Molecular Testing of Lymphomas

John GoodladDepartment of Pathology

Western General Hospital & University of EdinburghEdinburgh

john.goodlad@nhs.net

RCPath SymposiumMolecular Diagnosis on Tissues and Cells

Friday 25th November 2011

Molecular Techniques in Haematological Malignancy

Spectrum of disease•Lymphoma

•LeukaemiaLymphoidMyeloid

•Myelodysplasia

•Myeloproliferative disorders

Areas of application•Diagnosis / classification

•TherapyIdentification of specific targetsNewly tailored drugs

•Assessing response to treatment

Techniques available•Polymerase Chain Reaction

Standard PCRReverse transcriptase PCRReal time / Quantitative PCR (Q-PCR)

•Fluorescence in situ hybridisationInterphaseSpectral Karyotype Imaging (SKI)Fluorescence immunophenotyping & interphase cytogenetics (FICTION)

•Comparative Genomic Hybridisation

ConventionalArray based

•Gene expression profiling

•Massively parallel (next generation) sequencing

•Proteomics

Molecular Testing in Lymphoma

1. Establishing a diagnosis of lymphoma•What is the significance of clonality?

2. Classification of lymphoma

3. Discovery and future developments•Refining prognostic and diagnostic categories•Developing new therapeutic regimens

1. Clonality testing in lymphoma

Dominant clonality often used as a marker of lymphoid malignancy(Neoplastic versus benign lymphoproliferations)

Based on the premise that:•Neoplastic lymphocytes are clonal

•Reactive (‘benign’) populations of lymphocytes are polyclonal

PCR is method of choice for clonality assays:Strategies directed towards lymphocyte antigen receptor

•IG genes•TCR genes

Important to be aware of the limitations and pitfalls of this approach

IGH gene rearrangement

No encounter with antigen

DEATH

Encounter with appropriate antigen

SURVIVAL

Naïve B-cell

CD34+

Progenitor B cell

Pre-B cell

Mature B cell: IgM+/IgD+

Immature B cell: IgM+/IgD-

IGK+/-L gene rearrangement

Immunoglobulin gene rearrangements

5’ Vn D1 D2 D3 J1Dn J2 J3 Jn CCC C… 3’V2V1 V3

Immunoglobulin heavy chain gene rearrangement: generation of diversity

5’ Vn D1 D2 J2 J3 Jn CCC C… 3’V2V1 V3

1. D-J joining (incomplete DNA rearrangement)

5’ D2 J2 J3 Jn CCC C… 3’V2V1

2. V-DJ joining(complete DNA rearrangement)

3. Transcription

D2 J2 J3 Jn CCC C…V2 precursor IGH mRNA

4. RNA splicing

D2 J2 CV2 mature IGH mRNA

5. Translation

Gene segments IGH IGK IGL

V segments•Functional (family) 44 (7) 76 56•Rearrangeable (family) 66 (7)

D segments•Rearrangeable (family) 27 (7) - -

J segments•Functional 6 5 4•Rearrangeable 6 5 5

Van Dongen et al Leukemia 2003

Immunoglobulin heavy chain gene rearrangement:generation of diversity

Potential functional rearrangements of IGH = 44 x 27 x 6 = 1188Potentail functional rearrangements of IGK = 76 x 5 = 380Potential functional rearrangements of IGL = 56 x 4 = 224

Number of possible different IG molecules = 1188 x 380 x 224 = 101,122,560

B

T

BB

B

B

In the presence of antigen T- and B-lymphocytes combine to produce:

Plasma cells/specific antibody

An expanded clone of memory B-cells

A reactive lymphocyte proliferation is polyclonal;Each expanded clone has different gene re-arrangement

A neoplastic lymphocyte proliferation is clonal

•Same gene rearrangement•Same chromosomal abnormality

Polymerase Chain Reaction for IGH chain gene (and TCR gene) re-arrangement can be used to determine pattern of clonality within a lymphoid infiltrate

•Implication is that clonality = maligancy

primers

Products: Same size in monoclonal populationDifferent sizes in polyclonal population

Limitations and Pitfalls of Molecular Clonality Studies

1. Limited sensitivity

2. Clonality does not equate with malignancy

3. Ig & TCR re-arrangements are not markers of lineage

4. Pseudoclonality

5. Oligoclonality

6. False positive results

7. False negative results

How and when do we test for clonality

BIOMED 2: antigen receptor PCR targets for clonality studies

* V-J re-arrangements

•Number of possible IG molecules = 101,122,560

•Number of possible TCRA/B heterodimers = 2,979,236 •Number of possible TCRG/D heterodimers = 2880

