RQ-PCR Assay Research Articles

Is the JAK2V617F mutation detectable in healthy volunteers?

American Journal of HematologyVolume 85, Issue 4 p. 287-288 LetterFree Access Is the JAK2V617F mutation detectable in healthy volunteers?† Christophe Martinaud, Christophe Martinaud Hôpital d'Instruction des Armées, HIA Sainte-Anne, Toulon, FranceSearch for more papers by this authorPatrick Brisou, Patrick Brisou Hôpital d'Instruction des Armées, HIA Sainte-Anne, Toulon, FranceSearch for more papers by this authorMarie-Joelle Mozziconacci, Corresponding Author Marie-Joelle Mozziconacci mozziconaccimj@marseille.fnclcc.fr Biopathology Department, Institut Paoli-Calmettes, Marseille, FranceBiopathology Department, Institut Paoli-Calmettes, 232 Bd de Ste Marguerite, BP 156, 13273 Marseille Cedex 9, FranceSearch for more papers by this author Christophe Martinaud, Christophe Martinaud Hôpital d'Instruction des Armées, HIA Sainte-Anne, Toulon, FranceSearch for more papers by this authorPatrick Brisou, Patrick Brisou Hôpital d'Instruction des Armées, HIA Sainte-Anne, Toulon, FranceSearch for more papers by this authorMarie-Joelle Mozziconacci, Corresponding Author Marie-Joelle Mozziconacci mozziconaccimj@marseille.fnclcc.fr Biopathology Department, Institut Paoli-Calmettes, Marseille, FranceBiopathology Department, Institut Paoli-Calmettes, 232 Bd de Ste Marguerite, BP 156, 13273 Marseille Cedex 9, FranceSearch for more papers by this author First published: 23 December 2009 https://doi.org/10.1002/ajh.21627Citations: 26 † Conflict of interest: Nothing to report. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume85, Issue4April 2010Pages 287-288 ReferencesRelatedInformation

Current Diagnostic Approach in Acute Myeloid Leukemia in the Republic of Macedonia: A Proposal for Minimal Screening-Based Clinical Stratification

Full Abstract Introduction Acute myeloid leukemia (AML) represents a heterogeneous complex of disorders resulting from diverse mechanisms of leukemogenesis. Correct diagnosis of AML subtypes plays a central role for individual clinical risk stratification and therapeutic decisions. Motivated to improve and simplify the diagnosis and management of AML in the Republic of Macedonia, we conducted a prospective study at the University Clinic of Hematology. Patients and Methods A total of 76 adults (> 15 years of age; from an initial 77 tested) with acute leukemia who were consecutively admitted at the clinic were enrolled in the study. The aim of our study was to establish and standardize diagnostic approaches based on minimal screening tests that will facilitate risk-adapted therapy for each single AML patient. The diagnosis of acute leukemia was made by standard criteria of the French-American-British (FAB) Cooperative Study Group and confirmed by immunophenotyping bone marrow aspirates and/or peripheral blood samples following the criteria of the European Group for the Immunological Classification of Leukemia's (EGIL) and the British Committee for Standards in Hematology (BCSH). Results Our results showed that morphology and cytochemistry established lineage in 68 (89.4%) of patients. Immunophenotyping further revealed the exact lineage assignment of the blast cells in 11.8% of patients and also changed the lineage assignment in 3 patients and guided to implementation of specific molecular analyses for the further definition of some AML cases. Using an RQ-PCR assay we detected the presence of the fusion transcript PML/RARα in 5 patients and classified 10 more patients in the prognostic favorable genetic entity Core Binding Factor (CBF)-AML according to the presence of the abnormal fusion transcripts AML1—ETO, CBFβ—MYH11. Those results were essential for more appropriate single-patient therapeutic decisions. Conclusion Our multimodal diagnostic approach consisted of cytomorphology, cytochemistry, multiparameter flow cytometry, and molecular analyses improved the diagnosis and enabled individual clinical stratification in 38.2% of our AML patients.

Significance of Minimal Residual Disease in Patients with Chronic Myeloid Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract 3269Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for patients (pts) with chronic myeloid leukemia (CML). AlloHSCT is associated with long-term disease-free survival in 40% to 80% pts transplanted in early chronic phase of disease. The probability of relapse for pts transplanted in first chronic phase is 10% to 20% at 5 years, and is even higher (30% – 60%) for pts who received transplant in advanced phases of CML. The significance of minimal residual disease (MRD) in this clinical setting is uncertain. We enrolled 63 consecutive pts with CML who had received an alloHSCT between 1995 and 2007 and had BCR-ABL transcript quantity measured by RQ-PCR method on at least 2 occasions during follow-up in the period starting 6 months after alloHSCT. The reverse transcription was preformed using SuperScriptIII and random hexamers. Quantification of BCR-ABL was performed by RQ-PCR assay according to ‘Europe Against Cancer' protocol. BCR-ABL expression was normalized with endogenous control ABL gene and expressed as a ratio BCR-ABL/ABL. According to the amount of BCR-ABL transcript detected in blood or bone marrow after alloHSCT pts were allocated into 3 categories, including pts with no-detectable or stable very low-level of BCR-ABL transcripts (ratio BCR-ABL/ABL below 0.005%), pts with fluctuating-low level of BCR-ABL transcripts (0.005 – 0.01%) and pts with high-level of BCR-ABL transcripts (0.01 – 0.1%). We didn't find any relationships between different BCR-ABL levels after alloHSCT and clinical parameters at the time of CML diagnosis or transplantation, including Sokal, Hasford and Gratwohl scores. Median time from alloHSCT to molecular relapse (MR) was 38 months (range, 8.5 – 88.5 months). The 3-year progression rate into cytogenetic or hematological relapse of CML since MR was 70%. This progression occurred at a median time of 1.4 months (range, 0 – 3.2 months). We found strong correlation between the levels of BCR-ABL transcripts after alloHSCT and a risk of relapse. The incidence of MR was 0%, 26%, 71% for the low-level, fluctuating-low level and high-level of BCR-ABL transcript (p<.0001), respectively. Similarly the risk of cytogenetic and hematological relapse was 0%, 21%, 43% for these pts (p=.001), respectively. Five-year leukemia-free survival was 100%, 83.9% and 66.7% for the pts with low-level, fluctuating-low level and high-level BCR-ABL transcript (p=.003), respectively. There was no apparent relationship between the level of BCR-ABL transcript and overall survival. We conclude that pts with fluctuating-low and/or high levels of BCR-ABL transcripts are at higher risk of disease progression. Sequential RQ-PCR monitoring coupled with pre-emptive therapy can provide a valid strategy to reduce rates of relapse and development of a more individualized approach to management of pts with CML in major molecular response after alloHSCT. Disclosures:Warzocha:BMS: Consultancy, Honoraria; Celgene: Consultancy; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria.

