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1.
Ann Hematol ; 82(12): 743-9, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14513290

ABSTRACT

In follicular lymphoma the t(14;18) might be useful as a tumor marker in predicting the quality of the response to treatment. We investigated whether analyzing numbers of t(14;18)-positive cells in peripheral blood correlated with remission status in individual patients receiving a variety of treatments. Numbers of circulating t(14;18)-positive cells were determined by real-time polymerase chain reaction (PCR) technique. Disease parameters and response to treatment were related to the pre- and post-treatment numbers of circulating t(14;18)-positive cells for 53 follicular lymphoma patients. In these 53 patients, 70 treatment episodes were investigated. A content of more than 328 t(14;18)-positive cells per 75,000 cells prior to therapy correlated with the more advanced stage IV disease ( P=0.01), bone marrow involvement ( P<0.01), and overt leukemic lymphoma ( P=0.04). Therapy episodes that cleared circulation from t(14;18)-positive cells with more than one log resulted in a significantly longer progression-free survival than treatment episodes with less than one log decline (26 versus 12 months, respectively) ( P<0.01). After first-line treatment episodes, numbers of circulating t(14;18)-positive cells declined in fairly all cases, irrespective of the clinical response. However, for second or later lines of treatment, declining numbers of lymphoma cells correlated with a clinical remission, whereas increasing numbers of lymphoma cells were associated with clinically stable or progressive disease. From this, we conclude that quantitation of circulating t(14;18)-positive cells in peripheral blood is of only limited clinical significance in predicting treatment efficacy for the individual follicular lymphoma patient.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 18 , Lymphoma, Follicular/drug therapy , Lymphoma, Follicular/genetics , Neoplastic Cells, Circulating/drug effects , Translocation, Genetic , Cell Count , Chemotherapy, Adjuvant , Cytodiagnosis , Disease-Free Survival , Humans , Lymphoma, Follicular/diagnosis , Lymphoma, Follicular/pathology , Neoadjuvant Therapy , Neoplastic Cells, Circulating/pathology , Prognosis
2.
Leukemia ; 16(3): 362-7, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11896539

ABSTRACT

The pattern of X-chromosome inactivation (XCIP), or Lyonization, can be used to distinguish monoclonal from polyclonal cell populations in females. However, a skewed XCIP exists in hematopoietic cells in approximately 40% of healthy elderly females, interfering with interpretation of clonality assays. In hematopoiesis, an active stem cell pool is assumed to be present within a larger population of inactive stem cells, with a continuous exchange of cells between the two compartments. The assumption that the active stem cell pool size decreases with age may explain the phenomenon of acquired skewing occurring by chance and predicts the XCIP of this population to fluctuate. This fluctuation should be reflected in the XCIP of peripheral granulocytes. We examined the XCIP for fluctuations in time in peripheral granulocytes, monocytes and T cells of young, middle-aged and elderly healthy females. We used an optimized HUMARA PCR assay that eliminates unbalanced DNA amplification. We found no fluctuations in XCIP in any age group in up to 18 months follow-up. We conclude that acquired skewing arises gradually in life without fluctuations in XCIP and that analysis at multiple time points cannot distinguish monoclonal hematopoiesis from normal, skewed hematopoiesis.


Subject(s)
Dosage Compensation, Genetic , Hematopoiesis/genetics , X Chromosome/genetics , Adult , Aged , Aged, 80 and over , Aging/genetics , DNA/analysis , DNA Primers/chemistry , DNA, Neoplasm/analysis , Deoxyribonuclease HpaII/metabolism , Female , Granulocytes/cytology , Hematopoietic Stem Cells/cytology , Humans , Middle Aged , Monocytes/cytology , Polymerase Chain Reaction/methods , Receptors, Androgen/genetics , T-Lymphocytes/cytology
3.
J Endocrinol ; 172(3): 489-95, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11874697

