23/10/2017 1 Refining the diagnosis of clonal disorders of LGL Cancer Research Center (IBMCC), Department of Medicine and Service of Cytometry University of Salamanca & IBSAL - Salamanca, Spain ADVANCED HEMATOLOGY – ONCOLOGY SESSION. ESCCA CONFERENCE 2017. Thessaloniki (Greece) 24-27 September 2017 Definition of leukemias of Large Granular Lymphocytes (LGL) Initial description by Loughran et al: A rare chronic mature LPD of the T/NK cell lineage (Ann Intern Med. 1985; 102: 169) • Splenomegaly • Multiple autoantibodies with neutropenia • Lymphocytosis of LGL Loughran: 1985 1993 • Patients with T-LGL leukemia have clonal proliferations of CD3' LGL typically associated with chronic neutropenia and autoimmune features • NK-LGL leukemia is characterized by clonal CD3- LGL proliferation with an acute clinical presentation marked by massive hepatosplenomegaly and systemic illness. However, most patients with increased numbers of CD3- LGL do not have clinical features of NK-LGL leukemia and have a chronic clinical course. (Blood 1993; 82: 1) Two subtypes of LGL disorders were proposed: - T-LGL leukemia - Aggressive NK-cell leukemia Definition of leukemias of Large Granular Lymphocytes (LGL) Initial description by Loughran et al: A rare chronic mature LPD of the T/NK cell lineage (Ann Intern Med. 1985; 102: 169) • Splenomegaly • Multiple autoantibodies with neutropenia • Lymphocytosis of LGL Loughran: 1985 1993 • Patients with T-LGL leukemia have clonal proliferations of CD3' LGL typically associated with chronic neutropenia and autoimmune features • NK-LGL leukemia is characterized by clonal CD3- LGL proliferation with an acute clinical presentation marked by massive hepatosplenomegaly and systemic illness. However, most patients with increased numbers of CD3- LGL do not have clinical features of NK-LGL leukemia and have a chronic clinical course. (Blood 1993; 82: 1) 2001 Two subtypes of LGL disorders were proposed: - T-LGL leukemia - Aggressive NK-cell leukemia 2008 2016 • T-LGL leukemia • Aggressive NK-cell leukemia • T-LGL leukemia • Chronic lymphoproliferative disorders of NK-cells • Aggressive NK-cell leukemia WHO mature T- and NK-cell neoplasms Provisional entity in italics • T-LGL leukemia • Chronic lymphoproliferative disorders of NK-cells • Aggressive NK-cell leukemia Recognition of the 1993 classification scheme proposed by Loughran Mature T- and NK-cell neoplasms: WHO CLASSIFICATION (revised 2016) - T-cell prolymphocytic leukemia - T-cell LGL leukemia - Chronic LPD of NK cells - Agressive NK-cell leukemia - Systemic EBV+ T-cell lymphoma of childhood* - Hydroa vacciniforme-like lymphoproliferative disorder* - Adult T-cell leukemia/lymphoma - Extranodal NK/T-cell lymphoma, nasal type - Enteropathy-associated T-cell lymphoma - Monomorphic epitheliotropic intestinal T-cell lymphoma* - Indolent T-cell LPD of the GI tract* - Hepatosplenic T-cell lymphoma - Subcutaneous panniculitis-like T-cell lymphoma - Mycosis fungoides - Sezary syndrome - Primary cutaneous CD30+ T-cell LPD - Lymphomatoid papulosis - Primary cutaneous anaplastic large cell lymphoma - Primary cutaneous gamma-delta T-cell lymphoma - Primary cutaneous CD8+ agressive epidermotropic cytotoxic T-cell lymphoma - Primary cutaneous acral CD8+ T-cell lymphoma* - Primary cutaneous CD4+ small/medium T-cell LPD* - Peripheral T-cell lymphoma not otherwise specified (NOS) - Angioimmunoblastic T-cell lymphoma - Follicular T-cell lymphoma* - Nodal peripheral T-cell lymphoma with TFH phenotype* - Anaplastic large cell lymphoma, ALK+ - Anaplastic large cell lymphoma, ALK- * - Breast implant-associated anaplastic large-cell lymphoma* Provisional entities are shown in Italics * Changes from the 2008 