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Yaso Natkunam MD PhD Stanford University
An Update
Outline • WHO 2008: New strides in recognition of early lesions,
molecular subtypes and borderline categories
• In 2013: High throughput tools uncover novel targets with implications for diagnosis, prognosis and therapy
• Update on selected topics
– Improved diagnosis of small B-cell lymphomas – Aggressive B-cell lymphomas and borderline categories – Immunomodulation and the tumor microenvironment – NK cell biomarkers
Small B-Cell Lymphomas
• Follicular lymphoma • Marginal zone lymphoma • Lymphoplasmacytic lymphoma • Chronic lymphocytic leukemia • Mantle cell lymphoma
Anderson & Natkunam, 2010
“Differentiation-Linked Phenotype”
TdT+ CD5+ CD20+ sIgM sIgD
TdT+ CD10+
CD79a+ PAX5+
CD20+ CD10+ BCL6+
CD138+ cIgG cIgA
CD20+ CD10- BCL6-
A N T I G E N
Pro-B Pre-B Naïve B Plasma cell GC B Memory B Antigen-Independent Antigen-Dependent
B-Lymphoblastic
Post-GC
Marginal zone Lymphoplasmacytic
DLBCL Plasma cell myeloma
GC Follicular Burkitt DLBCL Hodgkin
TH
• Proliferation & clonal expansion • Somatic hypermutation • Class-switch recombination
B B
B-cell receptor signaling
FDC B-Cell
Lymphoma
Germinal Center
Follicular Lymphoma • Neoplasm of GC B-cells with at least
partially follicular growth pattern
• Hallmark t(14;18)(q21;q32) MBR/IgH translocation in 90% of FL
• t(14;18) or BCL2 over-expression alone is insufficient for neoplastic transformation
BCL2 - NL BCL2 - FL
Centrocyte
Centroblast
FL: Prognosis • Histologic Grading
– Grades 1 & 2 – indolent, incurable, diffuse areas irrelevant – Grade 3B and/or >25% diffuse areas - worse prognosis – Grading and proliferation – insufficient to capture heterogeneity
• Cytogenetics/progression of FL – > 6 chromosomal breaks – MYC, BCL6 rearrangements and inactivation of TP53 – Recurrent losses in 1p, 6q, 10q, 17p, trisomy 21, gain 1q
• GEP & MicroRNA Profiles – Importance of microenvironment – t(14;18) neg FL – late GC phenotype
• FL microenvironment – Macrophage subsets: CD68 and CD163 – T-cell subsets: FOXp3 (Treg), PD-1 (TFH)
• Perifollicular pattern of Treg a/w poor prognosis Horn et al. Haematologica 2011
Leich et al. Blood 2011 Koch et al. Hum Pathol 2012
HGAL
Lossos et al, Blood 2004 Natkunam et al, Blood 2005
Lu et al. Blood 2007 Temmins et al. AIMM 2011
Romero et al. Nature Commun 2012
• Sensitive/specific for GC B-cells
• HGAL+ DLBCL are low stage
• Regulates GC B-cell motility
• Regulates B-cell receptor signaling by Syk activation leading to lymphoid hyperplasia and massive amyloidosis in HGAL transgenic mice
Amyloidosis
WT SCA1-HGAL
HGAL
PAX5
HGAL Interfollicular
Component
• Positive in CD10- FL
• Not downregulated in interfollicular component
• Down regulated in bone marrow microenvironment
Younes et al. AJSP 2010
In situ Follicular Lymphoma BCL2 BCL2
BCL2
FLIS • Scattered strong BCL2+
follicles in normal NL – FL in subset – Colonization of pre-existing
follicles – A/W other neoplasia – marker
of genomic instability?
