The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories.

Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.

Epigenetic regulation of tumor suppressors in t(8:21)-containing AML.

The t(8;21) is perhaps the most frequent chromosomal translocation associated with acute myeloid leukemia. The translocation creates a fusion protein that consists of the DNA binding domain of the RUNX1 transcription factor fused to the MTG8 transcriptional co-repressor to create a potent transcriptional repressor. Here, we discuss the possibility that the t(8;21) fusion protein represses tumor suppressors that regulate the RAS signaling pathway and the p53 oncogenic checkpoint.

Degradable dU-based DNA template as a standard in real-time PCR quantitation.

Development of real-time quantitative PCR assays requires suitable positive controls. For assays with clinical applications, these controls may be difficult to obtain because some molecular aberrations are rare and patient material may be available in limited amounts. Because of the risk of introducing contaminations in the laboratory, cloned DNA is not a desirable alternative. We describe the use of dU-containing DNA as a positive control template in real-time quantitative PCR. dU-DNA constructs can be decontaminated by adding uracil N-glycosylase (UNG) to the reaction mixture. In addition, dU-DNA can be used for accurate quantification, because it allows quantification to be expressed in numbers of molecules. Since synthetic dU-DNA constructs can easily be quantitated spectroscopically, they provide a more accurate control than arbitrary cell line units. We applied this method for the detection of the E2A-Pbx1 gene fusion and show that UNG-containing reactions can be employed for diagnostics without loss of sensitivity, and that for positive and quantitative controls UNG negative reactions can be used. The use of dU-DNA provides a novel type of control template that can easily be integrated into existing PCR protocols.

A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.

Quantification of residual disease by real-time polymerase chain reaction (PCR) will become a pivotal tool in the development of patient-directed therapy. In recent years, various protocols to quantify minimal residual disease in leukemia or lymphoma patients have been developed. These assays assume that PCR efficiencies are equal for all samples. Determining t(14;18) and albumin reaction efficiencies for sixteen follicular lymphoma patient samples revealed higher efficiencies for blood samples than for lymph node samples in general. However, within one sample both reactions had equivalent efficiencies. Differences in amplification efficiencies between patient samples (low efficiencies) and the calibrator in quantitative analyses result in the underestimation of residual disease in patient samples whereby the weakest positive patient samples are at highest error. Based on these findings for patient samples, the efficiency compensation control was developed. This control includes two reference reactions in a multiplex setting, specific for the beta-actin and albumin housekeeping genes that are present in a constant ratio within DNA templates. The difference in threshold cycle values for both reference reactions, ie, the Ct(2) value, is dependent on the amplification efficiency, and is used to compensate for efficiency differences between patient samples and the calibrator. The beta-actin reference reaction is also used to normalize for DNA input. Furthermore, the efficiency compensation control facilitates identification of patient samples that are so contaminated with PCR inhibitory compounds that different amplification reactions are affected to a different extent. Accurate quantitation of residual disease in these samples is therefore impossible with the current quantitative real-time PCR protocols. Identification and exclusion of these inadequate samples will be of utmost importance in quantitative retrospective studies, but even more so, in future molecular diagnostic analyses.

Transcription of the MAGE-1 gene and the methylation status of its Ets binding promoter elements: a quantitative analysis in melanoma cell lines using a real-time polymerase chain reaction technique.

The human MAGE gene family comprises at least 12 highly homologous genes. This makes it very difficult to assess expression of a single member quantitatively by means of Northern blotting. In order to investigate expression of the MAGE-1 gene quantitatively we therefore used the recently developed real-time polymerase chain reaction (PCR), a novel fluorescence-based quantitative PCR technique. This powerful technique enables detection of expression levels which differ by as much as a factor of 10(5) in magnitude. MAGE-1 expression is known to correlate with demethylated status of the Ets binding sites of its promoter. In a panel of 19 melanoma and nine non-melanoma cell lines we were able to confirm the relationship between MAGE-1 expression and demethylation of the Ets binding promoter region. Five cell lines, however, showed only very slight expression, while the two essential Ets promoter elements were largely demethylated. Earlier studies have shown that treatment of MAGE-1-negative cell lines with the demethylating agent 5-aza-2'-deoxycytidine (DAC) is sufficient to induce MAGE-1 expression. We were able to induce clear MAGE-1 expression in two of the non-expressing cell lines by incubation with DAC, although this expression did not reach very high levels. Consistent with this low level of induction is the observation that the Ets binding sites of the MAGE-1 promoter were not completely demethylated in the DAC-treated cell populations. In conclusion, we show in this study that the real-time PCR technique is a very useful tool for the quantification of expression of highly homologous genes.

Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR.

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results.

In AML t(8;21) colony growth of both leukemic and residual normal progenitors is restricted to the CD34+, lineage-negative fraction.

In an earlier study we observed residual normal colonies in the CD34+, lineage-negative fraction in AML with a differentiated phenotype. The phenotype of both normal and leukemic progenitors in AML M2, t(8;21) was the subject of this study. The specific translocation enabled discrimination of normal and leukemic cells. Bone marrow samples from eight patients were evaluated for CD34 and the differentiation markers CD33, CD19 and CD56. Growth in all phenotypic fractions was measured in a single cell assay, which enabled quantification of plating efficiency, colony size and determination of progenitor cell origin. No growth was observed in the CD34-negative fraction. In the CD34+, lineage-positive fraction only clusters up to 20 cells were found in 6/8 samples. In 7/8 samples highly proliferative myeloid, erythroid and mixed colonies were cloned from the CD34+/CD56-CD19-CD33- fraction with a frequency between 1 and 12%. Such large colonies grew at a lower frequency (1-6%) from the CD34+/CD56 fraction (4/8 samples), the CD34+/CD56-CD19- fraction (5/8 samples) and from the CD34+/CD19- fraction (1/8 samples), respectively. Among the colonies consisting of more than 150 cells, only 3/45 evaluated were positive for the AML1/ETO fusion transcript. On the other hand, 8/19 colonies with less than 150 cells were AML1/ETO positive. This study shows that like normal progenitors leukemic progenitors are also present exclusively in the lineage-negative fraction in AML M2 t(8;21). A similar hierarchy of proliferation and differentiation was found for these leukemic progenitors, the smaller colony size fitting with their limited proliferation capacity. The frequency of leukemic progenitors was in the same range as their normal counterparts and detectable only after enrichment for the CD34+, lineage-negative population.

A low but functionally significant MDR1 expression protects primitive haemopoietic progenitor cells from anthracycline toxicity.

Pgp is expressed on normal haemopoietic progenitor cells. The significance of the efflux pump in protecting normal progenitors for anthracycline toxicity is not defined and is the subject of this study. Pgp was measured in CD34+ progenitors with a rhodamine efflux assay. A high efflux, modulated by verapamil, was only found in a distinct subpopulation (20-30%). Pgp measured by the monoclonal antibody antibody (MoAb) MRK-16 was low in the rhodamine dull, but significantly (P < 0.04) higher than in the rhodamine bright cells. Reverse transcriptase polymerase chain reaction (RT-PCR) of MDR1 mRNA showed a very weak signal in both populations. In a single-cell clonogenic assay, rhodamine dull cells appeared less sensitive to anthracyclines (IC50 daunorubicin 0.005 microg/ml; adriamycin 0.03 microg/ml) compared to rhodamine bright cells (IC50 daunorubicin 0.0025 microg/ml; adriamycin 0.01 microg/ml). Furthermore, verapamil significantly (P < 0.05) potentiated anthracycline toxicity only in the rhodamine dull cells, proving its Pgp-specific modulating effect. Rhodamine dull cells gave larger and more mixed colonies compatible with a more primitive origin. Although detection with MoAbs and RT-PCR revealed a low Pgp level, functionally this Pgp appeared to be very important in protecting primitive progenitors against anthracycline toxicity. This protection can be jeopardized by administration of Pgp modulators.

An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism.

The specificity of antisense oligonucleotides targeted to the mRNA breakpoint region of the Bcr-Abl oncogene, found in leukaemic cells from patients with chronic myeloid leukaemia, remains controversial due to non-specific effects. To prevent protein binding of oligonucleotides we designed and tested a methylphosphonate oligonucleotide with an attached 3' soluble phosphodiester tail. Growth of chronic myeloid leukaemia (CML) cell lines BV173, KCL-22 and cells of CML patients tested was inhibited by the b2a2 type antisense Bcr-Abl oligonucleotide and not with controls. Also the growth of control CD34+ cells of two healthy donors, control cell lines and cells from AML patients was only moderately affected or not affected. Bcr-Abl protein studies in combination with growth-determination experiments indicated that the antisense methylphosphonate Bcr-Abl oligonucleotide tested is a potent inhibitor of the growth of CML cells but works in a non-antisense manner.

Molecular Diagnosis of Acute Myeloid Leukemia with Maturation, FAB-Type M2.

Nonrandom chromosome abnormalities frequently are seen in particular subtypes of human leukemia and lymphoma. These abnormalities are considered to be involved in the neoplastic transformation and in tumor progression. The translocation (8;21) (q22;q22) is consistently associated with acute myeloid leukemia with maturation (French-American-British classification subtype M2; AML-M2). It accounts for 40% of pediatric type AML-M2 Molecular cloning of the chromosome 8-21 translocation breakpoint showed clustering on chromosome 21 within a limited region in the AML1 gene and on chromosome 8 within a limited region in the ETO gene (Eight Twenty One) also called MTG8 (Myeloid Translocation Gene on chromosome 8) (1-7). The t(8;21) results in a chimerical AML1/ETO gene on the der(8) chromosome. Rearrangement of AML1 was also detected in a patient with 8q- and only one chromosome 21, but without 21q+. This indicates that the molecular events on the der(8) chromosome leading to the chimerical AML1/ETO gene are more important than the events on the reciprocal 21q+ chromosome (8).

Phosphorothioate BCR-ABL antisense oligonucleotides induce cell death, but fail to reduce cellular bcr-abl protein levels.

The bcr-abl oncogene is a fusion gene resulting from a reciprocal translocation which forms the hallmark of chronic myeloid leukemia (CML). Antisense oligonucleotides complementary to the two possible mRNA breakpoints were found to inhibit cell growth of CML patient cells and cell lines, but doubt exists about their specificity. In order to test the specificity, phosphorothioate and 3' phosphorothioate capped antisense BCR-ABL oligonucleotides of different length were used. Stability, cellular uptake of oligonucleotides and effect on cell growth were studied in two CML cell lines, BV173 and LAMA-84. Phosphorothioate antisense BCR-ABL oligonucleotides were most stable, showed the highest uptake and induced cell death in BV173 but not in LAMA-84 cells. We selected the most effective antisense oligonucleotide for further analysis. The BV173 and LAMA-84 cell lines do not express the normal c-abl protein, we therefore used a c-abl specific monoclonal antibody for the detection of p210bcr-abl expression by flow cytometry. Dead cells found after treatment were gated out of analysis. Although BCR-ABL antisense oligonucleotides can induce apoptosis, no reduction of p210bcr-abl levels could be detected in living cells after treatment with antisense oligonucleotides. We conclude that antisense mediated inhibition of translation of mRNA into p210bcr-abl is not the mechanism responsible for the induction of apoptosis in cell line BV173.

Molecular diversity in AML1/ETO fusion transcripts in patients with t(8;21) positive acute myeloid leukaemia.

Patients with acute myeloid leukaemia with maturation (AML-M2) that carried the t(8;21) were tested for the presence of chimeric AML1/ETO mRNA. After RT-PCR, an expected band of 208 bp was observed on gel, as well as some slower migrating bands. The base composition of one of the additional products was determined and was found to contain a new 68-bp ETO sequence present at the fusion of AML1 and ETO genes. The derived protein sequence results in a truncated AML1 gene still containing the putative DNA binding domain. Molecular diversity in the AML1-ETO transcripts will have consequences for the detection of minimal residual disease and antisense studies.

Antisense BCR-ABL oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173.

BCR-ABL antisense oligonucleotides can specifically reduce colony formation of early hematopoietic progenitor cells from chronic myeloid leukemia (CML) patients. Little is known about the mechanism of this inhibition. We studied the inhibition of the bcr-abl oncogene using fluorescein-labeled phosphorothioate oligonucleotides in the Philadelphia chromosome-positive cell line BV173. Oligonucleotide stability, uptake, bcr-abl mRNA degradation, inhibition of cell proliferation, and cell death were studied. The oligonucleotide uptake was directly dependent on the extracellular concentration and was constant over the first 18 h of incubation. After that the uptake rate decreased. We detected a decrease in bcr-abl mRNA after 3 days of treatment with antisense oligonucleotides, but much less in controls. The controls used in the experiments were the sense oligonucleotide, equimolar amounts of sense and antisense, and an untreated control. Antisense oligonucleotides completely inhibited cell growth of BV173 cells and did not inhibit growth of HL-60 cells, whereas control oligonucleotides had no such effect on either cell line. An oligonucleotide specific for the other CML breakpoint was also effective in reducing cell growth of BV173. By the use of a DNA double staining technique to discriminate between necrotic and apoptotic cells, we detected a large number of apoptotic cells in antisense treated BV173 cultures after 5 days of treatment as compared to controls. We conclude that antisense BCR-ABL oligonucleotides reduce bcr-abl mRNA expression in BV173 cells mainly in a sequence-specific manner and induce apoptosis.