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Keywords:

  • chronic myeloid leukemia;
  • nilotinib;
  • leukemic stem cells;
  • CD34+ cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

BACKGROUND:

Compared with imatinib, nilotinib is a potent breakpoint cluster region/v-abl Abelson murine leukemia viral oncogene (bcr-abl) kinase inhibitor, and it induces higher rate and rapid complete cytogenetic response (CCyR), yet no clinical data are available regarding its efficacy against chronic myeloid leukemia (CML) stem cells. Earlier studies demonstrated that clusters of differentiation 34–positive, Philadelphia chromosome–positive (CD34+Ph+) cells are detectable in about 45% of patients with CML, despite being on long-term imatinib therapy and having achieved sustained CCyR.

METHODS:

CD34+ cells from bone marrow of de novo CML patients in the chronic phase (n = 24) treated with nilotinib (median duration of therapy, 22 months) were isolated and scored for BCR-ABL by fluorescent in situ hybridization (FISH) analysis. Similar analysis was also performed in 5 de novo CML chronic phase patients who achieved CCyR within 3 months of nilotinib therapy.

RESULTS:

FISH evaluation of a median of 100 CD34+ nuclei per patient revealed that only 1 of 20 (5%) evaluable patients showed residual Ph+ progenitor cells. In this patient, just 1 of 140 (0.7%) CD34+ interphase nuclei was found to be positive for BCR-ABL. Surprisingly, no CD34+Ph+ cells were found even in those 5 patients evaluated after 3 months of nilotinib treatment.

CONCLUSIONS:

This study assessed for the first time the persistence of CD34+Ph+ cells during nilotinib first-line treatment. Preliminary results showed that in patients in CCyR, even after short-term nilotinib therapy, residual leukemic progenitors are very rarely detected compared with imatinib-treated CCyR patients. It is yet to be determined if these findings will have an impact in the path to a cure of CML with tyrosine kinase inhibitors. Cancer 2012. © 2012 American Cancer Society.

Imatinib mesylate is highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML), inducing rapid hematologic and cytogenetic responses in the vast majority of patients. Although its efficacy has been widely confirmed, it has also been demonstrated that discontinuation of treatment is associated with molecular relapse in about 60% of patients, even if they had previously achieved a sustained complete molecular response (CMolR).1 The cause of CML reappearance could reside in the persistence of tyrosine kinase inhibitor (TKI)-resistant leukemic stem cells, representing a “quiescent” reservoir of the disease. In this regard, it has been reported that BCR-ABL–positive progenitor cells (identified as clusters of differentiation 34–positive, Philadelphia chromosome–positive [CD34+Ph+] cells) can still be detected in patients with complete cytogenetic response (CCyR) not only after short-term imatinib treatment2 but also after a stable, long-lasting CCyR.3 In fact, we had previously performed a study in which we evaluated the presence of residual bone marrow (BM) CD34+Ph+ cells in 31 CML patients in CCyR for a median time of 35 months during imatinib treatment. The study demonstrated that 45% of patients, the majority of whom were also in major molecular response (MMolR), still harbored a median of 1% (range, 1%-7%) of CD34+Ph+ CML cells in BM.3 Nilotinib, a second-generation TKI, has a greater potency and selectivity for BCR-ABL than does imatinib4 and was first approved for patients with CML in the chronic or accelerated phase who were resistant to or could not tolerate imatinib.5 Furthermore, a recent prospective study comparing nilotinib with imatinib as first-line treatment in patients with CML has confirmed considerable efficacy both in terms of CCyR and MMolR.6 Nilotinib appears to eradicate more rapidly the bulk of CML cells, at both dosages of 300 mg bid (twice daily) and 400 mg bid, inducing in early chronic phase patients a higher rate of CCyR at 6 months of treatment compared to imatinib (67% and 63% vs 45%, respectively).7 This superior efficacy has so far also been confirmed at 24 months of treatment with a CCyR rate of 87% and 85% versus 77% and a MMolR rate of 71% and 67% versus 44%, respectively.8 Despite these very promising results, to date, it is unknown if nilotinib would be more effective in eradicating CML CD34+ cells, hence, representing potential “curative” treatment for CML patients. On this matter, in vitro data showed that nilotinib, as well as imatinib, is unable to eliminate CML progenitors.9, 10 However, no reports have been published so far about the evaluation of persisting Ph+ progenitor cells in CML patients during first-line nilotinib treatment. Therefore, in order to compare the efficacy of this second-line TKI in eliminating CML precursors in comparison with imatinib, we investigated whether BM CD34+Ph+ progenitors could be still detected in a cohort of CML patients in CCyR following first-line nilotinib therapy.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

