Dr Prashant Tembhare

Transcription

1 Dr Prashant Tembhare

2 FCM very powerful technology in Identification and characterization of neoplastic plasma cells as it allows - simultaneous assessment of multiple antigens large numbers cells in short time Study low levels of plasma cells Simultaneous analysis of surface and intracytoplasmic antigens Quantitation of antigen density

3 Plasma cells are identified by expression of CD138++ Bright CD38++++

4

5

6 Blood 2014 Feb 27;123(9): Critical Reviews in Oncology/Hematology 88 (2013)

7 Normal plasma cells Specific markers- CD138, CD38 (strong), Strong CD229, Strong CD319 B cell lineage weak CD19, strong CD27, strong CD81 Polyclonal Moderate expression of CD45 Neoplastic plasma cells Aberrant expression- CD20, CD56, CD28, CD117, CD200 Loss of antigens CD19, CD27, CD45, CD81 Intracellular light chain restriction Rowstron AC et al. EMN report. Haematologica 2008 Mar; 93(3):

8

9 Normal PCs Abnormal PCs CD % CD % CD % CD % CD45+ mostly CD45+/- usually CD19+ >70% CD19- >95% CD % CD27+/ % CD56 - > 85% CD % CD20-100% CD % CD % CD % CD28-100% CD % CD81 bright CD81- Upto 70% CD200 Variable CD200 Stable Polyclonal 100% Monoclonal 100% Rowstron AC et al. EMN report. Haematologica 2008 Mar; 93(3):

10 Up to 50% CD19 can be negative in npcs Up to 25% CD56 can be positive in npcs CD45 is of little value in diff of apcs/npcs Paiva B et al. Cytometry Part B 2010;78B: Prashant Tembhare et al Leukemia Research 38 (2014) Peceliunas V et al. Part B 2011; 80B: Prashant Tembhare et al Leukemia Research 38 (2014) Some % of normal PCs can show abnormal expression of markers routinely used for immunophenotyping of PCs Hence routine markers like CD19, CD45, CD56 along with CD38 & CD138 are not enough may cause false positive results Light chain restriction is efficient but technically challenging Hence new markers like CD27, CD28, CD81, CD117 and CD200 are needed.

11 Normal Plasma cells - Immunophenotyping

12 Rapid, sensitive and specific Unusal morphology Reactive plasmacytosis B-NHL with Plasmacytic differentiation

13

14

15 % of Plasma cells in all nucleated cells = 38% with 99.9% abnormal clonal PCs

16 CD28 FITC 8c iclambda FITC 8c iclambda FITC CD19 APC CD19 APC CD56 PC7 SF bm_05_b-8.fcs FSC SSC PCs viability gate SF bm_05_b-8.fcs FSC SSC PCs viability SF12 gate 1393 bm_05_b-8.fcs FSC SSC PCs viability gate 0.00% 1393 bm_05_b-8.fcs FSC SSC PCs viability SF12 gate 1393 bm_05_b-8.fcs FSC SSC PCs viability SF12 gate 1393 bm_11_b-9 ic L K.fcs 8c B 19APC+38+ SF bm_11_b-9 ic L K.fcs CD19 neg PC 92.00% % % SF bm_05_b-8.fcs SINGLETS c PC 38v percp CD38 V % CD19 APC MM with CD28 expression % 0.91% 10 4 CD19 neg PCs tube B % 43.64% 54.73% CD45 V % 1.64% CD19 APC 0.27% % 0.36% % 0.36% CD20 AH % 2.41% 2.96% c ickappa PE 57.49% % 98.36% 1.64% CD19 APC 99.25% 0.34% 0.34% 0.07% c ickappa PE

17 MGUS vs MM immunophenotypic signature MGUS & SMM TTP to MM Good MM vs Bad MM new prognostic markers Quantitation clonal PCs in MM Prediction of Treatment response MRD Circulating PCs Role in prognostication Role in Plasmacytoma Role in Amyloidosis

18 MGUS -3% of population > 50 yrs of age Progress to MM with a 1% to 1.5% risk per year Very few PCs and difficult to establish clonality by BM Bx Many studies % of APCs in MGUS < 95% in 80-85% cases % of APCs in MM > 95 in 85-90% cases Complete negative CD45 - SMM & MM Strong CD56 expression SMM & MM Prashant Tembhare et al. Leukemia Research 38 (2014) Grassier M et al. Ann Biol Clin (Paris) May-Jun;71(3): Carulli G et al. Clin Ter. 2012;163(5):

