Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-b (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.
Summary Background and Methods: As a source of hematopoietic stem cells, cord blood (CB) is an alternative to bone marrow or peripheral blood stem cells (PBSC). The Mannheim CordBlood Bank has currently stored about 1,750 allogeneic CB units. Here we report our experiences and discuss future perspectives of CB banking. We analyzed CB units for nucleated cell (NC), mononucleated cell (MNC) and CD34+ cell count, volume, colony-forming units (CFU-GM) as well as ethnic background of the donor. Transplanted CB units were analyzed for patient and transplant characteristics and compared to stored CB units. Results: Only 25% of all collected CB units met storage criteria. Main reasons for exclusion were: i) insufficient volume (57.7%), ii) delayed arrival at the processing site (19.2%) and iii) little cell count (7.2%). Up to now 36 CB units have been released for transplantation mainly to children (62%). Transplant indications were hematological diseases, immune deficiencies and metabolic diseases. Transplanted CB units showed significantly higher cell counts compared to stored units (NC: 12.5 vs. 7.2 × 10 8 , MNC: 4.7 vs. 2.9 × 10 8 , CD34+ cells: 3.3 vs. 1.8 × 10 6 , mean; p < 0.001 each) and were found more often in ethnic minority groups (36 vs. 20%; p = 0.04). Conclusions: Even though cell count and volume are key parameters for the eligibility of CB units, our data indicate that the ethnic background of the donor also plays a major role. Collection and processing of CB should be optimized in order to gain maximum volume and cell counts.Schlüsselwörter Nabelschnurblut · Nabelschnurblut-Aufarbeitung · Stammzelltransplantation · Hämatopoetische Stammzellen Zusammenfassung Hintergrund und Methoden: Nabelschnurblut (CB) ist eine Alternative zur Gewinnung von hämatopoetischen Stammzellen aus Knochenmark oder peripheren Stammzellen. Die Mannheimer Nabelschnurblutbank hat bisher 1750 allogene Nabelschnurblutpräparate eingelagert. Wir berichten über unsere Erfahrungen und diskutieren Zukunftsperspektiven der Nabelschnurbluteinlagerung. Folgende CB-Parameter wurden untersucht: Volumen, nukleäre (NC), mononukleäre (MNC) und CD34+ Zellen, Kolonie bildende Einheiten (CFU-GM) sowie ethnische Herkunft des Spenders. Bei den transplantierten Präparaten wurden die Patienten-und Transplantatdaten ausgewertet und die Präparateeigenschaften mit eingelagerten CB verglichen. Ergebnisse: Nur 25% aller gesammelten Nabelschnurblutpräparate erfüllten die Einlagerungskriterien. Die Hauptursachen hierfür waren 1) zu niedriges Vollblutvolumen (57,7%), 2) zu spätes Eintreffen der Prä-parate im Verarbeitungszentrum (19,2%) sowie 3) zu niedrige Zellzahl (7,2%). Bisher wurden 36 Mannheimer Nabelschnurblutpräparate, vorwiegend an Kinder (62%) abgegeben. Die Transplantationsindikationen waren primär hämatologische Erkrankungen, Erkrankungen des Immunsystems und metabolische Erkrankungen. Die Zellzahlen der transplantierten Präparate waren signifikant höher, als die der eingelagerten (NC: 12,5 vs. 7,2 × 10 8 , MNC: 4,7 vs. 2,9 × 10 8 , CD34+ Zellen:...
