Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

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Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

BLM germline and somatic PKMYT1 and AHCY mutations: Genetic variations beyond MYCN and prognosis in neuroblastoma.

Neuroblastoma (NB) is the most common extra cranial solid tumor of childhood and often lethal in childhood. Clinical and biologic characteristics that are independently prognostic of outcome in NB are currently used for risk stratification to optimally the therapy. It includes age at diagnosis, International Neuroblastoma Staging System tumor histopathology and MYCN amplification. However, even in patients with theoretically good prognosis, such as localized tumor and non-amplified MYCN, either disease progress or recurrence may occur. Potential genetic determinants of this unfavorable behavior are not yet fully clarified. The presence of elevated expression of AHCY, PKMYT1, and BLM has accompanied poor prognosis MYCN-amplified neuroblastoma patients. Considering the potential implication of these genes on the clinical management of NB, we hypothesize that the identification of genetic variations may have significant impact during development of the recurrent or progressive disease. Using targeted DNA sequencing, we analyzed the mutation profiles of the genes PKMYT1, AHCY, and BLM in tumor samples of five patients with MYCN amplified and 15 MYCN non-amplified NB. In our study, BLM germline variants were detected in two patients with MYCN-non-amplified neuroblastoma. Our data allow us to hypothesize that, regardless of MYCN status, these mutations partially abolish BLM protein activity by impairing its ATPase and helicase activities. BLM mutations are also clinically relevant because BLM plays an important role in DNA damage repair and the maintenance of genomic integrity. We also found a novel variant in our cohort, PKMYT1 mutation localized in the C-terminal domain with effect unknown on NB. We hypothesize that this variant may affect the catalytic activity of PKMYT1 in NB, specifically when CDK1 is complexed to cyclins. The prognostic value of this mutation must be further investigated. Another mutation identified was a nonsynonymous variant in AHCY. This variant may be related to the slow progression of the disease, even in more aggressive cases. It affects the maintenance of the catalytic capacity of AHCY, leading to the consequent functional effects observed in the NB patients studied. In conclusion, our hypothesis may provide that mutations in BLM, AHCY and PKMYT1 genes found in children with MYCN-amplified or MYCN-non amplified neuroblastomas, may be associated with the prognosis of the disease.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

Dynamic expression of desmin, α-SMA and TGF-ß1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats.

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-ß1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT-PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-ß1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT-PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-ß1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT-PCR technique was more sensitive to expression changes than the semiquantitative method.

Investigation of human parvovirus B19 occurrence and genetic variability in different leukaemia entities.

Human parvovirus B19V (B19V) has been associated with various haematological disorders, but data on its prevalence in leukaemia are scarce. In this cross-sectional study, we investigated patients in Sao Paulo, Brazil with leukaemia to determine the molecular frequency of B19 variants and characterize the viral genetic variability by partial and complete sequencing of the coding of non-structural protein 1 (NS1)/viral capsid proteins 1 and 2 (VP1/VP2). The presence of B19V infections was investigated by PCR amplification of the viral NS1 gene fragment and confirmed by sequencing analysis. The NS1/VP1/VP2 and partially larger gene fragments of the NS1-positive samples were determined by overlapping nested PCR and direct sequencing results. The B19V NS1 was detected in 40 (16%) of 249 bone marrow samples including 12/78 (15.4%) acute lymphoblastic leukaemia, 25/155 (16.1%) acute myeloid leukaemia and 3/16 (18.7%) chronic myeloid leukaemia samples. Of the 40 participants, 25 (62.5%) were infected with genotype 1a and 15 (37.5%) with genotype 3b. The phylogenetic analysis of other regions revealed that 12/40 (30%) of the patients with leukaemia were co-infected with genotypes 1a and 3b. In addition, a new B19V intergenotypic recombinant (1a/3b) and an NS1 non-recombinant genotype 1a were detected in one patient. Our findings demonstrated a relatively high prevalence of B19V monoinfections and dual infections and provide, for the first time, evidence of inter-genotypic recombination in adults with leukaemia that may contribute to the genetic diversity of B19V and may also be a source of new emerging viral strains with future implications for diagnosis, therapy and efficient vaccine development.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas.

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99%) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99%) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

The performance of semi-quantitative differential PCR is similar to that of real-time PCR for the detection of the MYCN gene in neuroblastomas

Amplification of the MYCN gene in neuroblastomas is a potent biological marker of highly aggressive tumors, which are invariably fatal unless sound clinical management is applied. To determine the usefulness of semi-quantitative differential PCR (SQ-PCR) for accurate quantification of MYCN gene copy number, we evaluated the analytical performance of this method by comparing the results obtained with it for 101 tumor samples of neuroblastoma to that obtained by absolute and relative real-time PCR. Similar results were obtained for 100 (99 percent) samples, no significant difference was detected between the median log10 MYCN copy number (1.53 by SQ-PCR versus 1.55 by absolute real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). In the comparison of SQ-PCR and relative real-time PCR, SQ-PCR versus relative real-time PCR concordant results were found in 100 (99 percent) samples, no significant difference was found in median log10 MYCN copy number (1.53 by SQ-PCR versus 1.27 by relative real-time PCR), and the results of the two assays correlated closely (r = 0.8, Pearson correlation; P < 0.001). These findings indicate that the performance of SQ-PCR was comparable to that of real-time PCR for the amplification and quantification of MYCN copy number. Thus, SQ-PCR can be reliably used as an alternative assay in laboratories without facilities for real-time PCR.

Growth hormone and insulin-like growth factor I axis and growth of children with different sickle cell anemia haplotypes.

BACKGROUND: The purpose of this study was to examine the relationships between growth in children with sickle cell anemia and the different beta-globin haplotypes, as well as components of the insulin-like growth factor (IGF)/insulin-like growth factor binding protein (IGFBP) axis. PATIENTS AND METHODS: Growth parameters and plasma concentrations of growth hormone (GH), IGF-I, and IGFBP-3 were studied in 41 children with sickle cell anemia whose haplotypes were defined. RESULTS: Plasma concentrations of IGF-I (total, free, and free/total fraction) and IGFBP-3 were significantly reduced in all patients with sickle cell anemia compared with the healthy children. Patients with the CAR/CAR haplotype had significantly lower mean growth velocity compared with those with Ben/Ben. When the GH/IGF axis elements were compared in relation with the different haplotypes, total IGF-I levels in CAR/CAR patients were significantly lower compared with levels in patients with Ben/Ben. A positive correlation was found between hematocrit and total IGF-I and between fetal hemoglobin percentages and the z-scores for total IGF-I and IGFBP-3. There was a positive correlation between age, weight, height, bone age, and the various elements of the GH/IGF-I axis when all groups were considered, although the correlation was lost when the auxologic data were expressed as standard deviation score for age. Growth velocity and the z-score for growth velocity were not correlated with any element of the axis. CONCLUSIONS: The positive relationship between hematocrit and fetal hemoglobin percentages with total IGF-I, free/total IGF-I, and IGFBP-3 in patients with sickle cell anemia could show that the delayed growth of these patients may be linked to intrinsic factors of the disease, which also determine the low circulating concentrations of the various elements of the GH/IGF-I axis. It is reasonable to assume that decrease of total IGF-I concentrations in patients with CAR/CAR haplotype is secondary to the severity of the disease.

CK-19 expression by RT-PCR in the peripheral blood of breast cancer patients correlates with response to chemotherapy.

BACKGROUND: The recent introduction of sensitive RT-PCR-based techniques for the detection of epithelial antigen expression, such as CK-19, in the peripheral blood and bone marrow of breast cancer patients may provide an opportunity to evaluate tumor response at the molecular level, even in the absence of measurable disease while patients are still receiving chemotherapy. METHODS: We studied serially collected blood samples of 53 patients with breast cancer before, during, and after adjuvant, neoadjuvant, and palliative chemotherapy to evaluate its effects on the expression of CK-19 measured by RT-PCR. RESULTS: The percentage of CK-19 RT-PCR positivity decreased consistently from 43% (23/53) before chemotherapy to 14.3% (7/49), and to 18.9% (7/37) after 3 and 6 cycles, respectively (chi-square for linear trend = 7.948; p = 0.0048). Furthermore, there was a significant correlation between a negative CK-19 at three months and the response to chemotherapy (p = 0.024). CONCLUSION: We conclude that RT-PCR negativity for CK-19 expression at 3 months after the beginning of chemotherapy correlates with tumor response and, as treatment progresses, there is a significant trend for the occurrence of more negative RT-PCR results. Further studies are needed to confirm if this technique can be useful to assess response to chemotherapy in patients without measurable disease and if negativation of CK-19 expression while on chemotherapy is of prognostic significance.

