Allele-specific Oligonucleotide Primers Research Articles

Apolipoprotein E polymorphism in a Romanian population

Abstract Background: Apolipoprotein E (ApoE) is a ubiquitous protein involved in maintaining cholesterol homeostasis and lipoprotein clearance from circulation. It is coded by three alleles (ε2, ε3, ε4) with six genotypes (ε3/ε3, ε3/ε4, ε2/ε3, ε4/ε4, ε2/ε4, and ε2/ε2). Several studies have shown a relationship between ApoE-specific isoforms and different diseases. There are few data about the prevalence of ApoE polymorphism in the Romanian population. We aimed to assess the prevalence of Apolipoprotein E alleles in a healthy population from Romania and compare it with the data found in other countries of Europe. Methods: ApoE genotype was examined in 187 unrelated healthy volunteers (131 males and 56 females) from the region of southwest Romania (mean age 48.04 ± 8.35 years). Analysis of ApoE genotype variants was done using the PCR method with allele-specific oligonucleotide primers. Results: The frequency of heterozygote genotypes ε2/ε3, ε2/ε4, and ε3/ε4 was 4.8%, 1.6%, and 24.73%, respectively, while the frequency of homozygote genotypes ε2/ε2, ε3/ε3, ε4/ε4 was 0.5%, 66.12%, and 2.1%, respectively, with a mean frequency of ε2, ε3 and ε4 alleles of 6.9%, 96.25%, and 28.49%, respectively. East European countries have the lowest mean frequency of the ε4 allele (9.96%), and the highest frequency of the ε4 allele (23.38%) is in the Nordic Countries. Conclusions: The ε4 allele prevalence in this Romanian population is higher in comparison with other European countries, similar to Nordic countries of Europe.

Measurable residual disease in the treatment of chronic lymphocytic leukemia.

Treatment outcomes of chronic lymphocytic leukemia (CLL) have improved since chemoimmunotherapy and novel drugs became available for CLL treatment; therefore, more sensitive methods to evaluate residual CLL cells in patients are required. Measurable residual disease (MRD) has been assessed in several clinical trials on CLL using flow cytometry, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers, and high-throughput sequencing. MRD assessment is useful to predict the treatment outcomes in the context of chemotherapy and treatment with novel drugs such as venetoclax. In this review, we discuss major techniques for MRD assessment, data from relevant clinical trials, and the future of MRD assessment in CLL treatment.

Minimal Residual Disease in Chronic Lymphocytic Leukemia: A New Goal?

In chronic lymphocytic leukemia (CLL), there is a growing interest for minimal residual disease (MRD) monitoring, due to the availability of drug combinations capable of unprecedented complete clinical responses. The standardized and most commonly applied methods to assess MRD in CLL are based on flow cytometry (FCM) and, to a lesser extent, real-time quantitative PCR (RQ-PCR) with allele-specific oligonucleotide (ASO) primers of immunoglobulin heavy chain genes (IgH). Promising results are being obtained using droplet digital PCR (ddPCR) and next generation sequencing (NGS)-based approaches, with some advantages and a potential higher sensitivity compared to the standardized methodologies. Plasma cell-free DNA can also be explored as a more precise measure of residual disease from all different compartments, including the lymph nodes. From a clinical point of view, CLL MRD quantification has proven an independent prognostic marker of progression-free survival (PFS) and overall survival (OS) after chemoimmunotherapy as well as after allogeneic transplantation. In the era of mechanism-driven drugs, the paradigms of CLL treatment are being revolutionized, challenging the use of chemoimmunotherapy even in first-line. The continuous administration of ibrutinib single agent has led to prolonged PFS and OS in relapsed/refractory and treatment naïve CLL, including those with TP53 deletion/mutation or unmutated IGHV genes, though the clinical responses are rarely complete. More recently, chemo-free combinations of venetoclax+rituximab, venetoclax+obinutuzumab or ibrutinib+venetoclax have been shown capable of inducing undetectable MRD in the bone marrow, opening the way to protocols exploring a MRD-based duration of treatment, aiming at disease eradication. Thus, beside a durable disease control desirable particularly for older patients and/or for those with comorbidities, a MRD-negative complete remission is becoming a realistic prospect for CLL patients in an attempt to obtain a long-lasting eradication and possibly cure of the disease. Here we discuss the standardized and innovative technical approaches for MRD detection in CLL, the clinical impact of MRD monitoring in chemoimmunotherapy and chemo-free trials and the future clinical implications of MRD monitoring in CLL patients outside of clinical trials.

Separation-free single-base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites.

A separation-free single-base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele-specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5'-end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite-converted genomic DNA, and a DNA polymerase. A single base-extended primer (i.e., SBE product) that was 5'-Cy3- and 3'-Cy5-tagged was formed by incorporation of the Cy5-labeled terminator into the 3'-end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.

Mutation analysis of the PNPLA1 gene in a family with autosomal recessive congenital ichthyosis

Objective To identify a causative gene of autosomal recessive congenital ichthyosis (ARCI) in a Chinese family, and to analyze the genotype-phenotype correlation. Methods Peripheral blood samples were collected from the proband, his elder brother and parents, and genomic DNA was extracted from these blood samples. Genome-wide exome sequencing was conducted to determine the mutation site in the proband, and then allele-specific oligonucleotide primers were designed based on the mutation site. PCR was performed to detect the mutation site to further identify the causative gene of ARCI in the family. Results A new homozygous missense mutation was identified in exon 4 in 1 allele of the PNPLA1 gene in the proband, which led to a codon change from cytosine (C) to thymine (T) at position 700 (c.700C > T) and resulted in the substitution of proline by serine (p.pro234ser) . The same mutation was also detected in the proband′s brother, and his parents were the mutation carriers. No mutations were found in unrelated healthy Chinese individuals. Conclusion The missense mutation in the PNPLA1 gene (p.pro234ser) is associated with clinical symptoms of the patient with ARCI. Key words: Genetic diseases, inborn; Chromosome disorders; Mutation; Genes, PNPLA1; Autosomal recessive congenital ichthyosis

Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials

Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials

Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients

Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

Clinical Meanings of Quantitative PCR of Patient-Specific Immunoglobulin VDJ Regions Using Various Materials of Myeloma Patients

Introduction; The PCR with patient-specific, allele-specific oligonucleotide primers for individual IgH VDJ regions (ASO-PCR) is considered the most sensitive method to detect minimal residual disease (MRD) levels in patients with multiple myeloma (MM). In this study, we quantified the ASO-PCR products by using peripheral blood mononuclear cells (PBMNCs) as well as bone marrow mononuclear cells (BMMNCs). We also quantified the ASO-PCR products in mRNAs from CD20+38- B-cells in BM to examine whether there are clonogenic cells in relatively earlier B-cell fraction as well as cell-free DNAs from the sera to examine whether there are DNA fragments from MM cells in PB.Materials and methods; We have analyzed 30 MM cases. After an informed consent, patient-oriented PCR primers were designed from sequence information of immunoglobulin heavy chain (IgH) variable regions of myeloma cells of each patient.Results; The median age in this cohort was 64.5 years (36-83); the ratio of men to women was 16:14; and the numbers of IgG-, IgA-, and Bence Jones protein (BJP)- type patients were 18, 7, and 5, respectively. The patient-specific ASO primers could be designed in 25 cases, but not in 3 BJP-type cases or 2 IgG-type cases. We could quantify the ASO-PCR products in 20 of 30 BMMNCs samples at diagnosis. The ASO-PCR levels (IgH/β−actin levels)in BMMNCs correlated with those in PBMNCs, but not to the percent of plasma cells in BM or the values for M-protein. However, the ASO-PCR levels were decreased after the treatment and reflected tumor burden well individually. The ASO-PCR levels in PBMNCs showed a statistically significant correlation with those in BMMNCs at diagnosis (Spearman’s ρ= 0.98, P<0.001), 6 months (Spearman’s ρ= 0.83, P = 0.020) and 12 months (Spearman’s ρ= 1.90, P = 0.001). Therefore, ASO-PCR using PBMNCs as well as BMMNCs is suitable for MRD evaluation. We could detect the patient-specific IgH DNA sequences in cell-free DNA extracted from the sera and quantify the ASO-PCR products. The sequences of the ASO-PCR product were identical to the originally designed sequence, suggesting that detection of the ASO-PCR products in cell-free DNA could reflect the persistence of myeloma cells somewhere in body. The ASO-PCR products for CD20+CD38- B-cells in BM were relatively low but were clearly detected in 17 cases at diagnosis. The ASO-PCR levels in CD20+CD38- B-cells in BM showed good correlation with both values of ASO-PCR in BMMNCs (Spearman’s ρ= 1.35, P <0.001) and in PBMNCs (Spearman’s ρ= 1.09, P <0.001). Thus, the evaluation of relatively earlier B-cell stages of myeloma cells seems to be of interst, including for the possible existence of MM clones in CD20+CD38- B-cell population in BM.Discussion; Our findings that we could quantify the ASO-PCR products in PBMNCs, BM CD20+CD38- B-cells, cell-free DNA as well as BMMNCs strongly suggest wide sources of clinical materials to analyze. There were statistically significant correlations in values of the ASO-PCR products between BMMNCs and PBMNCs, therefore suggesting the possibility that clonogenic plasma cells or myeloma precursor cells might circulate in peripheral blood. We expect the possibility that PBMNCs will be a good source to monitor MRD. We could also detect and quantify the ASO-PCR products in cell-free DNA from the sera. To the best of our knowledge, this is the first report that IgH DNA fragments from MM cells circulate in the sera. Furthermore, we could detect and quantify the ASO-PCR products in CD20+CD38- B-cells in BM at diagnosis. These results indicated that clonogenic MM cells could exist not only in the CD20-CD38high plasma cell fraction but also in the CD20+CD38- B-cell fraction, which might include myeloma stem or initiating cells. Thus, we could consider the treatment strategies to include anti-CD20 antibodies against the clonogenic CD20+CD38- B-cell population. In conclusion, our ASO-PCR using various clinical materials is supposed to be useful for detecting MRD in the patients with MM as well as for clarifying the pathogenesis of MM. DisclosuresSata:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Shibayama:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Habuchi:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fukushima:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Fujita:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Ezoe:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Tadokoro:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Maeda:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Mizuki:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Oritani:Celgen: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Kanakura:Janssen: Research Funding; Takeda: Research Funding; Celgen: Research Funding.

Molecular-Based Classification of Acute Myeloid Leukemia and Its Role in Directing Rational Therapy

Acute myeloid leukemia (AML) is not a single pathologic entity but represents a heterogeneous group of malignancies. This heterogeneity is exemplified by the variable clinical outcomes that are observed in patients with AML, and it is largely the result of diverse mutations within the leukemic cells. These mutations range from relatively large genetic alterations, such as gains, losses, and translocations of chromosomes, to single nucleotide changes. Detection of many of these mutations is required for accurate diagnosis, prognosis, and treatment of patients with AML. As such, many testing modalities have been developed and are currently employed in clinical laboratories to ascertain mutational status at prognostically and therapeutically critical loci. The assays include those that specifically identify large chromosomal alterations, such as conventional metaphase analysis and fluorescence in situ hybridization, and methods that are geared more toward analysis of small mutations, such as PCR with allele-specific oligonucleotide primers. Furthermore, newer tests, including array analysis and next-generation sequencing, which can simultaneously probe numerous molecular aberrancies within tumor cells, are likely to become commonplace in AML diagnostics. Each testing method clearly has advantages and disadvantages, an understanding of which should influence the choice of test in various clinical circumstances. To aid such understanding, this review discusses both genetic mutations in AML and the clinical tests-including their pros and cons-that may be used to probe these abnormalities. Additionally, we highlight the significance of genetic testing by describing cases in which results of genetic testing significantly influence clinical management of patients with AML.

Sensitive and Specific Measurement of Minimal Residual Disease in Acute Lymphoblastic Leukemia

A sensitive and specific quantitative real-time polymerase chain reaction method, involving three rounds of amplification with two allele-specific oligonucleotide primers directed against an rearrangement, was developed to quantify minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL). For a single sample containing 10 microg of good quality DNA, MRD was quantifiable down to approximately 10(-6), which is at least 1 log more sensitive than current methods. Nonspecific amplification was rarely observed. The standard deviation of laboratory estimations was 0.32 log units at moderate or high levels of MRD, but increased markedly as the level of MRD and the number of intact marker gene rearrangements in the sample fell. In 23 children with ALL studied after induction therapy, the mean MRD level was 1.6 x 10(-5) and levels ranged from 1.5 x 10(-2) to less than 10(-7). Comparisons with the conventional one-round quantitative polymerase chain reaction method on 29 samples from another 24 children who received treatment resulted in concordant results for 22 samples and discordant results for seven samples. The sensitivity and specificity of the method are due to the use of nested polymerase chain reaction, one segment-specific and two allele-specific oligonucleotide primers, and the use of a large amount of good quality DNA. This method may improve MRD-based decisions on treatment for ALL patients, and the principles should be applicable to DNA-based MRD measurements in other disorders.

Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction

Lymphoma is the most common hematopoietic malignancy in dogs. Although a large proportion of dogs with lymphoma can achieve clinical remission by initial chemotherapy, most dogs die as a consequence of tumor relapse. We established a quantitative detection system for minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction (PCR). A canine T-cell lymphoma-derived cell line, namely, UL-1, was used to examine the specificity and sensitivity of the MRD detecting system. Allele-specific oligonucleotide primers and probes were designed based on the sequence of T-cell receptor γ chain (TCRγ) gene fragment of UL-1 cells in conjunction with its downstream sequence, which were obtained from the dog genome database. The real-time PCR system for plasmid DNA containing the TCRγ gene derived from UL-1 cells and the genomic DNA of UL-1 cells revealed that the system was accurate for 10–100,000 copies per reaction and its sensitivity was 1 cell per 10,000 cells. In order to monitor the kinetics of tumor cell number in canine lymphoma, we quantified the level of MRD in the peripheral blood of 7 dogs with lymphoma under chemotherapy. Since the lymphoma cells from the 7 patients were shown to be B-cell origin from the finding of clonal rearrangement of immunoglobulin heavy chain (IgH) gene, allele-specific oligonucleotide primers and probes were prepared based on the sequence of rearranged IgH gene in each case. The number of peripheral blood tumor cells measured by the real-time PCR was comparable to that estimated by conventional hematological examination in 2 cases of stage V lymphoma. MRD in the peripheral blood was detectable in all 7 cases, even in the complete remission (CR) phase. In the 7 lymphoma dogs, changes in the MRD levels of peripheral blood generally paralleled with the changes in the volumes of lymph nodes. Molecular CR, in which the MRD level was below the detection limit, was not observed in any of these 7 patients under chemotherapy. The MRD level detected by the real-time PCR method described here would be useful for investigating the kinetics of tumor cell growth and its regression in canine lymphoma patients.

Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)

POLYMORPHISM OF THE HUMAN TNF PROMOTER REGION IN BRAZILIAN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS.

INTRODUCTION: Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by multisystemic organ involvement, polyclonal B‐cell activation and the production of autoantibodies. Several family studies have shown convincing evidences of multiple genetic loci associated with SLE development. TNF is a potent proinflammatory cytokine that plays an important role in the pathogenesis of SLE, and certain alleles of the tumor necrosis factor (TNF) gene have been consistently associated with the disease, particularly the ‐308‐A/G functional promoter polymorphism. In the present study we analyzed the allelic distribution of TNF‐308A/G in Brazilian SLE patients.METHODS AND RESULTS: A total of 148 SLE patients and 130 matched healthy controls were typed for the TNF‐308A/G polymorphism by polymerase chain reaction using allele‐specific oligonucleotide primers. A significant increase of the TNF G/A 308 genotype was observed in SLE patients when compared with healthy controls (p &lt; 0.0001; OR = 7.156; 95% I. C.: 3.462–14.792).CONCLUSIONS: Since the Brazilian population is highly genetically diverse, the results reported here are in agreement with those reported for other populations, and corroborate the role of TNF in disease pathogenesis and the TNF‐308A/G genotype, which has been associated with high production of TNF, as conferring susceptibility to the disease.RESEARCH SUPPORT: CAPES

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F2 plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.

Consensus JH Gene Probes with Conjugated 3′-Minor Groove Binder for Monitoring Minimal Residual Disease in Acute Lymphoblastic Leukemia

Several approaches for the detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) have shown the importance of determining the level of MRD precisely. In the present study, we tested a new real-time quantitative polymerase chain reaction (RQ-PCR) strategy with minor groove binder (MGB) technology for immunoglobulin heavy chain gene rearrangements by positioning a MGB probe at the germline JH segments and one of the primers at the downstream introns in combination with an allele-specific oligonucleotide (ASO) primer complementary to the VH-DH or DH-JH junctional region. A MGB probe forms extremely stable duplexes with single-stranded DNA targets, allowing the use of shorter probes for hybridization-based assays. Therefore, it shows positional flexibility. We have designed two novel consensus MGB JH germline probes for analyzing all of the germline rearrangements registered in the V BASE database, and demonstrated that the MRD was detectable with the probes in 17 cases of childhood ALL. The actual copy number for the targets and dynamic changes before and after treatment were almost identical between the JH MGB probe and conventional non-MGB probes in each patient. MGB technology will undoubtedly contribute to MRD-PCR studies of childhood ALL.

