Mikrochimérismus – na hraně počínajícího relapsu AML?

Microchimerism – on the edge of incipient AML relapse?

The reoccurrence or increase in autologous hematopoiesis after allogeneic stem cell transplantation has been linked with incipient leukemia relapse, however, importance of such emergency regarding microchimerism (chimerism under 1% of autologous cells) remains controversial. We compared conventional PCR of minisatellite, microsatellite, or sex specific regions followed by fragment analysis (FA) with real-time quantitative PCR (RQ-PCR) of insertion/deletion and sex polymorphism. Chimerism values obtained from both analyses were compared with the status of the disease (continuous complete remission, molecular relapse, hematological relapse, HR). Whereas RQ-PCR analysis of microchimerism predicted full HR in 12/13 cases (92%) with median of 34 days (0-322 days), the conventional FA was successful only in 5/13 cases (38%) (median 0 days, interval 0-154 days; p = 0.002). RQ-PCR surpassed FA in 10 cases with median of earlier prediction of relapse of 35 days (interval 6-168 days). Besides this, RQ-PCR detected other 4/6 HR until one year after transplantation, beyond the sensitivity of FA. Further, RQ-PCR examination of peripheral blood in combination with bone marrow confirmed all 9 molecular relapses previously detected due to a specific molecular marker. We conclude, that RQ-PCR is sensitive and reliable method of microchimerism analysis that can predict impending relapse and allows therefore earlier clinical intervention in comparison with another, less sensitive method.

Key words:
chimerism, microchimerism, relapse, AML, RQ-PCR

Autoři: O. Horký; J. Mayer; L. Kablásková; M. Borský; M. Krejčí; D. Dvořáková
Působiště autorů: Centrum molekulární biologie a genové terapie, Interní hematoonkologická klinika LF MU a FN Brno
Vyšlo v časopise: Transfuze Hematol. dnes,15, 2009, No. 2, p. 97-102.
Kategorie: Souhrnné práce, původní práce, kazuistiky

Souhrn

Znovuobjevení či nárůst autologní krvetvorby po alogenní transplantaci hematopoetických buněk bývá spojen s počínajícím relapsem akutní myeloidní leukemie, nicméně význam mikrochimérismu (podílu autologních buněk pod hranicí 1 %) není jednoznačný. Byly porovnávány výsledky fragmentační analýzy (FA) a kvantitativní PCR v reálném čase (RQ-PCR). Hodnoty získané z obou analýz byly konfrontovány s klinickým stavem pacientů: kompletní remise, molekulární relaps, hematologický relaps. Zatímco RQ-PCR analýza periferní krve predikovala hematologický relaps ve 12 případech z 13 (92 %), s mediánem předpovědi 34 dnů (0–322 dní), byla FA úspěšná pouze v pěti případech (38 %) (medián předpovědi 0 dní, 0–154 dní) (p = 0,002). RQ-PCR předcházela FA v 10 případech, s mediánem dřívější předpovědi 35 dní (interval 6–168 dní). RQ-PCR dále zachytila dalších 4 z 6 časných hematologických relapsů (do jednoho roku po transplantaci), mimo dosah senzitivity FA. Kombinace vyšetření periferní krve a kostní dřeně navíc potvrdilo 9 molekulárních relapsů z 9. RQ-PCR se jeví jako citlivá a spolehlivá metoda stanovení mikrochimérismu, jehož dynamika umožňuje zachycení počátku relapsu a umožňuje tak časnou klinickou intervenci.

Klíčová slova:
chimérismus, mikrochimérismus, relaps, AML, RQ-PCR

Zdroje

1. Bader P, Niethammer D, Willasch A, Kreyenberg H, Klingebiel T. How and when should we monitor chimerism after allogeneic stem cell transplantation? Bone Marrow Transplant 2005; 35: 107–119.

2. Kern W, Haferlach C, Haferlach T, Schnittger S. Monitoring of Minimal Residual Disease in Acute Myeloid leukemia. Cancer 2008; 112: 4–16.

3. Lawler M, Humphries P, McCann S. Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction. Blood 1991; 77: 2504–2514.

4. Bader P, Hoelle W, Klingebiel T, et al. Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children. Bone Marrow Transplant 1997; 19: 697–702.

5. Bader P, Beck J, Frey A, et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant 1998; 21: 487–495.

6. Schattenberg A, de Witte T, Salden M, et al. Mixed hematopoietic chimerism after allogeneic transplantation with lymphocyte-depleted bone marrow is not associated with a higher incidence of relapse. Blood 1989; 73: 1367–1372.

7. Roy DC, Tantravahi R, Murray C, et al. Natural history of mixed chimerism after bone marrow transplantation with CD6-depleted allogeneic marrow: a stable equilibrium. Blood 1990; 75: 296–304.

8. Bertheas MF, Lafage M, Levy P, et al. Influence of mixed chimerism on the results of allogeneic bone marrow transplantation for leukemia. Blood 1991; 78: 3103–3106.

9. Suttorp M, Schmitz N, Dreger P, Schaub J, Löffler H. Monitoring of chimerism after allogeneic bone marrow transplantation with unmanipulated marrow by use of DNA polymorphisms. Leukemia 1993; 7: 679–687.

10. van Leeuwen JE, van Tol MJ, Joosten AM, Wijnen JTh, Meera Khan P, Vossen JM. Mixed T-lymphoid chimerism after allogeneic bone marrow transplantation for hematologic malignancies of children is not correlated with relapse. Blood 1993; 82: 1921–1928.

