Introduction
World Health Organisation (WHO) classification (5th edition) for Acute myeloid leukemia (AML) have AML with mutations in NPM1, CEBPA and RUNX1 and AML with cytogenetic abnormalities (eg. t (8;21), inv (16)) as separate entities.1 This stratification was done as each of these have their unique genetic profile with a varied prognosis. 2 C-kit and FLT3 mutations in AML have no diagnostic importance; however, they have prognostic significance. FLT3 mutation including both ITD and TKD has an overall bad prognosis across various cytogenetic abnormalities.1, 3, 4, 5, 6, 7, 8 Even though C-kit mutations has no stand-alone prognostic significance in inv(16), it has a worse prognosis in t(8;21). Multiple studies show contradictory results for C-kit mutation in relation to prognosis. While most of the studies concentrate on C- kit mutation status in core-binding factor (CBF) AML which includes AML with t(8;21) and AML with inv(16), it was not elucidated in AML patients with other cytogenetic abnormality and in AML with normal karyotype. Also, literature is lacking in terms of C-kit and FLT3 mutation status in Indian patients and their prognosis. 4, 9 We carried out our study to assess the correlation between French American British (FAB) AML classification and specific cytogenetic abnormalities with C-kit and FLT3 mutation in AML. Also, we wanted to evaluate the prognosis and survival of AML patients with respect to cytogenetic abnormalities and C-kit and FLT3 mutation status.
Materials and Methods
Retrospectively all AML cases in which C-kit and FLT3 mutation status was assessed were retrieved from hospital case records. C-kit D816V (Exon 17) mutation status was assessed by Real-time PCR; For FLT3 mutation, both Internal tandem duplication (ITD) and D835V mutation status were assessed using PCR and subsequent Gel electrophoresis (Figure 1, Figure 2).
The data regarding morphological with immunophenotypic diagnosis, conventional karyotyping, FISH for translocation 8;21 (t (8;21)) and inversion16 / translocation16;16 (t (16;16)) were also retrieved in all these cases along with follow-up from hospital records (Figure 2).
Statistical analysis
SPSS software (version 20) was used for statistical analysis; Chi-square test, ANOVA test, and t-test were used wherever necessary depending on the variables. p-value of <0.05 was taken as significant
Results
A total of 75 AML cases on which C-kit and FLT3 mutation was done were included in the study. The median age of the patients was 31 years with an age range of 2-64 years. Male-female (M:F) Ratio was 1.21:1 (41:34).
AML- M2 (34/75) was the commonest in the French American British classification (FAB) of AML followed by M1 (20/75) and M4 (12/75). There were eight cases of M5 and a single case of M0.
Among the Chromosomal abnormalities, excluding the single case in which karyotyping and fluorescent in-situ hybridization (FISH) was not done, t(8;21) was the commonest cytogenetic abnormality(18/74) with additional abnormalities of t(8;12;21) in one case and deletion of sex chromosome in 9 cases. Hyperdiploidy and trisomy 8 were the next common (both - 4/74) followed by inv(16) (3/74). Inv(16) had associated trisomy 22 in 2 cases. Other than this, two cases had monosomy 7, and one case each had t(5;11) and complex karyotype. Predominant cases were of normal karyotype (NK) (41/74) (Table 1).
Follow-up data was available for 43(57.3%) cases while 32 cases (42.7%) cases were lost to follow-up. Among these, 19 cases expired while on follow-up (26.3%) and 4 cases relapsed (5.3%) on follow-up. The Median follow-up period was 2.5 months.
Among the 18 cases of t(8;21), AML-M2 was the commonest(14/18), while for inv(16), there were 2 cases of M4 and a single case of M5a.
Two cases each of hyperdiploidy and trisomy 8 expired and none of these 8 cases attained remission post-induction therapy.
Both the cases with monosomy 7 were of AML- M2. One among them expired at 10-month follow-up and one other case which attained remission post induction was lost to follow-up. A single case of complex translocation had AML- M1 on morphology. This case did not attain remission post-induction therapy and expired at 10 months follow-up.
Ckit-D816V mutation
There were eight cases that were positive for C-kit – D816V mutation. AML- M2 was the commonest morphology identified (3/8) followed by M4(2/8). C-kit D816V mutation was associated equally with t(8;21) and NK (3 cases each). Two other cases had inv(16) with trisomy 22 as an additional abnormality in both cases. 62.5% of AML with C-kit mutation had associated cytogenetic abnormalities, while 37.5% of cases were associated with normal karyotype.
