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.

Homozygous deletion of RAG1, RAG2 and 5' region TRAF6 causes severe immune suppression and atypical osteopetrosis.

Mutations of several genes have been implicated in autosomal recessive osteopetrosis (OP), a disease caused by impaired function and differentiation of osteoclasts. Severe combined immune deficiencies (SCID) can likewise result from different genetic mutations. We report two siblings with SCID and an atypical phenotype of OP. A biallelic microdeletion encompassing the 5' region of TRAF6, RAG1 and RAG2 genes was identified. TRAF6, a tumor necrosis factor receptor-associated family member, plays an important role in T cell signaling and in RANKL-dependent osteoclast differentiation and activation but its role in human OP has not been previously reported. The RAG proteins are essential for recombination of B and T cell receptors, and for the survival and differentiation of these cells. This is the first study to report a homozygous deletion of TRAF6 as a cause of human disease.

Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?

The ALL IC-BFM 2002 protocol was created as an alternative to the MRD-based AIEOP-BFM ALL 2000 study, to integrate early response criteria into risk-group stratification in countries not performing routine PCR-based MRD testing. ALL IC stratification comprises the response to prednisone, bone marrow (BM) morphology at days 15 and 33, age, WBC and BCR/ABL or MLL/AF4 presence. Here, we compared this stratification to the MRD-based criteria using MRD evaluation in 163 patients from four ALL IC member countries at days 8, 15 and 33 and week 12. MRD negativity at day 33 was associated with an age of 1-5 years, WBC<20,000 microl(-1), non-T immunophenotype, good prednisone response and non-M3 morphology at day 15. There were no significant associations with gender or hyperdiploidy in the study group, or with TEL/AML1 fusion within BCP-ALL. Patients with M1/2 BM at day 8 tended to be MRD negative at week 12. Patients stratified into the standard-risk group had a better response than intermediate-risk group patients. However, 34% of them were MRD positive at day 33 and/or week 12. Our findings revealed that morphology-based ALL IC risk-group stratification allows the identification of most MRD high-risk patients, but fails to discriminate the MRD low-risk group assigned to therapy reduction.

Ewing Sarcoma tumor cells express CD34: implications for autologous stem cell transplantation.

The significance of tumor cell contamination in marrow and peripheral blood stem cell (PBSC) collections of patients with solid tumors remains controversial. Various methods have been developed to purge tumor cells from autologous stem cell products, including CD34+ selection. PBSC harvests from patients with Ewing family of tumors (EFT) were analyzed for contaminating tumor cells prior and after CD34+ selection using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FC) analyzes. The expression of CD34 was studied by RT-PCR and FC in 14 primary tumors and 13 PBSC harvests, respectively. Tumor cells were identified in the harvests by both methods. In two patients, contaminating tumor cells were evident by RT-PCR only after positive selection. FC analysis confirmed a higher level of tumor cells in the CD34+ fraction. In an attempt to explore this finding, expression of CD34 was detected in 93% of primary tumors and 67% of contaminated harvests. As CD34 is expressed on EFT cells, these cells may be enriched following CD34+ selection of harvests, although the total number of tumor cells is reduced. Other methods of purging, rather than CD34+ selection, should be explored in patients with EFT undergoing autologous stem cell transplantation.

Quantitation of alpha-fetoprotein messenger RNA for early detection of recurrent hepatocellular carcinoma: a prospective pilot study.

BACKGROUND: Alpha-fetoprotein (AFP) messenger RNA (mRNA) may be a potential marker of the dissemination of hepatocellular carcinoma (HCC) cells into the circulation. The aim of this prospective pilot study was to assess the prognostic value of quantitative levels of AFP mRNA in patients undergoing ablative treatment for HCC. METHODS: Peripheral blood samples were taken from seven patients before and after treatment for measurement of AFP mRNA levels by reverse-transcriptase polymerase chain reaction (RT-PCR). Patients were treated with percutaneous radiofrequency thermal ablation (n=3) or transarterial chemoembolization (n=4). The level of AFP mRNA in blood was serially determined, and the time course was related to the clinical course and disease outcome. The median duration of follow-up was 14 months (range, 9-16 months). RESULTS: HCC recurred locally in four patients, and lung metastases developed in two of them. Patients were divided into three groups on the basis of the pre- and post-treatment AFP mRNA status. Group 1 included four patients with consistently high serum AFP and AFP mRNA levels (pre- and post-treatment). These patients developed distant and local recurrence. Group 2 included a patient with serum-negative AFP mRNA and normal AFP levels at entry. Although serum AFP remained within normal range, mean AFP mRNA increased from 10 to 95 copies/microg RNA. This patient had no distant metastases, but his tumor markedly increased in size. In Group 3, AFP mRNA and serum AFP remained within normal range before and after treatment. These two patients did not develop either local or distant metastases during the follow-up period. CONCLUSIONS: Although this is a small sample size pilot study these findings imply that quantitative measurement of AFP-expressing cells in peripheral blood may serve as a marker of HCC recurrence.

Tumor cells are present in stem cell harvests of Ewings sarcoma patients and their persistence following transplantation is associated with relapse.

BACKGROUND: Tumor cells frequently contaminate autologous stem cell products in a variety of malignancies, but their clinical significance remains controversial. We retrospectively monitored tumor contamination in stem cell harvests from patients with Ewing family of tumors (EFT) all harboring the specific translocation EWS-FLI-1 that characterize these tumors. PROCEDURE: Twenty- seven harvests from 11 patients were included in the study. In addition, 6 and 19 bone marrow (BM) or peripheral blood (PBL) samples were available before and after transplantation, respectively, for RT-PCR and nested PCR analyzes. RESULTS: All 11 patients had contaminating tumor cells in their harvests. All samples prior to transplantation were RT-PCR positive. Two out of the 11 patients who underwent transplantation died of complications. Out of the remaining nine patients, two are alive and well 68 and 84 months from diagnosis, and are the only patients with no detectable tumor cells in their samples after transplantation. One of these patients harbored contaminating tumor cells in only one of the two harvests collected. Seven patients relapsed after transplant, and in four patients BM/PBL samples were available prior to the clinical relapse. All these samples harbored contaminating tumor cells. CONCLUSIONS: We suggest a possible correlation between the amount of contaminating cells in the harvest and relapse after transplantation. Quantitative RT-PCR studies of the chimeric transcripts are underway to explore this issue.

Molecular variants of the ATM gene in Hodgkin's disease in children.

Ataxia telangiectasia is an autosomal recessive disease with a striking predisposition of lymphoid malignancies. ATM mutations have been reported in adult sporadic lymphoma and leukaemia. The aim of this study was to investigate the possible involvement of the ATM gene in the carcinogenesis of Hodgkin disease in children. Tumours were obtained from 23 patients and were subjected to mutation screening and loss of heterozygosity analysis. Eight base substitutions were identified in seven patients. Of them, Y54Y, a silent change, was observed in two patients and a known polymorphism, D1853N, in three patients. Of the other two patients, one harboured a combined genotype P604S/F1463C, identified previously in two patients with Hodgkin lymphoma, and the other a novel missense mutation, V595A. The alterations were present in the germ line, and both had a more aggressive disease. In all, 100 matched normal ethnic controls were screened for these mutations and P604S/F1463C was identified in one healthy control. Loss of heterozygosity was identified in four patients and in three of them it was located centromeric to the ATM gene, and, in one, it spanned a large region, indicating the involvement of other tumour-suppressor genes in this disease. Missense variants of the ATM gene are a rare event in childhood Hodgkin disease.

Association between telomerase activity and outcome in patients with nonmetastatic Ewing family of tumors.

