To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive ( n =43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation ( n =13) revealed a 40-fold variation between patients (range, 0.027–1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1–2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3–17) of starting treatment, with a sensitivity of detection of up to 1 in 10 5 . One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

About 75% of patients with newly diagnosed chronic phase (CP) chronic myeloid leukemia (CML) treated initially with imatinib achieve complete cytogenetic remission (CCyR),1 and imatinib also induces Ph negativity, though less often, in patients treated in advanced phases of CML. Patients in CCyR may however still have as many as 1010 leukemia cells in their body, 2 and in CMLthe quantity of such “residual disease” can be monitored by quantitating BCR-ABL transcripts. 3 We have studied serially BCR-ABL transcript numbers in a series of CML patients treated with imatinib who achieved CCyR. The results confirm that they constitute a heterogeneous group whose individual prognoses may be very different. The failure to detect BCR-ABL transcripts on repeated testing was rare. We identified 42 CML patients (37 originally in CP, 5 in accelerated phase [AP]) treated with imatinib at 8 collaborating United Kingdom centers who were classified as having achieved CCyR on the basis of 100% Ph negativity in at least 20 marrow metaphases. At the time of starting imatinib the patients were newly diagnosed (n 15), chronic phase refractory to interferon- (n 24), or in relapse after stem cell transplantation (n 2). The median age was 52.3 years (range, 24.869.9 years), and median time to achieve CCyR was 5.5 months (range, 1.8-16.4 months). The daily dose of imatinib was 400 mg (CP) or 600 mg (AP). Serial quantitative polymerase chain reaction (Q-PCR) assays were performed at regular intervals at a single center using Taq-man technology (Applied Biosystems, Foster City, CA). 4 The median number of samples per patient was 5 (range, 3-15 samples). Results were expressed as a percentage ratio using ABL transcripts as the internal control. The failure to detect BCR-ABL by Taq-man assay was confirmed with a 2-step nested technique as previously described.5 The median BCR-ABL/ABL ratio was 0.19% (range, 0.0%-13.1%) for the first sample after achieving CCyR; only 2 patients had ratios above 2.0% (Figure 1). During serial monitoring thereafter 17 (40%) patients (15 originally in CP; 2 originally inAP) achieved a BCR-ABL/ ABLratio less than 0.02% on at least 2 occasions 4 or more weeks apart. Only 1 of the 17 patients who achieved a ratio less than 0.02% later lost the CCyR and progressed to hematologic relapse. Thirty-three patients always had detectable BCR-ABLtranscripts. In 7 cases transcripts were undetectable on at least one occasion but positive on others. Two patients had no detectable transcripts on serial monitoring for at least 6 months; in one case both Taq-man and nested PCR studies were negative on 3 occasions over 11 months, and in the other, 3 sequential specimens were negative over 6 months. In summary we have shown that Q-PCR values in CML patients in CCyR are very variable. The failure to detect any transcripts, as described by Barbany et al 6 in one patient, was a rare occurrence and frequently was not sustained. We suggest that the term these authors employed, “molecular remission,” is imprecise since it depends very much on the sensitivity of the assay employed. It would be preferable to refer to responding patients as having transcripts undetectable for a specified period. Our data support the recommendation that quantification of BCR-ABL transcripts is essential for optimum management of CMLpatients who achieve CCyR on imatinib. 7

In the May 1, 1999 issue of Blood, Faderl et al1 discuss the possible clinical role of polymerase chain reaction (PCR) analysis in the surveillance of residual disease in patients with chronic myelogenous leukemia (CML). Faderl et al1 raise questions regarding the significance of positive PCR reactions in patients who are in clinical and cytogenetic remission and express doubts about the possibility of using PCR analysis as a basis for therapy decisions. They indicate that a major drawback of current PCR studies is the lack of quantification of PCR data and that the short follow-up duration of published studies makes it difficult to interpret the results. The conclusions made are questionable in view of the fact that the investigators do not discuss any of the reports published by different groups on the monitoring of the dynamics of residual disease in CML patients by serial quantitative PCR analysis. 2-7 Numerous …

Lymphocyte transfusion from the marrow donor (DLT) is well established as an effective therapy for relapse of CML post allogeneic BMT. Reports thus far have been mostly limited to patients who received DLT from a matched sibling donor. We compared the efficacy and toxicity of DLT in 30 patients who were treated with cells from their HLA-identical sibling ( n = 18) or from their phenotypically HLA-matched unrelated marrow donor ( n = 12). The overall probability of obtaining a cytogenetic remission was 69% (95%CI: 51–83%) and was not significantly different between the two groups. The disease stage at the time of DLT was the only factor associated with cytogenetic remission by multivariate analysis; patients treated in cytogenetic or molecular relapse ( n = 11) were seven times more likely (RR = 7.4, 95%CI: 2.4–22.4, P = 0.0005) to respond compared to patients treated for hematologic relapse ( n = 19). There was a trend towards more acute GVHD II–IV in the unrelated donor group (58 vs 39%, P = 0.09), but the probability of developing extensive chronic GVHD was not significantly different (56 vs 39%, P = 0.4). We conclude that transfusion of donor cells from HLA-matched volunteer donors does not appreciably increase the risk of GVHD compared with transfusion of cells from HLA-identical siblings in patients with CML who relapse following allogeneic BMT. Conversely, there is no evidence for an increased graft-versus-leukemia effect after DLT from volunteer donors.

Interferon alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 398 samples from 163 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as internal control. The BCR-ABL/ABL ratios correlated with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

We have assessed the molecular response of 30 consecutive patients with chronic myeloid leukaemia (CML) treated for relapse after allogeneic bone marrow transplantation (BMT) by donor leucocyte transfusions (DLT). Response was evaluated by qualitative nested and quantitative competitive RT‐PCR for BCR‐ABL mRNA at various time intervals before and after DLT. The probability of attaining molecular remission at 2 years was 61% (95% CI 42–78%). Disease state at the time of DLT was significantly associated with response: molecular remission was achieved for 9/10 (90%) patients treated early (cytogenetic or molecular relapse) compared to only 8/20 (40%) patients treated late (haematological relapse; P=0.009). The Kaplan–Meier estimates of molecular remission at 2 years post DLT for patients treated in early or late relapse were 86.6% and 47.3% respectively (P=0.004). The median time interval from DLT to molecular remission was 11.0 months (range 2.5–32). Molecular remissions were durable for most (15/17) patients (median follow‐up 21.2 months; range 0–55). Two patterns of molecular response were found: a very rapid decline after an initial lag phase or a more gradual decline over a period of several months. We conclude that molecular monitoring is a sensitive indicator of response to DLT; different kinetics of molecular response may reflect disease heterogeneity or differences in the mode of action of DLT.

Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 305 samples from 133 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as an internal control. The BCR-ABL : ABL ratios correlated well with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

To test whether patients in remission after allogeneic bone marrow transplantation (BMT) possess a pool of chronic myeloid leukemia (CML) cells that do not express BCR-ABL mRNA, we have compared the results and sensitivity of amplification of BCR-ABL from genomic DNA with conventional reverse transcription-polymerase chain reaction (RT-PCR). Bubble PCR was used to amplify the genomic BCR-ABL translocation breakpoints from chronic-phase DNA of 10 patients with CML who subsequently underwent BMT. After cloning and sequencing of the amplification products, patient-specific ABL primers were synthesized and tested for both specificity and sensitivity in nested or heminested combinations with a variety of primers derived from the major breakpoint cluster region of the BCR gene. In all cases, combinations of primers were selected that enabled the detection of chronic-phase DNA from a specific patient at up to a 10(5)x dilution into DNA from a normal individual. Patterns of residual disease obtained by serial RT-PCR and DNA-PCR analyses of blood and bone marrow samples obtained after BMT were similar for most patients, including one treated for relapse by infusion of donor leukocytes. Of the 24 samples for direct comparison of RT-PCR and DNA-PCR, results were concordant in 19 (79%) cases. Five results were discordant. In two instances, RT-PCR was positive, while PCR from genomic DNA was negative; this discrepancy might have arisen due to the slightly greater sensitivity of RT-PCR compared with DNA-PCR. In three samples from three patients, two of whom had been transplanted in the accelerated phase, PCR from genomic DNA was positive while RT-PCR was negative; this could mean that some CML cells in these samples had a reduced or absent capacity to express BCR-ABL mRNA post-transplant. Of these three patients, one subsequently relapsed; and two are in remission at 21 and 24 months after the discordant result. Thus, the finding of a single DNA-PCR- positive, RT-PCR-negative results does not necessarily predict relapse. Because the great majority of samples (79%) gave concordant results with the two assays, we believe that patients in remission do not generally harbor a substantial pool of CML cells that do not express BCR-ABL mRNA.

One hundred and forty-three patients with p210 BCR-ABL-positive leukemia were studied for coexpression of p190 BCR-ABL mRNA. p190 mRNA was detected in 14 of 16 (88%) patients with chronic-phase chronic myeloid leukemia (CML) at diagnosis, in 10 of 10 (100%) CML patients in blast crisis, in 75 of 107 (70%) CML patients receiving interferon-alpha (IFN-alpha), and 10 of 10 (100%) patients with p210 BCR-ABL-positive acute lymphoblastic leukemia (ALL). Neither p210 nor p190 BCR-ABL transcripts were detected in normal healthy adults (n = 20). The numbers of p190 transcripts determined by competitive PCR in patients with CML were low compared with the numbers of p210 transcripts. The median numbers of p210 and p190 transcripts per unit volume of cDNA in positive samples were 1.0 x 10(5) (range, 15 to 1.4 x 10(6)) and 10 (range, 10 to 2.9 x 10(3)), respectively. The numbers of p190 and p210 transcripts were significantly correlated in individual samples (r = .65, P < .001). The median number of p210 BCR-ABL transcripts was significantly lower in samples negative for p190 BCR-ABL transcripts than in samples in which p190 BCR-ABL transcripts were identified (3.1 x 10(3)[n = 73] v 1.0 x 10(5)[n = 115]; P < .0001). The median ratio of p190 to p210 BCR-ABL mRNA was not significantly different between chronic phase CML (1.9 x 10(-4)) and CML in blast crisis (1.7 x 10(-4)). The median ratio in p210 ALL was also low (1.9 x 10(-3)) but significantly higher than that of CML. We conclude that pl90 BCR-ABL transcripts are frequently present at a low level in p210 BCR-ABL-positive leukemias. p190 mRNA may arise through alternative or missplicing and its presence is probably of no pathogenetic significance.

We report a 33-year-old man with Ph chromosome-positive CML who underwent an allogeneic BMT from an unrelated donor. DNA microsatellite studies showed complete donor chimaerism immediately after BMT followed by mixed chimaerism; by day + 45 haematopoiesis was exclusively of recipient origin. Throughout the first year post-transplant all marrow metaphases were Ph negative but with non-clonal rearrangements consistent with autologous recovery. Cytogenetic relapse of leukaemia was first detected 15 months post-transplant. This case is unusual in that non-malignant stem cells of recipient origin survived the transplant and reconstituted haematopoiesis very early after BMT. Later the leukaemic cells reasserted their 'proliferative' advantage.

We prospectively studied 98 chronic myeloid leukemia (CML) patients after bone marrow transplantation by competitive polymerase chain reaction to detect and quantify leukemia-specific BCR-ABL mRNA. Of 69 patients who had persistently undetectable, decreasing, or low BCR-ABL levels ( < 50 transcripts/microgram RNA) on sequential analysis, one (1%) subsequently relapsed. Of 29 patients who had increasing or persistently high BCR-ABL (> 50 transcripts/microgram RNA) on sequential analysis, 21 (72%) have relapsed (P < .00001). In 19 patients studied sequentially, a constant logarithmic increase in the number of BCR-ABL transcripts was found before relapse, indicating a constant BCR-ABL transcript doubling time. The doubling time for patients relapsing cytogenetically or into chronic phase (median, 24.7 days) was significantly longer than that of patients relapsing into advanced phases (median, 14.7 days; P = .005). Eight patients were treated for relapse by donor leukocyte transfusions. The doubling time of responders was significantly longer than that of nonresponders (P = .017). We conclude that quantification of BCR-ABL transcripts after bone marrow transplantation (BMT) is valuable in predicting relapse: a more rapid BCR-ABL transcript doubling time before relapse might indicate a more aggressive disease.

Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with chronic myelogenous leukemia (CML). We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive 2-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. A total of 250 peripheral blood and 55 bone marrow samples with 127 Philadelphia chromosome positive (Ph+) and 6 Ph-/BCR-ABL+ CML patients were investigated. Twenty-one patients were studied at diagnosis with IFN-alpha, 24 had a complete cytogenetic response, 21 a partial response, 12 a minor response, 26 no response, and 23 were unknown. Using nested RT-PCR, all 305 samples were positive for BCR-ABL transcripts. To standardize results for variability in RNA and cDNA quantity and quality, we quantified total ABL transcripts in each sample as internal control. The validity of ABL as internal control was shown by comparison with glucose-6-phosphate dehydrogenase transcript levels in 145 samples. The median BCR-ABL transcript numbers (and BCR- ABL/ABL ratios expressed as percentages) were 400/micrograms RNA (O.04%) in complete responders, 20,500/micrograms RNA (7.1%) in partial responders, 170,000/micrograms RNA (21.0%) in minor responders, and 430,000/micrograms RNA (58.7%) in nonresponders (P < .001). The cytogenetic results correlated with the BCR-ABL transcript numbers (r = .82; P < .001) and BCR-ABL/ABL ratios (r = .84; P < .001). Grouping the ratios BCR-ABL/ABL as less than 2%, 2% to 14% and greater than 14% to compare with cytogenetic complete response, partial response, and minor/nonresponse, the concordance between the two methods was 82% (chi2 P< .0001). We conclude that quantitative PCR with internal controls is as sensitive and reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

Technical modifications of the reverse-transcription/polymerase chain reaction (RT/PCR) amplification method now permit its use to detect amplified products from as few as one abnormal cell, either isolated or mixed with a larger number of normal cells. We studied the reproducibility of such results using as targets low numbers of cells from chronic myeloid leukaemia (CML) patients and CML cell lines in quintuplicate two-step RT/PCR designed to amplify BCR-ABL sequences. When one K562 or KYO1 cell was diluted in 10(3) non-CML HL60 cells, an amplification product was obtained in each test; at greater dilutions BCR-ABL transcripts were detected erratically. Titration of cDNA synthesised from 5 x 10(7) cells from four CML patients showed that whereas positive BCR-ABL sequences could be amplified in some tests starting with as little as a 1 in 10(7) dilution of cDNA template (corresponding to 5-10 cells), the dilution threshold for reproducible amplification was around 1 to 5 in 10(5) (100-500 cells). Quantitative PCR analysis revealed that reactions from 1 in 10(7) diluted cDNA contained less than 10 BCR-ABL transcripts as the starting template. The stochastic nature of the amplification from such small numbers of transcripts was illustrated by results of 10 replicate PCR tests on cDNA from a patient expressing both b3a2 and b2a2 transcripts: dilutions of cDNA up to 1 in 10(5) yielded dual transcript amplification in all 10 tests, but the 1 in 10(7) cDNA dilution resulted in b3a2 and b2a2 products in three tests, b3a2 only in three, b2a2 only in one and no amplification in three tests. We conclude that this 'sampling effect' may yield false-negative results and thus misinterpretation of data regarding assessment of gene expression when the quantity of target material available for study is very small.

Summary .A substantial minority of patients with chronic myeloid leukaemia (CML) achieve a complete response to treatment with interferon‐α (IFN‐α), defined as the disappearance of Philadelphia chromosome positive metaphases or, for patients who are Philadelphia chromosome negative but BCR‐ABL positive, the disappearance of the leukaemic clone as assayed by Southern blot. We have measured the levels of BCR‐ABL transcripts in 20 such patients by quantitative PCR. Results were standardized for both quality and quantity of cDNA by quantification of ABL as an internal control. All 20 patients had evidence of residual disease; the median number of transcripts was 750/μg RNA (range 10–22 000) and the median BCR‐ABL/ABL ratio was 0–17% (range 0–0008–3–6%). Our findings show that CML has not been eradicated in any patient and that the quantity of residual disease in complete responders may vary by as much as four orders of magnitude.