Interferon-alpha therapy increases type I insulin-like growth factor receptors expression on lymphoid cells from patients with chronic myelogenous leukemia.

Aiming to verify if insulin-like growth factor type I and its receptor (IGF-IR) are implicated on pathophysiology of chronic myelogenous leukemia (CML), we studied 35 patients with CML in chronic phase at diagnosis or during interferon-alpha (IFN-A) or hydroxyurea treatments. Cytometry flow analysis and reverse transcription PCR (RT-PCR) molecular assay for IGF-IR expression on peripheral blood cells from CML patients diagnosed didn't show statistical differences from the control group. Hydroxyurea treated patients had lower expression of IGF-IR in granulocytes, lymphocytes and monocytes (P<0.01). We found statistical higher percentage of T and B lymphocytes positive for IGF-IR on IFN-A treated patients (P<0.001). Also an increase of IGF-IR mRNA expression could be detected in this group when compared with patients in hydroxyurea therapy (P<0.05). Our study suggest that IGF-IR is not directly implicated on CML installation and that the increased expression of IGF-IR on lymphoid cells of IFN-A treated patients could contribute to the immune recognition of malignant cell clone by enhancing immunocompetent cell proliferation and action.

Deletion of the factor IX gene as a result of translocation t(X;1) in a girl affected by haemophilia B.

A balanced de novo translocation t(X;1) is described in a girl with severe haemophilia B. The translocated X was shown cytologically to be preferentially active, and methylation analysis of the DXS255 locus confirmed the skewed X-inactivation with the paternal allele being the active one. Cytogenetic and molecular analysis showed that this chromosomal rearrangement led to the deletion of at least part of the factor IX gene. Therefore, the girl was heterozygous for factor IX deficiency and expression of her clinical phenotype was the result of the inactivation of the normal maternal X chromosome. The localization of one of the X chromosome translocation breakpoints in YAC clone 957F9, that was demonstrated to map distally to the factor IX gene, revealed the complexity of this chromosomal rearrangement.

Mutational analysis of N-RAS and GAP-related domain of the neurofibromatosis type 1 gene in chronic myelogenous leukemia.

RAS mutations can be detected in a variable number of patients with myeloproliferative disorders such as myelodysplastic syndromes and acute myeloid leukemia, but are rare events in chronic myelogenous leukemia in chronic phase. However, there is good evidence supporting the involvement of RAS signalling pathway in CML and this could be due to alterations in RAS activity regulatory proteins. The neurofibromatosis (NF1) gene down-regulates the RAS signal transduction pathway through the inhibitory function of its GAP-related domain (GRD) on RAS protein. The loss or alteration of neurofibromin (the NF1 protein) may produce a disfunction similar to point mutations in the RAS gene resulting in the permanent stimulation of the RAS signal transduction pathway. Mutations involving the GRD region of the NF1 gene (GRD-NF1) have been described in a variety of tumors such as colon carcinoma and astrocytoma. Germline mutations and deletions in the NF1 gene, as seen in neurofibromatosis type 1, are also associated with certain myeloid disorders. In the present work, we sought to identify mutations in the codons 12/13 and 61 of RAS gene and in the Lys-1423 codon of GRD-NF1, which are well known hot spots in these genes, in a group of 36 adults and ten children with chronic myelogenous leukemia in chronic phase and blast crisis. Using the PCR-SSCP and the allele-specific restriction assay (ASRA) techniques, we were not able to observe any RAS or NF1 detectable mutation. These findings suggest that RAS and GRD-NF1 mutations are not involved either in chronic phase or in the progression to blast crisis in chronic myelogenous leukemia in adults and children.