Systemic mastocytosis is a clonal disorder associated with a constitutive activation of the c-kit tyrosine kinase based on point mutations and is characterized by mast cell infiltration of extracutaneous organs. Nilotinib is a novel aminopyrimidine which potently inhibits Bcr-Abl, as well as the PDGF-R, and c-kit tyrosine kinases. Preclinical data demonstrated the activity of nilotinib against D816V mutated c-kit in biochemical and cellular assays. This Phase II study was designed to evaluate the safety and efficacy of nilotinib administered at an oral dose of 400 mg twice daily to patients with systemic mastocytosis defined by specific disease criteria and with a clinical indication for treatment. Data are available for 60 patients (34 male, 26 female). The median age is 51 years (range, 29 to 79). Of the 60 patients 31 (52%) had extramedullary involvement at baseline. In 30/36 patients investigated (83%) D816V c-kit mutation was found by D-HPLC and/or conventional sequencing in bone marrow or extracutaneous organs. Two patients showed the c-kit I798I polymorphism. Treatment is ongoing for 38 (63%) patients; 22 (37%) have discontinued; ten (17%) for adverse events, seven (12%) withdrew consent, and one (2%) each for disease progression and lost to follow-up. There were two (3%) deaths related to disease progression. Based on investigators’ assessment of serum tryptase, bone marrow mast cell counts and improvement of clinical symptoms 12 patients (20%) had a documented clinical response including two (3%) complete, five (8%) incomplete, four (7%) minor, and one partial response. Adverse events occurring in >15% of patients included nausea in 28 (47%), headache in 26 (43%), fatigue in 25 (42%), vomiting in 22 (37%), diarrhea in 21 (35%), pruritis in 16 (27%), and rash in 15 (25%) patients, dizziness and muscle spasms in 14 (23%) patients each, bone pain in 12 (20%), pyrexia and myalgias in 11 (18%) patients each, and dyspnea, constipation, increased ALAT, and arthralgias in ten (17%) patients each. Most side effects occurred early after initiation of nilotinib therapy and were successfully treated with H1- and H2-blockers and/or corticosteroids, indicating a mast cell degranulation syndrome. Overall the most frequent Grade 3/4 adverse events included diarrhea in four (7%) patients, and thrombocytopenia and headache in three (5%) patients each. The data suggest that nilotinib has clinical activity and an acceptable safety and tolerability profile in patients with systemic mastocytosis with constitutive c-kit activation. Individual molecular characterization will help to guide targeted therapy in this disease.
Dasatinib (SPRYCEL®) has demonstrated significant efficacy in a high proportion of imatinib-resistant or -intolerant chronic phase CML patients (pts) concerning achievement of hematologic and cytogenetic responses. We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and achievement of major molecular response (MMR, BCR-ABL ≤0.1% according to International Scale, IS) after 12 months of dasatinib therapy in pts with chronic phase CML. We have investigated 1,605 peripheral blood samples from 202 pts (n=62 imatinib-intolerant, n=140 imatinib-resistant; 52% male, median age 60 yrs, range 21–78) who had been enrolled in an international phase II study (START-C study, CA180–013) investigating the activity of 70mg dasatinib BID after imatinib failure. Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Prior to dasatinib therapy, 34 different BCR-ABL mutations involving 29 amino acids were detected in 85/202 pts (42%) with a striking predominance in imatinib-resistant (77/140 pts, 55%) over imatinib-intolerant pts (8/62 pts, 13%). 75 pts showed one, 8 pts two and 2 pts three mutations. RQ-PCR data after 12 months of therapy was available from 154 pts (76%), samples from 48 pts (24%) were not available for monitoring after one year due to progressive disease. MMR was achieved in 28 imatinib-intolerant (45%) and 19 imatinib-resistant pts (14%, p<0.0001). The overall rate of imatinib-resistant pts with mutations was comparable in pts achieving MMR (group 1) vs pts not achieving MMR within 12 months (group 2; 53% vs 57%, p=0.80). Several mutations in imatinib-resistant pts are associated with differential response (group 1 vs group 2 response) and are presented with their IC50 values to dasatinib: H396R (n=0 vs n=6; IC50 0.6–1.3nM), M351T (n=1 vs n=7; 1.1nM), G250E/V (n=1 vs n=8; 1.8nM), Y253H (n=0 vs n=5; 1.3–10nM), L387M (n=0 vs n=2; 2nM), F359I/V (n=0 vs n=4; 2.2nM), E255K/V (n=1 vs n=4; 5.6–13nM), F317L (n=0 vs n=3; 7.4–18nM), T315I (n=0 vs n=3; >1,000nM). Of 85 pts without sufficient molecular response to dasatinib (BCR-ABL IS >5%) after 12 months (median, range 9–15) mutation analysis revealed the emergence of new mutations in 17 formerly imatinib-resistant pts (T315I, n=2; T315A, n=1; F317L, n=6; V299L, n=2; M351T, n=2; L248V, n=1; G250E, n=1; K271R, n=1; Y320C, n=1). We conclude that dasatinib is capable of inducing high rates of major molecular response after one year treatment particularly in imatinib-intolerant pts. Response dynamics depend on the individual type of mutation which may be a basis for individual dose adjustment according to the mutation pattern.
Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC kinases with preclinical activity against 20/21 imatinib resistant BCR-ABL mutations and clinical phase I/II efficacy in patients with chronic myelogenous leukemia (CML) and BCR-ABL positive acute lymphoblastic leukemia (ALL). We sought to establish a relationship between type of preexisting BCR-ABL mutations associated with imatinib resistance and efficacy of dasatinib in patients (pts) with CML and ALL. We have investigated 872 peripheral blood samples from 394 pts (53% male, median age 60 yrs, range 17–85) who had been enrolled in international phase II studies investigating the activity of 70mg dasatinib BID after imatinib failure (chronic phase, CP, n=198; accelerated phase, AP, n=78; myeloid blast crisis, MyBC, n=53; lymphoid blast crisis, LyBC, or ALL, n=65). Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. During follow up, pts were monitored in 3-monthly intervals by RQ-PCR for BCR-ABL mRNA transcripts and by mutation analysis to determine the quantitative course of the preexisting mutation or the emergence of new mutations. Hematologic and cytogenetic response data have been collected sequentially for a median of 8 months (range, 2–11) after start of therapy. Prior to dasatinib, 46 different BCR-ABL mutations involving 36 amino acids were detected in 202/394 pts (51%). 162 pts showed one, 33 pts two, 6 pts three, and 1 pt four mutations. Mutations were observed in 84 pts in CP (42%), 47 pts in AP (60%), 23 pts in MyBC (43%), and 48 pts in LyBC and ALL (74%). In patients with mutations, hematologic response was 91% in CP, 62% in AP, 41% in MyBC, and 34% in LyBC/ALL (p<0.01), major and complete cytogenetic response did not differ significantly (47% and 34% in CP, 35% and 27% in AP, 33% and 28% in MyBC, 53% and 51% in LyBC/ALL). Major cytogenetic response rates were comparable in pts bearing no mutations (44%), mutations within the P-Loop (43%), SH2 domain (47%), activation loop (56%), or other sites (49%), but not for T315I (0%, p<0.001). Sorting individual pts by the underlying mutation and cellular IC50 values of dasatinib revealed clearly higher hematologic and cytogenetic response rates in those with lower IC50 values. In line with the virtual insensitivity to dasatinib in vitro, none of the 17 pts carrying a T315I mutation showed any hematologic response. Two distinct patterns of response were observed: a parallel decrease of the BCR-ABL load and the mutated clone or a decrease of the BCR-ABL load followed by a decrease of the mutated clone after a delay of up to 4–6 months. Up until now 5 pts developed new mutations associated with dasatinib resistance (T315I, n=2, AP and MyBC pts; F317L+F486S, n=2, LyBC and MyBC pts; E507G, n=1, CP pt). We conclude that dasatinib is capable of inducing hematologic and cytogenetic remissions in a significant proportion of imatinib resistant pts harboring BCR-ABL mutations, except T315I. Response dynamics depend on the individual type of mutation which may be a basis for individual dose adaptation according to the mutation pattern.
The implantation of gentamicin loaded polymethylmethacrylate (PMMA) beats and other local antibiotic carriers is a common practice in the treatment of chronic osteomyelitis as is the use of local jet lavage débridement. This article presents the case of a patient with chronic osteomyelitis of the tibia, who had no complication after débridement, intramedullary reaming and pulse lavage without tourniquet but sustained a compartment syndrome 2 weeks later during a second procedure in which an intraoperative tourniquet and pulse lavage were combined.
The majority of patients (pts) with systemic mastocytosis (SM) is characterized by the presence of the transforming mutation D816V of the c-kit gene, resulting in a factor-independent activation of KIT, a receptor tyrosine kinase on the surface of mast cells. The mutation is regarded the causative event for the pathogenesis of the disease and a potential target for therapeutic intervention. Novel tyrosine kinase inhibitors, like dasatinib, nilotinib (AMN107) and midostaurin (PKC412) have inhibitory effects on c-kit D816V mutant cells according to recent in-vitro findings. However, the sensitivity of screening procedures for mutations by direct sequencing may be compromised by a small proportion of malignant cells in the bone marrow (BM) sample. We therefore sought to establish a sensitive strategy for the detection of c-kit mutations in BM and peripheral blood (PB) samples by D-HPLC (denaturing-high performance liquid chromatography) with fragment collection of the mutated proportion combined with direct sequencing. D-HPLC has been established using serial dilutions of c-kit D816V positive HMC-1 cells in a background of NB4 cells harboring wildtype c-kit and pts mRNA in control mRNA. D-HPLC was optimized for the detection of the D816V mutation down to 0.1–0.5% mutant clone (cell line and RNA dilution). In comparison, the detection limit for D816V mutations by conventional sequencing was 10 to 15%. The method was established for c-kit mutations in exon 17, but is applicable for mutations in exons 9, 11 and 13 as well. In case of a positive D-HPLC signal, the presence of the mutation was confirmed by direct sequencing of c-kit exon 17. Secondly, the D-HPLC eluates containing the mutated products were extracted by fragment collection, enriched by PCR and subsequently sequenced. The technique was applied to BM (n=81) and PB (n=38) samples from 95 pts (53 m, 42 f) meeting the WHO criteria for SM. Median age was 53 yrs (range 24–86). At diagnosis, D-HPLC was positive in 89% of the BM samples (72/81), whereas conventional sequencing revealed the D816V mutation in 69% of the pts (56/81), one patient was positive for the variant D816H mutation. However, the combination of fragment collection and direct sequencing improved the D816V mutation detection to 86% of pts (70/81) compared to sequencing only. The analysis of PB samples revealed D-HPLC positivity in 50% of pts (19/38) with a consecutive detection of the D816V mutation by direct sequencing alone in 34% of pts (13/38). Fragment collection followed by direct sequencing improved the D816V mutation detection to 50% (19/38). In conclusion,D-HPLC is a reliable and sensitive method for the screening of c-kit mutations in SM which is superior to conventional direct sequencing. In case of a positive DHPLC signal, sequencing is recommended to confirm and specify the mutation.The method is suitable for the surveillance of pts during therapy with novel tyrosine kinase inhibitors.Employing sensitive screening techniques, the D816V mutation could be detected in BM as well as in PB samples, but the reliability for a positive result was higher using BM.
