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Keywords:

  • chronic myeloid leukaemia stem cells;
  • minimal residual disease;
  • cytogenetics;
  • fluorescence-activated cell sorting;
  • quantitative polymerase chain reaction

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

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, CD34+ stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph+) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR4), 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 MR4 patients. Our findings show that residual Ph+ cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph+ 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 MR4, and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

Targeted therapy with tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myeloid leukaemia (CML) (O'Brien et al, 2003), and the majority of patients can now look forward to near-normal lives. After achieving complete cytogenetic remission (Hochhaus et al, 2009), patients are monitored using sensitive quantitative reverse transcription polymerase chain reaction (qPCR) assays specific for the BCR-ABL1 fusion transcript (Stentoft et al, 2001), which defines the achievement of major molecular response (MMR) and lower levels of residual disease (Hughes & Branford, 2009). However, the majority of patients who achieve the deepest measurable levels of molecular response (MR), i.e. 4-log reduction in BCR-ABL1 expression (MR4) or better (Cross et al, 2012), still harbour leukaemic cells that are capable of invoking relapse upon withdrawal of TKI treatment (Mahon et al, 2010). Consequently, new strategies are needed to unravel the true disease state in these patients. Both in vitro studies (Graham et al, 2002; Jørgensen et al, 2007) and data from patients in deep MR (Chomel et al, 2011) indicate that the persisting leukaemic cells in these patients are to be found within the most primitive undifferentiated cells.

Here we demonstrate that residual disease can be assessed at the stem cell level in patients in MR4, by evaluating sorted stem cells and progenitor cells with standard qPCR and interphase fluorescence in situ hybridization (iFISH) assays. Importantly, our results show that the rare remaining leukaemic stem cells are low BCR-ABL1 transcript producers, and that the latter DNA-based methodology therefore offers better disease detection in these patients. These findings provide a framework for improving follow-up and personalized decision-making in the increasingly large group of CML patients in deep MR.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

Patients

CML patients treated with TKIs for at least 1 year at the outpatient clinic at Aarhus University Hospital, and from whom we were able to obtain fresh bone marrow (BM) samples were studied. Samples were obtained after written informed consent from the patients, and approval by the Ethical Committee of the Central Denmark Region, Denmark.

Sample preparation and fluorescence-activated cell sorting

Mononuclear cells (MNC) were obtained by Lymphoprep separation (Axis-Shield plc, Dundee, Scotland) according to the manufacturers instructions. Before fluorescence-activated cell sorting (FACS), fresh cells were subjected to erythrocyte lysis (DAKO, Glostrup, Denmark), and separated into CD34+ enriched and CD34 cell fractions using the Human CD34 Selection kit (StemCell Technologies, Vancouver, BC, Canada) optimized for CD34+ cell yield. The median percentage of CD34+ cells in unseparated BM, the CD34+ enriched fraction and the CD34 fraction were 1·3% (range 0·36–2·1), 40% (4–74) and 0·072% (0·016–0·28) respectively. The CD34+ enriched cell fraction was stained with the following murine-anti-human antibodies: anti-CD38-fluorescein isothiocyanate (FITC) (clone AT 13/5; DAKO), anti-CD45-peridinin chlorophyll-cyanin 5.5 (PerCP-Cy5.5) (clone HI30; BioLegend, San Diego, CA, USA), anti-CD34-allophycocyanin (APC) (BIRMA-K3; DAKO) and anti-human inhibitory C-type lectin like receptor (hMICL)-phycoerythrin (PE) (clone HB3, prepared as previously described (Larsen et al, 2012)). For each sample, compensation was accomplished using a panel of non- and single-stained cells, and isotype controls were used for the antibodies that were essential for the sorting gates (FITC, PE and APC). Cells were sorted on a BD FACSAria™III (BD Biosciences, San Jose, CA, USA) into subsets of haematopoietic stem cells (HSC; defined as CD34+/CD38), hMICL+ progenitors (MpP; CD34+ CD38+ hMICL+), and hMICL progenitors (MnP; CD34+ CD38+ hMICL). Median purities of the sorted subsets were 99% (range 87–100), 92% (88–100), and 96% (94–100), respectively.

