Detection of a point mutation at codon 12 of the Kirsten-Ras [K-ras] oncogen in myelodysplastic syndrome

Mutations in ras genes have been observed in a variety of cancers and were found to play an important role in human leukemogenesis and in preleukemic disease as myelodysplastic syndrome [MDS]. The purpose of this study was to determine the prevalence of mutated K-ras oncogene in myelodysplastic syndrome [MDS]; with a special emphasis on their possible role in affecting clinical status, relation to karyotypic pattern; response to therapeutic measures; its impact on the fate of the disease and overall survival. Detection of point mutation of Kirsten-ras [K-ras] gene in 30 patients suffering from myelodysplastic syndrome was carried out using quantitative enriched polymerase chain reaction [QEPCR] and was confirmed by sequencing. QEPCR is a two- stage PCR procedure with modified primers that enriches mutant alleles, via restriction endonuclease digestion of normal alleles and enables identification of one mutant allele among 100,000 normal alleles. Activating mutations of the codon 12 of K-ras gene were detected in 7/30 [23.3%] cases of MDS, the most common mutation involved a substitution of aspartic acid for glycine [GGTGAT]. The incidence of K-ras mutations was found to be significantly associated with refractory anemia with excess blasts type II [RAEBII] and unclassified [UC] MDS than other subtypes [p=0.005], and was significantly associated with hypercellular bone marrow [p=0.04] showing marked dyserythropoitic changes. Furthermore, mutant K-ras gene was found to be significantly associated with abnormal karyotypes [p=0.04]. Patients with mutated K-ras gene were significantly associated with either high or intermediate risk according to International Prognostic Scoring System [IPSS] [p=0.001]. 6/7[85.7%] of those carrying the mutation showed poor response to treatment compared to non carriers with a statistical significant difference [p=0.009]. Five out of eight [62.5%] patients who were transformed to AML carried the mutant K-ras gene, their subtypes were RAEBII and unclassified MDS with abnormal cytogenetics mainly Monosomy 7. Overall survival was detected using Kaplan-Meier curve and the mean survival time of patients who carried K-ras mutations were significantly lower than those without the mutation [Log rank test=12.7; p=0.0004]. MDS patients bearing an mutated K-ras oncogene frequently showed poor response to treatment; leukemic progression of the disease and shorter overall survival, suggesting that an activated K-ras oncogene is a critical factor for prognostic evaluation; therapeutic decision and monitoring of response to treatment of MDS patients

Serine protease [TPS]: a diagnostic and prognostic marker in pediatric patients with acute non-lymphoblastic leukemia

The Serine protease, TPS [tryptase], is a specific marker for mast cells and mast cell-associated disorders. However, substantial amounts of TPS are also expressed in neoplastic myeloid, non-mast cell lineage. The aim of this study is determination and quantitation of TPS expression in patients with acute non-lymphoblastic leukemia [ANLL]; to evaluate its prognostic value and its relevance as a genetic marker for detection of minimal residual blast cells. Reverse transcriptase-polymerase chain reaction [RT-PCR] was used to detect levels of TPS from 30 newly diagnosed ANLL children and 10 normal children served as controls. TPS levels for positive cases were reevaluated after induction chemotherapy; after onset of relapse or by the end of the study. Our results showed that the gene transcripts were detected in 56.7% of patients but were not expressed by normal controls. The highest frequency of TPS was recorded in patients with M4 showing significantly higher levels compared to other FAB [French-American-British Classification] subtypes. TPS levels were directly correlated to TLC, absolute blast counts in peripheral blood, levels of CD34 and CD117. After induction chemotherapy, levels of TPS decreased significantly in those who achieved complete remission while it increased significantly in relapsed patients, with a risk estimate of relapse six times higher in positive cases than TPS negative patients. 84.6% of the patients negative for TPS achieved complete remission with better disease free survival. In conclusion, TPS is expressed in a group of patients with ANLL thus serves as a disease related marker; it could be used as a prognostic indicator in evaluating remission status and early relapse as its elevated levels are associated with high risk of relapse; again, it is useful in predicting disease outcome

Simultaneous identification, detection of Staphy lococcus aureus and methicillin resistance by polymerase chain reaction amplification of FEMB and MECA genes

Methicillin resistant Staphylococcus aureus [MRSA] is a major nosocomial pathogen and continuous to trigger outbreaks of infection. Simultaneous identification of Staph aureus and detection of methicillin resistance. The study included ninety five clinical specimens collected from intensive care unit [ICU] patients in Ain Shams University Hospitals. The method based on PCR detection of both femB gene and mecA gene. Prevalence of Staph aureus in ICU patient was 35%[29% MRSA, 6% MSSA] and Coagulase negative Staphylococci [CoNS] [5%]. Results of PCR revealed the presence of 22 femB-positive mecA-positive strains. Whereas, one strain was genotypically femB-positive mecA-negative. Of the 5 methicillin sensitive Staph aureus, 1 was femB-positive mecA-positive while the other 4 were femB-positive mecA-negative. Three of the CoNS were femB-negative mecA-negative while only 1 was phenotypically resistant but genotypically it was femB-negative mecA-positive. Simultaneous identification of Staph aureus and detection of methicillin resistance using PCR technique can be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner