Flow Cytometric Analysis of Cerebral Spinal Fluid Involvement by Leukemia or Lymphoma

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1 Flow Cytometric Analysis of Cerebral Spinal Fluid Involvement by Leukemia or Lymphoma Maryalice Stetler-Stevenson, M.D., Ph.D. Flow Cytometry Unit, Laboratory of Pathology, DCS, NCI, NIH DEPARTMENT OF HEALTH & HUMAN SERVICES

2 Involvement of CSF by B-Cell NHL Associated with high grade lymphoma Infrequent but portends poor prognosis Diagnosis based upon cytomorphologic evaluation of CSF- But 20-60% false negative Prophylactic treatment of CNS Decreases incidence of documented CNS involvement Increases toxicity and unnecessary in most patients Need new sensitive methods of detection Use of Flow Cytometry in past hampered by low cellularity

3 Diagnostic Flow Cytometric Analysis of CSF Specimens: Minimum Cellularity Requirements Study of minimal cellularity requirements for diagnosis: 43 CSF samples studied: 4 leukemia 9 multiple sclerosis, 8 headache, 4 meningitis, 5 hydrocephalus, 2 Parkinson s, 3 dementia, 5 brain neoplasia, 1 Guillain-Barre, 2 other 14 normal peripheral bloods Subira, D et al. Am J Clin Pathol 2002; 117:

4 Minimum Cellularity Requirements for Flow Cytometry Diagnosis of CSF Specimens Required Number of Cells/ L CSF: >5 cells/ L: Can detect CD3+T, CD4+T, CD8+T and B in 100% of specimens, Monocytes in 50% 1-5 cells/ L: Can detection CD3+T, CD4+T and CD8+T in 100% of specimens, B cells in 20%, Monocytes in 47% 1 cell/3 L: Can detect CD3+T and CD4+T in 100% of specimens, and CD8+T in 67%, Monocytes in 50%, No B cells. Subira, D et al. Am J Clin Pathol 2002; 117:

5 Minimum Cellularity Requirements for Flow Cytometry Diagnosis of CSF Specimens Minimum number of events required to define a cell population (B, T and NK cells): 13 or more clustered events sufficient to identify any population in all specimens studied <5 clustered events insufficient to identify populations

6 Diagnostic Flow Cytometric Analysis of CSF Specimens T lymphocytes were the most prevalent group with the exception of 1 patient with hydrocephalus and the 4 patients with B cell neoplasia B cells present in low numbers in nonneoplastic specimens (range % of cells)

7 Diagnostic Flow Cytometric Analysis of CSF Specimens Conclusion: Multicolor flow useful to detect lymphocyte subsets and malignancy in CSF despite low cellularity

8 Diagnostic Flow Cytometric Analysis of CSF Specimens WBC reference values in normal CSF: Subset Leukocytes Granulocytes Monocytes Lymphocytes T cells NKT cells B cells NK cells Immunological definition CD45 + CD45 +, SSC hi CD45 +, SSC im, FSC hi, CD4 dim CD45 +, SSC lo, FSC im CD3 + CD56 + CD3, 19 + CD3, 56 + Absolute number 1.12 ( ) 0.08 ( ) 0.23 ( ) 0.66 ( ) 0.62 ( ) 0.01 ( ) 0.00 ( ) 0.01 ( ) de Graaf MT et al. Clinical Cytometry 80B: 43-50, 2011

9 Utility of Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma Patients with aggressive B-cell lymphomas are at high risk of CNS involvement CSF evaluated in 51 patients with newly diagnosed aggressive B-cell lymphomas: Diffuse large B cell lymphoma (DLBCL) and Burkitt s lymphoma (BL) 23/ 51(45%) AIDS-related lymphoma 28/51 (55%) HIV negative- 6 Burkitts, 22 DLBCL CSF also evaluated in 9 patients with relapsed or treated aggressive B-cell lymphomas considered at high risk of CNS involvement Hegde, U et al. BLOOD 105 (2): , 2005

10 Examples of Flow Cytometry Results in Aggressive B-Cell Lymphoma A. Monoclonality: CD19+ B cells are Kappa+, Lambda - CD22 CD22 kappa B. Abnormal Antigen Pattern: CD19+ CD10+ B cells lambda CD19 CD10

11 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma: Results Flow Cytometry (FCM) more sensitive than Cytomorphology(CM) (p 2 =0.0020) FCM detected lymphoma in CSF in 22% of newly diagnosed and 33% of relapsed patients CM detected lymphoma in 2% of newly diagnosed and 11% of relapsed patients CM did not detect lymphoma in any Flow negative cases FCM detected lymphoma in many CM negative cases Low median percent of tumor cells (7%; range %) made detection by CM difficult but did not affect FCM detection Hegde, U et al. BLOOD 105 (2): , 2005

12 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma: Results CSF total protein, WBC/ l, and RBC/ l were not helpful Patient lactate dehydrogenase levels- not significant (P 2 =0.062) 2 or more extranodal sites was the only clinical finding associated with +CSF (P 2 =0.0057) The risk of + CSF increased with increasing extranodal sites Patients with increased extranodal sites and negative CSF should be considered for prophylactic treatment There was a trend between high international prognostic index (IPI) score and +CSF (P 2 =0.0057) Due to multiple extranodal sites Hegde, U et al. BLOOD 105 (2): , 2005

13 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma: Results 45% of patients with CSF positive by FCM relapsed in CNS and died despite having initially received active treatment Only 8% of patients with CSF negative by FCM relapsed in CNS and died despite only receiving less intensive prophylactic treatment Hegde, U et al. BLOOD 105 (2): , 2005

14 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma Conclusions: FCM more sensitive than CM, CSF total protein, CSF WBC and lactate dehydrogenase levels FCM detection of CSF involvement is a bad prognostic indicator Hegde, U et al. BLOOD 105 (2): , 2005

15 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma Study of sensitivity and specificity of a standardized Flow Cytometry panel for detecting neoplastic cells in stabilized CSF samples from patients with newly diagnosed aggressive B-NHL 11 Parameter- 6 colors, 1 tube Quijano, S, et al JCO 27(8), 2009

16 FLOW CYTOMETRIC ANALYSIS OF CSF SAMPLES FROM AGGRESSIVE B-NHLB Stabilised CSF samples (Transfix, Immunostep SL) Add PBS (4/mL) Centrifuge (1x) and concentrate (300mL) Cell staining of 1/3 sample FITC PE PERCPCy5.5 PECy7 APC APCCy7 CD8-sIgl CD56-sIgk CD4-CD19 CD3 CD20 CD45 Restaining (if negative) vs panel adapted to B-NHL phenotype Add PerfectCOUNT beads (Cytognos SL) Data acquisition (FACSCanto II; BDB) Kraan et al, CPC 2008

17 Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma 22% of cases (27/123) were aggressive B-NHL that were flow cytometry (FCM)+, 6% were cytomorphology (CM)+, 2% were CM suspicious All CM+ specimens were FCM+ except 1 case which was a false + Cases of FCM+CM+ had higher tumor cell number than FCM+CM- (p<0.0001) Cut off FCM+/CM- and FCM+/CM+ >20% tumor and >1 cell/ L Quijano, S, et al JCO 27(8), 2009

18 ABSOLUTE AND RELATIVE NUMBERS OF NEOPLASTIC B-CELLS IN FCM+ CSF SAMPLES % No. of cells/ml * % of cells 80% 60% 40% 20% 0% * Cytology+/FCM+ Cytology-/FCM+ Cytology+/FCM+ Cytology-/FCM+ *p<0.001 *Cut-off: <20% and <1 neoplastic B-cell/mL Quijano, S, et al JCO 27(8), 2009