PURPOSE: Telomerase is considered a molecular marker for malignancy. The aim of this study was to determine telomerase activity (TA) as a prognostic factor at diagnosis and as a marker for minimal residual disease during therapy and follow-up in nonmetastatic Ewing family of tumors (EFT). PATIENTS AND METHODS: Primary tumor specimens and 97 peripheral blood (PBL) samples from 31 EFT patients were analyzed for TA by the Telomeric Repeat Amplification Protocol (TRAP assay). The telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) gene expression was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and telomere length was determined by Southern blotting. The presence of the EFT chimeric transcripts was analyzed by RT-PCR. Correlations with progression-free survival were evaluated. RESULTS: At diagnosis, TA in primary tumors did not correlate with outcome. During therapy and follow-up, highly significant correlation was observed between high TA in PBL samples and adverse prognosis (P <.0001). None of the patients harboring low TA progressed, with a long follow-up (median, 60 months) and a progression-free survival (PFS) of 100%. In nine patients, high TA actually could predict relapse, long before overt clinical relapse. The group of patients with high TA and positive RT-PCR had the most adverse outcome; PFS of 20% (P =.0025). TA was found to be a better prognostic factor than RT-PCR and histopathologic response at surgery. CONCLUSION: The results suggest that TA is a significant prognostic variable, superior to the established clinical prognostic parameters during therapy and tumor surveillance. It could be used in combination with RT-PCR for a new risk classification.

Molecular cytogenetic parameters in Ewing sarcoma.

To evaluate possible genomic instability and possible random aneuploidy, we applied comparative genomic hybridization and fluorescence in situ techniques, and evaluated telomerase activity in 16 cases of Ewing sarcoma (EWS) and compared the results to 7 controls. Common secondary aberrations (gains of chromosomes 8 and 12) were found in the study group. There was a direct correlation between the detection of random aneuploidy and development of tumor relapse (P = 0.0047). Other detectable abnormal parameters (secondary) and high telomerase activity were also more common among the cases with relapse but did not reach a statistical significance (probably because of the small sample size). In EWS, the detection of random aneuploidy seems to be a sensitive parameter in the prediction of tumor relapse.

High frequency of loss of heterozygosity for 1p35-p36 (D1S247) in Wilms tumor.

We analyzed the loss of heterozygosity (LOH) for 1p in 18 Wilms tumors using a panel of 11 polymorphic markers. Loss of heterozygosity was identified in 56% of the tumors. The smallest region of overlap was defined for marker D1S247, underlying the 1p35-1p36.1 locus. This is the highest LOH frequency for 1p, or for the well-defined 11p13 and 11p15.5 loci. Based on the fact that tumors of all stages, with both favorable and unfavorable histology, exhibited LOH, we suggest that the 1p35-1p36.1 locus is involved in the etiology of Wilms tumor.

Clinical relevance of molecular diagnosis in childhood rhabdomyosarcoma.

Rhabdomyosarcoma may be divided into three subtypes--embryonal, alveolar, and undifferentiated sarcoma--which can be distinguished by molecular analysis. The authors applied reverse transcriptase-polymerase chain reaction analysis (RT-PCR) to analyze tumor samples from 14 children with rhabdomyosarcoma for the presence of the chimeric PAX3-FKHR transcript resulting from the translocation t(2;13)(q35,q14). Both fresh and paraffin-embedded tissues were used. In only nine specimens was the RNA intact for the analysis. The chimeric transcript was identified in seven samples: four alveolar type, one embryonal type, and two undifferentiated sarcoma. Histologic review was performed in the three samples with discordance between the molecular and histologic findings. A sample from a patient with a diagnosis of embryonal rhabdomyosarcoma on presentation and expression of PAX3-FKHR fusion transcript yielded a small focus of alveolar rhabdomyosarcoma and was reclassified as alveolar rhabdomyosarcoma. One of the samples from a patient with undifferentiated sarcoma was redefined as alveolar subtype; the diagnosis of the second undifferentiated sarcoma remained unchanged, in accordance with the histologic diagnosis. These findings further support the recommendation that molecular analysis be included in the diagnostic workup of childhood small round cell tumors to reach a more accurate diagnosis for tailoring of specific treatment.

Molecular analysis of childhood acute lymphoblastic leukemia in Israel.