MiRNA29a Regulates Oncogenic SKI Expression in Acute Myeloid Leukemia (AML).

Abstract 1968Poster Board I-991 Introduction:AML patients with deletion of chromosome 7 (−7) or deletion of 7q (−7q) have a poor prognosis. We have found that the nuclear oncogene SKI is overexpressed in AML, especially in AML with −7/−7q. SKI acts in AML as a repressor of retinoic acid induced myeloid differentiation (Ritter et al., (2006) Leukemia). As we found SKI up regulated in AML, we asked how SKI expression may be regulated. The aim of our study was to find a molecular background for increased SKI level. On chromosome 7 is a cluster of micro-RNAs (miRNAs) localized particularly around the fragile site 7q32 (Calin et al., (2003) PNAS). Therefore we investigated whether there exists a link between expression of miRNAs localized on chromosome 7 and up regulation of SKI expression in AML. Methods:We used micro RNA profiling analysis, FACS, Western blot, RQ-PCR and luciferase assays to determine the role of miRNA29a in regulating SKI expression. Results:We found that the expression of miRNA25, miRNA29a, miRNA183 and miRNA335 was downregulated in AML patients with -7/-7q. Transfection studies with these four miRNAs in HL60 cells revealed in FACS that miRNA29a inhibits SKI expression (60,4%) compared to nonsense control (100%) and other miRNAs (miRNA25: 91%, miRNA183: 101%, miRNA335: 93%). Western blot experiments confirmed that miRNA29a reduces SKI level in HL60 cells. In keeping, miRNA29a also represses expression of the SKI target gene Nr-CaM in IFB melanoma cells. Knock down of miRNA29a using miRNA29a inhibitor molecules induces SKI expression in the high miRNA29a and low SKI expressing cell line NW1539. Luciferase assays in NW1539 and HeLa transfected with 3′UTR-constructs and HeLa cells cotransfected with miRNA29a demonstrated that miRNA29a binds to 3′UTR of SKI in vitro. Furthermore, comparison of SKI and miRNA29a expression of AML patient samples indicates that miRNA29a expression is associated with low SKI level in vivo. Conclusion:Our data show that miRNA29a which is located on 7q32 regulates expression of the oncogene SKI in vitro and in vivo. We suggest the deletion of miRNA29a as mechanism for up regulation of SKI in AML with -7/-7q and thus propose that in AML, this effect may contribute to the tumor suppressive function of miRNA29a. Disclosures:No relevant conflicts of interest to declare.

CD44-Induced Differentiation of Acute Monoblastic Leukemia Cells Is Mediated by Prolonged Activation of ERK1/2 and by Cytokines.

Abstract 3103Poster Board III-40We have previously reported that specific monoclonal antibodies (mAbs) to the cell adhesion molecule CD44, trigger monocytic differentiation of blasts from patients with acute monoblastic leukemia (1) and that cytokines contribute this differentiation (2). To further analyze the mechanisms mediating CD44-induced monocytic differentiation of leukemic cells, we have used as a model the THP-1 leukemia cell line which differentiation has been induced by the Hermes-3 anti-CD44 mAb. Monocytic differentiation has been checked at day 3 by measuring, using flow cytometry, the increase in the expression levels of CD11b (myeloid-specific) and CD14 (monocyte-specific) differentiation antigens. Control THP-1 cells (untreated or treated with isotypic IgG) display only a little CD11b and do not express CD14.First, using western blot analysis, we show that CD44-ligation with Hermes-3 mAb rapidly triggers ERK1/2 MAP kinase phosphorylation, that persists for about 6-8 hours. This prolonged ERK1/2 phosphorylation is essential for CD44-induced differentiation, since its delayed inhibition (using the specific chemical inhibitor U0126) fully abrogates the differentiation process. Similar results have been obtained in Hermes-3 - treated primary blasts from patients with acute monoblastic leukemia. Interestingly, ERK1/2 signalling mediates normal monocytic differentiation induced by cytokines (3). Accordingly, our present results suggest that CD44-ligation might reactivate this physiological differentiation pathway persisting in monoblastic leukemia cells (THP-1 cells and primary leukemic monoblasts).Second, using real-time RQ-PCR and ELISA assays, we show that ERK1/2 activation leads to synthesis and secretion of a burst of cytokines, including IL-1b, IL-6, IL-8, GM-CSF and TNF-alpha. Among these cytokines, TNF-alpha- and IL-6, acting through an autocrine-paracrine pathway, play an important part in the CD44-induced differentiation process, since their functional inhibition with specific mAbs dramatically decreases the extent of this differentiation. TNF-alpha has a critical role in the increase of CD11b expression level. In addition, it cooperates with IL-6 for inducing CD14 expression. However, TNF-alpha- and IL-6 inhibition does not totally abrogate CD44-induced differentiation. This suggests that another unidentified cytokine is perhaps involved in the differentiation process. Alternatively, CD44-activated ERK1/2 pathway may directly reprogram the expression of monocytic differentiation genes. Interestingly, addition of exogenous IL-6 enhanced the level of CD14, suggesting that IL-6 and CD44-ligation may cooperate for differentiation therapy of acute monoblastic leukemia.1. Charrad et al. Nat. Med 5:669,1999 2. Delaunay et al. Leukemia 22:873, 2008 DisclosuresNo relevant conflicts of interest to declare.

Monozygotic Twins with Childhood ALL Demonstrate Prenatal Origin of the Philadelphia Chromosome and Post-Natal Ikaros Deletion.

Abstract 87Studies on twin pairs with concordant ALL have demonstrated the pre-natal occurrence of chromosomal aberrations associated to leukemia either for the rare ALL subsets with short latency, such as t(4;11), but also for more prevalent, later and less aggressive subtypes, such as t(12;21) or hyperdiploidy. SNP-array studies indicated that additional cooperative lesions occur in ALL, including hyperdiploidy, TEL/AML1 and t(9;22) subsets, and they functionally complement the pre-leukemic hit for the conversion to overt ALL. This seems to be true also for Ph+ ALL; although the very dismal prognosis indicates that the BCR/ABL fusion gene could be sufficient to drive the leukemia, SNP-array showed a frequent association with the deletion of the Ikaros gene (IKZF1), among others. However, so far a formal proof for the succession of events in Ph+ ALL was not available.We report a monozygotic twins pair (T1 and T2) with Ph+ ALL, diagnosed at the age of 32 and 36 months, respectively, and treated according to the AIEOP-BFM ALL2000 protocol. This twin pair was a model to demonstrate for the first time the pre-natal occurrence of the t(9;22) translocation and the post-natal origin of Ikaros gene deletion.A different pattern of Ig/TcR clonal rearrangements was found at diagnosis, although a ‘cross-test' identified a marker shared by the twins, being present in a small subpopulation in T2. Formal demonstration of common pre-natal origin of Ph+ ALL came from the cloning of the BCR/ABL genomic breakpoint, which was identical in both twins.The two twins had a different early response to treatment, being T1 prednisone good responder and T2 poor responder, both received BMT from the same sibling donor, and had different outcome: while T2 died, T1 is in CR after 6 years.Early MRD monitoring by using Ig/TcR markers in parallel with a specific genomic RQ-PCR assay indicated persistently high MRD levels in T2 by both methods. By contrast, a persistently high level of BCR/ABL genomic signal was present in the follow up of T1, despite morphological remission and Ig/TcR and immunophenotypic negativity at both day 33 and 78.With the aim to assess the nature of Ph+, Ig/TcR negative cells present in T1 remission sample, we performed FISH analysis on sorted bone marrow cells. We found that 51% of CD3+ and 45% of CD34-/19+/20+ (40% and 6% of total CD45 sorted population, respectively) were BCR-ABL+ and disomic, thus demonstrating that Ph+ cells were mature lymphoid cells. By contrast, the myeloid compartment was not affected, since CD34-/33+ and CD34+/19-/33+/38+ cells were all BCR-ABL negative.The Affymetrix GeneChip mapping analysis by 500K SNP array identified several and different additional abnormalities: T1 showed a subclone with high hyperdiploid karyotype, while T2 displayed deletion of IKZF1 (Δ4-7) and EBF1 genes. In addition, both twins showed identical deletions affecting ABL and BCR exons, confirming the common origin of the translocation. A patient-specific genomic RQ-PCR assay did not reveal the presence of the IKZF1 deletion in T1, demonstrating the post-natal occurrence of this lesion as a second genetic event in Ph+ cells of T2.In conclusion, the Ph chromosome originated in utero in a lymphoid committed precursor cell, generating the pre-natal non-leukemic Ph+ clone shared by the twins. After birth, a second independent hit transformed the Ph+ clone in T1 and T2, inducing hyperdiploidy in T1 and IKZF1 deletion in T2, with consequent different outcome. Residual pre-natal, non leukemic Ph+ cells persisted during normal lymphopoiesis.This twin pair represents a life model for the prenatal origin of the Ph chromosome as well as the occurrence of additional post-natal genetic events. Disclosures:No relevant conflicts of interest to declare.

Aggressive Systemic Mastocytosis Is Associated with a High Kit D816V Allele Burden in Peripheral Blood.

Abstract 2899Poster Board II-875According to 2008 WHO definitions, diagnosis of systemic mastocytosis (SM) is based on the presence of one major criteria (multifocal dense infiltrates of mast cells in bone marrow or organ biopsies) and at least one of the following minor criteria (i) >25% are atypical cells on bone marrow smears or are spindle-shaped in mast cell infiltrates of visceral organs, ii) KIT point mutation at codon 816 in the bone marrow or other extracutaneous organs, iii) mast cells express CD2 and/or CD25, iv) baseline serum tryptase concentration >20 ng/mL) or the presence of at least three minor criteria. Patients with aggressive SM (ASM) have to present with one or more C-findings (i) neutrophils <1,000/μL, Hb <10g/dL, or platelets <100,000/μL, ii) hepatomegaly with impaired liver function – elevated transaminases and/or bilirubin levels and/or hypoalbuminemia, iii) palpable splenomegaly with signs of hypersplenism, iv) malabsorption with significant hypoalbuminemia and/or significant loss. ASM and MCL are clearly associated with an inferior survival. The activating KIT D816V point mutation is thought to be pivotal for pathogenesis and potential targeted therapy with novel tyrosine kinase inhibitors. However, conventional sequencing (CS) of RNA/DNA extracted from bone marrow (BM) samples of SM patients reveals the mutation in less than 50-60% of patients while peripheral blood (PB) samples are frequently negative as consequence of low numbers of malignant cells and poor assay sensitivity. For better qualitative and additional quantitative assessment of the KIT D816V allele burden (expressed as ratio KIT D816V vs. KIT wildtype), we sought to establish (i) a D-HPLC (denaturing-high performance liquid chromatography) assay combined with direct sequencing in case of a positive D-HPLC signal and (ii) a LightCyclerTM based quantitative PCR (RQ-PCR). The assay sensitivities were calculated through serial cell (KIT D816V positive HMC-1 cells in NB4 cells) and RNA dilutions. The detection limit was estimated between 0.1 and 0.5% for both assays which was thus significantly improved in comparison to the detection limit of CS which was at 10–15%. Patient material was available from BM (n=134) and PB (n=93) from 173 patients (88 m, 85 f, median age 54 years) with diagnosis of SM including 10 patients with confirmed ASM (n=9) or aleukemic variant of MCL (n=1) according to WHO definitions. At diagnosis, D-HPLC, RQ-PCR and CS were positive in 78% (104/134), 84% (112/134) and 60% (80/134) of BM samples, respectively. Two patients had a KIT D816H and one patient had a KIT D816L mutation. In 54 cases with contemporaneously collected BM and PB samples, the KIT D816V mutation was found in PB of BM positive patients by D-HPLC, RQ-PCR and CS in 47% (22/46), 55% (27/49) and 38% (14/37) of cases, respectively. This information allowed the detection of the KIT D816V mutation in an additional 27 of 39 (69%) patients without available bone marrow biopsies. All ASM patients were KIT D816V positive in PB with a median KIT D816V allele burden of 36% (range 26–98%) vs. 7.8% (range 0.1–61%) in other stages of SM (p=0.0021). Eleven of 124 (8.9%) SM patients with a KIT D816V allele burden >20% are currently explored for the potential diagnosis of ASM or MCL. In conclusion, D-HPLC is a reliable and sensitive method for the screening of variable KIT mutations in BM and PB of SM patients and is clearly superior to CS. Application of RQ-PCR assays for the most common D816V mutation may overlook rare mutations but allows quantification of the KIT D816V allele burden which may be useful for diagnosis of ASM and monitoring of residual disease during treatment with novel tyrosine kinase inhibitors, e.g. midostaurin or dasatinib. Disclosures:No relevant conflicts of interest to declare.

Evaluation of the presence of IGF1R overexpression in wild-type and kinase mutant GI stromal tumors

10506 Background: Most adult GI stromal tumors have gain-of-function mutations in KIT (80%) or PDGFRA (5–7%). These pathogenetic mutations are the target for kinase inhibitor therapy. The pathogenesis of the 10–15% of GISTs lacking kinase mutations (WT GIST) is unknown; this includes most pediatric GISTs. Recently, Tarn et al. (PNAS 2008) identified IGF1R over-expression in all WT GIST in their series of cases, including one pediatric case. IGF1R may represent a novel therapeutic target for WT GISTs. Methods: We developed a quantitative RQ-PCR assay for IGF1R and GAPDH transcripts that is linear over 5 logs (R2 values of 0.99 and 0.98, respectively). We prepared cDNA from RNA isolated from 79 archival, formalin-fixed, paraffin-embedded GIST specimens and quantified IGF1R expression as the % ratio of IGF1R/GAPDH transcripts. We had previously genotyped the tumors to identify any kinase mutations. Results: Adult WT GIST had a 10–15 fold higher expression of IGF1R transcript than kinase mutant GISTs (WT GIST 13.7 ±18, n=35; KIT exon 11 mutant GIST 1.0 ± 1.1, n=26; PDGFRA mutant GIST 0.9 ± 1.0, n=8; KIT 9 or 13 mutation 0.8 ± 0.6, n=5). Interestingly, the WT GIST group could be further divided into tumors with low vs. high IGF1R expression. The low IGF1R expression group (mean 0.6, range 0.1–1.9, n=14), had similar IGF1R expression to kinase mutant GISTs. In contrast, the high expression group had more than 30-fold higher levels of IGF1R transcript (mean 22.5, range 7.3–76.5, n=21). We also examined IGF1R expression in 5 WT pediatric GISTs. The mean IGF1R expression in pediatric GISTs resembled that of the adult high expression group, with 4 of 5 cases showing IGF1R over-expression. Conclusions: WT GISTs are heterogeneous with regard to IGF1R expression and can be divided into two subgroups. One third of the WT GISTs overlap with kinase-mutant GISTs, while the remainder express IGF1R at more than 30-fold higher levels. These results have implications for evolving models of GIST pathogenesis and the clinical testing of IGF1R monoclonal antibodies for treatment of metastatic GIST. [Table: see text]

Post-Remission MRD Kinetics in Children with Acute Lymphoblastic Leukaemia Receiving Augmented BFM Consolidation Compared with Other Regimens: Results of the Medical Research Council Trial UKALL2003.

The BFM group (Blood Nov 2007; 110a: 585) has reported that monitoring of postconsolidation MRD kinetics identifies a very high risk (MRD VHR) group of children with ALL, who might benefit from novel therapy and first remission allogeneic transplantation. We have compared the effect of consolidation regimen on post-remission MRD kinetics using a standardised quality controlled RQ-PCR assay of Ig and TCR patient specific rearrangements with a reproducible quantitative range of 10−4 in an attempt to identify a MRD VHR group, similar to that reported by the BFM.MRC UKALL 2003 is a randomised trial testing whether treatment can be stratified by MRD at weeks 4 and 11 in children and young adults (up to age 25 years) with ALL from the UK and Ireland. Initially, patients are stratified by NCI criteria, cytogenetics and early morphological marrow response to receive three (Regimen A) or four (Regimen B, additional daunorubicin) drug induction. Patients with MRD >10−4 at end of induction (EOI) (MRD High Risk) are randomised to continue previously assigned therapy (regimen A or B) or receive the more intensive regimen C. The regimens are of escalating intensity (A→C) and include either CCG modified BFM (A), standard BFM (B) or augmented BFM (C) consolidation, the latter containing pegylated-asparaginase (Peg-ASP; Oncospar, Medac UK, 1000 units/m2 i.m. days 16 and 44) and vincristine (1.5mg/m2 i.v. days 16, 23, 44, 51) in addition to standard BFM consolidation. Only patients with high risk cytogenetics or day 28 BM3 marrow are eligible for CR1 transplant.Of the134 patients with MRD > 10–4 at EOI, 101 were MRD negative, and 33 had detectable disease post-consolidation therapy (16 low MRD positive; <5×10−4, 17 high MRD positive; >5×10−4). Patients who received augmented BFM consolidation had more rapid clearance of MRD (Neg = 85%, low pos =7%, high pos =8%) than those who received CCG modified or standard BFM consolidation (Neg 65%, low pos =17%, high pos =17%) at week 11 – 15 of treatment (P=0.03 independent of age and white cell count). With median follow-up of 27 months, ten patients have relapsed, 7 in the negative group (3 year relapse Free Survival [RFS] 91.2% se=3.5) 1 in the low positive group (RFS 92.9% se = 6.9) and 2 in the high positive group (RFS 85.7% se = 9.4). The latter two were recipients of the lower intensity Regimens A (isolated CNS relapse) and B (isolated marrow relapse). The small numbers of post-consolidation high-positive patients do not permit us to identify a VHR group with a worse outcome than those patients with lower MRD levels. However, the early relapse risk (<15% at 3 years) for such patients appears to be much lower than that reported by the BFM group (>50% at 3 years). Differences in treatment protocol before and after consolidation may account for this disparity.In patients treated on the current UK childhood ALL protocol, we are unable to identify a VHR group on the basis of post-consolidation MRD. Augmented BFM consolidation obtains more rapid clearance of MRD than other regimens which, with longer follow-up, might be expected to translate into a better EFS.

Minimal Residual Disease Assessed by NPM1 Mutation Specific RQ-PCR Is the Most Relevant Prognostic Parameter in NPM1-Mutated AML and Highly Useful to Guide Therapy

The aim of this study was to further evaluate the impact of minimal residual disease (MRD) in NPM1 mutated AML in comparison to other factors like FAB, cytogenetics, FLT3 mutations, NPM1 mutation type and age. In total 1002 samples of 219 NPM1 mutated (NPM1mut) patients (pts) were analysed at diagnosis, during, and after therapy. Pts were treated within different AML trials, and follow-up samples were referred to perform an NPM1 specific RQ-PCR for MRD. The cohort was comprised of 112 females and 107 males, median age was 58.8 years (range: 20–79 years). 207 had de novo AML (M0: n=5; M1: n=49; M2: n=55; M4=57; M5: n=28, M5: n=6; M7: n=1, nd: n=6), 4 s-AML and 5 t-AML. Cytogenetic data was available in 215 pts: 178 with normal (NK) and 37 with aberrant karyotypes (+4: n=4; +8: n=7; +21: n=2, two or more trisomies: n=4; -Y: n=4; del(7q): n=2; del(9q): n=3; del(20q): n=2; rare translocations: n=9). At diagnosis 87/219 pts (39.7%) had FLT3-ITDs in addition to the NPM1mut. FLT3-TKD status was available in 206 cases (14 mutated (6.7%) and 192 WT). The NPM1 mutation types were A (n=174), B (n=13), D (n=14), I: (n=4), L: (n=2), R: (n=4) and 8 with individual rare types. Univariate analysis for overall survival (OS) revealed unfavourable impact for age (p=0.049), and for FLT3-ITD (p=0.002), favourable impact for FLT3-TKD (p=0.046), and no impact for FAB, chromosomal aberrations or NPM1 mutation type. For MRD assessment for all 14 different NPM1 mutation types mRNA based RQ-PCR assays were established with sensitivities of 10,000–1,000,000. For each patient 2–17 samples (spls) were analyzed (median: 4) spanning a median follow up time of 252 days (range: 18–2347 days). Paired samples of diagnosis and relapse were available in 71 pts, in 8 pts also from second relapse. At relapse all cases had high NPM1 levels comparable to those at diagnosis. The FLT3-ITD status was mutated (+/+) at both time points in 25 pts and −/− in another 25 pts. 10 pts gained FLT3-ITD at relapse and 3 lost it. For 48 paired samples cytogenetics was available for both time points. A normal karyotype (NK) at both time points was detected in 36 pts, 7 cases showed a normal or aberrant karyotype (AK) at diagnosis and and AK at relapse (two of these gained additional aberrations at relapse), 2 different AK at both time points in were detected in 3 cases and a regression from AK to NK in 2 cases. These data show that NPM1 seems to be the primary genetic aberration in these cases and detection of NPM1 is more reliable to detect relapse than cytogenetics. To analyse the impact of NPM1 mutation levels on prognosis four different follow-up intervals were defined: interval 1: days 21–60 after start of therapy; interval 2: days 61–120; interval 3: days 121–365, 4: >365 days. First a set of 605 samples referred for analysis during first line treatment were analysed. Using Cox regression analysis a significant impact of MRD levels (as continuous variable) on EFS was detected for interval 2 (128 spls, p=0.008), interval 3 (214 spl; p<0.001), interval 4 (171 spls; p<0.001) but not for the early interval up to day 60 showing that early molecular response is not relevant for long time outcome. A multivariate analysis showed that MRD was the most significant prognostic parameter (p<0.001) (p-values for interval 3), followed by age (p=0.003), and pretreatment FLT3-ITD status (p=0.065). The same analysis was performed for a second set of 183 spls taken from 50 pts during salvage therapy after relapse. The most relevant interval for this group was between days 30–60 (26 spls; p=0.003). In a third set 87 spls from 28 pts after allogeneic bone marrow transplantation were analyzed. A prognostic impact of MRD could be shown for interval 2 (17 spls; p=0.005) and 3 (23 spls; p=0.006) (no samples from later intervals available). Of the total cohort 325 spls were analysed in parallel with RQ-PCR for NPM1 and genescan for the FLT3-ITD. A high correlation of both follow up markers was observed (r=0.807, p<0.001). Although the method for NPM1 detection is 3–4 log ranges more sensitive our data suggest parallel assessment for FLT3-ITD for high risk patients as many of them aquired FLT3-ITD as additional marker during progression. In conclusion, MRD is the most relevant prognostic marker in NPM1 mutated AML and it is a very useful tool to assess therapy response and to guide therapy.

Mathematical Simulation of BCR-ABL Real Time Quantitative Polymerase Chain Reaction (RQ-PCR) for Chronic Myeloid Leukemia (CML) Response Monitoring Provides Insight on the Basis of International Standardization.

BACKGROUND: Cytogenetic assessment to measure CML response to Glivec or Tasigna is expressed as a ratio of Ph+ CML to total cells. Molecular response determination for CML by RQ-PCR achieves a similar goal by obtaining a ratio of transcript levels of BCR-ABL expressed exclusively in Ph+ CML cells to the BCR or ABL control gene which is expressed in both Ph+ and normal cells. However when a RQ-PCR assay result is obtained, a different RQ-PCR ratio will be obtained for the same sample using BCR vs. ABL as control gene due to the intrinsic expression level differences in the two control genes. Experiments have demonstrated empirically that international standardization of RQ-PCR data obtained by assays using these different control genes is possible, thus allowing direct comparison of data. We have developed a mathematical simulation that explains the underlying basis for international standardization and this was experimentally confirmed to show how standardization is feasible in practice as a part of the Tyrosine Kinase Optimization and Selectivity (TOPS) trial.METHODS: We have mathematically simulated a correlation between theoretical cytogenetic response (Ph+ to total cell ratio) with the theoretical molecular response (BCR-ABL to control gene transcript ratio) by using the following assumptions; 1) BCR and BCR-ABL expression occur from similar promoters and likely have similar expression levels. 2) Ph+ cells have one normal BCR resulting in 1x level of BCR transcription and normal cells have two normal copies of BCR resulting in 2x level of BCR transcription (x=BCR transcription rate from one promoter). This simulation was extended to the ABL control gene as data of the relative expression level of ABL vs. BCR was available from samples in the TOPS trial, and the ABL transcript level is similar in both Ph+ and normal cells as the ABL PCR primers amplify both ABL and BCR-ABL transcripts. To experimentally test the simulation, RQ-PCR samples from patients in TOPS were exchanged between the study's regional RQ-PCR laboratories. Each sample was assayed at 2 labs, one using BCR and the other ABL as the control gene.RESULTS: Mathematical simulation suggests that RQ-PCR ratios with BCR but not ABL as control gene has a nonlinear relationship with the Ph+ to the total cell ratio across the entire range from 0 to 100%. However at clinically relevant RQ-PCR ratio of ≤10%, irrespective of whether BCR or ABL is used as a control gene, RQ-PCR ratios are linear but with different slopes due to higher ratios obtained with ABL as control gene. Despite the different slopes, RQ-PCR ratios obtained with either control gene can be easily inter-converted via a multiplicative factor to neutralize the bias between methods as is currently recommended in international standardization. Experimental testing in the TOPS trial showed that; 1) ABL transcript copy number is significantly lower than BCR transcript copy number leading to higher RQ-PCR ratios with ABL as control gene. 2) Mathematical correction of bias between BCR and ABL as control gene allows for direct comparison of RQ-PCR ratios with an 88.2% concordance when ratios are below 10%.CONCLUSIONS: Mathematical simulation shows that for BCR and ABL as the control gene, the basis for international standardization is a consistent linear relationship that can be easily inter-converted when PCR ratios are ≤ 10%. This can be experimentally demonstrated by the high level of concordance obtained when the same sample is analyzed using different control genes. For RQ-PCR ratios > 10%, standardization is not possible using the currently applied neutralization of bias method of conversion, but may be possible by using a formula that accounts for the non-linear relationship of BCR as a control gene. Simulation studies also highlight the importance of not only obtaining precise but also accurate RQ-PCR ratios thus enabling consistent standardization.

