FLT3 Internal Tandem Duplication(ITD) mutation in Acute Myeloid Leukemia and its clincopathological correlation – Study from a tertiary care centre in Southern India Dr.Ranjith.A.R, Post Graduate, Department of Pathology JIPMER, Pondicherry   Dr. Rajesh.N.G Associate Professor Department of Pathology Dr. Debdatta Basu Professor Department of Pathology Dr. Smita Kayal Assistant Professor Department of Medical Oncology Dr. Suresh Chandra Pradhan Senior Professor and head Department of Pharmacology

Disclosure Dr. Ranjith.A.R has no financial relationships with commercial entities to disclose

Background FLT 3 – Hematopoiesis FLT 3 ITD – Association with worse prognosis in acute myeloid leukemia

Background.. STUDY POPULATION OUTCOME Kottaridis et al. (2001) 850, Multicentric 27% , lower remission Meshinchi et al. (2001) Paediatric AML , 12% , FLT3 ITD worse than others Thiede et al. (2006) 979 patients , UK 20.4% , FAB M5 most common , least M2 Zhong et al(2012) China 20.3% , FAB M2 – MC Ghosh et al *(2012) India 19.4% Dunna et al *(2010) 20.3% , ass with low disease survival

Aim To assess the presence of FLT3 ITD mutation in newly diagnosed and relapsed cases of AML To identify the association of FLT3 ITD mutation status with FAB subtypes and other clinical parameters To identify the association of FLT3 ITD mutation status and the treatment response in AML.

Methodology Prospective clinical study Study period – January 2014 – June 2015 Inclusion – All newly diagnosed and relapsed cases Exclusion – AML cases in remission, Subleukemic presentation Sample size – 74 cases

DNA Extraction Sample – Pre chemotherapy peripheral blood DNA extracted using QIamp- DNA mini kit(manufactured by Qiagen) The quantity of DNA ranged from 12.5 – 7399 ng/μL

Primers Forward primer: 5'-GCAATTTAGGTATGAAAGCCAGC-3' Reverse primer: 5'-CTTTCAGCATTTTGACGGCAACC-3‘ Blasted using NCBI database Product size of 329 bp – Wild type FLT3 and anything >331 bp - Mutated FLT3

PCR conditions PCR conditions used were for 30 cycles – 94°C/40s, 56.5°C/30s, 72°C/40s, with a final 72°C/7mins * PCR amplicons were separated using agarose gel electrophoresis – 3%, 2 hours * Murphy et al. Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction . J Mol Diagn. 2003 May; 5(2): 96–102

Results 44 females, 30 males Adults – 59, Paediatric – 15

Results Total cases – 74 Positive cases – 11 14.8 % of cases of AML has FLT3 ITD mutation Age gender and fab follwd by cyto

Results… FLT3 ITD cases – 11 7 women, 4 men 22-60 years, none in paediatric age FAB subtype FLT 3 Positive M1(21) 5 M2(18) 2 M3(6) M4(19)

Sequencing Data All eleven cases – Insertion sequence in Exon 14-15 Base pair insertion varied from 11-84 bp

Haematological Parameters Parameters (Mean) FLT3 ITD + ve FLT3 ITD - ve p Value (2-tailed) Hemoglobin 6.2 g/ dl 6.27g/ dl 0.382 WBC count 50.8 X 109/L 32 X 109/L 0.022 Platelet 57.3 X 109/L 53.5 X 109/L 0.589 % blasts in PS 60 % 57% 0.714 % Blasts in BM 85% 74% 0.039 With mean value

Cup shaped nucleoli FLT3 +ve - 5/11 FLT3 –ve - 24/ 63

Immunophenotype CD 34, HLA DR, CD 117 and CD14, CD13 – 47 cases CD7 – 40 cases (9 positive) CD34 +ve – 67% of FLT3 +ve (vs) 81% of FLT3 -ve (p value – 0.046) CD7 was not significant Cd 34 was seen less in flt 3 –ve cases

Cytogenetics – 37 cases Risk category Number of cases Favourable (inv 16, PML RARA) 4 Intermediate (Normal, t(9,11), trisomy 8) 27 (25 normal) Poor (Complex, t(11,19), del 17) 3 unknown Three FLT3 positive cases – inv 16, complex karyotype and normal karyotype

Treatment details Follow up ranged from 2- 493 days All 74 patients 56 received treatment 36 patients achieved remission 49 died FLT3 positive (n=11) 9 received treatment 5 achieved remission 10 died

Kaplan – Meier survival curve OS - FLT3 positive – 55.6 days OS - FLT3 negative – 185.1 days FLT3 ITD patients have lesser overall survival rate (55.6 days) compared to patients who don’t have the mutation (185.1 days) in this study

Conclusion FLT3 ITD mutation was seen in 14.8% cases of AML in our centre and was associated with higher WBC count and higher blast percentage in marrow at diagnosis and a lower overall survival