Patient Population

The endpoint of the study was to evaluate the percentage of BM residual CD34+Ph+ cells in CML patients in CCyR during first-line nilotinib treatment. The patients studied were included in 2 clinical trials (GIMEMA CML0307 study, ClinicalTrials.gov number NCT00481052, and CAMN107A2303 study, ClinicalTrials.gov number NCT00471497), and the evaluation of residual leukemic stem cells was performed during a routine BM aspirate after receiving a specific patient informed consent. As in our previous CD34+Ph+ study during imatinib treatment,3 about 10 mL of BM per patient was collected in heparin-anticoagulated tubes. A small amount of sample was not further manipulated and was evaluated for standard fluorescent in situ hybridization (FISH) analysis and flow cytometry study. The rest of the sample was used for CD34+ isolation and subsequent FISH analysis of CD34+-purified cells.

Magnetic Labeling and Separation of CD34+ Progenitors

Bone marrow mononuclear cells (BMMCs) were isolated by density gradient separation, and CD34+ cells were selected from BMMCs using immunomagnetic column separation according to published methods and manufacturer instructions (Miltenyi Biotech, Auburn, Calif) and as previously published.3 Briefly, BMMCs were resuspended in buffer (phosphate-buffered saline; 0.5% fetal bovine serum; 2 mM ethylamine diamine tetraacetic acid) to obtain a concentration of 108 total cells/300 μL. Subsequently, 100 μL CD34 microbeads per 108 total cells and 100 μL/108 total cells of Fc receptor blocker were added to the cell suspension. After incubation for 30 minutes at 4°C, cell suspension was washed with buffer and applied to the immunomagnetic column at a concentration of approximately 108 total cells/500 μL. In order to achieve a highly purified CD34+ population, 2 rounds of magnetic separation were performed.

Flow Cytometry Analysis of CD34+ Cells

To determine the yield and the purity of sorted CD34+ cells, aliquots of whole BM were evaluated for CD34+ cells by flow cytometry. Similarly, aliquots of cells were analyzed after column separation. Flow cytometry analysis was performed by incubating each cell sample with anti-CD34, anti-CD38, and anti-CD45 fluorescent antibodies and subsequently by analyzing the samples on a FACScan flow cytometer (BD Biosciences, San Jose, Calif).

FISH Analysis of Whole BM Cells and Purified CD34+ Cells

FISH was performed on fixed cells according to conventional published methods and manufacturer specifications. Briefly, slides were denatured in 70% formamide/2× standard sodium citrate (SSC) for 3 minutes at 75°C and deydrated in ethanol solutions. About 10 μL of probes were hybridized on interphase cells overnight at 42°C, and washes were performed in 2× SSC and Tween/2× SSC, counterstained with 4′,6′-diamidine-2-phenylindole (DAPI). LSI BCR/ABL dual-color extra signal (ES), single fusion translocation was used as probe (Vysis, Downers Grove, Ill). Slides were analyzed with a Nikon 2 fluorescence microscope and images captured with a charge-coupled device camera using an image analysis system (Genikon). When conventional whole BM analysis was performed, at least 200 interphase cells were analyzed. In the case of CD34+-purified cells, 100 interphase nuclei was considered an adequate number for FISH analysis. Two observers independently scored only isolated cells in order to avoid possible false positive results (overlapping nuclei). In our experience, the LSI BCR/ABL ES probe has a false positive signal rate close to 0, and FISH negativity was defined as the complete absence of BCR-ABL fusion signal.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

Patient Population

Twenty-four patients were evaluated for residual CD34+Ph+ leukemic cells. The median age at CML diagnosis was 47 years (range, 29-80 years) with 18 males and 6 females. At the time of residual CD34+Ph+ evaluation, the median time of nilotinib treatment was 22 months (range, 9-30 months) with 17 patients (71%) receiving nilotinib 400 mg bid, 5 patients (21%) receiving 300 mg bid, whereas 2 patients (8%) were on 400 mg daily due to intolerance. All patients had been in CCyR for a median time of 17.5 months (range, 6-27 months); 20 of 24 patients (83%) had been in MMolR (ie, BCR-ABL/ABLIS ratio < 0.1%)11, 12 for a median time of 12 months (range, 1-27 months); 1 of 24 (4%) had been in CMolR (ie, BCR-ABL/ABLIS ratio < 0.01%)11, 12 for 12 months, whereas 3 of 24 (13%) had not yet achieved a MMolR. Patient characteristics are included in Table 1.