• Patients with this finding should be evaluated for FL but not treated
• High grade BCL2- cases – Clonal B-cells in normal
follicles – Circulating t(14:18)+ B-
cells in healthy individuals Bala et al. 2013 unpublished
• Most common chronic leukemia; typically in older adults • Monotonous small B-cells in PB, BM > LN • Admixed prolymphocytes and paraimmunoblasts
• CLL FISH panel • Del 13q14 (50%), Trisomy 12 (20%), del 11q22-23, 17p13, 6q21
• Two molecular subtypes • Ag-naïve: unmutated IgVH • Ag-experienced: mutated IgVH • ZAP70 surrogate
• Transformation • Large cell (Richters) • Prolymphocytic leukemia • Classical Hodgkin-like
Chronic Lymphocytic Leukemia
• Whole genome seq – Recurrent mutations a/w prognosis – NOTCH1, XPO1, MYD88
• 12% CLL, high stage & adverse outcome • Unmutated IgVH • Faster progression to DLBCL
• LEF1
– WNT/beta-catenin signaling – Normal: T-cells & pro-B, not mature B – CLL: 100% – Increased: Proimmunoblasts, Richters & DLBCL
• pERK – Aberrant expansion of anergic B-cells – Signaling inhibitors induce apoptosis
CLL – Signaling Molecules
Puente et a. Nature 2011 Gutierrez et al. Blood 2010
Tandon et al. Mod Pathol 2011 Apollonio et al. Blood 2013
LEF1 Richters
Lymphoplasmacytic Lymphoma ”Waldenstroms Macroglobulinemia”
• Closely overlaps with MZL in morphology and immunophenotype
• MYD88 mutation L265P in 91%
– Not exclusive
Treon et al, NEJM, 2012
Marginal Zone Lymphoma • Lymphoma of heterogeneous small B-cells
• Three categories: Extranodal, Nodal, Splenic
• Etiology varies with anatomic & geographic location
– H. pylori ~ 60-90% gastric – Chlamydia sp ~ 10-47% ocular – Hashimoto thyroiditis ~94% – Sjogren syndrome ~85%
• Challenges in diagnosis – No specific marker – Overlap with other small BCL – Refractory subtypes
Marginal zone
MNDA Marginal zone cells
Kanellis et al Leukemia 2009 Freud et al. 2013 unpublished
Aggressive B-Cell
Lymphoma
• Burkitt lymphoma • Diffuse large B-cell lymphoma, NOS • Primary mediastinal large B-cell • Intravascular large B-cell lymphoma • Primary effusion lymphoma
• BCL unclassifiable: DLBCL/Burkitt • BCL unclassifiable: DLBCL/CHL
• Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease
• ALK-positive large B-cell lymphoma • DLBCL a/w chronic inflammation • Lymphomatoid granulomatosis
DLBCL • Common • Heterogeneous • 40-50% cure rates
WHO 2008: Borderline Categories • Not distinct entities, but temporary categories
that await further study and refinement • Allows classification of cases not meeting criteria
for distinct diagnostic entities • Not intended as convenient waste baskets • Requires a multidisciplinary approach for patient
management 1. B-cell lymphoma, unclassifiable, with features
intermediate between DLBCL and Burkitt lymphoma
2. B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma
• Morphology – Intermediate between BL and
DLBCL • Immunophenotype
– CD10+ BCL6+ – Variable Ki-67 – Variable BCL2, often positive
• Genetics – MYC, BCL2/18q32, BCL6/3q27 – Double hit/triple hit – Complex karyotypes
• Clinical – Rare progression of FL – Aggressive, especially double hit
BCL Unclassifiable: DLBCL and Burkitt
Ki67
Dave et al. NEJM 2006 Hummel et al. NEJM 2006
Harris 2008
DLBCL ABC GC PM UN Bkt
• MYC rearrangement – IG-MYC common – Non IG-MYC sometimes
• Double-hit (Triple hit): MYC/8q24 in combination with BCL2/18q21 and/or BCL6/3q27
• MYC-complex karyotype - common • MYC-simple karyotype - rare • BCL2 or BCL6 without MYC - rare • Transformed FL (Blastic FL) - rare
BCLU: Genetic Features
“Double-Hit” B-Cell Lymphomas
Aukema et al, Blood 2010 Snuderl et al, AJSP 2010
Alizadeh et al, ASH, Dec 2010 Am. Soc Hematology, Dec 2010
• Incidence difficult to assess • Patients with poor prognostic factors
• High IPI, elevated LDH, BM and CNS involvement • Poor outcome despite high intensity chemo or RCHOP -
Does worse than Burkitt or DLBCL • Prognosis not explained by myc/8q24 alone
• Additional hits – BCL2 or karyotypic complexity • In Children, Ig/MYC breakpoints and simple
karyotypes are common and are best diagnosed as Burkitt lymphoma
• Aggressive mature B-cell lymphomas should be systematically studied using ancillary tests, in particular, FISH
• Features that are worrisome • GCB-immunophonotype with BCL2 expression • High growth fraction • BM or CNS involvement
