Blasts In Cerebrospinal Fluid Research Articles

Acute myeloid leukemia with central nervous system involvement successfully treated with gilteritinib

A 69-year-old woman was referred to our hospital due to hyperleukocytosis. We diagnosed acute myeloid leukemia and started induction therapy with the CAG regimen (aclarubicin, cytarabine and filgrastim). However, the patient was refractory to the initial treatment and developed quadriplegia, and a cerebrospinal fluid (CSF) test showed elevated blasts. We then performed intrathecal chemotherapy, and the number of blasts in CSF gradually decreased. But only two cycles of intrathecal therapy were possible due to severe methotrexate-induced mucositis. The leukemia cells had fms-like kinase 3-internal tandem duplication (FLT3-ITD), so we started treatment with oral gilteritinib. The patient then achieved hematological complete remission. Her paralysis was also resolving, and the CSF was clear of blasts for more than 6 months. Some reports show that gilteritinib may penetrate the CNS, and this case also supports the effectiveness of gilteritinib on CNS leukemia.

Multi-institutional analysis of outcomes in acute myeloid leukemia patients with central nervous system involvement

We evaluated outcomes of AML patients with central nervous system (CNS) involvement at two academic institutions. Fifty-two adult patients were identified. Neurologic symptoms were reported in 69% of patients, with headache the most common (33%). 84% (n = 42) of patients cleared their cerebrospinal fluid (CSF), with a median number of one dose of intrathecal (IT) chemotherapy. Of these patients, 21% (n = 9) had a CSF relapse, with 67% (n = 6) of those experiencing CSF relapse also having concurrent bone marrow relapse. Of the 36 patients with baseline neurologic symptoms, 69% had improvement in symptoms post-IT therapy. The median overall survival was 9.3 months and 3.5 months for patients with CNS involvement diagnosed before/during induction and at relapse, respectively. In this study, IT therapy was rapidly effective in clearing CSF blasts and improving neurologic symptoms in most patients. Few patients experienced CSF relapse, which predominantly occurred in the setting of concomitant bone marrow relapse.

A New and Cost-Effective Approach to Risk Stratification for Children with Precursor B-Cell Acute Lymphoblastic Leukemia Based on Competing Outcomes

Background: Acute lymphoblastic leukemia (ALL) is a malignancy of the white blood cells characterized by its rapid and aggressive progress that needs immediate treatment. ALL could affect both adults and children. Various patient- and disease-related factors may be involved in ALL patients’ prognosis. Therefore, it is critical to identify important risk factors related to the competing outcomes of patients with ALL. Objectives: This study aimed to stratify the risk of outcomes of children with precursor B-cell ALL using demographic characteristics, laboratory characteristics, and extramedullary diseases. To achieve this goal, we used the best competing risks model to make an appropriate decision for children's treatment according to this classification. Methods: In this retrospective cohort study, 393 patients with ALL were included. ALL with B cell origins (CD20, CD19, CD10, and CD22 positive markers) was differentiated using flow cytometry. Complete remission was defined by a lymphoblast count of less than 5% in the bone marrow, presence of no blasts in cerebrospinal fluid (CSF), as well as complete disappearance of clinical symptoms. Patients with ALL were treated based on Berlin-Frankfurt-Münster (BFM). Competing outcomes were first-relapse and non-relapse mortality, respectively. Risk factors affecting competing outcomes were assessed based on a fully specified sub-distribution model. Results: Five-year estimates for overall survival and event free survival were 75% (95% CI: 69 - 79%) and 71% (95% CI: 66 - 75%), respectively. Five-year incidence rates for first-relapse and non-relapse mortality in children were 11.4% (95% CI: 8.32 - 15.16%) and 17.6% (95% CI: 13.98 - 21.67%), respectively. Moreover, according to the results, children with WBC > 50000, hemoglobin 8, and tumor lysis syndrome for the first-relapse outcome, and children with central nervous system (CNS) involvement and tumor lysis syndrome for the non-relapse mortality (NRM) outcome were considered as high-risk groups. Conclusions: We found that extramedullary diseases could have a crucial role in the risk stratification of children with precursor B-cell ALL. Therefore, for a targeted and effective treatment of high-risk children, in addition to chemotherapy, using appropriate PI3K pathway inhibitors, JAK2 pathway inhibitors, and antibody-based immunotherapy is recommended to reduce minimal residual disease and, consequently, mortality rate.

Technological features of blast identification in the cerebrospinal fluid: A systematic review of flow cytometry and laboratory haematology methods.

