Annals of Oncology Advance Access originally published online on July 10, 2006
Annals of Oncology 2006 17(10):1553-1559; doi:10.1093/annonc/mdl150
© 2006 European Society for Medical Oncology
hematologic malignancies |
Real-time PCR analysis of PML-RAR
in newly diagnosed acute promyelocytic leukaemia patients treated with arsenic trioxide as a front-line therapy
Hematology, Oncology and BMT Research Center, Tehran University Medical Sciences, Tehran, Iran
*Correspondence to: Dr S. H. Ghaffari, Hematology, Oncology and BMT Research Center, Tehran University Medical Sciences, Shariaty Hospital, Tehran, Iran. Tel: +98-21-84902638; Fax: +98-21-8004140; E-mail: shghaffari2000{at}yahoo.com
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Abstract |
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Background: Recently, patients with acute promyelocytic leukaemia (APL) have experienced significant clinical gains after treatment with arsenic trioxide. However, the use of this agent as a front-line therapy for newly diagnosed patients is unclear.
Patients and methods: Of 95 newly diagnosed APL patients, 85 patients who achieved complete remission (CR) were sequentially evaluated during a 4–60 month period by conventional RT-PCR. A total of 30 patients (six relapsed and 24 in continued CR) were selected and monitored by quantitative real-time PCR (RQ-PCR) assay. The PML-RAR
fusion transcripts values were normalised to every 106 copies of G6PDH transcripts (NQ).
Results: RQ-PCR analyses showed a rapid rate of clearance of NQ levels during the courses of arsenic therapy. In the majority of patients in CR, the NQ levels were below 5 x 102 in peripheral blood (PB) samples. In all the relapsed cases with follow-up intervals of 1–6 months (median 3 months) clinical relapse was predictable by increasing NQ level above this threshold.
Conclusions: Our study highlights the usefulness of PB and the definition of threshold level for early prediction of relapse. The threshold level correlates well with risk of relapse; therefore, transcript ratio below the level should be regarded as a goal in the clinical management of this disease.
Key words:
arsenic trioxide, RQ-PCR, MRD, PML-RAR, APL
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Acute promyelocytic leukaemia (APL), AML-M3 subtype, was once considered the most devastating subtype of acute myeloid leukaemia because of its associated coagulopathy, fibrinolysis and rapid death due to internal bleeding. It is now the most treatable of all subtypes as a result of intensive research into the molecular pathogenesis. This research has led to a rational approach to treatment in which the use of differentiating agent all-trans-retinoic acid (ATRA) has proven to be an effective first-line treatment for inducing complete remission [1–4]. In spite of these advances, one-third of the patients would relapse and the majority of these patients would lose sensitivity to ATRA and chemotherapy [1, 3]. Recently, arsenic trioxide (As2O3) has been successfully added to therapeutic strategies for APL and it has been proved highly successful in the treatment of relapse, including patients resistant to other treatment modalities [4–7]. More recently there has been interest in determining whether this agent could have a role to play as potential first-line therapy [8, 9] or in combination with other agents, such as ATRA [10]. We have shown during the last 5 years that treatment of our APL patients with single-agent arsenic trioxide as a first-line therapy induces a high percentage of complete clinical remission (CR) and the majority of these patients remain in CR for long periods of time [11].
Molecular monitoring of residual leukaemia by qualitative and quantitative RT-PCR techniques has now provided important information about the effectiveness of treatment and the risk of recurrent disease as shown by minimal residual disease (MRD) analysis in patients who relapsed compared with those who stay in long CR. Several groups have published data regarding detection of PML-RAR using real-time RT-PCR in mostly ATRA-treated APL patients [12–17]. However, only limited data has been reported in arsenic-treated APL patients, especially in newly diagnosed patients [18, 19]. These studies were hampered by a limited number of cases and in very few cases a molecular relapse was demonstrated to precede the clinical relapse. Overall, the implications of the detection of residual disease after treatment of APL patients with arsenic trioxide are unclear. We conducted qualitative and quantitative real-time PCR studies to determine the predictive value of PML-RAR transcript levels at various time points after induction of CR. Our results suggest the existence of threshold above and below which patients generally have relapse or remain in CR, respectively.
