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Keywords:

  • FLT3 gene;
  • internal tandem duplications;
  • point mutations;
  • acute myeloid leukaemia;
  • cytogenetics

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. References

FLT3 gene mutations, either internal tandem duplication or point mutation type, are common in acute myeloid leukaemia (AML). We describe 21 AML cases with both types of gene mutations, so-called dual mutations, representing approximately 1% of all cases. Most newly diagnosed AML with FLT3 dual mutations had monocytic differentiation and a normal karyotype. Over the disease course, changes in FLT3 mutation status were seen in 89% of cases, and were associated with cytogenetic changes. We conclude that FLT3 dual mutations occur rarely in AML, and appear to be related to clonal evolution.

Two types of activating mutations of the fms-like tyrosine kinase-3 (FLT3) gene, internal tandem duplication (ITD) in the juxtamembrane domain and D835 point mutation (PM) in the activation loop of tyrosine kinase domain, have been identified in approximately 30% of acute myeloid leukaemia (AML) cases (Kiyoi et al, 1999; Abu-Duhier et al, 2000; Yamamoto et al, 2001; Schnittger et al, 2002; Thiede et al, 2002). These mutations are implicated in leukaemogenesis.

The simultaneous presence of both types of mutations, so-called FLT3 dual mutations, has been reported in 1–3% of AML patients (Karali et al, 2002; Thiede et al, 2002; Carnicer et al, 2004). To date, a systematic analysis of the features of AML carrying FLT3 dual mutations is absent in the literature. We searched our files for these cases, the study group.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. References

AML patients with greater than 5% bone marrow (BM) blasts were included between 1999 and 2004. Each newly diagnosed case was classified according to the World Health Organisation and the French–American–British classifications. Cytogenetic risk groups were stratified as high, intermediate or low (Lowenberg et al, 1999).

DNA was extracted from BM samples and the FLT3 gene was assessed for mutations of ITD and D835 PM type using polymerase chain reaction (PCR) assays (Yamamoto et al, 2001; Kiyoi & Naoe, 2002) followed by capillary electrophoresis. The forward primers were labelled with 6-carboxyfluorescein (FAM). For D835 PM analysis, PCR products were digested with EcoRV. In unmutated cases, digestion results in a 64 bp labelled fragment and a 48 bp unlabelled fragment, in contrast to a 112 bp fragment in mutated cases.

Results and discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. References

FLT3 gene mutations were identified in approximately 16% of AML patients (1600). This frequency was similar to that described in earlier studies. FLT3 dual mutations were detected in 21 patients (1·3%).

Several studies have described the clinicopathologic features of AML with either ITD or PM; these include marked leucocytosis, monocytic differentiation and normal karyotype or intermediate-risk cytogenetics (Schnittger et al, 2002; Thiede et al, 2002). The 14 newly diagnosed AML patients with FLT3 dual mutations in this study (Table I) frequently had marked leucocytosis (57%; >50 × 109/l), normal karyotype (69%) and monocytic differentiation (57%). FLT3 is persistently expressed during normal monocyte differentiation, which may explain why FLT3 gene mutations are preferentially associated with AML with monocytic differentiation (Gabbianelli et al, 1995).

Table I.  Summary of patients with FLT3 gene dual mutations.
CasesAge (years)/sexDiagnosis (WHO/FAB)CytogeneticsWBC*/BM blast (%)ITD/D835 PM
  1. AML, acute myeloid leukaemia; BM, bone marrow; CMML, chronic myelomonocytic leukaemia; nd; not done; ITD, internal tandem duplication of FLT3 gene; MM, myelomonocytic; RAEB, refractory anaemia with excess blasts; RARS, refractory anaemia with ringed sideroblasts; T-MDS, therapy-related myelodysplastic syndrome; WBC, white blood cell count; WHO/FAB, World Health Organisation/French–American–British classification.

  2. *Unit × 109/l.

  3. †The inv(9) alteration is a chromosomal polymorphism found in the general population with no known clinical significance.

  4. ‡Two differently sized ITD mutants were present initially. One mutant was lost in the subsequent (third and fifth) specimens.

  5. §Three differently sized ITD mutants were present initially. Two mutants were lost in the subsequent specimen.

  6. ¶Three neoplastic clones: 46, XY, +del(6), −12, −12, del(14), add(15), +18/48, XY, add(1), +8, +13/46, XY, add(6), del(9).

