Juxtaposition of the BCL11B Gene to a Novel Region at 17q by a t(14;17)(q32;Q21) in Childhood T-Cell Lymphoblastic Lymphoma.

Abstract T-cell acute lymphoblastic lymphoma/leukemia is frequently associated with recurrent genetic aberrations that result in the deregulation of transcription factors. In this respect, BCL11B plays a key role in the differentiation and survival during T-cell development. The 3′-located regulatory elements of BCL11B are juxtaposed to TLX3 by a cryptic t(5;14)(q35;q32) in approximately 20% of childhood T-ALL, which leads to inappropriate expression of TLX3. BCL11B can also fuse to TRDC through an inv(14)(q11.2q32.31) resulting in the expression of a BCL11B-TRDC fusion transcript in the absence of wild-type BCL11B. Moreover, a t(6;14) involving BCL11B and the 6q26 region has been described. We have identified a novel BCL11B rearrangement in a case of childhood T-cell lymphoblastic lymphoma. Cytogenetics detected a t(14;17)(q32;q21) and subsequent FISH analysis using BCL11B-spanning and BCL11B 3′-breakpoint-cluster-region flanking BAC clones revealed that BCL11B itself was not disrupted. However, a translocation breakpoint downstream of the BCL11B was observed suggesting the activation of a juxtaposed gene usually residing at 17q by the transcriptional regulatory elements of BCL11B. To narrow down the breakpoint at 17q a FISH-based chromosome-walking strategy using a set of chromosome 17q-specific BACs was employed. A BAC clone encompassing - from centromere to telomere - the genes RAB5C (a member of the RAS oncogene family), KCNH4 (potassium voltage-gated channel, subfamily H (eag-related), member 4), HCRT (hypocretin (orexin) neuropeptide precursor), GHDC (GH3 domain containing; LGP1), STAT5B (signal transducer and activator of transcription 5B), and the 5′-end of STAT5A showed a split signal indicating that one of these genes was juxataposed to the BCL11B enhancer. RAB5C, KCNH4, GHDC, and STAT5B are transcribed in a telomere-centromere orientation, whereas STAT5A shows the opposite transcriptional direction. Together with the FISH pattern observed these data suggested that STAT5A was the most likely candidate gene that might be inappropriately expressed via the regulatory elements of BCL11B. However, semi-quantitative expression analysis showed that neither STAT5A nor STAT5B were significantly upregulated in the affected lymph node as compared to normal bone marrow, peripheral blood, and thymus. In fact, compared to the expression levels in the other tissues STAT5A seemed to be expressed at lower levels. Thus, also the expression levels of RAB5C, KCNH4, and GHDC were analyzed. KCNH4 expression was almost undetectable in bone marrow, peripheral blood, and thymus and for all three genes no elevated expression was observed in the T-cell lymphoma. Owing to the unchanged expression of these genes also the transcription level of STAT3, which is localized further distal to the breakpoint determined by FISH was analyzed, and similar to STAT5A showed lower expression. However, depletion of STATs usually results in reduced cell viability and apoptosis. Together, our data suggest several scenarios: rearrangements of the region containing the remote enhancer of BCL11B are not necessarily accompanied by high expression of a gene juxtaposed into the close vicinity, expression levels of the juxtaposed gene may be just modulated rather than strongly enhanced, the presence of a more complex translocation undetectable by cytogenetics that results in the overexpression of a gene not obviously affected by the translocation or the generation of a fusion gene.

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Limited Reliability of Ig/TCR Based MRD Monitoring in BCR/ABL-Positive Childhood ALL: Comparison to Quantitative Fusion Transcript Detection.

Blood ◽

10.1182/blood.v108.11.2340.2340 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2340-2340

Author(s):

Katerina Krejcikova ◽

Katerina Muzikova ◽

Eva Fronkova ◽

Marketa Kalinova ◽

Leona Reznickova ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Fusion Gene ◽

False Negative ◽

Fusion Transcript ◽

Childhood All ◽

Expression Levels ◽

Diagnostic Samples ◽

Transcript Detection

Abstract Leukemias with the t(9;22) translocation resulting in BCR/ABL fusion protein expression comprise 3–5% of childhood ALL. Despite modern therapeutic regimens, their prognosis is inferior. Minimal residual disease (MRD) based on leukemia-specific immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements has become a tool influencing clinical decisions in many therapeutic trials for childhood ALL. The presence of BCR/ABL fusion gene offers a possibility of the fusion transcript detection - a faster and cheaper alternative to Ig/TCR-based MRD monitoring. Up to now, no direct comparison based on a sufficient number of samples has been done. We analyzed 350 follow-up samples from 16 children (aged 4–17 years) with BCR/ABL-positive ALL by Ig/TCR-based real-time quantitative PCR (RQ-PCR) and by reverse-transcriptase (RT) RQ-PCR for BCR/ABL transcripts. Beta-2 microglobulin housekeeping gene was used for cDNA quality normalization. WBC, age, immunophenotype and blast proportion in the bone marrow (BM) and peripheral blood (PB) showed no relation to the initial BCR/ABL level. All children expressed m-BCR/ABL transcript at the time of diagnosis; 3 of 16 children expressed both m-BCR/ABL and M-BCR/ABL transcripts representing the p190 and p210 variant of BCR/ABL protein, respectively. The expression levels of m-BCR/ABL in diagnostic samples differed up to 3 logs, being the lowest in patients expressing both variants of the fusion gene. In 38 samples from those patients, M-BCR/ABL expression was generally higher than m-BCR/ABL expression, being negative by m-BCR/ABL and positive by M-BCR/ABL in 13 samples. For further analysis we used the higher value of m- and M-BCR/ABL as the BCR/ABL MRD level. For the comparison with Ig/TCR-based method, MRD levels in follow-up samples were related to the expression levels in diagnostic samples, which were set to 1. In total, 133 (38%) and 127 (36%) samples were negative and positive by both methods, respectively. The quantitative levels differed by more than 1 log in 46 (36%) double-positive samples, being underestimated by Ig/TCR method in 25 cases and by m-BCR/ABL quantification in 21 cases. With the same sensitivity of both methods we found significantly more false-negative samples by Ig/TCR approach (70 samples) compared to BCR/ABL quantification (20 samples). Altogether, we tested 219 bone marrow (BM), 130 peripheral blood (PB) and 1 cerebrospinal fluid samples. The PB samples showed significantly worse correlation between the two methods compared to BM (p=0.02). Interestingly, some patients had higher MRD levels in PB compared to BM as shown by corresponding BM and PB samples. Our data suggest that BCR/ABL-positive childhood ALL is a biologically heterogeneous group. We show that all diagnostic samples should be screened for the simultaneous m- and M- BCR/ABL expression to avoid false-negativity when using m-BCR/ABL quantification only. In our hands, the quantification of BCR/ABL transcripts appears to be a more reliable method than the generally accepted Ig/TCR-based MRD monitoring as the number of false-negative samples by BCR/ABL quantification is significantly lower. This contention is further supported by our pilot data on transplanted patients where BCR/ABL positivity preceding transplantation seems to be a better predictor of subsequent relapse than Ig/TCR approach. Support: MSM0021620813, MZ00064203 and 62/2004 GAUK CR. KK and KM contributed equally to this work.

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A Human SIL-SCL Fusion Gene Displays a Development Block at the CD4− CD8− to CD4+CD8+ Transition in Transgenic Mice.

Blood ◽

10.1182/blood.v106.11.2833.2833 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2833-2833

Author(s):

Yue Cheng ◽

Zhenhua Zhang ◽

Chris Slape ◽

Peter D. Aplan

Keyword(s):

T Cell ◽

Fusion Gene ◽

Chromosomal Rearrangements ◽

Regulatory Elements ◽

Interstitial Deletion ◽

Lymphocytic Leukemia ◽

Bac Clone ◽

Cd8 Cells ◽

Human T Cell ◽

Bac Transgene

Abstract Chromosomal rearrangements leading to dysregulation of the SCL gene, located at chromosome 1p32, is a common event in the development of human T-cell acute lymphocytic leukemia (T-ALL). In the most common form of SCL gene rearrangement, an interstitial deletion of approximately 90 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SIL (SCL interrupting locus) gene, which is located directly upstream of SCL. To investigate the consequences of this event, we reproduced this gene alteration by using cre-mediated recombination. A BAC clone containing both human SIL and SCL genes was isolated and loxP sites were cloned into the intron 1 region of both the SIL and SCL genes, corresponding to the sites at which recombination leading to an interstitial deletion occurs in human T-ALL patients. This BAC clone was used to generate transgenic SILloxloxSCL mice. The SILloxloxSCL mice were bred to mice that express the cre recombinase in the thymus (Lck-cre mice). The BAC transgene was recombined between the two loxP sites in over 50% of the thymocytes from double transgenic Lck-cre/SILloxloxSCL mice, faithfully recapitulating the event seen in human T-ALL patients, and bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression was verified by RT-PCR. Using FACS analysis, we found that mice carrying both the SILloxloxSCL transgene and the Lck-cre transgene have decreased CD4+/CD8+, CD4+/CD8−, CD4−/CD8+ and increased CD4−/CD8− thymocytes compared to transgene-negative mice or mice that carried the SILloxloxSCL transgene but not the Lck-cre transgene. These findings were detected in mice from 6 to 15 months of age. Interestingly, increased numbers of CD44+ thymocytes can also been identified in SILloxloxSCL/Lck-cre mice. In the spleen, the SILloxloxSCL/Lck-cre mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results indicate an accumulation of immature T-cells in mice transgenic for both the SILloxloxSCL transgene and the Lck-cre transgene, and demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

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A Novel BACH2-BCL2L1 Fusion Gene in the Burkitt’s Lymphoma Derived Cell Line BLUE-1

Blood ◽

10.1182/blood.v112.11.4146.4146 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4146-4146

Author(s):

Seval Türkmen ◽

Mathias Riehn ◽

Stefan Mundlos ◽

Eckhard Thiel ◽

Thomas Burmeister

Keyword(s):

Transcription Factor ◽

T Cell ◽

Cell Line ◽

Leucine Zipper ◽

Fusion Gene ◽

Fusion Transcript ◽

Established Cell Line ◽

Basic Leucine Zipper ◽

Bac Clone ◽

Bcl2 Family

Abstract Abnormalities of the long arm of chromosome 6 are a common feature in various B-cell malignancies. However, in most cases the involved genes have not yet clearly been identified. We have molecularly characterized the recently established cell line BLUE-1 that has been derived from a relapsed sporadic Burkitt lymphoma. This cell lines carries a t(6;20)(q15;q11.2) rearrangement in addition to the typical t(8;14) with MYC-IgH fusion. The involved gene loci on chromosomes 6 and 20 were up to now unknown. To identify the involved gene loci on both chromosomes we applied a sequential BAC clone mapping strategy. BAC RP11-243J16 clone (20q11.21, bp 29,756,679–29,925,538) was found to hybridize with der(6) as well as der(20). BAC RP1-104D1 (6q15, bp 91,001,943–91,064,406) also covered the breakpoint region. Two of the involved genes in this region are the transcription factor BACH2 (basic leucine zipper transcription factor 2) on 6q15 and BCL2L1 (BCL-X) on 20q11, a member of the BCL2 anti-apoptosis gene family. We hypothesized that these two genes could be involved and by testing different primer combinations were able to amplify a BACH2-BCL2 fusion mRNA transcript using RT-PCR. In this fusion transcript the first (non-coding) exon of BACH2 was fused to the second (partially coding) exon of BCL2L1 thus effectively placing the BCL2L1 gene under the control of the BACH2 promotor. Western blot analysis showed a strong expression of BCL2L1. This is the first report of a fusion gene involving the genes BACH2 and BCL2L1. The prototype of the BCL2 family, BCL-2 on 18q21.3 is known to play a crucial role in various lymphomas but a clear role for the closely related BCL2L1 gene in lymphomas has not yet been established. BACH2 is known to be expressed in B-cells at various maturation stages and is believed to be involved in the machinery of class switch recombination (CSR). Bach2 −/− mice show increased IgM but decreased IgG and IgA levels and a deficient T cell-independent and T cell-dependent IgG response associated with defective CSR. In summary, this molecularly characterized translocation provides a new tool for studying recurrent 6q aberrations in lymphomas and for the action of the BCL2L1 antiapoptosis gene.

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A Novel FOXP1-PDGFRA Fusion Gene in A Case of Chronic Eosinophilic Leukemia.

Blood ◽

10.1182/blood.v120.21.2830.2830 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2830-2830

Author(s):

Yuka Sugimoto ◽

Akiko Sada ◽

Fumihiko Monma ◽

Kohshi Ohishi ◽

Masahiro Masuya ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Genomic Dna ◽

Low Dose ◽

Fusion Gene ◽

Fish Analysis ◽

Fusion Partner ◽

Rt Pcr ◽

Total Rna

Abstract Abstract 2830 Introduction Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 is a new major category in the 2008 WHO classification of myeloid malignancies. FIP1L1-PDGFRA fusion gene is currently the most common abnormality in this category, but there are some other fusion genes incorporating part of PDGFRA. In a case of myeloproliferative neoplasms (MPN) with eosinophilia and hepatosplenomegaly, karyotype by G-banding and fluorescence in situ hybridization (FISH) for 4q12 rearrangements indicated a PDGFRA rearrangement other than FIP1L1-PDGFRA, and a novel FOXP1-PDGFRA fusion gene was identified. Case presentation A 44-year-old male visited a clinic because of wet cough for one year. His peripheral blood showed leukocytosis of 43.15 × 109 /L with eosinophilia up to 57.5%, mild erythrocytosis (Hb 17.3 g/dL), and thrombocytopenia of 86 × 109 /L. CT scan of the abdomen revealed hepatosplenomegaly. He was referred to our hospital and received oral PSL (1 mg/kg) first, because pulmonary eosinophilic infiltration was suspected by follow-up CT findings. Pulmonary infiltration and his cough disappeared rapidly in a week, but his leukocytosis with eosinophilia was exacerbated again with PSL tapering. His bone marrow at the time of admission disclosed hypercellular marrow with myeloid hyperplasia and eosinophilia, of which karyotype was 46, XY, t(3:4)(p13;q12), inv(9)(p12q13) in all of 20 metaphases. FISH analysis with tricolor 4q12 rearrangement probe set indicated that PDGFRA was disrupted in 97.3% of his peripheral blood cells. These cytogenetic abnormalities of his bone marrow cells suggested involvement of PDGFRA fusion gene except for FIP1L1-PDGFRA and did not disappear after steroid administration for 2 weeks. After low-dose of imatinib (100 mg/day) was started, he achieved a hematological response within 5 days, and PSL could be gradually tapered off. 3 months after therapy, he obtained complete cytogenetic response (CCyR). He has been in CCyR and free of symptoms for more than 6 months with only low-dose imatinib. Methods and Results Genomic DNA and total RNA were isolated from white blood cells in his peripheral blood at diagnosis. Complementary DNA was synthesized from total RNA. FIP1L1-PDGFRA fusion transcript was proved to be negative by RT-PCR. Molecular cloning with 5′-RACE-PCR revealed a novel mRNA in-frame fusion between exon 23 of FOXP1 and a truncated PDGFRA exon12. Reciprocal PDGFRA-FOXP1 transcripts were confirmed by RT-PCR analysis and FOXP1-PDGFRA genomic DNA sequence was determined with genomic PCR. As in the case with FIP1L1-PDGFRA, the breakpoint of PDGFRA in FOXP1-PDGFRA was located between the two tryptophan (W) residues of the putative WW-domain. Meanwhile, the other breakpoint was near inverted repeat in intron 23 of FOXP1, which is presumed to be very fragile site. By FISH analysis after magnetic cell sorting with MicroBeads, the 4q12 abnormality attributed to FOXP1-PDGFRA fusion gene was detected in granulocytes, but not in CD19-positive B or CD3-positive T cells. Discussion In a case with chronic eosinophilia harboring 46, XY, t(3:4)(p13;q12), inv(9)(p12q13), novel FOXP1-PDGFRA fusion gene was identified. Similar karyotypic abnormality harboring t(3:4)(p13;q12) was reported in a case of MPN with chronic eosinophilia, but responsible fusion gene was not identified (Myint H, et al. Br J Haematol. 1995). FOXP1 is a transcription factor which is implicated in a variety of cellular processes and has a role in immune regulation and carcinogenesis (Wlodarska I, et al. Leukemia. 2005). As a fusion partner of FOXP1, PAX5 and ABL1 are reported in cases with acute lymphoblastic leukemia. Thus, this is a first report showing that FOXP1-PDGFRA fusion gene is involved in hematologic malignancy. It is likely that FOXP1-PDGFRA is constitutively activated tyrosine kinase, which does not depend on dimerization but on the disruption of an autoinhibitory juxtamembrane domain encoded by exon 12 of PDGFRA from its structure. Eosinophilia responded well to low dose of imatinib as observed in CEL with FIP1L1-PDGFRA. Conclusion FOXP1-PDGFRA was identified in CEL for the first time. This is the eighth reported fusion gene associated with PDGFRA in CEL so far. Our patient with FOXP1-PDGFRA promptly responded to low-dose of imatinib as same as other cases with PDGFRA abnormalities. Further investigation is still in progress. Disclosures: No relevant conflicts of interest to declare.

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A Novel Approach for Minimal Residual Disease Detection in Childhood T-Cell Lymphoblastic Lymphoma (T-LL): A Children’s Oncology Group Report.

Blood ◽

10.1182/blood.v110.11.3564.3564 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3564-3564 ◽

Cited By ~ 1

Author(s):

Elaine Coustan-Smith ◽

Minnie Abromowitz ◽

John T. Sandlund ◽

Dario Campana

Keyword(s):

Bone Marrow ◽

T Cell ◽

Minimal Residual Disease ◽

Induction Therapy ◽

Peripheral Blood ◽

Residual Disease ◽

Lymphoblastic Lymphoma ◽

Oncology Group ◽

Blood Samples ◽

Minimal Residual

Abstract Contemporary treatment of childhood T-LL produces 2 yr EFS of ≥ 85% (Reiter et al., Blood, 2000). To further improve cure rates without increasing treatment-related toxicities it is critically important to better understand the biology of the disease and identify prognostically important subgroups (Cairo et al., PBC, 2005). Approximately 15% of children with T-LL have disseminated disease at diagnosis as demonstrated by morphologic examination of bone marrow aspirates and biopsies. We developed a flow cytometric method that can detect 1 malignant T-cell among 10000 or more normal bone marrow and peripheral blood cells. The method relies on expression of CD3 and TdT, combined with the absence of B-cell and myeloid markers, to identify malignant T cells. We applied this method to study bone marrow samples collected at diagnosis from 70 patients enrolled in the Children’s Oncology Group A5971 study “Randomized Phase III Study for the Treatment of Newly Diagnosed Disseminated Lymphoblastic Lymphoma or Localized Lymphoblastic Lymphoma.” Of the 70 patients, 48 (68.6%) had CD3+/TdT+ lymphoma cells detectable in the bone marrow (median, 0.25%; range 0.01%–28%), a proportion considerably higher than estimates based on morphology. In 27 patients, we studied paired left and right iliac crest aspirates; in all cases, results were concordant (20 positive and 7 negative). The difference in percentages of CD3+/TdT+ cells measured among positive sample pairs was <1 log, suggesting homogeneous disease dissemination. Among the 70 patients, relapse has occurred to date in 11 patients, 3 in the primary site only, and 8 in other sites (with or without bone marrow involvement). All the 8 latter relapses occurred among the 32 patients with ≥ 0.1% CD3+/TdT+ cells at diagnosis in bone marrow; none was observed in the 38 patients with lower levels or no detectable disease (P = 0.001 by Fisher’s exact test). Because of findings in patients with T-cell acute lymphoblastic leukemia indicating that levels of minimal residual disease (MRD) in peripheral blood resemble those in bone marrow (Coustan-Smith et al., Blood, 2002), we hypothesized that blood could also be used for detecting disseminated T-LL. Paired marrow / blood samples from 61 patients were available for study. In 41 patients, similar levels of CD3+/TdT+ cells were detected in marrow and blood; in the remaining 20, CD3+/TdT+ cells were undetectable (<0.01%) in marrow, but detectable in 7 of the corresponding blood samples (range, 0.01%–0.09%). These results suggested that blood could be used to monitor early treatment response in patients with T-LL. Hence, Study A5971 was amended to determine the prevalence of MRD in blood during remission induction therapy. To date, 146 samples from 42 patients with T-LL have been studied; tests on days 7 and 28 are emerging as the most informative. Our findings provide new insights about disease dissemination in T-LL, and ways to measure early response to therapy and monitoring MRD in these patients. This may have potential implications in the future for alternative therapeutic strategies in the subset of children with T-LL with persistent MRD during induction therapy.

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Prognostic value of T-cell receptor γ rearrangement in peripheral blood or bone marrow of patients with peripheral T-cell lymphomas

Leukemia & Lymphoma ◽

10.1080/10428190701784409 ◽

2008 ◽

Vol 49 (2) ◽

pp. 237-246 ◽

Cited By ~ 4

Author(s):

Christian SchüTzinger ◽

Harald Esterbauer ◽

Gregor Hron ◽

Cathrin Skrabs ◽

Martin Uffmann ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

T Cell Receptor ◽

Peripheral Blood ◽

Prognostic Value ◽

Cell Receptor ◽

T Cell Lymphomas ◽

Peripheral T Cell Lymphomas

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Anti-CD19 Chimeric Antigen Receptor T Cell Treatment of Relapsed /Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Resistant to Bruton's Tyrosine Kinase (BTK) Inhibitor

Blood ◽

10.1182/blood-2020-143099 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Weihong Chen ◽

Xin Du ◽

Wenyujing Zhou ◽

Changru Luo ◽

Xiaoqing LI

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Therapy ◽

Lymph Nodes ◽

Peripheral Blood ◽

Serum Levels ◽

Car T Cell ◽

Car T ◽

Btk Inhibitor

CASE PRESENTATION: A 68-year-old male was diagnosed with CLL/SLL in November 2007. Bone marrow asp/bx: 36.5% lymphocytes, 78% CD19, 65% ATM (11q22 deleted) positive cells, 13.5% D13S25 (13q14.3 deleted). On December 10, 2009, the patient took FCR scheme for five cycles, followed by FR scheme for one cycle, and then a month of Chlorambucil. On September 5, 2013, the patient took BR scheme for four cycles with no effect. From March 2015 to Feb 2016, 420 mg of Ibrutinib was administered daily. On January 15, 2016, the patient developed swollen lymph nodes in his right neck with intermittent lumps, fever and nausea. He was admitted into the hospital at Feb 2, 2016. Test results: multiple swollen superficial lymph nodes over the body, with the biggest measuring 60×30mm on the right neck, with no tenderness. Supplementary tests: peripheral white blood cells (WBC) 11.94×10E9/L, lymphocyte 7.5×10E9/L, CD19 cells 6.73×10E9/L, bone marrow lymphocyte 62%, peripheral blood lymphocyte 52%. Immunophenotype: CD5, CD19, CD20dim, CD23, CD11b dim, HLA-DR expression, visible CD5+CD19+ cell clusters, and visible immunoglobulin cKappa with restricted expression. On March 10, 2016, peripheral blood platelet 60 × 10E9/L, CD19 cells 1.94×10E9/L, lactate dehydrogenase 460U/L, FER 115.6ng/ml, hepatitis B virus carrier. Diagnosis: CLL/SLL IV stage, ATM (11q22) deletion, D13S25 (13q14. 3) positive, CD19 positive. Relapse of CLL/SLL occurred again after four months and at this stage the patient was considered for therapy in a clinical trial of CD19-specific chimeric antigen receptor (CAR-) T cell therapy. Ethical approval and informed consent were obtained for anti-CD19 CAR T Cell treatment of ibrutinib resistance in relapsed/refractory CLL/SLL. We infused autologous T cells transduced with a CAR T 19 retroviral vector with CLL/SLL at doses of 3.3 × 10E8 CART19 cells on Mar. 16 2016. Patients were monitored for responses, toxic effects, and the expansion and persistence of circulating CART19 cells. After CART19 cells were infused, the patient experienced chills, fever, headache, weak, anorexia, nausea, shortness of breath, chest tightness, heart palpitation, hypotension and shock for 9 days. The serum levels of IFN-Υ were at their highest at day 7 after CAR T cells infusion. Serum interleukin 6 (IL-6) was at 680pg/ml and CD3+ cells were 97.5%, CD8+ cells 72.8% (18.7-32.8%), FER was 1529.5ng/ml (Normal No. 22-322ng/ml) 14 days after CAR-T cell infusion. The serum levels of IL-6 were at their highest at day14. The patient was diagnosed as having cytokine release syndrome. After the patient took the anti-IL-6R antibody and anti-TNF antibody, he began to recover gradually. Enlarge lymph nodes shrunk after being infused with CART19 cells for 7 days. The peripheral blood CD19 B lymphocytes were 0 on day 14 after infused with CAR T19 cells. Q-PCR was used to detect the amount of the peripheral blood CART19 cells, which stood at 5485 copies/μl, 924 copies/μl, 191 copies/μl respectively 2 weeks, 6 weeks and 3 months after infusing with CART19 cells. The peripheral blood CART 19 cells were not detectable 4 months after infusing with CART19 cells until present. The lymphadenopathy was decreased gradually after 14 days of infusion. The MRI test showed that lymphadenopathy reduced markedly or disappeared after 6 months of infusion. ATM (11q22 deleted) negative, D13S25 (13q14.3 deleted) negative. After treatment with CAR T 19 cell therapy for 53 months, the patient remained disease-free, the patient's lymph nodes, lymphocytes and I mmunoglobulins were normal. CONCLUSIONS : Cancer immunotherapy as a method of cancer treatment is the most effective after conventional treatments such as radiotherapy, chemotherapy, and surgery. For BTK Inhibitor resistance in relapsed and refractory CD19+ CLL/SLL, CD19 is a favorable target, because the expression of CD19 is limited to B cells and not present in other tissues or cells. Currently, the efficacy of this treatment in treating CLL/SLL remains to be seen. The effects of chemotherapy on the patient's B cell lymphoma are negligible, due to the fact that his CLL/SLL have become relapsed and refractory. As a result we chose the CAR T19 cell therapy genetic engineering technique as a method of treatment, to which the patient has responded well. Therefor, CAR T cell technology overcome the limitations of existing cancer therapies and has great potential for development and application. Disclosures No relevant conflicts of interest to declare.

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BCOR Mutation and TLS-ERG Expression in Acute Myeloid Leukemia with Monoclonal Immunoglobulinemia

Blood ◽

10.1182/blood-2019-122366 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5180-5180

Author(s):

Jian Huang ◽

Jingxia Jin ◽

Shuna Luo ◽

Xingnong Ye

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Plasma Cells ◽

Fusion Gene ◽

Next Generation Dna Sequencing ◽

School Of Medicine ◽

Immunofixation Electrophoresis

Acute myeloid leukemia(AML) originates from the abnormal clonal proliferation of myeloblast which often combined with clinical symptoms. Cytogenetic and molecular abnormalities are frequent in AML patience. To date, the driver genes for leukemia remain largely undiscovered. Monoclonal immunoglobulinemia is a group of diseases caused by excessive proliferation of plasma cells or immunoglobulin-producing lymphoid plasma cells and B lymphocytes. It can develop into malignant plasma cell disease. Herein, we report a AML patient was concomitant with monoclonal immunoglobulinemia, the patient was also accompanied by BCOR mutation and TLS-ERG fusion gene. A 55-year-old married female was admitted into our hospital due to repeated edema for 3 weeks. On admission, peripheral blood counts: PLT142×10^9/L, HB77g/L↓, WBC35.2×10^9/L.Bone marrow examination showed the mononuclear cell system proliferated actively, and the primitive infantile monocytes accounted for 86%. Cell morphology suggested M5b(Figure1A ). Fusion gene screening in bone marrow revealed that TLS-ERG expression. Immunophenotype of bone marrow cell:Abnormal myeloid primitive cells accounted for 96.39% of the nuclear cells,expressCD33, CD13, CD123, CD34, CD9, MPO(Figure 1D). Karyotype analysis of bone marrow cells showed in Figure 1B. Thus, AML was diagnosed. Next-generation DNA sequencing technology showed that BCOR (51.7%),PLCG1(49.9%),DIS3(48.4%),BRAF(51.6%), JAK2(45.1%) ,JAK3(49.0%) were mutated. Meanwhile, we found that Peripheral blood immunofixation electrophoresis showed that Gamma region is seen with a monoclonal light chain lambda component((Figure 1C.).Then, the patient underwent one cycle of IA(Idabisine hydrochloride 10mg d1-4, cytarabine 0.075g q12h d1-7). Twenty-five after chemotherapy onset, bone marrow examination showed that primitive and immature monocytes accounted for 3%. Chromosome become normal. Minimal residual disease(MRD):0.01%. The disease reached complete remission(CR). Peripheral blood immunofixation electrophoresis turned negative. Fusion gene detection showed that TLS-ERG turned negative. BCOR mutation was not detected by Next-generation DNA sequencing. Mutations of PLCG1,DIS3,BRAF,JAK2,JAK3 still exist. Monoclonal immunoglobulinemia and AML are both clonal diseases, but originated from different clones. This case has both malignant clones of granulocyte stem cell and malignant clones of B line, so it is worthy of discussion. By comparing CR before and after we found that while the patient's M protein turned negative, the TLS-ERG fusion gene and BCOR gene mutation also disappeared. The TLS-ERG fusion gene is formed by the rearrangement of TLS and ERG genes on chromosomes 16 and 21. The current study holds that the expression of this fusion gene indicates rapid disease progression and poor prognosis. BCOR mutations can be found in AML and often coincide with DNMT3 gene mutations, suggesting it may affect the occurrence of leukemia through epigenetics. BCOR is a newly discovered corepressor of BCL-6, which can play a supporting role when BCOR combines with DNA; when BCOR is overexpressed, it can enhance the inhibition of BCL-6. BCL-6 is highly expressed in tumor cells,it encodes transcriptional repressors which are required for the formation of germinal center and may affect apoptosis. We thinked that the monoclonal immunoglobulinemia of this patient may caused by the BCOR abnormal expression which increased the inhibitory effect of BCL-6 and affect the apoptosis of B cells, and B cells continue to secrete immunoglobulin. BCOR mutations are associated with poor prognosis. The patient with TLS-ERG fusion gene which is a poor prognosis gene.However, the BCOR gene mutation site is a non-hot spot mutation which has few clinical studies. Whether the BCOR gene mutation results in the combination of the two diseases requires further study. Acknowledgment:The research was supported by fundings of the public technology research projects of Yiwu,China (2016-S-05), the key medical discipline of Yiwu,China(Hematology,2018-2020),and the academician workstation of the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Email: [email protected] Figure 1 Disclosures No relevant conflicts of interest to declare.

