Vδ2-Jα rearrangements are frequent in precursor-B–acute lymphoblastic leukemia but rare in normal lymphoid cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3798-3804 ◽  
Author(s):  
Tomasz Szczepański ◽  
Vincent H. J. van der Velden ◽  
Patricia G. Hoogeveen ◽  
Maaike de Bie ◽  
Daniëlle C. H. Jacobs ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Gene Segment ◽  
Significant Proportion ◽  
High Sensitivity ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Patient Specific ◽  
Real Time Quantitative Pcr

Abstract The frequently occurring T-cell receptor delta (TCRD) deletions in precursor-B–acute lymphoblastic leukemia (precursor-B–ALL) are assumed to be mainly caused by Vδ2-Jα rearrangements. We designed a multiplex polymerase chain reaction tified clonal Vδ2-Jα rearrangements in 141 of 339 (41%) childhood and 8 of 22 (36%) adult precursor-B–ALL. A significant proportion (44%) of Vδ2-Jα rearrangements in childhood precursor-B–ALL were oligoclonal. Sequence analysis showed preferential usage of the Jα29 gene segment in 54% of rearrangements. The remaining Vδ2-Jα rearrangements used 26 other Jα segments, which included 2 additional clusters, one involv ing the most upstream Jα segments (ie, Jα48 to Jα61; 23%) and the second cluster located around the Jα9 gene segment (7%). Real-time quantitative PCR studies of normal lymphoid cells showed that Vδ2 rearrangements to upstream Jα segments occurred at low levels in the thymus (10–2 to 10–3) and were rare (generally below 10–3) in B-cell precursors and mature T cells. Vδ2-Jα29 rearrangements were virtually absent in normal lymphoid cells. The monoclonal Vδ2-Jα rearrangements in precursor-B–ALL may serve as patient-specific targets for detection of minimal residual disease, because they show high sensitivity (10–4 or less in most cases) and good stability (88% of rearrangements preserved at relapse).

Novel methods to assess cell-free circulating tumor DNA in acute lymphoblastic leukemia.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 10034-10034
Author(s):  
Meghan G Haney ◽  
Shilpa Sampathi ◽  
Yelena Chernyavskaya ◽  
Henry Moore ◽  
Tom C. Badgett ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Diagnostic Procedures ◽  
Cell Receptor ◽  
Response To Therapy ◽  
Patient Specific ◽  
Cns Disease

10034 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relatively high relapse rate, which is associated with poor prognosis. Currently, minimal residual disease (MRD) at the end of induction and consolidation therapy is the best predictor of patient relapse, however obtaining bone marrow aspirate is invasive and not always accurate. Another major concern in ALL is the presence of central nervous system (CNS) disease, which is often present long before clinical diagnosis can be made by flow cytometry. To circumvent these clinical challenges, we developed a new assay quantifying cell-free, circulating tumor (cfDNA) as a biomarker of disease progression, which can be correlated with MRD status as a predictor of relapse. cfDNA is frequently used to monitor progression of solid tumors, but pediatric leukemias lack common mutations that can be used to distinguish leukemic cfDNA from normal cfDNA. Methods: We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We developed a novel workflow for identifying VDJ rearrangements in leukemia cells and tracking their presence in cfDNA. We collected bone marrow, blood, and CSF samples from newly diagnosed patients, and cfDNA was isolated from blood and CSF samples throughout treatment. Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements of leukemic clones in genomic DNA. The MinION assay relies on patient-specific sequencing. We are also in the process of developing a universal assay that utilizes recurrent methylation changes in ALL to identify leukemic cfDNA in patient samples. Results: The MinION workflow was used to follow leukemic cfDNA throughout the course of treatment, and accurately identified MRD and CNS disease in patients. This workflow performed equivalent or better at detecting leukemic clones compared to MiSeq and droplet digital polymerase chain reaction (ddPCR), and is faster and less expensive than traditional Illumina sequencing. Methylation analysis of 865 ALL and 79 healthy samples yielded 55 regions and 19 specific methylation sites that were uniquely present in ALL samples. We are validating these sites by ddPCR to establish a panel of biomarkers to track ALL over time via cfDNA. Conclusions: The end goal of our study is provide a more sensitive and less invasive method for tracking MRD and CNS disease than current approaches. Results will ultimately be correlated with patient response to therapy, the presence of relapse or CNS disease, and overall outcomes determined by standard clinical diagnostic procedures.

scholarly journals Immature Immunoglobulin Gene Rearrangements Are Recurrent in B Precursor Adult Acute Lymphoblastic Leukemia Carrying TP53 Molecular Alterations

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 960
Author(s):  
Silvia Salmoiraghi ◽  
Roberta Cavagna ◽  
Marie Lorena Guinea Montalvo ◽  
Greta Ubiali ◽  
Manuela Tosi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Early Stage ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Disease Assessment ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Receptor ◽  
Molecular Alterations ◽  
Mutational Status

Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.

scholarly journals Detectable Molecular Residual Disease at the Beginning of Maintenance Therapy Indicates Poor Outcome in Children With T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1226-1232 ◽  
Author(s):  
Salvatore P. Dibenedetto ◽  
Luca Lo Nigro ◽  
Sharon Pine Mayer ◽  
Giovanni Rovera ◽  
Gino Schilirò
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Maintenance Therapy ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Strong Predictor ◽  
Blot Hybridization ◽  
Cell Receptor ◽  
Dot Blot ◽  
Poor Outcome

Abstract The aims of this study were twofold: (1) to assess the marrow of patients with T-lineage acute lymphoblastic leukemia (T-ALL) for the presence of molecular residual disease (MRD) at different times after diagnosis and determine its value as a prognostic indicator; and (2) to compare the sensitivity, rapidity, and reliability of two methods for routine clinical detection of rearranged T-cell receptor (TCR). Marrow aspirates from 23 patients with T-ALL diagnosed consecutively from 1982 to 1994 at the Division of Pediatric Hematology and Oncology, University of Catania, Italy, were obtained at diagnosis, at the end of induction therapy (6 to 7 weeks after diagnosis), at consolidation and/or reinforced reinduction (12 to 15 weeks after diagnosis), at the beginning of maintenance therapy (34 to 40 weeks after diagnosis), and at the end of therapy (96 to 104 weeks after diagnosis). DNA from the patients' marrow was screened using the polymerase chain reaction (PCR) for the four most common TCR δ rearrangements in T-ALL (Vδ1Jδ1, Vδ2Jδ1, Vδ3Jδ1, and Dδ2Jδ1) and, when negative, further tested for the presence of other possible TCR δ and TCR γ rearrangements. After identification of junctional rearrangements involving V, D, and J segments by DNA sequencing, clone-specific oligonucleotide probes 5′ end-labeled either with fluorescein or with [γ-32P]ATP were used for heminested PCR or dot hybridization of PCR products of marrows from patients in clinical remission. For 17 patients with samples that were informative at the molecular level, the estimated relapse-free survival (RFS) at 5 years was 48.6% (±12%). The sensitivity and specificity for detection of MRD relating to the outcome were 100% and 88.9% for the heminested fluorescence PCR and 71.4% and 88.9% for Southern/dot blot hybridization, respectively. Predictive negative and positive values were 100% and 90.7% for heminested fluorescence PCR, respectively. The probability of RFS based on evidence of MRD as detected by heminested fluorescence PCR at the time of initiation of maintenance therapy was 100% and 0% for MRD-negative and MRD-positive patients, respectively. Thus, the presence of MRD at the beginning of maintenance therapy is a strong predictor of poor outcome, and the molecular detection of MRD at that time might represent the basis for a therapeutic decision about such patients. By contrast, the absence of MRD at any time after initiation of treatment strongly correlates with a favorable outcome. The heminested fluorescence PCR appears to be more accurate and more rapid than other previously used methods for the detection of residual leukemia.

scholarly journals Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 692-702 ◽  
Author(s):  
EJ Steenbergen ◽  
OJ Verhagen ◽  
EF van Leeuwen ◽  
H van den Berg ◽  
AE von dem Borne ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Southern Blot ◽  
T Cell Receptor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
First Relapse ◽  
Minimal Residual

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.

scholarly journals Prospective monitoring and quantitation of residual blasts in childhood acute lymphoblastic leukemia by polymerase chain reaction study of delta and gamma T-cell receptor genes

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1892-1902 ◽  
Author(s):  
H Cave ◽  
C Guidal ◽  
P Rohrlich ◽  
MH Delfau ◽  
A Broyart ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell Receptor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
B Lineage ◽  
Minimal Residual

Abstract We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B- lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

The Repertoire of Gene Rearrangements in Acute Lymphoblastic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1464-1464
Author(s):  
Alexander A. Morley ◽  
Michael J. Brisco ◽  
Pamela J. Sykes ◽  
Sue Latham ◽  
Elizabeth Hughes ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Acute Lymphoblastic Leukemia ◽  
High Efficiency ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Gene Rearrangements ◽  
Childhood All ◽  
Repertoire Analysis ◽  
Specificity And Sensitivity