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patients and methods |
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patients and samples
A total of 95 APL patients (all new cases; 42 males and 53 females; age range 8–60 years) undergoing treatment from May 2000 to July 2005 were enrolled in this study at the time of diagnosis. The diagnosis of APL (AML-M3) was established on the basis of clinical presentation, morphological criteria of the FAB classification, cytogenetic evaluation for t(15; 17), fluorescence in situ hybridisation (FISH) for PML-RAR
translocation, and reverse transcription polymerase chain reaction (RT-PCR) analysis for PML-RAR transcripts. Patients received arsenic therapeutic protocol during the induction and consolidation periods. Written informed consent was obtained and the protocol was reviewed and approved by the institutional review board. Following diagnosis, 85 patients who achieved complete remission were monitored by conventional RT-PCR and from those 30 patients were selected and monitored by real-time PCR. Serial peripheral blood (PB) samples were analysed at presentation, after induction and consolidation therapy, and during remission.
treatment with arsenic trioxide
The arsenic trioxide (As2O3) solution was manufactured by the department of pharmacy affiliated with our research centre and was supplied in 10-ml vials containing 1 mg/ml. The drug was given intravenously daily (0.15 mg/kg of body weight) until the patient had achieved a complete remission or for a maximum of 60 days (induction). The patients were monitored carefully during these times. A second course was preformed after an interval of 28 days for a period of 28 days (consolidation). For more patients' characteristics, treatment details and survival rate, see Ghavamzadeh et al. [11].
RNA isolation and cDNA synthesis
Mononuclear cells (MNC) were isolated from PB by density gradient centrifugation on Ficoll-Hypaque. Total RNA was extracted from 107 MNC and from the NB4 cell line by TRIZOL (Gibco-BRL, Gaitherburg, MD). RNA quality was assessed visually by conformation of intact 28 S and 18 S ribosomal bands following agarose gel electrophoresis and ethidium bromide staining. The cDNA was synthesised using First Strand cDNA synthesis kit (Fermentas UAB, Lithuania). Briefly, 5 µl (1–2 µg) of RNA was RT into cDNA in a 20-µl reaction for 10 min at 25°C and 60 min at 42°C using 25 ng/µl random hexamer, 20 U of RNase inhibitor, 25 µm dNTP and 200 U of MMLV-RT according to the manufacturer's instructions.
PCR condition
Qualitative RT-PCR was routinely performed on all the patients' samples (450 samples) taken at the time of diagnosis and during the follow-ups. First and second round (nested) PCR amplification was performed using the procedure described by Biondi et al. [20]. Briefly, in a 20-µl reaction mixture containing 2 µl of the cDNA (100 ng of RNA equivalent), 1 U Taq DNA Polymerase (Fermentas), 20 pM of each first-round primers, 1 mM dNTP, 1.5 mM MgCl2 and 1x buffer (50 mM KCl, 10 mM Tris-HCl (PH 9.0) 1% Triton X-100). PCR was carried out for 35 cycles (30 s at 94°C, 45 s at 64°C and 45 s at 72°C). A second round of amplification (nested RT-PCR) using 1 µl of the first reaction product and a set of nested primers was performed using the same program.
The PCR products were analysed on 2% agarose gel and visualised by ethidium bromide staining. The integrity and the quality of RNA and the efficiency of cDNA synthesis were analysed by amplification of the ß2-microglobulin gene as an internal control. A positive control (cDNA derived from the NB4 cell line) and a negative control were included in each experiment. All tests were conducted at least in duplicate to confirm the results. Strict precautions were taken to prevent any contamination including the use of specifically PCR-designed UV flow cabinets, pipettes and filtered tips. All pre-PCR manipulation (RNA isolation, cDNA synthesis and PCR set-up) were performed in a room isolated from the post-PCR processing area.
real-time PCR using hybridisation probes
Quantification of PML-RAR and G6PDH (glucose-6-phosphate dehydrogenase) transcripts were carried out on MNC by real-time PCR with Roche LightCycler Instrument (Roche Diagnostics GmbH, Mannheim, Germany). All PCR reactions were performed using LightCycler t(15;17) Quantification Kit (Roche Diagnostics) in a total volume of 20 µl containing 1x Taq polymerase buffer, 4 mM MgCl2, 200 µM dNTP, 300 nM each primer, 200 nM hybridisation probes, 1.25 U FastStart Taq DNA polymerase and 2 µl of cDNA (100 ng of RNA equivalent). The thermocycler condition were 10 min at 95°C, followed by 45 cycles, each at 95°C for 10 s, 60°C 10 s and 72°C for 10 s. The primers for t(15;17) and the reference gene G6PDH provided by Roche are designed to span exon–intron boundaries to prevent occurrence of co-amplification of genomic DNA. One hybridisation probe is labelled at the 5'-end with Red 640 and modified at the 3'-end by phosphorylation, and the other probe is labelled at the 3'-end with fluorescein. The functional sensitivity of the assay is <10 copies of the PML-RAR fusion transcript positive cells in a background of 1 x 106 PML-RAR negative cells. The linear measuring range of the assay is 0.002–200 ng mRNA [21].
The PML-RAR fusion transcripts values obtained by RQ-PCR were normalised with respect to the number of G6PDH transcripts and expressed as PML-RAR fusion gene copy number every 106 copies of G6PDH (normalised quotient, NQ):
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patients analysed by RT-PCR
After a course of induction and consolidation therapy with arsenic trioxide, a complete remission was achieved in the 85 (89.5%) patients with APL. During the post-treatment follow-up (range 4–60; median 21), 20 patients relapsed (R1) between 4 and 33 months (median 17) after presentation; 15 of them achieved a second complete remission (CR2) after retreatment with arsenic. Five of these patients had a second relapse (R2) after 11–17 months (median 14.3) in remission; three of them achieved CR3; two patients had a third (R3) and one patient had a forth relapse (R4). All patients were RT-PCR positive at the time of diagnosis, the conversion to PCR negativity was observed 1–3 months after the start of therapy in the majority of patients as detected by conventional RT-PCR. Of 450 samples tested by conventional RT-PCR, beside presentation and relapse samples, only 20 samples became RT-PCR-positive. Of all the 28 relapse events (including R1–R4), only 14 (50%) relapses were preceded by PCR-positive results and in 14 relapse events the conventional PCR was uninformative.
patients analysed by real-time PCR
Of 85 APL patients, 30 patients (six relapsed and 24 in continued remission) were selected to study with quantitative real-time PCR. The selection for this study group was based solely on long follow-up period and availability of sequential samples; the RQ-PCR was retrospectively performed on stored samples of these patients. For the 24 patients who remained in CR and never had relapses, the median follow-up period was 29 months (range 20–60). Six patients had first (R1) relapse between 11–32 months (median 21) after presentation. Four of the six relapsed patients achieved CR2 after the second course of arsenic treatment in 30 days that was comparable with the time in patients who achieved a first CR. Three of these four patients had a second relapse (R2) after 11–17 months (median 13) in remission. One patient (no. 6) had third (R3) and fourth (R4) relapses. One patient (no. 3) also had a relapse in the central nervous system (CNS) with less than 5% promyelocytes in BMA and no detectable transcript in the PB sample; after 8 months he had a relapse in both of these sites. The clinical and haematological characteristics of the patients monitored with real-time PCR are shown in Table 1.
