Previously Unknown Stage of TCR Beta VDJ Recombination As a Novel Rich Source of Markers for Clonality Assessment and MRD Monitoring in T-ALL

Abstract Background. Genomic rearrangements of T and B cell receptor genes (BCR and TCR) have become the gold standard marker for clonality assessment and minimal residual disease monitoring (MRD) in acute lymphoblastic leukemia (ALL). B-ALL can bear both TCR and BCR rearrangements, whereas T-ALL contains mainly TCR rearrangements. The limitation on the number of potential markers decreases T-ALL cases suitable for MRD monitoring and its reliability. We discovered a missed stage of TRB locus VDJ-recombination during which rearrangement between D1 and D2 segments occurs. Here we examined this new type of rearrangement in pediatric T-ALL as a potential clonal marker for MRD diagnostics for the first time. Methods. Identification of TRB D1-D2 rearrangements was performed using targeted high-throughput sequencing on the Illumina MiSeq instrument [1]. Genomic libraries for sequencing were obtained using PCR with intronic primers flanking the TRBD1 and TRBD2 genes. Genomic DNA for the libraries was extracted from T-ALL patients' bone marrow and peripheral blood of healthy volunteers. DNA from normal CD4+ and CD8+ T-cells was used as a positive control. DNA from normal B-cells and the RMS cell line was used as a negative control. Extraction of TRB D1-D2 rearrangements from sequencing data was performed using modified MiXCR software. Post-analysis was performed using VDJtools software. Results. This study analyzed a cohort of 144 children aged 2-16 years with T-ALL and 12 healthy individuals aged 25-40 years. We identified TRB D1-D2 rearrangements in all analyzed samples except negative controls (B-cells and RMS cell line). The dominant TRB D1-D2 clone in all normal samples occupied 25-30% and had zero length of the D1-D2 junction. The clonal size of the second clone in the top was 3-8%. Based on this observation, we set a 30% cut-off for zero-length D1-D2 junction rearrangements and a 10% cut-off for others to identify abnormally proliferated T-ALL specific clones. We identified 221 different leukemia-related D1-D2 rearrangements in 124 T-ALL samples in total. We intersected them with those in normal samples to test the uniqueness of leukemia-related rearrangements as potential markers. 89% of analyzed rearrangements were specific for leukemia samples, so they can be used for tracking leukemic cells. 11% of rearrangements were present in at least one normal sample and can be used for clonality analysis but are useless for MRD monitoring due to its relatively high generation probability. Zero-length D-D junction was detected as a major clonal marker (25-100%) in 42 T-ALL samples. Nevertheless, 23 out of these 42 samples contained additional unique rearrangements useful for MRD. 29 out of the other 82 samples included one major rearrangement (clonal rate 80-90%), six samples had two major rearrangements with equal clonal rates (30-50%), and the rest of the samples contained 3-5 rearrangements representing oligoclonality. The length of the D-D junction among detected rearrangements in both leukemic and normal samples has a bimodal distribution which is probably linked to two different levels of TdT or exonuclease activity during D1-D2 recombination. Additionally, we observed a strong correlation (Mann-Whitney-U-test, p-value < 0.001) between the presence of TRB D1-D2 rearrangements in T-ALL samples and the CD117 negative immunophenotype of leukemic cells. Conclusions. Obtained results show that TRB D1-D2 rearrangements are a common feature of normal and malignant T-cells. Unique leukemia-related TRB D1-D2 rearrangements can be detected in 73% of pediatric T-ALL cases. It can be routinely detected by PCR with subsequent NGS and can be easily integrated into an existing multiplex system for T-ALL clonality and MRD analysis. References. Komkov et al. High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies. British Journal of Haematology. 2020 Mar. 188(5): 723-731. Acknowledgment. This work was supported by Russian Science Foundation grant № 20-75-10091 to AK. Disclosures No relevant conflicts of interest to declare.

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Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽

10.1182/blood.v114.22.1737.1737 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1737-1737

Author(s):

David M. Lucas ◽

Ellen J. Sass ◽

Ryan B. Edwards ◽

Li Pan ◽

Gerard Lozanski ◽

...

Keyword(s):

Drug Resistance ◽

T Cells ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Multi Drug Resistance ◽

Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

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A Mathematical Description of the Bone Marrow Dynamics of CAR T-Cell Therapy in B-cell Childhood Acute Lymphoblastic Leukemia

10.20944/preprints202105.0323.v1 ◽

2021 ◽

Author(s):

Álvaro Martínez-Rubio ◽

Salvador Chulián ◽

Cristina Blázquez Goñi ◽

Antonio Pérez Martínez ◽

Manuel Ramírez Orellana ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell Therapy ◽

Car T

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells and CAR T cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T cells and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load and tumor burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

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CD8+ T Cells Engineered to Express a ROR1-Specific Chimeric Antigen Receptor Specifically Recognize ROR1 Positive B Cell Tumors.

Blood ◽

10.1182/blood.v114.22.930.930 ◽

2009 ◽

Vol 114 (22) ◽

pp. 930-930

Author(s):

Michael Hudecek ◽

Thomas M Schmitt ◽

Sivasubramanian Baskar ◽

Wen-Chung Chang ◽

David G Maloney ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Cell Line ◽

Chimeric Antigen Receptor ◽

Cd8 T Cells ◽

Antigen Receptor ◽

Cell Receptor ◽

Surface Expression

Abstract Abstract 930 The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells. Disclosures: No relevant conflicts of interest to declare.

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A Mathematical Description of the Bone Marrow Dynamics during CAR T-Cell Therapy in B-Cell Childhood Acute Lymphoblastic Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22126371 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6371

Author(s):

Álvaro Martínez-Rubio ◽

Salvador Chulián ◽

Cristina Blázquez Blázquez Goñi ◽

Manuel Ramírez Ramírez Orellana ◽

Antonio Pérez Pérez Martínez ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell Therapy ◽

Car T

Chimeric Antigen Receptor (CAR) T-cell therapy has demonstrated high rates of response in recurrent B-cell Acute Lymphoblastic Leukemia in children and young adults. Despite this success, a fraction of patients’ experience relapse after treatment. Relapse is often preceded by recovery of healthy B cells, which suggests loss or dysfunction of CAR T-cells in bone marrow. This site is harder to access, and thus is not monitored as frequently as peripheral blood. Understanding the interplay between B cells, leukemic cells, and CAR T-cells in bone marrow is paramount in ascertaining the causes of lack of response. In this paper, we put forward a mathematical model representing the interaction between constantly renewing B cells, CAR T-cells, and leukemic cells in the bone marrow. Our model accounts for the maturation dynamics of B cells and incorporates effector and memory CAR T-cells. The model provides a plausible description of the dynamics of the various cellular compartments in bone marrow after CAR T infusion. After exploration of the parameter space, we found that the dynamics of CAR T product and disease were independent of the dose injected, initial B-cell load, and leukemia burden. We also show theoretically the importance of CAR T product attributes in determining therapy outcome, and have studied a variety of possible response scenarios, including second dosage schemes. We conclude by setting out ideas for the refinement of the model.

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RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

Journal of Biological Chemistry ◽

10.1074/jbc.m314273200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19523-19530 ◽

Cited By ~ 23

Author(s):

Benoit Guilbault ◽

Robert J. Kay

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Receptor ◽

B Cell Receptor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Wehi 231 ◽

Induced Apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-κB and Bcl-xL, which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-κB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-κB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-κB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.

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Establishment and characterization of a childhood T-cell acute lymphoblastic leukemia cell line, PER-255, with chromosome abnormalities involving 7q32-34 in association with T-cell receptor- beta gene rearrangement

Blood ◽

10.1182/blood.v74.1.369.bloodjournal741369 ◽

1989 ◽

Vol 74 (1) ◽

pp. 369-373

Author(s):

UR Kees ◽

R Lukeis ◽

J Ford ◽

OM Garson

Keyword(s):

T Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Cell Receptor ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

T Cell Receptor Beta ◽

Beta Gene ◽

Receptor Beta

A human leukemia cell line, PER-255, was established from the bone marrow of a 5-year-old boy with features typical of lymphomatous T- acute lymphoblastic leukemia (T-ALL). The leukemic origin of cell line PER-255 is indicated by its cytochemical and immunologic similarity to the patient's fresh leukemic cells, which correspond to immature cortical thymocytes. Southern blot analysis showed that the IgJH genes were in germline configuration, whereas both alleles of the T-cell receptor-beta (TCR-beta) gene were rearranged in PER-255 cells, with identical rearrangements present in the patient's leukemic cells. Cytogenetic analysis of the cell line revealed a single abnormal clone with the karyotype 46,XY,t(7;10)(q32–34;q24),t(9;12) (p22;p12–13). Reciprocal translocations involving chromosome bands 7q32–36, containing the gene for the TCR-beta chain, have been reported for a number of tumors of T-cell origin. Translocations involving the 7q32–36 region appear to be nonrandomly associated with childhood T-ALL, whereas abnormalities of 9p and 12p have been reported to be nonrandomly involved in ALL but not specifically associated with the T- cell phenotype.