Cytogenetics and interphase FISH analysis

Standard chromosome analysis was carried out as previously described (Veigaard et al, 2011). For iFISH analyses, CD34+ cell subpopulations were FACS sorted onto SuperFrost slides (Gerhard Menzel GmbH, Braunschweig, Germany) and allowed to air-dry. Slides were pretreated with pepsin for 3 min at 37°C according to the Histology FISH accessory kit (DAKO) to de-mask the nuclear DNA prior to hybridization. iFISH was performed by employing a BCR FISH DNA split signal probe (DAKO) according to the manufacturers instructions. Two hundred nuclei were evaluated in a blinded setup by two independent observers using epifluorescence microscopy as described (Kjeldsen & Roug, 2012). Cells were counted as positive if the fluorescent signals of the two probes were at least two signal widths apart. Based on our laboratory-specific cut-off value, samples with at least 4/200 cells with positive signals were considered Ph+. The laboratory-specific cut-off value was defined by employing the BCR split signal probe on cytogenetically prepared BM samples from five normal donors. Based on these data the upper boundary of a 99% reference range was calculated as mean number of cells with positive signal + 3 × standard deviations (SD) rounded to the nearest higher integer. Using this cut-off value, false positive rates were <1%. To rule out that the sorted cells samples differed from cytogenetically prepared samples, the BCR split signal probe was applied on both magnetically-sorted CD138+ cells (Human CD138 Positive Selection Kit, StemCell Technologies) from three multiple myeloma patients, and FACS-sorted CD34+ cell subsets from two normal donors and one patient with JAK2 positive chronic myeloproliferative neoplasm. All samples were negative by the laboratory-specific cut-off value. A total of nine positive cells out of 1600 counted cells in eight evaluated subsets were found (0·6%) in the FACS-sorted negative controls, with the number of positive cells per subset varying from 0/200 to 3/200. Representative images of positive and negative cells from FACS-sorted patient samples and negative controls are shown in Fig S1.

Quantitative PCR

Sorted CD34+ subfractions, the CD34 fraction and MNCs were stored at −80°C in MagNA Pure LC mRNA lysis buffer (Roche Diagnostics GmbH, Mannheim, Germany). For the standard qPCR assay, total RNA was isolated on a MagNA Pure LC or MagNA Pure 96 robot (Roche) according to the manufacturers instructions, and qPCR assay was performed as previously described (Stentoft et al, 2001). For the optimized qPCR assay, total RNA was isolated from 300 to 0·5 × 106 cells (Table SI) using the RNeasy micro kit or QIAmp RNA Blood mini kit (QIAGEN, Sollentuna, Sweden) and cDNA prepared using Moloney murine leukaemia virus Reverse Transcriptase or SuperScript VILO™ cDNA synthesis kits (Invitrogen, Paisley, UK). RNA from samples with cell counts above 0·75 × 109/l was measured on a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and diluted accordingly to ensure a maximum of 0·5 μg RNA per cDNA synthesis reaction to avoid inhibition during the qPCR reaction. The qPCR was performed as in our standard assay using either TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) or Brilliant II QPCR Master Mix (Agilent Technologies, Santa Clara, CA, USA) with added urasil-DNA glycosylase (UNG) enzyme (Applied Biosystems). Assay proficiency was evaluated by standard curves of each gene based on serial dilutions of the cell line K562 (Fig S2). BCR-ABL1 expression was displayed on the International Scale after conversion using our laboratory-specific conversion factor (Hughes et al, 2006), and samples classified as MMR or MR4 as recommended by Cross et al (2012). To ensure that at least 200 cells were evaluated in qPCR negative samples (corresponding to the number of cells counted by iFISH) qPCR negative samples with <200 cells/well (ie. <2400 sorted cells, n = 2) were excluded form further analyses.

Statistical analysis

Paired comparison of the two qPCR assays was carried out using Student's t-test for paired data (assay sensitivity) and in a 2 × 2 table using McNemar's test (detection of BCR-ABL1 expression). Comparisons of iFISH positivity between CD34+ subsets, detection of Ph+ cells in CD34+ subsets between qPCR and iFISH, and between patients with at least one iFISH+ CD34+ subset and at least one qPCR+ CD34+ subset were performed using logistic regression, allowing for clustering within patients. Comparison of qPCR positivity between MMR and MR4 patients was done in a 2 × 2 table using Fishers exact test. All analyses were conducted using stata (Version 11.2; StataCorp LP, College Station, TX, USA). P values <0·05 were considered significant.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

All studied patients were successfully responding to TKI therapy at the time of sampling, and had been treated for a median of 37 months (range 12–119). Eleven of the 17 studied patients had attained MR4 for a median of 30 months (11–88), with the remainder being in MMR. Full patient characteristics are listed in Table 1.

Table 1. Patient characteristics.
PatientSexAge (years)Time since Diagnosis (months)Current treatmentPrevious treatmentaTotal TKI treatment (months)Molecular responsebMR4 Duration (months)Last Cg analysisc
  1. Cg, cytogenetic; M, male; F, female; IM, Imatinib; NI, Nilotinib; DA, Dasatinib; IFN, Interferon-α; Ara-C, cytosine arabinoside; MMR major molecular response; MR4, 4-log reduction in BCR-ABL1 expression.

  2. a

    Parentheses denote duration of treatment.

  3. b

    Molecular response classified based on BCR-ABL1 expression in bone marrow (BM) as assessed by the standard quantitative polymerase chain reaction assay.

  4. c

    Number of Ph+ positve cells by interphase fluorescence in situ hybridization in BM in the most recently performed analysis. Parentheses denote interval between the analysis and the current study.

  5. d

    BM mononuclear cell sample not available. Classified as MMR based on peripheral blood BCR-ABL1 expression on the sample date.

1M4965NI 400 mg ×2IM (16 months) - NI (48 months)64MMR0% (0 months)
2M3714IM 400 mg ×1IM (14 months)14MMR9% (7 months)
3M3713NI 300 mg ×2NI (12 months)12MMRd0% (0 months)
4M5730NI 300 mg ×2IM (7 months) - NI (23 months)30MMR0% (0 months)
5M6034IM 300 mg ×1IM (32 months)32MMR0% (0 months)
6F62134NI 300 mg ×2INF (4 months) - INF+Ara-C (5 months) - IM (101 months) - NI (18 months)119MMR0% (83 months)
7M6752NI 300 mg ×2IM (10 months) - DA (9 months) - NI (26 months)46MR4300% (33 months)
8F7436IM 400 mg ×1IM (36 months)36MR4240% (27 months)
9M6224IM 400 mg ×1IM (1 month) - NI (3 months) - IM (18 months)23MR4110% (11 months)
10F4637NI 300 mg ×2IM (6 months) - NI (31 months)37MR4130% (0 months)
11F3764IM 400 mg ×1IM (64 months)64MR4380% (0 months)
12F45118IM 400 mg ×1IM (112 months)112MR4880% (13 months)
13M5191IM 400 mg ×1IM (3 months) - INF+IM (23 months) - IM (64 months)91MR4790% (64 months)
14F6637IM 400 mg ×1IM (36 months)36MR4183% (31 months)
15F8022IM 400 mg ×1IM (21 months)21MR4280% (16 months)
16F72165NI 200 mg ×2INF (21 months) - IM (53 months) - DA (18 months) - NI (48 months)119MR4453% (45 months)
17F30104IM 400 mg ×1IM (102 months)102MR4850% (88 months)

At the outset, we sought to define optimal conditions for the detection of the BCR-ABL1 fusion transcript. To this end, we compared our standard qPCR assay, which is based on our publications from the Europe against cancer group (Stentoft et al, 2001; Beillard et al, 2003), to a less automated one, in which manual RNA extraction and a more effective cDNA synthesis was employed. However, despite an improved sensitivity of 1·0 log (±0·15log; P < 0·001), the optimized assay only detected BCR-ABL1 mRNA in 2 additional samples compared to the standard assay (P = 0·63) (Fig 1). Consequently, the optimized assay revealed that a total of 7 patients classified as MR4 actually had attained MR5 (Table 2).

Table 2. Presence of residual CML cells as assessed by both iFISH and mRNA-based qPCR in all investigated cell populations
PatientBM MNCCD34HSCMnPMpP
qPCR, SAaqPCR, OAaqPCRaiFISHbqPCRaiFISHbqPCRaiFISHbqPCRa
  1. CML, chronic myeloid leukaemia, iFISH, interphase fluorescence in situ hybridization; qPCR, quantitative polymerase chain reaction; BM MNC, bone marrow mononuclar cells; HSC, haematopoietic stem cells; MnP, hMICL-negative progenitor cells; MpP, hMICL-positive progenitor cells; SA, standard assay; OA, optimized assay; BC, qPCR sensitivity below cut-off; ND, not determined; F, iFISH slide preparation failed.