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PML-RAR
isoformsThe PML-RAR
isoforms (L- and S-forms) were identified by RT-PCR in all 30 patients at presentation. The L-form (bcr1) was detected in 16 (53.3%) patients, 13 remain in CR and three relapsed. The S-form (bcr 3) was detected in 14 (46.7%) patients, 11 remain in CR and three relapsed. There was no statistically significant difference in the incidence of the PML-RAR isoform types between the relapsed and the CR group. The patients with bcr2 breakpoints were excluded from this study. The sensitivity of the assay was evaluated by generating standard curves from 10-fold dilution series of RNA concentrations. Standard curves were then constructed plotting the Ct versus the known copy number of each standard sample for L-, S- or G6PDH. The standard curves were saved in a standard-curve file. In all the following runs, a reference dilution was analysed and the standard curve loaded over this reference sample. Using the standard, as few as 10 copies of L- or S- form DNA could be reproducibly detected and the sensitivities achieved for S- and L-form were similar. The specificity of the assay was evaluated by testing 15 non-APL samples. No detectable signal was found with either L or S primer/probe set. The integrity of the RNA from each sample was confirmed by simultaneous G6PDH amplification.
evaluation of PML-RAR NQ levels at different phases of disease
The PML-RAR NQ level from different phases of APL were analysed in terms of samples obtained at the time of diagnosis (D), during the induction and consolidation therapy, complete remission (CR), up to the 6 months before relapse (BR) and at the time of relapse (R) (Figure 1). The differences in the level of NQ among the groups were highly significant (P <0.0001), as was determined by the Kruskal–Wallis test. The levels of NQ at the time of presentation or relapse were found to be in the similar range of 1 x 105 – 8 x 105 in PB. In more than 90% of the patients in continued remission, the level of NQ was found to be less than 5 x 102 in the PB samples (Figure 1).
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) and second (•) relapses. Each box represents a patient and numbers on the top of the box represent patients' number, the number on the left represent months before relapse. The values are expressed as PML-RARkinetics of reduction during the treatment
At presentation, the levels of PML-RAR
NQ were found to be in the range of 105–106 in PB. A log folds inter-individual difference in transcript levels was observed at diagnosis in patients. By more frequent sampling of some of the patients during the course of therapy, we observed a rapid decline in NQ level by more than one log after the induction therapy and more than 3–4 logs after the consolidation therapy in more than 90% of the patients. Figure 2 illustrates a typical example of such cases with a rapid reduction during the course of arsenic therapy. There was some variation in the degree and the rate of reduction during the induction time in some patients (not shown), presumably because of the hyperleukocytosis.
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PML-RAR
/G6PDH transcript level during follow-upThe level of MRD was analysed during the follow-up at various time points, usually at every 3-month interval. Serially monitored NQs of individual cases showed marked heterogeneity of pattern in PML-RAR
/G6PDH transcript expression level. In the patients who achieved remission, the NQ level generally declined to the lowest level after the consolidation treatment. However, the majority of patients continued to express detectable levels of fusion transcript even in long-term remission. Figure 3 illustrates the example of such cases that after a rapid reduction during the courses of arsenic therapy, the patient persisted in >25 months of CR, despite the intermittent NQ-positivity.
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early detection of relapse
By analysing the relapse incidence at different time points in the follow-up samples during the CR, we were able to divide the samples in two different groups based on NQ levels in order to identify high risk versus low risk groups for the possibility of a relapse. The 60 samples taken from patients in continued complete remission all had a NQ level less than 5 x 102. On the other hand, of the 11 samples taken from six relapsed patients during 1–6 months before relapse, nine samples had an NQ level greater than 5 x 102 (see Figure 1). Therefore, this data strongly suggest the existence of a cut-off point at approximately 5 x 102 above which a risk of relapse is high and beneath which shows the patients are in CR.
Figure 4 illustrates sequential monitoring of PML-RAR NQ levels in PB samples of patients who had first relapse (Figure 4A) and of those who had first and second relapses (Figure 4B). Figure 5 illustrates a patient (no. 6) who had a third and a fourth relapse (samples were not available from first and second relapses for RQ-PCR analysis). This patient had persistently high levels of transcript over the entire period between the third and fourth relapses (during the CR4) (Figure 5).