Rapid Detection of Invasive Fungal Infections in Hemato-Oncological Patients.

Invasive fungal infections (IFI) play an increasingly important role as life-threatening complications in immunocompromised patients. Recent surveys show that annually 450.000 patients suffer from invasive mycoses worldwide. Early application of antimycotic agents is an essential prerequisite for successful therapy. However, standardized diagnostic techniques permitting rapid, and sensitive screening for the clinically relevant fungi have been lacking. Moreover, the increasing incidence of invasive infections caused by hitherto uncommon fungal species requires diagnostic tests with very broad specificity. We developed a panfungal real-time PCR (RQ-PCR) screening assay targeting the highly conserved region of the 28S ribosomal multicopy gene. Universal primers and probes for two test panels in two separate PCR reactions were established facilitating the detection and quantitative assessment of a broad range of pathogenic fungi. The reaction of test panel I covers a range of moulds including species from the fungal genera Aspergillus, Fusarium, Penicillium, Cladosporium, and Scedosporium. The test panel of reaction II facilitates the detection of yeasts from the genera Candida, Cryptococcus, Malassezia, and Trichosporon as well as Zygomycetes (Mucor, Rhizopus, Rhizomucor, Absidia), and other emerging fungal pathogens. In total, more than 80 human pathogenic fungal species can be detected and quantified. The detection limit of the panfungal RQ-PCR assay in clinical specimens was shown to be in the range of 1 fg fungal DNA, which corresponds to a fraction of a single fungal genome. To assess the clinical applicability of the technique, more than 1.000 clinical specimens derived from 144 well-documented pediatric hemato-oncological patients at high risk of invasive fungal infection were analyzed. The specimens investigated included predominantly serial plasma samples (n>950), and a small number of other materials including bronchotracheal secretion, bronchoalveolar lavage, lung biopsy, liver biopsy, and cerebrospinal fluid. In about 65% of the patients analyzed, evidence of fungemia was found in serial specimens. In a large proportion of these patients (31%), both reactions revealed positive results indicating mixed fungal infections. Isolated positivity of reaction I or II was observed in 29% and 5%, respectively. The high percentage of patients with PCR-detectable fungal DNA in normally sterile clinical specimens raises questions about the interpretation and clinical relevance of the findings. The possible occurrence of contamination was largely excluded by the implementation of multiple controls at all stages of the diagnostic process. The great sensitivity of RQ-PCR analysis may permit the detection of low-level fungal DNAemia which may not generally reflect an imminent risk of severe fungal disease. Interpretation of the molecular findings therefore has to be performed with caution. Current evaluation of the RQ-PCR data in relation to the EORTC definitions of proven, probable and possible invasive fungal infection, which are based on clinical criteria, host factors, and microbiological criteria, revealed a very good correlation. Indeed, most patients with repeatedly positive RQ-PCR results, which had been generated in a double-blind fashion, received antifungal treatment on the basis of other findings. The assay presented is a powerful tool for rapid and economic screening of IFIs and ongoing analyses are expected to reveal how the technique could be implemented in clinical diagnostics to support timely onset and management of antifungal treatment.

Correlations of human herpesvirus 6B and CMV infection with acute GVHD in recipients of allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) and CMV reactivation were monitored in a cohort of 72 consecutive haematopoietic stem cell transplant (HSCT) patients using RQ-PCR and antigenaemia assay, respectively. The association between acute GVHD (aGVHD) and HHV-6B/CMV was evaluated. We found that on day 100 the cumulative incidence of grades I-IV aGVHD, grades II-IV aGVHD and grades III-IV aGVHD was 55.6, 27.8 and 13.9%, respectively. Multivariate analysis indicated that HHV-6B reactivation was closely correlated with a higher probability of grade II-IV aGVHD by day 30 (Hazard ratio (HR), 8.9; 95% confidence interval (CI), 2.6-31.0; P=0.0006), by day 50 (HR, 6.1; 95% CI, 2.1-17.8; P=0.0010) and by day 100 (HR, 4.8; 95% CI, 1.7-13.6; P=0.0028). However, CMV reactivation did not significantly affect the development of aGVHD by day 50 (HR, 0.8; 95% CI, 0.1-6.7; P=0.8236) and by day 100 (HR, 0.5; 95% CI, 0.1-4.4; P=0.5330) after HSCT. In conclusion, this study demonstrated that active HHV-6B infection, but not CMV, is significantly associated with an increased risk of aGVHD development after HSCT.

Monitoring of Cytomegalovirus Reactivation in Bone Marrow Transplant Recipients by Real-time PCR

Cytomegalovirus (CMV) has been recognized as the most important viral pathogen in persons undergoing bone marrow transplantation (BMT). The aim was to develop a quantitative PCR assay to quantify CMV DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation (BMT) patients. An in-house real-time PCR assay based on TaqMan technology was developed to monitor the quantity of CMV DNA in PBLs of the BMT recipients. Sequential blood samples (415 specimens) were collected from 43 patients as weekly intervals until day 100 after transplantation. The CMV DNA was quantified in parallel with the pp65 antigenemia assay in PBL samples. Viral reactivation occurred in 51% and 41.8% of the recipients as detected by RQ-PCR and antigenemia assays respectively. There was a significant correlation between both assays (P < 0.0001); however, the RQ-PCR was more sensitive than the antigenemia. CMV DNA was detected by the RQ-PCR by a median of 14 days earlier than the antigenemia. Preemptive therapy was implemented in the antigenemia positive cases. The administration of ganciclovir led to a rapid decrease in the viral load. After preemptive therapy, the antigenemia achieved a negative result earlier than the RQ-PCR assay (a median of 17.5 days). An increase of viral load in both quantitative assays and of cyclosporine serum level were identified as the most significant risk factors for CMV reactivation. The quantitative CMV PCR might be a useful tool for monitoring the CMV reactivation and guiding the efficacy of the CMV preemptive therapy in BMT recipients.

Evaluation of prenatal RHD typing strategies on cell‐free fetal DNA from maternal plasma

The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women. The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity. Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.