  2. a

    Results expressed on the International Scale (%). Samples with undetectable BCR-ABL1 expression are displayed as “<XX”, where XX denote the sensitivity level.

  3. b

    Results expressed as percentage of Ph+ cells.

  4. c

    BCR-ABL1 expression positive but not quantitative.

  5. d

    Separation of MnP and MpP not possible. Depicted results represent unseparated CD34+ CD38+ cells.

10·0430·0630·0444%BC0%<0·0723%<0·29
20·0410·0370·0613%0·0668%0·08910%0·047
3NDND0·0180%<0·212%d<0·0022dNDND
40·0290·0250·0166%<0·0414%0·050%<0·0014
50·0230·00940·018F<0·013F0·024F0·024
6<0·0220·00490·011ND16ND0·15ND0·34
7<0·01<0·00061<0·0004ND<0·77ND<0·0018ND0·0052
80·0072cND0·0066ND<0·930%<0·0080%<0·046
9<0·0066<0·000490·00632%<0·410%<0·0210%<0·25
10<0·0060·00610·0028NDBC0%<1·5NDBC
11<0·0060·00150·0020%<0·0840%<0·00250%<0·029
120·0054c<0·000480·00095c14%<0·238%<0·00160%<0·00072
13<0·0051<0·000660·00550%<0·592%<0·10%<0·23
140·005cND0·0013cND<0·310%<0·0040%<0·05
15<0·0042<0·00039<0·000822%<0·0410%<0·00160%<0·0012
16<0·0035<0·00040·008F<0·017F<0·00058F<0·00042
17<0·0032<0·00089<0·0012%<0·310%<0·00210%<0·00064
image

Figure 1. Quantitative BCR-ABL1 expression on the International Scale (IS) as assessed by a standard quantitative reverse transcription polymerase chain reaction (qPCR) assay and an optimized qPCR assay with improved sensitivity in CML patients in major molecular response (n = 5) and MR4 (n = 9). The number of samples with detectable BCR-ABL1 expression was compared using McNemar's test. MR4, 4-log reduction in BCR-ABL1 expression; MR5, 5-log reduction in BCR-ABL1 expression.

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Given that Ph+ stem cells have been shown to persist in patients in deep MR (Chomel et al, 2011), we next performed FACS sorting of CD34+ subpopulations as a vehicle for identifying the residual leukaemic cells. Here we also included the hMICL antigen, which has previously been reported to identify granulocyte/macrophage-committed progenitors (Marshall et al, 2004; van Rhenen et al, 2007), as a sorting criterion. From the BM samples we thus isolated a median of 0·13% HSCs, 0·4% MpPs, and 0·77% MnPs (Fig 2A). In one patient (Patient 3) our antibody failed to detect hMICL+ cells, and the CD34+ CD38+ fraction could not be separated into MpP and MnPs.

image

Figure 2. Detection of residual Ph+ cells by mRNA-based qPCR and iFISH in FACS sorted CD34+ stem- and progenitor cell subpopulations in CML patients in MMR and MR4. (A) Fluorescence-activated cell sorting (FACS) of haematopoietic stem cells (HSC: CD34+ CD38), hMICL-positive progenitors (MpP: CD34+ CD38+ hMICL+) and hMICL-negative progenitors (MnP: CD34+ CD38+ hMICL-). (B) Expression of BCR-ABL1 mRNA (on the International Scale (IS)) in the sorted CD34+ subsets. Error bars denote the median upper boundary of BCR-ABL1 expression (±95% confidence interval) in the cell subset (sensitivity levels of negative samples included as upper boundaries of BCR-ABL1 expression). (C) Frequencies of residual Ph+ cells as detected by interphase fluorescence in situ hybridization (iFISH) in the sorted CD34+ subsets. (D) Detection of residual Ph+ cells in sorted the subsets by iFISH and mRNA-based quantitative reverse transcription polymerase chain reaction (qPCR). Error bars denote standard error of the mean. (E) Combined qPCR and iFISH status of samples analysed by both modalities from patients with major molecular response (MMR) and MR4 (4-log reduction in BCR-ABL1 expression). Statistical significance was assessed using logistic regression allowing for within-patients clustering.

