definition of leukemias of large granular lymphocytes (lgl ... · -hydroa vacciniforme like...

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

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 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


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

2008

2016

• T-LGL leukemia• Aggressive NK-cell leukemia

• T-LGL leukemia• Chronic lymphoproliferative disorders of NK-cells• Aggressive NK-cell leukemiaW

HO

m

atu

reT-

and

NK

-cel

ln

eop

lasm

s

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 withSTAT3 and STAT5b mutations,

later (preliminarily) associatedwith more clinically aggesive

disease


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


A definitive diagnosis of LGL leukemia requires finding evidence of an

expanded clonal T- or NK-cell LGL proliferation









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►

►


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

No

rmal

PB


CD4/CD8 ratio = 2

CD20 + CD4 - PacBCD20 + CD4 - PacB CD5 - PerCP-Cy5.5

CD

3 -

AP

C

CD

8 +

anti

-sIgl

-FI

TC

CD

8 +

anti

-sIgl

-FI

TC


CD

3 -

AP

C

CD

8 +

anti

-sIgl

-FI

TC

CD

8 +

anti

-sIgl

-FI

TC

T-C

LPD

PB

Only mature lymphoid cells are shown

23/10/2017

4

Rapid immunophenotypic screening of T/NK lymphocytosis

No

rmal

PB

CD4/CD8 ratio = 2


CD

3 -

AP

C

CD

8 +

anti

-sIgl

-FI

TC

CD

8 +

anti

-sIgl

-FI

TC


CD

3 -

AP

C

CD

8 +

anti

-sIgl

-FI

TC

CD

8 +

anti

-sIgl

-FI

TC

T-C

LPD

PB

CD4/CD8 ratio = 0.32

Only mature lymphoid cells are shown

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


activated effector cytotoxic cells (g9+d2+)

Tgd LGLCD4+ T cellsCD8+ T cells


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

CD7 11

CD16 11

CD27 10

CD28 9

CD45RO 8

HLADR 7

CD45RA 18

CD57 16

CD45RO 13

CCR7 7

CD27 6

CD26 5

CD45RO 18

CD45RA 16

CD28 8

CyGranzyme 7

CD26 6

CCR7 4

CyGranzyme 9

CD57 9

CD94 9

CD16 9

CD28 7

CD45RA 7

Single clonal CD8+ T-CLPD cases

vs. normal CD8+ T-cells

Single clonal CD4-/CD8-/lo

T-CLPD cases

vs. normal CD4-/CD8-/lo T-cells

PC1

PC

2

PC1

PC1

PC

2

PC1

PC

2P

C2

EuroFlow T-CLPD panel: evaluation by PCA

Leukemia 2012; 26, 1908–1975

23/10/2017

5

Clonal CD56+ NK-cell cases Clonal CD56-/d NK-cell cases

Comparison of individual clonal CLPD-NK cases vs. normal NK-cells

Clonal CD56+ NK-cell cases Clonal CD56-/d NK-cell cases

CD56:PE Cy7 22.49

CD16:PacB 10.21

CD26:PE 10.13

HLADR:PacB 9.49

CD11c:APC 8.96

CD94:APC 7.32

CD56:PE Cy7 26.73

HLADR:PacB 14.62

CD7:FITC 14.09

CD94:APC 13.98

CD2:PB 7.58

cyPerforin:FITC 6.82

CD57:FITC 25.97

CD11c:APC 16.52

CD2:PacB 14.37

CD56:PE Cy7 13.63

CD16:PacB 6.45

HLADR:PacB 4.67

CD57:FITC 28.00

cyGranzB:PE 13.40

cyPerf:FITC 11.37

CD94:APC 7.42

CD56:PE Cy7 6.34

CD2:PacB 5.81

CD11c:APC 25.46

cyGranzB:PE 16.48

cyPerf:FITC 10.53

CD57:FITC 10.04

CD2:PacB 8.82

HLADR:PacB 8.62

CD57:FITC 37.03

cyPerf:FITC 11.19

cyGranzB:PE 8.96

CD94:APC 7.74

CD56:PE Cy7 7.22

CD7:FITC 4.45

HLADR:PacB 11.02

CD16:PacB 10.97

CD26:PE 10.94

CD94:APC 9.34

CD11c:APC 8.64

cyPerf:FITC 7.89

CD94:APC 19.00

HLADR:PacB 18.72

cyPerf:FITC 13.70

CD2:PacB 11.22

CD57:FITC 5.12

CD56:PECy7 5.00

CD57:FITC- 15.73

CD11c:APC 11.98

CD2:PacB 10.86

CD56:PE Cy7 9.91

HLADR:PacB 9.13

CD7:FITC 7.72

CD56:PE Cy7 25.85

CD7:FITC 12.08

CD57:FITC 11.31

cyGranzB:PE 10.09

HLADR:PacB 7.42

CD94:APC 7.28

CD57:FITC 32.83

cyGranzB:PE 12.52

CD7:FITC 10.30

CD56:PE Cy7 7.91

CD2:PacB 7.85

CD11c:APC 4.27

CD56:PE Cy7 27.10

CD7:FITC 16.38

CD57:FITC 15.90

cyGranzB:PE 8.16

CD94:APC 7.15

HLADR:PacB 3.83

g

PC1

PC

2

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


A definitive diagnosis of LGL leukemia requires finding evidence of an

expanded CLONAL T- or NK-cell LGL proliferation









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)

TCRαβ

CD4-

/CD8++

TCRαβ

CD4++/

CD8-/+d

TCRαβ

CD4++/

CD8-

TCRαβ

CD4++/

CD8++

TCRαβ

CD4-

/CD8-/+d

TCRγδ

CD4-

/CD8-/+d

CD56-/+d CD56+ CD56++

n=37 (55) n=10 (12) n=9 (18) n=1 (1) n=3 (3) n=14 (18) n=8 (11) n=12 (12) n=1 (2)

CLONAL

LGL n=66 (78) 29 (35) 10 (12) 1 (1) 2 (2) 11 (12) 6 (9) 6 (6) 1 (1)

No LGL* n=11 (13) 1 (2) 8 (8) 1 (1) 1 (2)

OLIGOCLONAL LGL n=4 (6) 3 (4) 1 (2)

POLYCLONAL

LGL Lymphocytosis

n=13 (13)4 (4) 1 (1) 2 (2) 6 (6)

Immunodeficiency

n=1 ( 2)(1) 1 (1)

Negative ctrols (cell

subsets)** (20)(9) (9) (1) (1)

CASES n= 95

(CELL POPULATIONS n= 132)

* 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


αβ CD4-/CD8++ 3/29 (10%) 10/35 (29%) 13/35 (37%) 0/35

αβ CD4++/CD8-/+d 0/6 0/12 0/12 1/12 (8%)

αβ CD4++/CD8++ 0/1 1/1 (100%) 1/1 (100%) NA

αβ CD4-/CD8-/+d 0/2 1/2 (50%) 1/2 (50%) 0/2

gδ CD4-/CD8-/+d 2/11 (18%) 4/12 (33%) 6/12 (50%) 0/11

TOTAL 5/49 (10%) 16/61 (26%) 21/61 (34%) 1/59 (2%)

CD56-/+d 0/5 2/8 (25%) 2/8 (25%) 0/8

CD56+ 0/6 1/6 (16,7%) 1/6 (17%) 0/6

CD56++ 0/1 0/1 0/1 0/1

TOTAL 0/12 3/15 (20%) 3/15 (20%) 0/15

CLO

NA

L LG

L T-

CEL

L P

OP

ULA

TIO

NS

C

LON

AL

LGL

NK

-CEL

L P

OP

ULA

TIO

NS

STAT3 gene mutationsSTAT5b gene

mutations

Ex20 Ex21 Ex21 or 21


αβ CD4-/CD8++ 3/29 (10%) 10/35 (29%) 13/35 (37%) 0/35

αβ CD4++/CD8-/+d 0/6 0/12 0/12 1/12 (8%)

αβ CD4++/CD8++ 0/1 1/1 (100%) 1/1 (100%) NA

αβ CD4-/CD8-/+d 0/2 1/2 (50%) 1/2 (50%) 0/2

gδ CD4-/CD8-/+d 2/11 (18%) 4/12 (33%) 6/12 (50%) 0/11

TOTAL 5/49 (10%) 16/61 (26%) 21/61 (34%) 1/59 (2%)

CD56-/+d 0/5 2/8 (25%) 2/8 (25%) 0/8

CD56+ 0/6 1/6 (16,7%) 1/6 (17%) 0/6

CD56++ 0/1 0/1 0/1 0/1

TOTAL 0/12 3/15 (20%) 3*/15 (20%) 0/15

CLO

NA

L LG

L T-

CEL

L P

OP

ULA

TIO

NS

C

LON

AL

LGL

NK

-CEL

L P

OP

ULA

TIO

NS


mutations


22/61 (36%)

*2m/1f

2 mut (n=2)


αβ CD4-/CD8++ 3/29 (10%) 10/35 (29%) 13/35 (37%) 0/35

αβ CD4++/CD8-/+d 0/6 0/12 0/12 1/12 (8%)

αβ CD4++/CD8++ 0/1 1/1 (100%) 1/1 (100%) NA

αβ CD4-/CD8-/+d 0/2 1/2 (50%) 1/2 (50%) 0/2

gδ CD4-/CD8-/+d 2/11 (18%) 4/12 (33%) 6/12 (50%) 0/11

TOTAL 5/49 (10%) 16/61 (26%) 21/61 (34%) 1/59 (2%)

CD56-/+d 0/5 2/8 (25%) 2/8 (25%) 0/8

CD56+ 0/6 1/6 (16,7%) 1/6 (17%) 0/6

CD56++ 0/1 0/1 0/1 0/1

TOTAL 0/12 3/15 (20%) 3/15 (20%) 0/15

CLO

NA

L LG

L T-

CEL

L P

OP

ULA

TIO

NS

C

LON

AL

LGL

NK

-CEL

L P

OP

ULA

TIO

NS


mutations


22/61 (36%)

None of the polyclonal cell populations, either from reactive lymphocytosis (n=13)

or residual normal T-cell subsets frompatients (n=20) showed STAT mutations

Normal and clonal CD8+ LGL cells show a similar (overlapping) phenotype (EF T-

CLPD panel)

LGL CD8+ Cases carrying STAT3 mutations

STAT3 mutated and non-mutated clonal CD8+ LGL cells also show an overlapping

phenotype (EF T-CLPD panel)

The only presence of clonal LGL cells in PB of otherwise healthy subjects do not mean that

the patient has a true leukemia

Persistent oligoclonal/clonal expansions of LGL in the elderly

Oligoclonal/clonal expansions of LGL induced by CMV and otherviruses

Transient Oligoclonal/clonal expansions of LGL after allogeneic HSCT, and solid organ (heart and renal) transplantation

Transient Oligoclonal/clonal expansions of LGL with treatment withdesatinib

T-LGL expansions are also found in patients with B-cell CLPD (MGUS, MBL, CLL, FL) and polyclonal hyper- or hypo-gammaglobulinemia

The hypothesis of chronic antigen stimulation has been proposed to be involved in the pathogenesis of oligoclonal/clonal LGL expansions

Lamy et al, Blood 2017

Clinical features of clonal T/NK LGL cases with and without STAT3 or STAT5b somatic mutations

Clonal LGL “mutated”

cases

(n=23)

Clonal LGL

“non-mutated” cases

(n=43)

P-value

Sex (male/female) 10/13 (44%/56%) 14/29 (33%/67%) NS

Age (years)* 65 (40-90) 66 (6-92) NS

Physical examination

Adenomegalies 2/16 (13%) 3/20 (15%) NS

Splenomegaly 4/18 (22%) 3/21 (14%) NS

Hepatomegaly 2/17 (12%) 2/21 (10%) NS

Skin lesions 0/14 (0%) 2/18 (11%) NS

Associated diseases

Other clonal-associateddiseases

2/13 (15%)a 6/18 (33%)b NS

Autoimmune diseases (other than cytopenias)

5/21 (24%) 1/20 (5%) NS

Deaths 0/11 (0%) 3/19 (16%) NS

Results expressed as number of cases (percentage) or * median (minimum-maximum values). NS: p-value >0.05

a CLL & MBL; b CLL, MBL, MBL, MGUS, mastocytosis & gastric carcinoma

23/10/2017

7

clonal LGL “mutated”

cases

(n=23)

Clonal LGL “no

mutated” cases

(n=41)

P-value

Absolute count of blood cells

Leucocytes (x109/L)* 6.4 (2-13.4) 7.9 (3.1-55) 0.05

Lymphocytes (x109/L)* 3.3 (0.3-10.4) 4.8 (1.1-49.5) NS (0.06)

Neutrophils (x109/L)* 1.5 (0.17-4) 2.6 (0.73-11) <0.0001

Eosinophils (x109/L)* 0.06 (0.01-0.37) 0.11 (0.01-0.3) 0.05

Hemoglobin (g/dL)* 13 (8.5-18.2) 13.4 (9-18.3) NS

Platelets (x109/L)* 227 (25-421) 236 (98-383) NS

Clonal LGL cells (x109/L)* 0.8 (0.07-4.4) 1.9 (0.05-50) NS

Leucocytosis (>10x109/L) 2/22 (18%) 12/35 (34%) NS

Lymphocytosis (>3.5x109/L) 11/22 (50%) 25/34 (74%) NS (0.09)

Cytopenias

Neutropenia (<1x109/L) 8/22 (36%) 1/34 (3%) 0.02

Severe neutropenia (<0.5x109/L) 3/22 (14%) 0/34 (0%) 0.05

Anemia (<10 g/dL) 1/20 (5%) 2/33 (6%) NS

Thrombocytopenia (<100x109/L) 1/18 (6%) 1/31 (3%) NS

Peripheral blood counts of clonal T/NK LGL cases with and without STAT3 or STAT5b somatic mutations



cases

(n=13)

