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Falko FendInstitute of Pathology and Neuropathology

Reference centre for haematopathologyUniversity Hospital Tübingen

Potential Application of New Technologies to Lymphoproliferative Diseases in GIT – With Special Reference to Marginal Zone Lymphoma

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Tissue - and microenvironment-specific manifestations of NHL

FL in BM

nodal MCL

CLL

MALT-Ly.stomach

Despite highly efficientrecirculation, extranodal lymphomas stay organ-confined for prolonged

time

Extranodal NHL usuallyderived from locallyantigen-experienced

cells

Secondary MALT

Intestinal T-NHLs

CTCL

Extensive dissemination Limited dissemination

Adhesion molecules and chemokine receptors govern lymhpocyte migration

Modified from Pals S T et al. Blood 2007;110:3102-3111

MucosaBM

Naive B/T-cells show broadrecirculation

Antigen contact andinteraction with accessorycells lead to reprogramming

Lymph node

The prototype: MALT lymphomaextranodal marginal zone B-cell lymphoma

Indolent B-cell lymphoma usually arising in acquired MALT, in a background of local chronic inflammation due to infection orautoimmune disease

May remain localized for prolonged time, late dissemination and relapses, often at other MALT sites (frequently different clones)

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Stomach: H. pylori

Thyroid: Hashimoto thyreoiditis

Salivary gland:Sjögrensyndrome,

Lung: Achromobacterxylosoxidans (?)*

Skin: Borrelia burgdorferi

Ocular adnexae: Chlamydiapsittaci

Regional differences in associationwith infectious agent

Pathogenetic role in partunconfirmed* Adam et al, BJH 2014

Gastric MALT lymphoma and Helicobacter

Initially 90% H.p. positivity, eradication leads to regression in 60-80% (including 50-66% H.p.+ DLBCL of early stage)

Both host and bacterial virulence, factors as well as nutrients play a role

Indirect stimulation of tumor growththrough variety of H.p. and T-cellmediated factors

Translocation of H.p. virulencefactor cagA into B-cells leads todirect activation of oncogenicsignalling pathways and associateswith response to eradication

Morgner et al, JCO 2001; Chen et al, JCO 2001; Chen JNCI 2005; Adam et al, BJH 2014; Raderer et al, Ann Hematol 2015; Kuo et al, Blood 2012, 2017; Govi et al, Blood 2011

Blaser et al, JCI 2004

Pathogenesis of gastric MALT lymphoma

6 Zucca et al, Clin Cancer Res 2014

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Decreasing incidencesince introduction oferadication therapy

Small series withsuccessful eradication in H. heilmanni infection

7 Khalil et al, Br J Haematol 2014http://treatment-of-diseases.net/

Gastric MALT lymphoma without H.p. infection

24/97 (25%) neg. by histology, serology and breath test

6/13 (46%) patients treated with antibiotics alone achieved objective response, 5/6 CCR

Other studies show H.p. negativity in 10-15% and response rates of 15-29%, lack ofpublished European data

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Raderer et al, Gut 2006; Ann Hematol 2015Zullo, J Clin Gastroenterol 2013; Asano, Tohoku J Exp Med 2012

Genetic alterations in gastric MALT-Lymphoma

Virtually always somatically hypermutated, evidence of antigen selectionOngoing mutations detected in subset of cases

Biased usage of VH3-30 and VH3-23 in gastric MALT lymphoma without t(11;18) and response to H.p. eradication; VH1-69, VH3-7

Recognition of autoantigens

t(11;18)(q21;q21) with formation of chimeric protein BIRC3/MALT1 in 30-50% (70% eradication-resistant und 0% sensitive MALT-lymphoma)

Trisomies 3,8,12,18,22 und mutations ofNFkB inhibitors

Zucca et al, Clin Cancer Res 2014

Translocations in MALT-lymphoma

10 Du M, Sem Cancer Biol 2016

Dichotomy in pathogenesis of MALT-Lymphoma

MZBCL of MALT-type DLBCL

t(11;18)-

t(11;18)+

+3q26-27additional

aberrations:

-5q21

-9p21

-13q14

-17p13

Transformed?NC

-6q

-5q21

-9p21

-13q14

-17p13

de novo?

NC

Starostik et al, Blood 2002; Flossbach et al, Int J Cancer; 2010; 2013

- Evidence for MALT origin of most gastric DLBCL (“blastic MALT lymphoma”)

- Increasing genetic complexity from small to large cell

- Frequent expression and translocation of BCL6 in DLBCL

Pathways to constitutive NFkB signaling in MALT lymphoma

12 Du M, Sem Cancer Biol 2016

Constitutive activation of BAFF signaling associated with therapy resistance and H.p. independence in t(11;18)- MALT lymphoma Kuo et al, J Pathol 2017

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How do we translate into the diagnostic setting?

13 14

100 125 150 175Size (nt)

100

112.78

120 140 160

-5,0E-010,0E+00

5,0E-01

1,0E+001,5E+002,0E+00

2,5E+00

3,0E+003,5E+00

4,0E+00

4,5E+00

20 25 30 35 40 45 50PCR Cy cle

TBPCy clin D1

Gene expression

Clonality Chromosomal translocations

Point mutations

Numerical aberrations

Epigeneticalterations

Nature 447, 433-440(24 May 2007)

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Morphology and immunophenotype

Geographic pattern with residual GC

Proliferation of lymphoid cells withirregular nuclei and variable, oftenclear cytoplasm

Small lymphocytic, centrocyte-like, monocytoid

frequent mono/polytypic plasma cells

Invasion of epithelia (lympho-epithelial lesions)

Reactive germinal centers, +/-colonization

Accompanying chronic/acuteinflammation

Non-specific immunophenotype

Differentation between lymphomaand inflammation difficult

Cytokeratin

CD20

Scoring of gastric infiltrates*

Score Diagnosis Histological features0 Normal Scattered plasma cells in lamina propria. No

lymphoid follicles1 Chronic active gastritis Small clusters of lymphocytes in lamina propria. No

lymphoid follicles. No lymphoepithelial lesions2 Chronic active gastritis with

florid lymphoid follicle formationProminent lymphoid follicles with surroundingmantle zone and plasma cells. No lymphoepitheliallesions

3 Suspicious lymphoid infiltrate, probably reactive

Lymphoid follicles surrounded by small lymphocytes that infiltrate diffusely in lamina propria and occasionally into epithelium

4 Suspicious lymphoid infiltrate, probably lymphoma

Lymphoid follicles surrounded by marginal zone cells that infiltrate diffusely in lamina propria and into epithelium in small groups

5 MALT lymphoma Presence of dense infiltrate of marginal zone cells in lamina propria with prominent lymphoepitheliallesions

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*modified from WotherspoonA et al, Lancet 1993

Is there a role for molecular primarydiagnosis?

Detection of B-cell clonality in biopsiesHigh rates of clonality initially reported (15-79%) in H.p. gastritis(especially in FFPE), associated with progression to MALT lymphoma

standard BIOMED-2 protocols and duplicates to avoid „pseudoclonal“ products show >90% clonality in MALT lymphoma, a subset (22%) ofclonal Wotherspoon 3/4 cases and lack of clonality in gastritis

Clonality = malignancy, but clonality very useful in appropriate settinguse all samples in case of multiple biopsies

Nakamura S et al, Am J Pathol 1998; Zucca et al, NEJM 1998; Hummel et al, Gut 2006

2 5 0 2 7 5 3 0 0

. A 0 3 _ 1 7 0 5 3 1 1 5 L 5

z e ( n t )

260

276.70

280 300

9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0

C 4 0 2 - 1 7 6 0 n g I g H - C . B 0 4 _ 1 7 0 5 3 1 1 5 L 2

S i z e ( n t )

90 100 120

137.65

140

FR2 FR3

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Molecular follow-up after H.p. eradication

Frequently prolonged persistence of residual infiltrates or clonalplasma cells

clonal B-cell rearrangement can be used as specific marker, but persistence not associated with recurrent disease

Histology with immunhistochemistry remains gold standard fordiagnosing recurrence

Fend et al, Leukemia 1994

The future of clonality deteminationNext generation sequencing – NGS

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

20%FR1

10%FR1

5% FR1 2% FR1 1% FR1 0.1%FR1

20%FR2

10%FR2

5% FR2 2% FR2 1% FR2 0.1%FR2

Granta Seq.

react. Clonotype 1

react. Clonotype 2

react. Clonotype 3

react. Clonotype 4

react. Clonotype 5

react. Clonotype 6

react. Clonotype 7

react. Clonotype 8

react. Clonotype 9

Granta dilution series

Exact identification and quantification of clonal sequenceHigh sensitivity for follow-upIn B-NHL somatic hypermutation

What else is there besides gastric MALT lymphoma?

