Standardization and application of quantitative PCR methods in patients with hematological malignancies

Using modem combination chemotherapy regimens, it has been possible to increase the rate of complete remission (CR) in acute lymphoblastic (ALL) and acute myeloid leukemia (AML). The major reason for treatment failure in acute leukemia (AL) is the resistance of leukemic blasts to chemotherapeutic agents, which is accompanied by an inability of the immune system to eradicate residual leukemic cells surviving initial treatment. Such cells are referred to as minimal residual disease (MRD), and are very difficult to detect against the background of regenerating bone marrow (BM).

The clinical definition of CR is traditionally based on conventional cytomorphological techniques, which have detection limits of 1-5%. Therefore more sensitive methods are needed for accurate evaluation of the presence of MRD. This thesis has been aimed at the standardization and assessment of molecular techniques for MRD detection in AL. A further goal has been the development of similar methods to gain quantitative information on the mRNA expression of various cytokines in lymph node biopsies from patients with Hodgkin's lymphoma (HL).

Experience from the cooperative projects "BIOMED-1 Concerted Action" (CA) and "Europe Against Cancer" (EAC) contributed to MRD evaluation in a standardized way. The collaborative BIOMED-1 study resulted in the standardization of primer sets, and conditions for RT-PCR, firstly for its use in clinical practice for the molecular classification of AL at diagnosis, and secondly for MRD detection during follow-up, in order to evaluate treatment effectiveness. The collaborative efforts of EAC resulted in the development of primers and probes for real-time quantitative (RQ)-PCR analysis for fusion gene (FG) and control gene (CG) transcripts (ABL, GUS, B2M). We applied RQ-PCR to investigate the differences over time in the stabilities of FG and CG transcripts, which may result in over- or under-estimation of MRD levels. The mean stability of most FG transcripts seemed to be comparable to those of the CG; however large differences were observed between patients expressing the same FG transcripts.

To compare MRD levels in follow-up BM samples from children with ALL two main methods were used: FC and semi-quantitative ASO-PCR techniques. Comparison between FC and ASOPCR showed significantly consistent results in 78% of the samples. Development of RQ-PCR permitted accurate quantification of PCR products during the exponential phase of the PCR amplification process, in complete contrast to classical PCR end-point quantification. We applied RQ-PCR and FC methods, to compare MRD levels in follow-up samples from 22 ALL patients. When results obtained by RQ-PCR and FC were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples. However, if a cutoff level of 0.01% was applied, the concordance was 89%.

Application of RQ-PCR techniques gains quantitative information on DNA and mRNA levels, and so we applied this approach to investigate cytokine mRNA expression in Hodgkin's Lymphoma (HL) samples and HL cell line. Reactive lymphatic tissue and peripheral blood mononuclear cells (PBMCs) from healthy donors were used as controls. The results demonstrated that expression levels of IL-13, IL-10, IL-1beta, IFNgamma, IL-15, and IL-12p35 in HL tissue were higher than those in PBMCs and reactive tissue. The highest mRNA levels for several of the cytokines were found in Epstein-Barr virus-positive samples.

In conclusion, this study provides the basis for rational and efficient molecular quantitative determination of MRD levels. These approaches are conducted whilst the patient is within the first months of treatment, and give information, which allows patient classification into low or high-risk groups. These groups may benefit from the possibility for group-specific treatment modalities, which minimize side effects and optimize treatment outcome. Moreover, the application of these methods can give valuable information on inherent biological variation between different lymphoma samples, and may help to unravel the complex cytokine network contributing to the clinical and biological heterogeneity of these diseases.

List of scientific papers

I. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, Gottardi E, Rambaldi A, Dotti G, Griesinger F, Parreira A, Gameiro P, Diaz MG, Malec M, Langerak AW, San Miguel JF, Biondi A (1999). Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 13(12): 1901-28.
https://pubmed.ncbi.nlm.nih.gov/10602411

II. van der Velden VH, Boeckx N, Gonzalez M, Malec M, Barbany G, Lion T, Gottardi E, Pallisgaard N, Beillard E, Hop WC, Hoogeveen PG, Gabert J, van Dongen JJ; Europe Against Cancer Program (2004). Differential stability of control gene and fusion gene transcripts over time may hamper accurate quantification of minimal residual disease--a study within the Europe Against Cancer Program. Leukemia. 18(4): 884-6.
https://pubmed.ncbi.nlm.nih.gov/14961029

III. Malec M, Bjorklund E, Soderhall S, Mazur J, Sjogren AM, Pisa P, Bjorkholm M, Porwit-MacDonald A (2001). Flow cytometry and allele-specific oligonucleotide PCR are equally effective in detection of minimal residual disease in ALL. Leukemia. 15(5): 716-27.
https://pubmed.ncbi.nlm.nih.gov/11368431

IV. Malec M, van der Velden VH, Bjorklund E, Wijkhuijs JM, Soderhall S, Mazur J, Bjorkholm M, Porwit-MacDonald A (2004). Analysis of minimal residual disease in childhood acute lymphoblastic leukemia: comparison between RQ-PCR analysis of Ig/TcR gene rearrangements and multicolor flow cytometric immunophenotyping. Leukemia. 18(10): 1630-6.
https://pubmed.ncbi.nlm.nih.gov/15295608

V. Malec M, Soderqvist M, Sirsjo A, MacNamara B, Lewin N, Sjoberg J, Bjorkholm M, Porwit-MacDonald A (2004). Real-time polymerase chain reaction determination of cytokine mRNA expression profiles in Hodgkins lymphoma. Haematologica. 89(6): 679-85.
https://pubmed.ncbi.nlm.nih.gov/15194535