Uma Dangi

Characteristics of BCR-ABL kinase domain mutations in chronic myeloid leukemia from India: not just missense mutations but insertions and deletions are also associated with TKI resistance

Abstract We document the characteristics of BCR-ABL kinase domain mutations (KDM) in the largest study from India comprising of 385 patients and demonstrate that more than half (51.9%) of these patients have detectable abnormalities in the KD both in adult and in pediatric chronic myelogenous leukemia (CML). These comprise singly occurring missense mutations (25.5%), polyclonal/compound point mutations (4.9%), and insertions/deletions (29.6%). Missense mutations were most commonly seen in the imatinib-binding region followed by the P-loop. The commonest mutation in our dataset was T315I. Other common missense mutations were Y253H, M244V, and F317L. A high prevalence of BCR-ABL exon7 deletion (p.R362fs*) was also seen (25.5% of the entire cohort), whereas the 35bpintron-derived insertion/truncation mutation detected in 12 patients. In the pediatric age group, 58.8% of patients harbored missense mutations, polyclonal/compound mutations as well as insertions and deletions. We detected 11 novel mutations (seven missense mutations and four insertions/deletions).

Immunogenetics of chronic lymphocytic leukemia

Introduction: Cytogenetic aberrations as well as presence of IGVH mutations are the underlying reason for clinical heterogeneity in Chronic Lymphocytic Leukemia (CLL). The presence of IGVH mutations as well as the predominant gene usage shows geographical variations. However, there is no study from India addressing immunogenetics of CLL. In a first Indian study we document the immunogenetics of CLL in a large tertiary hospital. Methods: We analyzed IGVH mutation status, VH gene usage, cytogenetic abnormalities using FISH, immunophenotyping data and correlated them with standard clinical variables in 84 patients of CLL. Results: Advanced Rai stage (Stage 3/4) was seen in 45% of our patients, where as 13q deletion was the commonest clonal cytogenetic abnormality detected in 48.4% of the cases. IGVH unmutated cases (55.2%) showed higher proportion expressing CD38 and CD49d, a preferential usage for VH1 and VH3 families (55.2%), presentation at an advanced Rai stage (52.8%) as well as more frequent presence of p53 deletions. As compared to the IGVH mutated cases greater proportion of IGVH unmutated patients (70%) required treatment. However, there was no significant difference in the time to treatment between mutated and unmutated cases which can be attributed to relatively short median follow up of 10 months. Conclusion: To summarize, we have seen a higher proportion of IGVH unmutated patients in our cohort (55.2%). The commonly used VH genes in the Indian population are IGVH 2-5, IGVH 1-2 and IGVH 1-69. Longer clinical follow up and a larger cohort is necessary to confirm the prognostic value of IGVH mutation analysis in Indian Patients with CLL.

Prevalence and patterns of cytomegalovirus DNAemia in adult patients with acute lymphoblastic leukemia on chemotherapy.