Table 1. Patient Characteristics, Disease Response to Nilotinib, and FISH Analysis Results at the Time of CD34+Ph+ Cell Evaluation
Patient No.Sex/Age, yMonths on NilotinibNilotinib Dosage (mg/day)Months of CCyRMMolRMonths of MMolRFISH CD34+ No. of Nuclei Ph+/No. of Nuclei Analyzed
  1. CCyR indicates complete cytogenetic response; CMolR, complete molecular response; FISH, fluorescent in situ hybridization; MMolR, major molecular response; NE, not evaluable.

1M/42158008No00/100
2F/351660013Yes110/60
3M/461660013Yes100/80
4M/581980014Yes10NE
5M/402180018No00/300
6M/562880025Yes220/100
7M/482180014Yes10/75
8M/772080017Yes140/200
9F/262080016Yes130/300
10M/552280017Yes140/100
11M/442880025Yes100/100
12M/402480021Yes211/140
13M/732760022Yes22NE
14F/582280015CMolR120/100
15M/801860015Yes150/100
16M/6496006Yes30/100
17M/502080017Yes120/300
18F/292940026Yes3NE
19F/423080027Yes270/65
20M/503080027Yes210/100
21F/442980026Yes260/86
22M/603080027Yes120/100
23M/473040027Yes180/100
24M/473080027No0NE

Yield and Immunophenotypic Analysis of CD34+ Population

Twenty-four BM samples were collected for CD34+Ph+ cell evaluation, harvesting a median volume of 10 mL (range, 8-14 mL). Total median cellularity was 20 × 106/mL (range, 5 × 106 to 42 × 106/mL), and the median percentage of CD34+ as measured by flow-cytometry was 0.64% (range, 0.03%-1.36%) of total BM cells. After immunomagnetic column separation, a median number of 4.5 × 105 (range, 0.9 × 105 to 1.5 × 106) of CD34+ cells was collected, thus resulting in a yield of about 50% of the expected number. Purity of CD34+ enriched population resulted in a median value of 89% (range, 43%-89%). Regarding CD38 antigen expression, a median of 86% (range, 23%-97%) were CD34+CD38high cells, whereas a median of 14% were CD34+CD38low.

FISH Analysis of Whole BM and CD34+ Selected Population

Standard FISH analysis was performed in all 24 patients on at least 200 nuclei of whole BM cells, and no Ph+ cells were identified. When we examined the CD34+ selected population, in 15 of 24 (63%) patients, FISH analysis was performed on a median of 100 nuclei (range, 100-300 nuclei), whereas in 5 of 24 (21%) patients, the median number of nuclei analyzed was 75 (range, 60-86 nuclei). In 4 of 24 (16%) patients, FISH analysis was not performed due to the scarce yield of BM purified CD34+ cells, and consequently, the inadequate number of evaluable nuclei (less than 50).

We found residual CD34+Ph+ cells only in 1 of 20 (5%) evaluated patients. Of note, in this patient, a total of 140 CD34+ interphase nuclei were analyzed and only 1 of them was found to be BCR-ABL–positive (0.7%).

Early CD34+Ph+ Evaluation During Nilotinib Treatment

In 5 additional patients, we were able to evaluate CD34+Ph+ cells after only 3 months of first-line nilotinib therapy. All 5 patients (3 males, 2 females; median age, 59 years; range, 43-67 years) were in the chronic phase at diagnosis and were enrolled in the CAMN107EIC01 study (EUDRACT code 2009-017775-19) receiving nilotinib 300 mg bid. Conventional cytogenetic analyses were performed after 3 months of nilotinib treatment, and all 5 patients had achieved CCyR. At the same time, FISH analysis on purified (median purity, 87%; range, 82%-87%) CD34+ cells was performed, and after a median of 100 nuclei were evaluated (range, 100-200 nuclei), none of the 5 patients showed residual CD34+Ph+ cells.

Patient Follow-Up

A total of 23 of 24 patients continued nilotinib and were monitored on a routine basis for cytogenetic and molecular residual disease, whereas 1 patient switched to imatinib due to nonhematological toxicity. After a median time of observation of 10 months (range, 6-16 months) from CD34+Ph+ cell evaluation, all 23 nilotinib-treated patients were still in CCyR. Regarding molecular response, only 3 of 23 (13%) patients did not achieve a MMolR, 11 of 23 patients (48%) were in MMolR with a median value of BCR-ABL/ABL ratio of 0.02% (range, 0.01%-0.05%), and 9 of 23 (39%) patients were in CMolR. The only patient in which CD34+Ph+ progenitor cells were still detectable was in MMolR for 21 months at the time of CD34+ evaluation and was in CMolR at the last follow-up.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

The introduction of the second-generation TKI nilotinib in the therapeutic setting of CML may change the future goal of disease treatment. In fact when compared to imatinib, first-line nilotinib treatment has shown a higher and earlier rate of MMolR and CMolR.6, 7 On the basis of these data, the next task could potentially be treatment discontinuation in “optimal responders” without their experiencing, as happens with imatinib, at least a 60% probability of molecular recurrence.1 In this regard, during imatinib treatment, persistence of residual CD34+Ph+ progenitor cells has been documented in vivo, even in patients with prolonged CCyR and MMolR.2, 3, 13 Because nilotinib appeared not to be superior to imatinib in inducing growth inhibition of CML progenitor cells in vitro,9 it could also be incapable of eradicating leukemic stem cells in vivo. However, no data are available thus far on the persistence of Ph+ progenitor cells during nilotinib treatment in patients with CML.