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With follow-up interval of 3 months or less, clinical relapses were detectable in all nine relapse events (including five R1, two R2, one R3 and one R4) by increasing NQ levels above the cut-off point. With a follow-up interval of 6 months or less, seven of nine relapses were detectable. A relapse was not detectable beforehand in one patient (Figure 4B, patient no. 5) with a follow-up interval of 4 months and 6 months before R1 and R2, respectively, and the NQ levels were below the threshold point. The time between molecular relapse and manifestation of clinical relapse was very short. The NQ level increased from 0 to 106 (6 logs) in 4 months and from 7 x 102 to 106 (3 logs) in 2 months during the first relapse, and with almost the same rate of disease recurrence during the second relapse.
multivariate analysis
The distribution of age, sex, WBC counts, platelet counts, Hb levels and isoform types (L/S) did not differ significantly between the group of patients who had subsequent relapses and those who remained in continuous CR (Fisher's exact and Mann–Whitney tests; P > 0.05) (Table 1). We also analysed the relationship of NQ levels and other factors at each time-period with the risk of relapse at presentation, CR and times before relapses using multiple logistic regression analysis. The results showed that the NQ levels 1–6 months before relapse were the only significant independent parameter for the early detection of relapse (P < 0.001). The statistical analyses were calculated by SPSS 11.0 software (SPSS, Chicago, IL).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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We have shown in the last 5 years the potential use of arsenic trioxide as a single-agent therapy for the treatment of newly diagnosed APL [11]. Our studies shows that most of newly diagnosed APL patients (
86%) can achieve durable CR after two courses of arsenic therapy and that many can achieve molecular remission as detected by conventional RT-PCR. However, qualitative analysis of PML-RAR was of limited value for predicting relapse in APL patients. In our hand, the RT-PCR assay with a sensitivity limit of 1 in 103–104 failed to detect residual disease in the majority of patients tested at the time before relapse. Approximately 50% of relapses were preceded by PCR-negative results. This could be a reflection of the relative insensitivity of the assay for analysis of APL cases that has been ascribed mostly to the relative low expression of PML-RAR [22, 23] and possibly to the use of PB in our cases. On the other hand, quantitative PCR has provided an excellent opportunity for specific and sensitive MRD detection in APL patients. In all of our follow-up cases, quantitative PCR was well correlated to clinical evolution and appeared to give earlier prediction of clinical outcome than the classical PCR.
Studying the kinetics of MRD during the therapy in PB of APL patients shows a significant reduction in the level of PML-RAR NQ level after induction and consolidation therapy with arsenic compared with the level at presentation. We have observed a decline in NQ level by more than one log after induction therapy and more than 3–4 logs after consolidation therapy (Figure 2). Our data and that of others indicate that reduction of PML-RAR fusion transcripts and achievement of PCR-negativity (by conventional RT-PCR) is an important therapeutic goal in APL since persistence of fusion transcripts until the end of consolidation therapy has been found to be highly predictive of relapse [24, 25]. On the other hand, the majority of our patients who ultimately relapsed have been tested RT-PCR negative at the end of consolidation therapy. In our follow-up study by RQ-PCR, all six patients who eventually relapsed had a decline in NQ level by more than 3–4 logs after consolidation therapy and were all negative by conventional RT-PCR. These observations presumably indicate that the rate of clearance of transcript levels and achievement of PCR negativity is not necessarily associated with long-term CR. This agrees with the notion that achievement of PCR negativity cannot be equated with cure and PCR assessment as this single time point cannot be relied upon to determine the optimum treatment approach in individual patients [24–26].