Quantitative Detection Using SYBR Green of Residual Disease in Myeloma Patients Treated with Revlimid or Velcade.

The treatment of multiple myeloma (MM) with new drugs like Velcade and Revlimid shows promise in the clinic. These two drugs give excellent partial to complete remissions, but the disease invariably returns indicating escape of the malignant clone. The current clinical practices for quantifying minimal residual disease (MRD), such as plasma cell counts, lack needed sensitivity. PCR based assays amplify the clonotypic IgH VDJ gene rearrangement in myeloma and identify all compartments of the malignant clone, providing a unique molecular signature to monitor disease. Every myeloma cell carries one copy of the specific IgH VDJ rearrangement and provides a quantitative measure of the number of malignant cells remaining in each patient. No previous work has quantified MRD after treatment with either Revlimid or Velcade. TaqMan real-time quantitative PCR (rqPCR) assays are able to quantify the level of disease burden but probes are expensive and difficult to design, making this assay unsuitable for routine clinical use. SYBR green, however, is an inexpensive alternative to probes and utilizes the same unique molecular VDJ signature with consistency and reliability (r2=0.9997). PCR product specificity is confirmed using melting curve analysis. This is the first report of the successful use of SYBR green in quantifying the level of malignant disease in MM. The specific clonal rearrangement is identified for each patient. Patient specific primers are designed and verified as being present in the majority of plasma cells using single cell PCR. A cloned IgH VDJ product for each patient provides a standard curve for each patient-specific rqPCR. A cloned B2Microglobulin PCR product provides an independent standard curve to quantify cell number. After correcting for copy number differences, the percentage of malignant cells is calculated by dividing the number of VDJ molecules by the number of B2M molecules. For all tests, the level of residual disease in bone marrow was measured using 150ng of DNA. In patients achieving complete clinical remission during treatment with Revlimid, the SYBR green method detected 0.104% clonal cells with a range of 0.014%–0.26%. In patients with a partial remission, 3.034% clonal cells were detected, with a range of 0.62%–7.79%. A subsequent bone marrow from one Revlimid treated patient revealed no clonal cells this using SYBR Green method. In patients treated with Velcade at the time of relapse, the remaining clonal cells averaged 1.042% with a range of 0.00075%–3.11%. When Velcade was used as a frontline therapy followed by a stem cell transplant, 0.10% clonal cells remained with a range of 0%–0.287%. In one dexamethasone treated patient who achieved a complete remission, 0.018% clonal cells were found. The SYBR green rqPCR assay for patient specific clonal VDJ was shown to reliably quantify residual disease. To date, our results suggest that frontline treatment with Velcade may lead to lower levels of residual disease than treatment at the time of relapse. In Revlimid treated patients, the extent of residual disease correlates with clinical classifications of complete or partial remission. The significance of these low numbers of malignant cells remains to be established, but the persistent occurrence of relapse suggests they are clinically relevant.

EEC Growth, JAK2 Mutation, PRV-1 Overexpression and X-Chromosome Inactivation Pattern: Predicting Values on Polycythemia Vera Evolution and Risk of Thrombosis in Idiopathic Thrombocytosis.

We have previously demonstrated that endogenous erythroid colony (EEC) growth could predict PV evolution in idiopathic thrombocytosis (IT) (Blood 83:744, 1994). We also showed that female patients with IT and monoclonal X-chromosome inactivation pattern (XCIP) had a high risk of vascular complications (Blood 100:1596, 2002). Recently, PRV-1 overexpression and JAK2V617F mutation have been described and implicated in the diagnosis of Ph(-) chronic myeloproliferative disorders. In a large cohort of patients with IT, we aimedto determine the correlation among the molecular markers of EEC growth, JAK2 mutation status, PRV-1 level and XCIP, andto assess the predicting values of these biomarkers on PV evolution and risk of thrombosis.Allele-specific PCR assay was used to detect JAK2V617F in peripheral blood granulocytes (n=178) or bone marrow cells (n=75) from patients with IT. EEC assay was performed on 235 patients in a serum-free culture system at initial diagnosis. PRV-1 mRNA expression in granulocytes from 187 patients was measured by real-time RQ-PCR assay and expressed as the copy number of PRV-1 normalized to GAPDH gene (NCN). Clonality analysis of XCIP with HUMARA-PCR assay was performed on 123 female patients. JAK2V617F was detected in 57.7% of patients with IT. Forty-one percent of IT patients had EEC growth at initial presentation. Of those analyzed for PRV-1 levels, 138 patients had not received platelet-lowering agents at time of analysis; 72 (38.5%) had PRV-1 overexpression with NCN ≥ 1 (NCN of 30 normal subjects, mean±SE: 0.32±0.04, range: 0.04–0.97). Seventy-six female patients had monoclonal XCIP and 28 had polyclonal XCIP; the remaining 19 patients had ambiguous or homozygous pattern of XCIP. EEC growth was strongly correlated with JAK2 mutation (P<0.0001) and PRV-1 overexpression (P<0.0001). PRV-1 overexpression was highly associated with JAK2 mutation (P<0.0001) and monoclonal XCIP (P=0.003). The frequency of JAK2 mutation or EEC growth was not different between monoclonal and polyclonal XCIP. Thirty-four patients subsequently developed PV (IT-PV), ranging from 1.5 months to 90 months. All 34 IT-PV patients had EEC growth and all but one harbored JAK2 mutation at initial presentation. Patients with IT-PV had a significantly higher frequency of EEC(+) (P<0.0001), JAK2(+) (P<0.0001), and PRV-1(+) (P<0.0001) than those without PV evolution, whereas there was no difference in age (P=0.914) or XCIP (P=0.550) between IT with and without PV evolution. One hundred and sixty-nine patients had all biomarkers of JAK2/EEC/PRV-1 analyzed, 57 patients were JAK2(+)/EEC(+)/PRV-1(+) and 55 were JAK2(-)/EEC(-)/PRV-1(-). Eighteen patients with all 3 biomarkers-positive developed PV later compared with none of patients with all 3 biomarkers-negative (P<0.0001). Eighty-two patients experienced thrombotic complications during their disease courses. The occurrence of thrombosis was strongly associated with EEC growth (P=0.0002), JAK2 mutation (P=0.007), PRV-1 level (P=0.004), and XCIP (P=0.034). The present study showed that EEC growth and JAK2 mutation were predictors of PV evolution; EEC growth, JAK2 mutation, PRV-1 overexpression and monoclonal XCIP predicted the risk of thrombosis in patients with IT.