ABSTRACT

Expression of human chorionic gonadotrophin (hCG) is associated with trophoblastic, testicular and other malignancies such as bladder, pancreatic, cervical, breast and prostate cancer. In the prostate, however, hCG expression, associated with neuroendocrine cells, is also found in normal tissue. Of the six highly homologous genes that all encode the beta-subunit of hCG, the beta 7 gene is reportedly the only gene expressed in several non-transformed tissues. The beta 3, 5 and 8 genes would be variably expressed in malignant tissue and placenta, but not in normal tissue. To assess to what extent this expression difference can also be found in the prostate, we compared the levels of the different hCG beta transcripts in concurrent normal and cancerous prostate tissues obtained from 17 patients. To this end, we developed a Taqman real-time fluorescent RT-PCR assay for hCG beta, and a quantitative assay specific for the beta 3, 5 and 8 genes, modified from the molecular beacon principle. This latter assay proved highly specific and capable of reliably distinguishing between these hCG beta transcripts that differ in only one nucleotide. Surprisingly, median expression levels of hCG beta were lower in prostate cancer when compared with normal tissue from the same patient. In contrast, hCG beta 3, 5 and 8 transcripts were found in normal tissue and did not differ in prostate cancer, arguing against a specific role of these transcripts in the development of prostate cancer.


Subject(s)
Biomarkers, Tumor/analysis , Chorionic Gonadotropin, beta Subunit, Human/analysis , Prostate/chemistry , Prostatic Neoplasms/chemistry , Case-Control Studies , Humans , Male , Reverse Transcriptase Polymerase Chain Reaction , Statistics, Nonparametric
4.
Leukemia ; 16(1): 13-21, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11840258

ABSTRACT

Using red cell phenotyping (RCP) and/or cytogenetics (CYT) we identified 19 patients with persisting mixed chimerism (MC) among 231 patients transplanted with partially T cell-depleted stem cell grafts from HLA-identical siblings. Persisting MC is defined as MC for more than 2 years in patients without any evidence of relapse. Median leukemia-free survival in these patients was 150 (range, 50-218) months. Diagnoses were ALL (n= 10); AML (n = 2); CML (n = 2); NHL (n = 2); MDS (n= 1); MM (n = 1) and SAA (n = 1). Purpose of this study was the long-term follow-up of MC and definition of patterns of chimerism in the various subsets of PBMCs and granulocytes. Using a PCR-STR technique CD3(+)/CD4(+) (T4 lymphocytes), CD3(+)/CD8(+) (T8 lymphocytes), CD45(+)/CD19(+) (B lymphocytes), CD45(+)/CD14(+) (monocytes), CD45(+)/CD15(+) (granulocytes) and CD3(-)/CD56(+) (NK-cells) were analyzed. The majority of patients with persisting MC were conditioned with a less intensive conditioning regimen and had little GVHD. Sequential monitoring of the chimerism resulted in a group of patients (n = 7) with very slow transient mixed chimerism that resulted in complete DC after median 7 years. Another nine patients had a relatively high percentage of persisting autologous cells for a median of 12 years and in three patients we observed a stable low percentage of autologous cells. Only two out of 19 patients (AML-CR1, CML-CP1) relapsed during follow-up. Both patients had a relatively high percentage of autologous cells. Chimerism in granulocytes and PBMC subsets was analyzed at a median of 8 years after SCT in nine patients. In five patients mixed chimerism simultaneously detected by RCP and CYT was associated with MC in all subsets. Within each individual patient the percentages of donor and recipient cells were very different between the different subsets. Two CML-CP1 patients were mixed chimera in only two subsets and in one patient these subsets represented pending relapse. In another two patients mixed chimerism with a very low number of autologous red cells was not found in the PBMCs because of the different sensitivity level of the RCP and the PCR-STR technique. We conclude that in patients with persisting mixed chimerism after partially T cell-depleted SCT a remarkable number of patients had lymphoid malignancies, the majority of the patients were conditioned with less intensive conditioning regimens and the mixed chimerism was not correlated with relapse. Chimerism in granulocytes and PBMC subsets did show great intra-individual differences in the subsets and these data correlated well with RCP and CYT data with the exception of the NK cells.


Subject(s)
Hematologic Neoplasms/therapy , Hematopoietic Stem Cell Transplantation , Adolescent , Adult , Cell Survival , Disease-Free Survival , Female , Follow-Up Studies , Graft Survival , Graft vs Host Disease/epidemiology , Graft vs Host Disease/etiology , Graft vs Host Disease/prevention & control , Hematologic Neoplasms/mortality , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunophenotyping , Lymphocyte Count , Lymphocyte Depletion , Lymphocyte Subsets , Male , Middle Aged , Myeloid Cells , Polymerase Chain Reaction , Sensitivity and Specificity , T-Lymphocytes , Transplantation Conditioning , Transplantation, Homologous
6.
Leukemia ; 15(12): 1962-5, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11753618

ABSTRACT

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.


Subject(s)
DNA Glycosylases , DNA/standards , Polymerase Chain Reaction/standards , DNA/chemistry , DNA/metabolism , Deoxyuracil Nucleotides/analysis , Deoxyuracil Nucleotides/metabolism , Homeodomain Proteins/genetics , Humans , N-Glycosyl Hydrolases/metabolism , Oncogene Proteins, Fusion/genetics , Reference Standards , Uracil-DNA Glycosidase
7.
Blood ; 98(4): 940-4, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11493436

ABSTRACT

In follicular lymphoma, the t(14;18) status of the peripheral blood and bone marrow analyzed by polymerase chain reaction (PCR) is assumed to correlate with disease activity in patients with relapsed disease. The clinical significance of quantitating circulating lymphoma cells by real-time PCR is reported in patients on first-line treatment. Thirty-four consecutive patients with previously untreated follicular lymphoma and detectable t(14;18)-positive cells in pretreatment peripheral blood samples were monitored. All patients were treated with standard chemotherapy in combination with interferon alfa-2b. Before and after induction therapy, blood samples were taken for quantitative analysis of t(14;18). At presentation, a median of 262 t(14;18)-positive cells per 75,000 normal cells was found (range, 1-75 000). Patients with lower numbers of circulating tumor cells more frequently had bulky disease (P =.02). Seventy-nine percent of the patients responded clinically to treatment. In 22 of 28 patients, including 4 patients in whom treatment had failed clinically, the number of circulating t(14;18)-positive cells decreased to undetectable or low levels after therapy. In the remaining responding patients, circulating tumor cells persisted after therapy. These quantitative data on circulating t(14;18)-positive cells call into question the usefulness of molecular monitoring of the blood in a group of patients with follicular lymphoma uniformly treated with a noncurative first-line regimen. T(14;18)-positive cells decreased in peripheral blood after treatment, irrespective of the clinical response. Therefore, the significance of so-called molecular remission should be reconsidered in follicular lymphoma. (Blood. 2001;98:940-944)


Subject(s)
Lymphoma, Follicular/drug therapy , Translocation, Genetic , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Blood Cell Count , Blood Cells/pathology , Blood Cells/ultrastructure , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 18/genetics , Cytogenetic Analysis , Female , Humans , Interferon alpha-2 , Interferon-alpha/administration & dosage , Lymphoma, Follicular/blood , Lymphoma, Follicular/genetics , Male , Middle Aged , Polymerase Chain Reaction , Prospective Studies , Recombinant Proteins , Treatment Outcome
8.
Thromb Haemost ; 86(6): 1435-9, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11776311

ABSTRACT

We have investigated the influence of the type of factor VIII deficient plasma used on the assay results of the Nijmegen modification of the Bethesda method for factor VIII inhibitors. Immuno depleted factor VIII deficient plasmas, lacking besides factor VIII also von Willebrand factor, gave decreased inhibitor titres compared to assay results with factor VIII deficient plasmas containing von Willebrand factor suggesting the need of the latter in the test system for the stability of factor VIII:C. Moreover the performance of the assay with immuno depleted plasma was contaminated in a certain type of this plasma by the presence of a factor VIII:C inhibitor. Chemically depleted factor VIII deficient plasma appeared to give falsely elevated titres when used in combination with other types of deficient plasmas as substrate plasma in the factor VIII:C assay due to the presence of activated factor Va in the preparation. Suggestions are described with respect to the observed limitations in order to obtain reliable results.


Subject(s)
Blood Coagulation Tests/standards , Factor VIII/antagonists & inhibitors , Hemophilia A/blood , Isoantibodies/blood , Buffers , Factor Va/analysis , False Positive Reactions , Humans , Hydrogen-Ion Concentration , Imidazoles/pharmacology , Immunoglobulin G/immunology , Reproducibility of Results , von Willebrand Diseases/blood , von Willebrand Factor/analysis , von Willebrand Factor/pharmacology
9.
Blood ; 96(1): 63-70, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10891431

ABSTRACT

Recently the Belgium-Dutch Hematology-Oncology group initiated a multicenter study to evaluate whether myeloma patients treated with intensive chemotherapy benefit from additional peripheral stem cell transplantation. To determine treatment response accurately, we decided to quantitate malignant cells. To test a consensus quantitation strategy, 5 centers independently determined the immunoglobulin heavy chain sequences of patient tumor cells and developed allele-specific oligonucleotides (ASO) and ASO-polymerase chain reaction (PCR). We compared the reproducibility of real-time quantitation with quantitation using limiting dilutions. We distributed DNA samples with a 4-log range of tumor cell concentrations and found average quantitation values deviating 74% and 42% from the input values with real-time PCR (1 center) and limiting dilutions (4 centers), respectively. Within single centers we found an average variation coefficient of 0.74, with limiting dilutions not significantly different from the average 0.82 center-to-center variation coefficient. Within a single center, real-time quantitation proved more reproducible (average variation coefficient, 0.36). Quantification was confirmed in 3 patients during treatment in the protocol. This report shows that real-time PCR or limiting dilution assays can be used for quantitation in a single multicenter trial. We present a consensus strategy that allows an accurate comparison of quantitation data generated in independent centers.


Subject(s)
Hematopoietic Stem Cell Transplantation , Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/immunology , Multiple Myeloma/therapy , Base Sequence , Belgium , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Calibration , Combined Modality Therapy , DNA Primers , Genes, Immunoglobulin , Humans , Immunoglobulin Heavy Chains/analysis , Molecular Sequence Data , Multiple Myeloma/pathology , Neoplasm Staging , Netherlands , Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity
10.
Br J Haematol ; 108(1): 116-25, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10651734

ABSTRACT

Fifteen consecutive patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) who relapsed from T-cell-depleted bone marrow transplantation (BMT) were successfully treated with donor leucocyte infusions (DLIs). Chimaerism was analysed using red blood cell phenotyping (RCP), and the results were compared with cytogenetic analysis and outcome of qualitative and quantitative polymerase chain reaction (PCR) for breakpoint molecules. In all patients, an increase in autologous erythrocytes and/or a decrease in donor red cells indicated relapse. Donor erythrocytes started to increase from 4 to 20 (median 12) weeks after DLI. At 6 and 12 months after DLI, complete donor chimaerism was found in 11 and 15 patients, respectively, and all patients were in cytogenic remission. A high percentage of autologous red cells at the time of DLI predicted pancytopenia. During relapse and after DLI, the percentage of autologous red cells was strongly correlated with the reappearance and disappearance of Ph-positive metaphases (r = 0.90; P < 0.001 and r = 0.96; P < 0.001 respectively). The same was true for the correlation between the percentage of autologous red cells and positivity/negativity in PCR for Bcr-Abl breakpoint molecules (r = 0.94; P < 0.001). A normalized Bcr-Abl dose of greater than 10-3 in real-time quantitative PCR correlated well with relapse and the presence of autologous red blood cells (r = 0.77; P < 0.001). We conclude that RCP is a sensitive, easy to perform and fast technique for the prediction of pending relapse after BMT for CML. RCP also predicts the response to DLI and the occurrence of bone marrow aplasia after DLI.


Subject(s)
Bone Marrow Transplantation/methods , Erythrocytes/physiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Adult , Aged , Female , Fusion Proteins, bcr-abl/genetics , Graft vs Host Disease/etiology , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukocyte Transfusion/methods , Male , Pancytopenia/etiology , Phenotype , Polymerase Chain Reaction/methods , Recurrence , Transplantation Chimera/genetics
11.
Melanoma Res ; 9(3): 213-22, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10465576

ABSTRACT

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.


Subject(s)
DNA Methylation , Melanoma/genetics , Neoplasm Proteins/genetics , Polymerase Chain Reaction/methods , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Transcription Factors/genetics , Antigens, Neoplasm , Binding Sites/genetics , Gene Expression Regulation, Neoplastic , Humans , Melanoma-Specific Antigens , Models, Genetic , Proto-Oncogene Proteins c-ets , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic , Tumor Cells, Cultured
13.
Br J Cancer ; 80(5-6): 883-91, 1999 May.
Article in English | MEDLINE | ID: mdl-10360670

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/genetics , Melanoma/blood , Melanoma/enzymology , Monophenol Monooxygenase/genetics , Neoplasm Proteins/genetics , Transcription, Genetic , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/blood , Female , Gene Amplification , Humans , Hydroxymethylbilane Synthase/genetics , MART-1 Antigen , Male , Melanoma/genetics , Middle Aged , Monophenol Monooxygenase/blood , Neoplasm Proteins/blood , Neoplastic Cells, Circulating/metabolism , Quality Control , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods
14.
J Exp Med ; 189(2): 301-8, 1999 Jan 18.
Article in English | MEDLINE | ID: mdl-9892612

ABSTRACT

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1-specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus-transformed B cells. The HB-1 gene-encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1-specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1-specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.


Subject(s)
Burkitt Lymphoma/immunology , HLA Antigens/immunology , HLA-B Antigens/genetics , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Base Sequence , Bone Marrow Transplantation/immunology , Cell Line , Clone Cells/immunology , Cloning, Molecular , DNA, Complementary/genetics , Female , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Graft vs Host Disease/immunology , Humans , Male , Minor Histocompatibility Antigens , Molecular Sequence Data , Pedigree , Peptide Fragments/immunology , Polymorphism, Genetic/genetics , Sequence Analysis
15.
Ann Oncol ; 10(11): 1349-54, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10631464

ABSTRACT

BACKGROUND: The capacity of the polymerase chain reaction (PCR) to detect very low numbers of cells bearing a t(14;18) translocation has led to its application in assessment of the results of treatment for follicular lymphoma, and suggestions that therapy might be guided by molecular studies. To test the reliability of PCR a collaborative study was undertaken to compare results from different laboratories in Europe and North America. METHODS: Twenty laboratories with records of publication in molecular diagnostics were sent blood from normal donors with varying numbers of t(14;18)-bearing cells added from a cell line with a translocation in the major breakpoint region (MBR) of the bcl-2 gene. Samples contained 1000, 100, 10, 1 or 0 cells per ml of whole blood and were sent blinded in duplicate. PCR methodology varied widely, with the total number of amplification cycles between 30 and 70, and 13 different primers used for the MBR region. Twelve laboratories used nested PCR and eight single round amplification. RESULTS: The sensitivity of nested and single round PCR was similar at 100 cells/ml but below this the nested method proved significantly more sensitive. The false positive rate was 28%, with 11 samples from 9 laboratories reported as positive when no t(14;18) cells were added. PCR product size and sequence analysis showed that false positives were due to contamination from cell-line DNA rather than background translocations in the donors. There was no significant difference in false positive rates between nested and single round techniques. CONCLUSION: The polymerase chain reaction to detect bcl-2-IgH rearrangements is presently carried out with widely disparate results. Further effort is required to bring forward a standard PCR protocol which can be re-tested in different laboratories to improve accuracy and reproducibility. The application of quantitative techniques such as real-time PCR may resolve many of the problems presently encountered.