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When analysing the sorted subsets, our optimized qPCR assay only detected BCR-ABL1 expression in 10/48 (21%) subsets (Fig 2B), despite a median input of 85 000 cells (range: 600–370 000; Table SI) in each reaction. When the sensitivity levels of negative samples were included as upper boundaries of expression, as suggested by Cross et al (2012), the median level of BCR-ABL1 transcript in the populations could be estimated to below 0·16% on the International Scale (95% confidence interval [CI]: 0·072%–0·54%) in the HSC, 0·014% (0·0043%–0·044%) in the MnP, and 0·021% (0·0051%–0·088%) in the MpP. Considering these data, we were surprised to find that iFISH detected Ph+ cells in 14/34 (41%) subsets in frequencies ranging from 2% to 14%, and most frequently in the HSC fraction (Fig 2C). Taken together, iFISH proved significantly better than qPCR in detecting residual Ph+ cells in the sorted fractions (P = 0·038), especially within the HSC fraction (P = 0·005) (Fig 2D). Moreover, in subsets where both methods were applied, all qPCR positive subsets were also iFISH positive (4/33) while 9/33 samples were qPCR/iFISH+, including all positive samples from MR4 patients (Fig 2E).

Combining the data from all sorted subsets within the individual patients (Fig 3A), iFISH detected Ph+ cells in at least one CD34+ fraction in 9/10 patients, while qPCR only found 5/15 positive (P = 0·013). Also, in eight of the 10 patients where qPCR was negative in all CD34+ subsets, we found detectable BCR-ABL1 mRNA (0·00094%–0·018%) in the CD34 fraction. Moreover, qPCR positivity was significantly less frequent in MR4 patients (1/10) compared to MMR patients (4/5, P = 0·017), while iFISH positivity was equally frequent (P = 1·0), thus indicating that low BCR-ABL1 producing Ph+ cells may be even more predominant in the MR4 patients. A more detailed evaluation of the cell types involved in the CML clone in the individual patients revealed that the HSC fraction most frequently contained detectable Ph+ cells (7/9 patients), either alone (n = 3) or in combination with Ph+ cells in one or both of the progenitor fractions (n = 4), while only 2 patients had detectable Ph+ solely within the progenitor fraction (Fig 3B).

image

Figure 3. Combined residual disease profile in the individual patients. (A) Detection of residual disease in the CD34- and in at least one of the analysed CD34+ compartments as assessed by both interphase fluorescence in situ hybridization (iFISH) and mRNA-based quantitative reverse transcription polymerase chain reaction (qPCR). (B) Profile of cell types involved in the persisting chronic myeloid leukaemia clones of the individual patients as detected by iFISH.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

Persistence of Ph+ stem- and progenitor cells has been demonstrated previously with similar techniques at diagnosis (Mustjoki et al, 2013) and after achieving MMR (Chu et al, 2011; Defina et al, 2012; Kumari et al, 2012; Minami et al, 2012), but only at deeper levels of MR using sensitive DNA-based qPCR (Ross et al, 2010) or long-term culture-initiating cell assays (Chomel et al, 2011). Thus, this is the first time that persisting CML stem cells and progenitor cells have been detected by iFISH in MR4 patients. Likewise, this is the first time that a discrepancy between DNA- and mRNA-based methods in the subgroup of patients eligible for TKI withdrawal attempts has been pinpointed.

Pre-selection of CD34+ stem- and progenitor cells unmasked residual disease, and enabled detection of Ph+ cells by iFISH in almost all analysed patients, including 5/6 of patients who had achieved at least MR4. Also, the frequent presence of Ph+ cells in the HSC subset shows that the persisting leukaemic cells in patients in deep MR are to be found among the most primitive cells, thus in agreement with previous reports (Chomel et al, 2011; Chu et al, 2011). However, we found persisting Ph+ cells with a FACS-based approach in patients with considerably longer durations of TKI treatments (up to 119 months) compared to the study reported by Chu et al (2011) Moreover, while persisting CML cells have been detected previously in intensely treated patients (Ross et al, 2010; Chomel et al, 2011), these studies did not allow an exact immunophenotypic identification of the persisting cells. Of note, Mustjoki et al (2010) failed to detect persisting Ph+ stem cells by iFISH in a similar group of patients. We speculate that differences in methodologies account for these discordant results. Thus, in our initial iFISH experiments we noticed large variation in the hybridization efficiency due to a significant part of the nuclei on the sorted slides being inaccessible for the probes. However, introduction of a pre-hybridization step with pepsin de-masked the nuclear DNA and increased both the signal intensity and the number of cells with signals to >95%. This, together with our observations on sorted myeloid progenitor cells from normal donors and non-CML patients, allowed us to reproducibly identify the residual Ph+ cells in the FACS-sorted cell samples.