classification * Highlight the discovery of STAT mutations described in 2012 (Koskele et al, NEJM 2012 & Jerez et al, Blood 2012) New subtypes recognized with STAT3 and STAT5b mutations, later (preliminarily) associated with more clinically aggesive disease Definition of leukemias of Large Granular Lymphocytes (LGL) A definitive diagnosis of LGL leukemia requires finding evidence of an expanded clonal T- or NK-cell LGL proliferation Lamy et al, Blood 2017; Oshimi, Intern Med 2017 DETECTION OF INCREASED NUMBER OF CIRCULATING LGL: • INITIALLY: Persistent (> 6mo) absolute count of LGL in PB >2 x 10 9 /L (normal value <0.3 x 10 9 /L) without a clear explanation (Semenzato et al, Blood 1997; WHO 2001) • However, it is frequent to find patients with <2 x 10 9 /L of clonally expanded LGLs showing similar clinical features to those with >2 x 10 9 LGLs/L. A lower number of PB clonal LGLs may be compatible with the diagnosis if: 1.- These cells are clonal & 2.- The patient show other clinical or hematological features such as rheumatoid artritis or cytopenias of unexplaind etiology Definition of leukemias of Large Granular Lymphocytes (LGL) A definitive diagnosis of LGL leukemia requires finding evidence of an expanded clonal T- or NK-cell LGL proliferation Lamy et al, Blood 2017; Oshimi, Intern Med 2017 DETECTION OF INCREASED NUMBER OF CIRCULATING LGL: • INITIALLY: Persistent (> 6mo) absolute count of LGL in PB >2 x 10 9 /L (normal value <0.3 x 10 9 /L) without a clear explanation (Semenzato et al, Blood 1997; WHO 2001) • However, it is frequent to find patients with <2 x 10 9 /L of clonally expanded LGLs showing similar clinical features to those with >2 x 10 9 LGLs/L. A lower number of PB clonal LGLs may be compatible with the diagnosis if: 1.- These cells are clonal & 2.- The patient show other clinical or hematological features such as rheumatoid artritis or cytopenias of unexplaind etiology
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23/10/2017
1
Refining the diagnosis of clonal
disorders of LGL
Cancer Research Center (IBMCC), Department of Medicine and Service of CytometryUniversity of Salamanca & IBSAL - Salamanca, Spain
Definition of leukemias of Large Granular Lymphocytes (LGL)
Initial description by Loughran et al:A rare chronic mature LPD of theT/NK cell lineage
(Ann Intern Med. 1985; 102: 169)
• Splenomegaly
• Multiple autoantibodies with neutropenia• Lymphocytosis of LGL
Loughran:
1985
1993• Patients with T-LGL leukemia have clonal proliferations of CD3'
LGL typically associated with chronic neutropenia andautoimmune features
• NK-LGL leukemia is characterized by clonal CD3- LGL proliferationwith an acute clinical presentation marked by massivehepatosplenomegaly and systemic illness. However, mostpatients with increased numbers of CD3- LGL do not haveclinical features of NK-LGL leukemia and have a chronic clinicalcourse.
(Blood 1993; 82: 1)
Two subtypes of LGL disorderswere proposed: - T-LGL leukemia- Aggressive NK-cell leukemia
Definition of leukemias of Large Granular Lymphocytes (LGL)
Initial description by Loughran et al:A rare chronic mature LPD of theT/NK cell lineage
(Ann Intern Med. 1985; 102: 169)
• Splenomegaly
• Multiple autoantibodies with neutropenia• Lymphocytosis of LGL
Loughran:
1985
1993• Patients with T-LGL leukemia have clonal proliferations of CD3'
LGL typically associated with chronic neutropenia andautoimmune features
• NK-LGL leukemia is characterized by clonal CD3- LGL proliferationwith an acute clinical presentation marked by massivehepatosplenomegaly and systemic illness. However, mostpatients with increased numbers of CD3- LGL do not haveclinical features of NK-LGL leukemia and have a chronic clinicalcourse.
(Blood 1993; 82: 1)
2001
Two subtypes of LGL disorderswere proposed: - T-LGL leukemia- Aggressive NK-cell leukemia
- Lymphomatoid papulosis- Primary cutaneous anaplastic large cell lymphoma
- Primary cutaneous gamma-delta T-cell lymphoma- Primary cutaneous CD8+ agressive epidermotropic cytotoxic T-cell lymphoma- Primary cutaneous acral CD8+ T-cell lymphoma*- Primary cutaneous CD4+ small/medium T-cell LPD*- Peripheral T-cell lymphoma not otherwise specified (NOS)- Angioimmunoblastic T-cell lymphoma- Follicular T-cell lymphoma*- Nodal peripheral T-cell lymphoma with TFH phenotype*- Anaplastic large cell lymphoma, ALK+- Anaplastic large cell lymphoma, ALK- *- Breast implant-associated anaplastic large-cell lymphoma* Provisional entities are shown in Italics
* Changes from the 2008 classification
* Highlight the discovery of STAT mutations described in
2012(Koskele et al, NEJM 2012 &
Jerez et al, Blood 2012)
New subtypes recognized withSTAT3 and STAT5b mutations,
later (preliminarily) associatedwith more clinically aggesive
disease
Definition of leukemias of Large Granular Lymphocytes (LGL)
A definitive diagnosis of LGL leukemia requires finding evidence of anexpanded clonal T- or NK-cell LGL proliferation
Lamy et al, Blood 2017; Oshimi, Intern Med 2017
DETECTION OF INCREASED NUMBER OF CIRCULATING LGL:
• INITIALLY: Persistent (> 6mo) absolute count of LGL in PB >2 x 109/L (normalvalue <0.3 x 109/L) without a clear explanation (Semenzato et al, Blood 1997; WHO 2001)
• However, it is frequent to find patients with <2 x 109/L of clonally expanded
LGLs showing similar clinical features to those with >2 x 109 LGLs/L.
A lower number of PB clonal LGLs may be compatible with the diagnosis if:
1.- These cells are clonal &
2.- The patient show other clinical or hematological features suchas rheumatoid artritis or cytopenias of unexplaind etiology
Definition of leukemias of Large Granular Lymphocytes (LGL)
A definitive diagnosis of LGL leukemia requires finding evidence of an
expanded clonal T- or NK-cell LGL proliferation
Lamy et al, Blood 2017; Oshimi, Intern Med 2017
DETECTION OF INCREASED NUMBER OF CIRCULATING LGL:
• INITIALLY: Persistent (> 6mo) absolute count of LGL in PB >2 x 109/L (normalvalue <0.3 x 109/L) without a clear explanation (Semenzato et al, Blood 1997; WHO 2001)
• However, it is frequent to find patients with <2 x 109/L of clonally expanded
LGLs showing similar clinical features to those with >2 x 109 LGLs/L.
A lower number of PB clonal LGLs may be compatible with the diagnosis if:
1.- These cells are clonal &
2.- The patient show other clinical or hematological features suchas rheumatoid artritis or cytopenias of unexplaind etiology
23/10/2017
2
How can multiparametric
flow cytometry
help for
diagnosis and classification
of LGL leukemia/lymphocytosis?
Sequential FCM strategy to diagnose and characterize T-CLPD
Leukemia 2012; 26, 1908–1975 (van Dongen et al on behalf of EuroFlow)
Lymphocytosis Screening Tube (LST)
Aim: DIAGNOSTIC SCREENING OF B, T & NK CELL CLPD
to identify the cell lineage involved (in the lymphocytosis), to detect the
most common aberrant phenotypes (CD5), and to assess B-cell clonality
Pacific Blue
PacificOrange
FITC PEPerCP Cy5.5
PECy7 APC APC-H7
CD20
CD4CD45
CD8
Anti-Igl
CD56
Anti-IgkCD5
CD19
Anti-TCRgdSmCD3 CD38
Markers aiming at identification of B-lineage cells, common B-cell aberrancies and toassess B-cell clonality
Markers aiming at the identification of T cells and T-cell subsets:.- TCRgd vs TCRab (non-TCRgd).- CD4+/CD8-; CD4-/CD8+; CD4+/CD8+; CD4-/CD8-; CD56+ subsets
and T-cell aberrancies
►
►
Markers aiming at identification of NK cells, and NK-cell subsets
Markers aiming at identification of non-hematopoietic cells►
►
Lymphocytosis Screening Tube (LST)
