Suitability of the PAXgene system to stabilize bone marrow RNA in imatinib-resistant patients with chronic myeloid leukemia.
BACKGROUND
Optimum sample quality is a crucial requirement for molecular monitoring of patients with chronic myeloid leukemia (CML) on therapy. Bedside RNA stabilization systems (e.g., PAXgene) have been developed to inhibit RNA degradation during shipment of samples from the clinical site to the specialized laboratory. In CML, blood but not bone marrow samples have been examined using RNA stabilization in previous studies. Therefore, we sought to investigate the applicability of the PAXgene system for bone marrow samples in CML.


METHODS
Simultaneously stabilized blood and bone marrow samples were obtained from 55 imatinib-resistant CML patients to compare RNA yield and purity, expression of two housekeeping genes (total ABL and beta-glucuronidase; GUS) by quantitative reverse-transcriptase polymerase chain reaction, BCR-ABL expression (ratios BCR-ABL/ABL and BCR-ABL/GUS), and BCR-ABL kinase domain mutations analyzed by denaturing high-performance liquid chromatography and direct sequencing.


RESULTS
RNA extraction revealed high-quality RNA derived from both stabilized blood and bone marrow samples. RNA yield was significantly higher in bone marrow (median 9.9 microg RNA/mL bone marrow) than in blood (median 4.3 microg RNA/mL blood) (p=0.0005). The number of housekeeping gene transcripts was comparable in blood and bone marrow (median ABL copies/2 microL cDNA 13,260 vs. 25,590; median GUS copies/2 microL cDNA 35,490 vs. 60,200; n.s.). Further, ratios BCR-ABL/ABL (blood vs. bone marrow, median 47% vs. 57%) and ratios BCR-ABL/GUS (blood vs. bone marrow, median 26% vs. 21%) were not significantly different. Results of mutation analysis corresponded in 51 out of 55 patients (93%), whereas moderate differences were observed in four patients.


CONCLUSIONS
We conclude that bone marrow can be effectively stabilized using the PAXgene system and shows concordance with blood in terms of BCR-ABL mRNA quantification and mutation analysis in imatinib-resistant CML patients.
Ernst
Thomas
T
III. Medizinische Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany.
Hoffmann
Jana
J
Erben
Philipp
P
Hehlmann
Rüdiger
R
Hochhaus
Andreas
A
Müller
Martin C
MC
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001549:Benzamides; D001853:Bone Marrow / Q000378:metabolism*; D004252:DNA Mutational Analysis / Q000379:methods; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D016044:Fusion Proteins, bcr-abl; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics; D010879:Piperazines / Q000494:pharmacology* / Q000627:therapeutic use; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology* / Q000627:therapeutic use; D020871:RNA Stability; D012334:RNA, Neoplasm / Q000097:blood / Q000378:metabolism*; D012015:Reference Standards
D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D012334:RNA, Neoplasm; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D005966:Glucuronidase