Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

Clinical and laboratory relevance of JAK2 V617F and BCR-ABL co-existence in Philadelphia positive CML patients.

Chronic Myeloid Leukaemia (CML) is characterized by BCR-ABL1 mutation. A number of research studies have published reports of concomitant JAK2-V617F mutation in BCR-ABL positive Chronic Myeloid Leukaemia. This study aims to investigate the frequency of JAK2-V617F mutation in BCR-ABL positive CML cases. After approval from ethical committee, participants were enrolled in the study. A total of 103 samples from CML patients were analysed for the presence of JAK2-V617F mutation using real-time polymerase chain reaction. Patients were monitored for treatment response using real-time quantitative PCR for BCR-ABL1 mutation. Out of 103 samples analysed, 2 patients tested positive for JAK2-V617F mutation. These two patients when treated with standard Tyrosine Kinase Inhibitors (TKI) therapy achieved molecular response and normalized the haemoglobin and white cell counts. However, one patient has sustained thrombocytosis. JAK2 remained positive throughout the treatment course. We could not follow the second patient till the end of the study. JAK2 mutation in BCR-ABL1 mutated CML appears to be rare. Treatment with TKI does not appear to reduce JAK2 mutation burden despite a decrease in BCR-ABL1 copy numbers.

Whole exome sequencing identifies a novel FANCD2 gene splice site mutation associated with disease progression in chronic myeloid leukemia: Implication in targeted therapy of advanced phase CML.

Tyrosine Kinase Inhibitors (TKIs) have significantly improved the clinical outcome of BCR-ABL+ Chronic Phase-Chronic Myeloid Leukemia (CP-CML). Nonetheless, approximately one-third of the CP-CML patient's progress to advanced phases of CML (accelerated and blast phase). Impaired DNA repair including mutations in Fanconi anemia (FA) pathway genes are responsible for progression of many cancers. Nevertheless, FA-pathways genes have never been reported in myeloid cancers. Hence, this study was aimed to discover DNA repair genes associated with CML progression. AP-CML patients were subjected to whole exome sequencing along with appropriate controls. A novel splice site FANCD2 mutation was detected. FANCD2 is a well-known FA-pathway gene with established role in DNA repair. This is first report of FA-pathway DNA repair genes in myeloid cancers that can serve as a novel marker of CML progression to clinically intervene CML progression. Further studies are needed to establish the functional role of FANCD2 in CML progression that can provide novel insights into CML pathogenesis. This study also indicates that a combination TKIs and Poly (ADP-ribose) polymerase (PARP) inhibitors like Olaparib (FDA approved anti-cancer drug for FA-pathway gene mutations) could improve the clinical outcome CML patients in accelerated and blast-crisis phases of the disease.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

OBJECTIVE: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. METHODOLOGY: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling  studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. RESULTS: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. CONCLUSION: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second  generation and third generation TKIs.

Long term outcome of chronic myeloid leukemia patients treated with imatinib: Report from a developing country.

The outcome of chronic myeloid leukemia has been greatly improved by the use of Imatinib (IM), a selective BCR/ABL kinase inhibitor. The aim of present study was to report long term follow-up & outcome of IM-treated CML patients along with their clinicopathological features, risk group stratification, adverse events and to compare it with CML patients reported from western countries. The mean follow-up of 123 CML patients was 5.5 years in present study, who were treated with frontline IM 400mg daily in a tertiary care hospital in Pakistan. Risk stratification scores, response to treatment (ELN guidelines) and survival outcomes estimated by Kaplan-Meier analysis. Mean age: 35 years (9-67 years) and M: F: 1.5:1, mean follow up time: 5.5 years (1-15 years). Overall survival (OS): at 5.5, 8, 10 and 12 years were 93%, 88%, 81% and 73%, respectively. Progressions free survival (PFS) was 95%, 83%, 83% and 78% at 5.5, 8, 10 and 12 years, respectively. OS estimate by Sokal score was significant (P-value: 0.0019). Additional chromosomal aberrations: 1.6%. Eighteen (14.6%) patients progressed to AP/BC. Adverse events were moderate and tolerable. We present findings from a long term follow up of CML patients treated with IM in a developing country. CML mean age at onset was considerably lower than the western populations. Furthermore, 5.5 years OS are comparable to western CML population. IM in our patients as frontline choice proved to be very effective. IM was found to be well tolerated, safe with manageable moderate side effects.

High frequency and poor prognosis of late childhood BCR-ABL-positive and MLL-AF4-positive ALL define the need for advanced molecular diagnostics and improved therapeutic strategies in pediatric B-ALL in Pakistan.

BACKGROUND: Fusion oncogenes (FOs) resulting from chromosomal abnormalities have an important role in leukemogenesis in pediatric B cell acute lymphoblastic leukemia (ALL). The most common FOs are BCR-ABL, MLL-AF4, ETV6-RUNX1, and TCF3-PBX1, all of which have important prognostic and drug selection implications. Moreover, frequencies of FOs have ethnic variations. We studied Pakistani frequencies of FOs, clinical pattern, and outcome in pediatric B-ALL. METHODS: FOs were studied in 188 patients at diagnosis using reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescent in situ hybridization (FISH). Data were analyzed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). RESULTS: FOs were detected in 87.2 % of patients. Mean overall survival was 70.9 weeks, 3-year survival was 31.9 %, and 3-year relapse-free survival was 18.1 %. Four patients died of drug toxicities. ETV6-RUNX1 (19.14 %) had better survival (110.9 weeks; p = 0.03); TCF3-PBX1 (2.1 %) was associated with inferior outcome and higher central nervous system (CNS) relapse risk; MLL-AF4 (18.1 %) was more common in the 8- to 15-year age group (24/34; p = 0.001) and was associated with organomegaly, low platelet count, and poor survival; and BCR-ABL (47.9 %) was associated with older age (7-15 years, 52/90), lower remission rates, shorter survival (43.73 ± 4.24 weeks) and higher white blood cell count. Overall, MLL-AF4 and BCR-ABL were detected in 66 % of B-ALL, presented in later childhood, and were associated with poor prognosis and inferior survival. CONCLUSIONS: This study reports the highest ethnic frequency of BCR-ABL FO in pediatric ALL, and is consistent with previous reports from our region. Poor prognosis BCR-ABL and MLL-AF4 was detected in two-thirds of pediatric B-ALL and is likely to be the reason for the already reported poor survival of childhood ALL in South-East Asia. Furthermore, MLL-AF4, usually most common in infants, presented in later childhood in most of the ALL patients, which was one of the unique findings in our study. The results presented here highlight the need for mandatory inclusion of molecular testing for pediatric ALL patients in clinical decision making, together with the incorporation of tyrosine kinase inhibitors, as well as hematopoietic stem cell transplantation facilities, to improve treatment outcome for patients in developing countries.

Feasibility, relevance and effectiveness of teaching and assessment of ethical status and communication skills as attributes of professionalism.

OBJECTIVE: To examine the feasibility and effectiveness of teaching and assessing professionalism in a developing country. METHODS: The pre-intervention and post-intervention study was conducted from January to August 2012 and comprised 7 workshops of three days each that were held at four teaching hospitals of Khyber Pakhtunkhwa province in Pakistan. Overall, there were 10 Objective Structured Clinical Examination stations and 10 written scenarios. After the pre-test, workshop was held on various aspects of professionalism which was considered 'intervention', and it was followed by a post-test similar to the pre-test at the end of day 3. Stata 12 was used for all statistical analyses. RESULTS: There were 136 postgraduate residents in the study. The correlation between Objective Structured Clinical Examination stations and written exam for pre-test was 0.42 (p<0.001), while for post-test the correlation was 0.17(p=0.046). Correlation between pre-test written and post-test Objective Structured Clinical Examination stations was 0.23 (p=0.001), but the correlation between pre-test Objective Structured Clinical Examination stations and post-test written was not significant (p>0.05).The standardised effect size for the adjusted regression was 0.37 for both comparisons (p<0.001).Mean pre-test scores were 38.13+/-13.13% vs. 76.50+/-14.4%for the post-test score (p<0.001). CONCLUSIONS: Although post-test scores increased significantly both for the Objective Structured Clinical Examination stations and the written scenarios, the former has shown a higher reliability compared to the written test. Furthermore, teaching and assessment of professionalism was found relevant, effective and feasible in resource-constrained countries. Teaching and assessment of professionalism has become globally relevant and is recommended to be included in the curricula of medical institutions.

Development and evaluation of standardized narrative cases depicting the general surgery professionalism milestones.

PURPOSE: Residency programs now are required to use educational milestones, which has led to the need for new methods of assessment. The literature suggests that narrative cases are a promising tool to track residents' progress. This study demonstrates the process for developing and evaluating narrative cases representing the five levels of the professionalism milestones. METHOD: In 2013, the authors identified 28 behaviors in the Accreditation Council for Graduate Medical Education general surgery professionalism milestones. They modified previously published narrative cases to fit these behaviors. To evaluate the quality of these cases, the authors developed a 28-item, five-point scale instrument, which 29 interdisciplinary faculty completed. The authors compared the faculty ratings by narrative case and specialty with the authors' initial rankings of the cases by milestone level. They used t tests and analysis of variance to compare mean scores across specialties. RESULTS: The authors developed 10 narrative cases, 2 for each of the 5 milestone levels. Each case contained at least 20 of the 28 behaviors identified in the milestones. Mean faculty ratings matched the milestone levels. Reliability was good (G coefficient = 0.86, phi coefficient = 0.85), indicating consistency in raters' ability to determine the proper milestone level for each case. CONCLUSIONS: The authors demonstrate a process for using specialty-specific milestones to develop narrative cases that map to a spectrum of professionalism behaviors. This process can be applied to other competencies and specialties to facilitate faculty awareness of resident performance descriptors and provide a frame of reference for milestones assessment.