Abstract Rearrangements of the immunoglobulin and T-cell receptor genes provide molecular markers for clones in acute lymphoblastic leukemia (ALL). Determination of the repertoire of gene rearrangements in ALL aids in understanding the clonal biology of the disease and provides molecular markers which can be used to quantify minimal residual disease (MRD). We have developed a sensitive PCR-based method for analysing the repertoire of immunoglobulin heavy chain (IgH) rearrangements in ALL. Multiple parallel quantitative PCR’s are performed in microplates using different segment-specific primers in different wells in order to determine the individual V (D) and J segments utilised by each rearrangement. The number of rearrangements detected in 18 children and 10 adults with ALL is shown in the table: VDJ rearrangements DJ rearrangements No. of rearrangements 1 2 3 4 1 2 Childhood ALL 0 11 3 2 1 1 Adult ALL 7 2 0 1 Since each PCR well contained only 2 ng of DNA, more sensitive repertoire analysis was also performed in samples from 10 of the children and 4 of the adults by using 100 ng of DNA in an initial preamplification, which involved a multiplexed PCR containing primers for all leader and J sequences of the IgH gene and which thus amplified all immunoglobulin sequences. The IgH repertoire of the amplified material was then analysed. This two-step approach should theoretically enable detection of clones which comprise down to approximately 10−4 the leukemic population. It detected all rearrangements previously detected by one-step repertoire analysis and, in addition, it detected 0–5 (mean 1.2) rearrangements marking small clones in childhood ALL and 0–3 (mean 1.0) rearrangements marking small clones in adult ALL. Sequencing showed that most, but not all, small clones had a lineage relationship to the dominant clone present in the leukemic population. Repertoire analysis of IgH rearrangements is a promising technique for identifying molecular markers for measurement of MRD in B-ALL, particularly childhood ALL, since: –it is conceptually simple and relatively quick –it detects IgH rearrangements with high efficiency, probably higher than that of current techniques. –IgH rearrangements are the best markers to use for measurement of MRD owing to the specificity and sensitivity that they provide –an enhanced ability to identify markers for both large and small leukemic clones may improve the identification of patients prone to relapse.

CpG Oligonucleotides Induce Anti-Leukemia Activity in a Syngeneic Murine Model of Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2830-2830
Author(s):  
Alix E. Seif ◽  
Marlo D. Bruno ◽  
Junior Hall ◽  
Valerie I. Brown ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Significant Proportion ◽  
Cpg Odn

Abstract Acute lymphoblastic leukemia (ALL) accounts for 80% of all pediatric leukemias and is the most common form of childhood cancer. While most children with ALL are cured by current therapies, refractory and relapsed ALL comprise a significant proportion of all pediatric cancers. Furthermore, nearly half of all ALL diagnoses occur in adults, who carry a much poorer prognosis, with the majority dying of relapsed disease. Relapsed ALL generally requires intensive therapy with significant associated morbidity and mortality. Development of novel therapies is essential to improving outcomes. DNA oligodeoxynucleotides containing CpG motifs (CpG ODN) stimulate anti-tumor immune activity via Toll-like receptor 9 (TLR9) activation and are currently in clinical trials for a variety of solid tumors. We have previously reported that CpG ODN stimulation alters antigen presentation by human ALL cells, enhancing allogeneic Th1 responses. In addition, we have shown that CpG ODN administration in vivo reduces the leukemic burden of primary human ALL xenografts in Nod-SCID mice, and that this activity is mediated in part by NK cells. To further the development of CpG ODN as a novel therapeutic agent for ALL, we have investigated the induction of anti-ALL activity by CpG ODN in a syngeneic ALL setting. CpG ODN did not exhibit direct toxicity against cell lines derived from leukemic Eμ-ret transgenic mice in vitro, nor did it alter CD40 or CD86 expression or cytokine production. However, using a flow cytometry-based in vitro killing assay we observed CpG ODN-induced elimination of leukemia cells when cultured with splenocytes or bone marrow cells from Eμ-ret transgene-negative mice (P=0.0388). The difference between CpG ODN-treated and untreated controls became more pronounced with increasing effector:target ratios (P<0.0001). Preliminary data show that depletion of NK cells markedly decreases the magnitude of the observed effect, supporting the hypothesis that this cell type is involved in targeted control of ALL in this model. The ability of CpG ODN to exert anti-leukemia activity in a syngeneic setting suggests that it may have utility as an adjuvant therapy. To test this hypothesis we administered CpG ODN (or PBS) to syngeneic leukemia-bearing mice 2 days after completion of a chemotherapy regimen used to reduce leukemia burden. When mice were sacrificed 3 weeks after treatment, we found significantly reduced leukemia burden in bone marrow (P=0.0019), spleen (P<0.00001) and blood (P=0.00028) of CpG ODN-treated mice. Cell-depletion and cytokine-neutralization assays are currently ongoing to define the mechanism of action of CpG ODN in these settings. To our knowledge, this is the first demonstration of CpG ODN-induced anti-ALL activity in a post-chemotherapy syngeneic model, suggesting that this agent has the potential to treat minimal residual disease and to reduce the incidence of relapse.

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