Ahmad Mukhtar Khalid

Interaction of anticancer drug methotrexate with DNA analyzed by electrochemical and spectroscopic methods.

Electrochemical DNA biosensor was used to study the interaction of methotrexate (MTX) with DNA immobilized on the bare surface of glassy carbon electrode (GCE). The binding mechanism of MTX with DNA was elucidated by using constant current potentiometric technique further supported by UV-Visible and FT-IR studies. The decrease in guanine peak area was used as an analytical signal for the interaction of drug with DNA in acetate buffer solution at pH 4.2 (20% ethanol). The binding constant (K) value calculated for MTX was 3.821×10(5)M(-1). UV-Visible studies indicated hyperchromic and hypsochromic shifts in the maximum absorption bands of MTX after interaction with DNA. FT-IR investigations of MTX-DNA interaction revealed significant changes in the characteristic IR absorption bands of all the bases and phosphate groups of DNA. Furthermore, the shift of characteristics bands of C=O, N-H, C-H and O-H groups of MTX endow evidence for the interaction of MTX with DNA supporting the intercalative binding between them.

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.

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.

One size fits all in prostate cancer: a story tale whose time has come and gone

The touchstone to evaluate accurately the aggressiveness and invasiveness of prostate cancer is something of a holy grail in the facet of urologic oncology. Gene expression and sequencing studies have improved our interpretations of the genetic determinants of the disease but are unsuccessful in the establishment of any unified classification to improve the molecular stratification. These questions addressing failure in rational drug design are difficult to answer in the multifaceted and heterogeneous pathogenesis of prostate cancer. In this review, we have developed a roadmap of the “recalcitrant prostate cancer proteome” to recognize the aspects of prostate cancer that may be helpful in effectively translating these findings to the clinic.

Case study of primary imatinib resistance and correlation of BCR-ABL multiple mutations in chronic myeloid leukemia

Background: The kinase inhibitor, imatinib (Gleevec™, Novartis) has proven to be an effective treatment for chronic myeloid leukemia, providing proof-of-principle for a molecularly targeted approach in oncology. Despite its success in BCR-ABL-positive chronic myeloid leukemia patients, primary imatinib resistance has emerged, resulting in disease relapse. Little is known about the underlying genetic causes of resistance, however, with emerging pharmacogenomic approaches, a more comprehensive picture is developing. In imatinib-resistant patients, point mutations have been detected in an ATP-binding domain of the ABL gene, which disturbs the binding of imatinib to this target leading to resistance. However, it remains to be determined whether these mutations confer primary imatinib resistance. Methods: Primary imatinib resistance was observed in a chronic myeloid leukemia patient with no hematological, cytogenetic and molecular response to 9 months treatment with 400 mg imatinib/day. A highly sensitive allele-specific oligonucleotide polymerase chain reaction was established to detect point mutations in BCR-ABL adenosine triphosphate-binding domain. Results: C944T and T1052C mutations were detected which cause complete and partial imatinib resistance, respectively. Conclusions: This is a case study of multiple point mutations conferring primary imatinib resistance in one patient at the same time. Elucidating the biological mechanisms of primary imatinib resistance will improve the ability to predict patient responsiveness and provide effective clinical management. Furthermore, the allele-specific oligonucleotide-polymerase chain reaction assay is very effective in detecting mutations related to imatinib resistance.

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