Original Article
Minimal Residual Disease in Childhood B-Lineage Lymphoblastic Leukemia — Persistence of Leukemic Cells during the First 18 Months of Treatment
N Engl J Med 1990; 323:448-455August 16, 1990
- Abstract
- Abstract
Background.
Whether patients in clinical remission for acute lymphoblastic leukemia (ALL) continue to harbor leukemic cells is not known, because methods of detecting residual malignant cells have not been sufficiently sensitive. This information might be useful for predicting recurrence and determining the duration of therapy.
Methods.
Using a sensitive new method — identifying complementarity-determining region III sequences with the polymerase chain reaction — we estimated the number of residual leukemic cells in the bone marrow of eight children with B-lineage lymphoblastic leukemia before and after remission.
Results.
Induction chemotherapy produced a 3-to-4-log reduction in the number of leukemic cells. In all samples obtained up to 18 months after diagnosis, however, 0.004 to 2.6 percent of bone marrow nucleated cells were residual leukemic cells. Among the four patients studied more than 18 months after diagnosis, three had no detectable leukemic cells in marrow samples. Despite this, one of them, who was no longer receiving therapy, had a central nervous system relapse. In one patient receiving maintenance chemotherapy, there was a 60-fold increase in leukemic cells three months before bone marrow relapse.
Conclusions.
The complete disappearance of leukemic cells (or their reduction below our method's threshold of detection, 1 in 100,000 cells) may be necessary to achieve a cure of ALL. The quantification of residual leukemic cells in serial marrow aspirates during therapy may allow the early detection of relapse. (N Engl J Med 1990; 323:448–55.)
Media in This Article
Figure 1
Southern Blot Analysis of Rearrangements of the Immunoglobulin Heavy-Chain Locus.Figure 2
Nucleotide Sequences of the CDRIII from the Leukemic Clones of Eight Patients with B-Lineage Acute Lymphoblastic Leukemia.Article Activity
- Article
HOW long patients with leukemia in clinical remission harbor leukemic cells is not known, nor is it known whether rare residual leukemic cells are capable of causing the recurrence of the disease. Until recently, the methods of detecting minimal residual leukemia have not been sufficiently sensitive to detect leukemic cells making up less than 2 to 5 percent of bone marrow mononuclear cells and have not been sufficiently specific to establish whether residual blasts were actually of leukemic origin.1 , 2 The identification and characterization of nonrandom chromosomal translocations at the molecular level3 4 5 6 and the use of the polymerase chain reaction7 , 8 to amplify these lymphoma- or leukemia-specific alterations in DNA or RNA9 10 11 12 13 14 have increased the sensitivity with which residual disease can be detected by three or four orders of magnitude. However, leukemia-specific chromosomal translocations are molecularly characterized in only a minority of patients, which limits the applicability of this approach.
During the differentiation of B and T lymphocytes, the rearrangement of the immunoglobulin and T-cell–receptor loci15 16 17 generates unique DNA sequences; in B lymphocytes, for example, the process generates a hypervariable sequence known as the complementarity-determining region III (CDRIII)17 of the immunoglobulin heavy chain. Since acute lymphoblastic leukemia (ALL) results from the clonal expansion of malignant B or T lymphocytes,16 , 18 these rearrangements can be exploited as specific markers for the leukemic clone. Consequently, several investigators have developed methods that use the polymerase chain reaction to detect residual malignant clones of the T-cell and B-cell lineages in lymphoblastic leukemias.19 20 21
We have recently described a rapid method to determine the CDRIII sequences of the leukemic cells in patients with B-lineage ALL.21 In the CDRIII—polymerase chain reaction method, the polymerase chain reaction amplifies the unique CDRIII sequences of the leukemic cells using primers homologous to consensus sequences in the variable (VH) and joining (JH) segments that flank the intervening diversity (D) segment in the rearranged heavy-chain immunoglobulin locus. The leukemia-specific CDRIII fragments amplified by the polymerase chain reaction are sequenced and used to generate diagnostic oligonucleotide probes that do not cross-hybridize with the CDRIII sequences of normal B lymphocytes.
Using this technique, we investigated the extent of minimal residual leukemia in a group of eight patients with B-lineage ALL for whom marrow samples were available after they had entered clinical remission.
