Khalid Tobal

Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission

Summary .The pericentric inversion of chromosome 16 [inv(16)(pl3q22)] and t(16;16)(pl3;q22) are chromosomal rearrangements frequently associated with AML FAB type M4Eo resulting in the production of a fusion gene CBFB/ MYH11. We studied 17 patients with a chromosome 16 abnormality (eight M4Eo, two Ml, one M2, three M4 without abnormal eosinophils, three MDS) for the presence of CBFB/MYH11 transcripts using an RT‐PCR technique. 10 AML patients with inv(16) tested RT‐PCR positive (eight at presentation, one in remission, one in remission and relapse). Three of these patients were originally reported by cytogenetic analysis to have del(16q22) but the positive RT‐PCR results prompted a cytogenetic re‐examination, resulting in the correction of the reports to inv(16). We show that although inv(16) is closely associated with AML M4Eo, it can also be detected in cases of AML M4 without abnormal eosinophils. Three cases of MDS with inv(16) were also RT‐PCR positive. Four patients with other chromosome 16 abnormalities were RT‐PCR negative. Four AML patients with inv(16) were studied in remission. All were RT‐PCR positive, including one patient in remission for 108 months and one 22 months post allogeneic bone marrow transplant. In the latter two remission patients, RT‐PCR evaluation was positive in bone marrow (BM) but not in peripheral blood, suggesting that BM may be the more informative. We conclude that this technique is valuable in the accurate molecular classification of AML, particularly as treatment options may be influenced by such information. Though RT‐PCR is highly sensitive in detecting CBFB/MYH11 fusion transcripts during remission, monitoring of minimal residual disease in patients with inv(16) remains to be established.

Prognostic significance of quantitative analysis of WT1 gene transcripts by competitive reverse transcription polymerase chain reaction in acute leukaemia

Summary. We have developed a sensitive, competitive, nested reverse transcription polymerase chain reaction (RT‐PCR) titration assay that quantifies the number of Wilms tumour (WT1) gene transcripts in bone marrow (BM) and peripheral blood (PB), coupled with a competitive RT‐PCR protocol for the ABL gene as control. We studied BM/PB samples from 107 acute myeloid leukaemia (AML) patients and 22 acute lymphoblastic leukaemia (ALL) patients at presentation and detected the WT1 gene in > 90% of patients by a qualitative assay. Quantitative analysis of WT1 transcript at presentation in 66 patients (52 AML, 14 ALL) correlated significantly with remission rate, disease‐free survival (DFS) and overall survival (OS) (P = 0·003). WT1 levels were normalized to 105ABL transcripts. Within good and standard cytogenetic risk groups, high WT1 levels correlated with poorer outcome. Serial quantification was performed in 35 patients (28 AML, seven ALL); those with less than 103 copies of WT1 after induction and second consolidation chemotherapy had significantly better DFS and OS. Fourteen patients have relapsed with a median complete remission duration of 12 (range 4–49) months. We detected a rise in WT1 levels in nine out of 14 patients, 2–4 months before the onset of haematological relapse, whereas in the remaining five patients, WT1 levels remained persistently high during the disease course. WT1 levels were lower in PB than in BM, but mirrored changes in the BM samples and were equally informative. We suggest that WT1 is a useful molecular target to monitor minimal residual disease in acute leukaemia, especially in cases without a specific fusion gene.

Persistence of RARa‐PML fusion mRNA detected by reverse transcriptase polymerase chain reaction in patients in long‐term remission of acute promyelocytic leukaemia

Summary. Acute promyelocytic leukaemia (APL) is characterized by t(15;17), which results in the formation of two chimaeric genes, PML‐RARa and RARa‐PML. PML‐RARa transcripts have been detected in all cases of APL whilst those of RARQ‐PML have been detected in only about 67% of cases. We have used reverse transcriptase polymerase chain reaction (RT‐PCR) to detect both fusion transcripts serially in 18 patients in remission of APL after chemotherapy and bone marrow transplantation. All patients were negative for PML‐RARa, whereas in six patients (remission 3‐9 years) RARa‐PML was consistently detected. Only one patient at remission showed the 5’breakpoint RARa‐PML, with the rest showing the 3’breakpoint 144 bp RARa‐PML. The level of sensitivity for detecting RARa‐PML was some 10‐fold higher than that for PML‐RARa.

