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http://www.nature.co...l/2403752a.html

Durability of molecular remission in chronic myeloid leukemia patients treated with imatinib vs allogeneic stem cell transplantation (and also healthy individual RT PCR comparison)

T Lange¹, T Bumm¹, M Mueller², S Otto¹, H-K Al-Ali¹, L Grommisch¹, S Musiol¹, C Franke¹, R Krahl¹, D Niederwieser¹ and M W N Deininger³

1. ¹Department of Hematology and Oncology, University of Leipzig, Germany
   
2. ²Department of Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Germany
   
3. ³Center for Hematologic Malignancies, Oregon Health & Science University, Portland, OR, USA
   

Correspondence: Dr MWN Deininger, Center for Hematologic Malignancies, Cancer Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Mailcode L592, Portland, OR 97239, USA. Fax: +1 503 494 1688; E-mail: deininge@ohsu.edu

Received 27 July 2004; Accepted 21 February 2005; Published online 28 April 2005.

TO THE EDITOR

In CML patients treated with allogeneic stem cell transplantation, achievement of molecular remission (MR), defined as undetectable BCR-ABL transcripts by nested RT-PCR, is associated with a low risk of relapse.¹ Imatinib induces high rates of complete cytogenetic response (CCR) in CML patients in chronic phase,² giving rise to hopes that stable MR may be achievable with nontransplant therapy. However, the reported incidence of MR in patients on imatinib varies greatly across different studies, likely reflecting differences in the sensitivity of the PCR assay, patient selection and possibly imatinib dose. For example, in the randomized trial that compared imatinib with interferon-alpha (IFN-alpha)/cytarabine, less than 4% of newly diagnosed patients with CCR became RT-PCR-negative on at least one occasion.³ Similar, in a study of 120 patients treated with imatinib in late chronic phase the rate of MR was 0%.⁴ A recently published study in a comparable cohort of patients reported an MR rate of 11.5%.⁵ Since none of these studies addressed the durability of imatinib-induced MR or provided a side-by-side comparison with allografting, we directly compared two cohorts of patients treated with imatinib or allografting who were monitored at a single center with identical assays. For the purpose of this study, MR was defined as the first negative nested RT-PCR test at least 100 days post transplant (in the case of allografting) or as the first negative nested RT-PCR test after initiating imatinib therapy. The duration of MR was defined as the time from the first MR to loss of MR or last follow-up sample.

We identified 33 patients who underwent allogeneic transplantation with a myeloablative preparative regimen between January 1999 and December 2002, at the University of Leipzig, Germany. Patients were monitored by nested RT-PCR for BCR-ABL at a median interval of 8.8 months (range 3.7-33.8). In all, 16 patients were excluded because of death less than 6 months after the transplant (n=7), failure to achieve MR (n=7), or loss of follow-up (n=2). The median follow-up of the remaining 17 patients is 24.3 months. The imatinib group consisted of 144 patients treated for CML in first chronic phase (newly diagnosed n=24, after IFN-alpha treatment n=80), second chronic phase (n=4) or accelerated phase (n=36) in Novartis-sponsored multi-institutional studies. The initial imatinib dose was 400 mg daily for patients with chronic and 600 mg for patients with accelerated phase. In all, 64 patients (44%) achieved CCR as determined by conventional cytogenetics. Furthermore, patients were also routinely monitored by quantitative RT-PCR (qPCR)⁶ at a median interval of 5.8 months (range 1.3-12.2). Negative qPCR results were followed with conventional nested PCR. All 19 patients with serial follow-up (Figure 1a) received continuous imatinib therapy between the first and last time points, and no changes to the dose were made during this interval with the exception of patient #15, who received 800 mg imatinib from month 6 to 15 for a rising platelet count.

Figure 1.

(a) Sequential RT-PCR results in 19 patients treated with imatinib who had undetectable BCR-ABL transcripts on at least one occasion. The dose of imatinib in these patients was constant throughout the observation time, with the exception of patient #15 who received 800 mg between months 6 and 15 for a rising platelet count. ( Sequential RT-PCR results in 17 patients who achieved MR after allogeneic transplant with a myeloablative preparative regimen. © Duration of molecular response in patients on imatinib and after allografting from the time of the first negative test. From the time of first MR, 43 samples were available from patients on imatinib and 32 from patients after allografting. The median follow-up was 11.8 (range 3.0-20.3) months in patients on imatinib and 18.0 (range 4-46) months in patients after allografting. The duration of MR was significantly shorter in patients on imatinib (P=0.0001, log rank). (d) Duration of RT-PCR negativity by replicate testing. After the first negative replicate test, 20 samples were available from nine patients on imatinib (median follow-up 11.1 (range 3.5-18.3) months) and 26 samples from 15 patients after allograft (median follow-up 26.0 (range 5.8-45.9) months). The duration of negativity by replicate testing was significantly shorter in patients on imatinib (P=0.0002, log rank).

Full figure and legend (79K)

Since the sensitivity of RT-PCR is dependent on the quality of the cDNA, we measured the expression of GAPDH as a control gene. The median level of BCR-ABL transcripts in the allografted patients was 4.5 10⁵/l cDNA (range 6 10³-2.1 10⁶) and 5 10⁵/l cDNA (range 1 10⁴-1.3 10⁶) in the patients on imatinib. In order to further increase sensitivity, samples negative by nested RT-PCR were further analyzed with 10 individual replicate nested RT-PCR reactions. Using this method, a sample was scored positive if at least one of 10 reactions was positive. The performance of the RT-PCR assays was tested in limiting dilution experiments, using K562 cells or peripheral blood white cells from two CML patients mixed with white blood cells from a healthy donor. At the time of this analysis, the two CML patients were being treated with imatinib, but their bone marrow was 100% Ph-positive by cytogenetics. While the detection limit of the nested RT-PCR assay for the dilution of K562 cells was in the range of 1:10⁶, the sensitivity for the dilutions of patient cells was only 1:10⁴ and 1:10³, respectively (Table 1). With the replicate tests, the sensitivity could be increased by one order of magnitude in the case of K562 cells and one patient.

Table 1 - Sensitivity of tenfold replicate PCR compared to nested PCR in K562 cells and two patients with CML CP in complete hematologic remission to imatinib.

Full table

In all, 19 patients on imatinib (all in CCR) achieved MR on at least one occasion, with the first negative result noted after a median of 16.8 (range 11.5-36.1) months of treatment (Figure 1a). Also, 17 patients achieved MR, at a median of 6.6 months after allografting (Figure 1b). The projected risk of molecular relapse at 12 months after the first negative RT-PCR result was 83% in patients on imatinib, but only 20% in patients after transplant (P=0.0001, Figure 1c). As it is conceivable that the likelihood of detecting residual disease may increase with the frequency of testing, we reasoned that the lower density of follow-up in the transplant cohort may exaggerate the difference between the two groups. However, the frequency of positive RT-PCR results after the first MR was overall higher in patients on imatinib than after allografting (25/43 vs 3/32, P=0.001, ² test), suggesting that the higher density of tests alone is unlikely to account for the difference. Only two patients (#6, #10) on imatinib remained in MR at 13.8 and 16.6 months (Figure 1a). While none of the three patients with molecular relapse after allograft lost CCR, one patient on imatinib (#15) progressed to cytogenetic relapse.

The higher rates of molecular relapse in patients on imatinib suggested a higher level of residual disease at the time of MR. The sensitivity of nested PCR can be increased by performing replicate PCR reactions from the same sample; with this technique BCR-ABL transcripts become detectable in some normal individuals.⁷ Experiments detailed in Table 1 confirmed this assumption in principle. However, it is worth noting that the sensitivity of the nested PCR in the dilutions of patient cells was 2-3 orders of magnitude lower than in dilutions of K562 cells. This is likely to reflect higher levels of BCR-ABL mRNA in K562 cells compared to patient cells, at least in the two individuals analyzed here who were in complete hematologic remission at the time of the experiment. We performed 10 replicate reactions (corresponding to a total of 10⁷ cells) on 45 samples from 17 patients after allografting, 36 samples from 19 patients on imatinib and 20 healthy controls. The replicate assay was positive in 18/36 samples (50%) from patients on imatinib, 8/46 (17.4%) after allografting and 4/20 (20%) from healthy individuals (Table 2). The differences were significant between patients on imatinib and after allografting (P=0.003, ² test) and between patients on imatinib and healthy individuals (P=0.005, ² test), but not between patients after allografting and healthy individuals (P=0.9, ² test), indicating a higher level of residual disease in patients in MR to imatinib than after allografting. In contrast, the frequency of positive samples in transplanted patients was not different from the background observed in healthy controls (20%). Similar rates have been seen in previous studies of healthy volunteers.⁷ Negativity by replicate testing was more stable in patients after allografting, although, even in these patients, positive replicate reactions continued to occur with longer follow-up (Figure 1d). As we did not serially follow normal volunteers, it is not known if this pattern would also be seen in the controls.

Table 2 - Results of replicate RT-PCR tests in patients treated with imatinib, allografting and in healthy individuals.

Full table

Our results demonstrate that MR is less durable in patients treated with imatinib than after allografting. Thus, from the time of first MR, the burden of residual disease may further decrease in transplanted patients, while it plateaus or increases in most patients on imatinib. Although these findings may be influenced by a higher density of BCR-ABL tests in the imatinib group, they are consistent with results from patients monitored by qPCR.³ In addition, more patients on imatinib than after allografting tested positive in replicate PCR reactions. Our data add to a growing body of evidence that disease eradication, at least with standard dose imatinib monotherapy, may be rare. Patients on imatinib should be made aware of the fact that a single negative RT-PCR test does not have the same significance as in patients after allografting. In addition, correct interpretation of the test results requires knowledge of the test's sensitivity and stringent controls for the quality and quantity of RNA assayed are crucial. It is indeed questionable whether in patients on imatinib a single negative RT-PCR test should be termed MR at all. It should be noted that we accepted a lower limit of GAPDH (6022 copies/l cDNA) expression than, for example, Scheuring et al, who used 20 000 copies/l as the cutoff.⁸ It is thus possible that the overall rate of positive results in our study would increase if samples with relatively low GAPDH expression were eliminated. However, given that GAPHDH expression is comparable between patients on imatinib and after allografting, this would not affect the comparison between the two groups. Prospective studies will be needed to determine how many sequential negative RT-PCR reactions predict for long-term negativity and long-term freedom from progression. Conversely, single positive tests may represent fluctuations due to technical reasons, for example, variations in the amount of input cDNA, and should thus not be over-interpreted.