Methods
Clinical Samples and DNA Analysis
Bone marrow samples from eight patients with B-lineage ALL were obtained at diagnosis and during chemotherapy (and from two of them at the end of treatment). Consent to use these specimens for research was given by the patients or their parents and by the Committee for Protection of Human Subjects at the Children's Hospital of Philadelphia. Table 1Table 1
Clinical and Laboratory Data of Patients with B-Lineage Acute Lymphoblastic Leukemia at Diagnosis. summarizes the patients' clinical characteristics. Peripheral blood was obtained from healthy volunteers as a source of a normal population of polyclonal B cells. Mononuclear cells were fractionated on a Ficoll–Hypaque gradient (d = 1.077)24; genomic DNA was isolated from mononuclear cells by established procedures25 or by rapid cell lysis with use of nonionic detergents.26 Rearrangements of the heavy-chain immunoglobulin gene were analyzed by means of Southern blotting.25 DNA was digested with EcoRI or HindIII restriction endonucleases (Boehringer–Mannheim, Indianapolis). The blots were probed with a 3.4-kb fragment from the joining region of the immunoglobulin heavy chain (JH),27 , 28 labeled with [α-32P]deoxycytidine triphosphate.29
Oligonucleotide Primers and Probes and the Polymerase Chain Reaction
Oligonucleotides were synthesized by solid-phase triester methods.30 The VH sense and the JH antisense primers for the CDRIII polymerase chain reaction have been described previously; the primers contain SalI and PstI cloning sites to allow ligation of the amplified sequences into recombinant vectors.21 The JH consensus (JC) probes that were used to detect all amplified CDRIII sequences consisted of a mixture of four 20-mer oligonucleotides derived from the sequences of J2, J3, J4, and J6 genes28 just 5′ to the JH antisense primer. The probes were end-labeled with [γ-32P]deoxyadenosine triphosphate by the kinase method.25
The polymerase chain reaction was carried out as described by Saiki et al.8 Precautions against the cross-contamination of amplified material were taken according to the recommendations of Kwok et al.31 and by handling marrow samples from the diagnostic and remission periods separately. An aliquot of the products of the polymerase chain reaction was analyzed by gel electrophoresis in 4 percent agarose (NuSieve, FMC, Rockland, Me.) and by Southern blotting with leukemia-specific oligonucleotide probes,21 with washing temperatures 1°C below the calculated melting points of the probes.32
Cloning and Sequencing of CDRIII Sequences from Diagnostic Marrow Samples
An aliquot of the material amplified by the polymerase chain reaction was digested with both SalI and PstI restriction endonucleases (Boehringer–Mannheim), purified by gel electrophoresis, and recovered from the gel by treatment with agarase33 (Calbio-Chem, San Diego, Calif.). Recovered DNA was ligated into Blue-script vector (Stratagene, La Jolla, Calif.) and transfected into Escherichia coli (strain JM 109).34 Transformants containing CDRIII DNA were detected by hybridization with 32P-labeled JC probes. Double-stranded DNA template from positive colonies was sequenced by the method of Sanger et al.35
Computer analysis of DNA-sequencing data was performed with the sequence-analysis software of the Genetics Computer Group (Release 5, University of Wisconsin) and a Micro Vax II computer (Digital Equipment, Maynard, Mass.).
Quantification of Residual Leukemic Cells
For an accurate quantitation of the percentage of leukemic CDRIII sequences among all the CDRIII sequences from B cells present in marrow samples from the remission period, a phage-quantitation assay was used. Gel slices corresponding to the area from 72 to 194 base pairs, which encompassed all the amplified CDRIII fragments, were excised and treated with agarase.33 According to our data, CDRIII fragments amplified from normal lymphocytes distribute from 79 to 151 base pairs (unpublished data). The recovered DNA was ligated into M13mp19 phage vector36 (Bethesda Research Laboratories, Gaithersburg, Md.) and transfected into E. coli strain DH5αF'IQ (Bethesda Research Laboratories). Recombinant plaques were lifted onto duplicate nitrocellulose filters. One set of filters was hybridized with 32P-labeled JC probes to detect all the CDRIII sequences amplified, and an identical set was hybridized with the patient's leukemia-specific CDRIII probe. For each sample, the percentage of leukemic CDRIII sequences was calculated as the number of plaques positive for the leukemia-specific probe divided by the number of plaques positive for JC probes and multiplied by 100. The value obtained represents the percentage of B-lineage ALL cells in the population of B lymphocytes. The percentage of leukemic cells in total marrow cells was estimated by multiplying the percentage of the leukemic CDRIII sequences by the ratio of lymphocytes present in the specific marrow sample as determined by morphologic analysis. For this method of quantification, it is assumed that all the lymphoid cells in the marrow differential are of the B lineage and have only one rearrangement of heavy-chain immunoglobulin. Therefore, the method probably overestimates slightly the amount of residual disease, since not all the marrow lymphoid cells are of the B lineage and some B cells have two rearrangements.
Results
Table 1 summarizes the clinical and laboratory data on the eight patients with B-lineage ALL at the time of diagnosis. The patients received treatment according to the Children's Cancer Study Group's risk-stratified protocols for ALL.22 , 23 As indicated in Table 2Table 2
Date of Marrow Sampling and Status of B Lineage in the Study Patients., marrow samples were obtained at diagnosis and during or after therapy; the clinical status of the patients at the time of the sampling is also shown. During the follow-up period (minimum, 14 months; maximum, 51 months), six patients remained in continuous remission, whereas the other two patients relapsed, either in the central nervous system (Patient C135, 42.7 months after diagnosis) or the bone marrow (Patient C52, 8.6 months after diagnosis).Rearrangements of the immunoglobulin heavy-chain locus were determined to confirm the diagnosis of B-lineage ALL and establish the number of rearranged alleles (and therefore the number of leukemia-specific CDRIII sequences present). Figure 1Figure 1
Southern Blot Analysis of Rearrangements of the Immunoglobulin Heavy-Chain Locus. shows that the leukemic cells of Patient C138 had both heavy-chain alleles rearranged, and the leukemic cells of three other patients (C137, C135, and C52) had only one allele rearranged. Leukemic cells from Patients C92, C111, and C136 also had one rearranged allele (data not shown). There was not a sufficient amount of DNA to perform this analysis for Patient C94. The CDRIII fragments of the rearranged immunoglobulin loci were sequenced after amplification with the polymerase chain reaction (Fig. 2Figure 2Nucleotide Sequences of the CDRIII from the Leukemic Clones of Eight Patients with B-Lineage Acute Lymphoblastic Leukemia.). The number of CDRIII sequences identified matched in six of the eight cases the number of rearrangements observed on Southern blot analysis. In two cases (Patients C135 and C52), however, in which only one rearranged allele was detected on Southern blotting, a predominant CDRIII sequence and one (in Patient C52) or two (in Patient C135) less frequent CDRIII sequences were also identified. According to the sequence data, leukemia-specific CDRIII diagnostic oligonucleotide probes (underlined in Fig. 2) were synthesized. For the three patients in whom more than one leukemic CDRIII sequence was identified, the one that was most frequently isolated from the marrow sample taken at diagnosis was used for the detection of residual disease.As shown in Figure 3Figure 3
Specificity of Leukemia-Specific Oligonucleotide Probes on Southern Blots of CDRIII Sequences Amplified by the Polymerase Chain Reaction.A, the products of the CDRIII polymerase chain reaction involving leukemic cells (lanes 1, 2, and 3) formed discrete bands when analyzed by gel electrophoresis, whereas the normal polyclonal lymphocytic population formed smear bands (lanes 4 through 9). When the products of the polymerase chain reaction were blotted and hybridized (Fig. 3B) with consensus probes for the J region (JC), signals were detected indicating that the visible bands seen in Figure 3A contained CDRIII sequences. However, when the leukemia-specific CDRIII probes were used in the hybridization analysis, positive signals were detected only with the corresponding leukemic marrow sample, confirming the high specificity of each of these probes for the corresponding leukemic clone (Fig. 3C through 3E).The presence of minimal residual disease in marrow samples taken from the eight patients during remission was preliminarily determined by amplification of the samples by CDRIII polymerase chain reaction and hybridization by Southern blotting with each patient's leukemia-specific CDRIII probes. We have previously shown that the limit of detection of CDRIII sequences by Southern blotting is about 1 malignant cell in 10,000.21 Figure 4 shows representative results in six patients. In four of these patients (C137, C138, C52, and C94) and in Patient C92 (data not shown), hybridization signals indicating residual disease were noted in at least one remission sample during the follow-up period. It is noteworthy that in the remission sample obtained from Patient C52 three months before a clinical relapse in the bone marrow, a dramatic increase in the intensity of the hybridization signal was observed (Fig. 4Figure 4
Detection by Southern Blot Analysis of Leukemia-Specific CDRIII Sequences Present in Marrow Amplified by Polymerase Chain Reaction from Six Patients with B-Lineage Acute Lymphoblastic Leukemia in Clinical Remission.), despite normal results on morphologic examination of his marrow.To bypass the problems of interpreting and quantitating questionable weak hybridization signals on Southern blots, we developed an alternative approach for estimating minimal residual disease. The CDRIII sequences amplified by the polymerase chain reaction from marrow B cells collected during remission were cloned into M13 phage and transfected into E. coli. The resulting phage plaques were transferred in duplicate to filters. One set of filters was hybridized with an oligonucleotide CDRIII probe specific for the leukemic sequences, and an identical set was hybridized with JH consensus probes (JC) that recognize every CDRIII sequence. The proportion of plaques positive for the leukemic CDRIII sequence in all plaques containing any CDRIII sequence approximates the proportion of B leukemic cells in all the marrow B cells in the remission samples. A calculation of the number of leukemic cells among all nucleated marrow cells can be made by extrapolating from the percentage of lymphoid cells in the marrow population as determined morphologically. A total of 5000 to 10,000 plaques with CDRIII inserts were screened for the marrow samples collected during remission from each of the eight patients. Figure 5Figure 5
Quantification of Residual Disease during Treatment. summarizes the quantitative data. The number above the bar at each sampling date represents the approximate percentage of leukemic B cells in all the B cells present in the marrow of each patient. The percentage of malignant cells in all the nucleated cells in the bone marrow was estimated as shown below the bar in bold letters. (For the diagnostic marrow samples only, the number below the bar represents the percentage of lymphoblasts as determined morphologically.)In all but one of the samples obtained up to 18 months after diagnosis, residual disease was detected at a low level, ranging from 0.004 to 0.43 percent leukemic cells. The only exception was a high level of 2.6 percent in the sample from Patient C52 obtained three months before a relapse in the bone marrow. Residual disease could not be detected in samples obtained more than 18 months after diagnosis in three of four patients (C111, C135, and C138). Even after a relapse in the central nervous system in Patient C135, residual disease remained undetectable in the bone marrow. In Figure 5, the data shown for Patient C135 represent results obtained with use of a leukemia-specific probe derived from the C135–87 CDRIII sequence (Fig. 2). A probe was also used that would hybridize to both of the other two CDRIII sequences (C135–112 and C135–113) isolated from this patient. In the marrow sample collected at diagnosis, approximately 10 percent of the phage plaques hybridized to this probe, whereas the samples taken when the patient had discontinued therapy, including the one obtained after the relapse in the central nervous system, did not have any residual disease detectable with this probe. In the three patients (C52, C92, and C137) for whom remission marrow was available during the first month of chemotherapy, there was a striking 3-to-4-log decrease in the number of leukemic cells. In the same three patients, subsequent maintenance therapy did not reduce residual disease in follow-up marrow samples obtained within six months of diagnosis.
