Juan F García. Linfoma de Hodgkin

Linfoma
de
Hodgkin
Juan F García
Hodgkin’s Lymphoma
Two diseases
Nodular lymphocyte predominant Hodgkin’s Lymphoma
Classical Hodgkin’s Lymphoma
•Nodular sclerosis HL
•Lymphocyte-rich classical HL
•Mixed cellularity HL
•Lymphocyte depletion HL
NLPHL
Formas clásicas de LH
•
•
Origen en células B del CG
Rasgos clínicos
• Niños y adultos jóvenes
• Estadios localizados
• Curso clínico indolente
• Puede progresar a linfoma difuso de
células grandes
Patología
• Patrón nodular
• Células tumorales de tipo L&H, con
fenotipo semejante a células B del CG
(CD20+, CD79a+, Bcl6+, OCT2+,
PAX5+, Ig+, Bcl2-,...)
• EMA+, CD30-, CD15• Background: linfocitos B maduros.
Células T CD57+
• No asociación con EBV
Origen en células B del CG
Rasgos clínicos
• Edad variable. Picos de incidencia en la
2ª y 5ª década
• Puede presentarse en estadios
avanzados
• Linfoma agresivo, de curso fatal sin
tratamiento
• Rara progresión
Patología
• Patrón variable según tipo histológico
• Células tumorales de tipo H&RS, con
ausencia de marcadores propios de
células B
• CD30+, CD15+
• Background: polimorfo: células T,
neutrófilos, eosinófilos,histiocitos, células
plasmáticas,...
• EBV 40-70%
Nodular lymphocyte predominant
Hodgkin’s Lymphoma (NLPHL)
Nodular Paragranuloma
NLPHL
Rasgos clínicos
• Niños y adultos jóvenes, estadios localizados, curso clínico indolente
• ...pero puede progresar a linfoma difuso de células grandes (confrecuencia
morfología tipo TCRBCL)
Morfología y fenotipo
• Patrón nodular: folículos irregulares con una reacción anormal del centro
germinal (transformación progresiva)
• Células tumorales de tipo L&H, con fenotipo semejante a células B del CG:
CD20+, CD79a+, Bcl6+, OCT2+, PAX5+, MUM1-/+, Ig+, Bcl2• EMA+, CD30-, CD15• Background: nódulos de linfocitos B maduros. Histiocitos. Células T CD57+
• No asociación con EBV
NLPHL
NLPHL
CD20
BCL2
IgD
OCT2
CD57
NLPHL
L&H cells
CD20
EMA
CD20
BCL6
OCT2
CD57+ TT -cells
NLPHL: pathology
• Nodular +/- diffuse pattern
• Neoplastic cells: L&H cells
• CD20+, CD79a+, BCL6+, BCL2-, OCT2+, PAX5+,
MUM1-/+, EMA+, CD30-, CD15-
• Background:
• B-cell nodules (PTGC), CD20+, IgD+, BCL2+, BCL6-,
CD10• CD57+ T-cells (rosettes).
• Histiocytes
• Variable fibrosis
Classical Hodgkin’s Lymphoma
Def.: primary lymphoid
malignancy in which tumour cells
(H&RS cells) usually represent a
minor population (<10%) within
the affected tissue, which mainly
consists of a heterogeneous
infiltrate of inflammatory cells.
• Origin: GC B-lymphocytes
• Phenotype:
• CD20-/+, CD79a-, Bcl6-, Igs-, OCT2-, PAX5+, MUM1+, Bcl2+ (30-40%)
• CD30+, CD15+ (70-80%), EBV(LMP1)+ (40-70%)
• Genetics:
• Rearranged and somatically mutated IgH genes
• Gains of 9p and 2p
CD30
> 90%
CD15
70-80%
CD79a
CD20
PAX5
BCL2
OveralSurvv
i al(Bc2
l)
1,1
1,0
,9
Bcl2
,8
,7
,6
Bcl2 +
,5
,4
,3
,2
,1
0,0
0
20
40
60
80
100
120
140
Months
Rassidakis GZ, et al. BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer
prognosis in patients treated with ABVD or equivalent regimens. Blood 2002;100(12):3935-41
Garcia JF, et al. Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle
checkpoints: analyses using tissue microarrays. Blood 2003;101(2):681-9
Brink AA, et al. Low p53 and high bcl-2 expression in Reed-Sternberg cells predicts poor clinical outcome for Hodgkin's
disease: involvement of apoptosis resistance? Mod Pathol. 1998;11:376-383
Classical HL: pathology
• Variable histological features:
subtypes NS, MC, LRCHL, LD
• Neoplastic cells: H&RS cells, and
variants: mononuclear (Hodgkin’s)
cells, lacunar cells, mummy cells.