30-50% of extranodal NHL are in the GI tract

75-85% of all GI-NHL in the stomach

What to look out for: Other primary extranodal lymphoma entities

DLBCLBurkitt lymhomaMantle cell lymphomaDuodenal follicular lymphomaEnteropathy-associated T-NHLMonomorphic epitheliotropic intestinal T-cell lymphoma

Premalignant/benign lymphoproliferations of GI tract

Indolent T-cell lymphoproliferative disorder of GI-tract25% of nodal NHL show GI involvement at primary DX

Strict criteria for diagnosis of primary GI lymphoma!

Complete phenotyping mandatory in primary diagnosis

Lympho-epithelial lesions can occur in other NHL

Cyclin D1

Lymphomatous polyposis

Distribution of subtypes among primary GI lymphomas

Koch et al.: Primary gastrointestinal Non-Hodgkin‘s lymphoma. German Multicenter Study JCO 19:3861-3873 (2001)

Primary intestinal/duodenal FL

• 63 patients, all stage IE• Uncharacteristic symptoms• Multiple warty polyps along descending part of duodenum• Limited to mucosa/submucosa in 19 of 20 cases• No ulcerations, no obstructive lesions• No involvement of stomach (n=61) and colorectum (n=39)

• Grade 1 in 60, Grade 2 in 3 cases• Typical immunophenotype (bcl-2+, bcl-6+, CD10+, low Ki-67,..)• t(14;18) by cytogenetics in 4/4 cases, no additional aberrations

Schmatz AI et al., J Clin Oncol 2011, 29:1445

Follicular lymphoma of the Duodenum

CD10(+)

Bcl-2(+)

Bcl-6(+)

H&E

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Schmatz AI et al., J Clin Oncol 2011, 29:1445Fend et al, J Hematop 2012; Uppsala workshop

Therapy n Complete regression

Stable lesions

Nodal dissem.

Follow-up

Watch&wait 24 7 17 2 55 (6-137)Radiation 19 19 0 0 37 (10-108)Rituximab 5 4 1 0 36 (29-118)Chemo +/-rad.

8 8 0 0 44 (15-99)

No evidence of large cell transformation at median follow-up of 77 mo.

Endoscopic appearance relative to therapy (n=56)

Primary intestinal follicular lymphoma

Schmatz AI et al., J Clin Oncol 2011, 29:1445

Why behaves DFL different from nodal FL?

Differential expression of CCL20 and MAdCAM between DFL and nFL, shared with MALT lymphoma

DFL shows similarities with in situ follicluar neoplasia and proablyrepresents a pre-malignant state in most cases

Role of local antigen stimulation currently unknown

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Hierarchical clustering based on GEP (Takata et al, J Cancer Sci 2017)

28Schmatz AI et al, JCO 2011

Indolent T-cell lymphoproliferativedisorder of GI tract

Rare disorder in adults

Diarrhia, dyspepsia, colics, intolerance tonutrients

Endoscopically multiple polyps or diffuse alteration of mucosa

The whole GI tract can be involved

Infiltration by small, inconspicuous T-cells withclonal rearrangement, CD8+>>CD4+

No response to chemotherapy, clinicalllyindolent with persistence of disease

29 Perry A et al, Blood 2013 30

CD4

CD3

02 5 0 0 05 0 0 0 07 5 0 0 0

1 0 0 0 0 01 2 5 0 0 01 5 0 0 0 01 7 5 0 0 02 0 0 0 0 0

6 0 7 0 8 0 9 0 1S i z e ( n t )

60 70 80

85.48

90

TCRγ

55-year old male with history of MALT lymphoma following eradication, extensive infiltrates in gastric and duodenal mucosa

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Is there a role for mutational analysis in MALT lymphoma?Oncogenically active mutations in the TOLL/IL-1 receptor adaptor molecule MYD88

Ngo et al, Nature 2011

High frequency of mutations activating NFkB pathway, especially MYD88 in DLBCL of ABC type, rarely in other lymphoma subtypes

Subtype Gene Frequency % RelevanceCLL MYD88

SF3B1NOTCH1

1.5-55-174-15

Mutated IGH, young, favourableNormal cytogenetics, progressionUnmutated IGH, progression