DNA sequences from several of the phage plaques that hybridized to the leukemia-specific probes in the samples collected during remission from some patients were determined and found to be identical to the CDRIII sequence determined at diagnosis.
Discussion
After the induction of remission, at least two years of continuous therapy are necessary to cure childhood ALL.37 38 39 This finding implies that most children harbor residual leukemia long after morphologically detectable blasts have disappeared from their blood and marrow. The measurement of terminal deoxynucleotidyltransferase, cytofluorimetric analysis with lineage-related monoclonal antibodies, and Southern blot analysis of heavy-chain-immunoglobulin rearrangements have proved only minimally or no better than morphology at detecting this residual disease.2 , 18 , 40 The blast-colony assay41 as modified by Estrov et al. may be more sensitive and specific than these techniques, but it is limited to cells that proliferate in vitro, and the number of residual leukemic cells cannot be quantitated.42
The CDRIII amplification by the polymerase chain reaction, with phage-library quantitation, as described here, can detect 1 leukemic cell among 100,000 normal cells, an order of sensitivity 2 to 3 logs higher than the other methods. Although the CDRIII-polymerase chain reaction technique is still technically demanding, it may be suitable for a cooperative group-reference laboratory. It can be used on stored, frozen samples and is applicable to peripheral blood, cerebrospinal fluid, and spinous-process bone marrow aspirates. A prospective trial involving a large number of patients, although it would require extensive staffing, is technically possible.
The CDRIII—polymerase chain reaction method is most feasible in cases of B-lineage ALL with one or two stable heavy-chain rearrangements, which fortunately appear to be the majority. For this reason we studied only patients whose leukemia, on the basis of Southern blot analysis, had only one or two rearranged bands. Nonetheless, in Patients C138, C135, and C52 the CDRIII—polymerase chain reaction method detected a number of CDRIII sequences that could not be explained by simple rearrangements of both heavy-chain alleles in a single clone of lymphoblasts. In Patient C138 the C138–105 and C138–111 clones may have come about through sequential VH rearrangements, as has been described in murine Blineage cancers43 , 44 and has been implicated in two of the multiple heavy-chain-immunoglobulin rearrangements in one case of ALL.45 In Patient C135, Southern blotting of the diagnostic specimen showed one rearranged band. However, the CDRIII—polymerase chain reaction method revealed one predominant sequence and two minor sequences that made up 10 percent of the specimen. The minor sequences differed by two point mutations and an additional base pair at the 3′ region of the VH gene. These may represent examples of somatic mutations, a phenomenon that occurs in B-cell lymphoma46 but has not been proved to occur in ALL.45
A second unanticipated finding was that the leukemia-specific probes hybridized with only 57 to 91 percent of the phage plaques containing CDRIII sequences, instead of more than 95 percent, as expected in view of the percentage of blasts in the specimens collected at diagnosis. Conceivably, the amplified CDRIII sequences that did not hybridize with the leukemia-specific probes were CDRIII sequences of normal B cells. Alternatively, they may have represented a limited number of different sequences derived from multiple heavy-chain-immunoglobulin rearrangements within the leukemic population,45 or mutated progeny of the predominant CDRIII sequence.
By far the most clinically provocative finding was the detection and quantitation of residual leukemia for up to 18 months after the patients had achieved clinical remission and the failure to detect it at the time they discontinued therapy. The only child who had an extramedullary relapse after therapy ended did not have residual marrow disease, suggesting that indeed the central nervous system was a sanctuary and that it had not yet reseeded the marrow. Ultimately, the ability to detect a clinically relevant level of residual leukemia may allow titration and individualizing of the duration of therapy. It may make it easier to evaluate various therapeutic maneuvers, such as delayed-intensification regimens.37 Studies of additional patients at the end of therapy may also reveal some who have detectable leukemia without a subsequent relapse, such as a cohort with sustained clonal remissions, as sometimes occurs in patients with acute or chronic myeloid leukemia.47 , 48 Surveillance marrow sampling was abandoned five years ago because it failed to detect residual or recurrent leukemia more than a few weeks before it became clinically apparent, and the detection of leukemia at that time had no effect on outcome.2 , 49 50 51 In our study, a large increase in the number of leukemic cells was detected more than three months before a clinical relapse, and this could justify the use of routine surveillance sampling if the outcome of the patients can be improved by the information provided.
Supported by grants (CA 10819 and CA 47983) from the National Cancer Institute.
Source Information
From the Wistar Institute (M.Y., B.A.R., G.R.), and the Division of Oncology, Children's Hospital of Philadelphia, and the School of Medicine, University of Pennsylvania (R.W., B.L., R.B.W.), all in Philadelphia. Address reprint requests to Dr. Rovera at the Wistar Institute. 36th and Spruce St., Philadelphia. PA 19104.
- References
References
1
Hagenbeek A, Löwenberg B, eds. Minimal residual disease in acute leukemia, 1986. Boston: Martinus Nijhoff, 1986.
2
Lange
Web of Science3
Groffen
CrossRef | Web of Science | Medline4
Shtivelman
CrossRef | Web of Science | Medline5
Tsujimoto
CrossRef | Web of Science | Medline6
Cleary
CrossRef | Web of Science | Medline7
Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. In: Wu R, ed. Methods in Enzymology. Vol. 155. Recombinant DNA, Part F. San Diego, Calif.: Academic Press, 1987: 335–50.
8
Saiki
CrossRef | Web of Science | Medline9
Lee
CrossRef | Web of Science | Medline10
Crescenzi
CrossRef | Web of Science | Medline11
Kawasaki
CrossRef | Web of Science | Medline12
Lee
Web of Science | Medline13
Roth
Web of Science | Medline14
Morgan
CrossRef | Web of Science | Medline15
Waldmann
CrossRef | Web of Science | Medline16
Waldmann
Full Text | Web of Science | Medline17
Tonegawa
CrossRef | Web of Science | Medline18
Wright
Web of Science | Medline19
D'Auriol
Web of Science | Medline20
Hansen-Hagge
Web of Science | Medline21
Yamada
CrossRef | Web of Science | Medline22
Meadows 23
Gaynon
CrossRef | Web of Science | Medline24
Boyum
Medline25
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989.
26
Higuchi R. Simple and rapid preparation of samples for PCR. In: Erlich HA, ed. PCR technology: principles and applications for DNA amplification. New York: Stockton Press, 1989:31–8.
27
Erikson
CrossRef | Web of Science | Medline28
Ravetch
CrossRef | Web of Science | Medline29
Feinberg
CrossRef | Web of Science | Medline30
Seliger 31
Kwok S, Mack DH, Kellogg DN, McKinney N, Faloona F, Sninsky JJ. Application of the polymerase chain reaction to the detection of human retroviruses. In: Erlich HA, Gibbs R, Kazazian HH Jr, eds. Polymerase chain reaction. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989:151–8.
32
Suggs SV, Hirose T, Miyake EH, et al. Use of synthetic oligodeoxyribonucleotides for the isolation of specifically cloned DNA sequences In: Brown DD, ed. Developmental biology using purified genes: proceedings of the 1981 ICN-UCLA Symposia on Developmental Biology Using Purified Genes, held in Keystone, Colorado, on March 15–20, 1981. Vol. 23. New York: Academic Press, 1981:683–93.
33
Bucan
Web of Science | Medline34
Hanahan
CrossRef | Web of Science | Medline35
Sanger
CrossRef | Web of Science | Medline36
Norrander
CrossRef | Web of Science | Medline37
Riehm H, Gadner H, Henze G, et al. Acute lymphoblastic leukemia: treatment results in three BFM studies (1970–1981). In: Murphy SB, Gilbert JR, eds. Leukemia research: advances in cell biology and treatment. New York: Elsevier Biomedical, 1983:251–63.
38
Bleyer 39
Treatment of acute lymphoblastic leukaemia: effect of variation in length of treatment on duration of remission. Report to the Medical Research Council by the Working Party on Leukaemia in Childhood . BMJ 1977; 2:495–7.
CrossRef | Web of Science40
Greaves
Web of Science | Medline41
Izaguirre
Web of Science | Medline42
Estrov
Full Text | Web of Science | Medline43
Reth
CrossRef | Web of Science | Medline44
Kleinfield
CrossRef | Web of Science | Medline45
Bird
CrossRef | Web of Science | Medline46
Cleary
CrossRef | Web of Science | Medline47
Gabert
CrossRef | Web of Science | Medline48
Fialkow
Full Text | Web of Science | Medline49
Rogers
CrossRef | Web of Science | Medline50
Komp
CrossRef | Web of Science | Medline51
Watson
CrossRef | Web of Science | Medline
- Citing Articles (61)
Citing Articles
1
J. Zhou, M. A Goldwasser, A. Li, S. E. Dahlberg, D. Neuberg, H. Wang, V. Dalton, K. D McBride, S. E. Sallan, L. B Silverman, J. G. Gribben, . (2007) Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium Protocol 95-01. Blood 110:5, 1607-1611
CrossRef2
Kevin J. Maher, Mary Ann Fletcher. (2005) Quantitative flow cytometry in the clinical laboratory. Clinical and Applied Immunology Reviews 5:6, 353-372
CrossRef3
Anna Butturini, John Klein, Robert Peter Gale. (2003) Modeling minimal residual disease (MRD)-testing. Leukemia Research 27:4, 293-300
CrossRef4
Carlos A. Scrideli, Simone Kashima, Rosana Cipolloti, Ricardo Defavery, José Eduardo Bernardes, Luiz G. Tone. (2002) Minimal residual disease in Brazilian children with acute lymphoid leukemia: comparison of three detection methods by PCR. Leukemia Research 26:5, 431-438
CrossRef5
Tomasz Szczepański, Thomas Flohr, Vincent H.J. van der Velden, Claus R. Bartram, Jacques J.M. van Dongen. (2002) Molecular monitoring of residual disease using antigen receptor genes in childhood acute lymphoblastic leukaemia. Best Practice & Research Clinical Haematology 15:1, 37-57
CrossRef6
Hitoshi Kiyoi, Tomoki Naoe. (2001) Immunoglobulin variable region structure and B-Cell malignancies. International Journal of Hematology 73:1, 47-53
CrossRef7
Brigitte Storch-Hagenlocher, J
rgen Haas, Maria Elisabeth Vogt-Schaden, Martin Bentz, Lisa Ann Hoffmann, Annette Biessmann, Brigitte Wildemann. (2000) Molecular analysis of the CDR3 encoding region of the immunoglobulin heavy chain locus in cerebrospinal fluid cells as a diagnostic tool in lymphomatous meningitis. Annals of Neurology 47:2, 211-217
CrossRef8
Cavé, Hélène , van der Werff ten Bosch, Jutte, Suciu, Stefan, Guidal, Christine, Waterkeyn, Christine, Otten, Jacques, Bakkus, Marleen, Thielemans, Kris, Grandchamp, Bernard, Vilmer, Etienne, Nelken, BrigitteFournier, MartineBoutard, PatrickLebrun, EmmanuelMéchinaud, FrançoiseGarand, RichardRobert, AlainDastugue, NicolePlouvier, EmmanuelRacadot, EvelyneFerster, AliceGyselinck, JanFenneteau, OdileDuval, MichelSolbu, GabrielManel, Anne-Marie. (1998) Clinical Significance of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia. New England Journal of Medicine 339:9, 591-598
Full Text9
S Faderl. (1998) The clinical significance of detection of residual disease in childhood ALL. Critical Reviews in Oncology/Hematology 28:1, 31-55
CrossRef10
Carlos A. Scrideli, Aguinaldo L. Simoes, Ricardo Defavery, José E. Bernardes, Maria Herbenia O. Duarte, Luiz G. Tone. (1997) Childhood B Lineage Acute Lymphoblastic Leukemia Clonality Study by the Polymerase Chain Reaction. Journal of Pediatric Hematology/Oncology 19:6, 516-522
CrossRef11
Gerald Martin, Robert J. Mairs. (1997) Synthesis of quantitation standards for nested competitive PCR for the determination of minimal residual disease in B-lineage acute lymphoblastic leukaemia. Leukemia Research 21:9, 807-809
CrossRef12
Nicolas Thiounn, François Saporta, Thierry A. Flam, Franck Pages, Marc Zerbib, Annick Vieillefond, Emmanuel Martin, Bernard Debré, Sylvie Chevillard. (1997) Positive prostate-specific antigen circulating cells detected by reverse transcriptasepolymerase chain reaction does not imply the presence of prostatic micrometastase s. Urology 50:2, 245-250
CrossRef13
Emina Torlakovic, David L Cherwitz, Jose Jessurun, John Scholes, Ronald McGlennen. (1997) B-cell gene rearrangement in benign and malignant lymphoid proliferations of mucosa-associated lymphoid tissue and lymph nodes. Human Pathology 28:2, 166-173
CrossRef14
Roberts, William Mark, Estrov, Zeev, Ouspenskaia, Maia V., Johnston, Dennis A., McClain, Kenneth L., Zipf, Theodore F., . (1997) Measurement of Residual Leukemia during Remission in Childhood Acute Lymphoblastic Leukemia. New England Journal of Medicine 336:5, 317-323
Full Text15
Greaves, Mel, . (1997) Silence of the Leukemic Clone. New England Journal of Medicine 336:5, 367-369
Full Text16
C. Harker Rhodes, Michael J. Glantz, Lisa Glantz, Andrew Lekos, George D. Sorenson, Charles Honsinger, Norman B. Levy. (1996) A comparison of polymerase chain reaction examination of cerebrospinal fluid and conventional cytology in the diagnosis of lymphomatous meningitis. Cancer 77:3, 543-548
CrossRef17
John A. Lust. (1996) Molecular genetics and lymphoproliferative disorders. Journal of Clinical Laboratory Analysis 10:6, 359-367
CrossRef18
Jorge E. Cortes, Hagop M. Kantarjian. (1995) Acute lymphoblastic leukemia a comprehensive review with emphasis on biology and therapy. Cancer 76:12, 2393-2417
CrossRef19
C. Scholten, M. Födinger, M. Mitterbauer, K. Laczika, G. Mitterbauer, O. A. Haas, P. Knöbl, I. Schwarzinger, R. Thalhammer, B. Purtscher, K. Geissler, C. Mannhalter, K. Lechner, U. Jaeger. (1995) Kinetics of minimal residual disease during induction/consolidation therapy in standard-risk adult B-lineage acute lymphoblastic leukemia. Annals of Hematology 71:4, 155-160
CrossRef20
G. Socie, M. Lawler, E. Gluckman, S.R. McCann, O. Brison. (1995) Studies on hemopoietic chimerism following allogeneic bone marrow transplantation in the molecular biology era. Leukemia Research 19:8, 497-504
CrossRef21
ANDRE BARUCHEL, JEAN-MICHEL CAYUELA, ELISABETH MACINTYRE, I ROLAND BERGER, FRANCOIS SIGAUX. (1995) Assessment of clonal evolution at Ig/TCR loci in acute lymphoblastic leukaemia by single-strand conformation polymorphism studies and highly resolutive PCR derived methods: implication for a general strategy of minimal residual disease detection. British Journal of Haematology 90:1, 85-93
CrossRef22
Gerald Martin, Robert Mairs, Jeremy Platt, Brenda Gibson. (1995) Rapid and sensitive non-radioactive assay for the detection of clonal gene rearrangements in B-lineage acute leukaemia. British Journal of Haematology 89:2, 417-420
CrossRef23
Denis C. Roy, Claude Perreault, Robert B
CrossRef24
Dario Campana. (1994) Monitoring minimal residual disease in acute leukemia: expectations, possibilities and initial clinical results. International Journal of Clinical & Laboratory Research 24:3, 132-138
CrossRef25
Makoto Takishita, Masaaki Kosaka, Tetsuya Goto, Shiro Saito. (1994) Cellular origin and extent of clonal involvement in multiple myeloma: genetic and phenotypic studies. British Journal of Haematology 87:4, 735-742
CrossRef26
Massimo Loda. (1994) Polymerase chain reaction-based methods for the detection of mutations in oncogenes and tumor suppressor genes. Human Pathology 25:6, 564-571
CrossRef27
Lambert Busque, Robert Ilaria, Ramana Tantravahi, Howard Weinstein, D.Gary Gilliland. (1994) Clonality analysis of childhood all in remission: No evidence of clonal hematopoiesis. Leukemia Research 18:2, 71-77
CrossRef28
Andrea Biondi, Alessandro Rambaldi. (1994) Polymerase chain reaction (PCR) approach for the evaluation of minimal residual disease in acute leukemia. Stem Cells 12:4, 394-401
CrossRef29
T. Carr, R.F. Stevens. (1994) Minimal residual disease in childhood acute lymphoblastic leukaemia. The Lancet 343:8891, 190
CrossRef30
M.J. Brisco, J. Condon, E. Hughes, S-H. Neoh, P.J. Sykes, R. Seshadri, A.A. Morley, I. Toogood, K. Waters, G. Tauro, H. Ekert. (1994) Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction. The Lancet 343:8891, 196-200
CrossRef31
Hitoshi Kiyoi, Tomoki Naoe, Tatsuya Yamauchi, Hisashi Fukatani, Kazuaki Kubo, Seiji Kojima, Ryuzo Ohno. (1993) Minimal residual disease status in pre-B acute lymphoblastic leukemia patients after chemotherapy and bone marrow transplantation: Assessment of the anti-leukemic effects of chemotherapy and BMT. Leukemia Research 17:8, 677-684
CrossRef32
K. Langlands, O. B. Eden, P. Micallef-Eynaud, A. C. Parker, R. S. Anthony. (1993) Direct sequence analysis of TCR Vδ2–Dδ3 rearrangements in common acute lymphoblastic leukaemia and application to detection of minimal residual disease. British Journal of Haematology 84:4, 648-655
CrossRef33
Claus R. Bartram. (1993) Detection of minimal residual leukemia by the polymerase chain reaction: potential implications for therapy. Clinica Chimica Acta 217:1, 75-83
CrossRef34
Ryuzo Ohno, Tohru Kobayashi, Mitsune Tanimoto, Akira Hiraoka, Kuniyuki Imai, Norio Asou, Masao Tomonaga, Kazuo Tsubaki, Isao Takahashi, Yoshihisa Kodera, Minoru Yoshida, Hirokazu Murakami, Tomoki Naoe, Masanori Shimoyama, Tetsuya Tsukada, Takaaki Takeo, Hirofumi Teshima, Yasusuke Onozawa, Koji Fujimoto, Kazutaka Kuriyama, Atsushi Horiuchi, Ikuro Kimura, Saburo Minami, Yasusada Miura, Shinichi Kageyama, Tohru Tahara, Tohru Masaoka, Shigeru Shirakawa, Hidehiko Saito. (1993) Randomized study of individualized induction therapy with or without vincristine, and of maintenance—intensification therapy between 4 or 12 courses in adult acute myeloid leukemia. AML-87 study of the Japan adult leukemia study group. Cancer 71:12, 3888-3895
CrossRef35
Raymond Liang, Vivian Chan, T. K. Chan, Thomas Wong, Edmond Chiu, Albert Lie, David Todd. (1993) Detection of immunoglobulin gene rearrangement in lymphoid malignancies of B-cell lineage by seminested polymerase chain reaction gene amplification. American Journal of Hematology 43:1, 24-28
CrossRef36
Ruey-Long Hong, Ssu-Wen Shen, Ming-Tseh Lin, Hwei-Fang Tien, Chin-Hsin Yang, Yao-Chang Chen. (1993) Lymphoblast colony-culture assay in acute lymphoblastic leukemia: A quantitative approach. Leukemia Research 17:5, 463-470
CrossRef37
Michael N. Potter, Colin G. Steward, Anthony Oakhill. (1993) The significance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia. British Journal of Haematology 83:3, 412-418
CrossRef38
Steffen Albrecht, Janet M. Bruner, Gary K. Segall. (1993) Immunoglobulin heavy chain rearrangements in primary brain lymphomas. A study using PCR to amplify CDR-III. The Journal of Pathology 169:3, 297-302
CrossRef39
William M. Crist, Ching‐Hon Pui. (1993) Clinical implications of cytogenetic and molecular analyses of pediatric acute lymphoblastic leukemia. Stem Cells 11:2, 81-87
CrossRef40
L. Peter Fielding, Cecilia M. Fenoglio-Preiser, Laurence S. Freedman. (1992) The future of prognostic factors in outcome prediction for patients with cancer. Cancer 70:9, 2367-2377