IGHA (FR1*)

IGHB (FR2*)

IGHC (FR3*)

IGHD (D-J)

IGHE (D-J

IGKA (V-J)

IGKB (Kde)

IGL (V-J)TCRBA (V-J)

TCRBB (V-J)

TCRBC (D-J)

TCRGA (V-J)

TCRGB (V-J)

TCRD (V-J)

Primer design / Multiplex PCR

•primers designed to cover maximum number of possible combinations for each re-arrangement

•Product size means effective with FFPE tissues (<300bp)

•use in multiplex reactions without cross annealing to each other

Majority of re-arrangements covered by:

•83 upstream primers•39 downstream primers•14 tubes (reaction mixtures)

WHAT WE USE

BIOMED 2 : Immunoglobulin gene re-arrangement:

•Different assays have different sensitivities

•Sensitivity of assay varies with lymphoma subtype (especially pre- or post GC)

•In 31 cases (20%) clonality demonstrated by only one assay

•Any one assay not suitable for all types of lymphoma

•Combination of assays should be performed to increase the sensitivity

Modified from Liu H et al. Br J Haematol 2007; 138:31-43

Pre-GC (%) GC & postGC (%)MCL SLL/CLL FL MALT DLBCL(n=4) (n=9) (n=30) (n=29) (n=24)

IGHA (FR1) 100 100 30 48 50

IGHB (FR2) 100 100 30 66 58

IGHC (FR3) 100 100 13 62 50

IGHD (D-J) 75 67 33 38 13

IGHE (D-J) 0 11 0 7 0

IGKA (V-J) 75 100 60 62 58

IGKB (Kde) 50 67 57 48 46

IGL (V-J) 75 44 23 28 8

ALL 100 100 94 97 96

BIOMED 2 in action: routine strategye.g. Liu et al Leukaemia 2007

DNA sample

DNA size ladder PCR

IGHB + IGKA+IGKB

IGHA + IGHC + IGHD

IGL + IGHE

TCRGA + TCRGB

TCRBA + TCRBB

TCRBC + TCRD

%+%+ with >1 reaction

58%

79%

91%

100%

99%

80%

%+

94%

100%

98%

%+ with >1 reaction

30%

73%

82%

DNA >300 bp

WHEN DO WE TEST?

1. Demonstration of clonality used as supportive evidence for neoplasia in morphologically or immunophenotypically abnormal lymphoproliferations

that do not fully fulfill criteria for malignancy.

N.B. Clonality does not equate with malignancy

Dominant clones can be found in many conditions that are not overtly malignant

Some clearly benign/reactive processes, e.g:•Reactive and progressively transformed germinal centres•Peripheral blood from patients infected with EBV or CMV•Any lymphoid proliferation in context of immunosuppression•Lichen planus•Lichen sclerosus et atrophicus•Drug hypersensitivity reactions•B-cutaneous lymphoid hyperplasia

Lymphoid proliferations that may be associated with progression to overt lymphoma in some, but by no means all cases, e.g:

•MGUS•Monoclonal B-lymphocytosis•“Cutaneous lymphoid dyscrasias”

•Pigmented purpuric dermatoses•Atypical lobular panniculitis•Pityriasis lichenoides

•“In situ lymphomas”:•Follicular •Mantle cell

Example 1: 55 year old male with peripheral blood lymphocytosis

Found to have infectious mononucleosis

Example 2: 65 year old female with breast carcinoma, axillary lymph node sample

“In situ follicular lymphoma”

2. Absence of clonality (polyclonal result) may help confirm a diagnosis.

Other haematolymphoid malignancies that should not have re-arranged IG or TCR genes, e.g:

•NK cell lymphomas•Myeloid sarcoma•Plasmacytoid dendritic cell neoplasms

Montypic but polyclonal lymphoid proliferations, e.g:•HHV8-asscociated Castlemans disease•HHV8(KSHV8)- and EBV- associated germinotropic lymphoproliferative disorder•Atypical marginal zone hyperplasia of MALT

Du et al Blood 2001, Du et al Blood 2002, Attygale et al Blood 2004

Example: 14 year old male with enlarged tonsils:

•Massively expanded marginal zones

•Lambda restricted population of cells on flow cytometry

•Polyclonal IG gene rearrangement (Fr1,2,3, IGK, IGL)

Atypical marginal zone hyperplasia

LYMPHOMA CLASSIFICATION: HISTORICAL PERSPECTIVE

1832: Thomas HodgkinOften accredited with first description of Hodgkin’s disease:

•"On Some Morbid Appearances of the Absorbent Glands and Spleen". Medico-Chirurgical Transactions, 17, 1832, 68–114

1666: Marcello Malpighi Publishes the first recorded description of any lymphoma (Hodgkin's disease)

•“De viscerum structuru exercitatio anatomica”

2. MOLECULAR TESTING AND LYMPHOMA CLASSIFICATION

An early indication of the limitations of early some lymphoma diagnoses/classifications

Original specimens of Thomas Hodgkin still preserved in Guy’s museum

Histological examination in 1926•3/7 cases diagnosed correctly•Tuberculosis•Other forms of lymphoma

Thomas Hodgkin’s diagnoses were based entirely on gross appearances

Jackson & Parker 1944

Lukes 1963

Rye 1965

Rappaport 1966

Lukes & Collins 1974

Kiel 1978

Working Formulation 1985

Updated Kiel 1988

Hodgkin’s disease

NHL

Primarily LN

Subsequent classification systems based purely on light microscopic appearances

First real lymphoma classification in 1944, followed rapidly by many others*

*all based entirely on light microscopic appearances•Giemsa

•Haematoxylin & eosin

Limitations of morphology based classifications:lymphomas with nodular/follicular growth pattern

Small cell lymphomas with nodular/follicular growth pattern circa 1980•centroblastic-centrocytic (small) & centrocytic: Kiel•follicular, predominantly small cleaved cell: W-F

Overall survival for this group of patients•Median = 6.93 years•5-year = approx 62%•10-year = 35.3%

This group of lymphomas contains subsets of cases with different chromosomal translocations

t(14;18)(q32;q21) t(11;14)(q13;q32)

BCL2 Cyclin D1

Follicular lymphoma Mantle cell lymphoma

Follicular lymphoma has much better outcome than mantle cell lymphoma

FL MCL

Median Survival: 8-10 years 3-4 years5-year OS: >70% <40%

Treatment dictated by classification:CHOP-like followed by myeloablative regimens and allogeneic stem cell transplant in younger patients

Wait and watch or symptomatic only

Fundamentals of modern lymphoma classification

The International Lymphoma Study Group

•Pathologists/haematopathologists•Clinicians

•US, Europe and rest of world

Nancy Harris - BostonElaine Jaffe - BethesdaHarald Stein - BerlinPeter Banks - San AntonioJohn Chan - Hong KongMichael Cleary - StanfordGeorge Delsol - ToulouseChris De Wolf-Peters - LeuvenBrunangelo Falini - PerugiaKevin Gatter - OxfordThomas Grogan - TucsonPeter Isaacson - LondonDaniel Knowles - CornellDavid Mason - OxfordKonrad Muller-Hermelink - WurzburgStefano Pileri - BolognaMiguel Piris - ToledoElizabeth Ralfkiaer - CopenhagenRoger Warnke - Stanford

1994:

A consensus list of lymphoid neoplasms that appear to be distinct clinical entities

All available information used to define entities• Morphology• Immunophenotype• Genetic features• Clinical features

Reproducibility proven in consistency studies (Blood. 1997 Jun 1;89(11):3909-18)

Clinical utility verified (Blood. 1997 Jun 1;89(11):3909-18)

Understanding that modifications would be required as knowledgeincrease

Internationally acceptable!

WHO 2008.

Classification of Tumours of Haematopoietic and Lymphoid Tissues

Treatment is dictated largely by the diagnostic category into which a tumour is placed

Breakapart Dual fusionIGH -IGK -IGL -

BCL2 IGH/BCL2 BCL6 -MALT1 IGH/MALT

AP12/MALT MYC MYC/IGH

CCND1 CCND1/IGHALK1

In Edinburgh:

1. Probes currently in routine use:

2. 3m tissue sections

3. Break-apart probes in first instance

4. Negative controls run on each test to determine cut-off value

5. Scoring on basis of number of abnormal versus normal signals

USE OF FISH +/- KARYOTYPING

1. Occasional as adjunct to clonality testing, often in atypical follicular proliferations;• IGH, IGL, IGK• BCL2• BCL6

2. Facilitate subclassification when pathological features inconclusive

3. All large B-cell lymphomas• Mandatory to make diagnosis of Burkitt lymphoma

MYC• Identify “double-hit” lymphoma

MYC, BCL2, BCL6, IGH, IGK, IGL

Example: 14 year old female with lesion on scalp

ALK1

•Small cell infiltrate in skin

•Relatively few ALK+ cells by IHC

•ALK translocation confirmed with breakapart probe

Anaplastic large cell lymphoma, small cell variant

•Techniques now available that permit analysis of thousands of genetic, epigentic and proteomic changes in tumours in relatively short space of time

•Array based technologies•Massively parallel sequencing

•Vast quantities of information can be obtained from a large number of samples in a relatively short period of time.

•Traditional methods allowed researchers to survey only a relatively small numbers of genes/abnormalities at any one time:

‘Chipping away at the coal face.’

‘Industrial strength processing’

IMPACT OF NEW HIGH THROUGHPUT TECHNOLOGIES

3. DISCOVERY AND FUTURE DIRECTIONS

Example 1: Diffuse large B-cell lymphoma

Advances in classification and treatment

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Alizadeh AA, et al. Nature 2000; 403: 503-511

Two main prognostic groups•Germinal centre B-like: good prognosis•Activated B-like; bad prognosis

Gene expression profiling studies on DLBCL show that ‘cell of origin’ is an important determinant of outcome

Gene expression profiling has identified a number of potential therapeutic targets

e.g./•GEP and other investigations show evidence of constitutive activation of NFkB pathway in ABL-DLBCL but not GCB-DLBCL

•Antiapoptotic effects of NFkB counteract action of conventional doxorubicin-based cytotoxic chemotherapy in DLBCL

•Inhibition of NFkB in ABL-DLBCL cell lines in vitro is toxic

•Inhibition of NFkB in vivo may sensitize tumour cells to chemotherapy and improve outcome

•Trial of bortezomib in conjunction with doxorubicin based chemotherapy in patients with relapsed/refractory DLBCL

Inactive NFkB exists as protein complex in cytoplasm

During NFkB activation, •IκB kinase (IKK) phosphorylates IκBα•IκBα dissociates from NF-κB•Freed NF-κB translocates to the nucleus and alters gene expression

Bortezomib blocks IκBα degradation

Prevents translocation of NF-κB to the nucleus

Bortezomib and NFkB Activation

Copyright ©2009 American Society of Hematology. Copyright restrictions may apply.

Dunleavy, K. et al. Blood 2009;113:6069-6076

Figure 2 Overall survival in patients with DLBCL

Bortezomib significantly improves survival in relapsed/refractory ABL-DLBCL but not GCB-DLBCL

Clinical trials already open to assess efficacy of Bortezomib (Velcade) as front line treatment in ABC-DLBCL, eg

UK: ISRCTN 51837425

A Randomized Evaluation of Molecular Guided Therapy for Diffuse Large B-Cell Lymphoma With

Bortezomib (REMoDL-B); ISRCTN 51837425.

GEP will be undertaken on samples of trial patients to stratify into GC and ABC type DLBCL

Example 2:Classic Hodgkin lymphoma; tumour cell genetics

impact on microenvironment to the benefit the tumour

Steidl et al Nature 2011.471.377

Common lymphoma associated translocations are rare in cHL

Whole transcriptome paired end sequencing (next-generation)•Genome wide mapping of base pair sequences

Translocation breakpointsMutationsGains and losses

Applied to two Hodgkin cell lines•KM-H2 (89.2 million base pair readings)•L428 (61.5 millon base pair readings)

C Steidl et al. Nature 000, 1-5 (2011) doi:10.1038/nature09754

CIITA–BX648577 gene fusion observed using paired-endmassively parallel whole transcriptome sequencing.

Found three translocations:•9q34.13 (BAT2LI) / 10q26.3 (MGMT)

•7p14.1-14.2 (ELMO1) / 15q26.1 (SLCO3A1)•15q21.3 (BX648577) / 16p13.13 (CIITA)

Studied incidence of CIITA translocations further by FISH; breakapart probe:

•15% classic Hodgkin lymphoma (8/55 cases)•38% primary mediastinal large B-cell lymphoma (29/77)•27% mediastinal grey zone lymphoma•3% diffuse large B-cell lymphoma (4/131)•11% testicular DLBCL•0% primary DLBCL of CNS

In cases of PMBCL presence of CTIIA correlates with:

•Poorer disease specific survival (63.0% vs 85.0% at 10 years)

Steidl et al, Nature 2011

CIITA: a major MHC class II transactivator

Fusion partners sought for CIITA using 3’ rapid amplification of cDNA ends (RACE)

•Several different partners•9p24 a frequent partner

Several genes at 9p24, including•JAK2•Programmed cell death ligand 1 (PD-L1) (CD273)•Programmed cell death ligand 2 (PD-L2) (CD274)

Breakpoints typically in region of CD273 and CD274 genes

HRS cells also shown to have copy number gains of 9p24•Green et al, Blood 2010; 116: 3268

TRANSLOCATIONS INVOLVING CIITA

CONSEQUENCES OF t(9;16)(q34.13; p13.13)CIITA / CD273 or CIITA / CD274

Translocation interferes with MHCII expression but upregulates PD-L expression

•Downregulation of MHCII•Upregulation of CD273 or CD274

Decreased MHCII expression correlates with poor survival in variety of lymphomas including cHL and DLBCL, eg

•Rimsza LM et al, Blood 2008•Diepstra A et al, JCO 2007•Roberts RA et al, Blood 2006

Upregulation of PD1 ligands correlates with inferior survival in several cancers

•Blank C et al, Cancer Immunol Immuntherapy

Effects mediated via modulation of anti-tumour immune response

Anti-tumour host immune response

Cytotoxic T-cells are critical in recognition and elimination of altered self antigens

•Virus infected cells•Tumour cells•MHC class I restricted - recognize antigen-MHC I complexes on specific target cells

Activated by Th1 cells•Recognize specific antigen-MHC II complexes

Th1Tc

MHC II MHC I

Downregulation of MHC II helps tumour cell evade recognition by tumour specific T-cells

Th1Tc

MHC II MHC I

MHC I MHC I

Programmed cell death 1 and its ligands

PD.1 expressed on a variety of cell types, including T-lymphocytes

Binding of PD.1 with one of its ligands (PD-L1 and PD-L2);•Inhibits activated T-cells(Induction of a resting state)•In some circumstances may facilitate apoptosis

Normally functions to induce self tolerance•prevent development of autoimmune disease

t(9;16)(q34.13; p13.13): a Novel Translocation

Recurrent genetic event with fusion that impacts through both sides of the translocation

First recurrent abnormality shown to favour tumour growth through effects on microenvironment, rather than tumour cell division, differentiation and death

Provides opportunity for therapeutic manipulation

Blocking PD.1:PD-L interactions may restore anti-tumour T-cell immunity•Specific PD.1 receptor blocking antibodies exist

• Already in clinical trials; lymphoma, carcinoma and melanoma•Gordon L et al, Ann Oncol 2011;22 (suppl4): iv102 (prelim report in DLBCL)

CONCLUSIONS (i)

Molecular testing is already well established in lymphoma diagnosis

•Differentiating reactive and neoplastic populations•Classification

Modern lymphoma classification systems define entities basis of shared biological and clinical characteristics, allowing them to be arranged into clinically relevant groupings

•Diagnostic category dictates treatment and likely prognosis

Molecular studies have changed our perception of cancer from that of a genetic disease to complex signaling network

•Highlight biological and clinical heterogeneity within disease categories•Identification of new prognostic markers•Identification of pathogenetic pathways of potential relevance•Identification of potential therapeutic targets

These advances will allow diagnostic categories to be refined and incorporated into updated lymphoma classifications

Ultimately may permit •Molecular diagnosis•Integration of diagnosis and therapeutics•Individually tailored treatment

CONCLUSIONS (ii)

John Goodlad, MD, FRCPathWestern General HospitalEdinburghScotland

Ahmet Dogan, MD, PhDMayo ClinicRochesterMinnesota, USA

Andrew Wotherspoon, MBChB, FRCPathRoyal Marsden NHS Foundation TrustLondonUK

Daphne de Jong, MD, PhDThe Netherlands Cancer InstituteAmsterdamThe Netherlands

1st EDINBURGH HAEMATOPATHOLOGY TUTORIAL:

“INTEGRATING TECHNOLOGICAL ADVANCES INTO DIAGNOSTIC PRACTICE”

JUNE 7-8, 2012

www.edinburgh-haematopathology.org.uk