealed LQT1, should lead the differential diagnosis in the setting of syncope/cardiac arrest during exercise and a normal resting QTc.