Identification and in silico analysis of noval alteration Arg420Gly in KIT proto oncogene among acute myeloid leukemia patients.

A number of studies have reported frequent incidence of c-kit gene mutations in association with core binding factor acute myeloid leukemia (CBF-AML). These genetic changes have become important prognostic predictors in patients with abnormal karyotype. Aim of this study was the detection of nucleotide alterations in newly diagnosed acute myeloid leukemia patients for three exons of c-kit gene, including cytogenetically normal patients. Thirty-one de novo AML patients were screened for any possible variations in exon 8, 11 and 17 sequences of c-kit proto-oncogene leading to amino acid substitutions or frame shift. Sanger sequencing method was employed followed by sequence analysis. Mutation data was then correlated with clinical and hematological parameters of patients and prognostic significance of genetic changes was assessed as well. The computational tools were then used to further understand the extent of damage caused by these mutations to c-kit protein. Fifteen (48.4%) mutant patients were observed with single, double or multiple mutations in one, two or all three exons studied. The analysis revealed eight new alterations which were not reported previously. Significant variation among mutant and non-mutant group of patients was observed with respect to FAB subtypes (x2 = 12.524, p = 0.029), Spleen size (x2 = 4.288, p = 0.038) and Red blood cell count (x2 = 8.447, p = 0.007). The survival analysis indicates poor overall and event free survival outcomes in mutant individuals. Furthermore, the in silico analysis suggests that changes in nucleotide sequences can possibly damage the protein structure and effect it's function. This study emphasizes the need to consider screening of c-kit gene alterations not only in CBF-AML but in cytogenetically normal AML patients as well. In current investigation the effect of mutation Arg420Gly on structure and function of c-kit protein was investigated, as this was the most observed substitution in present cohort. Various bioinformatics tools and techniques were employed, which determined that Arg420Gly is possibly non-pathogenic mutation.

Perceptions of oncology as a career choice among the early career doctors in Pakistan.

BACKGROUND: Lack of oncologists is a growing global concern. With the rise in cancer burden across the world, the supply-demand mismatch of the oncology workforce is projected to increase. Furthermore, oncology is a low-ranked field of choice among medical students, and without understanding the perceptions and concerns of early-career doctors regarding oncology, any investments made in cancer care will be futile. This study aims to determine the opinions of young doctors and the factors most affecting their preferences in order to devise focused strategies to attract more doctors into oncology. METHODS: A cross-sectional study was conducted on 300 early-career doctors across various public and private hospitals in Pakistan, from March to November 2019. A close-ended, self-administered questionnaire was used to assess their opinions in terms of the workplace environment, scope, and the emotional and financial aspects of oncology. Data was analyzed using SPSS version 23 and the influence (positive or negative) of the perceptions on the choice of oncology as a career was determined by binary logistic regression analysis. RESULTS: Almost three-quarters of the participants did not want a career in oncology. The top positive perceptions about oncology in descending order were: progressive field, gender-neutral, stable working hours, financially healthy, and work-family balance. Top negative perceptions were: lack of oncologic facilities in hospitals, radiation exposure, need for private practice, poor patient prognosis, high patient load, and depressing environment. Participants who attended private medical school (p < 0.10), planned to live abroad (p < 0.10), had an oncologist (p < 0.05), cancer survivor or death due to cancer in the family (p < 0.05), were more likely to adopt oncology as a career. Those who believed that poor patient prognosis can have an impact on career choice were less likely to prefer oncology (p < 0.05). CONCLUSION: Despite the rising cancer burden, early career doctors are reluctant to join oncology. Curricular, infrastructural and policy changes are needed at the level of medical school, oncology training and practice to recruit more young doctors and minimize the existing paucity of the oncologic workforce.

RUNX1 mutation and elevated FLT3 gene expression cooperates to induce inferior prognosis in cytogenetically normal acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) is a bone marrow malignancy having multiple molecular pathways driving its progress. In recent years, the main causes of AML considered all over the world are genetic variations in cancerous cells. The RUNX1 and FLT3 genes are necessary for the normal hematopoiesis and differentiation process of hematopoietic stem cells into mature blood cells, therefore they are the most common targets for point mutations resulting in AML. METHODS: We screened 32 CN-AML patients for FLT3-ITD (by Allele-specific PCR) and RUNX1 mutations (by Sanger sequencing). The FLT3 mRNA expression was assessed in all AML patients and its subgroups. RESULTS: Eight patients (25%) carried RUNX1 mutation (K83E) while three patients (9.37%) were found to have internal tandem duplications in FLT3 gene. The RUNX1 mutation data were correlated with clinical parameters and FLT3 gene expression profile. The RUNX1 mutations were observed to be significantly prevalent in older males. Moreover, RUNX1 and FLT3-mutated patients had lower complete remission rate, event-free survival rate, and lower overall survival rate than patients with wild-type RUNX1 and FLT3 gene. The RUNX1 and FLT3 mutant patients with up-regulated FLT3 gene expression showed even worse prognosis. Bradford Assay showed that protein concentration was down-regulated in RUNX1 and FLT3 mutants in comparison to RUNX1 and FLT3 wild-type groups. CONCLUSION: This study constitutes the first report from Pakistan reporting significant molecular mutation analysis of RUNX1 and FLT3 genes including FLT3 expression evaluation with follow-up. This provides an insight that aforementioned mutations are markers of poor prognosis but the study with a large AML cohort will be useful to further investigate their role in disease biology of AML.

Analysis of RAS gene mutations in cytogenetically normal de novo acute myeloid leukemia patients reveals some novel alterations.

Rat sarcoma gene (RAS) holds great importance in pathogenesis of acute myeloid leukemia (AML). The activated mutations in Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) and Kirsten rat sarcoma viral oncogene homolog (KRAS) confers proliferative and survival signals, deliberating numerous effects on overall survival and progression free survival in AML patients. In this study thirty one (31) blood samples of adult newly diagnosed AML patients were collected to identify possible incidence of mutations through amplification of KRAS (exon 1 and 2) and NRAS gene (exon 1 and 2) using polymerase chain reaction (PCR). Amplicons were then subjected to sequencing and were analyzed through Geneious Prime 2019. Five of thirty one (16.12%) patients had altered sites in either NRAS or KRAS. The NRAS mutations were observed in three AML patients (N = 3, 9.67%). A novel missense mutation NRAS-I36R (239 T > G) representing a substitution of single nucleotide basepair found in NRAS exon 1 while exon 2 was detected with heterozygous mutation NRAS-E63X (318G > T) and insertion (A), resulting in frameshift of the amino acid sequence and insertion of two nucleotide basepairs (TA) in two of the patients. KRAS mutations (N = 2, 6.45%) were found in exon 1 whereas no mutations in KRAS exon 2 were detected in our patient cohort. Mutation in KRAS Exon 1, KRAS-D30N (280G > A) was observed in two patients and one of them also had a novel heterozygous mutation KRAS-L16N (240G > C). In addition there was no statistically significant association of mutRAS gene of AML patients with several prognostic markers including age, gender, karyotyping, CD34 positivity, cytogenetic abnormalities, total leukocyte count, white blood cell count and French-American-British (FAB) classification. However, the presence of mutRAS gene were strongly associated (p = 0.001) with increased percentage of bone marrow blasts. The prevalence of mutations in correlation with clinical and hematological parameter is useful for risk stratification in AML patients.