• Proliferation index (Ki-67 IHC) is not a good indicator of aggressivity or double-hit cases
“Double-Hit” B-Cell Lymphomas Recommendations
Johnson et al, Blood 2009 Aukema et al, Blood 2010
TH
B B
B-cell receptor signaling
FDC B-Cell
Lymphoma
Host/Immune Microenvironment
• Co-stimulatory molecules • Immune microenvironment
Identification of Risk Groups Tumor Cell Biomarkers • LMO2 • HGAL
Two-Gene Model • LMO2 – Tumor cell • CD137 – Immune microenvironment
• Multiple GEP datasets on total of 787 DLBCL patients LMO2 green, CD79a red
Natkunam et al. Blood 2007 Natkunam et al. JCO 2008 Alizadeh et al. Blood 2011
Inducible Co-Stimulatory Target • Stimulation of CD137 enhances anti-lymphoma activity of anti-CD20 antibodies
• Synergy between anti-CD137 and anti CD20 may reduce or eliminate the need for chemotherapy
Kohrt et al. Blood 2011
750 Hematopoietic Neoplasms
CD137+
Follicular dendritic tumor 86%
Classical Hodgkin 86%
T-cell lymphoma 58%
Extranodal NK/TCL 37%
Anderson et al. AJP 2012
CD137
CD137 red CD3 green
CD137 red OCT2 green
CD137 Ligand
Zhao et al. AJSP 2012
CD137L red, CD137 green CD137L red, PAX5 green
Primary follicles
Secondary follicles
• Majority of B-cell lymphoma
• Not in classical Hodgkin lymphoma
• Good marker of mantle cell lymphoma
• Marker of memory B-cells
HRS Cells and Immune Microenvironment
Gascoyne EHA 2012 Natkunam et al. Blood 2007
Juszczynski et al. PNAS 2007 Green et al. Blood 2010
Steidl et al. NEJM 2010 Azambuja et al. Ann Onc 2012
Anderson et al. AJP 2012 Sanchez-Espiridion et al. Hematologica 2012
HGAL Galectin-1
9p24.1 amplification PD-L1/L2 & JAK2
TAM and CD68
CD137
Mature T and NK Lymphomas WHO 2008
• Predominantly Nodal – Peripheral TCL, NOS
– Angioimmunoblastic TCL
– Anaplastic large cell lymphoma, ALK+
– Anaplastic large cell lymphoma, ALK-
– Adult T-cell leukemia/lymphoma
• Predominantly Leukemic – T-cell LGL leukemia
– Chronic LPD of NK cells
– Aggressive NK-cell leukemia
– Sezary syndrome
• Predominantly Extranodal – NK/T-cell lymphoma, nasal type
– Enteropathy-associated TCL
– Hepatosplenic TCL
– Subcutaneous Panniculitis-like TCL
– Systemic EBV+ T-cell LPD of childhood
• Predominantly Cutaneous – Mycosis Fungoides
– Primary cutaneous CD30+ T-cell LPD
– Primary cutaneous gamma/delta TCL
• Natural killer (NK) cells are large granular lymphocytes
• 5-15% of blood lymphocytes; <1-2% of lymph node cells
• Traditionally considered part of innate immune system
• Recognize and kill malignant or infected cells
• Secrete chemokines, cytokines and other molecules to promote immunity
NK Cells
CD335 in NK Cells
CD2 CD7 CD56 CD16 CD57 CD161 KIR
CD2 CD7 CD56 CD16 CD57 CD161 KIR
CD2 CD56 CD16
CD2 CD7 CD56 CD16
CD56
NK cell T cell Monocyte Myeloid Plasma cell
CD335 CD335 CD335 CD335
CD56 CD335 CD335 CD335
• NK cells express pan-NK surface antigens and lack non-NK markers (CD3, CD19)
• These markers are not specific to NK cells
• CD335 (NKp46) is an activating receptor on mature NK cells
• It selectively labels CD3-CD56+ NK cells in normal adult blood
• CD335 was evaluated to assess potential diagnostic utility
(CD3-)
CD335 is a Specific Marker of NK Cells
CD56 CD335
CD335 CD335
(CD3-)
Normal adult blood · CD335 is a highly specific marker of human NK cells (CD3-CD56+) and is expressed in at least a subset of NK cell neoplasms (ENKL) · CD335 is aberrantly expressed in a subset (20%) of mature T cell neoplasms (T-LGL, ALCL, ALK+, MF, PTCL, NOS) · Flow cytometry and IHC data show promising utility of this marker in the diagnostic evaluation of NK and T cell neoplasia
Freud et al, 2013 unpublished
TH
B B
B-cell receptor signaling
FDC B-Cell Lymphoma
Understanding Disease Heterogeneity
Tumor Cell Biomarkers
• Co-stimulatory molecules • CD137-CD137L signaling • PD1-PD-L1/L2 signaling
• Tumor infiltrating cells • Regulatory T-cells • Th1, Th2, Th17
• Macrophage subsets • Dendritic cell subsets
• NK cells
Host/Immune Microenvironment
Stanford Pathology Oncology Roger Warnke Ronald Levy Shuchun Zhao Ash Alizadeh Matthew Anderson Ranjana Advani Caroline Temmins Holbrook Kohrt Aharon Freud Debra Czerwinski Dita Gratzinger Behnaz Taidi Ryan Metcalf Sheren Younes
Statistics & Informatics Robert Tibshirani Robert Marinelli
Acknowledgements
U Miami, Florida Izidore S Lossos Gerald Byrne
Rio de Janeiro, Brazil Denize Azambuja Nelson Spector
Guatemala City Hernan Molina Kirsch