Persistent lymphocytosis. Persistent lymphocytosis: are there prognostic indicators? Problem. Questions. Basic markers used to identify lymphocytes

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1 Persistent lymphocytosis Persistent lymphocytosis: are there prognostic indicators? Paul R. Avery VMD, PhD, DACVP Marjorie Williams, DVM Anne C. Avery VMD, PhD Clinical Immunology Laboratory Colorado State University 12 yr old MC golden retriever Possible seizure, lymphocytes; slightly larger than a neutrophil Treated with chlorambucil/pred/vincristine 474 days after diagnosis Arrests after surgery for laryngeal paralysis yr old FS Lethargy, wt. loss. Mild splenomegaly, mes. LNs 9,159 lymphocytes Treated with CHOP protocol 113 days after diagnosis Dies at home Problem Dog presents with mild lymphadenopathy and mild to moderate lymphocytosis Circulating lymphocytes described as reactive or small to intermediate-sized Flow cytometry submission form want to determine whether leukemia or Stage V lymphoma Questions Does lymphocyte phenotype predict prognosis? Is aberrant expression of phenotypic markers associated with a worse outcome? Can we distinguish Leukemia from Stage V Lymphoma? Basic markers used to identify lymphocytes 12 yr old dog; peripheral LNs CD4 B cell CD34: precursor cells CD45: pan-leukocyte T cell CD3 CD5 CD4 or 97 lymphs/ul mature 339 CD45 CD3 1

2 6 yr old canine mix -peripheral LNs palpable, decreased Plts 98.4% 8 yr old FS Lab -routine bloodwork 93% CD5 7.4% 8, lymphocytes/ul reactive 4 CD5 CD4 93% are T cells -Only 9% express CD4 or 1.4% Study Population Cases presented to the Clinical Immunology Service for flow cytometry 3-5 Inclusion Criteria 1. >5, circulating lymphocytes 2a. Homogeneous (>%) expansion of phenotypically normal lymphocytes 2b. Expansion (>%) of phenotypically normal lymphocytes with positive PCR result for clonality 3. Expansion of lymphocytes with an aberrant phenotype 4. Expansion of CD34+/Class II- cells Study Population 2 dogs met inclusion criteria CD5 CD4/8- CD34 Misc aberrant CD4 Phenotype Distribution % 14% 6% 5% 24% 31% Study Population 89/2 had sufficient follow-up data Date of death/euthanasia/last follow-up visit Treatment data Complete Blood Count at submission PE findings at submission Broken down into 4 main categories +, +, CD5+CD4/8-, CD34+ Survival proportions CD34 CD5+4/8-2

3 CD34+ Leukemias have a very poor outcome CD34 is considered a marker of pluripotent precursor cells Does not distinguish lymphoid vs. myeloid Median survival 16 days P<.1 compared to each of the other phenotypes 9/12 treated with multi-drug chemotherapy Does aberrant expression of surface molecules predict poor outcome? CD5+ CD4-/- T cells Loss of the pan-leukocyte antigen CD45 2/25 + 5/14 CD5+CD4-/- 6/ vs CD5+4/8- CD5+4/8- P=.72 CD45 Expression CD45pos CD45neg P=.35 Median Survival: Pos= 397 days, Neg= 285 days Are there cellular characteristics within the + lymphocytosis cases that influence outcome? Possible means of distinguishing CLL from Stage V Lymphoma? Lymphocyte Forward Scatter: Large P=.36 Median Survival: Large=115 days, =undefined 3

4 Large + Lymphocytosis Survival Days Lymph Percent + Other 25 18,7 81% Abd mass, abd LNs 56 7, 81% Peripheral LNs ,5 83% ,96 % Atypical circulating lymphs 212, 81% LN/BM cytology ,426 85% Peripheral LNs ,64 94% Peripheral LNs ,724 96% ,983 95% Persistent > 1 yr , % , 89% ,529 % Persistent > 1 yr , 84% Lymphocytosis: Multiple Drugs Prednisone/Chlorambucil Prednisone alone 11% 11% Large 13% 78% 87% Lymphocyte Forward Scatter: Large P= Large P= Are there cellular characteristics within the + lymphocytosis cases that influence outcome? Addressing issues relating to the concept of Blast Crisis? Initial Lymphocyte Count >,/ul <,/ul P=.8 Median Survival: >,/ul=131 days, <,/ul=98 days >,/ul <,/ul Lymphocytosis <,/ul Survival Days Lymph PCR CD45 E.canis Other ND Neg Neg 277 ND Pos Large cells, % Pos Neg Pos Pos 6 37 ND Pos Neg Persistent ND Neg 474 Pos Neg ND Pos Sm # CD5-4 Pos Pos Neg 631 Pos Pos 5 Neg Pos Neg Large cells, 85% Pos Pos Pos Pos Neg Persistent 4

5 Lymphocytosis: <,/ul 9% Initial Lymphocyte Count 18% 55% Multiple Drugs Prednisone/Chlorambucil Prednisone alone >,/ul ne 46% 8% 18% 23% 23% >,/ul <,/ul P=.17 Initial Lymphocyte Count >,/ul <,/ul P= Initial Lymphocyte Count small cells >, cells/ul <, cells/ul P= Lymphadenopathy 12/ + Lymphocytosis 5/12 dogs had <, lymphs/ul 16/ + lymphocytosis 5/16 dogs had small circulating cells Questions Does phenotype predict prognosis? CD34 expression is associated with a poor outcome Questions Is aberrant expression of phenotypic markers associated with a worse outcome? Loss of CD45 or CD4/8 expression does not appear to be of negative prognostic significance Aids in diagnosis of lymphoproliferative dz 5

6 Additional Questions Leukemia vs Stage V Lymphoma? cell + lymphocytosis equivalent to CLL/ lymphocytic leukemia in people? Are the + Leukemias with >, cells/ul the end stage of CLL or a more aggressive form T cell lymphoma? Canine persistent lymphocytosis Immunophenotype by flow cytometry Phenotypic homogeneity + T cells E.Canis - E.Canis + Mixed population of T cells, B cells + B cells cells Indolent CLL: long survival Monitor response to Tx. DDx >Thymoma > Hypoadrenocorticism > Rare parasitic diseases (e.g. Leishmania, Spirocerca lupi) Large cells Stage V lymphoma Prolymphocytic leukemia <, /µl >, lymphocytes/µl Indolent CLL: long survival Aggressive CLL: short survival Are there clinical parameters that influence outcome? Splenomegaly: Yes P=.9 Yes P= Median: 942 days, 75 days Lymphadenopathy: Yes P=. Yes P=.5 Median: 133 days, 666 days 6