Subtipo
Celularidad
neoplásica
EN
• Numerosas células
lacunares
• Variable número de
células de H/RS
CM
LHCRL
DL
Fondo
reactivo
Fibrosis
• Abundantes
eosinófilos
• Abundante
• Gruesos
septos que
dividen
nódulos
%
50-70%
• Numerosas células • Heterogéneo,
de H/RS y variantes
histiocitos,
eosinófilos y
• Células
células
mumificadas
plasmáticas
• Usualmente
ausente
• Escasas células de
H/RS,
mononucleares
• Nódulos de
linfocitos B
maduros
• Histiocitos
• Puede
existir
<5%
• Numerosa y
pleomórfica
• Escaso
• Ausente o
fibrosis
difusa
<5%
20-40%
Nodular Sclerosis HL
• Young females
• Mediastinum
• Usually EBV-
NS HL
H&RS + lacunar cells
CD30
Mixed Cellularity HL
• Males>Females
• Localized or
disseminated disease
• More frequent EBV+
• Common in HIV+
patients
MC HL
MC HL
Mummy cell
EBV LMP1
“Interfollicular” HL & Castleman-like features
CD30
Lymphocyte Rich
Classical HL
• Rare
• Usually stages I&II
disease
• Less aggressive
• EBV-/+
• Differential diagnosis
with NLPHL
LRCHL
LRCHL
CD20
CD30
EBV
LMP1
Lymphocyte Depleted HL
• Rare
• Older patients,
advanced stages
• Poor prognosis
• Some cases in HIV+
patients show LD
morphology
LD HL
Pathology
Neoplastic cells
Background
Fibrosis
Classical
HL
H&RS
Variants: lacunar
cells (NS),
mummy cells
(MC)
Polymorphous:
• Neutrophils
• Eosinophils
• Plasma cells
• T-cells
Broad septa in NS
Absent or Variable in MC,
LR, and LD
NLPHL
L&H
• B Lymphocytes +/• CD57+ T-cells
Phenotype
CD20
Classical
HL
NLPHL
CD79a OCT2 PAX5 MUM1 EMA CD30 CD15 EBV (LMP)
-/+
-
-/+
+
+
-/+
+
+/-
30-40%
+
+
+
+
-/+
+
-
-
-
Differential diagnosis between classical HL and DLBCL
Morphology !!!
Tumoral cells & reactive background
Phenotype
CD20 CD79a OCT2 PAX5 MUM1 BCL2 BCL6 CD30 CD15
HL
DLBCL
EBV
-/+
-
-/+
+
+
-/+
-
+
+/-
30-70%
LMP1+
+
+
+
+
-/+
+/-
+
-+
-
10-15%
LMP1-
Large B-Cell Lymphoma with
Hodgkin’s Features
Large B-Cell Lymphoma with
Hodgkin’s Features
CD20
PAX5
CD30
OCT2
CD15
CD79a
Large B-Cell Lymphoma with
Hodgkin’s Features
• ‘Grey zone’ lymphoma: neoplasms with
morphological and immunophenotypic features
intermediate between DLBCL and HL
• Usually young males with mediastinal
involvement, diagnosed at advanced stages
• Tumors with similar morphology and
immunophenotype may also present in
extramediastinal locations.
Patofisiología del Linfoma de Hodgkin
Cuestiones abiertas sobre el LH
Y la biología de la célula de H&RS
• Origen celular:
• linfocitos B del centro germinal
Rearranged and somatically mutated IgH genes in tumoral
cells of classical HL and NLPHL
Bräuninger et al, 1999a; Irsch et al, 1998; Kanzler et
al, 1996a, 1996b; Küppers et al, 1994; Marafioti et al,
1999, 2000; Müschen et al, 2000; Ohno et al, 1998;
Vockerodt et al, 1998,....
Cuestiones abiertas sobre el LH
Y la biología de la célula de H&RS
• Origen celular:
• linfocitos B del centro germinal
• Síntesis defectiva de inmunoglobulinas
• Déficit de factores de transcripción (OCT2, OCT1, BOB1)
• Mutaciones “crippling” de los genes de las Igs
CÉLULA PLASMÁTICA
CÉLULA B
PROGENITORA
IgG/A+
SELECCIÓN
ANTIGÉNICA
CÉLULA B
DE MEMORIA
IgG/A+
SIg+
HIPERMUTACIÓN
SOMÁTICA
IgM+/D+
CÉLULA B NAIVE
IGM+/D+
MANTO
IgM+/D-
IgM+/D-
Apoptosis
SIg-
Selección
negativa
CENTRO GERMINAL
Cuestiones abiertas sobre el LH
Y la biología de la célula de H&RS
• Origen celular:
• linfocitos B del centro germinal
• Síntesis defectiva de inmunoglobulinas
• Déficit de factores de transcripción (OCT2, OCT1, BOB1)
• Mutaciones “crippling” de los genes de las Igs
• Perdida de identidad linfoide B
Normal germinal centre B-cells (CD77+,
centroblasts) isolated from reactive tonsils.