To explore this issue, we checked the presence of residual CD34+Ph+ cells in a series of patients treated with nilotinib since diagnosis and in stable CCyR. Surprisingly, only 1 of 20 (5%) evaluable patients showed persistence of residual Ph+ progenitor cells at a negligible level, because just 1 of 140 (0.7%) CD34+ interphase nuclei analyzed by FISH was found to be BCR-ABL–positive. To our knowledge, this is the first time that the efficacy of nilotinib in reducing leukemic CD34+ cell burden has been evaluated in vivo. The results obtained in first-line nilotinib-treated patients are quite different from those we found previously in a fairly comparable series of CML patients in long-lasting CCyR during imatinib treatment. In fact, in the earlier study, about 45% of patients in CCyR for a median of 35 months still harbored a median of 1% of CD34+Ph+ cells.3 The difference we found in this study (1 of 20 patients with persisting CD34+Ph+ in the nilotinib-treated group vs 14 of 31 imatinib-treated patients) is significant, and it is even more remarkable considering that the median treatment length at the time of CML stem cell evaluation was much longer in the cohort of imatinib-treated patients compared with the nilotinib-treated cohort (39 vs 22 months). We do not necessarily imply that nilotinib fully eradicates CD34+Ph+ cells: our data may suggest that enhanced BCR-ABL kinase inhibition displayed by nilotinib induces a “deeper” suppression of CML progenitors and that it may be necessary to analyze a much greater number of CD34+ nuclei in order to reach a level of detectability.

For this purpose, it must be noted that the great majority of patients still presented residual disease at the molecular level. This may be explained by the persistence, however “undetectable,” of CD34+Ph+ cells and/or by the presence of more differentiated cells as the source of molecular disease. However, after a median of 10 additional months of treatment after CD34+Ph+ evaluation (ie, a median of 32 months of nilotinib therapy from diagnosis), only 13% of patients did not achieve MMolR, 56% of them were in stable MMolR, and 39% of them achieved CMolR.

In an additional 5 patients, we were able to evaluate CD34+Ph+ kinetics of reduction, because they were studied after only 3 months of nilotinib treatment. Surprisingly, together with achieving CCyR, all 5 patients already showed no detectable CD34+Ph+ cells. Despite the very limited number of patients studied, this may suggest that the fast inhibitory activity displayed by nilotinib on the bulk of CML affects stem cells as well. In conclusion, according to these preliminary results, the great majority of patients with CML who achieve CCyR with first-line nilotinb treatment do not show, unlike with imatinib-treated patients in CCyR, persistence of CD34+Ph+ cells. Even if these data appear in line with the overall better clinical results observed with nilotinib, their significance in the path to a cure for CML has yet to be determined. In fact, additional data and a longer follow-up are required to clarify if nilotinib is truly more efficient than imatinib in eliminating CML quiescent stem cells in vivo and if this will translate to a significantly higher number of nilotinib-treated patients who achieve CMolR and are able to discontinue the treatment without disease recurrence.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

We thank all additional authors who participated in the study: Michela Rondoni, Department of Hematology, University of Siena, Siena, Italy; Michele Baccarani, Department of Hematology/Oncology “L. and A. Seragnoli,” University of Bologna, Bologna, Italy; Alfonso Zaccaria, Santa Maria delle Croci Hospital, Ravenna, Italy; Renato Fanin, Hematology and Transplants Center, Udine, Italy; Valeria Santini, Hematology, AOU Careggi, University of Florence, Florence, Italy; Malgorzata Monika Trawinska, Department of Hematology, S. Eugenio Hospital, Tor Vergata University, Rome, Italy; and Giuliana Alimena, Department of Cellular Biotechnologies and Hematology, University of Rome “La Sapienza,” Rome, Italy. We thank Kathryn Smith for language revision.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURE

G. Rosti is a consultant with Novartis and Bristol-Myers Squibb. The remaining authors made no disclosure.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. FUNDING SOURCES
  8. REFERENCES
  • 1
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