haem - fend gi nhl belfast 2017 · fend et al, j hematop 2012; uppsala workshop therapy n complete...
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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!