1. 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

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

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

4. 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

5. 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.

6. 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

7. 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

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

9. 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 May; 5(2): 96–102

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

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

12. 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)

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

15. 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

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

17. 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

18. 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

19. 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

20. Kaplan – Meier survival curve
OS - FLT3 positive – 55.6 days
OS - FLT3 negative – 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

21. 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