Mean WBC count was in 30000/mm3 in C-kit positive cases while it was 45000/mm3 in negative cases. Mean Blast percentage in positive cases were 61%, while in negative cases was 56%. Both of these findings were not statistically significant. Remission post-induction therapy was attained in 37.5% (3/8) of C-kit positive cases which was not statistically significant from the overall remission rate post-induction (29.3%) (22/75 cases). While 50% of cases (4/8) did not attain remission in C-kit positive cases, 57.3% (43/75 cases) did not attain remission overall.
A single case of AML with C-kit mutation had relapse which had associated inv(16) with trisomy 22. Two cases of C-kit mutation are still on follow-up, one each with associated inv16+tri(22) and normal karyotype. No adverse events were noted in both these cases even at 3 years follow-up (Table 2).
FLT3 mutation
There were a total of 12 cases with FLT3 mutation, among which FLT3-TKD(D835) mutation was seen in 3 cases and FLT3-ITD mutation was seen in 9 cases. Mean WBC count and Blast percentage were not statistically significant. No specific association was seen with morphological FAB classification. AML-M1(4/9) is the commonest in cases with FLT3-ITD mutation and AML-M2(2/3) is the commonest in cases with FLT3-D835 mutation. The commonest karyotyping finding in these cases was NK (10/12) with one case each of hyperdiploidy and inv(16) with trisomy(22). Excluding 4 cases that were lost to follow-up, remission post-induction therapy was not attained in 80% (4/5) of FLT-3 ITD positive cases which was statistically significant (p-0.03) compared to the overall remission rate post-induction (29.3%) (22/75 cases). The one case which attained remission also relapsed during follow-up. Two of these cases expired (one each with hyperdiploidy and normal karyotype).
While in FLT-TKD mutated cases, 33.3% cases (1/3) did not attain remission in comparing 57.3% (43/75 cases) cases overall. This was not statistically significant. Among these three cases, one case relapsed on follow-up, while one case expired during induction chemotherapy; both of these cases had a normal karyotype (Table 3).
There was a single case with both C-Kit D816V mutation and FLT3 D835 mutation. Morphologically it was AML-M4. It had associated inv 16 with trisomy 22. This case attained complete remission post induction and the patient is event free at 3-year follow-up.
Figure 2
A: Cells show positivity for t(8;21) – FFOG (Dual colour dual fusion probe); B: Cells show positivity for inv (16)-FOG (Break apart probe for CBFB); C: Gel electrophoresis post restrictive endonuclease digestion showing one sample with FLT3 D835 haterozygous mutation; D: Gel electrophoresis showing one sample with FLT3 ITD mutation.
Table 1
Correlation between FAB classification and karyotyping
Table 2
Clinicopathological characteristics of C-kit-positive cases
Table 3
Clinicopathological characteristics of FLT3 positive cases
Discussion
Correlating with the general trend, AML occurs in adults (median age-31 years) with a wide age range (2-64 years). 10, 11, 12 AML- M2 was the commonest followed by AML M1 and M4 with a slight male preponderance (M:F ratio: 1.21:1). t(8;21) was the commonest cytogenetic abnormality accounting for 24%(18/75) of cases which was relatively higher than in published literature(5%).13, 14 Hyperdiploidy and trisomy 8 are both the second most common cytogenetic abnormalities with 4 cases each (5.3%) followed by inv16 (3/75) (4%). 15, 16
Coming to additional cytogenetic abnormalities, t(8;21) was commonly associated with loss of sex chromosomes(50%) and inv(16) had additional trisomy 22 in 66% cases. Both of these findings were concordant with the literature. Both hyperdiploidy and isolated Trisomy 8 had a bad prognosis showing a low complete remission rate and poor survival in our study similar to other studies. 15, 16
According to the literature, both C-kit and FLT3 mutation are associated with high tumor burden with more blast percentage and WBC count; However, this was not observed in our study. In our study, 66.6% (2/3) of FLT3 D835 mutation had either relapse or death, highlighting its worse prognosis across varying karyotypes. In addition, FLT3 ITD mutated cases showed a significantly low complete remission rate; Both findings are in concordance with the literature. 2, 3, 4, 5, 6
The single case of coexistent C-kit D816V mutation and FLT3-D835 mutation in AML with inv(16) has not been previously reported in the literature as far as our knowledge. Further studies are required to elucidate their incidence and prognostic significance which appears to be good in the single case found in our study.
Although C kit mutations are studied in detail in CBF AML in literature, 3, 6, 15 our study has highlighted the presence of C kit mutated AML cases in normal karyotype; Further studies are required to elucidate their prognosis and survival.
C-kit mutations do not appear to have reduced complete remission rate or poor survival in our study; This could be because of the small sample size; However, a study of large scale including more sample size is required to elucidate the exact survival data across different karyotypes.
Both FLT3 and C-kit mutations provide the potential for exploration of targeted RTK immunotherapy in AML cases in the future. 17, 18, 19, 20
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