MCL NOTCH1/2 10-12 Poor clinical outcome

FL EZH2MLL2CREBBP

14-278933

Chromatin modification, early eventLater event ?Early event

DLBCL EZH2CREBBPMLL2MYD88CD79a/b

7-1213-3220-3229-7016-60

GCB type, early eventGCB>ABC, early eventLater event ?ABC type, NFkB activation, CNSABC type, BCR signalling, CNS

LPL/WM MYD88 CXCR4

90-10028-36

L265PHigher disease activity

Burkitt ID3, TCF3 30-70 Transcription regulation, also in BCL-UHCL BRAF 99-100 V600E, not present in HCL-V

SMZL NOTCH2 25% Poor prognosis

AITL RHOA 50-70 G17V, may identify TFH-PTCLALCL ALK- DUSP22

P63308

Translocation, good prognosisTranslocation, poor prognosis

MYD88 L265P and small B-NHL – current status

High frequency in typical LPL/WM (79-100%)Treon NEJM 2011; Hunter Blood 2013; Xu Blood 2013; Gachard Leukemia 2013; Schmidt BJH 2015; Martinez-Lopez AJSP 2015; Hamadeh Mod Pathol 2015

Common in IgM MGUS (47%) Varettoni Blood 2013

Less common in non IgM LPL (<50%) Manasanch Leuk Lymphoma2014; King et al, AJCP 2016

Rare in splenic (6-15%) MZL associated with BM involvement and IgMparaprotein and nodal MZL (0-8%)

Parry CCR 2015; Hamadeh Mod Pathol 2015; Spina BLOOD 2016

Absent from most MALT-Ly (ocular adnexae 5%), gamma-heavy chaindisease and cold agglutinin disease

Hamadeh, Haematologica2014; Randen Haematologica 2014; Du 2016;

2-4% of CLL(mutated) in young patients with favourable outcomeMartinez-Trillos Blood 2014; Baliakas Leukemia 2015

CXCR4 mutations seem to be restricted to LPL, but few data33

Breast mass biopsyBreast mass biopsy

71-year-old woman

2013 Breast tumor with fast massenlargement

CD20 CD138

MUM1

p53

MIB1

2004 IgM MGUS (IgM 3730 mg/dl)

2009 Waldenströmmacroglobulinemia (IgM 5540 mg/dl) and lymphoplasmacytic lymphoma in bone marrow

Treated with 6 cycles ofRituximab/BendamustinPartial remission, stable

2012 Recurrence with WM (IgM 6470 mg/dl) and splenomegaly

L265P control

Breast mass 2013

Bone marrow 2009

MYD88 (L265P) mutation analysisMYD88 (L265P) mutation analysis

Identical clonal IGH rearrangement and TP53 exon 5 mutationHigh grade transformation of LPL/WM

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Enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-NHL (MEITL)

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CD56

EATL MEITLStrongly ass. with celiac disease,northern European

No association with celiac disease, Asians, Hispanic

pleomorphic monomorphicMucosal atrophy Mucosal infiltrationCD3+, CD103+, cytotoxic markers

CD3+, CD103+,cytotoxic markers

CD4-/CD8-, CD30+/-Usually αβ

CD8+, CD56+, γδ>αβ

9q+, 16q12.1- 9q+, 16q12.1-1q+, 5q+ 3p21.31-, MYC gainsOther genetic alterations?

Other genetic alterations?

MEITL

Mutational profile of MEITL

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Roberti et al, Nat Comm 2016; Nairismagi et al, Leukemia 2016; Kucuk et al, Nat Comm 2015;

Moffitt et al, J Exp Med 2017

Roberti et al, Nat Comm 2016

Molecular profiling defines MEITL as specific entityHigh frequency (92%) of SETD2 inactivation and subsequent

H3K36 trimethylation

High frequency of STAT5B (60%), JAK3 (46%) and SH2B3 (20%) mutations

Distinct, though in part overlapping genetic profile from EATL

39 Roberti et al, Nat Comm 2016

Summary

Clonality determination for the moment remains the most important molecular test for diagnosing GI tract lymphomas

NGS technologies and targeted mutational analysis help to better characterize/define entities and will play a bigger role for DD, prognostic assessment and follow-up

Analysis of cell-free DNA will be useful for monitoring patients with aggressive lymphoma, role in indolent tumors (e.g. MALT lymphomas) currently unclear

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Thank you for your attention!