Mudassar Iqbal

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.

Presence of novel compound BCR-ABL mutations in late chronic and advanced phase imatinib sensitive CML patients indicates their possible role in CML progression

ABSTRACT BCR-ABL kinase domain (KD) mutations are well known for causing resistance against tyrosine kinase inhibitors (TKIs) and disease progression in chronic myeloid leukemia (CML). In recent years, compound BCR-ABL mutations have emerged as a new threat to CML patients by causing higher degrees of resistance involving multiple TKIs, including ponatinib. However, there are limited reports about association of compound BCR-ABL mutations with disease progression in imatinib (IM) sensitive CML patients. Therefore, we investigated presence of ABL-KD mutations in chronic phase (n = 41), late chronic phase (n = 33) and accelerated phase (n = 16) imatinib responders. Direct sequencing analysis was used for this purpose. Eleven patients (12.22%) in late-CP CML were detected having total 24 types of point mutations, out of which 8 (72.72%) harbored compound mutated sites. SH2 contact site mutations were dominant in our study cohort, with E355G (3.33%) being the most prevalent. Five patients (45%) all having compound mutated sites, progressed to advanced phases of disease during follow up studies. Two novel silent mutations G208G and E292E/E were detected in combination with other mutants, indicating limited tolerance for BCR-ABL1 kinase domain for missense mutations. However, no patient in early CP of disease manifested mutated ABL-KD. Occurrence of mutations was found associated with elevated platelet count (p = 0.037) and patients of male sex (p = 0.049). The median overall survival and event free survival of CML patients (n = 90) was 6.98 and 5.8 y respectively. The compound missense mutations in BCR-ABL kinase domain responsible to elicit disease progression, drug resistance or disease relapse in CML, can be present in yet Imatinib sensitive patients. Disease progression observed here, emphasizes the need of ABL-KD mutation screening in late chronic phase CML patients for improved clinical management of disease.

Determinants of Antenatal Psychological Distress in Pakistani Women

INTRODUCTION An increasing number of evidence has demonstrated that poor antenatal psychological health can lead to adverse pregnancy outcomes. Studies conducted in various countries demonstrated a wide range of factors associated with psychological distress during pregnancy. METHODS A cross-sectional study was conducted between September 2011 and December 2012 in Peshawar, north-west Pakistan. A total of 230 women in their third trimester of pregnancy fulfilled the inclusion criteria. The antenatal psychological health status of women was measured using the Depression Anxiety Stress Scale. Relevant data regarding health and demographic-socioeconomic status were collected through personal interviews using standardized questionnaires. RESULTS Overall, 45% (n=104) of women exhibited symptoms for composite depression, anxiety, and stress (composite DAS). In the univariate analysis, maternal age, husband support, monthly income, family size, stressful life events, lack of confidence, domestic violence, and pregnancy-related concerns were strongly associated with antenatal composite DAS (p<0.01). The association of maternal composite DAS symptoms with age, monthly income, family size, and lack of confidence remained significant in the multivariate analysis (p<0.01). CONCLUSION A major proportion of women exhibited symptoms of antenatal composite DAS, and various factors were found to be related to their psychological distress. A young maternal age, low husband support, low income, large family size, adverse life events, lack of confidence, pregnancy-related concerns, and domestic violence were stronger determinants of poor antenatal psychological status. The study findings concluded that policymakers at the government level should launch special intervention programs to improve maternal perinatal mental and psychological health at the community level.