Clonal LGL “no

mutated” cases

(n=16)

P-value


Leucocytes (x109/L)* 6.5 (2.7-13.4) 8 (3.1-28) NS (0.07)

Lymphocytes (x109/L)* 3 (1.6-10.4) 5.6 (1.1-18) NS

Neutrophils (x109/L)* 1.6 (0.17-4) 2.2 (0.7-7.5) 0.03

Eosinophils (x109/L)* 0.6 (0.01-0.13) 0.08 (0.03-0.23) 0.03

Hemoglobin (g/dL)* 13.2 (11.4-18.2) 13.8 (9-18.3) NS

Platelets (x109/L)* 201 (121-421) 212 (98-383) NS

Clonal LGL cells (x109/L)* 0.82 (0.24-4.4) 0.61 (0.05-13) NS


Lymphocytosis (>3.5x109/L) 6/13 (46%) 11/14 (79%) NS

Cytopenias

Neutropenia (<1x109/L) 5/13 (39%) 1/14 (7%) NS (0.08)

Severe neutropenia (<0.5x109/L) 1/13 (8%) 0/14 (0%) NS

Anemia (<10 g/dL) 0/13 (0%) 2/14 (14%) NS

Thrombocytopenia (<100x109/L) 0/13 1/14 (7%) NS

Peripheral blood counts of clonal CD8+ TCRab LGL cases with and without STAT3 or STAT5b somatic mutations



cases

(n=6)

Clonal LGL “no

mutated” cases

(n=6)

P-value


Leucocytes (x109/L)* 5 (2-10) 7.2 (4-8) NS

Lymphocytes (x109/L)* 1.2 (0.3-5.3) 3.1 (2-3.7) NS

Neutrophils (x109/L)* 1.4 (0.3-1.9) 3.6 (2.6-3.8) 0.03

Eosinophils (x109/L)* 0.04 (0.01-0.4) 0.1 (0.02-0.18) NS

Hemoglobin (g/dL)* 12 (8.5.12.8) 12.5 (11-14.2) NS

Platelets (x109/L)* 231 (25-256) 302 (250-314) NS (0.08)

Clonal LGL cells (x109/L)* 0.62 (0.07-1.2) 0.24 (0.07-0.4) NS


Lymphocytosis (>3.5x109/L) 2/5 (40%) 1/4 (25%) NS

Cytopenias

Neutropenia (<1x109/L) 2/5 (40%) 0/4 (0%) NS

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

<2x109 clonal LGLs/L PLUSunexplained cytopenias, clinical evocative contextor STAT3/5b mutations

Modified from Lamy et al, Blood 2017, and van Dongen et al, Leukemia 2012

Concluding remarks: Proposal of an algorithm for the diagnosis of LGL leukemia

Clinico-biological contextLymphocytosis, atypical lymphocytes, hepato/splenomegaly, LN enlargement, unexplained cytopenias, eosinophilia, autoinmune conditions

Blood smearexaminationEF LST tube

Identification of an expanded and/or aberrant and/or suspected population of LGLs? YES

CONFIRMATION OF CLONALITY: TCRVb repertorie analysis by FCM and/or PCR*

* Previous step of FACSorting may be required

Polyclonal

Reactive LGL proliferation

Check viral infections(CMV, EBV, HIV) and

other reactive conditions (Trx, post-splecnectomy, etc.)

Monoclonal (oligoclonal?): EXTENDED PHENOTYPE (EF T-CLPD PANEL) & ifthe LGL phenotype is confirmed:(recommended) STAT mutations analyses*

“True” T-LGL leukemia / CLPD-NK

>2x109 persistentclonal LGLs/L

<2x109 (particularly0.5-1x109/L)

clonal/oligoclonalLGLs/L w/o cytopenias,

no STAT mut

No “true” LGL leukemia(“Monoclonal T-cell lymphocytosis”)

Periodic (annual?) evaluation

Prognostic value of STAT mutation has to be validated

EuroFlow consortium aims at innovation in flow cytometry

www.euroflow.org

FCM (the 8-color EuroFlow strategy) contributes to establish the T-cell lineage of T-CLPD, to distinguish normal/reactive from

clonal/aberrant T cells and to a more precise classification into WHO categories

THE CIC/USAL-IBSAL TEAM

23/10/2017

8

1% of leucocytes

TCRgd+ T-CELL LYMPHOCYTOSIS TCRgd+ T-CELL LYMPHOCYTOSIS OF UNCERTAIN SIGNIFICANCE?

definition of leukemias of large granular lymphocytes (lgl ... · -hydroa vacciniforme like...

Documents