Th e use of immunosuppressive agents such as high-dose steroids and anti-metabolites for prolonged periods can predispose patients with acute lymphoblastic leukemia (ALL) to develop cytomegalovirus (CMV) reactivation and disease, more so in countries with a background of high CMV seropositivity ( 90%) such as India [1]. Th ere are abundant data on the pattern and time course of CMV reactivation or primary infection in allogeneic stem cell transplant recipients, and well-defi ned strategies have been developed for prophylaxis, screening and pre-emptive treatment [2]. However, there is a remarkable paucity of data regarding CMV reactivation in ALL. Reactivation can lead to prolonged cytopenias, fever and other manifestations, which are often misdiagnosed and treated empirically with antimicrobials. Delays in diagnosis and therapy can compromise the dose intensity of chemotherapy, a key to achieving cure in ALL. Th ese facts underlie the importance of understanding the pattern, time of occurrence and clinical manifestations of CMV reactivation in ALL. To address this, we analyzed adult patients with ALL ( 14 years) who were treated with multi-agent chemotherapy (MCP-841) between July 2009 and July 2011 at Tata Memorial Hospital, India to understand the clinical features and pattern of CMV reactivation. CMV DNA was estimated in blood with real time quantitative polymerase chain reaction (PCR) (Roche; CMV DNA Quant Kit) in patients with clinical suspicion of CMV reactivation. Th is included the presence of persistent fever alone (fever of unknown origin), hepatomegaly and/or splenomegaly (relapse of ALL being ruled out), cytopenias (absolute neutrophil count less than 1000/mm 3 , platelet count less than 100 000/mm 3 , hemoglobin less than 8 g/dL) unexplained by the previous chemotherapy, respiratory symptoms (cough, coryza, infi ltrates on chest radiography), gastrointestinal symptoms (abdominal pain, anorexia, loose stools), unexplained liver dysfunction, signifi cant weight loss and skin rashes. Th e lowest detection limit with this method was 392 copies/mL. CMV serology was not done at baseline prior to ALL therapy given the high prevalence of CMV seropositivity in our population. We did not carry out active surveillance for CMV reactivation. Th ese patients were not given any antibacterial, antifungal or antiviral prophylaxis except co-trimoxazole. Patients with neutropenic and non-neutropenic fever were investigated with blood (bacterial/fungal), sputum and urine culture, computed tomography (CT) of the thorax, ultrasound scan of the abdomen and pelvis, bronchoscopy with culture of bronchoalveolar lavage in cases with pneumonia (if there was no response to antibacterial and antifungal therapy), and were included if no cause was found or if there was no response to therapy. Patients with deranged liver function tests were included if they had normal imaging, negative serology for hepatitis viruses and no history of hepatotoxic drug usage. None of the patients was subjected to biopsy to prove CMV disease due to low platelets and coagulopathy. Patients with raised CMV DNA copy numbers and clinical features suggestive of CMV infection (after excluding all other causes) were treated with ganciclovir. CMV DNA copy numbers were monitored until they became negative. Isolation of CMV DNA in whole blood along with clinical or laboratory features consistent with CMV infection, response to ganciclovir based therapy and temporal association of response to declining CMV DNA copy numbers was labeled as CMV reactivation. Case records were analyzed for demographics, chemotherapy details and the type of therapy prior to CMV reactivation, clinical features, laboratory parameters, viral load, antiviral therapy and response.

978PCORRELATION OF PHARMACOKINETICS OF 6MP AND ITS METABOLITES WITH HAEMATOLOGICAL TOXICITY: STEP TOWARDS DEVELOPMENT OF PHARMACOKINETICS BASED MODEL FOR OPTIMAL 6 MP DOSING IN ADULT ALL

ABSTRACT Aim: Dosing of 6MP has remained empirical in acute lymphoblastic leukemia (ALL) with leucocyte count and thiopurine methyl transferase (TPMT) being the main determinants. However, TPMT alone cannot predict for marked inter-individual differences. In our experience, adult ALL patients tolerate standard 6- MP doses poorly, resulting in frequent dose interruptions or subtherapeutic doses. To address this issue we are conducting a study to develop pharmacokinetic model (PK) for optimal 6 MP dosing. Herein we report the preliminary data for correlationof PK parameters of 6 MP and its metabolites with toxicity (dose interruptions and dose reduction). Methods: Adult ALL patients (18 years or more) who were receiving 6 MP during interim maintenance (IM)or maintenance (M) phase of MCP841 protocol were enrolled after informed consent. Plasma samples were collected on day 1 for measuring 6-mercaptopurine levels using HPLC. TPMT activity was calculated by measuring 6-mMP (6-methylmercaptopurine) and 6-methyl thioguanine nucleotides (6-mTGN) levels in erythrocytes on day 1.Levels for 6-mMP and 6-TGN were also measured in erythrocytes using HPLC on day 8 and day 22 of the IM and on day 1 of M phase upto 6 months post enrollment. Statistical analysis was performed with receiver operating characteristic curve to assess the discrimination potential of level of 6-mp, 6-mMP (day 8 and day 22), 6-TGN (day 8 and day 22), 6-mMP and 6-mTGN levels (day 1-IM) for toxicity. Mean AUC has been compared for the above parameters using Mann-Whitney test. Binomial logistic regression was performed to assess correlation of various covariates with the toxicity. Results: 29 patients (males-24, females-5) with median age of 28 years (range,16-50) were enrolled.14 patients experienced dose interruptions (median-1, range1-2)) and 10 required dose reduction due to grade 4 cytopenias. OnDay 22 levels, a concentration of 0.315 ug/ml for 6-mMP (AUC = 0.73, p = 0.2) and 0.17 ug/ml for 6-TGN (AUC = 0.92,p value =0.01)in erythrocytes could successfully discriminate between patients who suffered from toxicity. Day 8 levels were not discriminatory. Hemoglobin (p= 0.05) and TPMT activity (p = 0.08) were found to be significant covariates for toxicity. Conclusions: Preliminary data does suggest the value of PK variables on 6 MP toxicity. Disclosure: All authors have declared no conflicts of interest.