Assessment of Mycelia Extract from Trichoderma harzianum for its Antifungal, Insecticidal and Phytotoxic Importance

Trichodermaharzianum was cultured on potato dextrose broth media at 20°C in an incubator and mycelial cells were extracted with ethyl acetate to obtain the organic extract for in-vitro bio-activities including antifungal, insecticidal and phytotoxicity. Different strains of fungal pathogens including Aspergillusflavus, Rhizopusstolonifer and Pythiumultimum were used to assess the antifungal potential of T. harzianum extract. The inhibitory effect was found 82% for A. flavus , 77% for P. ultimum and 73% for R. stolonifer when compared with positive and negative control experiments. Aphids (Macrosiphumrosae ) as a test insects were used to perform the insecticidal activity that showed potent activity with LC50 (38.88 µgml-1). The herbicidal potential was evaluated against duck weed (Lemna minor ) which showed that by using very high concentration (1000 µgml-1) only 60% lethality was achieved. This pilot study revealed that the organic extract obtained from T. harzianum contains useful compounds having potential to be utilized in the development of fungicides and pesticides for the improvement of agricultural sector of the country.

Fabrication of silver nanoparticles from mycological flora and their importance against agro-pathogens: Towards green nano-pesticides

A special appreciation and sincere gratitude to my advisor Prof. Adham Ramadan, for being a tremendous mentor and for his continuous support, patience, and immense knowledge. His guidance helped so much throughout the research, and am really grateful to him for accepting me to work under his supervision. My sincere gratitude to the huge technical support, provided by Mr. Mahmoud Abdel Moez, Mr. Victor and Mr. Emad Farag. I am also very grateful to Mr. Ahmed Omaya for being a supportive friend in my research work and for facilitating a lot of technicalities. Without their help, I wouldn’t have conducted this work. Would like to acknowledge the academic, and grant support provided by the American University in Cairo. I would like to acknowledge the support of my graduate professors, their educational material and advices. I would like to thank my big family: parents, grandparents, sister, brother, aunt, and uncles. Each one of them provided me with emotional support, they were patient and standing out there for me all along. Their prayers and encouraging is what sustained me that far. Last but definitely not least, my colleagues who turned to be best friends ever, who made this experience more fun and smooth. Words are not enough to express how supportive they were throughout my research work, career and on a personal level. Thanks for each and every one of them: Zahraa, Raghda, Alshaima, Yomna, Ruaa and Woroud............................................................................................................................... 15 Declaration .......................................................................................................................... 16 Copyright statement........................................................................................................... 17 Dedication ........................................................................................................................... 18 Acknowledgements ............................................................................................................. 19 Rational for submitting in alternative format ................................................................. 20 Thesis outline ...................................................................................................................... 21 Chapter One ....................................................................................................................... 22The direct synthesis of hydroxyapatite─1,8-octan-bisphosphonic acid (HAp─BISPH) nanocrystals has been carried out in presence of increasing amounts of BISPH in solution, by hydrothermal method at 120 °C for 15 h. XRD, IR, NMR-MAS (31P, 1H and 13C), TEM, AFM, TGA and chemical analysis were used to characterize the structure, morphology and composition of the products. X-ray powder diffraction patterns show that the incorporation of bisphosphonate moieties induces a significant loss of the material crystallinity and a clear decrease of the crystallite size. TEM and AFM images show that the precipitated apatite particles prepared in the presence of this bisphosphonic acid are nanosized. The IR and NMR-MAS 1H spectroscopy show that the BISPH can replace the OH− groups of the apatitic structure.

Antioxidant Activity of Morchella conica Collected from Swat Valley

5 Abstract 6 7 The present study was conducted University of Agriculture Peshawar, KPK, during the year 8 20142015. Mushroom phenolic considered as an excellent antioxidant and synergist. The main 9 objective of this study was to determine the antioxidant activity of Morchella conica commonly 10 called black morels at different concentration. For this purpose the Morchella conica extract was 11 used to determining the free radical scavenging activity. The results showed that increase in the 12 concentration increases the antioxidant activity. The antioxidant activity of Morchella conica 13 was recorded 44.86 ± 0.94, 62.72± 1.64, 74.14 ± 2.63, 83.35 ± 0.313, 85.01 ± 0.80, 86.53 ± 0.22, 14 88.03 ± 0.35, 88.56 ± 0.37 and 93.53 ± 0.01 respectively at 100 ppm, 150 ppm, 200 ppm, 250 15 ppm, 300 ppm, 500 ppm, 1000 ppm, 2000 ppm, 3000 ppm. 16