CrossRef41
L. Lennard. (1992) The clinical pharmacology of 6-mercaptopurine. European Journal of Clinical Pharmacology 43:4, 329-339
CrossRef42
Jeffrey Sklar, Janina Longtine. (1992) The clinical significance of antigen receptor gene rearrangements in lymphoid neoplasia. Cancer 70:S4, 1710-1718
CrossRef43
M.N Potter. (1992) The detection of minimal residual disease in acute lymphoblastic leukaemia. Blood Reviews 6:2, 68-82
CrossRef44
Raymond Liang, Vivian Chan, T. K. Chan, Thomas Wong, David Todd. (1992) Detection of immunoglobulin gene rearrangement in B-cell lymphomas by polymerase chain reaction gene amplification. Hematological Oncology 10:3-4, 149-154
CrossRef45
Daniel H. Ryan. (1992) Phenotypic heterogeneity in acute leukemia. Clinica Chimica Acta 206:1-2, 9-23
CrossRef46
Johannes Drach, Doris Drach, Herta Glassl, Claus Gattringer, Heinz Huber. (1992) Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease. Cytometry 13:8, 893-901
CrossRef47
Russell M. Lebovitz, Steffen Albrecht. (1992) Molecular Biology in the Diagnosis and Prognosis of Solid and Lymphoid Tumors. Cancer Investigation 10:5, 399-416
CrossRef48
Ernest S. Kawasaki. (1992) The Polymerase Chain Reaction: Its use in the Molecular Characterization and Diagnosis of Cancers. Cancer Investigation 10:5, 417-429
CrossRef49
Kenneth Lucas, Mary Jean Gula, Julie Blatt. (1992) Relapse in Acute Lymphoblastic Leukemia as a Function of White Blood Cell and Absolute Neutrophil Counts During Maintenance Chemotherapy. Pediatric Hematology-Oncology 9:2, 91-97
CrossRef50
ROBERT S. NEGRIN. (1992) Use of the Polymerase Chain Reaction for the Detection of Tumor Cell Involvement of Bone Marrow and Peripheral Blood: Implications for Purging. Journal of Hematotherapy 1:4, 361-368
CrossRef51
John E. Dick, Tsvee Lapidot, Francoise Pflumio. (1991) Transplantation of Normal and Leukemic Human Bone Marrow into Immune-Deficient Mice: Development of Animal Models for Human Hematopoiesis. Immunological Reviews 124:1, 25-43
CrossRef52
Raymond Liang, Vivian Chan, T. K. Chan, Thomas Wong, Edmond Chiu, David Todd. (1991) Detection of immunoglobulin gene rearrangement in acute and chronic lymphoid leukemias of B-cell lineage by polymerase chain reaction gene amplification. American Journal of Hematology 38:3, 189-193
CrossRef53
R.P. Gale, A. Buttunni. (1991) Maintenance chemotherapy and cure of childhood acute lymphoblastic leukaemia. The Lancet 338:8778, 1315-1318
CrossRef54
Y. Nizet, P. Martiat, J. L. Vaerman, M. Philippe, C. Wildmann, J. P. Staelens, G. Cornu, A. Ferrant, J. L. Michaux, G. Sokal. (1991) Follow-up of residual disease (MRD) in B lineage acute leukaemias using a simplified PCR strategy: evolution of MRD rather than its detection is correlated with clinical outcome. British Journal of Haematology 79:2, 205-210
CrossRef55
M. J. Brisco, J. Condon, P. J. Sykes, S. H. Neoh, A. A. Morley. (1991) Detection and quantitation of neoplastic cells in acute lymphoblastic leukaemia, by use of the polymerase chain reaction. British Journal of Haematology 79:2, 211-217
CrossRef56
Minoru Asada, Toshiyuki Miyashita, Fumio Bessho, Noboru Kobayashi, Shuki Mizutani. (1991) Malignant Cell Detection in Burkitt's Lymphoma Using Third-complementarity-determining Region (CDRIII), Clone-specific Probe Developed by Sequencing DNA from Stored Slides. Cancer Science 82:7, 848-853
CrossRef57
Jacques J.M.ban Dongen, Ingrid L.M. Wolvers-Tettero. (1991) Analysis of immunoglobulin and T cell receptor genes. Part II: Possibilities and limitations in the diagnosis and management of lymphoproliferative diseases and related disorders. Clinica Chimica Acta 198:1-2, 93-174
CrossRef58
(1991) Detection of Minimal Residual Disease in Childhood Leukemia with the Polymerase Chain Reaction. New England Journal of Medicine 324:11, 772-775
Full Text59
Miriam Deane, John D. Norton. (1991) Immunoglobulin gene ‘fingerprinting’: an approach to analysis of B lymphoid clonality in lymphoproliferative disorders. British Journal of Haematology 77:3, 274-281
CrossRef60
Miriam Deane, John D. Norton. (1991) Detection of Immunoglobulin Gene Rearrangement in B Cell Neoplasias by Polymerase Chain Reaction Gene Amplification. Leukemia & Lymphoma 5:1, 9-22
CrossRef61
J. Drach, C. Gattringer and, H. Huber. (1991) Combined flow cytometric assessment of cell surface antigens and nuclear TdT for the detection of minimal residual disease in acute leukaemia. British Journal of Haematology 77:1, 37-42
CrossRef