Concomitant p53 mutation and MYCN amplification in neuroblastoma.

The MYCN oncogene is amplified in 20% of childhood neuroblastoma and is associated independently with poor prognosis. Alteration of the p53 tumor supressor gene, in contrast, occurs infrequently in these tumors. In this report, we described a 3-year-old girl with stage IV neuroblastoma. Molecular analysis revealed, both MYCN gene amplification and a point mutation of the p53 tumor supressor gene. To our knowledge, this is the first reported case of neuroblastoma with genetic alterations of both these genes.

Reverse transcriptase-polymerase chain reaction analysis of cytokeratin 19 expression in the peripheral blood mononuclear cells of normal female blood donors.

BACKGROUND: Early detection of haematogenous dissemination of epithelial tumours afforded by the analysis of epithelial antigen expression in the peripheral blood mononuclear fraction (PBMN) and bone marrow may confer a worse prognosis to patients with carcinoma. Cytokeratin 19 is a protein normally expressed by epithelial cells including normal and malignant mammary cells. Previous studies have demonstrated that analysis of cytokeratin 19 expression by the reverse transcriptase-polymerase chain reaction (RT-PCR) can detect one epithelial cell in as many as 10(5)-10(7) haematopoetic cells. Despite its sensitivity concern has been voiced recently about the specificity of this technique owing to the detection of cytokeratin 19 expression in the PBMN of normal volunteers and the bone marrow of patients with haematological malignancies. AIMS: To assess the sensitivity and specificity of RT-PCR detection of cytokeratin 19 in PBMN of normal female blood donors. METHODS: Blood was taken from 52 normal female blood donors and PBMN separated through Fycol gradient centrifugation. Cytokeratin 19 was measured using a two step nested RT-PCR assay. RESULTS: No amplification was found in the first step for any of the samples studied, whereas in the second step amplification was observed in 10 of the 52 samples. Both steps could detect one MCF-7 cell (the cytokeratin 19 positive control) in 10(6) CEM (cytokeratin 19 negative control) cells. CONCLUSIONS: As both PCR steps are sensitive to the 10(-6) level, performing only the first amplification step may decrease the non-specificity of this method. Further studies are needed to define the specificity and sensitivity of this technique in blood and bone marrow specimens of women with breast cancer.

Molecular and immunohistochemical analysis of P53 in phaeochromocytoma.

We searched for mutations of the p53 gene in 25 phaeochromocytomas using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the entire conserved region of the gene, encompassing exons 4-8; expression of the p53 protein was assessed by immunohistochemistry. No mutations were found, while a polymorphism in codon 72 was observed. Immunohistochemistry revealed nuclear p53 overexpression in one tumour sample. We conclude that mutations of the 'hotspot' region of the p53 gene do not seem to play a role in the pathogenesis of phaeochromocytoma.

Further evidence for the lack of correlation between the breakpoint site within M-BCR and CML prognosis and for the occasional involvement of p53 in transformation.

The actual significance of the type of BCR-ABL rearrangement in chronic myeloid leukemia (CML) prognosis remains controversial. Also, the molecular events that lead to CML progression are largely unknown. We analyzed the M-BCR breakpoint position in 64 CML patients by Southern blot and correlated the molecular findings with the cytogenetic, hematologic, and clinical data. No statistically significant differences were found with respect to the clinical and hematologic data presented at diagnosis or in the median duration of chronic phase (CP) and survival between the groups of patients with 5' and 3' breakpoints. We also studied by PCR-SSCP and direct sequencing the p53 gene in patients with specimens available in both chronic phase and blast crisis. We identified p53 mutations in 17% of the blast crisis samples analyzed, whereas no abnormalities were found in CP. This finding suggests that only in a minor fraction of cases are lesions in the p53 gene involved in transformation. Given the present findings, along with previous reports, we believe that a novel mechanism to explain the heterogeneity of CML should be postulated and actively pursued, as should the identification of secondary molecular events more consistently involved in progression.