19 Utility of Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma Normal Subsets in CSF % Monocytes increased in CSF samples negative for lymphoma-(p<0.001) Number of CD3+ (p=0,001), CD4+CD3+(p<0.0001), CD8+CD3+(P=0.007) and NK cells (p=0.02) higher in CSF+for lymphoma The following were more prevelant in patients with CSF FCM+ than FCM-: neurological Sx (p<0.0001); cranial nerve Sx (p=0.09); lower performance status (p=0.02), extranodal involvement (p=0.03); BM involvement; (p=0.04); serum 2-microglobulin(p=0.04) Quijano, S, et al JCO 27(8), 2009

20 Utility of Flow Cytometry in Diagnosis of CSF Involvement by Aggressive B-Cell Lymphoma Conclusions: FCM is more sensitive than CM Cut-off level for CM >20% tumor and/or 1 cell/ L A standard single tube panel can be used for detection of B-NHL, regardless of subtype Normal subsets may be useful Quijano, S, et al JCO 27(8), 2009

21 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma Study of 1054 samples from 219 patients with proven diagnosis of hematologic malignancy DLBCL (55), Precursor B cell (37), Burkitt (8), other B-NHL (50), AML (40), CML (7), other (22) FCM and CM diagnoses compared Criteria for FCM diagnosis was based upon number of cells in cluster with immunophenotype c/w malignancy- Positive >25 events Suspicious events Negative < 10 events Bromberg, JEC et al. Neurology 68:

22 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma 27% or 60/219 patients CSF + for tumor (FCM or CM) 80% of the patients with +CSF had clinical features suspicious for involvement Only 17% of the patients with +CSF had + MRI Although + CSF more likely with pleocytosis and increased CSF protein, +CSF occurred with normal cell count and protein Bromberg, JEC et al. Neurology 68:

23 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma 48 patients had + first CSF sample Flow Cytometry Cytomorphology Negative Suspicious Positive Negative Suspicious Positive Total Total Patients with +CM had increased pleocytosis (84%) and symptoms (95%) and trend to decreased progression free survival-? Increased tumor load Multiple Samples: In 12/60 patients additional samples required for diagnosis Bromberg, JEC et al. Neurology 68:

24 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma Conclusions: FCM more sensitive than CM CM is important too Additional samples increase sensitivity of detection Bromberg, JEC et al. Neurology 68:

25 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma: Technical Aspects Samples should be placed into stabilization media as soon as possible to avoid deterioration of cells due to the rapid in vitro cytotoxic effects of CSF on leukocytes Stabilization medium 1: Complete RPMI containing 5% FBS-stabile up to 18 hours at 4ºC Stabilization medium 2: Transfix (Cytomark Ltd, Buckingham, MK18 2LR, UK)-stabile up to 48 days at 4ºC Kraan et al. Current Protocols in Cytometry 2008; Chapter 6:unit 6.25

26 Flow Cytometry in Diagnosis of CSF Involvement by Leukemia or Lymphoma: Technical Aspects Unacceptable cell loss with multiple washes Minimal immunoglobulin in CSF- just adding 3-5 ml of PBS to CSF and centrifuging is enough for cytophilic antibody Aspirate, not decant Red cell lysis can be a problem with diffuse large B cell lymphoma Kraan et al. Current Protocols in Cytometry 2008; Chapter 6:unit 6.25

27 Conclusions: FCM can be successfully performed on very small samples >5 cells/ L and >13 clustered events enough for populations 1 cell/3 L and >5 clustered events enough for some populations CSF is + for malignant cells by FCM in 22% of aggressive B-NHL FCM is the most sensitive method for detecting B cell non-hodgkin s lymphoma in CSF CM requires higher levels of tumor cells for detection Lactate dehydrogenase, WBC and CSF protein not useful Flow cytometric evaluation of CSF for diagnosis is vital in aggressive B-NHL and has prognostic importance FCM+ patients markedly increased incidence of relapse and death despite active treatment Normal subsets in CSF may have prognostic importance