The lack of detectable fusion transcript mRNA in the sorted stem cells and progenitor cells could be interpreted merely as evidence of the scarcity of CML cells at this point of disease. However, also taking the iFISH data into account, these results clearly indicate the persistence of low BCR-ABL1 producing Ph+ stem cells and progenitor cells in these patients, thus extending recent findings. (Kumari et al, 2012). However, the latter study (Kumari et al, 2012) did not include patients in MR4, and therefore not those candidates eligible for TKI withdrawal. In fact, our data demonstrated that low BCR-ABL1 producing clones may be even more predominant among the residual CML cells in patients in MR4 or above compared to MMR patients. Relying solely on mRNA-based methods may therefore underestimate the true level of residual disease, and hamper our ability to assess residual disease in the most successfully treated patients.

Intriguingly, we found detectable BCR-ABL1 mRNA in the differentiated CD34 fraction in the majority of patients where the fusion transcript was undetectable in all CD34+ subsets. While the superior sensitivity obtained in the analysis on the abundant CD34 cells probably account for this, it does show that the low levels of detectable fusion transcript in routine samples from deep MR patients mainly originate from differentiated cells, rather than from a small population of high BCR-ABL1 producing stem cells and progenitor cells. Moreover, it demonstrates that the persisting CML stem cells and progenitor cells are functionally capable of maintaining a population of differentiated Ph+ cells.

Both the selection of candidates for TKI discontinuation and the evaluation of future stem cell targeted treatments would greatly benefit from methods that assess levels of persisting CML stem cells. Our results reveal that DNA-based techniques are required to accomplish this, and to define the next level of response beyond the achievement of undetectable BCR-ABL1 mRNA. Compared to previously employed methods (Ross et al, 2010; Chomel et al, 2011; Kumari et al, 2012), the current approach constitutes a more clinically applicable manner of assessing residual disease in CML patients in deep MR. Notably, it requires neither the tailoring of patient-specific reagents, nor the employment of time-consuming culture assays. If the prognostic value of the level of low BCR-ABL1 producing Ph+ stem cells could be confirmed in a prospective study, iFISH based assessment of FACS-sorted stem cells and progenitor cells would provide us with a powerful tool for the early and safe identification of candidates for TKI withdrawal.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank our patients for donating samples for this study, Prof. Dr. Jesper Stentoft for facilitating the collection of patient samples, Karin Brændstrup and Pia Skov Kristensen for invaluable technical assistance, and Prof. Marianne Hokland for access to the sorters at the FACS Core Facility. This work was supported by grants to P.H. from The Danish Cancer Society, the Danish MRC, the John and Birthe Meyer Foundation, and the Karen Elise Jensen Foundation.

Author contribution

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

P.B.v.K.N. designed parts of the study, performed the experiments, analysed and discussed the data and wrote the paper; C.C.P. assisted with the FACS sorting and analysis of flow cytometric data; C.G.N. designed the molecular analyses; H.B.O. assisted with the statistical analysis; A.S.R. provided the anti-hMICL antibody; L.N. performed part of the experiments; P.H. designed the study, discussed the data and wrote the paper; E.K. performed the iFISH analyses and discussed the data. All authors contributed to revising the paper.

Conflict of interest

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information

P.H. received research funding from Novartis. The remaining authors have no competing interests.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contribution
  8. Conflict of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
bjh12589-sup-0001-TabS1-FigS1-S2.pdfapplication/PDF1935K

Fig S1.Representative images of Philadelphia positive (BCR split signal +) and negative (BCR split signal −) cells from (A) FACS sorted CML patients samples and (B) FACS sorted negative controls.

Fig S2. Standard curves of the qPCR assay for each gene based on serial 5-fold dilutions of cDNA from the K562 cell line.

Table S1. Details of analyses performed on the sorted cell subsets.

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