Single sample
 129/FAML-MM/M446, XX65/67+/+
 240/FAML-MM/M446, XX75/75+/+
 355/FAML-MM/M446, XX228/79+/+
 462/MCMML to AML/M446, XY, inv(9)†43/74+/+
 531/FAML with inv(16)/M446, XX, inv(16)71/80+/+
 666/MRefractory AML45, XY, t(3;3), −713/7+/+
 774/MRelapsed AML/M446, XY, t(11;22)60/82+/+
 863/MRelapsed AML46, XY, t(13;16)19/82+/+
 982/MAML from RARS/M446, XY27/66+/+
 1062/MAML with inv(16)/M447, XY, del(7), +8, inv(16), −17, −19, +2mar19/76+/+
 1177/MAML with no maturation/M146, XY84/91+/+
 1255/FRelapsed persistent AML46, XX, t(11;19)13/95+/+
Multiple samples
Gain of point mutation
 1359/MRelapsed AML46, XY, t(7;12); 46, XY, del(7)2·5/10+/−
Persistent AML46, XY, t(7;12); 45, XY, −72·5/27+/+
Persistent AML46, XY, t(7;12)2·0/35+/+
 1446/MAML from T-MDS/M246, XY121/68+‡/−
Relapsed AMLnd1·0/35+/+
Persistent AMLnd35/78+/+
Persistent AMLcomplex, 3 neoplastic clones¶31/59+/+
Persistent AMLnd14/78+/+
 1524/MRelapsed AMLnd0·3/10+/−
Persistent AML43–46, XY, del(2)1·8/44+/+
 1684/MAML-MM/M4nd130/84+§/−
Persistent AML47, XY, +42·8/83+/+
 1728/FAML with maturation/M246, XX4·2/32+/−
Minimal diseasen.d.0·7/3−/+
Relapsed AML47, XX, +81·7/3·5+/+
Gain of dual mutations
 1848/MAML without maturation/M146, XY1·1/73
Persistent AML46, XY5·6/16
Relapsed AML46, XY, t(5;8)1·5/78+/+
Loss of point mutation
 1965/FAML from T-MDS/M146, XX, del(14)0·9/71+/+
Minimal disease46, XX4·7/3+/−
 2070/FRAEB to AML/M246, XX, inv(14)9·9/30+/+
Persistent AML46, XX, inv(14)7·8/43+/−
Persistent AMLnd7·9/38+/−
Persistent AMLnd5·2/39+/−
Persistent dual mutations
 2120/FRelapsed AML/M446, XX, del(9); 47, XX, +mar2·1/77+/+
Persistent AML46, XX, del(9); 45, XX, add(4), del(6), del(9), −70·5/94+/+

Fifteen patients had FLT3 dual mutations in the initial specimen analysed. There was considerable heterogeneity in the levels of mutant. Most patients had low levels of the mutant, indicating that mutations may be present in only a minority of leukaemic cells. Two patients had high levels of mutations (either ITD or PM), which would be more indicative of biallelic mutation or loss of a normal allele.

Variation of FLT3 mutations over the disease course

Nine patients had multiple samples analysed over the disease course (Table I). In the interval from initial analysis to detection of change in mutational status, five patients were treated with idarubicin and cytarabine, two with fludarabine and cytarabine, one with clofarabine and one with hydroxyurea. Two patients (cases 14 and 21), previously treated with fludarabine and cytarabine, and idarubicin and cytarabine respectively failed and received the FLT3 kinase inhibitor, CEP-701.

FLT3 gene mutations were unstable over the disease course (Fig 1). Three patients acquired PM, two of which initially had multiple ITD and then acquired PM while retaining 1 ITD. One patient acquired ITD and PM. Two patients lost PM. One patient had no change. Thus, changes in PM were common (8/9), whereas changes in ITD were infrequent (3/9). This variation led to six patients acquiring an additional mutation to reach dual mutational status.

image

Figure 1. AML with internal tandem duplication (ITD) and D835 point mutation (PM) of FLT3 gene (case 20). Initial sample was obtained on 17 October 2002, and the second post-therapy sample was obtained on 6 January 2003. ITD persisted (indicated by arrow) and PM was lost (no evidence of 112 bp band, indicated by arrowhead).

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Changes in FLT3 mutational status correlate with cytogenetic changes

Nine patients had multiple samples analysed, seven of these patients had cytogenetic analysis (Table I). In three relapsed cases (cases 14, 17 and 18), a change of mutation was accompanied by cytogenetic changes. In three patients with persistent AML, one patient who acquired PM also showed cytogenetic changes (case 13), one had no change in FLT3 status despite cytogenetic changes (case 21), and one patient had loss of PM but no cytogenetic change (case 20). One patient with minimal disease (case 19) lost PM with reversion to normal karyotype. Overall, five of seven patients who had changes in FLT3 mutation status also had cytogenetic changes. Furthermore, FLT3 dual mutations were detected in four of five samples with multiple cytogenetic subclones. Thus, changes in FLT3 mutation status appear to correlate with cytogenetic changes. As AML is known to be genetically unstable, the gain or loss of FLT3 gene mutations probably reflects clonal evolution over the disease course.

During this study, the FLT3 gene was analysed in approximately 400 patients with myelodysplastic syndrome (MDS). Interestingly, none of these patients had FLT3 dual mutations. The finding of dual mutations exclusively in AML supports the notion that progression of MDS to AML is associated with step-wise acquisition of genetic aberrations (Shih et al, 2004).

In summary, this is the largest study of AML with FLT3 dual mutations to date. This phenomenon is uncommon and appears to be a manifestation of clonal evolution. FLT3 mutations are unstable over the disease course, with frequent changes in PM, and infrequent changes in ITD.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. References
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