The major accomplishment of our study was that we were able to establish a threshold of transcript level in the post-consolidation period. The PML-RAR NQ level of 5 x 102 was defined as the threshold level; above this level suggests that patients are in high risk of relapse and below this level shows patients are in CR. In all relapse cases with follow-up intervals of 1–6 months (median 3) a clinical relapse was predictable by increasing NQ levels above the threshold level. Data analysis by Spearman's correlation also shows that higher levels at the time of increase are correlated with a more rapid manifestation of the haematological relapse (P = 0.007). Therefore, the transcript ratio below the threshold level should be regarded as a goal in the clinical management of this disease; above this value the patient should be considered for therapeutic intervention. Indeed, it has been shown that pre-emptive administration of salvage therapy at the time of molecular relapse can result in a second molecular remission [27]. Thus, by frequent sampling and RQ-PCR monitoring, patients at high risk of relapse can be identified and therapeutic intervention can be started at an early stage when the tumour burden is still low.
In patients who achieved remission, serially monitored NQs of individual cases showed marked heterogeneity of pattern in PML-RAR transcript expression. The transcript level generally declined to the lowest levels after the consolidation treatment (Figure 3). However, only half of the samples during this period tested negative by the RQ-PCR assay and the majority of patients continue to express a detectable level of fusion transcripts even in long-term remission. While most studies have found a strong correlation between PCR-positivity and relapse on APL patients, our data and a few recently published data on treatment of APL with ATRA [16, 28] show a high frequency of MRD-positive patients who do not relapse. An explanation of this may be the difference in sensitivity of the MRD detection method applied. This is of interest because it suggest that patients with APL who are considered to be cured of their disease may still have evidence of residual disease, in accordance with data previously acquired in patients with t(8;21)-associated AML [29–31]. This supports the notions that the interplay between residual APL disease and the host is a dynamic process [16]; the immunological mechanisms may play a role in maintaining remission in patients with APL [29] and relapse occurs from a subpopulation of APL cells that undergoes further change(s) leading to clonal escape from host control mechanisms [16].
Molecular monitoring of patients with frequent clinical relapses shows that some of these patients may never achieve molecular remission between their relapses and that their NQ levels persist above the threshold level. Figure 5 illustrates the example of such a case; this patient appeared to have acquired drug resistance and to have become refractory not only to arsenic but also to ATRA and chemotherapy. This indicates that although a patient with a relapsed APL has a good chance of achieving second, third or fourth remissions with arsenic, frequent disease recurrence in some patients may be associated with high frequency of refractory leukaemia and with a shorter CR period.
In summary, this study shows that most of newly diagnosed APL patients can achieve durable CR after two courses of arsenic therapy and that many can achieve molecular remission as detected by conventional RT-PCR. The RQ-PCR study shows that there is a rapid kinetic of clearance of PML-RAR NQ level (3–4 logs reduction) during the courses of arsenic therapy. Serial monitoring of MRD showed that in all patients in continued remission the level of PML-RAR NQ was always below 5 x 102 in the PB samples. This level of NQ was defined as the ‘threshold’ level in the post-consolidation period, which was highly correlated with risk of relapse. In the majority of the relapsed cases with follow-up intervals of 1–6 months (median 3) a clinical relapse was predictable by increasing the NQ level above the threshold. This study also indicates that PB is a reliable source of MRD monitoring in APL patients and can be performed more easily and frequently with less patient discomfort.
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Acknowledgements |
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We would like to thank all clinicians and nurses who participated in this study and provided material for this investigation. In particular we acknowledge Drs Jahani, Mosavi, Bibordi, Bahar and Irvani. We would also like to thank Dr Hossaini for assistance in manufacturing arsenic trioxide and Ms Ashori and Drs Shamshiri and Totonchi for assistance in the statistical analysis.
This study was supported by grants from Hematology, Oncology and BMT Research Center, Tehran University Medical Sciences.
Received for publication March 17, 2006. Revision received May 9, 2006. Accepted for publication May 22, 2006.
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  S. H. Ghaffari, N. Shayan-Asl, A. H. Jamialahmadi, K. Alimoghaddam, and A. Ghavamzadeh Telomerase activity and telomere length in patients with acute promyelocytic leukemia: indicative of proliferative activity, disease progression, and overall survival Ann. Onc., June 20, 2008; (2008) mdn394v1. [Abstract] [Full Text] [PDF]
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