Ninety-two Israeli children with acute lymphoblastic leukemia (ALL) (67 B-lineage and 25 T-lineage) were analyzed for the immunological antigen receptor gene configuration. Thirty-nine of the patients (27 B-lineage and 12 T-lineage) relapsed. The incidence of the identified rearrangements within the immunoglobulin heavy chain (IgH) and T-cell receptor (TCR)beta, gamma and delta genes, at diagnosis, was in accordance with previous studies from other countries. Furthermore, the clinical relevance of bi/oligoclonal status, at diagnosis, and clonal selection was determined in this long-term follow-up study (median 112 months). A similar relapse rate was observed among the B-lineage patients with bi/oligoclonal and monoclonal patterns indicated by IgH gene rearrangement. Based on our results, we suggest that bi/oligoclonality has no prognostic significance (P=0.8533). Clonal variations between diagnosis and subsequent relapses were detected in 60% (12/20) of the patients; 64% (7/11) B-lineage and 55% (5/9) T-lineage. Clonal selection significantly correlated with shorter duration of remission and earlier recurrence (P=0.0025).

A novel germ-line mutation in the noncoding region of the p53 gene in a Li-Fraumeni family.

We identified a novel germ-line p53 mutation in the noncoding, nonsplicing regions of a Li-Fraumeni family. Patients belonging to this family included pediatric medulloblastoma and rhabdomyosarcoma patients and a breast carcinoma patient. Three positions in the p53 gene were analyzed for loss of heterozygosity (LOH). One of the three loci retained heterozygosity, whereas the other two exhibited LOH. Sequence analysis of the third locus identified a change of 5'-CCGGGTGA-3' to 5'-CCAGGTTGGA-3', 63 bp downstream of exon 6. The mutation was identified in the germ line of the two pediatric patients and in each of the related parents. We excluded any additional mutation in the entire coding region of the p53 gene, including splice-site intronic sequences. Strong positive nuclear staining of the p53 protein was detected in both normal and tumor paraffin-embedded tissues. Eighty-five normal persons were negative for this alteration, which thus supports it as a mutation. These results may indicate that genetic changes within the noncoding region of the p53 gene may serve as an alternative mechanism of activating this gene. Mutations in the noncoding region of this gene should be further studied.

Mutations of the adenomatous polyposis coli and p53 genes in a child with Turcot's syndrome.

Turcot's syndrome is a rare heritable complex that is characterized by an association between a primary neuroepithelial tumor of the central nervous system and multiple colonic polyps. The aim of this study was to analyze genetic alterations in a case of Turcot's syndrome in a 10.5-year-old boy in whom a colorectal tumor developed 3.5 years following astrocytoma. An APC germline non-sense mutation at codon 1284 leading to a truncated protein was identified, as was a somatic p53 mutation in the colorectal carcinoma in exon 7, codon 244. The latter was not identified in the primary astrocytoma. However, immunohistochemistry revealed high p53 protein expression in both tumors, suggesting an additional p53 mutation in the primary astrocytic tumor. The diverse p53 mutations observed in this unique syndrome in two different sites and stages of the disease may shed light on the multistep progression of the malignant events.

p53 mutation as the second event in juvenile chronic myelogenous leukemia in a patient with neurofibromatosis type 1.

BACKGROUND: Young patients with neurofibromatosis type 1 (NF1) are at increased risk of developing various malignancies, most of which are myeloid disorders. The observed loss of NF1 allele in the myeloid malignancies of NF1 patients suggests a role of NF1 as a tumor suppressor gene. Loss of 17p was found to be quite frequent in neural crest tumors from patients with NF1, raising the possibility of p53 tumor suppressor gene involvement in other NF1-related tumors. METHODS: The authors studied mutations in the NF1 and p53 genes, using loss of heterozygosity, single strand conformation polymorphism, heteroduplex and sequencing analyses. RESULTS: An NF1 germline mutation was identified in exon 31 of a child who developed juvenile chronic myelogenous leukemia (JCML). The mutation was segregated within the proband's family. A 14bp deletion at exon 6 of the p53 gene was observed when JCML was diagnosed, and the wild-type p53 allele was lost during progression of the disease. No loss of the normal NF1 allele could be detected. CONCLUSIONS: A germline mutation in the NF1 gene and sequential inactivation of p53 alleles in the malignant clone of JCML raise the possibility of a correlation between NF1 and p53 genes in the tumorigenesis of JCML.

Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia.

Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 B-lineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. p53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No p53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the p53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL.