Molecular MRD Research Articles

Highly Sensitive Monitoring of Minimal Residual Disease in FLT3-itd-Positive Acute Myeloid Leukemia Patients with a Mutation Specific Quantitative-PCR Method,

Abstract 3530Minimal residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) can predict relapse clearly in advance and therefore allows early therapeutic intervention. Moreover, recent studies have highlighted the significance of personalized treatment on the basis of MRD status for improving outcome in AML.The FLT3 internal tandem duplication (FLT3 -ITD) is one of the most frequent mutations in AML patients occurring in 15–35% of all cases and approximately in 20–30% of cytogenetic normal (CN) AML. Clinically, FLT3 -ITDs have been strongly associated with high leukocytosis, high blast counts, normal karyotype and, most importantly, poor clinical outcome. However, due to the high sequence variability of individual FLT3 -ITD commonly a universal PCR approach is applied which has a low sensitivity (approx. 1: 5×102). For this reasons FLT3 -ITD is regarded as not suitable for longitudinal MRD measurements.The aim of this study was to develop a novel cDNA-based, highly sensitive quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay for the detection of the FLT3 -ITD mutation level. Furthermore, the prognostic value of FLT3 -ITD-based MRD detection in AML patients was compared with other genetic markers like NPM1 mutations, MLL -partial tandem duplication (MLL -PTD) and the expression of PML /RARα fusion gene or WT1 over-expression.To design patient-specific qRT-PCR, FLT3 -ITDs were amplified with universal primers, purified corresponding bands from agarose gel and directly sequenced. On the basis of the FLT3 -ITD, individual mutation-specific primers were designed. The expression of FLT3 -ITD was determined using complementary DNA samples at different points in time diagnosis and subsequent treatment. For estimation of the sensitivity and specificity of this approach we used a dilution of FLT3 -ITD cDNA in a pool of FLT3 -unmutated cDNA.From a total of 394 newly diagnosed AML patients 55 (14%) were FLT3 /ITD positive. Retrospectively we analyzed ITD mutation of FLT3 in 39 available cases. The length of ITD ranged from 3 to 144 base pairs (median 46). Nine patients had extra insertions of 2–38 base pairs between two repeats. For the FLT3 -ITD quantification we developed patient-specific qRT-PCR for 29 individuals with mutation-specific forward primers and studied 83 peripheral blood and 61 bone marrow samples. Three cases with WT1, fifteen with NPM1, three with MLL -PTD and five with PML /RARα fusion genes expression levels were compared with FLT3 -ITD expression levels. 26 of 29 assays (90%) were highly specific (1:104 − 1:105) and yielded similar results when compared to other high sensitive assays for molecular markers like NPM1 or PML /RARα. In three cases (10%) a co-amplification of the wild-type could not be avoided resulting in lower sensitivity (1:103). We could show that FLT3 -ITD positivity reliably predicted relapse up to 10 months in advance. 92% patients, who achieved FLT3 -ITD negativity with our assay, did not relapse. Furthermore we compared paired PB and BM samples at diagnosis and after induction therapy in 5 cases. The difference in FLT3 -ITD expression were not statistically significant (p=0.8) which is in line with recent studies.We conclude that highly sensitive detection of individual FLT3 -ITD posses equal prognostic power in AML like established molecular MRD markers. Using this approach MRD guided treatment decisions appear to be justified and should be incorporated in future studies. Disclosures:Hallek:Hoffmann-la Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kreuzer:Alexion: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Shire: Honoraria, Research Funding.

Treatment of Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) with Concurrent Chemotherapy and Imatinib Mesylate.

Imatinib mesylate as a single agent has modest activity in refractory/relapsed Ph-positive ALL. Use of concurrent chemotherapy and imatinib mesylate in newly diagnosed Ph-positive ALL was explored. Nineteen patients (pts) with de novo Ph-positive ALL were included in a prospective trial. Induction chemotherapy consisted of imatinib mesylate (400 mg/d, p.o.) and VCR (1.5mg/m2/wk), DNR (60 mg/m2/wk) and PDN (60mg/m2/d) for 4 weeks if adequate bone marrow response (<5% blast cells) at day +14 was observed. If not mitoxantrone (12 mg/m2, d15, 16, 17) and HD-ARAC (1,000mg/m2/12h d 18, 19) were administered. Consolidation chemotherapy (C1) included imatinib mesylate and MP, HD-MTX (1.5 g/m2), VM-26 and ARA-C. Pts with a HLA-identical family or MUD donor were submitted to SCT. IF no MUD donor was found a second consolidation cycle (C2) with imatinib mesylate, VCR, DNR, DXM and CPM was administered. Pts without family donor or with no MUD donor after 6 mo. of active search were submitted to autologous SCT. After SCT imatinib mesylate was administered from the sustained hematological recovery to the eventual relapse or at least up to 1 yr. of continuous molecular remission. Sequential MRD study was performed by cytofluorometry and quantitative RT-PCR. Up to July 2004, 19 pts (10 males, mean [SD] age 43[12] yr., range 17–62, p190bcr-abl in 75% of the cases) were included. One pt is still on treatment, 1 died in induction because of infection, 1 was refractory and the remaining 16 (89%) attained CR. Slow bone marrow response was observed in 4 out of 14 evaluable pts. Three patients have relapsed before SCT, 8 are on consolidation therapy and SCT was performed in 5 (3 from family donor and 2 MUD). 1 pt died after SCT and no relapses after SCT have been observed to date (range 1–6 months from SCT). With a median follow-up of 5 (0.2–14) mo., 3 pts have died, 2 are alive in second CR and 13 are in first CR, with a median DFS of 8 mo. and an OS probability of 66±30%. Levels of BCR-ABL/GUS x100 less than 0.05 were observed in 6/12 (50%) pts at the end of induction therapy. There was a further 1–2 log median reduction of BCR/ABL transcripts at the end of C1, (BCR-ABL/GUS x100 less than 0.05 in 5/6 cases). No additional reduction of transcripts was observed between C1 and SCT. A good correlation between molecular and cytofluorometric MRD data was observed. Use of concurrent chemotherapy and imatinib mesylate in newly diagnosed Ph-positive ALL pts is feasible and the preliminary data are promising in terms of high rate of clinical and molecular CR. Continued accrual and longer follow-up of the current cohort is needed.