International Standardized Approach to Molecular and Flow Cytometric Residual Disease Monitoring in CLL.

Conventional therapy in CLL is not curative, partly because minimal residual disease (MRD) is usually detectable after therapy. Recent therapeutic approaches achieve higher complete remission (CR) rates, and often aim for MRD eradication as this has repeatedly been shown to predict improved outcome. However, many different MRD techniques have been used to assess response, making it extremely difficult to interpret and compare different clinical trials. The aim of this international collaboration is to develop standardized flow cytometric and PCR approaches to MRD monitoring in CLL that are broadly applicable; to assess their relative sensitivity and specificity in a variety of laboratories; and to establish a standardized reporting convention to facilitate the interpretation of clinical trials. These techniques may be used as a benchmark for assessing response and comparing the efficacy of different therapeutic approaches. PCR approaches using consensus primers to the immunoglobulin heavy chain (IgH) gene are straightforward and frequently used, but have highly variable sensitivity between patients. In the proposed standardized method, the IgH gene is amplified using the BIOMED-2 primers (van Dongen et al, Leukemia 2003, 17:2257) and an allele-specific CDR3 primer is designed. CLL cells are identified by RQ-PCR using the allele-specific oligonucleotide (ASO) primer coupled with intronic JH primers and consensus reporter probes. The amount of PCR product is compared to a single copy housekeeping gene (albumin) to identify the number of CLL cells in the sample. This approach reproducibly quantitates as few as 1 CLL cell in 10,000 other leucocytes, and may provide qualitative information below this level. Multi-parameter flow cytometric analysis with combinations such as CD19/CD5/CD79b/CD20 is reported to have a sensitivity approaching that of ASO-PCR. However, such approaches are less effective in situations where normal B-cells lack CD20, e.g. in patients treated with combinations including rituximab and in normal bone marrow. To circumvent these problems, the group has tested 58 individual four-colour combinations of antibodies identified from protein expression profiling and previously published studies. CD79b/CD43/CD19/CD5, CD81/CD22/CD19/CD5, and CD20/CD38/CD19/CD5 provided the least inter-laboratory variation, independent of methodology. They show low contamination by non-CLL cells and were therefore selected as a core panel for MRD analysis. Dilutional studies indicate that a quantitative sensitivity equivalent for detection of 1 CLL cell in 10,000 leucocytes is achievable; the practical sensitivity is being tested in blinded dilutional analyses. The proposed flow cytometry approach is applicable to all sample types and all therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD negative status in real time. The PCR approach is highly sensitive but more complex, and is most suited to retrospective response assessment of clinical trials. We have identified the appropriate standardized analytical methods for both techniques and are currently validating these findings. These approaches will allow direct comparison of efficacy between different clinical trials, allowing more rapid international progress towards identifying a curative approach.

Immunomagnetic enrichment of CD138 positive cells from weakly infiltrated myeloma patients samples enables the determination of the tumor clone specific IgH rearrangement.

In multiple myeloma, the polymerase chain reaction (PCR) of the Ig heavy chain with allele-specific oligonucleotide (ASO) primers is a common and well-described method of identifying the tumor clone in peripheral blood (PB), bone marrow (BM) or leukapheresis products (LA). A factor which is crucial to the detection of clonal Ig rearrangements lies in the 'purity' of the tumor tissue used for the consensus PCR. We describe the application of a method to enrich CD138 positive myeloma cells derived from weakly infiltrated PB-, BM- and LA-samples. These are subjected to immunomagnetic enrichment with the MACS system, using an CD138 antibody directly conjugated to magnetic beads to obtain an enriched tumor cell population and the subsequent amplification of tumor specific IgH rearrangements. We investigated 29 samples (ten PB, ten BM, nine LA) with a median myeloma cell content of 0.5%. The approach led to a median enrichment factor of 118. Tumor-specific rearrangements could be amplified reproducibly from samples containing less than 0.1% myeloma cells.

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia

AbstractTumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B-cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 104 to 106 normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH VH gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 × 104 normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution.

Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia

Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B-cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 104 to 106 normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH VH gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 × 104 normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution.