Jean Gabert

Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35–45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n=278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action ‘Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation’ with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A ↔ B) and nested PCR (internal primers C ↔ D) as well as for ‘shifted’ PCR with a primer upstream (E5′ primer) or downstream (E3′ primer) of the external A ↔ B primers. The ‘shifted’ E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10−2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10−4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – a Europe against cancer program

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects

Detection of minimal residual disease (MRD) has prognostic value in many hematologic malignancies, including acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, non-Hodgkins lymphoma, and multiple myeloma. Quantitative MRD data can be obtained with real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T-cell receptor gene rearrangements, breakpoint fusion regions of chromosome aberrations, fusion-gene transcripts, aberrant genes, or aberrantly expressed genes, their application being dependent on the type of disease. RQ-PCR analysis can be performed with SYBR Green I, hydrolysis (TaqMan) probes, or hybridization (LightCycler) probes, as detection system in several RQ-PCR instruments. Dependent on the type of MRD-PCR target, different types of oligonucleotides can be used for specific detection, such as an allele-specific oligonucleotide (ASO) probe, an ASO forward primer, an ASO reverse primer, or germline probe and primers. To assess the quantity and quality of the RNA/DNA, one or more control genes must be included. Finally, the interpretation of RQ-PCR MRD data needs standardized criteria and reporting of MRD data needs international uniformity. Several European networks have now been established and common guidelines for data analysis and for reporting of MRD data are being developed. These networks also include standardization of technology as well as regular quality control rounds, both being essential for the introduction of RQ-PCR-based MRD detection in multicenter clinical treatment protocols.

Should Adolescents With Acute Lymphoblastic Leukemia Be Treated as Old Children or Young Adults? Comparison of the French FRALLE-93 and LALA-94 Trials

PURPOSE To compare pediatric and adult therapeutic practices in the treatment of acute lymphoblastic leukemia (ALL) in adolescents. PATIENTS AND METHODS From June 1993 to September 1994, 77 and 100 adolescents (15 to 20 years of age) were enrolled in the pediatric FRALLE-93 and adult LALA-94 protocols, respectively. Among the different prognostic factors, we retrospectively analyzed the effect of the trial on achieving complete remission (CR) and event-free survival (EFS). RESULTS Patients were younger in the FRALLE-93 than in the LALA-94 protocol (median age, 15.9 v 17.9 years, respectively), but other characteristics were similar, including median WBC count (18 x 10(9) cells/L v 16 x 10(9) cells/L), B/T-lineage (54 of 23 v 72 of 28 patients), CD10-negative ALL (13% v 15%), and poor-risk cytogenetics (t(9;22), t(4;11), or hypodiploidy less than 45 chromosomes: 6% v 5%). The CR rate depended on WBC count (P =.005) and trial (94% v 83% in FRALLE-93 and LALA-94, respectively; P =.04). Univariate analysis showed that unfavorable prognostic factors for EFS were as follows: the trial (estimated 5-year EFS, 67% v 41% for FRALLE-93 and LALA-94, respectively; P <.0001), an increasing WBC count (P <.0001), poor-risk cytogenetics (P =.005), and T-lineage (P =.01). The trial and WBC count remained significant parameters for EFS in multivariate analysis (P <.0001 and P =.0004). Lineage subgroup analysis showed an advantage for the FRALLE-93 trial for CR achievement (98% v 81%; P =.002) and EFS (P =.0002) in B-lineage ALL and for EFS (P =.05) in T-lineage ALL. Age was not a significant prognostic factor in this population of adolescents. CONCLUSION This studys findings indicate that adolescents should be included in intensive pediatric protocols and that new trials should be designed, inspired by pediatric protocols, for the treatment of young adults with ALL.

Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene

The human poliovirus (PV) receptor (PVR) is a member of the immunoglobulin (Ig) superfamily with unknown cellular function. We have isolated a human PVR-related (PRR) cDNA. The deduced amino acid (aa) sequence of PRR showed, in the extracellular region, 51.7 and 54.3% similarity with human PVR and with the murine PVR homolog, respectively. The cDNA coding sequence is 1.6-kb long and encodes a deduced 57-kDa protein; this protein has a structural organization analogous to that of PVR, that is, one V- and two C-set Ig domains, with a conserved number of aa. Northern blot analysis indicated that a major 5.9-kb transcript is present in all normal human tissues tested. In situ hybridization showed that the PRR gene is located at bands q23-q24 of human chromosome 11.

Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA-94 trial.

Fifty-four percent of adults with acute lymphoblastic leukemia (ALL) who entered the LALA-94 trial experienced a first relapse. We examined the outcome of these 421 adult patients. One hundred and eighty-seven patients (44%) achieved a second complete remission (CR). The median disease-free survival (DFS) was 5.2 months with a 5-year DFS at 12%. Factors predicting a better outcome after relapse were any transplant performed in second CR (P<0.0001), a first CR duration >1 year (P=0.04) and platelet level >100 × 109/l at relapse (P=0.04). Risk groups defined at diagnosis and treatment received in first CR did not influence the outcome after relapse. The best results were obtained in a subset of patients who were eligible for allogeneic stem cell transplantation (SCT). Geno-identical allogeneic SCT was performed in 55 patients, and 3 patients received donor lymphocyte infusions. Forty-four transplantations were performed from an unrelated donor (of which four were cord blood). We conclude that most adult patients with recurring ALL could not be rescued using current available therapies, although allogeneic SCT remains the best therapeutic option.

Responder and nonresponder patients exhibit different peripheral transcriptional signatures during major depressive episode.

To date, it remains impossible to guarantee that short-term treatment given to a patient suffering from a major depressive episode (MDE) will improve long-term efficacy. Objective biological measurements and biomarkers that could help in predicting the clinical evolution of MDE are still warranted. To better understand the reason nearly half of MDE patients respond poorly to current antidepressive treatments, we examined the gene expression profile of peripheral blood samples collected from 16 severe MDE patients and 13 matched controls. Using a naturalistic and longitudinal design, we ascertained mRNA and microRNA (miRNA) expression at baseline, 2 and 8 weeks later. On a genome-wide scale, we detected transcripts with roles in various biological processes as significantly dysregulated between MDE patients and controls, notably those involved in nucleotide binding and chromatin assembly. We also established putative interactions between dysregulated mRNAs and miRNAs that may contribute to MDE physiopathology. We selected a set of mRNA candidates for quantitative reverse transcriptase PCR (RT-qPCR) to validate that the transcriptional signatures observed in responders is different from nonresponders. Furthermore, we identified a combination of four mRNAs (PPT1, TNF, IL1B and HIST1H1E) that could be predictive of treatment response. Altogether, these results highlight the importance of studies investigating the tight relationship between peripheral transcriptional changes and the dynamic clinical progression of MDE patients to provide biomarkers of MDE evolution and prognosis.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Clinical variations modulate patterns of gene expression and define blood biomarkers in major depression

The aim of the study is to compare the expression level of candidate genes between patients suffering from a severe major depressive episode (MDE) and controls, and also among patients during MDE evolution. After a comprehensive review of the biological data related to mood disorders, we initiated a hypothesis-driven exploration of candidate mRNAs. Using RT-qPCR, we analyzed peripheral blood mononuclear cells (PBMCs) mRNA obtained from a homogeneous population of 11 patients who suffered from severe melancholic MDE. To assess the evolution of MDE, we analyzed PBMC mRNAs that were collected on Day 1 and 8 weeks later. Data from these patient samples were analyzed in comparison to age- and sex-matched healthy controls. Among 40 candidate genes consistently transcribed in PBMCs, 10 were differentially expressed in at least one comparison. We found that variations of mRNA levels for NRG1, SORT1 and TPH1 were interesting state-dependent biological markers of the disease. We also observed that variations in other mRNA expression were associated with treatment efficacy or clinical improvement (CREB1, HDAC5, HSPA2, HTR1B, HTR2A, and SLC6A4/5HTT). Significantly, 5HTT exhibited a strong correlation with clinical score evolution. We also found a state-independent marker, IL10. Moreover, the analysis of 2 separate MDEs concerning a same patient revealed comparable results for the expression of CREB1, HSPA2, HTR1B, NRG1 and TPH1. Overall, our results indicate that PBMCs obtained at different time points during MDE progression represent a promising avenue to discover biological markers for depression.