Kirsten Merx

Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha

The degree of tumor load reduction as measured by cytogenetic response is an important prognostic factor for chronic myelogenous leukemia (CML) patients on therapy. We sought to determine whether BCR-ABL transcript levels can predict chromosomal response. Residual disease was evaluated in 120 CML patients in chronic phase (CP) treated with the selective tyrosine kinase inhibitor imatinib after resistance or intolerance to interferon α (IFN). Median time of therapy was 401 days (range 111–704). BCR-ABL and total ABL transcripts were measured in 486 peripheral blood (PB) specimens with a real time RT-PCR approach using fluorescent-labeled hybridization probes (LightCycler technology) and results were expressed as the ratio BCR-ABL/ABL. Cytogenetic response was determined in 3-monthly intervals: From 101 evaluable patients, 42 achieved a complete (CR, 0% Philadelphia chromosome (Ph)- positive metaphases), 18 a partial (PR, 1–34% Ph+), 13 a minor (MR, 35–94% Ph+), and 26 no response (NR, >94% Ph+). All PB samples were RT-PCR positive. The proportion of Ph+ metaphases and simultaneous BCR-ABL/ABL ratios correlated with r = 0.74, P < 0.0001. In order to investigate whether early molecular analysis may predict cytogenetic response, quantitative RT-PCR data obtained after 1 and 2 months of therapy were compared with cytogenetic response at 6 months. BCR-ABL/ABL ratios after 1 month were not predictive, but results after 2 months correlated with the consecutive cytogenetic response (P = 0.0008). The probability for a major cytogenetic response was significantly higher in patients with a BCR-ABL/ABL ratio <20% after 2 months of imatinib therapy. We conclude that: (1) quantitative determination of residual disease with real time RT-PCR is a reliable and sensitive method to monitor CML patients on imatinib therapy; (2) BCR-ABL/ABL ratios correlate well with cytogenetic response; (3) in IFN-pretreated patients all complete responders to imatinib have evidence of residual disease with the limited follow-up available; and (4) cytogenetic response at 6 months of therapy in CP patients is predictable with real time RT-PCR at 2 months.

Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon α/ara-C

We sought to determine dynamics of BCR-ABL mRNA expression levels in 139 patients with chronic myelogenous leukemia (CML) in early chronic phase, randomized to receive imatinib (n=69) or interferon (IFN)/Ara-C (n=70). The response was sequentially monitored by cytogenetics from bone marrow metaphases (n=803) and qualitative and quantitative RT-PCR from peripheral blood samples (n=1117). Complete cytogenetic response (CCR) was achieved in 60 (imatinib, 87%) vs 10 patients (IFN/Ara-C, 14%) after a median observation time of 24 months. Within the first year after CCR, best median ratio BCR-ABL/ABL was 0.087%, (imatinib, n=48) vs 0.27% (IFN/Ara-C, n=9, P=0.025). BCR-ABL was undetectable in 25 cases by real-time PCR, but in only four patients by nested PCR. Median best response in patients with relapse after CCR was 0.24% (n=3) as compared to 0.029% in patients with continuous remission (n=52, P=0.029). We conclude that (i) treatment with imatinib in newly diagnosed CML patients is associated with a rapid decrease of BCR-ABL transcript levels; (ii) nested PCR may reveal residual BCR-ABL transcripts in samples that are negative by real-time PCR; (iii) BCR-ABL transcript levels parallel cytogenetic response, and (iv) imatinib is superior to IFN/Ara-C in terms of the speed and degree of molecular responses, but residual disease is rarely eliminated.

Molecular monitoring of response to imatinib (Glivec®) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission

A significant proportion of chronic myeloid leukemia (CML) patients achieve a major cytogenetic remission (MCR) to imatinib therapy after failing interferon (IFN) α-based protocols. We sought to determine levels of residual disease in patients with MCR using various molecular methods and to establish a relation between residual BCR-ABL transcript levels and rate of relapse in complete cytogenetic remission (CCR). Response was measured by conventional cytogenetic analysis, hypermetaphase and interphase fluorescence in situ hybridization (HM-FISH, IP-FISH) of bone marrow (BM) cells, qualitative nested and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for BCR-ABL transcripts. We investigated 323 peripheral blood (PB) and BM samples from 48 CML patients who achieved a complete (Ph+ 0%; n=41) or partial (Ph+ 1–34%; n=7) cytogenetic remission after 3–20 months of imatinib therapy. Prior to imatinib, 35 patients were in chronic phase (CP), eight in accelerated phase (AP), four in myeloid and one in lymphoid blast crisis. HM-FISH results correlated with ratios BCR-ABL/ABL in PB and BM. In patients with CCR, residual disease was detectable by HM-FISH (31%), IP-FISH (18%), and RT-PCR (100%). During follow-up, BCR-ABL became undetectable in two patients (one CP, one AP) by both nested and quantitative RT-PCR. CCR is ongoing in 30 evaluable patients, 11 patients have relapsed. At the time of best response, median ratios BCR-ABL/ABL were 2.1% (range 0.82–7.8) in patients with subsequent relapse and 0.075% (range 0–3.9) in patients with ongoing remission (P=0.0011). All 16 CP patients, who achieved ratios BCR-ABL/ABL <0.1% as best molecular response are in continuous remission, while 6/13 patients (46%) with ratios ⩾0.1% have relapsed (P=0.0036). We conclude that: (i) in patients with CCR to imatinib, HM-FISH and RT-PCR usually reveal residual BCR-ABL+ cells; (ii) RT-PCR results derived from PB and BM are comparable in CP CML; and (iii) low levels of residual disease with ratios BCR-ABL/ABL <0.1% are associated with continuous remission.

Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization.

The sensitivity of assays designed to monitor minimal residual disease (MRD) by RT-PCR in leukemia depend on quality and quantity of RNA derived from peripheral blood (PB) and bone marrow (BM) leukocytes. Shipment of material may lead to RNA degradation resulting in a loss of sensitivity and, potentially, false negative results. Furthermore, degradation may lead to inaccurate estimates of MRD in positive specimens. We sought to determine feasibility and efficacy of a novel blood collection and processing system which is based on integrated RNA stabilization at the time of phlebotomy (PAXgene Blood RNA Kit) by comparison with standard methods of RNA extraction (cesium chloride gradient ultracentrifugation and RNeasy Mini Kit) using unstabilized EDTA anticoagulated PB. In 26 patients with chronic myelogenous leukemia (CML) on therapy, PB was processed after a storage time at room temperature of 2 and 72 h according to these protocols. BCR-ABL, total ABL and glucose-6-phosphate dehydrogenase (G6PD) mRNA transcripts of PB samples were quantified as a measure for response to therapy and RNA integrity. RNA yield expressed as the ratio of ABL transcripts after a storage time of 72 h/ABL transcripts after a storage time of 2 h at room temperature was significantly higher with the stabilizing method (median 0.40) compared to the RNeasy method using unstabilized PB (median 0.13, P = 0.01). Furthermore, ratios BCR-ABL/ABL after 72 vs 2 h still correlated well using the PAXgene method (r = 0.99, P < 0.0001) in contrast to the standard method which did not (r = 0.65, P = 0.03). Even investigation of complete cytogenetic responders with very low tumor burden showed a good correlation of ratios BCR-ABL/ABL compared to the reference method. Comparable results were achieved using G6PD transcripts as standard. We conclude that the new PAXgene stabilization method could improve RNA quality and the comparability of molecular monitoring within and between multicenter trials.

Standardization of Preanalytical Factors for Minimal Residual Disease Analysis in Chronic Myelogenous Leukemia

Optimal sample quality is a prerequisite to generate valid data in the detection of minimal residual disease (MRD) in leukemias. Thus, the risk of obtaining ‘false’-negative results is increased when both quality and quantity of RNA are suboptimal. Factors which affect the sensitivity and consequently the validity of MRD results are reviewed. RNA degradation in unstabilized peripheral blood (PB) samples does not play a major role in samples being processed on the day of blood collection. However, the simulation of sample shipping at room temperature with a delay of sample processing of up to 3 days causes a dramatic loss of intact RNA. RNA degradation can be prevented by the use of a bedside RNA stabilization system. Additionally, the stabilizing procedure is capable of keeping real-time quantitative polymerase chain reaction (RQ-PCR) results comparable whether the sample is processed immediately or with a delay of up to 3 days. Consistent quantitative data cannot be obtained with unstabilized blood samples. Furthermore, the optimum volume of PB required for MRD diagnostics in patients with BCR-ABL-positive chronic myelogenous leukemia in complete cytogenetic remission is revisited. Ten milliliters of PB is sufficient for processing on the day of blood collection whereas the use of only 5 ml PB may result in false-negative results. Standardization of preanalytical and analytical factors is necessary to provide a comparability of RQ-PCR results from different laboratories within multicenter studies. The definition of ‘undetectable BCR-ABL’ in an individual patient should take these preanalytical parameters into consideration.

Does the Addition of Cetuximab to Radiochemotherapy Improve Outcome of Patients with Locally Advanced Rectal Cancer? Long-Term Results from Phase II Trials

Purpose. The addition of cetuximab to radiochemotherapy (RCT) failed to improve complete response rates in locally advanced rectal cancer (LARC). We report the long-term results in patients treated within two sequential clinical trials. Methods. Patients receiving neoadjuvant RCT using capecitabine and irinotecan (CapIri) within a phase I/II trial or CapIri + cetuximab within a phase II trial were evaluated for analysis of disease-free survival (DFS) and overall survival (OS). KRAS exon 2 mutational status had been analyzed in patients receiving cetuximab. Results. 37 patients from the CapIri trial and 49 patients from the CapIri-cetuximab treatment group were evaluable. Median follow-up time was 75.2 months. The 5-year DFS rate was 82% (CapIri) and 79% (CapIri-cetuximab) (P = 0.62). The median OS was 127.4 months. 5-year OS was 73% for both groups (CapIri and CapIri-cetuximab) (P = 0.61). No significant difference in DFS (P = 0.86) or OS (P = 0.39) was noticed between patients receiving CapIri and those receiving CapIri-cetuximab with KRAS wild-type tumors. Conclusions. As the addition of cetuximab did not improve neither DFS nor OS it should not play a role in the perioperative treatment of patients with LARC, not even of patients with (K)RAS WT tumors.

Molecular Surveillance of Chronic Myeloid Leukemia Patients in the Imatinib Era – Evaluation of Response and Resistance

Residual disease in chronic myeloid leukemia patients may be assessed by various molecular methods. After imatinib treatment a significant proportion of patients achieve complete cytogenetic remission (CCR) and a sensitive method is necessary to monitor treatment response and to detect early signs of relapse. Reverse-transcriptase polymerase chain reaction (RT-PCR) is by far the most sensitive approach to assess residual disease in this group of patients. Qualitative PCR methods give only limited information about the residual leukemic mass. Quantitative RT-PCR (Q-PCR) assays enable to monitor the kinetics of residual BCR-ABL transcripts over time in patients with a good response to imatinib. Early Q-PCR results on imatinib treatment can help to identify individuals who are likely to have a good response. In chronic phase patients after CCR, Q-PCR may identify patients who are likely to continue with their CCR or to relapse and may help to optimize treatment for this group of patients. The definition of molecular surrogate endpoints beyond CCR for studies which are currently planned demands standardization of the nomenclature and of technologies to measure these targets.

Aktuelle Therapiekonzepte bei chronischer myeloischer Leukämie Die Studie IV der Deutschen CML-Studiengruppe

Zum ThemaDie chronische myeloische Leukämie (CML) stellt eine Modellerkrankung für Diagnostik und Therapie neoplastischer Erkrankungen dar. Die zu Grunde liegende zytogenetische Aberration, die Philadelphia-Translokation, mit der BCR-ABL-Genfusion sowie der mehrstufige Verlauf mit der stabilen, therapeutisch gut zu beeinflussenden chronischen Phase, der Akzelerationsphase und der Blastenphase ermöglichen die Übertragung molekularzytogenetischer Erkenntnisse in die klinisch-therapeutische Anwendung.Standardtherapien der CML sind Interferon-α (IFN) in Verbindung mit Hydroxyurea, sowie die allogene Stammzelltransplantation. Die Auswahl individuell geeigneter Therapien sollte unter Beachtung der modernen Risikoindices (IFN-Score und EBMT-Score) erfolgen. Eine Reduktion der Dosierung der Konditionierungstherapie ermöglicht die Stammzelltransplantation auch bei älteren Patienten. Auf der Grundlage der molekularen Aufklärung der Pathogenese der CML erfolgte die Entwicklung des selektiven Inhibitors der BCR-ABL-Tyrosinkinase Imatinib. Vielversprechende präklinische Daten wurden in Phase-I- bis -III-Studien bestätigt. Der Stellenwert der einzelnen Therapiebausteine wird im Rahmen der Studie IV der Deutschen CML-Studiengruppe geprüft.

Association of changes in D-dimer and other coagulation markers with changes in Marder score after treatment of acute venous thrombosis.

AbstractAim: Coagulation markers are sensitive tools to assess ongoing thrombus formation. An association between changes in these markers and changes in venographic Marder scores in patients with acute deep vein-thrombosis treated with low-molecular-weight (LMWH) or unfractionated heparin (UFH) has not been reported. Methods: We investigated differences in coagulation parameters before and at the end of a twelve days the treatment of patients with an improvement versus no improvement of the venographic findings at the end of the treatment with LMWH (n = 48) and UFH (n = 41). Results: Patients with lower values in the Marder score had lower D-dimer levels at day 12 compared to entry treated with UFH and LMWH (p < 0.001). Not improved Marder scores paralleled unchanged D-dimer levels at end of both treatments. Higher values of factor-Xa inhibition and Heptest assay (p < 0.001) were measured at the end of treatment in LMWH- in contrast to UFH-patients. Thrombin inhibition was lower and unchanged at day 12 in patients treated with LMWH and UFH, respectively. Thrombin generation inhibition and release of tissue-factor pathway inhibitor remained unchanged in both groups. Conclusion: An improved Marder score is associated with a decrease of D-dimer during UFH and LMWH treatment of deep vein-thrombosis.

Prognostic Impact of mRNA Expression Levels of HER1–4 (ERBB1–4) in Patients with Locally Advanced Rectal Cancer

Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC) undergoing perioperative chemoradiotherapy (CRT). Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases EGFR (HER1, ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67) mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63) treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1) had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy.