scholarly journals CD79a Is Associated with Central Nervous System Infiltration of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 386-386 ◽  
Author(s):  
Lennart Lenk ◽  
Fotini Vogiatzi ◽  
Michela Carlet ◽  
Christian Vokuhl ◽  
Gunnar Cario ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Bone Marrow ◽  
Nervous System ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Mrna Levels ◽  
Cell Precursor ◽  
Cns Involvement ◽  
Nsg Mice

Abstract Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research. In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo. To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches. To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL. Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD79a promotes CNS-infiltration and leukemia engraftment in pediatric B-cell precursor acute lymphoblastic leukemia

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lennart Lenk ◽  
Michela Carlet ◽  
Fotini Vogiatzi ◽  
Lea Spory ◽  
Dorothee Winterberg ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Leukemia Cells ◽  
Cell Precursor ◽  
Cns Involvement ◽  
Xenograft Models

AbstractCentral nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and –relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.

scholarly journals Central Nervous System Involvement Detected By Flow Cytometry Is a Risk Factor for Relapse in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 657-657 ◽  
Author(s):  
Maria Thastrup ◽  
Hanne Vibeke Marquart ◽  
Mette Levinsen ◽  
Kathrine Grell ◽  
Jonas Abrahamsson ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Bone Marrow ◽  
Nervous System ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cox Model ◽  
Lymphoblastic Leukemia ◽  
Cns Involvement ◽  
Fcm Analysis ◽  
Risk Of Relapse

Abstract Background: Central nervous system (CNS) involvement is common in childhood acute lymphoblastic leukemia (ALL). Today all patients receive intensive prophylactic CNS-directed therapy, which is associated with short- and long-term neurotoxicity. The conventional method for diagnosing CNS leukemia is by microscopic examination of cytospin preparations of cerebrospinal fluid (CSF). Both high (CNS3: ≥5 x 109 cells/L) and low level CNS disease (CNS2: <5 x 109 cells/L) indicate intensified CNS-directed therapy. The CNS is involved in 30-40% of relapses, and most occur in patients classified as CNS negative by cytospin (CNS1). We investigated if sensitive flow cytometric (FCM) analysis of CSF at diagnosis improves detection of CNS involvement and prediction of relapse risk. Methods: Cytospin results were collected from local oncology clinics for patients treated according to the Nordic Society of Pediatric Hematology (NOPHO) ALL2008 protocol from July 2008 to December 2017 (N=1841). CSF samples for FCM were obtained from patients enrolled from September 2012 to December 2017 at 17 participating centers (N=669). CSF samples were collected into Transfix® tubes (Intermedico Ltd., Hellerup, Denmark) and shipped to Rigshospitalet, Copenhagen, Denmark for centralized analysis as previously described (Levinsen at al., Pediatr Blood Cancer 2016). FCM positivity required detection of ≥10 cells with a leukemia-associated phenotype. CSF FCM results were blinded to the treating physician. Time to relapse was analyzed using Cox proportional hazards models with delayed entry at remission and with death and second malignant neoplasm as competing events. Results: At diagnosis, 241 of 1841 (13.1%; CNS2: 8.9% and CNS3: 4.2%) patients were positive by cytospin, whereas 167 of 669 (25.0%) patients were positive by FCM. FCM positivity was not associated with shipment duration (median: 2 days). In total, 191 of the 669 patients (28.6%) were CNS positive by cytospin and/or FCM. CNS positivity was associated with higher white blood cell (WBC) count at diagnosis, T-cell ALL, lack of t(12;21), and traumatic lumbar puncture (TLP) (all comparisons: p < 0.001). However, patients with CNS positivity did not differ in their end of induction bone-marrow minimal residual disease (MRD) levels compared to the remaining patients (cytospin: median MRD 1.2 x 10-4 vs. 0.9 x 10-4, p = 0.058; FCM: median MRD 2.0 x 10-4 vs. 1.7 x 10-4, p = 0.62). During follow-up, 30 patients relapsed with nine of the relapses involving the CNS and 16 relapses isolated to the bone-marrow. The 4-year cumulative incidence of any relapse was higher for patients who were CNS positive by cytospin (16.1% vs. 8.9%), FCM (18.6% vs. 6.0%), and combined cytospin/FCM (19.0% vs. 5.5%) (Figure 1a). Simple Cox regressions yielded relapse hazard ratio (HR) estimates of CNS positivity at diagnosis of 2.4 for cytospin (95% CI 1.0-5.7, p = 0.04), 3.5 for FCM (95% CI 1.7-7.2, p < 0.001), and 4.1 for combined cytospin/FCM (95% CI 2.0-8.7, p < 0.001). Cytospin and/or FCM was associated with increased risk of both isolated bone-marrow relapse (16 events, HR 3.1, 95% CI 1.2-8.4, p = 0.024) and of any CNS relapse (9 events, HR 4.9, 95% CI 1.2-19.5, p = 0.025). In a multiple Cox model stratified by immunophenotype and risk group, predictors of relapse were age (per year: HR 1.1, 95% CI 1.0-1.2, p = 0.003), WBC at diagnosis (per doubling: HR 1.3, 95% CI 1.1-1.6, p = 0.005), and cytospin and/or FCM (HR 3.2, 95% CI 1.4-7.2, p = 0.006) (Figure 1b). Cytospin and/or FCM was associated with a significantly higher risk of relapse for BCP-ALL (N=581; 23 relapses; HR 4.5, 95% CI 1.9-10.4, p = < 0.001). For T-cell ALL no significant association was observed (N=85; 7 relapses; HR 1.6, 95% CI 0.3-8.2, p = 0.58), however the number of T-cell ALL was too small to allow reliable conclusions. In a simple Cox model, TLP was associated with a significantly higher risk of relapse than no TLP (HR 2.7, 95% CI 1.3-5.9, p = 0.011), but this was only the case for patients who were positive by FCM (FCM pos: HR 2.7, 95% CI 1.0-7.0, p = 0.043; FCM neg: 0.7, 95% CI 0.1-5.0, p = 0.68). Conclusion: FCM markedly increases the detection rate for CNS involvement at diagnosis, and distinguish between patients at high and low risk of relapse. Diagnosis of CNS leukemia by combined cytospin and FCM analysis should be the standard for accurate classification of CNS status, which will enable better stratification of CNS-directed and systemic therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Assessment of Central Nervous System Involvement in Pediatric Acute Lymphoblastic Leukemia Patients Using Next-Generation Sequencing Method

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4981-4981
Author(s):  
John K Choi ◽  
Sima Jeha ◽  
Jianbiao Zheng ◽  
Victoria Carlton ◽  
Malek Faham ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Bone Marrow ◽  
Nervous System ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Cns Involvement ◽  
Employment Equity ◽  
Pediatric All ◽  
Equity Ownership ◽  
Generation Sequencing

Abstract Background: Successful outcome in childhood acute lymphoblastic leukemia (ALL) relies upon appropriate central nervous system (CNS)-directed therapy for treatment of subclinical or overt CNS leukemia. Patients with leukemia blasts detected in the cerebrospinal fluid (CSF) at diagnosis have poorer survival compared with patients without blasts in the CSF and require intensified intrathecal therapy to avert a higher rate of relapse. Traditionally, CSF assessment is performed by morphological analysis of CSF smears and the blasts confirmed by immunohistochemistry or flow cytometry. The confirmation tests are done at few institutions and rely on subjective operator judgment for identification of leukemic blasts. A precise definition of CNS involvement is necessary to avoid over- or under- treatment. Next-generation sequencing (NGS) is currently being used for minimal residual disease (MRD) assessment in lymphoid malignancies (Faham et al., Blood 2012). In this study, we assessed whether the NGS method could be used to detect leukemic clonotypes in CSF samples from 89 newly diagnosed pediatric ALL patients. Methods: Diagnostic bone marrow samples and paired CSF samples were obtained from 89 patients. CSF samples were collected in one tube and fractionated for Wright stained cytospins (1ml), TdT immunohistochemistry (1ml), and the remainder (3-5ml) for NGS-based MRD studies (Adaptive Biotechnologies, South San Francisco, CA). Briefly, using universal primer sets, we amplified variable, diversity, and joining gene segments from immunoglobulin (Ig) heavy chain (IGH), Ig kappa chain (IGK), and T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) loci from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then quantitated in CSF (cell pellet and supernatant) samples obtained at diagnosis. A quantitative and standardized measure of leukemic clonotype level per million leukocytes in each follow-up sample was determined. Results: Diagnostic bone marrow samples from 89 pediatric ALL patients were used to identify the tumor-specific clonotypes. At least one tumor-specific clonotype was identified in 86/89 (97%) of the patients. The IGH-VDJ assay was the most frequent gene rearrangement: at least one IGH-VDJ clonal rearrangement was detected in 66 of the diagnostic ALL samples. TRD was the second most informative receptor with a clonal rearrangement found in 43 patients, followed by TRG in 38 patients, IGK in 31 patients, TCB in 16 patients and IGH-DJ in 13 patients. The level of tumor burden associated with each leukemic clonotype was then assessed in CSF samples drawn at diagnosis from each of the 86 patients. All 40 CNS1 patients had no detectable or minimal (<10) leukemic cell equivalents. Of the 24 patients with CNS2 but TDT negative status, only 2 had >10 leukemic cell equivalents in the CSF. By contrast, 8 of 20 (40%) patients with CNS2 and TDT positive status and both patients with CNS3 had CSF with >10 leukemic cell equivalents (Figure 1). Conclusions: This study demonstrates that the NGS-based method can be used to assess CNS involvement in pediatric patients with ALL. Based on these results, NGS-based CNS assessment will be used for patient stratification in future clinical trials for pediatric ALL. Figure 1. Figure 1. Disclosures Zheng: Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Carlton:Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

Mitoxantrone Improves the Outcome of Children with Central Nervous System (CNS) Involvement at First Relapse of Acute Lymphoblastic Leukemia (ALL)-Results of the International ALLR3 Study

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3303-3303
Author(s):  
Ashish Narayan Masurekar ◽  
Catriona Anne Parker ◽  
Satarupa Choudhuri ◽  
Carly Leighton ◽  
Jeremy Hancock ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Bone Marrow ◽  
Nervous System ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Cns Involvement ◽  
Cns Disease ◽  
Off Label ◽  
Cns Relapse ◽  
Time To Relapse

Abstract Abstract 3303 Introduction: Despite improvement in frontline therapy in childhood acute lymphoblastic leukemia (ALL), central nervous system (CNS) relapse remains a significant clinical problem. The ALLR3 trial (ISCRTN 45724312) was designed specifically to address this issue with the use of drugs known to penetrate the CNS. The trial incorporated a randomization between Mitoxantrone and Idarubicin during induction. Mitoxantrone showed an early benefit in all patients resulting in closure of the randomization in December 2007 (ASH Annual Meeting Abstracts, Nov 2009; 114:3390). Subsequently all patients now receive Mitoxantrone. Here we report on the outcome of patients with isolated CNS relapse (iCNSr) or combined CNS relapse (involvement of CNS and bone marrow, cCNSr). Methods: CNS involvement was defined as ≥5 WBC/μl with morphological evidence of blasts in the cerebrospinal fluid (CSF). Combined relapse (cCNSr) was defined as CNS disease with ≥ 5% blasts in the bone marrow. Time to relapse was classified as, Very Early: within 18 months of first diagnosis; Early: after 18 months of first diagnosis but within 6 months of stopping therapy and Late: more than 6 months after stopping therapy. All patients received 3 blocks of chemotherapy. Subsequently, allogenic stem cell transplant (allo-SCT) was offered to all very early relapses (iCNSr & cCNSr), early iCNSr (irrespective of immunophenotype), all T-cell cCNSr (irrespective of time to relapse) and early or late pre-B cCNSr that had a minimal residual disease level of ≥ 104 at the end of induction. All other patients were offered chemotherapy and cranial radiotherapy. Results: Of a total of 330 relapsed patients, 102 (31%) had CNS involvement. Of these 63 (62%) had iCNSr and 39 (38%) had cCNSr. The incidence of CNS disease was higher in males (M:F, CNS relapses 2.5:1 vs all relapses 1.5:1). CNS relapses had a higher proportion of T-cell disease (pre B:T CNS relapses 3.6:1 vs all relapses 7.8:1]. The number of patients presenting in very early, early and late phases were 19 (19%), 55 (54%) and 28 (27%) respectively. All late iCNSr patients were males. Almost all late relapses (iCNSr and cCNSr) (27/28) were of a pre B phenotype. At the end of induction phase, 91/102 (89%) achieved complete remission (CR) and 82/102 (80%) remained in CR after 3 blocks of chemotherapy. The estimated 3-year overall survival (OS) and progression free survival (PFS) for all patients with CNS disease was 45.5% (95%CI 32.9, 58.0) & 43.4% (95%CI 32.0, 54.7) respectively. There were no significant differences in survival with respect to site of the disease (combined vs isolated), gender or immunophenotype (pre B vs T). As shown in Table 1, CNS relapse patients who received Mitoxantrone had a significantly improved outcome when compared to those who received Idarubicin. This was most evident in those who had i) iCNSr, ii) pre-B phenotype and iii) allo-SCT, when analyzed on an intention to treat basis. This represents a considerable improvement in outcome compared to the results obtained in these sub-groups of patients in the previous UK ALLR2 study (Roy A et.al. Br. J. Haem. 2005;130:67-75). Conclusion: Mitoxantrone is highly effective in children with relapsed pre B ALL who have CNS involvement. As there were no other differences between patients treated on Mitoxantrone or Idarubicin, effective systemic therapy is as important as CNS directed therapy, if not more, in treating patients with CNS relapse. Disclosures: Off Label Use: Most drugs used in this protocol are off label as the majority of drugs used in childhood ALL are not liscensed for use in children.

scholarly journals CENTRAL NERVOUS SYSTEM INVOLVEMENT IN ADULT ACUTE LYMPHOBLASTIC LEUKEMIA: DIAGNOSTIC TOOLS, PROPHYLAXIS AND THERAPY

2014 ◽  
Vol 6 (1) ◽  
pp. e2014075 ◽  
Author(s):  
Maria Ilaria Del Principe ◽  
Luca Maurillo ◽  
Francesco Buccisano ◽  
Giuseppe Sconocchia ◽  
Mariagiovanna Cefalo ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Intrathecal Injection ◽  
False Negative ◽  
Diagnostic Tools ◽  
Positive Finding ◽  
Cns Involvement ◽  
Hematopoietic Stem

In adult patients with acute lymphoblastic leukemia (ALL), Central Nervous System (CNS) involvement is associated with a very poor prognosis. The diagnostic assessment of this condition relies on the use of neuroradiology, conventional cytology (CC) and flow cytometry (FCM). Among these approaches, which is the gold standard it is still a matter of debate. Neuroradiology and CC have a limited sensitivity with a higher rate of false negative results. FCM demonstrated a superior sensitivity over CC, particularly when low levels of CNS infiltrating cells are present. Although prospective studies of large series of patients are still awaited, a positive finding by FCM appears to anticipate an adverse outcome even if CC shows no infiltration. Current strategies for adult ALL CNS-directed prophylaxis or therapy involve systemic and intrathecal chemotherapy and radiation therapy. Actually, early and frequent intrathecal injection of cytostatic combined with systemic chemotherapy is the most effective strategy to reduce the frequency of CNS involvement. In patients with CNS overt ALL, at diagnosis or upon relapse, allogenic hematopoietic stem cell transplantation might be considered. This review will discuss risk factors, diagnostic techniques for identification of CNS infiltration and modalities of prophylaxis and therapy to manage it. 

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