Cell-free tumour DNA testing for early detection of cancer - a potential future tool.

In recent years, detection of cell-free tumour DNA (ctDNA) or liquid biopsy has emerged as an attractive noninvasive methodology to detect cancer-specific genetic aberrations in plasma, and numerous studies have reported on the feasibility of ctDNA in advanced cancer. In particular, ctDNA assays can capture a more 'global' portrait of tumour heterogeneity, monitor therapy response, and lead to early detection of resistance mutations. More recently, ctDNA analysis has also been proposed as a promising future tool for detection of early cancer and/or cancer screening. As the average proportion of mutated DNA in plasma is very low (0.4% even in advanced cancer), exceedingly sensitive techniques need to be developed. In addition, as tumours are genetically heterogeneous, any screening test needs to assay multiple genetic targets in order to increase the chances of detection. Further research on the genetic progression from normal to cancer cells and their release of ctDNA is imperative in order to avoid overtreating benign/indolent lesions, causing more harm than good by early diagnosis. More knowledge on the sources and elimination of cell-free DNA will enable better interpretation in older individuals and those with comorbidities. In addition, as white blood cells are the major source of cell-free DNA in plasma, it is important to distinguish acquired mutations in leukocytes (benign clonal haematopoiesis) from an upcoming haematological malignancy or other cancer. In conclusion, although many studies report encouraging results, further technical development and larger studies are warranted before applying ctDNA analysis for early cancer detection in the clinic.

Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia.

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Impact of malignant stem cell burden on therapy outcome in newly diagnosed chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38-) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1-100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P=0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P=0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

The frequency and prognostic impact of dic(9;20)(p13.2;q11.2) in childhood B-cell precursor acute lymphoblastic leukemia: results from the NOPHO ALL-2000 trial.

The dic(9;20)(p13.2;q11.2) is reported to be present in ∼2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, it easily escapes detection by G-banding analysis and its true prevalence is hence unknown. We performed interphase fluorescence in situ hybridization analyses-in a three-step manner-using probes for: (i) CDKN2A at 9p21, (ii) 20p and 20q subtelomeres and (iii) cen9 and cen20. Out of 1033 BCP ALLs diagnosed from 2001 to 2006, 533 were analyzed; 16% (84/533) displayed 9p21 deletions, of which 30% (25/84) had dic(9;20). Thus, dic(9;20)-positivity was found in 4.7% (25/533), making it the third most common genetic subgroup after high hyperdiploidy and t(12;21)(p13;q22). The dic(9;20) was associated with a female predominance and an age peak at 3 years; 18/25 (72%) were allocated to non-standard risk treatment at diagnosis. Including cases detected by G-banding alone, 29 dic(9;20)-positive cases were treated according to the NOPHO ALL 2000 protocol. Relapses occurred in 24% (7/29) resulting in a 5-year event-free survival of 0.69, which was significantly worse than for t(12;21) (0.87; P=0.002) and high hyperdiploidy (0.82; P=0.04). We conclude that dic(9;20) is twice as common as previously surmised, with many cases going undetected by G-banding analysis, and that dic(9;20) should be considered a non-standard risk abnormality.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Vascular endothelial growth factor gene expression in middle cerebral artery occlusion in the rat.

BACKGROUND: Focal cerebral ischemia induces up-regulation of angiogenic growth factors such as vascular endothelial growth factor (VEGF), which may have both beneficial and harmful effects to the ischemic brain. Vascular endothelial growth factor is up-regulated in models of brain ischemia, but the underlying mechanisms in vivo remain unclear. In the present report we have investigated the concomitant changes in VEGF and glyceraldehyde dehydrogenase (GAPDH) mRNA expression in a model of permanent and transient cerebral ischemia. METHODS: Male Sprague-Dawley rats were exposed to permanent or transient (2 h) middle cerebral artery occlusion (PMCAO, TMCAO). Brain samples were collected at survival times ranging from 6 h to 1 week, and the levels of VEGF164 and GAPDH mRNA were determined using reverse-transcriptase real-time polymerase chain reaction (RT-PCR). RESULTS: The VEGF mRNA levels decreased gradually over the observation period in a similar manner in both PMCAO and TMCAO. Maximum levels, seen at early observation time points, did not significantly deviate from sham controls. No statistically significant changes in GAPDH mRNA levels were observed, but there was a tendency towards a postischemic decrease with subsequent return to control levels over time. The VEGF/GAPDH ratio followed a pattern of decrease similar to VEGF mRNA alone. CONCLUSION: The VEGF mRNA levels at 6 h after MCAO remain near baseline and thereafter decline, regardless of whether the occlusion is permanent or transient (2 h). The findings raise the question of other than transcriptional regulation of VEGF in cerebral ischemia.

Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles.

Single-nucleotide polymorphisms (SNPs) have the potential to be particularly useful as markers for monitoring of chimerism after stem cell transplantation (SCT) because they can be analyzed by accurate and robust methods. We used a two-phased minisequencing strategy for monitoring chimerism after SCT. First, informative SNPs with alleles differing between donor and recipient were identified using a multiplex microarray-based minisequencing system screening 51 SNPs to ensure that multiple informative SNPs were detected in each donor-recipient pair. Secondly, the development of chimerism was followed up after SCT by sensitive, quantitative analysis of individual informative SNPs by applying the minisequencing method in a microtiter plate format. Using this panel of SNPs, we identified multiple informative SNPs in nine unrelated and in 16 related donor-recipient pairs. Samples from nine of the donor-recipient pairs taken at time points ranging from 1 month to 8 years after transplantation were available for analysis. In these samples, we monitored the allelic ratios of two or three informative SNPs in individual minisequencing reactions. The results agreed well with the data obtained by microsatellite analysis. Thus, we conclude that the two-phased minisequencing strategy is a useful approach in the following up of patients after SCT.

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 promyelocytic 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.

Beta-globin mRNA increases rapidly during erythropoietin treatment.

Recombinant human erythropoietin (r-HuEpo) has an important role in the treatment of anaemic patients. Because of the high cost of r-HuEpo treatment, an early indicator of whether a patient is responding to the therapy would be valuable. Although measurement of gene expression is a promising new tool, it has not yet been established in clinical practice. The response pattern of a possible new marker, beta-globin mRNA, is compared with reticulocyte count, levels of haemoglobin, transferrin receptor and ferritin after r-HuEpo treatment. Eight healthy volunteers were stimulated with erythropoietin three times a week for four weeks and compared with five untreated control subjects. Blood samples were collected before each erythropoietin injection. Quantitative measurement of beta-globin mRNA was performed by poly(A) selection onto a manifold plastic support, coated with oligo(dT). The mRNA was reverse transcribed, followed by quantitative analysis using PCR via the 5' nuclease assay. The individuals treated with rHuEpo showed a more distinct increase in beta-globin mRNA levels than all other laboratory measurements. Beta-globin mRNA levels are therefore promising as a marker for the response to treatment with Epo.

RNA-templated DNA ligation for transcript analysis.

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.

Manifold-assisted reverse transcription-PCR with real-time detection for measurement of the BCR-ABL fusion transcript in chronic myeloid leukemia patients.

BACKGROUND: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML). METHODS: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system. RESULTS: The detection limit of the method was one positive K562 cell among 10(5) negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data. CONCLUSIONS: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.

Enhanced detection and distinction of RNA by enzymatic probe ligation.

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe-target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.

Expression profiling across many samples via manifold-assisted mRNA processing.

Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented

Molecular genetic applications of streptavidin-coated manifold supports.

Practical problems of handling large numbers of samples limit the application of molecular genetic procedures in clinical settings and in research. In the present review we describe a multipronged manifold support, coated with streptavidin, that offers distinct advantages in preparative and diagnostic applications. In order to increase the surface available on the manifold, porous Sepharose particles conjugated with streptavidin were attached to the plastic support. This procedure increased the surface by almost three orders of magnitude, permitting sufficient streptavidin to be coupled to the support for most routine applications. The manifold supports have been used for sample preparation and in a number of genetic assays, including allele discrimination assays and DNA sequencing, In all these assay formats the manifold supports allow large numbers of samples to be processed in parallel.