Abstract 564: Favorable prognostic features at diagnosis along with young age, early remission, high relapse rate and poor survival of TCF3-PBX1 positive adult ALL necessitates the need for differential genetic diagnosis and treatment using pediatric ALL protocols

Introduction: ALL involves many genetic abnormalities, many of which lead to formation of fusion oncogenes (FGs) causing leukemogenesis1. Role of FGs in prognosis and drug selection is established in pediatric ALL but is unclear in adult AL 2 except BCR-ABL and MLL-AF4. Therefore, objective our was to find out the association of common FGs with clinical pattern. Methods: We studied 5most common FGs by RT-PCR3 and interphase FISH at diagnosis and their association with clinical features and treatment outcome in 208 adult ALL patients (2002-2012) treated with MRC UKALL XII protocol. Results: FGs were detected in 78.8% patients. Overall survival (OS) was 26.17 months (mo) and relapse free survival (RFS) was 11.147 mo. SIL-TAL1+ ALL (35.36%) manifested lymphadenopathy, frequent organomegaly, low platelets count and poor survival. BCR-ABL+ (20.3%) ALL manifested high TLC, prominent spleenomegaly, low platelets count, poor survival (OS & RFS 9.3 & 6.3 mo, respectively) and 10% less chances of 4-6 week remission as compared to BCR-ABL negative. MLL-AF4 (12.19%) was associated with elevated TLC, organomegaly, frequent CNS involvement and poor clinical outcome (OS 8.8 mo). ETV6-RUNX1 (t 12;21) (4.8%), was associated with low TLC, uncommon organomegaly, high CR rates and with higher OS (30.2 mo). Long term survival of ETV6-RUNX1 was negatively affected by frequent late relapses. Unexpectedly, TCF3-PBX1 (16.3%) was significantly higher than globally reported (3%). It was associated with younger age (59% with 15-29 years), lower TLC (82%), platelet count higher than 50×109/l (71%), less common hepatomegaly (12%), less common spleenomegaly (18%), early CR (65%), all indicative of good prognosis. Despite that, very surprisingly, high relapse rate (76.1%) and shorter OS (11.6 months) were observed in TCF3-PBX1+ patients. Discussion / Conclusions: Favorable prognosis, younger age, high 4-week CR (65%), higher relapse rates and shorter OS highlight in TCF3-PBX1+ ALL necessitate differential genetic diagnosis and intensified and treatment with pediatric protocols in this subgroup4,5. Lymphadenopathy in SIL-TAL1 can help in differential diagnosis of this subgroup (t-cell ALL) ALL in poor countries. These findings along with lower ETV6-RUNX1 frequency and higher MLL-AF4 reflect ethnic differences in disease biology and treatment outcome of adult ALL5. Overall high percentage of poor prognostic FGs may be the strongest reasons of overall poor outcome of adult ALL in our country. Therefore, routine molecular testing for ALL FGs at diagnosis and its implication in prognostic stratification and drug selection during various phases of treatment are recommended. References: 1. Harrison & Foroni, 2002. 2. Moorman et al., 2007 3. Van Dongen et al, 1998. 4. Foa et al., 2003. 5. Burmeister et al., 2010. Citation Format: Zafar Iqbal*, Tanveer Akhtar, Noreen Sabir, Tashfeen Khalid Awan, Salman Basit, Aamer Aleem, Ahmad Mukhtar Khalid, Mudassar Iqbal, Mahmood Rasool. Favorable prognostic features at diagnosis along with young age, early remission, high relapse rate and poor survival of TCF3-PBX1 positive adult ALL necessitates the need for differential genetic diagnosis and treatment using pediatric ALL protocols. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 564. doi:10.1158/1538-7445.AM2014-564