11. van Leeuwen JEM, van Tol MJD, Joosten AM, et al. Persistence of host-type hematopoiesis after allogeneic bone marrow transplantation for leukemia is significantly related to the recipient’s age and/or the conditioning regimen, but it is not associated with an increased risk of relapse. Blood 1994; 83: 3059–3067.

12. Choi SJ, Lee KH, Lee JH, et al. Prognostic value of hematopoietic chimerism in patients with acute leukemia after allogeneic bone marrow transplantation: a prospective study. Bone Marrow Transplant 2000; 26: 327–332.

13. Schaap N, Schattenberg A, Mensink E, et al. Long-term follow-up of persisting mixed chimerism after partially T cell-depleted allogeneic stem cell transplantation. Leukemia 2002; 16: 13–21.

14. Wäsch R, Bertz H, Kunzmann R, Finke J. Incidence of mixed chimaerism and clinical outcome in 101 patients after myeloablative conditioning regimens and allogeneic stem cell transplantation. Br J Haematol 2000; 109: 743–750.

15. Formánková R, Honzátková L, Sieglová Z, Starý J, Sedláček P, Brdička R. Detailed monitoring of hematopoietic chimerism in a child treated by adoptive immunotherapy for high risk of relapse after BMT for acute myeloid leukemia. Bone Marrow Transplant 2000; 25: 453–456.

16. Mattsson J, Uzunel M, Tammik L, et al. Leukemia lineage specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001; 15: 1976–1985.

17. Schaap N, Schattenberg A, Mensink E, et al. Long-term follow-up of persisting mixed chimerism after partially T cell depleted allogeneic stem cell transplantation. Leukemia 2002; 16: 13–21.

18. Pérez-Simón JA, Caballero D, Diez-Campelo M, et al. Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation. Leukemia 2002; 16: 1423–1431.

19. Barrios M, Jimenez-Velasco A, Roman-Gomez J, et al. Chimerism status is a useful predictor of relapse after allogeneic stem cell transplantation for acute leukemia. Haematologica 2003; 88: 801–810.

20. Formánková R, Sedláček P, Kršková L, Říhová H, Šrámková L, Starý J. Chimerism-directed adoptive immunotherapy in prevention and treatment of post-transplant relapse of leukemia in childhood. Haematologica 2003; 88: 117–118.

21. Scheffold C, Kroeger M, Zuehlsdorf M, et al. Prediction of relapse of acute myeloid leukemia in allogeneic transplant recipients by marrow CD34+ donor cell chimerism analysis. Leukemia 2004; 18: 2048–2050.

22. Lamba R, Abella E, Kukuruga D, et al. Mixed hematopoietic chimerism at day 90 following allogeneic myeloablative stem cell transplantation is a predictor of relapse and survival. Leukemia 2004; 18: 1681–1686.

23. Fernandez-Aviles F, Urbano-Ispizua A, Aymerich M, et al. Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeat-markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood. Leukemia 2003; 17: 613–620.

24. Huisman C, de Weger RA, de Vries L, Tilanus MGJ, Verdonck LF. Chimerism analysis within 6 month of allogeneic stem cell transplantation predict relapse in acute myeloid leukemia. Bone Marrow Transplant 2007; 39: 285–291.

25. Antin JH, Childs R, Filipovich AH, et al. Establishment of complete and mixed donor chimerism after allogeneic lymphohematopoietic transplantation: recommendations from a workshop at the 2001 Tandem Meetings of the International Bone Marrow Transplant Registry and the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant 2001; 7: 473–485.

26. Lion T. Summary: Reports on quantitative analysis of chimerism after allogeneic stem cell transplantation by PCR amplification of microsatellite markers and capillary electrophoresis with fluorescence detection. Leukemia 2003; 17: 252–254.

27. Horký O, Dvořáková D, Mayer J. Kvantitativní analýza chimerismu po alogenní transplantaci hematopoetických buněk. Trans Hemat dnes 2004; 10: 70–75.

28. Fehse B, Chukhlovin A, Kuhlcke K, et al. Real-time quantitative Y chromosome-specific PCR (QYCS-PCR) for monitoring hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation. J Hematother Stem Cell Res 2001; 10: 419–425.

29. Alizadeh M, Bernard M, Danic B, et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002; 99: 4618–4625.

30. Jiménez-Velasco A, Barrios M, Román-Gómez J, et al. Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alles and insertion/deletion polymorphisms. Leukemia 2005; 19: 336–343.

31. Koldehoff M, Steckel NK, Hlinka M, Beelen DW, Elmaagacli AH. Quantitative analysis of chimerism after allogeneic stem cell transplantation by real time polymerase chain reaction with single nucleotide polymorphism, standard tandem repeats, and Y chromosome-specific sequences. Am J Hematol 2006; 81: 735–746.

32. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988; 16: 1215.

33. Masmas TN, Madsen HO, Petersen SL, et al. Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction. Biol Blood Marrow Transplant 2005; 11: 558–566.

34. Horký O, Mayer J, Dvořáková D. Stanovení chimérismu pomocí polymerázové řetězové reakce v reálném čase. Přehled a první vlastní zkušennosti. Trans Hemat dnes 2007; 13: 73–78.

35. Gabert J, Beillard E, van der Velden VHJ, et al. 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. Leukemia 2003; 17: 2318–2357.