Aim: DIAGNOSTIC SCREENING OF B, T & NK CELL CLPD
to identify the cell lineage involved (in the lymphocytosis), to detect the
most common aberrant phenotypes (CD5), and to assess B-cell clonality
Pacific Blue
PacificOrange
FITC PEPerCP Cy5.5
PECy7 APC APC-H7
CD20
CD4CD45
CD8
Anti-Igl
CD56
Anti-IgkCD5
CD19
Anti-TCRgdSmCD3 CD38
Markers aiming at identification of B-lineage cells, common B-cell aberrancies and toassess B-cell clonality
Markers aiming at the identification of T cells and T-cell subsets:.- TCRgd vs TCRab (non-TCRgd).- CD4+/CD8-; CD4-/CD8+; CD4+/CD8+; CD4-/CD8-; CD56+ subsets
and T-cell aberrancies
►
►
Markers aiming at identification of NK cells, and NK-cell subsets
Markers aiming at identification of non-hematopoietic cells►
►
Normal Large Granular Lymphocytes (LGL) belong to diverse T/NK-cell lineages
TCRgd+ T-cellsTCRab+ T-cells NK-cells
CD3-
CD4-
CD8-/lo
CD3+/hi
CD4-
CD8-/lo
CD3+
CD4-
CD8+
CD3+
CD4-
CD8-
CD3+
CD4+
CD8-/lo
EARLY EFFECTORS
CENTRAL MEMORY
CCR7:PE-LOGICAL
CD45RA:APC-LOGICAL
CD
27
Phenotypic dissection of human CD8++/CD4-
TCRab T-cell maturation subsets
Courtesy: M Perez-Andres, University of Salamanca (modified)
PERIPHERALMEMORY
NAÏVE
TERMINALEFFECTORS
TRANSITIONAL MEMORY
23/10/2017
3
Phenotypic characterization of human CD8++/CD4- TCRab T-cell maturation subsets
67% of CD8+ T cells
Phenotypic characterization of human CD8-/lo/CD4- TCRgd T-cell maturation subsets
Phenotypic dissection of human CD4+ T-cellmaturation subsets
NAÏVE
CYTOTOXICEFFECTOR
CELLS
CENTRAL MEMORY
CCR7:PE-LOGICAL
CD45RA:APC-LOGICAL
CD
27
EFFECTORMEMORY
Courtesy: M Perez-Andres, University of Salamanca (modified)
3% ofCD4 T cells
Phenotypic characterization of human cytotoxic CD4+ T cells
Peripheral memory & effector cells
Phenotypic characterization of human NK cells
Rapid immunophenotypic screening of T/NK lymphocytosis
Lymphocytosis Screening Tube (LST)T-cell large granular leukemia derives fromdifferent cytotoxic T-cell counterparts and shows a phenotype of activated effector cytotoxic cells
Normal Peripheral blood (adult donor)Total T cells
CD4+ T cellsCD8+ T cells
CLONAL T cellsCD4+ T cells CD8+ T cells
Tab CD8+/CD4- LGL
T-cell large granular leukemia derives fromdifferent cytotoxic T-cell counterparts and shows a phenotype of
activated effector cytotoxic cells
No
rmal
PB
CD4+ T cells
CD8+ T cells
CLONAL T cells
CD4+ T cellsCD8+ T cells
Tab
CD
8+ /
CD
4-LG
L
T-cell large granular leukemia derives fromdifferent cytotoxic T-cell counterparts and shows a phenotype of
activated effector cytotoxic cells (g9+d2+)
Tgd LGLCD4+ T cellsCD8+ T cells
T-cell large granular leukemia derives fromdifferent cytotoxic T-cell counterparts and shows a phenotype of
activated effector cytotoxic cells
CD4+ TCRab LGL leukemia / lymphocytosis
ClonalCD4+ T cells
Normal residual CD4+ T cells
CD7 FITC
CD
28 A
PC
“Low-count CD4+ T-LGL clonal lymphocytosis” is a common finding in the elderly, without cytopenias or autoinmune phenomena (driven bt CMV) Lima, Almeida et al, Am J Pathol 2003
Leukemia 2012; 26, 1908–1975 (van Dongen et al on behalf of EuroFlow)
Comparison of individual clonal CLPD-NK cases vs. polyclonal expanded NK-cells
Definition of leukemias of Large Granular Lymphocytes (LGL)
A definitive diagnosis of LGL leukemia requires finding evidence of an
expanded CLONAL T- or NK-cell LGL proliferation
Lamy et al, Blood 2017; Oshimi, Intern Med 2017
DETECTION OF INCREASED NUMBER OF CIRCULATING LGL:
• INITIALLY: Persistent (> 6mo) absolute count of LGL in PB >2 x 109/L (normalvalue <0.3 x 109/L) without a clear explanation (Semenzato et al, Blood 1997; WHO 2001)
• However, it is frequent to find patients with <2 x 109/L of clonally expanded
LGLs showing similar clinical features to those with >2 x 109 LGLs/L.