Subject(s)
Immunoglobulin Heavy Chains/genetics , Lymphoma, Follicular/genetics , Polymerase Chain Reaction/methods , Proto-Oncogene Proteins c-bcl-2/genetics , Translocation, Genetic , Adult , Aged , Analysis of Variance , Base Sequence , Female , Humans , Lymphoma, Follicular/diagnosis , Male , Middle Aged , Molecular Sequence Data , Probability , Sensitivity and Specificity
16.
Leukemia ; 12(11): 1782-8, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9823954

ABSTRACT

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.


Subject(s)
Antigens, CD34/immunology , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Hematopoietic Stem Cells/pathology , Leukemia, Myeloid, Acute/genetics , Translocation, Genetic , Base Sequence , Cell Division , Cell Separation , DNA Primers , Hematopoietic Stem Cells/immunology , Humans , Immunophenotyping , Leukemia, Myeloid, Acute/pathology , Reverse Transcriptase Polymerase Chain Reaction
19.
Leukemia ; 12(8): 1295-302, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9697887

ABSTRACT

Bone marrow plasma cells constitute the bulk of malignant cells in multiple myeloma patients. B-lymphocytes having immunoglobulin heavy chain gene rearrangements identical to those of the malignant clone (clonally related B-lymphocytes) may function as malignant plasma cell precursors. We and others proposed the use of anti-idiotypic antibodies to isolate and study clonally related B-lymphocytes. This strategy failed until now because anti-idiotypic antibodies raised by conventional hybridoma techniques proved to react frequently with epitopes shared by different idiotypes. Recently, we succeeded in selecting specific single chain Fv antibodies from phage libraries. To select single chain Fv bearing phages specifically directed against the immunoglobulin idiotype expressed by myeloma tumor cells we panned a semisynthetic phage library against purified myeloma paraprotein Fab fragments. The selection was performed in the presence of soluble polyclonal immunoglobulin as a competitor. Three independent selections for three myeloma patients yielded 10-26 clones. Between two and seven of the selected clones were reactive with patient Fab and not with polyclonal immunoglobulin in enzyme-linked immunosorbent assays. Five out of six anti-idiotypic single chain Fvs were able to specifically stain fixed monoclonal plasma cells in myeloma bone marrow. Idiotype specificity of these single chain Fvs was confirmed by flow cytometry since they did not react with monoclonal plasma cells of other patients, a panel of nine myeloma cell lines, isolated polyclonal bone marrow plasma cells and cultured B-lymphocytes. Using these anti-idiotypic reagents we were able to detect 25 myeloma plasma cells in a background of 50000 immunoglobulin isotype-matched cells of the myeloma cell line UM-1 or 50000 donor bone marrow cells (sensitivity 0.05%). This paper shows that highly specific anti-idiotypic single chain Fv antibody fragments selected from a phage display library can be used to detect rare idiotypic cells in patient samples.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Multiple Myeloma/immunology , Muscle Proteins , Plasma Cells/immunology , Animals , Antibody Specificity , B-Lymphocytes/immunology , Bacteriophage M13 , Cells, Cultured , Connectin , Fibroblasts/metabolism , Genes, myc , Humans , Immunoglobulin Fab Fragments/immunology , Mice , Myeloma Proteins/immunology , Peptide Library , Tumor Cells, Cultured , Vesicular stomatitis Indiana virus
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