In eosinophilia-associated myeloproliferative disorders with rearrangements of PDGFRA or PDGFRB, molecular diagnosis of the respective fusion genes and monitoring of minimal residual disease (MRD) during treatment with imatinib are compromised by the heterogeneity of the fusion partners. We therefore sought to establish a rapid and reliable quantitative RT-PCR assay (RQ-PCR) using the LightCycler technology for the detection and quantification of PDGFR fusion transcripts by universal amplification of regions which are located downstream to known breakpoint cluster regions within PDGFRA exon 12 and between intron 9 and 11 of PDGFRB. Diagnostic analyses were performed in cDNAs derived from bone marrow or peripheral blood samples of 39 patients (pts) with FIP1L1-PDGFRA (n=31), BCR-PDGFRA (n=1), CDK5RAP2-PDGFRA (n=1), H4-PDGFRB (n=2), ETV6-PDGFRB (n=2), GIT2-PDGFRB (n=1) and GPIAP1-PDGFRB (n=1) fusion genes (36m, 3f, median age 56 ys, range 20 – 73). Except in FIP1L1-PDGFRA positive cases, all patients revealed involvement of chromosome bands 4q12 (PDGFRA) or 5q31–33 (PDGFRB) in chromosomal aberrations identified by conventional cytogenetics. As external standards for quantification, serial dilutions of plasmids containing normal PDGFRA and PDGFRB sequences were employed. ABL transcripts were quantified as internal control and results were expressed as ratios PDGFRA/ABL or PDGFRB/ABL. A cut-off point for overexpression of PDGFRA and PDGFRB (mean+/−2SD) was determined by analysis of a series of 30 healthy volunteers. In healthy individuals, PDGFRA is expressed at very low levels if at all, whereas PDGFRB is expressed at comparable levels to ABL. Serial dilutions of the FIP1L1-PDGFRA positive EOL1 cell line in HL60 cells and of mRNAs derived from patients with known fusion genes (FIP1L1-PDGFRA and H4-PDGFRB) in control mRNA revealed an assay sensitivity of up to 1:1,000 for both fusion genes which was two logs lower than the sensitivity of the specific nested RT-PCRs (1:100,000). At diagnosis, all pts with PDGFR fusion genes showed significantly increased transcript levels compared to healthy controls. The transcript levels ranged within 4 orders of magnitude for PDGFRA (ratio PDGFRA/ABL 0.03–51) and one order of magnitude for PDGFRB fusion genes (ratio PDGFRB/ABL 190–1350). Serial quantification for MRD monitoring during treatment with imatinib has been performed in 21 pts with PDGFRA (100–400 mg/d) and 5 pts with PDGFRB (400mg/d) fusion genes, respectively. In PDGFRA cases, RQ-PCR for PDGFRA transcripts became negative in 21 of 21 patients after a median of 13 weeks (range, 8–67) which was confirmed by fusion gene specific nested RT-PCR in 19 of 21 cases after a median of 21 weeks (range, 28–67). In PDGFRB cases, RQ-PCR for PDGFRB became negative in 4 of 5 patients after a median of 44 weeks (range 16–72 weeks) which was confirmed by fusion gene specific nested RT-PCR in 1 of 5 patients after 40 weeks. We conclude that the universal quantification of regions which are located downstream to known breakpoint cluster regions of PDGFRA and PDGFRB is a sensitive and reliable assay for the routine screening of constitutive activation of PDGFRA or PDGFRB and for monitoring of residual disease during treatment with tyrosine kinase inhibitors.
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