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