Muhammad Absar

Five most common prognostically important fusion oncogenes are detected in the majority of Pakistani pediatric acute lymphoblastic leukemia patients and are strongly associated with disease biology and treatment outcome.

BACKGROUND AND OBJECTIVES Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. METHOD We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. RESULTS Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival (43.7±4.24 weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). CONCLUSIONS This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, associated with poor overall survival. Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.

Prognostically Significant Fusion Oncogenes in Pakistani Patients with Adult Acute Lymphoblastic Leukemia and their Association with Disease Biology and Outcome

BACKGROUND AND OBJECTIVES Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. METHODS We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. RESULTS Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL positive patients had frequent organomegaly and usually presented with a platelets count of less than 50 x10(9)/l. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. CONCLUSIONS This is the first study from Pakistan which investigated the frequency of 5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequencies of some of the oncogenes were different from those reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This information will help in the prognostic stratification and risk adapted management of adult ALL patients.

Sensitive Detection of Pre-Existing BCR-ABL Kinase Domain Mutations in CD34+ Cells of Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia Patients Is Associated with Imatinib Resistance: Implications in the Post-Imatinib Era

Background BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. Methods We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. Results Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8–48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. Conclusion Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.

In-vitro susceptibility of methicillin-resistant Stayphylococcus aureus to honey

Wound infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is becoming much complicated and costly to treat as antimicrobial resistance is quite common. Twenty five MRSA strains isolated from infected wounds and three ATCC reference strains were evaluated for their susceptibility to locally produced black seed (Nigella sativa), beri (ZiziphusJujuba) and shain honey (Plectranthus rugosus wall) by agar incorporation assay. Medically graded manuka honey (UMF 21+) was included as control. Locally produced black seed honey inhibited all clinical isolates at mean MIC of 5.5% (v/v), whereas manuka honey at mean MIC of 4.4% (v/v). The other two locally produced honey; beri and shain honey inhibited these isolates at 6.4% and 10.4% (v/v) respectively. The result of the study has demonstrated that indigenous black seed honey has comparable antibacterial activity to manuka honey and thus offers a good new addition to the existing honey resource for the treatment of wound infections.

Low Frequency of ETV6-RUNX1 (t 12; 21) in Saudi Arabian Pediatric Acute Lymphoblastic Leukemia Patients: Association with Clinical Parameters and Early Remission.

BACKGROUND Pediatric acute lymphoblastic leukemia (pALL) patients at King Abdulaziz Medical City represent a pure Saudi Arabian population. ETV6-RUNX1 positive pALL patients have good prognosis as compared to ETV6-RUNX1 negative counterparts. Therefore, frequencies of these two patient groups have a huge consideration in treatment strategies of pALL in a given population. Different geographical locations have been reported to have different frequencies of ETV6-RUNX1 ranging from 10% in Southeast Asia to 30% in Australia. AIM Therefore, the objective of this study was to establish the ETV6-RUNX1 status of Saudi Arabian pALL patients and its association with clinical parameters and early remission. MATERIALS AND METHODS Clinical parameters and ETV6-RUNX1 status (using FISH technique) of pALL patients attending the Pediatric Oncology Clinic, King Abdulaziz Medical City, Riyadh from 2006 to 2011 were studied. Comparisons between ETV6-RUNX1 positive and negative groups were accomplished using chi-square test or Fishers exact test. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC). RESULTS Out of 54 patients, 33 were male and 21 were females (ratio 1.57:1). B- and T-cell lineages were found in 47 (87%) and 7 (13%) patients respectively. Only 5 (9.3%) patients were ETV6-RUNX1 positive while 49(80.7%) were ETV6-RUNX1 negative. All ETV6-RUNX1 patients (100%) were of B-cell lineage and 80% (4/5) were in the 3-7 year age group. None of the ETV6-RUNX11 patients had ≥ 5% blasts (no remission) at day 14 as compared with 9% in the ETV6-RUNX1 negative group (Figure 1). CONCLUSIONS Frequency of ETV6-RUNX1 positive patients (less than 10%) in our pALL patients is much lower than reported for most European countries, North America, Australia and Japan while it is in accordance with ETV6-RUNX1 frequencies from Egypt (11.6%), Pakistan (10%), Spain (2%) and India (5-7%). This shows ethnic differences in genetics of pALL as well as higher frequencies of ETV6-RUNX1 positive pALL mostly in more industrialized countries, probably due to some industrial pollutants or westernized lifestyle.

Comparison of Molecular and Phenotypic Methods for the Detection and Characterization of Carbapenem Resistant Enterobacteriaceae.

In recent years, there has been a rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE). This study aimed to compare phenotypic and molecular methods for detection and characterization of CRE isolates at a large tertiary care hospital in Saudi Arabia. This study was carried out between January 2011 and November 2013 at the King Khalid University Hospital (KKUH) in Saudi Arabia. Determination of presence of extended-spectrum beta-lactamases (ESBL) and carbapenem resistance was in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Phenotypic classification was done by the MASTDISCS(TM) ID inhibitor combination disk method. Genotypic characterization of ESBL and carbapenemase genes was performed by the Check-MDR CT102. Diversilab rep-PCR was used for the determination of clonal relationship. Of the 883 ESBL-positive Enterobacteriaceae detected during the study period, 14 (1.6%) isolates were carbapenem resistant. Both the molecular genotypic characterization and phenotypic testing were in agreement in the detection of all 8 metalo-beta-lactamases (MBL) producing isolates. Of these 8 MBL-producers, 5 were positive for blaNDM gene and 3 were positive for blaVIM gene. Molecular method identified additional blaOXA gene isolates while MASTDISCS(TM) ID detected one AmpC producer isolate. Both methods agreed in identifying 2 carbapenem resistant isolates which were negative for carbapenemase genes. Diversilab rep-PCR analysis of the 9 Klebsiella pneumoniae isolates revealed polyclonal distribution into eight clusters. MASTDISCS(TM) ID is a reliable simple cheap phenotypic method for detection of majority of carbapenemase genes with the exception of the blaOXA gene. We recommend to use such method in the clinical laboratory.