A lower number of PB clonal LGLs may be compatible with the diagnosis if:
1.- These cells are clonal &
2.- The patient show other clinical or hematological features suchas rheumatoid artritis or cytopenias of unexplaind etiology
Assesment of T/NK clonality
EVIDENCE OF T-LGL CLONALITY:
Analysis of TCR gene rearrangement by PCR (or deep sequencing):
May require a previuos step of FACSorting to purify cells
TCRVb repertoire analysis at the protein level by FCM:
- The IOT panel covers around 75% of the TCR-Vb spectrum
- High correlation between FCM and PCR (Lima, Almeida et al, Am J Pathol 2001)
EVIDENCE OF NK-LGL CLONALITY:NK cells do not express receptors generated by gene recombination!
- Cytogenetic abnormalities
- EBV-DNA clonally incorporated to cells
- NK-cell receptor polymorphisms
- X-linked molecular studies (HUMARA assay)
There is not an universal method for assessing
NK-cell clonality
Assesment of T/NK clonality
EVIDENCE OF T-LGL CLONALITY:
Analysis of TCR gene rearrangement by PCR (or deep sequencing):
May require a previuos step of FACSorting to purify cells
TCRVb repertoire analysis at the protein level by FCM:
- The IOT panel covers around 75% of the Vb spectrum
- High correlation between FCM and PCR (Lima, Almeida et al, Am J Pathol 2001)
EVIDENCE OF NK-LGL CLONALITY:NK cells do not express receptors generated by gene recombination!
- Cytogenetic abnormalities (but only present in very few cases)
- EBV-DNA clonally incorporated to cells (only for ANKL)
- NK-cell receptor polymorphisms (it should require a large panel of Ab)
- X-linked molecular studies (HUMARA assay)
There is not an universal method for assessing
NK-cell clonality(only for heterocygous females& usually requires sorting of suspected cells)
Assessment of T/NK clonality
Potential role of STAT somatic mutations
Recurrent activating somatic mutations of the STAT3 gene weredemonstrated in 28% to 40% of T-LGL leukemia and around 30%of CLPD-NK
Mutations are primarily located in exons 20 and 21 encoding the Src homology 2domain, which drives the dimerization and activation of the Stat protein
STAT mutations have being associated with more clinicallyaggressive disease, BUT association with specific clínico-
biological features remains uncertain
(Koskele et al, NEJM 2012 & Jerez et al, Blood 2012)
Activating somatic mutations of the STAT5b gene were found in CD8+ T-LGL leukemia and CLPD-NK, but at very low frequency(<2%) and in 6/11 cases of CD4+ T-LGL leukemias
(Rajala et al, Blood 2013; Anderson et al, Blood 2016)
* Diverse T-cell chronic lymphoproliferative disorders other than LGL disorders** Phenotypically normal T-cell subpopulations from patients with clonal LGL expansions
Analysis of somatic mutations in the STAT3 or STAT5b genes in FACSorted T/NK-cell populations
23/10/2017
6
Analysis of somatic mutations in the STAT3 or STAT5b genes in FACSorted T/NK-cell populations
Severe neutropenia (<0.5x109/L) 2/5 (50%) 0/4 (0%) NS
Anemia (<10 g/dL) 1/4 (25%) 0/4 (0%) NS
Thrombocytopenia (<100x109/L) 1/3 (33%) 0/4 (0%) NSResults expressed as number of cases (percentage) or * median (minimum-maximum values). NS: p-value >0.05
Peripheral blood counts of clonal TCRgd LGL cases with and without STAT3 or STAT5b somatic mutations