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

Abstract Abstract 2576 Cerebrospinal fluid (CSF) involvement by leukemic blasts occurs in fewer than 10 % of adult patients with newly diagnosed acute lymphoblastic leukemia/lymphoma (ALL). Leukemic meningitis is diagnosed by microscopic detection of blasts in the CSF. Flow cytometry is a highly sensitive tool for detection of aberrant cells. We sought to analyze the additional benefit flow cytometry might provide for the diagnosis of leukemic meningitis. Between 11/2007 and 8/2011, 80 patients were diagnosed with ALL and treated at Emory University. 800 CSF samples were available for analysis 80 of which were collected from a diagnostic lumbar puncture (LP), 689 from follow-up LPs and 31 from LPs obtained at the time of relapse. As shown in the table, flow cytometry confirmed the presence of leukemic blasts in one, four and five samples diagnosed with leukemic meningitis by cytology at diagnosis, different stages of treatment and relapse, respectively. One and three samples were positive for leukemic blasts by cytology but negative by flow cytometry during different treatment stages and relapse respectively. We conclude that flow cytometry provided no additional benefit to cytology in the diagnosis of leukemic meningitis. Table: CSF Cytology and Flow Cytometry in 80 Adult ALL patients: CSF samples New Diagnosis N=80 Induction/Consolidation/Intensification/Maintenance/Remission/Post-transplant N = 689 Systemic relapse N = 31 N Cytology N = 80 Flow cytometry N = 66 Cytology N = 689 Flow cytometry N = 188 Cytology N = 31 Flow cytometry N = 13 Negative 79/80 65/66 684/689 184/188 23/31 8/13 Positive 1/80 1/66∼ 5/689 4/188∼* 8/31 5/13∼** ∼ CSF samples positive by flow cytometry were also positive by cytology * One CSF sample was positive by cytology but negative by flow cytometry ** CSF flow cytometry was not done in 3/8 positive CSF samples by cytology Disclosures: No relevant conflicts of interest to declare.

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Sensitivity and specificity of cerebrospinal fluid flow cytometry for the diagnosis of leukemic meningitis in acute lymphoblastic leukemia/lymphoma

Leukemia & Lymphoma ◽

10.3109/10428194.2013.852667 ◽

2014 ◽

Vol 55 (7) ◽

pp. 1498-1500 ◽

Cited By ~ 11

Author(s):

Zahi Mitri ◽

Momin T. Siddiqui ◽

Fuad El Rassi ◽

Jeannine T. Holden ◽

Leonard T. Heffner ◽

...

Keyword(s):

Flow Cytometry ◽

Cerebrospinal Fluid ◽

Fluid Flow ◽

Acute Lymphoblastic Leukemia ◽

Sensitivity And Specificity ◽

Lymphoblastic Leukemia ◽

Cerebrospinal Fluid Flow ◽

Leukemic Meningitis

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Long-Term Follow up of Chinese Children with Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Detection

Blood ◽

10.1182/blood.v112.11.4877.4877 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4877-4877

Author(s):

Yongmin Tang ◽

Xiaojun Xu ◽

Hongqiang Shen ◽

Hua Song ◽

Shuwen Shi ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Prognostic Factor ◽

Relapse Rate ◽

Lymphoblastic Leukemia ◽

Chinese Children ◽

Rank Test ◽

Log Rank Test ◽

Minimal Residual

Abstract Introduction and objective: Recent studies from resource-rich countries have showed that minimal residual diseases (MRD) were the most important independent prognostic factor for children with acute lymphoblastic leukemia (ALL). However, experience from Chinese children with this disease has not been reported in the English literature. In this study, we reported our experience in small series of children with ALL based on multi-parameter flow cytometric detection of MRD to study the relationship between MRD levels and the long term survivals in Chinese children with ALL. Methods: Using multi-parameter flow cytometry and at least one patient specific leukemia associated immunophenotype (LAIP) to determine the MRD levels in 71 Chinese children with ALL expressing leukemia associated immunophenotype (LAIP) from January 2002 to December 2003. Bone marrow aspirates were collected on day 15 and the 1st, 3rd, 6th, 12th, 18th, 24th, 30th month after chemotherapy. Leukemia associated Immunophenotype for each patient was identified according to the patients’ initial immunophenotypes determined by multi-parameter flow cytometry using a panel of more than 30 antibodies. Results: The one, two and three year disease free survival (DFS) for patients with a MRD level less than 10−2 on day 15 were (88.3±6.4) %, (84.3±7.2) % and (76.3±8.5) %, respectively, whereas those for patients with MRD levels ≥10−2 on day 15 were 100%, 0% and 0%, respectively (log rank test, P=0.004). The 5-year DFS for patients with MRD levels <10−2 and ≥10−2 on day 15 were (83.8±6.1) % and 0%, respectively (P<0.01). The 5-year DFS were significantly different between patients with MRD levels <10−4 and those ≥10−4 on the 6th or 12th month. Out of 71 patients, the 5-year DFS for patients with MRD levels <10−3 was 100%, however, the 1-year, 3-year and 5-year DFS for patients with MRD levels ≥10−3 were (87.3±5.3) %, (71.4±7.3) % and (68.0±7.7) %, respectively (log rank test, P=0.004). And the 1-year, 3-year and 5-year relapse rates for the latter group were 10.5%, 13.5% and 15.8%, which showed a tendency of difference in terms of relapse rate when compared to patients with MRD levels <10−3 (5-year relapse rate: 15.8% vs. 0%, P=0.08). Conclusions: MRD detected by multi-parameter flow cytometry is also an independent prognostic factor in Chinese children with ALL.

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Use Of Rearranged Immune Receptor Sequencing To Measure Minimal Residual Disease (MRD) In Bone Marrow, Cerebrospinal Fluid and Testes In Relapsed Childhood Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.4984.4984 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4984-4984

Author(s):

Norman J. Lacayo ◽

Li Weng ◽

Charles Gawad ◽

Malek Faham ◽

Gary V Dahl

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cerebrospinal Fluid ◽

Acute Lymphoblastic Leukemia ◽

Deep Sequencing ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Minimal Residual

Abstract Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012). Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy and kappa chain (IGH and IGK), as well as T-cell receptor beta, delta and gamma. The assay was performed on genomic DNA isolated from cells from the bone marrow, cerebrospinal fluid, or testes. The test was first done at the time of relapse to identify the malignant clonotype, which was monitored at subsequent time points. The patients were ineligible for clinical trials and concurrently underwent MRD testing using flow cytometry. The sequencing assays were performed to show feasibility of the approach. Results Patient one was a 14 y/o ALL relapse patient who was not in morphologic remission after standard re-induction therapy. The malignant clonotype was identified on a bone marrow aspirate from relapse; follow-up MRD tests were done using both flow cytometry and deep sequencing five times throughout salvage therapy with 5-aza-2'-deoxycytidine, suberoylanilide hydroxamic acid and high dose cytarabine over 75 days; the last two MRD data points showed 0.6% and 6% by ClonoSIGHT MRD and 0.4% and 1.3% by flow cytometry MRD. Morphologic remission with count recovery was used as the criteria to direct this patient to SCT. Patient two was a 9 y/o with ALL, for whom MRD was used to test for relapsed disease in multiple tissues. This patient experienced three isolated testicular relapses (M1 marrow and no CNS involvement) at the time of each relapse. The ClonoSIGHT assay was used on tissue from a testicular biopsy to identify the malignant clone(s). Testing of the bone marrow and cerebrospinal fluid did not detect the malignant clones in those sites. This patient underwent therapeutic orchiectomy and 4-week systemic re-induction resulting in a fourth complete remission and now is under evaluation for consolidation therapy with a SCT. A third patient was an 8 y/o with a combined bone marrow and testicular ALL relapse, who was in morphologic remission in the marrow after re-induction therapy and testicular radiotherapy. Prior to undergoing SCT the patient had negative MRD by flow cytometry but had 0.008% MRD using the ClonoSIGHT MRD assay. The fourth patient was a 15-yo with ALL relapse at 9 years from first remission, treated with a four-drug re-induction and Berlin-Frankfurt-Münster based consolidation and maintenance therapy. This patient was MRD negative by both flow cytometry and ClonoSIGHT MRD at end of re-induction as well as end of consolidation and remains in remission. Conclusions We have shown the feasibility of using sequencing-based tests for monitoring MRD in children with relapsed ALL in medullary (bone marrow) and extramedullary compartments (testes and CSF). Further studies are needed to establish the prognostic value of MRD detected by the ClonoSIGHT assay in both medullary and extramedullary sites that are below the limit of detection of PCR and flow cytometry. These sequencing-based tests may provide a useful tool to develop risk stratification schemas for drug development in relapsed childhood ALL. Disclosures: Weng: Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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