Survey on perceptions and skills amongst postgraduate residents regarding breaking bad news at teaching hospitals in Peshawar, Pakistan.

OBJECTIVE: To assess the perception, skills and comfort level of postgraduate residents with respect to breaking bad news. METHODS: Five workshops were conducted on communication skills, including the task of breaking bad news, at three teaching hospitals of Peshawar, Pakistan. Teaching methods included interactive lectures, video presentations, role play and small group discussions. Pre- and post-workshop data was collected from all the 97 participants to assess their previous training, comfort level and perceptions regarding the subject and any improvement after attending the workshop. RESULTS: Of the total participants, 92 (95%) residents had not received any training in communication skills at the undergraduate level. Only 64 (66%) residents had witnessed bad news being broken by a consultant. Before the workshop, 83 (85%) residents felt either not comfortable or somewhat comfortable while breaking bad news compared to 36% post-workshop (p < 0.0000). Besides, 64 (66%) residents reported breaking bad news to be extremely stressful or very stressful before the workshop versus 25% post-workshop (p < 0.0000). Before the workshop, 18 (19%) residents said they would withhold the information from the patient on family's insistence despite the patient's wish to be informed, compared to 6% post-workshop (p < 0.007). Regarding the utility of the workshop, 91 (94%) residents said it had changed their perceptions to a major extent, while 92 (95%) residents rated the workshop as extremely useful or very useful. CONCLUSION: Formal structured training in breaking bad news is lacking both at undergraduate and postgraduate levels in Pakistan. Structured training programmes for residents can do the task effectively.

Safety of cytotoxic chemotherapy during pregnancy.

OBJECTIVE: To To present an experience and results of treatment of pregnant cancer patients with cytotoxic chemotherapy from second trimester of pregnancy. METHODS: Eighteen consecutive pregnant patients treated at Khyber Teaching Hospital, Peshawar between December 2000 and August 2006 for different types of malignancies are reported. Six patients (33%) had breast cancer, four (22%) had chronic myeloid leukaemia, two (11%) had Hodgkin's disease, two (11%) had acute myeloid leukaemia and one each had recurrent ovarian carcinoma (5.7%), soft-tissue sarcoma (5.7%), acute lymphoblastic leukaemia (5.7%) and non-Hodgkin's lymphoma (5.7%). Various chemotherapeutic protocols were administered from second trimester of pregnancy onwards. Detailed obstetrical examinations and high risk foetal ultrasound monitoring were performed regularly. RESULTS: Two patients were lost to follow-up after one course of chemotherapy while two patients chose to have therapeutic abortion. Out of the remaining 14 patients, one patient had spontaneous abortion while one patient had an intra-uterine death of foetus during chemotherapy. Remaining 12/14 (86%) patients gave birth to live, healthy babies and no foetal malformations were observed. Six out of 140 breast cancer patients (4.3%) during the study period had concomitant pregnancy. Four patients with breast cancer had modified radical mastectomy with axillary dissection during pregnancy (median gestational age 22 weeks) and no operative or post-operative complications were noted. Three out of four breast cancer patients (75%) had hormone receptor negative tumours. CONCLUSION: Chemotherapy during the second and third trimester of pregnancy can be safe if proper obstetric and radiologic monitoring is performed. Long term side-effects in these children need to be studied with extended periods of follow-up.

Plasma cell leukemia: case report of a rare and aggressive variant of multiple myeloma.

Plasma cell leukemia (PCL) is a rare disease and is the least common variant of multiple myeloma accounting for 2-3% of all plasma cell dyscrasias. We report a patient who presented with history of high grade fever, weakness, palpitations, loss of appetite, bone pains and mental confusion for twenty days. Initial evaluation revealed plasmacytosis with blood plasma cell count of 5184/cumm. His hemoglobin (Hb) was 11.3 gm/dl, platelets were 75000/cumm and total leucocyte count (TLC) was 21600/cumm (24% plasma cells). Bone marrow examination revealed >60% plasmablasts. Serum LDH was high at 3117 U/L and serum calcium was also elevated at 13.9 mg/dl. A diagnosis of PCL was made and the patient was started on treatment for hypercalcaemia with Melphalan/Prednisolone regime along with supportive care. Patient deteriorated very rapidly despite treatment and died on the eighth day. A detailed report of this case and a review of PCL is presented here.