Involvement of the central nervous system (CNS) by acute leukemias (ALs) has important implications for risk stratification and disease outcome. The clinical laboratory plays an essential role in assessment of cerebrospinal fluid (CSF) specimens from patients with ALs at initial diagnosis, at the end of treatment, and when CNS involvement is clinically suspected. The two challenges for the laboratory are 1) to accurately provide a cell count of the CSF and 2) to successfully distinguish blasts from other cell types. These tasks are classically performed using manual techniques, which suffer from suboptimal turnaround time, imprecision, and inconsistent inter-operator performance. Technological innovations in flow cytometry and hematology analyzer technology have provided useful complements and/or alternatives to conventional manual techniques. We performed a PRISMA-compliant systematic review to address the medical literature regarding the development and current state of the art of CSF blast identification using flow cytometry and laboratory hematology technologies. We searched the peer reviewed medical literature using MEDLINE (PubMed interface), Web of Science, and Embase using the keywords "CSF or cerebrospinal" AND "blasts(s)". 108 articles were suitable for inclusion in our systematic review. These articles covered 1) clinical rationale for CSF blast identification; 2) morphology-based CSF blast identification; 3) the role of flow cytometry; 4) use of hematology analyzers for CSF blast identification; and 5) quality issues. 9 /L, which is much lower than the original machine count and platelet transfusion was warranted. 1) Clinical laboratory testing plays a central role in risk stratification and clinical management of patients with acute leukemias, most clearly in pediatric ALs; 2) studies focused on other patient populations, including adults and patients with AML are less prevalent in the literature; 3) improvements in instrumentation may provide better performance for the classification of CSF specimens. Current challenges include: 1) more precisely characterizing the natural history of AL involvement of the CNS, 2) improvements in automated cell count technology of low cellularity specimens, 3) defining the role of flow MRD testing of CSF specimens and 4) improved recognition of specimen quality by clinicians and laboratory personnel.

Flow Cytometric Detection of Malignant Blasts in Cerebrospinal Fluid: A Biomarker of Central Nervous System Involvement in Childhood Acute Lymphoblastic Leukemia.

Despite the excellent prognosis for children and adolescents with acute lymphoblastic lymphoma (ALL), the involvement of the central nervous system (CNS) represents a major therapeutic challenge. Patients who develop CNS relapse have a very poor prognosis, and since current methods cannot reliably identify patients with CNS involvement or patients at high risk of CNS relapse, all children with ALL receive CNS-directed treatment. The current golden standard for detecting CNS involvement is the assessment of cytomorphology on cytospin slides of cerebrospinal fluid (CSF). This technique is inadequate due to low sensitivity and reproducibility. Flow cytometric analysis of CSF represent a novel, highly specific and sensitive technique for the detection of leukemic cells in the CNS. In prospective studies, CSF flow cytometry demonstrated two to three times higher rates of CNS involvement at diagnosis of childhood ALL than conventional cytospin, and especially demonstrated superior sensitivity in detecting low-level CNS disease. CNS involvement determined via flow cytometry has been linked to a higher risk of CNS relapse and poor outcomes in several studies. In this review, we discuss the central analytical concepts of CSF flow cytometry and summarize the current evidence supporting the use of flow cytometric detection of malignant blasts as a biomarker of CNS involvement in childhood ALL.

Applicability and Prognostic Significance of Flow Cytometric Cerebrospinal Fluid Investigation in Children with Acute Lymphoblastic Leukemia

Conventionally central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is found by blasts detection in cerebrospinal fluid (CSF) via cytospin slides microscopy. It was shown earlier, that low levels of tumor cells, undetectable by cytomorphology (CM), could be found in several ALL cases using more sensitive techniques such as multicolor flow cytometry (MFC) (A. Popov et al, ASH-2011). Nevertheless prognostic value of these low levels of blasts in CSF is still unclear. The aim of present study was to evaluate the clinical significance of MFC application for initial diagnostics of leukemic cells in CSF of children with ALL.Patients and methods. Since December 2008 till November 2014 in 155 consecutive children older than 1 year with ALL initial CNS involvement was investigated by CSF flow cytometry. Study group included 65 girls and 90 boys aged from 1 to 16 years (median age 4 years) treated according to ALL MB-2008 protocol. 134 patients (86.5%) suffered from various types of B-cell precursor ALL (BCP-ALL), while remaining 21 cases (23.5%) were T-lineage ALL. According to the protocol stratification system 59 patients belonged to standard risk group (SRG), 87 - to intermediate risk group (ImRG) and 9 - to high risk group. CSF lesion was studied in parallel by microscopy of cytospin slides and by MFC. CNS status was classified as CNS1 (no blast cells in CSF), CNS2 (<5 WBC/ul CSF with blast cells), or CNS3 (≥5 WBC/ul CSF with blast cells, or other signs of CNS involvement). Antibodies' combinations for cytometric CSF assessment were developed on the basis of leukemic cells immunophenotype defined by initial diagnostic bone marrow investigation. Treatment outcome was estimated by cumulative incidence of relapse (CIR). Median of follow up was 5 years.Results. In 5 patients CNS3 status was defined. These patients were excluded from the further analysis due to obvious neuroleukemia signs. CNS2 was found in 23 of 150 remaining cases (15.3%) while 127 (84.7) had CNS1. Totally 53 out of 150 studied children (35.3%) had MFC-detectable CSF lesion. In 32 CNS1-patients leukemic blasts were found only by MFC. Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by MFC (median = 418, range 8-158171 and median = 34, range 5-2762 respectively, p<0.001). Thus MFC allows detecting tumor cells in CSF much more frequently (p<0.001) than conventional CM. This difference could be explained mainly by higher MFC sensitivity. Both CM and MFC presented results, valuable for outcome prediction. Children with CNS2 (n=23) had higher relapse incidence that patients with CNS1 (n=127): CIR 0.38(0.11) and 0.16(0.05) respectively, p=0.005. 53 MFC-positive patients displayed higher CIR compared to 97 MFC-negative ones: 0.26(0.06) and 0.15(0.05) respectively, p=0.019. Nevertheless MFC lacked prognostic impact in clinically relevant patients' groups. Among CNS1 cases the CIR difference between MFC-positive (n=32) and MFC-negative (n=95) groups was not significant: 0.19(0.07) and 0.15(0.05) respectively, p=0.148. SRG patients, who could be restratified according to the results of CSF inspection, also haven't displayed any difference in relapse incidence between 51 MFC-negative patients (CIR 0.18(0.07)) and 8 MFC-positive ones (CIR 0.27(0.17), p=0.313). In ImRG after exclusion of T-ALL cases cytometric CSF assessment also lacked its prognostic impact: MFC-negative group (n=33) had similar outcome to MFC-positive patients (n=33): CIR 0.08(0.06) and 0.17(0.07) respectively, p=0.239. In 37 initially MFC-positive cases follow-up CSF samples were also studied. Patients with fast clearance of CNS burden (n=24) displayed better outcome compared to children who remained MFC-positive (n=13), although these differences didn't achieve statistical significance: CIR 0.32(0.10) and 0.54(0.14) respectively, p=0.116.Conclusions. Our data shows that MFC is able to find in CSF even small number of leukemic blasts, which are undetectable by conventional cytospin slides microscopy. Despite of this advantage of cytometric CSF inspection, the prognostic value of MFC data remains controversial. In contrast, larger tumor cells populations that could be detected in CSF also by cytomorpology are more strictly associated with high incidence of relapse. Nevertheless this data should be confirmed on larger patients' cohort in multicenter trial. DisclosuresNo relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia Cells Bypass the Blood Brain Barrier to Enter the CNS Via a Neuronal Developmental Pathway

Background: In the absence of prophylactic treatment, CNS disease involvement will occur in 30-70% of acute lymphoblastic leukemia (ALL) patients. CNS metastasis is observed in all ALL subtypes, suggesting that lymphoblasts utilize a conserved mechanism to invade the CNS. CNS ALL relapse predicts a poor outcome, but treatment options remain limited due to a minimal understanding of the biologic processes involved.PI3K regulates in many cell activities, including proliferation, migration, and apoptosis. The δ isoform of PI3K is preferentially expressed in immune cells. The PI3Kδ inhibitor, idelalisib, is approved for use in combination with rituximab for treatment of chronic lymphocytic leukemia. Previously, the PI3Kd tool compound, GS-649443 (443), which has more favorable murine pharmacokinetic properties, was evaluated in in vivo models of acute lymphoblastic leukemia (Yao H, et al. ASH meeting, 2016). Here we report on the mechanisms of ALL CNS invasion and the potential for PI3Kd inhibition to block CNS disease.Results: Our prior study of 443 in Nalm-6 (N6) B-ALL mice demonstrated that PI3Kd inhibition prevents ALL CNS disease progression. All control mice showed symptoms of CNS involvement including hind limb paralysis, but only 17% of 443-treated mice developed paralysis at their clinical endpoint. There was no difference in ALL burden or apoptotic rate in bone marrow (BM) or spleen of treated vs. control mice at matched time points. In contrast, cerebrospinal fluid (CSF) blast counts were significantly decreased in 443-treated mice.To confirm our results were not isolated to the N6 model, we tested the efficacy of PI3Kδ inhibition in mice engrafted with primary human ALL (hALL). Treated mice demonstrated a modest decrease in peripheral disease burden. Similar to the results in N6 mice, 443 markedly decreased CNS disease burden in the hALL model (Fig. 1).Examination of 443 pharmacokinetics as well as mitotic and apoptotic indices of CSF blasts isolated from 443 vs. vehicle-treated mice indicated that PI3Kd inhibition did not directly impact growth of ALL in the CNS. We therefore investigated whether 443 prevented migration of blasts from the periphery into the CNS. We found that 443 decreased Transwell® (TW) migration of N6 and hALL cells toward SDF-1 or human CSF. We next interrogated the signaling pathways downstream of PI3K that are implicated in cell motility and found that 443 suppressed the phosphorylation of myosin light chain 2 in ALL cells. To identify candidate regulatory molecules upstream of this pathway, we performed a microarray analysis of N6 cells isolated from GS-443 vs. vehicle-treated mice. This showed that multiple genes within focal adhesion and contractility pathways were downregulated in vivo in 443 treated blasts, including integrin α6.α6, a subunit of cell surface laminin receptors, is expressed in the majority of ALL cases. Laminin deposition is found in the extracellular matrix (ECM) of CNS microvessels and meninges, where it coordinates α6-dependent neuronal progenitor cell pathfinding. Using confocal microscopy, we determined that N6 and hALL cells in circulation are unable to breach the blood brain barrier. Rather, we identified that ALL cells migrate into the CNS along vessels that pass directly between vertebral or calvarial BM and the subarachnoid space, and that the ECM of these bridging vessels is enriched in laminin (Fig. 2). In in vitro invasion assays, we confirmed that ALL migration to CSF is enhanced by laminin in an α6-dependent manner. Lastly, the in vivo effects of α6 were investigated in the N6 model. Mice were treated with a6 neutralizing antibodies beginning on post-engraftment day 1 through the development of clinical symptoms requiring sacrifice. Anti-α6-treated mice showed cachexia at their clinical endpoint but none had severe CNS symptoms, whereas 100% of isotype control-treated mice showed hind limb paralysis. No difference in ALL burden or apoptotic rate was observed in the BM or spleen of anti-α6 vs. isotype control groups. In contrast, there was a significant decrease in the number of blasts harvested from the CSF of anti-α6-treated mice (Fig. 3).Conclusion: Our results identify a novel ALL metastasis mechanism. Expression of integrin α6, common in ALL, allows cells to co-opt neural migratory pathways to invade the CNS. The potential for PI3Kδ inhibition to block development of CNS ALL warrants further investigation in a clinical study. [Display omitted] DisclosuresYao:Gilead Sciences, Inc.: Research Funding. Warner:Gilead Sciences, Inc.: Research Funding. Price:Gilead Sciences, Inc.: Research Funding. Olivere:Gilead Sciences, Inc.: Research Funding. Cantelli:Gilead Sciences, Inc.: Research Funding. Ridge:Gilead Sciences, Inc.: Research Funding. Ngo:Gilead Sciences, Inc.: Research Funding. Ravichandran:Gilead Sciences, Inc.: Research Funding. Tannheimer:Gilead Sciences, Inc.: Employment. Sipkins:Gilead Sciences, Inc.: Research Funding.

In childhood mature B-NHL with CNS disease, patients with blasts in cerebrospinal fluid are at higher risk of failure

AbstractTo identify the factors influencing outcome in childhood mature B-cell non-Hodgkin lymphoma and acute leukemia (B-NHL/AL) with central nervous system (CNS) disease (CNS+), we analyzed patients <18 years with newly diagnosed B-NHL/AL registered in 3 Lymphomes Malins B studies in France between 1989 to 2011. CNS+ was diagnosed on fulfillment of ≥1 of the following criteria: any L3 cerebrospinal fluid (CSF) blasts (CSF+), cranial nerve palsy, isolated intracerebral mass but also clinical spinal cord compression, and cranial or spinal parameningeal extension. Two hundred seventeen out of 1690 patients (12.8%) were CNS+. CNS+ was significantly associated with male gender, head/neck locations, Burkitt histology, high initial lactate dehydrogenase (LDH) level, and bone marrow involvement. CSF+ was the most frequent pattern of CNS+ (45%). For the 217 CNS+ patients, the 5-year event-free survival (EFS) and overall survival rates (95% confidence interval) were 81.5% (75.8% to 86.1%) and 83.9% (78.4% to 88.2%), respectively. In multivariate analysis, among CNS+ patients, low EFS was associated with CSF+, high initial LDH level, and poor response to cyclophosphamide, oncovin (vincristine), prednisone prephase. These findings have been considered for patient's stratification in the international randomized phase 3 trial Inter-B-NHL-ritux 2010 for children and adolescents with high-risk B-NHL/AL with CNS+ CSF+ patients only receiving intensified chemotherapy.

Successful application of anti-CD19 CAR-T therapy with IL-6 knocking down to patients with central nervous system B-cell acute lymphocytic leukemia.

Few studies have described chimeric antigen receptor–modified T cell (CAR-T) therapy for central nervous system (CNS) B-cell acute lymphocytic leukemia (B-ALL) patients due to life-threatening CAR-T-related encephalopathy (CRES) safety issues. In this study, CAR-Ts targeting CD19 with short hairpin RNA (shRNA)-IL-6 gene silencing technology (ssCART-19s) were prepared. We conducted a phase 1 clinical trial (ClinicalTrials.gov number, NCT03064269). Three patients with relapsed CNS B-ALL were enrolled, conditioned with the fludarabine and cyclophosphamide for lymphocyte depletion and infused with ssCART-19s for three consecutive days. Clinical symptoms and laboratory examinations were monitored. After ssCART-19 treatment, three patients' symptoms resolved almost entirely. Brain leukemic infiltration reduced significantly based on magnetic resonance imaging (MRI), and there were no leukemic blasts in cerebrospinal fluid (CSF), which was confirmed by cytological and molecular examinations. Additionally, increases in the levels of cytokines and immune cells were observed in the CSF of all patients. Only grade 1 cytokine release syndrome (CRS) manifesting as fever was noted in patients. In conclusion, CAR-Ts with shRNA-IL-6 gene knockdown migrated into the CNS, eradicated leukemic cells and elevated cytokines in CSF with mild, acceptable side effects.

Treatment of Testicular Relapse of B-cell Acute Lymphoblastic Leukemia With CD19-specific Chimeric Antigen Receptor T Cells

BackgroundIrradiation has been a standard treatment for testicular relapse but is associated with severe hypogonadism. Because CD19-specific chimeric antigen receptor T (CAR-T) cells can eradicate leukemic blasts in cerebrospinal fluid, a pharmacologic sanctuary site, we tested the efficacy of this therapy in 7 boys with isolated testicular relapse of B-cell acute lymphoblastic leukemia. Patients and MethodsCD19-specific CAR-T cells were generated with the use of autologous T cells transduced with a lentiviral vector to express a CAR molecule containing anti-CD19 scFv derived from the HI19α murine monoclonal antibody, human CD8α hinge, and human 4-1BB (CD137) and CD3ζ costimulatory signaling transmembrane domains. After the conditioning regimen, which consisted of intravenous fludarabine and intravenous cyclophosphamide, 7 patients with a median age of 9 years (range, 2-10 years) with isolated testicular relapse received a single infusion of CD19 CAR-T cells at a total dose of 5 × 106 all T cells per kilogram. ResultsAll 7 patients achieved complete remission with normal testes. Six patients remained in second remission for 5 to 23 months (median, 14 months), and 1 patient subsequently relapsed in the bone marrow. The probability of event-free survival for all patients at 12 months of follow-up was 83.3% ± 15.2% (standard error). The treatment was well-tolerated, with grade 1 cytokine-release syndrome developing in 5 patients. ConclusionThese results suggest that CAR-T cell therapy is a treatment option for patients with testicular relapse.

Flow cytometric detection of leukemic blasts in cerebrospinal fluid predicts risk of relapse in childhood acute lymphoblastic leukemia: a Nordic Society of Pediatric Hematology and Oncology study

Central nervous system (CNS) involvement by cytospin is associated with increased risk of relapse in childhood acute lymphoblastic leukemia. We investigated if flow cytometric analysis of cerebrospinal fluid (CSF) at diagnosis improves the prediction of relapse. This prospective cohort study included patients (1.0-17.9 years) treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol. CSF flow cytometry samples were obtained at 17 centers, preserved with Transfix®, and analyzed at a central laboratory. One-hundred and seventy-one (25.4%) of 673 patients were positive by flow cytometry (CNSflow+). The 4-year cumulative incidence of relapse was higher for patients with cytospin positivity (CNScyto+) (17.1% vs. 7.5%), CNSflow+ (16.5% vs. 5.6%), and cytospin and/or flow positivity (CNScomb+) (16.7% vs. 5.1%). In Cox regression analysis stratified by immunophenotype and minimal residual disease day 29 and adjusted by sex, predictors of relapse were age (hazard ratio [HR] 1.1, 95% CI 1.1-1.2, P < 0.001), white blood cell count at diagnosis (HR 1.4, 95% CI 1.1-1.6, P < 0.001), and CNScomb+ (HR 2.2, 95% CI 1.0-4.7, P = 0.042). Flow cytometric analysis of CSF improves detection of CNS leukemia, distinguishes patients with high and low risk of relapse, and may improve future risk stratification and CNS-directed therapy.

Absolute count of leukemic blasts in cerebrospinal fluid as detected by flow cytometry is a relevant prognostic factor in children with acute lymphoblastic leukemia.

Usually, central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed by cytomorphology (CM) of cerebrospinal fluid (CSF) on cytospin slides. Multicolor flow cytometry (MFC) provides the opportunity to detect low numbers of leukemia cells undetectable by CM. The present study aimed at evaluating the clinical significance of MFC for the diagnosis of CNS involvement at initial manifestation of childhood ALL. In 155 children with ALL, CSF samples were studied in parallel by CM and MFC. Patients were treated according to protocol ALL-MB-2008 for childhood ALL. The prognostic impact of the leukemia burden in CSF was determined categorizing the findings as positive/negative. In addition, the absolute blast cell count per 1ml of CSF was studied as a continuous variable. CSF positivity was significantly more frequent using MFC compared with CM (35.3% vs. 15.3% of patients). The outcome of MFC-positive and MFC-negative patients was not different in clinically relevant patient risk groups-CNS1, standard and intermediate-risk groups. Using the quantitative approach, at the threshold level of 20 blasts per ml of CSF, patients could be divided into two groups with a significantly different outcome, irrespective of the clinical risk group, the type of CNS-directed therapy, and the CNS status determined by CM. Our data do not support the concept of re-stratification and modification of therapy based on qualitative CSF investigation by MFC. However, MFC is a highly sensitive technique of CSF investigation improving the definition of CNS involvement in childhood ALL, and quantitative measurement of blast cells in CSF, if well-organized, can be a useful additional tool for stratification of patients in clinical trials.

Recommendations for cerebrospinal fluid examination in acute leukemia

Cytological identification of blasts in cerebrospinal fluid in acute leukemia, lymphoid or myeloid, in adult and child, at diagnosis or during follow up lead to the diagnosis of leukemic meningitidis. Suitable CNS therapy based on a defined "CNS status" following an international standardized classification, lead to decrease cerebrospinal relapses. Established in 1993, this classification allows to treat patients based on their CNS status. Based on the red blood cells count, nucleated cells count and presence of blasts, it requires a standard technical procedure that guarantees the comparability of results coming from different medical laboratory. To improve the quality of cerebrospinal fluid analysis, in acute leukemias, preanalytical guidelines (turn around time), analytical guidelines (cytocentrifugation, adding serum protein, speed and duration of cytocentrifugation) and postanalytical guidelines (duration of conservation) are set by the Groupe francophone d'hématologie cellulaire.

Clinical characteristics of patients with central nervous system relapse in BCR-ABL1-positive acute lymphoblastic leukemia: the importance of characterizing ABL1 mutations in cerebrospinal fluid

We investigated the frequency, predictors, and evolution of acute lymphoblastic leukemia (ALL) in patients with CNS relapse and introduced a novel method for studying BCR-ABL1 protein variants in cDNA from bone marrow (BM) and cerebrospinal fluid (CSF) blast cells. A total of 128 patients were analyzed in two PETHEMA clinical trials. All achieved complete remission after imatinib treatment. Of these, 30 (23%) experienced a relapse after achieving complete remission, and 13 (10%) had an isolated CNS relapse or combined CNS and BM relapses. We compared the characteristics of patients with and without CNS relapse and further analyzed CSF and BM samples from two of the 13 patients with CNS relapse. In both patients, classical sequencing analysis of the kinase domain of BCR-ABL1 from the cDNA of CSF blasts revealed the pathogenic variant p.L387M. We also performed ultra-deep next-generation sequencing (NGS) in three samples from one of the relapsed patients. We did not find the mutation in the BM sample, but we did find it in CSF blasts with 45% of reads at the time of relapse. These data demonstrate the feasibility of detecting BCR-ABL1 mutations in CSF blasts by NGS and highlight the importance of monitoring clonal evolution over time.

Immunophenotyping of the cerebrospinal fluid as a prognostic factor at diagnosis of acute lymphoblastic leukemia in children and adolescents

ABSTRACTThis study aimed at evaluating the use of immunophenotyping (IMP) in the identification of blast cells in the cerebrospinal fluid (CSF) of children and adolescents with acute lymphoblastic leukemia (ALL). Sixty-seven patients aged 18 years or younger were included. Fifty-five CSF samples were analyzed at initial diagnosis and 17 at the time of relapse. A cytological analysis (CA) was performed in all 72 samples, while IMP was done in 63. Blasts were identified in only three samples by CA, whereas all three samples were found negative by IMP, one of which had no isolation of nucleated cells after centrifugation. Among the samples analyzed by IMP, 11 showed a positive blast count, two of which had been inconclusive using CA. No equivalence was found between CA and IMP results (p = 0.55). CSF IMP positivity was not associated with other risk factors for ALL relapse. Among the 55 patients included at the time of diagnosis of ALL, eight relapsed during follow-up. Considering the cases of central nervous system (CNS) relapse, one of the patients belonged to the CSF IMP-positive group (11%) at diagnosis, and the other two cases, to the IMP-negative (5%) group. Detection of CSF blast cells using IMP was associated with a worse overall (p < 0.0001) and event-free survival (p < 0.0001). These results show that CSF IMP may be a useful additional method to conventional CA in the diagnosis of CNS involvement in ALL, and for the identification of high-risk subgroups that would benefit from an intensified therapy.

Prognostic Significance of Cerebrospinal Fluid Lymphoblasts at Initial Presentation with Acute Lymphoblastic Leukemia Attending at Bangabandhu Sheikh Mujib Medical University, Bangladesh

Prognostic Significance of Cerebrospinal Fluid Lymphoblasts at Initial Presentation with Acute Lymphoblastic Leukemia Attending at Bangabandhu Sheikh Mujib Medical University, Bangladesh

Phase Ib Trial of the mTOR Inhibitor Everolimus Given in Combination with Multiagent Chemotherapy in Relapsed Acute Lymphoblastic Leukemia

Introduction: Treatment of relapsed childhood acute lymphoblastic leukemia (ALL) remains a significant clinical challenge with a need for novel, more effective therapies. Pre-clinical studies have demonstrated that mTOR inhibitors have single agent activity in ALL and are synergistic with other chemotherapy agents typically used to treat ALL, including vincristine, corticosteroids, doxorubicin and L-asparaginase.Methods: Dana-Farber Cancer Institute Protocol 11-237 tested the combination of everolimus (given orally on days 1-32) with a standard reinduction regimen of prednisone (40 mg/m2/daily orally on days 4-32), weekly vincristine (1.5 mg/m2/dose, max 2 mg, on days 4,11,18 and 25), PEG-asparaginase (2,500 U/m2/dose IV on days 5 and 18) and doxorubicin (30 mg/m2/dose on days 4 and 5) with dexrazoxane pretreatment (300 mg/m2/dose on days 4 and 5). Intrathecal cytarabine was administered on day 1 and intrathecal methotrexate, cytarabine and hydrocortisone (IT-MAH) was administered on days 18 and 32; patients (pts) with cerebrospinal fluid blasts at study entry received additional IT-MAH on days 11 and 25. Pts with first bone marrow relapse ALL occurring greater than 18 months after continuous remission were eligible for participation. The primary aim was to identify a maximum tolerated dose of everolimus in combination with this backbone utilizing a classic "3 + 3" dose escalation design. Three dose levels (DL) of everolimus were tested (2-, 3-, and 5-mg/m2/day). 5 mg/m2/day (DL3) was the highest dose tested because it was the recommended phase 2 dose (RP2D) of single-agent everolimus in children with solid tumors. Secondary aims included assessment of second complete remission (CR2) rate and levels of end-reinduction minimal residual disease (MRD) assessed by allele-specific oligonucleotide PCR. On-target activity of everolimus was assessed 6 hours after the first dose of everolimus by examining phosphorylated S6 (pS6) levels in peripheral blood blasts by flow cytometry.Results: Thirteen pts with first relapse B-ALL were enrolled. (Table 1) Median age at enrollment was 10 yrs (range: 2-22 yrs). Median duration of first CR was 3.7 yrs (range 1.5-7.7 yrs). One pt treated at DL 3 stopped study drug early due to Grade 4 infection (meningitis due to Rothia mucilaginosa) and was inevaluable for dose limiting toxicity (DLT) assessment. Table 2 displays the most common Grade 2 or higher adverse events (AE's) attributable to everolimus. There were no Grade 5 events and no episodes of interstitial pneumonitis. No DLTs were observed at DL1 or DL2. One of 6 evaluable pts treated at DL3 experienced a DLT (Grade 4 hyperbilirubinemia). 12 of the 13 pts achieved CR (92%). Of the 12 pts who achieved CR, 9 (75%) had low end-reinduction MRD (²0.001). (Table 1) Of the 7 pts with evaluable samples (2 at DL1, 1 at DL2 and 4 at DL3), 6 (86%) had decrease in pS6 in peripheral blasts (>20% decrease in mean fluorescence intensity), consistent with on-target mTOR inhibition. Only 1 pt (treated at DL1) did not have evidence of pS6 inhibition; this pt achieved CR2 with low MRD.Discussion: Everolimus given in combination with a standard 4-drug reinduction was well tolerated at all dose levels tested in pediatric pts with first relapse ALL. CR2 rates were comparable to published re-induction regimens, with a promising proportion of pts with low end-reinduction MRD. An expansion cohort is currently enrolling to further assess toxicity of this combination, particularly infectious risks, in order to define the RP2D.Table 1Patients and Response to TherapyPt #Dose LevelAge (yrs)Duration of CR1 (yrs)DLT?CR?MRD0011107.7NoYesHigh0021203.5NoYesLow003183.1NoYesLow004221.5NoYesHigh0052225.1NoNoN/A006253.7NoYesLow0073125.7NoYesLow0083103.0N/AYesLow0093114.3NoYesLow0103214.8YesYesHigh011373.4NoYesLow0123135.0NoYesLow0133173.0NoYesLowTable 2Grade 2 or higher Adverse Events Attributable to Everolimus (Possible, Probable, Definite) at all dose levels; N=13 patientsToxicityGrade 2Grade 3Grade 4Grade 5Bacteremia0300Bacterial meningitis0010Invasive fungal disease0100Febrile Neutropenia0700Mucositis2200Anorexia3100AST increase1520ALT increase1520Bilirubin increase0210Hypophosphatemia2510 DisclosuresPauly:Seattle Genetics, Inc.: Research Funding. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Detection of central nervous system involvement in childhood acute lymphoblastic leukemia by cytomorphology and flow cytometry of the cerebrospinal fluid.

Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement based on cytomorphological examination of the cerebrospinal fluid (CSF) alone is not as sensitive with low cell counts as flow cytometric immunophenotyping (FCI) of the CSF. However, the importance of low CSF blasts counts at diagnosis is uncertain. We sought to determine the significance of FCI in relation to conventional morphological examination. We retrospectively compared FCI of the CSF with cytomorphology at diagnosis or relapse of childhood ALL. All patients were diagnosed 2000-2012 in Stockholm or Umeå, Sweden. Clinical data were collected from medical records and the Nordic leukemia registry. Treatment assignment was based on morphological examination only. The cohort was comprised of 214 patients with ALL. CSF involvement was detected by both methods in 20 patients, in 17 by FCI alone, and in one patient by cytomorphology alone. The relapse rate was higher for patients with negative cytology but positive FCI compared to those without CNS involvement using both methods. The difference was especially marked in the current protocol. However, none of the patients with negative CSF cytology but positive FCI had a CNS relapse. FCI of the CSF increased the detection rate of CNS involvement of ALL approximately two times compared to cytomorphology. Patients with low-level CNS involvement may benefit from additional intensified systemic or CNS-directed therapy, but larger studies are needed.

Differentiating Burkitt Lymphoma from Burkitt-like Lymphoma in Pediatrics: Report from the Japanese Pediatric Leukemia/Lymphoma Study Group B-NHL03 Study

Introduction: Pediatric mature B-cell non-Hodgkin lymphoma (B-NHL) is mostly classified as Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). It has a predominantly germinal center origin. In pediatric BL, some cases are difficult to distinguish from DLBCL, which has been referred to as atypical BL or Burkitt-like lymphoma (BLL). In the recent World Health Organization classification of 2008, B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (IDB) was defined as a new but heterogeneous category. This group has morphological and genetic features of both DLBCL and BL. Therefore, IDB includes atypical BL or BLL in children. To the best of our knowledge, little is known about the exact difference between pediatric BL and IDB, and a distinction needs to be made between the clinical aspects of these two groups. In this study, we performed a clinicopathological study using the cohort from Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) B-NHL03 study, which was the nation-wide prospective study for pediatric B-NHL in Japan, to understand the meaning of the classification of BL in children.Methods: From November 2004 to January 2011, 346 cases of newly diagnosed pediatric B-NHL were enrolled in the B-NHL03 study. Of these, 208 cases were diagnosed in the central pathology review system with reference laboratory results. Seventy-eight cases were subclassified as BL (37.5%), 42 as IDB (20.2%), 57 as DLBCL (27.4%), and 31 as not subclassified (14.9%). Finally, 120 cases of BL or IDB were included in our study cohort. The pathological features and diagnostic and prognostic factors of those cases were analyzed.Results: The median age was 9.0 years (range, 1.9–15.5) in BL patients and 9.1 years (3.0–15.8) in IDB patients. Male (M) predominance was observed in both groups and the M/female (F) sex ratio was 5.5 (M/F 66/12) in BL and 9.5 (38/4) in IDB cases. According to the Murphy staging system, 7.7%/24.4%/39.7%/28.2% of BL patients and 14.3%/40.5%/26.2%/19.0% IDB patients were at stage I/II/III/IV, respectively. The frequency of the cases with central nervous system diseases and cerebrospinal fluid (CSF) blasts were 10.3% and 5.1% in BL and 7.1% and 2.4% in IDB, respectively. In addition, the number of the cases with elevated lactate dehydrogenase (LDH) levels (more than twice the institutional upper limit) was 43.6% for BL and 33.3% for IBD. 4-year overall survival and event-free survival (EFS) were 93.6% and 91.0% for BL and 95.2% and 85.7% for IBD, respectively. In general, there is little statistical difference in clinical presentation between both groups. Pathological examination showed that BL has a significantly high frequency of C-myc translocation (57.1% vs. 33.3%, p = 0.03). Immunohistochemistry demonstrated that most cases in both groups were CD10 positive (BL 97.4%, IBD 95.1%), Bcl-6 positive (both groups 100%), and overexpressed Ki67 (≥90%; BL 93.6%, IBD 92.9%). Moreover, 11.1% of BL and 5.7% of IBD patients were Bcl-2 positive. Because a large difference was not observed in the clinicopathological presentation of the two patient groups, we combined both to analyze prognostic factors. To determine the effects of various risk factors of EFS, log-rank testing with stratification for age (≥10 or <10), sex (M or F), stage (I & II or III & IV), CSF blasts level (±), LDH level (more than or less than twice the institutional upper limit), C-myc translocation(±), immunohistochemical staining of CD10 (±), Bcl-2 (±), Bcl-6 (±), and Ki67 (≥ or <90%) were examined. High staging (83.3% vs. 97.9%, p = 0.012), CSF blast+ levels (40.0% vs. 91.3%, p = 0.001), and high LDH levels (77.1% vs. 97.2%, p = 0.001) significantly correlated with inferior outcome.Conclusions: We conclude that in our cohort, the distinction between BL and IDB does not have much meaning in B-NHL in children. Furthermore, our study shows that the prognostic factors in childhood B-NHL identified in clinical trials in Western countries are identified in patients with the broad BL in Japan. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fluid of Children with Acute Lymphoblastic Leukemia

Background: Central nervous system (CNS)-directed treatment has reduced risk of CNS relapse for childhood acute lymphoblastic leukemia (ALL), which accounts for 30-40% of initial relapses. Compared with CNS-negative patients, patients with CNS leukemia (>5 leukocytes/µL cerebrospinal fluid (CSF) and lymphoblasts) suffer from more CNS+ relapses. Conventional cytology (CC) is specific (>95%), but nonsensitive (<50%), since leukocytes in CSF decay within few hours after lumbar puncture.Method: We prospectively assessed centralised multi-parameter flow cytometry (FCM) of fixated CSF versus local CC in Nordic/Baltic childhood ALL. Diagnostic samples from 172 children aged 0-18 years with de novo and eight children with relapsed ALL were investigated in 297 CSF samples from 180 patients. Kinetics of disappearance of leukemic cells in the CSF was evaluated until day 15. Antibody-combinations reflected the immunophenotype of leukemic blasts in bone marrow.Result: Of 172 newly diagnosed patients, 51 (30%) had CSF involvement by FCM, while CC was positive in 16 patients (9%) (p<0.001). CSF involvement was detected by both FCM and CC in four of eight patients with relapse (50%). Among newly diagnosed patients, samples positive by FCM and CC had higher leukemic blast count compared to samples positive by FCM only (medians: 0.545 (range: 0.005-4.801) versus 0.016 (range: 0.003-1.38) leukemic blasts/µL; p<0.001). Among newly diagnosed patients with samples positive by FCM and CC, the CSF blast count was related to the CSF leukocyte count (rs=0.82; p=0.001). Compared to newly diagnosed patients who were FCM-negative, those with FCM-positivity had higher WBC (median: 37 versus 9 x 109/L; p<0.001), were younger (medians: 3 versus 5 years, p=0.04), and more often had T-cell ALL (12/51 (24%) versus (6/121 (5%), p<0.001). Five (16%) of 31 newly diagnosed patients with FCM detected blasts at diagnosis and available data at day 15 still had CSF leukemic blasts on day 15. So far the two patients who later developed CNS and/or bone marrow relapse were positive by flow (1.38 and 0.178 blasts/µL), but CNS negative by CC or had CSF leukocyte count <5/µL and lymphoblasts on CC, respectively.Conclusion: Leukemic blasts are present in spinal fluid of one third of newly diagnosed ALL. CSF involvement is associated with other higher risk characteristics. The prognostic value of these findings awaits prospective evaluation. DisclosuresNo relevant conflicts of interest to declare.