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High-Throughput Sequencing of T-Cell Receptor Gene Loci for Minimal Residual Disease Monitoring in T Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2545.2545 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2545-2545

Author(s):

David Wu ◽

Anna Sherwood ◽

Stuart S. Winter ◽

Kimberly Dunsmore ◽

Mignon L Loh ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Post Treatment ◽

Pre Treatment ◽

Higher Sensitivity

Abstract Abstract 2545 There is increasing evidence for the utility of minimal residual disease (MRD) assessment in predicting clinical outcomes of patients with T cell lymphoblastic leukemia (T-ALL). Evaluation of MRD by PCR-based analysis of T-cell receptor (TCR) genes has a sensitivity of 10−5, but requires the use of individualized patient-specific primers, which is laborious, expensive and difficult to implement for real-time, clinical decision-making. Multi-parametric flow cytometry is currently limited to a sensitivity of 10−4, requires viable cells, and is poorly standardized. High-throughput DNA sequencing offers the potential to equal or surpass the higher sensitivity of PCR-based MRD testing with reduced cost, improved turn-around time, and better standardization. Paired samples of pediatric T-ALL from 14 patients enrolled on Children's Oncology Group AALL0434 were obtained at diagnosis and at day 29 post-induction therapy. The complementarity determining regions (CDR3) regions of TCRB and TCRG were sequenced for all 28 specimens using an Illumina GA2 platform as previously described (see Blood, 114(19):4099–4107, 2009 and Sci Transl Med. 3(90):90ra61, 2011). Pre-treatment samples were used to obtain unique TCR sequences for the leukemic clone, and post-treatment samples were assessed for the frequency of each TCR sequence as a percentage of the total. The frequency of each sequence was also enumerated in post-treatment samples from all other patients to evaluate specificity. These results were compared to MRD results obtained by 9-color flow cytometry per trial protocol. Eleven of 14 pre-treatment samples (78.6%) had a detectable clonal population based on TCRG sequence analysis, and 10 of these also had a clonal TCRB sequence. Five samples exhibited an additional unique TCRG sequence, consistent either with rearrangement of both TCRG loci or the presence of two clonal subpopulations. Two of 3 cases without a detectable clonal TCR gene sequence had the immunophenotype of early thymic precursor (ETP) T-ALL and would be expected to have germline TCRB and TCRG genes. No other cases were ETP. Clones were found in all 5 informative post-treatment samples positive for MRD by flow cytometry, as well as at a low level in 3 additional patients without MRD by flow cytometry, suggesting superior sensitivity for sequencing. The background sequence frequencies were very low (0–10−5) in other patient post-treatment samples, being slightly higher for TCRG than for TCRB, consistent with germline sequence diversity. We demonstrate the potential of high-throughput sequencing for analysis of MRD in pediatric T-ALL. The number of cases in which the assay is informative (78.6%) is similar to that seen with standard PCR MRD methods, but evaluation of more cases is needed. MRD by sequencing appears to have a higher sensitivity than current flow cytometric methods, although direct comparison of MRD frequencies from the two techniques is problematic and will require normalization. The strong association of ETP status and lack of clonal TCR sequence identification at diagnosis suggests utility in identifying this poor outcome subset of T-ALL. Disclosures: Sherwood: Adaptive TCR, Seattle, WA: Employment, Equity Ownership. Wood:Becton, Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive TCR, Seattle, WA: Consultancy, Equity Ownership, Patents & Royalties.

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Establishment and characterization of a childhood T-cell acute lymphoblastic leukemia cell line, PER-255, with chromosome abnormalities involving 7q32-34 in association with T-cell receptor- beta gene rearrangement

Blood ◽

10.1182/blood.v74.1.369.369 ◽

1989 ◽

Vol 74 (1) ◽

pp. 369-373 ◽

Cited By ~ 22

Author(s):

UR Kees ◽

R Lukeis ◽

J Ford ◽

OM Garson

Keyword(s):

T Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Cell Receptor ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

T Cell Receptor Beta ◽

Beta Gene ◽

Receptor Beta

Abstract A human leukemia cell line, PER-255, was established from the bone marrow of a 5-year-old boy with features typical of lymphomatous T- acute lymphoblastic leukemia (T-ALL). The leukemic origin of cell line PER-255 is indicated by its cytochemical and immunologic similarity to the patient's fresh leukemic cells, which correspond to immature cortical thymocytes. Southern blot analysis showed that the IgJH genes were in germline configuration, whereas both alleles of the T-cell receptor-beta (TCR-beta) gene were rearranged in PER-255 cells, with identical rearrangements present in the patient's leukemic cells. Cytogenetic analysis of the cell line revealed a single abnormal clone with the karyotype 46,XY,t(7;10)(q32–34;q24),t(9;12) (p22;p12–13). Reciprocal translocations involving chromosome bands 7q32–36, containing the gene for the TCR-beta chain, have been reported for a number of tumors of T-cell origin. Translocations involving the 7q32–36 region appear to be nonrandomly associated with childhood T-ALL, whereas abnormalities of 9p and 12p have been reported to be nonrandomly involved in ALL but not specifically associated with the T- cell phenotype.

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