The presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. However, detection is complicated by the existence of hundreds of potential frameshift insertions, clonal heterogeneity, and absence of sequence information when the NPM1 mutation is identified using capillary electrophoresis. Thus, some patients are ineligible for NPM1 MRD monitoring. Furthermore, a subset of patients with NPM1-mutated AML will have false-negative MRD results because of clonal evolution. To simplify and improve MRD testing for NPM1, we present a novel digital PCR technique composed of massively multiplex pools of insertion-specific primers that selectively detect mutated but not wild-type NPM1. By measuring reaction end points using digital PCR technology, the resulting single assay enables sensitive and specific quantification of most NPM1 exon 12 mutations in a manner that is robust to clonal heterogeneity, does not require NPM1 sequence information, and obviates the need for maintenance of hundreds of type-specific assays and associated plasmid standards.

Objective: Our aim was to identify factors favoring long term survival in patients presenting with stage IV epithelial ovarian cancer.Methods: We did retrospective analysis of thirty patients with stage IV epithelial ovarian cancer diagnosed and treated at Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan from 2006 to 2013. Patient’s demographics, clinical data and histopathology were abstracted from cancer registry department of our hospital. Chi-square test was used to find the association between clinic-pathological variables and long term survival. Result: All patients received chemotherapy and surgery as per ovarian cancer guidelines. Of the thirty patients, eleven patients survived greater than four years median survival was recorded as thirty five months. Absence of co-morbidities and good performance status indicated good results of therapy however did not have statistically significant impact on survival. Higher CA-125 at presentation i.e.>1000(normal range : <21 U/ml), response to initial chemotherapy, interval cytoreductive surgery and complete response after induction therapy were significantly associated with long term survival (P<0.05).Conclusion: Prognosis of patients presenting with stage IV epithelial ovarian cancer remains poor. Very high values of CA-125 (>1000) at presentation, response to initial chemotherapy, interval surgical resection and complete remission after induction therapy, appear to be significant prognostic factors for long term survival. Further studies exploring molecular profiling and immunological factors are warranted.

Objective: Our aim was to identify factors favoring long term survival in patients presenting with stage IV epithelial ovarian cancer.Methods: We did retrospective analysis of thirty patients with stage IV epithelial ovarian cancer diagnosed and treated at Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan from 2006 to 2013. Patient’s demographics, clinical data and histopathology were abstracted from cancer registry department of our hospital. Chi-square test was used to find the association between clinic-pathological variables and long term survival. Result: All patients received chemotherapy and surgery as per ovarian cancer guidelines. Of the thirty patients, eleven patients survived greater than four years median survival was recorded as thirty five months. Absence of co-morbidities and good performance status indicated good results of therapy however did not have statistically significant impact on survival. Higher CA-125 at presentation i.e.>1000(normal range : <21 U/ml), response to initial chemotherapy, interval cytoreductive surgery and complete response after induction therapy were significantly associated with long term survival (P<0.05).Conclusion: Prognosis of patients presenting with stage IV epithelial ovarian cancer remains poor. Very high values of CA-125 (>1000) at presentation, response to initial chemotherapy, interval surgical resection and complete remission after induction therapy, appear to be significant prognostic factors for long term survival. Further studies exploring molecular profiling and immunological factors are warranted.

Introduction: Acute myelogenous leukemia (AML) is a fatal disease with dismal outcomes in which most patients relapse and ultimately succumb to their disease. The presence of minimal residual disease (MRD) in patients has been shown to be predictive of relapse and can thereby improve clinical decision-making. NPM1 exon 12 frameshift mutations (NPM1mut) are found in ~50% of cytogenetically normal AML and represent a well-established molecular MRD marker typically assessed by RQ-PCR. While assays are readily available for the most common NPM1mut, detection is complicated by hundreds of potential frameshift insertions, reports of NPM1mut type-switching, and a lack of sequence data in patients diagnosed for NPM1mut by capillary electrophoresis. To simplify the deployment and increase the robustness of NPM1mut MRD detection, we developed a digital droplet PCR assay comprised of multiplex primer pools capable of detecting >95% of NPM1mut subtypes without requiring prior knowledge of NPM1 sequence. To assess its potential in patient care, we tested sensitivity and concordance with established type-specific assays and its robustness across different NPM1mut types. We further demonstrate its use in patients lacking NPM1mut sequence information. Results: Detection of 200+ subtypes was evaluated. The assay demonstrated excellent concordance with subtype-specific assays (ρc = 0.97-0.99) as determined by testing in patient samples, cell lines, and synthetic cDNA bearing rare single and multiplex NPM1mut. OCI-AML3/MV-4-11 dilution experiments revealed sensitivity comparable with standard assays (1-2 x 10-5) while retaining specificity for mutant NPM1. Multiplex primer pools produced lower coefficient of variation across digital PCR partitions relative to single primers containing deoxyinosine or 5-nitroindole. To illustrate practical implementation where NPM1mut levels could not otherwise be monitored, we performed longitudinal retrospective MRD analysis of a patient who presented to our center without NPM1mut sequence data having been diagnosed for NPM1mut by capillary electrophoresis at an outside clinic. Monitoring was performed over the course of 356 days. To determine the actual NPM1 subtype, we performed ultra-deep targeted mRNA-seq during a transient spike in NPM1mut levels during remission at day 28. We identified 48/14,271 reads (0.34%) supporting the NPM1 subtype as type D (c.863_864insCCTG) with the incidental finding of IDH1-R132H mutation previously unknown in this patient. Subsequent re-evaluation with the subtype-specific assay (type D) demonstrated agreement with the massively multiplex assay. Steady increases in NPM1mut/104 ABL1 ratios were observed to peak at day 196 when the patient presented 22% blasts in the bone marrow and >28,000 NPM1mut/104ABL1 and re-entered remission with subsequent care. Targeted next generation sequencing at this time further corroborated the NPM1 type D and IDH1-R132H mutation. Conclusions: The new assay demonstrates sensitive and robust quantification of MRD in a variety of NPM1+ AML. Patients lacking in NPM1 sequence information or who harbor rare subtypes with current standard method would greatly benefit from the assay. Moreover, deployment of the NPM1 MRD testing is simplified by covering >95% of NPM1 mutated patients in a single test without requiring plasmid standards or custom assays. Additionally, we show that MRD measurements with the multiplex assay can be used to guide the timing of deep sequencing to capture potential information about imminent relapse and to facilitate intervention at earlier stages in the disease. Disclosures Ritchie: Celgene: Speakers Bureau; Pfizer: Honoraria; Novartis: Honoraria; Arian: Speakers Bureau; Incyte: Speakers Bureau. Guzman:Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy.

Background and Purpose: Chronic Myeloid Leukemia (CML) is a common hematological malignancy. The characteristic molecular abnormality is the presence of Philadelphia chromosome or BCR-ABL fusion gene which is the result of 9:22 translocation. Tyrosine kinase inhibitors (TKIs) form the main stay of treatment in CML with excellent responses. The purpose of this study was to determine the impact of additional chromosomal abnormalities on outcomes in CML.Methods: This is a retrospective chart review of all patients who were diagnosed with CML in chronic phase (CP) with additional chromosomal abnormalities (ACAs) over a period of 5 years from 2010 to 2015 at Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan. Results: A total of 283 patients were diagnosed with CML from January 2010 to January 2015. 31 patients out of these were found to have additional chromosomal abnormalities at the time of diagnosis in addition to BCR-ABL fusion gene or Philadelphia chromosome detection. Out of these 31 patients, 23 (74.2%) were males whereas 8 (25.8%) were females. 13 (41.9%) were in the age group of 31 to 50 years whereas the other two groups that is 18 to 30 years and 51 to 70 years had 9 patients each. After approval from the government which usually takes a standard 2-3 weeks’ time, these patients were started on tyrosine kinase inhibitors which was Imatinib in 30 (96.8%) and Nilotinib in 1 (3.2%) patient. Conventional cytogenetic analysis performed for each patient at the time of diagnosis revealed that 11 (35.5%) of patients had variant Philadelphia chromosome followed by 7 patients (22.6%) with trisomy 8. 5 patients (16.1%) had multiple chromosomal abnormalities including trisomy 8, deletion 1 and isochrome 17q. 2 patents each had isochrome 17q, inversion 3 and deletion 9 abnormalities. 1 patient had deletion 7 whereas 1 had variant Philadelphia chromosome with other chromosomal abnormalities. Conclusion: It was evident that frequently occurring ACAs In our CML population were Variant Philadelphia chromosome and trisomy 8.