UZ Leuven

Fusion gene and Ig/TCR gene rearrangements are complementary but infrequent targets for PCRbased detection of minimal residual disease in acute myeloid leukaemia. Boeckx N, Willemse M, Szscepanski T, Langerak TW, van der Velden VHJ, Vandekerckhove P, van Dongen JJM. Leukemia 2002;16:368-375. Abstract: PCR-based monitoring of minimal residual disease (MRD) in acute leukemias can be achieved via detection of fusion gene transcripts of chromosome aberrations or detection of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements. We wished to assess whether both PCR targets are complementary in acute myeloid leukemia (AML). We investigated 105 consecutive AML cases for the presence of fusion gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR): AML1-ETO associated with t(8;21), CBFB-MYH11 with inv(16), PML-RARA with t(15;17), BCR-ABL with t(9;22), and MLL-AF4 with t(4;11). In 17 out of 105 AML cases (16%), fusion gene transcripts were found. Ninety-five of these AML patients (13 with fusion gene transcripts) were also investigated for the presence of IGH, IGK, TCRG and TCRD rearrangements by Southern blot and/or PCR heteroduplex analysis and sequencing. In nine out of 95 patients (9.5%), such rearrangements were found. Combined data revealed that only one patient with a fusion gene transcript had a coexistent Ig/TCR rearrangement. The nine AML patients with Ig/TCR rearrangements, as well as five additional AML patients from a previous study were investigated in more detail, revealing that Ig/TCR rearrangements in AML are immature and unusual. The presence of Ig/TCR rearrangements in AML did not correlate with RAG gene expression levels as determined by real-time quantitative PCR. In conclusion, fusion gene transcripts and Ig/TCR rearrangements are infrequent, but complementary MRD-PCR targets in AML.

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program. Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, Barbany G, Cazzaniga G, Cayuela JM, Cave H, Pane F, Aerts JL, De Micheli D, Thirion X, Pradel V, Gonzalez M, Viehmann S, Malec M, Saglio G, van Dongen JJM.  Leukemia 2003;17: 1-40. Abstract: Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytic leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Detection of micrometastatic disease and monitoring of perioperative tumor cell dissemination in primary operable breast cancer patients using realtime quantitative reverse transcription-PCR. Ismail MS, Wynendaele W, Aerts JL, Paridaens R, Gaafar R, Shakankiry N, Khaled HM, Christiaens MR, Wildiers H, Omar S, Vandekerckhove P, Van Oosterom AT. Clin Cancer Res 2004;10:196-201. Abstract: PURPOSE: We previously found a statistically significant number of cytokeratin 19 (CK19)+ cells in peripheral blood (PB) of stage IV breast cancer (BC) patients compared with those of healthy volunteers, using a quantitative real-time reverse transcription- PCR. We aimed to apply the technique on bone marrow (BM) of primary operable BC patients. Preand postoperative PB samples of these patients were further analyzed to investigate possible shedding of CK19+ cells during the operation. EXPERIMENTAL DESIGN: In 54 primary operable BC patients, we analyzed 50 BM samples taken preoperatively and 297 PB samples. PB samples were collected before surgery; immediately after surgery; on the first, second, and fifth day postoperatively; and one month postoperatively. RESULTS: In BM of controls and BC patients, we detected a median of 28 and 568 CK19+ cells/5 x 10(6) leukocytes, respectively (P < 0.001). In preoperative blood (B-1) samples, we measured a median of 109 CK19+ cells. Using the upper limit of 95% confidence interval of controls as cutoff, 74% and 52% of BM and (B-1), respectively were considered CK19+. There was no significant correlation between CK19+ cells in BM and (B-1) and classical prognostic factors. We found no significant difference between blood samples at different time points with respect to the average CK19+ cells. CONCLUSIONS: In primary BC patients, we detected high numbers of CK19+ cells in BM and PB (B-1) samples compared with controls. However, no significant correlation between the presence of CK19+ cells in BM and PB and classical prognostic factors was found. We detected no statistically significant influence of surgical manipulation on CK19+ cells.

Detection of minimal residual disease in acute leukemia. van der Velden VHJ, Boeckx N, van Wering ER, van Dongen JJM. Review. J Biol Regul Homeost Agents 2004;18:146-154. Abstract: Detection of minimal residual disease (MRD) has prognostic value in acute lymphoblastic leukemia and acute myeloid leukemia. Particularly the evaluation of early treatment response has high prognostic value, because this allows identification of true low-risk and high-risk patients, who may profit from treatment reduction or treatment intensification, respectively. Consequently, monitoring of MRD is now being incorporated in many clinical protocols. Analysis of MRD in acute leukemia is currently mainly performed using flowcytometric immunophenotyping, real-time quantitative (RQ-)PCR analysis of fusion gene transcripts, and RQ-PCR analysis of rearranged immunoglobulin and/or T-cell receptor genes. These three techniques differ in their applicability and sensitivity and it should be noted that MRD results obtained by one method cannot yet easily be compared with MRD results obtained by another method. Also between laboratories applying the same method, significant variations in MRD results can be present. Consequently, multicenter clinical studies with MRD-based treatment intervention need standardization of MRD techniques and quality control of MRD results. 

High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin (Mylotarg®) treatment in acute myeloid leukemia patients. van der Velden VHJ, Boeckx N, Jedema I, te Marvelde JG, Hoogeveen PG, Boogaerts M, van Dongen JJM. Leukemia 2004;18:983-988. Abstract: Gemtuzumab ozogamicin (Mylotarg) induces remission in approximately 30% of relapsed AML patients. We previously demonstrated that gemtuzumab infusion results in near-complete CD33 saturation in peripheral blood, and that saturating gemtuzumab levels result in continuous binding and internalization of gemtuzumab due to renewed CD33 expression. We now demonstrate that a high CD33-antigen load in peripheral blood is an independent adverse prognostic factor, likely due to peripheral consumption of gemtuzumab. Indeed, CD33 saturation in bone marrow is significantly reduced (40-90% saturation) as compared with CD33 saturation in corresponding peripheral blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related with reduced cell kill. Apparently, high CD33-antigen loads in blood consume gemtuzumab and thereby limit its penetration into bone marrow. Consequently, CD33 saturation in bone marrow is reduced, which hampers efficient cell kill. Therefore, gemtuzumab should be administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by classical chemotherapy.

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