CD77+
5 well characterised HL-derived cell lines:
L540
L428
HDLM2
KMH2
L1236
Two independent cultures from each line
L1236
L428
HDLM2
L540
KMH2
Gene expression profile of
Hodgkin’s Lymphoma cell lines
Gene expression profile of Hodgkin’s Lymphoma cell lines:
Inactivation of B-cell receptor signalling and B-cell differentiation program
CD22
TNFRSF17
MHC class II (B-cell maturation factor)
CD19
B-CELL RECEPTOR
CD20
CD79B LYN
CD79A
BTK
SYK
VAV
BRDG1
HCLS1
PKC
IRF4
BCL6
FOS
CD40
CD10
Cuestiones abiertas sobre el LH
Y la biología de la célula de H&RS
• Origen celular:
• linfocitos B del centro germinal
• Síntesis defectiva de inmunoglobulinas
• Déficit de factores de transcripción (OCT2, OCT1, BOB1)
• Mutaciones “crippling” de los genes de las Igs
• Perdida de identidad linfoide B
• Diferentes mecanismos de supervivencia
• Activación de NF-kappaB (CD30, EBV, JAK2, STATs, IkBa ,...)
• Programa B defectivo
• Desregulación de los sistemas de control del ciclo celular y de
la apoptosis
Identification of a gene expression signature
associated with unfavourable treatment
response in Hodgkin’s Lymphoma
Abel Sánchez-Aguilera
Lymphoma Group
Molecular Pathology Programme
Spanish National Cancer Centre (CNIO)
Gene expression profile of Hodgkin’s Lymphoma
Tissue samples from HL patients with well-defined treatment response (29 samples)
Stringent selection criteria:
• Classical HL (nodular sclerosis or mixed cellularity), HIV-,
• Advanced stages (IIB, III, IV).
• Treated with standard chemotherapeutic protocols (ABVD or variants, containing adriamycin).
• Availability of frozen tissue from pre-treatment biopsy.
• Follow-up of at least 2 years.
• Good treatment response (14) = sustained complete remission.
• Poor treatment response (15) = no complete remission, or relapse within 12 months after treatment.
RNA extraction
Control samples:
• 5 reactive lymph node samples.
• 5 HL-derived cell lines.
• Normal centroblasts.
RNA amplification by T7 in vitro transcription
Fluorescent labelling and hybridisation vs Universal Reference RNA (Stratagene)
CNIO OncoChip v2 (13471 human clones)
CE
CeLL
Ly ntroLIN
mp bla ES
h n sts
od
es
HODGKIN’S
LYMPHOMAS
Good
Poor
response response Rel.
Hierarchical clustering of genes
associated with treatment response
CLUSTER 1
Immune response
CD8B1 ALDH1A1
CD3D
LYZ
SH2D1A STAT1
ITM2A
CLUSTER 2
Extracellular matrix
Adhesion
Cell-cell signalling
FZD4
PTGS1
CR1
HSPG2
CCL26
TIMP4
HLA-DRB3
CLUSTER 3
Apoptosis
Signal transduction
CYCS
CASP14
PDCD10
STK17A
CLUSTER 4
Cell cycle
TOP2A
PCNA
RRM2
TYMS
PRKACB
PPP1CA
RAB27A
CDH1
MAD2L1
CDC2
CHEK1
STK6
Role of T and NK-cells
Alvaro T, et al. Outcome in Hodgkin's lymphoma can be predicted
from the presence of accompanying cytotoxic and regulatory T cells.
Clin Cancer Res 2005. 11(4):1467-73.
Role of T and NK-cells
Alvaro T, et al. Clin Cancer Res 2005. 11(4):1467-73.
Comparison of survival time (OS, EFS, DFS)
according to TIA-1+ cells and FoxP3+:
(a)
tumor infiltrate with a low level of TIA-1+
cells and a high level of FoxP3+ cells,
(b)
tumor infiltrate with a high level of TIA-1+
cells and a low level of FoxP3+ cells,
(c)
tumor infiltrate with intermediate levels of
TIA-1+ and FoxP3+ cells.
CE
CeLL
Ly ntroLIN
mp bla ES
h n sts
od
es
HODGKIN’S
LYMPHOMAS
Good
Poor
response response Rel.
Hierarchical clustering of genes
associated with treatment response
CLUSTER 1
Immune response
CD8B1 ALDH1A1
CD3D
LYZ
SH2D1A STAT1
ITM2A
CLUSTER 2
Extracellular matrix
Adhesion
Cell-cell signalling
FZD4
PTGS1
CR1
HSPG2
CCL26
TIMP4
HLA-DRB3
CLUSTER 3
Apoptosis
Signal transduction
CYCS
CASP14
PDCD10
STK17A
CLUSTER 4
Cell cycle
TOP2A
PCNA
RRM2
TYMS
PRKACB
PPP1CA
RAB27A
CDH1
MAD2L1
CDC2
CHEK1
STK6
Identification of genes involved in HL progression:
Genes related with genome integrity checkpoints and G2/M
checkpoint
Analysis of centrosomes and spindle checkpoint in H&RS cell lines:
disrupted transition through mitosis in Hodgkin cells
untreated
untreated
20 h
20 h
hyper-G2/M
48 h
48 h
30
70
25
60
20
50
apoptosis
40
15
30
10
20
5
10
0
0
PBL
L1236
HDLM2
L428
PBL
L1236
HDLM2
L428
Validation of the
expression data in
an independent
series of patients.
235 cases
IHC on TMAs
Linfoma de Hodgkin
Dr. Juan F García