High level of microsatellite instability but not hypermethylation of mismatch repair genes in therapy‐related and secondary acute myeloid leukaemia and myelodysplastic syndrome

Summary. Microsatellite instability (MSI) is associated with defects in the DNA mismatch repair (MMR) system, such as mutation or epigenetic silencing of the genes by promoter hypermethylation. We investigated the presence of MSI and promoter hypermethylation of hMLH1 and hMSH2 genes in 82 patients (68 acute myeloid leukaemia, AML; 14 myelodysplastic syndromes, MDS). Twelve separate microsatellite loci, including three mononucleotide repeat markers, were used. Mutator phenotype (RER+) was detected in 20 AML (29·4%) and 3 MDS (21·4%) patients. RER+ rate was much higher in the therapy‐related and secondary cases compared with the de novo cases. Three out of 7 (42·9%) secondary (s‐AML) and 8 out of 17 (47·1%) therapy‐related (t‐AML) showed RER+ in comparison with 9 out of 44 (20·5%) de novo cases. Similar rates were detected in MDS patients (2/2 therapy‐related and 1/12 de novo). The promoter hypermethylation was found in three hMLH1 (3·7%) and two hMSH2 (2·4%) genes. All these five patients had AML and were older than 60 years of age. Two of them had s‐AML and one had t‐AML. RER+ was detected in three of these five patients. Our data suggest that genetic instability is associated with AML and MDS, especially t‐AML and s‐AML. In addition, our results indicate that the hMSH2 and hMLH1 promoter hypermethylation is not a common event in these malignancies, but may play a role in the development of AML in elderly patients.

Detection of t(8;21) by reverse transcriptase polymerase chain reaction in patients in remission of acute myeloid leukaemia type M2 after chemotherapy or bone marrow transplantation

Seven patients with acute myeloid leukaemia (AML) type M2 and t(8;21), treated with intensive chemotherapy followed in two cases by allogeneic or autologous bone marrow transplant (BMT), were tested for the presence of transcripts of the characteristic chimaeric gene AML1/ETO by reverse transcriptase polymerase chain reaction (RT-PCR) at serial intervals during remission. Six patients, including both BMT patients, demonstrated persistence of t(8;21), one being consistently negative in peripheral blood but bone marrow positive. Bone marrow may, therefore, be more reliable than peripheral blood for detecting residual t(8;21) cells. Our results show persistence of t(8;21) cells in AML-M2 patients following chemotherapy or BMT, some of whom were in long-term remission.

MOLECULAR MONITORING OF MINIMAL RESIDUAL DISEASE IN ACUTE MYELOBLASTIC LEUKEMIA WITH T(8;21) BY RT-PCR

The t(8;21) is one of the most common translocations in acute myeloid leukaemia (AML) occurring in approximately 20% of adult and 40% of paediatric AML-M2. This translocation fuses the AML1 gene on chromosome 21q to the MTG8 (ETO) gene on chromosome 8q to produce the fusion gene AML1-MTG8. Transcripts for the AML1-MTG8 fusion gene have been detected in the majority of patients in remission by qualitative RT-PCR methods. Thus for such patients these methods are unsuitable for monitoring minimal residual disease (MRD). Furthermore, the diverse form of transcripts for this fusion gene was found in patients at different phases of their disease, which rules out the usefulness of the expression of any particular set of transcripts as a marker for monitoring MRD in those patients. On the other hand a quantitative RT-PCR method we developed, was able to assess the effectiveness of treatment and predict relapse up to four months before the onset of haematological relapse. This method should distinguish patients in stable remission from those at high risk of relapse and therefore identify patients who would require additional or new treatment such as BMT.

Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21)

Patients in long‐term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT‐PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi‐solid culture and picked individual CFU‐GM and BFU‐E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT‐PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU‐GM and between two and 21 BFU‐E colonies were analysed from each patient sample; 0–23% CFU‐GM and 0–17% BFU‐E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.