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CD8 Expression on B Cells in Chronic Lymphocytic Leukemia

Archives of Pathology & Laboratory Medicine ◽

10.5858/2000-124-1361-ceobci ◽

2000 ◽

Vol 124 (9) ◽

pp. 1361-1363

Author(s):

Anwarul Islam ◽

Adrian O. Vladutiu ◽

Theresa Donahue ◽

Selina Akhter ◽

Amy M. Sands ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Tract Infection ◽

Respiratory Tract ◽

Respiratory Tract Infection ◽

Peripheral Blood ◽

Lymphocytic Leukemia ◽

Cell Antigen

Abstract The expression of CD8, a restricted T-cell antigen, on B cells in B chronic lymphocytic leukemia is rare, and its significance, if any, remains unknown. We report herein a patient with B chronic lymphocytic leukemia in whom CD8 was strongly expressed on all B cells, both in the bone marrow and peripheral blood. The patient required no therapy for 6 years after being diagnosed as having B chronic lymphocytic leukemia. Then, when the disease progressed, he was treated with conventional doses of fludarabine phosphate (25 mg/m2 daily for 5 days), but unlike other patients with B chronic lymphocytic leukemia he tolerated this therapy poorly. He received a total of only 4 series of fludarabine therapy, and following each course of treatment, he developed considerable myelosuppression. After the fourth course of therapy, his bone marrow failed to show any evidence of regeneration, and he died as a result of intercurrent respiratory tract infection 1 month after his last dose of fludarabine was given.

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Human T-cell antigens defined by monoclonal antibodies. Absence of T65 on committed myeloid and erythroid progenitors

Blood ◽

10.1182/blood.v56.5.943.943 ◽

1980 ◽

Vol 56 (5) ◽

pp. 943-946 ◽

Cited By ~ 13

Author(s):

R Taetle ◽

I Royston

Keyword(s):

Bone Marrow ◽

T Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Myelogenous Leukemia ◽

Erythroid Progenitors ◽

Normal Peripheral Blood ◽

Human T Cell ◽

Complement Dependent Cytotoxicity ◽

T Cell Antigens

Abstract The complement-dependent cytotoxicity of monoclonal T-cell antibody (T101) for normal and abnormal hemopoietic progenitors was assessed. T101 demonstrated toxicity for normal T-colony-forming cells from peripheral blood and bone marrow. Cytotoxicity was absent for normal peripheral blood and bone marrow granulocytes/macrophage (CFU-C) and erythroid (BFU-E) progenitors. The antibody was also not toxic for peripheral blood blast progenitors from patients with acute myelogenous leukemia (AML). These studies indicate the absence of the antigen defined by T101 (T65) from normal progenitor cells and from blast progenitors in patients with AML. T101 may be used in the treatment of T-cell malignancies and in the prevention of graft-versus-host disease (GVHD) without damage to normal progenitor cells.

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