Tanveer Akhtar

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.

Genetic characterization of the Makrani people of Pakistan from mitochondrial DNA control-region data

To estimate genetic and forensic parameters, the entire mitochondrial DNA control region of 100 unrelated Makrani individuals (males, n=96; females, n=4) living in Pakistan (Turbat, Panjgur, Awaran, Kharan, Nasirabad, Gwadar, Buleda, Karachi and Burewala) was sequenced. We observed a total of 70 different haplotypes of which 54 were unique and 16 were shared by more than one individual. The Makrani population showed a high genetic diversity (0.9688) and, consequently, a high power of discrimination (0.9592). Our results revealed a strongly admixed mtDNA pool composed of African haplogroups (28%), West Eurasian haplogroups (26%), South Asian haplogroups (24%), and East Asian haplogroups (2%), while the origin of the remaining individuals (20%) could not be confidently assigned. The results of this study are a valuable contribution to build a database of mtDNA variation in Pakistan.

Distinct gene mutations, their prognostic relevance and molecularly targeted therapies in Acute Myeloid Leukemia (AML)

Acquired genetic alterations which include balanced and unbalanced chromosome aberrations and submicroscopic gene mutations and changes in gene expression strongly influenced by pretreatment clinical features and prognosis of adults patients with acute myeloid leukemia (AML). Cytogenetic profiling separate AML patients into three broad prognostic groups: favorable, intermediate and adverse. The cytogenetic risk classifications vary to some extent for younger adult patients and for those aged 60 years or older. In many cases, patients with specific cytogenetic rearrangement such as those with a normal karyotype or those with either RUNX1-RUNX1T1 or CBFB-MYH11 feature of core-binding factor (CBF) can be further subdivided into prognostic categories depend on the presence or absence of specific gene mutations or changes in gene expression. Advancement in the understanding of cancer genetic and discovery of recurrent mutations in AML provide opportunity to develop targeted therapies and improve the clinical outcome. The identified gene mutations, mainly targetable lesions are gain of function mutations of JAK2 and cKIT and FLT3 in APL have been associated with clinical features and/ or outcome of patients with these AML subtypes. These data emphasize the significance of genetic testing for common translocations for diagnosis, prognosis and increasingly targeted therapy in acute leukemia. Notably, these several molecular genetic alterations constitute a variety of diverse new targets for salvage therapies. These approaches intend to develop targeted treatment concepts that depend on interference with molecular genetics or epigenetic mechanisms. This report provides an overview on characteristic gene mutations, discuss their biological functions and Prognostic significance, which serve as basis for selected therapy approaches now or might represent options for such approaches in the future and expected to have a role in treating AML subtypes with characteristic molecular alterations

Mitochondrial DNA control region sequences study in Saraiki population from Pakistan

The analysis of mitochondrial DNA (mtDNA) control region was carried in 85 unrelated Sariki individuals living in the different provinces of Pakistan. DNA was extracted from blood preserved in EDTA vacutainers. Hypervariable regions (HV1, HV2 & HV3) were PCR amplified and sequenced. Sequencing results were aligned and compared with revised Cambridge reference sequence (rCRS). The sequencing results showed presence of total 63 different haplotypes, 58 of them are unique and 05 are common haplotypes shared by more than one individual. The most common haplotype observed was (W6) with a frequency 12.9% of population sample. The Saraiki population was detected with genetic diversity (0.9570) and power of discrimination (0.9458). This study will be beneficial for forensic casework.

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.

Hematological changes in the hydatidosed male sheep after experimental inoculation of Echinococcus granulosus eggs

T gondii is an intracellular parasite, causing a severe disease in immunocompromised humans and having an economic impact in domestic animals. One of the transmission routes to humans is the consumption of raw or undercooked meat from livestock, such as pork. Therefore, an experimental infection study was performed to identify the immunological parameters in pigs, induced by the innate or adapted immune system of the host and to associate the immune response with the presence of the parasite in the tissues. In the present study two groups of seronegative 5-week-old pigs were inoculated at d0 with 6000 tissue cysts of T. gondii strain IPB-LR or IPB-Gangji. The PBMC’s were collected two-weekly and cultured for 72 hours upon in vitro stimulation with heterologous antigens from RH-strain, fractionated by continuous-elution electrophoresis into separate pools. The cells were triple-stained for the flow cytometry and based on the expression of the membrane markers, divided into T-lymphocyte subsets (CD3+CD4+CD8α-, CD3+CD4+CD8αdim and CD3+CD4-CD8αbright), followed by the intracellular IFN-γ staining. Additionally, the supernatant from the cultured cells was tested in IFN-γ ELISA. Four or six months post infection the animals were euthanized and the parasite was detected in tissues by bio-assay and qPCR. In our study we detected a strong increase in the cytokine production, induced by the isolated fractions of the native antigens and confirmed by flow cytometry and cytokine ELISA. Interestingly, this increase was time and strain dependent, being related with the amount of the parasitic DNA detected in the tissue samples.

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