Relapse-Risk Stratification in Acute Promyelocytic Leukemia Patients by PML-RARa Transcript Quantification.

The detection of PML-RARa by real-time PCR (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) patients; however, the clinical value of using a RQ-PCR approach remains to be determined. Our aim was to analyze the correlation between relapse risk and MRD levels assessed by RQ-PCR at different phases of treatment, and to compare these data with conventional qualitative RT-PCR. Follow-up samples from 145 APL patients treated with the PETHEMA protocols were analyzed by RQ-PCR protocol from Europe Against Cancer program and by RT-PCR method from European BIOMED-1 Concerted Action. After induction therapy, no association was found between the presence of a RQ-PCR positive result and relapse. After the third consolidation course, three cases remained positive by RQ-PCR assays and two of them (67%) relapsed. By contrast, only 16 out of 119 (13%) patients with negative RQ-PCR relapsed. During maintenance therapy and out-of treatment, a significant correlation between a RQ-PCR positive result and a poor relapse-free survival was observed (p < 0.0001). All patients with > 10 PML-RARa normalized number copies (NCN) at maintenance or out-of therapy relapsed (n=19), while all patients with < 1 NCN within out-of treatment group remained in hematological remission (n=75). 8 patients with < 1 NCN during maintenance therapy finally relapsed, but all of them appeared beyond the end of treatment. In addition, in the intermediate group of patients (NCN 1–10) (n=18) relapse probability of 40% at 5 years was observed. All hematological relapses were predicted if a follow-up sample had been obtained within the previous 4 months. Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients.

Sensitivity and Applicability of Six-Color Flow Cytometry Is Comparable to ASO-Primer-Real-Time PCR (RQ-PCR) for Minimal Residual Disease (MRD) Monitoring in Adult Acute Lymphoblastic Leukemia (ALL) - A Comparative Analysis in 70 Patients from the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL).

Although the quantification of MRD by PCR or flow cytometry (FC) is of proven prognostic value in ALL, both methods were never compared in adults. Since 3- to 4-color FC showed equal or inferior sensitivity to PCR in childhood ALL, we herein prospectively compared six-color FC (6C-FC) at initial diagnosis and during early follow-up in adult ALL to RQ-PCR as reference method.Diagnostic samples from 70 consecutive ALL patients (pts) (22 T-, 48 B-lineage; 18 to 71 yrs) were screened by 14 different multiplex immungene consensus PCRs as published (Bruggemann et al., Blood, 2006) and simultaneously by 8 different 6C-FC combinations which included a total of 29 different antibodies. At least one of the consensus PCRs revealed monoclonality in 64/70 pts (91 %), whereas immunophenotypic aberrations could distinguish ALL from benign hematopoiesis in 67/70 cases (96 %). 6C-FC was positive in all PCR negative samples, while PCR was successful in all samples lacking immunophenotypic aberrations. The standardized 6C-FC method revealed a median of 4 immunophenotypic aberrations per pt (range 1 to 8). The most frequently observed aberrations in B-lineage ALL included CD58++ (76 %), CD11a++(50%), CD22++ (44 %), low CD38 (50%), and KORSA+ (35 %). Tdt+cytoplasmatic(cy)CD3+ hallmarked 86% of T-lineage ALL cases which were additionally characterized by CD7+ (100%), surface(s) CD3- (90%), CD99+ (52 %), and CD1a+ (38 %).Up to now, RQ-PCR assays have been established for 38 pts (median sensitivity 10−4; range 10−5 to 10−2) thus allowing for comparisons of MRD assessments to 6C-FC in 155 follow-up samples. 110/155 (71 %) samples were collected during the first 4 therapy months. 6C-FC marker combinations for follow-up always included CD34/CD10/CD19/CD45 in B-lineage and TdT/cyCD3/sCD3/CD5 in T-lineage ALL. Stainings were individualized by adding 2 pt-specific markers each to up to 3 tubes. A total of 155 follow-up specimens comprised 13 FC-/RQ-PCR+ (8 %), 4 FC+/RQ-PCR- (3 %), 88 concordantly negative (57 %), and 50 concordantly positive (32 %) samples. Due to extremely low MRD, in 11/13 FC-/RQ-PCR+ samples (85 %) the results of RQ-PCR were qualitatively positive only, but not accurately quantifiable.The minimum MRD level detectable by 6C-FC was 3x10−5, as proven by a concordantly positive RQ-PCR result. MRD levels obtained by both methods correlated well (Spearman r = 0.85; p < 0.0001) in follow-up samples that were positive both by RQ-PCR and by 6C-FC. The median ratio between 6C-FC and RQ-PCR MRD results was 0.48 (range 0.012 to 113). In 11% of samples the ratio between MRD levels obtained by the two methods differed more than tenfold.Our novel standardized 6C-FC approach is highly sensitive for MRD assessments in adult ALL. Our results suggest the applicability of 6C-FC in a multicenter setting, an excellent specificity, a good quantitative correlation and a similar sensitivity when compared to RQ-PCR. Longer follow-up and the inclusion of more samples in particular from later time points are required to decide whether or not the two methods are of comparable clinical significance.

BCR-ABL mRNA levels at and after the time of a complete cytogenetic response (CCR) predict the duration of CCR in imatinib mesylate–treated patients with CML

AbstractAlthough most patients with chronic myeloid leukemia (CML) treated with imatinib mesylate achieve a complete cytogenetic response (CCR), some patients will relapse. To determine the potential of real-time quantitative BCR-ABL reverse transcriptase–polymerase chain reaction (RT-PCR) to predict the duration of continued CCR, we monitored 85 patients treated with imatinib mesylate who achieved a CCR. With a median follow-up of 13 months after CCR (29 months after imatinib mesylate; median 6 RQ-PCR assays), 23 patients (27%) had disease progression (predominantly loss of CCR). Compared with the median baseline level of BCR-ABL mRNA, 42% of patients achieved at least a 2-log molecular response at the time of first reaching CCR. Failure to achieve a 2-log response at the time of CCR was an independent predictive marker of subsequent progression-free survival (hazard ratio = 5.8; 95% CI, 1.7-20; P = .005). After CCR, BCR-ABL mRNA levels progressively declined for at least the next 15 months, and 42 patients (49%) ultimately achieved at least a 3-log reduction in BCR-ABL mRNA. Patients failing to achieve this 3-log response, at any time during therapy, had significantly shorter progression-free survival (hazard ratio = 8.1; 95% CI, 3.1-22; P < .001). The achievement of either a 2-log molecular response at the time of CCR or a 3-log response anytime thereafter is a significant and independent prognostic marker of subsequent progression-free survival.