Susanne Saussele

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 105cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-α (n = 219), allogeneic BMT (n = 17), chemotherapy (n = 11), or at diagnosis (n = 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n = 201, r = 0.90, P < 0.0001); (2) the proportion of philadelphia chromosome positive metaphases determined by cytogenetics (n = 81, P < 0.0001); and (3) the bcr ratio determined by southern blot analysis (n = 122, P < 0.0001). we conclude that real-time pcr with hybridization probes is a reliable and sensitive method to monitor cml patients after therapy. the major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete pcr analysis takes less than 60 min.

Predicting complete cytogenetic response and subsequent progression-free survival in 2060 patients with CML on imatinib treatment: the EUTOS score

The outcome of chronic myeloid leukemia (CML) has been profoundly changed by the introduction of tyrosine kinase inhibitors into therapy, but the prognosis of patients with CML is still evaluated using prognostic scores developed in the chemotherapy and interferon era. The present work describes a new prognostic score that is superior to the Sokal and Euro scores both in its prognostic ability and in its simplicity. The predictive power of the score was developed and tested on a group of patients selected from a registry of 2060 patients enrolled in studies of first-line treatment with imatinib-based regimes. The EUTOS score using the percentage of basophils and spleen size best discriminated between high-risk and low-risk groups of patients, with a positive predictive value of not reaching a CCgR of 34%. Five-year progression-free survival was significantly better in the low- than in the high-risk group (90% vs 82%, P = .006). These results were confirmed in the validation sample. The score can be used to identify CML patients with significantly lower probabilities of responding to therapy and survival, thus alerting physicians to those patients who require closer observation and early intervention.

Double induction strategy including high dose cytarabine in combination with all- trans retinoic acid: effects in patients with newly diagnosed acute promyelocytic leukemia

A prospective multicenter study was performed to investigate the clinical and molecular results of intensified double induction therapy including high-dose cytarabine (ara-C) in combination with ATRA in newly diagnosed acute promyelocytic leukemia (APL), followed by consolidation and 3 years maintenance therapy. Fifty-one patients, diagnosed and monitored from December 1994 to June 1999, were evaluated. The median age was 43 (16–60) years. The morphologic diagnosis was M3 in 40 (78%) and M3v in 11 (22%) patients. In 15 (30%) patients the initial white blood cell counts were ⩾5 × 109/l. The cytogenetic or molecular proof of the translocation t(15;17) was a mandatory prerequisite for eligibility. The diagnosis was confirmed by karyotyping in 46 and by RT-PCR of the PML/RARα transcript in 45 cases. The rate of complete hematological remission was 92% and the early death rate 8%. Monitoring of minimal residual disease by RT-PCR of PML/RARα (sensitivity 10−4) showed negativity in 29 of 32 (91%) evaluable cases after induction, in 23 of 25 (92%) after consolidation, and in 27 of 30 (90%) during maintenance, after a median time of 2, 4 and of 18 months after diagnosis, respectively. After a median follow-up of 27 months, the estimated actuarial 2 years overall and event-free survival were both 88% (79, 97), and the 2 years relapse-free survival 96% (90, 100). The high antileukemic efficacy of this treatment strategy is demonstrated by a rapid and extensive reduction of the malignant clone and by a low relapse rate. The results suggest that the intensity of the induction chemotherapy combined with ATRA is one of the factors which may have a critical influence on the outcome of APL. A randomized trial should assess the value of an induction therapy including ATRA and high-dose ara-C in comparison to standard-dose ara-C.

Allogeneic hematopoietic stem cell transplantation (allo SCT) for chronic myeloid leukemia in the imatinib era: evaluation of its impact within a subgroup of the randomized German CML Study IV

The role of allogeneic stem cell transplantation in chronic myeloid leukemia is being reevaluated. Whereas drug treatment has been shown to be superior in first-line treatment, data on allogeneic hematopoietic stem cell transplantation (allo SCT) as second-line therapy after imatinib failure are scarce. Using an interim safety analysis of the randomized German CML Study IV designed to optimize imatinib therapy by combination, dose escalation, and transplantation, we here report on 84 patients who underwent consecutive transplantation according to predefined criteria (low European Group for Blood and Marrow Transplantation [EBMT] score, imatinib failure, and advanced disease). Three-year survival after transplantation of 56 patients in chronic phase was 91% (median follow-up: 30 months). Transplantation-related mortality was 8%. In a matched pair comparison of patients who received a transplant and those who did not, survival was not different. Three-year survival after transplantation of 28 patients in advanced phase was 59%. Eighty-eight percent of patients who received a transplant achieved complete molecular remissions. We conclude that allo SCT could become the preferred second-line option after imatinib failure for suitable patients with a donor. The study is registered at the National Institutes of Health, http://clinicaltrials.gov: NCT00055874.

Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: long-term observation of 1151 patients from the randomized CML Study IV

The prognostic relevance of additional cytogenetic findings at diagnosis of chronic myeloid leukemia (CML) is unclear. The impact of additional cytogenetic findings at diagnosis on time to complete cytogenetic (CCR) and major molecular remission (MMR) and progression-free (PFS) and overall survival (OS) was analyzed using data from 1151 Philadelphia chromosome-positive (Ph(+)) CML patients randomized to the German CML Study IV. At diagnosis, 1003 of 1151 patients (87%) had standard t(9;22)(q34;q11) only, 69 patients (6.0%) had variant t(v;22), and 79 (6.9%) additional cytogenetic aberrations (ACAs). Of these, 38 patients (3.3%) lacked the Y chromosome (-Y) and 41 patients (3.6%) had ACAs except -Y; 16 of these (1.4%) were major route (second Philadelphia [Ph] chromosome, trisomy 8, isochromosome 17q, or trisomy 19) and 25 minor route (all other) ACAs. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), -Y, minor- and major-route ACAs, the 5-year PFS was 90%, 81%, 88%, 96%, and 50%, and the 5-year OS was 92%, 87%, 91%, 96%, and 53%, respectively. In patients with major-route ACAs, the times to CCR and MMR were longer and PFS and OS were shorter (P < .001) than in patients with standard t(9;22). We conclude that major-route ACAs at diagnosis are associated with a negative impact on survival and signify progression to the accelerated phase and blast crisis.

Activated innate lymphoid cells are associated with a reduced susceptibility to graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders but is often complicated by graft-versus-host disease (GVHD), which causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radiochemotherapy, and allogeneic HSCT on composition, phenotype, and recovery of circulating innate lymphoid cells (ILCs) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2, and NCR(-)ILC3 was slow compared with that of neutrophils and monocytes. NCR(+) ILC3 cells, which are not present in the circulation of healthy persons, appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILCs before transplantation, as well as donor ILCs after transplantation, expressed activation (CD69), proliferation (Ki-67), and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion of ILCs expressing these markers was associated with a decreased susceptibility to therapy-induced mucositis and acute GVHD. Taken together, these data suggest that ILC recovery and treatment-related tissue damage are interrelated and affect the development of GVHD.

Safety and efficacy of imatinib in CML over a period of 10 years: data from the randomized CML-study IV

Tyrosine kinase inhibitors (TKI) have changed the natural course of chronic myeloid leukemia (CML). With the advent of second-generation TKI safety and efficacy issues have gained interest. The randomized CML - Study IV was used for a long-term evaluation of imatinib (IM). 1503 patients have received IM, 1379 IM monotherapy. After a median observation of 7.1 years, 965 patients (64%) still received IM. At 10 years, progression-free survival was 82%, overall survival 84%, 59% achieved MR5, 72% MR4.5, 81% MR4, 89% major molecular remission and 92% MR2 (molecular equivalent to complete cytogenetic remission). All response levels were reached faster with IM800 mg except MR5. Eight-year probabilities of adverse drug reactions (ADR) were 76%, of grades 3–4 22%, of non-hematologic 73%, and of hematologic 28%. More ADR were observed with IM800 mg and IM400 mg plus interferon α (IFN). Most patients had their first ADR early with decreasing frequency later on. No new late toxicity was observed. ADR to IM are frequent, but mostly mild and manageable, also with IM 800 mg and IM 400 mg+IFN. The deep molecular response rates indicate that most patients are candidates for IM discontinuation. After 10 years, IM continues to be an excellent initial choice for most patients with CML.

European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia

Most reports on chronic myeloid leukaemia (CML) treatment with tyrosine kinase inhibitors (TKIs) focus on efficacy, particularly on molecular response and outcome. In contrast, adverse events (AEs) are often reported as infrequent, minor, tolerable and manageable, but they are increasingly important as therapy is potentially lifelong and multiple TKIs are available. For this reason, the European LeukemiaNet panel for CML management recommendations presents an exhaustive and critical summary of AEs emerging during CML treatment, to assist their understanding, management and prevention. There are five major conclusions. First, the main purpose of CML treatment is the antileukemic effect. Suboptimal management of AEs must not compromise this first objective. Second, most patients will have AEs, usually early, mostly mild to moderate, and which will resolve spontaneously or are easily controlled by simple means. Third, reduction or interruption of treatment must only be done if optimal management of the AE cannot be accomplished in other ways, and frequent monitoring is needed to detect resolution of the AE as early as possible. Fourth, attention must be given to comorbidities and drug interactions, and to new events unrelated to TKIs that are inevitable during such a prolonged treatment. Fifth, some TKI-related AEs have emerged which were not predicted or detected in earlier studies, maybe because of suboptimal attention to or absence from the preclinical data. Overall, imatinib has demonstrated a good long-term safety profile, though recent findings suggest underestimation of symptom severity by physicians. Second and third generation TKIs have shown higher response rates, but have been associated with unexpected problems, some of which could be irreversible. We hope these recommendations will help to minimise adverse events, and we believe that an optimal management of them will be rewarded by better TKI compliance and thus better CML outcomes, together with better quality of life.

High dose ara-C in the treatment of newly diagnosed acute promyelocytic leukemia: long-term results of the German AMLCG.

The objective of this study for newly diagnosed acute promyelocytic Leukemia (APL) was to evaluate the efficacy of an intensified double induction chemotherapy including high dose ara-C (HD) and all-trans retinoic acid (ATRA) followed by consolidation and 3 years maintenance therapy. In contrast to APL studies stratifying therapy according to pretreatment white blood cell (WBC) count < and ⩾10 × 109/l (low/intermediate and high risk according to the Sanz score), our patients received uniform therapy. From 1994 to 2005, 142 patients (age, 16–60 years) were enrolled. In the low/intermediate (n=105) vs high (n=37) WBC group, the rates of complete remission were 95.2 vs 83.8%, of induction death were 4.8 vs 16.2% (P=0.05) and of molecular remission were 87.5 vs 91.3% (P=1). Long-term overall survival was 84.4 vs 73.0% (P=0.12), event free survival was 78.3 vs 67.3% (P=0.11), relapse free survival was 82.1 vs 80.0% (P=0.83) and the cumulative incidence of relapse was 7.4 vs 11.4% (P=0.46). No relapse or death occurred after 4.7 years. ATRA and intensified chemotherapy including HD ara-C followed by prolonged maintenance therapy reduced the relapse risk in high risk patients. Pretreatment WBC count ⩾10 × 109/l count was no relevant prognostic factor for relapse.

Clinical trials underestimate the age of chronic myeloid leukemia (CML) patients. Incidence and median age of Ph/BCR-ABL-positive CML and other chronic myeloproliferative disorders in a representative area in Germany

Clinical trials underestimate the age of chronic myeloid leukemia (CML) patients. Incidence and median age of Ph/BCR-ABL-positive CML and other chronic myeloproliferative disorders in a representative area in Germany