19

20

21

22 Risk factor associated with progression of MGUS SMM-MM % of abnormal PCs Abnormal PCs > 95% - high risk Abnormal PCs < 95% - low risk Perez-Persona et al, Blood 2007, 110: Ratio of abnormal to normal plasma cells (A/N ratio) > 4 high risk a/w with probability of progression to MM of 35% at 5 yrs < 4 low risk a/w with probability of progression to MM of 3% at 5 yrs Jerez A et al. Ann Med. 2009;41: Aneuploidy DNA cycle analysis by FCM Perez-Persona et al, Blood 2007, 110:

23 In MGUS high risk factors for progression 95% or greater of apc/bmpc by flow DNA aneuploidy by flow Blood. 2007;110:

24 Prognostication Markers like expression of CD19, CD81, CD28 & CD117, CD200 prognosis CD117 - longer PFS and OS CD28 - Increased proliferative activity, greater BM & organ/tissue involvement high-risk cytogenetic combination of CD28+ & CD117- : shorter PFS CD19 positivity poor prognosis CD81 positivity poor prognosis Mateo et al J Clin Oncol : Paiva et al. Leukemia (2012) Hyperdiploidy (DNA content >1.06) - better clinical outcome San Miguel JF, et al. Leuk Lymphoma.1996;23: S-phase > 2% - Shorter disease-free and overall survival San Miguel JF et al, Blood. 1995;85:

25 CD19+ MM vs CD19- MM CD28-CD117+ MM CD28-CD117- MM & CD28+CD117+ MM CD28+CD117- MM Mateo et al J Clin Oncol :

26 Leukemia (2012)

27 Med G1 PCs = Med G1 Ly = DI = 1.32 Tembhare P et al. Cytometry A Dec 15.

28 Paiva B et al GEM/PETHEMA group MM > 5% normal PCs - Longer PFS & OS MM < 5% normal PCs - Shorter PFS & OS

29 MM < 95% APCs Good Prognosis

30 Blood :

31 Leukemia (2013) 27,

32

33

34 Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation Paiva et al on behalf of GEM/PETHEMA

35 Determining response: IMWG response criteria Consensus recommendations for the uniform reporting of clinical trials: report of the IMW Consensus Panel 1 IMWG response criteria Sr/Ur M-protein by electrophoresis & IFX, % APCs in BM by IHC or FCM Sr free light chains (sflc ratio)

36 MRD in MM >/= 0.01% APCs Post transplant Day 100 MRD MRD highly predictive of treatment response and clinical out-come British Journal of Haematology, 128, , Paiva B et al. Blood 2008;112: San Migual JF Haematologica 2005; 90: Rowstron AC et al. J Clin Oncol 31:

37 MRD = 0.18%

38 Circulating PCs detected by slide immunofluorescence risk factor in newly diagnosed MM (Blood. 1996;88: ) shortened progression-free survival in SMM (BJH. 1994;87: ) indicates high risk MGUS & SMM (JCO. 2005;20;23: & Leukemia. 2013;27:680-5) Slide-based immunofluorescence assay - complex and time & labor intensive, and requires fluorescence microscopy. Flow cytometric analysis is rapid and readily available and good correlation with the immunofluorescence-based method. (Rawston et al BJH1997;97:46-55.)

39 1997: Rowstron et al - Br J Haematol Apr;97(1): Absolute numbers CPC decreased - responding to treatment, remained elevated - refractory disease, increased - undergoing relapse. CPCs was independent of β2- microglobulin, albumin, and C- reactive protein except a weak correlation between the degree of BM involvement

40 Gonsalves et al - Leukemia Oct;28(10): (Mayo) 400 cpcs / events was associated with - higher plasma cell (PC) proliferation and - adverse cytogenetics. - High median time-to-next-treatment and overall survival (OS) Gonsalves et al Br J Haematol Nov;167(4): Quantification of clonal circulating plasma cells in relapsed multiple myeloma

41 solitary lytic lesion of plasma cells on skeletal survey in an otherwise asymptomatic patient. A bone marrow examination from an uninvolved site contains less than 10% of abnormal plasma cells. Median follow-up of 3.7 years, 28 of 50 patients have progressed with a median TTP of 18 months. Progression was documented in 72% of patients with apcs vs 12.5% without apcs median TTP, 26months vs not reached; p = Blood. 2014;124(8):

42

43

44

45 Flow cytometric immunophenotyping is extremely useful in Diagnosis Treatment response monitoring Prognostication of MM and precursor conditions as well as other plasma cell neoplasms

46

47 One Year Advanced flow cytometry and molecular training certified course for technologist/technicians