Premium
Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR‐ABL 1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission
Author(s) -
Kooten Niekerk Peter B.,
Petersen Charlotte C.,
Nyvold Charlotte G.,
Ommen Hans B.,
Roug Anne S.,
Nederby Line,
Hokland Peter,
Kjeldsen Eigil
Publication year - 2014
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/bjh.12589
Subject(s) - stem cell , fluorescence in situ hybridization , progenitor cell , cell sorting , minimal residual disease , cd34 , interphase , microbiology and biotechnology , cancer research , real time polymerase chain reaction , reverse transcriptase , myeloid leukemia , flow cytometry , biology , medicine , polymerase chain reaction , immunology , bone marrow , gene , biochemistry , chromosome
Summary The exact disease state of chronic myeloid leukaemia ( CML ) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction ( qPCR ) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD 34 + stem cell and progenitor cell subpopulations were isolated by fluorescence‐activated cell sorting ( FACS ), and their content of residual Philadelphia positive ( P h + ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4‐log reduction in BCR ‐ ABL 1 expression ( MR 4 ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization ( iFISH ). Despite evaluating fewer cells, iFISH proved superior to mRNA ‐based qPCR in detecting residual Ph + stem cells ( P = 0·005), and detected Ph + stem‐ and progenitor cells in 9/10 patients at frequencies of 2–14%. Moreover, while all qPCR + samples also were iFISH + , 9/33 samples were qPCR ‐/ iFISH + , including all positive samples from MR 4 patients. Our findings show that residual Ph + cells are low BCR ‐ ABL 1 producers, and that DNA ‐based methods are required to assess the content of persisting P h + stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR 4 , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.